血清乙肝病毒DNA煮沸抽提法的建立和应用

Establishment and application of boiling method to extract HBV DNA from serum sample

目的 建立一种简便的血清HBV DNA抽提方法一煮沸抽提法,并将其应用于巢式PCR扩增。方法 选取16份CHB血清,分别应用直接煮沸法和异硫氰酸胍-酚/氯仿法进行HBV DNA抽提后,进行荧光定量PCR测定;并以煮沸法抽提样品为模板,进行HBV DNA P区部分片段巢式PCR扩增。结果 将上述两种抽提方法的荧光定量PCR结果取对数后进行直线相关分析和配对样本t检验,结果显示:两种方法的定量PCR结果之间存在良好的相关性(r=0.696,P=0.003),且二者定量PCR结果无差异(P=0.663)。16份标本巢式PCR扩增均为阳性,PCR产物分子量大小与预期值相符。结论 直接煮沸法抽提HBV DNA,简便、快速、效果好,适用于HBV的科研和临床检测工作。

Objective To establish a new method to extract HBV DNA from serum sample for nested PCR.Methods The DNA templates from 16 serum samples with different HBV DNA concentration were prepared by boiling serum to release HBV DNA from the virus particles and purifying the nucleic acid from serum. Then, the difference of HBV DNA quantitative were valued by fluorescence quantitative PCR. Finally, the DNA templates prepared by boiling method were used to nested PCR for amplifying segment of HBV P gene. Results The HBV DNA quantitative values by fluorescence quantitative PCR using the templates prepared by the two methods were positively correlative ( r = 0.696, P = 0 .003) and not significantly different ( P - 0.663) . The results of nested PCR of 16 DNA templates by boiling method were positive. Conclusions The boiling method to extract HBV DNA from serum sample for PCR is convenient, rapid and reliable, and can be applied to scientific research and clinical detection of HBV.