Molecular Method for Detection of Total Coliforms in Drinking Water Samples

Abstract

e This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culturebased methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality.

Detection of Microbiological Content in Water as Indicator of Its Ecological State − Mini‐Review

“…PCR is largely used as the most accurate diagnostic technique, which provides for detection of low microbial titers, can predict the number of target organisms (qPCR), simultaneously registers different microorganisms with multiple sets of primers . Evaluation of microbial diversity with PCR is performed by whole‐genome sequencing , target gene sequencing , or such generalized community fingerprinting techniques as terminal restriction fragment length polymorphism, T‐RELP, denaturing gradient gel electrophoresis (DGGE), and temperature gradient gel electrophoresis (TGGE), based on the size of the amplification products . The traditional gene target for bacterial identification is 16S rRNA.…”

“…The traditional gene target for bacterial identification is 16S rRNA. When three gene targets ( lacZ , wecG , and 16S rRNA) were used in PCR assays of well water quality, the following results were obtained: 79.5% of the samples tested by culture‐based methods and 77.9, 67.2, and 80.3% of the samples tested by PCR‐based methods, respectively, contained TC, which illustrates advantages of the 16S rRNA technique . Yet, in some cases 16S rRNA gene target shows limitations in differentiating bacterial taxa, distinct in plasmid structure or else .…”

Real-Time TaqMan PCR for Rapid Detection and Quantification of Coliforms in Chilled Meat

“…Moreover, DNA-based methods offer the option of storing samples until analysis which is most useful in field sampling. Now, there are many polymerase chain reaction (PCR)-based molecular methods to detect coliforms targeting the 16S to 23S ribosomal RNA (rRNA) (Soejima et al 2012), 16S rRNA (Kämpfer et al 2008;Maheux et al 2014), and β-galactosidase (lacZ) gene (Bej et al 1990;Martín et al 2010;Tantawiwat et al 2005) nucleotide sequences. However, since the copy number of the 16S rRNA and 16S-23S rRNA can vary from one to as many as hundred copies (Dick and Field 2004), total bacterial counts evaluated by the number of 16S rRNA or16S-23S rRNA may fluctuate according to the differences of microbiota in food matrix.…”

Molecular detection by analysis of the 16S rRNA gene of fecal coliform bacteria from the two Korean Apodemus species (Apodemus agrarius and A. peninsulae)

“…The bacterial 16S rRNA gene has been used to detect bacterial pathogens from various environmental sources such as wildlife feces (Maciel et al, 2011), drinking water (Maheux et al, 2014;Zhang et al, 2015), agricultural vegetables (Gorski et al, 2011), and soil (Gorski et al, 2011). In South Korea, however, there have been few studies detecting pathogenic bacteria from fecal coliforms of wild mice using molecular markers such as the 16S rRNA gene.…”