鉴别猪伪狂犬病病毒gI/gE基因部分缺失疫苗株和野毒株的TaqMan实时荧光定量PCR检测方法的建立

Establishment of a TaqMan Real-Time Quantitative PCR Detection Method to Differentiate gI/gE Gene Partially Deleted Vaccine Strains and Wild Strains of Swine Pseudorabies Virus

为建立一种鉴别猪伪狂犬病病毒(PRV)gI/gE基因部分缺失疫苗株和野毒株的TaqMan实时荧光定量PCR检测方法,本试验依据PRV HB-98株和HB2000株的gE和gI基因缺失片段设计特异性引物和探针,优化反应体系,绘制标准曲线,进行特异性试验和敏感性试验,对临床样本和疫苗样本进行检测,并与商品化试剂盒的检测结果进行对比。结果显示,本试验建立的TaqMan实时荧光定量PCR检测方法的标准曲线R^(2)为0.9971;该方法特异性强,能区分PRV与其他常见的猪病原体;敏感性高,最低检测限为10 copies/μL;对临床样本的检测结果与商品化试剂盒的检测结果一致;能够区分PRV gI/gE基因部分缺失的疫苗株和野毒株。结果表明,本试验建立了一种特异性强、灵敏度高、适用范围广的鉴别诊断PRV gI/gE基因部分缺失疫苗株和野毒株的TaqMan实时荧光定量PCR检测方法。

In order to establish a method to differentiate between gI/gE gene partially deleted vaccine strains and wild strains of pseudorabies virus(PRV)in swine,a TaqMan real-time quantitative PCR detection method was developed.Specific primers and probes were designed based on the gE and gI gene fragments of PRV strains HB-98 and HB2000.The reaction system was optimized,a standard curve was drawn,and specificity and sensitivity tests were conducted.Clinical and vaccine samples were tested,and the results were compared with those obtained using commercial kits.The results showed that the standard curve of the established TaqMan real-time quantitative PCR detection method had an R^(2)value of 0.9971.This method demonstrated strong specificity,distinguishing PRV from other common swine pathogens,and high sensitivity with a minimum detection limit of 10 copies/μL.The detection results for clinical samples were consistent with those obtained using commercial kits.Moreover,this method could differentiate between PRV strains with partially deleted gI/gE genes and wild strains.In conclusion,this study successfully established a TaqMan real-time quantitative PCR detection method with high specificity,sensitivity,and broad applicability for the differential diagnosis of PRV gI/gE gene partially deleted vaccine strains and wild strains.