畜牧兽医学报2024，Vol.55Issue(5)：2259-2266,8.DOI:10.11843/j.issn.0366-6964.2024.05.043
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 Detection Method for Enzootic Nasal Tumor Virus of Goats
摘要
Abstract
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 × 102-6.30 × 107 copies·μL-1,there is a good linear relationship within the range,with a correlation coefficient of R2 of 0.996 5,amplification efficiency of 110％,slope of the linear equation of-2.953,and minimum detection limit of 6.30×101 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.关键词
山羊地方性鼻内肿瘤病毒/羊内源性逆转录病毒/TaqMan/荧光定量RT-PCRKey words
enzootic nasal tumor virus of goats/endogenous retroviruses/TaqMan/Real-time quantitative RT-PCR分类
农业科技引用本文复制引用
李鹏飞,高桂琴,周广青,吴锦艳,颜新敏,曹小安,何继军,袁莉刚,尚佑军..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[J].畜牧兽医学报,2024,55(5):2259-2266,8.基金项目
国家现代农业产业技术体系项目(CARS-39-04B) （CARS-39-04B）
白银市2023年度高层次人才(团队)引进项目(BYTIP2023-10) （团队）
甘肃省科技特派团专项(22CX8NA012) （22CX8NA012）
甘肃农业大学实践项目(GSAU-JSFW-2023-07) （GSAU-JSFW-2023-07）
