Design and Optimization of PCR Assay for Rapid Detection of Enterotoxigenic and Enteroaggregative Pathotypes of E. coli

Escherichia coli is one of the most important causative agents of acute diarrheal disease in the world. Diarrheal diseases are one of the main causes of mortality among children in developing countries. The aim of this study was to develop a PCR technique for diagnosis of specific virulence genes of Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC).
In this study, specific genes ST and LT were used for diagnosis of ETEC pathotype and AAF/II specific gene was used for diagnosis of EAEC pathotype. The PCR technique was designed and optimized for these three genes. The reaction products were cloned in pTZ57R/T vector as positive control. The primers’ sensitivity was determined by measurement of detection limit, and primers’ specificity was evaluated using other bacterial genomes.
According to the results of the PCR products analysis using agarose gel electrophoresis, the bands for AAF/II, LT, and ST were obtained 384, 459, and 150 bp, respectively. Also, the reaction of the genomes of control bacteria using the designed primers was negative. The detection limit based on copy number for ST, LT, and genes, was obtained 390, 1500, and 2500 copies, respectively.
Considering the findings of this study, design of these assays in Iran is a progress in the improving rapid and accurate diagnosis of pathogens and detection of E. coli pathotypes.