Molecular Detection of Seafood-Borne Human Pathogenic Bacteria - Aquaculture Microbiology and Biotechnology - page 114

has been found between the presence of the trh gene and urease production
among clinical isolates, which is considered to be an unusual characteristic
of V. parahaemolyticus. Kaufman et al. (2002) suggested that the tdh, trh and
urease test can be used to identify and track potentially virulent strains in
oysters.
Sensitivity of V. Parahaemolyticus to Low Temperatures
The sensitivity of V. parahaemolyticus to low temperatures is well
documented. Temyo (1966) observed a 3 log reduction of V. parahaemolyticus
at 4°C in peptone broth containing 3% NaCl. Similar observations have
been reported when the organism was held at low temperatures in shrimp
(Bradshaw et al ., 1974; Vanderzant and Nickelson, 1972) oysters (Johnson
and Liston, 1973; Thomson and Thacker, 1973; Goatcher et al., 1974;
homogenized fi sh fi llets (Matches et al ., 1971) and crabmeat (Johnson and
Liston, 1973).
Detection and Isolation of V. Parahaemolyticus Enrichment
Cultivation
The U.S.F.D.A.(United States Food and Drug Administration) Manual
(Elliot et al., 1998) recommends blending food samples in 3% NaCl or
phosphate buffered saline (PBS), enriching into alkaline Peptone Water
(1% NaCl) or APS (3% NaCl), and then streaking onto TCBS agar ( Table
5.1 ). Typical colonies are bluish green. Isolates such as V. alginolyticus
produce yellow colonies due to the fermentation of sucrose. The inability
of V. parahaemolyticus isolates to ferment sucrose is a primary differential
characteristic. Twedt (1989) listed 13 liquid media and 11 agar media for
selective cultivation of V. parahaemolyticus .
Use of the Polymerase Chain Reaction for Detection of
V. Parahaemolyticus
Tada et al . (1992) established PCR protocols for the specifi c detection of
the tdh and trh genes of V. parahaemolyticus. The selection of primers ( Table
5.2 ) took into consideration that the tdh and trh genes are known to have
sequence divergence of up to 3.3% and 16% respectively.
Bej et al . (1999) developed a multiplex PCR assay for total and virulent
strains of V. parahaemolyticus based on the amplifi cation of a 450-bp
sequence (Brasher et al., 1998) of the thermolabile hemolysin gene ( tlh ),
a 269-bp sequence of the thermostable direct hemolysin gene ( tdh ), and a
500-bp sequence of the thermostable direct hemolysin-related ( trh ) gene
(Table 5.4). All 111 V. parahaemolyticus isolates studied yielded the tlh
amplicons. However, only 60 isolates yielded the tdh amplicon, and 43
yielded the trh amplicon.