摘要
目的 对不同核酸提取方法在荧光定量聚合酶链反应 (FQ PCR)检测血清乙型肝炎病毒核酸 (HBVDNA)含量中应用进行评价 ,为临床方法学选择和实验结果的评价提供理论依据。方法 选用直接煮沸裂解法、NP 4 0煮沸裂解法、沉淀煮沸裂解法、磁珠核酸提取法等 4种核酸提取方法提取乙型肝炎血清标本和含不同含量肝素的血清标本中HBVDNA模板 ,用荧光实时定量PCR法对其进行准确定量 ,从提取效率、抗干扰能力方面进行评价。结果 4种核酸提取方法中磁珠核酸提取法提取效率为最高 ,对数换算后 ,以此相对提取效率为 10 0 % ,则直接煮沸裂解法为最低 81% (P <0 0 5 )、NP 4 0煮沸裂解法为 95 % (P >0 .0 5 )、沉淀煮沸裂解法为 88.7% (P <0 .0 5 ) ;从抗干扰能力方面 ,当肝素含量为 5 0 0U/ml时 ,磁珠核酸提取法抗干扰能力最强 ,沉淀煮沸裂解法次之、直接煮沸裂解法和NP 4 0煮沸裂解法为最差 ,其HBVDNA含量抑制率分别为 9.4 %、13.5 %、35 .4 %、5 2 .7%。结论 本实验结果认为磁珠核酸提取法为实验室首选的核酸提取方法。
Objective To investigate the effects of the nuclei acid extraction methods, including direct?0.4% NP 40?precipitation boiling extraction and magnetic beads extraction methods, on HBV DNA detection by fluorescence real time quantitative PCR (Taqman). Methods Using the four kinds of nuclei acid extraction methods and fluorescence real time quantitative PCR (Taqman), nucleic acid of HBV DNA were extracted and detected in hepatitis B patients serum samples, witch contain different concentration of heparin (0～1 250 U/ml), but with high HBV DNA concentration. The nucleic acid extraction methods were evaluated for the efficiency of extraction and the ability of anti interference. Results The extraction efficiency of magnetic beads nuclei acid extraction method was the highest (relative 100%) and the direct boiling extraction method was the lowest (relative 81%, P <0.05). The ability of anti interference of magnetic beads nuclei acid extraction method was also the strongest, direct and 0.4% NP 40 boiling extraction methods were the weakest. When heparin concentration in plasma was 500 U/ml, the inhibitory rates were 9.4%?13.5%?35.4%?52.7% respectively. Conclusion The results suggested that magnetic beads nuclei acid extraction method is the best one. The direct and 0.4% NP40 boiling extraction methods were not suitable for the HBV DNA template extraction for fluorescence real time quantitative PCR (Taqman).
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2002年第4期212-214,共3页
Chinese Journal of Laboratory Medicine
