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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cobasTaq 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 HBV primer probe suitable for HBV epidemic in China and establish a method for the quantitative detection of HBV fluorescence. Methods Nucleotide sequences of HBV, China and complete genome were input into Nucleicides of NCBI to find the HBV sequences in China. After the comparison, the primers were designed in the conserved sequences. The amplified PCR products were cloned into plasmid pDD-19 Standard sequence sequencing and sequencing, calculate the copy number of the plasmid, the plasmid standard was diluted into different concentrations of plasmid standard, third-party standard plasmid plasmid calibration, draw the HBV standard curve, and Probit analysis to calculate the lower limit of verification Sensitivity, specificity and more. The established method of fluorescence quantitative detection of HBV was detected in 100 cases and the correlation was analyzed with the results of Roche cobas Taq Screen MPX Test, version 2.0 test kit. Results The primers were specific and the plasmid sequencing results were in good agreement with the target product. The curve of plasmid standard was R ~ 2> 0.999, and the amplification efficiency was 95.43%. The linear range (2 × 10 ~ 2-2.5 × 10 ~ 9) / m L, the detection limit of 95% was 16.2 IU / m L, the intra-assay repetitive coefficient of variation was 0.15% -1.11%, and the inter-assay coefficient of variation was 0.91% -4.67%. Correlation with Roche test results (R = 0.96). Conclusion A pair of primer probes suitable for the epidemiological characteristics of HBV in China were successfully designed. The established method of fluorescence quantitative detection of HBV has the advantages of high sensitivity, good specificity and good repeatability. In order to detect and monitor the blood HBV DNA load in China, Provided technical support.