【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。更多还原

【Abstract】 Most laboratory detection methods for animal diseases are based on one single target pathogen pattern,but the prevalence of these animal diseases is usually characterized by mixed infection with more than one pathogens.In order to overcome this shortcoming,one new simultaneous detection assay of multiplex ligation-dependent probe amplification(MLPA)was described here,followed by our previous study on simultaneous detection of five bovine virus by MLPA.Five pairs of specific probe targeted at blue tongue virus(BTV),bovine viral diarrhea/mucosal disease virus(BVDV),infectious bovine rhinotracheitis virus(IBRV),enzootic bovine leukemia virus(EBLV),foot and mouth disease virus(FMDV)were designed,respectively in this study.The mixture of five standard RNA/DNA was used as template,together with the mixture of these probes as probe and the PCR universal primer,one MLPA method for simultaneous detection of the five bovine viruses was developed.The specificity test showed that the designed probes have good specificity and each probe pair only amplified its target from the template mixture,without any cross-reaction among these probe pairs and viruses.The sensitivity test showed that the detection limit is up to 2 000 copies in one MLPA reaction.The clinical sample test showed that the newly five-plex-MLPA can identify all of 50 negative and 86 positive samples and the agreement with the current real time PCR is 100%.All the results showed that the developed five-plex-MLPA method in this study realized simultaneous detection of five viruses in one reaction,which indicates MLPA technology maybe an alternative to simultaneous detection of multi-target pathogens in veterinary medicine.更多还原