乙型肝炎病毒DNA标准物质的研究

目的研制国内用于HBV DNA扩增检测的血清标准物质。方法用HBV DNA阴性血浆将阳性血浆稀释至含量约1.00×106拷贝/ml,0.5 ml/支分装,然后进行真空冷冻干燥。检测方法采用实时荧光定量PCR方法和罗氏公司的PCR内标定量方法。检测室温14 d、37℃7 d、2～8℃6个月和-20℃时制备物的HBV DNA含量变化,确定其在不同条件下的稳定性。2年内不定期检测8次-20℃和-70℃保存时制备物的HBV DNA含量变化,确定其长期保存的稳定性。取30份样本分别检测其HBV DNA含量,并与混合后样本的HBV DNA含量进行比较,确定制备物的均匀性。制备标准品的HBV DNA含量通过与国际标准物质比对及根据国际标准物与制备标准物的吸光度值建立的标准曲线计算获得。结果该标准物质HBV DNA定值为(1.03±0.21)×106U/ml。稳定性实验表明制备物在室温、37℃和2～8℃时的HBV DNA含量与对照组(-20℃)比较,差异无统计学意义(t值分别为0.152、0.949、1.300,P值均>0.05);-20℃保存2年的制备物HBV DNA含量与对照组(-70℃)比较,差异无统计学意义(t=0.449,P>0.05)。均一性检测结果表明瓶间不精密度为4.46%,批内精密度和总精密度差异无统计学意义(F=1.0250,P>0.05)。结论该制备物达到了国家一级标准物质的要求,可以作为用于核酸扩增检测的HBV DNA标准物质。

Establishment of the first national standards for nucleic acid amplification technology assay for HBV DNA

OBJECTIVES: To establish a Chinese national standard for a nucleic acid test (NAT) for HBV DNA. METHODS: The candidate sample of HBV DNA positive plasma was diluted with HBV-negative human plasma. The sample was lyophilised with a concentration of approximately 300,000 copies/ml. The measurement methods used included Roche Amplicor assay (version 2.0) and real-time PCR. The lyophilised preparation was calibrated by the international standard (NIBSC code: 97/746) from NIBSC. RESULTS: The quantity of this lyophilised preparation was (1.29+/-0.24) x 10(5)IU/ml in comparison with the international standard for HBV DNA 97/746. The stability test indicated that the sample was stable at room temperature (20 to 25 degrees C) for 2 weeks and at 37 degrees C for at least 1 week. Long-term stability was observed at 2 to 8 degrees C for 6 months and at -20 degrees C for more than 2 years with no significant changes. The vial-to-vial imprecision rate was 3.53%. CONCLUSION: Based on the results of this study, our lyophilized sample can be used as a standard in China for the nucleic acid test (NAT) for HBV DNA.