WIT Press


Development Of PCR Protocol For Detection Of Escherichia Coli In Drinking Water

Price

Free (open access)

Paper DOI

10.2495/WRM130201

Volume

171

Pages

10

Page Range

225 - 234

Published

2013

Size

515 kb

Author(s)

J. Imtiaz, I. Hashmi, A. Saeed, I. A. Qazi & M. Arshad

Abstract

Escherichia coli is a pathogenic microorganism that may cause severe gastrointestinal illness in humans. This pathogen may be transmitted in a variety of ways, including food and water. As with most waterborne pathogens E. coli is difficult to detect and enumerate with accuracy in drinking waters due to methodological limitations. The aim of this study was to develop a PCR protocol for the detection of E. coli O157:H7 and E. coli virulence gene SLT-I (Shiga like toxin) in drinking water using a double enrichment step. This method is comprised of bacterial DNA purification using Genomic DNA extraction kit followed by PCR detection. The PCR optimization was done with E. coli O157:H7 strain EDL 933 (ATCC 43895). The oligonucleotide primers Rfb and SLT-I were used for targeting O157 and SLT-I genes respectively. The specific PCR product of these primers were obtained at 292 bp and 210 bp for Rfb and SLT-I respectively, which were visualized by gel electrophoresis and ethidium bromide staining. In spiked water samples, PCR results showed high sensitivity (<100 CFU/L) for Escherichia coli. The results obtained showed that the developed protocol would be utilized as a routine analysis for monitoring drinking water contamination. Furthermore, the simple and rapid protocol of the proposed technique provides results at a fraction of time required by the traditional culture techniques (24 hours compared to 2–6 days). Keywords: Escherichia coli, PCR, drinking water, O157:H7, SLT-I, Rfb, enrichment.

Keywords

Keywords: Escherichia coli, PCR, drinking water, O157:H7, SLT-I, Rfb, enrichment.