山羊地方性鼻内肿瘤病毒TaqMan荧光定量RT-PCR检测方法的建立及应用

Establishment and Application of TaqMan Fluorescence Quantitative RT-PCR DetectionMethod for Enzootic Nasal Tumor Virus of Goats

山羊地方性鼻内肿瘤(enzootic nasal tumor, ENT)是山羊的病毒性传染病,由山羊地方性鼻内肿瘤病毒(enzootic nasal tumor virus of goats, ENTV-2)所引起,感染后可以导致山羊的鼻道筛骨上皮细胞发生不可逆的癌变,已在世界范围内广泛存在,对山羊养殖业造成严重的经济损失。为建立快速、准确且能定量分析ENTV-2的检测方法,本研究根据GenBank上发布的ENTV-2 env基因(MT254061.1)保守区域设计引物和TaqMan探针,建立ENTV-2 TaqMan探针荧光定量RT-PCR检测方法,并对其特异性、灵敏性、重复性与临床检测效果进行验证。结果显示,重组质粒标准品模板浓度为6.30×10^(2)~6.30×10^(7) copies·μL^(-1)呈现良好的线性关系,相关系数R^(2)为0.996 5,扩增效率为110%,线性方程的斜率为-2.953,最低检测限度为6.30×10^(1) copies·μL^(-1);组内变异系数和组间变异系数均小于2%,重复性好;与口蹄疫病毒((foot-and-mouth disease virus, FMDV)、小反刍兽疫病毒(peste des petits ruminants virus, PPRV)、蓝舌病毒(bluetongue virus)、羊内源性逆转录病毒(endogenous retroviruses)等均无交叉反应,表明该方法具有很好的特异性。利用本研究所建立的TaqMan探针实时荧光定量RT-PCR检测方法对29份临床样品进行检测,该方法较普通PCR方法的检出率更高。综上表明,本研究成功建立了ENTV-2 TaqMan探针实时荧光定量RT-PCR检测方法,为ENTV-2的快速诊断和流行病学调查提供技术手段。

Enzootic nasal tumor is a viral transmitted infection of goats,which is caused by enzootic nasal tumor virus(ENTV-2)of goats.After infection,it can lead to irreversible canceration of goat nasal meatus ethmoid epithelial cells,which has been widespread in the world,causing serious economic losses to the goat breeding industry.there is an urgent need to establish a fast,accurate,and quantitative detection method for ENTV-2,we designed primers and TaqMan probes based on the conserved region of the ENTV-2 env gene published on GenBank,and developed a TaqMan probe real-time fluorescence quantitative RT-PCR detection method(qRT-PCR)for ENTV-2,and then verified its specificity,sensitivity,reproducibility,and clinical detection effectiveness.The results showed that the concentration of the recombinant plasmid standard template was 6.30×10^(2)-6.30×10^(7) copies·μL^(-1),there is a good linear relationship within the range,with a correlation coefficient of R^(2) of 0.9965,amplification efficiency of 110%,slope of the linear equation of-2.953,and minimum detection limit of 6.30×10^(1) copies·μL^(-1);The intra group and inter group coefficients of variation are both less than 2%,indicating good reproducibility;There was no cross reaction with Foot-and-mouth disease virus(FMDV),peste des petits ruminants virus(PPRV),Endogenous retroviruses(ERVs),etc.,indicating that this method has good specificity.The qRT-PCR established in this study was used to detect 29 clinical samples,and the detection rate of this method is higher than that of ordinary PCR methods.The above results indicate that this study has successfully established a qRT-PCR for ENTV-2,providing a technical means for rapid diagnosis and epidemiological investigation of ENTV-2.