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

doi:10.11843/j.issn.0366-6964.2024.05.043

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (5): 2259-2266.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

李鹏飞^1,2, 高桂琴³, 周广青², 吴锦艳², 颜新敏², 曹小安², 何继军², 袁莉刚^1*, 尚佑军^2*

1. 甘肃农业大学动物医学院, 兰州 730070;
2. 中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室, 兰州 730046;
3. 白银市农业综合行政执法队, 白银 730900

收稿日期:2023-07-06 出版日期:2024-05-23 发布日期:2024-05-27
通讯作者: 袁莉刚，主要从事基础兽医学研究，E-mail:yuan2918@126.com ；尚佑军，主要从事家畜疫病流行病学与诊防技术研究，E-mail:shangyoujun@caas.cn
作者简介:李鹏飞(1990-)，男，甘肃会宁人，博士生，主要从事动物传染病研究，E-mail:1054175341@qq.com
基金资助:
国家现代农业产业技术体系项目(CARS-39-04B)；白银市2023年度高层次人才(团队)引进项目(BYTIP2023-10)；甘肃省科技特派团专项(22CX8NA012)；甘肃农业大学实践项目(GSAU-JSFW-2023-07)

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

LI Pengfei^1,2, GAO Guiqin³, ZHOU Guangqing², WU Jinyan², YAN Xinmin², CAO Xiaoan², HE Jijun², YUAN Ligang^1*, SHANG Youjun^2*

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2. State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
3. The Agricultural Comprehensive Administrative Law Enforcement Team of Baiyin, Baiyin 730900, China

Received:2023-07-06 Online:2024-05-23 Published:2024-05-27

摘要/Abstract

摘要： 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6.30×10²～6.30×10⁷ copies·μL^-1呈现良好的线性关系，相关系数R²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6.30×10¹ 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。

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-PCR

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.

Key words: enzootic nasal tumor virus of goats, endogenous retroviruses, TaqMan, Real-time quantitative RT-PCR

中图分类号:

S858.275.3

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.

LI Pengfei, GAO Guiqin, ZHOU Guangqing, WU Jinyan, YAN Xinmin, CAO Xiaoan, HE Jijun, YUAN Ligang, SHANG Youjun. Establishment and Application of TaqMan Fluorescence Quantitative RT-PCR Detection Method for Enzootic Nasal Tumor Virus of Goats[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2259-2266.

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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

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