赤羽病病毒荧光定量RT-PCR检测方法的建立及应用

Establishment and Application of a Fluorescence Quantitative RT-PCR for Akabane Disease Virus

为建立一种快速、灵敏的赤羽病病毒(Akabanediseasevirus,AKAV)核酸检测方法,以AKAV S基因为靶基因,设计特异性引物和Taq Man探针,通过优化反应体系和条件,建立了AKAV荧光定量RT-PCR检测方法,随后开展了敏感性、特异性及重复性评估,并利用该方法与AKAV检疫行业标准推荐方法同时对临床采集的80份牛羊血液样品进行检测,以检验方法的临床应用效果。结果显示:本研究建立的检测方法敏感性较高,最低检测限为13.4 copies/μL;特异性良好,与口蹄疫病毒、牛冠状病毒、牛结节性皮肤病病毒、牛病毒性腹泻病毒、传染性牛鼻气管炎病毒、蓝舌病病毒、牛白血病病毒等均无交叉反应;组内试验变异系数为0.93%~2.47%,组间试验为1.25%~2.76%,重复性良好;利用建立的方法从80份临床样品中检出AKAV阳性样品21份,该检测结果与AKAV检疫行业标准推荐方法的符合率为98.75%。结果表明,本研究建立的AKAV荧光定量RT-PCR检测方法敏感性高,特异性及重复性好,适用于赤羽病临床检测,为赤羽病病原学监测和诊断技术标准开发提供了技术支撑。

In order to establish a rapid and sensitive nucleic acid detection method for Akabane disease virusAKAVa fluorescence quantitative RT-PCR assay for AKAV was developed targeting at AKAV S gene,through designing specific primers and Taq Man probe and optimizing reaction system and conditions,followed by evaluation on its sensitivity,specificity and reproducibility,then 80 clinical bovine and ovine blood samples were simultaneously detected using the established method and that recommended by AKAV quarantine industry standard.The results revealed that the established method was highly sensitive with the lowest detection limit of 13.4 copies/μL;good specificity was observed without cross-reaction with foot-and-mouth disease virus(FMDV),bovine coronavirus(BCV),lumpy skin disease virus(LSDV),bovine viral diarrhoea virus(BVDV),infectious bovine rhinotracheitis virus(IBRV),bluetongue virus(BTV)and bovine leukaemia virus(BLV);good reproducibility was represented by intra-and-inter group coefficients of variation of 0.93%-2.47%and 1.25%-2.76%,respectively;and 21 out of 80 clinical samples were positive as detected by the method,98.75%coincident with that by the recommended method by AKAV quarantine industry standard.In conclusion,the established method,with high sensitivity,good specificity and reproducibility,was appropriate to detect Akabane disease(AKD),technically supporting the development of pathogenetic monitoring and diagnostic technology standards for AKAV.