A型塞尼卡病毒与O型、A型、亚洲Ⅰ型口蹄疫病毒多重TaqMan荧光定量RT-PCR检测方法的建立及应用  

Development and Application of A Multiplex TaqMan Real-time RT-PCR for Differential Detection of Senecavirus A and Serotypes O,A,Asia Ⅰ of Foot-and-Mouth Disease Virus

作　　者：施开创[1] 谢守玉 赵晶 莫胜兰[1] 刘惠心 尹彦文[1] 司红彬[2] 屈素洁[1] 陆文俊[1] 冯淑萍[1] 粟艳琼[1] SHI Kaichuang;XIE Shouyu;ZHAO Jing;MO Shenglan;LIU Huixin;YIN Yanwen;SI Hongbin;QU Sujie;LU Wenjun;FENG Shuping;SU Yanqiong(Guangxi Center for Animal Disease Control and Prevention,Nanning 530001,China;College of Animal Science and Technology,Guangxi University,Nanning 530005,China)

机构地区：[1]广西动物疫病预防控制中心,南宁530001 [2]广西大学动物科学技术学院,南宁530005

出　　处：《中国动物传染病学报》2022年第2期84-92,共9页Chinese Journal of Animal Infectious Diseases

基　　金：广西科技重大专项(桂科AA17204057);广西水产畜牧科技项目(桂渔牧科201528017);广西农业科技项目(Z201954)。

摘　　要：为建立鉴别检测A型塞尼卡病毒(SVA)与O型、A型、亚洲Ⅰ型口蹄疫病毒(FMDV)的方法,针对SVA 3D基因与O型、A型、亚洲Ⅰ型FMDV VP1基因,分别设计特异性引物和TaqMan探针,经优化反应条件,建立了同时检测SVA与O型、A型、亚洲Ⅰ型FMDV的TaqMan荧光定量RT-PCR方法。所建立的方法能特异性扩增SVA及O型、A型、亚洲Ⅰ型FMDV,与其他主要猪源病毒无交叉反应;对SVA与O型、A型、亚洲Ⅰ型FMDV质粒标准品的检出下限分别为2.50×10^(1)、2.50×10^(2)、2.50×10^(2)、2.50×10^(2)copies/μL;组内与组间重复性试验的变异系数均小于2%。应用所建立方法检测2019年来自广西的30份临床疑似样品,SVA和O型FMDV的检出阳性率分别为20%和70%,而未能检出A型和亚洲Ⅰ型FMDV。结果表明,本研究所建立的多重荧光定量RT-PCR方法为SVA与O型、A型、亚洲Ⅰ型FMDV的临床检测和流行病学调查提供了特异、敏感、高效的技术手段。To develop an assay for differential detection of Senecavirus A(SVA)and serotypes O,A,Asia Ⅰ of Foot-and-mouth disease virus(FMDV),four specific primers and TaqMan probes were designed for SVA 3D gene and VP1 genes of FMDV serotypes O,A,Asia Ⅰ.The TaqMan real-time RT-PCR assay was developed after optimizing several reaction conditions.The results showed that the assay only specifically detected individual SVA or FMDV serotypes O,A,Asia Ⅰ but not with other porcine viruses.The limit detections of the standard plasmids were 2.50×10^(1) copies/μL for SVA,2.50×10^(2),2.50×10^(2) and 2.50×10^(2) copies/μL for FMDV serotypes O,A,Asia I,respectively.The assay had good reproducibility with intra-and inter-assay coefficients of variation at less than 2%.The method was used to test 30 suspicious clinical samples collected in 2019 from Guangxi province and the positive rates of SVA and FMDV serotype O were 20% and 70%,respectively.There were no positive samples for FMDV serotypes A,Asia Ⅰ.The results indicated that the PCR assay developed here provided a specific,sensitive and efficient differential method for detection and epidemiological investigation of SVA and FMDV serotypes O,A,Asia I.

关 键 词：A型塞尼卡病毒 口蹄疫病毒 荧光定量RT-PCR 检测方法 

分 类 号：S852.4[农业科学—基础兽医学] S852.65[农业科学—兽医学]