Abstract
Wastewater surveillance of SARS-CoV-2 has become a promising tool to estimate population-level changes in community infections and the prevalence of COVID-19 disease. Although many studies have reported the detection and quantification of SARS-CoV-2 in wastewater, remarkable variation remains in the methodology. In this study, we validated a molecular testing method by concentrating viruses from wastewater using ultrafiltration and detecting SARS-CoV-2 using one-step RT-qPCR assay. The following parameters were optimized including sample storage condition, wastewater pH, RNA extraction and RT-qPCR assay by quantification of SARS-CoV-2 or spiked human coronavirus strain 229E (hCoV-229E). Wastewater samples stored at 4 °C after collection showed significantly enhanced detection of SARS-CoV-2 with approximately 2-3 PCR-cycle threshold (Ct) values less when compared to samples stored at -20 °C. Pre-adjustment of the wastewater pH to 9.6 to aid virus desorption followed by pH readjustment to neutral after solid removal significantly increased the recovery of spiked hCoV-229E. Of the five commercially available RNA isolation kits evaluated, the MagMAX-96 viral RNA isolation kit showed the best recovery of hCoV-229E (50.1 ± 20.1%). Compared with two-step RT-qPCR, one-step RT-qPCR improved sensitivity for SARS-CoV-2 detection. Salmon DNA was included for monitoring PCR inhibition and pepper mild mottle virus (PMMoV), a fecal indicator indigenous to wastewater, was used to normalize SARS-CoV-2 levels in wastewater. Our method for molecular detection of SARS-CoV-2 in wastewater provides a useful tool for public health surveillance of COVID-19.
Keywords: PMMoV; RT-qPCR; SARS-CoV-2; Wastewater; hCoV-229E.
【저자키워드】 SARS-CoV-2, RT-qPCR, wastewater, PMMoV, hCoV-229E., 【초록키워드】 COVID-19, viruses, public health, coronavirus, Variation, Infection, virus, COVID-19 disease, RNA extraction, DNA, SARS-CoV-2 detection, sensitivity, RT-qPCR, wastewater, Viral, PCR, Surveillance, cycle threshold, qPCR, HCoV, Isolation, Community, Mild, HCoV-229E, Viral RNA, methodology, molecular, threshold, quantification, disease, 229E, Neutral, Ultrafiltration, fecal, followed by, best, community infections, parameter, FIVE, significantly increased, human coronavirus, was used, significantly, reported, evaluated, provide, less, changes in, prevalence of COVID-19, RNA isolation kit, Salmon, 【제목키워드】 molecular, quantification,