Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.
Payment Certification Agency, Ministry of Health, Phnom Penh, Cambodia.
Sci Rep. 2022 Feb 14;12(1):2419. doi: 10.1038/s41598-022-04952-2.
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.
本研究旨在开发可行且有效的通过 Sanger 测序策略对显著 SARS-CoV-2 变体进行通用筛查的策略，然后在日本广岛实际应用于大规模筛查。对来自广岛的 734 例 COVID-19 确诊病例的样本进行了显著 SARS-CoV-2 变体（B.1.1.7、B.1.351、P.1、B.1.617.2、B.1.617.1、C.37、B.1.1.529 等）的筛查。使用内部设计的引物对 hCoV-Spike-A 和标准扩增方案，通过巢式 RT-PCR 扩增靶向刺突区。此外，还使用引物对 hCoV-Spike-B 和 hCoV-Spike-C 随机扩增 96 个样本。对于未扩增的样本，按体积增加方案进行第二次扩增尝试。之后，使用相应的引物对扩增产物进行 Sanger 测序。使用标准方案时，引物对 hCoV-Spike-A、hCoV-Spike-B 和 hCoV-Spike-C 的阳性扩增率分别为 87.3%、83.3%和 93.8%，而通过体积增加方案进行第二次扩增尝试后，其阳性扩增率分别提高至 99.6%、95.8%和 96.9%。基因组序列的准备率分别为 96.9%、100%和 100%。在 48 个突变株中，26 株为 B.1.1.7（Alpha），7 株为 E484K 单突变株，其余为其他类型的突变株。此外，在广岛首次报告了 5 例带有 N501S 单点突变的聚集性病例。本研究表明 Sanger 测序在筛查大量显著 SARS-CoV-2 变体样本时具有可靠性和有效性。与下一代测序（NGS）相比，我们的方法为识别新兴变体提供了一种可行、普遍适用且实用的工具，成本更低，耗时更短，尤其适用于那些无法实际应用 NGS 的国家。我们的方法不仅可以识别已有的变体，还可以检查其他罕见类型的突变或新出现的变体，这对于大流行的预防和控制至关重要。
