Detection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification

Abstract

A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.Vibrio cholerae is the etiological agent of epidemic cholera, which causes watery diarrhea that can result in the rapid dehydration and death of infected persons. Coastal waters are an important reservoir of V. cholerae, and cholera is generally transmitted to humans via contaminated water or seafood (12, 13). Of more than 200 known serogroups of V. cholerae, the two well-known serogroups O1 and O139 have been associated with epidemic cholera (11). The serogroup O1 can be divided into three serotypes, Inaba, Ogawa, and Hikojima, and each serotype can be divided into two biotypes, classical and El Tor (30). The other serogroups of V. cholerae, collectively referred to as non-O1 and non-O139 serogroups, have not been associated with epidemics but have been associated with occasional outbreaks of cholera-like disease. These other serogroups are usually isolated from patients with mild diarrhea or from the environment (27,39). Vibrio species such as V. parahaemolyticus, V. cholerae, and V. vulnificus are found in blue mussels harvested along the coastline of Norway (5).The pathogenesis of cholera is a complex process, and the major virulence factors of V. cholerae are the cholera toxin (CT) encoded by the ctxAB genes and the toxin-coregulated pilus (TCP) encoded by the tcpA gene (30). CT leads to increased intestinal secretion of electrolytes and water into the lumen. TCP is a ...

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