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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(TNA) 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.

SUBMITTER: Mondal S

PROVIDER: S-EPMC8262391 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Current technologies for targeted characterization and manipulation of viral RNA primarily involve amplification or ultracentrifugation with isopycnic gradients of viral particles to decrease host RNA background. The former strategy is non-compatible for characterizing properties innate to RNA strands such as secondary structure, RNA-RNA interactions, and also for nanopore direct RNA sequencing involving the sequencing of native RNA strands. The latter strategy, ultracentrifugation, causes loss in genomic information due to its inability to retrieve unassembled viral RNA. To address this, we developed a novel application of current nucleic acid hybridization technologies for direct characterization of RNA. In particular, we modified a current enrichment protocol to capture whole viral native RNA genomes for downstream RNA assays to circumvent the abovementioned problems. This technique involves hybridization of biotinylated baits at 500 nucleotides (nt) intervals, stringent washes and release of free native RNA strands using DNase I treatment, with a turnaround time of about 6 h 15 min. RT-qPCR was used as the primary proof of concept that capture-based purification indeed removes host background. Subsequently, capture-based purification was applied to direct RNA sequencing as proof of concept that capture-based purification can be coupled with downstream RNA assays. We report that this protocol was able to successfully purify viral RNA by 561- to 791-fold. We also report that application of this protocol to direct RNA sequencing yielded a reduction in human host RNA background by 1580-fold, a 99.91% recovery of viral genome with at least 15× coverage, and a mean coverage across the genome of 120×. This report is, to the best of our knowledge, the first description of a capture-based purification method for assays that involve direct manipulation or characterisation of native RNA. This report also describes a successful application of capture-based purification as a direct RNA sequencing strategy that addresses certain limitations of current strategies in sequencing RNA viral genomes.

Project description:Insensitivity and technical complexity have impeded the implementation of high-throughput nucleic acid sequencing in differential diagnosis of viral infections in clinical laboratories. Here, we describe the development of a virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) that increases the sensitivity of sequence-based virus detection and characterization. The system uses ~2 million probes that cover the genomes of members of the 207 viral taxa known to infect vertebrates, including humans. A biotinylated oligonucleotide library was synthesized on the NimbleGen cleavable array platform and used for solution-based capture of viral nucleic acids present in complex samples containing variable proportions of viral and host nucleic acids. The use of VirCapSeq-VERT resulted in a 100- to 10,000-fold increase in viral reads from blood and tissue homogenates compared to conventional Illumina sequencing using established virus enrichment procedures, including filtration, nuclease treatments, and RiboZero rRNA subtraction. VirCapSeq-VERT had a limit of detection comparable to that of agent-specific real-time PCR in serum, blood, and tissue extracts. Furthermore, the method identified novel viruses whose genomes were approximately 40% different from the known virus genomes used for designing the probe library. The VirCapSeq-VERT platform is ideally suited for analyses of virome composition and dynamics. IMPORTANCE : VirCapSeq-VERT enables detection of viral sequences in complex sample backgrounds, including those found in clinical specimens, such as serum, blood, and tissue. The highly multiplexed nature of the system allows both the simultaneous identification and the comprehensive genetic characterization of all known vertebrate viruses, their genetic variants, and novel viruses. The operational simplicity and efficiency of the VirCapSeq-VERT platform may facilitate transition of high-throughput sequencing to clinical diagnostic as well as research applications.

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A direct capture method for purification and detection of viral nucleic acid enables epidemiological surveillance of SARS-CoV-2.

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