高敏乙型肝炎病毒脱氧核糖核酸定量检测在低病毒载量患者治疗监测中的应用
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摘要
目的对高敏乙型肝炎(乙肝)病毒脱氧核糖核酸(HBV DNA)定量检测进行方法学评价,并探讨该方法在低病毒载量的乙肝患者治疗监测中的应用价值。方法采用全自动核酸提纯及荧光聚合酶链式反应分析系统检测HBV DNA,评价该方法的线性范围、精密度、最低定量检测限、防污染能力和最低检测限等性能指标,采用电化学发光法检测HBV血清学标志物和全自动生化仪检测丙氨酸氨基转移酶(ALT),并分析两者之间的相互关系。结果高敏HBV DNA定量检测方法的线性范围为50~5.0×10~8 IU/ml,最低定量检测限为50 IU/ml,批内精密度与批间精密度均<5%,最低检测限为20 IU/ml,防污染能力强。在抗病毒治疗的中,HBV DNA载量<500 IU/ml的乙肝患者占总数的46.40%;HBVDNA载量为30~500 IU/ml的HBeAg阳性或阴性患者的ALT异常率明显高于HBV DNA载量<30 IU/ml的患者(P=0.012、0.001),但不同HBV DNA载量与两对半血清学模式差异无统计学意义(P=0.179)。结论基于全自动核酸提纯及荧光PCR分析系统的高敏HBV DNA定量检测方法各性能指标良好,适于低病毒载量的乙肝患者治疗监测,临床应加强该类乙肝患者的治疗监测。
Objective To evaluate the performance of highly sensitive HBV DNA quantitative detection and discuss its application in the treatment monitoring of hepatitis B patients with low viral load. Methods Automatic nucleic acid extraction and fluorescence PCR analysis system was used to detect HBV DNA, the linear range, precision, limit of detection(LOD), limit of quantitative detection and anti.pollution capability of the method were evaluated. HBV serological markers were detected by electrochemiluminescence immunoassay. ALT was detected by automatic biochemical analyzer. And the relationship between HBV DNA, serological markers and ALT were analyzed. Results The result showed that the linear range of high sensitivity HBV DNA quantitative detection was between 50 IU/ml and 5.0× 10~8 IU/ml. The LOD of the method reached 30 IU/ml. The precision of inter and intra.run CV were all less than 5%. And the method had a strong anti.pollution capability. The patients with HBV DNA load <500 IU/ml accounted for 46.4%. In HBeAg positive or negative group, the ALT abnormal rate of patients with HBV DNA load ranging from 30 IU/ml to 500 IU/ml was significantly higher than those with HBV DNA load <30 IU/ml(P=0.012,P=0.001), but different HBV DNA load had no relationship with serological markers patterns(P=0.179). Conclusion Automatic nucleic acid extraction and fluorescence PCR analysis system based highly sensitive HBV DNA quantitative detection has good performance, which is suitable for the treatment monitoring of patients with low hepatitis B viral load. And the treatment monitoring of such hepatitis B patients should be enhanced.
Objective To evaluate the performance of highly sensitive HBV DNA quantitative detection and discuss its application in the treatment monitoring of hepatitis B patients with low viral load. Methods Automatic nucleic acid extraction and fluorescence PCR analysis system was used to detect HBV DNA, the linear range, precision, limit of detection(LOD), limit of quantitative detection and anti.pollution capability of the method were evaluated. HBV serological markers were detected by electrochemiluminescence immunoassay. ALT was detected by automatic biochemical analyzer. And the relationship between HBV DNA, serological markers and ALT were analyzed. Results The result showed that the linear range of high sensitivity HBV DNA quantitative detection was between 50 IU/ml and 5.0× 10~8 IU/ml. The LOD of the method reached 30 IU/ml. The precision of inter and intra.run CV were all less than 5%. And the method had a strong anti.pollution capability. The patients with HBV DNA load <500 IU/ml accounted for 46.4%. In HBeAg positive or negative group, the ALT abnormal rate of patients with HBV DNA load ranging from 30 IU/ml to 500 IU/ml was significantly higher than those with HBV DNA load <30 IU/ml(P=0.012,P=0.001), but different HBV DNA load had no relationship with serological markers patterns(P=0.179). Conclusion Automatic nucleic acid extraction and fluorescence PCR analysis system based highly sensitive HBV DNA quantitative detection has good performance, which is suitable for the treatment monitoring of patients with low hepatitis B viral load. And the treatment monitoring of such hepatitis B patients should be enhanced.
引文
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[14]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.DOI:10.3969/j.issn.1673-4130.2014.02.049.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.DOI:10.3969/j.issn.1673-4130.2014.02.049.
[2]王贵强,王福生,成军,等.慢性乙型肝炎防治指南(2015年更新版)[J].临床肝胆病杂志,2015,31(12):1941-1960.DOI:10.3969/j.issn.1001-5256.2015.12.002.Wang GQ,Wang FS,Cheng J,et al.The guideline of prevention and treatment for chronic hepatitis B:a 2015 update[J].J Clin Hepa,2015,31(12):1941-1960.DOI:10.3969/j.issn.1001-5256.2015.12.002.
[3]李敏伟.HBV-DNA及HBsAg定量对低病毒载量慢性乙型肝炎的临床意义[D].杭州:浙江大学,2014.Li MW.Serum HBV DNA and hepatitis B surface antigen levels help predict diseases progression in patients with low hepatitis B virus loads[D].Hangzhou:Zhejiang University,2014.
[4]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.DOI:10.3877/cma.j.issn.2095-5820.2013.01.008.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(version2013)[J].Chin J Clin Lab Manag,2013,1(1):36-40.DOI:10.3877/cma.j.issn.2095-5820.2013.01.008.
[5]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.DOI:10.19485/j.cnki.issn1007-0931.2015.12.035.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 J Prev Med,2015,27(12):1292-1293,1296.DOI:10.19485/j.cnki.issn1007-0931.2015.12.035.
[6]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.DOI:10.3760/cma.j.issn.1009-9158.2013.03.022.Shen T,Long L,Deng ZP,et al.Evaluation of novel domestic hrpstitis B viral DNA quantitative fluorescence diagnostic kit[J].Chin J Lab Med,2013,36(3):280-285.DOI:10.3760/cma.j.issn.1009-9158.2013.03.022.
[7]Han MS,Park Y,Nah H,et al.Comparison of the QIAGEN artus HBV QS.RGQ assay with the roche COBAS/COBAS TaqMan HBV assay for quantifying viral DNA in sera of chronic hepatitis B patients[J].Ann Lab Med,2017,37(3):248-253.DOI:10.3343/alm.2017.37.3.248.
[8]Fourati S,Pawlotsky JM.Recent advances in understanding and diagnosing hepatitis B virus infection[J].F1000 Res,2016,5:2243.DOI:10.12688/f1000research.8983.1.
[9]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.DOI:10.3760/cma.j.issn.1009-9158.2012.02.004.Jiang SZ,Lu FM,Zhuang H.The clinical significance of quantitative detection of HBV DNA in the chronic infected patients[J].Chin J Lab Med,2012,35(2):117-121.DOI:10.3760/cma.j.issn.1009-9158.2012.02.004.
[10]Lin CL,Kao JH.New perspectives of biomarkers for the management of chronic hepatitis B[J].Clin Mol Hepatol,2016,22(4):423-431.DOI:10.3350/cmh.2016.0069.
[11]World Health Organization.Guidelines for the prevention,care and treatment of persons with chronic hepatitis B infection[R].Geneva:WHO,2015.
[12]Lampertico P,Maini M,Papatheodoridis G.Optimal management of hepatitis B virus infection.EASL Special Conference[J].J Hepatol,2015,63(5):1238-1253.DOI:10.1016/j.jhep.2015.06.026.
[13]Liaw YF,Kao JH,Piratvisuth T,et al.Asian.Pacific consensus statement on the management of chronic hepatitis B:a 2012update[J].Hepatol Int,2012,6(3):531-561.DOI:10.1007/s12072-012-9365-4.
[14]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.DOI:10.3969/j.issn.1673-4130.2014.02.049.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.DOI:10.3969/j.issn.1673-4130.2014.02.049.