三种HBV DNA提取方法对荧光定量PCR检测结果影响的比较

目的 研究3种核酸提取方法对黄疸、溶血和脂血标本HBV DNA荧光定量结果的影响,为进一步提高临床检验质最和改进检测技术提供理论依据.方法 采用目前临床常用的多聚糖病毒沉淀浓缩法(PEG法)、碱液直接裂解和微量核酸释放剂(Micro-Nucleic-Releaser,MNR)等3种核酸提取方法,对含有HBV的黄疸、溶血和脂血标本进行荧光定量PCR检测,研究不同标本对荧光PCR定量结果和扩增效率的影响.选择酚一氯仿提纯法作为核酸提取对照方法.结果 黄疸、溶血和脂血标本,对3种不同核酸提取方法的荧光PCR定量结果和扩增效率都产生不同程度的影响,其中完全溶血的标本对PCR扩增效率的影响最大,如定量值为4×103拷贝/ml的低拷贝完全溶血标本,MNR法测定结果为2.21×103拷贝/ml,PEG法为1.02×103拷贝/ml,碱性直接裂解法的定量值为阴性,临床自然溶血较重的标本对荧光定量检测影响较小,脂血和黄疸标本对荧光定量PCR扩增效率有明显抑制作用,但对实际定最值无明显影响;其中,MNR法在不同标本的定量重复性和荧光PCR扩增效率最好,碱液直接裂解法较差.酚.氯仿提纯法虽然获得较高的扩增效率,但存在大约有1个数量级的核酸丢失.结论 基于MNR核酸提取方法的荧光定量PCR检测,对不同的临床标本均可获得较好的扩增效率、敏感性和重复性,操作简便、快速且无核酸丢失和污染,适合临床检验应用.

The comparison of three different nucleic acid isolation methods in hepatitis B virus DNA detection with real-time quantitative PCR

Objective To investigate the influence of different HBV DNA extraction methods with Micro-Nucleic-Releaser (MNR), polysaccharide deposition method and boiling method in real-time quantitative and provide reference data for clinical lab regarding the PCR kit selection. Methods Three different HBV DNA isolation methods including MNR, polysaccharide deposition method and boiling method were used to study the extraction efficiency and anti-interference ability when HBV DNA virus in lipemia, haemolysis or jaundice serum samples was detected with real-time PCR. The traditional HBV DNA isolation method of Hydroxybenzene-Chloroform was used as control method. Results The quantitative results and amplification efficiency differed among different extraction methods. Complete hemolytic sample brought biggest influence, modest hemolytic sample give minimal influence, whereas the lipemia and jaundice did not bring significant influence to detection. The MNR give the best amplification efficiency and quantitativereproducibility. The extraction efficiency of boiling method was lowest. The fluorescent efficiency of real-timePCR from Hydroxybenzene-Chloroform methods was better than other methods, but it suffered from losingHBV DNA. Conclusions Real-time PCR system of HBV DNA isolation by MNR can provide an accurate,highly sensitive, and rapid method to quantify Hepatitis B virus. It is suitable to use in clinical laboratory.