Quantitative PCR for determining the infectivity of bacteriophage MS2 upon inactivation by heat, UVB and singlet oxygen: advantages and limitations of an enzymatic treatment to reduce false-positive results

Health risks posed by waterborne viruses are difficult to assess because it is tedious or impossible to determine the infectivity of many viruses. Recent studies hypothesized that quantitative PCR (qPCR) could selectively quantify infective viruses if preceded by an enzymatic treatment (ET) to reduce confounding false-positive signals. The goal of this study was to determine if enzymatic treatment qPCR (ET-qPCR) can be used to accurately quantify the infectivity of the human viral surrogate, bacteriophage MS2, upon partial inactivation by three treatments (72°C, singlet oxygen, and UV). Viruses were inactivated in buffered solutions and a lake water sample, and assayed with culturing, qPCR, and ET-qPCR. To ensure that inactivating genome damage was fully captured, primer sets that covered the entire coding region were used. The susceptibility of different genome regions and the maximum genomic damage after each inactivating treatment was compared. We found that 1) qPCR alone caused false-positive results for all treatments, 2) ET-qPCR significantly reduced (up to >5.2 log units), but did not eliminate, the false-positive signals, 3) the elimination of false-positive signals differed between inactivating treatments. By assaying the whole coding region, we demonstrated that genome damage only partially accounts for virus inactivation. The possibility of achieving complete accordance between culture- and PCR-based assays is therefore called into doubt. Despite these differences, we postulate that ET-qPCR can track infectivity given that decreases in infectivity were always accompanied by dose-dependent decreases in ET-qPCR signal. By decreasing false-positive signals ET-qPCR facilitated the detection of infectivity loss compared to qPCR.