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Transia Plate Salmonella Gold for the detection of Salmonella spp. in foods and feeds
Abstract A method for the detection of Salmonella spp. in foods and feeds is presented. This method, which is based on a two step enrichment followed by a sandwich-type immunoassay, produces a presumptive positive result within 36 to 40 hours of analysis. The inclusivity and exclusivity of the test were assessed on 129 Salmonella strains from 101 different serovars and 72 strains from 50 non-Salmonella species; these results demonstrated the high levels of inclusivity (96.1 %) and exclusivity (100 %) of the antibodies used in the assay. The overall method agreement calculated on confirmed results between Transia Plate Salmonella Gold (TPSG) and the ISO 6579:2002 methods was 98.8 %. With a sensitivity rate of 99.3 % and a specificity rate of 97.4 % the Transia Plate Salmonella Gold method appeared to give comparable results to the reference method. The high specificity of the antibodies used in the method was confirmed by the absence of false positive reactions, as all the positive results obtained with the Transia test were confirmed by streaking the RVS enrichment broth onto selective agars. The ruggedness of this method was studied in this work and showed that the length of the heat inactivation step, the reagents volumes and incubation parameters could be exceptionally modified without any significant effect on the results. Method Authors
Patrice Arbault, V. Buecher, S. Poumerol and M.-L. Sorin
Diffchamb S.A., 8 rue Saint-Jean de Dieu, 69007 Lyon, France Submitting Company Diffchamb AB, F O Petersons Gata 32, SE-421 31 Västra Frölunda, Sweden Independent Laboratory Campden & Chorleywood Food Research Association, Chipping Campden, Gloucestershire, GL55 6LD, United Kingdom Reviewers Michael Brodsky (Brodsky Consultants, Canada) Joseph Odumeru (University of Guelph, Canada) Thomas Hammack (FDA CFSAN, USA)
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1. Scope of Method
1.1 Target organisms – Salmonella spp. 1.2 Tested matrices Cooked chicken, raw milk, cantaloupe, sausages, raw shrimps, yogurt, mayonnaise, shell eggs, frozen red berries & currants, bean sprouts, raw ground beef, smoked fish (trout), fresh pasta, milk chocolate, ground black pepper, cake mix, dry milk-based infant formula, dry food for cat, raw ground turkey and Brie cheese. 1.3 Summary of validated performance claims The Transia Plate Salmonella Gold assay for detection of Salmonella spp. in foods and feeds has been tested internally and externally and has shown 99.3 % sensitivity and 97.4 % specificity. The external study demonstrated that the Transia Plate Salmonella Gold method is equivalent to the ISO 6579:2002 reference method. The use of Transia Plate Salmonella Gold assay allows the user to get a presumptive result of analysis within a total time of 36 to 46 hours, which is a significant improvement compared with the time needed by the reference method (minimum one day more) to give the same kind of information. Indeed, quicker reliable results allow food business operators to release fresh products from production one day earlier in case of negative result. In case of positive result these operators can react earlier and thus limit the possible outcomes of the contamination. The excellent specificity of the antibodies used in the assay minimises the risk of false positive reactions and therefore improves the reliability of the screening by reducing the risks of inopportune and non-relevant production chains shutdown or product recalls.
2. Definitions
2.1 Inclusivity: Inclusivity rate = 100 x (No. of pure Salmonella cultures positive with Transia Plate) /
(Total No. of pure Salmonella cultures tested). 2.2 Exclusivity: Exclusivity rate = 100 x (No. of pure non-Salmonella cultures negative with Transia
Plate) / (Total No. of pure non-Salmonella cultures tested).
3. Principle
Transia Plate Salmonella Gold is a sandwich-type immunoassay relying on a polyclonal antibody coated onto the solid phase (wells of a microtitre plate with divisible strips) and a monoclonal antibody labeled with a peroxydase. The monoclonal antibody recognizes specifically a lipopolysaccharide determinant. The sample (enriched broth) is added to the well after a heat inactivation step to release the Salmonella antigens from the culture possibly present in the sample. After a three (3) step protocol whose total incubation time is 105 minutes, the reading of the microtitre plate is performed with a spectrophotometer at 450 nm.
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4. General Information Salmonella is the leading foodborne bacteria for human enteritis worldwide through the consumption of a variety of contaminated foods, such as eggs, meats and seafood. As Salmonella is isolated from many different matrices, a very large number of food producers are looking for the bacteria in their raw material, manufacturing lines and in their finished products. Today, Salmonella accounts for the largest number of foodborne pathogen analysis worldwide and represents a significant workload. The detection methods for Salmonella are numerous and rely on different technologies, from the cultural methods to the immunoassays and genomic tests. However, some of these methods have not been adequately developed for the routine screening of many food samples. The drawbacks are many and method related. For example, the cultural methods are time-consuming and require a lot of plating with subjective result interpretation. On the other hand PCR-based assays offer a quicker answer, but require a tedious sample preparation followed by cumbersome amplification and detection steps. Immunoassays have been developed for years but some require long enrichment protocols or are known to generate false positive answers due to a lack of specificity.
5. Test Kit Information
5.1 Kit Name: Transia Plate Salmonella Gold. The kit includes 1 microtitre plate (96 wells in 12 divisible 8 wells strips) or 10 microtitre plates (960 wells in 12 divisible 8 wells strips).
5.2 Catalog Number: SA0180 (96 wells), SA0190 (960 wells) 5.3 Ordering Information:
From the U.S.: Diffchamb Inc. c/o RAISIO STAEST US, INC. 325 Deming Way Summerville, SC 29483 USA Phone +843 873 3007 (ext. 7103) or 1-866 -DIFFCHAMB Fax +843 873 3545 [email protected] Website address: www.diffchamb.com
From abroad: Diffchamb AB F O Petersons Gata 32 421 31 Västra Frölunda Sweden Phone +46 31 742 33 50 Fax +46 31 58 33 70 [email protected]
5.4 Test Kit Reagents:
5.4.1 Negative control – ready to use – 1 x 6 mL (SA0180), 2 x 15 mL (SA0190). 5.4.2 Positive control: LPS (lipopolysaccharide) – ready to use – 1 x 3 mL
(SA0180), 2 x 8 mL (SA0190). 5.4.3 Conjugate: anti-Salmonella antibodies conjugated to peroxydase – ready to
use – 1 x 15 mL (SA0180), 4 x 30 mL (SA0190). 5.4.4 Washing buffer – concentrated 20X – 1 x 60 mL (SA0180), 1 x 800 mL
(SA0190). 5.4.5 Substrate: Urea-H2O2 – 1 x 10 mL (SA0180), 3 x 30 mL (SA0190). 5.4.6 Chromogen: TMB (Tetramethylbenzidine) – 1 x 10 mL (SA0180), 3 x 30 mL
(SA0190).
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5.4.7 Stop solution: H2SO4 – ready to use – 1 x 15 mL (SA0180), 5 x 30 mL (SA0190).
6. Additional Media and Reagents
6.1 Enrichment: 6.1.1 Distilled water. 6.1.2 Buffered peptone water. 6.1.3 Rappaport-Vassiliadis Soya broth (RVS).
6.2 Confirmation of positive results: 6.2.1 Isolation: Selective agar plate such as xylose lysine deoxycholate (XLD) or
brilliant green agar (BGA). 6.2.2 Identification: Biochemical identification gallery (such as API 20E,
BioMérieux, Art. No. 20100 or Microbact 24E, Medvet, Art. No. 1074).
7. Equipment
7.1 Scales (1 to 500 g ± 0.1g) and weighing vessels. 7.2 Homogeniser or mixer (Stomacher Seward 400C). 7.3 Filter-fitted stomacher bags. 7.4 Tubes (20 mL) for the subcultures. 7.5 Magnetic stirrer. 7.6 Air Incubator capable of maintaining 37 ± 1°C. 7.7 Air Incubator capable of maintaining 41.5 ± 0.5°C (or preferably a water bath with
circulating water). 7.8 Vortex. 7.9 Screw-cap tubes (5 or 10 mL) resistant to +100°C. 7.10 Water bath 100°C (boiling water). 7.11 Micropipette 100-1000 µL.
7.12 Micropipette 10-100 µL (optional). 7.13 Wash bottle (or preferably an automatic microplate washer). 7.14 Absorbent paper. 7.15 Multipipette with 2.5 and 5 mL tips.
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7.16 Microplate reader – single or double beam reading 450 nm filter and reference filter ≥595 nm.
7.17 Sterile transfer pipettes (1 mL ± 0.05 mL)
8. Standard Reference Material
ISO 6579:2002, Microbiology of Food and Animal Feeding Stuffs – Horizontal Method for the Detection of Salmonella spp. Feldsine, P., Abeyta, C., & Andrews W.H. (2002) AOAC International Methods Committee Guidelines for Validation of Qualitative and Quantitative Food Microbiological Official Methods of Analysis, J. AOAC Int. 85, 1187-1200.
9. Safety Precautions
Good laboratory practice should be employed when using the kit. Safety clothing should be worn and skin contact with reagents avoided. Material safety data sheet available on request. Contaminated material should be disposed according to local, state and federal regulations.
10. Sample Preparation
10.1 Homogenise 25 g or 25 mL of the sample with 225 mL of buffered peptone water in a stomacher bag. Use of a filter-fitted stomacher bag is recommended.
10.2 Incubate at 37 ± 1°C for 16-20 hours. 10.3 Homogenize and inoculate 0.1 mL of the pre-culture broth in 10 mL of RVS. 10.4 Incubate for 18-24 hours at 41.5 ± 0.5°C. 10.5 Heat 1 to 2 mL of the enrichment broth in a tube in a water bath at 100°C (boiling
water) for 20 minutes, then cool to room temperature. Keep the rest of the enrichment broth at 3 ± 2°C should confirmation of a positive result prove necessary later.
11. Analysis
The test should be performed at room temperature (18-25°C). Remove the reagents from the box and allow them to come to room temperature at least one hour before use. Have all reagents and samples ready for use so that the various materials can be added to the wells without delay. Shake each sample manually or using a Vortex before use.
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11.1 Attach the required number of strips to the plate: two wells for the negative control, one for the positive control and one well for each sample. Return the unused strips to the zip-lock bag with dehydrating agent and close it tightly. Write the position of the controls and samples on the working sheet.
11.2 Distribute 100 µL of the controls and the samples into the assigned wells. Cover the
plate.
11.3 Incubate at room temperature (18-30°C) for one hour. Prepare the washing buffer. 11.4 Holding the plate firmly, shake out the contents of the plate over paper towelling by
briskly flicking the wrist. Rinse each well; keep the washing buffer in the wells for one minute (for the first two washings). Empty the plate over a container and then remove the washing buffer by inverting the plate on a paper towel and tapping the plate firmly several times. Repeat the washing five times.
11.5 Add 100 µL of conjugate (vial 4) to each well. Be careful not to touch the wells with
the dispensing tip. Cover the plate. 11.6 Incubate at room temperature (18-30°C) for 30 minutes. Just before the end of this incubation stage, prepare the required volume of
substrate/chromogen mixture using the following formula: required volume = (60 µL substrate + 60 µL chromogen) x number of wells used]. 11.7 Wash the microplate 5 times as indicated in paragraph 11.4. 11.8 Add 100 µL of the substrate/chromogen mixture to each well using a multipipette and
cover the plate. Discard any unused mixture. The chromogen and substrate can be added without prior mixing - add 50 µL of
substrate (vial 5) and 50 µL of chromogen (vial 6) to the wells.
Controls and samples
Conjugate
Substrate/chromogen mixture
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11.9 Incubate at room temperature (18-30°C) for 15 minutes. 11.10 Add 100 µL of stop solution (vial 7) to each well following the same order used when
the substrate/chromogen were added. Mix the contents of the wells thoroughly to ensure complete color conversion. The blue turns to yellow.
11.11 Read the optical densities at 450 nm using a microtiterplate reader (blank on air), use a
reference filter ≥595 nm for double beam reading. 12 Result interpretation
12.1 Validation of the test The optical density of the positive control (PC) must be higher than or equal to 0.700. The optical density of the negative control (NC) must be lower than or equal to 0.150
(for double beam reading) or 0.200 (for single beam reading). If the controls do not meet these requirements, the results are invalid. 12.2 Positive threshold The positive threshold is calculated as the average of the negative controls plus 0.11: [(NC1 + NC2) / 2] + 0.11 12.3 Negative threshold The negative threshold is calculated as the positive threshold multiplied by 0.9 12.4 Positive samples A sample is considered positive for Salmonella if its optical density is equal to or
higher than the positive threshold 12.5 Negative samples
A sample is considered negative for Salmonella if its optical density is lower than the negative threshold
12.6 Doubtful samples
A sample is considered doubtful if its optical density is lower than the positive threshold but equal to or higher than the negative threshold
12.7 Confirmation of a doubtful or positive result A positive or doubtful result should be confirmed by streaking the non-heated RVS
broth onto selective media plates followed by biochemical identification (see section 6.2, Confirmation of positive results).
Stop solution
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13 Internal Validation Studies Internal validation studies were conducted at the R&D Centre of the Diffchamb Group (Diffchamb S.A. – Lyon, France). 13.1 Inclusivity Testing One hundred twenty nine Salmonella strains from 101 different serovars were analysed
to determine the inclusivity of the Transia Plate Salmonella Gold kit (see Table 1).
13.1.1 Methodology From a 20-hour culture in BPW (buffered peptone water) at 37 ± 1oC, each
Salmonella strain was subcultured (0.1 mL into 10 mL) in RVS (Rappaport Vassiliadis soya broth) at 41.5 ± 0.5oC for 18-24 hours. Following enrichment, 1 mL of each broth was taken and heat-treated in a water bath at 100oC for 20 minutes. The heat-treated samples were then processed as described in the Transia Plate Salmonella Gold test kit insert.
Each unboiled enrichment broth was serially diluted in PBS (Phosphate buffer saline broth) and appropriate dilutions were spread-plated onto TSYEA (Trypticase soya yeast extract agar). The plates were incubated at 37 ± 1oC for 24 ± 3 h.
13.1.2 Results
Inclusivity results are summarised in Table 3. Among the 129 strains tested, Transia Plate Salmonella Gold correctly detected 124, resulting in an overall inclusivity of 96.1 %.
13.2 Exclusivity Testing
Seventy-two (72) strains from 50 different species belonging to non-Salmonella genera (Table 2) were analyzed to determine the exclusivity of the kit.
13.2.1 Methodology
From a 20-hour culture in BPW (buffered peptone water) at 37 ± 1oC, each non-Salmonella strain was subcultured (0.1 mL into 10 mL) in BPW at 37 ± 1oC for 18-24 hours. Following enrichment, 1 mL of each broth was taken and heat-treated in a water bath at 100oC for 20 minutes. The heat-treated samples were then processed as described in the Transia Plate Salmonella Gold test kit insert.
Each unboiled enrichment broth was serially diluted in PBS (Phosphate buffer saline broth) and appropriate dilutions were spread-plated onto TSYEA (Trypticase soya yeast extract agar). The plates were incubated at 37 ± 1oC for 24 ± 3 h.
13.2.2 Results
Exclusivity results are summarised in Table 3. All 72 strains tested have been detected as negative with Transia Plate Salmonella Gold, resulting in an overall exclusivity of 100 %.
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13.3 Method Comparison Study
The purpose of this study was to compare the Transia Plate Salmonella Gold method with the ISO 6579:2002 method for the detection of Salmonella spp in eighteen (18) different matrices. 13.3.1 Methodology
13.3.1.1 Selected strains
As agreed with AOAC R.I. the following combinations matrix/strain was tested:
Matrix Total count (cfu/g)
Strain Reference Origin
Cooked chicken 2 x 104 Salmonella Kentucky 305 AFSSA – France
Raw milk 5.4 x 106 Salmonella Dublin 1236 Dairy – Denmark
Cantaloupe 3.5 x 105 Salmonella Poona 781 Pasteur Institute Paris – France
Sausages 4.4 x 107 Salmonella Bredeney 1656 Pasteur Institute Lille – France
Raw shrimps 8.1 x 108 Salmonella Montevideo 1449 NCTC 5747
Yogurt 1 x 102 Salmonella Typhimurium 1654 Pasteur Institute Lille – France
Mayonnaise < 102 Salmonella Heidelberg 1689 Isolated from chicken gizzard – France
Shell eggs 2.7 x 104 Salmonella Enteritidis 1535 Isolated from cocoa - France
Frozen red berries & currants 2 x 103 Salmonella Newport 1442 Isolated from chicken wings – France
Bean sprouts 4.5 x 108 Salmonella Infantis 1305 Service laboratory – Australia
Raw ground beef 1.5 x 109 Salmonella Anatum 1622 Service laboratory – UK
Smoked fish (trout) 6.9 x 107 Salmonella Virchow 1451 Isolated from chicken filet – France
Fresh pasta < 102 Salmonella IIIb 61:r:1.5 1575 Isolated from lamb tongue – France
Milk chocolate 1.2 x 102 Salmonella Senftenberg 1486 Dairy – UK
Ground black pepper 6.6 x 104 Salmonella Indiana 1428 Isolated from cow liver – France
Cake mix 8.2 x 103 Salmonella Thompson 306 Food Inspection Services – France
Dry milk-based infant formula < 102 Salmonella Blockley 1100 Isolated from chicken gizzard – France
Dry food for cat 7 x 102 Salmonella Mbandaka 1618 Service laboratory – UK
13.3.1.2 Inoculum preparation
Each strain was cultured in M broth (Merck; Darmstadt, Germany), incubated overnight at 37°C. 0.1 mL was subcultured into 5 mL of M broth, for about 4-6 hours at 37°C. This culture was then serially diluted in PBS. Dilutions were used for moist foods contamination. Strains used for the inoculation of processed foods were stressed by heat (10 minutes at 50°C), prior to dilution in PBS.
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For dry foods contamination, a freeze-dried bacterial culture was grinded to a fine powder and added to 200-500 g of the matrix. After storage at room temperature for 1-2 weeks, the cell density was estimated, using serial dilutions and plating on Tryptone Soya Yeast Extract Agar, by difference between the Total Plate Count of the inoculated and uninoculated matrices. This preparation was used to inoculate two 1000 g portions of each dry food matrix.
13.3.1.3 Inoculation procedure Two 1000 g portions of each matrix were inoculated with a dilution of the
culture or an aliquot of dry food, estimated to give, for the first one, an inoculum level of 1-10 cfu/25g (3 cfu/25g was aimed) and for the second one, an inoculum level of 10-50 cfu/25g (30 cfu/25g was aimed), after 3 days of storage.
A further 500 g portion of each food remained uninoculated. The inoculated and uninoculated food portions were stored for 48-72 hours at 2-8°C for moist foods and at room temperature for dry foods, prior to testing.
After storage, 20 x 25 g samples from the two inoculated food portions and 5 x 25 g samples from the uninoculated food were weighed out for each matrix. Also, further samples were taken for MPN analysis (3 x 100 g, 3 x 10 g, 3 x 1 g and 3 x 0.1 g). Samples were then tested by the ISO 6579:2002 method for the detection of Salmonella spp and by the Transia Plate Salmonella Gold Test, as briefly described in the next paragraph.
13.3.1.4 ISO 6579:2002 method for the detection of Salmonella spp. For the pre-enrichment of all food matrices, samples (including MPN
samples) were, after homogenization with a stomacher, enriched in Buffered Peptone Water (BPW, Biokar BM01008) and incubated at 37°C for 18 ± 2h (see Appendix 1).
Following pre-enrichment, 1ml of each sample was subcultured into 10ml Muller-Kaufmann Tetrathionate-novobiocin broth (MK, Oxoid CM343) and 0.1ml into 10ml Rappaport Vassiliadis Soya Peptone broth (RVS, Biokar BM07408). RVS broths were incubated at 41.5°C for 21-24h and MKTTn broths were incubated at 37°C for 24 ± 3h.
After incubation, a loopful of each selective enrichment broth was
streaked onto Xylose Lysine Desoxycholate Agar (XLD, Oxoid CM469) and Brilliant Green Agar (BGA, Biokar BK091HA). Plates were incubated at 37°C for 24h ± 3h. Plates were then examined for typical Salmonella colonies.
Upto five (5) typical Salmonella colonies per sample were confirmed by
testing the presence of Salmonella O-antigens by slide agglutination using Salmonella O antisera and biochemically using Microbact 24E system (Medvet Australia, article nr 1074).
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The MPN of Salmonella present in the initial samples on the day of analysis was determined by referring to the MPN tables in the Bacteriological Analytical Manual (8th edition).
13.3.1.5 Transia Plate Salmonella Gold Test The Transia Plate Salmonella Gold Test was performed on the RVS
selective enrichment broths used in the ISO method (see Appendix 1). Following 21-24h incubation, 1 to 2ml of each sample was taken and heat-treated in a water bath at 100°C for 20 minutes. The heat-treated samples were then processed as described in the Transia Plate Salmonella Gold test kit insert. Samples found to be positive were confirmed according to the ISO method; typical Salmonella colonies observed on XLD and BGA plates streaked from unboiled RVS broths were confirmed by testing the presence of Salmonella O-antigens by slide agglutination using Salmonella O antisera and biochemically using Microbact 24E system (Medvet Australia, article nr 1074).
13.3.2 Results
All the results are summarized in Table 4 and shown full in Appendix 2 to 19. None of the uninoculated samples were found to contain Salmonella by the two methods. TPSG gave totally equivalent detection results compared to ISO method for all matrices contaminated with low levels of Salmonella except for raw milk. Indeed, for raw milk samples, TPSG gave six positive and two false negative samples, compared to ISO method. ISO method gave five positive and three false negative samples, compared to the Transia method. In total, the two methods gave eight positive samples for raw milk.
13.3.3 Conclusion The Transia Plate Salmonella Gold test successfully detected low levels of Salmonella in the eighteen (18) food matrices and globally results between the two methods are similar. For all food matrices, except the raw milk, Transia Plate Salmonella Gold test and ISO method gave totally identical results. A few disaccording results were observed with raw milk but are not significant due to the extremely low inoculation level (1 cfu/25 g).
13.4 Lot-to-lot and Stability Study
13.4.1 Lot-to-lot consistency The lot-to-lot consistency was assessed by testing in duplicate three (3)
negative controls and three (3) dilutions of two (2) different Salmonella cultures for three (3) different kit lots. One lot was tested at the date of release, the second 40 days after release and the third 9 months after release.
13.4.1.1 Methodology Positive test strains: From a 24-hour culture at 37 ± 2º C in BPW (Buffered Peptone Water), each Salmonella strain (No. 1431 and 1435 from the Diffchamb collection) was subcultured (0.2 mL into 10 mL) in
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RVS (Rappaport Vassiliadis Soya broth) for 24 h ± 2 hours at 41.5 ± 0.5oC. The RVS was then split in two parts: - one was serially diluted in sterile PBS (Phosphate Buffer Saline) and
successively 1 mL of each appropriate dilution and 15 mL of TSYE agar (cooled to 45 ± 1º C) were poured in a Petri dish (3 replicates per dilution). After homogenization and solidification of the agar, the Petri dishes were inverted and incubated for 24 to 48 hours at 37 ± 2º C before enumeration.
- the second part was immediately inactivated (20 minutes at 100º C) and refrigerated.
After reading of the Petri dishes and estimation of the cell count, the inactivated subculture of Salmonella was adjusted to the requested levels (1x105, 5x105 and 1x106 cells/mL) by dilution in RVS before being processed as described in the Transia Plate Salmonella Gold test kit insert. Negative test strains: From a 24-hour culture in BPW, each non-Salmonella strain (No. 1693, 1694 and 1771 from the Diffchamb collection) was subcultured (0.2 mL into 10 mL) in BPW for 24 h ± 2 hours at 41.5 ± 0.5oC. The cell count was then estimated by reading the optical density at 470 nm and the cultures were adjusted to 109 cells/mL by dilution in BPW before a heat inactivation of 20 minutes at 100°C.
13.4.1.2 Results
The results from the lot-to-lot consistency study are summarised in Table 5. They show clearly that the three (3) lots analysed gave similar results. The strain nr 1431 (Salmonella Heidelberg) was found negative in all cases at 1x105 cells/mL but always positive at the higher levels. The strain nr 1435 (Salmonella Enteritidis) was found slightly positive (just above the positive threshold) in all cases at 1x105 cells/mL and always significantly positive at the higher levels. The non-Salmonella strains did not generate any signal. In every case the interpretation was the same within the duplicates.
13.4.2. Stability
The stability was assessed by following a kit lot over a 31-day period. Testing was performed on the release date and then 14 and 31 days after, in duplicate with a negative control (non-Salmonella cultured broth), and three (3) dilutions of two (2) different Salmonella cultures. This stability study allowed us to compare 2 different storage temperature conditions for kits stored at 37 ± 2°C and 5 ± 3°C for a period of 14 and 31 days. Tests were performed in parallel on the same set of samples.
13.4.2.1. Methodology
Positive test strains: From a 24-hour culture at 37 ± 2º C in BPW (Buffered Peptone Water), each Salmonella strain (No. 1305 and 1435 from the Diffchamb collection) was subcultured (0.2 mL into 10 mL) in
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RVS (Rappaport Vassiliadis Soya broth) for 24 h ± 2 hours at 41.5 ± 0.5oC. The RVS was then split in two parts: - one part was serially diluted in sterile PBS (Phosphate Buffer Saline)
and successively 1 mL of each appropriate dilution and 15 mL of TSYE agar (cooled to 45 ± 1º C) were poured in a Petri dish (3 replicates per dilution). After homogenization and solidification of the agar, the Petri dishes were inverted and incubated for 24 to 48 hours at 37 ± 2º C before enumeration.
- the second part was immediately inactivated (20 minutes at 100º C) and refrigerated.
After reading of the Petri dishes and estimation of the cell count, the inactivated subculture of Salmonella was adjusted to the requested levels by dilution in RVS before being processed as described in the Transia Plate Salmonella Gold kit insert. Negative test strain: From a 24-hour culture in BPW, a Citrobacter diversus strain (No. 1163 from the Diffchamb collection) was subcultured (0.2 mL into 10 mL) in BPW for 24 h ± 2 hours at 41.5 ± 0.5oC. The cell count was then estimated by reading the optical density at 470 nm and the cultures were adjusted to 109 cells/mL by dilution in RVS before a heat inactivation of 20 minutes at 100°C.
13.4.2.2. Results The results of the stability study are summarised in Table 6. They show that the results obtained with all the positive and negative controls used led to the same interpretation even after 31-day storage at 37 ± 2 oC. These conditions, which were selected in order to generate accelerated stability data, did not create significant differences compared with the normal refrigerated storage conditions (5 ± 3°C).
13.5 Ruggedness study
13.5.1 Influence of the heat-treatment time The Transia Plate Salmonella Gold assay requires a 20 minute-heat
inactivation of the enriched RVS broth in order to damage the cell structure and improve the availability of the antigens to the antibodies. This leads also to a sterilisation of the broth which is an important safety factor for the ELISA test. It may happen that the duration of this step is not fully controlled by the user and we found interesting to study the effect of a shortened or increased heat-treatment time on the final response of the assay. In this study heat-treatment times of 15, 20 and 25 minutes were tested; they correspond to the time requested in the package insert, increased or decreased by 5 minutes. This analysis was performed three (3) times, using three (3) different kit lots.
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13.5.1.1 Methodology
13.5.1.1.1. Positive strains: From a 20-hour culture at 37°C in BPW (Buffered Peptone Water), two Salmonella strains (Salmonella Enteritidis No. 1834 and Salmonella Typhimurium No. 1654 from the Diffchamb collection) were subcultured (0.1 mL into 10 mL) in RVS broth (Rappaport Vassiliadis Soya) for 24 hours at 41.5°C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100°C. Three (3) dilutions were tested by ELISA.
13.5.1.1.2. Negative strain: From a 20-hour culture at 37°C in BPW (Buffered
Peptone Water), a non-Salmonella strain (Citrobacter freundii No. 1694 from the Diffchamb collection) was subcultured (0.1 mL into 10 mL) in RVS broth (Rappaport Vassiliadis Soya) for 24 hours at 41.5°C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100°C. The pure culture was tested by ELISA.
13.5.1.2 Results
The results of this study are summarized in Table 7. They show that the interpretation of the test is the same regardless the heat-treatment time (15, 20 or 25 minutes).
13.5.2. Influence of the variation of sample or conjugate volume Another possible deviation that may be introduced by the user includes non-compliance with the specified volumes. In this study, the effects of different volume distribution of sample and conjugate were separately examined.
13.5.2.1. Variation of the sample volume The Transia Plate Salmonella Gold assay requires a sample volume of 100 µL. In this study, the effects of a variation of this volume were examined. The negative strain and three (3) dilutions of the positive strains were tested five (5) times, using three (3) different sample volumes: 80, 100 and 120 µL. These volumes correspond respectively to the sample volume requested in the package insert, increased or decreased by 20 µL. This analysis was performed three (3) times, using three (3) different kit lots.
13.5.2.1.1. Methodology
13.5.2.1.1.1. Positive Strains: Positive strains: From a 20-hour culture at 37°C in BPW (Buffered Peptone Water), two Salmonella strains (Salmonella Enteritidis No. 1834 and Salmonella Typhimurium No. 1654 from the Diffchamb collection) were subcultured (0.1 mL into
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10 mL) in RVS broth (Rappaport Vassiliadis Soya) for 24 hours at 41.5°C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100°C. Three (3) dilutions were tested by ELISA.
13.5.2.1.1.2. Negative strain:
From a 20-hour culture at 37°C in BPW (Buffered Peptone Water), a non-Salmonella strain (Citrobacter freundii No. 1694 from the Diffchamb collection) was subcultured (0.1 mL into 10 mL) in RVS broth (Rappaport Vassiliadis Soya) for 24 hours at 41.5°C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100°C. The pure culture was tested by ELISA.
13.5.2.1.2. Results
The results of this study are summarized in Table 8. They show that the interpretation of the test is the same regardless the sample volume (80, 100 or 120 µL).
13.5.2.2. Variation of the conjugate volume
The Transia Plate Salmonella Gold assay requires a conjugate volume of 100 µL. In this study, the effects of a variation of this volume were examined.The negative strain and three dilutions of the positive strains were tested five (5) times, using three different conjugate volumes: 80, 100 and 120 µL. These volumes correspond respectively to the conjugate volume requested in the package insert, increased or decreased by 20 µL. This analysis was performed three (3) times, using three (3) different kit lots.
13.5.2.2.1. Methodology
13.5.2.2.1.1. Positive strains: From a 20-hour culture at 37°C in BPW (Buffered Peptone Water), two Salmonella strains (Salmonella Enteritidis No. 1834 and Salmonella Typhimurium No. 1654 from the Diffchamb collection) were subcultured (0.1 mL into 10 mL) in RVS broth (Rappaport Vassiliadis Soya) for 24 hours at 41.5°C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100°C. Three (3) dilutions were tested by ELISA.
13.5.2.2.1.2. Negative strain: From a 20-hour culture at 37°C in BPW
(Buffered Peptone Water), a non-Salmonella strain (Citrobacter freundii No. 1694 from the Diffchamb collection) was subcultured (0.1 mL into 10 mL) in RVS broth (Rappaport
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Vassiliadis Soya) for 24 hours at 41.5°C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100°C. The pure culture was tested by ELISA.
13.5.2.2.2. Results
The results of this study are summarized in Table 9. They show that the interpretation of the test is the same regardless the conjugate volume (80, 100 or 120 µL).
14 Independent Validation Studies AOAC Research Institute approached CCFRA to carry out an independent validation of the Transia Plate Salmonella Gold Test (Diffchamb) against the ISO 6579:2002 method for the detection of Salmonella spp., in two (2) food matrices (raw ground turkey and Brie cheese).
14.1. Materials and methods The following food matrices were tested: Raw ground turkey Brie cheese 14.1.1 Strain details
For the raw ground turkey study, a single strain of Salmonella enterica subsp. enterica serovar Enteritidis PT4 (CCFRA code 1004) was used. This strain was isolated from chicken and was obtained from the Central Public Health Laboratory, Colindale, London, United Kingdom.
For the Brie cheese study, a single strain of Salmonella enterica subsp. enterica serovar Typhimurium PT195 (CCFRA code 1009) was used. This strain was isolated from milk and was obtained from the Central Public Health Laboratory, Colindale, London, United Kingdom.
14.1.2 Preparation of inoculum Each strain of Salmonella was cultured in 10ml Nutrient Broth (NB, Oxoid CM1) incubated overnight at 37oC. Cultures were then serially diluted in Maximum Recovery Diluent (MRD, Lab M Lab103).
14.1.3 Inoculation procedure
One 1500g portion of each food matrix was inoculated with a dilution of the appropriate culture estimated to give an inoculum level of 1-10 cfu/25g. A further 500g portion of each food remained uninoculated. The inoculated and uninoculated food portions were stored at 2-8oC for 72h prior to testing.
After chilled storage, 20 x 25g samples from the inoculated food portion and 5 x 25g samples from the uninoculated food were weighed out, for each matrix. Also, further samples were taken from the inoculated portion of each
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matrix for MPN analysis (3 x 100g, 3 x 10g, 3 x 1.0g and 3 x 0.1g portions). Samples were then tested by the ISO 6579:2002 method for the detection of Salmonella spp., and by the Transia Plate Salmonella Gold Test, as briefly described overleaf.
14.1.4 ISO 6579:2002 method for the detection of Salmonella spp. For the pre-enrichment of both food matrices, samples (including MPN
samples) were enriched in Buffered Peptone Water (BPW, Oxoid CM1049) and incubated at 37 ± 1oC for 18 ± 2h (see Appendix 1).
Following pre-enrichment, 1ml of each sample was subcultured into 10ml
Muller-Kauffmann Tetrathionate-novobiocin broth (MKTTn, Oxoid CM1048) and 0.1ml into 10ml Rappaport-Vassiliadis Soya Peptone broth (RVS, Oxoid CM866). MKTTn broths were incubated at 37 ± 1oC for 24 ± 3h and RVS broths were incubated at 41.5 ± 0.5oC for 21-24h.
After incubation, a loopful of each selective enrichment broth was streaked
onto Xylose Lysine Desoxycholate Agar (XLD, Oxoid CM469) and Brilliant Green Agar (BGA, Oxoid CM263). Plates were incubated at 37 ± 1oC for 24 ± 3h. Plates were then examined for typical Salmonella colonies.
Up-to five (5) typical Salmonella colonies per sample were confirmed by
testing for the presence of Salmonella O- and H-antigens by slide agglutination using Salmonella polyvalent O and H antisera (Mast Assure M14294 and M14317) and biochemically using API 20E strips (bioMerieux 20100).
The MPN of Salmonella present in the initial samples on the day of analysis
was determined by referring to the MPN tables in the FDA-BAM manual (8th Edition).
14.1.5 Transia Plate Salmonella Gold test The Transia Plate Salmonella Gold Test was performed on the RVS selective
enrichment broths used in the ISO method (see Appendix 1). Following 21-24h incubation in RVS, 1 to 2ml of each sample was taken and heat-treated in a waterbath at 100oC for 20 minutes. The heat-treated samples were then processed as described in the Transia Plate Salmonella Gold kit insert.
Samples found to be assay positive by the Transia Plate Salmonella Gold test,
were confirmed by the ISO method, as both were derived from the same 25g sample. Where appropriate, typical Salmonella colonies observed on XLD and BGA plates streaked from unboiled RVS broths were used to confirm both the ISO method and Transia Plate Salmonella gold.
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14.2 Results
14.2.1 MPN results The results obtained using the MPN procedure indicated a contamination
level in the raw ground turkey to be 6 cfu/25g and in the Brie cheese 2.3 cfu/25g. The 95% confidence intervals on these estimations were 1.45-24.875 and 0.55-9.725 respectively.
14.2.2 Raw ground turkey The TPSG and ISO results from raw ground turkey are summarized in Table
4 and shown in full in Appendix 20. None of the uninoculated raw ground turkey samples were found to contain Salmonella by the Transia Plate Salmonella Gold test or by the ISO culture method. The TPSG gave equivalent detection rates compared to the ISO culture method for raw ground turkey contaminated with low levels of Salmonella.
14.2.3 Brie cheese The TPSG and ISO results from Brie cheese are summarized in Table 4 and
shown in full in Appendix 21. None of the uninoculated Brie cheese samples were found to contain Salmonella by the Transia Plate Salmonella Gold test or by the ISO culture method. The TPSG gave equivalent detection rates compared to the ISO culture method for Brie cheese contaminated with low levels of Salmonella.
14.3 Conclusion
The Transia Plate Salmonella Gold test successfully detected low levels of Salmonella in raw ground turkey and Brie cheese. For both food matrices, the Transia Plate Salmonella Gold test gave the same Salmonella detection rate as the ISO culture method.
15. Discussion In this study, the inclusivity and exclusivity of the Transia Plate Salmonella Gold test were examined by testing the method with pure cultures of various Salmonella and non-Salmonella strains. Overall, the test performed very well with inclusivity and exclusivity rates of 96.1 % and 100 %, respectively. The accuracy of the test as compared with the ISO 6579:2002 reference method on 400 samples from 20 matrices performed identically, showing a sensitivity of 99.3 % and a specificity of 97.4 % (see Appendix 22). Despite the low spiking level of the samples (between 1 and 10 CFU/25 g.) the overall agreement between the two (2) methods is excellent with an obtained value of 98.8 %. Transia Plate Salmonella Gold test did not give any false positive signal; when the ELISA test gave a positive or doubtful result, the presumptive test result was then always confirmed, after streaking of the RVS selective broth on selective agars, by the isolation and biochemical confirmation of a Salmonella strain. The ruggedness of Transia Plate Salmonella Gold has been documented by studying the effects of variations in sample or conjugate volume and heat inactivation time. Although important
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variations of these parameters might affect significantly the optical densities, the test interpretation was not modified and the accuracy of the analysis was preserved. 16. Conclusion Our evaluation of the Transia Plate Salmonella Gold demonstrated that this method is equivalent to the ISO 6579:2002 reference method for detection of Salmonella spp. in all foods and feeds tested. This study demonstrated the consistent sensitivity and specificity of the Transia Plate Salmonella Gold assay for the detection of Salmonella spp. in food and feed matrices.
17. References
(1) Haeghebaert, S., Le Querrec, F., Vaillant, V., Delarocque Astagneau E. & Bouvet, P. (1998) Les Toxi-Infections Alimentaires Collectives en France en 1997. BEH, 177-81
(2) Anonymous (2000) Preliminary Food Net Data on the Incidence of Foodborne
Illnesses Selected Sites, United States. MMWR (Morbidity & Mortality, Weekly Report), 49, 201-205
(3) FSIS (1999). Salmonella Serotypes Isolated from Raw Meat and Poultry.
Electronic memorandum available at www.fsis.usda.gov/OPHS/haccp/serolyr.htm (4) U.S. Food and Drug Administration (2003) Bacteriological Analytical Manual
Online, January 2001, Chapter 5, Salmonella, http://www.cfsan.fda.gov/~comm/bam-5.html
(5) Tsang, R.S.W., Schlecht, S., Aleksia, S., Chan, K.H. & Chau P.Y. (1991) Lack of
the α-1,2-linked N-acetyl-D-glucosamine Epitope in the Outer Core Structures of Lipopolysaccharides from certain O Serogroups and Subspecies of Salmonella enterica. Res. Microbiolol. 142, 521-533.
(6) Maijala, R., Johansson, T. & Hirn, J. (1992) Growth of Salmonella and
Competing Flora in Five Commercial Rappaport-Vassiliadis (RV) Media. Int. J. of Food Microbial 17, 1-8.
(7) Peterz, M., Wiberg, C. & Norberg, P. (1989). The effect of Incubation
Temperature and Magnesium Chloride Concentration on Growth of Salmonella in Home-Made and in Commercially Available Dehydrated Rappaport-Vassiliadis Broths J. Appl. Bacterial. 66, 523-528
(8) Owen, P. & Meehan, M. (1994) Immunochemistry of Bacterial Antigens,
Immunochemistry, C.J. Van Oss & M.H.V. Van Regenmortel (Eds), Marcel Dekker, New-York, pp 393-453.
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(9) Feldsine, P., Abeyta, C. & Andrews W.H. (2002) AOAC International Methods Committee Guidelines for Validation of Qualitative and Quantitative Food Microbiological Official Methods of Analysis, J. AOAC Int. 85, 1187-1200.
(10) Sharp, J. M. (2002) Development of Immunomagnetic Capture (IMC) Based
Techniques for the Detection of Salmonella on Poultry Carcasses, Thesis, Faculty of the Virginia-Maryland Regional College of Veterinary Medicine, pp 3-5.
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Table 1 – Inclusivity: Salmonella strains
Ref. Serotype Group OD Result Ref. Serotype Group OD Result
1716 S. Abaetetuba F 0,541 + 1738 S. Cubana G2 0,475 +
1623 S. Agama B 0,841 + 1692 S. Derby B 0,543 +
1607 S. Agona B 1,811 + 1236 S. Dublin D1 >3 +
712 S. Alabama D1 1,963 + 1604 S. Dublin D1 2,457 +
1622 S. Anatum E1 1,215 + 1466 S. Dublin D1 2,375 +
1685 S. Anatum E1 0,455 + 332 S. Eimsbuttel atypical C4 >3 +
713 S. Antarctica D1 >3 + 1478 S. Emek C3 >3 +
1483 S. Arizonae II Rough 2,185 + 688 S. Enteritidis D1 1,771 +
787 S. Arizonae IIIa 48 : z4, z23:- Y 0,056 - 1195 S. Enteritidis D1 2,013 +
1655 S. Arizonae IIIa 48 : z4, z23:- Y 0,071 - 1214 S. Enteritidis D1 >3 +
1148 S. Arizonae IIIb 61 :-:- non-motile 61 0,803 + 1535 S. Enteritidis D1 1,751 +
1329 S. Arizonae IIIb 61 :-:- non-motile 61 0,505 + 668 S. Gallinarum pullorum D1 1,942 +
1117 S. Arizonae IIIb 61 :-:- non-motile 61 1,155 + 1609 S. Give E1 0,748 +
1308 S. Arizonae IIIb 61 :i:- 61 1,103 + 299 S. Gloucester B 1,851 +
1273 S. Arizonae IIIb 61 :i: z53 61 1,134 + 1487 S. Goldcoast C2 2,120 +
1591 S. Arizonae IIIb 61 :k:1,5,7 61 0,909 + 1475 S. Goverdham D >3 +
1575 S. Arizonae IIIb 61 :r:1,5 61 1,170 + 1453 S. Hadar C2 1,816 +
1481 S. Babelsberg M 2,407 + 1458 S. Hadar C2 >3 +
1691 S. Banana B 0,543 + 1482 S. Haifa B 1,951 +
1644 S. Bergen X 0,061 - 1621 S. Havana G2 0,719 +
1100 S. Blockley C2 >3 + 1695 S. Havana G2 0,613 +
1444 S. Bovismorbificans C2 >3 + 1431 S. Heidelberg B 1,147 +
708 S. Braenderup C1 >3 + 1689 S. Heidelberg B 0,915 +
1351 S. Brandenburg B 1,527 + 1473 S. Hull I 1,842 +
1283 S. Bredeney B 1,453 + 1467 S. Hvittingfoss I 2,168 +
1656 S. Bredeney B 1,668 + 1663 S. I 1,3,19:z27:- E4 0,543 +
1474 S. Brunei C3 >3 + 836 S. Idikan G2 0,651 +
1737 S. Caracas H 0,634 + 1381 S. Indiana B 1,996 +
1129 S. Cerro K >3 + 1428 S. Indiana B 2,327 +
862 S. Chester B >3 + 1305 S. Infantis C1 1,636 +
1251 S. Coeln C1 2,335 + 1306 S. Infantis C1 1,232 +
1066 S. Corvallis C3 1,192 + 1476 S. Johannesburg R 0,595 +
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Table 1 – Inclusivity: Salmonella strains (continued)
Ref. Serotype Group OD Result Ref. Serotype Group OD Result
715 S. Kapemba D1 2,097 + 1241 S. Poona G1 2,075 +
1111 S. Kedougou G2 0,841 + 838 S. Quentin D2 1,733 +
1613 S. Kedougou G2 1,441 + 311 S. Ramatgan N 0,668 +
305 S. Kentucky C3 2,034 + 919 S. Reading B 2,154 +
1617 S. Kentucky C3 1,574 + 1238 S. Rissen C1 1,478 +
1477 S. Koketime V 0,535 + 1412 S. Saint-Paul B 1,299 +
1471 S. Kottbus C2 1,893 + 1415 S. Saint-Paul B 1,313 +
736 S. Lagos B 1,645 + 1698 S. Salford I 0,412 +
296 S. Lille C1 2,006 + 1479 S. Schleissheim B 1,651 +
1645 S. Livingstone C1 0,626 + 900 S. Schwarzengrund B 1,670 +
859 S. London E1 0,968 + 1697 S. Senegal F 0,582 +
1560 S. Manhattan C3 1,417 + 888 S. Senftenberg E4 0,735 +
1618 S. Mbandaka C1 1,557 + 1406 S. Senftenberg E4 1,046 +
1746 S. Mbandaka C1 1,049 + 1486 S. Senfentberg E4 1,421 +
1261 S. Meleagridis E1 0,427 + 904 S. Stanleyville B 1,645 +
1612 S. Mikawasima C1 1,604 + 702 S. Stuivenberg E4 0,525 +
303 S. Minnesota L 0,913 + 1276 S. Tennesse C1 1,553 +
1213 S. Montevideo C1 1,461 + 306 S. Thompson C1 2,117 +
1449 S. Montevideo C1 1,219 + 763 S. Tripoli B 1,023 +
1328 S. Muenchen C2 1,722 + 730 S. Typhi D1 2,383 +
1210 S. Newport C2 1,926 + 293 S. Typhimurium B 1,698 +
1442 S. Newport C2 >3 + 924 S. Typhimurium B 1,600 +
783 S. Nima M 0,484 + 1024 S. Typhimurium B 1,189 +
1614 S. Norton C1 2,337 + 1654 S. Typhimurium B 1,448 +
1207 S. Ohio C1 1,744 + 784 S. Urbana N 0,104 -
1188 S. Oranienburg C1 1,259 + 780 S. Veneziana F 1,420 +
1701 S. Orion E1 0,557 + 1194 S. Virchow C1 2,033 +
837 S. Ouakam D2 1,542 + 1451 S. Virchow C1 1,548 +
1159 S. Panama D1 1,790 + 1468 S. Virginia C3 1,411 +
731 S. paratyphi A A 2,015 + 681 S. Wien B 1,732 +
732 S. paratyphi B B 1,318 + 295 S. Zanzibar E1 2,123 +
1002 S. paratyphi B B 1,287 + 786 Sal. I 47 : z4, z23 : - X 0,037 -
781 S. Poona G1 2,533 +
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Table 2 – Exclusivity: Non-Salmonella strains
Ref. Bacteria species OD Result Ref. Bacteria species OD Result 567 Aeromonas hydrophila 0,064 - 1183 E. coli O111 0,107 - 641 Bac. Cereus 0,077 - 1184 E. coli O126 0,083 - 664 Bac. Cereus 0,085 - 1404 E. coli O157 0,081 - 383 Bac. sphaericus 0,077 - 1536 E. coli O157 H7 0,082 -
BA16* Bac. licheniformis 0,072 - 1156 E. coli O26 H11 0,081 - LE3* Candida albicans 0,069 - ESC15* E. Hermanii 0,053 - 735 Cit. amalonaticus 0,077 - 1391 Hafnia alvei 0,083 -
1409 Cit. braakii 0,081 - 1414 Hafnia alvei 0,082 - 1677 Cit. braakii 0,039 - 451 Klebsiella oxytoca 0,083 - 433 Cit.braakii 0,043 - 1079 Klebsiella pneumoniae 0,080 - 433 Cit.braakii 0,057 - 1162 Klebsiella pneumoniae 0,084 - 795 Cit.braakii 0,041 - 1507 List. monocytogenes 0,085 - 933 Cit. diversus 0,078 - 926 Proteus mirabilis 0,079 -
1163 Cit. diversus 0,077 - 436 Proteus morganii 0,062 - 1082 Cit. freundii 0,038 - 917 Proteus vulgaris 0,066 - 1082 Cit. freundii 0,055 - 133 Pseudo. aeruginosa 0,072 - 1085 Cit. freundii 0,039 - 522 Pseudo. fluorescens 0,076 - 1289 Cit. freundii 0,080 - LE6* Rhodotorula rubra 0,096 - 1348 Cit. freundii 0,102 - LE5* Saccharomyces cerevisiae 0,079 - 1438 Cit. freundii 0,080 - 927 Serratia liquefaciens 0,067 - 1043 Cit. freundii 0,079 - 429 Serratia marcescens 0,078 - 1694 Cit. freundii 0,086 - 328 Shigella flexneri 0,082 - 1660 Cit. youngae 0,046 - SHI16* Shigella sonnei 0,085 - 1703 Cit. youngae 0,047 - 572 Staph. aureus 0,059 - 815 Ent. agglomerans 0,068 - 574 Staph. aureus 0,076 - 764 Ent. Amnigenus 0,071 - 1069 Staph. aureus 0,080 - 937 Ent. cancerogenus 0,079 - 1132 Staph. aureus A + 0,077 - 769 Ent. cloacae 0,084 - 1138 Staph. aureus A + 0,084 - 427 Ent. cloacae 0,065 - 637 Staph. epidermis 0,079 -
1699 Ent. cloacae 0,083 - 588 Staph. haemoliticus 0,067 - 1078 Ent. intermedium 0,078 - 852 Staph. hominis 0,064 - 1693 Ent. nimipressuralis 0,068 - ST12* Staph. hyicus 0,080 - 839 Ent. sakazakii 0,081 - ST6* Staph. Saprophyticus 0,057 - 17* Erwinia spp. 0,074 - 570 Yersinia enterocolitica 0,080 - 765 Escherichia adecarboxylata 0,068 - 428 Yersinia enterocolitica 0,079 -
1702 E. coli 0,048 - 430 Yersinia pseudotuberculosis 0,071 -
* reference from Pasteur Institute Lille (SERMHA)
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Table 3 - Inclusivity/Exclusivity Study Results
Strains TPSG results Total
Positive Negative
Salmonella 124 5 129
Non-Salmonella 0 72 72
Total 124 77 201 Inclusivity = 124 / 129 = 96.1 % Exclusivity = 72 / 72 = 100.0 %
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Table 4 : Summary results comparing the detection of Salmonella by the TPSG and ISO methods Positive with
TP Salmonella Gold Sensivityb, % False negativec, %
Specificityd, %
False positivee, % Test sample Level MPN
/ 25 g
Total No. of test
portions Presumptive Confirmed
Positive with ISO 6579
χ²a TPSG ISO
6579 TPSG ISO 6579 TPSG TPSG
Agreement %
Low 6 20 16 16 16 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Cooked chicken Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 7 20 19 19 19 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Cantaloupe Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 4 20 18 18 18 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Sausages Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 6 20 15 15 15 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Raw shrimps Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 14 14 14 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Yogurt Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 6 6 6 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Mayonnaise Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 13 13 13 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Shell eggs Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 4 20 13 13 13 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Frozen berries Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 17 17 17 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Bean sprouts Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 2 20 17 17 17 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Raw ground beef Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 2 20 14 14 14 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Smoked fish Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 19 19 19 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Uncooked noodles Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 2 20 17 17 17 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Milk chocolate Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 10 20 14 14 14 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Black pepper Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 6 20 19 19 19 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Cake mix Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 10 10 10 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Infant formula Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 2 20 11 11 11 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Dry pet food Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 6 6 5 0 75.0 62.5 25.0 37.5 80.0 20.0 75.0 Raw milk Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 6 20 17 17 17 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Raw ground turkeyg Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 2 20 12 12 12 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Brie cheeseg Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0
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a χ² ,as defined by McNemar is (|a - b| - 1)² / (a + b) where a = test portions positive by TPSG and negative by ISO method and b = test portions negative by TPSG and positive by ISO method. A χ² value greater than 3.84 indicated significance at P < 0.05.
b Sensitivity rate was defined as 100 times the total number of analyzed positive test portions among “known” positive tests portions divided by the total number of “known” test portions, where “known” positive was defined as test portions confirmed positive by the reference method.
c Incidence of false negatives was 100 – sensitivity rate. d Specificity rate was defined as 100 times the total number of analyzed negative test portions among “known” negative test portions divided by
the total number of “known” negative test portions, where known negative was defined as test portions confirmed negative by the reference method and negative controls.
e Incidence of false positives was 100 – specificity rate. f Statistical analysis was not applicable, methods gave equivalent results. g Matrix included in the independent validation studies.
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Table 5 – Lot-to-lot consistency study
Release date 10 MAY 04 06 APR 04 26 NOV 03
Lot 21A 12A 58Z
Period of testing Release (newly manufactured) Release + 40 days Release + 9 months
Test parameters
NC1 : 0.050 NC2 : 0.051 PC : 2.820
Negative threshold : 0.144 Positive threshold : 0.160
NC1 : 0.040 NC2 : 0.042 PC : 2.148
Negative threshold : 0.136 Positive threshold : 0.151
NC1 : 0.052 NC2 : 0.051 PC : 2.181
Negative threshold : 0.145 Positive threshold : 0.161
Salmonella Heidelberg ( strain 1431) 1x105 cells/mL 0.132 (-) 0.138 (-) 0.087 (-) 0.095 (-) 0.080 (-) 0.084 (-) 5x105 cells/mL 0.320 (+) 0.322 (+) 0.234 (+) 0.227 (+) 0.203 (+) 0.211 (+) 1x106 cells/mL 0.536 (+) 0.523 (+) 0.400 (+) 0.402 (+) 0.426 (+) 0.416 (+) Salmonella Enteritidis ( strain 1435) 1x105 cells/mL 0.161 (+) 0.165 (+) 0.156 (+) 0.165 (+) 0.163 (+) 0.174 (+) 5x105 cells/mL 0.480 (+) 0.461 (+) 0.391 (+) 0.397 (+) 0.387 (+) 0.391 (+) 1x106 cells/mL 0.827 (+) 0.837 (+) 0.617 (+) 0.601 (+) 0.573 (+) 0.569 (+) Enterobacter nimipressuralis ( strain 1693) 109 cells/mL 0.057 (-) 0.061 (-) 0.044 (-) 0.042 (-) 0.052 (-) 0.058 (-) Citrobacter freundii ( strain 1694) 109 cells/mL 0.064 (-) 0.052 (-) 0.041 (-) 0.053 (-) 0.061 (-) 0.053 (-) Escherichia coli (strain 1771) 109 cells/mL 0.059 (-) 0.061 (-) 0.048 (-) 0.050 (-) 0.056 (-) 0.058 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation
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Table 6 – Stability
Lot 26W – Release date : 26 JUL 2004
Period of testing Release (newly manufactured)
Release + 14 days at 5 ± 3oC
Release + 14 days at 37 ± 2oC
Test parameters
NC1 : 0.056 NC2 : 0.055 PC : 2.426
Negative threshold : 0.149 Positive threshold : 0.165
NC1 : 0.046 NC2 : 0.049 PC : 2.179
Negative threshold : 0.141 Positive threshold : 0.157
NC1 : 0.079 NC2 : 0.081 PC : 1.279
Negative threshold : 0.171 Positive threshold : 0.190
Salmonella Infantis ( strain 1305) 1x106 cells/mL 0.219 (+) 0.224 (+) 0.178 (+) 0.178 (+) 0.205 (+) 0.212 (+) 2.5x106 cells/mL 0.356 (+) 0.366 (+) 0.295 (+) 0.289 (+) 0.355 (+) 0.347 (+) 5x106 cells/mL 0.496 (+) 0.502 (+) 0.468 (+) 0.472 (+) 0.560 (+) 0.595 (+) Salmonella Enteritidis ( strain 1435) 1x105 cells/mL 0.220 (+) 0.211 (+) 0.193 (+) 0.194 (+) 0.200 (+) 0.197 (+) 5x105 cells/mL 0.679 (+) 0.664 (+) 0.605 (+) 0.603 (+) 0.597 (+) 0.607 (+) 1x106 cells/mL 1.048 (+) 1.109 (+) 0.937 (+) 0.926 (+) 0.963 (+) 0.935 (+) Citrobacter diversus ( strain 1163) 109 cells/mL 0.071 (-) 0.075 (-) 0.051 (-) 0.052 (-) 0.090 (-) 0.093 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation
Lot 26W – Release date : 26 JUL 2004
Period of testing Release (newly manufactured)
Release + 31 days at 5 ± 3oC
Release + 31 days at 37 ± 2oC
Test parameters
NC1 : 0.056 NC2 : 0.055 PC : 2.426
Negative threshold : 0.149 Positive threshold : 0.165
NC1 : 0.052 NC2 : 0.055 PC : 2.571
Negative threshold : 0.147 Positive threshold : 0.163
NC1 : 0.089 NC2 : 0.085 PC : 1.080
Negative threshold : 0.177 Positive threshold : 0.197
Salmonella Infantis ( strain 1305) 1x106 cells/mL 0.219 (+) 0.224 (+) 0.177 (+) 0.186 (+) 0.210 (+) 0.204 (+) 2.5x106 cells/mL 0.356 (+) 0.366 (+) 0.296 (+) 0.309 (+) 0.326 (+) 0.326 (+) 5x106 cells/mL 0.496 (+) 0.502 (+) 0.457 (+) 0.463 (+) 0.424 (+) 0.438 (+) Salmonella Enteritidis ( strain 1435) 1x105 cells/mL 0.220 (+) 0.211 (+) 0.187 (+) 0.183 (+) 0.204 (+) 0.209 (+) 5x105 cells/mL 0.679 (+) 0.664 (+) 0.553 (+) 0.555 (+) 0.527 (+) 0.523 (+) 1x106 cells/mL 1.048 (+) 1.109 (+) 0.884 (+) 0.906 (+) 0.885 (+) 0.866 (+) Citrobacter diversus ( strain 1163) 109 cells/mL 0.071 (-) 0.075 (-) 0.059 (-) 0.060 (-) 0.097 (-) 0.097 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation
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Table 7 - Ruggedness Study - Variation of the heat treatment time
Lot 2A
Heat treatment time 15 minutes 20 minutes 25 minutes
Test parameters
NC1 : 0.077 NC2 : 0.077 PC : 1.968
NEG threshold: 0.168 POS threshold: 0.187
NC1 : 0.082 NC2 : 0.085
PC : 2.19 NEG threshold: 0.174 POS threshold: 0.194
NC1 : 0.083 NC2 : 0.083 PC : 2.207
NEG threshold: 0.174 POS threshold: 0.193
S. Typhimurium ( strain 1654) 2.5x105 cells/mL 0.133 (-) 0.141 (-) 0.144 (-) 2.5x106 cells/mL 0.359 (+) 0.355 (+) 0.384 (+) 2.5x107 cells/mL 0.912 (+) 0.873 (+) 0.896 (+) S. Enteritidis ( strain 1834) 7.6x103 cells/mL 0.115 (-) 0.124 (-) 0.123 (-) 7.6x104 cells/mL 0.275 (+) 0.344 (+) 0.376 (+) 7.6x105 cells/mL 1.194 (+) 1.278 (+) 1.423 (+) Citrobacter freundii (strain 1694) 109 cells/mL 0.088 (-) 0.096 (-) 0.093 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation O.D. written in Italic are average values of 5 replicates
Lot 8A
Heat treatment time 15 minutes 20 minutes 25 minutes
Test parameters
NC1 : 0.077 NC2 : 0.079 PC : 2.624
NEG threshold: 0.169 POS threshold: 0.188
NC1 : 0.100 NC2 : 0.096 PC : 2.802
NEG threshold: 0.187 POS threshold: 0.208
NC1 : 0.089 NC2 : 0.083 PC : 2.661
NEG threshold: 0.176 POS threshold: 0.196
S. Typhimurium ( strain 1654) 2.5x105 cells/mL 0.132 (-) 0.127 (-) 0.128 (-) 2.5x106 cells/mL 0.378 (+) 0.347 (+) 0.383 (+) 2.5x107 cells/mL 0.898 (+) 0.865 (+) 0.884 (+) S. Enteritidis ( strain 1834) 7.6x103 cells/mL 0.103 (-) 0.106 (-) 0.110 (-) 7.6x104 cells/mL 0.231 (+) 0.234 (+) 0.233 (+) 7.6x105 cells/mL 0.798 (+) 0.770 (+) 0.870 (+) Citrobacter freundii (strain 1694) 109 cells/mL 0.089 (-) 0.107 (-) 0.098 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation O.D. written in Italic are average values of 5 replicates
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Lot 12A
Heat treatment time 15 minutes 20 minutes 25 minutes
Test parameters
NC1 : 0.113 NC2 : 0.103 PC : 3.181
NEG threshold: 0.196 POS threshold: 0.218
NC1 : 0.092 NC2 : 0.091 PC : 2.870
NEG threshold: 0.181 POS threshold: 0.202
NC1 : 0.096 NC2 : 0.100 PC : 2.897
NEG threshold: 0.187 POS threshold: 0.208
S. Typhimurium ( strain 1654) 2.5x105 cells/mL 0.134 (-) 0.145 (-) 0.146 (-) 2.5x106 cells/mL 0.398 (+) 0.407 (+) 0.437 (+) 2.5x107 cells/mL 1.027 (+) 1.027 (+) 1.060 (+) S. Enteritidis ( strain 1834) 7.6x103 cells/mL 0.133 (-) 0.110 (-) 0.112 (-) 7.6x104 cells/mL 0.253 (+) 0.227 (+) 0.217 (+) 7.6x105 cells/mL 0.770 (+) 0.621 (+) 0.665 (+) Citrobacter freundii (strain 1694) 109 cells/mL 0.121 (-) 0.113 (-) 0.107 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation O.D. written in Italic are average values of 5 replicates
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Table 8 - Ruggedness Study - Variation of the sample volume
Lot 2A
Sample volume 80 µL 100 µL 120 µL
Test parameters
NC1 : 0.085 NC2 : 0.119 PC : 2.395
NEG threshold: 0.191 POS threshold: 0.212
NC1 : 0.086 NC2 : 0.084 PC : 2.463
NEG threshold: 0.176 POS threshold: 0.195
NC1 : 0.080 NC2 : 0.081 PC : 2.481
NEG threshold: 0.171 POS threshold: 0.191
S. Typhimurium ( strain 1654) 3.9x105 cells/mL 0.139 (-) 0.135 (-) 0.136 (-) 3.9x106 cells/mL 0.404 (+) 0.442 (+) 0.424 (+) 3.9x107 cells/mL 1.105 (+) 1.110 (+) 1.100 (+) S. Enteritidis ( strain 1834) 9.2x103 cells/mL 0.121 (-) 0.117 (-) 0.116 (-) 9.2x104 cells/mL 0.254 (+) 0.244 (+) 0.249 (+) 9.2x105 cells/mL 1.123 (+) 1.091 (+) 1.099 (+) Citrobacter freundii (strain 1694) 109 cells/mL 0.101 (-) 0.093 (-) 0.094 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation O.D. written in Italic are average values of 5 replicates
Lot 8A
Sample volume 80 µL 100 µL 120 µL
Test parameters
NC1 : 0.097 NC2 : 0.117 PC : 3.063
NEG threshold: 0.195 POS threshold: 0.217
NC1 : 0.091 NC2 : 0.091 PC : 3.099
NEG threshold: 0.181 POS threshold: 0.201
NC1 : 0.090 NC2 : 0.116 PC : 2.874
NEG threshold: 0.192 POS threshold: 0.213
S. Typhimurium ( strain 1654) 3.9x105 cells/mL 0.137 (-) 0.143 (-) 0.147 (-) 3.9x106 cells/mL 0.465 (+) 0.439 (+) 0.477 (+) 3.9x107 cells/mL 1.204 (+) 1.254 (+) 1.240 (+) S. Enteritidis ( strain 1834) 9.2x103 cells/mL 0.114 (-) 0.126 (-) 0.138 (-) 9.2x104 cells/mL 0.254 (+) 0.282 (+) 0.263 (+) 9.2x105 cells/mL 1.428 (+) 1.360 (+) 1.258 (+) Citrobacter freundii (strain 1694) 109 cells/mL 0.097 (-) 0.105 (-) 0.110 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation O.D. written in Italic are average values of 5 replicates
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Lot 12A
Sample volume 80 µL 100 µL 120 µL
Test parameters
NC1 : 0.101 NC2 : 0.114 PC : 2.539
NEG threshold: 0.196 POS threshold: 0.218
NC1 : 0.089 NC2 : 0.103 PC : 3.079
NEG threshold: 0.185 POS threshold: 0.206
NC1 : 0.084 NC2 : 0.083 PC : 2.342
NEG threshold: 0.174 POS threshold: 0.194
S. Typhimurium ( strain 1654) 3.9x105 cells/mL 0.130 (-) 0.130 (-) 0.138 (-) 3.9x106 cells/mL 0.404 (+) 0.417 (+) 0.437 (+) 3.9x107 cells/mL 1.229 (+) 1.241 (+) 1.258 (+) S. Enteritidis ( strain 1834) 9.2x103 cells/mL 0.135 (-) 0.129 (-) 0.128 (-) 9.2x104 cells/mL 0.330 (+) 0.281 (+) 0.346 (+) 9.2x105 cells/mL 1.553 (+) 1.431 (+) 1.420 (+) Citrobacter freundii (strain 1694) 109 cells/mL 0.127 (-) 0.102 (-) 0.113 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation O.D. written in Italic are average values of 5 replicates
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Table 9 - Ruggedness Study - Variation of the conjugate volume
Lot 2A
Conjugate volume 80 µL 100 µL 120 µL
Test parameters
NC1 : 0.076 NC2 : 0.083 PC : 2.165
NEG threshold: 0.171 POS threshold: 0.190
NC1 : 0.079 NC2 : 0.080 PC : 2.133
NEG threshold: 0.171 POS threshold: 0.190
NC1 : 0.081 NC2 : 0.085 PC : 2.306
NEG threshold: 0.174 POS threshold: 0.193
S. Typhimurium ( strain 1654) 3.9x105 cells/mL 0.141 (-) 0.148 (-) 0.150 (-) 3.9x106 cells/mL 0.530 (+) 0.542 (+) 0.500 (+) 3.9x107 cells/mL 1.406 (+) 1.494 (+) 1.514 (+) S. Enteritidis ( strain 1834) 9.2x103 cells/mL 0.109 (-) 0.104 (-) 0.109 (-) 9.2x104 cells/mL 0.199 (+) 0.209 (+) 0.227 (+) 9.2x105 cells/mL 0.961 (+) 1.021 (+) 1.126 (+) Citrobacter freundii (strain 1694) 109 cells/mL 0.075 (-) 0.081 (-) 0.088 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation O.D. written in Italic are average values of 5 replicates
Lot 8A
Conjugate volume 80 µL 100 µL 120 µL
Test parameters
NC1 : 0.077 NC2 : 0.075 PC : 2.190
NEG threshold: 0.167 POS threshold: 0.186
NC1 : 0.076 NC2 : 0.077 PC : 2.588
NEG threshold: 0.168 POS threshold: 0.187
NC1 : 0.087 NC2 : 0.138 PC : 2.550
NEG threshold: 0.200 POS threshold: 0.223
S. Typhimurium ( strain 1654) 3.9x105 cells/mL 0.151 (-) 0.161 (-) 0.174 (-) 3.9x106 cells/mL 0.546 (+) 0.581 (+) 0.567 (+) 3.9x107 cells/mL 1.459 (+) 1.497 (+) 1.468 (+) S. Enteritidis ( strain 1834) 9.2x103 cells/mL 0.101 (-) 0.105 (-) 0.116 (-) 9.2x104 cells/mL 0.220 (+) 0.227 (+) 0.256 (+) 9.2x105 cells/mL 1.090 (+) 1.140 (+) 1.176 (+) Citrobacter freundii (strain 1694) 109 cells/mL 0.081 (-) 0.085 (-) 0.103 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation O.D. written in Italic are average values of 5 replicates
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Lot 12A
Conjugate volume 80 µL 100 µL 120 µL
Test parameters
NC1 : 0.083 NC2 : 0.104 PC : 2.506
NEG threshold: 0.183 POS threshold: 0.204
NC1 : 0.078 NC2 : 0.079 PC : 2.660
NEG threshold: 0.170 POS threshold: 0.189
NC1 : 0.092 NC2 : 0.090 PC : 2.602
NEG threshold: 0.181 POS threshold: 0.201
S. Typhimurium ( strain 1654) 3.9x105 cells/mL 0.151 (-) 0.157 (-) 0.160 (-) 3.9x106 cells/mL 0.505 (+) 0.526 (+) 0.496 (+) 3.9x107 cells/mL 1.426 (+) 1.444 (+) 1.453 (+) S. Enteritidis ( strain 1834) 9.2x103 cells/mL 0.143 (-) 0.131 (-) 0.159 (-) 9.2x104 cells/mL 0.222 (+) 0.214 (+) 0.229 (+) 9.2x105 cells/mL 0.915 (+) 0.964 (+) 0.913 (+) Citrobacter freundii (strain 1694) 109 cells/mL 0.087 (-) 0.087 (-) 0.098 (-)
NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation O.D. written in Italic are average values of 5 replicates
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Appendix 1: Flow diagram showing the ISO 6579:2002 method and the Transia Plate Salmonella Gold test for the detection of Salmonella spp.
Salmonella cultured in M broth or Nutrient broth incubated overnight at 37°C
(or contamination by a freeze-dried bacterial culture)
Food matrix inoculation at 1-10 cfu/25g
Storage of food portions at 2-8°C or 18-25°C for 72h
MPN analysis : Weigh of 3 x 100g, 3 x 10g, 3 x 1g and 3 x 0.1g from the inoculated and uninoculated food portions; pre-enrichment in BPW at 37 ± 1°C for 16-20h.
Samples : Weigh of 5 x 25g uninoculated samples and 20 x 25g inoculated samples; pre-enrichment with 225ml of BPW at 37 ± 1°C for 16-20 h.
0.1ml BPW into 10ml RVS. Incubation at 41.5 ± 1 °C for 18-24 h
1ml BPW into 10ml MKTTn. Incubation at 37 ± 1 °C for 21-27 h.
0.1ml BPW into 10ml RVS. Incubation at 41.5 ± 0.5 °C for 18-24 h .
1ml BPW into 10ml MKTTn. Incubation at 37 ± 1°C for 21-27 h.
Streaking onto XLD and BGA plates. Incubation at 37 ± 1 °C for 21-27 h
Heat-treatment of 1 or 2ml RVS at 100°C for 20 minutes in a boiling water. Cool to room temperature
Transia Plate Salmonella Gold Confirmation of presumptive positive colonies by biochemical (API 20E or Microbact 24E) and serological tests (poly O and poly H), on upto 5 colonies per sample
ISO method TPSG
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Appendix 2: Comparison of TPSG and ISO results from uninoculated and inoculated cooked chicken.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
8 0,129 - NT - NT 15 0,113 - NT - NT 27 0,120 - NT - NT 32
0 cfu/25g 0,126 - NT - NT
36 0,124 - NT - NT Total 0/5 0/5 0/5
6 0,783 + + + + 7 1,031 + + + +
12 0,393 + + + + 13 0,997 + + + + 14 0,774 + + + + 16 1,230 + + + + 18 0,854 + + + + 20 0,805 + + + + 21 0,758 + + + + 22 0,841 + + + + 23 0,507 + + + + 25 0,117 - NT - NT 26 1,135 + + + + 28 0,122 - NT - NT 31 0,718 + + + + 35 0,828 + + + + 39 0,803 + + + + 40 0,839 + + + + 41
6 cfu/25g
Salmonella Kentucky
0,117 - NT - NT 42 0,115 - NT - NT
Total 16/20 16/20 16/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 2.915 Negative control 1 = 0.085 Negative control 2 = 0.093 Positive threshold = (0.085 +0.093)/2 + 0.11 = 0.199 Negative threshold = 0.199 x 0.9 = 0.180 OD value for a positive sample ≥ 0.199 OD value for a negative sample < 0.180
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Appendix 3: Comparison of TPSG and ISO results from uninoculated and inoculated cantaloupe.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation
Presumptive positive colonies
Confirmation
209 0,116 - NT - NT 217 0,111 - NT - NT 218 0,097 - NT - NT 236
0 cfu/25g 0,104 - NT - NT
246 0,100 - NT - NT Total 0/5 0/5 0/5
206 0,398 + + + + 207 0,449 + + + + 212 0,490 + + + + 215 0,435 + + + + 219 0,471 + + + + 221 0,400 + + + + 222 0,416 + + + + 224 0,440 + + + + 226 0,495 + + + + 230 0,421 + + + + 231 0,461 + + + + 232 0,495 + + + + 234 0,538 + + + + 238 0,413 + + + + 242 0,395 + + + + 243 0,458 + + + + 244 0,396 + + + + 247 0,106 - NT - NT 248
7 cfu/25g
Salmonella Poona
0,403 + + + + 250 0,425 + + + +
Total 19/20 19/20 19/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 2.856 Negative control 1 = 0.086 Negative control 2 = 0.096 Positive threshold = (0.086 +0.096)/2 + 0.11 = 0.201 Negative threshold = 0.201 x 0.9 = 0.181 OD value for a positive sample ≥ 0.201 OD value for a negative sample < 0.181
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Appendix 4: Comparison of TPSG and ISO results from uninoculated and inoculated sausages.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
310 0,093 - NT - NT 316 0,103 - NT - NT 323 0,117 - NT d - 332
0 cfu/25g 0,099 - NT - NT
341 0,100 - NT - NT Total 0/5 0/5 0/5
306 1,287 + + + + 309 1,436 + + + + 312 0,111 - NT - NT 313 1,518 + + + + 315 1,603 + + + + 317 1,297 + + + + 320 1,184 + + + + 324 1,527 + + + + 325 1,496 + + + + 328 1,708 + + + + 329 1,632 + + + + 331 1,854 + + + + 334 0,095 - NT - NT 337 1,685 + + + + 340 1,662 + + + + 343 1,643 + + + + 346 1,567 + + + + 347 1,711 + + + + 349
4 cfu/25g
Salmonella Bredeney
1,799 + + + + 350 1,687 + + + +
Total 18/20 18/20 18/20
NT = not tested + = positive - = negative d = doubtful presumptive positive colonies Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.079 Negative control 1 = 0.083 Negative control 2 = 0.088 Positive threshold = (0.083 +0.088)/2 + 0.11 = 0.196 Negative threshold = 0.1955 x 0.9 = 0.176 OD value for a positive sample ≥ 0.196 OD value for a negative sample < 0.176
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Appendix 5: Comparison of TPSG and ISO results from uninoculated and inoculated raw shrimps.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
409 0,109 - NT - NT 419 0,108 - NT - NT 428 0,111 - NT - NT 439
0 cfu/25g 0,098 - NT - NT
446 0,097 - NT - NT Total 0/5 0/5 0/5
406 2,085 + + + + 407 0,104 - NT - NT 410 2,169 + + + + 411 0,109 - NT - NT 413 0,113 - NT - NT 415 0,113 - NT - NT 418 2,475 + + + + 421 2,310 + + + + 422 2,318 + + + + 423 2,084 + + + + 427 2,041 + + + + 430 2,178 + + + + 432 0,097 - NT - NT 435 1,853 + + + + 437 1,906 + + + + 441 1,828 + + + + 442 1,620 + + + + 445 2,250 + + + + 449
6 cfu/25g
Salmonella Montevideo
2,272 + + + + 450 2,259 + + + +
Total 15/20 15/20 15/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.269 Negative control 1 = 0.096 Negative control 2 = 0.089 Positive threshold = (0.096 +0.089)/2 + 0.11 = 0.203 Negative threshold = 0.203 x 0.9 = 0.182 OD value for a positive sample ≥ 0.203 OD value for a negative sample < 0.182
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Appendix 6: Comparison of TPSG and ISO results from uninoculated and inoculated yogurt.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
508 0,097 - NT - NT 522 0,104 - NT - NT 532 0,094 - NT - NT 541
0 cfu/25g 0,095 - NT - NT
542 0,100 - NT - NT Total 0/5 0/5 0/5
506 1,357 + + + + 507 0,099 - NT - NT 510 1,334 + + + + 513 0,104 - NT - NT 515 1,414 + + + + 517 1,398 + + + + 520 1,386 + + + + 523 1,364 + + + + 527 1,414 + + + + 528 0,093 - NT - NT 531 0,098 - NT - NT 533 0,090 - NT - NT 535 1,381 + + + + 538 1,424 + + + + 539 1,467 + + + + 540 0,095 - NT - NT 543 1,345 + + + + 546 1,344 + + + + 548
1 cfu/25g
Salmonella Typhimurium
1,265 + + + + 550 1,296 + + + +
Total 14/20 14/20 14/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 2.788 Negative control 1 = 0.084 Negative control 2 = 0.085 Positive threshold = (0.084 +0.085)/2 + 0.11 = 0.195 Negative threshold = 0.195 x 0.9 = 0.175 OD value for a positive sample ≥ 0.195 OD value for a negative sample < 0.175
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Appendix 7: Comparison of TPSG and ISO results from uninoculated and inoculated mayonnaise.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confimation Presumptive positive colonies
Confirmation
1811 0,120 - NT - NT 1817 0,130 - NT - NT 1823 0,129 - NT - NT 1828
0 cfu/25g 0,128 - NT - NT
1835 0,119 - NT - NT Total 0/5 0/5 0/5
1806 0,117 - NT - NT 1807 0,130 - NT - NT 1812 0,119 - NT - NT 1813 1,824 + + + + 1814 0,143 - NT - NT 1818 1,724 + + + + 1819 0,129 - NT - NT 1822 0,125 - NT - NT 1825 0,136 - NT - NT 1827 0,122 - NT - NT 1830 0,148 - NT - NT 1832 1,809 + + + + 1833 0,128 - NT - NT 1836 0,124 - NT - NT 1839 0,131 - NT - NT 1840 0,144 - NT - NT 1843 1,599 + + + + 1845 1,838 + + + + 1847
1 cfu/25g
Salmonella Heidelberg
1,833 + + + + 1849 0,142 - NT - NT
Total 6/20 6/20 6/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 2.785 Negative control 1 = 0.097 Negative control 2 = 0.103 Positive threshold = (0.097 +0.103)/2 + 0.11 = 0.210 Negative threshold = 0.210 x 0.9 = 0.189 OD value for a positive sample ≥ 0.210 OD value for a negative sample < 0.189
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Appendix 8: Comparison of TPSG and ISO results from uninoculated and inoculated shell eggs.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
709 0,114 - NT - NT 710 0,137 - NT - NT 726 0,118 - NT - NT 733
0 cfu/25g 0,125 - NT - NT
749 0,100 - NT - NT Total 0/5 0/5 0/5
706 2,591 + + + + 707 1,719 + + + + 708 0,105 - NT - NT 711 2,523 + + + + 714 0,108 - NT - NT 715 2,862 + + + + 719 2,249 + + + + 720 0,106 - NT - NT 721 2,299 + + + + 723 2,037 + + + + 728 2,918 + + + + 729 2,995 + + + + 730 0,112 - NT - NT 731 2,930 + + + + 735 2,034 + + + + 736 0,109 - NT - NT 740 2,565 + + + + 742 2,662 + + + + 746
1 cfu/25g
Salmonella Enteritidis
0,107 - NT d - 747 0,110 - NT - NT
Total 13/20 13/20 13/20
NT = not tested + = positive - = negative d = doubtful presumptive positive colonies Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.203 Negative control 1 = 0.084 Negative control 2 = 0.083 Positive threshold = (0.084 +0.083)/2 + 0.11 = 0.194 Negative threshold = 0.210 x 0.9 = 0.174 OD value for a positive sample ≥ 0.194 OD value for a negative sample < 0.174
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Appendix 9: Comparison of TPSG and ISO results from uninoculated and inoculated frozen berries.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
2909 0,106 - NT - NT 2918 0,098 - NT - NT 2926 0,103 - NT - NT 2942
0 cfu/25g 0,113 - NT - NT
2959 0,112 - NT - NT Total 0/5 0/5 0/5
2906 1,065 + + + + 2910 0,101 - NT - NT 2912 1,151 + + + + 2913 0,101 - NT - NT 2919 1,086 + + + + 2920 0,113 - NT - NT 2924 1,175 + + + + 2927 1,095 + + + + 2929 1,134 + + + + 2930 0,105 - NT - NT 2934 0,108 - NT - NT 2935 0,106 - NT - NT 2938 1,136 + + + + 2939 1,166 + + + + 2940 1,127 + + + + 2947 1,282 + + + + 2948 1,118 + + + + 2951 1,142 + + + + 2952
4 cfu/25g
Salmonella Newport
0,114 - NT - NT 2958 1,207 + + + +
Total 13/20 13/20 13/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 2.552 Negative control 1 = 0.094 Negative control 2 = 0.096 Positive threshold = (0.094 +0.096)/2 + 0.11 = 0.205 Negative threshold = 0.205 x 0.9 = 0.185 OD value for a positive sample ≥ 0.205 OD value for a negative sample < 0.185
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Appendix 10: Comparison of TPSG and ISO results from uninoculated and inoculated bean sprouts.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
908 0,098 - NT - NT 915 0,098 - NT - NT 916 0,115 - NT - NT 929
0 cfu/25g
0,104 - NT - NT 942 0,116 - NT - NT
Total 0/5 0/5 0/5 906 2,030 + + + + 907 0,098 - NT - NT 909 1,671 + + + + 910 2,306 + + + + 911 2,282 + + + + 921 2,232 + + + + 922 1,627 + + + + 923 0,100 - NT d - 926 2,264 + + + + 928 0,117 - NT - NT 932 2,534 + + + + 933 2,402 + + + + 934 2,629 + + + + 935 2,308 + + + + 936 1,809 + + + + 943 1,456 + + + + 945 2,268 + + + + 947 2,375 + + + + 948
1 cfu/25g
Salmonella Infantis
2,417 + + + + 949 2,137 + + + +
Total 17/20 17/20 17/20
NT = not tested + = positive - = negative d = doubtful presumptive positive colonies Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.149 Negative control 1 = 0.098 Negative control 2 = 0.113 Positive threshold = (0.098 +0.113)/2 + 0.11 = 0.216 Negative threshold = 0.216 x 0.9 = 0.194 OD value for a positive sample ≥ 0.216 OD value for a negative sample < 0.194
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Appendix 11: Comparison of TPSG and ISO results from uninoculated and inoculated raw ground beef.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
1009 0,118 - NT - NT 1020 0,106 - NT d - 1021 0,105 - NT - NT 1034
0 cfu/25g
0,107 - NT - NT 1041 0,105 - NT - NT
Total 0/5 0/5 0/5 1006 0,105 - NT - NT 1011 2,091 + + + + 1012 1,900 + + + + 1014 2,005 + + + + 1015 1,926 + + + + 1017 1,919 + + + + 1022 2,073 + + + + 1026 0,110 - NT d - 1027 1,599 + + + + 1028 1,885 + + + + 1029 2,079 + + + + 1030 2,075 + + + + 1032 2,137 + + + + 1035 0,098 - NT d - 1036 1,955 + + + + 1037 2,012 + + + + 1039 2,020 + + + + 1042 2,012 + + + + 1048
2 cfu/25g
Salmonella Anatum
2,045 + + + + 1050 2,104 + + + +
Total 17/20 17/20 17/20
NT = not tested + = positive - = negative d = doubtful presumptive positive colonies Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 2.847 Negative control 1 = 0.088 Negative control 2 = 0.095 Positive threshold = (0.088 +0.095)/2 + 0.11 = 0.202 Negative threshold = 0.202 x 0.9 = 0.181 OD value for a positive sample ≥ 0.202 OD value for a negative sample < 0.181
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Appendix 12: Comparison of TPSG and ISO results from uninoculated and inoculated smoked fish.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
1111 0,115 - NT d - 1122 0,112 - - d - 1127 0,120 - NT - NT 1133
0 cfu/25g
0,125 - NT - NT 1147 0,142 - NT - NT
Total 0/5 0/5 0/5 1106 0,109 - NT - NT 1107 2,058 + + + + 1112 0,111 - NT - NT 1114 2,127 + + + + 1117 2,158 + + + + 1123 2,522 + + + + 1124 2,091 + + + + 1125 2,742 + + + + 1129 0,119 - NT - NT 1130 0,114 - NT - NT 1134 2,042 + + + + 1135 1,999 + + + + 1136 0,131 - NT d - 1137 2,032 + + + + 1138 0,114 - NT d - 1139 2,211 + + + + 1140 2,472 + + + + 1148 1,843 + + + + 1149
2 cfu/25g
Salmonella Virchow
1,756 + + + + 1150 2,438 + + + +
Total 14/20 14/20 14/20
NT = not tested + = positive - = negative d = doubtful presumptive positive colonies Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.018 Negative control 1 = 0.098 Negative control 2 = 0.108 Positive threshold = (0.098 +0.108)/2 + 0.11 = 0.213 Negative threshold = 0.213 x 0.9 = 0.192 OD value for a positive sample ≥ 0.213 OD value for a negative sample < 0.192
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Appendix 13: Comparison of TPSG and ISO results from uninoculated and inoculated uncooked noodles.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
2808 0,107 - NT - NT 2811 0,123 - NT - NT 2814 0,115 - NT - NT 2815
0 cfu/25g
0,112 - NT - NT 2823 0,116 - NT - NT
Total 0/5 0/5 0/5 2806 0,962 + + + + 2807 0,907 + + + + 2809 0,793 + + + + 2810 0,859 + + + + 2812 0,886 + + + + 2813 0,546 + + + + 2816 0,830 + + + + 2817 0,774 + + + + 2818 0,799 + + + + 2819 0,925 + + + + 2820 0,812 + + + + 2821 0,790 + + + + 2822 0,785 + + + + 2824 0,976 + + + + 2825 0,702 + + + + 2826 0,903 + + + + 2827 0,119 - NT - NT 2828 0,767 + + + + 2829
1 cfu/25g
Salmonella IIIb 61:z:1.5
1,186 + + + + 2830 0,439 + + + +
Total 19/20 19/20 19/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.077 Negative control 1 = 0.145 Negative control 2 = 0.147 Positive threshold = (0.145 +0.147)/2 + 0.11 = 0.256 Negative threshold = 0.256 x 0.9 = 0.230 OD value for a positive sample ≥ 0.256 OD value for a negative sample < 0.230
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Appendix 14: Comparison of TPSG and ISO results from uninoculated and inoculated milk chocolate.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
2313 0,128 - NT - NT 2318 0,112 - NT - NT 2324 0,127 - NT - NT 2335
0 cfu/25g
0,123 - NT - NT 2341 0,112 - NT - NT
Total 0/5 0/5 0/5 2306 2,523 + + + + 2307 2,693 + + + + 2308 2,760 + + + + 2314 2,684 + + + + 2316 2,695 + + + + 2319 2,558 + + + + 2320 2,641 + + + + 2325 2,690 + + + + 2326 2,792 + + + + 2327 2,767 + + + + 2329 0,120 - NT - NT 2332 2,671 + + + + 2333 0,142 - NT - NT 2336 2,620 + + + + 2337 2,303 + + + + 2342 2,826 + + + + 2345 1,896 + + + + 2347 1,941 + + + + 2349
2 cfu/25g
Salmonella Senftenberg
0,128 - NT - NT 2350 2,016 + + + +
Total 17/20 17/20 17/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 2.477 Negative control 1 = 0.119 Negative control 2 = 0.114 Positive threshold = (0.119 +0.114)/2 + 0.11 = 0.227 Negative threshold = 0.227 x 0.9 = 0.204 OD value for a positive sample ≥ 0.227 OD value for a negative sample < 0.204
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Appendix 15: Comparison of TPSG and ISO results from uninoculated and inoculated black pepper.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
1408 0,121 - NT - NT 1418 0,121 - NT - NT 1429 0,123 - NT - NT 1443
0 cfu/25g
0,123 - NT - NT 1444 0,123 - NT - NT
Total 0/5 0/5 0/5 1406 0,117 - NT - NT 1407 0,121 - NT - NT 1410 0,129 - NT - NT 1411 2,683 + + + + 1414 2,927 + + + + 1415 0,118 - NT - NT 1421 0,116 - NT - NT 1422 2,615 + + + + 1423 2,846 + + + + 1427 2,864 + + + + 1432 2,639 + + + + 1434 3,001 + + + + 1436 2,700 + + + + 1438 2,890 + + + + 1440 3,107 + + + + 1441 3,038 + + + + 1446 2,812 + + + + 1447 2,514 + + + + 1449
10 cfu/25g
Salmonella Indiana
0,132 - NT - NT 1450 2,787 + + + +
Total 14/20 14/20 14/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.254 Negative control 1 = 0.113 Negative control 2 = 0.101 Positive threshold = (0.113 +0.101)/2 + 0.11 = 0.217 Negative threshold = 0.217 x 0.9 = 0.195 OD value for a positive sample ≥ 0.217 OD value for a negative sample < 0.195
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Appendix 16: Comparison of TPSG and ISO results from uninoculated and inoculated cake mix.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
2419 0,143 - NT - NT 2424 0,142 - NT - NT 2432 0,145 - NT - NT 2440
0 cfu/25g
0,151 - NT - NT 2443 0,158 - NT - NT
Total 0/5 0/5 0/5 2406 2,066 + + + + 2407 2,316 + + + + 2408 0,986 + + + + 2409 2,018 + + + + 2412 1,918 + + + + 2415 2,272 + + + + 2420 2,302 + + + + 2421 0,154 - NT - NT 2425 2,271 + + + + 2429 1,983 + + + + 2430 1,855 + + + + 2434 2,359 + + + + 2435 2,055 + + + + 2437 2,145 + + + + 2441 2,626 + + + + 2442 2,375 + + + + 2445 2,158 + + + + 2446 2,431 + + + + 2447
6 cfu/25g
Salmonella Thompson
2,177 + + + + 2449 2,741 + + + +
Total 19/20 19/20 19/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.364 Negative control 1 = 0.126 Negative control 2 = 0.132 Positive threshold = (0.126 +0.132)/2 + 0.11 = 0.239 Negative threshold = 0.239 x 0.9 = 0.215 OD value for a positive sample ≥ 0.239 OD value for a negative sample < 0.215
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Appendix 17: Comparison of TPSG and ISO results from uninoculated and inoculated infant formula.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
2212 0,111 - NT - NT 2216 0,119 - NT - NT 2224 0,135 - NT - NT 2227
0 cfu/25g
0,129 - NT - NT 2235 0,148 - NT - NT
Total 0/5 0/5 0/5 2207 0,837 + + + + 2208 0,963 + + + + 2209 0,939 + + + + 2220 0,960 + + + + 2221 0,801 + + + + 2222 0,909 + + + + 2223 0,124 - NT - NT 2225 0,134 - NT - NT 2226 0,126 - NT - NT 2228 0,908 + + + + 2229 0,923 + + + + 2230 0,133 - NT - NT 2238 0,971 + + + + 2239 0,142 - NT - NT 2240 0,920 + + + + 2244 0,105 - NT - NT 2246 0,129 - NT - NT 2248 0,132 - NT - NT 2250
1 cfu/25g
Salmonella Blockley
0,136 - NT - NT 2207 0,127 - NT - NT
Total 10/20 10/20 10/20
NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 2.556 Negative control 1 = 0.097 Negative control 2 = 0.094 Positive threshold = (0.097 +0.094)/2 + 0.11 = 0.206 Negative threshold = 0.206 x 0.9 = 0.185 OD value for a positive sample ≥ 0.206 OD value for a negative sample < 0.185
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Appendix 18: Comparison of TPSG and ISO results from uninoculated and inoculated dry pet food.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
2608 0,112 - NT - NT 2610 0,102 - NT - NT 2615 0,101 - NT - NT 2616
0 cfu/25g
0,096 - NT - NT 2629 0,106 - NT - NT
Total 0/5 0/5 0/5 2611 0,107 - NT - NT 2612 2,219 + + + + 2617 0,100 - NT - NT 2620 0,110 - NT - NT 2621 2,599 + + + + 2622 2,528 + + + + 2624 0,103 - NT - NT 2626 0,112 - NT - NT 2627 0,101 - NT - NT 2628 2,840 + + + + 2633 2,053 + + + + 2634 0,103 - NT d - 2635 2,426 + + + + 2636 2,252 + + + + 2638 2,272 + + + + 2640 2,471 + + + + 2642 2,439 + + + + 2644 0,115 - NT - NT 2645
2 cfu/25g
Salmonella Mbandaka
2,228 + + + + 2649 0,117 - NT - NT
Total 11/20 11/20 11/20
NT = not tested + = positive - = negative d = doubtful presumptive positive colonies Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.284 Negative control 1 = 0.082 Negative control 2 = 0.089 Positive threshold = (0.082 +0.089)/2 + 0.11 = 0.196 Negative threshold = 0.196 x 0.9 = 0.176 OD value for a positive sample ≥ 0.196 OD value for a negative sample < 0.176
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Appendix 19: Comparison of TPSG and ISO results from uninoculated and inoculated raw milk.
Transia Plate Salmonella Gold
ISO Culture method
Sample code
Contamination level / strain
Optical density (OD)
Results Confirmation Presumptive positive colonies
Confirmation
2708 0,126 - NT d - 2711 0,129 - NT d - 2729 0,141 - NT d - 2744
0 cfu/25g
0,122 - NT - NT 2747 0,133 - NT - NT
Total 0/5 0/5 0/5 2709 0,200 + / - + d - 2710 1,989 + + + + 2714 0,237 + + + + 2715 0,129 - NT d - 2716 0,141 - NT d - 2717 0,214 + / - + + + 2718 2,177 + + - NT 2719 0,130 - NT d - 2723 1,628 + + d - 2725 0,175 - NT - NT 2727 0,125 - NT d - 2731 0,137 - NT - NT 2732 0,129 - NT d - 2738 0,190 - NT + + 2739 0,142 - NT d - 2742 0,143 - NT - NT 2743 0,139 - NT d + 2745 0,158 - NT - NT 2746
1 cfu/25g
Salmonella Dublin
0,144 - NT d - 2749 0,143 - NT d -
Total 6/20 6/20 5/20
NT = not tested + = positive - = negative +/- = doubtful sample d = doubtful presumptive positive colonies Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.629 Negative control 1 = 0.097 Negative control 2 = 0.117 Positive threshold = (0.097 +0.117)/2 + 0.11 = 0.217 Negative threshold = 0.217 x 0.9 = 0.195 OD value for a positive sample ≥ 0.217 OD value for a negative sample < 0.195
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Appendix 20. Comparison of TPSG and ISO results from uninoculated raw ground turkey
Transia Plate Salmonella Gold ISO Culture Method Sample code Optical Density Results Presumptive Confirmed
27 0.112 - - NT
30 0.115 - - NT
33 0.104 - - NT
44 0.103 - - NT
48 0.107 - - NT
Total 0/5 0/5 0/5
NT = Not tested + = Positive - = Negative Transia Plate Salmonella Gold test parameters: Optical Density (OD) values of kit controls: Positive control = 2.672
Negative control 1 = 0.109 Negative control 2 = 0.094
Positive threshold = ((0.109+0.094)/2) + 0.11 = 0.2115 Negative threshold = 0.2115 x 0.9 = 0.190 OD value for a positive sample ≥0.2115 OD value for a negative sample <0.190
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Appendix 20 (continued). Comparison of TPSG and ISO results from raw ground turkey contaminated with low levels (6 cfu/25g) of Salmonella Enteritidis (CCFRA 1004)
Transia Plate Salmonella Gold ISO Culture Method Sample code Optical Density Results Presumptive Confirmed
26 1.607 + + +
28 1.671 + + +
29 0.606 + + +
31 0.124 - - NT
32 1.300 + + +
34 1.479 + + +
35 1.377 + + +
36 1.630 + + +
37 1.541 + + +
38 0.114 - - NT
39 1.492 + + +
40 1.628 + + +
41 1.509 + + +
42 0.104 - - NT
43 1.354 + + +
45 0.959 + + +
46 1.550 + + +
47 1.333 + + +
49 1.604 + + +
50 1.622 + + +
Total 17/20 17/20 17/20
NT = Not tested + = Positive - = Negative Transia Plate Salmonella Gold test parameters: Optical Density (OD) values of kit controls: Positive control = 2.672
Negative control 1 = 0.109 Negative control 2 = 0.094
Positive threshold = ((0.109+0.094)/2) + 0.11 = 0.2115 Negative threshold = 0.2115 x 0.9 = 0.190 OD value for a positive sample ≥0.2115 OD value for a negative sample <0.190
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Appendix 21. Comparison of TPSG and ISO results from uninoculated Brie cheese
Transia Plate Salmonella Gold ISO Culture Method Sample code Optical Density Results Presumptive Confirmed
6 0.115 - - NT
8 0.108 - - NT
9 0.109 - - NT
14 0.109 - - NT
19 0.102 - - NT
Total 0/5 0/5 0/5
NT = Not tested + = Positive - = Negative Transia Plate Salmonella Gold test parameters: Optical Density (OD) values of kit controls: Positive control = 2.857
Negative control 1 = 0.105 Negative control 2 = 0.105
Positive threshold = ((0.105+0.105)/2) + 0.11 = 0.215 Negative threshold = 0.215 x 0.9 = 0.194 OD value for a positive sample ≥0.215 OD value for a negative sample <0.194
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Appendix 21 (continued). Comparison of TPSG and ISO results from Brie cheese contaminated with low levels (2.3 cfu/25g) of Salmonella Typhimurium (CCFRA 1009)
Transia Plate Salmonella Gold ISO Culture Method Sample code Optical Density Results Presumptive Confirmed
1 0.904 + + +
2 0.710 + + +
3 0.122 - - NT
4 0.831 + + +
5 0.771 + + +
7 0.117 - - NT
10 0.105 - - NT
11 0.849 + + +
12 0.714 + + +
13 0.107 - - NT
15 0.113 - - NT
16 0.107 - - NT
17 0.561 + + +
18 0.557 + + +
20 0.571 + + +
21 0.698 + + +
22 0.735 + + +
23 0.106 - - NT
24 0.106 - - NT
25 0.630 + + +
Total 12/20 12/20 12/20
NT = Not tested + = Positive - = Negative Transia Plate Salmonella Gold test parameters: Optical Density (OD) values of kit controls: Positive control = 2.857
Negative control 1 = 0.105 Negative control 2 = 0.105
Positive threshold = ((0.105+0.105)/2) + 0.11 = 0.215 Negative threshold = 0.215 x 0.9 = 0.194 OD value for a positive sample ≥0.215 OD value for a negative sample <0.194
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Appendix 22: Calculation of performance indicators after generalized categorization of test samples Calculations applied on all results obtained on samples contaminated at the low level Transia Plate Salmonella Gold
method result
Positive Negative Total Positive N11 = 284 N12 = 2 N1● = 286 ISO 6579:2002
method result Negative N21 = 3 N22 = 111 N2● = 114 Total N●1 = 287 N●2 = 113 Total = 400
Chi-square = (( | N12 – N21 | ) – 1 )2 / ( N12 + N21 ) = (( | 2 – 3 | - 1 )2 / ( 2 + 3 ) = ( 1 – 1 )2 / 5 = 0 Sensitivity rate = N11 / N1● = 284 / 286 = 99.3 % Specificity rate = N22 / N2● = 111 / 114 = 97.4 % False negative rate = N12 / N1● = 2 / 286 = 0.7 % False positive rate = N21 / N2● = 3 / 114 = 2.6 %