Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes have been detected in wastewater worldwide. However, the assessment of SARS-CoV-2 infectivity in wastewater has been limited due to the stringent requirements of biosafety level 3. The main objective of this study is to investigate the applicability of capsid integrity RT-qPCR for the selective detection of intact SARS-CoV-2 in wastewater. Three capsid integrity reagents, namely ethidium monoazide (EMA, 0.1-100 μM), propidium monoazide (PMA, 0.1-100 μM), and cis-dichlorodiammineplatinum (CDDP, 0.1-1000 μM), were tested for their effects on different forms (including free genomes, intact and heat-inactivated) of murine hepatitis virus (MHV), which was used as a surrogate for SARS-CoV-2. CDDP at a concentration of 100 μM was identified as the most efficient reagent for the selective detection of infectious MHV by RT-qPCR (CDDP-RT-qPCR). Next, two common virus concentration methods including ultrafiltration (UF) and polyethylene glycol (PEG) precipitation were investigated for their compatibility with capsid integrity RT-qPCR. The UF method was more suitable than the PEG method since it recovered intact MHV (mean ± SD, 38% ± 29%) in wastewater much better than the PEG method did (0.013% ± 0.015%). Finally, CDDP-RT-qPCR was compared with RT-qPCR alone for the detection of SARS-CoV-2 in 16 raw wastewater samples collected in the Greater Tokyo Area. Five samples were positive for SARS-CoV-2 when evaluated by RT-qPCR alone. However, intact SARS-CoV-2 was detected in only three positive samples when determined by CDDP-RT-qPCR. Although CDDP-RT-qPCR was unable to determine the infectivity of SARS-CoV-2 in wastewater, this method could improve the interpretation of positive results of SARS-CoV-2 obtained by RT-qPCR.
Keywords: Capsid integrity RT-qPCR; SARS-CoV-2; concentration method; intact virus; wastewater.
【저자키워드】 SARS-CoV-2, Wastewater., Concentration method, Capsid integrity RT-qPCR, intact virus, 【초록키워드】 severe acute respiratory syndrome coronavirus 2, coronavirus, Genome, severe acute respiratory syndrome Coronavirus, virus, RT-qPCR, wastewater, qPCR, Interpretation, genomes, MHV, murine hepatitis virus, Concentration, EMA, Polyethylene glycol, Ultrafiltration, PMA, Tokyo, determined by, biosafety level, level 3, glycol, acute respiratory syndrome, acute respiratory syndrome coronavirus, reagents, positive result, selective, applicability, positive, surrogate, positive sample, capsid, Effect, heat-inactivated, Biosafety Level 3, IMPROVE, tested, was used, collected, investigated, evaluated, form, determine, Area, 【제목키워드】 selective, capsid,