鹿流行性出血热病毒实时荧光RT-RPA检测方法的建立

Development of a real-time RT-RPA assay for the detection of epizootic hemorrhagic disease virus

为建立一种快速检测鹿流行性出血热病毒(EHDV)的实时荧光RT-RPA检测方法,本研究根据EHDV保守的非结构蛋白NS1基因(segment 5)设计特异性引物和探针,建立了敏感、快速的EHDV核酸实时荧光RT-RPA检测方法。结果显示,该方法特异性强,能准确检测EHDV核酸,与蓝舌病病毒(BTV)、牛病毒性腹泻病毒(BVDV)、口蹄疫病毒(FMDV)等病原核酸无交叉反应;敏感性高,本试验中最低可检测到8×10~1copies/μL;检测时间短,仅需15 min;重复性好。采用所建立方法对115份样品进行检测,结果与OIE推荐的实时荧光RT-PCR法检测结果相同。上述结果表明,本研究建立的实时荧光RT-RPA快速检测法操作简便,可用于基层实验室快速检测。

To establish a rapid assay for the detection of epizootic hemorrhagic disease virus(EHDV),a real-time fluorescent RT-PCR assay was developed with specific primers and probes designed according to the conservative genome segment 5 encoding nonstructural protein 1(NS1) of EHDV.In result, the RT-RPA assay was specific for EHDV and did not cross react with other antigen nucleic acid,such as BTV,BVDV and FMDV.The RT-RPA assay was rapid and sensitive.The limit of detection of RT-RPA for EHDV was 8×10~1 copies/μL in this test.A total of 115 samples were tested by the RT-RPA assay,and the results was the same as the real-time RT-PCR recommended by OIE.In conclusion,the RT-RPA assay for rapid detection of EHDV can be used in primary laboratories.