Detection of etiological agent for cholera by PCR protocol.
BACKGROUND
PCR protocol for Vibrio cholerae, the causative agent of the diarrheal disease cholera has been described in this report. We report the detection of Vibrio species in 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, wherein monitoring of target organisms is very crucial.


METHODS
Single step thermocycling programs have been reported for amplification of specific target loci. We have demonstrated that a gradient multi-step thermocycling program is more efficient in improving the sensitivity of detection by PCR. The method for preparation of template DNA from environmental sample uses filtration of water followed by harvesting the possible bacterial residue. This was further treated with proteinase K and heat to yield DNA compatible for PCR. The protocol was optimized for amplification of target loci from standard strains as well as from environmental water samples.


RESULTS
The method can simultaneously detect two different loci of Vibrio cholerae in a single reaction. The sensitivity of detection achieved for the pathogen was 100 cells per reaction. The specificity of the primers was demonstrated by spiking the reaction with non-specific template. The developed protocol was successfully extended to environmental samples.


CONCLUSIONS
The developed duplex PCR protocol allows the simultaneous detection of two genetic loci of the target pathogen from water samples. This enhances the efficiency of detection and provides a sensitive tool for the rapid detection of the pathogen that can be useful in epidemic conditions where the time factor involved in the identification of target pathogen is very crucial.
National Environmental Engineering Research Institute, Nagpur, India.
A
A
Kapley
H J
HJ
Purohit
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D002771:Cholera / Q000382:microbiology*; D002772:Cholera Toxin / Q000235:genetics; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D036781:Fimbriae Proteins; D005798:Genes, Bacterial; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic; D014734:Vibrio cholerae / Q000235:genetics / Q000302:isolation & purification*
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; C085807:TcpA protein, Vibrio cholerae; D036781:Fimbriae Proteins; D002772:Cholera Toxin