S基因部分序列测序用于新冠病毒变异株快速检测

Partial sequencing of S-gene for the identification of SARS-CoV-2 variants

目的建立一种简便快速鉴定新冠病毒变异株的方法。方法根据新冠病毒变异株的棘突蛋白(spike protein,S)基因序列,在高度保守区域设计特异性引物,从实时荧光RT-PCR阳性样本中提取RNA采用一步法RT-PCR扩增新冠病毒核酸检测阳性的鼻咽拭子样本,用PCR产物直接测序。将测得序列与新冠病毒参考序列的S基因序列进行比对,确定突变位点,再确定变异株类型;同时通过全球共享所有流感数据倡议(Global Initiative of Sharing All Influenza Data,GISAID)数据库进行同源性分析,根据吻合度最高的病毒序列确定变异株类型,与二代测序结果比较,评价方法的准确性。结果20份经荧光定量RT-PCR检测确认为新冠病毒核酸阳性的鼻咽拭子样本,其中13份用一步法RT-PCR扩增到新冠病毒S基因部分序列,PCR产物全部测序成功,确定的变异株类型与二代测序获得全序列后进行的病毒毒株分类完全吻合。结论S基因部分序列一代测序可准确检测新冠病毒的主要突变株,为实验室提供快速、简便的新冠病毒变异监测策略。

Objective To develop a method for the identification of SARS-CoV-2 variants based on sequencing partial spike protein(S)gene.Methods According to the sequence of SARS-CoV-2 S-gene,the primer set was designed based on the highly conserved region.RNA extracted from Real-time RT-PCR positive samples was amplified by using designed primers with one-step RT-PCR,the products of PCR were sequenced.The mutant sites were identified by compared with reference sequence of SARS-CoV-2 strain.Meanwhile,the strains of positive samples were confirmed by making sequence analysis and homology blast in GISAID website.Compare the consistency of the identification of SARS-CoV-2 variants based on partial S-gene sequencing and whole next generation genome sequencing.Results The partial S gene was amplified and sequenced successfully from 13 of 20 positive samples confirmed by Real-time PCR.The identification of SARS-CoV-2 variants based on partial S gene sequencing was identical with whole genome sequencing results.Conclusion Sequencing partial S-gene can accurately identify the main SARS-CoV-2 variants.The method is rapid and simple enough to be implemented in any SARS-CoV-2 diagnostic laboratory.