CAJ | 학술논문

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103拷贝/μl、102拷贝/μl和102拷贝/μl.103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.
목적 초보건립백령병독속Luminex다중액상심편핵산검측방법.방법 설계립부특곡열병독(RVFV)、발열반혈소판감소종합정병독(SFTSV)화합특란덕병독(HLV)특이성인물화탐침,채용파서렬부집다중PCR기술(Tem-PCR)대기파서렬진행확증,병건립Luminex다중액상심편핵산검측방법.이용RVFV、SFTSV화HLV파서렬Tem-PCR산물,이급한탄병독(HTNV)등7충출혈열병독핵산확증산물평개기특이성,이용체외전록병독RNA삼고품평개기민감성화은정성.결과 건립적백령병독속병독Luminex다중액상심편핵산검측방법가특이성검측RVFV、SFTSV화HLV파서렬,차여HTNV등7충출혈열병독무교차반응.대체외전록획득적RVFV、SFTSV화HLV적RNA삼고품,가분별검출103고패/μl、102고패/μl화102고패/μl.103고패/μl이상각농도RNA삼고품검측치적조내화조간변이계수균소우15％.결론 초보건립료백령병독속Luminex다중액상심편핵산검측방법,가유효검측RVFV、SFTSV화HLV,위상관전염병적실험실진단전정료기출.
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 103 copies/μl,102 copies/μ1 and102copies/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 103 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.