牛病毒性腹泻病毒Nano-PCR检测方法的建立及初步应用  

Establishment and preliminary application of Nano-PCR for the detection of bovine viral diarrhea virus

作　　者：李健友 刘泽余[1] 刘占悝 李家伟 张淑琴 王超[1] 郭利[1] LI Jian-you;LIU Ze-yu;LIU Zhan-kui;LI Jia-wei;ZHANG Shu-qin;WANG Chao;GUO Li(Key Loboratory of Special Animal Epidemic Disease ,Ministry of Agricultural ,Institute of Special Economic Animal and Plant Sciences ,Chinese Academy of Agricultural Sciences , Changchun 130112 ,China;Northeast Agricultural University, Harbin 150030, China;Animal Husbandry and Veterinary Station of Antu ,Antu 133600, China)

机构地区：[1]中国农业科学院特产研究所农业部经济动物疫病重点实验室特种动物分子生物学重点实验室,吉林长春130122 [2]东北农业大学,黑龙江哈尔滨150030 [3]安图县畜牧兽医总站,吉林安图133600

出　　处：《中国兽医科学》2018年第5期580-585,共6页Chinese Veterinary Science

基　　金：吉林省科技厅产业技术创新战略联盟项目(20170309002NY);吉林省科技厅重点科技成果转化项目(20150307024NY);国家重点研发计划项目(2016YFD0500907);中国农业科学院“创新工程”项目

摘　　要：为建立一种特异、灵敏、快速检测牛病毒性腹泻病毒（BVDV）的分子检测方法,本研究利用金纳米颗粒材料与传统PCR技术相结合,针对BVDV 5′UTR保守序列设计引物,建立了BVDV Nano-PCR新型分子检测方法,并与RT-PCR及商品化IDEXX试剂盒BVDV抗原检测方法分别对临床样品进行对比研究。特异性与敏感性试验结果显示,建立的Nano-PCR只对BVDV特异,而对牛传染性鼻气管炎病毒（IBRV）、牛呼吸道合胞体病毒（BRSV）、牛副流感3型病毒（BPIV3）均无交叉反应;Nano-PCR敏感性是普通PCR的10倍,最低核酸检测量为6.84×10~2copies/L。临床检测结果显示,建立的Nano-PCR方法对38份临床样品的阳性检出率为34.21%,是RT-PCR的2.6倍,是IDEXX试剂盒的1.5倍。本研究结果表明,建立的BVDV Nano-PCR方法可以用来对临床样品进行快速诊断和鉴定,具有特异性高、灵敏度好、快速稳定的优点。In order to establish a specific,sensitive and rapid molecular detection method for bovine viral diarrhea virus（BVDV）,in this study,a novel nano-PCR for BVDV was established based on the combination of gold nanoparticle and traditional PCR technology with primers designed for BVDV 5′UTR conserved sequences.A comparative study of clinical samples was performed on the results of RT-PCR and the commercial IDEXX BVDV antigen Test Kit.The specificity and sensitivity tests showed that the Nano-PCR was only sensitive to BVDV,without cross reaction to the other viruses,such as infectious bovine rhinotracheitis virus,bovine respiratory syncytial virus and bovine parainfluenza type-3 virus.In the sensitivity test,the Nano-PCR showed 10 times sensitivity than that of the traditional PCR technology,with the minimum detection of 6.84 ×103 copies/L.The clinical test results showed that the coincidence rate of this technique could reach to 100%.The positive detection rate of 38 clinical samples was 34.21%,which was 2.6 times that of RT-PCR and also the 1.5 times of the IDEXX kit.In conclusion,the established BVDV Nano-PCR can be used for the rapid diagnosis and identification ofclinical samples,and has the advantages of high specificity,good sensitivity,fast and stable.

关 键 词：牛病毒性腹泻病毒 Nano-PCR RT-PCR IDEXX试剂盒 

分 类 号：S852.659.6[农业科学—基础兽医学]