Rapid Methods for Foodborne

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Rapid Methods for Foodborne Bacterial Pathogens
Major pathogens
Campylobacter jejuni Salmonella E. coli
O157 Listeria
Conventional biochemical tests
Kit-based, miniaturized
Automated
Profile generation
Growth enrichment
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Miniaturized Kits
Biochemical reactions
API 20E Enterobacteriaceae 18-24
h Generate profile
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Miniaturized Kits
Biochemical reactions
ENTEROTUBE II
12 tests O/N
MICRO-ID MICRO-ID Listeria
15 tests 4 h
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Microbiology International
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Large Lab-based Systems
System Incubation (h) /test
Vitek GN1 2-12 5.35
Vitek2 IDGNB 3 7.15
Microscan Neg 2 16-20 12.79
Microscan R Neg 3 2.3 14.39
Trek Sensititre GNID 5-24 9. 41
Conductance-based Malthus Salmonella, Listeria,
E. coli, Listeria
Impedance-based
Bactometer
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Evaluation of Accuracy and Repeatability of
Identification of Food-Borne Pathogens by
Automated Bacterial Identification Systems
Joseph A. Odumeru,1 Marina Steele,1 Lynne
Fruhner,1 Carolyn Larkin,1 Jiangdong Jiang,1
Elroy Mann,3 and W. Bruce McNab2
JCM 1999
Evaluated 5 systems Vitek - biochemical Microsc
an - bochemical MIS - fatty
acids Replianalyzer - biochemical Microlog - C
oxidation
4/5 - specificities gt 97.5
5/5 - sensitivity gt80 for Salmonella Listeria
Repeatability 30-100
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Genera Species of Enterobacteriaceae Over Time
140
Genera
120
Species
100
Number
80
60
40
20
0
1939
1952
1962
1974
1986
1995
2003
YEAR
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Fast systems are mostly DNA- or Antibody-based
Pre-enrichment is usually needed
DNA approaches
Probes target rRNA target virulence genes
PCR single gene detection?
Phage highly specific marker
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DNA-based Test Systems for Listeria
Hybridization
Accu-Probe Test from primary culture SS
DNA probe chemiluminescent label rRNA (L.
monocytogenes) Read in luminometer lt 50
minutes Sensitivity 100 Specificity 99.7
Similar system for Campylobacter
Gene-Trak Listeria Gene-Trak Listeria
monocytogenes (Also Salmonella O157H7)
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Home-made Duffy et al.
20 h
(polycarbonate)
(48 h)
DNA hybridization
A rapid technique for detection of pathogens in
food products
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DNA-based Test Systems for Listeria
PCR
BAX Overnight enrichment Primers,
polymerase, dNTPs tablet Fluorescent signals
Does melting curve analysis 100 sensitivity
specificity (Listeria, L. monocytogenes,
O157H7, Salmonella, Campylobacter,
Cryptosporidium)
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DNA-based Test Systems for Listeria
PCR
PROBELIA U instead of T Uracil-D-Glycosylase
Heated, denatured Internal control 0.5
CFU/assay (L. monocytogenes, Salmonella,
O157H7, Campylobacter)
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Antibody-based methods
ELISA Campy, Salmonella, E. coli O157H7,
Listeria
LA Campy, Salmonella, E. coli O157H7,
Listeria
PPT. Salmonella, E. coli O157H7, Listeria
IMS Salmonella, E. coli O157H7, Listeria
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ELISA
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ELISAs for Listeria
Transia plate
Sandwich ELISA (Listeria, L. monocytogenes, Salmo
nella, O157H7, VTEC, Campylobacter)
Reveal
Tecra
Listeria-Tek
Assurance EIA
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ELISAs for Listeria
Variations on the theme
VIDAS - ELFA
EIAFoss IMS ELISA
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Latex Agglutination E. coli O157
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BLUSPOT Salmonella test
1. Pick 1 colony from a plate
2. Suspend in a glass tube contain- ing 0,250
ml of reagent 1 Mix well. Let stand 5 min at RT
3. Add 0.8 ml of reagent 2 Mix well
4. Put 200 µl of the sample on the membrane and
let completely soak
5. Add 250 µl conjugate let completely soak
6. Add 250 µl chromogen let completely soak
7. Add 100 µl of stop solution and let
completely soak
8. Read visually or with BLUSPOT reader.
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Precipitation
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Precipitation
Conventional
Non-selective pre-enrichment (1 d) Selective
enrichment (1-2 d) Plating on differential
selective agars (1-2 d)
Singlepath Salmonella
Non-selective pre-enrichment (1 d) Selective
enrichment (1 d) Singlepath test 1 h
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Gold-labelled immunosorbent assay
104 107 bacteria/ml
Boil
Control
Binding zone 2nd Ab
Sample gold-labelled Ab
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IMS
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PCR-ELISA (Exptl)
Hong et al. 2003. AEM. 693492-99
Campylobacter Salmonella
Carcass rinse Sediment bacteria Extract DNA
7h, 3
PCR
invA, ceuE
Digoxigenin (DIG) label
Biotin molecule on capture probe
ELISA
Plates coated with streptavidin
Detection with DIG kit
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PCR-ELISA (Exptl)
Hong et al. 2003. AEM. 693492-99
Detection Campylobacter limit 40 CFU/ml
31/32 ve (32/32) sensitivity 0.88,
specificity 0.98 linear correlation between OD
counts (R 0.792)
Detection Salmonella limit 200 CFU/ml
26/120 ve (30/120) linear correlation
between OD counts (R 0.924)
Sensitivity 0.67 specificity 0.93
No Enrichment
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Luminex LabMAP system
Unique microspheres
Target conserved 23S rRNA gene (rrl )
Amplicons 86 109 bp biotin labelled
Capture oligo probes coupled to spheres
PCR product - 96-well plate
Probe-coupled spheres added Hybridize 15 20 min
Spin
Re-suspend with streptavidin-R-phycoerythrin
React for 5-10 min
Read in Luminex machine
Dunbar et al. J. Microbiol Meth 53245-52
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Biosensors
Example
Liposome
Antibody on the outside
Dye on the inside
Said to detect in 8 minutes
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Use of Tests in Food Industry
1999 (USA) 144.3 million microbiology
tests 5,979 processing plants
Avg. 464 tests/week 24,000/y
16.3 (23.5 million) pathogen-specific Cost -
53.4 million (2.27 labour OH)
Dairy 31.8
Processed 36.2
Fruit Veg 9.7
Meat 22.3
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Thats it!