Guodong Zhang, Ph.D.1
Li Maria Ma, Ph.D.2
1Center for Food Safety and Applied Nutrition
Food and Drug Administration
U.S. Department of Health & Human Services
2National Institute for Microbial Forensics & Food and Agricultural Biosecurity
Oklahoma State University
Rapid Detection Technologies for
Foodborne Bacterial Pathogens
Mention of trade names or commercial
products in the presentation is solely for the
purpose of providing scientific information and
does not imply recommendation or
endorsement by the U. S. Food and Drug
Administration.
Pathogens Monitored by Foodborne Diseases
Active Surveillance Network (FoodNet)
BacteriaSalmonella
Campylobacter
Shiga toxin-producing (STEC) O157
non-O157 STEC
Listeria
Shigella
Vibrio
Yersinia
Parasites➢ Cryptosporidium
➢ Cyclospora
Pre-enrichment (Most cases)
Enrichment
Plating on selective media
Confirmation
Traditional Culture Detection of
Foodborne Bacteria
(Still the golden standard)
Major Breakthrough in recent years
Conventional selective medium vs chromogenic medium
Fig. Shigella sonnei colonies on different media. Shigella colonies are colorless on XLD and
mauve on Rainbow™ agar. Left, XLD; Middle, Rainbow™ agar; Right, Rainbow™ agar
supplemented with streptomycin.
PreEnrichment
and Enrichment
Key for shortening detection time
Be realistic (biological limit)
Detection of Bacteria in Foods/Environments
Culture Methods
• Pre-enrichment
• Enrichment
• Plating
• Confirmation
Immunological Methods
• ELISA
• IMS
• Bio-sensor
Nucleic acid Methods
• PCR RNA, DNA
• Isothermal
• Sequencing
• Bio-sensor
• Spectroscopy
• Microarrays
Morphology
• Cell size
• Cell shape
• Flagella
• Endospores
• Colony morphology
Serology, Biochemistry
Selective media
• Agglutination test
• API
• BIOLOG ID
• VITEK ID
• Chromogenic media
DNA-based Technology
• PCR
• DNA hybridization
• MicroArray
• 16S/28S rRNAgene sequencing
• Whole genome sequencing
Bacterial Typing
Early on
• Serotyping, Phage Typing
70’s
• Serotyping, Phage Typing
• RFLP
80’s, 90’s
• Serotyping, Phage Typing
• RFLP
• RAPD, AFLP, PFGE
2000-
Serotyping, Phage Typing
RFLP
RAPD, AFLP, PFGE
MLVA, MLST, DNA Arrays, MS, Ribotyping, Sequencing
Future
• Serotyping
• Sequencing
• Whole Genome Sequencing
Rapid
Sensitive
Simple, easy-to-operate
Accurate, quantitative detection method
Live and dead cells
Portable
Future Directions for Detection
Biomeme
ExcitePCR is developing the
FireflyDXTM family of pathogen detection
systems, portable devices offering rapid sample-
to-result detection in less than 30 minutes using
real-time polymerase chain reaction (PCR)
chemistry. FireflyDX devices will be capable of
multiplex assays and utilize single-use,
disposable cartridges for lab-quality results at
the Point-of-Need and Point-of-Care (PON /
POC). The FireflyDX systems combine sample
lysis, purification, realtime PCR analysis, and
reporting of results. The initial, primary use of
these systems are expected to be in clinical and
molecular diagnostic settings, agricultural
screening in domestic sectors and developing
countries, PON / POC monitoring of pathogenic
outbreaks, and for the detection of biological
agents associated with weapons of mass
destruction.https://www.excitepcr.com/
TWO G E N E R A T I O N S A H E A D O F T H E C O M P E T I T I O N
1 1 Y E A R S O F I N N O V A T I O N
PORTABLE FOOD TESTING SYSTEM (Aptamer)
S T A RT
Analyzer Test Str ips C ibusDx App Data Repos i tory
https://rokabio.com/products/atlas-system/
DNA / RNA Isothermal Amplification Technologies
Genie II (L)
Genie III (R)
Evaluation of 3M Molecular
Detection System and ANSR
Pathogen Detection System for
rapid detection of Salmonella
from egg products
SamplesEgg nog
Liquid Egg white
Liquid Egg yolk
Egg custard
Egg custard soft cake
Deviled egg halves
Egg salad
Egg rolls cookies
Macaroni & Cheese
Protein plus elbow pasta
Extra wide egg noodles
Amish extra wide egg
noodles
Extra broad egg noodles
Egg matzos
Whole powdered eggs
Egg white powder
Egg yolk powder
Scrambled eggs with
bacon
Egg drop Asian soup mix
Egg crystals
Evaluation of of 3M MDS and ANSR PDS Salmonella LAMP assays using
preenrichment egg product samples in comparison with culture methods
BrothCulture
Method3M1 ANSR2
Fisher’s exact
Test 3
3M vs Culture
Fisher’s exact
Test
ANSR vs Culture
LB4 63/805 61/80 60/80 0.8501 0.7080
BPW6 64/80 64/80 64/80 1.0000 1.0000
Total 127/160 125/160 124/160 0.8914 0.7859
Uninoculated
control0/40 0/40 0/40
1 3M, 3M Molecular Detection System. 2 ANSR, ANSR Pathogen Detection System. 3 Fisher’s exact test was conducted using SAS 9.3_M 1.4 LB, lactose broth.5 Number of positive samples/ numbers of total samples tested.6 BPW, buffered peptone water.
Hu, L., et.al. 2017. Poultry Science, Volume 96, Issue 5: 1410–1418, https://doi.org/10.3382/ps/pew399
Design of a New LAMP Assay
for Salmonella ser. Enteritidis
[Comparison with PCR and Culture
Methods]
Primers for our Salmonella ser. Enteritidis LAMP method
Primer Type Sequence (5' ----------> 3') Length (bp)
F3 Forward outer GTGAGGGCGAGGTTTGAAC 19
B3 Backward outer GGTCATGGTCGGGTATTCGA 20
FIP (F1c + F2) Forward innerTCCTCCACTGACAGGATTCCCA-
AAGTGGAGCAGCTGAGCA40
BIP (B1c + B2) Backward innerCAAACTGAACGTTTGGCTGCCT-
GCAGTGACTGGCATCAAGAA42
Hu, L. et. al.. Submitted to Food Control.
Isolates No. Positive/ Total No. Tested
LAMP PCR
Salmonella Enteritidis111/114 111/114
Non Salmonella Enteritidis
Salmonella 0/34 0/34
Non-Salmonella0/35 0/35
Inclusivity and Exclusivity
Hu, L. et. al.. Submitted to Food Control.
Strains ID Source No. of StrainsLAMP
results
Real-time PCR
results
CDC_2010K_1441 egg outbreak USA 20101
CDC_2010K_1543 egg outbreak USA 20101
CVM_N202 chicken breast1
GP raw egg shell1
SAL1568 -1
SAL1569 -1
SE 10 chicken ovary1
SE 12 egg follicle1
SE 22 poultry environment1
SE 26 chicken viscera1
S4-1 egg process plant1
S4-2 egg process plant1
III-4 food 1
02-0062 -1
042-2 eggs1
13-2 chicken1
13183 chicken breast1
17905 chicken1
17917 human 1
17918 human 1
17923human 1
17927 human 1
17929 human1
17931 human 1
18088 chicken breast1
18093 ground turkey 1
18509 human 1
18510 human 1
18511human 1
18512 human 1
18514 human 1
18516 human 1
18518 human 1
18567 poultry 1
18569poultry 1
18570poultry 1
18571poultry 1
18572poultry 1
18573 poultry 1
18574poultry 1
18575poultry 1
50027 chicken 1
50190 Schwann's ice cream1
5575 patient 1
5657 patient 1
5794 patient 1
5833 food, kim chee1
5834 food, pancake1
5835 food, fish1
5836 food, vegetable1
5837 food, pancake1
5839 food, fish1
5840 food, fish1
6058 patient 1
91-24857 swine1
91-24982 cattle1
91-25711 cattle1
91-29096 cattle1
92-123 nursing home outbreak1
92-14247 chicken 1
92-160 dog 1
92-2178 clinical (feces)1
92-2189 clinical (feces)1
92-4 water1
92-604 clinical (feces)1
92-609 chicken meat1
95-657-613 ice cream1
95-657-940 ice cream1
95-571-660 ice cream1
18577 poultry1
18578 poultry 1
18579poultry 1
18580poultry 1
18581poultry 1
22510 chicken - animal1
22532 chicken - animal1
22599 human 1
22600 human 1
22601human 1
22606human 1
22619 human 1
22621human 1
22689 chicken breast1
22690 chicken breast1
22704 ground turkey 1
22705 ground turkey 1
22706 ground turkey 1
23a -1
23b -1
236 -1
23690human 1
23703 human 1
23711human 1
23716human 1
30658 chicken breast1
30661 chicken breast1
30663 ground turkey 1
33944 chicken breast1
31 eggs1
35 egg process plant1
415 -1
419C -1
420 -1
434 ice cream1
493-1 eggs1
50000 chicken ovary 1
50001 human 1
50002 butterscotch pudding1
50004 chicken 1
50005 egg slurry1
50006 pasta, human1
50007 human, lobster 1
50009 pasta shells1
50010 human, pasta1
Salmonella ser. Enteritidis
isolates (114) used for
inclusivity test
Strain ID Genus. Species/ Serotype. Subspecies No. of StrainsLAMP
resultsReal-time PCR results
ATCC 14870 Jonesia denitrificans1
N/A Listeria innocua1
ATCC 51780 (1/2b) Listeria monocytogenes1
ATCC 15313 (4b) Listeria monocytogenes1
ATCC 19120 Listeria grayi1
ATCC 25401 Listeria grayi(murrayi)1
ATCC 35897 Listeria welshimeri1
ATCC 19119 Listeria ivanovii1
ATCC 35967 Listeria seeligeri1
ATCC 43888 E. coli O157:H71
EC P1334 E. coli O91:H21
1
EC CL-15 E. coli O113:K75:H21
1
ATCC 55055 Bacillus cereus1
6A16 Bacillus cereus
1
3A16 Bacillus subtilis, lactipan
1
ATCC 19268 Bacillus thuringiensis1
3476 Bacillus amyloliquifaciens1
ATCC 27853 Pseudomonas aeruginosa1
ATCC 25923 Staphylococcus aureus1
SHI0168 Shigella sonnei
1
670 Shigella flexneri
1
973 Shigella dysenteriae
1
970 Shigella boydii
1
11629 Vibrio cholerae
1
7708 Vibrio metschnikovii
1
17802 Vibrio parahaemolyticus
1
29306 Vibrio vulnificus
1
66N Proteus vulgaris
1
NES 14 Klebsiella pneumoniae
1
47N Citrobacter Freundii
1
8645 Erwinia mallotivora
1
1391 Brenneria nigrifluens
1
E604 Cronobacter sakazaki
1
E265 Cronobacter malonaticus
1
E464Cronobacter dublinensis, lactaridi
1
Strains ID SerotypeNo. of Strains LAMP results Real-time PCR results
607310-1 Heidelberg 1
579082-8 Heidelberg 1
607309-34 Heidelberg 1
02-0105 Heidelberg
1
Sal 0723 Typhimurium 1
Sal 0728 Typhimurium 1
02-0115 Typhimurium
1
02-0061 Newport
1
2433 Typhi
1
2105 H Saphra
1
1465 H Rubislaw
1
2069 H Michigan
1
2308 H Urbana
1
885 H Vietnam
1
3030 H Tornow
1
768 H Gera
1
1941 H Fresno
1
3029 H Brisbane
1
4000 H Agona
1
1501 H Muenchen
1
1097 H Senftenberg
1
1108480-16#1 Senftenberg
1
1250 H Muenster
1
1 H Montevideo
1
1108480-16#2 Montevideo
1
1070 H Johannesburg
1
2080 H Javiana
1
3170 H Inverness
1
1061 H Cubana
1
1158 H Cerro
1
1988 H Alachua
1
1108447-08 Give
1
CM 01 Poona
1
CM 02 Saintpaul
1
Isolates (69) used in exclusivity test
Comparison of LAMP and RT-PCR with
Culture Method for the Detection of Salmonella
ser. Enteritidis
Samples
No. of Salmonella ser. Enteritidis
positive/ total
Culture LAMPReal-time
PCR
Inoculated 54/160 54/160 54/160
Non-inoculated
(Control) 0/40 0/40 0/40
ACKNOWLEDGMENT
Dr. Lijun Hu
Dr. Eric W. Brown
Mr. Thomas S. Hammack
Dr. Xiaohua He
Dr. Shimin Zheng
