多重连接探针扩增对5种牛病毒同步检测方法的建立

发布时间：2018-09-05 11:55

【摘要】：现有的动物疫病诊断技术多是针对单一病原检测,而动物疫病的流行通常是多种病原混合感染,目前的诊断技术不能很好地满足国境口岸快速、高通量检疫的需求,一直以来多重检测技术的研究是动物疫情监测、疫病控制领域关注的焦点。本试验在前期猪病混合感染诊断研究基础上,进一步利用多重连接探针扩增(MLPA)技术,以蓝舌病病毒(BTV)、牛传染性鼻气管炎病毒(IBRV)、牛病毒性腹泻病毒(BVDV)、牛地方流行性白血病病毒(EBLV)和口蹄疫病毒(FMDV)为研究对象,分别设计这5种牛病毒的MLPA探针,将这5种探针混合,建立可以同时检测这5种病毒的MLPA扩增方法。特异性试验表明:每种病毒的探针都是特异的,只能识别各自的目的片段,不能扩增出其他相关病毒,探针之间也不存在交叉反应;敏感性试验结果表明:5重MLPA扩增的最低检测限可以达到单个病毒核酸的2 000个拷贝。经过对136份临床样品的检测,该方法与现有荧光PCR方法的符合率达到100%,能够正确地检测出每份阴、阳性样品及混合感染样品。本研究建立的5种病毒MLPA检测方法可以实现1次采样,1次分析,同时检测5种牛病毒的目的,该技术特异性强、敏感性高,加之其多重性检测的优势,有望成为未来疫病检测的新方向。
[Abstract]:Most of the existing diagnostic techniques for animal epidemic diseases are aimed at the detection of a single pathogen, and the prevalence of animal epidemic diseases is usually a mixture of various pathogens. The current diagnostic techniques cannot well meet the needs of rapid and high-throughput quarantine at border ports. The research of multiple detection techniques has been the focus of animal epidemic surveillance and epidemic control. On the basis of the previous studies on the diagnosis of mixed infection of swine disease, the multiplex (MLPA) technique was further used in this experiment. The five bovine infectious rhinotracheitis virus (BTV),) bovine infectious rhinotracheitis virus (BTV), bovine viral diarrhea virus (BVDV), bovine endemic leukemia virus (EBLV) and foot-and-mouth disease virus (FMDV) were studied. A MLPA amplification method for simultaneous detection of these five viruses was established. The specificity test showed that the probes of each virus were specific and could only identify their target fragments and could not amplify other related viruses and there was no cross reaction between the probes. The sensitivity test results showed that the minimum detection limit of MLPA amplification was 2 000 copies of a single virus nucleic acid. The coincidence rate between this method and the existing fluorescent PCR method was 100, and each negative, positive and mixed infected sample could be detected correctly. In this study, the MLPA detection method of five viruses can be used to detect five kinds of bovine viruses at the same time. The method has the advantages of high specificity, high sensitivity and multiple detection. It is expected to be a new direction of disease detection in the future.
【作者单位】：北京出入境检验检疫局;宁夏出入境检验检疫局;
【基金】：国家质检总局科技计划资助项目(2014IK251)
【分类号】：S852.653

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