基于非洲猪瘟病毒<em>MGF100</em>-<em>1L</em>基因的荧光定量PCR方法的建立与评价

doi:10.3969/j.issn.1674-7968.2026.02.017

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摘要非洲猪瘟(African swine fever, ASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)引发的一种高度传染性疾病,其致死率极高。本研究旨在开发一种特异、灵敏的ASFV TaqMan荧光定量PCR (real-time PCR, qPCR)检测方法。对ASFV多基因家族(multigene families, MGFs)中的MGF100-1L基因(GenBank No. MK333180.1)序列进行分析,针对该基因保守区设计并筛选出特异性引物和探针组合。通过构建包含该基因片段的重组质粒,作为标准品用于优化qPCR反应体系,建立ASFV qPCR检测方法,并对方法的特异性、灵敏性、重复性、符合率进行评价。用该检测方法对90份临床样品进行检测,并与世界动物卫生组织(World Organisation for Animal Health, WOAH)推荐的检测方法进行比对。结果表明,标准曲线线性方程为Y=-3.304X+38.793,相关系数为0.99,表明线性良好。灵敏性分析发现,所建立的qPCR方法最低检出限(limit of detection, LOD)为0.87拷贝/µL,灵敏性与WOAH方法(0.94拷贝/μL)基本一致。特异性分析验证了该方法与猪瘟病毒(Classical swine fever virus, CSFV)、伪狂犬病病毒(Pseudorabies virus, PRV)、猪细小病毒(Porcine parvovirus, PPV)、猪圆环病毒2型(Porcine circovirus type 2, PCV2)及口蹄疫病毒(Foot-and-mouth disease virus, FMDV)等均无交叉反应。临床样品检测中,该方法与WOAH推荐方法相比,一致性高(Kappa=0.903, P<0.01),且对弱阳性样本的检出率显著提升(比WOAH方法多检出4个样本)。本研究成功构建了基于ASFV MGF100-1L基因的qPCR检测体系,该体系展现出优异的特异性、灵敏性,且与现有经典方法检测结果高度一致。该方法为临床ASFV检测,特别是早期诊断和防控提供了更灵敏可靠的技术手段。

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	董睿
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	曾巧英
	朱紫祥

关键词 ：非洲猪瘟病毒, MGF100-1L基因, 荧光定量PCR

Abstract：African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious disease with extremely high mortality. This study aimed to develop a specific and sensitive real-time PCR (qPCR) assay for detecting ASFV. The MGF100-1L gene (GenBank No. MK333180.1), belonging to the multigene families (MGFs) of ASFV, was analyzed to design and screen specific primers and probes targeting conserved regions. A recombinant plasmid containing the target gene fragment was constructed as a standard for optimizing the qPCR reaction system. The established ASFV qPCR method was evaluated for specificity, sensitivity, reproducibility, and agreement. A total of 90 clinical samples were tested and compared with the method recommended by the World Organisation for Animal Health (WOAH). Results showed that the standard curve linear equation was Y=-3.304X+38.793 with a correlation coefficient of 0.99, indicating good linearity. The limit of detection (LOD) was 0.87 copies/µL, demonstrating sensitivity comparable to the WOAH method. Specificity analysis confirmed no cross-reactivity with Classical swine fever virus (CSFV), Pseudorabies virus (PRV), Porcine parvovirus (PPV), Porcine circovirus type 2 (PCV2), or Foot-and-mouth disease virus (FMDV). In clinical sample testing, the method showed high agreement with the WOAH method (Kappa=0.903, P<0.01) and significantly improved detection rates for weakly positive samples (4 additional samples detected compared to the WOAH method). This study successfully established a qPCR detection system based on the ASFV MGF100-1L gene, exhibiting excellent specificity, sensitivity, and high consistency with existing standard methods. This method provides a more sensitive and reliable technical tool for clinical ASFV detection, particularly for early diagnosis and control.

Key words： African swine fever virus (ASFV) MGF100-1L gene Real-time PCR

收稿日期: 2025-06-12

中图分类号： S851.33

基金资助:国家重点研发计划(2021YFD1800100); 广东省“十四五”农业科技十大重点领域关键技术攻关项目(2024KJ14); 中央高校基本科研业务费(24CXNA055); 甘肃省联合研究基金(24JRRA813); 甘肃省创新群体项目(23JRRA546); 国家生猪产业技术体系(CARS-35); 国家生猪技术创新中心项目(NCTIP-XD/C03); 中国农业科学院科技创新工程(CAAS-CSLPDCP-202302; CAAS-ASTIP-2025-LVRI)

通讯作者: ^*zhuzixiang@caas.cn

引用本文:

董睿, 芮弦, 石正旺, 潘阳阳, 包世俊, 曾巧英, 朱紫祥. 基于非洲猪瘟病毒MGF100-1L基因的荧光定量PCR方法的建立与评价[J]. 农业生物技术学报, 2026, 34(2): 444-455.
DONG Rui, RUI Xian, SHI Zheng-Wang, PAN Yang-Yang, BAO Shi-Jun, ZENG Qiao-Ying, ZHU Zi-Xiang. Establishment and Evaluation of Fluorescence Quantitative PCR Method Based on the African swine fever virus MGF100-1L Gene. 农业生物技术学报, 2026, 34(2): 444-455.

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https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2026.02.017 或 https://journal05.magtech.org.cn/Jwk_ny/CN/Y2026/V34/I2/444

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