Rapid Method for Species-Specific Identification of Vibrio cholerae Using Primers Targeted to the Gene of Outer Membrane Protein OmpW

Abstract

The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 (∼98%) of 233 were positive for toxR. None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW- andtoxR-specific probes in DNA dot blot assay. While theV. cholerae strains reacted with ompW probe, only one (V. mimicus) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus,V. splendidus, V. alginolyticus, V. fluvialis, V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxAgenes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.

Detection, Isolation, and Identification of Vibrio cholerae from the Environment

“…This protocol (Nandi et al, 2000) can be used in place of Basic Protocol 4, since it detects the V. cholerae-specific ompW sequence. It also allows for the detection of the ctxA gene of the cholera toxin.…”

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

“…Preliminary identification of the strains had been performed on the bases of colony morphology on TCBS, sucrose utilization, oxidase test, salt requirement (growth on 0%, 3%, 8% and 10% NaCl medium), resistance to vibriostatic O129 (10 and 150 lg), string test, Voges-Proskauer, cellobiose utilization and reactions on triple sugar iron -TSI -agar slant (Oliver and Kaper 1997;Ripabelli et al 1997). Strains presumptively identified as V. parahaemolyticus and V. alginolyticus had been confirmed by 16S rRNA gene sequencing (Marchesi et al 1998;Oakey et al 2003), while presumptive V. cholerae strains had been confirmed by PCR targeted to the ompW gene (Nandi et al 2000).…”

Dependent population dynamics between chironomids (nonbiting midges) and Vibrio cholerae

“…Yellow colonies from TCBS were subcultured onto a nonselective medium such as Luria agar, and then tested for oxidase (1% tetramethyl p-phenylenediamine, Sigma, St Louis, MO) and string test (0.5% sodium deoxycholate, Sigma). The identity of the isolates that were suspected to be V. cholerae was further verified using the multiplex PCR assay according to Nandi et al (2000), by a simultaneous addition of primer pairs for ompW and ctxA (Table 1), in the same reaction mixture. All the isolates that were identified as V. cholerae were examined further to determine whether they were members of the O1 and O139 serogroups by slide agglutination by using two specific antiserums; (1) a poly antiserum specific for O1 surface antigen inaba or ogawa (Difco), and (2) an antiserum specific for O139 surface antigen (The Ministry of Health, Israel).…”