全自动核酸提取平台高敏HBV DNA 定量检测的性能评价
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摘要
目的验证Natch S全自动核酸提取平台高敏HBV DNA定量检测的性能。方法参考美国临床实验室标准化协会批准指南(CLSI-EP),采用Natch S全自动核酸提取平台及实时荧光定量聚合酶链反应分析系统检测高敏HBV DNA,评价该方法的精密度、正确度、线性范围、检测能力和抗污染能力等性能指标,采用化学发光微粒子免疫检测法检测HBV标志物和IFCC速率法检测丙氨酸氨基转移酶(ALT),并分析两者之间的相互关系。结果精密度方面,高(10~7 IU/ml)、中(10~3 IU/ml)和低(10~2 IU/ml)浓度样本的批内与批间精密度CV值均≤5.0%;正确度方面,参加2017-2018年全国临床检验中心室间质评正确率100%,且实测值与靶值偏倚均<±0.20 lg IU/ml;线性范围方面,在(1.0×10~2～5.0×10~9)IU/ml范围内有良好的线性关系(Y=0.4+0.97X,R~2=0.999≥0.98,P<0.05);检测能力方面,检出限30 IU/ml和定量限100 IU/ml的检出率100%,且定量限100 IU/ml的CV值为3.21%;抗污染能力方面,与高浓度(>10~6 IU/ml)阳性样本间隔放置的阴性样本均未检出阳性结果;临床应用评价,566例慢性乙型肝炎(乙肝)患者血清样本高敏HBV DNA载量分布情况以<30 IU/ml组为主,占45.94%,与其他组ALT异常构成比差异有统计学意义(P均≤0.043),同一HBV DNA载量水平HBeAg>1.0 S/Co组与HBeAg≤1.0 S/Co组ALT异常构成比差异无统计学意义(P均≥0.61)。结论基于Natch S全自动核酸提取平台的高敏HBV DNA定量检测各项分析性能指标均表现良好,符合PCR检测要求,且对低病毒载量慢性乙肝患者抗病毒治疗监测具有重要意义。
Objective To evaluate the performance of the Natch S automatic nucleic acid extraction platform for high-sensitivity HBV DNA quantitative detection. Methods Based on the American standard CLSI-EP, the Natch S automatic nucleic acid extraction platform and PCR analysis system was applied to detect the high-sensitivity HBV DNA. The precision, accuracy, linear range, detection ability and anti-pollution ability of this method was analyzed as well as the correlation with HBV DNA, ALT and HBeAg. Results Both the intra-assay and inter-assay precision CV values were ≤5.0%.The correct rate of EQA was 100% from 2017 to 2018.The linear range was good in the range of( 1.0×10~2-5.0×10~9) IU/ml(Y=0.4+0.97X, R~2=0.999≥0.98, P<0.05).The detection rate of 30 IU/ml LOD and 100 IU/ml LOQ was 100%, and the CV value of 100 IU/ml LOQ was 3.21%; No positive results were detected in the negative samples in terms of anti-pollution ability.The distribution of HBV DNA in 566 patients with chronic hepatitis B was mainly in the group of <30 IU/ml(45.94%).There was a statistically significant difference in the ratio of ALT abnormalities between the<30 IU/ml group and other groups(P≤0.043).There was no significant difference in the ratio of ALT abnormalities between HBeAg>1.0 S/Co group and the HBeAg≤1.0 S/Co group at the same HBV DNA level(P≥0.61). Conclusions Based on the Natch S automatic nucleic acid extraction platform,the high-sensitivity HBV DNA quantitative detection performance indicators are all good for monitoring of anti-viral treatment of chronic hepatitis B patients with low levels of HBV DNA.
Objective To evaluate the performance of the Natch S automatic nucleic acid extraction platform for high-sensitivity HBV DNA quantitative detection. Methods Based on the American standard CLSI-EP, the Natch S automatic nucleic acid extraction platform and PCR analysis system was applied to detect the high-sensitivity HBV DNA. The precision, accuracy, linear range, detection ability and anti-pollution ability of this method was analyzed as well as the correlation with HBV DNA, ALT and HBeAg. Results Both the intra-assay and inter-assay precision CV values were ≤5.0%.The correct rate of EQA was 100% from 2017 to 2018.The linear range was good in the range of( 1.0×10~2-5.0×10~9) IU/ml(Y=0.4+0.97X, R~2=0.999≥0.98, P<0.05).The detection rate of 30 IU/ml LOD and 100 IU/ml LOQ was 100%, and the CV value of 100 IU/ml LOQ was 3.21%; No positive results were detected in the negative samples in terms of anti-pollution ability.The distribution of HBV DNA in 566 patients with chronic hepatitis B was mainly in the group of <30 IU/ml(45.94%).There was a statistically significant difference in the ratio of ALT abnormalities between the<30 IU/ml group and other groups(P≤0.043).There was no significant difference in the ratio of ALT abnormalities between HBeAg>1.0 S/Co group and the HBeAg≤1.0 S/Co group at the same HBV DNA level(P≥0.61). Conclusions Based on the Natch S automatic nucleic acid extraction platform,the high-sensitivity HBV DNA quantitative detection performance indicators are all good for monitoring of anti-viral treatment of chronic hepatitis B patients with low levels of HBV DNA.
引文
[1] Ott JJ,Stevens GA,Groger J,et al.Global epidemiology of hepatitis B virus infection:new estimates of age-specific HBsAg seropre valence and endemicity[J].Vaccine,2012,30(12):2212-2219.
[2] Lozano R,Naghavi M,Foreman K,et al.Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010:a systematic analysis for the Global Burden of Disease Study 2010[J].Lancet,2012,380(9859):2095-2128.
[3] Goldstein ST,Zhou F,Hadler SC,et al.A mathematical model to estimate global hepatitis B disease burden and vaccination impact[J].Int J Epidemiol,2005,34(6):1329-1339.
[4] Wang FS,Fan JG,Zhang Z,et al.The global burden of liver disease:the major impact of China[J].Hepatology,2014,60(6):2099-2108.
[5] Chinese Society of Hepatology and Chinese Society of Infectious Disease,Chinese Medical Association.The guideline of prevention and treatment for chronic hepatitis B:2015 update version[J].Zhongguo Bingdubing Zazhi,2015,5(6):401-424.(in Chinese)中华医学会肝病学分会,中华医学会感染病学分会.慢性乙型肝炎防治指南(2015年更新版)[J].中国病毒病杂志,2015,5(6):401-424.
[6] Tu T,Budzinska MA,Shackel NA,et al.HBV DNA integration:molecular mechanisms and clinical implications[J].Viruses,2017,9(4):75.
[7] Jiang SZ,Lu FM,Zhuang H.The clinical significance of quantitative detection of HBV DNA in the chronic infected patients[J].Zhonghua Jianyan Yixue Zazhi,2012,35(2):117-121.(in Chinese)蒋素贞,鲁凤民,庄辉.慢性乙型肝炎病毒DNA定量检测的临床意义[J].中华检验医学杂志,2012,35(2):117-121.
[8] Clinical and Laboratory Standards Institute.Evaluation of precision of quantitative measurement procedures[S].Wayne,PA,USA:CLSI,2014.
[9] Clinical and Laboratory Standards Institute.User verification of precision and estimation of bias[S].Wayne,PA,USA:CLSI,2014.
[10] Clinical and Laboratory Standards Institute.Evaluation of the linearity of quantitative analytical methods[S].Wayne,PA,USA:CLSI,2003.
[11] European Association for the Study of the Liver.EASL clinical practice guidelines management of chronic hepatitis B virus infection[J].J Hepatol,2012,57:167-185.
[12] Liu WW,Guan M.Interpretation of the guidance on the application of accreditation criteria for the medical laboratory quality and competence in the field of gene amplification testing(version 2013)[J].Zhonghua Linchuang Shiyanshi Guanli Dianzi Zazhi,2013,1(1):36-40.(in Chinese)刘维薇,关明.2013版《医学实验室质量和能力认可准则在基因扩增检验领域的应用说明》改版解读[J].中华临床实验室管理电子杂志,2013,1(1):36-40.
[13] Shen T,Long L,Deng ZP.Evaluation of a novel domestic hepatitis B viral DNA quantitative fluorescence diagnostic kit[J].Zhonghua Jianyan Yixue Zazhi,2013,36(3):280-285.(in Chinese)沈弢,龙璐,邓中平,等.新型国产乙型肝炎病毒核酸定量检测试剂的质量评价[J].中华检验医学杂志,2013,36(3):280-285.
[14] Zha Y,Wang XL,Zhu SY,et al.The extraction effect of nucleic acid in HBV DNA fluorescence detection by boiling cracking method and magnetic bead method[J].Zhejiang Yufang Yixue,2015,27(12):1292-1293,1296.(in Chinese) 查瑶,王小灵,朱诗艳,等.煮沸裂解法与磁珠法在HBVDNA荧光定量检测中的核酸提取效果[J].浙江预防医学,2015,27(12):1292-1293,1296.
[15] Liu ZY,Wang N,Liu H,et al.Association of hepatitis B serological marker detection and quantitative detection of HBV DNA[J].Int J Lab Med,2014,35(2):233-235.(in Chinese)刘志勇,王娜,刘浩,等.乙型肝炎血清学标志物定量检测与HBV DNA定量检测结果的相关性分析[J].国际检验医学杂志,2014,35(2):233-235.
[2] Lozano R,Naghavi M,Foreman K,et al.Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010:a systematic analysis for the Global Burden of Disease Study 2010[J].Lancet,2012,380(9859):2095-2128.
[3] Goldstein ST,Zhou F,Hadler SC,et al.A mathematical model to estimate global hepatitis B disease burden and vaccination impact[J].Int J Epidemiol,2005,34(6):1329-1339.
[4] Wang FS,Fan JG,Zhang Z,et al.The global burden of liver disease:the major impact of China[J].Hepatology,2014,60(6):2099-2108.
[5] Chinese Society of Hepatology and Chinese Society of Infectious Disease,Chinese Medical Association.The guideline of prevention and treatment for chronic hepatitis B:2015 update version[J].Zhongguo Bingdubing Zazhi,2015,5(6):401-424.(in Chinese)中华医学会肝病学分会,中华医学会感染病学分会.慢性乙型肝炎防治指南(2015年更新版)[J].中国病毒病杂志,2015,5(6):401-424.
[6] Tu T,Budzinska MA,Shackel NA,et al.HBV DNA integration:molecular mechanisms and clinical implications[J].Viruses,2017,9(4):75.
[7] Jiang SZ,Lu FM,Zhuang H.The clinical significance of quantitative detection of HBV DNA in the chronic infected patients[J].Zhonghua Jianyan Yixue Zazhi,2012,35(2):117-121.(in Chinese)蒋素贞,鲁凤民,庄辉.慢性乙型肝炎病毒DNA定量检测的临床意义[J].中华检验医学杂志,2012,35(2):117-121.
[8] Clinical and Laboratory Standards Institute.Evaluation of precision of quantitative measurement procedures[S].Wayne,PA,USA:CLSI,2014.
[9] Clinical and Laboratory Standards Institute.User verification of precision and estimation of bias[S].Wayne,PA,USA:CLSI,2014.
[10] Clinical and Laboratory Standards Institute.Evaluation of the linearity of quantitative analytical methods[S].Wayne,PA,USA:CLSI,2003.
[11] European Association for the Study of the Liver.EASL clinical practice guidelines management of chronic hepatitis B virus infection[J].J Hepatol,2012,57:167-185.
[12] Liu WW,Guan M.Interpretation of the guidance on the application of accreditation criteria for the medical laboratory quality and competence in the field of gene amplification testing(version 2013)[J].Zhonghua Linchuang Shiyanshi Guanli Dianzi Zazhi,2013,1(1):36-40.(in Chinese)刘维薇,关明.2013版《医学实验室质量和能力认可准则在基因扩增检验领域的应用说明》改版解读[J].中华临床实验室管理电子杂志,2013,1(1):36-40.
[13] Shen T,Long L,Deng ZP.Evaluation of a novel domestic hepatitis B viral DNA quantitative fluorescence diagnostic kit[J].Zhonghua Jianyan Yixue Zazhi,2013,36(3):280-285.(in Chinese)沈弢,龙璐,邓中平,等.新型国产乙型肝炎病毒核酸定量检测试剂的质量评价[J].中华检验医学杂志,2013,36(3):280-285.
[14] Zha Y,Wang XL,Zhu SY,et al.The extraction effect of nucleic acid in HBV DNA fluorescence detection by boiling cracking method and magnetic bead method[J].Zhejiang Yufang Yixue,2015,27(12):1292-1293,1296.(in Chinese) 查瑶,王小灵,朱诗艳,等.煮沸裂解法与磁珠法在HBVDNA荧光定量检测中的核酸提取效果[J].浙江预防医学,2015,27(12):1292-1293,1296.
[15] Liu ZY,Wang N,Liu H,et al.Association of hepatitis B serological marker detection and quantitative detection of HBV DNA[J].Int J Lab Med,2014,35(2):233-235.(in Chinese)刘志勇,王娜,刘浩,等.乙型肝炎血清学标志物定量检测与HBV DNA定量检测结果的相关性分析[J].国际检验医学杂志,2014,35(2):233-235.