Design and testing of a PCR assay to quantify coliform bacteria in drinking water

Here we present an enhanced lacZ qPCR assay targeting {beta}-galactosidase, encoded by lacZ, to assess coliforms in drinking water. Coliform bacteria are a diverse group of over 80 species, commonly found in enteric environments, and act as indicators of water supply contamination. Due to the significant variation among coliforms, and the size of the group designing a single target was challenging, as previous studies have demonstrated. To address this, a coliform lacZ sequence database was created, and the phylogeny of the group was reviewed. The degree of phylogenetic differences both between and among different coliform genera is indeed large, raising questions about their current definition. Using the database, current and new primer sets were tested for specificity and coverage both in silico and in vitro. The de novo primer set, LZ1, was found to be the most effective for qPCR. When compared directly with traditional culture-based methods and flow cytometry total cell counts, the LZ1 lacZ qPCR assay quantified E. coli in drinking water down to a concentration of 1.82 x 103 cfu/100mL, equivalent to 360 cfu per qPCR reaction. ImportanceColiform bacteria and E. coli are key indicators of faecal contamination in drinking water. Compliance with U.K., U.S.A., or European regulations requires that drinking water be free of these organisms. However, the culture-based methods used to quantify these bacteria have remained unchanged for over 100 years, relying on the culturability of the organisms, sampling only a small volume at a single time point, and lacking direct correlation to pathogenic microbes. There is a pressing need for a rapid, high-throughput analysis capable of detecting coliforms more representatively, without relying solely on culturability. By analysing larger volumes or more replicates, this qPCR assay could potentially be used to assess the risk of drinking water contamination over longer periods of time, offering a more comprehensive evaluation compared to traditional, culture-dependent methods.