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为了建立检测口蹄疫的快速实用分子生物学技术,本研究借助新兴的环介导等温扩增技术,建立了一种灵敏、特异、快速的分子生物学检测方法-体外等温扩增检测方法。设计了6条针对FMDV3D基因的8个位点的特异性引物,建立了一套从核酸抽提到检测仅需要75min的快速检测技术。所建立的检测方法灵敏,与实时荧光RT-PCR灵敏度相当;且具有特异性,对其他水泡性疾病病原体检测均为阴性;而且简单,不需要复杂的仪器,常规水浴锅在63.5℃即可进行,肉眼可直接观察检测结果。该方法是适合基层和现场检测而无需送至中心实验室的快速灵敏实用的分子生物学检测方法。
In order to establish a rapid and practical molecular biology technique for detection of foot-and-mouth disease, this study established a sensitive, specific and rapid molecular biological detection method - in vitro isothermal amplification detection method with the aid of the emerging ring-mediated isothermal amplification technique. Six specific primers targeting FMDV3D gene were designed, and a set of rapid detection technology was established which only takes 75 min from nucleic acid extraction to detection. The established detection method is sensitive and has the same sensitivity as real-time fluorescence RT-PCR, and has the specificity of being negative for the detection of other vesicular disease pathogens. Moreover, the detection method is simple and does not require complicated instruments. A conventional water bath pot can be performed at 63.5 ° C. , The naked eye can directly observe the test results. This method is a fast and sensitive molecular biological detection method suitable for grassroots and field testing without having to be sent to a central laboratory.