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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 | 2021 Nov

REPOSITORIES: biostudies-literature

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

Navarro Anna A Gómez Livia L Sanseverino Isabella I Niegowska Magdalena M Roka Eszter E Pedraccini Rosalba R Vargha Marta M Lettieri Teresa T

The Science of the total environment 20210714

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 quantifi ...[more]

PMID: 34298359

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Project description:Wastewater-based genomic surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus shows promise to complement genomic epidemiology efforts. Multiplex tiling PCR is a desirable approach for targeted genome sequencing of SARS-CoV-2 in wastewater due to its low cost and rapid turnaround time. However, it is not clear how different multiplex tiling PCR primer schemes or wastewater sample matrices impact the resulting SARS-CoV-2 genome coverage. The objective of this work was to assess the performance of three different multiplex primer schemes, consisting of 150-bp, 400-bp, and 1,200-bp amplicons, as well as two wastewater sample matrices, influent wastewater and primary sludge, for targeted genome sequencing of SARS-CoV-2. Wastewater samples were collected weekly from five municipal wastewater treatment plants (WWTPs) in the Metro Vancouver region of British Columbia, Canada during a period of increased coronavirus disease 19 (COVID-19) case counts from February to April 2021. RNA extracted from clarified influent wastewater provided significantly higher genome coverage (breadth and median depth) than primary sludge samples across all primer schemes. Shorter amplicons appeared to be more resilient to sample RNA degradation but were hindered by greater primer pool complexity in the 150-bp scheme. The identified optimal primer scheme (400 bp) and sample matrix (influent) were capable of detecting the emergence of mutations associated with genomic variants of concern, for which the daily wastewater load significantly correlated with clinical case counts. Taken together, these results provide guidance on best practices for implementing wastewater-based genomic surveillance and demonstrate its ability to inform epidemiology efforts by detecting genomic variants of concern circulating within a geographic region. IMPORTANCE Monitoring the genomic characteristics of the SARS-CoV-2 virus circulating in a population can shed important insights into epidemiological aspects of the COVID-19 outbreak. Sequencing every clinical patient sample in a highly populous area is a difficult feat, and thus sequencing SARS-CoV-2 RNA in municipal wastewater offers great promise to augment genomic surveillance by characterizing a pooled population sample matrix, particularly during an escalating outbreak. Here, we assess different approaches and sample matrices for rapid targeted genome sequencing of SARS-CoV-2 in municipal wastewater. We demonstrate that the optimal approach is capable of detecting the emergence of SARS-CoV-2 genomic variants of concern, with strong correlations to clinical case data in the province of British Columbia. These results provide guidance on best practices on, as well as further support for, the application of wastewater genomic surveillance as a tool to augment current genomic epidemiology efforts.

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:BackgroundThe COVID-19 has put emphasis on pivotal needs for diagnosis and surveillance worldwide, with the subsequent shortage of diagnostic reagents and kits. Therefore, it has become strategic for the countries to access diagnostics, expand testing capacity, and develop their own diagnostic capabilities and alternative rapid accurate nucleic acid diagnostics that are at lower costs. Here, we propose a visual SARS-CoV-2 detection using a one-step fast multiplex reverse transcription-PCR (RT-PCR) amplification coupled to lateral flow immunoassay detection on a PCRD device (Abingdon Health, UK).MethodsWe developed various simplex fast-PCRs for screening sets of primer pairs newly designed or selected from literature or from validated WHO diagnostics, targeting S, N, E, RdRp or ORF1ab genes. We retained primers showing specific and stable amplification to assess for their suitability for detection on PCRD. Thus, fast RT-PCR amplifications were performed using the retained primers. They were doubly labeled with Fam and Biotin or Dig and Biotin to allow visual detection of the labeled amplicons on the lateral flow immunoassay PCR Detection (PCRD) device, looking at lack of interaction of the labeled primers (or primer dimers) with the test-lines in negative or no RNA controls. We set up all the assays using RNAs isolated from patients' nasopharyngeal swabs. We used two simplex assays, targeting two different viral genomic regions (N and E) and showing specific detection on PCRD, to set up a one-step fast multiplex RT-PCR assay (where both differently labeled primer pairs were engaged) coupled to amplicons' detection on a PCRD device. We evaluated this novel assay on 50 SARS-CoV-2 positive and 50 SARS-CoV-2 negative samples and compared its performance to the results of the quantitative RT-PCR (RT-qPCR) assays used for diagnosing the patients, here considered as the standard tests.ResultsThe new assay achieved a sensitivity of 88% (44/50) and a specificity of 98% (49/50). All patients who presented Ct values lower than 33 were positive for our assay. Except for one patient, those with Ct values above 33 returned negative results.ConclusionOur results have brought proof of principle on the usefulness of the one-step fast multiplex RT- PCR assay coupled to PCRD as a new assay for specific, sensitive, and rapid detection of SARS-CoV-2 without requiring costly laboratory equipment, and thus, at reduced costs in a format prone to be deployed when resources are limited. This assay offers a viable alternative for COVID-19 diagnosis or screening at points of need.

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

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