New approach for discrimination of Vibrio vulnificus by real-time PCR before and after gamma-irradiation

The effects of gamma-irradiation on the destruction of Vibrio vulnificus by real-time PCR were studied. gamma-irradiation was found to result in extensive reduction in the molecular size of DNA. Irradiation of viable cells (1 x 10(6) CFU/ml) at 1.08 KGy resulted in 100% destruction determined by plate counts, with most of the DNA from the irradiated cells having a bp-length of less than 1000. The use of a pair of primers to amplify a 1000-bp sequence of DNA from cells exposed to 1.08 KGy failed to yield amplification. In contrast, primers designed to amplify sequences of 700, 300, and 70-bp yielded amplification with C(t) values resulting in 13.4, 27.6, and 45.4% detection of genomic targets. When viable cells of V. vulnificus were exposed to 1.08, 3.0, and 5.0 kGy, the average molecular size of genomic DNA visualized in an agarose gel decreased with increasing dose, corresponding to an increased probability of amplification with primers targeting sequences of decreasing size.