伪狂犬病病毒微滴数字PCR定量检测方法的建立

发布时间：2018-05-27 14:07

本文选题：伪狂犬病病毒 + 微滴数字PCR　；参考：《畜牧兽医学报》2017年09期

【摘要】：为了建立伪狂犬病病毒(PRV)微滴数字PCR(droplet digital PCR,ddPCR)的检测方法,以伪狂犬病病毒gB基因为靶基因,建立了可对其准确定量的微滴数字PCR方法。对ddPCR反应中的引物浓度、探针浓度、退火温度进行了优化。对建立的ddPCR方法的特异性和敏感性也进行测定,并且应用于临床样品的检测。结果表明,ddPCR的最佳引物浓度为0.9μmol·L~(-1),探针浓度为0.25μmol·L~(-1),退火温度为60℃。ddPCR方法线性相关系数(R2)为0.998,显示呈良好的线性关系。检测限为6.1copies·μL~(-1),比荧光定量PCR(Real Time PCR,qPCR)的检测限低,变异系数为2.81%,与其他病毒无交叉反应。通过对临床样品的检测,与病毒分离比较,ddPCR方法的敏感性为87.5%,特异性为96.2%,符合率为97%。结果表明新建立的ddPCR方法,敏感性高、特异性强,可用于伪狂犬病病毒的定量检测。
[Abstract]:In order to establish the detection method of pseudorabies virus (PRV) microdrop digital PCR (droplet digital PCR, ddPCR), the pseudorabies virus gB gene was used as the target gene, and the precise quantitative microdrop digital PCR method was established. The primer concentration, probe concentration and annealing temperature in the ddPCR reaction were optimized. The specificity of the ddPCR method for the ddPCR was established. The results showed that the best primer concentration of ddPCR was 0.9 Mu mol / L~ (-1), the concentration of the probe was 0.25 mol. L~ (-1), the annealing temperature was 60 C and the linear correlation coefficient (R2) was 0.998, showing a good linear relationship. The detection limit was 6.1copies. The detection limit of CR (Real Time PCR, qPCR) is low, the coefficient of variation is 2.81%, and there is no cross reaction with other viruses. By comparing the clinical samples, the sensitivity of the ddPCR method is 87.5% and the specificity is 96.2%. The coincidence rate is 97%. results show that the newly established ddPCR method is highly sensitive and specific, and can be used for pseudorabies virus. Quantitative detection.
【作者单位】：中国动物疫病预防控制中心OIE猪繁殖与呼吸综合征参考实验室;中国农业大学动物医学院;
【基金】：科技部科技基础性工作专项(2013FY113300-6)
【分类号】：S852.65

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