Capsid integrity RT-qPCR for the selective detection of intact SARS-CoV-2 in wastewater

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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.

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Journal

Journal ID (nlm-ta): Sci Total Environ

Journal ID (iso-abbrev): Sci Total Environ

Title: The Science of the Total Environment

Publisher: Published by Elsevier B.V.

ISSN (Print): 0048-9697

ISSN (Electronic): 1879-1026

Publication date PMC-release: 8 June 2021

Publication date (Electronic): 8 June 2021

Electronic Location Identifier: 148342

Affiliations

[a ]Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

[b ]Graduate School of Agricultural and Life Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8657, Japan

Author notes

[* ]Corresponding authors.

Article

Publisher Item ID: S0048-9697(21)03413-6 Publisher ID: 148342

DOI: 10.1016/j.scitotenv.2021.148342

PMC ID: 8184355

PubMed ID: 34139497

SO-VID: bc1f6847-5e09-4664-99a5-943c6129099c

License:

Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

History

Date received : 7 May 2021

Date revision received : 4 June 2021

Date accepted : 5 June 2021

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