Immunological and DNAmethods Immunological methods Antibodybased ELISA immunoprecipitation

Immunological and DNAmethods

Immunological methods • Antibody-based – ELISA, immunoprecipitation, lateral flow • Antibodies recognize foreign particles (antigens); • Recognize antibodies from other species; • Can conjugate antibodies to colored particles http: //www. nwfsc. noaa. gov www. immei. de

http: //www. neogen. com/Food. Safety/R_Index. html

Lateral flow analysis (LFA) medicine. nevada. edu

Lateral flow assay medicine. nevada. edu

Advantages/disadvantages • Advantages – 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)

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

A G T C www. biologycorner. com

DNA strands are “complementary” Heat, high p. H http: //en. wikipedia. org/wiki/Image: GC_DNA_base_pair. svg

Polymerase Chain Reaction

Ingredients for PCR • Template (ie. DNA from food sample) • Two primers; – ~20 base pair pieces of DNA – single stranded DNA • Deoxynucleotide triphosphates (d. ATP, d. TTP, d. CTP, d. GTP) • DNA polymerase (Taq)

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

1 5’ 3’ 3’ 5’ Heat, then cool 3’ 5’ 2 “primers” 3’ 3’ + Taq polymerase + 3 http: //www. youtube. com/watch? v=HMC 7 c 2 T 8 f. Vk&feature=fvwrel 5’

1 2 3

Power of PCR Number of cycles amplification 20 1, 000 30 1, 000, 000 40 1, 000, 000

Analyzing the reaction http: //www. biochem. arizona. edu/classes/bioc 471/pages/Lecture 2/AMG 1. 12. gif

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

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

The good and bad of PCR • Advantages: – 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)