摘要
牛肠道病毒（Bovine enterovirus,BEV）是2011年在我国部分牛场新发现的一种消化道病毒病病原,因此亟需建立针对BEV快速、有效的检测方法。本研究根据BEV 3D基因保守序列,设计并合成一对特异性引物,建立了检测BEV的SYBR Green I实时荧光定量RT-PCR方法。该方法与引起牛的病毒性腹泻的其它几种牛的病毒均无交叉反应,检出敏感度达7.13×10^1拷贝/μL,比常规RT-PCR检测方法高10倍。应用该方法检测了3个规模化奶牛场送检的41份奶牛腹泻样本和3份气溶胶样本,腹泻样品阳性检出率为39.02%（16/41）;3份气溶胶样本均为阳性,表明该方法灵敏度高、特异性强、重复性好,可同时检测大量临床样本,本方法为BEV的早期快速诊断和定量分析提供了技术支撑。
The bovine enterovirus （BEV） is a pathogen found the digestive tracts of cattle. Recently, the BEV was discovered in cattle in a province in China. A rapid and effective detection method for the BEV is essential. An assay was carried out using two specific primers designed to amplify a highly conserved se- quence of the 3D gene. A recombinant plasmid containing the target gene 3D was constructed as a standard control. The limit of detection of the reaction was 7.13 × 10^1 plasmid copies/μL of initial templates, which was tenfold more sensitive than the conventional reverse-transcription-polymerase chain reaction （RT- PCR）. Moreover, the assay was highly specific because all negative controls and other viruses of clinical relevance did not develop positive results. Assay performance on field samples was evaluated on 44 （41 di- arrhea and 3 aerosol） samples and compared with the conventional RT-PCR assay. Sixteen diarrhea sam- ples were positive （16/41, 39.02 %） and 3 aerosol samples were positive （3/3, 100%）. Preliminary results for clinical detection showed that the SYBR Green I real-time PCR assay was highly sensitive, specific and reproducible. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications for epidemics and in BEV research.
出处
《病毒学报》
CAS
CSCD
北大核心
2015年第5期488-493,共6页
Chinese Journal of Virology
基金
现代农业(奶牛)产业技术体系科学家岗位(Cars-37
何洪彬)
泰山学者海外特聘专家(tshw20100417)
国家自然科学基金(31272586
31302095
31302129)
山东省农业重大应用技术创新课题(何洪彬
仲跻峰
王洪梅)
兽医生物技术国家重点实验室开放课题基金(SKLVBF201510)
山东省自然科学基金培养基金(ZR2014CP029)
山东省农业科学院科技创新重点项目(2014CXZ08)子课题(2014CXZ08-3)
山东省农业科学院重大成果培育项目(2015CGPY02)
