不同方法抽提血清HBVDNA得率的比较  

Comparison of seven isolation methods for HBV DNA

作　　者：徐文胜[1] 缪晓辉[1] 潘怡[1] 吴文雅[1] 孔宪涛[1] 

机构地区：[1]上海第二军医大学长征医院感染科,200003

出　　处：《肝脏》2001年第4期219-221,共3页Chinese Hepatology

摘　　要：目的　比较不同的HBVDNA抽提方法对PCR产物量的影响。方法　将HBVDNA阳性血清及投入了HBVDNA质粒的HBVDNA阴性血清 ,分别采用 7种不同方法抽提核酸。抽提物作PCR后 ,产物行琼脂糖凝胶电泳 ,并对阳性扩增条带进行密度定量。结果　血清直接煮沸法、经典的蛋白酶裂解加酚 /氯仿抽提法、蛋白酶裂解加酚 /氯仿抽提法省缺乙醇沉淀和柱抽提法的HBVDNA抽提得率分别为 75 .2 %、13.8%、2 1.9%和 31.0 %;用碱变性裂解和蛋白酶裂解后煮沸所得上清液 ,以及血清直接作为模板 ,行PCR后不能得到阳性扩增条带。结论　核酸抽提方法选择不当能直接影响基因检测的灵敏度 ,导致基因定量准确性下降。血清直接煮沸法抽提HBVDNA得率高、操作简便、省时和经济 ,值得推荐。Objective To compare the recovery of HBV DNA by different methods of template preparation. Methods Two kinds of serum sample were used in the experiment: (1) HBV DNA plasmid were added to HBV DNA-negative serum, (2) HBV DNA positive serum detected by dot hybridization. The samples were prepared with seven methods for isolation of HBV DNA and amplified by PCR. PCR products were analyzed by agarose gel electrophoresis and density-quantified with SIXING image system.Results Four methods (serum boiling, protease plus phenol: chloroform: isoamyl alcohol with ethanol sedimentation, protease plus phenol: chloroform: isoamyl alcohol without ethanol sedimentation, DNA isolation kit) of template preparation were shown to be positive for HBV DNA amplicon after PCR, with the recovery of 75.2%, 13.8%, 21.9% and 31.0% respectively. HBV DNA could not be detected when the templates are prepared as follows: alkaline-denatured serum, serum cleaved with protease and boiling, and serum without any preparation.Conclusion Above results strongly suggested that the method of HBV DNA isolation can be one of the factors to the sensitivity of PCR, especially for quantitative analysis. With many advantages such as high recovery, less time and material consuming and convenience, serum boiling may be the best choice of template preparation for PCR.

关 键 词：乙型肝炎病毒 基因检测 聚合酶链反应 抽提方法 HBV-DNA 

分 类 号：R446.5[医药卫生—诊断学]