TaqMan PCR for Detection of Vibrio cholerae O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Oysters, and Synthetic Seawater

Abstract

Vibrio cholerae is recognized as a leading human waterborne pathogen. Traditional diagnostic testing for Vibrio is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, a TaqMan PCR assay is presented for quantitative detection of V. cholerae in pure cultures, oysters, and synthetic seawater. Primers and probe were designed from the nonclassical hemolysin (hlyA) sequence of V. cholerae strains. This probe was applied to DNA from 60 bacterial strains comprising 21 genera. The TaqMan PCR assay was positive for all of the strains of V. cholerae tested and negative for all other species of Vibrio tested. In addition, none of the other genera tested was amplified with the TaqMan primers and probe used in this study. The results of the TaqMan PCR with raw oysters and spiked with V. cholerae serotypes O1 and O139 were comparable to those of pure cultures. The sensitivity of the assay was in the range of 6 to 8 CFU g ؊1 and 10 CFU ml ؊1 in spiked raw oyster and synthetic seawater samples, respectively. The total assay could be completed in 3 h. Quantification of the Vibrio cells was linear over at least 6 log units. The TaqMan probe and primer set developed in this study can be used as a rapid screening tool for the presence of V. cholerae in oysters and seawater without prior isolation and characterization of the bacteria by traditional microbiological methods.Vibrio cholerae is a waterborne pathogen that causes gastrointestinal disorders with a wide range of clinical manifestations, including vomiting and rice-like diarrhea (24). The association of human illness with consumption of V. choleraecontaminated oysters, seawater, and other shellfish is well documented (29, 37). Consumption of raw oysters correlates strongly with gastrointestinal infections, and several Vibrio species, including strains of Vibrio parahaemolyticus, Vibrio vulnificus, and V. cholerae, have been implicated as the causative agents.Coastal areas with brackish waters and estuarine regions are niches for many Vibrio species, including strains of toxigenic O1 V. cholerae. Epidemic cholera strains are endemic in several regions, including the U.S. gulf coast and Australia, and are occasionally involved in illnesses in these regions (24). Because Vibrio species attach to material suspended in water, shellfish and mollusks that are in these environments can be expected to consume Vibrio during feeding (24).Traditional identification methods currently used are time-consuming and laborious, requiring prolonged incubation and selective enrichment to reduce the growth of background flora. Vibrio cells may also enter a viable but not culturable (VBNC) state, caused by nutrient starvation and physical stress. This may explain the failure of traditional culture techniques to isolate this organism from contaminated water and food samples implicated in food-borne outbreaks (10,27,47). Several investigator...

Role of Indole Production on Virulence of Vibrio cholerae Using Galleria mellonella Larvae Model

“…It is of interest that indole was increased at the concentration of NaCl 5 and 6 % whereas the growth rate of V. cholerae started decreasing at the concentration of NaCl between 1 and 3 %. It has been reported that 3 % NaCl could stress V. cholerae to exist in a viable but non-culturable cells [30]. Fig.…”

Sensitive and Rapid Detection of Cholera Toxin-Producing Vibrio cholerae Using Loop-Mediated Isothermal Amplification

“…Cholera toxin (CT) is a major virulence determinant of V. cholerae. This bacterium is indigenous to fresh and blackish water environments in tropical, subtropical and temperate areas worldwide, the threat of epidemic cholera is restricted primarily to developing countries with warm climates [1][2][3]. V. cholerae causes seafood borne infection, water-borne outbreaks and epidemics in terrestrial environments [1,3].…”

“…V. cholerae causes seafood borne infection, water-borne outbreaks and epidemics in terrestrial environments [1,3]. Therefore, ingestion of raw or undercooked seafood such as shrimp and drinking water contaminated with V. cholera are risk factors in humans [1][2][3]. Most V. cholerae isolates from the environ-ment do not produce CT, nor do they possess the genetic potential to produce CT. V. cholerae O1 and O139 are the major seroytpes associated with illness, and some V. cholerae non-O1 and non-O139 isolates produce CT.…”

Biodiversity of Vibrios

“…DNA extracted directely from water, food, or fecal samples is used as the template for PCR. PCR products are detected with a double-stranded DNA dye, e.g., SYBR Green I (127, 302), or probes (54,242) and occurs in about 1 h. The sensitivity of this technique is high, and it can detect as few as 10 V. cholerae CFU per ml of water sample (242). Reliable identification of V. cholerae, V. parahaemolyticus, and V. vulnificus is apparent when a combination of 20 species-specific genes from different human pathogens are used (127).…”