RAA-LFD检测RHdV-2方法的建立

Establishment and development of RAA-LFD method for detection of RHdV-2

【目的】建立基于重组酶介导的核酸等温扩增(RAA)的侧流层析试纸条(LFD)方法,用于快速诊断兔出血症病毒(RHdV)2型(RHdV-2)。【方法】采用生物信息学软件分析RHdV-2、RHdV 1型(RHdV^(-1))基因组,筛选RHdV-2特异且保守的片段,设计RAA-LFD探针与特异性引物,以筛选最适引物及其浓度;对RAA-LFD检测的最适反应温度和时间进行优化,并对该方法的灵敏性、特异性、重复性进行评价及临床样品检测。【结果】同源性分析表明,RHdV-2的VP60基因存在特异且保守的序列。基于VP60基因设计3对引物,发现上游引物序列为5'-TCATTTGTACCCTTCAGCGGTACCACCATCC-3'、下游引物序列为5'-CCCGGCTGTTGTCTGGTTTATGCCATTT-3'的扩增效果最好,引物与探针混合液的最适浓度为5.000μmol·L^(-1)。用LFD-RAA方法检测RHdV-2的最适反应温度为39℃,最适反应时间为30 min。敏感性检测结果显示:以RHdV-2 VP60重组质粒为模板的最低检出量为6.7×103 ng·μL^(-1)。用建立的RAA-LFD方法对RHdV^(-1)的检测结果呈阴性,对RHdV-2的检测结果呈阳性,表明该方法的特异性良好。重复性检测结果显示,同一批次和不同批次试剂盒的检测结果均一致,表明重复性好。31份临床样品检测结果显示,5份为RHdV-2阳性样品。【结论】建立的检测RHdV-2的RAA-LFD方法具有简捷、易操作的特点,适合养殖场对RHdV-2的快速诊断。

【Objective】Lateral flow dipstick(LFD)based on recombinase aided amplification(RAA)was established and developed for the fast detection of rabbit hemorrhagic disease virus(RHdV)serum type 2(RHdV-2).【Method】The specific and conserved gene of RHdV-2,screened by bioinformatics analysis of RHdV^(-1)and RHdV-2 genomes,was used for the design and screening of RAA-LFD probes and specific primers.Then the RAA-LFD method was optimized by varying reaction time and temperature,and its sensitivity,specificity,repeatability as well as application on clinical samples were evaluated.【Result】Part of the VP60 gene of RHdV-2 was specific and conserved by homology analysis.With the optimal primer pair(5'-TCATTTGTACCCTTCAGCGGTACCACCATCC-3',5'-CCCGGCTGTTGTCTGGTTTATGCCATTT-3'),the optimal reaction condition of the RAA-LFD method was to create a mixing concentration of probe and primer at 5μmol·L^(-1),and react for 30 min at 39℃.Sensitivity test revealed that the minimal detection limit of the optimized RAA-LFD method was 6.7×103 ng·μL^(-1)with recombinant plasmid of VP60 as the template,and the negative detection of RHdV^(-1)indicated its good specificity.This method also showed good repeatability with consistent results within the same batch as well as between different batches of kits.Using this method,5 out of 31 clinical samples were positive for RHdV-2.【Conclusion】The established RAA-LFD method for RHdV-2 is simple and easy to operate,and is suitable for rapid diagnosis of RHdV-2 on farms.