Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR

Abstract

The nucleotide sequence of pR72H cloned from Vibrio parahaemolyticus 93 was determined. We examined all V. parahaemolyticus gene sequences published in the GenBank-EMBL databases for homology and found that no other DNA sequence of V. parahaemolyticus was highly homologous to the sequence reported in this study. A pair of primers, VP33-VP32, derived from a pR72H fragment were selected to detect V. parahaemolyticus. The sensitivity of PCR detection for a pure culture of V. parahaemolyticus was 10 cells from crude bacterial lysates. Furthermore, a detection level of 2.6 fg, equivalent to 1 cell, was obtained by using purified chromosomal DNA as the template. The expected PCR products were obtained from all V. parahaemolyticus strains tested (n ‫؍‬ 124), while no PCR amplicons were found in other vibrios or related genera (n ‫؍‬ 50). High levels (10 6 to 10 10 CFU/ml) of Escherichia coli cells did not affect the PCR assay sensitivity. The presence of 10 8 V. parahaemolyticus cells or 10 9 E. coli cells in the PCR mixtures completely inhibited the PCR. When oyster samples were inoculated with V. parahaemolyticus 93 and cultured in tryptic soy broth containing 3% NaCl for 3 h at 35؇C, an initial sample inoculum level of 9.3 CFU/g was detected in a PCR assay with crude bacterial lysates. The PCR assay with enrichment culturing in salt polymyxin broth was compared with the conventional method for naturally contaminated shellfish and fish samples. We conclude that this PCR assay with enrichment culturing is a good alternative method for the detection of V. parahaemolyticus.

Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species: validation by DNA–DNA hybridization

“…If the R72H fragment was ampli¢ed, then one of two PCR products, approximately 320 or 387 bp in length, was obtained, for clinical strains and strains from seafood and seawater, regardless of their origin. The 387-bp amplicon was described in a previous report [15]. The di¡erence in the size of the fragment seems to be independent of the source of the strain.…”

“…The e⁄ciency of extraction was assessed by electrophoresis of DNA samples in a 1% (w/v) agarose gel. Oligonucleotide primers speci¢c for the R72H fragment [15] and the tl gene [14] were used for PCR, under the conditions speci¢ed by the authors, to con¢rm the correct identi¢cation of strains as V. parahaemolyticus or V. alginolyticus by biochemical means. PCR-ampli¢ed DNA (10 Wl) was subjected to electrophoresis in a 2% (w/v) agarose gel (Gibco BRL, Cergy Pontoise, France).…”

“…Firstly, Bej et al [14] suggested that PCR ampli¢cation of the tl gene, which encodes a thermolabile hemolysin, was V. parahaemolyticus-speci¢c and could be used as a species-speci¢c marker. Secondly, Lee et al [15,16] described the R72H DNA sequence, which is highly conserved in V. parahaemolyticus strains and can be used for species-speci¢c detection, although its function is unknown. Analysis of the sequences £anking the V. parahaemolyticus R72H fragment showed that this fragment was located after an rRNA operon and was composed of a non-coding region and a phosphatidylserine synthetase gene.…”

“…R72H nucleotide sequence of reference strain 93[15] compared with 387-bp (V. parahaemolyticus type strain 75.2T) and 320-bp (V. parahaemolyticus strain L56 from seawater) ampli¢cation product sequences. *, deleted nucleotides;^, identical nucleotides; underlining indicates the relative positions of primers.…”

Comparison of different biochemical and molecular methods for the identification of Vibrio parahaemolyticus

“…52% between V. parahaemolyticus and Vibrio cholerae). Other targets proposed for PCR detection were a cloned HindIII DNA fragment of V. parahaemolyticus chromosomal DNA, namely the pR72H fragment (Lee et al 1995;Robert-Pillot et al 2002) and a thermolabile haemolysin gene (tl), which is not associated with illness in humans. This haemolysin was shown to be present in all of the V. parahaemolyticus tested (Taniguchi et al 1985(Taniguchi et al , 1986 and was therefore used to detect this species in shellfish (Bej et al 1999).…”