Novel Multiplexed Amplicon-Based Sequencing to Quantify SARS-CoV-2 RNA from Wastewater

Gaofeng Ni; Ji Lu; Nova Maulani; Wei Tian; Lin Yang; Ivon Harliwong; Zhiyao Wang; Jochen Mueller; Bicheng Yang; Zhiguo Yuan; Shihu Hu; Jianhua Guo

doi:10.1021/acs.estlett.1c00408

Novel Multiplexed Amplicon-Based Sequencing to Quantify SARS-CoV-2 RNA from Wastewater

Environ Sci Technol Lett. 2021 Jul 8;8(8):683-690. doi: 10.1021/acs.estlett.1c00408. eCollection 2021 Jan 10.

Authors

Gaofeng Ni¹, Ji Lu¹, Nova Maulani¹, Wei Tian², Lin Yang², Ivon Harliwong², Zhiyao Wang¹, Jochen Mueller³, Bicheng Yang², Zhiguo Yuan¹, Shihu Hu¹, Jianhua Guo¹

Affiliations

¹ Advanced Water Management Centre, The University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia.
² BGI Australia, 300 Herston Road, Herston, Brisbane, QLD 4006, Australia.
³ Queensland Alliance for Environmental Health Sciences (QAEHS), The University of Queensland, 20 Cornwall Street, Woolloongabba, QLD 4103, Australia.

PMID: 37566375
DOI: 10.1021/acs.estlett.1c00408

Abstract

The application of wastewater-based epidemiology (WBE) to support the global response to the COVID-19 pandemic has shown encouraging outcomes. The accurate, sensitive, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in municipal wastewater is critical for WBE. Here, we present a novel approach based on multiplexed amplicon-based sequencing, namely the ATOPlex platform, for detecting SARS-CoV-2. The ATOPlex platform is capable of quantifying SARS-CoV-2 RNA at concentrations that are at least 1 order of magnitude lower than the detection limit of reverse transcription quantitative polymerase chain reaction (RT-qPCR). Robust and accurate phylogenetic placement can be done at viral concentrations 4 times lower than the detection limit of RT-qPCR. We further found that the solid fraction in wastewater harbors a considerable amount of viral RNA, highlighting the need to extract viral RNA from the solid and liquid fractions of wastewater. This study delivers a highly sensitive, phylogenetically informative, and high-throughput analytical workflow that facilitates the application of WBE.