Reliability of non-culturable virus monitoring by PCR-based detection methods in environmental waters containing various concentrations of target RNA.

Owing to the lack of practical cell culture system for human noroviruses (HuNoV), various detection methods based on conventional reverse transcription-PCR (RT-PCR) and the quantitative real-time PCR have been major tools for monitoring environmental water safety. In this study, we showed that the proportion of water sample concentrates used for one-step RT-PCR significantly influences false-negative findings of the non-culturable viruses. In total, 59 archived samples of previously analyzed water concentrates were reexamined for HuNoV RNA by the one-step RT-PCR and semi-nested PCR. Using new aliquots for RNA extraction for every trial, up to 20 PCR trials were performed for each archive to determine whether the crosscheck results supported the previous determinations. We reconfirmed that 27.6% (8/29) of the samples were HuNoV-positive samples: 6.7% (1/15) from groundwater, 33.3% (3/9) from river water, and 80% (4/5) from treated sewage effluent (TSE). These results corresponded to the ratio of previously negative HuNoV samples now identified as positive (8/30): 6.7% (1/15) from groundwater, 20% (1/5) from river water, and 60% (6/10) from TSE. To elucidate the cause of these results, 16 different concentrations of murine norovirus (MNV) RNA (from 2×10(2) to 8×10(3) copies, divided into 10 tubes for each concentration) were subjected to one-step RT-PCR. The detection frequency and reproducibility decreased sharply when the number of MNV RNA copies fell below threshold levels. These observations suggest that the proportion of water concentrate used for PCR-based detection should be considered carefully when deciding viral presence in certain types of environmental water, particularly in regard with legal controls.