Molecular Detection of Seafood-Borne Human Pathogenic Bacteria - Aquaculture Microbiology and Biotechnology - page 120

Fukushima et al. (2003) developed a series of duplex Rti-PCR assays
for detection of 17 species of food and waterborne pathogens including
V. cholerae in stools utilizing SYBR Green as the fl uorescent reporter
molecule. The primers rtxC-F/rtxC-R ( Table 5.2 ) amplifi ed a 265-bp
sequence of the rtx toxin gene and were used to detect V. cholerae strains O1
and O139 as well as non-O1 strains, except for the classical V. cholerae O1
strains. The primers CT-F/CT-R ( Table 5.2 ) amplifi ed a 308-bp sequence
of the ctx gene and were used to detect V. cholerae O139 Bengal (Nair et
al., 1994). Without enrichment of seeded stool samples, the detection level
with DNA purifi cation was about 10 5 CFU/g of stool. The protocol for
detection of less than 10 4 CFU/g required overnight enrichment.
Blackstone et al. (2007) developed a Rti-PCR assay for detection of
V. cholerae harboring the ctxA gene. The primers ctxA-F/ctxA-R ( Table 5.2 )
amplifi ed a sequence of the ctxA gene. The probe ctxAP ( Table 5.2 ) was
labeled at the 5'-end with FAM or TET and at the 3'-end with a black hole
quencher (BHQ). Shellfi sh tissue from Mobile Bay (3 oyster samples and
3 clam samples) were homogenized in APW and subjected to overnight
enrichment incubation at 42°C. A 1-ml aliquot of enrichment was boiled
for 10 min and 2 to 2.5 ml incorporated into Rti-PCR assays. All six
shellfi sh samples were positive for V. cholerae and harbored the ctxA gene.
The detection limit was 0.8 CFU per Rti-PCR reaction with clams and was
less then 10 CFU per Rti-PCR with oysters.
Lalitha et al. (2008) developed a PCR assay specifi c for all strains of
V. cholerae including O1, 0139, and non-O1/non-O139 serogroups and
biotypes. The primers VHMF/VHA-AS5 ( Table 5.2 ) amplifi ed a 519-bp
sequence of the lolB gene that encodes an outer membrane lipoprotein. All
45 V. cholerae strains (34 O1 El Tor, one classical, four O139, and fi ve non-1/
non-O139) were found to harbor the lolB gene while 40 other Vibrio species
and 56 enteric Gram-negative reference strains of other genera did not
harbor the gene. The diagnostic sensitivity and specifi city with 633 clinical
rectal swab samples were 98.5% and 100% respectively.
Mendes et al. (2008) developed a multiplex single-tube PCR assay for
detection of the V. cholerae serotype. The ctxA gene was targeted with a pair
of external primers and a pair of internally nested primers that yielded a
fi nal amplicon of 302-bp ( Table 5.2 ). In addition, a pair of primers ( Table
5.2 ) was added that amplifi ed a 198-bp sequence of the rfbN gene that
encodes the O1 serotype.
CLOSTRIDIUM BOTULINUM
Overview
C. botulinum is a Gram-positive obligately anaerobic spore-forming rod
of which there are seven types A-G based on serological distinction of the