Development of a simple, rapid multiplex PCR tool kit by using the 16S rRNA gene for the identification of faecal and non-faecal coliforms in drinking water

A multiplex method for the detection of faecal and non-faecal coliforms in drinking water was developed using three primers from the V2, V3 and V9 variable regions of 16S rRNA gene. 194F, 474F and 1436R are the three primers designed for specific amplification of V2, V3, V9 hyper variable regions of 16S rRNA gene. Multiplex PCR allowed for differentiation of the total coliform from faecal coliform by specific amplicons: 1,285 bp of amplicon is specific for 6 non-faecal coliform genera and 1,009 bp of amplicon is specific for faecal coliform ie. E. coli. If the drinking water was contaminated with both faecal and non-faecal coliforms then two amplicons of 1,285 bp and 1,009 bp by combination of three primers are observed. The multiplex PCR assay based on 16S rRNA gene should be a beneficial tool kit for the rapid identification of the total coliforms in the large number of water samples compared with traditional methods. Results can be acquired within 3 hrs of time as compared with classic method of MPN (3–4 days). This assay will be useful in diversification and detection of seven genera of total coliforms by using variable regions of 16S rRNA.