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

ABSTRACT: 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.

SUBMITTER: Navarro A

PROVIDER: S-EPMC8278834 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The ongoing pandemic caused by the emergence of SARS-CoV-2 has resulted in millions of deaths worldwide despite the various measures announced by the authorities. Wastewater-based epidemiology has the ability to provide a day-to-day estimation of the number of infected people in a fast and cost-effective manner. However, owing to the complex nature of wastewater, wastewater monitoring for viral genome copies is affected by the extensive viral fragmentation that takes place all the way to the sewage and the analytical lab. The aim of this study was to evaluate different methodologies for the concentration and extraction of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. We compare 5 different concentration methods and 4 commercially available kits for the RNA extraction. To evaluate the performance and the recovery of these, SARS-CoV-2 isolated from patients was used as a spike control. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and N3). Using spiked samples, recoveries were estimated 2.1–37.6% using different extraction kits and 0.1–2.1% using different concentration kits. It was found that a direct capture-based method, evaluated against a variety of concentration methods, is the best in terms of recovery, time and cost. Interestingly, we noticed a good agreement between the results provided by RT-qPCR and RT-ddPCR in terms of recovery. This evaluation can serve as a guide for laboratories establishing a protocol to perform wastewater monitoring of SARS-CoV-2. Overall, data presented here reinforces the validity of WBE for SARS-CoV-2 surveillance, uncovers potential caveats in the selection of concentration and extraction protocols and points towards optimal solutions to maximize its potential. Graphical abstract Image 1

Project description:Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

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

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