Liu Yan, Zhu Zhen, Du Jige, Zhu Xiaojie, Pan Chenfan, Yin Chunsheng, Sun Weidong
Animal Laboratory, China Institute of Veterinary Drug Control, Beijing, China.
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Front Vet Sci. 2024 Jul 10;11:1337690. doi: 10.3389/fvets.2024.1337690. eCollection 2024.
Coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), canine coronavirus (CCoV), and feline infectious peritonitis virus (FIPV), have the potential for interspecies transmission. These viruses can be present in complex environments where humans, dogs, and cats coexist, posing a significant threat to both human and animal safety.
In this study, we developed a novel multiplex TaqMan-probe-based real-time PCR assay for the simultaneous detection and differentiation of SARS-CoV-2, CCoV, and FIPV. Specific primers and TaqMan fluorescent probes were designed based on the N region of SARS-CoV-2 and FIPV, as well as the S region of CCoV, which demonstrated a remarkable sensitivity and specificity toward the targeted viruses, as few as 21.83, 17.25 and 9.25 copies/μL for SARS-CoV-2, CCoV and FIPV, respectively. The standard curve constructed by the optimized method in our present study showed a high amplification efficiency within or near the optimal range of 91% to 116% and R(2) values were at least 0.95 for the abovementioned coronaviruses. A total of 91 samples, including six plasmid mixed mock samples, four virus fluid mixing simulated samples, and 81 clinical samples, were analyzed using this method. Results demonstrated strong agreement with conventional approaches.
By enabling the simultaneous detection of three viruses, this method enhances testing efficiency while decreasing costs. Importantly, it provides a valuable tool for the prevalence and geographical distribution of suspected and co-infected animals, ultimately contributing to the advancement of both animal and public health.
冠状病毒，包括严重急性呼吸综合征冠状病毒2（SARS-CoV-2）、犬冠状病毒（CCoV）和猫传染性腹膜炎病毒（FIPV），具有跨物种传播的可能性。这些病毒可存在于人类、狗和猫共存的复杂环境中，对人类和动物安全构成重大威胁。
在本研究中，我们开发了一种基于新型多重TaqMan探针的实时荧光定量PCR检测方法，用于同时检测和区分SARS-CoV-2、CCoV和FIPV。根据SARS-CoV-2和FIPV的N区域以及CCoV的S区域设计了特异性引物和TaqMan荧光探针，对目标病毒显示出显著的敏感性和特异性，SARS-CoV-2、CCoV和FIPV的检测限分别低至21.83、17.25和9.25拷贝/微升。本研究通过优化方法构建的标准曲线在91%至116%的最佳范围内或接近该范围时显示出高扩增效率，上述冠状病毒的R(2)值至少为0.95。使用该方法对总共91个样本进行了分析，包括6个质粒混合模拟样本、4个病毒液混合模拟样本和81个临床样本。结果表明与传统方法高度一致。
通过能够同时检测三种病毒，该方法提高了检测效率，同时降低了成本。重要的是，它为疑似和共同感染动物的流行情况和地理分布提供了有价值的工具，最终有助于动物和公共卫生的发展。
