1 Immunological and DNA-methods

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

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4 Lateral flow analysis (LFA)medicine.nevada.edu

5 Lateral flow assay medicine.nevada.edu

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8 Advantages/disadvantagesAntibodies 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” (www.bact.wisc.edu)

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 conditionsDenature (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 methodsSYBR Green fluorescent dye BAX PCR detection method Blue light Blue light No fluorescence Green light

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21 PCR speeds up detection1-3 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)