目的 建立并验证能同步检测免疫猪血浆中非洲猪瘟病毒（African swine fever virus，ASFV）、猪细小病毒（porcine parvovirus，PPV）和伪狂犬病病毒（pseudorabies virus，PRV）的荧光PCR法。方法 参考上述3种病毒的国家标准，设计特异性的引物、探针和标准质粒，对重组质粒进行鉴定后，建立ASFV、PPV、PRV荧光PCR方法，并验证方法的线性、特异性、灵敏度和重复性。用建立的方法对送检的491份免疫猪血浆进行检测。结果 ASFV、PPV、PRV-糖蛋白H和糖蛋白E基因的标准曲线决定系数分别为0.999 1、0.999 8、0.999 5、0.999 9，阴阳性符合率均为100%；针对猪血浆样本的ASFV、PPV、PRV单检检测限均为25拷贝/μL，5混检检测限均为125拷贝/μL，批内和批间的重复性变异系数均＜5%。送检的491份免疫猪血浆样本中未检出ASFV和PRV，有极少数样本存在PPV。结论 建立的ASFV、PPV、PRV荧光PCR方法具有良好的线性、高特异性、高灵敏度和良好的重复性。

Objective To develop and validate a fluorescent PCR assay capable of synchronously detecting African swine fever virus (ASFV), porcine parvovirus (PPV) and pseudorabies virus (PRV) in immune swine plasma.Methods Specific primers and probes and standard plasmids were designed according to national standards of the above 3 viruses. The recombinant plasmids were identified and standard curves were plotted using these plasmids. The linearity, specificity, sensitivity and reproducibility of the developed method were validated and tested on 491 clinical samples of immune swine plasma.Results The coefficients of determination of the standard curves for ASFV, PPV, as well as PRV-glycoprotein H and PRV-glycoprotein E genes were 0.999 1, 0.999 8, 0.999 5,and 0.999 9, respectively, and the negative-positive compliance rate was 100%. The limits of detection for pig plasma samples were all 25 copies/μL, the detection limit of 5-sample pooled testing were 125 copies/μL for all targets, and the coefficients of variation for intra- and inter-batch were all <5%. No ASFV or PRV was detected in 491 samples, and PPV was detected in very few samples.Conclusion The fluorescent PCR assay developed in this study has good linearity, high specificity, high sensitivity and good reproducibility.