TaqMan荧光定量PCR检测猪流行性腹泻病毒方法的建立与初步应用  

DEVELOPMENT AND PRELIMINARY APPLICATION OF A TAQMAN-BASED REAL-TIME PCR FOR DETECTION OF PORCINE EPIDEMIC DIARRHEA VIRUS

作　　者：刘邓[1,2] 袁秀芳[2] 冉多良[1] 徐丽华[2] 牛登元[3] 王朝文[3] 王一成[2] 

机构地区：[1]新疆农业大学,乌鲁木齐830052 [2]浙江省农业科学院畜牧兽医研究所,杭州310021 [3]甘肃农业大学,兰州730070

出　　处：《中国动物传染病学报》2010年第1期28-34,共7页Chinese Journal of Animal Infectious Diseases

基　　金：浙江省科技计划重点项目(2007C22057)

摘　　要：根据GenBank中公布的猪流行性腹泻病毒Ⅳ基因序列，设计了一对特异性引物和探针，扩增长度为186bp的片段。以克隆Ⅳ基因的质粒作为阳性标准品，建立了一种快速检测猪流行腹泻病毒含量的TaqMan荧光定量PCR方法。该方法在10^9-10^1copies／μL范围内具有良好的线性关系，可检测到初始模板中10copies／μL的质粒DNA，以猪圆环病毒、猪乙脑病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病毒和猪细小病毒为模板时，均检测不到荧光信号，而重复性实验中其变异系数均小于2％，表明此方法具有很好的特异性和重复性。应用此方法对采集的60份，陆床样品进行检测，其阳性检出率为92％，而用常规RT-PCR方法进行检测，阳性检出率仅为80％。表明荧光定量RT-PCR方法的敏感性明显高于常规PcR检测方法。A 186 bp fragment was amplified using the specific primers and TaqMan probe designed according to PEDV-N gene sequence deposited in GenBank. Subsequently, a real-time quantitative PCR for detection of PEDV was developed with standard plasmid DNA from the cloned PEDV-N gene. The sensitivity of the assay was 10 copies/μt L plasmid DNA The assay was specific for PEDV because no amplification was found for PCV, JEV, SFV, PRRSV, PRV and PPV. The coefficient variation （CV） of intra/inter-assay for the same DNA sample was less than 2%. The results indicated that the standard curves were shown to be of high linearity, specificity, sensitivity and reproducibility.

关 键 词：猪流行性腹泻病毒 荧光定量RT-PCR TaqMan荧光探针 

分 类 号：S858.282.659.6[农业科学—临床兽医学] Q7[农业科学—兽医学]