白蛉病毒属病毒Luminex多重液相芯片核酸检测方法研究  

Development of a multiplex luminex assay for nucleic acid detection of Phlebovirus

作　　者：李阿茜 何程程[1] 张硕[1] 李川[1] 张全福[1] 梁米芳[1] 李德新[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《中华实验和临床病毒学杂志》2015年第2期165-167,共3页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的 初步建立白蛉病毒属Luminex多重液相芯片核酸检测方法.方法 设计立夫特谷热病毒（RVFV）、发热伴血小板减少综合征病毒（SFTSV）和哈特兰德病毒（HLV）特异性引物和探针,采用靶序列富集多重PCR技术（Tem-PCR）对其靶序列进行扩增,并建立Luminex多重液相芯片核酸检测方法.利用RVFV、SFTSV和HLV靶序列Tem-PCR产物,以及汉滩病毒（HTNV）等7种出血热病毒核酸扩增产物评价其特异性,利用体外转录病毒RNA参考品评价其敏感性和稳定性.结果 建立的白蛉病毒属病毒Luminex多重液相芯片核酸检测方法可特异性检测RVFV、SFTSV和HLV靶序列,且与HTNV等7种出血热病毒无交叉反应.对体外转录获得的RVFV、SFTSV和HLV的RNA参考品,可分别检出10^3拷贝/μl、10^2拷贝/μl和10^2拷贝/μl.10^3拷贝/μl以上各浓度RNA参考品检测值的组内和组间变异系数均小于15％.结论 初步建立了白蛉病毒属Luminex多重液相芯片核酸检测方法,可有效检测RVFV、SFTSV和HLV,为相关传染病的实验室诊断奠定了基础.Objective To establish a multiplex Luminex assay for nucleic acid detection of Phlebovirus.Methods Specific primers and probes of Rift Vally fever virus（RVFV）,Severe fever with thrombocytopenia syndrome bunyavirus （SFTSV） and Heartland virus （HLV） were designed.Target fragments of the three viruses were amplified using target enriched multiplex （Tem） PCR,and then a multiplex Luminex assay of nucleic acid detection was established.The specificity of the developed Luminex assay was evaluated withTem-PCR products of RVFV,SFTSV,HLV and the amplification products of 7 hemorrhagic fever viruses （HFVs） including Hantand virus（HTNV）,and its sensitivity and reproducibility was evaluated with in vitro transcribed viral RNA standards of gradient dilution.Results Using the developed multiplex Luminex assay of nucleic acid detection,the target sequences of RVFV,SFTSV and HLV could be detected specifically,and there was no cross-reaction with 7 HFVsincluding HTNV.In vitro transcribed RNA of 10^3 copies/μl,10^2 copies/μ1 and10^2copies/μl could be detected for RVFV,SFTSV and HLV detection,respectively.The coefficient of variation of detection values were all less than 15% in each concentration more than 10^3 copies/μl of RNA standards for both intra-assays and inter-assays.Conclusion A multiplex Luminex assay of nucleic acid detection for phlebovirus was preliminary established to effectively detect RVFV,SFTSV and HLV,laying the foundation for the laboratory diagnosis of related infectious diseases.

关 键 词：出血热 病毒性 白蛉病毒 液相芯片 聚合酶链反应 

分 类 号：R373[医药卫生—病原生物学]