HBV DNA高敏检测试剂盒的性能验证和临床应用评价
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摘要
目的对基于Pre-NAT全自动核酸提取系统的乙型肝炎病毒(hepatitis B virus,HBV)DNA高敏检测试剂盒(以下简称高敏试剂)进行性能验证和临床应用评价。方法根据美国临床实验室标准化协会(Clinical and Laboratory Standards Institute,CLSI)制定的试剂盒性能评价标准,采用高敏试剂分别对HBV标准品、临床HBV DNA高、中、低值样本及阴性样本进行定量检测,评价高敏试剂的精密度、正确度、分析测量范围、临床可报告范围和最低检测限等指标。同时采用高敏试剂和COBAS AmpliPrep/COBAS TaqMan 48高敏病毒载量系统检测44份乙型肝炎患者血清,进行方法学比较并分析高敏试剂与COBAS高敏检测系统的相关性。结果高敏试剂检测高、中、低值标本的批内不精密度s_r分别为0.069、0.085、0.059,室内不精密度s_l分别为0.083、30.138、0.117。偏倚标准差为0.25,小于厂家声明的偏倚标准差0.45。分析测量范围广,在25～(1.0×109)IU/mL范围内成线性。样本的最大稀释度为1∶80,功能灵敏度为10 IU/mL,临床可报告范围为10～(8.0×1010)IU/mL。最低检测限为10 IU/mL,低于厂家声明的检测下限20 IU/mL。高敏试剂与COBAS AmpliPrep/COBAS TaqMan 48病毒载量系统的相关性好(y=0.987 6 x+0.189 4,r=0.975 4,P<0.05)。结论所评价的高敏试剂的精密度和正确度高,分析测量范围和临床可报告范围宽,最低检测限低,与COBAS高敏检测系统的相关性好,具有良好的临床应用价值。
Objective To verify performance and evaluate clinical application of HBV DNA highsensitivity detection kit based on Pre-NAT automatic nucleic acid extraction system. Methods According to the kit performance evaluation standard developed by the Clinical and Laboratory Standards Institute(CLSI),high-sensitivity reagents were used to quantitatively detect HBV standards,clinical HBV DNA high,medium and low samples and negative samples. The precision,accuracy,analytical measurement range,clinical reportable range and minimum detection limit of high-sensitivity reagents were evaluated. At the same time,44 serum samples of hepatitis B patients were detected by high-sensitivity reagent and COBAS AmpliPrep/COBAS TaqMan 48 high-sensitivity viral load system,and the correlation between high-sensitivity reagent and COBAS high-sensitivity detection system were analyzed. Results The intra-assay imprecision sr of high-,medium-,and low-value specimens was 0.069,0.085,and 0.059,respectively,and the indoor imprecision sl were 0.083,30.138,and 0.117,respectively by HBV DNA high-sensitivity quantitative detection kit based on Pre-NAT system. The standard deviation of bias is 0.25,which is less than the manufacturers stated standard deviation of 0.45. The analytical measurement range is wide and linear in the range of 25～(1.0×10~9)IU/mL.The maximum dilution of the sample is 1∶80,the functional sensitivity is 10 IU/mL,and the clinical reportable range is 10～(8.0 × 10~(10))IU/mL. The minimum detection limit is 10 IU/mL,which is below the manufacturers stated lower limit of 20 IU/mL. The high-sensitivity reagent correlated well with the COBAS AmpliPrep/COBAS TaqMan 48 viral load system(y=0.987 6 x+0.189 4,r=0.975 4,P<0.05). Conclusion The HBV DNA high-sensitivity detection kit based on Pre-NAT automatic nucleic acid extraction system has high precision and correctness. The analytical measurement range and clinical reportable range are wide,and the minimum detection limit is low. This system has a good correlation with COBAS high-sensitivity detection system and has good clinical application value.
Objective To verify performance and evaluate clinical application of HBV DNA highsensitivity detection kit based on Pre-NAT automatic nucleic acid extraction system. Methods According to the kit performance evaluation standard developed by the Clinical and Laboratory Standards Institute(CLSI),high-sensitivity reagents were used to quantitatively detect HBV standards,clinical HBV DNA high,medium and low samples and negative samples. The precision,accuracy,analytical measurement range,clinical reportable range and minimum detection limit of high-sensitivity reagents were evaluated. At the same time,44 serum samples of hepatitis B patients were detected by high-sensitivity reagent and COBAS AmpliPrep/COBAS TaqMan 48 high-sensitivity viral load system,and the correlation between high-sensitivity reagent and COBAS high-sensitivity detection system were analyzed. Results The intra-assay imprecision sr of high-,medium-,and low-value specimens was 0.069,0.085,and 0.059,respectively,and the indoor imprecision sl were 0.083,30.138,and 0.117,respectively by HBV DNA high-sensitivity quantitative detection kit based on Pre-NAT system. The standard deviation of bias is 0.25,which is less than the manufacturers stated standard deviation of 0.45. The analytical measurement range is wide and linear in the range of 25～(1.0×10~9)IU/mL.The maximum dilution of the sample is 1∶80,the functional sensitivity is 10 IU/mL,and the clinical reportable range is 10～(8.0 × 10~(10))IU/mL. The minimum detection limit is 10 IU/mL,which is below the manufacturers stated lower limit of 20 IU/mL. The high-sensitivity reagent correlated well with the COBAS AmpliPrep/COBAS TaqMan 48 viral load system(y=0.987 6 x+0.189 4,r=0.975 4,P<0.05). Conclusion The HBV DNA high-sensitivity detection kit based on Pre-NAT automatic nucleic acid extraction system has high precision and correctness. The analytical measurement range and clinical reportable range are wide,and the minimum detection limit is low. This system has a good correlation with COBAS high-sensitivity detection system and has good clinical application value.
引文
[1]应盛,董飞波,孙毅,等.COBAS TaqMan系统检测血清HBV-DNA的临床价值探讨[J].中华医院感染学杂志,2017,27(2):305-308.
[2]王创俊,王庆,曹治家,等.NGS检测乙型肝炎病毒耐药基因突变情况及其临床意义[J].分子诊断与治疗杂志,2017,9(1):28-32.
[3]张春娇,仲崇明.核酸免提取HBV-DNA检测方法性能评价[J].实验与检验医学,2018,36(4):513-515.
[4]Liaw YF,Kao JH,Piratvisuth T,et al.Asian-Pacific consensus statement on the management of chronic hepatitis B:a 2012 update[J].Hepatol Int,2012,6(3):531-561.
[5]EASL clinical practice guidelines:Management of chronic hepatitis B virus infection[J].J Hepatol,2012,57(1):167-185.
[6]Clinical and Laboratory Standards Institute.User verification of performance for precision and trueness:approved guideline-second edition[S].EP15-A2,CLSI,2005.
[7]Clinical and Laboratory Standards Institute.Evaluation of the linearity of quantitative measurement procedures:a statistical approach.approved guideline[S].CLSI documents EP6-A.Wayne,PA:CLSI,2003.
[8]Moretti M,Sisti D,Rocchi MB,et al.CLSI EP17-Aprotocol:a useful tool for better understanding the low end performance of total prostate-specific antigen assays[J].Clin Chim Acta,2011,412(11-12):1143-1145.
[9]Clinical and Laboratory Standards Institute.Method comparison and bias estimation using patient samples;approved guideline-second edition[S].CLSI documents EP9-A2.Wayne,PA:CLSI,2002.
[10]吕允凤.乙型肝炎病毒核酸定量检测试剂性能评价要点[J].中国药事,2013,27(10):1072-1074.
[11]高春芳,吴孟超.乙型肝炎病毒感染标志物的检测现状和思考[J].中华检验医学杂志,2015,38(3):145-147.
[12]张辉,吴冬生,方敏,等.荧光定量PCR法与酶联免疫吸附法在乙型肝炎病毒检测中的应用[J].解放军预防医学杂志,2017,35(11):1459-1461.
[13]齐林,刘永芳.乙型肝炎病毒DNA与血清标志物的关系[J].国际检验医学杂志,2017,38(1):130-131.
[14]张宇,李冬,戴燕,等.罗氏LightCycler Nano荧光定量PCR仪性能评价[J].检验医学与临床,2015,12(2):145-146.
[15]秦绪珍,孙江燕,韩建华,等.COBAS Taqman HBV DNA的方法学验证[J].现代检验医学杂志,2008,23(1):29-31.
[16]庞志宇,谢在春,刘玥,等.HBV-DNA定量检测试剂盒性能验证[J].检验医学与临床,2015,12(17):2530-2532.
[17]林健聪,王红翠,吴英松,等.乙肝标志物定量测定试剂盒的性能验证及临床应用评价[J].分子诊断与治疗杂志,2018,10(1):47-55.
[18]柳直英,哈斯朝鲁.一种乙型肝炎病毒-DNA实时荧光定量聚合酶链反应试剂的质量评价[J].实用医技杂志,2018,25(8):826-829.
[19]药监总局通告[2013]3号.乙型肝炎病毒脱氧核糖核酸定量检测试剂注册技术审查指导原则.http://samr.cfda.gov.cn/WS01/CL0087/81119.html,2013-05-17.
[20]Allice T,Cerutti F,Pittaluga F,et al.COBAS AmpliPrep-COBAS TaqMan hepatitis B virus(HBV)test:a novel automated real-time PCR assay for quantification of HBV DNA in plasma[J].J Clin Microbiol,2007,45(3):828-834.
[2]王创俊,王庆,曹治家,等.NGS检测乙型肝炎病毒耐药基因突变情况及其临床意义[J].分子诊断与治疗杂志,2017,9(1):28-32.
[3]张春娇,仲崇明.核酸免提取HBV-DNA检测方法性能评价[J].实验与检验医学,2018,36(4):513-515.
[4]Liaw YF,Kao JH,Piratvisuth T,et al.Asian-Pacific consensus statement on the management of chronic hepatitis B:a 2012 update[J].Hepatol Int,2012,6(3):531-561.
[5]EASL clinical practice guidelines:Management of chronic hepatitis B virus infection[J].J Hepatol,2012,57(1):167-185.
[6]Clinical and Laboratory Standards Institute.User verification of performance for precision and trueness:approved guideline-second edition[S].EP15-A2,CLSI,2005.
[7]Clinical and Laboratory Standards Institute.Evaluation of the linearity of quantitative measurement procedures:a statistical approach.approved guideline[S].CLSI documents EP6-A.Wayne,PA:CLSI,2003.
[8]Moretti M,Sisti D,Rocchi MB,et al.CLSI EP17-Aprotocol:a useful tool for better understanding the low end performance of total prostate-specific antigen assays[J].Clin Chim Acta,2011,412(11-12):1143-1145.
[9]Clinical and Laboratory Standards Institute.Method comparison and bias estimation using patient samples;approved guideline-second edition[S].CLSI documents EP9-A2.Wayne,PA:CLSI,2002.
[10]吕允凤.乙型肝炎病毒核酸定量检测试剂性能评价要点[J].中国药事,2013,27(10):1072-1074.
[11]高春芳,吴孟超.乙型肝炎病毒感染标志物的检测现状和思考[J].中华检验医学杂志,2015,38(3):145-147.
[12]张辉,吴冬生,方敏,等.荧光定量PCR法与酶联免疫吸附法在乙型肝炎病毒检测中的应用[J].解放军预防医学杂志,2017,35(11):1459-1461.
[13]齐林,刘永芳.乙型肝炎病毒DNA与血清标志物的关系[J].国际检验医学杂志,2017,38(1):130-131.
[14]张宇,李冬,戴燕,等.罗氏LightCycler Nano荧光定量PCR仪性能评价[J].检验医学与临床,2015,12(2):145-146.
[15]秦绪珍,孙江燕,韩建华,等.COBAS Taqman HBV DNA的方法学验证[J].现代检验医学杂志,2008,23(1):29-31.
[16]庞志宇,谢在春,刘玥,等.HBV-DNA定量检测试剂盒性能验证[J].检验医学与临床,2015,12(17):2530-2532.
[17]林健聪,王红翠,吴英松,等.乙肝标志物定量测定试剂盒的性能验证及临床应用评价[J].分子诊断与治疗杂志,2018,10(1):47-55.
[18]柳直英,哈斯朝鲁.一种乙型肝炎病毒-DNA实时荧光定量聚合酶链反应试剂的质量评价[J].实用医技杂志,2018,25(8):826-829.
[19]药监总局通告[2013]3号.乙型肝炎病毒脱氧核糖核酸定量检测试剂注册技术审查指导原则.http://samr.cfda.gov.cn/WS01/CL0087/81119.html,2013-05-17.
[20]Allice T,Cerutti F,Pittaluga F,et al.COBAS AmpliPrep-COBAS TaqMan hepatitis B virus(HBV)test:a novel automated real-time PCR assay for quantification of HBV DNA in plasma[J].J Clin Microbiol,2007,45(3):828-834.