Efficient quantification of somatic coliphages in water: Development and validation of the Enumera® Rapid kit

The integration of somatic coliphage analysis into water quality regulations has driven the development of more streamlined, easier, and faster detection methods. These include the Bluephage method, initially designed for the qualitative assessment of coliphages in 100 mL water samples. In the present study this technique was adapted for quantitative analysis using the most probable number method, enabling quantification of somatic coliphages in 100 mL water samples within 6.5 h of incubation. Early readings were optimised using an algorithm developed from an extensive dataset of over 400 environmental samples. This new quantitative method has been refined and commercialised as the Enumera® Rapid kit (BPF-SE, Bluephage S.L.), which includes a new reference somatic coliphage, strain GR8. After sequencing and further characterisation, it was confirmed that the new strain belongs to the same species as the reference somatic coliphage of the ISO 10705-2 method. The Enumera® Rapid kit was then validated by assessing its efficacy across 151 samples from various water matrices. The method provided a mean recovery rate of 103 %, ranging from 95 % for drinking water to 116 % for wastewater, at concentrations from 0 to 300 PFU/100 mL. In comparison, ISO 10705-2 applied after membrane filtration yielded a mean recovery rate of 63.8 %, with the highest rate observed for wastewater (88.9 %). The results obtained with the Enumera® Rapid kit did not differ significantly compared to the reference method after adjusting for potential losses during the concentration stage outlined in ISO 10705-3. The new method also demonstrated a robust accuracy of 0.16 logs, good linearity, and achieved a limit of detection and quantification of 1 MPN/100 mL. The Enumera® Rapid kit enables rapid and straightforward quantification of somatic coliphages in 100 mL water samples within a single working day. Its streamlined workflow serves as a useful tool for microbial water quality monitoring.