磁珠分离纯化技术在实时荧光定量PCR检测HBVDNA中的应用  

Study on the Magnetic Beads Separation Purification Technology in Real-time Fluorescent Quantitative PCR for Detecting HBV DNA

作　　者：李成德[1] 岑丽莲[1] 陈金强 黄玉环[1] 梁梅芳[1] 梁洁玲[1] 黄晓佳[1] 陈雄毅[1] 李海珠[1] 

机构地区：[1]肇庆市第一人民医院检验科,广东肇庆526021 [2]肇庆市端州区妇幼保健院检验科,广东肇庆526040

出　　处：《现代检验医学杂志》2011年第3期68-71,共4页Journal of Modern Laboratory Medicine

基　　金：基金项目：广东省肇庆市科技创新计划项目（2010E286）.

摘　　要：目的评价磁珠分离纯化技术（简称磁珠法）提取核酸的重复性、提取效率一致性、线性范围、灵敏度及抗干扰性，初步探讨其在实时荧光定量PCR检测HBVDNA中的应用价值。方法采用磁珠法提取HBVDNAlog值分别为4。16，5．23和7．04的质控血清进行重复性试验，HBVDNA阳性血清10倍梯度稀释的系列标本评价其提取效率一致性，HBVDNA强阳性血清作10倍系列稀释后检测其线性范围，HBVDNA标准血清与舍干扰成分（溶血、黄痘和脂血）的HBVDNA阴性血清，按4：0，3：1，2：2，1：3和0：4比例分别混合成5个不同浓度标本用于千扰试验。以提取方法为沉淀煮沸法的PCR荧光诊断试剂作对照，应用磁珠法及配套扩增试剂检测288份乙型肝炎患者血清HBVDNA含量，并比较测定结果。结果磁珠法批内和批间平均变异系数分别为3．66％和4．75％；HBVDNA含量不同的血清提取效率均在98％以上；在1．28×10^2 IU／L～1．28×10^9IU／L之阍有良好的线性关系；最低检出限为60．5IU／L；不同浓度的黄疸和脂血对扩增曲线和实验结果无明显影响；血清中血红蛋白浓度至50g／L时，HBVDNA结果相差约0．5～1．0个数量级。磁珠法和沉淀煮沸法的阳性率分别为88．19％和82．64％。当沉淀煮沸法检测结果log值处在2．699～4．000之间时，两法检测相应标本结果差异有统计学意义。结论磁珠法高效、快速和易于自动化，应用于HBVDNA检测对乙型肝炎患者的临床诊断和疗效监测将有十分重要的意义。Objective To investigate the repeatability,the consistency of extraction efficiency,the linear range,the sensitivity and the anti-jamming performance of the magnetic beads separation and purification technology （referred to as the magnetic beads method） by which the nucleic acid were extracted ,and preliminarily evaluate the application value of the magnetic beads method in HBV DNA detection by the real-tlme fluorescence quantitative PCR （RTFQ-PCR）. Methods The quality control serums which the log value of HBV DNA were 4.16,5.23 and 7.04 respectively were extracted by the magnetic beads method for repeatability experiment, 10-fold dilution for a series of specimens with a HBV DNA positive serum were used to evaluate the extraction efficiency of consistency,and a HBV DNA strongly positive serum was selected after 10-fold serial dilutions for the linear range. A HBV DNA standard serum and HBV DNA negative serums containing interference （hemoglobin, bilirubin and lipin） were mixed according to 4 ： 0,3 ： 1,2 ： 2,1 ： 3 and 0 ： 4 proportion respectively into five different concentrations specimens for disturbance experiment. A fluorescent PCR diagnostic reagents taking the precipitation boiling method as extraction method as control, 288 serums HBV DNA content in hepatitis B patients which templates were prepared by the magnetic beads method were detected,then compared the test results. Results The inter-and intra coefficient of variation of the magnetic beads method were 3.66% and 4. 75% ,respectively. The extraction efficiency of the magnetic beads method in detecting serums with different content of HBV DNA were 98% or more. The magnetic beads method had a good linear relationship in the 1.28 × 10^2IU/L- 1.28 × 10^9 IU/L scope,and the minimum detection limit was 6.05 × 10^1 IU/L. The ieterus and the lipin with different concentrations had no effect on the amplification curve and the experimental result detected by RTFQ-PCR. When Hb content was 50 g/L ,the results had a difference of about 0.

关 键 词：磁珠分离纯化技术 乙型肝炎病毒 脱氧核糖核酸 实时荧光PCR 

分 类 号：R512.62[医药卫生—内科学] Q503[医药卫生—临床医学]