Simple and Accurate Detection of Vibrio Cholera Using Triplex Dot Blotting Assay

Cholera outbreak is more common in developing countries. The causative agent of the disease is Vibrio cholerae strains O1 and O139. Traditional diagnostic testing for Vibrio is not always reliable, because Vibrio can enter a viable but non cultivable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this investigation, a triplex dot blotting assay has been developed for accurate and simple detection of V. cholerae using cholera toxin (ctxA, ctxB) and outer membrane protein (ompW) genes. The target genes were amplified using specific primers during monoplex polymerase chain reaction (PCR) and the amplicons were blotted on a nylon membrane. DIG-labeled PCR products in size of 219 (ctxA), 317 (ctxB) and 498 (ompW) bp, were amplified by a triplex PCR and used in a hybridization step as a probe. The positive signal was detected by applying anti-DIG HRP conjugate and chromogenic substrate. The results showed that the assay is sensitive enough to detect 10 cfu of V. cholerae O1. Also, the assay is specific enough to differentiate V. cholera from enterotoxigenic E. coli. The triplex dot blotting on clinical samples showed that it is more sensitive than monoplex and triplex PCR. In conclusion, we introduce a new rapid, sensitive and specific method for diagnosis of V. cholera in clinical specimens.