Rapid visual detection of diarrhoea-associated eaeA gene using polymerase spiral reaction (PSR) from clinical samples

Abstract

Diarroeagenic Escherichia coli including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) is associated with acute diarrhoea in children and young animals. The virulence is associated with attaching and effacing lesions encoded by eaeA gene which is considered as marker for EPEC and EHEC. Diagnosis of EPEC and/or EHEC is traditionally done by conventional PCR assay, which sometimes provide false-negative result and also costly as well as difficult to perform at field level. A novel isothermal polymerase spiral reaction (PSR) is developed for detection of eaeA gene in E. coli isolated from diarrhoeic pig, cattle, sheep and goat. Samples were collected from diarrhoeic animals for isolation and identification of the E. coli. eaeA gene was detected by conventional PCR and PSR assays. A total of 39 isolates were recorded positive for eaeA gene by conventional PCR, whereas 52 isolates were recorded positive for eaeA gene by PSR assay. PSR technique was 100 times more sensitive than conventional PCR. PSR techniques was found to be more sensitive, specific, cost effective, user friendly and reliable technique over conventional PCR. The PSR technique developed in the present study can be applied for screening of the clinical isolates for confirmation of EPEC and/or EHEC.