☆ 4.7 Article

SARS-CoV-2 detection in wastewater using multiplex quantitative PCR

SCIENCE OF THE TOTAL ENVIRONMENT (2021)

Journal

SCIENCE OF THE TOTAL ENVIRONMENT

Volume 797, Issue -, Pages -

Publisher

ELSEVIER

DOI: 10.1016/j.scitotenv.2021.148890

Keywords

SARS-CoV-2; Wastewater; Multiplex assay; RT-qPCR; Environmental surveillance

Funding

Exploratory Research program under the project NanoMicrobials

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Abstract

A multiplex RT-qPCR method was developed for simultaneous detection of different SARS-CoV-2 genes. The results showed that the multiplex assay was comparable to single assay in detecting SARS-CoV-2, but faster and cost-effective.

A multiplex reverse transcription quantitative PCR (RT-qPCR)-based method was designed for the simultaneous detection of different SARS-CoV-2 genes. In this study, we used three target genes encoding for the nucleocapsid 1 and 3 (N1, N3), and the spike (S) proteins, all commonly used in the detection of SARS-CoV-2 in human and environmental samples. The performance of the multiplex assay, compared to the single assay was assessed for the standard calibration curve, required for absolute quantification, and then, for the real environmental samples to detect SARS-CoV-2. For this latter, four environmental samples were collected at a local wastewater treatment plant (WWTP). The results showed that the cycle threshold (Ct) values of the multiplex were comparable to the values obtained by the singleplex PCR. The amplification of the three target genes indicated the presence of SARS-CoV-2 in the four water samples with an increasing trend in February and these results were confirmed in the multiplex approach, showing the robustness of this method and its applicability for the relative abundance analysis among the samples. Overall, both the laboratory and field work results demonstrated that the multiplex PCR assay developed in this study could provide a method for SARS-CoV-2 detection as robust as the single qPCR, but faster and cost-effective, reducing by three times the number of reactions, and consequently the handling time and reagents. (c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Circulation of SARS-CoV-2 Omicron sub-lineages revealed by multiplex genotyping RT-qPCR assays for sewage surveillance

Jiahui Ding, Xiaoqing Xu, Yu Deng, Xiawan Zheng, Tong Zhang

Summary: Sewage surveillance is an important tool in combating the COVID-19 pandemic, and this study establishes a rapid and high-throughput detection workflow to track the circulation of Omicron BA.4/BA.5 variants in Hong Kong. The multiplex RT-qPCR assays used in this study accurately discriminate and quantitate target genotypes, even in the presence of multiple variants in sewage samples. The study demonstrates the feasibility and applicability of multiplex RT-qPCR assays for monitoring epidemic trends and tracking variant displacement dynamics in sewage samples.

SCIENCE OF THE TOTAL ENVIRONMENT (2023)