Co-detection of three species of water-borne bacteria by multiplex PCR

Abstract

Monitoring of water-borne pathogens is important to safeguard public health. In view of various limitations inherent in the traditional culture methods, the feasibility of using the polymerase chain reaction (PCR) to monitor water-borne pathogens was investigated. The STN enterotoxin gene of Salmonella typhimurium, the STO enterotoxin gene of Vibrio cholerae, the LTI and LTII enterotoxin genes of Escheuichia coli, and the housekeeping genes, ARO-A and PHO-A of S. typhimurium and E. coli, respectively, were used as gene targets for PCR detection of toxigenic and general strains of these organisms. Six pairs of oligonucleotide primers were chosen to amplify internal fragments of the respective genes, and the identity of the PCR products was confirmed by restriction endonuclease digestion. The specificity of individual primer pairs in PCR was evaluated on DNA templates of 54 different bacterial isolates. The results showed that the LTI, LTII and STO primer sets were highly specific for toxigenic strains of E. coli H10407 (LTI+), E. coli SA53 (LTII+) and V. cholerae NRT (STO+), respectively. The PHO-A primers showed species-specific amplification products for all nine E. coli isolates examined, while the STN and ARO-A primer sets yielded species-specific amplification products for the 10 S. typhimurium isolates tested. Detection sensitivity of the ARO-A and PHO-A primer sets for S. typhlmurium and E. coli, respectively, was estimated at 103 CFU. Using three different combinations of the above primer sets, multiplex PCR was performed to detect toxigenic and non-toxigenic strains of V. cholerae, S. typhimurium and E. coli in seawater samples artificially spiked with the organisms. The technique, which showed positive co-detection of the respective target genes in each case, only required a turnaround time of 5 h. Results of the present study indicate that the multiplex PCR is a potentially powerful technique for the rapid codetection of enteropathogenic bacteria in routine water quality monitoring.