Evaluation of propidium monoazide and long-amplicon qPCR as an infectivity assay for coliphage

Standardized and rapid assays for viable viral pathogens are needed to inform human health risk assessments. Conventional qPCR is designed to enumerate the gene copies of an organism in a sample, but does not identify those that originated from a viable pathogen. This study was undertaken to evaluate modified qPCR methods as infectivity assays for the enumeration of infectious MS2 coliphage. Propidium monoazide (PMA) treatment coupled with long-amplicon qPCR assays were assessed for their ability to quantify infectious MS2 in pure cultures and following inactivation by a range of UV light exposures and chlorine doses. The qPCR results were compared to the plaque assay, which was used as the standard to indicate the level of infectious MS2 in each sample. For pure cultures, PMA-qPCR results were not significantly different from the plaque assay (p>0.05). At >4 log inactivation, combined PMA and long-amplicon qPCR assays overestimated the level of infectious MS2 remaining (p<0.05). The most accurate long-amplicon qPCR infectivity assay targeted a 624-bp region at the 5' end of the genome. Modified qPCR approaches may be useful tools to monitor the loss of infectivity as a result of disinfection processes.