磁珠快速提取乙型肝炎病毒DNA方法的建立及其应用研究

To establish a magnetic beads based nucleic acid extraction method and apply for quantitative detection of hepatitis B virus

目的建立一种基于磁珠法的快捷、简便、高纯度血液HBV DNA提取方法（以下简称磁珠法），为用于临床自动化定量检测的核酸诊断试剂研发提供技术支持。方法用科华生物公司内部5份HBV强阳性血清标本，先通过磁珠的磁性、悬浮性、HBV DNA提取效果等对4种磁珠（A～D磁珠）进行筛选，并对磁珠用量（1～1．75μL／m1）、裂解液pH值（4～9）、洗涤液中是否加入乙醇和洗脱液带磁珠扩增等条件进行系统优化；并对建立的磁珠法线性范围、精密度、灵敏度、抗污染和抗干扰能力等技术性能进行评价。然后，分别用磁珠法与国内成熟的煮沸法定量检测100份HBV血清标本，用磁珠法与国际“金标准”Roche COBAS TaqManTest（以下简称Roche法）定量检测50份HBV血清标本，并比较分析3种方法的相关性。结果通过实验条件优选试验确定超顺磁A磁珠在物理性能和DNA提取效果都优于其他3种磁珠，磁珠最佳使用量为1．25μl／ml，裂解液最佳pH值为7—8，洗涤液中可去除乙醇成分，并可直接带磁珠进行扩增反应。通过平行检测5份公司内部HBV强阳性血清标本以及梯度稀释的血清标本确定磁珠法的线性范围为4×101～4×108 IU／ml，煮沸法为4×102-4×108 IU／ml；磁珠法检测灵敏度为30IU／ml，高于煮沸法（100IU／ml）；在低浓度HBV血清标本检测中，磁珠法的精密度（CV为6．0％）优于煮沸法（CV为11．9％）；磁珠法检测的假阳性率为0，而煮沸法假阳性率为3．1％，磁珠法抗污染能力优于煮沸法；煮沸法在标本中肝素钠含量达到62．5U／ml时，检出血清中HBV DNA含量明显下降，当肝素钠达1000U／ml时，无法检出HBV DNA，而肝素钠对磁珠法几乎无影响。同时，磁珠法和煮沸法平行检测100份临床HBV血清标本，磁珠法的阳性率（86％）高于煮沸法（74％），且2种方法的相关性较好（R2=0．948，P〈0．05）；磁珠法和Roche法平行检测50份临床HBV血清标本，磁珠法检出阳性率[95％（49／50）]与Roche法[100％（50／50）]接近，2种方法间有显著相关性（R2=0．963，P〈0．05）。结论建立的磁珠法定量检测HBV DNA的得率高，具有较宽的检测范围、定量准确、操作简单，可作为实验室首选的核酸提取方法用于临床乙型肝炎诊断。

Objective To facilitate the establishment of fully automatic clinical diagnostic nucleic acid assay, a study was carried out for developing a fast and convenient method of extracting highly purified viral nucleic acid from human blood with magnetic beads. Method Five KHB （ Shanghai Kehua Biological Engineering Company Limited） internal strong positive HBV serum samples were used in the process, for the method development, screening and selecting the magnetic beads based on magnetism, suspension power and extraction effects of HBV DNA, the optimization of the selected key components of extraction reagents were performed, which including the quantity of magnetic beads（ 1 - 1.75 μl/ml） , the buffer pH of lysis （4 - 9 ）, washing conditions with or without alcohol, elution for proliferation with or without magnetic beads, and etc. The evaluation for the extraction method was carried out including linearity, precision, sensitivity, specificity, anti-interference and resistance to contamination. The magnetic beads method was compared with domestic mature boiling method in a quantitative test with 100 HBV serum samples, the magnetic beads method was also compared with the interntional ＂ Gold standard＂ Roche COBAS Tagman test method （Roche method） in a quantitative test with 50 HBV serum samples and the results were statistically compared for their linear regression. Results Optimization experiment result showed superparamagnetic beads A was the best among the candidates （C, B, D） in terms of their physical properties and extraction effects of DNA. The optimum condition of the assay were as follow：magnetic beads best used at 1.25 μl/ml, buffer pH 7 -8 of lysis, ethanol was eliminated from washing buffer, proliferation can be carried out with magnetic beads. The parallel comparison experiments with five KHB internal strong positive HBV serum sample and gradient diluted serum sample between beads method and boiling method were carried out. The results showed that the linear range of beads method was 4×101 -4×108 IU/ml and that of boiling method was 4×102 -4×108 IU/ml. The sensitivity for beads method was 30 IU/ml and that of boiling method was 100 IU/ml. The precision CV of beads method was 6.0% and boiling method was 11.9%. The false positive rate was 0 for beads method vs 3.1% for boiling method. The anti-interference ability experiment showed that when sample contained sodium heparin concentration exceed 62. 5 U/ml, there was significant drop of HBV DNA detection with boiling method and no HBV DNA was detected when sodium heparin concentration in the sample reached 1000 U/ml. However, sample sodium heparin concentration had no effect on HBV DNA detection with beads method. The comparison study between beads method and boiling method was carried out by use of 100 clinical HBV positive serum samples. The result showed that the sensitivity for beads method was 86% vs boiling method 74%. The correlation between the two method was R2 = 0. 948, P 〈 0. 05. The comparison study between beads method and Roche method was carried use of 50 clinical HBV DNA positive serum samples and the result showed that sensitivity for beads method was 95% （49/50） vs Roche method was 100% （50/50）. The correlation was R2 = 0. 963, P 〈 0.05. Conclusions The established method for automatic nucleic acid extraction with magnetic bead displayed characteristic of high yield nucleic acid extraction, broader testing range, accurate quantification and convenient in operation. This can be one choice for nucleic acid extraction and applicable for clinical automatic diagnostic system.