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A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern.

ABSTRACT: The global spread of new SARS-CoV-2 variants of concern underscore an urgent need of simple deployed molecular tools that can differentiate these lineages. Several tools and protocols have been shared since the beginning of the COVID-19 pandemic, but they need to be timely adapted to cope with SARS-CoV-2 evolution. Although whole-genome sequencing (WGS) of the virus genetic material has been widely used, it still presents practical difficulties such as high cost, shortage of available reagents in the global market, need of a specialized laboratorial infrastructure and well-trained staff. These limitations result in SARS-CoV-2 surveillance blackouts across several countries. Here we propose a rapid and accessible protocol based on Sanger sequencing of a single PCR fragment that is able to identify and discriminate all SARS-CoV-2 variants of concern (VOCs) identified so far, according to each characteristic mutational profile at the Spike-RBD region (K417N/T, E484K, N501Y, A570D). Twelve COVID-19 samples from Brazilian patients were evaluated for both WGS and Sanger sequencing: three P.2, two P.1, six B.1.1 and one B.1.1.117 lineage. All results from the Sanger sequencing method perfectly matched the mutational profile of VOCs and non-VOCs RBD's characterized by WGS. In summary, this approach allows a much broader network of laboratories to perform molecular surveillance of SARS-CoV-2 VOCs and report results within a shorter time frame, which is of utmost importance in the context of rapid public health decisions in a fast evolving worldwide pandemic.

SUBMITTER: Bezerra MF

PROVIDER: S-EPMC8105304 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern.

Bezerra Matheus Filgueira MF Machado Lais Ceschini LC De Carvalho Viviane do Carmo Vasconcelos VDCV Docena Cássia C Brandão-Filho Sinval Pinto SP Ayres Constância Flávia Junqueira CFJ Paiva Marcelo Henrique Santos MHS Wallau Gabriel Luz GL

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 20210508

The global spread of new SARS-CoV-2 variants of concern underscore an urgent need of simple deployed molecular tools that can differentiate these lineages. Several tools and protocols have been shared since the beginning of the COVID-19 pandemic, but they need to be timely adapted to cope with SARS-CoV-2 evolution. Although whole-genome sequencing (WGS) of the virus genetic material has been widely used, it still presents practical difficulties such as high cost, shortage of available reagents i ...[more]

PMID: 33975021

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Project description:This study aimed to develop the feasible and effective universal screening strategy of the notable SARS-CoV-2 variants by Sanger Sequencing Strategy and then practically applied it for mass screening in Hiroshima, Japan. A total of 734 samples from COVID-19 confirmed cases in Hiroshima were screened for the notable SARS-CoV-2 variants (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.1, C.37, B.1.1.529, etc.). The targeted spike region is amplified by nested RT-PCR using in-house designed primer set hCoV-Spike-A and standard amplification protocol. Additionally, randomly selected 96 samples were also amplified using primer sets hCoV-Spike-B and hCoV-Spike-C. The negative amplified samples were repeated for second attempt of amplification by volume-up protocol. Thereafter, the amplified products were assigned for Sanger sequencing using corresponding primers. The positive amplification rate of primer set hCoV-Spike-A, hCoV-Spike-B and hCoV-Spike-C were 87.3%, 83.3% and 93.8% respectively for standard protocol and increased to 99.6%, 95.8% and 96.9% after second attempt by volume-up protocol. The readiness of genome sequences was 96.9%, 100% and 100% respectively. Among 48 mutant isolates, 26 were B.1.1.7 (Alpha), 7 were E484K single mutation and the rest were other types of mutation. Moreover, 5 cluster cases with single mutation at N501S were firstly reported in Hiroshima. This study indicates the reliability and effectiveness of Sanger sequencing to screen large number of samples for the notable SARS-CoV-2 variants. Compared to the Next Generation Sequencing (NGS), our method introduces the feasible, universally applicable, and practically useful tool for identification of the emerging variants with less expensive and time consuming especially in those countries where the NGS is not practically available. Our method allows not only to identify the pre-existing variants but also to examine other rare type of mutation or newly emerged variants and is crucial for prevention and control of pandemic.

Dataset Information

A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern.

Publications

A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern.

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