BACKGROUND: PCR protocol for Vibrio cholerae, the causative agent of thediarrheal disease cholera has been described in this report. We report the detection of Vibrio speciesin drinking water samples in a duplex PCR reaction. The target loci used in the study were ctxA and tcpA.The sensitivity and efficiency of detection of this protocol can be applied in epidemic conditions, whereinmonitoring of target organisms is very crucial.
MATERIAL AND METHODS: Single step thermocycling programshave been reported for amplification of specific target loci. We have demonstrated that a gradient multi-stepthermocycling program is more efficient in improving the sensitivity of detection by PCR. The methodfor preparation of template DNA from environmental sample uses filtration of water followed by harvestingthe possible bacterial residue. This was further treated with proteinase K and heat to yield DNA compatiblefor PCR. The protocol was optimized for amplification of target loci from standard strains as well asfrom environmental water samples.
RESULTS: The method can simultaneously detect two different loci ofVibrio cholerae in a single reaction. The sensitivity of detection achieved for the pathogen was 100cells per reaction. The specificity of the primers was demonstrated by spiking the reaction with non-specifictemplate. The developed protocol was successfully extended to environmental samples.
CONCLUSIONS: Thedeveloped duplex PCR protocol allows the simultaneous detection of two genetic loci of the target pathogenfrom water samples. This enhances the efficiency of detection and provides a sensitive tool for the rapiddetection of the pathogen that can be useful in epidemic conditions where the time factor involved inthe identification of target pathogen is very crucial.

Keywords: Vibrio, PCR, Waterborne pathogens, ctxAB, tcpA