人偏肺病毒的逆转录重组酶介导等温扩增荧光检测方法的建立  

Establishment of a reverse transcriptase-mediated isothermal amplification fluorescence assay for detection of human metapneumovirus

作　　者：焦素黎[1] 谢蕾[1] 毛洋 倪红霞[1] 李永东[1] JIAO Su-li;XIE Lei;MAO Yang;NI Hong-xia;LI Yong-dong(Ningbo Municipal Center for Disease Control and Prevention,Ningbo 315010,China)

机构地区：[1]浙江省宁波市疾病预防控制中心,宁波315010

出　　处：《中国人兽共患病学报》2023年第8期780-783,共4页Chinese Journal of Zoonoses

基　　金：宁波市医疗卫生品牌学科资助(No.PPXK2018-10)。

摘　　要：目的建立一种基于逆转录重组酶介导的等温扩增(reverse transcription recombinase-aided isothermal amplification,RT-RAA)方法的人偏肺病毒快速分子诊断技术,并对其检测效果进行初步分析。方法从NCBI数据库中下载人偏病毒基因组序列,利用Mega软件比对分析基因组序列,确定高度保守序列作为分子检测靶标。以人偏肺病毒N蛋白基因保守序列作为靶序列,设计引物和荧光探针。并以宁波地区人偏肺病毒基因组RNA为模板,利用实时荧光定量PCR对荧光RT-RAA检测体系进行评价。以构建含有N蛋白基因序列的不同浓度重组质粒为模板进行RT-RAA荧光扩增,分析其检测灵敏性;利用建立的RT-RAA荧光法分析检测人偏肺病毒、呼吸道合胞病毒、腺病毒和博卡病毒基因组,评价其检测特异性。结果成功建立了人偏肺病毒RT-RAA扩增荧光检测方法,可在30 min内完成偏肺病毒的特异性扩增检测。以重组质粒为检测模板,RT-RAA荧光检测法的最低检出限为102拷贝/μL重组质粒,且均在15 min内出现阳性特异性扩增。与实时荧光定量PCR检测一致性达98.6%,且与其他3种病毒无交叉反应特性。结论成功建立了一种基于RT-RAA扩增的人偏肺病毒荧光检测方法,该方法灵敏度高、特异性好,在人偏病毒快速现场检测中具有潜在应用价值。A rapid molecular diagnostic technique for detection of human metapneumovirus(HmPV)based on RT-RAA was established,and its detection ability was preliminarily analyzed.HmPV genome sequences were downloaded from the NCBI database and compared and analyzed in Mega software.On the basis of sequence alignment,the conserved sequence of the HmPV N gene was selected as the target sequence,and primers and fluorescent probes were designed.The genomes of HmPV,respiratory syncytial virus,adenovirus and Boca virus were used as templates,and the RT-RAA detection system was evaluated through comparison with real-time fluorescence quantitative PCR(qRT-PCR)results.RT-RAA fluorescence was determined with different concentrations of recombinant plasmids containing the N gene sequence as templates,and the detection sensitivity was analyzed.The established RT-RAA fluorescence method was used to analyze the genomes of respiratory syncytial virus,adenovirus and Boca virus.The RT-RAA fluorescence detection method for human metapneumovirus was successfully established and could be completed within 20 minutes.With the recombinant plasmid used as the detection template,the minimum detection limit of RT-RAA fluorescence was 102 copies/μL.The consistency with qRT-PCR results was 98.6%,and no cross reaction with the other three viruses was observed.A fluorescence detection method of human metapneumovirus based on RT-RAA amplification was successfully established.This method has high sensitivity and specificity,and potential application value in the rapid field detection of human metapneumovirus.

关 键 词：人偏肺病毒 重组酶介导的等温扩增 分子检测 检测效果 

分 类 号：R373.1[医药卫生—病原生物学]