用于传染性支气管炎病毒核酸检测的N蛋白基因装甲RNA质控品的制备

doi:10.11843/j.issn.0366-6964.2025.11.034

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (11): 5770-5777.doi: 10.11843/j.issn.0366-6964.2025.11.034

用于传染性支气管炎病毒核酸检测的N蛋白基因装甲RNA质控品的制备

熊曜宇¹^,²(), 刘丹¹(), 高建帅¹, 李慧彤¹, 张博源¹, 丁家波¹, 蒋卉¹, 范学政¹, 沈青春¹^,*()

1. 中国农业科学院北京畜牧兽医研究所农业农村部动物生物安全风险预警及防控重点实验室(北方), 北京 100193
2. 甘肃农业大学动物科学技术学院, 兰州 730070

收稿日期:2024-09-06 出版日期:2025-11-23 发布日期:2025-11-27
通讯作者: 沈青春 E-mail:616203596@qq.com;1465512558@qq.com;shenqingchun@caas.cn
作者简介:熊曜宇(2004-)，男，湖北武汉人，本科生，主要从事动物科学研究，E-mail: 616203596@qq.com
刘丹(2001-)，女，河南信阳人，硕士生，主要从事预防兽医学研究，E-mail: 1465512558@qq.com
第一联系人：
熊曜宇与刘丹为同等贡献作者
基金资助:
重大外来动物疫病阻断与防控技术研发项目(2022YFD1800500)

Preparation of N-protein Gene Armor RNA Control for Infectious Bronchitis Virus Nucleic Acid Detection

XIONG Yaoyu¹^,²(), LIU Dan¹(), GAO Jianshuai¹, LI Huitong¹, ZHANG Boyuan¹, DING Jiabo¹, JIANG Hui¹, FAN Xuezheng¹, SHEN Qingchun¹^,*()

1. Key Laboratory of Animal Biosafety Risk Prevention and Control (North) of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences of CAAS, Beijing 100193, China
2. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China

Received:2024-09-06 Online:2025-11-23 Published:2025-11-27
Contact: SHEN Qingchun E-mail:616203596@qq.com;1465512558@qq.com;shenqingchun@caas.cn

摘要/Abstract

摘要：

旨在研制可应用于传染性支气管炎病毒(Infectious bronchitis virus, IBV)核酸检测的质控品。本研究选取IBV较为保守的N蛋白基因作为目标靶序列，将其与MS2噬菌体衣壳蛋白基因、包装位点序列等克隆到载体pET-28a(+)中，经诱导表达和纯化，获得内部包含有IBV的N蛋白基因序列RNA的类病毒颗粒，用电镜观察其物理特征，数字PCR对拷贝数进行精确定量，使用现行国家标准GB/T23197—2022中的实时荧光定量RT-PCR方法对纯化后的装甲RNA进行了检测验证，对RNase酶抵御能力以及保存稳定性也进行了检验。综上，本研究成功构建、表达组装出了包含IBV N基因的装甲RNA，耐RNase酶的降解，可在37℃下可稳定保存15 d以上。制备的装甲RNA具备成为IBV阳性质控品的潜力，可实现对该RNA病毒检测全过程的质控。

关键词: 禽传染性支气管炎, N蛋白, 质控样品, 核酸检测, 装甲RNA

Abstract:

The objective of this study is to develop a quality-controled product that can be used in the RNA detection of infectious bronchitis virus (IBV). The relatively conserved N protein gene of IBV was selected as the target sequence, and it was cloned into the vector pET-28a(+) with the MS2 phage capsid protein gene and packaging site sequence, and the RNA containing the N protein gene sequence of IBV was obtained by induction expression and purification, and its physical characteristics were observed by electron microscopy, and the copy number was accurately quantified by digital PCR. The real-time RT-PCR method introduced in the national standard GB/T23197—2022 was used to verify the detection of purified armor RNA, and the RNase enzyme resistance and storage stability were verified as well. The armored RNA containing IBV N gene was successfully constructed, which was resistant to the degradation of RNase enzyme and could be stably stored at 37℃ for above 15 days. The developed armor RNA has the potential to become a positive control product for IBV, which can realize the quality control of the whole process of RNA virus detection.

Key words: avian infectious bronchitis, protein N, quality control sample, nucleic acid detection, armored RNA

中图分类号:

S852.659.2

熊曜宇, 刘丹, 高建帅, 李慧彤, 张博源, 丁家波, 蒋卉, 范学政, 沈青春. 用于传染性支气管炎病毒核酸检测的N蛋白基因装甲RNA质控品的制备[J]. 畜牧兽医学报, 2025, 56(11): 5770-5777.

XIONG Yaoyu, LIU Dan, GAO Jianshuai, LI Huitong, ZHANG Boyuan, DING Jiabo, JIANG Hui, FAN Xuezheng, SHEN Qingchun. Preparation of N-protein Gene Armor RNA Control for Infectious Bronchitis Virus Nucleic Acid Detection[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5770-5777.

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差异源 Source of difference	平方和 SS	自由度 df	均方 MS	F值 F-value	P值 P-value	F临界值 F crit
组间Between groups	0.009 12	2	0.004 56	0.670 26	0.529 726	3.885 294
组内Within groups	0.081 64	12	0.006 803
总计Total	0.090 76	14	/	/	/	/

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用于传染性支气管炎病毒核酸检测的N蛋白基因装甲RNA质控品的制备

Preparation of N-protein Gene Armor RNA Control for Infectious Bronchitis Virus Nucleic Acid Detection

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