Zhang Zichuang, Li Jing, Zhang Chengqi, Bai Xue, Zhang Tie
Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China.
The College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China.
Animals (Basel). 2024 Dec 18;14(24):3663. doi: 10.3390/ani14243663.
Feline calicivirus (FCV) is one of the most common viral pathogens in domestic cats worldwide, which mainly causes upper respiratory tract infections in felines and seriously threatens the health of felines. Consequently, it is crucial to establish a rapid detection method to efficiently take control and prevent the spread of FCV. To construct the Cas13a-RAA-LFD reaction system, this study specifically designed recombinase-aided amplification (RAA) primers added with a T7 promoter and CRISPR RNA (crRNA), which were both based on the FCV relatively conserved sequence. The Cas13a protein cleaved the reporting probes only when crRNA recognized the target sequence. The results could be directly observed by lateral flow dipsticks (LFDs). To evaluate this system, factors such as RAA amplification time, Cas13a protein concentration, crRNA concentration, and CRISPR reaction time were optimized. Then, a comparison of the coincidence rate for clinical samples between this method and the polymerase chain reaction (PCR) agarose electrophoresis method was performed to evaluate the reliability of the method. Eventually, the results indicated that the target gene could be effectively amplified by the Cas13a-RAA-LFD method, and the results could be visually observed by LFD. The method could detect FCV specifically, whilst having no cross-reaction with other common viruses which infect felines, such as feline parvovirus (FPV), feline coronavirus (FCoV) and feline herpesvirus (FHV). This method is extremely sensitive and has been validated to detect viral nucleic acids down to 10 copies/μL. The good reproducibility and stability of the method were also verified by this study. Testing of clinical samples proved that the coincidence rate of clinical detection reached 96.39%. In summary, this study established a simplistic, efficient, accurate, and visualized FCV detection method, which can be utilized for early prevention and control of FCV.
猫杯状病毒（FCV）是全球家养猫中最常见的病毒病原体之一，主要引起猫的上呼吸道感染，严重威胁猫的健康。因此，建立一种快速检测方法以有效控制和预防FCV的传播至关重要。为构建Cas13a-RAA-LFD反应体系，本研究基于FCV相对保守序列，专门设计了添加T7启动子的重组酶辅助扩增（RAA）引物和CRISPR RNA（crRNA）。只有当crRNA识别靶序列时，Cas13a蛋白才会切割报告探针。结果可通过侧向流动试纸条（LFD）直接观察。为评估该体系，对RAA扩增时间、Cas13a蛋白浓度、crRNA浓度和CRISPR反应时间等因素进行了优化。然后，将该方法与聚合酶链反应（PCR）琼脂糖电泳法对临床样本的符合率进行比较，以评估该方法的可靠性。最终，结果表明Cas13a-RAA-LFD方法可有效扩增靶基因，结果可通过LFD直观观察。该方法可特异性检测FCV，与其他感染猫的常见病毒，如猫细小病毒（FPV）、猫冠状病毒（FCoV）和猫疱疹病毒（FHV）无交叉反应。该方法极其灵敏，已验证可检测低至10拷贝/μL的病毒核酸。本研究还验证了该方法具有良好的重复性和稳定性。临床样本检测证明临床检测符合率达到96.39%。综上所述，本研究建立了一种简单、高效、准确且可视化的FCV检测方法，可用于FCV的早期防控。
