Publications
Environmental Science & Technology LettersJul 2021 DOI:
10.1021/acs.estlett.1c00408

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

Ni, Gaofeng; Lu, Ji; Maulani, Nova; Tian, Wei; Yang, Lin; Harliwong, Ivon; Wang, Zhiyao; Mueller, Jochen; Yang, Bicheng; Yuan, Zhiguo; Hu, Shihu; Guo, Jianhua
Product Used
NGS
Abstract
Most previous studies of SARS-CoV-2 monitoring in wastewater involved an initial step to remove the solid fraction of wastewater and focus the extraction and analysis of viral concentration solely on the basis of the liquid fraction.(4,13,35,36) This neglects the fact that organic matter and suspended solids in the wastewater matrix are likely to offer the surface for adsorption and protection for viral particles.(37−39) Our current study revealed that despite the inferred concentration being extremely low, a consistently stronger viral signal was detected from the solid fraction of wastewater rather than the liquid fraction. Multiple studies also pointed out the possibility that particles or solid-state substances in municipal wastewater might harbor a considerable amount of virus.(30−33) For instance, by investigating the adsorption and inactivation kinetics among enveloped viruses [murine hepatitis virus (MHV) and _Pseudomonas_ phage Φ6] and non-enveloped viruses (_Enterobacteria_ phage MS2 and T3), Ye and colleagues reported that ≤26% of the two enveloped viruses attached to the solid fraction of wastewater, while 6% of the non-enveloped viruses was attached to the solid fraction.(33) Balboa et al. thoroughly investigated the presence of SARS-CoV-2 viruses in Spanish WWTPs and suggested that most SARS-CoV-2 viral particles were concentrated in the sludge lines, indicating that the solid fraction of wastewater is a potential hot spot for detecting SARS-CoV-2.(32) Moreover, a systematic assessment of the adsorption behavior of waterborne viruses to solid interfaces has pointed out that electrostatics and the hydrophobic effect of different surfaces favor adsorption of the virus onto different solids.(40) Our findings support these previous studies,(3,8,30−32) which suggest that inclusion of the solid phase in the analysis of wastewater will maximize recovery of viral signals in wastewater and improve its usefulness as a surveillance tool for WBE. It should be noted that methods of RNA extraction could affect downstream analysis, and its optimization is an ongoing research and development area.(41,42) The total RNA extraction kit (i.e., PowerMicrobiome) used in this study is optimized toward solid fraction extraction, as the liquid fraction of wastewater might dilute the initial lysing buffer. To minimize systematic biases from different extraction kits, we chose a single extraction method for both solid and liquid fractions. This might lead to variation in the extraction efficiency caused by a difference in the physical state of the samples (liquid vs solid), which should be assessed in the future.
Product Used
NGS

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