2022—2024年中国部分地区牛病毒性腹泻病毒分子检测及基因分型

doi:10.11843/j.issn.0366-6964.2025.10.050

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (10): 5328-5334.doi: 10.11843/j.issn.0366-6964.2025.10.050

• 研究简报 • 上一篇

2022—2024年中国部分地区牛病毒性腹泻病毒分子检测及基因分型

安乐乐¹^,²(), 任晓婷¹, 蓝秋菊¹^,², 莫永利¹, 曹小安³, 丁玉林⁴, 马忠仁¹, 马晓霞¹^,²^,*()

1. 西北民族大学生物医学研究中心生物工程与技术国家民委重点实验室，兰州 730030
2. 西北民族大学生命科学与工程学院，兰州 730100
3. 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 730030
4. 内蒙古农业大学兽医学院动物疾病临床诊疗技术重点实验室，呼和浩特 010018

收稿日期:2025-01-09 出版日期:2025-10-23 发布日期:2025-11-01
通讯作者: 马晓霞 E-mail:1803180130@qq.com;maxiaoxia956@163.com
作者简介:安乐乐(1997-)，男，河南郸城县人，硕士生，主要从事病毒基因工程研究，E-mail：1803180130@qq.com
基金资助:
甘肃省自然科学基金(23JRRA715)；西北民族大学引进人才科研项目(xbmuyjrc202225)

Molecular Detection and Genotyping of Bovine Viral Diarrhea Virus in Some Areas of China from 2022 to 2024

AN Lele¹^,²(), REN Xiaoting¹, LAN Qiuju¹^,², MO Yongli¹, CAO Xiaoan³, DING Yulin⁴, MA Zhongren¹, MA Xiaoxia¹^,²^,*()

1. Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China
2. College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730100, China
3. State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730030, China
4. College of Veterinary Medicine, Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Inner Mongolia Agricultural University, Hohhot 010018, China

Received:2025-01-09 Online:2025-10-23 Published:2025-11-01
Contact: MA Xiaoxia E-mail:1803180130@qq.com;maxiaoxia956@163.com

摘要/Abstract

摘要：

牛病毒性腹泻病毒(BVDV)是引起牛群中重要经济损失的病毒之一，尤其对幼牛的健康和发展造成了严重影响。本试验旨在建立通用型BVDV SYBR Green Ⅰ实时荧光定量PCR(BVDV SYBR Green Ⅰ qPCR)方法并对中国部分地区散养牛群样本进行病原学检测，掌握中国部分地区散养牛群BVDV感染情况、流行基因型及亚型。依据GenBank不同基因亚型BVDV 5′UTR序列建立通用型BVDV SYBR Green Ⅰ qPCR方法。使用该方法对2022年6月—2024年12月采集的712份散养牛血清样本及咳嗽、发烧、拉稀症状犊牛组织样本进行检测及遗传进化分析，同时对犊牛样本进行病毒分离、间接免疫荧光(IFA)鉴定和苏木精-伊红染色法(HE染色)观察。结果显示，建立的BVDV SYBR Green Ⅰ qPCR方法标准曲线的线性关系良好、敏感性高、特异性强和重复性好。散养牛血清样本阳性率为19.10%，并成功分离BVDV-24GS毒株。在5′UTR基因系统进化树分析中，BVDV基因亚型包括1c、1m、1o、1i、1q、1b和2。以上结果表明，成功建立通用型BVDV SYBR Green Ⅰ qPCR方法并实施初步检测，中国部分地区散养牛群中均存在不同程度BVDV感染，病毒基因分型鉴定丰富了BVDV的分子流行病学基础数据，为BVDV的科学防控、疫苗研制和免疫策略优化提供了依据。

关键词: 牛病毒性腹泻病毒, SYBR Green Ⅰ实时荧光定量PCR, 分子检测, 基因分型鉴定

Abstract:

Bovine viral diarrhea virus (BVDV) is one of the major pathogens responsible for significant economic losses in cattle herds, particularly affecting the health and development of calves. This study aimed to establish a universal SYBR Green I-based real-time quantitative PCR (qPCR) assay for BVDV detection and to investigate the epidemiology, genotypes, and subtypes of BVDV in samples collected from free-range cattle herds in selected regions of China. A universal BVDV SYBR Green I qPCR assay was developed based on the 5′ untranslated region (5′UTR) sequences of various BVDV genotypes obtained from GenBank. Using this method, a total of 712 serum samples from free-range cattle, as well as tissue samples from calves exhibiting symptoms such as coughing, fever, and diarrhea collected between June 2022 and December 2024, were tested and subjected to phylogenetic analysis. Additionally, virus isolation, indirect immunofluorescence assay (IFA), and hematoxylin-eosin (HE) staining were performed on calf samples. The established BVDV SYBR Green I qPCR assay demonstrated high sensitivity, strong specificity, good linearity, and excellent reproducibility. The positive detection rate in serum samples was 19.10%, and the BVDV-24GS strain was successfully isolated. Phylogenetic analysis of the 5′UTR gene sequences revealed that the BVDV strains belonged to subtypes 1c, 1m, 1o, 1i, 1q, 1b, and 2. These findings indicate the successful development and application of a universal BVDV SYBR Green I qPCR assay, and the presence of varying degrees of BVDV infection among free-range cattle in parts of China. The identification of viral genotypes enriches the molecular epidemiological data of BVDV and provides a scientific basis for effective disease control, vaccine development, and optimization of immunization strategies.

Key words: bovine viral diarrhea virus, SYBR Green I real-time quantitative PCR, molecular detection, genotype identification

中图分类号:

S852.653

安乐乐, 任晓婷, 蓝秋菊, 莫永利, 曹小安, 丁玉林, 马忠仁, 马晓霞. 2022—2024年中国部分地区牛病毒性腹泻病毒分子检测及基因分型[J]. 畜牧兽医学报, 2025, 56(10): 5328-5334.

AN Lele, REN Xiaoting, LAN Qiuju, MO Yongli, CAO Xiaoan, DING Yulin, MA Zhongren, MA Xiaoxia. Molecular Detection and Genotyping of Bovine Viral Diarrhea Virus in Some Areas of China from 2022 to 2024[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5328-5334.

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质粒拷贝数/ (Copies·μL^-1) Copy No.	组内重复性试验Intra-group reproducibility tests		组间重复性试验Inter-group reproducibility tests
质粒拷贝数/ (Copies·μL^-1) Copy No.	$\bar x \pm s$	交异系数/% CV	$\bar x \pm s$	交异系数/% CV
1.80×10⁸	13.330±0.074	0.555	13.060±0.308	2.358
1.80×10⁷	17.120±0.036	0.210	16.860±0.298	1.767
1.80×10⁶	21.290±0.033	0.155	21.440±0.461	2.150
1.80×10⁵	25.470±0.095	0.371	25.470±0.214	0.840

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2022—2024年中国部分地区牛病毒性腹泻病毒分子检测及基因分型

Molecular Detection and Genotyping of Bovine Viral Diarrhea Virus in Some Areas of China from 2022 to 2024

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