Contamination of surface and potable water in South Asia by Salmonellae: culture-independent quantification with molecular beacon real-time PCR.

Low numbers (15-100CFU) of Salmonella in food or water may pose a public health risk. The management of infections caused by Salmonella spp. during outbreaks or forecasting of contamination of aquatic resources largely depends on rapid, sensitive and accurate diagnostic in few hours. In this study, a real-time PCR assay in Molecular-Beacon format was developed and culture-independent quantitative enumeration of Salmonella spp. in surface and potable water is being reported for the first time from northern part of India. Molecular Beacon was designed in highly conserved region of invA gene (present in wide range of Salmonella serotypes including all subspecies) encoding an essential component of the invasion associated specialized type Ø protein secretion apparatus for detection of Salmonella spp. in water. The assay could detect directly 10 and 1 genomic equivalent of Salmonella typhimurium ATCC 14028 per PCR with detection probability of 100 and 20%, respectively. Further, the assay could detect 10CFU/PCR or more of reference strain (S. typhimurium ATCC 14028) without any enrichment in the presence of 10(8)CFUml(-1) of non-pathogenic E. coli (E. coli DH5alpha) with 100% detection probability. The assay could enumerate Salmonella spp. in surface (n=40) and potable waters (n=10) directly (without enrichment). Results indicate that northern India is at high risk of developing Salmonella borne infections. Further, real-time PCR assay in Molecular Beacon format can be used for identification of critical contamination points in natural water resources and potable water distribution systems, necessary to implement vaccination plan timely for prevention of waterborne outbreaks caused by Salmonella spp.