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

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.