牛诺瓦病毒和牛轮状病毒双重RAA-LFD快速检测方法的建立及应用

Establishment and Application of Rapid Detection Method for Bovine Norovirus and Bovine Rotavirus Dual RAA-LFD

为建立能同时检测牛诺瓦病毒(bovine norovirus,BNoV)和牛轮状病毒(bovine rotavirus,BRV)的重组酶辅助扩增-侧流层析试纸条(recombinase aided amplification-lateral flow dipstick,RAA-LFD)快速检测方法。本试验按照RAA引物探针设计原则,分别针对BNoV RdRp基因和BRV VP7基因的保守区设计引物和探针,制备标准重组质粒,建立并优化RAA-LFD反应体系,同时对该检测方法进行特异性、敏感性及重复性评估,用建立的RAA-LFD法与PCR法、qPCR法分别对采集的168份腹泻犊牛的临床样本进行平行检测。结果显示,该方法可在20 min,39℃的条件下扩增目标片段,对BNoV和BRV敏感度均为103 copies·μL^(-1),与PCR的检测结果基本一致,且与牛冠状病毒(bovine coronavirus,BCoV)、牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)和牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)无交叉反应,虽低于qPCR检测结果,但可通过肉眼直接观察试纸条的检测结果。综上所述,RAA-LFD法灵敏度较高、且简便、快速、同时不依赖于仪器、专业操作人员,适用于现场检测。

This study aimed to establish a recombinase aided amplification-lateral flow dipstick(RAA-LFD)method for simultaneous detection of bovine norovirus(BNoV)and Bovine rotavirus(BRV).According to the principle of RAA primer probe design,this test first designed primers and probes targeted the conserved regions of BNoV RdRp gene and BRV VP 7 gene,second prepared the standard recombinant plasmids,then established and optimized the RAA-LFD reaction system,finally the specificity,sensitivity and repeatability of the detection method were also evaluated.At the same time,168 clinical samples of calves with diarrhea were tested in parallel by the RAA-LFD,PCR and qPCR method.The results showed that the method could amplify the target fragment in 20 minutes at 39℃.The sensitivity of this method is 103 copies·μL^(-1)for both BNoV and BRV,and is basically consistent with the detection results of PCR.It has no cross-reaction with bovine coronavirus(BCoV),bovine viral diarrhea virus(BVDV)and infectious bovine rhinotracheitis virus(IBRV).Although the test strip result may be lower than that of the qPCR test,it can be easily observed with the naked eye.In summary,the RAA-LFD method has high sensitivity,and is simple,fast,and does not require specialized equipment or operators,making it ideal for on-site detection.