磁珠吸附法定量检测乙型肝炎病毒的效能

The efficiency of magnetic bead adsorption for quantitative detection of hepatitis B virus DNA

目的建立磁珠吸附定量检测HBV DNA的方法,并评价该方法的检测效率。方法将病毒载量为10~7IU/mL的血清样本,倍比稀释成不同浓度的HBV DNA作为血清标准品（理论值）,分别用磁珠吸附定量法（试剂1）、磁珠法（试剂2）、煮沸法（试剂3）3种方法提取HBV DNA,从灵敏度、定量线性关系方面比较本实验方法与国产磁珠法核酸自动提取法和传统煮沸法的提取效果,并对本实验方法进行稳定性验证。结果灵敏度：HBV DNA定量的检测下限理论值为10^1IU/mL,试剂1为3.520×10^1 IU/mL,试剂2为9.123×10^3 IU/mL,试剂3为6.195×10^1 IU/mL。相关性：试剂1、试剂2、试剂3提取HBV DNA定量与理论值相关性分析的r值分别为0.986、0.950、0.979（均P〈0.01）。稳定性：3种方法检测的相对偏差均值分别为0.243±0.405（试剂1）、1.189±0.855（试剂2）、-0.439±0.618（试剂3）,试剂1对同一样本在不同时间检测结果的相对偏差均值为0.505±0.659。结论本实验所设计的血清HBV DNA提取方法灵敏,定量线性关系好,结果稳定准确,为定量检测HBV DNA奠定了基础。

Objective To establish a magnetic bead adsorption method to quantitatively detect hepatitis B virus（HBV）DNA,and to evaluate its efficiency.Methods Serum template of 107 IU/ml HBV virus was diluted into different concentrations as serum standard panels（theoretical value）,HBV DNA in which were extracted by our magnetic bead adsorption method（reagent 1）,domestic automatic nucleic acids extraction with magnetic beads（reagent 2）and boiling method（reagent 3）,respectively.Comparisons in extraction efficiency among those 3 methods were carried out by analyzing sensitivity,quantitative linear relationship and stability.Results Theoretical value of lower HBV DNA detection limit was101 IU/mL,and the extraction results were 3.520×10^1 IU/mL by reagent 1,9.123×10^3 IU/mL by reagent 2 and 6.195×10^1 IU/mL by reagent 3.Correlation analysis between extraction results and theoretical values showed r values as 0.986（reagent 1,P〈0.001）,0.950（reagent 2,P=0.001）and 0.979（reagent 3,P〈0.001）,respectively,which revealed statistically significant differences.Average relative deviations of the 3 methods were 0.243±0.405（reagent 1）,1.189±0.855（reagent 2）,-0.439±0.618（reagent 3）,respectively.In addition,the average relative deviation of reagent 1 for same sample at different times was 0.505±0.659.Conclusion Magnetic bead adsorption method for HBV DNA extraction has good sensitivity,quantitative linear relationship,stability and accuracy,which might be a reliable method for the quantitative detection of HBV DNA.