一种适合中国地区HBV流行特征的荧光定量PCR检测方法的初探

Establishment of a preliminary real-time quantitative RT-PCR and nucleic acid test method to detect HBV based on nationalistic characteristics

目的设计1套适合中国地区HBV流行特征的高灵敏度、高特异性的HBV引物探针,建立HBV荧光定量检测的方法。方法在NCBI的Nucletide中输入HBV、China、complete genome等关键词找到中国地区的HBV序列,经比对后在保守序列设计引物探针,引物扩增的PCR产物经PMD-19载体连接制备成质粒标准品并测序验证序列,计算质粒拷贝数,将质粒标准品稀释成不同浓度的质粒标准品,用第三方标准品对质粒标准品做校正,绘制HBV标准曲线,并用Probit分析计算检测下限,验证灵敏度、特异性等。将所建立的HBV荧光定量检测方法检测100例标本,并与罗氏cobasTaq Screen MPX Test,version 2.0检测试剂盒检测结果做相关性分析。结果引物探针特异性好,质粒测序结果与目的产物完全吻合,质粒标准品曲线R^2>0.999,扩增效率为95.43%,线性范围(2×10~2-2.5×10~9)IU/m L,95%检测下限为16.2 IU/m L,批内重复变异系数为0.15%-1.11%,批间变异系数为0.91%-4.67%。与罗氏检测结果相关性高(R=0.96)。结论成功设计出1对适合中国HBV流行特征的引物探针,所建立的HBV荧光定量检测的方法灵敏度高、特异性好、重复性好,为在对中国地区开展血液HBV DNA载量检测及监控提供了技术支撑。

Objective To design a highly sensitive and highly specific real-time quantitative RT-PCR nucleic acid test method to detect HBV based on Chinese characteristics. Methods HBV sequences with prevalence in China with the key words ＂ HBV＂, ＂ China＂, ＂ complete genome＂ and others were searched in the National Center for Biotechnology Information（ NCBI） nucleotide database. A sequence identity comparison was then performed by DNAMAN software. Primers and probe in the conserved regions were designed. The PCR amplified products were prepared to make HBV fragment DNA plasmid with PMD-19 vector. A standard curve was drawn after plasmid verification. The limits of detection after calibration in the plasmid by the third-party standard HBV inactivated virus was calculated. Finally,the method to test 100 plasma samples which have been tested by Roche cobas v2. 0 system was implemented. Results The primers and probe were sensitive and specific. The plasmid sequencing results were completely consistent with the HBV DNA sequence. The plasmid standard curve showed R^2 0. 999. The amplification efficiency was 95. 43%. The linear range was from 2 × 10~2 to 2. 5 × 10~9 IU / m L.The 95% detection limitation was 16. 2 IU / m L. The intra-assay variable coefficient varied from 0. 15% to 1. 11%,while the inter-assay variable coefficient varied from 0. 91% to 4. 67%. The results of the samples were in accordance with previous test results. Conclusion A nucleic acid test method with high sensitivity,high specificity and high stability was established to detect HBV-DNA by real-time quantitative RT-PCR,which is useful to monitor blood safety.