口蹄疫病毒实时荧光RT-RPA快速检测方法的建立

Development of the real-time reverse transcription recombinase polymerase amplification for rapid detection of foot-and-mouth disease virus

为建立一种简单、快速、特异的口蹄疫病毒(FMDV)分子检测方法,本研究基于FMDV的3D基因,设计特异性引物和exo探针,建立了用于FMDV快速检测的等温实时荧光反转录重组酶聚合酶(Real-time RT-RPA)方法。该方法采用便携式等温扩增仪-Genie Ⅲ实时监测扩增结果,40℃20 min即可完成检测。结果显示,FMDV RT-RPA方法能够特异性扩增FMDV 3D基因,而对水泡性口炎病毒、猪繁殖与呼吸综合征病毒、猪瘟病毒、脑心肌炎病毒等均无扩增;以体外转录的FMDV 3D RNA作为模板,该方法在95%置信区间检出下限为1.0×10~2拷贝/μL,组内和组间变异系数均小于1%;使用43份含有灭活FMDV的模拟临床样品分析显示,RT-RPA对FMDV的检出率为34.9%(15/43),略低于荧光定量RT-PCR的检出率(39.5%,17/43),而两种方法的符合率为95.3%(41/43)。RT-RPA能够在6 min～16 min内完成检测,而实时荧光RT-PCR则需要30 min～51 min。本研究所建立的实时荧光RT-RPA方法反应快速,特异性强,灵敏性高,操作简单,结合便携式具有荧光检测功能的等温扩增仪Genie Ⅲ,组建了FMDV快速检测平台,在基层兽医部门,尤其在野外及疫情现场的FMDV检测中具有极大的应用潜力,为设备仪器有限的实验室和田间对FMDV的快速检测提供了一种有效的方法,对于条件有限地区FMD的防控具有重要意义。

To develop a simple, rapid and specific method for the foot-and-mouth disease virus(FMDV) detection, the real-time reverse-transcription recombinase polymerase amplification assay(real-time RT-RPA) was developed using primers targetting the conserved region of the 3D gene, and the amplification was performed at 40℃ in the portable Genie Ⅲ scanner device for 20 min under the real-time monitoring. The amplification of real-time RT-RPA was specific for FMDV, but not for VSV,PRRSV, CSFV, EMCV, PRV and PCV2, displaying a high specificity. Using the in vitro transcribed FMDV RNA as template, the analytical sensitivity was 10~2 copies at 95% cases. The co-efficient of variations in intra-and inter-assays were both less than 1%.The assay performance was evaluated by testing 43 clinical samples by the real-time RT-RPA and a real-time RT-PCR assay. Fifteen samples were FMDV positive in RT-RPA(34.9%, 15/43), while the positive rate was 39.5%(17/43) in real-time RT-PCR. The diagnostic agreement between the two assays was 95.3%(41/43). It took 6-16 min in the RT-RPA assay to obtain the positive results, while the real-time RT-PCR took about 30 min to 51 min. The developed FMDV RT-RPA assay is rapid, highly specific,highly sensitive and easy to perform, and the FMDV rapid detection platform was established with Genie Ⅲ, which provides a rapid and reliable diagnostic tool for FMD in the field, and is of great significance in FMD provention.