Rapid Culture-Independent Quantitative Detection of Enterotoxigenic Escherichia coli in Surface Waters by Real-Time PCR with Molecular Beacon

摘要

Rapid and reliable detection of enterotoxigenic Escherichia coli (ETEC) is critical for the management of the waterborne diseases threatening human lives worldwide. In this study, a culture-independent real-time PCR assay, in molecular beacon format, was designed and validated for detection and quantitative enumeration of ETEC harboring LT1 gene (encoding heat labile toxin) in surface waters contaminated by fecal pollutants of human and animal origin. It was observed that the assay was able to detect 2 CFU/mL of ETEC (r = 0.997; PCR efficiency = 99.8%) from water samples spiked by a reference organism (E. coli MTCC 723). In the presence of 10~6 CFU/mL of nonpathogenic ￡ coli(E. coli DH5cc), the lowest detection limit from spiked water samples was 4 CFU/mL. The assay was 500 times more sensitive than conventional PCR using the same oligomers (Student's t test p < 0.05). The assay could specifically detect and quantify ETEC (1.2 x 103 to 1.4 x 10~6 CFU/100 mL) in polluted surface waters of river Gomti. The rapid culture-independent assay developed in this study for detection and quantitative enumeration of ETEC can be used for preliminary monitoring of surface waters to prevent waterborne outbreaks.