牛诺瓦病毒和牛轮状病毒双重RAA-LFD快速检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2024.01.039

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (1): 406-412.doi: 10.11843/j.issn.0366-6964.2024.01.039

牛诺瓦病毒和牛轮状病毒双重RAA-LFD快速检测方法的建立及应用

李惠惠^1,2, 董可儿^1,2, 刘馨博^1,2, 张春晓^1,2, 马超³, 陈利苹⁴, 钟旗⁵, 姚刚^1,2*, 马雪连^1,2*

1. 新疆农业大学动物医学学院, 乌鲁木齐 830052;
2. 动物保健与畜产品质量安全研究实验室, 乌鲁木齐 830052;
3. 乌鲁木齐市动物园, 乌鲁木齐 830001;
4. 深圳市真瑞生物有限公司, 深圳 518000;
5. 新疆畜牧科学院兽医研究所, 乌鲁木齐 830000

收稿日期:2023-04-04 出版日期:2024-01-23 发布日期:2024-01-24
通讯作者: 马雪连,主要从事动物保健与畜产品质量安全研究,E-mail:maxuelian@xjau.edu.cn;姚刚,主要从事动物生长发育与畜产品质量安全研究,E-mail:yg@xjau.edu.cn
作者简介:李惠惠(1996-),女,新疆阜康人,硕士生,主要从事动物保健与畜产品质量安全研究,E-mail:1343481646@qq.com
基金资助:
国家自然科学基金（32002251）：肉生高效健康饲养技术集成研究与示范（2021A02003-1）

Establishment and Application of Rapid Detection Method for Bovine Norovirus and Bovine Rotavirus Dual RAA-LFD

LI Huihui^1,2, DONG Keer^1,2, LIU Xinbo^1,2, ZHANG Chunxiao^1,2, MA Chao³, CHEN Liping⁴, ZHONG Qi⁵, YAO Gang^1,2*, MA Xuelian^1,2*

1. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China;
2. The Research Team of Animal Healthcare and Quality & Safety of Livestock Products, Urumqi 830052, China;
3. Urumqi Zoo, Urumqi 830001, China;
4. Shenzhen Zhenrui Biological Co., LTD, Shenzhen 518000, China;
5. Institute of Veterinary Medicine, Xinjiang Academy of Animal Science, Urumqi 830000, China

Received:2023-04-04 Online:2024-01-23 Published:2024-01-24

摘要/Abstract

摘要： 为建立能同时检测牛诺瓦病毒（bovine norovirus， BNoV）和牛轮状病毒（bovine rotavirus， BRV）的重组酶辅助扩增-侧流层析试纸条（recombinase aided amplification-lateral flow dipstick， RAA-LFD）快速检测方法。本试验按照RAA引物探针设计原则，分别针对BNoV RdRp基因和BRV VP7基因的保守区设计引物和探针，制备标准重组质粒，建立并优化RAA-LFD反应体系，同时对该检测方法进行特异性、敏感性及重复性评估，用建立的RAA-LFD法与PCR法、qPCR法分别对采集的168份腹泻犊牛的临床样本进行平行检测。结果显示，该方法可在20 min，39℃的条件下扩增目标片段，对BNoV和BRV敏感度均为10³ copies·μL^-1，与PCR的检测结果基本一致，且与牛冠状病毒（bovine coronavirus， BCoV）、牛病毒性腹泻病毒（bovine viral diarrhea virus， BVDV）和牛传染性鼻气管炎病毒（infectious bovine rhinotracheitis virus， IBRV）无交叉反应，虽低于qPCR检测结果，但可通过肉眼直接观察试纸条的检测结果。综上所述，RAA-LFD法灵敏度较高、且简便、快速、同时不依赖于仪器、专业操作人员，适用于现场检测。

关键词: 牛诺瓦病毒, 牛轮状病毒, 重组酶介导扩增, 侧向流试纸条

Abstract: This study aimed to establish a recombinase aided amplification-lateral flow dipstick (RAA-LFD) method for simultaneous detection of bovine norovirus(BNoV) and Bovine rotavirus(BRV). According to the principle of RAA primer probe design, this test first designed primers and probes targeted the conserved regions of BNoV RdRp gene and BRV VP7 gene, second prepared the standard recombinant plasmids, then established and optimized the RAA-LFD reaction system, finally the specificity, sensitivity and repeatability of the detection method were also evaluated. At the same time, 168 clinical samples of calves with diarrhea were tested in parallel by the RAA-LFD, PCR and qPCR method. The results showed that the method could amplify the target fragment in 20 minutes at 39 ℃. The sensitivity of this method is 10³ copies·μL^-1 for both BNoV and BRV, and is basically consistent with the detection results of PCR. It has no cross-reaction with bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV). Although the test strip result may be lower than that of the qPCR test, it can be easily observed with the naked eye. In summary, the RAA-LFD method has high sensitivity, and is simple, fast, and does not require specialized equipment or operators, making it ideal for on-site detection.

Key words: bovine norovirus, bovine rotavirus, recombinant enzyme-mediated amplification, lateral flow test strips

中图分类号:

李惠惠, 董可儿, 刘馨博, 张春晓, 马超, 陈利苹, 钟旗, 姚刚, 马雪连. 牛诺瓦病毒和牛轮状病毒双重RAA-LFD快速检测方法的建立及应用[J]. 畜牧兽医学报, 2024, 55(1): 406-412.

LI Huihui, DONG Keer, LIU Xinbo, ZHANG Chunxiao, MA Chao, CHEN Liping, ZHONG Qi, YAO Gang, MA Xuelian. Establishment and Application of Rapid Detection Method for Bovine Norovirus and Bovine Rotavirus Dual RAA-LFD[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 406-412.

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牛诺瓦病毒和牛轮状病毒双重RAA-LFD快速检测方法的建立及应用

Establishment and Application of Rapid Detection Method for Bovine Norovirus and Bovine Rotavirus Dual RAA-LFD

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[1]	孙吉, 岳华, 汤承. 检测牛5种腹泻病毒的多重RT-PCR方法的建立及应用[J]. 畜牧兽医学报, 2022, 53(1): 209-218.
[2]	刘梦瑶, 王展慧, 吴浩, 谷月, 吴文学. 牛星状病毒、牛病毒性腹泻病毒1型、牛冠状病毒和牛轮状病毒四重实时荧光定量RT-PCR检测方法的建立[J]. 畜牧兽医学报, 2021, 52(7): 1942-1952.
[3]	李然, 汤承, 罗雪, 岳华. 一株牦牛源G6P[11]型牛轮状病毒的分离鉴定及部分基因的序列分析[J]. 畜牧兽医学报, 2019, 50(3): 592-601.