新型冠状病毒巢式PCR检测方法的建立与初步应用  

Establishment of a nested PCR assay for the detection of 2019 novel coronavirus and its preliminary application

作　　者：石伟先[1] 冯兆民 崔淑娟[1] 潘阳[1] 钱城[1] 辛若雷[1] 杨鹏[1] 王全意[1] 张代涛[1] 高志勇[1] Shi Weixian;Feng Zhaomin;Cui Shujuan;Pan Yang;Qian Cheng;Xin Ruolei;Yang Peng;Wang Quanyi;Zhang Daitao;Gao Zhiyong(Institute for Infectious Disease and Endemic Disease Control,Beijing Center for Disease Prevention and Control,Beijing 100013,China)

机构地区：[1]北京市疾病预防控制中心传染病地方病控制所,北京100013

出　　处：《中华实验和临床病毒学杂志》2022年第2期214-218,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：北京市百千万人才工程项目(2020A051);首都卫生发展科研专项(首发2021-1G-3012)。

摘　　要：目的建立一种检测新型冠状病毒的巢式PCR方法,作为实时荧光PCR法检测新型冠状病毒的补充,并探讨该方法在临床样本检测中的初步应用价值。方法根据新型冠状病毒基因保守序列,利用在线软件设计N、S基因引物,构建巢式PCR反应体系,N基因巢式PCR及产物测序用于定性检测,S基因PCR产物测序用于型别初步鉴定。检测梯度稀释新型冠状病毒阳性样本评价检测灵敏度,检测流感病毒和人冠状病毒OC43、229E、HKU1、NL63等样本评价其特异性。分别检测Ct值>33与Ct值<33的各15份新型冠状病毒阳性样本,对扩增产物进行测序及比对分析,对检测方法进行验证。结果所建立的巢式PCR法可扩增出355 bp N基因片段及449 bp S基因片段的特异性条带,冠状病毒229E、OC43、HKU1、NL63、H3N2亚型流感病毒、甲型H1N1流感病毒、乙型流感病毒阳性样本无扩增条带。N基因片段最低检测限可达Ct值37.21。30份新冠核酸阳性样本中,巢式PCR检测的N基因阳性符合一致率达100%(30/30);S基因阳性符合一致率达60%(18/30)。28份样本N基因片段测序成功,BLAST与新型冠状病毒相似性100%。12份样本S基因片段可获得位点突变特征结果。结论建立了可特异检测新型冠状病毒的巢式PCR方法,并可通过对PCR扩增产物测序分析其位点突变特征,可作为实时荧光PCR法的补充。Objective To establish a nested PCR method to detect the 2019 novel coronavirus(2019-nCoV),as a supplement to the real-time fluorescent PCR method,and discuss the preliminary application value of this method in clinical diagnosis.Methods According to the conservative sequences of the 2019-nCoV gene,the nested PCR primers including N gene and S gene,were designed on line.By optimizing the nested PCR reaction systems,the qualitative detection was established by testing N gene and sequencing its PCR product while the preliminary type identification was established by testing S gene and sequencing its PCR product.The sensitivity was evaluated by the gradient dilution of 2019-nCoV positive samples’nucleic acid and the specificity was evaluated by detecting the human coronavirus OC43,229E,HKU1,NL63,influenza virus positive samples.The established method was applied to 15 samples with Ct>33 and 15 samples with Ct<33 screened by real-time fluorescent PCR,and the positive amplification result were sequenced and analyzed to verify the result.Results The established nested PCR method could amplify specific bands of 355 bp N gene fragment and 449 bp S gene fragment.No amplifications occurred in other human coronaviruses samples including 229E、OC43、HKU1、NL63 or in influenza virus samples including H3N2,H1N1(pdm)and B.The minimum detection limit of the N gene fragment could reach Ct value about 37.21.Among the 30 COVID-19 positive samples,the N gene positive coincidence rate detected by nested PCR was 100%(30/30);the S gene positive coincidence rate reached 60%(18/30).28 samples’sequences of N gene fragment were completely consistent with 2019-nCoV by BLAST,and the characteristic result of site mutations of 12 samples’S gene was obtained.Conclusions A nested PCR method for the specific detection of 2019-nCoV was established,and some characteristic mutations on S gene could be analyzed by sequencing the PCR amplified products.It could be used as a supplement to the real-time fluorescent PCR method.

关 键 词：新型冠状病毒 实时荧光PCR法 巢式PCR法 

分 类 号：R440[医药卫生—诊断学]