磁珠核酸提取方法在血浆EBV-DNA实时荧光定量PCR检测中应用的性能评价

Performance evaluation of magnetic bead nucleic acid extraction in realtime fluorescence quantitative PCR detection system for plasma EBV-DNA

目的评价磁珠核酸提取方法在血浆EB病毒DNA(EBV-DNA)实时荧光定量PCR检测中应用的性能。方法采用自动化磁珠法提取核酸,并结合实时荧光定量PCR测定EBV-DNA,利用第三方定值参考物质评价该方法的检测性能。同时选取100例鼻咽癌患者,分别用磁珠法与煮沸法定量检测其血浆EBV-DNA,比较分析两种方法的相关性。结果基于自动化磁珠法的实时荧光定量PCR系统检测EBV-DNA,其低、中、高水平的重复性精密度均<3/5 TEa(TEa=靶值±0.4 lg),中间精密度<4/5 TEa;在5.4×101~5.4×10~5U/mL范围内具有良好的线性;最低检测限约为3.334×10~1U/mL;样本中Hb干扰物浓度低于8.667 g/L,对磁珠法检测结果无明显影响。磁珠法与煮沸法的检测结果呈线性相关,阴阳性符合率为83%。结论采用自动化磁珠核酸提取方法结合实时荧光定量PCR检测血浆EBV-DNA,具备良好的检测性能。

Objective To evaluate the application performance of magnetic bead nucleic acid extraction method in realtime fluorescence quantitative PCR detection for plasma Epstein-Barr virus（EBV） DNA. Methods Automated magnetic bead method was used to extract nucleic acid, and combined real-time fluorescence quantitative PCR assays for quantitative EBV-DNA. The detection performance of the method was evaluated by using the third-party reference materials.In addition, 100 patients with NPC were selected, and the plasma EBV-DNA was quantitatively detected by magnetic beads method and boiling method. The correlation between the two methods was analyzed. Results In the automated magnetic bead method-based real-time fluorescence quantitative PCR detection system for EBV-DNA, the repeatability precision of the low, medium and high were all 3/5 TEa（TEa = target ± 0.4 lg）, and the intermediate precision were all 4/5 Tea. In the range of 5.4×101-5.4×10^5U/mL, there had good linear relationship. The minimum detection limit was about 3.334 ×10^1U/mL. And the concentration of Hb interference in plasma was lower than 8.667 g/L, the results of magnetic bead test were not significantly affected. The detection results between magnetic bead method and boiling method were linearly related, and the qualitative coincidence rate was 83%. Conclusion The detection of plasma EBVDNA by automated magnetic bead nucleic acid extraction method can achieve good detection performance.