Septaplex PCR assay for rapid identification of Vibrio cholerae including detection of virulence and int SXT genes.
In this study, we describe a septaplex PCR assay for rapid identification of Vibrio cholerae including detection of the virulence and intsxt genes. Conditions were optimized to amplify fragments of ISRrRNA (encoding for 16S-23S rRNA gene, Intergenic spacer regions), O1rfb (O1 serogroup specific rfb), O139rfb (O139 serogroup specific rfb), ctxA (cholera toxin subunit A), tcpA (toxin coregulated pilus), and intsxt (sxt integron) simultaneously in a single PCR. The septaplex PCR was evaluated using 211 strains of V. cholerae and six water samples for in situ testing. PCR results were correlated with genotype data obtained by individual PCR and slot-blot assays. The one-step PCR described here can be used to identify V. cholerae accurately and rapidly. Also, the virulence and intsxt genes can be simultaneously detected, providing a useful method for monitoring pathogenic, intsxt-positive and nonpathogenic, intsxt-negative V. cholerae serogroups both in the environment and clinical settings.
Mantri
Chinmay K
CK
Institute of Life Sciences, Nalco Square, Bhubaneswar, India.
Mohapatra
Saswat S
SS
Ramamurthy
Thandavarayan
T
Ghosh
Raikamal
R
Colwell
Rita R
RR
Singh
Durg V
DV
D005814:Genes, Viral; D006801:Humans; D032023:Integrons / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic / Q000382:microbiology*; D012680:Sensitivity and Specificity; D014734:Vibrio cholerae / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics* / Q000302:isolation & purification
D011933:Reagent Kits, Diagnostic; D037521:Virulence Factors