【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。更多还原

【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.更多还原