基于逆转录-酶促重组等温扩增原理的西尼罗病毒基因检测方法的建立

A reverse transcriptase aid-enzymatic recombinase isothermal amplification-based method for detection of West Nile virus

目的建立一种可用于检测西尼罗病毒特异性基因片段的逆转录-酶促重组等温扩增(RT-ERA)方法。方法以西尼罗病毒NS5基因的高度保守序列作为靶序列,根据ERA反应原理设计、合成引物及荧光探针,建立荧光RT-ERA反应体系。分别以不同拷贝数(10^(4)、10^(3)、10^(2)、10、1拷贝/μl)的含120 bp靶基因片段的重组质粒及不同浓度(10、1、10^(-1)、10^(-2)、10^(-3)ng/μl)西尼罗病毒RNA为模板进行荧光RT-ERA法扩增,评价该方法的敏感度;分别以森林脑炎病毒、基孔肯雅病毒、Ⅰ型登革病毒、西尼罗病毒、乙型脑炎病毒、黄热病毒和甲型流感病毒H2N1的RNA为模板进行荧光RT-ERA法检测,评价该方法的特异性。结果建立的荧光RT-ERA法可在39℃、20 min内特异性扩增西尼罗病毒RNA。敏感度检测结果显示,以重组质粒为模板,荧光RT-ERA法最低可检出的质粒拷贝数为1拷贝/μl;以RNA为模板,荧光RT-ERA法最低可检测浓度为10^(-3)ng/μl。特异性检测结果显示,以森林脑炎病毒、基孔肯雅病毒、登革病毒I型、乙型脑炎病毒、黄热病毒和甲型流感病毒H2N1的RNA为模板,荧光RT-ERA法检测结果均为阴性,仅西尼罗病毒RNA扩增阳性。结论成功建立了一种可用于检测西尼罗病毒RNA的荧光RT-ERA法,该方法操作简便,敏感度和特异性均较好。

Objective To establish a reverse transcription-enzymatic recombinase isothermal amplification (RT-ERA) assay for detection of specific gene segment of West Nile virus.Methods The highly conserved region of NS5 gene (120 bp) was selected as the target gene fragment to be detected.The primers and fluorescence probes were designed and synthesized based on the isothermal amplification principle to establish a fluorescence RT-ERA assay system.The fluorescence RT-ERA assay was performed to detect serial diluted recombinant plasmids (10~4,10~3,10~2,10,1 copy/μl) containing target gene fragments and West Nile virus RNA at different concentrations (10,1,10^(-1),10^(-2),10^(-3)ng/μl) to determine the sensitivity.Further,this assay was applied to detect the genomic RNA of Tick-Borne Encephalitis virus,Chikungunya virus,Yellow fever virus,Dengue virus type I,Japanese Encephalitis virus and Influenza A virus (H2N1) to evaluate the specificity.Results A fluorescence RT-ERA assay was successfully established,which was effective in amplifying the specific gene fragments of West Nile virus within 20 min at 39℃.The minimum detectable limit of the fluorescence RT-ERA assay was 1 copy/μl using recombinant plasmids as templates and 10^(-3)ng/μl using West Nile virus RNA samples as templates.The results of the fluorescent RT-ERA assays were all negative for detecting the genomic RNA from tick-borne encephalitis virus,chikungunya virus,yellow fever virus,dengue virus type I,Japanese encephalitis virus,and Influenza A virus (H2N1).Conclusion A fluorescence RT-ERA assay for detection of West Nile virus RNA is successfully established,which is easy to use,sensitive and specific.