Development of PCR protocol for detection ofEscherichia coliin drinking water

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).

Validation of a Multiplex PCR for the Simultaneous Detection of E. coli and Class 1 Integron

“…The product of PCR was subsequently digested to identify the specific class of Integron present. The oligonucleotide primers Rfb and SLT-1 were used for targeting O157 and SLT-I genes of E. coli, respectively in previous studies (Imtiaz et al 2013). In addition, multiplex PCR conditions were optimized for the detection of integrons in E. coli producing plasmid-mediated AmpC β-lactamases (Perez et al 2002).…”

Shiga toxin-producing Escherichia coli O157:H7 among diarrheic patients and their cattle in Amhara National Regional State, Ethiopia

“…Amplification of rfb O157 and flic H7 are genes encoding for the O157 somatic and H7 flagellar antigens respectively. These were estimated by using adapted primers [ 31 , 32 ] ( Table 1 ). The amplification mixture contains 5μl platinum super II green master mix (Taq DNA polymerase, dNTPs, MgCl 2, and reaction buffer at optimal buffer concentration for efficient amplification of DNA templates by PCR, 1.25μl purified DNA, 0.25μl of each forward and reverse primer, then the volume was completed to 3.25μl by deionized water.…”

“…Amplification of rfb O157 and flic H7 genes. Isolates were confirmed as STEC O157:H7 by using specific primers for rfb O157 (292 bp) [30,31] and flic H7 genes (625bp) [30,32]. Amplification of rfb O157 and flic H7 are genes encoding for the O157 somatic and H7 flagellar antigens respectively.…”

Detection of Salmonella in Wastewater Irrigated Farms in Meknes City, Morocco

“…The Polymerase Chain Reaction (PCR) has proven to be one of the reliable and highly sensitive microbiological methods for the detection and identification of pathogenic bacteria [14]. PCR tests have been developed for the detection of pathogens in water, food and other environmental samples [15]. This study combined conventional and molecular methods to detect Salmonella in irrigation water, irrigated vegetables, and soil.…”