Quantitative PCR for Determining the Infectivity of Bacteriophage MS2 upon Inactivation by Heat, UV-B Radiation, and Singlet Oxygen: Advantages and Limitations of an Enzymatic Treatment To Reduce False-Positive Results

Abstract

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 ET with qPCR (ET-qPCR) can be used to accurately quantify the infectivity of the human viral surrogate bacteriophage MS2 upon partial inactivation by three treatments (heating at 72°C, singlet oxygen, and UV radiation). 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 were compared. We found that (i) qPCR alone caused false-positive results for all treatments, (ii) ET-qPCR significantly reduced (up to >5.2 log units) but did not eliminate the false-positive signals, and (iii) 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 improved the detection of infectivity loss relative to qPCR.Water-and food-borne viruses are a major worldwide source of gastroenteritis, and thus the detection and inactivation of infective viruses are important public health priorities. Cell culture can be employed to quantify the infectivity of certain viruses. However, culturing can take days to weeks to yield results, and many viruses important to public health, e.g., hepatitis A and noroviruses, are either difficult to culture or are nonculturable (3,19,38). Immunological and spectrometryand microscopy-based methods have also been developed, but none of them has provided a satisfactory alternative (5,26,43,45).The advent of PCR and quantitative PCR (qPCR) in environmental microbiology had raised hopes that these limitations would be overcome. While PCR lives up to its promise to provide rapid, sensitive, and specific detection of environmental viruses, its ability to differentiate infective from inactivated viruses has not been realized. This is mainly due to the persistence of genomes of inactivated viruses that remain either partially or fully intact. During PCR, intact genomic regions of inactivated viruses are amplified and produce confounding false-positive PCR signals (3,11,37,38,41). These false positives inhibit our ability to use qPCR to obtain quantitative inform...

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