FINAL PROGRAMME ABSTRACTS OF LECTURES AND POSTERS … of abstracts RME2014.pdf · FINAL PROGRAMME &...

RME2014 1 31 March – 2 April 2014, the Netherlands

FINAL PROGRAMME & ABSTRACTS OF LECTURES AND POSTERS

THE RME CONFERENCE SERIES

9TH CONFERENCE

RME2014

Food Feed Water Analysis innovations and breakthroughs!

31 March – 2 April 2014 Noordwijkerhout, the Netherlands

2 RME2014 31 March – 2 April 2014, the Netherlands

THE RME CONFERENCE SERIES – 9TH CONFERENCE

SILVER SPONSOR

EXHIBITORS BaseClear, the Netherlands BioDot Ltd., UK Bio-Rad Laboratories, France Charm Sciences, Inc., USA Innova Biosciences, UK MAITRE Megazyme, Ireland MP Biomedicals, France Qiagen Benelux, the Netherlands Randox Food Diagnostics, UK R-Biopharm AG, Germany Scienion AG, Germany Sigma-Aldrich, Switzerland

SUPPORTER

SECRETARIAT Bastiaanse Communication P.O. Box 179 NL-3720 AD Bilthoven the Netherlands T +31 30 2294247 F +31 30 2252910 [email protected] www.bastiaanse-communication.com


CONTENTS Conference history 4 Welcome 5 Programme 6-17 Programme at a glance 6 Conference programme 7-14 Workshop & hands-on-training programme 16-17 Abstracts of lectures 18-79 Monday 31 March 2014 Plenary keynote lectures 18-20 Plenary meeting Challenges for rapid methods – an industry point of view 21-24

Tuesday 1 April 2014 Plenary keynote lecture 25 Parallel session 1 Advances in microbial sampling and analysis 26-35 Parallel session 2 Advances in contaminant analysis 36-42 Parallel session 3 Advances in microbial analysis 43-49 Parallel session 4 Advances in contaminant analysis 50-56 Wednesday 2 April 2014 Parallel session 5 Future developments in the area of food and environmental analysis 57-67 Parallel session 6 RME2014 meets MoniQA 68-75 Plenary meeting Food authenticity after Burgergate and Fishgate…what is next? 76-79 Abstracts of posters 80-146 Index 80-86 Abstracts 87-146 Key to the abstracts of lectures and posters:

abstracts of lectures and posters are grouped separately;

lectures are grouped according to the daily programme;

posters are grouped in an alphabetical order according to the corresponding author. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of RME2014. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liabil ity, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein.


THE RME CONFERENCE SERIES CONFERENCE HISTORY In 2004, the RME conference series started as ‘Rapid Methods Europe’, providing a reference source for anyone interested in the rapid detection of biological and chemical contaminants in food, feed and the environment. During the years it has strengthened its position as an important meeting point for academia and industry. The term ‘rapid’ is used variously in discussions of rapid methodology and instrumentation. It should be noted that ‘rapid’ is not a goal in itself. In addition to increased speed, rapid methods must also take account of other criteria such as sampling and sample preparation, multitarget screening, lower detection limits, accuracy and sensitivity, data analysis, total costs proportionate to the benefits, etc., eventually leading to methods best suited for use. The RME conference series focuses on the developments in the shift from slower, traditional measurements to more rapid methods for microbial and chemical analysis of food, feed and water analysis ensuring safety and quality. ADVISORY COMMITTEE Dr. Aart van Amerongen Food & Biobased Research, Wageningen UR, the Netherlands Dr. Daniel Barug Ranks Meel, the Netherlands Helena B. Bastiaanse Bastiaanse Communication, the Netherlands Prof.dr. Sarah De Saeger Department of Bio-analysis, Ghent University, Belgium Hans Dijk Scienion, Germany Bram van der Gaag INCAS3, the Netherlands Dr. Gerrit Keizer Prionics Lelystad, the Netherlands Dr. Kitty Maassen National Institute for Public Health and the Environment,

the Netherlands Dr. Roland Poms MoniQA Association / International Association for Cereal

Science and Technology, Austria Dr. Bert Popping Eurofins Scientific Group, UK Dr. Michele Suman Barilla, Italy


THE RME CONFERENCE SERIES – 9TH CONFERENCE WELCOME Continuing the successful series of Rapid Methods Europe conferences, RME2014 takes place in Noorderwijkerhout, the Netherlands, 31 March – 2 April 2014. RME2014 is the 9th in a series of conferences dedicated to innovations and breakthroughs in microbiological and chemical analysis of food, feed and water. This series aims to further strengthen the academia-industry relations and to further disseminate advanced research towards practical applications in food, feed and water analysis. RME2014 presents new and cutting-edge technologies focusing on multitarget screening, lower detection limits, wider range of matrices and shorter time, with an eye toward looking for unknown compounds and moving tests out of the laboratory. RME2014 features:

Plenary lectures & parallel sessions

Reviews and case studies

Poster sessions

Workshops & hands-on-training

Instrument & manufacturers exhibition You are cordially invited to take part in the discussions with participants from different disciplines and to meet business relations in your area. We wish you an active and fruitful meeting! On behalf of the Advisory Committee,

Dr. Daniel Barug


PROGRAMME AT A GLANCE Monday 31 March 2014

12:45 – 13:00 Opening of RME2014

Instrument & manufacturers exhibition

13:00 – 14:00 Plenary keynote lectures

14:00 – 15:15 Plenary meeting Challenges for rapid methods – an industry point of view

15:45 – 16:45 Workshops & hands-on-training

17:00 – 19:00 Excursion to the Keukenhof

19:30 – 21:00 RME’s Lounge Party

Tuesday 1 April 2014

08:30 – 09:00 Plenary keynote lecture

Instrument & manufacturers exhibition

09:00 – 09:30 Plenary lightning talks (short presentations by exhibitors)

09:39 – 12:30 Parallel session 1 Advances in microbial sampling and analysis

Parallel session 2 Advances in contaminant analysis

12:30 – 13:30 Workshops & hands-on-training Poster viewing

13:30 – 16:35 Parallel session 3 Advances in microbial analysis

Parallel session 4 Advances in contaminant analysis

16:35 – 17:00 Speed presentations (selected posters)

Speed presentations (selected posters)

17:00 – 18:30 Poster viewing & drinks

20:00 Conference dinner (reservations only)

Wednesday 2 April 2014

08:30 – 10:45 Parallel session 5 Future developments in the area of food and environmental analysis

Parallel session 6 RME2014 meets MoniQA

Instrument & manufacturers exhibition 11:15 – 12:45 Plenary meeting

Food authenticity – after Burgergate and Fishgate...what is next?

12:45 Closing of RME2014

13:00 – 15:00 MoniQA Association General Assembly (by invitation only)


CONFERENCE PROGRAMME MONDAY 31 MARCH 2014 12:45 Opening of RME2014

Plenary keynote lectures 13:00 Reducing the phytosanitary risks in production systems and global trade of plant material: how rapid and reliable methods can help both business and inspection agencies John van Ruiten, Director, Naktuinbouw, Netherlands Inspection Service for Horticulture, the Netherlands 13:30 Proficiency testing: ticking the box for my accreditation or educational tool? Dr. Kate Wilkinson, The Food & Environment Research Agency, UK

Plenary meeting: Challenges for rapid methods – an industry point of view The advent of rapid measurement devices provides exciting opportunities to access simple tools to support verification and quality control processes. However, before selecting and approving these technologies, the capabilities of each system must be examined carefully, separating the facts from the marketing claims. Some of the requirements that would be expected from a rapid measurement device to be accepted for use in a global market place will be explored. Chair: Dr. Aart van Amerongen, Food & Biobased Research, Wageningen UR, the

Netherlands 14:00 The search for Spock’s tricorder: a perspective from a major FMCG company

on the technical requirements for the adoption of a rapid procedure Dr. Ashley Scribbins, Corporate Quality & Food Safety, Mars Incorporated, UK

14:25 Rapid microbiological methods: a food industry perspective

Dr. Balamurugan Jagadeesan, Nestlé Research Center, Switzerland 14:50 Rapid methods for the quality control of drinking water

Dr. Vicente Catalán, Labaqua, Aqualogy Environment, Spain 15:15 Networking break & exhibition 15:45 – 16:45 Workshops & hands-on-training (see pages 16-17)

o R-Biopharm: Innovative sample clean-up methods for mycotoxin analysis o Qiagen: Viability PCR – the next level in using PCR as a detection technology for

microorganisms o BioDot: Dispensing technology impact on test line intensity and width o MAITRE: Hands-on-training – communicating food science research

17:00 – 19:00 Excursion to the Keukenhof: get inspired! The Keukenhof is an open air flower exhibition organised for the first time in 1949. This has expanded to an annually recurring event that has always drawn great numbers of visitors from all over the world. The park is 32 hectares wide and filled with blooming tulips, hyacinths, daffodils and other spring bulbs. 19:30 – 21:00 RME’s Lounge Party


TUESDAY 1 APRIL 2014

08:30 Plenary keynote lecture Rapid microbiology methods and The Coca-Cola Company Dr. Colm Scully, Coca-Cola Services, Belgium

09:00 Plenary lightning talks (short presentations by exhibitors)

o Advances in rapid analyses for microbial water quality and for mycotoxin detection in feedstuff and milk – Wilbert Kokke, Charm Sciences, the Netherlands

o Rapid microbial genome sequencing – Dr. Derek Butler, BaseClear, the Netherlands o Fastprep-24, flexibility in sample preparation – Serges Pouedet, MP Biomedicals, France o Quantitative certified reference microorganisms – Ivo Siegrist, Sigma-Aldrich, Switzerland o Measuring enzymes and carbohydrates in foods and feeds – Barry McCleary, Megazyme

International, Ireland o Bio-Rad overview: Food Science Division – Gerrit Dijkstra, Bio-Rad Laboratories, the

Netherlands o Innova Biosciences conjugation technology for enhanced assay development – Tom Speedy,

Innova Biosciences, UK

Parallel session 1: Advances in microbial sampling and analysis The future of rapid methods in microbiology is very bright with many new developments and applications, but sampling is often the forgotten beginning. Chair: Dr. Gerrit Keizer, Prionics Lelystad, the Netherlands 09:30 Implications of bacterial clustering for sampling plans

Dr. Ursula Gonzales-Barron, Centro de Investigação de Montanha, Escola Superior Agrária – Instituto Politécnico de Bragança, Portugal

09:55 Sampling and detection technologies of pathogens for safe drinking water: a review

Prof.dr. Marc Desmulliez, School of Engineering and Physical Sciences, Heriot-Watt University, UK

10:20 Contributed paper: Remote-controlled microbiological auto-sampling and on-line

monitoring of chemo-physical parameters and enzymatic activities at drinking water resources: event & time series analysis Dr. Hermann Stadler, Department for Water Resources Management, Joanneum Research, Austria

10:45 Networking break & exhibition 11:15 Viability PCR – a toolbox for live/dead differentiation of microorganisms

Dr. Marcia Armstrong, Qiagen, USA 11:40 Advances in DNA-based biosensors for rapid microbes detection in food and water:

looking for highly specific probes Dr. Lise Barthelmebs, Institut de Modélisation et d’Analyse en Géo-Environnement et Santé, Université de Perpignan Via Domitia, France

12:05 Simple field tests for the diagnosis of livestock diseases: is this an achievable goal?

Dr. Donald King, Vesicular Disease Reference Laboratory, The Pirbright Institute, UK 12:30 – 13:30 Networking break & exhibition

Poster viewing Workshops & hands-on-training (see pages 16-17)


TUESDAY 1 APRIL 2014

08:30 Plenary keynote lecture Rapid microbiology methods and The Coca-Cola Company Dr. Colm Scully, Coca-Cola Services, Belgium

09:00 Plenary lightning talks (short presentations by exhibitors)

o Advances in rapid analyses for microbial water quality and mycotoxin detection in feedstuff and milk – Wilbert Kokke, Charm Sciences, the Netherlands

o Rapid microbial genome sequencing – Dr. Derek Butler, BaseClear, the Netherlands o FastPrep-24, flexibility in sample preparation – Serges Pouedet, MP Biomedicals, France o Quantitative certified reference microorganisms – Ivo Siegrist, Sigma-Aldrich, Switzerland o Measuring enzymes and carbohydrates in foods and feeds – Barry McCleary, Megazyme

International, Ireland o Bio-Rad overview: Food Science Division – Gerrit Dijkstra, Bio-Rad Laboratories, the

Netherlands o Innova Biosciences conjugation technology for enhanced assay development – Tom Speedy,

Innova Biosciences, UK

Parallel session 2: Advances in contaminant analysis Advanced technologies provide many opportunities for improved detection. Novel technologies aside, a closer look needs to be taken at upstream sample preparation steps, even as they are applied to traditional detection technologies. Chair: Dr. Michele Suman, Barilla, Italy 09:30 Less is more – generic sample preparation in residue analysis

Dr. Bjorn Berendsen, RIKILT Wageningen UR, the Netherlands 09:55 Speeding up sample preparation in food analysis

Dr. Lourdes Ramos, Department of Instrumental Analysis and Environmental Chemistry, Institute of General Organic Chemistry of the Spanish National Research Council, Spain

10:20 Current GMO sample preparation and analysis approaches by the EU Reference

Laboratory for GM Food & Feed Dr. Maddalena Querci, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Italy

10:45 Networking break & exhibition 11:15 Single extraction method for multimycotoxin analysis

Julie Brunkhorst, Trilogy Analytical Laboratory, USA 11:40 Contributed paper: Potential of front-face fluorescence to monitor acrylamide and colour of potato crisps

Dr. Pierre Lacotte, Spectralys Innovation, France 12:05 The potential of ‘omics’ in chemical analyses of food, feed and water

Prof.dr. Hanspeter Nägeli, Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Switzerland

12:30 – 13:30 Networking break & exhibition

Poster viewing Workshops & hands-on-training (see pages 16-17)


TUESDAY 1 APRIL 2014 Parallel session 3: Advances in microbial analysis This session provides an insight into selected areas of microbial analysis where there have been recent advances or where the pathogen itself has some topicality. Chair: Dr. Kitty Maassen, National Institute for Public Health and the Environment, the

Netherlands 13:30 Laser optical sensor, a label-free rapid on-plate pathogen screening tool

Prof.dr. Arun Bhunia, Department of Food Science, Purdue University, USA 13:55 Rapid methods for detection of botulinum toxin-producing clostridia and their toxins:

the state of the art in Europe and in the world Dr. Fabrizio Anniballi, Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Italy

14:20 An integrated and ultrasensitive biosensor for the detection of pathogens

Dr. Mohammed Zourob, Center of Biomedical Engineering, Cranfield University, UK 14:45 DNA microarrays as versatile tools for freshwater quality testing

Dr. Holger Eickhoff, Scienion AG, Germany 15:15 Networking break & exhibition 15:45 Contributed paper: Rapid lateral flow detection of Legionella-specific RNA and

DNA amplicons Dr. Heleen van den Bosch, Food & Biobased Research, Wageningen UR, the Netherlands

16:10 Characterisation of movement at the nanoscale: a fast determination of bacterial

resistance to antibiotics Dr. Giovanni Longo, Laboratory of the Physics of Living Matter, École polytechnique fédérale de Lausanne, Switzerland


o Applications of polymer microarrays to water purification (P53) Sesha Venkateswaran, School of Chemistry, University of Edinburg, UK

o Development and validation of a nucleic acid microarray immunoassay and a nucleic acid lateral flow microarray immunoassay for the detection of Plasmodium species (P2) Dr. Aart van Amerongen, Food & Biobased Research, Wageningen UR, the Netherlands

o On-site isothermal LAMP assays for bacteria, viroid and phytoplasma detection (P30) Dr. Polona Kogovšek, Department of Systems Biology and Biotechnology, National Institute of Biology, Slovenia

17:00 – 18:30 Poster viewing & drinks 20:00 – 22:30 Conference dinner (reservations only)


TUESDAY 1 APRIL 2014 Parallel session 4: Advances in contaminant analysis Selected areas of contaminant analysis where there have been recent advances or where the contaminant itself has some topicality are highlighted. Chair: Prof.dr. Sarah De Saeger, Ghent University, Belgium 13:30 Mycohunt: a rapid biosensor for the detection of deoxynivalenol in wheat dust

Melanie Sanders, Department of Bio-analysis, Ghent University, Belgium 13:55 Droplet digital PCR, a cutting edge technology for quantitative analysis of food and

feed samples: GMOs as a case study Dr. Dany Morisset, Department of Biotechnology and Systems Biology, National Institute of Biology, Slovenia and CropDesign, Belgium

14:20 Analysis and risk of fullerenes and other carbon-based nanomaterials in the total

environment Dr. Marinella Farré, Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research of the Spanish National Research Council, Spain

14:45 Contributed paper: A simple and sensitive screening assay for antiviral neuraminidase inhibitors in poultry

Dr. Mariel Pikkemaat, RIKILT Wageningen UR, the Netherlands 15:15 Networking break & exhibition 15:45 Quantitative targeted residue and retrospective data analysis of relevant pesticides,

antibiotics and mycotoxins in bakery raw materials and food commodities by LC- HRMS Exactive™ (Orbitrap Technology) Dr. Michele Suman, Food Research Labs, Barilla, Italy

16:10 Collect data, not samples: application of field portable GC-MS for on-site food

sampling and analysis Dr. Joeri Vercammen, IS-X, Interscience, Belgium


o Fluorescence polarisation immunoassays for rapid determination of mycotoxins in foodstuffs (P36) Dr. Vincenzo Lippolis, Institute of Sciences of Food Production, National Research Council of Italy, Italy

o Metabolomic investigations to detect biomarkers associated with changes in meat maturity (P18) Dr. James Donarski, The Food and Environment Research Agency, UK

o First reference materials for GM rapeseed powder mixtures (P17) Dr. Blagica Dimitrievska, Institute for Reference Materials and Measurements, Joint Research Centre, European Commission, Belgium

17:00 – 18:30 Poster viewing & drinks 20:00 – 22:30 Conference dinner (reservations only)


WEDNESDAY 2 APRIL 2014 Parallel session 5: Future developments in the area of food and environmental analysis Coming soon? Chair: Bram van der Gaag, INCAS3, the Netherlands 08:30 Point-of-care device for rapid detection of microorganisms in food

Dr. Maria Carmen Morant-Miñana, Microsensors Unit, CIC microGUNE, Spain 08:50 Airborne bacteria: from microbial physics to rapid identification

Prof.dr. Paul D. Maguire, Nanotechnology and Integrated Bio-Engineering, University of Ulster, UK

09:10 Contributed paper: Hydrochip Life+, development of a molecular chip for rapid

determination of the quality of surface water by using diatoms as indicator organisms Marije IJszenga, Vitens laboratory, the Netherlands

09:30 Fiber optic biosensing for allergen and pathogen screening in food

Dr. Steven Vermeir, Department of Biosystems, KU Leuven, Belgium 09:50 Using nature’s genius for pollutant detection

Dr. Johannes Raff, Helmholtz Institute Freiburg for Resource Technology, Helmholz-Zentrum Dresden-Rossendorf, Germany

10:10 Ultrasensitive surface enhanced Raman spectroscopy sensor and potential

applications in environmental monitoring Ali Özhan Altun, Department of Mechanical and Process Engineering, ETH Zurich, Switzerland

10:30 The single cell future of MALDI-TOF mass spectrometry

Dr. Gerold de Valk, BiosparQ, the Netherlands 10:45 Networking break & exhibition 11:15 – 12:45 Final plenary meeting: Food authenticity – after Burgergate and

Fishgate...what is next? see page 14


WEDNESDAY 2 APRIL 2014 Parallel session 6: RME2014 meets MoniQA MoniQA is an international and interdisciplinary network of professionals from institutions working in food research, regulatory bodies and trade, providing solutions to promote a safer and secure food supply worldwide. Chair: Dr. Roland Poms, MoniQA Association / International Association for Cereal Science

and Technology, Austria Hans P. van Egmond, M.Sc., RIKILT Wageningen UR, the Netherlands

08:30 Looking for the best compromise in rapid mycotoxins tests: speed, sensitivity,

precision, accuracy Dr. Anton J. Alldrick, Science Division, Campden BRI, UK

08:50 Performance criteria for rapid screening methods to detect mycotoxins

Dr. Jörg Stroka, Institute for Reference Materials and Measurements, Joint Research Centre, European Commission, Belgium

09:10 Isothermal amplification of DNA as a novel approach for the rapid detection of DNA

in food samples: a simple test for the food allergen celery Dr. Kurt Brunner, Department IFA-Tulln, BOKU Vienna, Austria

09:30 Towards the production of reference materials for food allergen and gluten-free

analysis for improved food safety Dr. Roland Poms, MoniQA Association / International Association for Cereal Science and Technology, Austria

09:50 Rocket fuel for media headlines: the perchlorate story

Dr. Bert Popping, Eurofins Scientific Group, UK 10:10 Validation of binary methods – challenges and models

Dr. Christoph von Holst, Institute for Reference Materials and Measurements, Joint Research Centre, European Commission, Belgium

10:30 Novel quantitative real-time and multiplex PCR for allergen detection

Ronald Niemeijer, R-Biopharm, Germany 10:45 Networking break & exhibition 11:15 – 12:45 Final plenary meeting: Food authenticity – after Burgergate and

Fishgate...what is next? see page 14


WEDNESDAY 2 APRIL 2014 Final plenary meeting: Food authenticity – after Burgergate and Fishgate...what is next? Authenticity yesterday, today and tomorrow! Chair: Dr. Bert Popping, Eurofins Scientific Group, UK 11:15 Introduction to the topic: Authenticity issues in the food industry

Dr. Bert Popping, Eurofins Scientific Group, UK 11:30 Faith in food: fraud detection by novel analytical techniques

Prof.dr. Saskia van Ruth, RIKILT Wageningen UR, the Netherlands 11:55 Meat for the masses: protein mass spectrometry for the authentication of meat

products Dr. Jens Brockmeyer, Institute of Food Chemistry, University of Münster, Germany

12:20 Labelfish – a European project to combat fraud in the fish industry

Dr. Carmen González Sotelo, Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Spain

12:45 Closing of RME2014 13:00 – 15:00 MoniQA Association General Assembly (by invitation only)


LOOKING FORWARD TO

SEEING YOU AGAIN NEXT YEAR!

RME2015

SPRING 2015

THE NETHERLANDS


WORKSHOP & HANDS-ON-TRAINING PROGRAMME R-BIOPHARM Monday 31 March 2014 15:45 – 16:45 Tuesday 1 April 2014 12:30 – 13:30 Workshop 1: Innovative sample clean-up methods for mycotoxin analysis In the past years, we have seen how different mycotoxins have affected cereal crops all over the world. One year maize in, e.g., the USA is contaminated with high concentrations of deoxynivalenol, the next year the crop is contaminated with aflatoxins, followed by a year with relative low amounts of mycotoxins. The same pattern is seen all over the world, however, with different mycotoxins and different commodities at different times. For a mycotoxin testing lab this means it has to be able to test significant numbers of samples in short time (harvest time), probably on multiple mycotoxins. Although recent developments have improved the efficiency of the analytical part, sample preparation and clean-up can be still time- and labour-consuming. In this workshop, we will address methods for single extraction of multiple mycotoxins prior to HPLC and LC-MS/MS and a fully automated on-line sample clean-up method in combination with HPLC or LC-MS/MS.

QIAGEN Monday 31 March 2014 15:45 – 16:45 Tuesday 1 April 2014 12:30 – 13:30 Workshop 2: Viability PCR – the next level in using PCR as a detection technology for microorganisms Real-time PCR (rt-PCR) provides a fast and powerful tool to analyze samples for the presence of potentially harmful microbes. However, there is a risk of false positives through the detection of DNA from harmless dead cells. Live/dead differentiation can play an important role in procedures, such as hygiene testing (success of decontamination processes), water testing (distinguishing between live and dead Legionella for regulatory compliance) and human diagnostics (monitoring medication efficiency in pathogen killing). Viability rt-PCR utilizes the DNA-masking compound propidium monoazide (PMA), which is able to enter dead and membrane-compromised pathogen cells and to intercalate into DNA, rendering the DNA from dead cells inaccessible to and thus not detectable by rt-PCR. In this workshop, Qiagen will demonstrate a universally applicable toolbox centering on PMA, together with a new illumination device designed to catalyze the PMA reaction.


BIODOT Monday 31 March 2014 15:45 – 16:45 Tuesday 1 April 2014 12:30 – 13:30 Workshop 3: Dispensing technology impact on test line intensity and width BioDot will demonstrate the new ceramic tip and in-line degasser used on the XYZ range of movement platforms. This innovation has been produced to further ensure the quality of lines is improved and meeting customer's expectations when diagnostic tests are being read in the ever increasing area of quantification and the demanding requirements the reader has on line quality and position. In addition, the contact and none contact Frontline and Biojet HR will be demonstrated to show potential differences between the two dispense technologies and their impacts on different nitrocellulose suppliers.

MAITRE Throughout all conference breaks on Monday 31 March, Tuesday 1 April and Wednesday 2 April 2014 Hands-on-training: Communicating food science research The project MAITRE (Media Action for International Training of Researchers), funded by the European Union, enters into the debate of science communication and education. The project aims to facilitate the dialogue between scientists and journalists by enriching the exchange and collaboration of food researchers and media. During the conference, short TV interviews (1-2 min) will be taken on spot with the participants. How to behave in front of the camera and how to handle an interview with journalists, including mistakes made, will be explained from the taken interview. Use the opportunity to get tips and hints from experienced media professionals!


LECTURES REDUCING THE PHYTOSANITARY RISKS IN PRODUCTION SYSTEMS AND GLOBAL TRADE OF PLANT MATERIAL: HOW RAPID AND RELIABLE METHODS CAN HELP BOTH BUSINESS AND INPSECTION AGENCIES Plenary keynote lecture John van Ruiten Naktuinbouw, Netherlands Inspection Service for Horticulture, the Netherlands [email protected] Phytosanitary problems in crop cultivation, landscaping and forestry are from all times, but it can be seen that spread of plant diseases still causes great losses and threats of environmental conditions. With an ongoing increase in global trade of plant material (seed, young plants, trees) and other plant products, new pathogens continuously occur, sometimes in crops in which their existence was not known before. There is a continuous evolution between hosts (crops/commodities) and their pathogens. In the past, new outbreaks remained isolated events. Today, they can spread very rapidly and make plant material with increasing volumes more prone to diseases. There are various options to combat these threats. Traditionally ‘quarantine measures’ (do not allow certain imports or keep imports isolated/separated for a certain period) are amongst the first reactions that countries apply. Although this approach has certainly helped slow down the introduction speed of pathogens, it cannot be the only answer to the issue. And if not supported by additional actions, such ‘blocking’ strategies will certainly harm national and international trade in seeds/plants and plant products. A step forward has been made in creating policies of developing acknowledged pest-free production sites or locations combined with certified pathogen-tested plant material. Also, in an international FAO context, the International Plant Protection Convention (IPPC) is active in globally developing International Standards for Phytosanitary Measures (ISPM standards) for phytosanitary safeguarding production and trade of healthy seeds and plants for planting. In the European Union (EU), an intensification of phytosanitary strategies is visible, too. With the recent proposal of a new EU Phytosanitary Regulation, the European Commission advocates a system that provides better guaranties for marketing healthy (disease-free) material. Another major development is that pest control with the use of crop protection compounds is under critical observation. There is a very clear tendency (worldwide) that the use of chemical pesticides has to be brought down to much lower levels. This requires the development of a renewed strategy for crop protection and hygiene systems. The global list of harmful (quarantine) organisms in plants is exceptionally long (estimated to go over 1000 pathogens). The global list of relevant crop damaging pathogens for which crop protection strategies are required, is even much longer. In pathogen detection not only seed and plant material must be tested and evaluated. To produce and market material in clean systems methods for monitoring (clean) water, equipment, people and air also need to be developed. These detection systems are for many pathogens still in their infancy. Naktuinbouw operates programmes like NAL and Naktuinbouw Elite, in which a systems approach for disease detection is applied: on the one hand, site intensive, ultrasensitive and reliable (and expensive) testing of individual plants to be used for the start of mass propagation programmes and, on the other hand, big scale, cheaper routine testing techniques for controlling and monitoring material to be marketed in high volumes.


And the third major point of attention is found in the necessity to have the possibility to fully ‘track and trace’ plant material and foods created from them from ‘farm to fork’. So with adequate labelling, this traceability (and possibilities for recall and forward actions) has to be improved. Better methods for identifying and checking origin of products is urgently needed. Technologies for tracking and tracing in the agricultural sector are now under attention. Isotope analysis, metabolite measurements or other ‘source identifying methods’ have still to prove their value. These three developments require company quality systems, official control, and adequate inspection and detection methods for accreditation and certification. Of course these methods must be quick, reliable and robust, and properly priced. And, even more important, the methods used need to be internationally harmonised and validated.


PROFICIENCY TESTING; TICKING THE BOX FOR MY ACCREDITATION OR EDUCATIONAL TOOL? Plenary keynote lecture Kate Wilkinson The Food & Environment Research Agency, UK [email protected] Proficiency testing (PT) for analytical chemistry and microbiology in the food and environmental sectors is well established. Many national and international schemes have been providing valuable assurance in the quality of results for laboratories and their customers for many years. However, the motivation for many laboratories to participate is still the need to satisfy their accreditation body for maintenance of their ISO 17025 accreditation. The requirement to “have a quality control procedure, such as participation in a PT scheme if a suitable scheme exists” sometimes drives participation without understanding the full benefits or relevance of the scheme chosen. Knowing how to understand both the benefits and limits of a PT can help you get the most out of the cost you must incur to take part. Using examples from the Food Analysis Performance Assessment Scheme (FAPAS), the suitability, frequency and timing of PT use will be discussed, particularly when developing new methods, improving existing methods or responding to emergencies, such as meat authenticity. Additionally, the use to the analytical community of the data generated by PTs will be examined.


THE SEARCH FOR SPOCK’S TRICORDER: A PERSPECTIVE FROM A MAJOR FMCG COMPANY ON THE TECHNICAL REQUIREMENTS FOR THE ADOPTION OF A RAPID PROCEDURE Ashley Scribbins Corporate Quality & Food Safety, Mars Incorporated, UK [email protected] Cost reduction pressures, globalisation of raw material sourcing, food fraud, food safety concerns, increasing regulation, twenty-four hour globalized media, brand protection, speed to market: these are just some of the common everyday pressures faced by major FMCG companies. The QA/QC laboratory plays an essential role in providing data that informs the supply chain, informing thousands of critical decisions globally every day in a multinational company. However, traditional laboratory analysis is seen as time consuming and therefore costly, and every production manager would like to have access to immediate information. Hence the appeal of a rapid method is strong and it is easy to be seduced by claims of simple, rapid analysis that can lead to cost savings through reduced warehousing or demurrage costs. Before selecting and investing in a rapid technology, a FMCG company must carefully evaluate a number of key criteria in the selection method. The rationale is simple: no FMCG organization can afford to have methods which might provide incorrect or inconsistent data, leading them to take the wrong decision. A market recall for a leading branded business can have a catastrophic impact on consumer trust and the longevity of the company. In this presentation, I will explore some of the key requirements of a rapid measurement device in ensuring suitability for a global market place, providing insights on requirements for each stage of the evaluation process, from validation to support models.


RAPID MICROBIOLOGICAL METHODS: A FOOD INDUSTRY PERSPECTIVE Balamurugan Jagadeesan Nestlé Research Center, Switzerland [email protected] In the food industry, microbiological methods are used to ensure raw material quality, and perform line sample, environmental and finished product testing. Rapid methods are often employed to obtain faster results and thus enable quicker decisions during the food manufacturing process. Commercially available detection methods are mostly culture-, antibody- or nucleic acid-based assays. Improvements to the existing assays are constantly being made to increase the method sensitivity and specificity, ease of handling, automation, etc. In spite of the developments in the detection component of a method, improvements to sample preparation of food matrices has to be still adequately addressed to maximise the benefit from the technological advances. Validation of a new method using ISO 16140 or the Official Methods of Analysis (OMA) of AOAC International guidelines is a prerequisite for a method to be considered for day-to-day use. In addition to the official validation studies, partial validation is often required to demonstrate the performance of a method in a specific food matrix prior to implementation. Besides meeting the performance criteria, a method should also be user friendly, easy to implement and robust enough in a routine set up.


RAPID METHODS FOR THE QUALITY CONTROL OF DRINKING WATER Vicente Catalán, A. Yáñez, E. Soria and J. Llorca Labaqua, Aqualogy Environment, Spain [email protected] Ensuring chemical and microbial quality of water is a worldwide ever-increasing issue. Water must be treated and monitored on an ongoing basis following current regulations and at each stage of the integral water cycle to ensure a continuous supply of quality water. Since water pollution can originate from many different sources, a variety of pollution prevention and control measures IS needed. The standard methods used for the detection of pollutants and pathogenic bacteria in water are labour-intensive and the time needed to obtain results is generally long. Therefore, there is a need to speed up regular monitoring and have new analytical systems for specific, rapid and on-line detection OF pollutants in water allowing quick decisions. The implementation of early warning systems (EWS) could be an important tool to prevent or mitigate the impacts of an intentional pollution event, and should be focused on the detection of both microorganisms and chemical compounds. EWS emerge as good candidates to be part of safe drinking water programmes, although at the moment some technical constraints and regulatory issues remain. An essential fact to consider is that detection of contaminants, as part of EWS, may not be based on conventional methods usually applied in laboratories. Because of this, facilities and diagnostic laboratories are facing a new challenge that forces them to transition to in situ control systems, with implementation of automated systems, in situ analysis, and eventually overcoming an on-line laboratory. In this sense, the industry has responded by offering automated systems for the laboratory that improve efficiency, consistency and greatly reduce response time-to-results. Most of the laboratory processes (pre-analytical, analytical and post-analytical) are being automated, to meet market requirements. In microbiology, some processes such as culture media preparation, colony counting or sample concentration have been automated to reduce time-to-results and improve the culture-based methodology accepted as a gold standard. As an alternative to conventional methods, new technologies have been developed for the fast detection of microorganisms, such as molecular methods based on qPCR, ATP detection, advanced microscopy, flow cytometry, immunoassays, etc. Most of them have already been implemented as laboratory and/or ‘in situ’ methods. Based on all these technologies, different devices have been developed to get ‘on-line’ detection. However, at the moment they are at very early stages and need intensive and detailed validation to be feasible candidates for implementation as routine methodologies in the potable water distribution network. There is a group of emerging technologies that could be good candidates to fulfil the requirements becoming EWS for the detection of pathogens, such as DNA microarrays, molecular imprinted polymers, microcantilever systems, photoluminescent biochips, surface plasmon resonance, etc. Finally, a critical issue in environmental detection of bacteria is the limit of detection; small amounts of cells should be detected. Therefore, detection technologies that are part of EWS must be combined with sample concentration systems. Some of these under evaluation are hollow fibre ultrafiltration, hydroxyapatite whole cell capture, and lectin and carbohydrate affinity. The conventional process to analyse organic compounds in waters involves sample preparation to obtain two improvements: avoiding matrix interferences and increasing the sensitivity to obtain the required limit of quantification (LOQ). In the last decades, laboratories have been implementing new on-lab methods that allow substituting the classical methods of


extraction and sampling preparation (liquid-liquid extraction (LLE) to optimise these processes. The development of methods such as solid phase extraction (SPE) has allowed for improvements towards a complete on-lab automation of the sample preparation process. On the other hand, other techniques such as solid phase microextraction (SPME) or stir bar sorptive extraction (SBSE) have not only allowed to reduce the matrix effect but also to increase the sensitivity and operation of the process due to the fact that they are solvent-free techniques. Some of these sample preparation techniques have just been adapted onto passive/integrative samplers. These devices allow continuous monitoring of the pollutant as prior step to on-line devices. Different methods such as enzyme-based detection, microchip surface acoustic wave (SAW) and different kinds of biosensors could be good candidates to be implemented in the rapid quality control of chemicals compounds. The emerging technologies will improve efficiency, consistency and greatly reduce response time-to-results but, for successful implementation, is mandatory that they should be in accordance with the regulations requirements and their cost should be easily assumable by the end users that are demanding them.


RAPID MICROBIOLOGY METHODS AND THE COCA-COLA COMPANY Plenary keynote lecture Colm Scully Coca-Cola Services, Belgium [email protected] Classical microbiological methods have traditionally been used in the Coca-Cola Company for product release, environmental monitoring and troubleshooting. As demand grows for more varied and natural products this has in turn increased complexity of the company’s portfolio, supply chain routes and new ingredients. In addition to this, economic pressures to release product safely to the consumer without increasing the risk either of spoilage microbes or public safety has highlighted the need for rapid microbiological methods that can offer more than the classical approach. To meet the demands, The Coca-Cola Company and bottling partners have begun to investigate and invest in rapid microbiological methods. These methods should offer similar performance, test for organisms not easily cultured by existing methods or offer time savings to improve release of product to the market. This presentation will focus on the following aspects:

an overview of the technologies currently deployed by the Coca-Cola Company;

the microorganisms of concern to the Coca-Cola Company in different beverage, ingredient and environmental matrices;

the validation expectations and the requirements for the Coca-Cola Company; and

the current challenges faced by the company in selecting rapid methods.


IMPLICATIONS OF BACTERIAL CLUSTERING FOR SAMPLING PLANS Ursula Gonzales-Barron Centro de Investigação de Montanha, Escola Superior Agrária – Instituto Politécnico de Bragança, Portugal [email protected] Sampling and testing for microorganisms in foods is a risk management strategy used to evaluate whether a food safety system achieves the appropriate level of control. Whether sampling plans to test microorganisms in foods are derived to meet the consumer’s or producer’s quality requirements, the statistical methods proposed for their design have been entirely based on quality control statistics. Reason as to why two simplifying assumptions have been traditionally adopted: (i) that the log microbial concentrations among food units produced in a batch can be represented by a normal distribution, and (ii) that its measure of spread (i.e., standard deviation) is constant among batches of production. Furthermore, from these assumptions, it is implicitly understood that the normal distribution is adequate for any contamination level (i.e., low counts or high counts), and that the measure of spread is independent of the contamination level. Nevertheless, recent work has demonstrated that the normality assumption only holds for high microbial concentrations. Heterogeneous Poisson and zero-modified distributions have been proven to be more appropriate to characterise bacterial clustering, low microbial concentrations and microbial data consisting of many zero counts. On the other hand, the within-batch measure of spread is in many cases dependent on the contamination level and associated with the within-batch mean concentration. This phenomenon is very likely to be an effect of the physical agglomeration of bacterial cells. The importance of the choice of the most appropriate distribution to represent bacterial clustering resides on its critical effect on the acceptance probability for the design of sampling plans. Furthermore, the derivation of operating characteristic (OC) curves should ideally take into account the shifts in the within-batch measure of spread for the acceptance probability calculation as we move along the x-axis values of within-batch mean concentration. It has been shown that the Poisson-gamma assumption allowing for clustering produces stricter sampling plans than both the lognormal and the Poisson distribution for the same level of safety. Unlike the manufacturing process of non-food items that, when under control, has been proven to operate with only chance (or non-assignable) causes of variation that are an intrinsic part of the process, and therefore leading to a stable variance, in the production of food items, the batch-to-batch variability in the microbiological quality of the food product is expected to be more unstable due to phenomena inherent to the biological systems. To account for the effect of the batch-to-batch variability, a novel approach to jointly model the within-batch and between-batch variability in microbial counts based on a random-effects Poisson-gamma model has been proposed to characterise low and high microbial counts from many batches of production. Thus, in the derivation of an OC curve, the uncertainty around the within-batch spread measure for a given within-batch mean can be propagated by simulation to the probability of batch acceptance. In this way, OC curves are obtained with confidence intervals representing the uncertainty about the spread measure arising from the between-batch variability. The OC curves obtained as such lead to more conservative sampling plans than using classical statistics based on the normal distribution. Thus, new modelling trends based on more realistic assumptions are examined that consider the clustering of bacteria, the intrinsic variability among food batches and the use of past monitoring microbial data.


SAMPLING AND DETECTION TECHNOLOGIES OF PATHOGENS FOR SAFE DRINKING WATER: A REVIEW Marc Desmulliez School of Engineering and Physical Sciences, Heriot-Watt University, UK [email protected] The alternative to the monitoring of waterborne pathogens through the detection of faecal indicators is based on the direct identification of particular pathogens. The extraordinary difficulty to detect, let alone to determine the species, viability and infectivity of low concentrations of pathogens in large water volumes, demands the use of sophisticated sampling and detection technologies. These technologies will be reviewed in the presentation with emphasis on molecular methods of detection. Sampling technologies The sampling technologies of pathogens present in drinking, irrigation and food processing water rely on a number of considerations related to the significance and relevance of a specific volume of water and the influence of the water quality to the sampling procedure. Large volumes of water ranging from 40 to 100 litres need to be analysed to achieve detection of low concentration of pathogens. In that respect various filtration techniques exist which reduce the water volume while retaining the microorganisms. The available filtration methods (US-EPA, 2001, 2005) to concentrate waterborne pathogens are either too costly for studies requiring large numbers of samples, limited to small sample volumes, or not very portable for routine field applications. Other filtration techniques include packed glass wool, NanoCeramTM filters, hollow-fibre ultrafilters, and, more recently, xylem tissue in sapwood for the filtration of bacteria. Glass wool filtration is a cost-effective and easy-to-use method to retain viruses, bacteria and protozoa, but its efficiency and reliability are still under study. NanoCeramTM are cost effective newly developed electropositive plated microporous filters composed of microglass filaments coated with nanoalumina fibres. Most bacteria are large enough to be mechanically filtered or retained while smaller particles such as viruses are retained principally by electro-adhesion, which has proved useful for the isolation of viruses from water. Hollow fibre ultra-filters normally have a cut-off of ~30KDa and will retain parasites, bacteria and viruses. They are disposable (cost ~€ 25), thus avoiding the risk of cross contamination and filtration can either be achieved by coupling the filter directly to a tap or pumping an environmental water sample through the filter. They have mainly been used for filtration of tap water but have also been used for surface waters. In all the techniques presented above, the microorganisms need to be eluted from the filters. Filtration, as well as elution, needs to be optimised to give a sufficient recovery of a control panel of model organisms representing bacteria, viruses and parasites, from treated as well as untreated ground and surface waters. Secondary concentration procedures will differ between the kingdoms, preventing thereby the delivery of a universal protocol. Moreover significant amounts of organic and inorganic inhibitors are known to be enriched during the concentration of water samples; this might perturb the molecular detection and quantification of present pathogens. Detection technologies The review will concentrate mainly on molecular detection technologies, which include nucleic acids amplification, microarrays, optical methods such as FISH, lab-on-chip microfluidic platforms or the use of biosensors in case of ATP monitoring. Nucleic acid amplification is covered only in this abstract. A wide range of PCR and real-time PCR systems have been developed for the detection of pathogens in food and water with various platforms (Taqman, LightCycler, PriProEt, etc.) showing high specificity and sensitivity in the


detection of pathogens Compared with the classical gel-based single, nested and ELISA-based test systems, real-time PCR assays offer: (i) faster and higher throughput, (ii) one-step amplification set-up, (iii) on-line detection of the product minimising the risk of lab contamination and consequently (iv) higher automation potential. Other amplification systems such as NASBA, LAMP, and RPA do not require thermal denaturation of templates and can operate at a low and constant temperature. They therefore offer an easy and affordable technology for the integration in combined filtration, detection and output signal devices relative to other amplification methods. Recombinase polymerase amplification (RPA) has been developed for automated analysis of antibiotic resistance genes providing results in 20 min with a limit of detection of less than ten copies. Multiplex detection uses multiple primers to allow amplification of multiple templates within a single reaction is also an attractive proposition. By broadening one analysis to several targets, such as the most commonly waterborne viruses in a water sample, the speed and cost of diagnostics can be decreased significantly. Such multiplex systems need to be benchmarked however against standard monoplex PCR and real time PCR with respect to sensitivity and specificity. The table below provides a small snapshot of current molecular detection technologies available that will be targeted in the EU project Aquavalens.

Platform Detection Technique

Targets/ pathogens

Localisation Advantages and progress

Multiplex PCR RT-qPCR, qPCR, RPA

Three most relevant viruses, bacteria, parasites and MST- organisms respectively

Water quality control laboratories

Transfer of single detection protocols to multiplex assays (WP7). Integration for automatic sampling (WP8)

AqµaTAS PCR AdV, NoV, HAV Field sampling Water quality control laboratories

Low cost, rapid in-field testing

Biosensor Microbial activity

ATP On-line distribution network

Early warning, automated and adapted to new test beds (chlorinated water).

SPC FISH Antibody

Bacteria Cryptosporidium

Water quality control laboratories

Improved Campylobacter, V. cholerae and Cryptosporidium detection

Vermicon FISH Bacteria, Water quality control laboratories

Autofocus function on the automatic scanning and output signal

Luminex PCR (VOCMA) All targets identified in cluster 1

Research / Water quality control laboratories

Flexibility and multiplexity of detection. Low cost per target organism. For extended control of raw water, water safety plans and during outbreaks.

Automated Platforms with sample preparation units

Mb-online / Bacterial enzyme activity

E. coli, enterococci

On-line water treatment plants

Early warning, indication for event sampling. Enterococci more relevant indicator for viruses and parasites

Shaw/fluorescence detection

Parasites On-line water treatment plants, Water quality control laboratories

Full Sample preparation modules. Automatic scanning and output signal


Conclusions Ideally, the development of technological platforms for sampling and detection of pathogens should be considered as the ultimate goal to bring rapid and robust solutions, thereby reducing the cost of such analysis, and eliminate some of the human handling time and potential for contamination. Such platforms should possess the following qualities: (1) low limit of detection, (2) potential for speciation, (3) robustness of detection, (4) low manufacturing and operational costs, (5) potential for simultaneous analysis of multiple pathogens, (6) fit to market demand, (7) retrofit with existing water management systems, (8) high system reliability, (9) size and portability of the system and (10) potential for automation. It is fair to say that, today, no such platforms exist for the sampling and detection of the three kingdoms (protozoa, bacteria and viruses). Acknowledgements The author would like to acknowledge the financial support of the EU-funded Integrated Project Aquavalens (FP7-311846) addressing the work programme area 2.2.5 Environmental impacts and total food chain KBBE.2012.2.5-01. Most of the contents of the abstract presented here stem from the original proposal of Aquavalens.


REMOTE-CONTROLLED MICROBIOLOGICAL AUTO-SAMPLING AND ON-LINE MONITORING OF CHEMO-PHYSICAL PARAMETERS AND ENZYMATIC ACTIVITIES AT DRINKING WATER RESOURCES: EVENT & TIME SERIES ANALYSIS Contributed paper Hermann Stadler1 and A. Farnleitner2 1Institute for Water, Energy and Sustainability, Department for Water Resources Management, Joanneum Research, Austria and 2Institute of Chemical Engineering, Department of Applied Biochemistry and Gene Technology, Vienna University of Technology, Austria [email protected] Water resources from alpine and other mountainous karst aquifers play an important role for water supply in many countries. Worldwide approximately 25% of the population are fed by drinking water from karst aquifers. These karst regions cover 12.5% of the land surface [1]. In Austria, 25% of the people are supplied with karst water, including Vienna [2]. As regulated in the Water Framework Directive (WFD), karstic catchments require a sustainable protection. The increasing impact to such regions and the different utilization in the watersheds of karst springs are important reasons to establish early warning systems and quality assurance networks in water supplies. These systems rely heavily on in situ measurements and on-line and near real-time availability of the data. With a satellite-based networking of measuring and sampling stations it was possible to carry out precipitation-triggered event monitoring campaigns at different karst springs [3] combining on-line measurements of hydrological parameters with field-laboratory-based analyses of microbial faecal indicators [4] and enzymatic in situ measurements [5]. The targets in the study were (i) to investigate the dynamic of microbial faecal pollution indicators, chemical parameters and environmental isotopes at a high resolution time scale during hydrological events, and (ii) to evaluate the in previous investigations established parameter SAC254 (spectral absorption coefficient at 254nm) as an appropriate real-time pollution proxy for optimised spring water abstraction management within an early warning. System description ORBCOMM low earth orbiting (LEO) satellite system was chosen. It is a ‘little-LEO’ system, with 30 servicing satellites in 6 orbit planes of 800 km altitude. It provides bi-directional ‘short message’ data-transfer at 2.4/4.8 kbps, with data blocks preferably less than some 100 Bytes. ORBCOMM operates at frequencies about 140 MHz, providing large satellite footprints, and requires only low-cost/low-power equipment. The ORBCOMM modem transmits its data to the satellite, from where down-link transmission is performed either directly to one of the gateway earth stations (GES). The GES emails the data to the receiver via internet or re-transmits it to any ‘nomadic’ ORBCOMM modem again via satellite. The precipitation station (PS) is located in the catchment area of the spring, where the event sampling will be carried out. It is equipped with a tipping bucket, a data logger and a LEO-Satellite modem. It can be supplemented with additional meteorological sensors and sampling devices. The monitoring and sampling site at the spring (spring sampling station (SSS)) is equipped with an additional data logger, a pressure probe to register the changing of discharge, two automatic sampling units (one for the reference sample and one for the periodic samples) and a LEO-Satellite modem for real-time control and data transmission. It can be supplemented with additional hydrological or meteorological sensors. References 1. Martin, J.B. and White, W.B. (eds.), 2008. Frontiers of karst research – Special Publication 13.

Karst Waters Institute, Leesburg, VA, USA.


2. Zwahlen, F. (ed.), 2003). Vulnerability and risk mapping for the protection of carbonate (karst) aquifers. Scope - Goals - Results. COST Action 620, European Commission, DG Science, Research and Development, Luxemburg, 39 pp.

3. Stadler H., Klock, E., Skritek, P., Mach, R.L., Zerobin, W. and Farnleitner, A.H., 2010. The spectral absorption coefficient at 254 nm as a near real time early warning proxy for detecting faecal pollution events at alpine karst water resources. Water Science and Technology 62: 1898-1906.

4. Stadler H., Skritek, P., Sommer, R., Mach, R., Zerobin, W. and Farnleitner, A.H., 2008. Microbiological monitoring and automated event sampling at karst springs using LEO-satellites. Water Science and Technology 58: 899-909.

3. Stadler H., Klock, E., Skritek, P., Mach, R.L., Zerobin, W. and Farnleitner, A.H., 2010. The spectral absorption coefficient at 254 nm as a near real time early warning proxy for detecting faecal pollution events at alpine karst water resources. Water Science and Technology 62: 1898-1906.

5. Ryzinska-Paier, G., Lendenfeld, T., Correa, K., Stadler, H., Blaschke, A.P., Kirschner, A.K.T., Mach, R.L. and Farnleitner, A.H. Field testing of a novel concept for automated on-line monitoring of β-D-glucuronidase activity in water resources. Water Science and Technology (in press).


VIABILITY PCR – A TOOLBOX FOR LIVE/DEAD DIFFERENTIATION OF MICROORGANISMS A. Nocker-Einsiedler, K. Wolf, S. Fakih, Marcia Armstrong, S. Luley and R. Peist Qiagen, USA [email protected] Nucleic acid detection methods, such as real-time PCR (q-PCR) are fast and powerful tools to analyze samples for the presence of potentially harmful microbes, but also hold the risk of false positives by detecting nucleic acid from harmless dead cells. Viability PCR utilizes the DNA-masking compound propidium monoazide (PMA) which enters dead/membrane-compromised cells and modifies their DNA, resulting in a strong reduction in amplifiability. The presentation gives an overview of the development of PMA, its performance with different downstream analysis methods, applications in different fields, and ways to adjust the efficiency of dead cell exclusion. Different bacterial species (typically pathogens) exposed to increasingly lethal stress gradient or defined mixtures of live and killed cells were prepared and treated with PMA. Following activation of the compound by illumination with a specific wavelength, the reagent is irreversibly bound to DNA of dead cells, masking it so that it is not PCR visible anymore. Efficient suppression of amplification of such modified DNA allows preferential detection of live cells. Real time PCR results representing full workflow data with and without pathogen relevant matrices are presented showing PMA efficiency and introducing a new tool box concept. Data obtained with live and killed pathogens treated and not-treated with PMA demonstrate the extent of suppression of amplification. PMA exposure time, incubation temperature and amplicon length were found to be efficient and easy-to-adjust parameters to tailor the assay to different microbial species and adjust the efficiency of dead cell exclusion. Live/dead differentiation can play an important role in procedures, such as hygiene testing (success of decontamination processes), water testing (distinguishing between live and dead Legionella for regulatory compliance) and human diagnostics (monitoring medication efficiency in pathogen killing).


ADVANCES IN DNA-BASED BIOSENSORS FOR RAPID MICROBES DETECTION IN FOOD AND WATER: LOOKING FOR HIGHLY SPECIFIC PROBES Lise Barthelmebs1, N. Paniel1, Julia Baudart2,3 and Thierry Noguer1 1Institut de Modélisation et d’Analyse en Géo-Environnement et Santé, Université de Perpignan Via Domitia, France, 2Observatoire Océanologique, UMPC Université Paris, France and 3CNRS, Observatoire Océanologique, France [email protected] The increasing concerns about microorganisms and public health, food and environmental safety have boosted the need for more rapid, specific, robust and highly sensitive detection methods. Although normalised microbiological methods are available, they need well-trained personnel, are labour-intensive and not adapted to on-site measurement for high frequency of analysis. For this purpose, a lot of research has been directed towards the development of alternative methods, such as immunological methods, PCR based methods, and biosensor methods. Biosensors have become attractive alternatives for monitoring the microbiological quality of waters and food due to their simplicity, versatility, portability, low cost and potential for accurate, real-time detection. Among the various biorecognition elements reported for biosensor development, antibodies have been mainly used. These elements can directly and rapidly detect pathogenic bacteria with high affinity and specificity. However, major drawbacks associated with antibodies include their production, instability and post-production modification that limit their application. Recent evolutions in biotechnology, nanotechnology and surface chemistry have created the possibility to develop novel affinity-based recognition elements which could overcome the limitations encountered with immunologic methods. Nucleic acids have appeared as promising recognition elements for analytical applications. They present many advantages compared to antibodies, such as their stability, high temperature resistance, ease of modification, combined with their fast, low cost and animal-friendly production avoiding batch-to-batch variations. In biosensors for microbial detection, nucleic acids can be used based on two different approaches.

In the genosensor approach, single stranded DNA (ssDNA) is used as the capture probe element, targeting DNA or RNA in a sample that is detected through the hybridization reaction. The probe design is the main key step that will define the specificity of the genosensor, because the specificity of the hybridization reaction is dependent on the biorecognition properties of the capture probe. By consequence, the design of the capture probe must be performed with a great attention, and different considerations must be taken, such as the specificity and structure of the probes designed, and the accessibility of the sequence target. The potential of DNA genosensors as a sensitive, rapid and portable tool for several applications in environmental and food analysis has been demonstrated. Such devices are of considerable interest due to their promise for obtaining sequence-specific information in a faster, simpler and cheaper manner compared to traditional nucleic acid assays.

In the aptasensor approach, ssDNA or RNA are used as the receptor element, named aptamer, which is able to bind the targeted microbe molecule with an affinity and a specificity similar to those of antibody. Aptamers are engineered through an in vitro process called SELEX, which involves iterative cycles of selection and amplification starting from an ss random DNA or RNA library. Cell membranes specific to pathogenic microorganisms or live cells can be targeted in the SELEX. Aptamers appear as alternative recognition tools to replace antibodies as binding reagents in diagnostic assays. The development of aptamer-based biosensors is one of the most


active research topics in the past decade; but detection of microorganisms using aptasensors is a promising area currently underdeveloped. All the recent studies on this topic have demonstrated the great potential of aptasensors for microorganisms screening. With the identification of new aptamer structures for specific bacteria targets, their use in biosensors will increase, and aptasensor approach may find its place in the market to favourably compete with the expensive immunological methods that are the main commercial rapid methods used at the present time.

These two approaches towards DNA-based sensors will be illustrated with several examples with a particular attention to recent advances and trends in selection of biorecognition elements, DNA immobilization strategies and sensing formats.


SIMPLE FIELD TESTS FOR THE DIAGNOSIS OF LIVESTOCK DISEASES: IS THIS AN ACHIEVABLE GOAL? Donald P. King, B. Armson, V. Mioulet and V. Fowler Vesicular Disease Reference Laboratory, The Pirbright Institute, UK [email protected] Rapid and accurate diagnostic tests make an important contribution to programmes to monitor and eradicate infectious diseases that infect animals. Using foot-and-mouth disease (FMD) as an example, this presentation outlines recent progress to develop new field tools for detection of the agents that cause high-impact livestock diseases. The principal driver for this work is to develop tools that can be used locally to assist in decision making. Advances in this area have (i) validated simple-to-use lateral-flow devices for the detection of FMD virus, (ii) evaluated new hardware platforms to allow PCR testing for FMD viral RNA by non-specialists in the field, and (iii) developed new isothermal molecular assays that could be deployed into field settings. Using generic solutions, it is now possible to imagine a new paradigm for how the collection and testing of samples to monitor the spread of important livestock diseases might be achieved in European countries, as well as in the developing world where many of these diseases are endemic.


LESS IS MORE – GENERIC SAMPLE PREPARATION IN RESIDUE ANALYSIS Bjorn J.A. Berendsen, L.A.M. Stolker and M.W.F. Nielen RIKILT Wageningen UR, the Netherlands [email protected] Sample preparation is not a goal in itself, but merely a logical consequence of the detection technique applied, so we see a clear relation between the selectivity of applied sample preparation procedures and the selectivity of detection techniques available. Technology available to residue testing laboratories has advanced significantly with for example the development of high resolution and accurate mass spectrometric (MS) techniques that can be combined with orthogonal separation techniques like ultra-high performance liquid chromatography (UHPLC) and ion mobility spectrometry (IMS). These technological innovations have triggered the development of new analytical multi-residue and even multi-class screening solutions, confirmatory methods and even the ability to discover unexpected (un)known residues. These new methods are improving the effectiveness and efficiency of residue testing programs. With the increasing number of compounds that are analysed simultaneously, analytical methods, including the sample-preparation procedure, have to be applicable to compounds having very different physico-chemical properties. Multiresidue protocols therefore include very generic sample preparation procedures to obtain sufficient recovery for all compounds. As a prerequisite, high sample throughput and therefore low analytical costs should be achieved. The most frequently reported generic sample preparation methods – sometimes including more than 150 different compounds – are a solvent extraction only (SE), solid-phase extraction (SPE) and a quick, easy, cheap, effective, rugged, and safe (QuEChERS) approach. In the simultaneous analysis of multiple classes of veterinary drugs in products of animal origin, SE without further purification is frequently reported as the method of choice. Here, SE includes conventional liquid-liquid extraction of liquid matrices and the liquid extraction of homogenized tissues (e.g. muscle, liver and kidney). To obtain optimal results, the extraction solvent has to be selected in such a way that efficient extraction of the target compounds is obtained, whereas the extraction of matrix constituents remains limited in order to prevent excessive matrix effects. The selection of the solvent therefore depends not only on the target compounds, but also on the matrix. SPE is frequently used as a clean-up technique in the simultaneous analysis of multiple classes of veterinary drugs in products of animal origin. Three types of SPE can be distinguished: (i) on-column SPE, in which the extract is applied onto an SPE cartridge to retain the compounds of interest, followed by a wash step to remove matrix interferences and the subsequent elution of the compounds from the cartridge; (ii) SPE applying a highly organic raw extract onto an SPE cartridge and immediately collecting the eluent for further analysis; and (iii) dispersive SPE (dSPE) by adding sorbent material to a raw extract, followed by shaking and centrifugation and subsequent isolation of the supernatant which is part of the QuEChERS protocol. In general, MEs can be significantly reduced by using an on-column SPE procedure. However, to use an SPE approach for a broad range of compounds, having different physico-chemical properties, the choice of the interaction mechanism is limited to reversed phase or mixed-mode materials. To retain even the polar target compounds, a low organic content of the extract that is applied onto the SPE cartridge is essential, so the extraction solvent should have a high water content or additional procedures have to be applied to change the solvent of the raw extract prior to SPE, which


are usually time consuming. Even though the use of a highly organic extraction solvent (e.g. ACN or ACE) is favourable for high SE recoveries, aqueous solvents are frequently applied if the extraction is followed by an SPE procedure. In other cases, the organic solvent is evaporated before SPE, which is time consuming, or the organic phase is diluted with an aqueous solvent. QuEChERS procedures are well established in the field of pesticide residue analysis and are becoming more popular in veterinary drug residue analysis. A QuEChERS procedure comprises extraction with an organic solvent, phase separation using a high salt content and is usually followed by dSPE. Here especially the type and amount of drying material and dSPE sorbent material are critical factors and depend both on the compounds included in the method and the matrix. In summary, by using these straightforward methods that are usually less time consuming, more compounds can be analysed within a single run. However, as a result of applying a generic, inherently non-selective sample-preparation procedure, the final extract contains a significant amount of matrix constituents that might interfere in the detection and overall method selectivity can be compromised. One should therefore be aware of the pronounced matrix effects and the limited selectivity of the sample-preparation procedure, which might result in more interfering signals and thus a less selective procedure, higher LODs, more variation in the quantitative result and a higher maintenance frequency. To put things into perspective, besides the continuation of the development of generic non-selective sample-preparation methods and the automation of these straightforward procedures, an expected parallel and opposite future trend is towards highly selective sample preparation to produce precise quantitative results at low levels and to be able to comply with regulations regarding confirmation of the identity of a compounds as is mandatory when analysing some compounds having a chiral centre (e.g. chloramphenicol).


SPEEDING UP SAMPLE PREPARATION IN FOOD ANALYSIS Lourdes Ramos Department of Instrumental Analysis and Environmental Chemistry, Institute of General Organic Chemistry of the Spanish National Research Council, Spain [email protected] In food analysis, as in many other application fields, the use of powerful and sophisticated instrumental techniques for final identification and quantitation of the target analytes contrast sharply with the rather conventional techniques in use for previous extraction, purification and concentration steps. This is particularly true in the case of trace analysis, where the low concentrations at which the analytes should accurately be determined obliges to their exhaustive extraction from relatively large amounts of sample to ensure proper detectability. The efforts carried out during the last two to three decades in the field of sample preparation have yielded new, and frequently miniaturized, analytical approaches that have facilitated some degree of integration and automation in the treatment of (relatively simple and/or pristine) liquid matrices. The advances achieved in solid-phase extraction and solid-phase microextraction and recent developments in the field of selective sorbents have been key aspects in this context. On the contrary, advances in the treatment of (semi-)solid foodstuffs have been much more limited. The requirement of performing a quantitative extraction of the target compounds from the (usually very complex) matrix in which they are entrapped have typically forced the use of exhaustive (i.e. non-selective) extraction techniques. For this purpose, solid-liquid extraction and Shoxlet extraction are still widely accepted and used for routine applications and/or for reference purposes. The non-selective nature of these techniques makes the subsequent purification of the obtained extracts before instrumental analysis mandatory. These treatments usually involve highly manipulative multistep procedures where automation or even partial integration are still more the exception than the rule. In this presentation, the feasibility of several modern analytical techniques for exhaustive and efficient, but also faster and environmental friendly, extraction of trace pollutants from fat-containing biological tissues will be evaluated using selected classes of persistent organic compounds (POPs) as model compounds. Approaches based on the use of matrix-solid phase dispersion (MSPD) with cosorbent and enhanced extraction techniques, such as pressurized liquid extraction (PLE) and ultrasonic assisted extraction (UAE), will be discussed and their relative merits and shortcomings evaluated. Special attention will be paid to the case of USE with ultrasonic tip followed by disposable pipette purification due to the rapidity of the approach (complete sample preparation was done in 15 min), minimum amount of reagents (1.5 ml of n-hexane and 0.8 g of acidic silica) consumption and potential for automation. Acknowledgments The Author thanks MINECO for project CTQ2012-32957 and CM for programme ANALISYC-II (S-2009/AGR-1464).


CURRENT GMO SAMPLE PREPARATION AND ANALYSIS APPROACHES BY THE EU REFERENCE LABORATORY FOR GM FOOD & FEED Maddalena Querci Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Italy [email protected] The use of genetically modified organisms (GMOs), their release into the environment, their cultivation, importation and, in particular, their utilisation as food, food ingredients and animal feed, is regulated in the European Union (EU) by a strict legal frame. The underlying principle is that any use, from cultivation to commercialization of GMOs is allowed in the European territory only after Community authorization based on an accurate risk assessment and issued on a case-by-case basis. Within the European legal frame, a key role is played by the European Commission Joint Research Centre (JRC), Institute for Health and Consumer Protection (IHCP), Molecular Biology and Genomics Unit, which hosts the EU Reference Laboratory for GM Food & Feed (EU-RL GMFF). The EU-RL GMFF undertakes, by legal mandate, a series of activities to assure that the legal system is enforced uniformly and effectively across the EU, and that the critical measures required within the process of implementation are available. The principal legal duties and tasks of the EU-RL GMFF, as defined by Regulation (EC) No 1829/2003, include the testing and validation of event specific detection methods for identification and quantification of transformation events in food or feed and the preparation, storage and distribution to national reference laboratories of the required positive and negative control samples. Indeed, the authorisation of a GMO within the EU is only possible if the applicant has provided an event-specific, quantitative detection method that is validated by the EU-RL GMFF according to internationally accepted standards. These validated methods are the basis for the control and monitoring of the authorised GM events in the distribution chain, because they can unambiguously distinguish and quantify the GMO of interest (i.e. the event) from all other possible GMOs and non-GMOs. In the context of Regulation (EC) No 1829/2003, the EU-RL GMFF is assisted in this task by an expert network of regulatory control laboratories established in the EU Member States, known as the European Network of GMO Laboratories (ENGL). This network plays a crucial role in the definition, harmonisation and standardisation of minimum performance criteria that methods for the detection, identification and quantification of GMOs must meet in order to be valid for regulatory use. The EU-RL GMFF and the ENGL, via the establishment of ad-hoc working groups, are constantly engaged in better understanding and improving the technical aspects of GMO sample preparation and analytical approaches. As outcome of this ongoing task, a series of Guidance Documents have been prepared in the past and are currently under elaboration or update, including on sample preparation procedures and method performance requirements. In combination with the well-established polymerase chain reaction (PCR) based methods, the EU-RL GMFF is constantly exploring, developing and/or adopting novel methods and analytical approaches to further improve power and throughput of detection, identification and/or quantification of GMOs. The presentation will provide an overview of such approaches currently under study or already in use at the JRC's Molecular Biology and Genomics unit: from the introduction of a pre-amplification step to cope with limited DNA recovery in difficult samples, to the development of multi-target detection systems to provide control laboratories with a tool to cope with the increasing number and complexity of GMO analyses, to the introduction of digital PCR and of next generation sequencing to enlarge the analytical and discrimination power of currently used techniques.


EVALUATION OF A SINGLE EXTRACTION METHOD FOR USE IN MULTIMYCOTOXIN ANALYSIS Julie L. Brunkhorst, B.R. Malone, J.S. Bierbaum, J.B. Buhr, T. Clarke, S. Dennler-Sima and N.A. McKernan Trilogy Analytical Laboratory, USA [email protected] The use of a single extraction method for the analysis of multi mycotoxins within a single sample can be problematic. Typically at least two separate solvents are required to extract aflatoxins, fumonisins, zearalenone, ochratoxin, deoxynivalenol, T-2 toxin and HT-2 toxin from a matrix. An evaluation was performed to determine if a single extraction solvent could be utilized to efficiently extract the most commonly analyzed mycotoxins. Naturally contaminated mycotoxin reference materials were extracted with various ratios of solvents to determine the one solvent that would extract all the major mycotoxins with the highest recoveries. Acceptable extraction efficiency for all the mycotoxins was obtained using the extraction solvent of acetonitrile/water (80/20).


POTENTIAL OF FRONT-FACE FLUORESCENCE TO MONITOR ACRYLAMIDE AND COLOUR OF POTATO CRISPS Contributed paper A. Acharid, J. Rizkallah, I. Birlouez-Aragon and Pierre Lacotte Spectralys Innovation, France [email protected] In 2002, acrylamide has been identified as a major contaminant in some severely heat-treated food products. This is particularly the case for potato products, and especially potato crisps. Currently, the most common method to measure acrylamide content in potato crisps is liquid or gas chromatography coupled to mass spectrometry. But such techniques are expensive and time-consuming. Rapid and simple methods are needed to be implemented near the manufacturing plants to comply with European Commission guidance values. In addition, rapid techniques can help developing acrylamide mitigating strategies that preserve the sensorial profile of the product. We present an analyser based on a new front-face fluorescence technology, and demonstrate its potential to simultaneously predict colour and acrylamide in potato crisps. Starting from 3 different varieties of potatoes with various levels of asparagine and reducing sugars, we managed to establish satisfying calibration models over acrylamide content, thanks to multilinear regression between PARAFAC decomposition scores and the chromatographically measured acrylamide or colorimetric data. 100 samples were analyzed over 9 months for water content, colour and acrylamide using both ELISA and LC-MS/MS. The three varieties were stored for different times and were from different geographical origin. The frying process was carried out on 3 different frying tunnels, so that the total variability was high. A first PCA allowed identifying the structure of the variables and building some groups essentially driven by the potato variety. One model per variety was firstly built with satisfactory prediction quality. It will be discussed how these models can be merged to develop a general model and the level of robustness of the prediction by analyzing the prediction error observed on new samples. The colour was similarly predicted, allowing comparison of acrylamide levels for a given target colour. In conclusion, front face fluorescence seems a powerful tool to predict in real time colour and acrylamide content of potato crisps for routine quality control at the production line.


THE POTENTIAL OF ‘-OMICS’ IN CHEMICAL ANALYSES OF FOOD, FEED AND WATER Hanspeter Naegeli Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse [email protected] Cell-based assays provide a wide range of functional sensors (or ‘cytosensors') for the detection of chemical hazards. In fact, taking advantage of their immense adaptive plasticity, living cells respond with characteristic reaction patterns to potentially adverse chemical stimuli. Therefore, the fundamental hypothesis underlying this cytosensor strategy is that toxicant-specific changes of cell morphology, function and subcellular or molecular composition reveal the presence of chemical insults. In my presentation, I will discuss the predictive power of cytosensors for the identification of contaminants or residues and the future significance of this cell-based strategy for risk assessments in the food production chain. It is expected that broad-spectrum ‘fingerprinting’ assays (transcriptomics, proteomics, metabolomics or other ‘-omics’) will gain importance in the evaluation of mixture effects, the functional analysis of food additives or supplements, the analysis of risk/benefits of novel foods, the monitoring of hazards associated with food storage or processing and the detection of emerging or unexpected contaminants. Biotechnological advances and flexible instrumental platforms provide powerful methods (for example reporter gene assays, low- or high-density microarrays, multi-channel flow cytometry, tandem mass spectrometry) to measure such molecular fingerprints, thus providing new opportunities for an effect- and risk-based control of food quality and safety. Future developments will increase the cytosensor responsiveness by integrating multiple genetic constructs coding for dedicated receptors. Miniaturized chip culture formats will be developed towards a multi-contaminant screening platform and cancer cell lines will be progressively replaced by immortalized primary cells that reflect more faithfully the biologic response of whole organisms. In particular, 3-D tissue culture systems facilitate key signalling processes such as communication between cells and interactions with the extracellular matrix and, hence, mimic in vivo responses that should be adopted to enhance the performance of cytosensors. Another predicted development is the use of induced pluripotent stem cells, which provide a rich biological source with many advantages for cytosensor applications, as they can be differentiated into different cell types or even complex tissues.


LASER OPTICAL SENSOR, A LABEL-FREE RAPID ON-PLATE PATHOGEN SCREENING TOOL Arun K. Bhunia Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, USA [email protected] Rapid pathogen testing tools are in high demand. The advances in biophysical methods in diagnostics have created an increasing interest in light scattering technologies, such as spectroscopy, surface plasmon resonance, and optical forward scattering [1]. We have developed a novel, label-free on-plate colony screening tool using light scattering technology, called BARDOT (bacterial rapid detection using optical scattering technology) for pathogens from food, clinical specimens or environmental samples. When a red laser is illuminated in the centre of the bacterial colony, it generates unique scatter signature for each phylogeny. For detection and identification of unknown pathogens or non-pathogenic bacteria, the scatter patterns are compared with the scatter image libraries [2-4]. There is a strong correlation between bacterial genotype and phenotype, thus chromogenic differential media or highly selective pathogen-specific media can be used for screening of pathogens at the genus, species or even at the serovar level. After a brief enrichment in broth, or immunomagnetic separation, test samples are plated on selective/differential agar media for colony growth to a diameter of 1.1 ± 0.2, which is then screened by BARDOT. It has been shown to be effective for detection of Vibrio [3], Salmonella [4], Escherichia coli O157:H7, Bacillus, etc., from dairy, meat, vegetable and seafoods within 24 h [2]. This technology is based on the gold-standard culturing method and is suitable for detection of 1 cell/g. BARDOT positive colonies can be further verified by PCR, genetic fingerprinting, matrix-assisted laser desorption/ionization (MALDI), immunoassays or whole or partial genome sequencing. Besides, BARDOT can be used for bacterial community analysis on products and also for process verification and hygiene monitoring in a food processing plant. Acknowledgements Funding: US Department of Agriculture project number 1935-42000-072-02G. References 1. Bhunia, A.K., 2011. Rapid pathogen screening tools for food safety. Food technology 65: 38-43. 2. Banada, P.P., Huff, K. Bae, E.,Rajwa, B., Aroonnual, A., Bayraktar, B., Adil, A., Robinson, J.P.,

Hirleman, E.D. and Bhunia, A.K., 2009. Label-free detection of multiple bacterial pathogens using light-scattering sensor. Biosensors and Bioelectronics 24: 1685-1692.

3. Huff, K., Arronnual, A., Littlejohn, A.E.F., Rajwa, B., Bae, E., Banada, P.P., Patsekin, V., Hirleman, E.D., Robinson, J.P., Richards, G.P. and Bhunia, A.K., 2012. Light-scattering sensor for real-time identification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate. Microbial Biotechnology 5: 607-620.

4. Singh, A.K., Bettasso, A.M., Bae, E., Rajwa, B., Dundar, M.M., Forster, M.D., Liu, L., Barrett, B., Lovchik, J., Robinson, J.P., Hirleman, E.D. and Bhunia, A.K., 2014. Laser optical sensor, a label-free on-plate Salmonella enterica colony detection tool. mBio 5: e01019-01013.


RAPID METHODS FOR DETECTION OF BOTULINUM TOXIN-PRODUCING CLOSTRIDIA AND THEIR TOXINS: THE STATE OF THE ART IN EUROPE AND IN THE WORLD Fabrizio Anniballi National Reference Centre for Botulism, Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Italy [email protected] The species Clostridium botulinum and some strains of Clostridium baratii and Clostridium butyricum are classified as botulinum neurotoxin-producing clostridia. The common feature of these organisms is the capability of producing the most poisonous toxins known to humans. Minute amount of botulinum neurotoxins are sufficient to cause botulism and even death. The consumption of small quantity of food in which botulinum neurotoxin-producing clostridia have grown can results in botulism intoxication. A single gram of crystalline toxin, when evenly dispersed and inhaled, would kill one million people. Because of their high toxicity and relative ease in production, botulinum toxins have been represented as a biological weapon and bioterrorism threat agent. Therefore, the detection of botulinum neurotoxins and neurotoxigenic microorganisms is crucial for public health. The current gold standard method for detection of botulinum toxins relies on the mouse bioassay, which presents several drawbacks, such as the need of up to 96 h to obtain results, the use of large number of animals, as well as the availability of high skilled personnel and special facilities. The availability of rapid and specific methods for testing of botulinum neurotoxins and botulinum neurotoxin-producing clostridia is a prerequisite for establishing prompt public health response and monitoring programmes to track and trace these agents. With the aim of reducing and replacing animals intended for diagnostic use, during the last two decades several rapid and alternative methods have been developed, although none of them are yet fully capable to supplant mouse bioassay. Performances, strengths, weakness as well as diagnostic applicability of ELISA, Endopep-MS, cell-based assays, and PCR-based methods will be discussed.


AN INTEGRATED AND ULTRASENSITIVE BIOSENSOR FOR THE DETECTION OF PATHOGENS M. Safavieh1, M.U. Ahmed2 and Mohammed Zourob3

1Institut national de la recherche scientifique, Université du Québec, Canada, 2Faculty of Science, Universiti Brunei Darussalam, Brunei Darussalam and 3Center of Biomedical Engineering, Cranfield University, UK [email protected] Nucleic acid tests are well established rapid methods that provide high-sensitive and specific results. The most commonly used technique is PCR, which has been used for more than two decades now. However, the requirements for precise thermal cycling make it complicated to engineer point-of-care (POC) devices based on this technique. Instead, isothermal amplification techniques, such as recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA) and loop-mediated isothermal amplification (LAMP), have been developed operating at a single temperature. However, despite providing many advantages, some of these techniques need multiple enzymes for operation, while others need elaborated methods, such as designing molecular probes, to incresase specificity. Here, we present a compact device based on colorimetric and real-time electrochemical monitoring of LAMP for detection of pathogenic bacteria. The device can be fabricated at low production cost using simple methods and easy-to-access materials on a flexible substrate. After explaining the device assembly, the colorimetric and real-time electrochemical monitoring using hydroxynapthtol blue (HNB) and a dipyridophenazine (DPPZ)-type osmium complex, respectively, will be discussed. The device is capable of detecting, for example, 30 cfu/ml Escherichia coli. References 1. Su, L., Jia, W., Hou, C. and Lei, Y., 2011. Microbial biosensors: a review. Biosensors and

Bioelectronics 26: 1788-1799. 2. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T.,

2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28: e63. 3. Goto, M., Honda, E., Ogura, A., Nomoto, A. and Hanaki, K., 2009. Colorimetric detection of loop-

mediated isothermal amplification reaction by using hydroxy naphthol blue. BioTechniques 46: 167-172.

4. Kivlehan, F., Mavré, F., Talini, L., Limogesand, B. and Marchal, D., 2011. Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids. Analyst 136: 3635-3642.

5. Safavieh, M., Ahmed, M.U., Tolba, M. and Zourob, M., 2012. Microfluidic electrochemical assay for rapid detection and quantification of Escherichia coli. Biosensors and Bioelectronics 31: 523-528.

6. Safavieh, M., Ahmed, M.U., Sokullu, E., Ng, A., Braescu, L. and Zourob, M., 2014. A simple

cassette as point-of-care diagnostic device for naked-eye colorimetric bacteria detection. Analyst 139: 482-487.

7. Safavieh, M., Ahmed, M.U., Ng, A. and Zourob, M., 2014. High-throughput real-time electrochemical monitoring of LAMP for pathogenic bacteria detection (in press; doi:10.1016/j.bios.2014.02.002).


DNA MICROARRAYS AS VERSATILE TOOLS FOR FRESHWATER QUALITY TESTING Holger Eickhoff, W. Weigel and H. Dijk Scienion AG, Germany [email protected] µAQUA is the acronym for the EU research project ‘Universal microarrays for the evaluation of fresh-water quality based on detection of pathogens and their toxins’, which has been funded by the 7th Framework Programme of the European Commission. 12 partners from eight countries are committed to the project (http://microqaqua.eu). The overall project goal is to develop efficient, sensitive, robust, rapid and inexpensive aquatic biosensors to monitor various aspects of water quality as part of the strategy for control and prevention of diseases caused by waterborne pathogens and algal toxins. μAQUA aims to design and develop a universal microarray chip for a water-based high-throughput detection of known and emerging pathogens (bacteria, viruses, protozoa and cyanobacteria). This information is used to assess water quality by monitoring the presence of select bio-indicators. The present chip version allows for simultaneous testing of almost 50 organisms. These organisms include pathogens that are considered to be potentially most dangerous for human health. The biochip content represents the standard pathogens whose presence is tested by all national water authorities in Europe. In addition to pathogen specific capture sequences, the microarray is able to detect several diatom algal species that are known to react rapidly and sensitively to water quality changes and are used universally as biomarkers for water quality assessment.


RAPID LATERAL FLOW DETECTION OF LEGIONELLA-SPECIFIC RNA AND DNA AMPLICONS Contributed paper Heleen van den Bosch Food & Biobased Research, Wageningen UR, the Netherlands [email protected] Legionella outbreaks have great sanitary, economic and social implications. The currently used diagnostic reference method to detect Legionella spp. is based on bacterial culturing, which could take more than a week to obtain conclusive results. The European 7th Framework Programme project ‘PINVIALEG’ addressed the detection of Legionella pneumophila type 1 within a couple of hours. NASBA and PCR amplicons were detected by a lateral flow readout platform (nucleic acid lateral flow immunoassay; NALFIA). NASBA was chosen to be able to identify viable Legionella only. However, the RNA template and the NASBA amplicon are sensitive to degradation and, therefore, difficult to work with. Hence, in this project a test based on PCR (with DNA templates and amplicons) was developed as well. In the PCR-NALFIA, a primer set specific for Legionella spp. was taken as a control for comparison with conventional methods. Furthermore, this primer set can detect disease causing Legionella species other than L. pneumophila. The combination of molecular methods with lateral flow results in a rapid, on-site test for the detection of Legionella spp. and L. pneumophila type 1. NASBA specific for L. pneumophila type 1 was developed yielding single strand anti-sense RNA amplicons. To detect such amplicons in NALFIA, two oligonucleotide probes were designed that hybridise specifically with these amplicons. The probes were labelled with biotin and digoxigenin, respectively. Consequently, the final product could be sandwiched between an anti-digoxigenin antibody immobilised on the nitrocellulose membrane and neutravidin bound onto the surface of carbon nanoparticles (NASBA-NALFIA). Furthermore, a multiplex PCR for Legionella spp. and L. pneumophila was developed. Both tests (NASBA-NALFIA and PCR-NALFIA) have been evaluated successfully within the PINVIALEG consortium; they show good specificity, inclusivity and sensitivity. Possible applications are analysis of cooling tower waters and tap waters from a variety of sources (e.g. hotels, swimming pools).


CHARACTERISATION OF MOVEMENT AT THE NANOSCALE: A FAST DETERMINATION OF BACTERIAL RESISTANCE TO ANTIBIOTICS Giovanni Longo Laboratory of the Physics of Living Matter, École polytechnique fédérale de Lausanne, Switzerland [email protected] The importance of the characterisation of movement in biological samples ranges from the fields of biology and microbiology to pharmaceuticals and drug development. For instance, the movement of living systems can deliver useful information regarding the metabolism of the specimen under investigation and can be used to define their response to external stimuli. In this presentation, I will show how nanomechanical sensors can be used to characterize the nano-sized fluctuations of specimens of interest in the fields of biology, microbiology and pharmacology. I will show how these devices can be extremely fast tools (minutes, compared to hours or days) to characterize bacterial resistances (Figure 1). Antibiotics represent one of humanity’s most important medical inventions, yet antibiotic resistance has emerged as a very significant health care problem due to the extensive use and misuse of antibiotics in human and veterinary medicine. It will be demonstrated how this system can quantitatively determine, in less than 30 minutes, the response to antibiotics of bacterial species including slow-growing microorganisms. Such extremely fast characterizations have been exploited to study Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) species (Figure 2) and preliminary results will be presented of the application of this technique to study mycobacteria.

Figure 1. The nanomotion sensor detects movements of samples deposited on its surface. Bacteria on the sensor’s surface cause its fluctuation.

Figure 2. Characterisation of bacterial resistances. Response of Escherichia coli to the exposure to antibiotics and reconstruction of a complete antibiogram.


Since the technique is versatile and simple, it can be applied to several systems of interest in the fields of medicine, drug development or microbiology, including yeasts and fungi, plant and mammalian cells and, finally proteins and protein complexes. These studies have defined how this new diagnostic tool can be used to characterise biological samples and how this information can be used to understand better their metabolic pathways. The speed and sensitivity of the technique will have a massive impact, with applications in general and molecular biology, microbiology, drug development and medicine. Its versatility allows also foreseeing its possible future use to identify and characterize life in hostile environments. References 1. Longo, G., Alonso-Sarduy, L., Marques Rio, L., Bizzini, A., Trampuz, A., Notz, J., Dietler, G. and

Kasas, S., 2013. Rapid detection of bacterial resistance to antibiotics using AFM cantilevers as nanomechanical sensors. Nature Nanotechnology 8: 522-526.

2. Kasas, S., Longo, G., Dietler, G. and Alonso-Sarduy, L. Nanoscale motion detector. Patent no. PCT/IB2011054553.

3. Aghayee, S., Benadiba, C., Notz, J., Kasas, S., Dietler, G. and Longo, G., 2013. Combination of fluorescence microscopy and nanomotion detection to characterize bacteria. Journal of Molecular Recognition 26: 590-595.

4. Alonso-Sarduy, L., Longo, G., De Los Rios, P., Benedetti, F., Vobornik, D., Dietler, G. and Kasas, S. Real-time measurement of protein ligand-induced conformational changes using nanomechanical detection. Nature Communications (under review).

5. Kasas, S., Alonso, L., Tournu, H., Notz, J., Dietler, G. and Longo, G. A nanoscale life detector (in preparation).


MYCOHUNT: A RAPID BIOSENSOR FOR THE DETECTION OF DEOXYNIVALENOL IN WHEAT DUST Melanie Sanders1, Y. Guo1, A. Galvita1, A. Wootsch2, A. Uderszky2, A. Ágoston2, F. Martens3, J. Jantra4 , G. Haesaert3, M. Hedström4, B. Mattiasson4 , M. Eeckhout3, S. De Saeger1 and E. Karacsonyi2 1Department of Bio-analysis, Faculty of Pharmaceutical Sciences, Ghent University, Belgium, 2MFKK Invention and Research Center Ltd., Hungary, 3Department of Applied Biosciences, Faculty of Bioscience Engineering, Ghent University, Belgium and 4Department of Biotechnology, Lund University, Sweden [email protected] Mycotoxins are secondary metabolites produced by several fungi, e.g. Aspergillus ssp., Penicillium ssp., and Fusarium ssp., under natural conditions. The contamination of food and feed with mycotoxins poses a serious threat to the health of humans and animals. Mycotoxicoses are diseases caused by mycotoxin ingestion, inhalation or skin contact. The effects of mycotoxins in animals are diverse, varying from immune suppression to death in severe cases, depending on toxin-related (type of mycotoxin consumed, level and duration of intake), animal-related (animal species, sex, age, breed, general health and immune status) and environmental (farm management, hygiene, temperature) factors. Despite extensive efforts during crop growth, harvesting or storage, it is not possible to completely avoid mycotoxin contamination. Fusarium toxins called trichothecenes form the most commonly found mycotoxin group in Europe, among them deoxynivalenol occurs at highest rate. The mycotoxin deoxynivalenol, also known as DON or vomitoxin, is one of about 150 related compounds of trichothecenes and is of particular relevance to the wheat crop. Proposers of the MycoHunt project expressed their need of a fast, on-site, effective sampling method by obtaining and testing the air and light/low weight particles, such as dust, which is created when tons of bulk grains are moved and transported through closed systems. Wheat dust and air can be considered as a representative sample, as it is generated by all sections of the wheat pile. However, so far the measurement of mycotoxin-contaminated dust has been addressed in occupational health-related topics. Testing of wheat dust and air gives a relatively low amount of sample, which requires a highly sensitive, DON-specific measurement methodology. Consequently, MycoHunt proposed to use an amperometric immunoassay technology to which monoclonal antibodies were developed. The anti-DON monoclonal antibodies were immobilized on CNBr-activated Sepharose beads and a direct competitive flow immunoassay was carried out using the automatic flow injection system named VersAFlo (Capsenze HB, Sweden) coupled to an amperometric detector. Acknowledgements This work was executed thanks to the contribution of HGFA (Hungary), SEEDYZ (Greece), CESFAC (Spain), ASEMAC (Spain), Synagra (Belgium), EASRET (Greece), Impuls Ltd. (Poland), OSV Srl., (Italy), Dunagabona Ltd. (Hungary), Dimitriaki S.A. (Greece), ETIA (France), Bioforum S.A. (Greece), EST Ltd. (UK), Veluwe Granen (the Netherlands). The research leading to these results has received funding from the European Community’s 7th Framework Programme (FP7/2007-2013) under grant agreement no. 243633.


DROPLET DIGITAL PCR, A CUTTING-EDGE TECHNOLOGY FOR QUANTITATIVE ANALYSIS OF FOOD AND FEED SAMPLES: GMOS AS A CASE STUDY Dany Morisset1,2, D. Štebih1, K. Gruden1 and J. Žel1

1Department of Biotechnology and Systems Biology, National Institute of Biology, Slovenia and 2CropDesign N.V. - A BASF Plant Science Company, Belgium [email protected] In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed. We have investigated the performance of droplet digital PCR (ddPCR) to quantify the GMO contents in food samples. In addition to the quantitative aspects, sensitivity and behaviour of ddPCR with difficult samples containing inhibitors of nucleic acid amplification or with samples with very low amount of target were evaluated.


ANALYSIS AND RISK OF FULLERENES AND OTHER CARBON-BASED NANOMATERIALS IN THE TOTAL ENVIRONMENT Marinella Farré1, J. Sanchís1, M. Olmos1 and D. Barceló1,2 1Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research of the Spanish National Research Council, Spain and 2Catalan Institute of Water Research Spain [email protected] Natural sources of nanoparticles (NPs) in the atmosphere include natural event, such as volcanic eruptions, forest fires, hydrothermal vent systems and biological processes. However, the natural background of NPs in the atmosphere is low in comparison to those caused by anthropogenic process, such as diesel- and gasoline-fuelled vehicles and stationary combustion sources, which for many years have contributed to the particulate material in the atmosphere in a wide size range, including NPs. Carbon based nanomaterials (NMs) of different kinds have also been reported to occur in ordinary hydrocarbon flames [1, 2] and emitted from common heat sources [1,3]. It has been assessed that the amount of incidental NPs in the atmosphere due to human activity is more than 36% of the total particulate number concentrations, and the forecast for the next coming years is that there will be a strong increase due to emissions from nanotechnology industry. Fullerenes have attracted considerable interest in many fields of research and have found numerous applications, which will be dramatically increased during the following years. Therefore, it is essential to determine the risk that these materials may pose to human health and the environmental [4,5]. Most of the current environmental research on the impact of carbon-based NMs has been directed to elucidate ecotoxicological aspects and only few quantitative analytical methods for measuring NPs in natural systems are available, which results in a serious lack of information about their occurrence in the environment. To better understand the whole environmental risk associated to nano-sized pollution, their relations with other contaminants and their fate and behaviour, it is of high importance to evaluate their presence in the environment. In this presentation, different analytical approaches based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) will be introduced and environmental results of the investigation of the occurrence of fullerenes (C60, C70, C76, C78 and C84 fullerene, C60 pyrrolidine tris-acid ethyl ester, (6,6)-phenyl-C61 butyric acid butyl ester and (6,6)-thienyl C61 butyric acid methyl ester) in different environmental matrices [6-8] (suspended materials of wastewater and river water, air borne particulate, soils and sediments) will be discussed. Preliminary data on the characterization of nanomaterials under real environmental scenarios of exposure and the toxicity of fullerene soot and other carbon based nanomaterials, such as multiwall carbon nanotubes and graphene, will be presented. Preliminary data on their synergistic/antagonistic effects with representative xenobiotics susceptible to be present in the same environmental compartments will be as well presented and discussed. Acknowledgements This work was supported by the by the Nano-Trojan (CTM-2011-24051).

References 1. Murr, L.E. and Soto, K.F., 2005. A TEM study of soot, carbon nanotubes, and related fullerene

nanopolyhedra in common fuel-gas combustion sources. Materials Characterization 55: 50–65. 2. Murr, L.E. and Garza, K.M., 2009. Natural and anthropogenic environmental nanoparticulates:

their microstructural characterization and respiratory health implications. Atmospheric Environment 43: 2683-2692.


3. Murr, L.E., Soto, K.F., Guerrero, P.A., Lopez, D.A. and Ramirez, D.A., 2004. TEM observations of carbon nanotubes and related nanocrystal aggregates collected from domestic and commercial fuel gas combustion sources. Microscopy and Microanalysis 10 Supplement S02: 410-411.

4. US Environmental Protection Agency, 2007. Nanotechnology White Paper. Washington, DC, USA.

5. Sanchís, J., Farré, M. and Barceló, D., 2012. Analysis and fate of organic nanomaterials in environmental samples. Comprehensive Analytical Chemistry 59:131-168.

6. Sanchís, J., Berrojalbiz, N., Caballero, G., Dachs, J., Farré, M. and Barceló, D., 2012. Occurrence of aerosol-bound fullerenes in the Mediterranean Sea atmosphere. Environmental Science and Technology 46: 1335-1343.

7. Farré, M., Pérez, S., Gajda-Schrantz, K., Osorio, V., Kantiani, L., Ginebreda, A. and Barceló, D., 2010. First determination of C60 and C70 fullerenes and N-methylfullero-pyrrolidine C60 on the suspended material of wastewater effluents by liquid chromatography hybrid quadrupole linear ion trap tandem mass spectrometry. Journal of Hydrology 383: 44-51.

8. Sanchís, J., Božović, D., Al-Harb, N., Silva, L.F., Farré, M. and Barceló, D., 2013. Quantitative trace analysis of fullerenes in river sediment from Spain and soils from Saudi Arabia. Analytical and Bioanalytical Chemistry 405: 5915-5923.


A SIMPLE AND SENSITIVE SCREENING ASSAY FOR ANTIVIRAL NEURAMINIDASE INHIBITORS IN POULTRY Contributed paper Mariel G. Pikkemaat1, F. Ligtermoet1, B.J. Berendsen1, R.S. Wegh1, J. Peters1 and S. Yongkiettrakul2 1RIKILT Wageningen UR, the Netherlands and 2Protein-Ligand Engineering and Molecular Biology Laboratory, BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand [email protected] Neuraminidase inhibitors like oseltamivir (Tamiflu) and zanamivir (Relenza) are an important class of antiviral drugs for treatment of influenza in human medicine. After the outbreak of avian influenza A (H5N1) in Asia (2004), the WHO strongly requested to ban the use of antiviral drugs in animals to prevent development of drug resistance. The recent H7N9 outbreak raised the alert levels again. To detect abuse of these antivirals in poultry, a simple high-throughput bioassay screening method was developed. The assay uses a recombinantly expressed neuraminidase enzyme fragment [1] and is based on an endpoint fluorescence measurement employing a fluorogenic substrate, 2’-(4-methylumbelliferyl)-α-D- N-acetylneuraminic acid (MUNANA). Active neuraminidase will hydrolyse MUNANA to a fluorescent product, so the presence of neuraminidase inhibitors will subsequently yield a reduced fluorescence signal compared to an uncontaminated reference sample. Poultry tissue fluid is analyzed without any additional sample preparation and the assay can be scaled to a high-throughput microtiter format, requiring only as little as 10 µl sample and a turnaround time of less than 90 minutes. The assay was validated according to 2002/657/EC using blank and spiked poultry muscle samples, as well as incurred material from an animal experiment. A total of 100 routine monitoring poultry samples were analyzed to determine a baseline fluorescence signal. This yielded a fluorescence cut off value of <69% (compared to a negative control without muscle matrix) below which samples were considered non-compliant. To determine the detection capacity, the assay was performed with poultry spiked at several concentrations of zanamivir and oseltamivir carboxylate. Both oseltamivir carboxylate and zanamivir spiked tissue samples already showed significant inhibition at 1 µg/kg, while these compounds could be detected with 95% reliability at 5 µg/kg. The results with the incurred tissue confirmed this very low detection capacity, showing that this new method is convenient, fast and extremely sensitive. References 1. Yongkiettrakul, S., Boonyapakron, K., Jongkaewwattana, A., Wanitchang, A., Leartsakulpanich,

U., Chitnumsub, P., Eurwilaichitr, L. and Yuthavong, Y., 2009.Avian influenza A/H5N1neuraminidase expressed in yeast with a functional head domain. Journal of Virological Methods 156: 44-51.


QUANTITATIVE TARGETED RESIDUE AND RETROSPECTIVE DATA ANALYSIS OF RELEVANT PESTICIDES, ANTIBIOTICS AND MYCOTOXINS IN BAKERY RAW MATERIALS AND FOOD COMMODITIES BY LC-HRMS EXACTIVE™ (ORBITRAP TECHNOLOGY) Michele Suman1, E. De Dominicis2, E. Gritti2 and D. Catellani1 1Barilla Food Research Labs, Italy and 2R&D Department Chelab-Silliker, Mérieux NutriSciences Company, Italy [email protected] In a global food market, the industry requires not only ‘traditional’ multiresidues MRM mass spectrometry techniques to quantify a list of targeted compounds. In fact, there is a growing interest for analytical strategies devoted to cross categories of chemical contaminants and non-targeted approaches capable of detecting new molecules potentially hazardous. We have developed a reliable method for the simultaneous quantitative determination of various categories of relevant food contaminants at trace levels in both raw materials and finished products, thanks to a straightforward sample preparation together with a multi-target approach based on UHPLC coupled with LC-HRMS Exactive™, a non-hybrid single stage Orbitrap mass spectrometer (used at resolution FWHM 50.000). The full validation of all the target molecules in bakery products has been performed: linearity in the matrix, LOD, LOQ, recovery at two concentration level (LOQ and 10xLOQ) and precision in terms of repeatability and reproducibility. Extracts of blank, naturally contaminated and fortified minicakes, prepared through a combined use of industrial and pilot plant production lines, were analysed at different concentration levels. Good quantitative results have been obtained for most of the tested molecules (pesticides, aflatoxins, zearalenone, trichothecenes, sulphonamides and phenicol antibiotics). This method has been validated for bakery matrices, but the scope can be extended to other food commodities. The potential presence of further molecules that can have toxicological implications (such as 3-acetyl-deoxynivalenol or deoxynivalenol-3-glucoside) have been evaluated exploiting retrospective investigations through post-acquisition processing techniques. References 1. De Dominicis, E., Commissati, I. and Suman, M., 2012. Targeted screening of pesticides,

veterinary drugs and mycotoxins in bakery ingredients and food commodities by liquid chromatography-high-resolution single-stage Orbitrap mass spectrometry. Journal of Mass Spectrometry 47: 1232-1241.

2. De Dominicis, E., Suman, M., Gritti, E. and Catellani, D. 2013. In: Proceedings of AOAC Europe - ASFILAB International Symposium, 18-19 April 2013, Paris, France.


COLLECT DATA, NOT SAMPLES: APPLICATION OF FIELD PORTABLE GC-MS FOR ON-SITE FOOD SAMPLING AND ANALYSIS Joeri Vercammen IS-X, Belgium [email protected] The Tridion-9 GC-TMS is the smallest fully functional field portable GC-MS system currently available on the market. It is a robust device, which unites all recent technological advances with respect to field sampling, ultrafast GC and miniature MS in one single system. On-site analysis is becoming increasingly important, particularly when time, money and/or human health and safety are at stake. The IS-X team has completed several studies with the Tridion GC/MS focusing on a variety of applications in petrochemistry, forensics, environmental and food analysis. In this presentation we will briefly highlight the various technologies applied in the system and illustrate its possibilities by means of several real-life applications, such as shipping freshness, contamination to long time storage, and source determination.


POINT-OF-CARE DEVICE FOR RAPID DETECTION OF MICROORGANISM IN FOOD Maria Carmen Morant-Miñana CIC microGUNE, Spain [email protected] The needed of doing laboratory operations on a small scale using point of care (POC) devices is inviting the research community to develop diagnostic technologies for global public health [1]. A relevant aspect in that issue is food, which is one of the most important resources, but also causes important illness in the human being. In 2010, a total of 212,064 Campylobacter cases in humans were reported in Europe [2]. This represents an increase for the fifth consecutive year with 7% more cases compared to 2009. Several strategies have been applied for their identification and diagnosis. Despite current diagnosis methods are time-consuming, very few examples of portable diagnosing tools are available. The main reason is that the device should be small, cheap and capable of performing near-real-time analysis. Lab on a chip (LOC) has been described as a perfect platform to accomplish these requirements [3]. In this context, a new polymer-device involving disposable components with multiple chamber and complex microfluidics control elements has been reported [4]. The device is a complete real-time polymerase chain reaction assay (RT-PCR) with improved sensitivity in just over 30 minutes. The microfluidic operation process of the device consists of the next steps: (i) clinical sample preparation; (ii) thermal cell lysis and magnetic particle target concentration; and (iii) DNA amplification by RT-PCR. The major drawback of measuring the resultant fluorescent signal of the thermally cycled mixture as a detection system is that the dye will bind to any double-stranded DNA in the reaction, which will result in an overestimation of the target concentration. As array technologies based on the immobilisation of multiple specific DNA fragments onto solid surfaces and the detection of DNA duplexes resulting from hybridisation with complementary target DNA appeared, electrochemistry has become an interesting approach for its applications in the field of sensors for DNA hybridisation. The main advantages of DNA electrochemical biosensors are that they are faster, simpler and cheaper than traditional diagnostic methods. Thiolated DNA can be well ordered onto a gold surface by using of spacer molecules between DNA and a thiol moiety [5]. Here, we report the development of a biosensor part of a complete sensing microdevice applied to the detection of Campylobacter jejuni. The analytical method relies of the deposition of thin-film based gold microelectrodes on polymeric substrates and subsequent modification of the surface with a DNA-thiolated probe. This is a first approach for the fabrication of a lab-on-a-chip biodevice integrating DNA sensor technology into a microfluidic system, which is believed to perform an automated and complete assay, including sample preparation, PCR amplification, and electrochemical detection. References 1. Daw, R. and Finkelstein, J., 2006. Lab on a chip. Nature 442: 367. 2. European Food Safety Authority, 2012. The European Union summary report on trends and

sources of zoonoses, zoonotic agents and food-borne outbreaks in 2010. EFSA Journal 10: 2597. 3. Yoon, J.-Y. and Kim, B., 2012 Lab-on-a-chip pathogen sensors for food safety. Sensors 12:

10713-10741. 4. Verdoy, D., Barrenetxea, Z., Berganzo, J., Agirregabiria, M., Ruano-López, J.M., Marimón, J.M.

and Olabarria, G., 2012. A novel real-time micro-PCR based point-of-care device for Salmonella detection in human clinical samples. Biosensors and Bioelectronics 32: 259-265.

5. Paleček, E. and Bartošik, M., 2012. Electrochemistry of nucleic acids. Chemical Reviews 112: 3427-3481.


AIRBORNE BACTERIA: FROM MICROBIAL PHYSICS TO RAPID IDENTIFICATION Paul Maguire1, E. Bennet2, D. Diver2, C. Mahony1, D. Mariotti1, D.A. McDowell3, H. Potts2 and D. Rutherford1,3 1Nanotechnology & Integrated Bio-Engineering, University of Ulster, UK, 2Department of Physics, University of Glasgow, UK and 3Food Microbiology Research Group, University of Ulster, UK [email protected] New methods of detecting pathogenic bacteria which avoid the limitations of current culture, biosensor or spectrometric approaches are under active investigation and a successful breakthrough would have a significant impact in many arenas that directly affect human health and welfare, such as bio-security, airborne hospital-acquired infections, clinical diagnosis, and food safety. The threat of pathogenic agent dispersal has exercised governments worldwide in the past decade and rapid advances in biotechnologies are creating new vulnerabilities. Advances in medical therapies, e.g. chemotherapy, have led to greater numbers of immune-compromised patients who are highly susceptible to dangerous hospital-acquired opportunistic infections. While detection strategies, instrumentation and protocols vary considerably depending on application, the search for an ‘instant’ pathogen recognition system provides scientists with an exciting goal and, of course, an immense challenge. We would also like such a system to be inexpensive, autonomous, robust and reliable and although it may not be available soon, it is worth looking at new approaches that might get us there. New technologies often follow either a fast/portable or a quantitative/accurate path but advances in microfluidics and lab-on-chip platforms for multiplexed detection aim to blur this distinction. Rapid detection strategies currently under investigation or development involve multichannel immunoassays, real-time PCR, electrokinetic microarrays, conjugate siderophores, pathogen-specific antibodies and bioluminescence, e.g. CANARY, organism and whole cell-based biosensors, natural and synthetic biosensor receptors, among others. New biosensor transduction techniques include terahertz spectroscopy, leaky waveguides and acoustic resonators as examples. From this vibrant research field we can expect on-going innovation and new product breakthroughs. There is considerable scientific interest in gaining greater insight into the physical properties and mechanisms involved in biological processes and organisms; the importance of forces in biology or the physics of infection are examples of current areas of exploration where research in the physical, chemical and biological sciences might overlap at a fundamental level. These new directions will of course require the application of new measurement techniques and while these will be focussed on precise and controlled laboratory experimentation, there is potential scope for their development into the rapid methods arena. The ‘biosensor’ can be defined in simple terms as a biological ‘material’ integrated with a physiochemical transducer. Alternative detection strategies involve measurements related to the physical properties of the species of interest. These include, most notably, mass spectrometry, vibrational spectroscopy and, latterly, field-flow fractionation. However in the case of bacteria, properties such as mass are not measured directly but are inferred through e.g. bio-/chemical rules, associative pattern recognition and/or database feature matching and this has implications for development of rapid and autonomous instrumentation. The fundamental physical properties of any object can be listed. Examples include size, shape, mass, mechanical (hardness, elasticity etc), electrical (total, charge, polarisation, distribution, etc.), optical (absorption, colour, refractive index, etc.), and chemical. In the case of bacteria in general, these properties are not well-characterised. It is not likely that any


single property could be used as a robust differentiator of bacterial species but measurement of multiple characteristics simultaneously could provide a way forward. This may imply the characterisation of individual cells rather than ensembles and will require the development of methods to capture/isolate, stimulate and measure the species of interest in situ. Advances in microfabrication, plasmonics and charge manipulation/detection will be required for future instrument or sensor development. In this talk, we will discuss current trends and options within the arena of physical measurement and report on a recently commenced project to capture and manipulate individual bacteria-laden aerosol droplets and bacterial cells with the ultimate aim of applying forces and determining individual responses.


HYDROCHIP LIFE+, DEVELOPMENT OF A MOLECULAR CHIP FOR RAPID DETERMINATION OF THE QUALITY OF SURFACE WATER BY USING DIATOMS AS INDICATOR ORGANISMS Contributed paper Marije IJszenga, A. Atsma, S. in ’t Veld and B. de Graaf Vitens laboratory, the Netherlands [email protected] A good quality of the surface water is essential for drinking water production, fishery, recreation and also has an important value in itself. The quality of the surface water is under a lot of pressure by for example the emission of heavy metals, fertilizers and pesticides. A current method to determine the quality of surface water is the use of the composition of diatom populations. Diatoms are present in surface waters and the presence or absence of certain types of diatoms gives an indication of the quality of water. Current ecological monitoring is based on the collection and analysis of numerous field samples by well-trained specialists and is labour-, time- and cost-intensive. A less expensive and more objective approach for fast identification of biota in field samples would be a beneficial upgrade of currently applied methods. Because of these reasons, STOWA, Vitens, TNO, Waternet and Hoogheemraadschap Hollands Noordkwartier started a project to develop a new rapid method to determine diatom populations, based on molecular techniques. This project started in 2012 is called Hydrochip Life+ and will take a period of 4 years. The Hydrochip Life+ project demonstrates a new measuring device called Hydrochip and provides the opportunity to monitor diatoms in an innovative way. The Hydrochip is a microarray device which contains specific biomarkers based on the 18S rRNA gene of the different species of diatoms. 18S rRNA probes for the various diatom taxa on the chip were collected from available data and through a single-cell approach. By using these biomarkers it is easy to ascertain what kinds of diatoms are present in the water and translate this to the ecological state of the water. In addition, the platform is fast, cost effective, flexible and can be adapted changing the biomarker composition. The result is obtained within 6 hours, while the results from the microscopic analysis can take up to 6 months. The goal of the project is to demonstrate and validate the use of Hydrochip as a method to measure ecological water quality parameters to the same standard as the classical microscopic methods. In this research, we further developed the Hydrochip and started analysing surface water samples and comparing the results with the classic standard light microscopic analysis. The first tests show that the Hydrochip yields reliable results in part of the trophic spectrum. These results demonstrate that this tool could be useful for the water boards to ascertain water quality by means of diatoms, with the benefit that we can more rapidly analyse samples.


FIBER OPTIC BIOSENSING FOR ALLERGEN AND PATHOGEN SCREENING IN FOOD F. Delport, K. Knez, I. Arghir, N. Marien, D. Spasic, Steven Vermeir and J. Lammertyn BIOSYST-MeBioS, KU Leuven, Belgium [email protected] Point-of-care (POC) diagnostic tests for allergen analysis are expected to deliver fast and sensitive detection of DNA and/or protein targets. However, nowadays, detection of molecular targets alone, without their quantification and/or identification is often insufficient [1,2]. Although, different assays have been developed to meet these requirements, including quantitative PCR (qPCR) [3] followed by high resolution melting analysis as well as ligation assays [4], most of them are still largely incompatible with the concept of POC testing due to their cost or complexity. Furthermore, antibody sandwich assays, such as ELISA and lateral flow tests, are the industry standard, but lack kinetics and they need multiple handling steps and another detection setup. In this work, we present the fiber optic (FO) SPR sensor as a flexible platform for multiplex, real-time monitoring of DNA amplification and detection of single nucleotide polymorphisms (SNPs) in a single sample. Furthermore, we report for the first time the selection of aptamers against one of the most important peanut allergens, Ara h1 and the comparison to the antibody based detection (Figure 1). Three approaches of an immunoassay to detect Ara h1 peanut allergens in chocolate candy bars were compared; a label-free assay, a secondary antibody sandwich assay and a nanobead enhanced assay (Figure 1A) [5]. Although label-free detection is the most convenient, our results illustrate that functionalized nanobeads can offer a refined solution to improve the fiber SPR detection limit. By applying magnetite nanoparticles as a secondary label, the detection limit of the SPR bioassay for Ara h1 was improved by two orders of magnitude from 9 to 0.09 g/ml (Figure 1B). The SPR fibers could be regenerated easily and one fiber could be reused for up to 35 times without loss of sensitivity. The results were benchmarked against a commercially available polyclonal ELISA kit with an excellent correlation, but a longer linear dynamic range. In addition, with the SPR fiber we could measure the samples twice as fast as compared to the fastest ELISA protocol. Several Ara h1 DNA aptamers were selected using capillary electrophoresis (CE)-SELEX [6]. The selected aptamers specifically recognized Ara h1 and did not significantly bind with other proteins, including another peanut allergen Ara h2. Furthermore, the selected aptamer was used for bioassay development on the FO-SPR biosensor platform for detecting Ara h1 protein in both buffer and food matrix samples demonstrating its real potential for the development of novel, more accurate aptamer-based biosensors.

(A) (B)

Figure 1. (A) Overview of the Ara h1 immunoassay strategies on the fiber optic SPR biosensor. (B) Comparison of the three presented immunoassays on the fiber optic SPR sensor for different Ara h1 concentrations. The error bars indicate standard deviations (n = 3).


The aforementioned FO-SPR set-up (Figure 2A) has been used for monitoring the DNA melting profile by implementing DNA functionalized gold nanoparticles (Au NP) as labels (Figure 2B), which allowed discriminating SNPs at high resolution [7,8]. However, to realise simultaneous quantification and cycle-to-cycle identification of the reaction products, the FO-SPR melting assay was combined either with the PCR or with ligase chain reaction (LCR). The FO-SPR LCR assay amplifies DNA in an exponential manner through multiple ligation cycles that progress through 3 succeeding phases: hybridization, ligation and melting phase (Figure 2C). The detection limit of the FO-SPR melting assay was drastically improved using the LCR, whereas capacity for SNP detection was preserved (Figure 2D,E). Furthermore, to achieve detection of multiple targets within the same sample, FO-SPR melting assay was combined with solution phase PCR using two sets of hybridization probes. The DNA targets in this study were used to discriminate Mycobacterium bovis from Mycobacterium avium subsp. paratuberculosis. These two bacteria, which are frequently encountered in life stock, were used in a proof-of-concept study that showed the capacity of FO-SPR melting assay for multiplex DNA detection (Figure 2F), thereby further emphasizing the potential of this platform in POC test development.

Figure 2. (A) Schematic representation of the FO-SPR setup with all components. (B) Schematic representation of a FO-SPR melting assay (top panel) with DNA target (1) and DNA probes immobilized on the FO-SPR sensor (2) and on Au NP (3). Gene probes hybridize at the FO surface, and are subsequently melted off by a gradual increase of the temperature, resulting in the FO-SPR sensorgram (bottom panel). (C) Schematic overview of the FO-SPR LCR: (1) Different components of the reaction. (2) LCR reaction where the forward and reverse probes are ligated only in the presence of the target sequence, resulting in an exponential amplification during multiple cycles. (3) The forward LCR product can, during the LCR reaction, form a complex with two complementary probes immobilized on the FO-SPR sensor and on Au NPs, allowing real-time monitoring of the reaction. (D) The derived calibration curve with Ct values from the FO-SPR LCR spans 7 orders of magnitude for DNA concentrations. (E) Obtained signals for WT and MM target DNA using FO-SPR LCR assay (right panel). (F) FO-SPR multiplex PCR, which allows resolving the melting point of the two targets.


References 1. Cornett, E.M., Campbell, E.A., Gulenay, G., Peterson, E., Bhaskar, N. and Kolpashchikov, D.M.,

2012. Molecular logic gates for DNA analysis: detection of rifampin resistance in M. tuberculosis DNA. Angewandte Chemie International Edition 51: 9075-9077.

2. Murray, C.J. and Salomon, J.A., 1998. Modeling the impact of global tuberculosis strategies. Proceedings of the National Academy of Sciences of the USA 95: 13881-13886.

3. Cheng, J.C., Huang, C.L., Lin, C.C., Chen, C.C., Chang, Y.C., Chang, S.S. and Tseng, C.P., 2006. Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR. Clinical chemistry 52: 1997-2004.

4. Sando, S., Abe, H. and Kool, E.T., 2004. Quenched auto-ligating DNAs: multicolor identification of nucleic acids at single nucleotide resolution. Journal of the American Chemical Society 126: 1081-1087.

5. Pollet, J., Delport, F., Janssen, K.P., Tran, D.T., Wouters, J., Verbiest, T. and Lammertyn, J., 2011. Fast and accurate peanut allergen detection with nanobead enhanced optical fiber SPR biosensor. Talanta 83: 1436-1441.

6. Tran, D.T., Knez, K., Janssen, K.P., Pollet, J., Spasic, D. and Lammertyn, J., 2012. Selection of aptamers against Ara h 1 protein for FO-SPR biosensing of peanut allergens in food matrices. Biosensors and Bioelectronics 43: 245-251.

7. Pollet, J., Janssen, K., Knez, K. and Lammertyn, J., 2011. Real-time monitoring of solid-phase PCR using fiber-optic SPR. Small 7: 1003-1006.

8. Knez, K., Janssen, K.P., Spasic, D., Declerck, P., Vanysacker, L., Denis, C., Tran, D.T. and Lammertyn, J., 2013. Spherical nucleic acid enhanced FO-SPR DNA melting for detection of mutations in Legionella pneumophila. Analytical Chemistry 85: 1734-1742.


USING NATURE'S GENIUS FOR POLLUTANT DETECTION Johannes Raff1,2, U. Weinert2, N. Nikolaus3, T. Guenther2, B. Strehlitz3 and K. Pollmann2

1Institute of Resource Ecology, Helmholtz-Zentrum Dresden-Rossendorf, Germany, 2Helmholtz Institute Freiberg for Resource Technology, Helmholtz-Zentrum Dresden-Rossendorf, Germany and 3Centre for Environmental Biotechnology, Helmholtz Centre for Environmental Research, Germany [email protected] In all environments, single cell organisms such as bacteria are directly affected by changing and sometimes extreme environmental conditions. This includes not only pH, temperature and salinity but also the presence of toxic ions, compounds and complexes. It is thus essential for these microorganisms to possess a robust and selectively permeable cell surface. For this purpose, many bacteria form a proteinaceous cell envelope, the so called

surface layer (S-layer). This cell envelope has different functions in different organisms for example the binding of toxic metals and metalloids to protect cells from being damaged by these elements. On other cells, S-layers may act as immobilisation matrix for exoenzymes, as molecular sieve or as ion and molecule trap or they protect the cell from being affected by other bacteria or by lytic enzymes. S-layers are composed of identical protein or glycoprotein monomers, which are able to self-assemble to highly ordered monomolecular layers. They form paracrystalline sheets in suspension, on interfaces and on surfaces (Figure 1). Furthermore, on the surface of such a layer different functional groups are available, which can be modified without the loss of its structure. The protein layers are in general mechanically and chemically highly stable. These properties make S-layers very interesting building blocks for the construction of new bioinspired nanomaterials and nanocoatings. Using the two-dimensional protein arrays in combination with layer-by-layer technique different kinds of technical

surfaces can be functionalised. This technique is used to design a new kind of sensory layers which will allow detecting small amounts of analytes with high selectivity. This sensory device will consist of an S-layer coating, a selective receptor and two fluorescence dyes. Aptamers were used as compound specific receptors. These are short, single stranded nucleic acid oligomers that meet the requirements for a more selective and sensitive detection of pollutants in nature, medicine and industry. Aptamers are able to recognise almost all classes of substrates and bind them in analogy to antigen-antibody interactions. By using an in vitro selection and amplification technique aptamers can be developed for pollutants like heavy metals, pharmaceuticals but also for proteins and complex targets like viruses and microbial spores. Currently, several aptamers for different antibiotics have been selected and experiments confirmed their high selectivity for single antibiotics or groups of antibiotics. As third component two fluorescence dyes allowing a FRET as signal transducer system will be coupled onto the S-layer lattice. This setup can be combined in different ways to optimise the sensitivity and selectivity of the sensor. Comparable to the binding of exoenzymes on S-layer carrying cells the three sensor components can also be linked to S-layer coated technical surfaces. In first experiments, we coupled the model aptamer, anti-thrombin-aptamer, on S-layer proteins and proved its functionality after being linked to the protein. Furthermore, we modified S-layer proteins with

Figure 1: Transmission electron micrograph of the S-layer from L. sphaericus JG-A12. The insert is a correlation averaged reconstruction of the protein lattice.


a FRET pair and proved the energy transfer between them. We used a FRET pair containing a green and red fluorescence dye and succeed to detect a FRET between the S-layer linked fluorescence dyes. The FRET efficiency was 40 %. Because of the regular arranged functional groups on the S-layer lattice, coupling of the sensor components can be done in a defined and reproducible way. In further work we will combine all components, aptamers and fluorescence dyes, on the S-layer proteins (Figure 2). The aptamer will bind the specific analyte, affecting also the fluorescence dyes and disturbing FRET because of their close proximity to each other. In result a sensory layer is developed which uses the high specificity of aptamers and fluorescence dyes for an easy detection due to an optical signal. Additionally, in the future other techniques such as phage surface display will be used to select also peptide based binding molecules. Hopefully, this will allow detecting even more and also other kinds of pollutants not being bound by aptamers.

Figure 2. Scheme of a possible sensory set-up and how it operates.


ULTRA-SENSITIVE SURFACE ENHANCED RAMAN SPECTROSCOPY SENSOR AND POTENTIAL APLICATIONS IN ENVIRONMENTAL MONITORING Ali Ozhan Altun, T.C. Bond and H.G. Park Department of Mechanical and Process Engineering, ETH Zurich, Switzerland [email protected] Today, water resources around urban areas are in a great threat due to the degree of contamination in terms of micropollutants. Increasing use of pesticides, pharmaceuticals and cosmetic products release more micropollutants in the wastewater stream day by day. Their resistance to environmental degradation enables them to transport in a long range, eventually accumulating in human and animal tissues inducing potential serious impacts. The term micropollutant comes from the fact that they are generally present in water at low concentrations from pico- to nanogram per litre. Tracking the amount of these molecules at such low concentrations is crucial for water quality monitoring. Therefore, it is of great importance to develop a practical and fieldable technique to overcome this challenge. Recently, we have developed a metal/dielectric/vertically aligned CNT sandwich structure for chemical and biomolecular detection using the science of surface enhanced Raman spectroscopy (SERS). High sensitivity is accomplished with the insertion of a nanolayer in between plasmonic metal (gold) and the metallic wire template (multiwalled carbon nanotubes (CNT)). The rational design is twofold: (i) inserting of a screening material against electron transfer between gold and CNT to prevent otherwise quenched local surface plasmon resonance; and (ii) creating a ‘kissing nanowire’ effect to the gold-hafnia-CNT nanowires to enhance the SERS-based, chemical detection sensitivity. Experiments verified our nanoarchitecture design by demonstrating femtomolar detection of BPE (1,2 bis-(4-pyridyl)-ethylene). These outstanding results of ultrahigh sensitivity for BPE motivated us to apply our sensor to micropollutant detection. We used our SERS sensor on a number of micropollutants, such as atenolol, antipyrine, benzotriazol, cabamazepin, diclofenac, and pyridine at low concentrations. As all the SERS measurements were performed in a liquid cell environment, we believe that our sensor has a potential for field applications as a molecular sensor to detect low-concentrations micropollutants.


THE SINGLE CELL FUTURE OF MALDI-TOF MASS SPECTROMETRY Gerold C. de Valk BiosparQ BV, the Netherlands [email protected] A major obstacle for rapid and cost-effective microbial analysis is the need for culture to allow identification of microorganisms. Culture is laborious, time consuming and requires considerable laboratory infrastructure. Molecular detection using PCR has proven to be effective in a range of applications but requires relatively large resources, is still time consuming and interpretation of the test results can be difficult. The introduction of MALDI-TOF MS-based bacterial identification has been one of the most rapid and successful transformations of the microbiology laboratory in the last decades. This method combines the MALDI technique with a time-of-flight (TOF) mass spectrometer and applies it to (microbial) cells. Part of the peptides, proteins and other elements contained in the cell become visible as peaks in a so-called mass spectrum. It appears that the combination of peak locations is highly specific for the genus/species of the microbe, allowing identification. However, the rate-limiting step in diagnosis using MALDI-TOF still is the requirement of a pure culture. As a result, appropriate follow-up actions are delayed and in-line application in food production is not possible. BiosparQ has developed a groundbreaking technology to solve these problems by the development of a novel platform, the Cirrus next generation MALDI-TOF, capable of instant diagnosis of samples without the need for culture. The Cirrus performance is not only unique in terms of limited time to results, its design offers also full microbial profiling capability, which is of much more value than just ‘detecting the bacteria one is looking for’. The Cirrus development strongly depends on a trans-disciplinary approach, integrating single cell dispensing, single cell MALDI TOF mass spectrometry, and single cell-based mass spectrum sorting algorithms. To overcome the requirement of culturing and isolation, in the Cirrus platform the traditional target-plate based transport of the sample to the mass spectrometry sensor is replaced by an aerosol-based transport. Instead of depositing samples onto a target plate, a suspension containing the sample as well as the matrix is 'printed' into an air stream. Subsequently the aerosol flow is dried and the resulting particles are transported to the ion source, using a three stage nozzle-skimmer vacuum inlet. In the ion source, the particle stream is optically interrogated. Based on the outcome of this interrogation, individual particles may be ionized by illumination with the UV laser. In this approach, a mass spectrum is generated for every particle. Using a library of classifiers, each individual spectrum is provided with a probability that it matches with one of the classes in the library. Hence, samples of mixed origin can be analyzed with the Cirrus implementation of MALDI-TOF mass spectrometry, enabling direct analysis of microbial samples. This opens new, fieldable applications for MALDI TOF mass spectrometry. Initial tests using Staphylococcus epidermidis, Bacillus thuringiensis and Escherichia coli strains prove that this groundbreaking approach of single cell MALDI-TOF mass spectrometry indeed works and that from individual bacterial cells relevant proteomic information can be extracted that allows identification. Based on results obtained with the Cirrus prototype, as little as 10 to 20 organisms will be sufficient to classify the organism under consideration with a usable level of confidence.


LOOKING FOR THE BEST COMPROMISE IN RAPID MYCOTOXINS TESTS: SPEED, SENSITIVITY, PRECISION, ACCURACY Anton J. Alldrick Campden BRI, UK [email protected] Mycotoxins provide additional challenges to food businesses in terms of successful management of food-safety management systems. These reflect, in part, an unusually high dependency on the activities of others in the supply chain to ensure that levels of contamination remain within set limits. Consequently, analyses for mycotoxins by food businesses are primarily commissioned for one or a combination of two reasons: to determine compliance with regulatory or commercial standards or as part of an exercise to verify the efficacy of the businesses food-safety management systems and in particular those associated with supplier quality assurance (SQA). Given the regulatory/commercial implications, the standard of evidence needed to demonstrate (non)compliance will be the greater than that needed for simple verification. Consequently decisions relating to matters of regulatory or commercial arbitration need to be based on agreed and well defined methods of analysis, which are normally laboratory based. These data are also often sufficient to be used to verify food-safety management systems. However, supply conditions, e.g. adverse weather conditions, may predicate the need for increased levels of verification, in particular with regard to SQA; rapid mycotoxin test kits have the potential to both meet this need and also satisfy the requirements of statistical process control. Such test kits have to be compatible with the commercial environment in which they are to be used. Frequently, there are contractual obligations for a customer to accept or reject a shipment within a short period of time (often in the order of 20-30 minutes) and therefore rapid test kits must be both ‘rapid’ and operator-friendly. Commercial specifications set by customers often set lower limits for mycotoxins than those set out in legislation. Furthermore, within the certain jurisdictions, e.g. European Union, food businesses have an obligation to report food and feed which does not comply with the regulations to the relevant regulatory body. Given the potential actions that might arise from data obtained with such test kits they must therefore be demonstrably reliable in terms of sensitivity, precision and accuracy. Deployment of such kits cannot therefore be considered to be a ‘turnkey’ exercise and, as in the case of laboratory-based assays, care must taken in the validation and subsequent verification of the use of such kits for a given material being used within a particular food business establishment. In particular, this means demonstrating under local conditions that results from the use of these kits are comparable to those that would be obtained using official or reference methods.


PERFORMANCE CRITERIA FOR RAPID SCREENING METHODS TO DETECT MYCOTOXINS Jörg Stroka Institute for Reference Materials and Measurements, Joint Research Centre, European Commission, Belgium [email protected] A need for screening methods that allow an early detection of potentially with mycotoxins contaminated-commodities has been claimed since more than a decade. It has been proposed that such screening methods have the highest potential if used in a combined strategy with confirmatory methods, thus allowing a rapid judgement of suspect goods. Such an approach will allow stakeholders to focus confirmatory analytical efforts on a small fraction of ‘suspect’ commodities and releasing those deemed as safe with ease. However, due a lack of generally accepted or prescribed performance criteria to judge the suitability of screening methods they will suffer acceptances. Various producers of test kits as well as multimycotoxin method developers have published performance data for their methods. However, neither a universal scheme that allows a comparison of method performance was used between products nor a general rule (suitable for screening methods) that allows a classification of the method as fit-for-purpose was used. Upon request of the Directorate-General for Health and Consumers (DG SANCO), a small group of experts (mycotoxin reference laboratories, academia and end user testing labs) started drafting performance criteria rules to develop a guidance document. Currently, the defined rules are at a stage to be implemented in European Union legislation, which will give them the necessary formal character to be used in official control. In summary, these rules focus on screening methods that quantify the target mycotoxins. Spectroscopic methods that concern other physical characteristics related to other properties or substances as contamination indicators are not considered (at this stage). The rules are tailored specifically for mycotoxin control, however, methodology independent. The main criterion for method evaluation is the determination of a sufficient low rate of false negatives (<5%) by defining a cut-off value specific for the method tested, below which results can be classified as compliant. The presentation shows by means of examples the concept of minimum performance criteria for screening methods and discusses the strengths and boundaries of the evaluation concept.


ISOTHERMAL AMPLIFICATION OF DNA AS A NOVEL APPROACH FOR THE RAPID DETECTION OF DNA IN FOOD SAMPLES: A SIMPLE TEST FOR THE FOOD ALLERGEN CELERY Kurt Brunner Department IFA-Tulln, BOKU Vienna, Austria [email protected] DNA is commonly used as the specific analytical target for the detection of – frequently undesired – organisms, such as pathogens, GMOs or allergens in food samples. However, PCR methods are laborious procedures, requiring expensive equipment, such as thermal cyclers and skilled personnel. Furthermore, the high energy demand of thermal cyclers for efficient heating and cooling makes the production of battery-operated devices difficult. All these facts limit the application of standard PCR methods for on-site tests. An alternative DNA amplification method is the loop-mediated isothermal amplification (LAMP), which was developed in 2000. Four oligonucleotides binding six distinct regions of the target sequence and a BstI polymerase with strand displacement activity undergo cyclic amplification steps and produce various sized DNA strands with inverted repeats of the target DNA sequence. Based on the lack of a denaturation step, the assay can be carried out under isothermal conditions, eliminating the need for a thermal cycler and thus making this method highly cost- and time-efficient as well as field-applicable and easy-to-perform. Celery (Apium graveolens L.) is a widely used ingredient in seasonings, sauces, boullions and instant meals. Allergy to celery is a rather common food allergy in Europe causing digestive disorders, respiratory distress and skin reactions when ingested. Avoiding the allergen containing food is the only option to prevent an allergic reaction, which may often be difficult due to the contamination of food products with allergenic ingredients. The contaminants can be introduced by food processing machines, storage and shipping or are added deliberately for flavouring purposes. Since 2005, celery and celery products have to be labelled according to Directive 2003/89/EC due to their allergenic potential. This legislation raises the demand for specific and sensitive methods for the detection of allergens in food products. Although the characterisation of celery allergens via mass spectrometry has been reported, currently, the two most prominent analytical techniques for allergen detection in food are protein-based enzyme linked immunosorbent assays (ELISA) and nucleic acid-based polymerase chain reaction (PCR) assays. However, to date, no ELISA assay is available for the specific detection of celery due to cross reactions with parsley, carrot and other closely related members of the Apiaceae family. So far, several real-time PCR assays have been developed targeting different celery specific genes. To provide a DNA-based, rapid and simple detection method suitable for high throughput analysis, a LAMP assay for the detection of celery was developed and optimized. The assay was tested for specificity for celery. It could be demonstrated that the LAMP reaction does not amplify closely related plant species, although the DNA sequences are highly similar. The limit of detection (LOD) for spiked food samples was found to be in the low mg celery per kg food range. A robustness test was performed to show reliable detection independent from the instrument used for amplification (thermal cyclers or different heating blocks). We also evaluated various signal detection methods including real-time fluorescence measurements, agarose gel electrophoresis or nucleic acid staining with intercalating dyes. The analysis of ten commercial food samples representing diverse and complex food matrices clearly demonstrated the potential of LAMP for a rapid and simple detection of traces of celery even in complex food matrices. In general, the performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products and especially its insensitivity to co-isolated PCR inhibitors makes this assay a promising tool for food analysis in the future.


TOWARDS THE PRODUCTION OF REFERENCE MATERIALS FOR FOOD ALLERGEN AND GLUTEN-FREE ANALYSIS FOR IMPROVED FOOD SAFETY MANAGEMENT Roland Poms International Association for Cereal Science and Technology (ICC), Austria and International Association for Monitoring and Quality Assurance in the Total Food Supply Chain (MoniQA), Austria [email protected] Effective allergenic risk assessment and management are important to protect allergic consumers and to comply with allergen labelling regulations. Such approaches require reliable analytical tools for the detection of allergens in food. In recent years, various allergen-detection methods have been published, and immune-chemical test kits have become commercially available and widely used. Alternatively, methods using specific DNA fragment identification – usually based on polymerase chain reaction (PCR) have also been employed. More recent developments use also liquid chromatography-mass spectrometry (LC-MS) to analyse for allergen or gluten specific peptides in food products. Due to the nature of the analytes and their susceptibility to various processing effects, reliability and comparability of results have posed a great challenge. Both reference methods and reference materials are urgently needed here. It is one of the aims of MoniQA (Monitoring and Quality Assurance in the Total food Supply Chain (www.moniqa.org), the global food safety network registered as MoniQA Association (initially funded by the European Commission as a Network of Excellence, 2007-2012)) to provide guidelines for method validation, reference materials and assess the reliability of methods and obtained results through validation studies and proficiency testing schemes. MoniQA developed incurred reference materials for the analysis of milk and egg allergens; recently, these materials were used in an international ring trial. Additionally, MoniQA worked on the development of LC-MS based reference methods for gluten-free and food allergen analysis. Currently, the specific requirements and the design of global gluten-free standard materials and food allergen reference/testing materials are discussed by an international group under MoniQA’s leadership, involving representatives from universities and food industries, the European Prolamin Working Group, AOAC, Health Canada, ICC, RMI Australia, and others. The aim of MoniQA’s initiative is the publication of a guidance document on the special requirements and production of allergen reference materials, following a similar effort by the same group in 2010 publishing a globally harmonised guidance and best practice document on validation procedures for quantitative food allergen ELISA methods [1]. The new document shall allow everyone who is able to fulfil the described requirements to produce and to offer reference material. This material shall receive a certain ‘certification or labelling’ that identifies the material as such, produced according to this ‘guidance paper’ (in effect a practical guide to implement ISO Guide 35 applied to the special issue of allergens and gluten). In a recent meeting in Washington, Missouri, USA (March 2013), MoniQA established a task force that identified the first priority commodities: gluten, milk, egg, peanut, hazelnut, and soy. The plan is to provide well-characterized commodity materials for the eventual production of incurred reference materials, spiked samples and extracts. The need for appropriate analytical tools to assure food safety for allergic consumers requires the collaboration of various disciplines, such as food technology, clinical research, consumer and social sciences, analytical chemistry and others, and various stakeholder groups bridging from research and training at the university and industry level, to


international organisations (Codex, CEN, ISO, amongst others) and regulators. References 1. Abbott, M., Hayward, S., Ross, W., Godefroy, S.B., Ulberth, F., Van Hengel, A.J., Roberts, J.,

Akiyama, H., Popping, B., Yeung, J.M., Wehling, P., Taylor, S., Poms, R.E. and Delahaut, P., 2010. Validation procedures for quantitative food allergen ELISA methods: community guidance and best practices. Journal of AOAC International 93: 442-450.


ROCKET FUEL FOR MEDIA HEADLINES: THE PERCHLORATE STORY Bert Popping Eurofins Scientific Group, UK [email protected] The number of food scares appear to have increased over the past few years. After Burgergate, Fishgate and the mycotoxin-contaminated animal feed, a new food scare hit the headlines: perchlorate. Is it a real increase of incidents or is additional attention paid to this area? Do detection technologies get more sensitive? Let us focus on perchlorate. This was ‘re’discovered in early 2013 and subsequently found predominantly in fruits and vegetables. Media reported numerous positive samples significantly exceeding the recommended level. These products were found in stores of several well-known retailers. But what is perchlorate and how is it used? Perchlorate is a very small molecule which occurs naturally but can also be manmade. Perchlorate is used in rocket fuels and is found in drinking water. But how does it get into foods and water? And how does it affect health? Which levels are dangerous? The presentation will look at the discovery of perchlorate, its usage across food and non-food, as well as government actions taken at national and European levels to protect consumers' health and balance this to still allow trade of fresh produce. The presentation will also look at the way media presented the new food scare, consumer perception and the role of NGOs. From the perspective of the routine food testing laboratory, it will look at customer needs, implementation of a new detection method for perchlorate, validation and proficiency testing.


VALIDATION OF BINARY METHODS – CHALLENGES AND MODELS Christoph von Holst Institute for Reference Materials and Measurements, Joint Research Centre, European Commission, Belgium [email protected] Binary methods express the results of analysis in terms of ‘present’ or ‘not present’, often in conjunction with a legal limit of the target analyte. In such a case, these methods are often used as screening method, where samples with a negative result are considered as compliant, whereas positive samples require the application of a confirmatory method to establish whether the sample is compliant or not. Screening methods are routinely used in different fields, such as official control but also internal quality control conducted by industry. Moreover, quite different formats of screening methods are available. For instance, there are methods that are based on a visual inspection, such as a dipstick or those where the analytical response is evaluated with a reading device, thus producing a numerical value that can be compared against a previously set cut-off value of the response. Though both types lead to a binary result, they require significantly different data treatment within the frame of method validation. The most important performance characteristics of binary methods are sensitivity and specificity. The sensitivity is frequently expressed as rate of correct positive results of samples containing the target analyte at a specific concentration. Alternatively, the lack of sensitivity is given as rate of false negative results. Plotting the rate of positive results against the corresponding analyte concentration indicates the level at which samples containing the analyte at this concentration are detected with a predefined probability, e.g. 95%. If this concentration is below or at the limit to be monitored, sufficient sensitivity has been demonstrated. It is also important that the test classifies samples that do not contain the target analyte at levels below the legal limit correctly as negatives. The latter characteristic is the specificity of a method. The purpose of the talk is to present current examples for the validation of screening methods, applying different types of statistics. By using real world examples, the challenges of the validation of binary methods are shown.


NOVEL QUANTITATIVE REAL-TIME AND MULTIPLEX PCR FOR ALLERGEN DETECTION Ronald Niemeijer R-Biopharm AG, Germany [email protected] Food allergen management has become of increasing importance for the food industry in many countries. Since avoidance of the allergenic food is still the most effective means of preventing an allergic reaction in susceptible individuals, informing the consumer about the presence of potential allergenic ingredients in products is of great importance. For quality control and verification of the food allergen management plans reliable test methods are needed, as well as for verification of compliance with legal requirements on food allergen labelling and the identification of allergenic cross-contaminants. Several detection methods for these food allergens have been developed and are commercially available, the most frequently applied and accepted technique has been the enzyme-linked immunosorbent assay (ELISA). A different approach to specifically detect an allergenic food component is to detect a specific sequence of the DNA. Several studies have demonstrated good correlation between protein-based and DNA-based methods in the detection of allergenic foods at the low mg/kg level. Thus, DNA was shown to be an alternative molecular marker to identify the presence of allergenic food components in unprocessed as well as processed foods. PCR potentially has an excellent specificity and is based on a fully defined chemistry. Multiplex detection as well as quantification of various allergenic foods by PCR has been demonstrated. Apart from the detection of milk and egg, specific and sensitive PCR methods are commercially available for the most relevant allergenic foods. Like with all other allergen detection methods, sample preparation methods are very important in order to avoid false negative or a-specific results. For the PCR method, special attention should be paid on using an efficient DNA extraction and clean-up method, particular in processed or complex food materials. DNA-based methods offer a different but effective approach in allergen management. Depending on the analytical requirements and foods to be tested, DNA-based methods can be the right choice for the industry for allergen detection.


INTRODUCTION TO THE TOPIC: AUTHENTICITY ISSUES IN THE FOOD INDUSTRY Bert Popping Eurofins Scientific Group, UK [email protected] How long have food authenticity issues been around? 10 years? 50 years? 100 years? Food fraud is by no means a new business, but it gets more and more sophisticated, and the high complexity of the food chain makes it often more difficult to detect it. But what is actually authentic? Is a Black Forest ham (‘Schwarzwälder Schinken’) authentic if its animal was raised in Poland and only the ham smoked in the Black Forest region? What about Basmati and Texmati? Does it make a difference where the rice is grown as long as the variety is the same? What is the difference between protected designation of origin (PDO) and protected geographical indication (PGI)? And what is a traditional specialty guaranteed (TSG) regime? And what is the incentive of food fraud? Is money always the trigger? Which products are most at risk? Wines, juices, fish, spices, herbs? The presentation will explain the history of food fraud to the present day, the innovations made to commit fraud and keep it undiscovered, as well as the innovations made to discover new fraudulent activities. It will give some brief case studies and discuss the latest issues, including Burgergate.


FAITH IN FOOD: FRAUD DETECTION BY NOVEL ANALYTICAL TECHNIQUES Saskia van Ruth, M. Alewijn, S. Heenan, E. Capuano, G. van der Veer, V. Acierno, I. Silvis and M. Muilwijk RIKILT Wageningen UR and Wageningen University, the Netherlands [email protected] Several large cases of food fraud came to light in 2013: horse meat, fish species, organic eggs, etc. Foods are nowadays sourced around the globe, the food supply chain has become an extensive and fragile network, and food fraud reaches to every dinner table in the world. Fraud may relate to the composition of foods, and then especially to substitution and extension of constituents. Examples are adulteration of olive oil with hazelnut oil, replacement of more expensive fish by cheaper species, horse meat instead of beef, dilution of juice/wine with water, and melamine in milk powders. The second category of food fraud involves the history of the food product, e.g. production practice, geographical origin, or technology applied. Food fraud results in unfair competition and ruins consumer confidence. Management systems, certification, and paper trailing are all very useful to prevent and detect food fraud. Analytical tools can support and complement these processes. In this paper, various novel analytical techniques that can be used for rapid screening or confirmation will be presented and illustrated with several examples.


MEAT FOR THE MASSES: PROTEIN MASS SPECTROMETRY FOR THE AUTHENTICATION OF MEAT PRODUCTS Jens Brockmeyer Institute of Food Chemistry, University of Münster, Germany [email protected] Fraudulent substitution of meat species in food is highly relevant as demonstrated by the ‘horse meat scandal’ where at least 50,000 tons of beef products contained horse meat. Besides the economical aspect, the undeclared blending of food with pork or horse is a severe problem for consumers that have ethical or religious concerns about the consumption of this species. We therefore developed a sensitive mass spectrometrical approach for the detection of trace contaminations of horse and pork in beef by use of species-specific peptides. These biomarkers were identified by a shotgun proteomic approach using tryptic digests of protein extracts from horse meat, pork, and beef. For each species, about 5,000 peptides and approximately 500 proteins were identified and specificity of potential biomarker peptides was assessed by targeted analysis of 21 samples from beef, horse, pork, chicken, and lamb. For the most sensitive peptides, a multiple reaction monitoring (MRM) method was developed that allows for the detection of 0.55% horse or pork in a beef matrix. To enhance sensitivity, we applied MRM3 experiments and were able to detect down to 0.13% pork contamination in beef. To the best of our knowledge, we present here the first rapid and sensitive mass spectrometrical method for the detection of horse and pork by use of MRM and MRM3 that is readily applicable in routine laboratories.


LABELFISH – A EUROPEAN PROJECT TO COMBAT FRAUD IN THE FISH INDUSTRY Carmen G. Sotelo Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Spain [email protected] Fraud refers to deliberate actions intended for the misleading of consumers in different ways. In the case of seafood, most of the time the term fraud involves the substitution of valuable species by others of lower price, therefore meaning an economic loss for consumers, but also mislabelling can hide other malpractices, such as illegal capture procedures. Traceability of fish and seafood is mandatory since 2005 within the EU. Full implementation requires an adequate management of information and also the availability of techniques that allow the verification of the information transmitted. These are essential tools to combat food fraud. However, recent cases have shown that, although legislation and techniques are available, there are still some crises related with food fraud that merit a deep evaluation and analysis of the problem. LABELFISH is a project funded by the Atlantic Area Programme and includes participants of six countries in Europe, mainly from the Atlantic area, which are characterized by an intense economic and social relationship with marine resources (partners: B. Boufana, University of Salford, UK; D. Calvo Dopico, University of La Coruña, Spain; A. Griffiths, University of Salford, UK; M. Jérôme, IFREMER, France; K. Kappel, Max Rubner-Institut, Germany; J. Maguire, Indigo Rock Marine Research Station, Ireland; S. Mariani, University of Salford, UK; R. Mendes, Portuguese Institute for the Sea and Atmosphere, Portugal; R. Pérez-Martín, Instituto de Investigaciones Marinas CSIC, Spain; U. Schröder, Max Rubner-Institut, Germany; M. Shorten, Indigo Rock Marine Research Station, Ireland; H. Silva, Portuguese Institute for the Sea and Atmosphere, Portugal; C. Smith, Indigo Rock Marine Research Station, Ireland; C.G. Sotelo and A. Velasco, Instituto de Investigaciones Marinas CSIC, Spain; V. Verrez-Bagnis, Ifremer, France). One of the main aims of LABELFISH is the establishment of a network of laboratories and national control bodies with experience and interest in seafood labelling and traceability. The objectives include the level of implementation of traceability schemes in most important European seafood value chains, the analysis and detection of possible examples of seafood fraud across Europe, the consumers’ perception about seafood labelling, the current methodologies used for controlling the veracity of seafood labels, and how to propose harmonised methodologies for the adequate control of seafood labelling in the European Union. Some of these aspects and preliminary results of LABELFISH will be presented in this talk.


POSTERS P1 Bead-based suspension array for subtyping avian influenza infections in

chickens René Achterberg, J. Post, R. Bossers-de Vries, S. Pritz-Verschuren, R. Bouwstra, V. Broks, C. Overbeek, G. Koch and F.J. van der Wal Central Veterinary Institute, Wageningen UR, the Netherlands

P2 Development and validation of a nucleic acid microarray immunoassay and a

nucleic acid lateral flow microarray immunoassay for the detection of Plasmodium species G.A. Posthuma-Trumpie

1, A.P.H.A. Moers

1, C.J. Sutherland

2, R. Burrow

2, H.D.F.H. Schallig

3,

H.M. de Vogel-van den Bosch1 and Aart van Amerongen

1

1Biomolecular Sensing and Diagnostics, Wageningen UR, the Netherlands,

2Department of

Immunology and Infection, London School of Hygiene and Tropical Medicine, UK and 3Unit

Parasitology, Department Biomedical Research, Royal Tropical Institute , the Netherlands P3 Viability PCR: an evaluation of propidium monoazide for live/dead

differentiation of microorganisms in turbid solutions K. Wolf, S. Fakih, C. Küppers, A. Bogdoll, S. Schlappa, Marcia Armstrong, S. Luley and R. Peist Qiagen, Germany

P4 Salmonella and Listeria monocytogenes detection in complex food matrices

using a fully automated pathogen purification and detection system Marcia Armstrong, S. Fakih, S. Schlappa, C. Kueppers, P. Cornelius and S. Luley Qiagen, Germany

P5 Analysis of antibiotics in complex food matrices using a solid phase extraction

sorbent based on molecularly imprinted polymers D. Derrien

1, C. Perollier

1, O. Lépine

1, Michel Arotçaréna

1, K. Naraghi

1, K. Haupt

2 and

S. Bayoudh1

1Polyintell, France and

2Compiegne University of Technology, France

P6 Experiences with regard to the introduction of rapid methods in a drinking

water laboratory Adrie Atsma, S. in ’t Veld, M. IJszenga and B. de Graaf. Vitens laboratory, the Netherlands

P7 The CoSYPS Path Food project: development of a system for detection and identification of food-borne pathogenic bacteria present in carcass samples based on a ‘combinatory qPCR’ technology Elodie Barbau-Piednoir

1,2,3, J. Mahillon

1, N. Roosens

2 and N. Botteldoorn

3

1Laboratory of Food and Environmental Microbiology, Université catholique de Louvain,

Belgium, 2Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health,

Belgium and 3Food Pathogens, Scientific Institute of Public Health, Belgium

P8 Innovative analytical tools for β-triketone herbicides monitoring

E. Rocaboy, T. Noguer and Lise Barthelmebs Institut de Modélisation et d’Analyse en Géo-Environnement et Santé, Université Perpignan Via Domitia, France

P9 A novel and rapid approach for monitoring enzymatic protein hydrolysis using infrared spectroscopy

N.K. Afseth, T. Aspevik, F. Lundby, B. Narum, Å. Oterhals and Ulrike Böcker

Nofima – Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway


P10 Isothermal DNA amplification approaches for the detection of genetically modified maize C. Zahradnik

1, C. Kolm

1, R. Martzy

1, R.L. Mach

2, A.H. Farnleitner

2, R. Krska

3 and Kurt

Brunner1

1Institute for Analytical Chemistry, Vienna University of Technology, Austria,

2Research Group

Environmental Microbiology and Molecular Ecology, Institute for Chemical Engineering, Vienna University of Technology, Austria and

3Institute for Chemical Engineering, Department

IFA-Tulln, BOKU Vienna, Austria P11 A sensitive and robust method for automated on-line monitoring of enzymatic

activities in water and water resources P. Stadler

1, T. Lendenfeld

2, G. Ryzinska-Paier

3,6, A.P. Blaschke

5,6, H. Stadler

4, R. Mach

3,6,

Kurt Brunner3,6

, J. Appels7, M Zessner

1,8 and A.H. Farnleitner

1,3,6

1Centre for Water Resource Systems, Vienna University of Technology, Austria, ²mbOnline

GmbH, and WSB Labor GmbH, Austria, ³Research Group Environmental Microbiology and Molecular Ecology, Institute of Chemical Engineering, Vienna University of Technology, Austria,

4Department of Water Resources Management, Institute for Water, Energy and

Sustainability, Joanneum Research, Austria, 5Institute of Hydraulic Engineering and Water

Resources Management, Austria, 6Interuniversity Cooperation Centre for Water and Health,

Vienna University of Technology, Austria, 7microLAN BV, the Netherlands and

8Institute for

Water Quality, Vienna University of Technology, Vienna P12 Assessing the probabilistic relationship between colony forming units (cfu) and

most probable number (MPN) estimates Vasco Cadavez and U. Gonzales-Barron CIMO Mountain Research Centre, School of Agriculture, Polytechnic Institute of Braganza, Portugal

P13 Using new fast methods for microbiological water analysis – a safety issue for

the consumer M. Claessens-van der Wiel, T. Lijzenga, A. Douma and G. Wubbels WLN, the Netherlands

P14 Challenges in the development of water-based extraction methods for

screening of mycotoxins in food and feed Barbara Cvak Romer Labs Division Holding GmbH, Austria

P15 Production of MIP particles for multi-mycotoxin analysis

Gilke De Middeleer1,2

, P. Lenain1, P. Dubruel

2 and S. De Saeger

1

1Laboratory of Food Analysis, Department of Bio-analysis, Ghent University, Belgium and

2Polymer Chemistry and Biomaterials Group, Department of Organic Chemistry, Ghent

University, Belgium P16 Biomarkers as accurate tool for the assessment of mycotoxin exposure at

individual levels in Belgium Ellen Heyndrickx

1, I. Sioen

2, J. Diana Di Mavungu

1, S. De Henauw

2, A. Callebaut

3

and S. De Saeger1

1Laboratory of Food Analysis, Ghent University, Belgium,

2Department of Public Health, Ghent

University, Belgium and 3Toxins and Natural Components, CODA-CERVA, Belgium

P17 First reference materials for GM rapeseed powder mixtures

Blagica Dimitrievska, H. Emteborg, A.M. Kortekaas, J. Charoud-Got, J. Seghers and S. Trapmann Institute for Reference Materials and Measurements, Joint Research Centre, European Commission, Belgium


P18 Metabolomic investigations to detect biomarkers associated with changes in meat maturity

James Donarski, M. Harrison, S. Jones and A. Charlton Food and Environment Research Agency, UK

P19 Analysis of mycotoxins in grains using efficient SPE clean-up and superficially

porous HPLC columns E. Barrey

1, K. Espenshied

1, M. Ye

1, O. Shimelis

1 and Christine Dumas

2

Sigma-Aldrich, USA and Sigma-Aldrich Sàrl, France

P20 Extraction and analysis of neonicotinoid pesticides using QuEChERS approach K. Stenerson

1, O. Shimelis

1 and Christine Dumas

2

1Sigma-Aldrich, USA and

2Sigma-Aldrich Sàrl, France

P21 Development of a direct competitive ELISA for the detection of furazolidone

metabolite in foodstuff of animal origin and honey Kseniya M. Filippova, E.S. Vylegzhanina, I.S. Nesterenko, A.A. Komarov and A.N. Panin The All-Russian State Center for Quality and Standardization of Veterinary Drugs and Feeds, Russia

P22 Development of an immunochemical method for boldenone detection

Lucie Fojtíková, S. Göselová, B. Holubová, M. Blažková, L Fukal and O. Lapčík Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Czech Republic

P23 A breakthrough in food allergen testing – development of a one-minute

extraction procedure coupled to a fast ELISA assay Lukas Frank

1, E. Halbmayr-Jech

1, A. Rogers

2, M. Röder

3 and T. Hein

3

1Romer Labs Division Holding GmbH, Austria,

2Romer Labs UK Ltd., UK and

3ifp Institut für

Produktqualität GmbH, Germany P24 Update on the revision and validation of EN-ISO 11290-1 & 2 standard methods

for detection and enumeration of Listeria monocytogenes Nathalie Gnanou-Besse

1, P. Rollier

2 and B. Lombard

1

1Laboratoire de Sécurité des Aliments, Anses, France and

2ACTALIA Cecalait, France

P25 Rapid immunoassay for the detection of anabolic steroid methandienone

Sandra Göselová, L. Fojtíková, B. Holubová, O. Lapčík and L. Fukal Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Czech Republic

P26 On-line sample preparation method performance for multiresidue food contaminant analysis Laszlo Hollosi

1 and M. Godula

2

1Thermo Fisher Scientific, Germany and

2Thermo Fisher Scientific, Czech Republic

P27 Construction and characterization of recombinant bispecific single-chain

daibody and green fluorescent protein fusion for multi-analyte analysis of fluoroquinolones and sulfonamides K. Wen

1, M. Chen

1, Z. Wang

1.2 and Haiyang Jiang

1,2

1College of Veterinary Medicine, China Agricultural University, China and

2National Reference

Laboratories for Veterinary Drug Residue, China


P28 From farm-to-fork : Merck Millipore Singlepath® Direct Campy Poultry rapid test kit for farm-based direct detection of Campylobacter spp. in caecal-type samples from live chicken Lisa John

1, J. Slaghuis

1, M. Wadl

2, M. Wagner

3, T. Seliwiorstow

4, J. Baré

4,

M. Uyttendaele5, L. De Zutter

4 and C. Lindhardt

1

1Merck Millipore, LBR-Applications, Merck KGaA, Germany, ²Unit for Surveillance,

Department for Infectious Disease Epidemiology, Robert Koch-Institute, Germany, 3Department for Veterinary Public Health and Food Safety, University of Veterinary Medicine,

Austria, 4Faculty of Veterinary Medicine, Veterinary Public Health and Food Safety, University

of Ghent, Belgium and 5Faculty of Bio-Science Engineering, Department of Food Safety and

Food Quality, University of Ghent, Belgium

P29 Concentration of large volumes of water for waterborne pathogen detection

using a tangential flow filtration based system Abdelfateh Kerrouche, S. Wilhelm, H. Bridle and M. Desmulliez MicroSystems Engineering Centre, Heriot-Watt University, UK

P30 On-site isothermal LAMP assays for bacteria, viroid and phytoplasma detection P. Kogovšek

1, N. Boonham

3, M. Dermastia

1, M. Dickinson

4, T. Dreo

1, J. Hall

3,

J. Hodgetts3, R. Lenarčič

1, P. Llop

2, N. Mehle

1, D. Morisset

1, P. Nikolić

1, M. Pirc

1,

N. Prezelj1, A. Rotter

1 and M. Ravnikar

1

1National Institute of Biology, Slovenia,

2Biosistemika, Research and Development LLC,

Slovenia, 3The Food and Environment Research Agency, UK and

4School of Biosciences,

University of Nottingham, UK P31 Evaluation of a lateral flow test for quantitative detection of aflatoxin M1 at

50 ng/l in raw commingled milk Wilbert Kokke

1 and R. Salter

2

1Kentron Microbiology BV, the Netherlands and

2Charm Sciences, Inc., USA

P32 Coliphage assay to detect faecal contamination in water in 8 hours Wilbert Kokke

1 and R. Salter

2



P33 Water-based extraction of mycotoxins from animal feedstuffs Wilbert Kokke

1, J. Jabor

2 and M. Tess

2



P34 Authentication of Basmati rice using SSR-PCR and the QIAxcel Advanced

system R. Cassier

1 and Mirjana Kozulic

2

1Laboratoire AdGène, France and

2Qiagen Instruments AG, Switzerland

P35 Characterisation of infant formula quality parameters using the FAST method

and the Amaltheys® analyser A. Liogier de Sereys, S. Muller, A. Acharid, V. Fogliano, Pierre Lacotte and I. Birlouez-Aragon Spectralys Innovation, France

P36 Fluorescence polarisation immunoassays for rapid determination of

mycotoxins in foodstuffs Vincenzo Lippolis

1, M. Pascale

1, M. Suman

2 and A. Visconti

1

1Institute of Sciences of Food Production , National Research Council of Italy, Italy and

2Food

Research Labs, Barilla SpA, Italy


P37 Rapid test systems for detection and quantification of microorganisms in food and beverages: Listeria and Salmonella Kathleen Merx, K. Vetter, U.-M. Kohlstock and M. Zachlod Becit GmbH, Germany

P38 The identification of new species of bacteria from the genus Cronobacter using biochemical tests, polymerase chain reaction and mass spectrometry

Denisa Mihalová, B. Javůrková, M. Blažková, P. Rauch and L. Fukal Department of Biochemistry and Microbiology, Institute of Chemical Technology in Prague, Czech Republic

P39 Rapid multi-target detection of pathogens causing mastitis Antoine P.H.A. Moers,

M. Koets, L. van Zeeland, T. Posthuma and A. van Amerongen

Biomolecular Sensing and Diagnostics, Wageningen UR, the Netherlands P40 Detection of single nucleotide polymorphisms in Plasmodium falciparum by

PCR primer extension and lateral flow immunoassay Antoine P.H.A. Moers

1, C.J. Sutherland

2, R.L. Hallet

2, R. Burrow

2, H.D.F.H. Schallig

3 and

A. van Amerongen1

1Biomolecular Sensing and Diagnostics, Wageningen UR, the Netherlands,

2Department of

Immunology and Infection, London School of Hygiene and Tropical Medicine, UK and 3Parasitology Unit, Biomedical Research, Royal Tropical Institute the Netherlands

P41 Evaluation of the Evidence Investigator platform from Randox for the screening of drug residues in milk matrices A. Beck and Claudia Mujahid Nestlé Research Center, Switzerland

P42 Subtyping of avian influenza infection using a protein microarray

Truus Posthuma1, J. Post

2, R. Bouwstra

2, G. Koch

2 and A. van Amerongen

1

1Biomolecular Sensing and Diagnostics, Wageningen UR, the Netherlands and

2Central

Veterinary Institute, Wageningen UR, the Netherlands

P43 Evaluation of alternative methods for the extraction and fractionation of bioactive carbohydrates L. Ruiz-Aceituno, B. Alonso-Rodríguez, M.L. Sanz and Lourdes Ramos Department of Instrumental Analysis and Environmental Chemistry, IQOG-CSIC, Spain

P44 A novel device for on-site pathogens detection in food Pedro Razquin

1, G. Cebrián

1, X. Arias

1, L. Mata

1, P. Aldamiz-Echevarría

2, J. Escobal

2,

J. Berganza2, G. Diez

2, J.I. Valpuesta

3, A. Perez

3 and J. Fernandez de Mendiola

3

1Zeulab, Spain,

2Centro Tecnológico Gaiker, Spain and

3CTL-TH Packaging, Spain

P45 Developing a rapid in-farm diagnostic test for Campylobacter M. Rosario Romero

1, M. D’Agostino, N. Cook

1, S. Robles

1, C. Fernández

1 and M.P. Andreou

2

1The Food and Environment Research Agency, UK and

2Optisense Ltd., UK

P46 Quantification of phomopsin A: biosynthesis of 15N6-labelled internal standard Svenja Schloss, I. Wedell, M. Koch and R. Maul Federal Institute for Material Research and Testing, Germany

P47 Microbial quality control of beer by RNA

K. Vetter1, K. Merx

1 and Jvo Siegrist

2

1BECIT GmbH, Germany and

2Sigma-Aldrich, Switzerland

P48 Validation of a new chromogenic media for Clostridium perfringens

M. Manafi1 and Jvo Siegrist

2

1Medical University Vienna, Austria and

2Sigma-Aldrich, Switzerland


P49 Surface plasmon resonance (SPR) assay for the detection and quantification of total amount of okadaic acid, DTX1 and DTX2 in shellfish Gurmit Singh

1, B. Brady

1, T. Koerner

1, A.-C. Huet

2 and P. Delahaut

2

1Food Research Division, Bureau of Chemical Safety, Health Canada, Canada and

2Centre

d’Economie Rurale, Health Department, Belgium

P50 Pathatrix Auto™ – the first AFNOR-approved real-time PCR method for detecting Salmonella in pooled food samples J. Wall

1, D. Sohier

2, R. Conrad

1 and Katrien Vanhonacker

1

1Thermo Fisher Scientific, Inc., USA and

2Adria, France

P51 Campylobacter detection system ready for field application

Jos M.B.M. van der Vossen, E. Lommen, H. Rahaoui, F.H.J. Schuren and R.C. Montijn Microbiology and Systems Biology, TNO, the Netherlands

P52 Bead-based suspension array for detection and identification of tick-borne Borrelia species R.P. Achterberg

1, C.B. van Solt-Smits

1, M.E. Hovius

2, H. Sprong

3, M. Fonville

3, F.M. De Bree

1

and Fimme J. van der Wal1

1Infection Biology, Central Veterinary Institute, the Netherlands,

2Companion Animal Hospital ‘t

Heike, the Netherlands and 3Laboratory for Zoonoses and Environmental Microbiology,

National Institute of Public Health and the Environment, the Netherlands

P53 Applications of polymer microarrays to water purification Sesha Venkateswaran School of Chemistry, University of Edinburg, UK

P54 Quantification errors using qPCR

Lucie Vondrakova, J. Pazlarova and K. Demnerova Department of Biochemistry and Microbiology, Institute of Chemical Technology in Prague, Czech Republic

P55 Simultaneous determination of free and conjugated Alternaria toxins in various

matrices by LC-MS/MS Jeroen Walravens

1, H. Mikula

2, M. Rychlik

3, S. Asam

4, J. Diana Di Mavungu

1, A. Van

Landschoot5, L. Vanhaecke

6 and S. De Saeger

1

1Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Belgium,

2Institute of Applied Synthetic Chemistry, Vienna University of Technology, Austria,

3Bioanalytik Weihenstephan, ZIEL Research Center for Nutrition and Food Sciences, TU

München, Germany, 4Chair of Analytical Food Chemistry, TU München, Germany,

5Associated Faculty of Applied Bioscience Engineering, Biochemistry-Brewery Research

Group, HoGent, Belgium and 6Laboratory of Chemical Analysis, Faculty of Veterinary

Medicine, Ghent University, Belgium

P56 Quad-fluorescence polarisation immunoasay for simultaneous quantitative high-throughput screening of multiple antibacterial residues in milk T. Mi1 and Zhanhui Wang1,2

1College of Veterinary Medicine, China Agricultural University, China and

2National Reference

Laboratories for Veterinary Drug Residues, China

P57 A rapid fluorescence polarisation immunoassay for the determination of six

macrolide antibiotics in milk H. Zhang1 and Zhanhui Wang1,2 1College of Veterinary Medicine, China Agricultural University, China and

2National Reference

Laboratories for Veterinary Drug Residues, China


P58 Lanthanoid complexes as specific detection principle in silica sol-gel immunoassays Jan H. Wichers

1, X. Liu

2, E. Bouwman

2 and A. van Amerongen

1

1Biomolecular Sensing and Diagnostics, Wageningen UR, the Netherlands and

2Leiden

Institute of Chemistry, Gorlaeus Laboratories, Leiden University, the Netherlands P59 An independent evaluation of the fast detection and confirmation of Listeria

species in food by Singlepath® Listeria and Singlepath® L'mono lateral flow test Heike Wulff

1, M. Goll

2, T. Miller

2, F. Düker

2, C. Spielvogel

1, R. Ossmer

1, L. John

1 and M. Bülte

2

1Merck Millipore, Germany and

2Justus-Liebig-Universität Giessen, Germany

P60 Fast detection of viable Legionella pneumophila cells in water using flow cytometry M. Adela Yáñez

1, L. Martínez

1, E. Soria-Soria

1, R. Múrtula

1, A. Parker

2 and V. Catalán

1

1Aqualogy, Labaqua, Spain and

2 MemTeq Ventures, Spain


P1 BEAD-BASED SUSPENSION ARRAY FOR SUBTYPING AVIAN INFLUENZA INFECTIONS IN CHICKENS René Achterberg, J. Post, R. Bossers-de Vries, S. Pritz-Verschuren, R. Bouwstra, V. Broks, C. Overbeek, G. Koch and F.J. van der Wal Central Veterinary Institute, Wageningen UR, the Netherlands [email protected] Avian Influenza (AI) is caused by the influenza A virus. Subtyping of AI is based on the surface proteins haemagglutinin (H) and neuraminidase (N). Most AI viruses cause only mild symptoms in poultry. However, AI viruses of the subtype H5 and H7 can evolve into high pathogenic avian influenza (HPAI) by only few mutations. Since HPAI viruses cause severe disease and high mortality, AI caused by H5 and H7 subtypes is a notifiable disease for which immediate control action is compulsory. In the Netherlands, monitoring of AI is, among others, based on serology, where each flock is tested with ELISA for the presence of antibodies against influenza nucleoprotein. AI-positive samples are subsequently tested in the haemagglutination inhibition test using H5 and H7 antigens. As this is an elaborate test for which relatively large volumes of serum are required, the current scheme does not allow for complete subtyping, thereby preventing full insight in the introduction of subtypes and their transmission. With a comprehensive panel of antigens complete subtyping would be enabled, beneficial for both veterinary and human medicine. To enable comprehensive subtyping of AI virus infections in poultry, a serological multiplex assay was set up in the format of an automatable bead-based suspension array. To this end, recombinant H and N proteins (H1-16, N1-9; 1-6 variants per subtype) were coupled to Luminex beads using standard chemistry. The first results with a set of reference sera showed that the assay is capable of correct subtyping. Further evaluation with field samples is in progress. Acknowledgements This research was funded by the Dutch Ministry of Economic Affairs.


P2 DEVELOPMENT AND VALIDATION OF A NUCLEIC ACID MICROARRAY IMMUNO-ASSAY AND A NUCLEIC ACID LATERAL FLOW MICROARRAY IMMUNOASSAY FOR THE DETECTION OF PLASMODIUM SPECIES Selected for speed presentation G.A. Posthuma-Trumpie1, A.P.H.A. Moers1, C.J. Sutherland2, R. Burrow2, H.D.F.H. Schallig3, H.M. de Vogel-van den Bosch1 and Aart van Amerongen1

1Biomolecular Sensing and Diagnostics, Wageningen UR, the Netherlands, 2Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, UK and 3Unit Parasitology, Department Biomedical Research, Royal Tropical Institute , the Netherlands [email protected] Microscopy is frequently used for malaria diagnosis, but at low parasitaemia it becomes less sensitive and time-consuming. On the other hand, molecular tools allow for specific and sensitive diagnosis, but current formats, such as PCR combined with gel electrophoresis and qPCR, are difficult to implement in resource poor settings. Therefore, we developed two simple detection systems, the nucleic acid microarray immunoassay (NAMIA), a microarray test on nitrocellulose pads mounted on a microscopic slide, and the nucleic acid lateral flow microarray immunoassay (NALMIA, max. 30 min run time). Visualisation was achieved by using black carbon nanoparticles with NeutrAvidin-alkaline phosphatase (NAMIA) or NeutrAvidin (NALMIA) adsorbed at their surface. In the case of NAMIA a first incubation with carbon-NeutrAvidin-alkaline phosphatase (30 min) was followed by a short incubation (10 min) with enzyme substrate to further enhance the spot-signal by the precipitating dye formed. A multiplex PCR was used to amplify DNA specific for all Plasmodium species (Pan-Plasmodium), P. vivax, P. falciparum and the human gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that served as an internal amplification control. Specificity was obtained through primer sets with discriminating tags on the forward primers and biotin on all reverse primers. A validation study was performed with 110 human samples. Reference was expert microscopy and in some cases rt-PCR at the PHE Malaria Reference Laboratory. For the NAMIA test platform, the kappa values were 0.91 and 0.88 for P. falciparum and P. vivax, respectively. In the case of the NALMIA test platform, these k values were 0.70 and 0.93. These kappa values indicate a good to excellent agreement with the reference method qPCR. Both formats provide a sensitive and specific test on the presence of Plasmodium species. PCR-NAMIA and PCR-NALMIA are fast and low-cost test formats. After a brief instruction, health workers can perform these tests in low-resource environments. Acknowledgements This work was partly funded by the European Commission FP7 grant contract 201889 ‘Multi-drug resistance in malaria under combination therapy: assessment of specific markers and development of innovative, rapid and simple diagnostics’ (MALACTRES), and by the Wageningen UR Kennisbasis cluster KB-17 ‘New technologies’, theme 002.02 ‘Diagnostics and biosensors’, project 003 ‘Miniaturised, multi-analyte and on-site diagnostic platforms’.


P3 VIABILITY PCR: AN EVALUATION OF PROPIDIUM MONOAZIDE FOR LIVE/DEAD DIFFERENTIATION OF MICROORGANISMS IN TURBID SOLUTIONS K. Wolf, S. Fakih, C. Küppers, A. Bogdoll, S. Schlappa, Marcia Armstrong, S. Luley and R. Peist Qiagen, Germany [email protected] Real-time PCR (rt-PCR) provides fast and powerful tools to analyse samples for the presence of potentially harmful microbes, but cannot differentiate between DNA from live and dead cells, which can be required for applications such as the determination of viability in probiotic bacteria or hygiene monitoring. Viability PCR utilises the DNA-masking compound propidium monoazide (PMA), which is able to enter dead and membrane-compromised cells, modifying the DNA on photo-activation, resulting in a strong inhibition of PCR amplification. One concern with the use of a photo-activated compound is suboptimal performance in the presence of turbid or coloured matrices. The aim of this study was to evaluate the effect of such matrices on the performance of viability PCR centering on PMA and a new illumination device. Live or heat-killed pathogens were prepared (Salmonella spp. and Listeria spp.) and treated with/without PMA. Samples were prepared in a 2.5% solution of Nutella matrix. Samples of a Gram-positive probiotic bacterium were studied in a milk matrix. Pathogen mixtures were illuminated using the illumination device at a specific wavelength to irreversibly bind the reagent to dead cell DNA, rendering it no longer PCR visible. Efficient suppression of amplification of such modified DNA allows preferential rt-PCR detection of live cells. The rt-PCR performance data from these cell mixtures with or without PMA treatment demonstrates the extent of the masking effect of PMA on dead cells. Dead cells treated with PMA show a ~6-7.5 higher Ct value than their non-PMA treated counterpart (X2<0.05). The data using turbid/coloured matrices demonstrates that the PMA activating light is sufficient to penetrate such samples, allowing successful DNA intercalation and subsequent live/dead detection even in the presence of potentially interfering matrices. Live/dead differentiation can play an important role in procedures such as hygiene monitoring (success of decontamination processes), water testing (distinguishing between live and dead Legionella for regulatory compliance) and human diagnostics (monitoring medication efficiency in pathogen killing).


P4 SALMONELLA AND LISTERIA MONOCYTOGENES DETECTION IN COMPLEX FOOD MATRICES USING A FULLY AUTOMATED PATHOGEN PURIFICATION AND DETECTION SYSTEM Marcia Armstrong, S. Fakih, S. Schlappa, C. Kueppers, P. Cornelius and S. Luley Qiagen, Germany [email protected] The global food industry has been presented with increasing food safety concerns and a resulting demand for rapid, accurate, and easy-to-use pathogen detection systems. Real-time PCR represents a powerful tool for specific and rapid detection of foodborne pathogens. The complexity of food matrix cultures can limit the universal adoption of such methods. The purpose of the current study was to develop a single universal automated method for the purification of Gram-negative (Salmonella spp.) and Gram-positive (Listeria monocytogenes) bacteria from such matrices. A fully automated purification method for both Gram-negative and Gram-positive pathogens was developed on the QIAsymphony Rotor-Gene Q platform. For the purposes of this study, the capacity of the automated DNA extraction and real-time PCR detection system to deal with high inhibitor concentrations was tested by preparation of pathogen enrichment cultures with different food:medium ratios. Starting with the established 1:10 ratio (25 g food in 225 ml enrichment medium), the food matrix background was constantly increased up to 1:6 ratio (25 g food in 125 ml enrichment medium). For Salmonella spp., the increase in matrix background was tested with chocolate, peanut butter, whole milk, and ground beef in BPW. Similarly, 25 g samples of 3.5% fat milk, sliced turkey and smoked salmon were added to decreasing volumes of ONE Broth medium plus supplement, spiked with Listeria monocytogenes. The resulting enrichment cultures for both bacteria types were directly processed on a QIAsymphony SP module using the QIAsymphony Bacteria Kit. The protocol is based on a chemically aided heat lysis, with specific depletion of food-derived inhibitors and DNA purification with magnetic silica beads. The resulting DNA solution was assayed for the presence of pathogen using real-time PCR based pathogen detection assays on the Rotor-Gene Q. Reliable pathogen DNA recovery and detection for both Gram-negative and Gram-positive bacteria were observed over all tested matrix:medium ratios, suggesting an efficient background depletion during DNA isolation. For all tested ratios, Salmonella detection showed stable CT values between 18 (whole milk) and 30 (ground beef). For Listeria, CT values ranged between 24 (3.5% fat milk) and 34 (sliced turkey). The fully automated pathogen purification and detection system presented here provides a powerful, reproducible, and sensitive method for the purification and detection of Salmonella spp. and Listeria monocytogenes in difficult food matrices using a universal one-for-all purification system.


P5 ANALYSIS OF ANTIBIOTICS IN COMPLEX FOOD MATRICES USING A SOLID PHASE EXTRACTION SORBENT BASED ON MOLECULARLY IMPRINTED POLYMERS D. Derrien1, C. Perollier1, O. Lépine1, Michel Arotçaréna1, K. Naraghi1, K. Haupt2 and S. Bayoudh1

1Polyintell, France and 2Compiegne University of Technology, France [email protected] Broad spectrum antibiotics such as tetracyclines (TCs) are widely used in veterinary medicine in food-producing animals. These antibiotics and their degradation products may be present in food of an animal origin and cause harmful effects on consumers. So, an adequate withdrawal time is required to ensure the safety of food and prevent the occurrence of these antibiotics in tissues from slaughtered animals or in milk. Therefore, the presence of these molecules in foodstuff is regulated with a maximum residue levels. Other broad spectrum antibiotics, such as chloramphenicol, are prohibited from use in food-producing animals in the European Union, the USA and many other countries due to major human health concerns. No residue must be found in food. To prevent the consumption of contaminated food, reliable analytical methods are needed to ensure the control of the food chain. Thanks to the technological improvement of analytical devices, very low concentrations of these residues can be detected. However, even with these highly performing equipments, sample pre-treatment remains a key factor for the success of the analysis by reducing interferences and theirs subsequent consequences, the ion suppression effects and the detector fouling. This poster shows sample pre-treatments for the analysis of tetracyclines (oxytetracycline, tetracycline, chlortetracycline and their epimers) and chloramphenicol in complex food matrices. The method is based on solid phase extraction (SPE) cartridges based on molecularly imprinted polymer (MIP). MIPs are affinity columns made with polymers that are very stable to aqueous or organic solvents as well as temperature. These cost-effective products are widely used for clean-up and preconcentration applications. High recovery yields were obtained after a fast and efficient clean-up. Moreover these MIP could be used for a direct detection of analytes in food and environmental samples with a sensor [1]. References 1. Ton, X.A., Acha, V., Haupt, K. and B. Tse Sum Bui, B., 2012. Biosensors and Bioelectronics 36:

22-28. Acknowledgments EU – IRMED project.


P6 EXPERIENCES WITH REGARD TO THE INTRODUCTION OF RAPID METHODS IN A DRINKING WATER LABORATORY Adrie Atsma, S. in ’t Veld, M. IJszenga and B. de Graaf. Vitens laboratory, the Netherlands [email protected] Vitens laboratory performs bacteriological analyses on more than 200,000 water samples a year. Most of these analyses are based on cultivation. A disadvantage of cultivation is that a large population of the target organisms in a water sample is not culturable. Besides that, the culturing time is relatively long, up to 7 days. To ascertain a good drinking water quality it is important to know this as quickly as possible. Therefore a lot of rapid methods are being developed, most of them based on polymerase chain reaction (PCR)-related methods. The advantage of introducing PCR-based methods is the relatively short processing time of PCR (a few hours); and PCR is selective, flexible, easy to automate and with a PCR method non-culturable species can also be detected. A point of discussion is that no prediction about the viability of the detected microorganisms can be made. In this presentation, our experiences in relation to this issue will be presented. Two developed rapid methods, the EccoPCR and PMA-qPCR, are introduced here. The EccoPCR is used as an ‘early warning’ screening for the presence of culturable Escherichia coli and coliforms in water samples. With this method, the processing time is reduced to 8 hours. The PMA-qPCR is used by Vitens laboratory for measuring intact Legionella (pneumophila) species in drinking water. The use of PMA-qPCR has led to a reduction in analysis time from 7 days to 5 hours. Both PCR-related methods are currently being applied in the laboratory. Vitens laboratory sees the development and introduction of rapid PCR-related methods as an opportunity to replace or as an addition to traditional culture-related methods, providing a fast answer for the drinking water companies about water quality.


P7 THE COSYPS PATH FOOD PROJECT: DEVELOPMENT OF A SYSTEM FOR DETECTION AND IDENTIFICATION OF FOOD-BORNE PATHOGENIC BACTERIA PRESENT IN CARCASS SAMPLES BASED ON A ‘COMBINATORY QPCR’ TECHNOLOGY Elodie Barbau-Piednoir1,2,3, J. Mahillon1, N. Roosens2 and N. Botteldoorn3 1Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, Belgium, 2Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health, Belgium and 3Food Pathogens, Scientific Institute of Public Health, Belgium [email protected] In foodborne pathogens control laboratories, the official detection methods are ISO methods. These methods are mainly culture-based methods, which are time consuming (several days), labour-intensive and monotarget. Therefore, there is a need to develop methods for rapid detection and identification of living pathogens in foodstuffs. In this study, a combinatory SYBR®Green qPCR screening system for rapid and simultaneous detection of Listeria spp. and Salmonella spp. in carcasses swabs samples, named CoSYPS Path Food, was developed and validated. This system is based on the method developed by the Scientific Institute of Public Health (WIV-ISP) to detect the presence of genetically modified organisms in food samples using the combinatory SYBR®Green qPCR Screening (CoSYPS) [1,2]. Two sets of SYBR®Green qPCR assays have been developed and validated for the detection and discrimination at species and/or subspecies level of Listeria spp. and Salmonella spp. [3,4]. All steps, from food sample to strains isolation and confirmation, were then developed. The entire process is called complete CoSYPS Path Food workflow. Finally, the system was validated according to ISO 16140 (comparison with the ISO methods [5,7]) on spiked artificial swabs contaminated with one or both targets, and at different concentrations. Due to its multitargets strategy, the CoSYPS Path Food system is a reliable system. Indeed, a lower number of false negative is expected compared to monotarget qPCR detection systems. This multilevel detection also gives additional information on the detected strains (species, subspecies). Using the complete CoSYPS Path Food workflow, bacterial strains were detected in two days, which considerably reduces the time of analysis compared to the conventional ISO methods that require four to five days. The limit of detection of the complete CoSYPS Path food workflow was the same as the reference ISO methods: 2 to 4 cfu Listeria/swab and 1 to 2 cfu Salmonella/swab, even when both pathogens are present in the same sample. In conclusion, the complete CoSYPS Path Food workflow is a reliable and fast alternative to the conventional culture-based methods. Moreover, it gives additional information on the detected the strains (both at the species and subspecies levels). References 1. Van den Bulcke, M., Lievens, A., Leunda Casi, A., Mbongolo Mbella, G., Barbau-Piednoir, E. and

Sneyers, M. (inventors), 2008. PCT/EP2008/051059WO/2008/092866. 2. Van den Bulcke, M., Lievens, A., Barbau-Piednoir, E., Mbongolombella, G., Roosens, N.,

Sneyers, M. and Leunda Casi, A., 2009. Analytical and Bioanalytical Chemistry 396: 2113-2123. 3. Barbau-Piednoir, E., Botteldoorn, N., Yde, M., Mahillon, J. and Roosens, N.H., 2013. Applied

Microbiology and Biotechnology 97: 4021-4037. 4. Barbau-Piednoir, E., Roosens, N.H., Bertrand, S., Mahillon, J. and Botteldoorn, N., 2013. Applied

Microbiolology and Biotechnology 97: 9811-9824. 5. International Organization for Standardization (ISO), 1996. ISO 11290-1:1996. 6. International Organization for Standardization (ISO), 2002. ISO 6579:2002. 7. International Organization for Standardization (ISO), 2005. ISO 11290-1/A1:2005.


P8 INNOVATIVE ANALYTICAL TOOLS FOR Β-TRIKETONE HERBICIDES MONITORING E. Rocaboy, T. Noguer and Lise Barthelmebs Institut de Modélisation et d’Analyse en Géo-Environnement et Santé, Université Perpignan Via Domitia, France [email protected] Since atrazine was banned in several European countries, new selective herbicides, such as those from the β-triketone family, have been developed and introduced onto the market today. They are mainly represented by sulcotrione and mesotrione, registered in Europe in 1993, and in 2000 respectively. The molecular target site of these compounds is the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD), involved in the biosynthesis of carotenoids in plants and which inhibition results in bleaching of the foliage. In the frame of TRICETOX, a French research project funded by Agence National de la Recherche, we aim to develop new analytical tools, as alternative methods to chromatographic chemical analysis, for in-field β-triketone herbicides monitoring. These methods have to be easy to handle, in accordance with the critical value for β-triketone herbicide detection (5 µM) and not to require expensive instrumentation and highly trained personnel. In a first strategy, we have developed a whole cell colorimetric bioassay based on HPPD inhibition using a recombinant Escherichia coli expressing a vegetal HPPD. The principle of the bioassay is based on the ability of the recombinant E. coli clone, immobilised in sol-gel matrix in a 96 wells microplate format, to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenyl-pyruvate (HPP) and homogentisate (HGA). Addition of sulcotrione was shown to decrease the production of the pigmentation. The limit of detection obtained for mesotrione, tembotrione and sulcotrione was 0.069 µM, 0.051 µM and 0.038 µM, respectively, allowing to validate the whole cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. In the second strategy, the inhibitory capacity of β-triketone herbicides towards HPPD was also used to propose an enzymatic biosensor based on an electro-chemical transduction method. HPPD purified from the recombinant E. coli clone catalyses the transformation of HPP into HGA, which can be oxidized electrochemically at an appropriate potential. Electrochemical detection was performed, using home-made carbon screen-printed electrodes (CSPE) as the transducer. CSPE offer many advantages, such as small size, mass and cost-effective production, that allows using them as disposable electrodes. Our results showed that addition of sulcotrione led to the diminution of the measured current. Several methods are currently assessed for HPPD immobilisation. It is expected that the developed biosensor will allow attaining lower limits of detection than the bioassays, due to the inherent sensitivity of the transduction, and could be used for environmental applications requiring high capacity monitoring, such as environmental risk assessment or bioremediation process surveillance.


P9 A NOVEL AND RAPID APPROACH FOR MONITORING ENZYMATIC PROTEIN HYDROLYSIS USING INFRARED SPECTROSCOPY N.K. Afseth, T. Aspevik, F. Lundby, B. Narum, Å. Oterhals and Ulrike Böcker

Nofima – Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway [email protected] Enzymatic protein hydrolysis of by-products of marine and animal origin is an emerging industry world-wide. In both the animal and marine sector, enzymatic protein hydrolysis has been a subject of focus for several years, and currently there are several commercial companies that produce protein hydrolysates from by-products of various origins. One of the main challenges regarding industrial enzymatic protein hydrolysis is to provide stable and optimal product quality based on raw materials of highly variable composition. Rapid and robust monitoring of raw material quality, the hydrolysis process and end product quality is thus of great importance. There is, however, currently a lack of techniques to serve these purposes. A crucial point in the hydrolysis is to decide when to terminate the process and inactivate the enzymes. There are numerous techniques based on chemical reference methods to serve this purpose, but the techniques are generally time-consuming and not feasible for industrial use. Infrared spectra are known to be sensitive probes for protein structure, and feasibility studies using rapid infrared and near-infrared sensors for monitoring of hydrolysis reactions have shown promising results. The limiting factor for these analyses is, however, related to sensitivity issues related to low concentrations of the analytes in aqueous solutions, and interferences from water in the protein region of the infrared spectra. Thus, we have recently developed an alternative approach for infrared analysis based on thin dried films of protein hydrolysates. Using this methodology, we are now able to monitor the enzymatic hydrolysis of byproducts from salmonoid and chicken production based on spectral changes in the protein regions of the infrared spectra. Moreover, the information is quantitative and can be directly related to the degree of hydrolysis as measured by standard chemical reference methodology. The approach is rapid, easy to use, could be automated, and can potentially be implemented in low-cost instrumentation.


P10 ISOTHERMAL DNA AMPLIFICATION APPROACHES FOR THE DETECTION OF GENETICALLY MODIFIED MAIZE C. Zahradnik1, C. Kolm1, R. Martzy1, R.L. Mach2, A.H. Farnleitner2, R. Krska3 and Kurt Brunner1 1Institute for Analytical Chemistry, Vienna University of Technology, Austria, 2Research Group Environmental Microbiology and Molecular Ecology, Institute for Chemical Engineering, Vienna University of Technology, Austria and 3Institute for Chemical Engineering, Department IFA-Tulln, BOKU Vienna, Austria [email protected] In conjunction with the required mandatory labelling for genetically modified organisms (GMOs), the European Directives (EG) 1139/98 and 49/2000 introduced a threshold limit of 1%. Above this level, the declaration ‘contains genetically modified organisms’ is obligatory for food products. Although validated polymerase chain reaction (PCR) assays are available and meet the requirements that are needed for the detection and evaluation of agricultural commodities, this method is time-consuming, expensive and requires not only costly instruments but also trained personnel. These facts make a rapid, on-site testing for GMOs rather difficult. Recently, several amplification methods that can be performed under isothermal conditions have been developed. All these techniques use DNA polymerases with strand displacement activity. The time- and energy-consuming DNA denaturation step of conventional PCR assays can be omitted with this class of enzymes. A complex temperature profile is not needed anymore and the reactions can be conducted with a simple heating block instead. An additional simplification is accomplished by visual detection of amplification products with agarose gel electrophoresis or direct staining with SYBR Green I at the end of the amplification reaction. In this study, four different isothermal amplification methods, i.e. loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR) and rolling circle amplification (RCA), have been evaluated to detect the 35S promotor used in numerous genetically modified plants. Each approach was tested for specificity and sensitivity; furthermore, the operability of the method was also taken into account. Results were verified by comparison of the isothermal methods with validated PCR assays using certified reference material as samples. It could be demonstrated that LAMP and HDA can be used for future screening application whereas the other methods failed to specifically amplify the target sequence.


P11 A SENSITIVE AND ROBUST METHOD FOR AUTOMATED ON-LINE MONITORING OF ENZYMATIC ACTIVITIES IN WATER AND WATER RESOURCES P. Stadler1, T. Lendenfeld2, G. Ryzinska-Paier3,6, A.P. Blaschke5,6, H. Stadler4, R. Mach3,6, Kurt Brunner3,6, J. Appels7, M Zessner1,8 and A.H. Farnleitner1,3,6

1Centre for Water Resource Systems, Vienna University of Technology, Austria, ²mbOnline GmbH, and WSB Labor GmbH, Austria, ³Research Group Environmental Microbiology and Molecular Ecology, Institute of Chemical Engineering, Vienna University of Technology, Austria, 4Department of Water Resources Management, Institute for Water, Energy and Sustainability, Joanneum Research, Austria, 5Institute of Hydraulic Engineering and Water Resources Management, Austria, 6Interuniversity Cooperation Centre for Water and Health, Vienna University of Technology, Austria, 7microLAN BV, the Netherlands and 8Institute for Water Quality, Vienna University of Technology, Vienna [email protected] On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring at water resources (using glucuronidases, galactosidases, esterases, etc.). Due to the possible short measuring intervals, enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes the successful realisation of a novel concept for automated on-line monitoring of enzymatic activities. During a two years field study at karstic and porous ground water resources, the new method proved sensitive and robust for enzymatic on-line determination. It could also be demonstrated that rapid enzymatic on-site determination can also be operated at an agricultural influenced catchment. The tested device can be adapted for any enzymatic reaction with diagnostic relevance for microbial water quality monitoring (for which substrates are available). The option of automated filtration of up to 5 litres enables a highly sensitive quantification of enzymatic activities. Internet-based data transfer, using internal control parameters for verification and a dynamic determination of the limit of quantification, enables reliable enzymatic on-line monitoring. Although surface waters are a big challenge for automated detection devices due to high sediment load during event conditions, it is shown that rapid and on-site enzymatic detection can successfully be operated from a technical point of view. Selection of the type of measured enzymatic activities has to be done on a catchment-specific basis and further work is needed to learn more about its detailed information characteristics in different habitats. The results of this study highlight that automated on-line monitoring devices for microbial or biochemical parameters to support water quality monitoring is a realistic task. The application of such automated field systems will likely become increasingly important for sustainable and pro-active water management in the near future. References 1. Ryzinska-Paier, G., Lendenfeld, T., Correa, K., Stadler, P., Blaschke, A.P., Mach, R.L., Stadler,

H., Kirschner, A.K.T. and Farnleitner, A.H. A sensitive and robust method for automated on-line monitoring of enzymatic activities in water and water resources. Water Science Technology (in press).


P12 ASSESSING THE PROBABILISTIC RELATIONSHIP BETWEEN COLONY FORMING UNITS (CFU) AND MOST PROBABLE NUMBER (MPN) ESTIMATES Vasco Cadavez and U. Gonzales-Barron CIMO Mountain Research Centre, School of Agriculture, Polytechnic Institute of Braganza, Portugal [email protected] Serial dilution assays and plate counting are two common procedures for estimating microbial concentration in foods, with results reported as most probable number (MPN) and colony forming unit (cfu) estimates of the true (unknown) concentration. As the MPN technique is one of the preferred methods for quantifying pathogen concentration, such as Escherichia coli O157:H7 and Salmonella, in many food commodities, it is desirable to assess its performance based on the more ‘precise’ plate counting technique. Thus, the objective of this study was to derive a theoretical probabilistic relationship between the probability distribution of three- and five-tube MPN patterns, and the observed cfu, based on a common underlying true microbial concentration in the food sample. This relationship was derived considering the intrinsic variabilities of the two procedures, that is the observed cfu is a realisation of the Poisson distribution with unknown mean microbial concentration c; the MPN pattern or number of positive tubes in a three- or five-tube serial dilution originates from a binomial distribution (n, pi) with n=3 or 5, respectively; and pi is the Poisson-based probability of occurrence of at least one microbial cell present in each of the tubes at dilution i. The probabilistic relationship was modelled in MS Excel using the ModelRisk 5.1 simulation package add-on. Expected values, mode and 95% confidence interval (CI) of MPN were estimated for a given observed cfu concentration in the range of 100 to 8,000 cfu per g. The probabilistic model allowed understanding the reasons why the CIs of the MPN estimates are wider than those of the cfu estimates. It also showed that the discrete nature of the MPN procedure was the cause of having a poor resolution to distinguish contamination over wide ranges of cfu values. For instance, for a three-tube dilution assay and plate count measurements between 700-1,300 cfu/g, the most likely corresponding MPN value would be 920 MPN/g. As the microbial contamination increased, the MPN resolution became even poorer. Thus, for a higher value of 4,300 MPN/g, the possible corresponding plate count measurements would be anywhere between 2,900-6,900 cfu/g. While the use of a five-tube serial dilution assay slightly improved the resolution of the technique, its main difference with the three-tube dilution was the considerable reduction of the MPN confidence intervals for a given cfu. For instance, at an observed plate count measurement of 2,000 cfu/g, a three-tube assay might produce discrete values between 310-7,500 MPN/g (95% CI), while values of 680-7,000 MPN/g could be observed with a single five-tube MPN test. This theoretical probabilistic model demonstrated the low correlation between cfu and MPN estimates. Moreover, as this model only took into account the intrinsic variability, the lack of correspondence between the two measurements might be further affected by extrinsic variability, such as the food matrix and physical aggregation of microbial cells.


P13 USING NEW FAST METHODS FOR MICROBIOLOGICAL WATER ANALYSIS – A SAFETY ISSUE FOR THE CONSUMER M. Claessens-van der Wiel, T. Lijzenga, A. Douma and G. Wubbels WLN, the Netherlands [email protected] Dutch drinking water is the safest and cleanest drinking water of the world. Drinking water can be produced out of groundwater or out of surface water after extensive purification. The drinking water is transported in a distribution network without adding any disinfectants. Surface water normally contains a lot of bacteria and chemicals that are prohibited in drinking water according to the Dutch Law. Therefore, the surface water production plants have complex purification steps to remove inter alia pesticides, herbicides, pharmaceuticals and also pathogenic microorganisms. For the reason that there is no disinfectant in the distribution network to kill unwanted bacteria or viruses, it is important to make safe drinking water and to contain it safe in the distribution pipes. Frequently, new pipes are introduced in the system and sadly enough old pipes sometimes break by accident causing unwanted leakages. In those cases, there is a risk that pathogens can enter the distribution network and contaminate the drinking water. During and after a correction to the pipeline, the drinking water is microbiological monitored for the presence of faecal indicators Escherichia coli and enterococci. It takes a minimum of 18 hours before it is known if the water is safe to consume (free of pathogens). It is not always possible to shut down the drinking water for that long time, so it has to be delivered to the consumer with a boiling advice and a risk of contamination. Therefore, it is very important to know as soon as possible if there is any faecal contamination. Normally, water is monitored by culturing techniques that consume a lot of time. In the last decennium, WLN introduced new techniques, such as RT-PCR, MALDI-TOF and flow cytometry to improve the speed and quality of the monitoring methods. For direct analysis of E. coli and enterococci, WLN developed two reverse transcriptase PCR (RT-PCR) methods. Development of these two new methods was quite a challenge, as they had to be very sensitive. WLN has succeeded to make the RT-PCRs as sensitive as the reference culture methods that can detect 1 colony forming unit (cfu)/100 ml. The use of RT-PCR for the detection of indicators of faecal pollution minimises the risk of drinking contaminated drinking water by the consumer.


P14 CHALLENGES IN THE DEVELOPMENT OF WATER-BASED EXTRACTION METHODS FOR SCREENING OF MYCOTOXINS IN FOOD AND FEED Barbara Cvak Romer Labs Division Holding GmbH, Austria [email protected] Approximately 25% of the worldwide cereal harvest is contaminated with mycotoxins, naturally occurring toxins produced by fungi. Therefore, existing and well-known fast and easy screening methods, such as simple strip tests (lateral flow devices) or ELISA tests, have to be enhanced and optimised steadily. Fast and simple extraction of the toxins from agricultural commodities is a crucial step in the development of such a test system. This is usually performed with organic solvents, such as methanol or acetonitrile, due to insolubility of most mycotoxins in polar solvents. If cereals are extracted on-site, untrained people are exposed to danger by using these substances. Moreover, these substances are harmful to the environment, at least as big amounts of solvent are used for sample extraction. Therefore, the reduction of organic solvent consumption or complete replacement is an issue, and of great interest and importance for the future. This presentation will demonstrate the challenges as well as our latest results in implementing new extraction methods by employing less toxic solvents and newly developed buffer systems on an aqueous basis for the detection of mycotoxins by using lateral flow devices.


P15 PRODUCTION OF MIP PARTICLES FOR MULTI-MYCOTOXIN ANALYSIS Gilke De Middeleer1,2, P. Lenain1, P. Dubruel2 and S. De Saeger1

1Laboratory of Food Analysis, Department of Bio-analysis, Ghent University, Belgium and 2Polymer Chemistry and Biomaterials Group, Department of Organic Chemistry, Ghent University, Belgium [email protected] To guarantee food safety, controlling microbiological and chemical hazards is a prerequisite for the food and feed industry. Within the large variety of contaminants, mycotoxins are important and naturally occurring in food and feed. They are secondary metabolites produced by various fungal species. Although mycotoxins are only present in very low concentrations (mg/kg – μg/kg range), they can cause toxic effects in humans and animals, and the economic consequences of contaminations should not be underestimated. A sensitive, accurate and rapid analysis of these toxins is highly needed [1]. Mycotoxin analysis includes rapid screening and confirmatory methods. In the past ten years, interest moved towards a multi-analyte approach. Therefore, selective recognition elements that bind with different target analytes are required. Antibodies represent the most commonly used recognition elements in mycotoxin analysis, but alternatives are being developed since antibodies suffer from some disadvantages including a limited stability [2]. The aim of this research was to use molecularly imprinted polymers (MIP) as alternative recognition elements for multimycotoxin analysis. MIP of different sizes (nm-µm) against ergot alkaloids were produced by using precipitation or emulsion polymerisation or by the core-shell technique. To produce MIP particles, 2,2-azobisisobutyronitrile (initiator), metergoline (template), methacrylic acid, ethylene glycol dimethacrylate, divinylbenzene, trimethylolpropane trimethacrylate and acetonitrile were used. Additionally, different ratios of crosslinking agents have been evaluated. By using MIP particles as molecular recognition elements, target analytes can be selectively extracted from a sample. Such selective recognition elements are difficult to combine with multimycotoxin analysis. Therefore, MIP for different mycotoxins can finally be combined to develop multimycotoxin screening tests and new sample preparation methods by using solid phase extraction (SPE) columns. Very interestingly, the proposed strategy may result in more efficient multimycotoxin analysis. Acknowledgements The authors would like to thank the agency for Innovation by Science and Technology (IWT) for the financial support. References 1. Hoogland, H. and Lelieveld, H., 2007. In: Nollet, L.M.L. and Toldrá, F., eds. Advances in Food

Diagnostics, Blackwell Publishing, Ames, IA, USA, pp. 347-358. 2. Fodey, T., Leonard, P., O'Mahony, J., O’Kennedy, R. and Danaher, M., 2011. Trends in Analytical

Chemistry 30: 254-269.


P16 BIOMARKERS AS ACCURATE TOOL FOR THE ASSESSMENT OF MYCOTOXIN EXPOSURE AT INDIVIDUAL LEVELS IN BELGIUM Ellen Heyndrickx1, I. Sioen2, J. Diana Di Mavungu1, S. De Henauw2, A. Callebaut3 and S. De Saeger1

1Laboratory of Food Analysis, Ghent University, Belgium, 2Department of Public Health, Ghent University, Belgium and 3Toxins and Natural Components, CODA-CERVA, Belgium [email protected] Mycotoxins are considered to be an important risk factor in our food chain. Therefore, it is relevant to estimate the exposure of our population to these toxins. Mycotoxin exposure assessment is currently indirect, based on combining occurrence data in food with food consumption data. The accuracy of this approach is limited due to the heterogeneous distribution of mycotoxins in food, the presence of masked mycotoxins and the under- and overestimation in food consumption data. Therefore, biomarkers have been proposed as a suitable alternative. Biomarkers of the most common mycotoxins have been validated. The individual variation in absorption, distribution, metabolisation and excretion is integrated into the formation of the biomarker, whereby a more accurate assessment of exposure at the individual level can be performed. Thanks to the high sensitivity of the recently developed analytical instruments, biomarkers can be easily measured in different matrices at very low levels. In this study, a quantitative assessment of mycotoxin exposure associated with the dietary intake was performed based on the direct measurement of biomarkers of exposure in morning urine of 300 adults and 100 children. The urine was gathered across Belgium according to a designed study protocol over the different seasons in 2013. Every participant completed a food frequency questionnaire to assess the consumption of some relevant foodstuffs of both the day before the urine collection and the previous month. The protocol was approved by the ethical committee of the University Hospital of Ghent. Validated multi-toxin LC-MS/MS methods were used to analyse aflatoxins, fumonisins, ochratoxin A, trichothecenes, zearalenone and their metabolites in morning urine. Correlations between the biomarker concentrations measured and the food consumption reported was estimated, to explore whether the exposure can be explained by the consumption of certain foods. Study design, methods and results of the whole sampling period are to be discussed. Acknowledgements This research was financially supported by Federal Public Service Health, Food Chain Safety and Environment (Project RT 11/2).


P17 FIRST REFERENCE MATERIALS FOR GM RAPESEED POWDER MIXTURES Selected for speed presentation Blagica Dimitrievska, H. Emteborg, A.M. Kortekaas, J. Charoud-Got, J. Seghers and S. Trapmann Institute for Reference Materials and Measurements, Joint Research Centre, European Commission, Belgium [email protected] The Institute for Reference Materials and Measurements (IRMM) released the first rapeseed powder material certified for a GMO mass fraction worldwide. This certified reference material will help GMO testing laboratories to test for the presence of 73496 rapeseed and to implement the European labelling threshold once the 73496 GM rapeseed event has been authorised for the European market. The prerequisite for any GMO reference material development is to produce stable and homogenous powder mixtures. The high-fat content of rapeseed caused a real challenge. Therefore, new grinding and mixing techniques had to be developed allowing the preparation of powder materials, which can be used for calibration and quality control without the need for a further grinding or mixing step by the analytical laboratory. Like other GMO certified reference materials (CRMs) offered by IRMM, the rapeseed CRMs were prepared according to ISO Guides 34 by gravimetrically mixing of the dried GM and non-GM rapeseed powders. This poster informs analytical laboratories about the development and the benefits of the worldwide first CRM for GM rapeseed powder mixtures. It outlines the application of these new reference materials for the low level presence legislation (EC) 619/2011 and the implementation of the food and feed labelling legislation (EC) 1829/2003, thereby contributing to accurate food and feed analysis.


P18 METABOLOMIC INVESTIGATIONS TO DETECT BIOMARKERS ASSOCIATED WITH CHANGES IN MEAT MATURITY Selected for speed presentation James Donarski, M. Harrison, S. Jones and A. Charlton Food and Environment Research Agency, UK [email protected] The study aimed to determine if the 1H NMR spectroscopic profile of beef was altered upon wet aging and whether it was possible to observe differences in the profile dependant on the wet aging temperature conditions used. Wet aging is a process for the maturation of meat where a sample is aged in a vacuum-sealed bag at a reduced temperature. This process is different to the traditional process of dry-aging meat where carcasses/cuts of meat are hung, uncovered, in a refrigerated environment. Dry aging is usually only used for highest quality meat sold in premium restaurants, whereas most supermarket meat undergoes the wet aging process. The maturation age of beef is often used as a quality indicator where beef that has been matured for longer is seen as ‘best’. Premium products available from UK retailers are often labelled as having a maturation age of either 21 or 28 days. A comparison of the price of sirloin steak from three retail outlets showed that products with a specific labelling claim relating to their maturation age were, on average, 42% more expensive than those without. This price differential could therefore lead to fraudulent labelling to increase profits. The cut of meat used for these experiments was the Striploin/Porterhouse (Longissimus dorsi). Two striploins were procured providing approximately 16 kg of meat in total. In brief, each striploin was dissected into 66 steaks, individually vacuum -sealed and labelled. One sample from each striploin was removed and analysed in triplicate by 1H NMR spectroscopy. The remaining samples were split and stored at –20 (frozen), 4 (ideal) or 20 °C (ambient). One steak from each striploin under each of the storage conditions (i.e. 6 steaks per day) was analysed in triplicate on consecutive weekdays up to a maturation age of 28 days. The resulting 1H NMR spectral data were analysed using both univariate and multivariate statics. A statistical model was generated that was able to successfully discriminate beef aged using ideal conditions (21 days and 28 days) versus samples stored incorrectly. Further investigation of the data revealed that the concentration of a series of analytes was shown to linearly correlate with meat maturity.


P19 ANALYSIS OF MYCOTOXINS IN GRAINS USING EFFICIENT SPE CLEAN-UP AND SUPERFICIALLY POROUS HPLC COLUMNS E. Barrey1, K. Espenshied1, M. Ye1, O. Shimelis1 and Christine Dumas2 Sigma-Aldrich, USA and Sigma-Aldrich Sàrl, France [email protected] Mycotoxins are toxic secondary metabolites produced by fungi, which can exist in food as a result of fungal infection of crops. Their strong resistance to decomposition and digestion cause mycotoxins to remain in the food chain in meat and dairy products. The analysis of mycotoxins in food and animal feed has been a challenge mainly due to the complexity of food matrices and desired low detection limits. Immunoaffinity solid phase extraction (SPE) cartridges have high selectivity for mycotoxins, but they are expensive and the procedure involves multiple steps during cleanup. In this study, we investigated a line of new materials that are designed for sample preparation of mycotoxins in complex food matrices, such as grains and grain products. The materials are stable and rugged under common laboratory conditions. The proposed methods require homogenisation of the matrix, blending of the sample with extraction solvent, and then passing an aliquot of supernatant through SPE cartridge. Performance of the SPE products was evaluated for aflatoxins as compared to immunoaffinity clean-up. Recoveries ranging from 101-108% were obtained for the SPE product and 78-101% for the immunoaffinity column. The analysis for deoxynivalenol (DON) was also conducted using an SPE product with maize, wheat, and peanut matrices. Recoveries for DON were 79-90% for the various matrices. Ochratoxin was extracted with an SPE product and average recovery of 108% was produced. These products have demonstrated a faster and easier alternative sample preparation method for the quantitative analysis of mycotoxins in various grains and grain products.


P20 EXTRACTION AND ANALYSIS OF NEONICOTINOID PESTICIDES USING QUECHERS APPROACH K. Stenerson1, O. Shimelis1 and Christine Dumas2

1Sigma-Aldrich, USA and 2Sigma-Aldrich Sàrl, France [email protected] Pesticide compounds of the neonicotinoid class have been used extensively in crop protection. The advent of the die-off of honey bees due to colony collapse disorder (CCD) has spawned investigation into pesticide exposure as the cause. Some studies have indicated that exposure of honey bees to neonicotinoids has detrimental effects on their behaviour. Recently, the European Union has adopted a regulation restricting the use of three of these pesticides, i.e. clothianidin, thiamethoxam, and imidacloprid. Here, a quick easy cheap effective rugged and safe (QuEChERS) approach was used to develop a method for extraction of neonicotinoid pesticides from matrices with which honey bees come most in contact, flower blossoms. Seven neonicotinoids were extracted from cherry, apple, and dandelion blossoms and analysed via LC-MS/MS. A number of sorbent mixtures were tested for the cleanup method, and a mixture of PSA and C18 sorbents provided the best recoveries and matrix removal. At a spiking level of 50 ng/g, recoveries of >89% were achieved for all tested neonicotinoids. Good reproducibility was achieved with the method, as indicated by RSD values of less than 7% for spiked replicates.


P21 DEVELOPMENT OF A DIRECT COMPETITIVE ELISA FOR THE DETECTION OF FURAZOLIDONE METABOLITE IN FOODSTUFF OF ANIMAL ORIGIN AND HONEY Kseniya M. Filippova, E.S. Vylegzhanina, I.S. Nesterenko, A.A. Komarov and A.N. Panin The All-Russian State Center for Quality and Standardization of Veterinary Drugs and Feeds, Russia [email protected] Furazolidone (N-(5-nitro-2-furfurylidene-3-amino)-2-oxazolidinone) is a synthetic broad-spectrum antibiotic used widely as a feed additive for the prevention and treatment of gastrointestinal infections caused by Escherichia coli and Salmonella in swine, poultry and cattle. This medical is also used for the treatment of foulbrood disease caused by Streptococcus pluton, Bacillus paraalvei and Bacillus alvei in honey bees. The use of furazolidone has been prohibited completely in food animal production in the European Union (EU) since 1995 due to its carcinogenicity and mutagenicity; however, it is still widely used in Russia. Furazolidone is characterised by its rapid metabolism to 3-amino-2-oxazolidinone (AOZ) in vivo in less than a few hours. Furthermore, the AOZ residue is stable in tissues and persists for at least 6 weeks in pig tissues after drug withdrawal. As a result, effective analytical detection of furazolidone could be carried out by the determination of bound furazolidone metabolite AOZ. In Russia, the MRL for nitrofuran metabolite residues is 1 μg/kg. Thus, residue screening techniques are the most efficient ways for risk assessment of furazolidone abuse. A polyclonal antibody-based direct competitive enzyme-linked immunosorbent assay (ELISA) was developed for the determination of furazolidone metabolite 3-amino-2-oxazolidone (AOZ). The highly specific antibody was targeted for CPAOZ, the carboxylic derivative of AOZ, conjugated with keyhole limpet hemocyanin (KLH). A new adjuvant FloravitTM was used for the production of polyclonal antibodies against CPAOZ-KLH. FloravitTM is a bioactive additive based on cultural medium of Fusarium sambucinum. For the ELISA sample preparation was performed by derivatization with 2-nitrobenzaldehyde and the following ethyl acetate extraction. The 50% inhibition values (IC50) was 8 μg/l for 2-np-AOZ (the benzaldehyde derivative of AOZ) which corresponded to 3.5 μg/l for AOZ. The limits of detection were 1 μg/kg. The coefficients of variation were less than 20% over the range of AOZ concentrations studied. The linear detection range was between 0.7 and 60 μg/l. All results were submitted by HPLC-MS. The results suggest that the direct ELISA is a specific, accurate and sensitive method of detecting AOZ residues in foodstuff of animal origin.


P22 DEVELOPMENT OF AN IMMUNOCHEMICAL METHOD FOR BOLDENONE DETECTION Lucie Fojtíková, S. Göselová, B. Holubová, M. Blažková, L. Fukal and O. Lapčík Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Czech Republic [email protected] Androsta-1,4-diene-17-ol-3-one, also called boldenone, is an androgenic anabolic steroid. Its structure is very similar to the male hormone testosterone, which differs from testosterone by only one double bond at the C-1,2 position. Boldenone and its esters are illegally used (especially by bodybuilders and athletes) to enhance physical condition and increase body mass and muscle strength. Due to the serious health risks associated with the use of anabolic steroids, it is desirable to develop tools that allow rapid and simple analysis. The standard methods for analysis of steroids are chromatographic methods. These techniques are expensive, require specialised instrumentation and do not allow the rapid analysis of a large number of samples. Immunochemical methods represent a suitable alternative overcoming the problems mentioned above, especially enzyme-linked immunosorbent assay (ELISA). The aim of the work was the development of an immunochemical method for the detection of boldenone. The competitive format of ELISA was developed using polyclonal antibodies and two immobilisation conjugates. Firstly, the protocols of the methods were optimised; suitable conditions of the detection procedures were chosen (combination of immunoreagents, and their concentrations, the composition of dilution buffers, times and temperatures of incubations). Two formats of indirect competitive ELISA were built as a combination of immobilisation conjugate BOL-3-OVA and antibody PL 131 (with detection limit 17±7 pg/ml) and immobilisation conjugate BOL-17-OVA and antibody PL 128 (with detection limit 14±7 pg/ml). The reactivity with structurally similar substances was tested to verify the specificity of assembled formats. Analytical abilities of developed ELISAs were verified on series of samples of matrix artificially contaminated with analyte. Recovery of the format with antibody PL 131 achieved 98% and of the variant with PL 128 103%. Acknowledgments This work was supported by the Grant of Ministry of the Interior of Czech Republic (no. VG20122015075).


P23 A BREAKTHROUGH IN FOOD ALLERGEN TESTING – DEVELOPMENT OF A ONE-MINUTE EXTRACTION PROCEDURE COUPLED TO A FAST ELISA ASSAY Lukas Frank1, E. Halbmayr-Jech1, A. Rogers2, M. Röder3 and T. Hein3

1Romer Labs Division Holding GmbH, Austria, 2Romer Labs UK Ltd., UK and 3ifp Institut für Produktqualität GmbH, Germany [email protected] Today, around 2-3% of the adult population and 5-8% of children are affected by food allergies. That is an impropriate response of the immune system to certain contents of food and drinks – mostly proteins – that are mistakenly believed to be harmful. Since already very low amounts of allergen can cause allergic reactions, which may lead to anaphylactic shock in severe cases, allergic persons must strictly avoid the consumption of allergen containing food. With allergens being the largest single cause of global product recalls, food manufacturers are seeking for fast and reliable methods ensuring the correct labelling of their products and preventing product recalls. Meeting these requirements, a new fast allergen ELISA assay was developed and validated for 6 different allergens, namely almond, casein, egg, hazelnut, macadamia nut and peanut. All six allergen ELISA test kits incorporate an extremely fast extraction procedure – using only extraction capsules containing a proprietary powdered buffer and hot water – of only 1 minute and short incubation times in the ELISA assay of only 10 minutes. Besides a low cross reactivity, the kits also showed a good performance in intra and inter-assay precision validation with variation being below 15% in almost every kit. The limit of detection (LOD) was calculated based on the mean blank value of 19 blank extractions plus three-fold standard deviation. All kits have LODs ranging in the low mg/kg level (almond, 0.5 mg/kg; casein, 0.2 mg/kg; egg, 0.5 mg/kg; hazelnut, 1 mg/kg; macadamia nut, 1 mg/kg; peanut, 0.5 mg/kg). Recoveries were ranging from 64-130% when kits were challenged to recover spiked allergens in difficult food matrices, such as chocolate, milk drinks, cookies and ice cream. The kits also showed an equivalent performance in recovery when being compared to established ELISA assays from the market. The validation showed that these new developed allergen ELISA assays are not only capable providing results in an extreme fast way, but also yield accurate results that you can rely on, making them suitable tools for the detection of allergens in every kind of foodstuff.


P24 UPDATE ON THE REVISION AND VALIDATION OF EN-ISO 11290-1 & 2 STANDARD METHODS FOR DETECTION AND ENUMERATION OF LISTERIA MONOCYTOGENES Nathalie Gnanou-Besse1, P. Rollier2 and B. Lombard1

1Laboratoire de Sécurité des Aliments, Anses, France and 2ACTALIA Cecalait, France [email protected] The reference methods for the detection and enumeration of Listeria monocytogenes in food (standards EN-ISO 11290-1 and 2) are under revision. A CEN ad hoc working group ‘Listeria’ works on this revision with 3 main objectives: simplification of the confirmation step, extension of the scope to include Listeria species, replacement of phosphatidyl inositol by soya lecithin. Other items are also discussed: preparation of the homogenate and resuscitation deletion (enumeration), modification of enrichment duration (detection), choice of reference strains, extension of the scope to include environmental samples, correction of several errors and inconsistencies in the standards/amendments. The draft prISO/TS 11290-1 and 2, established by this working group, was launched as new work item proposal (NWIP) vote at ISO level at the end of 2011. In parallel to this, CEN/TC 275/WG 6, in charge of standardisation in food microbiology at European level, has received a mandate from the European Commission to standardise and/or validate by interlaboratory studies (ILS) a set of reference methods in food microbiology, including the reference methods for the detection and enumeration of L. monocytogenes in food (standards EN-ISO 11290-1 and 2, under revision). The interlaboratory validation studies have been performed in 2013 and publication of the standards have to be completed by the end of 2016. The Food Safety Laboratory (Laboratoire de Sécurité des Aliments, Anses), is the project leader for that topic, i.e. the organizer of the ILS in 4 matrices (cold-smoked salmon, milk powder, vegetables, environment), in collaboration with ACTALIA Cecalait, the co-project leader for a dairy product matrix (fresh cheese). The objective of this presentation is to make an update on the revision of the Listeria standards EN ISO 11290 parts 1 and 2 and of its validation (CEN mandate M381).


P25 RAPID IMMUNOASSAY FOR THE DETECTION OF ANABOLIC STEROID METHANDIENONE Sandra Göselová, L. Fojtíková, B. Holubová, O. Lapčík and L. Fukal Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Czech Republic [email protected] Anabolic androgenic steroids (AAS) are widely used compounds in sport today. AAS are synthetic derivatives of the male hormone testosterone. Their use is illegal and prohibited without medical supervision. Top sportsmen or common sportsmen can come across steroids as a doping in various forms of tablets, injections or food supplements where steroids are illegally added. Methandienone, sometimes known as dianabol, is one of the most commonly abused anabolic steroid. Methandienone accelerates the growth of weight primarily as muscle and increases physical fitness and stamina. Side effects include aggression, increased blood pressure, acne, hair loss and disorders of the reproductive tract. Traditional methods for analysis of steroids (LC-MS and GC-MS) are highly sensitive and reliable. They involve multiple steps during sample preparation and analysis, require expensive equipment and skilled analysts, Therefore, these instrumental methods are unsuitable for routine analysis of a large number of samples or on-site determinations, in contrast to immunoassays, which could be portable and cost-effective with an adequate sensitivity, high selectivity, and a simple sample extraction process. The aim of this work was to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of methandienone in food supplements. Conjugates of methandienone were synthesised and conjugated with bovine serum albumin (BSA) and ovalbumin. Conjugate with BSA was used as an immunogen to obtain polyclonal rabbit antibodies, and conjugate with ovalbumin was used for coating microtiter plates. ELISA was optimised and the analytical parameters were obtained from the calibration curve. Under optimal experimental conditions, the most sensitive assay achieved IC50 and the limit of detection reached values of 1.45 and 0.17 ng/ml, respectively. Specificity was verified by the testing the cross-reactivity; the reactivity was examined with 44 standards of anabolic steroids. Furthermore, different matrices of dietary supplements and sample preparation methods were tested. Finally, the method was successfully applied to real samples. Acknowledgments This work was supported by the Grant of Ministry of the Interior of the Czech Republic (no. MV0 VG20112015045).


P26 ON-LINE SAMPLE PREPARATION METHOD PERFORMANCE FOR MULTIRESIDUE FOOD CONTAMINANT ANALYSIS Laszlo Hollosi1 and M. Godula2

1Thermo Fisher Scientific, Germany and 2Thermo Fisher Scientific, Czech Republic [email protected]

Liquid phase on-line sample preparation techniques possess lot of benefits over off-line sample preparation techniques. Through on-line coupling, advantages of solid phase extraction (sample clean-up and pre-concentration) and direct injection (sample analysis) techniques can be achieved. Due to elimination of the most significant error source (human factor) from the sample handling process, on-line sample preparation techniques can significantly increase overall efficiency by reducing sample preparation time and operational costs, and simultaneously increasing both method repeatability and reproducibility values. On-line coupling of sample preparation (trap) and conventional HPLC or (U)HPLC columns can be performed in many different ways. A wide variety of column materials and separation principles are available and can be combined to achieve effective clean-up and/or enrichment on the trap column (conventional SPE, size exclusion, turbulent flow, etc.) and further, complete separation on the coupled analytical column. Orthogonality is a key parameter for efficient and selective online system performance, however, it is often difficult to apply or ignored resulting in complicated or even impossible method set-up or optimisation. On-line sample preparation methods are recently widely used in clinical laboratories, however, only few applications are known for food safety relevant compounds. This presentation aims to fill a gap and report on special hardware set-up making even non-orthogonal online systems capable to perform according to the current legislation requirements for food safety relevant to multiresidue applications. Methods developed for three high relevant food contamination classes – pesticides, veterinary drugs and mycotoxins – in different liquid or solid matrices will be shown and established method performance parameters (matrix effect, detection limit, intermediate precision, and method bias) presented.


P27 CONSTRUCTION AND CHARACTERIZATION OF RECOMBINANT BISPECIFIC SINGLE-CHAIN DAIBODY AND GREEN FLUORESCENT PROTEIN FUSION FOR MULTI-ANALYTE ANALYSIS OF FLUOROQUINOLONES AND SULFONAMIDES K. Wen1, M. Chen1, Z. Wang1.2 and Haiyang Jiang1,2

1College of Veterinary Medicine, China Agricultural University, China and 2National Reference Laboratories for Veterinary Drug Residue, China [email protected] A new fusion of binder and tracer, consisting of a recombinant bispecific single-chain diabody (scDb) to fluoroquinolones (FQs) and sulfonamides (SAs) and a fluorescent protein from Aequorea coerulescens (AcGFP), was successfully constructed and expressed in Escherichia coli. The fusion was constructed by linking the recombinant scDb and the AcGFP with a novel flexible helical linker of 20 amino acids in the format of scDb-linker-AcGFP. The C-terminally c-myc and 6×histidines tagged fusion was expressed in soluble functional form in the periplasm of E. coli. The expressed fusion was further characterized with indirect competitive enzyme-linked immunosorbent assay (icELISA), showing that the affinity and specificity of the fusion antibodies were fully retained from both of the two parental antibodies, capable of binding FQs and SAs simultaneously. Based on the fusion, a fast, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for determination of FQs and SAs was developed. The 50% inhibition concentration (IC50) values of the optimised FLISA were 0.45 ng/ml for FQs and 0.75 ng/ml for SAs. Moreover, the cross-reactivity profiles of this fusion were 2-137% for 19 FQs and 10-230% for 13 SAs, which were highly consistent with their parental antibodies. The proposed FLISA was able to rapidly detect 19 FQs and 13 SAs below the maximum residue limits (MRLs) level in buffer, which indicated that this fusion could be applied for monitoring of FQs and SAs in real samples.


P28 FROM FARM-TO-FORK : MERCK MILLIPORE SINGLEPATH® DIRECT CAMPY POULTRY RAPID TEST KIT FOR FARM-BASED DIRECT DETECTION OF CAMPYLOBACTER SPP. IN CAECAL-TYPE SAMPLES FROM LIVE CHICKEN Lisa John1, J. Slaghuis1, M. Wadl2, M. Wagner3, T. Seliwiorstow4, J. Baré4, M. Uyttendaele5, L. De Zutter4 and C. Lindhardt1 1Merck Millipore, LBR-Applications, Merck KGaA, Germany, ²Unit for Surveillance, Department for Infectious Disease Epidemiology, Robert Koch-Institute, Germany, 3Department for Veterinary Public Health and Food Safety, University of Veterinary Medicine, Austria, 4Faculty of Veterinary Medicine, Veterinary Public Health and Food Safety, University of Ghent, Belgium and 5Faculty of Bio-Science Engineering, Department of Food Safety and Food Quality, University of Ghent, Belgium [email protected] The 2012 EFSA Scientific Opinion on meat inspection [1] proposed testing the Campylobacter status of live broiler flocks ≤3 days prior to slaughter, to identify the ‘high shedding’ flocks and allow segregation from low-shedding at slaughter, thereby avoiding cross-contamination of carcasses and reducing human consumption of Campylobacter spp. Such a strategy requires on-farm testing and a method that requires no specialised equipment or laboratory-trained personnel. Lateral flow technology fulfils this requirement and offers a reliable, fast, user-friendly, alternative detection method to the laboratory-based cultural reference methods. The purpose of this study was to develop and evaluate a qualitative immunochromatographic assay for direct (non-enrichment) detection of high shedding (≥7.5 log10 cfu/g of caecal-type sample) Campylobacter jejuni and Campylobacter coli broiler chicken flocks, within 2 hours of sampling, as a rapid and farm-based alternative to standard cultural reference methods to monitor Campylobacter status of flocks and assist slaughter scheduling. A sandwich lateral flow assay was developed, using gold labelled specific antibodies for Campylobacter spp. A non-enrichment sample preparation protocol was developed to enable a time-to-result of within 1 hour of sampling. Evaluation was by field studies conducted both on-farm (caecal droppings) and at slaughterhouse (caecal contents) using a cross-seasonal representative set of broiler chicken caecal-type samples. Reference method comparison was with ISO 10272 cultural method. In a field trial of caecal droppings collected on farm (n=60), Singlepath® Direct Campy Poultry achieved a sensitivity of 96% (% correctly classified positive) and a specificity of >99% (% correctly classified negative) based on a limit of detection of ≥7.5 log10 cfu/g of caecal content. In a field trial of caecal contents collected at slaughter (n=60), the test kit achieved a sensitivity of 96% (% correctly classified positive) and a specificity of >99% (% correctly classified negative) based on a limit of detection of ≥7.5 log10 cfu/g of caecal content. The Singlepath® Direct Campy Poultry Rapid Test Kit provides a unique, alternative, fast and simple method for detection of high shedding (≥7.5 log10 cfu/g of caecal-type sample) C. jejuni and C. coli broiler chicken flocks, on-farm or at slaughter, and can assist in monitoring Campylobacter spp. status of flocks and in slaughter scheduling. References 1. European Food Safety Authority, 2012. EFSA Journal 10: 2741.


P29 CONCENTRATION OF LARGE VOLUMES OF WATER FOR WATERBORNE PATHOGEN DETECTION USING A TANGENTIAL FLOW FILTRATION BASED SYSTEM Abdelfateh Kerrouche, S. Wilhelm, H. Bridle and M. Desmulliez MicroSystems Engineering Centre, Heriot-Watt University, UK [email protected] Waterborne pathogens are a major concern in both developing and developed countries for the supply of safe drinking water used for consumption or food preparation. The concentration of the pathogens in water is extremely low. So, sample preparation is an important stage, which is required for the detection of pathogens, as large volumes of sample need to be processed. A sample processing device for medium scale manufacturing has been developed. The system concentrates and enriches water containing Cryptosporidium parvum and Giardia lamblia (oo)cysts from a large volume of water in order to enable further detection processes. The device module concentrates a large sample volume (e.g. 50 l, 500 l) into a small volume (around 580 ml) while retaining the biological species. This system is based on a tangent hollow fibre filter (module E 1m2 150kD PAN) manufactured by NBS Biological. Fluid and particles smaller than the filter pore size (800 nm) escape the circulation to a drain connection until the desired amount of input water is processed. However, the system batch processes only 90 l in an hour, exceeding the 1000 l in 24 hours recommended by the US Environmental Protection Agency (EPA) 1623 method. Controlled experiments have been performed using fluorescent particles (latex beads) with diameters of 1μm and 5μm provided by Cospheric. A high recovery rate of 70% was confirmed.


P30 ON-SITE ISOTHERMAL LAMP ASSAYS FOR BACTERIA, VIROID AND PHYTOPLASMA DETECTION Selected for speed presentation Polona Kogovšek1, N. Boonham3, M. Dermastia1, M. Dickinson4, T. Dreo1, J. Hall3, J. Hodgetts3, R. Lenarčič1, P. Llop2, N. Mehle1, D. Morisset1, P. Nikolić1, M. Pirc1, N. Prezelj1, A. Rotter1 and M. Ravnikar1 1National Institute of Biology, Slovenia, 2Biosistemika, Research and Development LLC, Slovenia, 3The Food and Environment Research Agency, UK and 4School of Biosciences, University of Nottingham, UK [email protected] Increased global trade with plant material facilitates spread of plant pathogens. Early detection of the infected material at the entry points, such as ports, airports and border crossings, or at the production sites can prevent pathogen spread and possible consequent economic loss. Of particular importance are pests with a broad range of host plants. We have developed and validated specific and rapid on-site detection methods for selected quarantine plant pathogenic bacteria, viroid and phytoplasma. Isothermal loop-mediated amplification (LAMP) proved to be a fast and simple method. It gives results in 20-30 minutes, including sample preparation and amplification reaction. The isothermal reaction and reading of the final result can be done in a simple heater/reader or performed in a device that detects amplification in real-time based on fluorescence. The major advantage of LAMP being its speed and portability, we have optimised both the reaction itself and the sample processing for on-site use. Depending on the pathogen concentration and type of plant material, simple boiling of crude plant extract is often enough due to LAMP’s relative resistance to inhibitors. If necessary, plant sample can be homogenised on-site manually or with a simple device. The use of intercalating dye enables an additional control of amplification through melting curve analysis. In general, the LAMP assays are approximately ten times more sensitive than conventional PCR and ten times less sensitive than real-time PCR. The method can also be run and monitored on a regular qPCR cycler and is, as such, suitable as a fast screening test in diagnostic laboratories. The whole development of the procedure was done within the European projects Q-detect and Vitisens.


P31 EVALUATION OF A LATERAL FLOW TEST FOR QUANTITATIVE DETECTION OF AFLATOXIN M1 AT 50 NG/L IN RAW COMMINGLED MILK Wilbert Kokke1 and R. Salter2

1Kentron Microbiology BV, the Netherlands and 2Charm Sciences, Inc., USA [email protected] In the spring of 2013, when milk from south eastern Europe tested positive for aflatoxin M1 (AFM1) at the EU maximum level (ML), there was dairy stakeholder demand for easy and rapid raw milk screening tests validated to detect the EU ML of 50 ng/l. Charm Sciences, Inc. (CSI) makes a rapid one step assay (ROSA) lateral flow test for AFM1 (MRLAFMQ test), which is quantitative in a range from 15 to 75 ng/l and qualitatively interprets positive and negative below 50 ng/l in 15 minutes using a 40 ng/l limit. The method uses existing equipment in dairies, which is used for screening farm tanks and truck samples for antibiotic residues prior to unloading at dairies. CSI contracted the ILVO T&V laboratory (Belgium) to perform a validation according to the model of the 2010 EU screening test guidelines to determine detection capability, robustness and applicability to truck and farm raw milk samples. This study evaluated fortified AFM1 raw milk samples and determined a qualitative detection capability of 50 ng/l with a low false positive rate at 25 ng/l of <3.5%, a false negative rate of <2% at 50 ng/l. Over 200 producer milk samples from Belgium were evaluated in the spring/summer of 2013 with a false positive rate of 0.3%. The quantitative reading had a mean positive bias of 2 to 6 ng/l at 50 ng/l with a standard deviation of about 5 to 8 ng/l at three fortified study concentrations of 25, 50 and 75 ng/l. The limit of detection, defined as three times the standard deviation of a negative sample, was 14 to 19 ng/l and the limit of quantification, defined as ten times the standard deviation of negative samples, was 45 to 50 ng/l, using two different reader models in commercial dairy use. Results are obtained in 15 minutes without the need for milk dilution or a sample preparation step using dairy testing equipment in routine use for antibiotic residue analysis. The test is appropriate for testing raw commingled milk AFM1 screening at the EU ML prior to milk purchase and unload at dairies. Using the MRLAFMQ quantitative features, the method could be useful for early farm to fork AFM1 identification and sustainable remediation of milk producing regions at risk for AFM1 outbreak. Further collaborative work is needed to determine method precision parameters and compare results of natural incurred samples detected by screening methods, and their quantification and confirmation using official reference methods.


P32 COLIPHAGE ASSAY TO DETECT FAECAL CONTAMINATION IN WATER IN 8 HOURS Wilbert Kokke1 and R. Salter2

1Kentron Microbiology BV, the Netherlands and 2Charm Sciences, Inc., USA [email protected] Coliphages are viruses to coliform bacteria and are considered an indicator of faecal contamination in ground water. Public health officials debate coliphage detection and whether there is correlation with regulated Escherichia coli contamination, but as a viral surrogate, coliphage detection provides an opportunity to verify log reduction of viruses in water production processes. In addition, coliphage offers a working day faecal detection breakthrough. This work describes an 8 hour qualitative detection of a single plaque forming unit in large volumes of water, 100 ml – 10 l. The Fast Phage method (Charm Sciences, Inc.) was approved by the US Environmental Protection Agency (EPA) in May 2013 [1]. The same day positive prediction is 95% correlative to plaque formation. The detection utilises an E. coli host enriched transfer to trigger β-galactosidase expression in coliphage lysing host cells. The early detection of coliphage presence is with the fluorometric enzyme substrate MUG-GAL [2]. The method was collaborative studied in a national Tier 2 study design using primary waste water spiked ground water [3]. Detection was verified to be equivalent to EPA Method 1601 [4]. Recently, the method was modified for larger volume water samples with the use of electrostatic filters. The pre-enrichment step was performed in filter housings of filtered 10 l samples to detect 1-2 plaque forming units (pfu/10 l). This is an exciting breakthrough, because coliphage concentrations in primary human waste are generally 103. The absence of coliphage in 10 l or greater volumes offers the ability to verify 5-6 log reduction of viruses in water reuse and recharge processes, such as in drinking water produced from ground water from aquifers supplemented with reclaimed brown waters. In addition, the genetic code triggering host lysis and the resulting biochemistry have the potential to be tied with rapid detection instrumentation and automation to further reduce the time of detection. More public health research is needed to understand the detection of coliphage in water sources, as it relates to the incidence of water and foodborne disease. Microbiologists may want to consider coliphage genetics and biochemistry in the development of real-time detection systems for the quality of drinking and bathing water. Coliphage as a process control indicator in water management systems including filtration, waste water recharge, and water used in food production offers great promise in achievable sensitivity and speed to detect in macro-scale volumes of water. References 1. US Federal Register, May 31, 2013. US EPA OW. New Methods (MS-4606 M) EPA 815-F-13-

002. 2. Salter, R.S., Durbin, G.W., Conklin, E., Rosen, J. and Clancy, J., 2010. Applied and

Environmental Microbiology 76: 7803-7810. 3. Salter, R.S. and Durbin, G.W., 2012. Journal - American Water Works Association 104: E480-

E488. 4. EPA Method 1601. Coliphage in water by two-step enrichment procedure, April 2001.


P33 WATER-BASED EXTRACTION OF MYCOTOXINS FROM ANIMAL FEEDSTUFFS Wilbert Kokke1, J. Jabor2 and M. Tess2

1Kentron Microbiology BV, the Netherlands and 2Charm Sciences, Inc., USA [email protected] Mycotoxins are toxic secondary metabolites produced by moulds that can grow in moisture and disease susceptible grains and feedstuff. Contaminated feedstuffs, if consumed, pose health concerns in animals and humans. Susceptible feedstuffs are therefore screened for mycotoxins to prevent animal consumption. Mycotoxins are typically tested in feedstuff via organic solvent extraction. These organic solvents provide occupational hazards due to their flammability and toxicity; organic extraction is an obstacle to moving screening away from central laboratory analysis and closer to a more economical and sustainable farm-to-fork prevention programme. An innovative breakthrough, water extraction technology (WET) (Charm Sciences, Inc., USA) uses a non-hazardous extraction powder and water to extract mycotoxins into an aqueous solvent. This extraction method combined with easy-to-use lateral flow assay format creates an effective and non-hazardous test method to rapidly and accurately analyze feedstuffs in the field. The water-based extraction can be disposed as normal waste, provided that positive mycotoxin samples do not violate local disposal regulations. The WET extraction powder can be used as a universal extraction of number of mycotoxins, including aflatoxin, fumonisin, zearalenone, T-2 and H-2 toxins; ochratoxin can be extracted using an alternate extraction powder and deoxynivalenol (DON) is water-soluble without the need for any extraction powder. For analysis, the extract is diluted into the detectable range of the screening test without any additional clean-up steps. The diluted extract is added to the lateral flow test, which is incubated and analysed in a quantitative reader calibrated to the feedstuff. Precision and accuracy using WET has received a number of official approvals from the USDA GIPSA, because it demonstrates comparable results to HPLC reference methods. CVs typically seen at levels of 5, 20, and 100 μg/kg for aflatoxin in maize are 11%/19%, 9%/10%, and 9%/13% for HPLC vs. WET, respectively. CVs typically seen at levels of 1, 2, and 5 mg/kg fumonisin in maize are 5%/10%, 8%/7%, and 5%/7% for HPLC vs. WET, respectively. Lateral flow quantitative mean determinations were within 10% of the reference method determinations in the GIPSA submission studies. The simplified test method can eliminate user error by reducing the number of steps required to complete the assay. Extraction powder packets are much easier to handle and store compared to organic solvents and provide a mycotoxin screening option greener than organic extraction-based tests. Water-based extraction allows an early to farm, more sustainable and environmentally safe mycotoxin avoidance programme for feedstuffs. There is no organic waste storage, hazard or disposal. In addition, the elimination of organic solvents allows a DOT-free test-kit shipping with a smaller space footprint.


P34 AUTHENTICATION OF BASMATI RICE USING SSR-PCR AND THE QIAXCEL ADVANCED SYSTEM R. Cassier1 and Mirjana Kozulic2

1Laboratoire AdGène, France and 2Qiagen Instruments AG, Switzerland [email protected]

Basmati rice is one of the most popular types of rice worldwide, representing approximately 40% of the dry rice market. Import of Basmati rice has to comply with various regulations (EC 1234/2007, EC 1785/2003, EC 972/2006) and requires an import certificate. Various analytical methods, such as examining grain dimension, amylose content, cooking elongation and aroma, have been developed, but they are too time-consuming or unreliable. SSRs (simple sequence repeats) are genetic markers that can be used for Basmati rice identification. However, the interpretation of results can be difficult, especially for samples containing more than 3 individuals. Accurate DNA fragment sizing, optimally within 2 bp, is difficult to achieve with classical electrophoresis, which is also very time- consuming, especially when performing simplex analyses. Therefore, we have developed an SSR-PCR protocol for routine analysis of Basmati rice using the QIAxcel Advanced system. Genomic DNA was purified with the QIAsymphony DSP DNA mini kit (Qiagen) from homogenised grains. Amplification was performed as duplex PCR with 8 SSR markers: RM1+RM72, RM44+RM55, RM241+RM202, and RM348+RM171. Capillary electrophoresis was performed with the QIAxcel System in combination with High Resolution Kit. Our own programmed Excel sheets were used for fragment identification. The 8 SSR markers demonstrated a high discrimination power among Basmati and non-Basmati (adulterated) rice, tested with 13 Basmati and non-Basmati rice samples. The results show that each rice has a characteristic stable and reproducible fragment profile. The reproducibility and stability of the profiles were monitored by repeating the analysis 11 times with a Basmati sample (Taraori variety).This result will allow us to validate rice’s SSR data coming from public databases. The limit of detection was 0.1% for non-Basmati rice in Basmati rice. The limit of quantification was 1.0% non-Basmati rice in 99.0% Basmati rice. The global uncertainty of the analysis is 5.2% and the uncertainty at >95% Basmati rice is 1.9%. The quantification has an accuracy of 0.2% and a dispersion of 2.0% based on 133 measurements made on 37 different mixtures of rice. Accurate, reliable and time-saving identification and quantification of Basmati rice and its adulterants can be made using the SSR-PCR protocol, where duplex SSRs were analysed with the QIAxcel Advanced system and the ScreenGel software or Excel sheets. The High Resolution Kit makes it possible to determine the size of the SSRs with an accuracy of 2 bp - 3 bp, thus providing a useful tool in routine Basmati rice authentication analysis.


P35 CHARACTERISATION OF INFANT FORMULA QUALITY PARAMETERS USING THE FAST METHOD AND THE AMALTHEYS® ANALYSER A. Liogier de Sereys, S. Muller, A. Acharid, V. Fogliano, Pierre Lacotte and I. Birlouez-Aragon Spectralys Innovation, France [email protected] Infant formulas’ composition is strictly regulated at European level. Following the 2008 melamine adulteration scandal in China, monitoring of infant formulas quality parameters has become of paramount importance for industrials and final consumers. A set of 4 quality indicators based on fluorescence was developed to answer the need for rapid and non-destructive assessment of infant formulas’ quality parameters, including the rehydration ability of the powder and protein nutritional quality. These indicators are measured using the Amaltheys fluorescence analyser. This analyser proposes to assess whey soluble and denatured proteins and extent of Maillard reaction in 5 min, according to the patented FAST method. The principle of the method is to prepare a soluble supernatant at pH 4.6 containing the pure soluble whey proteins and measure protein autofluorescence resulting from native tryptophan and neoformed advanced Maillard products. The first fluorescence is measured at excitation/emission 280/340 nm and is strongly correlated to Kjeldahl non-caseic nitrogen fraction; the second one at 340/430 nm is mainly correlated with carboxymethyl lysine (CML). Calibration and validation of the measurement is achieved using certified whey standard and reference material. We have evidenced that the Amaltheys analyser enables accurate assessment of whey protein denaturation rate and Maillard reaction products, with good correlation to conventional indicators. The amount of denatured whey proteins is obtained by subtracting the soluble whey proteins from the total whey content in the recipe. We showed that the level of denatured whey protein is the main predictor for powder solubility and dispersibility. The FAST index was indicative of the heat charge applied during the process in relation to the extent of the Maillard reaction. The FAST index was well correlated with furosine and CML in the case of high lactose content. However, oxidation of long-chain polyunsaturated fatty acids (LC-PUFA) in the presence of transition metal ions had a stronger impact on the FAST index, especially when the lactose concentration was low. A benchmarking study was performed to evaluate the potential of the FAST method to discriminate 24 commercial powdered infant formulas according to their quality parameters. The three quality indicators measured with the analyser, i.e. soluble, denatured whey proteins and the FAST index, were coupled to another fluorescence signal, particle scattering at 280 nm, to discriminate and classify the formulas using principal component analysis (PCA). We demonstrated that the combination of the four fluorescence signals allowed discriminating three main classes of infant formulas according to their nutritional properties and rehydration parameters.


P36 FLUORESCENCE POLARISATION IMMUNOASSAYS FOR RAPID DETERMINATION OF MYCOTOXINS IN FOODSTUFFS Selected for speed presentation Vincenzo Lippolis1, M. Pascale1, M. Suman2 and A. Visconti1 1Institute of Sciences of Food Production, National Research Council of Italy, Italy and 2Food Research Labs, Barilla SpA, Italy [email protected] Mycotoxins are naturally occurring toxic metabolites produced by filamentous fungi under a wide range of climatic conditions on different agricultural crops during growth, drying and subsequent storage. Monitoring, control, risk assessment and prevention of mycotoxins in foods are important issues worldwide associated with public health, agricultural production, food processing and trade. For these reasons the European Commission has set recommended levels or maximum permitted levels for mycotoxins of major concern in a wide range of foodstuffs. Analytical methods for the determination of mycotoxins in foods are commonly based on chromatographic techniques (GC, HPLC or LC-MS). Although these methods permit a sensitive and accurate determination of the analyte, they require skilled personnel and are time-consuming, expensive, and unsuitable for screening purposes. Simple, rapid, and more effective screening methods for mycotoxins determination are highly demanded. Fluorescence polarisation immunoassay (FPIA) is a homogenous assay that measures competition between a fluorescently labelled antigen (tracer) and unlabelled antigen in solution for binding a specific antibody. The FP signal is inversely related to the antigen content that competes with the tracer, and it increases when the binding of specific antibody to the tracer increases. Unlike most immunoassays (e.g. ELISA), the main advantage of this format is that additional manipulation steps, as multiple washing steps or separation of free from antibody-bound analyte, are not necessary. The selection of the appropriate antibody-tracer combination determines the speed, accuracy, precision and sensitivity of a FPIA. Incubation times, cross-reactivity, compatibility with organic solvents and matrix effects are analytical parameters to be evaluated and optimized in the development of a FPIA. We have recently developed several FPIAs for the determination of mycotoxins in cereals and processed products, including deoxynivalenol in wheat and derived products, ochratoxin A in wheat, T-2 and HT-2 toxins in wheat, barley, oats and oat flakes [1-3]. An accurate validation of these assays has been performed on each tested matrix using either artificially and naturally contaminated samples and reference materials. These FPIAs are rapid, easy-to-use, readily automated, and suitable for high-throughput screening as well as for the quantitative determination of mycotoxins in foodstuffs at levels below regulatory levels. Acknowledgements Parts of the work have been supported by the Italian AGER (Agro-Food and Research, project ‘From seed to pasta’) and the Italian Ministry of Education, University and Research, MIUR (P.O.N. Ricerca and Competitività 2007-2013), project no. 02_00186_341751212792 - S.I.Mi.S.A. ‘New strategies for improvement of food safety: prevention, control, correction’. References 1. Valenzano, S., Lippolis, V., Pascale, M., De Marco, A., Maragos, C.M., Suman, M. and Visconti,

A., 2014. Food Analytical Methods 7: 806-813. 2. Lippolis, V., Pascale, M., Valenzano,S., Porricelli, A.C.R., Suman, M. and Visconti, A., 2014. Food

Analytical Methods 7: 298-307. 3. Lippolis, V., Pascale, M., Valenzano, S., Pluchinotta, V., Baumgartner, S., Krska, R. and Visconti,

A., 2011. Analytical and Bioanalytical Chemistry 401: 2561-2571.


P37 RAPID TEST SYSTEMS FOR DETECTION AND QUANTIFICATION OF MICROORGANISMS IN FOOD AND BEVERAGES: LISTERIA AND SALMONELLA Kathleen Merx, K. Vetter, U.-M. Kohlstock and M. Zachlod Becit GmbH, Germany [email protected] Salmonella and Listeria can cause serious and potentially fatal diseases by consumption of contaminated food. The use of gold standard methods for identification and quantification via pre-culturing, enrichment, plating and at the end often confirmation by PCR are tedious and not suitable for testing foodstuff with short shelf lives. The established technology allows to significantly reduce the pre-cultivation step to 24 hours. The identification and quantification is based on the detection of target molecules from the microorganism of interest by means of specific capture and detection probes in a so-called sandwich hybridisation. The target molecules of the microorganisms contained in the sample are captured in a specific microwell binding plate with the help of the capture probe. In addition to the capture probe, a detection probe is coupled to the target molecule. An enzyme is attached afterwards in a subsequent incubation step. After several washing steps, reaction with a colour substrate gives a blue colouration, which changes to yellow after the addition of a stop solution. The yellow colour enables highly sensitive photometric measurement at 450 nm. Comparison is made with the standard solutions contained in the test kit. The Salmonella test system contains specially designed probes for the identification of bacteria of the genus, including the detection of the most relevant Salmonella serovars like S. enteritidis, S. typhimurium, S. typhi and S. paratyphi. To detect Listeria, two rapid test systems are available. One to quantify Listeria spp. (including the detection of the most relevant species L. monocytogenes) and a second with a species-specific probe for L. monocytogenes. The rapid test system has significant time- and labour-saving benefits over traditional methods. It also has benefits over PCR and real-time PCR, which, although highly sensitive, are susceptible to experimental interferences, such as template inhibition from insufficient purification [1], and lack quantification accuracy due to biases associated with PCR and reverse transcription reactions (a general accepted error connected to these methods). In contrast, our method is nearly independent of the influences of sample matrix and detects only living cells. References 1. Bustin, S.A., 2000. Journal of Molecular Endocrinology 25: 169-193.


P38 THE IDENTIFICATION OF NEW SPECIES OF BACTERIA FROM THE GENUS CRONOBACTER USING BIOCHEMICAL TESTS, POLYMERASE CHAIN REACTION AND MASS SPECTROMETRY Denisa Mihalová, B. Javůrková, M. Blažková, P. Rauch and L. Fukal Department of Biochemistry and Microbiology, Institute of Chemical Technology in Prague, Czech Republic [email protected] Bacteria of the genus Cronobacter are known as dangerous opportunistic pathogens causing severe infectious diseases of neonates and immunodeficient individuals. Among these diseases, we count above all meningitis, necrotising enterocolitis and sepsis. The occurrence of these infections is sporadic, however, fatal in many cases. Fast and accurate identification of these pathogens can prevent the fatal consequences of their infections. The Cronobacter genus was created in 2008 by reclassification of the former species Enterobacter sakazakii. Its taxonomy has a complicated history, with several species transferred to this genus till now. At this time, the Cronobacter genus is divided into ten species and three subspecies. The recent changes in taxonomy have been carried out last year; three new species C. helveticus, C. pulveris and C. zurichensis were incorporated into the genus Cronobacter. Some species within the genus are associated with neonate infections and have a higher risk of virulence. For this reason, attention is focused on the precise species and subspecies identification. The aim of this work was to characterise the strains belonging to new species of the genus Cronobacter, namely C. helveticus, C. pulveris and C. zurichensis, using biochemical tests, polymerase chain reaction and MALDI-TOF mass spectrometry. Initially, genus- and species-specific PCR was performed. For this purpose, the rpoB gene was used, which is suitable for phylogenetic analysis of closely related species. Eleven specific biochemical test tubes and BioMérieux commercial kit ID 32E were performed for the biochemical characterisation. Finally, MALDI-TOF mass spectra of strains belonging to the new species were measured and evaluated using mMass 3.8 software. The measured mass spectra were compared with a database of reference spectra Maldi Biotyper 2.0 (Bruker Daltonics). The results were compared with other representatives of the genus Cronobacter. The results of the biochemical characterisation showed that the new species differed greatly from other representatives of the genus Cronobacter. The obtained results were confirmed by PCR analysis of the gene rpoB and by mass spectra. Acknowledgements This work was supported by the Czech Grant Agency (project no. 13-23509S) and Specific University Research (MSMT No. 21/2013, MSMT No.20/2014).


P39 RAPID MULTI-TARGET DETECTION OF PATHOGENS CAUSING MASTITIS Antoine P.H.A. Moers, M. Koets, L. van Zeeland, T. Posthuma and A. van Amerongen Biomolecular Sensing and Diagnostics, Wageningen UR, the Netherlands [email protected] Bovine mastitis is one of the most costly diseases of dairy cattle, resulting in a great deal of economic losses, mostly because of a reduction of milk yield, decreased milk quality, and higher production costs [1]. Mastitis can be caused by a wide range of pathogens; well over 100 different pathogens have been identified as a cause of mastitis. These pathogens can be divided in major pathogens causing clinical mastitis, including Staphylococcus aureus, Streptococcus spp., Escherichia coli, Klebsiella spp. and Mycoplasma bovis, and minor pathogens that normally cause subclinical and less frequently clinical mastitis as Corynebacterium bovis in some herds [2]. Here, we present rapid multi-target detection platforms to identify the most common mastitis-causing pathogens: Staphylococcus aureus, the Streptococcus spp. S. agalactiae, S. dysgalactiae, and S. uberis, Corynebacterium bovis, and Mycoplasma bovis. A rapid species-specific PCR with tagged-primers was combined with a nucleic acid microarray immunoassay in which carbon nanoparticles were being used as detection principle. The double-tagged dsDNA molecules were sandwiched between tag-specific antibodies at the assay surface and neutravidin at the carbon nanoparticles detection molecules. The assay can be performed as a lateral flow assay, in nitrocellulose pads mounted on glass slides and in 96-wells microtiter plates, enabling the high-throughput and automated screening of many samples. In the case of slides and microtiter plates, the signal can be enhanced through the conversion of a substrate to a precipitable dye by enzyme molecules bound onto the carbon nanoparticles. The PCR procedure coupled to the lateral flow format is designed to be used on-site to reduce the time-to-answer for treatment and, therefore, may improve dairy economics. Acknowledgements This work was financially supported by the Wageningen UR Kennisbasis cluster KB-17 ‘New technologies’, theme 002.02 ‘Diagnostics and biosensors’, project 003 ‘Miniaturised, multi-analyte and on-site diagnostic platforms’. References 1. Lee, K.-H., Lee, J.-W., Wang, S.-W., Liu, L.-Y., Lee, M.-F., Chuang, S.-T., Shy, Y.-M., Chang, C.-

L., Wu, M.-C. and Chi, C.-H. 2008. Journal of Veterinary Diagnostic Investigation 20: 463-471. 2. Hamadani, H, Khan, A.A., Banday, M.T., Ashraf, I., Handoo, N., Bashir, A. and Hamadani, A.,

2013. International Journal of Livestock Research 3: 42-55.


P40 DETECTION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN PLASMODIUM FALCIPARUM BY PCR PRIMER EXTENSION AND LATERAL FLOW IMMUNOASSAY Antoine P.H.A. Moers1, C.J. Sutherland2, R.L. Hallet2, R. Burrow2, H.D.F.H. Schallig3 and A. van Amerongen1

1Biomolecular Sensing and Diagnostics, Wageningen UR, the Netherlands, 2Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, UK and 3Parasitology Unit, Biomedical Research, Royal Tropical Institute the Netherlands; coordinator of the EU MALACTRES project [email protected] Resistance of Plasmodium falciparum strains to particular drugs is linked to single nucleotide polymorphisms (SNPs). To be able to administer the proper drug combination a simple, cheap and fast method is required. In addition, the method should be suited to be performed at point-of-care, i.e. in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum. The SNPs are positioned at amino acids 86, 184 and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1), and amino acid 76 of the P. falciparum chloroquine resistance transporter gene (pfcrt). PEXT products were visualised by a nucleic acid lateral flow immunoassay (NALFIA) in which carbon nanoparticles were used as detection labels. PCR-PEXT-NALFIAs were successfully developed for genotyping of pfmdr1-86, pfmdr1-184, pfmdr1-1246 and pfcrt-76 and showed good correlation to the reference qPCR method in a small set of 17 field samples. Subsequently, the methods were evaluated in a blind study design and with a set of 150 field samples. High specificity values of 0.97-1.00 were found for all 4 genotyping assays. Sensitivity values ranged from 0.67 to 0.91, except for the PCR-PEXT-NALFIA for pfcrt-76T that had a sensitivity of 0.42. The methods showed a good to perfect agreement compared to qPCR (κ=0.64-0.90). A moderate agreement was calculated for pfcrt-76T genotyping (κ=0.42). The use of PCR and PEXT for amplification of SNPs and NALFIA as the readout system for the detection of SNPs in P. falciparum compared well to qPCR as the reference method. Further optimisation of these assays can be made by multiplexing of the different methods into one assay. The methods are cheap, simple and rapid, which make them a valuable tool for the point-of-care detection of resistant P. falciparum strains in developing countries. Acknowledgments This work was partly funded by the European Commission FP7 grant contract 201889 ‘Multi-drug resistance in malaria under combination therapy: assessment of specific markers and development of innovative, rapid and simple diagnostics’ (MALACTRES).


P41 EVALUATION OF THE EVIDENCE INVESTIGATOR PLATFORM FROM RANDOX FOR THE SCREENING OF DRUG RESIDUES IN MILK MATRICES A. Beck and Claudia Mujahid Nestlé Research Center, Switzerland [email protected] The Evidence Investigator platform from Randox Food Diagnostics is a semi-automated bench top analyser that uses biochip array technology and immunological detection similar to competitive ELISA and delivers semi-quantitative results using external calibration curve. The platform was evaluated for the screening of a large range of veterinary drugs in raw milk and milk derivatives. Five test arrays were selected for regulated antibiotics (array ß-lactams, array I sulfonamides, array II tetracyclines, quinolones and others, and array IV amino-glycosides and macrolides), two for banned antibiotics (array III chloramphenicol milk only and array III chloramphenicol and nitrofurans), and one for non-antibiotic veterinary drugs (anthelmintics array). In total 78 veterinary drug compounds were tested using six different arrays in this evaluation. Out of 78 target analytes, 67 were detectable at concentrations between 0.5 and 10 µg/kg. The result for key compounds, such as penicillin, could also be confirmed in milk derivatives, such as lactose and whey powders. Penicillin was detected at 1 µg/kg for all matrices. In our hands, the anthelmintics array was not reproducible for milk matrices. Overall, the results of the evaluation showed a broad coverage of all key antibiotics and several banned veterinary drugs for milk. A good sensitivity was obtained with minimal sample preparation for milk matrices (raw milk and milk fractions).


P42 SUBTYPING OF AVIAN INFLUENZA INFECTION USING A PROTEIN MICROARRAY Truus Posthuma1, J. Post2, R. Bouwstra2, G. Koch2 and A. van Amerongen1

1Biomolecular Sensing and Diagnostics, Wageningen UR, the Netherlands and 2Central Veterinary Institute, Wageningen UR, the Netherlands [email protected] Avian influenza (AI) outbreaks in poultry cause huge economic losses and are a major threat to public health worldwide. Migratory water birds form a natural reservoir of many different AI virus subtypes and pose a risk for introduction of these viruses to poultry. AI virus infections in reservoir hosts are usually asymptomatic and when introduced in poultry normally cause only a mild or no disease, indicated as low pathogenicity avian influenza (LPAI). LPAI viruses of the H5 and H7 subtypes, however, can evolve to high pathogenicity (HPAI) viruses and therefore are notifiable. Measures to control these subtypes have been included in European Union (EU) legislation and, consequently, surveillance programmes have been implemented by EU member states. In the Netherlands a part of the surveillance programme is based on subtyping monitoring, where each commercial poultry flock is tested with ELISA for the presence of antibodies against nucleoprotein. The subtypes of the AI virus are characterised by two envelope proteins, i.e. haemagglutinin and neuraminidase. Although for control purposes detection of only H5 and H7 subtypes is required, it is important from a public health point of view to determine all subtypes (for instance H6N1, H9N2 and H10N8). Therefore, gene constructs representing all H and N subtypes were produced using an in vitro expression system. Proteins produced were used to develop an influenza microarray. A first initiative of a similar microarray detecting some human influenza viruses was described in a paper by Koopmans et al. [1]. Here we will present an extension of this platform that also includes the avian haemagglutinin and neuraminidase subtypes (H1-H16 and N1-N9). A carefully selected control panel of reference sera has been used to develop and validate the microarray. In addition, some field sera were tested to evaluate the practical applicability of the test. Data of both the control panel and the field samples will be shown. Acknowledgements The financial support of the Wageningen UR Kennisbasis cluster KB-17 ‘New technologies’, theme 002.02 ‘Diagnostics and biosensors’, project 003 ‘Miniaturised, multi-analyte and on-site diagnostic platforms’ and of WOT project WOT-01-001-003b ‘Protein array for subtyping of influenza antibodies’, Dutch Ministry of Economic Affairs, is highly appreciated. References 1. Koopmans, M., De Bruin, E., Godeke, G.J., Friesema, I. Van Gageldonk, R., Schipper, M., Meijer,

A., Van Binnendijk, R., Rimmelzwaan, G.F., De Jong, M.D., Buisman, A., Van Beek, J., Van de Vijver, D. and Reimerink, J., 2012. Clinical Microbiology and Infection 18: 797-807.


P43 EVALUATION OF ALTERNATIVE METHODS FOR THE EXTRACTION AND FRACTIONATION OF BIOACTIVE CARBOHYDRATES

L. Ruiz-Aceituno, B. Alonso-Rodríguez, M.L. Sanz and Lourdes Ramos Department of Instrumental Analysis and Environmental Chemistry, IQOG-CSIC, Spain [email protected]; [email protected] Interest in biological activities of carbohydrates is currently increasing in different research areas, such as food, pharmaceuticals, and environmental science. Among them, inositols exhibit different activities, mainly connected to insulin-related diseases [1]. Different approaches have been followed for the extraction of these compounds from natural sources [2]. However, the development of new more economical and less time-consuming procedures is relevant, especially from the food industry point of view. Pressurised liquid extraction (PLE) or microwave assisted extraction (MWAE) are efficient techniques, generally providing a significant reduction in extraction times and solvent consumption as compared with conventional, i.e. non-solvent-enhanced, extraction procedures. During inositols extraction, other sugars that could interfere in their bioactivity are usually co-extracted. This fact makes the subsequent fractionation of the obtained extracts mandatory. However, this is a challenging process, considering the similarity of these compounds structures and their divergent range of concentrations. Conventional techniques, such as ion-exchange chromatography [2] or activated charcoal treatment [3], are tedious and low effective. Meanwhile, the use of alternative techniques, such as yeast treatment and the use of ionic liquids (ILs), which have not been applied before to this purpose, are of great interest. In this work, PLE and MWAE have been optimised and applied to the extraction of inositols from agro-industrial by-products, such as artichoke (Cynara scolymus) leaves. Alternative fractionation treatments, such as the use of Saccharomyces cerevisiae and ILs, have also been evaluated. Mono-, di-, trisaccharides and inositols (myo-, scyllo- and chiro-inositol) were efficiently extracted from artichoke external leaves in only 13 minutes for PLE and 3 minutes for MWAE. The use of yeast allowed the preservation of inositols content while removing other co-extracted low molecular weight carbohydrates. Regarding the possibility of selective extraction of inositols using ILs, a first approach was done using carbohydrate standards. Solubility values for inositols and a selected set of relevant carbohydrates were determined for the first time [4]. Some of the investigated ILs were found to allow an appropriate separation of bioactive inositol from relevant carbohydrates in binary mixtures of carbohydrates. These extraction and fractionation processes are valuable analytical alternatives that can easily extended to other by-products of the food industry due to their analytical potential to be exploited, due to their inherent social and economic benefits. Acknowledgments Authors thank MINECO for project CTQ2012-32957 and CM for program ANALISYC-II (S-2009/AGR-1464). References 1. Ruiz-Aceituno, L., Rodríguez-Sánchez, S., Ruiz-Matute, A.I., Ramos, L., Soria, A.C. and Sanz,

M.L., 2013. Journal of the Science of Food and Agriculture 93: 2797-2803. 2. Macias Camero, B. and Sanjuan Merino, C., 2004. US patent number 6699511. 3. Streeter, J.G., 2001. Crop Science 41: 1985-1987. 4. Carrero-Carralero, C., Ruiz-Aceituno, L., Moreno, F.J. and Sanz, M.L. Green Chemistry

(submitted).


P44 A NOVEL DEVICE FOR ON-SITE PATHOGENS DETECTION IN FOOD Pedro Razquin1, G. Cebrián1, X. Arias1, L. Mata1, P. Aldamiz-Echevarría2, J. Escobal2, J. Berganza2, G. Diez2, J.I. Valpuesta3, A. Perez3 and J. Fernandez de Mendiola3

1Zeulab, Spain, 2Centro Tecnológico Gaiker, Spain and 3CTL-TH Packaging, Spain [email protected] Food industry needs fast and simple analytical tools to ensure the safety of processed foods. Therefore, pathogens detection is a priority for this sector. Available methods can be fast, simple, specific and sensitive. However, they can also be time-consuming and require expensive equipment or qualified personnel. Ready-to-use and integrated systems (lab-in-a-box) would help the industry to take decisions for early release of end products. In this project, we have developed a disposable device for on-site analysis of pathogens in food. The system combines sample preparation, enrichment, detection and inactivation in one device. Specific ready-to-use media, test and inactivation reagent are included in different compartments and together in the same container. External actuators are implemented, so there is no sample transfer neither risk of contamination. The method requires minimum equipment, as only an incubator at 37ºC is needed. The system can be currently applied for Salmonella and Listeria detection in less than 24 or 48 hours, respectively. A preliminary validation study showed satisfactory results for both bacteria in dairy and meat products. This device will allow food producers to easily run in-house tests in a short time, with no risk of contamination and at a reasonable cost.


P45 DEVELOPING A RAPID IN-FARM DIAGNOSTIC TEST FOR CAMPYLOBACTER M. Rosario Romero1, M. D’Agostino1, N. Cook1, S. Robles1, C. Fernández1 and M.P. Andreou2

1The Food and Environment Research Agency, UK and 2Optisense Ltd., UK [email protected] Campylobacter is the most common cause of acute bacterial gastroenteritis in humans, causing around 600,000 cases of illness and 100 deaths annually in the UK, and an estimated cost of £ 600M. Reduction of Campylobacter incidence in the food chain is a priority in Europe, as part of a wider strategy to achieve a secure food chain. A major source of Campylobacter infection is poultry. Current systems to monitor infection in-farm involve sending samples to the laboratory, with the associated delays and costs. Rapid and robust tests are required that can be implemented on site to promptly inform actions. Involving the farmers directly with monitoring makes them part of the solution to the problem. This type of tests will enable farmers to react quickly and apply effective biosecurity measures, fulfilling an important need of the poultry industry. We are using a combination of DNA loop-mediated isothermal amplification (LAMP) and antibody technologies as a rapid assay for detection of Campylobacter jejuni and C. coli in poultry faeces. Specific antibodies are used for bacteria isolation and concentration, precluding the need for bacterial culturing. Once the bacteria are concentrated on antibody-coated magnetic beads, they are transferred to a small, portable instrument where the LAMP reaction takes place, with fluorescence detection for assay read-out. A kit is being designed containing all the required consumables and reagents, from sample preparation to results. We are aiming to reduce manual intervention to a minimum, thus minimising risks of contamination and allowing results to be obtained in about one hour after sample collection. We have developed a LAMP assay for C. jejuni and C. coli that shows good specificity and high sensitivity. In addition, an immunocapture protocol has been developed for sample enrichment and clean-up. This step has been optimised in turkey faecal samples (farm boot swabs) and it enables concentration of the bacteria and elimination of substances that might interfere with the DNA amplification reaction. We have shown that the bacteria isolated from the faeces using this method can be transferred directly to the LAMP instrument, without the need for previous DNA extraction, and be detected in around 40 minutes. 33 genome copies can be detected with a probability of 95%; this level of sensitivity indicates the ultimate utility of the approach. Current work includes determination of the limit of detection of the combined antibody/LAMP assay in farm samples and final integration of the assay kit and instrument.


P46 QUANTIFICATION OF PHOMOPSIN A: BIOSYNTHESIS OF 15N6-LABELLED INTERNAL STANDARD Svenja Schloss, I. Wedell, M. Koch and R. Maul Federal Institute for Material Research and Testing, Germany [email protected] Among the European grain legumes, sweet lupines have the highest protein content and possess a very valuable protein composition. With respect to both protein content and composition, they are superior to field beans and field peas. Moreover, cultivation of lupines has a fertilisation effect for subsequent crops due to nitrogen fixation. The seeds of various lupine species have been used both as food and feed for over 2,000 years. Particularly in Australia, lupines are widely used, whilst this plant has been moved to the scientific and agricultural focus in the European Union only a few years ago. Along with a growing interest in the lupine and products thereof, concerns have been raised about typical fungal contaminations. Diaporthe toxica (formerly referred to as Phomopsis leptostromiformis) grows parasitically or saprophytically on field lupines (e.g. Lupinus luteus, L. albus, and L. angustifolius) [1]. The hepatotoxic metabolites phomopsin A and B are responsible for the field mycotoxicosis of sheep known as lupinosis [2]. The aim of the present study was to develop a sensitive and robust LC-MS/MS quantification method for phomopsin A. As co-elution of matrix components may lead to ion suppression or ion enhancement effects in LC-MS analysis, the use of isotope-labelled internal standards is advisable. To date, no isotope standard is available for phomopsin A. Thus, in the first instance, the biosynthesis of 15N6 isotope labelled phomopsin A was established using the known phomopsin A producing strain D. toxica. By cultivation of the fungus on defined media containing Na15NO3 and 15N-labelled yeast extract as the only nitrogen sources, fully 15N6-labeled phomopsin A could be obtained. The identity of 15N6-phomopsin A and the absence of native phomopsin A were confirmed by hrMS. The stable isotope-labelled standard enables the further optimisation of sample extraction and enhanced quantitative LC-MS/MS method development. References 1. Lanigan, G.W., Payne, A.L. , Smith, L.W., Wood, P.McR. and Petterson, D.S., 1979. Applied and

Environmental Microbiology 37: 289-292. 2. Culvenor, C.C.J., Beck, A.B., Clarke, M., Cockrum, P.A., Edgar, J.A., Frahn, J.L., Jago, M.W.,

Lanigan, G.W., Pane, A.L., Peterson, J.E., Smith, L.W. and White, R.R., 1977. Australian Journal of Biological Sciences 30: 269-277.

Figure 1. Phomopsin A.


P47 MICROBIAL QUALITY CONTROL OF BEER BY RNA K. Vetter1, K. Merx1 and Jvo Siegrist2

1BECIT GmbH, Germany and 2Sigma-Aldrich, Switzerland [email protected] Beer-spoiling microorganisms cause an increase of turbidity and unpleasant sensory changes in beer. Since improved process technology in modern breweries has resulted in significant reduction of oxygen content in the final product, the role of strictly anaerobic bacteria, such as Pectinatus and Megasphaera, have increased. The detection of these organisms takes a weak or longer as done traditionally by incubation on culture medium. Thus, the development of a rapid and specific method for the detection of beer spoilage organisms is needed. A total of 178 beer samples with alcohol contents between 2.5 and 6.7 % were tested with a molecular biological test system based on hybridisation of rRNA with biotin-labelled capture probe and a DIG-labelled detection probe (sandwich hybridisation). This system detects all known beer spoiling microorganism. The beer samples were spiked with Lactobacillus brevis, L. coryniformis, L. lindneri, Pediococcus damnosus, Megasphaera cerevisiae, and Pectinatus frisingensis in a range between 103 and 106 cfu/sample. Additionally, 32 real brewery samples were also examined with this system. The results were controlled on NBB, MRS, or PYF agar. For the development of further genera and species, specific test system experiments were performed with 17 pure bacterial cultures of most common beer spoiling microorganism. The results show that the test system is able to detect the spiked beer-spoiling microorganisms in all beer samples. The detection limit differs for Lactobacillus spp. and the other mentioned beer spoiling genera between 104 and 105 cfu/sample. For a more sophisticated analysis, several other test systems Pectinatus spp./Megasphaera spp., Pediococcus spp., L. brevis, L. lindneri, L. collinoides, L. rossiae and L. backii were developed. These optimised systems were able to detect specific bacterial counts between 2.0 x 104 and 5.5 x 108 cfu/sample without any significant cross reaction. In conclusion, the results of the presented test system are comparable to classical cultivation methods. Development and optimisation of new group- and species-specific test systems offer the ability to detect the most known beer spoiling organism in one test platform in a time and cost-saving manner.


P48 VALIDATION OF A NEW CHROMOGENIC MEDIA FOR CLOSTRIDIUM PERFRINGENS M. Manafi1 and Jvo Siegrist2 1Medical University Vienna, Austria and 2Sigma-Aldrich, Switzerland [email protected]

The European Directive on drinking water quality has included mCP agar as the reference method for recovering Clostridium perfringens from drinking waters. mCP agar is difficult to handle, the reaction is reversible and the cells are killed by the ammonia evaporation. In the present study, three media (mCP, TSCF and CP ChromoSelect Agar) were evaluated for recovery of C. perfringens in different surface water samples. Out of 139 water samples using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for C. perfringens in at least one of the culture media. Green-coloured colonies on CP ChromoSelect Agar (CCP agar) were counted as presumptive C. perfringens isolates. Out of 483 green colonies on CCP agar, 96.3 % (465 strains, indole-negative) were identified as C. perfringens, 15 strains (3.1%) were indole-positive and were identified as C. sordelli, C. bifermentans or C. tetani. Only 3 strains (0.6 %) gave false positive results and were identified as C. fallax, C. botulinum, and C. tertium. Variance analysis of the data obtained shows statistically no significant differences in the counts obtained between media employed in this work. The mCP method is very onerous for routine screening and bacterial colonies could not be used for further biochemical testing. The colonies on CCP and TSC were easy to count and subculture for confirmation tests. TSC detects sulfite-reducing clostridia, including species other than C. perfringens, and in some cases excessive blackening of the agar frustrated counting of the colonies. If the contamination was too high, TSC did not consistently produce black colonies and as a consequence, the colonies were white and gave false negative results. In conclusion, the identification of typical and atypical colonies isolated from all media demonstrated that CCP agar was the most useful medium for C. perfringens recovery in water samples.


P49 SURFACE PLASMON RESONANCE (SPR) ASSAY FOR THE DETECTION AND QUANTIFICATION OF TOTAL AMOUNT OF OKADAIC ACID, DTX1 AND DTX2 IN SHELLFISH

Gurmit Singh1, B. Brady1, T. Koerner1, A.-C. Huet2 and P. Delahaut2

1Food Research Division, Bureau of Chemical Safety, Health Canada, Canada and 2Centre d’Economie Rurale, Health Department, Belgium [email protected] Okadaic acid (OA) and the chemical related structures dinophysistoxins (DTX1, DTX2) are lipophilic marine biotoxins. Okadaic acid and DTX1 are the main diarrhoeic shellfish poisoning (DSP) toxins due to their strong diarrhoetic activity. The mouse bioassay is widely used to detect DSP toxins, but animal welfare concerns have prompted researchers to seek alternative methods of detection. A surface plasmon resonance biosensor immunoassay has been developed for the rapid determination of the total amount of OA, DTX1 and DTX2 in mussel tissue. The assay is very sensitive with detection limits for OA, DTX1 and DTX2 at 12.9, 29.6 and 13.3 ng/g, respectively. The resulting limits of detection (LODs) of this single-laboratory validated method were well below the existing regulatory limit set by Health Canada (200 ng/g, under review) and the European Commission (160 ng/g) for OA and its analogues. Repeatability of total OA, DTX1 and DTX2 spiked in mussel tissue were assessed at 30.0 (spiked 10.0 ng of each toxin) and 60.0 ng/g (spiked 20.0 ng of each toxin) resulting in a coefficient of variation of 5.8 and 15.5%, respectively. The recoveries at these spiking levels ranged from 76 to 123%. Certified reference materials were used to estimate assay precision, confirm recovery and establish method accuracy based on data compliance with assigned values.


P50 PATHATRIX AUTO™ – THE FIRST AFNOR-APPROVED REAL-TIME PCR METHOD FOR DETECTING SALMONELLA IN POOLED FOOD SAMPLES J. Wall1, D. Sohier2, R. Conrad1 and Katrien Vanhonacker1

1Thermo Fisher Scientific, Inc., USA and 2Adria, France [email protected] The Pathatrix Auto™ pathogen isolation platform provides a workflow that is able to process as many as ten individual food enrichments in the same sample pool. This sampling format has never been approved in the European Union (EU) market, and would require extensive validation efforts by an expert testing lab to evidence that the approach is not only possible, but practical. To validate this product for food safety testing in the EU, this workflow would need to demonstrate a relevant relative detection limit, show statistical similarity to the ISO 16140 reference through accuracy, sensitivity, and specificity, and prove its robustness and practicability in the field. Adria Developpement was selected to perform the evaluation to ascertain the Pathatrix Auto’s ability to detect Salmonella in pooled food sample types by real-time PCR and selective agar plating. A ring trial proficiency study with 15 independent food safety testing laboratories was also conducted to verify that the workflow was functional and accurate with minimal training. In both the Adria Developpement study and the ring trial, the candidate and reference methods were found to be statistically similar. Of the 202 different food sample types tested during this evaluation, a relative accuracy of 93.1%, a relative sensitivity of 87.7%, and a relative specificity of 96.7% was attained. The relative detection limit was determined to be 0.4-1.5 log cfu/25 g of sample, which was statistically similar to the reference. The selected ring trial laboratories demonstrated 100% proficiency and accuracy in performing the workflow. These results were satisfactory for approval by the AFNOR committee, and yielded the first validated method for sample pooling in the EU. The demonstrated robustness, accuracy, and ease of use of this workflow allows the user to rapidly screen for rare contamination events with high confidence, with up to a 90% cost savings over other PCR-based platforms.


P51 CAMPYLOBACTER DETECTION SYSTEM READY FOR FIELD APPLICATION Jos M.B.M. van der Vossen, E. Lommen, H. Rahaoui, F.H.J. Schuren and R.C. Montijn Microbiology and Systems Biology, TNO, the Netherlands [email protected] The current gold standard for Campylobacter detection is culturing, which is time-consuming and requires a dedicated laboratory. Molecular technologies, such as real-time PCR, can reduce the analysis time significantly, but routine application of this technique on site is currently not feasible. The multiple days period involved in culturing will only allow for retrospective information. Therefore, traditional analysis technology prohibits the implementation of direct managerial measures, which can be taken to prevent the entry of Campylobacter-contaminated poultry flocks into the food chain. Because of lack of speed, traditional analysis is also of limited value for the further understanding of possible factors involved in flocks getting infected with Campylobacter. We are aiming at a simple and rapid test, gaining results within less than a few hours. By using such a rapid test, flocks carrying Campylobacter in faecal samples can be excluded from fresh meat production lines at the entrance of broiler meat processing plants. Risk assessment studies have indicated that human health risks are anticipated to decrease under such a regime. Contaminated flocks could be directed towards a dedicated line in the plant for further heat processing to reduce human health risks. At TNO, we have developed a new Campylobacter detection test based on isothermal DNA amplification. The test includes sampling of bacterial material from cloaca, faeces and meat, subsequent concentration of bacteria and cell lysis, followed by the collection and purification of DNA and finally isothermal detection. The test is targeting generically the Campylobacter spp. 16S rDNA sequence. The performance of the test has been investigated and showed excellent detection of poultry relevant Campylobacter species, including C. jejuni and C. coli, the most prevalent ones. The performance of the test is comparable to traditional culturing and real-time PCR results and moreover, results are available within one hour. With a basic training the test can be performed by anyone at poultry production sites.


P52 BEAD-BASED SUSPENSION ARRAY FOR DETECTION AND IDENTIFICATION OF TICK-BORNE BORRELIA SPECIES R.P. Achterberg1, C.B. van Solt-Smits1, M.E. Hovius2, H. Sprong3, M. Fonville3, F.M. De Bree1 and Fimme J. van der Wal1 1Infection Biology, Central Veterinary Institute, the Netherlands, 2Companion Animal Hospital ‘t Heike, the Netherlands and 3Laboratory for Zoonoses and Environmental Microbiology, National Institute of Public Health and the Environment, the Netherlands [email protected] In the Netherlands, screening for tick-borne pathogens is performed using real-time PCR on DNA extracted from ticks, followed by sequencing of positive samples, thereby disallowing identification in case of double infections. Until recently, testing was performed with several reverse line blots (RLBs), each containing ~10 different probes. Advantages of RLBs are their capabilities to differentiate between bacterial species and to detect double infections, disadvantages are that RLBs are fastidious, elaborate, and time-consuming. The aim of this work was to ‘test drive’ bead-based suspension arrays for the simultaneous detection and identification of tick-borne Borrelia species in DNA extracted from ticks, using the flow cytometry-based multi-analyte profiling (xMAP) technology from Luminex. For xMAP of DNA, fluorescent nanoparticles (beads) are used to build multiplex assays simply by mixing different bead sets that are covered with specific probes. Each bead set contains a different ratio of red and infrared fluorophores, which enables identification of beads. A third fluorophore on the target molecule, i.e. a labelled amplicon, is used to monitor if targets are hybridised to the beads. Using standard chemistry, aminated oligonucleotides (probes) were covalently linked to carboxylated Luminex beads, resulting in various bead sets with general and specific probes for the 23S-5S intergenic spacer (IGS) of Borrelia species. Detection and identification of Borrelia was performed by using a mixture of the various bead sets, for a 30 minute hybridisation of biotinylated amplicons, followed by labelling with streptavidin-phycoerythrin (SAPE) and subsequent investigation by flow cytometry on a dedicated Luminex instrument. To evaluate the resulting suspension array, DNA extracts from over 1000 ticks were used to generate biotinylated amplicons with a universal PCR targeting the 23S-5S IGS of Borrelia spp. All PCR reactions were then subjected to a direct hybridisation assay in presence of a mixture of probe-containing beads, labelled with SAPE, and analysed by flow cytometry. Out of 1056 ticks, 185 were found to carry one or more Borrelia species (131 and 54, respectively). The most prevalent species was B. afzelii, followed by B. burgdorferi sensu stricto, B. garinii, B. sensu lato (unidentified species) and B. valaisiana. These results show the viability of bead-based suspension arrays for (relatively) rapid detection and identification of multiple species. Currently, confirmation of these results by next generation sequencing is in progress. Acknowledgements This work was partly funded by the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement Number 222633 (WildTech).


P53 APPLICATIONS OF POLYMER MICROARRAYS TO WATER PURIFICATION Selected for speed presentation Sesha Venkateswaran School of Chemistry, University of Edinburg, UK [email protected] Polymer microarrays offer a high-throughput approach to the screening and assessment of a large number of polymeric materials. Using this technology the Bradley research group collaborate with various leading national and international researchers and discover novel polymers to fulfil many applications in areas as diverse as food hygiene, water safety, coatings for biomedical devices and substrates for cellular control. Tying in this technological approach with Helen Bridle to study protozoan ‘polymer interactions’, we identified materials that either trap waterborne protozoan parasites Cryptosporidium parvum and Giardia Lamblia, or prevent their adhesion, both of which have major practical applications. Material properties, including polymer composition, wettability and surface chemistry, allowed the identification of binding/polymer structure function relationships. Understanding these binding interactions could assist in improved water treatment processes. The identified polymers have been scaled-up for application in water filter media.


P54 QUANTIFICATION ERRORS USING QPCR Lucie Vondrakova, J. Pazlarova and K. Demnerova Department of Biochemistry and Microbiology, Institute of Chemical Technology in Prague, Czech Republic [email protected] Molecular genetic methods based on nucleic acid detection are indisputably very reliable and fast tools used to identify various pathogens that pose a threat to public health. However, main disadvantage of these methods is the inability to distinguish signals originating from different sources (live, dead or compromised cells), which is a significant limitation, especially when used for quantification of live infective agents. The aim of this study was to compare results of quantification obtained using qPCR when different DNA pre-treatments were applied. Influence of two DNA-intercalating dyes (EMA/PMA) was examined and cultures of thermotolerant Campylobacter spp. were quantified. Acknowledgements Financial support from specific university research (MSMT No 21/2013 and MSMT No. 21/2014).


P55

SIMULTANEOUS DETERMINATION OF FREE AND CONJUGATED ALTERNARIA TOXINS IN VARIOUS MATRICES BY LC-MS/MS Jeroen Walravens1, H. Mikula2, M. Rychlik3, S. Asam4, J. Diana Di Mavungu1, A. Van Landschoot5, L. Vanhaecke6 and S. De Saeger1 1Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Belgium, 2Institute of Applied Synthetic Chemistry, Vienna University of Technology, Austria, 3Bioanalytik Weihenstephan, ZIEL Research Center for Nutrition and Food Sciences, TU München, Germany, 4Chair of Analytical Food Chemistry, TU München, Germany, 5Associated Faculty of Applied Bioscience Engineering, Biochemistry-Brewery Research Group, HoGent, Belgium and 6Laboratory of Chemical Analysis, Faculty of Veterinary Medicine, Ghent University, Belgium [email protected] The fungal genus Alternaria contains numerous species that can contaminate a wide variety of crops in the field and cause post-harvest decay of various fruits, grains and vegetables. In addition to causing economic losses, Alternaria spp. can form mycotoxins under certain conditions. Due to the possible risk for public health related to the presence of Alternaria toxins in food, the European Food Safety Authority (EFSA) stipulated that additional quantitative occurrence data are urgently needed to refine exposure assessment. Furthermore, Alternaria toxins can in line with other xenobiotics be partly metabolised, which may lead to the formation of conjugated metabolites in plants. These masked mycotoxins are of human health concern, as they may be capable to release their native precursors in the digestive tract of organisms. Therefore, a fast and sensitive UPLC-ESI+/--MS/MS method for the determination of free (AOH, AME, ALT, TeA, TEN and ATX I) and conjugated (AOH- and AME-3-sulphate, AOH- and AME-3-glucoside) Alternaria toxins in multiple matrices, such as cereal products (rice, oat flakes), beer, fruit and vegetable juices (carrot, apple and grape juice), tomato products, lentils, sesame and sunflower oils/seeds was developed and validated (in agreement with the criteria mentioned in Regulation No 401/2006/EC; Commission Decision 2002/657/EC). The method, applying isotopically labelled internal standards, allowed for the simultaneous determination of ten Alternaria toxins in a one-step chromatographic run (7 minutes). Minimal to no sample clean-up was carried out on the different matrices, making it possible to perform sampling, analysis and finally detection and quantification in less than 24 hours. Yet, low limits of quantification (<1-5 µg/kg) were obtained for all matrices. Subsequently, 200 samples of a variety of commercially available foodstuffs will be analysed between February 2013 and March 2014. Acknowledgements This study was funded by the Federal Public Service of Health, Food Chain Safety and Environment (Contract RF12/6261-ALTER).


P56 QUAD-FLUORESCENCE POLARISATION IMMUNOASAY FOR SIMULTANEOUS QUANTITATIVE HIGH-THROUGHPUT SCREENING OF MULTIPLE ANTIBACTERIAL RESIDUES IN MILK T. Mi1 and Zhanhui Wang1,2

1College of Veterinary Medicine, China Agricultural University, China and 2National Reference Laboratories for Veterinary Drug Residues, China [email protected] Many kinds of antibacterial agents have been extensively applied in veterinary treatment. Thus, it is necessary to monitor these residues in food samples to obtain a better food safety assurance. Conventional ELISA is constrained as a result of antibody recognition capability, multiple washing steps and a long-time immunoreaction required. Fluorescence polarisation immunoassay (FPIA) is a promising and reliable homogeneous immunoassay to overcome those shortcomings, because homogeneous immunoreaction in solution phase can reach equilibrium in minutes or even seconds, and no separation or washing steps are required. In this work, four groups of widely used antibacterial agents, including streptomycin (STR), sulfathiazole (ST), enrofloxacin (ENR) and cephalexin (CEP) were selected as the model analytes. And four commercial fluorophores, including fluorescein, tetramethylrhodamine, Texas Red and CF633 with different fluorescence spectra easily distinguishable from each other, were selected to synthesize fluorescent conjugates (tracers). And all four tracers were prepared by linking amino-reactive fluorescent label derivatives with these antibacterial agents to obtain the corresponding tracers. Quad-fluorophore FPIA platform was developed by combination of four tracers with corresponding specific monoclonal antibodies, and applied to simultaneously determine four antibacterial residues in milk. Optimised quad-fluorophore FPIA exhibited a satisfactory compatibility for the multiple analysis, and showed a limit of detection (LOD) of 7.3 ng/ml for STR, 2.7 ng/ml for ST, 2.5 ng/ml for ENR, and 3.8 ng/ml for CEP. All four antibacterial agents are highly soluble in water, and could be simultaneously extracted from milk and determined in one assay system. Recoveries for four antibacterial agents fortified milk were from 59.8-88.1%, with relative standard deviation lower than 25.5%.


P57 A RAPID FLUORESCENCE POLARISATION IMMUNOASSAY FOR THE DETERMINATION OF SIX MACROLIDE ANTIBIOTICS IN MILK H. Zhang1 and Zhanhui Wang1,2

1College of Veterinary Medicine, China Agricultural University, China and 2National Reference Laboratories for Veterinary Drug Residues, China [email protected] Macrolide antibiotics are widely used to treat infections caused by Gram-positive bacteria and some Gram-negative cocci in both human and animal medicine. Their residues in food of animal origin have caused great worldwide attention. To meet the requirement of regulation of macrolides, there is urgency to develop high-throughput and multi-analyte detection methods. In this work, a rapid and quantitative high-throughput fluorescence polarisation immunoassay was developed for the determination of six macrolide antibiotics in milk. Five fluorescein-labelled tracers of erythromycin (ERY) were synthesised and purified by thin layer chromatography (TLC), including ERY-DTAF, ERY-FITC, ERY-CMO-4’-AMF, ERY-sulforhodamine 101 and ERY-AF647. Four of these tracers showed high binding ability with three anti-ERY monoclonal antibodies, except for ERY-sulforhodamine 101. Under optimal conditions, the FPIA developed based on the tracer ERY-FITC and antibody 5B2 showed the high sensitivity with IC50 values of 3.5 to 20.59 ng/ml for ERY, dirithromycin, erythromycin, roxithromycin, erythromycylamine, erythromycin ethyl succinate and clarithromycin. An extraction procedure using 20% acetonitrile was applied to extract macrolide antibiotics in this FPIA immunoassay. The average recoveries of macrolide antibiotics-spiked milk samples at 50, 100, 150 µg/l ranged from76.4 to 127.2% with CV values less than 17%. The fluorescence polarisation assay in the work provides a rapid and high-throughput screening method for multi-analyte detection.


P58 LANTHANOID COMPLEXES AS SPECIFIC DETECTION PRINCIPLE IN SILICA SOL-GEL IMMUNOASSAYS Jan H. Wichers1, X. Liu2, E. Bouwman2 and A. van Amerongen1

1Biomolecular Sensing and Diagnostics, Wageningen UR, the Netherlands and 2Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, the Netherlands [email protected] Lanthanoids, also known as rare-earth elements, can be used as part of a signal principle in diagnostics. When bound to specific chelating ligands, these metals provide, via the antenna-effect, strong luminescent signals that can be detected in a time-resolved fluorescence (TRF) mode. The fluorescence lifetime of the specific signal is several orders of magnitude longer than that of the non-specific background. This enables us to measure the response of the label at a time when the background (e.g. due to auto-fluorescence of a sample) has already decayed. The large Stokes’ shift, i.e. the difference between excitation and emission wavelengths, and the narrow emission peak further contribute to an excellent signal-to-noise ratio. We explored the potential of covalently incorporating chelating ligands in porous silica sol-gels. Lanthanoid complexes do not suffer from self-quenching and, therefore, it is possible to pack the chelating ligands at high density inside such sol-gels without quenching the fluorescence of the final complexes. Silica nanoparticles having capture antibodies immobilised at their surface, are entrapped in the sol-gel in close proximity of the chelating ligands. Following incubation in a competitive format between a fixed amount of a lanthanoid-labelled tracer analyte and the analyte from the sample, the remaining bound lanthanoid metal ions will be released. The intensity of fluorescence upon interaction of these ions with the chelating ligand molecules is a measure of the concentration of the free analyte in the sample. Suitable lanthanoid ions are europium, samarium, terbium and dysprosium. The europium ion, Eu3+, is most often used. Terbium, Tb3+, has been introduced as a second choice, and together with europium dual labelling is possible. Triple and quadruple labelling can be done with samarium, Sm3+, or dysprosium, Dy3+. Porous silica sol-gels, functionalised with chelating ligands, can be moulded into almost any frame and onto many (‘silicon-based’) sensor surfaces, which renders them suitable for application in different platforms (e.g. microfluidic chip, column/cuvette, microsieve). We will present our initial results for this silica sol-gel immunoassay detection system. Acknowledgements The authors acknowledge the financial contribution from the programme NanoNextNL (Project 05A.01 Microbial Food Quality and Safety).


P59 AN INDEPENDENT EVALUATION OF THE FAST DETECTION AND CONFIRMATION OF LISTERIA SPECIES IN FOOD BY SINGLEPATH® LISTERIA AND SINGLEPATH® L'MONO LATERAL FLOW TEST Heike Wulff1, M. Goll2, T. Miller2, F. Düker2, C. Spielvogel1, R. Ossmer1, L. John1 and M. Bülte2 1Merck Millipore, Germany and 2Justus-Liebig-Universität Giessen, Germany [email protected] Listeria monocytogenes is known to be a human and animal pathogen. The presence of Listeria, in particular of L. innocua, is an indicator for critical hygienic conditions in the production process. Therefore, hygiene-status monitoring procedures and risk-related quality controls in foods should be performed for the Listeria genus. Singlepath® L’mono and Singlepath® Listeria, based on lateral flow technology, offer a reliable, fast and user-friendly test method for confirmation. Singlepath® L’mono and Singlepath® Listeria screening methods indicate the presence or absence of Listeria spp./L. monocytogenes after 48 hours of enrichment, 50% faster than the standard ISO method. The purpose of this study was to independently evaluate the screening protocol for Singlepath® L’mono on foods in comparison to the standard reference method and the confirmation protocol of flagellated Listeria by Singlepath® Listeria and L. monocytogenes by Singlepath® L’mono. For the screening protocol for Singlepath® L’mono 4 foods (smoked salmon, minced meat, paté and cheese, each n=10 samples) were artificially contaminated with L. monocytogenes at spiking levels of 7 to 250 cfu/10g. Additionally, 19 samples of smoked salmon from retail were tested. The samples were enriched according to DIN EN ISO 11290-2005 and tested by Singlepath® L’mono. Presumptive colonies of a total of 61 samples were confirmed by Singlepath® Listeria after incubation for 5 hours at 30°C and a total of 40 strains were confirmed by Singlepath® L’mono after incubation for 1 hour at 37°C in Full Fraser or TSB broth. For screening by Singlepath® L’mono, 95% of samples were detected positive, after a total enrichment time of 48 hours, compared to the standard method. 10 out of 19 smoked salmon samples from retail were tested positive by Singlepath® L’mono, being conform to the reference method. Confirmation by Singlepath® L’mono and Singlepath® Listeria resulted in a sensitivity of 100%. In conclusion, Singlepath® Listeria and Singlepath® L’mono provide a fast confirmation of Listeria spp. and L. monocytogenes with easy handling. The screening protocol of Singlepath® L’mono can be used as an alternative method being faster than the standard method.


P60 FAST DETECTION OF VIABLE LEGIONELLA PNEUMOPHILA CELLS IN WATER USING FLOW CYTOMETRY M. Adela Yáñez1, L. Martínez1, E. Soria-Soria1, R. Múrtula1, A. Parker2 and V. Catalán1

1Aqualogy, Labaqua, Spain and 2 MemTeq Ventures, Spain [email protected] Legionella pneumophila is a pathogenic bacterium that has found an appropriate ecological niche in several manmade aquatic environments, such as potable water systems, cooling towers, evaporative condensers, and wastewater systems. Worldwide, it is responsible for numerous outbreaks of legionellosis, which can be fatal. The most common method used to study L. pneumophila in environmental samples is culture isolation. Nevertheless, this methodology is labour-intensive, does not permit the isolation of viable but non culturable bacteria (VBNC) nor the isolation of L. pneumophila from samples with a high level of microbiota and the minimum time to obtain the final results is between 10 and 14 days. Because of these limitations, one of the greatest challenges is to develop rapid methods capable of differentiate between dead and live cells. In this sense, flow cytometry has become a promising tool in food and environmental microbiology. The recent availability of ‘on-the-bench’ and portable new generation cytometers has enhanced the application of this tool to diverse fields in microbiology, including environmental diagnostics. In this work, we have evaluated flow cytometric assay (FCA) as a quantitative and fast detection method of L. pneumophila cells in water samples coupled to a new system to concentrate the sample in shorter time, maintaining sample integrity and representativeness. Natural samples from potable water systems and cooling towers as well as samples spiked with L. pneumophila were analysed in parallel by three detection methods: (i) flow cytometric assay (FCA); (ii) culture isolation under ISO 11731; and (iii) PMA (propidium monoazide) qPCR detection. The procedure for the FCA assay includes a sample concentration step using CellTrap technology (MemTeq), staining of the cells with FITC-conjugated L. pneumophila specific antibody and subsequent analysis of the sample using flow cytometry (Accuri, BD) and propidium iodide as a viability marker. The detection of L. pneumophila cells in environmental samples using flow cytometry coupled to a previous concentration step using CellTrap is emerging as a good candidate for rapid results with the added value of portability, because the system can be transported to the facility on risk for on-site analysis.

Date post:	11-Jun-2020
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