Dataset Information

Direct RT-qPCR assay for SARS-CoV-2 variants of concern (Alpha, B.1.1.7 and Beta, B.1.351) detection and quantification in wastewater.

ABSTRACT: Less than a year following the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, variants of concern have emerged in the form of variant Alpha (B.1.1.7, the British variant) and Beta (B.1.351, the South Africa variant). Due to their high infectivity and morbidity, it has become clear that it is crucial to quickly and effectively detect these and other variants. Here, we report improved primers-probe sets for reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) for SARS-CoV-2 detection including a rapid, cost-effective, and direct RT-qPCR method for detection of the two variants of concern (Alpha, B.1.1.7 and Beta, B.1.351). All the developed primers-probe sets were fully characterized, demonstrating sensitive and specific detection. These primer-probe sets were also successfully employed on wastewater samples aimed at detecting and even quantifying new variants in a geographical area, even prior to the reports by the medical testing. The novel primers-probe sets presented here will enable proper responses for pandemic containment, particularly considering the emergence of variants of concern.

SUBMITTER: Yaniv K

PROVIDER: S-EPMC8262398 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Direct RT-qPCR assay for SARS-CoV-2 variants of concern (Alpha, B.1.1.7 and Beta, B.1.351) detection and quantification in wastewater.

Yaniv Karin K Ozer Eden E Shagan Marilou M Lakkakula Satish S Plotkin Noam N Bhandarkar Nikhil Suresh NS Kushmaro Ariel A

Environmental research 20210707

Less than a year following the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, variants of concern have emerged in the form of variant Alpha (B.1.1.7, the British variant) and Beta (B.1.351, the South Africa variant). Due to their high infectivity and morbidity, it has become clear that it is crucial to quickly and effectively detect these and other variants. Here, we report improved primers-probe sets for reverse transcriptase quantitative polymerase chain reaction (RT-qP ...[more]

PMID: 34245731

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Project description:Emerging SARS-CoV-2 (SC-2) variants with increased infectivity and vaccine resistance are of major concern. Rapid identification of such variants is important for the public health decision making and to provide valuable data for epidemiological and policy decision making. We developed a multiplex reverse transcriptase quantitative PCR (RT-qPCR) assay that can specifically identify and differentiate between the emerging B.1.1.7 and B.1.351 SC-2 variants. In a single assay, we combined four reactions-one that detects SC-2 RNA independently of the strain, one that detects the D3L mutation, which is specific to variant B.1.1.7, one that detects the 242 to 244 deletion, which is specific to variant B.1.351, and the fourth reaction, which identifies the human RNAseP gene, serving as an endogenous control for RNA extraction integrity. We show that the strain-specific reactions target mutations that are strongly associated with the target variants and not with other major known variants. The assay's specificity was tested against a panel of respiratory pathogens (n = 16), showing high specificity toward SC-2 RNA. The assay's sensitivity was assessed using both in vitro transcribed RNA and clinical samples and was determined to be between 20 and 40 viral RNA copies per reaction. The assay performance was corroborated with Sanger and whole-genome sequencing, showing complete agreement with the sequencing results. The new assay is currently implemented in the routine diagnostic work at the Central Virology Laboratory, and may be used in other laboratories to facilitate the diagnosis of these major worldwide-circulating SC-2 variants. IMPORTANCE This study describes the design and utilization of a multiplex reverse transcriptase quantitative PCR (RT-qPCR) to identify SARS-COV-2 (SC2) RNA in general and, specifically, to detect whether it is of lineage B.1.1.7 or B.1.351. Implementation of this method in diagnostic and research laboratories worldwide may help the efforts to contain the COVID-19 pandemic. The method can be easily scaled up and be used in high-throughput laboratories, as well as small ones. In addition to immediate help in diagnostic efforts, this method may also help in epidemiological studies focused on the spread of emerging SC-2 lineages.

Project description:SARS-CoV-2 variants of concern, demonstrating higher infection rate and lower vaccine effectiveness as compared with the original virus, are important factors propelling the ongoing COVID-19 global outbreak. Therefore, prompt identification of these variants in the environment is essential for pandemic assessment and containment efforts. One well established tool for such viral monitoring is the use of wastewater systems. Here, we describe continuous monitoring of traces of SARS-CoV-2 viruses in the municipal wastewater of a large city in Israel. By observing morbidity fluctuations (during three main COVID-19 surges) occurring in parallel with Pfizer-BioNTech COVID-19 vaccine vaccination rate, compromised immunity was revealed in the current morbidity peak. RT-qPCR assays for the Original (D614G), Alpha and Beta variants had been previously developed and are being employed for wastewater surveillance. In the present study we developed a sensitive RT-qPCR assay designed for the rapid, direct detection of Gamma and Delta variants of concern. Sensitive quantification and detection of the various variants showed the prevalence of the original variant during the first morbidity peak. The dominance of the Alpha variant over the original variant correlated with the second morbidity peak. These variants decreased concurrently with an increase in vaccinations (Feb-March 2021) and the observed decrease in morbidity. The appearance and subsequent rise of the Delta variant became evident and corresponded to the third morbidity peak (June-August 2021). These results suggest a high vaccine neutralization efficiency towards the Alpha variant compared to its neutralization efficiency towards the Delta variant. Moreover, the third vaccination dose (booster) seems to regain neutralization efficiency towards the Delta variant. The developed assays and wastewater-based epidemiology are important tools aiding in morbidity surveillance and disclosing vaccination efforts and immunity dynamics in the community.

Project description:ObjectivesThe clinical outcomes of the Beta (B.1.351) variant of concern (VOC) of the SARS-CoV-2 virus remain poorly understood. In early 2021, northeastern France experienced an outbreak of Beta that was not observed elsewhere. This outbreak slightly preceded and then overlapped with a second outbreak of the better understood VOC Alpha (B.1.1.7) in the region. This situation allowed us to contemporaneously compare Alpha and Beta in terms of the characteristics, management, and outcomes of critically ill patients.MethodsA multicenter prospective cohort study was conducted on all consecutive adult patients who had laboratory confirmed SARS CoV-2 infection, underwent variant screening, and were admitted to one of four intensive care units (ICU) for acute respiratory failure between January 9th and May 15th, 2021. Primary outcome was 60-day mortality. Differences between Alpha and Beta in terms of other outcomes, patient variables, management, and vaccination characteristics were also explored by univariate analysis. The factors that associated with 60-day death in Alpha- and Beta-infected patients were examined with logistic regression analysis.ResultsIn total, 333 patients (median age, 63 years; 68% male) were enrolled. Of these, 174 and 159 had Alpha and Beta, respectively. The two groups did not differ significantly in terms of 60-day mortality (19 vs. 23%), 28-day mortality (17 vs. 20%), need for mechanical ventilation (60 vs. 61%), mechanical ventilation duration (14 vs. 15 days), other management variables, patient demographic variables, comorbidities, or clinical variables on ICU admission. The vast majority of patients were unvaccinated (94%). The remaining 18 patients had received a partial vaccine course and 2 were fully vaccinated. The vaccinated patients were equally likely to have Alpha and Beta.ConclusionsBeta did not differ from Alpha in terms of patient characteristics, management, or outcomes in critically ill patients.Trial registrationClinicalTrials.gov, identifier: NCT04906850.

Project description:Effective wastewater surveillance of SARS-CoV-2 RNA requires the rigorous characterization of the limit of detection resulting from the entire sampling process - the process limit of detection (PLOD). Yet to date, no studies have gone beyond quantifying the assay limit of detection (ALOD) for RT-qPCR or RT-dPCR assays. While the ALOD is the lowest number of gene copies (GC) associated with a 95% probability of detection in a single PCR reaction, the PLOD represents the sensitivity of the method after considering the efficiency of all processing steps (e.g., sample handling, concentration, nucleic acid extraction, and PCR assays) to determine the number of GC in the wastewater sample matrix with a specific probability of detection. The primary objective of this study was to estimate the PLOD resulting from the combination of primary concentration and extraction with six SARS-CoV-2 assays: five RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab (CCDC N and CCDC ORF1ab), and E_Sarbeco RT-qPCR, and one RT-dPCR assay (US CDC N1 RT-dPCR) using two models (exponential survival and cumulative Gaussian). An adsorption extraction (AE) concentration method (i.e., virus adsorption on membrane and the RNA extraction from the membrane) was used to concentrate gamma-irradiated SARS-CoV-2 seeded into 36 wastewater samples. Overall, the US CDC N1 RT-dPCR and RT-qPCR assays had the lowest ALODs (< 10 GC/reaction) and PLODs (<3,954 GC/50 mL; 95% probability of detection) regardless of the seeding level and model used. Nevertheless, consistent amplification and detection rates decreased when seeding levels were < 2.32 × 103 GC/50 mL even for US CDC N1 RT-qPCR and RT-dPCR assays. Consequently, when SARS-CoV-2 RNA concentrations are expected to be low, it may be necessary to improve the positive detection rates of wastewater surveillance by analyzing additional field and RT-PCR replicates. To the best of our knowledge, this is the first study to assess the SARS-CoV-2 PLOD for wastewater and provides important insights on the analytical limitations for trace detection of SARS-CoV-2 RNA in wastewater.

Dataset Information

Direct RT-qPCR assay for SARS-CoV-2 variants of concern (Alpha, B.1.1.7 and Beta, B.1.351) detection and quantification in wastewater.

Publications

Direct RT-qPCR assay for SARS-CoV-2 variants of concern (Alpha, B.1.1.7 and Beta, B.1.351) detection and quantification in wastewater.

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