CHOLERA-TOXIN GENE POLYMERASE CHAIN-REACTION FOR DETECTION OF NON-CULTURABLE VIBRIO-CHOLERAE O1

Cholera enterotoxin is a major antigenic determinant for virulence of Vibrio cholerae O1 which can enter into a viable but non-culturable (N -C) state, not detectable by conventional culture methods, yet remain capable of producing enterotoxin and potentially pathogenic. PCR was a pplied in the current study to detect the cholera toxin (ctx) gene of N-C cells, thus eliminating the necessity of culture. Sets of oligonuc leotide primers were designed, based on the ctxAB operon of V. cholera e O1, to detect the presence of the ctx gene. DNA from both culturable and N-C cells of V. cholerae O1 was amplified by PCR using sets of pr imers flanking 302-, 564- and 777-bp fragments of the ctx gene. The PC R method employed was capable of detecting the ctx gene in N-C V. chol erae in aquatic microcosms and in diarrheal stool samples from three p atients who had distinct clinical symptoms of cholera but were culture -negative for V. cholerae O1 and non-O1 and enterotoxigenic Escherichi a coli. Forty cycles of a two-step reaction (30 s each at 94 and 60-de grees-C) were optimal and more time efficient than a three-step PCR de scribed previously. The procedure, from the point of heating microcosm s or broth culture samples to observation on gels, requires < 4 h to c omplete.