用于非洲猪瘟病毒多靶标核酸检测的DNA假病毒制备及定量

doi:10.11843/j.issn.0366-6964.2025.07.047

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (7): 3555-3560.doi: 10.11843/j.issn.0366-6964.2025.07.047

• 研究简报 • 上一篇

用于非洲猪瘟病毒多靶标核酸检测的DNA假病毒制备及定量

邓俊花¹^,²(), 李昊轩¹^,², 陈冬杰¹^,², 吕继洲¹^,², 王晶晶¹^,², 张舟¹^,², 袁向芬¹^,², 魏方¹^,², 吴绍强¹^,²^,*()

1. 中国检验检疫科学研究院，北京 100176
2. 国家市场监督管理总局技术创新中心(动植物产品质量与安全)，北京 100176

收稿日期:2024-09-03 出版日期:2025-07-23 发布日期:2025-07-25
通讯作者: 吴绍强 E-mail:dengqifei-198825@163.com;sqwu@sina.com
作者简介:邓俊花(1980-)，女，山东高唐人，副研究员，硕士，主要从事重大动物疫病检测、标准物质研究，E-mail: dengqifei-198825@163.com；Tel：010-53897668
基金资助:
国家重点研发技术项目(2022YFD1800605)

Preparation and Quantification of DNA Pseudovirus with Multi-Target Nucleic Acid Detection for African Swine Fever Virus

DENG Junhua¹^,²(), LI Haoxuan¹^,², CHEN Dongjie¹^,², LV Jizhou¹^,², WANG Jingjing¹^,², ZHANG Zhou¹^,², YUAN Xiangfen¹^,², WEI Fang¹^,², WU Shaoqiang¹^,²^,*()

1. Chinese Academy of Inspection and Quarantine, Beijing 100176, China
2. Technology Innovation Center of Animal and Plant Product Quality, Safety and Control, State Administration for Market Regulation, Beijing 100176, China

Received:2024-09-03 Online:2025-07-23 Published:2025-07-25
Contact: WU Shaoqiang E-mail:dengqifei-198825@163.com;sqwu@sina.com

摘要/Abstract

摘要：

本研究旨在研制一种全程质控非洲猪瘟病毒(ASFV)核酸检测、避免质粒交叉污染的DNA假病毒。将ASFV B646L/CD2v/MGF505-1R/MGF360-12L 4种检测关键基因及EGFP基因共构建于杆状病毒的转移载体pFastBac^TM Dual中，转化形成重组Bacmid pFastBac-EGFP-ASFV，转染至sf21细胞，包装产生ASFV重组杆状病毒。结果显示：通过荧光倒置显微镜观察，表达的EGFP蛋白绿色荧光信号指示成功获取AcMNPV-EGFP-ASFV病毒；重组杆状病毒基因组溯源分析，ASFV B646L/CD2v/MGF505-1R/MGF360-12L基因序列满足现行检测方法质控需求；ASFV质控品均匀、稳定，-20 ℃保存至少可稳定24个月。采用ddPCR定量为4.09×10³ copies·μL^-1。本研究非洲猪瘟病毒DNA假病毒的制备，为非洲猪瘟疫情监测提供了新的技术支撑。

关键词: 非洲猪瘟病毒, DNA假病毒, 定量

Abstract:

The study aims to develop a DNA pseudovirus with full quality control and avoiding plasmid cross-contamination in nucleic acid detection for African swine fever virus (ASFV). The B646L/CD2v/MGF505-1R/MGF360-12L key-genes of ASFV and EGFP gene were co-combined into the baculovirus transfer vector pFastBac^TMDual. The Bacmid pFastBac-EGFP-ASFV was formed by transformation. The Bacmid was transfected into sf21 cells and packaged to produce AcMNPV-EGFP-ASFV. The green fluorescence signal of EGFP protein expressed indicated that AcMNPV-EGFP-ASFV was preparated successfully. Genome traceability analysis of the AcMNPV-EGFP-ASFV showed that the ASFV target gene sequence was met the requirements of current detection methods. The ASFV quality control product is uniform and stable, and is stably stored at -20 ℃ for at least 24 months. The quantification is 4.09×10³ copies·μL^-1 using ddPCR. The preparation of ASFV DNA pseudovirus provides a new technical support for the monitoring of the African swine fever.

Key words: African swine fever virus, DNA pseudovirus, quantification

中图分类号:

S852.659.1

邓俊花, 李昊轩, 陈冬杰, 吕继洲, 王晶晶, 张舟, 袁向芬, 魏方, 吴绍强. 用于非洲猪瘟病毒多靶标核酸检测的DNA假病毒制备及定量[J]. 畜牧兽医学报, 2025, 56(7): 3555-3560.

DENG Junhua, LI Haoxuan, CHEN Dongjie, LV Jizhou, WANG Jingjing, ZHANG Zhou, YUAN Xiangfen, WEI Fang, WU Shaoqiang. Preparation and Quantification of DNA Pseudovirus with Multi-Target Nucleic Acid Detection for African Swine Fever Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3555-3560.

图/表 3

参考文献 13

1	ZHOU X , LI N , LUO Y , et al. Emergence of African swine fever in China, 2018[J]. Transbound Emerg Dis, 2018, 65 (6): 1482- 1484.
2	GE S , LI J , FAN X , et al. Molecular characterization of African swine fever virus, China, 2018[J]. Emerg Infect Dis,, 2018, 24 (11): 2131- 2133.
3	SUN E C , ZHANG Z J , WANG Z L , et al. Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020[J]. Life Sci, 2021, 64 (5): 752- 765.
4	SUN E C , HUANG L Y , ZHANG X F , et al. Genotype Ⅰ African swine fever viruses emerged in domestic pigs in China and caused chronic infection[J]. Emerg Microb Infect, 2021, 10 (1): 2183- 2193.
5	ZHAO D M , SUN E C , HUANG L Y , et al. Highly lethal genotype Ⅰ and Ⅱ recombinant African swine fever viruses detected in pigs[J]. Nat Commun, 2023, 14 (1): 3096.
6	吴绍强, 邓俊花, 冯春燕, 等. 非洲猪瘟病毒荧光PCR检测试剂盒(2019)[Z]. 新兽药证字74号, 2019.
	WU S Q, DENG J H, FENG C Y, et al. The real-time PCR detection kit for African swine fever virus (2019)[Z]. New Veterinary Drug Certificate No. 74, 2019. (in Chinese)
7	邓俊花, 吴绍强, 王彩霞, 等. 非洲猪瘟病毒MGF-505R基因荧光PCR检测试剂、试剂盒及应用(ZL202010045654.1)[P]. 2022.09.02.
	DENG J H, WU S Q, WANG C X, et al. The real-time PCR detection reagents, kits and applications for MGF-505R gene of African swine fever virus(ZL202010045654.1)[P]. 2022.09.02. (in Chinese)
8	郭振华, 邢广旭, 翁茂洋, 等. 非洲猪瘟病毒野毒株与基因缺失株三重荧光定量PCR鉴别检测方法的建立及应用[J]. 中国兽医学报, 2022, 42 (4): 619- 625.
	GUO Z H , XING G X , WENG M Y , et al. Development and application of a triplex real-time PCR method for detection and differentiation of gene-deleted and wide-type African swine fever virus[J]. Chinese Journal of Veterinary Science, 2022, 42 (4): 619- 625.
9	韩玉, 王涛, 潘力, 等. 基于非洲猪瘟病毒CD2v基因TaqMan荧光定量PCR检测方法的建立[J]. 中国兽医学报, 2021, 41 (5): 847- 852.
	HAN Y , WANG T , PAN L , et al. Establishment of a TaqMan real-time PCR assay targeting the African swine fever virus CD2v gene[J]. Chinese Journal of Veterinary Science,, 2021, 41 (5): 847- 852.
10	吴晓东, 李林, 胡永新, 等. 非洲猪瘟诊断技术(GB/T 18648—2020) [S]. 国家市场监督管理总局, 2020.12.14.
	WU X D, LI L, HU Y X, et al. Diagnostic Techniques for African Swine Fever (GB/T 18648—2020) [S]. State Administration for Market Regulation, 2020.12.14. (in Chinese)
11	WOAH. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2024. Chapter 3.8.1 African swine fever[M/OL]. https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.08.01_ASF.pdf. (2024-09-03)
12	农业农村部.关于进一步严厉打击违法研制生产经营使用非洲猪瘟疫苗行为的通知(农办牧[2020] 39号)附件-非洲猪瘟病毒流行株与基因缺失株鉴别检测规范[EB].2020. https://www.gov.cn/zhengce/zhengceku/2020-08/25/5537334/files/4a606841b20b48e9a6d97905db500a25.pdf(2024-09-03).
	Ministry of Agriculture and Rural Affairs of the People's Republic of China. Attachment: Specifications for Differentiation and Detection of Epidemic Strains and Genetically Deficient Strains of African Swine Fever Virus//[EB] Notice on further strengthening the punishment of illegal activities in research, production, distribution and use of African swine fever vaccines (Nong Ban Mu[2020] No. 39). https://www.gov.cn/zhengce/zhengceku/2020-08/25/5537334/files/4a606841b20b48e9a6d97905db500a25.pdf (2024-09-03).(in Chinese)
13	农业农村部.养猪场非洲猪瘟变异株监测技术指南[EB]. http://www.moa.gov.cn/govpublic/xmsyj/202103/t20210322_6364254.htm. (2024-09-03).
	Ministry of Agriculture and Rural Affairs of the People's Republic of China. Guidelines for monitoring African swine fever variant strains in pig farms[EB]. http://www.moa.gov.cn/govpublic/xmsyj/202103/t20210322_6364254.htm.(2024-09-03).(in Chinese)

[1]	王飞燕, 刘超凡, 张亚楠, 周晓甜, 任静, 袁晨, 李潭清, 宋勤叶. 猪IL-15单克隆抗体的制备及双抗体夹心ELISA方法的建立[J]. 畜牧兽医学报, 2025, 56(7): 3442-3452.
[2]	卢会鹏, 曹世诺, 吴植, 陈长春, 陈文玉, 成玉婷, 周晓慧, 孙怀昌, 朱善元. 表达非洲猪瘟病毒基因B602L-B646L的重组病毒免疫原性的初步分析[J]. 畜牧兽医学报, 2025, 56(6): 2816-2825.
[3]	陈长春, 吴植, 任冠宇, 陈文玉, 曹世诺, 朱睿, 张力, 成玉婷, 朱善元, 卢会鹏. 表达非洲猪瘟pp62与Hsp70蛋白重组腺病毒对小鼠的免疫原性分析[J]. 畜牧兽医学报, 2025, 56(6): 3027-3031.
[4]	侯中一, 王宝维, 张名爱, 孔敏, 张晶, 王秉翰, 岳斌, 鲁秀, 凡文磊. 基于蛋白质组学解析鹅肥肝形成的脂质代谢调控机制[J]. 畜牧兽医学报, 2025, 56(5): 2182-2193.
[5]	韩阳瑞, 夏炉明, 李瑞芳, 张曼玉, 杜晶莹, 孙卿, 李增强, 赵洪进, 王权. 弓形虫抗体镧系微球免疫层析试纸条方法的建立[J]. 畜牧兽医学报, 2025, 56(5): 2353-2363.
[6]	张越, 茹毅, 郝荣增, 杨锐, 赵陇和, 李亚军, 杨洋, 张荣, 蒋成辉, 郑海学. 非洲猪瘟病毒H108R蛋白的制备及其免疫原性评价[J]. 畜牧兽医学报, 2025, 56(3): 1344-1354.
[7]	马晓莉, 李段, 曾道平, 刘燕玲, 王晓敏, 彭国良, 宋长绪, 王磊, 徐铮. 非洲猪瘟病毒p72蛋白抗体全自动化学发光酶免疫检测方法的建立[J]. 畜牧兽医学报, 2025, 56(3): 1355-1365.
[8]	王纯, 王晴, 吴晓倩, 胡婷, 崔卫涛, 裴洁, 宋铁平, 胡思顺, 张万坡, 李自力, 周祖涛. 基于荧光定量PCR检测和原位杂交技术分析湖北省蛋鸡滑液支原体的感染情况[J]. 畜牧兽医学报, 2025, 56(3): 1396-1407.
[9]	于江玮, 程慧敏, 林健, 杨宝琳, 黄程, 杨志远, 胡格. 鸭瘟病毒TaqMan荧光定量PCR检测方法的建立及应用[J]. 畜牧兽医学报, 2025, 56(2): 765-773.
[10]	张素, 孙丽芳, 李兰兰, 吴琳娇, 陈磊清, 吴允昆. 非洲猪瘟病毒结构蛋白与宿主蛋白相互作用研究进展[J]. 畜牧兽医学报, 2025, 56(1): 95-106.
[11]	王星月, 杨志远, 林健, 张紫萱, 周雨婷, 程慧敏, 刘月焕, 胡格. 鸽圆环病毒荧光定量PCR检测方法的建立及应用[J]. 畜牧兽医学报, 2025, 56(1): 335-342.
[12]	冯露, 田宏, 郑海学, 石正旺, 罗俊聪, 张晓阳, 尉娟娟, 周静, 廖焕程, 王婉莹. 基于酶促重组酶扩增的非洲猪瘟病毒检测方法[J]. 畜牧兽医学报, 2024, 55(9): 4226-4231.
[13]	周扬, 吴炜姿, 曹伟胜, 王福广, 许秀琼, 钟文霞, 吴立炀, 叶健, 卢受昇. 基于Nanopore测序技术的非洲猪瘟病毒全基因组测序方法建立[J]. 畜牧兽医学报, 2024, 55(5): 2080-2089.
[14]	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.
[15]	江锦秀, 张靖鹏, 林裕胜, 刘维巍, 胡奇林, 万春和. 基于Hsp70基因的绵羊肺炎支原体TaqMan检测方法的建立及其遗传演化分析[J]. 畜牧兽医学报, 2024, 55(4): 1684-1695.

用于非洲猪瘟病毒多靶标核酸检测的DNA假病毒制备及定量

Preparation and Quantification of DNA Pseudovirus with Multi-Target Nucleic Acid Detection for African Swine Fever Virus

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 3

参考文献 13

相关文章 15

编辑推荐

Metrics

本文评价