塔希纳病毒的重组酶介导扩增快速检测方法

Establishment of a reverse transcription recombinase-mediated amplification assay for rapid detection of Tahyna virus

目的采用反转录重组酶介导核酸扩增(RT-RAA)方法建立塔希纳病毒(TAHV)检测方法。方法在TAHV S节段保守区设计引物和探针。用构建含检测目的基因片段的质粒和病毒细胞培养物对TAHV的RT-RAA检测方法的灵敏性进行评价,通过检测黄病毒属、甲病毒属、正布尼亚病毒属和东南亚十二节段病毒属共7种病毒验证方法的特异性。并选用2018年采集自宁夏回族自治区的30批次蚊虫样本来评价该检测方法。结果该方法对构建的质粒标准品的检测下线为100拷贝/反应,病毒培养物最低检出限为10空斑形成单位/反应,与其他7种虫媒病毒无交叉反应。在检测蚊虫样本时,该方法与实时定量PCR方法检测结果的一致性为100%。结论建立了快速、特异以及灵敏的TAHV的RT-RAA检测方法,并适用于标本中TAHV核酸的现场检测。

Objective To establish a reverse transcription recombinase-mediated nucleic acid amplification(RT-RAA)assay for the rapid detection of Tahyna virus nucleic acid.Methods Primers and probes were designed in the conserved region of S segment of Tahyna virus.The sensitivity of the new RT-RAA assay was evaluated by constructing plasmids containing target gene fragments and cultured virus.The specificity of the assay were tested by seven viruses of flavivirus,alphavirus,Orthobunyavirus and Seadornavirus.Additionally,30 batches of mosquito samples were used to evaluate the performance of the RT-RAA assay.Results The limit detection of the assay was 100 copies per reaction for the constructed plasmid standard and 1 plaque-forming units(PFU)per reaction of Tahyna virus titers.There was no cross-reaction with other arboviruses.Conclusion A highly sensitive,specific,and easy-to-use assay was established for the rapid detection of Tahyna virus.