梅迪-维斯纳病毒和羊痘病毒多联实时定量PCR检测方法的建立及初步应用  

Establishment of a Multiplex Real-Time Fluorescence Quantitative PCR Assay for Detection of Brucella and Mycobacterium tuberculosis

作　　者：徐军[1] 孙志华[2] 刘娟[2] 孟茹 戴莉[1] 段晓东[1] 叶志辉[1] 

机构地区：[1]新疆阿拉山口出入境检验检疫局,阿拉山口833418 [2]石河子大学动物科技学院,石河子832003 [3]青海省西宁市畜牧兽医总站,西宁810000

出　　处：《石河子大学学报（自然科学版）》2012年第4期448-451,共4页Journal of Shihezi University(Natural Science)

基　　金：国家质量监督检验检疫总局科技项目(2009IK287)

摘　　要：利用多联实时荧光定量PCR技术建立了一种梅迪-维斯纳病毒和羊痘病毒快速鉴别诊断方法。分别设计并合成梅迪-维斯纳病毒和羊痘病毒基因的引物,建立多联实时定量PCR快速鉴别诊断方法;对所建立的方法进行稳定性、特异性和敏感性试验;并用所建立的方法对临床样品进行检测。结果显示:设计的引物敏感性和特异性较好,该多联实时荧光定量PCR方法中梅迪-维斯纳病毒Tm值为89～90℃,羊痘病毒Tm值为91～92℃,对其他供试的菌株则为阴性,并且该方法对梅迪-维斯纳病毒的DNA最低检出量为25拷贝/μL,羊痘病毒为40拷贝/μL,两病原都存在时为80拷贝/μL。研究结果表明本实验建立的方法可用于同时检测梅迪-维斯纳病毒和羊痘病毒,为动物检疫提供了一种有效的检测方法。To establish a method of multiplex real-time fluorescence quantitative PCR assay for fast diagnosis of Maedi-Visna virus（MVV） and Capripox virus（CPV）.We designed and synthesized primers of MVV and CPV genes according to gene sequence published in GenBank,established multiplex RTFQ-PCR,then tested its stability,specificity and sensitivity,and detected the clinic and imitation samples with this method.The Tm of multiplex RT-PCR to amplify brucella and mycobacterium tuberculosis was 89~90 ℃ and 91~92 ℃.But the results of the amplification of other bacteria were negative.The lowest detection limit for DNA of Brucella,MVV and CPV was 25 copies/μL,40 copies/μL,80 copies/μL,respectively.In conclusion,the assay could be used to detect CPV simultaneously.

关 键 词：梅迪-维斯纳病毒 羊痘病毒 多联实时荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]