鉴别非洲猪瘟病毒和猪瘟病毒野毒株二重TaqMan MGB实时荧光定量PCR检测方法的建立及初步应用

旨在建立特异、敏感的实时荧光定量PCR（FQ-PCR）方法，用于非洲猪瘟病毒（ASFV）和猪瘟病毒（CSFV）野毒株的快速鉴别检测。针对ASFV的P72基因和CSFV野毒株的5'UTR非编码区序列的保守区域分别设计1对特异性引物和1条探针，经优化反应条件，建立一种基于TaqMan MGB探针技术的FQ-PCR方法，验证方法的敏感性、特异性和稳定性，对50份临床样品进行检测，并与猪瘟国标方法及OIE推荐的非洲猪瘟检测方法进行比较分析。结果显示：建立的鉴别ASFV和CSFV野毒株二重FQ-PCR检测方法在10⁰~10⁶拷贝·μL^-1模板范围内有良好的线性关系；对ASFV和CSFV基因出现阳性扩增信号，但对猪瘟病毒疫苗株、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型、猪细小病毒、猪乙型脑炎病毒、副猪嗜血杆菌等病原对照未出现扩增；批内、批间试验变异系数在1.18%~2.08%，重复性良好；对ASFV和CSFV的最低检测模板浓度均为10拷贝·μL^-1；利用建立的二重FQ-PCR方法对50份临床样品进行检测，检测结果与猪瘟国标方法及OIE推荐的非洲猪瘟检测方法结果完全一致。本研究成功建立了鉴别ASFV和CSFV野毒株二重TaqMan MGB FQ-PCR方法，为ASFV和CSFV野毒株的鉴别诊断提供了快速、敏感、特异且能满足临床检测需求的检测方法。

Establishment and Application of a Duplex TaqMan MGB FQ-PCR for Differential Detection of African Swine Fever Virus and Classical Swine Fever Virus Wild Strains

To detect the African swine fever virus (ASFV) and Classical swine fever virus (CSFV) wild strains rapidly, a specific and sensitive real-time fluorescence quantitative PCR (FQ-PCR) method was established by using the TaqMan MGB probe technique. Specific primers and probes were designed based on the P72 gene of ASFV and 5' non-coding region of the CSFV wild strain to establish the duplex FQ-PCR assay based on TaqMan MGB probe technique. The sensitivity, specificity and stability of FQ-PCR were determined, and 50 clinical samples were simultaneously detected by the FQ-PCR assay, the national standard of CSFV and the OIE recommended detection method for ASFV. The results indicated that the duplex FQ-PCR assay was successfully established and showed a good linear relationship at a template range of 10⁰-10⁶ copies·μL^-1; the specificity of duplex FQ-PCR assay revealed that amplifications were showed synthetic on ASFV and CSFV genes, but other pathogens have no amplifications; variation coefficient of intra and inter assay were 1.18%-2.08%, respectively; the sensitivity of the assay was 10 copies·μL^-1; Fifty clinical samples was detected by the duplex FQ-PCR, the results was consistent with detection of CSFV by national standard and detection of ASFV by OIE recommended method. The duplex TaqMan MGB FQ-PCR assay of ASFV and CSFV wild strain was specific, sensitive, rapid and suitable for identify detection of ASFV and CSFV wild strains.