两种核酸提取方法对血清HBV DNA荧光定量检测结果的影响  

The influence of two different nucleic acid isolation methods on serum HBV DNA detection with real-time quantitative PCR

作　　者：范公忍[1] 韩聚强[1] 胡学玲[1] 陈天宝[1] 胡海[1] 曹建彪[1] 

机构地区：[1]北京军区总医院肝病治疗中心,北京100700

出　　处：《中国卫生检验杂志》2013年第9期2110-2112,2114,共4页Chinese Journal of Health Laboratory Technology

摘　　要：目的评价两种HBV DNA提取方法对定量检测结果的影响。方法 178例临床诊断为慢性乙型肝炎患者的血清样本分别采用磁珠法和煮沸法提取HBV DNA,经荧光定量PCR方法平行检测HBV DNA载量,比较两种提取方法检测结果的准确性、灵敏度及特异性;对两种方法检测结果不一致的样本采用罗氏公司COBAS试剂作标准进行复检,比较两种提取方法同COBAS试剂检测结果的相关性。结果 178例血清样本用两种方法提取DNA荧光定量PCR检测结果均阳性67例(37.6%),均阴性97例(54.5%);14例磁珠法检测阳性、而煮沸法检测结果阴性(7.9%)。经统计学分析两种提取方法检测结果具有显著性差异(χ2=7.22,P<0.05)。比较磁珠法提取样本PCR与罗氏COBAS试剂的定量结果具有很好一致性(r=0.936,P<0.01)。结论磁珠法提取核酸检测结果比传统的煮沸法结果显示出较好的线性范围和准确性;通过该提取方法可使全部核酸样本参与到PCR反应过程中,最大限度地减少了核酸丢失,结果准确性高、敏感性好,对于监测乙型肝炎患者血液中低载量HBV DNA水平具有重要意义。Objective To evaluate the influence of two HBV DNA extracting methods on quantitative test results.Methods HBV DNA was extracted from serum samples of 178 chronic hepatitis B patients by magnetic beads method and boiling method respectively and detected with real-lime quantitative PCR.Comparing the accuracy,sensitivity and specificity of the HBV DNA test results of the two methods,the inconsistent samples were re-examined with the Roche COBAS standard reagent and the relevance of the results was compared.Results Among the 178 serum samples,the DNA fluorescent quantitative PCR results of 67 cases（37.6%） were positive and 97 cases（54.5%） were negative by the two DNA extracting methods;Moreover,14 cases were positive by the magnetic beads method,but negative by boiling method（7.9%）.The statistical analysis of test results of the two extraction methods had significant differences（χ2= 7.22,P !0.05）.The quantitative PCR results of the magnetic beads extraction had good agreement（r = 0.936,P !0.01） with the Roche COBAS reagent.Conclusion Compared with the traditional boiling method,the nucleic acid detection results of magnetic bead extraction showed a better linear range and accuracy.By the isolating method,all the nucleic acid samples involved in the process of PCR reaction could minimize the lost of nucleic acid and obtain the results with high accuracy and sensitivity.It has important significance in monitoring blood of HBV patients with low HBV DNA load.

关 键 词：肝炎病毒 乙型 核酸检测 分析灵敏度 

分 类 号：R512.62[医药卫生—内科学]