Application of Reverse Transcriptase –PCR (RT-PCR) for rapid detection of viable Escherichia coli in drinking water samples

Abstract

BackgroundPolymerase chain reaction (PCR) is preferred to other methods for detecting Escherichia coli (E. coli) in water in terms of speed, accuracy and efficiency. False positive result is considered as the major disadvantages of PCR. For this reason, reverse transcriptase-polymerase chain reaction (RT-PCR) can be used to solve this problem. The aim of present study was to determine the efficiency of RT-PCR for rapid detection of viable Escherichia coli in drinking water samples and enhance its sensitivity through application of different filter membranes.Materials and methodsSpecific primers were designed for 16S rRNA and elongation Factor II genes. Different concentrations of bacteria were passed through FHLP and HAWP filters. Then, RT-PCR was performed using 16srRNA and EF –Tu primers. Contamination of 10 wells was determined by RT-PCR in Arak city. To evaluate RT-PCR efficiency, the results were compared with most probable number (MPN) method.ResultsRT-PCR is able to detect bacteria in different concentrations. Application of EF II primers reduced false positive results compared to 16S rRNA primers. The FHLP hydrophobic filters have higher ability to absorb bacteria compared with HAWB hydrophilic filters. So the use of hydrophobic filters will increase the sensitivity of RT-PCR.ConclusionRT-PCR shows a higher sensitivity compared to conventional water contamination detection method. Unlike PCR, RT-PCR does not lead to false positive results. The use of EF-Tu primers can reduce the incidence of false positive results. Furthermore, hydrophobic filters have a higher ability to absorb bacteria compared to hydrophilic filters.

Molecular monitoring of disinfection efficacy of E. coli O157:H7 in bottled purified drinking water by quantitative PCR with a novel dye

“…The filter-sterilized (Millipore, pore diameter of 0.5 microns) water samples were artificially inoculated with E. coli O157:H7 to achieve two concentrations, 10 3 or 10 4 CFU/100 ml. Cell collection and preparation were same to the methods described by Molaee, Abtahi, Ghannadzadeh, Karimi, and Ghaznavi-Rad (2015) with some modifications.…”

The microbiome of translocated bacterial populations in patients with and without inflammatory bowel disease

“…12,13 Using these techniques, O'Brien et al investigated the microbiome of mesenteric lymph nodes and intestinal mucosa of the resection specimens of patients with IBD and controls. 20 For example, bacterial RNA is used to determine the presence of viable bacteria in drinking water, 21 and in the manufacture of probiotics, detection of bacterial RNA demonstrates the presence of living, albeit dormant, organisms weeks after storage, despite an inability to culture them. The microbiome of the regional lymph nodes correlated closely with that found at nearby mucosal sites.…”

“…18 This turnover makes bacterial RNA a surrogate marker for the presence of living and unstressed bacterial cells, 19 and it is regularly used as a proxy for the detection of living bacteria. 20 For example, bacterial RNA is used to determine the presence of viable bacteria in drinking water, 21 and in the manufacture of probiotics, detection of bacterial RNA demonstrates the presence of living, albeit dormant, organisms weeks after storage, despite an inability to culture them. 22 Furthermore, the presence of RNA identifies unculturable bacteria, such as latent Mycobacterium tuberculosis, that are in a dormant state due to metabolic stress.…”

Tracking bacterial virulence: global modulators as indicators

“…In contrast to tedious and time-consuming classical methodologies such as the Multiple Tube Fermentation technique, approaches such as the PCR, are rapid, highly specific and sensitive 35 36 . Different genes, such as 16S rDNA, EF-Tu 37 , uidA or yaiO 38 have been used to detect E. coli by PCR in environmental samples. For the detection of specific E. coli pathotypes, combinations of primers specific for virulence factors are designed 39 40 41 .…”