三种核酸提取方法用于检测血浆EBV—DNA的性能及应用评价

Evaluation of performance and application of three nucleic acid extraction methods for quantification of plasma Epstein-Barr virus DNA

目的评价并比较用于检测血浆EBV-DNA的3种核酸提取方法的分析性能及临床应用价值。方法分别采用硅膜离心柱法、浓缩煮沸裂解法或自动化磁珠吸附法平行提取核酸，并通过实时荧光定量PCR测定EBV-DNA。利用第三方定值参考物质，比较三种方法的检测性能，并配对检测100例NPC患者及100名健康体检者血浆，评估3种方法的临床应用价值。结果3种方法提取并检测EBV-DNA的正确度、不精密度均符合要求，且对临床样本的测定结果均呈线性相关。但磁珠法的重复性不精密度和中间不精密度较离心柱法与煮沸法均小且稳定（均〈3%）；最低检测限（约3.334×101 IU/ml）较离心柱法（4.159×101 IU/ml）与煮沸法（8.511×101 IU/ml）稍灵敏；线性范围（5.4×101～5.4×105 IU/ml）较煮沸法（5.4×102～5.4×105 IU/ml）稍宽；抗Hb干扰能力优于煮沸法；而且对NPC样本检测的阳性率及平均病毒载量（95%，8.342×103 IU/ml）均显著高于离心柱法（84%，4.707×103 IU/ml）与煮沸法（78%，2.571×103 IU/ml）（P均〈0.05）。 结论采用自动化磁珠吸附法提取检测血浆EBV-DNA可以实现更好的检测性能，并具备更高的临床应用价值。

ObjectiveTo evaluate and compare the analytical performances and application values of three nucleic acid extraction methods for quantification of plasma Epstein-Barr Virus （EBV） DNA.MethodsIt used silica membrane spin column, boiling and automated magnetic bead method to extract viral nucleic acid in parallel, and combined real-time fluorescence quantitative PCR assays for quantitative EBV-DNA quantification.The performances of three methods were determined and compared by using the third-party reference materials, and the clinical values were analyzed by pairing detecting 100 NPC patients and 100 healthy subjects in pair.ResultsThe accuracy and imprecision of three methods were all in line with requirements, and the results of clinical samples were linearly correlated. But actually the reproducibility and intermediate imprecision of the magnetic bead method were smaller and stable than those of the spin column method and the boiling method （all 〈3%）; the limit of detection for the magnetic bead method was 3.334×101 IU/ml, better than that of spin column method （4.159×101 IU/ml） and boiling method （8.511×101 IU/ml）; the linear range of the magnetic bead method was 5.4×101-5.4×105 IU/ml, slightly wider than that of the boiling method（5.4×102-5.4×105 IU/ml）; the ability of anti - Hb interference ability of magnetic bead method is better than that of boiling method; and the positive rate and the mean viral load of the NPC samples measured with the magnetic bead method were significantly higher （95%, 8.342×103 IU/ml） than those measured with the spin column method （84%, 4.707×103 IU/ml） and the boiling method （78%, 2.571×103 IU/ml） （P all〈0.05）.ConclusionThe automated magnetic bead nucleic acid extraction method offered better analytical performance and higher clinical value for EBV DNA quantification in plasma.（Chin J Lab Med, 2018, 41： 59-65）