热灭活处理对新型冠状病毒鼻咽拭子核酸检测质量的影响  

Effect of heat inactivation treatment on detection quality of 2019 novel coronavirus nucleic acid in nasopharyngeal swabs

作　　者：曹娴 浦晓红 许晔琼 赵一琳 俞建峰 陈洁 宛传丹 Cao Xian;Pu Xiaohong;Xu Yeqiong;Zhao Yilin;Yu Jianfeng;Chen Jie;Wan Chuandan(Quality Administrative Office,Changshu Medical Examination Institute,Changshu 215500,Jiangsu,China)

机构地区：[1]常熟市医学检验所质量管理科,江苏常熟215500

出　　处：《实用检验医师杂志》2022年第3期263-266,共4页Chinese Journal of Clinical Pathologist

基　　金：江苏省常熟市科技发展计划(社会发展)项目(CS202013,CS202113)。

摘　　要：目的探讨新型冠状病毒(2019-nCoV)检测前热灭活处理对核酸稳定性和检测质量的影响。方法收集2020年2—7月常熟市医学检验所接收的2019-nCoV核酸阳性标本,检验操作严格按照三级生物安全防控进行前处理分装。标本分为未灭活组(20份)和热灭活组(30份),未灭活组根据不同待检时长分为2 h组、8 h组、24 h组、48 h组,每组5份;热灭活组分为热灭活组1(56℃,30 min)、热灭活组2(56℃,60 min)、热灭活组3(56℃,2 h),每组10份。采用全自动核酸提取仪提取核酸后,以双重荧光反转录聚合酶链反应(RT-PCR)检测2019-nCoV的ORF1ab基因和N基因片段。阳性将呈典型S形曲线扩增,记录并比较各组基线与曲线指数增长拐点所对应的循环数(CT值)。结果热灭活组1、热灭活组2、热灭活组3的ORF1ab基因和N基因CT值均明显高于未灭活组(ORF1ab基因:30.5±0.4、32.3±0.4、36.1±0.5比27.3±0.3,N基因:30.1±0.6、34.1±0.5、37.2±0.5比27.2±0.4,均P<0.05)。未灭活组待检时长2 h、8 h、24 h、48 h组的ORF1ab基因和N基因CT值比较差异均无统计学意义(ORF1ab基因:28.3±0.5、29.5±0.3、29.4±0.3、30.2±0.7,N基因:26.5±0.3、27.3±0.4、27.1±0.5、28.7±0.7,均P>0.05)。热灭活组1、2、3对高浓度核酸(5×10^(5)个拷贝/mL)的CT值均明显高于未灭活组(ORF1ab基因:31.5±0.3、33.3±0.4、37.3±0.6比26.3±0.3,N基因:31.1±0.7、34.2±0.5、38.8±0.3比25.2±0.4,均P<0.05)。结论热灭活处理可影响2019-nCoV核酸的稳定性,降低ORF1ab基因和N基因的扩增CT值。48 h内待检时长对ORF1ab和N基因的扩增CT值无显著影响。2019-nCoV核酸筛查应加强三级防护,避免热灭活处理,有利于提高2019-nCoV阳性样本的筛查效率。Objective To explore the influence of heat inactivation treatment on nucleic acid stability and detection quality of 2019 novel coronavirus(2019-nCoV)before laboratory detection.Methods Received in Changshu Medical Examination institute,the positive samples of 2019-nCoV from February to July 2020 were collected,and pre-processing was conducted and sub-packaging was done in strict accordance with the level-Ⅲbiosafety prevention.The samples were divided into non-inactivation group(20 samples)and heat inactivation group(30 samples).The non-inactivation group was then divided into 2 hours,8 hours,24 hours and 48 hours groups according to different time to be detected,with 5 samples in each group.The heat inactivation group was divided into heat inactivation group 1(56℃,30 minutes),heat inactivation group 2(56℃,60 minutes)and heat inactivation group 3(56℃,2 hours),with 10 samples in each group.After the nucleic acid was extracted by an automatic nucleic acid extractor,the ORF1ab gene and N gene fragments of 2019-nCoV were detected by double fluorescent reverse transcription polymerase chain reaction(RT-PCR).Positive gene will be amplified as a typical S-shaped curve.The cycle threshold(CT value)corresponding to the inflection point of baseline and curve exponential growth in each group was recorded and compared.Results CT values of ORF1ab gene and N gene in heat inactivation group 1,heat inactivation group 2 and heat inactivated group 3 were higher than those in non-inactivation group(ORF1ab gene:30.5±0.4,32.3±0.4,36.1±0.5 vs.27.3±0.3,N gene:30.1±0.6,34.1±0.5,37.2±0.5 vs.27.2±0.4,all P<0.05).There was no statistically significant difference between CT values of ORF1ab gene and N gene in the non-nactivation group for 2 hours,8 hours,24 hours and 48 hours(ORF1ab gene:28.3±0.5,29.5±0.3,29.4±0.3,30.2±0.7,N gene:26.5±0.3,27.3±0.4,27.1±0.5,28.7±0.7,all P>0.05).Heat inactivation 1,2,3 groups increased the CT value significantly in high concentration nucleic acid(5×10^(5) copies/mL)compared with the n

关 键 词：新型冠状病毒 热灭活 核酸检测 反转录聚合酶链反应 

分 类 号：R446.5[医药卫生—诊断学]