非洲猪瘟病毒核酸荧光定量PCR检测方法研究

Study on the fluorescent quantitative PCR method for the detection of African swine fever virus nucleic acid

中文关键词:  非洲猪瘟病毒  荧光定量PCR  微滴式数字PCR

英文关键词:African swine fever virus  fluorescent quantitative PCR  droplet digital PCR

中文摘要:

      目的 建立非洲猪瘟病毒核酸的荧光定量PCR检测方法, 并与微滴式数字PCR检测方法的优缺点进行比较。方法 根据非洲猪瘟病毒保守基因VP72和K205设计合成引物和探针, 探究荧光定量PCR检测方法的反应条件和方法的特异性、线性、灵敏性和重复性, 及其与微滴式数字PCR检测方法灵敏度差异。结果 本研究建立的非洲猪瘟病毒核酸荧光定量PCR检测方法检出限达到0.1 fg/反应, 在0.1~500.0 fg/反应范围内呈现良好的线性关系, 检测重复性变异系数均低于1%, 不与猪瘟病毒、猪蓝耳病毒发生交叉反应。结论 本研究建立的荧光定量PCR法灵敏度高、特异性强, 适用于猪肉及其制品中非洲猪瘟病毒的检测。

英文摘要:

      Objective To establish a fluorescent quantitative PCR method for the detection of African swine fever virus nucleic acid, and compare its advantages and disadvantages with droplet digital PCR detection method. Methods Primers and probes were designed and synthesized based on the conservative genes VP72 and K205 of the African swine fever virus, the reaction conditions, the specificity, linearity, sensitivity and repeatability of fluorescent quantitative PCR detection method were explored, and the sensitivity differences with the parameters of the droplet digital PCR detection method were obtained. Results The limit of detection of the African swine fever virus nucleic acid fluorescent quantitative PCR detection method established in this study was 0.1 fg/reaction, the results showed good linearity within the range of 0.1?500.0 fg/reaction, and the detection repeatability coefficients of variation were less than 1%, and did not cross-react with classic swine fever virus, porcine reproductive and respiratory syndrome virus. Conclusion The fluorescent quantitative PCR method established in this study has high sensitivity and specificity, which is suitable for the detection of African swine fever virus in pork and its products.