Dataset Information

Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern.

ABSTRACT: Rapid identification and continuous surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are critical for guiding the response to the COVID-19 pandemic. Whole-genome sequencing (WGS) is a preferred tool for this aim, but many laboratories suffer from a lack of resources to support population-level sequencing. Here, we describe two PCR strategies targeting spike protein mutations to identify the Alpha, Delta, and Omicron variants. Signature mutations were selected using a dedicated bioinformatic program. The selected mutations in Alpha and Delta variants were detected using multicolor melting curve analysis (MMCA). Thirty-two mutations of the Omicron variant were targeted using the MeltArray approach in one reaction, which was able to detect the Omicron subvariants BA.1, BA.2, BA.3, and BA.4/5. The limits of detection varied from five to 50 copies of RNA templates/reactions. No cross-reactivity was observed with nine other respiratory viruses, including other coronaviruses. We validated the MMCA and MeltArray assays using 309 SARS-CoV-2 positive samples collected at different time points. These assays exhibited 98.3% to 100% sensitivity and 100% specificity compared with WGS. Multiplexed real-time PCR strategies represent an alternative tool capable of identifying current SARS-CoV-2 VOCs, adaptable for emerging variants and accessible for laboratories using existing equipment and personnel. IMPORTANCE Rapid detection and mutation surveillance of SARS-CoV-2 VOCs is crucial for COVID-19 control, management, and prevention. We developed two rapid molecular assays based on the real-time PCR platform to identify important variants of concern, including the Omicron variant with a large number of mutations. Signature mutations were selected by an R program. Then, MMCA assays were established for Alpha and Delta variants, and a MeltArray assay targeting 32 mutations was developed for Omicron variant. These multiplexed PCR assays could be performed in a 96-well real-time PCR instrument within 2.5 h, offering a high-throughput choice for dynamic monitoring of SARS-CoV-2 VOCs in a standard microbiology laboratory.

SUBMITTER: Yan T

PROVIDER: S-EPMC9603638 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern.

Yan Ting T Xu Ye Y Zheng Rongrong R Zeng Xiaohong X Chen Zehui Z Lin Su S Xia Zihan Z Liao Yiqun Y Zhang Yongyou Y Li Qingge Q

Microbiology spectrum 20220915 5

Rapid identification and continuous surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are critical for guiding the response to the COVID-19 pandemic. Whole-genome sequencing (WGS) is a preferred tool for this aim, but many laboratories suffer from a lack of resources to support population-level sequencing. Here, we describe two PCR strategies targeting spike protein mutations to identify the Alpha, Delta, and Omicron variants. Signature mutations were selected ...[more]

PMID: 36106882

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Project description:Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide. Many variants of SARS-CoV-2 have been reported, some of which have increased transmissibility and/or reduced susceptibility to vaccines. There is an urgent need for variant phenotyping for epidemiological surveillance of circulating lineages. Whole-genome sequencing is the gold standard for identifying SARS-CoV-2 variants, which constitutes a major bottleneck in developing countries. Methodological simplification could increase epidemiological surveillance feasibility and efficiency. We designed a novel multiplex real-time reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2 variants with S gene mutations. This multiplex PCR typing method was established to detect 9 mutations with specific primers and probes (ΔHV 69/70, K417T, K417N, L452R, E484K, E484Q, N501Y, P681H, and P681R) against the receptor-binding domain of the spike protein of SARS-CoV-2 variants. In silico analyses showed high specificity of the assays. Variants of concern (VOC) typing results were found to be highly specific for our intended targets, with no cross-reactivity observed with other upper respiratory viruses. The PCR-based typing methods were further validated using whole-genome sequencing and a commercial kit that was applied to clinical samples of 250 COVID-19 patients from Taiwan. The screening of these samples allowed the identification of epidemic trends by time intervals, including B.1.617.2 in the third Taiwan wave outbreak. This PCR typing strategy allowed the detection of five major variants of concern and also provided an open-source PCR assay which could rapidly be deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, P.1, and B.1.617.2 variants and of four Omicron mutations on the spike protein (ΔHV 69/70, K417N, N501Y, P681H). IMPORTANCE COVID-19 has spread globally. SARS-CoV-2 variants of concern (VOCs) are leading the next waves of the COVID-19 pandemic. Previous studies have pointed out that these VOCs may have increased infectivity, have reduced vaccine susceptibility, change treatment regimens, and increase the difficulty of epidemic prevention policy. Understanding SARS-CoV-2 variants remains an issue of concern for all local government authorities and is critical for establishing and implementing effective public health measures. A novel SARS-CoV-2 variant identification method based on a multiplex real-time RT-PCR was developed in this study. Five SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, and Omicron) were identified simultaneously using this method. PCR typing can provide rapid testing results with lower cost and higher feasibility, which is well within the capacity for any diagnostic laboratory. Characterizing these variants and their mutations is important for tracking SAR-CoV-2 evolution and is conducive to public infection control and policy formulation strategies.

Dataset Information

Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern.

Publications

Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern.

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