Evaluation of existing lacZ primers and de novo design of an optimized qPCR assay to quantify coliform bacteria in drinking water

Abstract

Aims This study aimed to evaluate existing and de novo lacZ primers using in silico and experimental validation to develop a quantitative polymerase chain reaction (qPCR) assay capable of reliably quantifying coliforms and differentiating them from non-coliform Enterobacteriaceae as currently defined. Methods and results A comprehensive lacZ sequence database was compiled to define coliform and non-coliform targets. Both published and de novo primers were assessed for specificity and coverage. The de novo primer set LZ1 (F: CCGWGYRTKATCATCTGGTC, R: TSATCSACGCGSGCGTACAT; 173 bp amplicon) showed 87.5% coverage of the test panel and was optimal for qPCR. Compared with culture-based methods and flow cytometry, LZ1 quantified Escherichia coli in drinking water at 1 × 103 cfu 100 ml-1. The limit of quantification indicated an 80% probability of detecting 100 copies with > 3 replicates. Existing primer LZ3 best distinguished coliforms from non-coliforms and is a promising target for identification. Conclusions We present a validated qPCR assay targeting the lacZ gene, supported by in silico and experimental validation, and a phylogenetic analysis of lacZ and 16S rRNA sequences, highlighting the challenges associated with coliform detection.