A real-time multiplex PCR for the identification and typing of Vibrio cholerae

David R Greig; Tadgh J Hickey; Megan D Boxall; Hafsah Begum; Amy Gentle; Claire Jenkins; Marie A Chattaway

doi:10.1016/j.diagmicrobio.2017.11.017

A real-time multiplex PCR for the identification and typing of Vibrio cholerae

Diagn Microbiol Infect Dis. 2018 Mar;90(3):171-176. doi: 10.1016/j.diagmicrobio.2017.11.017. Epub 2017 Dec 1.

Authors

David R Greig¹, Tadgh J Hickey², Megan D Boxall³, Hafsah Begum⁴, Amy Gentle⁵, Claire Jenkins⁶, Marie A Chattaway⁷

Affiliations

¹ Gastrointestinal Bacteria Reference Unit, Public Health England, 61 Colindale Avenue, London NW9 5HT, UK. Electronic address: [email protected].
² Gastrointestinal Bacteria Reference Unit, Public Health England, 61 Colindale Avenue, London NW9 5HT, UK; University of Hertfordshire, School of Life and Medical Sciences, College Lane, Hatfield, AL10 9AB, UK. Electronic address: [email protected].
³ Gastrointestinal Bacteria Reference Unit, Public Health England, 61 Colindale Avenue, London NW9 5HT, UK; Coventry University, Faculty of Health and Life Sciences, Priory Street, Coventry CV1 5FB, UK. Electronic address: [email protected].
⁴ Gastrointestinal Bacteria Reference Unit, Public Health England, 61 Colindale Avenue, London NW9 5HT, UK; Coventry University, Faculty of Health and Life Sciences, Priory Street, Coventry CV1 5FB, UK. Electronic address: [email protected].
⁵ Gastrointestinal Bacteria Reference Unit, Public Health England, 61 Colindale Avenue, London NW9 5HT, UK. Electronic address: [email protected].
⁶ Gastrointestinal Bacteria Reference Unit, Public Health England, 61 Colindale Avenue, London NW9 5HT, UK. Electronic address: [email protected].
⁷ Gastrointestinal Bacteria Reference Unit, Public Health England, 61 Colindale Avenue, London NW9 5HT, UK. Electronic address: [email protected].

PMID: 29274667
DOI: 10.1016/j.diagmicrobio.2017.11.017

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.

Keywords: Biotype; Real-time PCR; Serogroup; Toxin encoding genes; Vibrio cholerae.

Publication types

Validation Study

MeSH terms

Adolescent
Adult
Aged
Bacterial Proteins / genetics
Child
Cholera / diagnosis*
Cholera / microbiology
Cholera Toxin / genetics
DNA-Binding Proteins / genetics
Female
Fimbriae Proteins / genetics
Humans
Male
Middle Aged
Multiplex Polymerase Chain Reaction / methods*
Real-Time Polymerase Chain Reaction / methods*
Serotyping
Transcription Factors / genetics
Vibrio cholerae / classification*
Vibrio cholerae / genetics*
Vibrio cholerae / isolation & purification
Young Adult

Substances

Bacterial Proteins
DNA-Binding Proteins
TcpA protein, Vibrio cholerae
Transcription Factors
toxR protein, Vibrio cholerae
Fimbriae Proteins
Cholera Toxin