Application of duplex-PCR in rapid and reliable detection of toxigenic Vibrio cholerae in water samples in Thailand

摘要: Toxigenic Vibrio cholerae, the cause of cholera, is a native flora aquatic environment which transmitted through drinking water and still remains leading morbidity mortality in many developing countries including Thailand. The culture method (CM), routinely used for assessing quality, has not proven as efficient molecular methods because notorious pathogen survives mostly non-culturable state. We employed duplex-polymerase chain reaction (duplex-PCR) detection tcpA ctxA genes toxigenic V. compared PCR with CM various waters Khon Kaen Municipality, also evaluated effect different pre-PCR conditions on results including: 1) filtered enriched alkaline peptone (APW) 3 h before PCR, 2) without enrichment 3) use only APW 6 PCR. Of 96 samples (taken from waste-water, potable waste-water patients' houses, rivers) tested, 48 (50%) were positive by duplex-PCR, whereas 29 (30%) cholerae CM. isolated CM, 2 (7%) belonging to serovar O1, while rests non-O1/ non-O139. Results revealed, therefore, that tcpA-targeted duplex more sensitive than detected much less non-toxigenic cholerae. Template DNA low 100 fg or 23 cells sample was Pre-PCR filtration followed significantly increase efficiency duplex-PCR