病毒灭活处理对2019新型冠状病毒核酸检测弱阳性结果的影响  

Effect of virus inactivation on weak positive results of nucleic acid test for 2019 novel coronavirus

作　　者：段秀枝[1] 王旭楚 俞攀 刘伟伟[1] 李翔 张乐乐 张巩 唐沪强[1] 陈勤[1] 吴先国[1] 陶志华[1] Duan Xiuzhi;Wang Xuchu;Yu Pan;Liu Weiwei;Li Xiang;Zhang Lele;Zhang Gong;Tang Huqiang;Chen Qin;Wu Xianguo;Tao Zhihua(Department of Laboratory Medicine,the Second Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou 310009,China)

机构地区：[1]浙江大学医学院附属第二医院检验科,杭州310009

出　　处：《中华检验医学杂志》2020年第4期358-363,共6页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金青年科学基金(81902156)。

摘　　要：目的:探讨病毒灭活处理对2019新型冠状病毒核酸检测弱阳性结果的影响。方法:回顾性研究浙江大学医学院附属第二医院2020年1至2月2019新型冠状病毒不同浓度的3例实时荧光定量聚合酶链反应(RT-PCR)核酸检测阳性患者的鼻咽拭子标本。采用不灭活处理和56℃30 min水浴、56℃60 min干浴及60℃30 min干浴3组灭活处理方式对鼻咽拭子保存液标本进行病毒灭活,提取RNA,然后3种商品化新型冠状病毒核酸实时荧光RT-PCR试剂分别进行检测,用循环阈值(Ct)评价病毒灭活对2019新型冠状病毒核酸检测结果影响。结果:灭活前ORF1ab基因Ct值分别为23.28±0.28、25.25±0.25、28.93±0.44、32.06±0.47、35.20±0.38、32.89±0.38、36.24±0.23、33.30±0.46,灭活后ORF1ab基因Ct值分别为灭活组1:23.60±0.20、27.29±0.30、31.83±0.51、37.41±0.46,灭活组2:24.25±0.34、27.18±0.42、31.84±0.61、34.99±1.01、34.89±0.45,灭活组3∶23.37±0.17、26.89±0.52、32.05±0.50;灭活前N基因Ct值分别为24.38±0.09、26.64±0.11、30.35±0.12、33.29±0.33、36.93±0.11、34.50±0.12、35.63±0.12,灭活后N基因Ct值分别为灭活组1:24.66±0.11、28.52±0.14、32.71±0.14、37.00±0.13,灭活组2:25.41±0.10、28.79±0.15、33.29±0.28,灭活组3:23.37±0.11、28.68±0.11、33.54±0.13、37.18±0.23。灭活前后扩增Ct值比较差异无统计学意义(ORF1ab基因:t=-1.416;N基因:t=-1.379,P均>0.05),3组灭活处理组间Ct值差异也无统计学意义(ORF1ab基因:t=-0.460;N基因:t=-0.132,P均>0.05)。但不同稀释倍数下的灭活组(1,2,3)与未灭活组Ct值比较,未灭活组和灭活组(1,2,3)Ct值差异有统计学意义。10×稀释:ORF1ab Ct值:未灭活组:25.25±0.25,灭活组1,2,3分别为27.29±0.30、27.18±0.42和26.89±0.52,t(ORF1ab)=-7.327,P<0.01;N基因Ct值:未灭活组:26.64±0.11,灭活组1,2,3分别为28.52±0.14、28.79±0.15和28.68±0.11,t(N)=-19.340,P<0.01。100×稀释:ORF1abCt值:未灭活组:28.93±0.44,灭活组1,2,3分别为31.83±0.Objective To investigate the effect of virus inactivation on weak positive result of 2019 novel coronavirus(2019-nCoV)nucleic acid test.Methods A retrospective study was conducted on the nasopharyngeal swabs of three patients with positive PCR nucleic acid test for 2019-nCoV at different concentrations in the Second affiliated Hospital of Zhejiang University Medical College from January to February 2020.The virus in nasopharyngeal swab specimens were inactivated by water bath at 56℃for 30 min,dry bath at 56℃for 60 min and dry bath at 60℃for 30 min respectively.After treatment,these samples RNA were extracted and then detected by three new commercial quantitative real-time polymerase chain reaction reagent kits for 2019-nCoV.Cycle threshold(Ct)value was used to evaluate the effect of virus inactivation on nucleic acid detection of 2019-nCoV.Results There was no significant difference between the groups before and after inactivation.Ct values of ORF1ab gene before inactivation were 23.28±0.28,25.25±0.25,28.93±0.44,32.06±0.47,35.20±0.38,32.89±0.38,36.24±0.23,33.30±0.46,and those after inactivation were,group 1:23.60±0.20,27.29±0.30,31.83±0.51,37.41±0.46,group 2:24.25±0.34,27.18±0.42,31.84±0.61,34.99±1.01,34.89±0.45,group 3:23.37±0.17,26.89±0.52,32.05±0.50.Ct value of N gene before inactivation were 24.38±0.09,26.64±0.11,30.35±0.12,33.29±0.33,36.93±0.11,34.50±0.12,35.63±0.12,those after inactivation were,group 1:24.66±0.11,28.52±0.14,32.71±0.14,37.00±0.13;group 2:25.41±0.10,28.79±0.15,33.29±0.28;group 3:23.37±0.11,28.68±0.11,33.54±0.13,37.18±0.23(ORF1ab gene:t=-1.416;N gene:t=-1.379,P>0.05).There was no significant difference among the three inactivation groups,the specific Ct values are shown above(ORF1ab gene:t=-0.460;N gene:t=-0.132,P>0.05).However,the Ct values of the inactivated groups(1,2,3)and the non-inactivated group at different dilution times were different(10×:Ct value of ORF1ab was 25.25±0.25 in the non-inactivated group,and 27.29±0.30,27.18±0.42 and 26.89�

关 键 词：冠状病毒属 病毒灭活 核酸类 实时聚合酶链反应 

分 类 号：R4[医药卫生—临床医学]