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

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 (R2) 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. (Figure presented.)