36B-5 |
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S. WANG, Dept. of Food Science, Univ. of Massachusetts, 100 Holdsworth Way, Amherst, MA 01003 and R. E. Levin, Dept. of Food Science, Univ. of Massachusetts, Amherst, 346 Chenoweth Lab., Box 31410, Amherst, MA 01003-1410. V. vulnificus is an estuarine bacterium capable of causing a rapidly fatal infection in humans. The vvh gene of the pathogen encodes the hemolysin toxin. Competitive PCR is used to reduce experimental variations via quantitative PCR and is based on an internal standard (IS) in the PCR reaction which competes for reaction reagents with chromosomal DNA of the target organism. The objective of this work is to develop a rapid method for quantification of V. vulnificus in seafood. A hybrid primer and a reverse primer were used for generation of a 508-bp DNA fragment by PCR for use as the IS in the competitive PCR. Different amounts of chromosomal DNA of V. vulnificus from seafood then competed with a constant amount of IS in the same PCR reaction to generate a standard curve between CFU and the ratio of the florescent intensity of the DNA bands. The forward primer, reverse primer and hybrid primer were designed by our lab; the specificity of the primers was proven. The optimized constant content of IS was 0.844 pg in a 50 ul PCR reaction. A minimum of 220 cells from a pure culture was detected in a PCR reaction. In 10 g clam tissue, 270 cells per gram of tissue and 7 cells per gram of tissue were detected respectively without enrichment and with 10 h enrichment in TSB+. Fresh raw seafood is becoming increasing popular in the world. The safety of the uncooked seafood is very important to consumers. The established quantitative competitive PCR can be used to determine the pathogen in seafood within 24 h.
Session 36B, Biotechnology: General
2005 IFT Annual Meeting, July 15-20 - New Orleans, Louisiana |