Beijing Institute of Biological Products Company Limited, Beijing, 100176, China.
Mol Biol Rep. 2024 Aug 7;51(1):892. doi: 10.1007/s11033-024-09799-6.
Background The emergence and rapid spread of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), poses a significant threat to human health and public safety. While next-generation sequencing (NGS) is capable of detecting and tracking new COVID-19 variants for disease diagnosis and prevention, its high cost and time-consuming nature limit its widespread use. In this study, our aim was to develop a highly adaptable and accurate RT-PCR method for identifying the Delta or BA.1 variants in inactivated COVID-19 vaccine. We devised three two-plex RT-PCR methods targeting specific mutation sites: S: Δ156-157, S: N211-, L212I, and S: Δ142-144, Y145D. The RT-PCR method targeting the S: Δ156-157 mutation site was able to distinguish the Delta variant from other COVID-19 virus strains, while the RT-PCR methods targeting the S: N211-, L212I or S: Δ142-144, Y145D mutation sites were able to distinguish the BA.1 variant from other COVID-19 virus strains. We separately validated these three two-plex RT-PCR methods, and the results demonstrated good linearity, repeatability, reproducibility, and specificity for each method. Moreover, all three methods can be applied in the production of SARS-CoV-2 variant inactivated vaccines, enabling the identification of Delta or BA.1 variants in virus cultures as well as in inactivated vaccine stocks. This study presents a systematic approach to identify COVID-19 variants using multiple RT-PCR methods. We successfully developed three two-plex RT-PCR methods that can identify Delta and BA.1 variants based on specific mutation sites, and we completed the validation of these three methods.
由严重急性呼吸系统综合征冠状病毒 2 型（SARS-CoV-2）引起的 2019 年冠状病毒病（COVID-19）的出现和迅速传播对人类健康和公共安全构成了重大威胁。虽然下一代测序（NGS）能够检测和跟踪用于疾病诊断和预防的新的 COVID-19 变体，但它的高成本和耗时性质限制了其广泛应用。在这项研究中，我们的目的是开发一种高度适应和准确的 RT-PCR 方法，用于鉴定灭活 COVID-19 疫苗中的 Delta 或 BA.1 变体。我们设计了三种针对特定突变位点的两重 RT-PCR 方法：S：Δ156-157、S：N211-、L212I 和 S：Δ142-144、Y145D。针对 S：Δ156-157 突变位点的 RT-PCR 方法能够将 Delta 变体与其他 COVID-19 病毒株区分开来，而针对 S：N211-、L212I 或 S：Δ142-144、Y145D 突变位点的 RT-PCR 方法能够将 BA.1 变体与其他 COVID-19 病毒株区分开来。我们分别验证了这三种两重 RT-PCR 方法，结果表明每种方法都具有良好的线性、重复性、重现性和特异性。此外，这三种方法都可以应用于 SARS-CoV-2 变体灭活疫苗的生产，能够在病毒培养物以及灭活疫苗库存中识别 Delta 或 BA.1 变体。本研究提出了一种使用多种 RT-PCR 方法识别 COVID-19 变体的系统方法。我们成功开发了三种两重 RT-PCR 方法，可以根据特定的突变位点识别 Delta 和 BA.1 变体，并完成了这三种方法的验证。
