Yin Dongdong, Xu Shuangshuang, Liu Yayun, Guo Hao, Lan Mengdie, Yin Lei, Wang Jieru, Dai Yin, Shen Xuehuai, Zhan Kai, Pan Xiaocheng
Anhui Provincial Key Laboratory of Livestock and Poultry Product Safety, Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Sciences, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province, Hefei 230031, China.
College of Veterinary Medicine, Anhui Agricultural University, Hefei 230036, China.
Microorganisms. 2025 Jul 3;13(7):1573. doi: 10.3390/microorganisms13071573.
Since its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), making timely and precise diagnosis a considerable challenge. To address this, we established a TaqMan-based multiplex real-time quantitative PCR (qPCR) assay capable of simultaneously detecting ASFV, CSFV, PRRSV, PRV, and PCV2. Specific primer-probe sets were developed targeting conserved genomic regions: the ASFV gene, CSFV region, PRRSV , PCV2 gene, and PRV gene. After thorough optimization, the assay demonstrated robust analytical performance, exhibiting strong target specificity with no cross-detection of non-target pathogens. The detection threshold was determined to be 10 copies/μL per virus, indicating high assay sensitivity. Repeatability analysis revealed low variability, with intra- and inter-assay coefficient of variation values remaining below 2.3%. When applied to 95 clinical samples, the multiplex assay yielded results that were fully consistent with those obtained using commercially available singleplex qPCR kits. In conclusion, the multiplex TaqMan qPCR method developed in this study is characterized by high specificity, sensitivity, and reproducibility. It provides a reliable and efficient diagnostic tool for the simultaneous detection and differential diagnosis of ASFV and other clinically similar viral infections in swine, thereby offering robust technical support for swine disease surveillance and control.
自2018年非洲猪瘟病毒（ASFV）在中国出现以来，因其高传播性和死亡率，对养猪业构成了严重威胁。ASFV感染的临床症状常与其他猪病毒如经典猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）、伪狂犬病病毒（PRV）和猪圆环病毒2型（PCV2）引起的症状重叠，这使得及时、准确的诊断成为一项巨大挑战。为解决这一问题，我们建立了一种基于TaqMan的多重实时定量PCR（qPCR）检测方法，能够同时检测ASFV、CSFV、PRRSV、PRV和PCV2。针对保守基因组区域设计了特异性引物-探针组：ASFV基因、CSFV区域、PRRSV、PCV2基因和PRV基因。经过全面优化，该检测方法展现出强大的分析性能，对目标具有高度特异性，未出现非目标病原体的交叉检测。确定每种病毒的检测阈值为10拷贝/μL，表明该检测方法具有高灵敏度。重复性分析显示变异率较低，批内和批间变异系数值均低于2.3%。当应用于95份临床样本时，多重检测结果与使用市售单重qPCR试剂盒获得的结果完全一致。总之，本研究开发的多重TaqMan qPCR方法具有高特异性、灵敏度和可重复性。它为同时检测和鉴别诊断猪的ASFV及其他临床相似病毒感染提供了可靠、高效的诊断工具，从而为猪病监测和防控提供了有力的技术支持。
