基于RAA荧光法快速检测呼吸道腺病毒的研究

Study of a method for rapid detection of respiratory adenovirus using RAA fluorescence assay

目的利用重组酶介导核酸扩增技术(RAA)建立快速检测呼吸道腺病毒的方法。方法根据NCBI基因库中呼吸道腺病毒hexon基因保守序列设计引物及探针,建立RAA检测方法。构建携带腺病毒hexon基因片段的重组质粒,梯度稀释,评价RAA方法的灵敏性;用建立的RAA方法检测人偏肺病毒、人博卡病毒、呼吸道合胞病毒、人鼻病毒、甲型流感病毒、乙型流感病毒,验证方法的特异性。结果用不同拷贝数hexon基因片段的重组质粒进行RAA扩增,在20min内即可得到扩增结果,最低扩增拷贝数为10拷贝/μl;与人偏肺病毒、人博卡病毒、呼吸道合胞病毒、人鼻病毒、甲型流感病毒、乙型流感病毒无交叉反应。结论建立的RAA方法灵敏度和特异性高,反应快速、操作简单,适用于呼吸道腺病毒的快速检测。

Objective To establish a quick assay for respiratory adenovirus detection by using recombinase aided amplification(RAA)method.Methods To design universal primers and probes according to the conserved hexon gene sequence of respiratory adenovirus in NCBI database,and then a recombinant plasmid containing hexon gene was constructed.The recombinant plasmid of gradient dilution was used as templates to evaluate the sensitivity of the method.The specificity of the method was evaluated by using genomic DNAs of human metapneumovirus,human bocavirus,respiratory syncytial virus,human rhinovirus,influenza A virus and influenza B virus as templates.Results Recombinant plasmids of gradient dilution were amplified by using RAA method within 20 minutes,the detection limit was 10 copies/μl.No cross reactions was observed with human metapneumovirus,human bocavirus,respiratory syncytial virus,human rhinovirus,influenza A virus and influenza B virus.Conclusion The established RAA assay has high sensitivity and specificity,and it is rapid,ready-to-hand,suitable for rapid detection of respiratory adenovirus.