猪伪狂犬病病毒TaqMan荧光定量PCR方法的建立

Establishment of a TaqMan quantitative polymerase chain reaction assay for pseudorabies virus

针对猪伪狂犬病病毒(pseudorabies virus,PRV)gE基因保守区的核苷酸序列设计1对特异性引物和TaqMan探针,建立并优化了一种可快速、定量检测PRV野毒的TaqMan实时荧光定量PCR方法。应用该方法检测猪常见病毒性病原(猪瘟病毒、猪细小病毒、猪圆环病毒2型),PRV gE基因缺失株,以及健康猪的组织,结果均为阴性,证明该方法特异性良好。该检测方法能够检到的阳性质粒模板最低浓度为254 copies/μL,最低病毒浓度为4.22 TCID50/100μL,比常规PCR敏感性高10倍;重复性试验结果表明该方法重复性良好;用TaqMan实时荧光定量PCR方法和常规PCR方法同时检测37份临床样品,其中前者检出阳性病料22份,阳性检出率为59.64%,后者检出阳性病料15份,阳性检出率为40.54%,2种方法的符合率为81.08%。综上所述,该方法的建立为PRV的实验室诊断及流行病学调查提供了快速、准确的检测手段。

To develop a rapid and effective method for PR diagnose,a pair of primers and probes specific for the conserved region of PRV gE gene was designed,and a TaqMan real-time quantitative polymerase chain reaction(TaqMan qPCR)method was generated.The TaqMan qPCR method generated here was able to detect PRV wild strains but was negative to detect the other common viruses prevalent in pigs such as CSFV,PPV,and PCV2 as well as DNA from the tissues of PRV-negative pigs.These findings suggest a good specificity for the method.Sensitivity tests showed that the minimum concentration of the DNA or the virus that the TaqMan qPCR is able to detect was 254 copies/μL and 4.22 TCID50/100μL respectively,and the method was found to be at least 10-fold more sensitive than the regular PCR currently being used.The total coincidence rate between the TaqMan qPCR and the regular PCR method was 81.08%.The TaqMan qPCR generated here is able to detect PRV wild strains rapidly and accurately,which might be used as an effective method for PR diagnosis in clinic settings.