Pretreatment with propidium monoazide/sodium lauroyl sarcosinate improves discrimination of infectious waterborne virus by RT-qPCR combined with magnetic separation

摘要

AbstractRT-qPCR allows sensitive detection of viral particles of both infectious and noninfectious viruses in water environments, but cannot discriminate non-infectious from infectious viruses. In this study, we aimed to optimize RT-qPCR-based detection of chlorine-inactivated human norovirus (NoV) and pepper mild mottle virus (PMMoV) in suspension by pretreatment with an optimal combination of a monoazide and a detergent that can efficiently penetrate damaged viral capsids. Four methods were compared to determine the efficacy of chlorine disinfection (at 1, 3, and 5 min mg/L): (A) RT-qPCR alone, (B) RT-qPCR assay preceded by magnetic bead separation for enrichment of viral particles (MBS-RT-qPCR), (C) MBS-RT-qPCR assay with pretreatment with propidium monoazide (PMA-MBS-RT-qPCR), and (D) PMA-MBS-RT-qPCR assay with pretreatment with sodium lauroyl sarcosinate (INCI-PMA-MBS-RT-qPCR). On the basis of a PMA optimization assay, 200 and 300 μM PMA were used in subsequent experiments for NoV GII.4 and PMMoV, respectively. Optimal INCI concentrations, having minimal influence on NoV GII.4 and PMMoV, were found to be 0.5% and 0.2% INCI, respectively. For NoV GII.4, there were significant differences (P < 0.05) in log10genome copies between the PMA-treated and the INCI + PMA-treated samples (log10genome copies differed by 1.11 and 0.59 log10for 3 and 5 min mg/L of chlorine, respectively). For PMMoV, INCI induced differences in log10genome copies of 0.92, 1.18, and 1.86, for 1, 3, and 5 min mg/L of chlorine, respectively. Overall, the results of this study indicate that an optimal combination of PMA and INCI could be very useful for evaluating disinfection methods in water treatment strategies.Graphical abstractDisplay OmittedHighlights•Propidium monoazide (PMA) pretreatment improves RT-qPCR detection of NoV and PMMoV.•Sodium lauroyl sarcosinate (INCI) pretreatment further improves virus detection.•Chlorine disinfection (1–5.0 min mg/L) effectively reduces NoV and PMMoV titers.•PMA and INCI might help evaluate disinfection methods in water treatment strategies.