基于 TSP 的 PCR 快速鉴定 SARS-CoV-2 的 L 和 S 型毒株。
TSP-based PCR for rapid identification of L and S type strains of SARS-CoV-2.
机构信息
Indian Council of Medical Research-Regional Medical Research Centre for NE Region, Bokel, Dibrugarh, Assam, 786010, India.
Department of Clinical Microbiology, Sher-I Kashmir Institute of Medical Sciences, Soura, Srinagar, Jammu & Kashmir, India.
出版信息
Indian J Med Microbiol. 2021 Jan;39(1):73-80. doi: 10.1016/j.ijmmb.2021.01.003. Epub 2021 Jan 15.
BACKGROUND
In the initial few months of the COVID-19 pandemic, two distinct strains of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) were identified (L and S strain) based on a tightly linked SNP between two widely separated nucleotides at location 8782 (ORF1ab T8517C) and position 28,144 (ORF8: C251T, codon S84L).
MATERIALS AND METHODS
A Type Specific Primer based one step RT-PCR (TSP-PCR) test to distinguish the L and S type strains of SARS-CoV-2 without the need for viral genome sequencing, was developed. The study also analyzed 18,221 whole genome sequences (WGS) available up to April 2020 to know the prevalence of L and S type of strains. Phylogenetic and recombination analysis of SARS-CoV-2 genome with nearest animal and human coronaviruses were analyzed using MEGA X and SimPlot version 3.5.1 software respectively.
RESULTS
The rapid TSP-PCR distinguished the L and S type strains of SARS-CoV-2 by amplifying a specific 326 bp and 256 bp fragment of the L and S type strain respectively. The test was used to analyzed 120 random SARS-CoV-2 positive samples from Assam, India among which 118 were found to be of L-type strains only. On analysis of 18,221 WGS, it was found that L type was the predominant strain with an overall prevalence ∼90%. However, pockets of high prevalence of S-type strains (>35%) were still in circulation in Washington region in April 2020. The study did not detect any significant recombination events between closely related coronavirus and SARS-CoV-2.
CONCLUSION
TSP-based PCR for identification of circulating strains of SARS-CoV-2, will add in rapid identification of strains of COVID-19 pandemic to understand the spread of the virus, its transmissibility and adaptation into human population. Though, the S-type strains have decreased drastically across the globe since April 2020, the role of TSP-PCR in geographical niches where such strains are still prevalent may help in rapidly distinguishing the strains and study its evolution.
背景
在 COVID-19 大流行的最初几个月中，根据位置 8782（ORF1ab T8517C）和位置 28144（ORF8：C251T，密码子 S84L）处两个相距较远的核苷酸之间紧密连锁的 SNP，鉴定出两种截然不同的严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2）菌株（L 株和 S 株）。
材料和方法
开发了一种基于型特异性引物的一步 RT-PCR（TSP-PCR）测试，无需病毒基因组测序即可区分 SARS-CoV-2 的 L 株和 S 株。该研究还分析了截至 2020 年 4 月可用的 18221 个全基因组序列（WGS），以了解 L 株和 S 株的流行情况。使用 MEGA X 和 SimPlot 版本 3.5.1 软件分别对 SARS-CoV-2 基因组与最近的动物和人类冠状病毒进行了系统发生和重组分析。
结果
快速 TSP-PCR 通过分别扩增 L 株和 S 株的特定 326bp 和 256bp 片段来区分 SARS-CoV-2 的 L 株和 S 株。该测试用于分析来自印度阿萨姆邦的 120 个随机 SARS-CoV-2 阳性样本，其中 118 个样本仅为 L 株。对 18221 个 WGS 的分析发现，L 株是主要流行株，总体流行率约为 90%。然而，2020 年 4 月，华盛顿地区仍有高流行率（>35%）的 S 株流行。该研究未检测到密切相关的冠状病毒与 SARS-CoV-2 之间存在任何明显的重组事件。
结论
基于 TSP 的 PCR 用于鉴定 SARS-CoV-2 的循环株，将有助于快速鉴定 COVID-19 大流行的病毒株，以了解病毒的传播、传播力和适应性进入人群。尽管自 2020 年 4 月以来，全球 S 株的流行率急剧下降，但 TSP-PCR 在这些菌株仍然流行的地理区域中的作用可能有助于快速区分菌株并研究其进化。
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