A Direct Capture Method for Purification and Detection of Viral Nucleic Acid Enables Epidemiological Surveillance of SARS-CoV-2

Abstract

Studies have demonstrated that SARS-CoV-2 RNA can be detected in the feces of infected individuals. This finding spurred investigation into using wastewater-based epidemiology (WBE) to monitor SARS-CoV-2 RNA and track the appearance and spread of COVID-19 in communities. SARS-CoV-2 is present at low levels in wastewater, making sample concentration a prerequisite for sensitive detection and utility in WBE. Whereas common methods for isolating viral genetic material are biased toward intact virus isolation, it is likely that a relatively low percentage of the total SARS-CoV-2 RNA genome in wastewater is contained within intact virions. Therefore, we hypothesized that a direct unbiased total nucleic acid extraction method could overcome the cumbersome protocols, variability and low recovery rates associated with the former methods. This led to development of a simple, rapid, and modular alternative to existing purification methods. In an initial concentration step, chaotropic agents are added to raw sewage allowing binding of nucleic acid from free nucleoprotein complexes, partially intact, and intact virions to a silica matrix. The eluted nucleic acid is then purified using manual or semi-automated methods. RT-qPCR enzyme mixes were formulated that demonstrate substantial inhibitor resistance. In addition, multiplexed probe-based RT-qPCR assays detecting the N1, N2 (nucleocapsid) and E (envelope) gene fragments of SARS-CoV-2 were developed. The RT-qPCR assays also contain primers and probes to detect Pepper Mild Mottle Virus (PMMoV), a fecal indicator RNA virus present in wastewater, and an exogenous control RNA to measure effects of RT-qPCR inhibitors. Using this workflow, we monitored wastewater samples from three wastewater treatment plants (WWTP) in Dane County, Wisconsin. We also successfully sequenced a subset of samples to ensure compatibility with a SARS-CoV-2 amplicon panel and demonstrated the potential for SARS-CoV-2 variant detection. Data obtained here underscore the potential for wastewater surveillance of SARS-CoV-2 and other infectious agents in communities.

Evaluation of Methods and Processes for Robust Monitoring of SARS-CoV-2 in Wastewater

“…Ultimately, the only two methods that efficiently concentrated both SARS-CoV-2 and PMMoV were Centricon units coupled with Qiagen extraction and either of two tested kit versions from Promega (Wizard Enviro TNA Kit and Maxwell RSC Enviro TNA Kit). These conclusions are in accordance with previous similar studies (Mondal et al, 2021;Pecson et al, 2021), where they also point out that the Promega kits offered higher throughput in comparison to Centricon Filters. The two methods perform similarly for the detection of SARS-CoV-2, but there is a preferentially higher concentration efficiency of PMMoV with the Promega kit (Fig.…”

Bam-readcount - rapid generation of basepair-resolution sequence metrics

“…It has been adopted as a lightweight variant caller, finding known mutations in pre-leukemic phenotypes and used for detecting therapy-altering mutations from cell-free DNA (Wyatt et al, 2016;Xie et al, 2014). Viral researchers have tracked nucleotide changes across samples to understand diversity in Varicella Zoster Virus Encephalitis and to perform epidemiological surveillance in wastewater of SARS-CoV-2 (Depledge et al, 2018;Mondal et al, 2021). Those with RNA-sequencing data have found it useful for identifying allele-specific expression in cancer, or for enabling copy-number detection in single-cell RNA sequencing by retrieving allele frequencies (Cancer Genome Atlas Research Network et al, 2013;Müller et al, 2018).…”