Quadruplex PCR for simultaneous detection of serotype, biotype, toxigenic potential, and central regulating factor of Vibrio cholerae

Abstract

A quadruplex PCR was developed for the simultaneous detection of genes specific for Vibrio cholerae O1 and/or O139 serogroup (wbe and/or wbf), cholera toxin A subunit (ctxA), toxin-coregulated pilus (tcpA), and central regulating protein ToxR (toxR) in a single tube reaction. This is a simple, rapid, and accurate approach for the detection of toxigenic V. cholerae O1 and/or O139 and can prevent the rapid spread of the disease by early detection.

FIG. 1.

Ethidium bromide-stained agarose gel electrophoresis of quadruplex PCR discriminates biotype El Tor from the Classical serogroup and discriminates O1 from O139 and simultaneously detects ctxA and toxR genes. Lane M, 100-bp DNA ladder (Bangalore Genei); quadruplex PCR product, lane 1, ctxA, tcpA, (Classical), toxR, and wbe (O1) gene-positive V. cholerae O1 biotype Classical 569B; lane 2, ctxA, tcpA, (El Tor), toxR, and wbe (O1) gene-positive V. cholerae O1 biotype El Tor strain 20 (VC20); lane 3, JP1; lane 4, Pu1372 (from watery stool); lanes 5 through 7, ctxA, tcpA (El Tor), toxR, and wbf gene-positive V. cholerae O139 strains SG24, KH3, and Pu1416 (from watery stool), respectively; lane 8, ctxA, tcpA, (El Tor), toxR, wbe (O1), and wbf (O139) gene-positive V. cholerae mix infection Pu1442; simplex PCR product, lanes 9 through 14, ctxA, tcpA (El Tor), wbe, wbf, toxR, and tcpA (Classical) genes, respectively; multiplex PCR product, lane 15, ctxA and tcpA (El Tor) gene-positive VC20; lane 16, ctxA and wbe gene-positive VC20; lane 17, ctxA and wbf gene-positive V. cholerae O139 strain SG24; lane 18, ctxA, wbe, and wbf gene-positive V. cholerae mix infection Pu1442; lane M, 100-bp DNA ladder (Bangalore Genei).