A Novel Molecular Quantitative Method for Rapid and Sensitive Detection of Escherichia coli from Roof-Harvested Rainwater
Harvesting of roof-top rainwater is commonly practiced either for potable or other household purposes. We report a rapid method for concentration of microbial cells coupled with DNA extraction procedure that we subjected to xanQ-based quantitative PCR (abbreviated as CoDEX-qPCR, [Concentration of microbial cells, DNA Extraction and XanQ-based qPCR]) purported for sensitive detection of E. coli from spiked and roof-harvested rainwater samples. The CoDEX-qPCR assay was compared with MI agar method, uidA, yaiO, tuf genes based methods in terms of specificity and sensitivity. The sensitivity of the primers targeting the xanQ gene was evaluated using 79 bacterial species, which indicated 100% sensitivity of primers for detection of E. coli. The limit of detection (LOD) of CoDEX-qPCR method was estimated to be 2.4 CFU/100mL (2.18×104 gene copies). The optimized CoDEX-qPCR assay was then applied to 110 roof-harvested rainwater samples in which E. coli was detected in 79 (71.81%) samples whereas the MI agar method detected the presence of E. coli only in 60 (54.54%) samples. It was observed that the sensitivity of CoDEX-qPCR for detection of E. coli was 17.27% more than the MI agar method. Moreover, universal 16S rRNA sequencing of isolates from rainwater samples revealed the presence of Escherichia coli, Escherichia hermannii, Enterobacter cloacae, Enterobacter ludwigii, Enterobacter pulveris and Pseudomonas mosselii. Consequently, the new CoDEX-qPCR method showed sensitive and rapid detection (3 hours) of E. coli which can be potentially employed for evaluating the microbiological quality of not only rainwater samples but also of environmental water samples from equivalent source.