Application of SYBR Green Real-Time PCR for Detection of Toxigenic Vibrio cholerae O1 in the Aquatic Environment
Aschana Tirapattanun, Chariya Chomvarin, Warawan wongboot and Boonnapa Kanoktippornc* Author for corresponding; e-mail address: chariya@kku.ac.th
Volume: Vol.42 No.3 (JULY 2015)
Research Article
DOI:
Received: 5 March 2014, Revised: -, Accepted: 8 July 2014, Published: -
Citation: Tirapattanun A., Chomvarin C., Wongboot W. and Kanoktippornc B., Application of SYBR Green Real-Time PCR for Detection of Toxigenic Vibrio cholerae O1 in the Aquatic Environment, Chiang Mai Journal of Science, 2015; 42(3): 588-598.
Abstract
Vibrio cholerae O1 is the causative agent of cholera disease and its natural habitat is the aquatic environment. V. cholerae can become a "viable but non-culturable" (VBNC) organism resulting in unsuccessful isolation from aquatic environments; therefore, sensitive, rapid and accurate detection of VBNC taken from an aquatic environment is needed. The aim of this study was to develop real-time SYBR green PCR compared to the conventional PCR and culture methods for detection of V. cholerae O1 in water samples. The SYBR green real-time PCR assays were developed using specific primers targeting genes for the outer membrane protein (ompW), cholera toxin A (ctxA), rfbO1 (serogroup O1) and rfbO139 (serogroup O139). The respective sensitivity of uniplex (ompW, rfbO139) and duplex (ctxA and rfbO1) SYBR green real-time PCR was 102 CFU/ml (3 CFU/PCR reaction) and 103 CFU/ml (25 CFU/PCR reaction). V. cholerae O1 was detected in 31.8% (27/85) of samples and all were ctxA positive by SYBR green real-time PCR and conventional PCR, vs. 3.5% (3/85 samples) by culture method. Our results indicate that both PCR-based assays have similar efficiency for detecting ompW, ctxA, rfbO1 and rfbO139 genes and could be applied for rapid detection of V. cholerae O1 in environmental water samples.