Cao Zhi, Xu Hang, Zhao Xinru, Zhang Ke, Yin Dehua, Ma Shuai, Li Wenling, Li Siyu, Ren Jianwei, Wen Jianxin
College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China.
Shandong Collaborative Innovation Center for Development of Veterinary Pharmaceuticals, Qingdao, 266109, China.
BMC Vet Res. 2025 Jan 14;21(1):18. doi: 10.1186/s12917-024-04349-5.
Aleutian mink disease, mink viral enteritis and canine distemper are known as the three most serious diseases that cause great economic loss in the mink industry. In clinical practice, aleutian mink disease virus (AMDV), mink enteritis virus (MEV) and canine distemper virus (CDV) are common mixed infections, and they have similar clinical clinical signs, such as diarrhoea. Therefore, a rapid and accurate differential diagnosis method for use on mink ranches is essential for the control of these three pathogens. Here, we developed multiplex one-step real-time quantitative PCR (RT‒qPCR) assays for the simultaneous detection and quantification of AMDV, MEV and CDV by using three primers and probes based on the conserved NS1, VP2 and N genes, respectively.
The results showed that the established method can not cross-react with other mink pathogens, with a detection sensitivity of 25 copies/µL and a coefficient of variation less than 3.51%. Moreover, the interference experiment showed that the presence of AMDV, MEV and CDV templates at different concentrations would not interfere with the detection results. Furthermore, two hundred clinical samples of mink with diarrhoea were simultaneously analysed using multiplex RT‒qPCR and single RT‒qPCR, the Kappa values were all greater than 0.921, indicating that there was a high degree of coincidence between the two detection methods.
In conclusion, multiplex RT‒qPCR exhibited high specificity, sensitivity, and reproducibility, indicating that this method can be used as a reliable and specific tool for the differential detection and quantification of AMDV, MEV and CDV.
阿留申水貂病、水貂病毒性肠炎和犬瘟热被认为是给水貂养殖业造成巨大经济损失的三种最严重疾病。在临床实践中，阿留申水貂病病毒（AMDV）、水貂肠炎病毒（MEV）和犬瘟热病毒（CDV）常混合感染，且它们具有相似的临床症状，如腹泻。因此，一种用于水貂养殖场的快速准确的鉴别诊断方法对于控制这三种病原体至关重要。在此，我们开发了多重一步实时定量PCR（RT-qPCR）检测方法，通过分别基于保守的NS1、VP2和N基因使用三种引物和探针，同时检测和定量AMDV、MEV和CDV。
结果表明，所建立的方法不会与其他水貂病原体发生交叉反应，检测灵敏度为25拷贝/μL，变异系数小于3.51%。此外，干扰实验表明，不同浓度的AMDV、MEV和CDV模板的存在不会干扰检测结果。此外，使用多重RT-qPCR和单重RT-qPCR同时分析了200份腹泻水貂临床样本，Kappa值均大于0.921，表明两种检测方法之间具有高度一致性。
总之，多重RT-qPCR表现出高特异性、灵敏度和可重复性，表明该方法可作为一种可靠且特异的工具用于AMDV、MEV和CDV的鉴别检测和定量。
