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  

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

作　　者：王淑娟[1] 班付国[1] 王东方[1] 刘影 赵雪丽[1] 谢彩华[1] 王翠[1] 马震原[1] 杨海波[1] 柴茂 闫若潜[1] WANG Shujuan;BAN Fuguo;WANG Dongfang;LIU Ying;ZHAO Xueli;XIE Caihua;WANG Cui;MA Zhenyuan;YANG Haibo;CHAI Mao;YAN Ruoqian(Henan Centre for Animal Disease Control&Prevention,Zhengzhou 450008,China)

机构地区：[1]河南省动物疫病预防控制中心,郑州450008

出　　处：《畜牧兽医学报》2021年第1期177-184,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：河南省科技创新领军人才(204200510012)。

摘　　要：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10~0~10~6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。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 P 72 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 10 0-10 6copies·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.

关 键 词：非洲猪瘟病毒 猪瘟病毒野毒株 二重TaqMan MGB FQ-PCR 检测 

分 类 号：S852.659.1[农业科学—基础兽医学] S852.651[农业科学—兽医学]