Molecular Detection of Seafood-Borne Human Pathogenic Bacteria - Aquaculture Microbiology and Biotechnology - page 119

166 clinical and environmental isolates of V. cholerae. All 166 isolates were
01 El Tor, O139 or non-O1 serotypes and all harbored the rtxA and rtxC
genes which are considered specifi c for all V. cholerae isolates. Only the
non-O1 serogroups failed to harbor the ctxB gene.
Nandi et al. (2000) assessed the distribution of genes for an outer
membrane protein (OmpW) and a regulatory protein ToxR) among 254
V. cholerae isolates. The primers ompW-F/ompW-R ( Table 5.2 ) amplifi ed
a 588-bp sequence of the ompW gene. The primers toxR-F/toxr-R ( Table
5.2 ) amplifi ed an 883-bp sequence of the t oxR gene. The primers ctxA-F/
ctxA-R ( Table 5.2 ) amplifi ed a 301-bp sequence of the ctxA gene. All 254
isolates were found to harbor the ompW gene while 229 (98%) were found to
harbor the toxR gene. None of the other 40 strains belonging to other Vibrio
species produced amplicons with either ompW - or toxR -specifi c primers
nor did 80 strains from other bacterial genera. Restriction fragment length
polymorphism (RFLP) analysis and nucleotide sequence data revealed
that the ompW gene sequence is highly conserved among V. cholerae
strains belonging to different biotypes and/or serogroups. The authors
concluded that their observations suggested that the ompW gene can be
targeted for the species-specifi c identifi cation of V. cholera e strains and are
more suitable than the toxR gene. They then developed a multiplex PCR
involving both the ompW and ctxA genes for screening both toxigenic and
nontoxigenic strains of clinical and environmental isolates of V. cholerae .
V. cholerae 01 and 0139 serotypes are considered to cause noninvasive
epidemic cholera in developing countries, but non-01/non0-0139
serotypes may be invasive and cause systemic bacteremia and septicemia.
Namdari et al. (2000) reported on the consumption of raw clams by a healthy
individual in Maryland, USA followed 18 h later by the development of
severe profuse watery diarrhea, nausea, and vomiting with complete
recovery after 72 h. Blood cultures were positive for a non-01 and non-
0139 strain of V. cholerae that was cytotoxic to Hep-2 cell cultures. PCR
confi rmed that the isolate did not harbor the ctxA gene.
Lee et al. (2003) developed a multiplex PCR assay linked to a microwell
sandwich assay for detection of Salmonella and three Vibrio species
including V. cholerae in seeded oyster homogenates. The primers L-ctx/R-
ctx ( Table 5.2 ) amplifi ed a 302-bp sequence of the ctxA gene of V. cholerae .
Individual capture probes were then added and covalently bound to the
wells. The phosphorylated capture probe PP-ctx ( Table 5.2 ) was used for
V. cholerae . Multiplex amplicons were denatured in the wells and incubated
for hybridization to the immobilized probes. The biotinylated probe BP-
ctx ( Table 5.2 ) was then added followed by an alkaline phosphatase-avidin
conjugate and then enzyme substrate added for color development.
Enrichment of seeded oyster homogenates in APW allowed the detection
of 10 2 CFU/g of tissue.