一种可用于OBI检测的高灵敏度核酸提取方法的建立与验证

Establishment and preliminary verification of a highly sensitive nucleic acid extraction method for OBI detection

目的 建立和验证1种可用于隐匿性HBV感染(OBI)检测的大体积高灵敏度核酸提取方法,并将其应用于低病毒载量OBI标本的定量检测。方法 参考罗氏核酸检测试剂盒核酸提取方法,建立1种大体积核酸提取方法。将HBV标准品分别配置成10 000 IU/mL、1 000 IU/mL、100 IU/mL、10 IU/mL和1 IU/mL浓度,采用磁珠法从10 mL相应浓度的标准品中提取核酸,采用荧光定量PCR法检测各浓度的CT值,每个浓度梯度进行平行双份检测,以病毒浓度的对数为X轴,2次检测CT值的平均值为Y轴,构建荧光定量标准曲线和回归方程。通过3次重复实验验证该方法的稳定性。应用本方法提取低病毒载量OBI标本核酸并进行荧光定量。结果 3次HBV病毒荧光定量标准曲线扩增效率(E)均在90%~105%,回归方程(R2)均>0.99。不同浓度标准品CT值的变异系数(CV)分别为0.63%、0.78%、1.52%、1.36%和0.78%。本方法可以提取出病毒载量低至1 IU/mL的OBI标本核酸并进行定量。结论 本方法HBV核酸定量检测系统检测下限可达1 IU/mL,并且具有较强稳定性和灵敏度,可用于低病毒载量OBI的定量检测。

Objective To establish and verify a new nucleic acid extraction method for OBI detection with large volume and high sensitivity,and apply it in the quantitative determination of OBI samples with low viral load.Methods The method for nucleic acid extraction with large volume was established based on the method of Roche nucleic acid detection kit.HBV standards were configured into 10000 IU/mL,1000 IU/mL,100 IU/mL,10 IU/mL and 1 IU/mL respectively,and nucleic acid was extracted from the 10 mL standards by magnetic beads.CT values of each concentration were detected by fluorescence quantitative PCR and each concentration gradient was detected in parallel duplicates.The logarithm of virus concentration was taken as the X-axis and the average CT values of two tests were taken as the Y-axis to construct the fluorescence quantitative standard curve and regression equation.Three repeated experiments were conducted to verify the stability of the method.This method was used to extract nucleic acid from OBI samples with low viral load,and fluorescence quantification was performed.Results The amplification efficiency of fluorescence quantitative standard curves ranged from 90%to 105%,and the regression equation was greater than 0.99.The variation coefficients of variation of CT values were 0.63%,0.78%,1.52%,1.36%and 0.78%,respectively.This method can extract nucleic acid from OBI samples with viral load of 1 IU/mL for quantification.Conclusion The detection limit of HBV nucleic acid quantitative detection system can reach 1 IU/mL,and it has strong stability and high sensitivity,which can be used for the quantitative detection of OBI with low viral load.