A novel molecular quantitative method for rapid and sensitive detection of Escherichia coli from roof-harvested rainwater

Abstract

The harvesting of roof-top rainwater is commonly practiced either for potable or other household purposes. We report a rapid method for the concentration of microbial cells coupled with a 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]) which purports the sensitive detection of E. coli in roof-harvested rainwater samples. The CoDEX-qPCR assay was compared with the MI agar method and uidA, yaiO, and 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 a 100% sensitivity of the primers for the detection of E. coli. The limit of detection (LOD) of the CoDEX-qPCR method was estimated to be 2.4 CFU/100 mL (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 in only 60 (54.54%) samples. It was observed that the sensitivity of CoDEX-qPCR for the detection of E. coli was 17.27% more than that of the MI agar method. Moreover, the universal 16S rRNA gene sequencing of the 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 a 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 sources.