猪传染性胃肠炎病毒荧光RT-RAA方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2023.05.041

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (5): 2208-2214.doi: 10.11843/j.issn.0366-6964.2023.05.041

• 研究简报 • 上一篇

猪传染性胃肠炎病毒荧光RT-RAA方法的建立及初步应用

吕荞^1,2,3, 赵钟毅^2,3, 尹德玮^2,3, 刘宇梦², 汪伟¹, 郑敏³, 胡诗悦⁴, 赵宸辰¹, 张昕宇¹, 雷晓哓¹, 陆景仪¹, 孙文超^1*, 兰添^1*

1. 温州大学病毒学研究所温州市病毒学与免疫学重点实验室, 温州 325035;
2. 广西大学动物科学技术学院, 南宁 530003;
3. 广西动物疫病预防控制中心, 南宁 530004;
4. 长春市动物疫病预防控制中心, 长春 130061

收稿日期:2022-06-07 出版日期:2023-05-23 发布日期:2023-05-20
通讯作者: 孙文超,主要从事动物传染病学研究,E-mail:sunwenchao131@163.com;兰添,主要从事动物传染病学研究,E-mail:827002151@qq.com
作者简介:吕荞(1997-),男,浙江衢州人,硕士生,主要从事动物传染病学研究,E-mail:2993448350@qq.com
基金资助:
温州市基础性科研项目(N20190005;N20180010)

Establishment and Preliminary Application of a Real-time RT-RAA for Detection of Transmissible Gastroenteritis Virus

Lü Qiao^1,2,3, ZHAO Zhongyi^2,3, YIN Dewei^2,3, LIU Yumeng², WANG Wei¹, ZHENG Min³, HU Shiyue⁴, ZHAO Chenchen¹, ZHANG Xinyu¹, LEI Xiaoxiao¹, LU Jingyi¹, SUN Wenchao^1*, LAN Tian^1*

1. Wenzhou Key Laboratory for Virology and Immunology, Institute of Virology, Wenzhou University, Wenzhou 325035, China;
2. College of Animal Science and Technology, Guangxi University, Nanning 530003, China;
3. Guangxi Center for Animal Disease Prevention and Control, Nanning 530004, China;
4. Changchun Center for Animal Disease Control and Prevention, Changchun 130061, China

Received:2022-06-07 Online:2023-05-23 Published:2023-05-20

摘要/Abstract

摘要： 旨在建立一种简便、高效的猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)的检测方法。本研究针对TGEV N基因设计了特异性的引物和探针,并构建重组质粒标准品TGEV-N,经过筛选及优化后建立TGEV荧光逆转录重组酶介异等温扩增(reverse-transcription recombinase-aid amplification,RT-RAA)检测方法。结果显示,该方法在42 ℃恒温条件下进行,20 min即可完成检测,与猪流行性腹泻病毒、猪繁殖与呼吸综合征病毒、猪丁型冠状病毒、猪肠道α冠状病毒等猪源病毒均无交叉反应;将TGEV-N稀释后,以10⁰~10⁶拷贝·μL^-1作为模板,进行敏感性试验,结果表明,该方法最低检出限可以达到6.62×10¹拷贝·μL^-1;采用该方法对50份猪源样品进行检测,阳性率为6%(3/50),检测结果与RT-PCR一致。本试验建立的TGEV荧光RT-RAA检测方法简便、高效、特异、灵敏,为TGEV的临床筛查提供可行技术。

关键词: 猪传染性胃肠炎病毒, 荧光RT-RAA, 临床检测

Abstract: In order to diagnose transmissible gastroenteritis virus(TGEV)efficiently and easily, specific primers, probes and recombinant standard plasmids TGEV-N were designed. By screening the primers and optimizing the reaction conditions, a real-time reverse-transcription recombinase-aid amplification (RT-RAA) assay was established. The assay can be completed within 20 min at 42℃ and there was no cross-reactivity with porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, porcine deltacoronavirus and porcine enteric alphacoronavirus. Sensitivity test conducted with serial dilutions of TGEV-N ranging from 10⁰-10⁶ copies·μL^-1 showed the minimum detection was 6.62×10¹copies·μL^-1. 50 porcine samples were tested by this assay and the positive rate of TGEV was 6%, which was consistent with RT-PCR. The TGEV real-time RT-RAA assay was specific, sensitive and efficient and could be used for clinical screening of TGEV.

Key words: transmissible gastroenteritis virus, real-time RT-RAA, clinical detection

中图分类号:

S852.651

吕荞, 赵钟毅, 尹德玮, 刘宇梦, 汪伟, 郑敏, 胡诗悦, 赵宸辰, 张昕宇, 雷晓哓, 陆景仪, 孙文超, 兰添. 猪传染性胃肠炎病毒荧光RT-RAA方法的建立及初步应用[J]. 畜牧兽医学报, 2023, 54(5): 2208-2214.

Lü Qiao, ZHAO Zhongyi, YIN Dewei, LIU Yumeng, WANG Wei, ZHENG Min, HU Shiyue, ZHAO Chenchen, ZHANG Xinyu, LEI Xiaoxiao, LU Jingyi, SUN Wenchao, LAN Tian. Establishment and Preliminary Application of a Real-time RT-RAA for Detection of Transmissible Gastroenteritis Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2208-2214.

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猪传染性胃肠炎病毒荧光RT-RAA方法的建立及初步应用

Establishment and Preliminary Application of a Real-time RT-RAA for Detection of Transmissible Gastroenteritis Virus

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