【摘要】 为建立禽肾炎病毒（ANV）的荧光重组酶介导的等温扩增（RT-RAA）快速检测方法，本研究根据ANV ORF 1b基因保守序列设计3对特异性引物和探针，在探针中间分别标记FAM荧光基团、BHQ1淬灭基团。提取ANV的总RNA反转录为cDNA作为模板，以PEGBF/R作为引物，采用PCR扩增ANV ORF 1b基因保守序列，并克隆于pMD18-T载体中，构建重组质粒标准品pANV-ORF1b并采用双酶切和测序鉴定正确后作为模板，采用矩阵法筛选确定最优的引物和探针组合，并对反应条件进行优化。结果显示：在39℃恒温，引物和探针浓度分别为0.64μmol/L和0.48μmol/L条件下20 min内可实现对目的片段的有效扩增，并且扩增结果可直接观察，由此初步建立了ANV荧光RTRAA检测方法。利用建立的方法检测ANV、鸡星状病毒、传染性支气管炎病毒、禽流感病毒和禽腺病毒等16种禽类常见病原，结果显示该方法仅能检测到ANV，与其他病原均无交叉反应；以10倍倍比稀释后的重组质粒标准品pANV-ORF1b作为模板，采用本研究建立的荧光RT-RAA方法扩增，分析该方法的敏感性，结果显示对pANV-ORF1b的检测限为1.5×10~1拷贝/μL；对3个不同浓度的p ANV-ORF1b在同一时间和3个不同时间点利用建立的荧光RT-RAA进行批内和批间重复性试验，结果显示批内和批间重复性试验的变异系数均小于5%。对200份采自广西南宁不同地区的临床样品采用本实验建立的荧光RT-RAA方法以及RT-PCR和RT-qPCR方法检测。荧光RT-RAA方法检测结果显示：阳性样品为43份（21.5%），阴性样品为157份（78.5%）;RT-q PCR检测结果显示，阳性样品为45份（22.5%），阴性样品为155份（77.5%）;RT-PCR检测结果显示，阳性样品为38份（19%），阴性样品为162份（81%），荧光RT-RAA检测结果与RT-qPCR和RT-PCR的总符合率分别为95.55%和88.37%。综上所述，本研究建立的ANV荧光RT-RAA检测方法特异性强、敏感性高、重复性好，适用于ANV的临床大规模检测，为ANV所致疫病的监控提供了有力的技术支持。更多还原

【Abstract】 In order to develop a rapid detection method for avian nephritis virus (ANV),Three specific primers and probes pairs were designed based on the conserved sequence of the ANV ORF 1b gene.The FAM fluorescent group and BHQ1 quenching group were labeled in the middle of the probe respectively.Using the square matrix method,the optimal combination of primers and probes were determined through screening using the square matrix method and then the reaction conditions were optimized.The results showed that the target ANV gene fragment could be successfully amplified at a temperature of 39℃,with primer and probe concentrations set at 0.64μmol/L and 0.48μmol/L,respectively,within 20 minutes,facilitating on-site observation.Therefore,a preliminary fluorescence RT-RAA detection method for ANV was established.This method is specific to ANV and does not crossreact with sixteen other common avian pathogens,including chicken astrovirus,infectious bronchitis virus,avian influenza virus,and avian adenovirus.Sensitivity evaluation using a 10-fold diluted recombinant plasmid standard pANV-ORF1b showed a minimum detection concentration of 1.5×10~1 copies/μL intra-assay and inter-assay repeatability tests on three different concentrations of p ANV-ORF1b plasmid standard at the same time and three different time points showed coefficient of variations less than 5%.Testing 200clinical samples from Nanning,Guangxi using the established fluorescent RT-RAA method in this experiment and RT-qPCR showed43 positive (21.5%) and 157 negative samples (78.5%) by fluorescence RT-RAA,and 45 positive (22.5%) and 155 negative samples(77.5%) by RT-qPCR.SPSS analysis indicated a 95.55%consistency between the two methods.This study successfully established a rapid and accurate fluorescence RT-RAA method for ANV detection,and characterized by good specificity,sensitivity and repeatability,providing a new technical tool for large-scale clinical differentiation and detection of ANV.更多还原