MOLECULAR CHARACTERIZATION, DETECTION AND QUANTIFICATION OF ESCHERICHIA COLI STRAINS ISOLATED FROM DIARRHOEIC LAMBS USING BOTH GRADIENT PCR AND SYBR GREEN REAL TIME PCR.

There are different types of pathogenic Escherichia coli (E. coli) carrying various types of genes. E. coli has universal stress protein (usp) genes which are species specific and confirm its presence. The uspA gene is important for survival of E. coli during cellular growth, adhesion and motility. Likewise, enteropathogenic Escherichia coli (EPEC) are an important cause of infantile diarrhoea which colonizes the small intestinal epithelial lining and causes lesions in the microvilli. There are several different well-characterized and putative adhesive factors involved in this process including bundle forming pilin (BFP) protein, and well defined bfpA gene. In the present study, E. coli were isolated from a total of 100 diarrhoeic lambs of Udaipur region located in southern part of Rajasthan. These isolates were screened by a series of biochemical tests and then subjected to amplification of the genomic DNA using universal stress protein A (uspA) gene for E. coli resulting into a single amplicon of 884 bp. These isolates were then subjected to characterization of enteropathogenic E. coli (EPEC) using bundle forming pilin protein A (bfpA) gene. The amplification of bfpA gene resulted into a single amplicon of 326 bp. A normal polymerase chain reaction (PCR) was performed to amplify genes and quantification of amplified products of both the genes were obtained by using fluorescent probes or fluorescent DNA-binding dyes and real-time PCR instruments that measure fluorescence while performing the thermal cycling for PCR. [ABSTRACT FROM AUTHOR]