Wang and Levin (2006) developed a Rti-PCR assay for quantifi cation of
Vibrio vulnifi cus seeded into clam tissue homogenates using the primers of
Panicker et al. (2004). Without enrichment, the limit of detection was 1 x
10 2 CFU/g of tissue with a linear detection range of 1 x 10 2 to 1 x 10 8 CFU/g.
With a 5 h non-selective enrichment, the limit of detection was 1 CFU/g
of tissue with a linear detection range of 1 to 1 x 10 6 CFU/g of tissue. A 10-
fold higher detection limit with seeded clam tissue homogenates occurred
compared to a pure culture. After 5 h of non-selective enrichment the
detection limit with a pure broth culture and seeded tissue homogenates
were identical at 1 CFU/ml, however, the Ct (thermal cycles required
for initial detection of amplifi cation) value with tissue homogenates was
about 3 cycles higher than with a pure culture refl ecting some level of PCR
inhibition from the tissue.
Lee and Levin (2008) developed a novel method for discriminating
Vibrio vulnifi cus by Rti-PCR before and after γ-irradiation based on
the observation that γ-irradiation results in extensive reduction in the
molecular size of DNA. Irradiation of viable cells (1 x 10 6 CFU/ml) at 1.08
KGy [KiloGray (radiation unit measure)] 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 amplifi cation. In contrast, primers designed to amplify sequences
of 700, 300, and 70-bp yielded amplifi cation with Ct values resulting in
13.4, 27.6, and 45.4% detection of genomic targets. When viable cells of V.
vulnifi cus 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 amplifi cation with
primers targeting sequences of decreasing size.
RAPD (Random Amplifi ed Polymorphic DNA)
Radu et al . (1998) subjected 26 biotype 1 and 10 biotype 2 isolates to
RAPD analysis using two random primers designated Gen 1-50-03 and
with primer Gen 1-50-03, with all six RAPD types represented by one or
more strains of biotype 1. With biotype 2 strains, only three of these RAPD
types were distinguished. With primer Gen 1-50-09, a total of fi ve RAPD
types were distinguished, with all fi ve RAPD types represented by one
or more strains of biotype 1. With biotype 2 strains, only four of these
RAPD types were generated. Results also indicated that certain biotype
1 and biotype 2 strains yielded identical RAPD profi les with both RAPD
primers, indicating a high degree of DNA sequence similarity between
such strains of the two biotypes.