1. Immunological and DNA-methods

2. Immunological methods
Antibody-based
ELISA, immunoprecipitation, lateral flow
Antibodies recognize foreign particles (antigens);
Recognize antibodies from other species;
Can conjugate antibodies to colored particles

4. Lateral flow analysis (LFA)
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5. Lateral flow assay
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8. Advantages/disadvantages
Antibodies are highly specific;
Antibodies are rather straight-forward to generate;
Lateral flow is extremely simple and inexpensive.
Disadvantages
For bacteria detection, requires ~104 organisms in original sample (enrichment needed often);
Inhibitors present in food matrix;
Works best with large molecules (proteins, etc)

9. DNA-based methods (PCR)
“DNA” or deoxyribonucleic acid
The genetic information
Image from Ken Todar’s “The Microbial World” (

10. A G T C

11. DNA strands are “complementary”
Heat, high pH

12. Polymerase Chain Reaction

13. Ingredients for PCR Template (ie. DNA from food sample) Two primers;
~20 base pair pieces of DNA
single stranded DNA
Deoxynucleotide triphosphates (dATP, dTTP, dCTP, dGTP)
DNA polymerase (Taq)

14. PCR reaction conditions
Denature (94 oC for 45 seconds)
Annealing (45-72 oC for 30 seconds)
Extension (72 oC, 1 minute per kb product)
Depending upon product size, and number of cycles run, reaction takes ~1-3 hours.
25-40 times

15. 1 2 3 5’ 3’ 3’ 5’ Heat, then cool 5’ 3’ “primers” 5’ 3’
+ Taq polymerase + 3

16. 1 2 3

17. Power of PCR Number of cycles amplification 20 1,000,000
,000,000
,000,000,000
,000,000,000,000

18. Analyzing the reaction

19. Fluorescence-based methods
SYBR Green fluorescent dye
BAX PCR detection method
Blue light
Blue light
No fluorescence
Green light

21. PCR speeds up detection
1-3 h PCR assay
4-24 h enrichment
Primers to toxin gene or other gene unique to specific bacterium

22. The good and bad of PCR Advantages: Disadvantages:
Has potential to be most sensitive technique available.
Easy to develop in-house, plus many commercially available kits
Newer PCR methods allow “real-time” monitoring of reaction for increased speed.
Disadvantages:
Can’t determine if organisms are alive.
Very sensitive technique, susceptible to cross contamination.
Inhibitors present in food products (PCR is an enzymatic reaction)