Development of a Novel Quantitative PCR Assay for Somatic Coliphages to Advance Water Safety Diagnostics

Abstract Somatic coliphages are recognized reliable indicators of faecal contamination in water. Their routine monitoring relies on time-consuming, labour-demanding culture-based methods, which are limited to detecting total somatic coliphages. Here, we developed and validated a novel family-specific quantitative PCR (qPCR) assay targeting four major families of somatic coliphages— Myoviridae , Podoviridae , Siphoviridae , and Microviridae —to enhance sensitivity, specificity, and throughput. The assays were designed using in silico primer screening against reference genomes. The qPCR assay was specific to the phage families (100% assay specificity within the tested prototypes) and had a LOD and LOQ of 10.4 copies/µl, 2.7 copies/µl, 26.5 copies/µl, and 3.3 copies/µl for Myoviridae, Microviridae, Podoviridae and Siphoviridae , respectively. Efficiencies between 90 and 110% were achieved. Coefficients of determination ( R 2 ) were 0.993–0.996. Comparison of ISO (culture-based method) and qPCR methods revealed significant correlations ( p < 0.05), showing that the methods are comparable. The qPCR method was tested on a range of environmental waters to demonstrate wide application of the assay. This showed season and source-specific patterns in phage family distribution, with Podoviridae and Siphoviridae significantly enriched in winter-collected farm water samples, and Myoviridae dominating in farm summer samples, and a more even phage-family relative abundance in wastewater with no significant differences. We suggest that this qPCR assay could be used as a screening tool to complement standard protocols for regulatory monitoring, ensuring drinking water safety. It is also a valuable research tool for investigating the environmental ecology of somatic coliphages and understanding sources of faecal pollution.