A real-time multiplex PCR for the identification and typing of Vibrio cholerae |
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Authors: | David R Greig Tadgh J Hickey Megan D Boxall Hafsah Begum Amy Gentle Claire Jenkins Marie A Chattaway |
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Institution: | 1. Gastrointestinal Bacteria Reference Unit, Public Health England, 61 Colindale Avenue, London NW9 5HT, UK;2. University of Hertfordshire, School of Life and Medical Sciences, College Lane, Hatfield, AL10 9AB, UK;3. Coventry University, Faculty of Health and Life Sciences, Priory Street, Coventry CV1 5FB, UK |
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Abstract: | We report the development and validation of a duo-triplex real-time polymerase chain reaction (PCR) for the rapid identification and typing of Vibrio cholerae. The PCR assay targets a species-specific toxR gene present in all strains of V. cholerae and used as a marker for the species wbeO1 and wbfO139, encoding the O1 and O139 somatic antigens, and ctxA, encoding cholera toxin (CT). The two tcpA variants associated with the classical and El-Tor biotypes are used to infer biotype. The assay was evaluated using 178 isolates comprising eight different Vibrio species, including 122 isolates of V. cholerae. The PCR results of 171/178 (96.1%) isolates were concordant with the serotyping, biotyping, and expected CT results. Variants of toxR (n = 3), nonfunctional wbeO1 (n = 1), and CT-negative isolates of V. cholerae O1 (n = 3) were likely explanations for the mismatched results. This duo-triplex real-time PCR is a reproducible and robust assay for the rapid identification and typing of V. cholerae belonging to the highly pathogenic, pandemic lineages. |
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Keywords: | PCR polymerase chain reaction GBRU Gastrointestinal Bacteria Reference Unit PHE Public Health England CT cholera toxin TCP toxin co-regulated pilus Real-time PCR Serogroup Biotype Toxin encoding genes |
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