Culture-free detection and enumeration of STEC in water

Shiga toxin-producing Escherichia coli (STEC) causes worldwide outbreaks of food and waterborne diseases. Rapid identification of causative agents is critical for early intervention in the case of widespread diarrheal epidemics to prevent mortality. In this study, a Molecular-Beacon targeting stx2 gene (highly associated with human illness) was designed to develop a culture-independent real-time PCR assay for detection and quantification of STEC in water samples. The assay could detect lowest 10 genomic equivalent (GE) of the reference strain (E. coli I.T.R.C.-18) per PCR or 100 GE/mL. The presence of 10(6)CFU/mL of non-pathogenic E. coli DH5α has no impact on sensitivity of the assay. The assay could successfully enumerate STEC in surface water (collected from a sewage impacted river) and potable water samples collected from Lucknow city without prior enrichment. The assay will be useful in pre-emptive monitoring of surface/potable waters to prevent waterborne outbreaks caused by STEC.