Framework for Using Quantitative PCR as a Nonculture Based Method To Estimate Virus Infectivity

Measuring the efficiency of virus disinfection with quantitative PCR (qPCR) has been criticized as inadequate due to the production of false-positive signals. Such a claim, however, presupposes an understanding of the theoretical qPCR response. Many studies have assumed that the loss in qPCR signal upon disinfection should equal the loss in infectivity, without accounting for the fact that qPCR typically assays only a fraction of the viral genome. This study aimed to develop a theoretical framework to relate viral infectivity with genome damage measured by qPCR. The framework quantified damage to the entire genome based on the qPCR amplification of smaller sections, assuming single-hit inactivation and a Poissonian distribution of damage. The framework was tested and modified using UV(254) inactivation studies with bacteriophage MS2 (culturing and qPCR of approximately half the genome). Genome regions showed heterogeneous sensitivities to UV(254) treatment, thus deviating from the assumption of Poissonian damage. We offered two modifications to account for these deviations and confirmed that the qPCR-based framework accurately estimated virus infectivity. This framework offers the potential to monitor the infectivity of viruses that remain nonculturable (norovirus). While developed for UV(254)-inactivated virus, the framework should apply to any disinfection technique that causes inactivation via single genomic lesions.