论文部分内容阅读
目的:研究模板纯化与否对血浆HBV DNA实时荧光定量PCR检测线性范围、重复性、灵敏度的影响。方法:采用国产离心柱法DNA纯化试剂盒及实时定量PCR试剂盒提供的直接煮沸裂解法处理107～10110倍浓度梯度稀释的血浆样本和标准品及乙肝窗口期患者的血清标本,用国产HBV PCR荧光定量扩增试剂盒检测,比较其检测结果线性范围、重复性、灵敏度及乙肝窗口期患者HBV DNA阳性检出率。结果:离心柱纯化组和煮沸裂解组的检测线性范围分别为107IU/ml～101IU/ml和107IU/ml～103IU/ml;灵敏度分别为24 IU/ml～48 IU/ml和1200 IU/ml～2400 IU/ml;104IU/ml、103IU/ml、102IU/ml样品的批内变异系数(CV)离心柱纯化组为2.5%、2.3%、1.8%,煮沸裂解组为1.9%、1.6%、12%,批间变异系数(CV)离心柱纯化组为2.3%、2.9%、5.4%,煮沸裂解组为1.7%、4.5%、7.7%;离心柱纯化组和煮沸裂解组的窗口期患者血清标本阳性检出率分别为83.9%和3.6%。结论:HBV血清样品经离心柱纯化可以极大地改善乙型肝炎病毒DNA实时荧光定量PCR检测结果。
Objective: To study the influence of template purification on the linear range, repeatability and sensitivity of plasma HBV DNA real-time PCR. Methods: Serum samples from 107 to 10110-fold dilutions of plasma samples and standard samples and hepatitis B patients were directly processed by the domestic centrifugal column DNA purification kit and real-time quantitative PCR kit. Fluorescence quantitative amplification kit was used to detect the linear range, repeatability, sensitivity and positive detection rate of HBV DNA in patients with hepatitis B during the test. Results: The linear range of the column purification group and the boiling lysis group were 107IU / ml ~ 101IU / ml and 107IU / ml ~ 103IU / ml respectively; the sensitivity was 24 IU / ml ~ 48IU / ml and 1200IU / The intra-assay coefficient of variation (CV) for the samples of 104 IU / ml, 103 IU / ml, and 102 IU / ml for the 2400 IU / ml was 2.5%, 2.3%, 1.8% for the purified column and 1.9% for the boiled lysis group %, Respectively. The coefficient of variation (CV) in the centrifugation column was 2.3%, 2.9% and 5.4% in the purified group and 1.7%, 4.5% and 7.7% in the boiling lysate group. The positive detection rates were 83.9% and 3.6% respectively. Conclusion: The purification of HBV serum samples by centrifugation column can greatly improve the detection of hepatitis B virus DNA by real-time fluorescence quantitative PCR.