CO-DETECTION OF 3 SPECIES OF WATER-BORNE BACTERIA BY MULTIPLEX PCR

Monitoring of water-borne pathogens is important to safeguard public h ealth, In view of various limitations inherent in the traditional cult ure methods, the feasibility of using the polymerase chain reaction (P CR) to monitor water-borne pathogens was investigated, The STN enterot oxin gene of Salmonella typhimurium, the STO enterotoxin gene of Vibri o cholerae, the LTI and LTII enterotoxin genes of Escherichia coli, an d the housekeeping genes, ARO-A and PHO-A of S. typhimurium and E. col i, respectively, were used as gene targets for PCR detection of toxige nic and general strains of these organisms. Six pairs of oligonucleoti de primers were chosen to amplify internal fragments of the respective genes, and the identity of the PCR products was cofirmed by restricti on endonuclease digestion. The specificity of individual primer pairs in PCR was evaluated on DNA templates of 54 different bacterial isolat es, The results showed that the LTI, LTII and STO primer sets were hig hly specific for toxigenic strains of E. coli H10407 (LTI+), E. coli S A53 (LTII+) and V. cholerae NRT (STO+), respectively The PHO-A primers showed species-specific amplification products for all nine E. coli i solates examined, while the STN and ARO-A primer sets yielded species- specific amplification products for the 10 S. typhimurium isolates tes ted. Detection sensitivity of the ARO-A and PHO-A primer sets for S. t yphimurium and E. coli, respectively, was estimated at 10(3) CFU. Usin g three different combinations of the above primer sets, multiplex PCR was performed to detect toxigenic and non-toxigenic strains of V. cho lerae, S. typhimurium and E. coli in seawater samples artificially spi ked with the organisms. The technique, which showed positive co-detect ion of the respective target genes in each case, only required a turna round time of 5 h, Results of the present study indicate that the mult iplex PCR is a potentially powerful technique for the rapid co-detecti on of enteropathogenic bacteria in routine water quality monitoring.