血清HBV-DNA模板不同提取方法的比较和选择

Comparison of different serum HBV-DNA extraction methods

目的比较直接煮沸法、碱裂解法、浓缩碱裂解法和碱裂解3倍量提取法四种提取法提取样本核酸的差异;选择简便、可靠的检测乙肝病毒DNA(HBV-DNA)提取方法。方法运用中山医科大学达安基因股份有限公司生产的HBV-DNA试剂盒中推荐的浓缩碱裂解法,与直接煮沸法、碱裂解法、碱裂解3倍量提取法,分别提取血清样本核酸模板,用美国MJ实时荧光定量PCR仪检测HBV-DNA,对结果进行对比分析。结果共89例乙肝表面抗原(HB-sAg)阳性患者血清,浓缩碱裂解法和碱裂解3倍量提取法检测,两者均有62例HBV-DNA阳性(>1000拷贝/ml定性为阳性)阳性率69.6%,阳性拷贝/ml均值用对数形式表示分别为(6.652±1.301)和(6.601±1.282),两者间结果无明显差异(P>0.05);直接煮沸法检测出47例阳性(均在上述62例中)阳性率52.8%;碱裂解法测出57例阳性(均在上述62例中)阳性率64.0%,阳性拷贝/ml均值与浓缩碱裂解法和碱裂解3倍量提取法相比较,阳性检出率较低,有非常显著性差异(P<0.01)和显著性差异(P<0.05)。结论碱裂解3倍量提取法操作简单,省时、省材料,重复性好,阳性检出率与浓缩碱裂解法一致,建议使用碱裂解3倍量提取法检测HBV-DNA。

Objective To compare different HBV - DNA extraction methods including boiling, alkaline lysis, high concentration alkaline lysis, and 3 times sampling after alkaline lysis, in order to select the most simple and reliable method to extract HBV - DNA from serum. Methods Serum DNA was extracted with high concentration alkaline lysis procedure recommended by the manufacturer of the DNA extraction kit （ Daan Gene Limited Company, Zhongshan Medical University）, boiling, alkaline lysis, and 3 times sampling after alkaline lysis respectively. HBV - DNA was examined by fluorescent quantitation PCR, the results obtained with different DNA extraction methods were compared and analyzed. Resuits Of the 89 HBV - DNA positive serum samples, 62 samples （69.6%） showed positive （ 〉 1000 copies/ml） with both high concentration alkaline lysis method and the method of 3 times sampling after alkaline lysis, and the means of positive sampleg copyies/ml of the two methods didn t show difference （ P 〉0.05）. However, 47 （52.8%） and 57 （64.0%） positive samples were detected with boiling method and alkaline lysis method respectively. The positive samples detected with boiling method and alkaline lysis method were all detected by high concentration alkaline lysis method and big sampling method after alkaline lysis, but the mean copyies/ml of the positive samples and the positive rate were significantly lower （ P 〈 0.01 and P 〈 0.05 respectively）. Conclusion The method of 3 times sampling after alkaline lysis is suggested for HBV - DNA ex traction from serum for its simple, economic and high reproducibility.