Li Jing, Zhang Zichuang, Zhang Zongshu, Chen Xi, Wang Chunguang, Zhai Xianghe, Zhang Tie
College of Veterinary Medicine, Hebei Agricultural University, Baoding, China.
Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, China.
Front Vet Sci. 2024 Aug 16;11:1424238. doi: 10.3389/fvets.2024.1424238. eCollection 2024.
Avian leukemia virus (ALV) is one of the main pathogens of poultry tumor diseases, and has caused significant economic losses to the poultry industry since its discovery. Therefore, establishing a rapid detection method is essential to effectively prevent and control the spread of ALV. In this study, specific CRISPR RNA (crRNA) and recombinase-aided amplification (RAA) primers with T7 promoter were designed based on the relatively conserved sequence of avian leukemia virus. When crRNA recognized the target sequence, Cas13a protein was activated to cut the reporting probes, and then the detection results were read by using lateral flow dipstick (LFD). The RAA-CRISPR/Cas13a-LFD reaction system was constructed. The RAA amplification time, Cas13a protein concentration, crRNA concentration and CRISPR reaction time were optimized to evaluate the specificity, sensitivity and reproducibility of the system. Finally, RAA-CRISPR/Cas13a-LFD method was compared with Polymerase chain reaction (PCR)-Agarose electrophoresis method and qPCR method in the detection of clinical samples, and the reliability of RAA-CRISPR/Cas13a-LFD method was evaluated. The results showed that the RAA-CRISPR/Cas13a-LFD method could effectively amplify the target gene at 37°C for 40 min, and the test results could be determined by LFD visual observation. The method had good specificity and no cross-reaction with Marek's disease virus (MDV), Fowl adenovirus (FAdV), Infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), Infectious laryngotracheitis virus (ILTV), and Infectious bronchitis virus (IBV). The minimum detection limit of the method was 10 copies/μL, and it had good repeatability and stability. The coincidence rate of clinical detection reached 97.69% and 99.23%. In summary, this study established a simple, efficient, accurate and visualized ALV detection method, which can be used for the prevention and rapid clinical diagnosis of avian leukosis (AL).
禽白血病病毒（ALV）是家禽肿瘤疾病的主要病原体之一，自发现以来给家禽业造成了重大经济损失。因此，建立快速检测方法对于有效预防和控制ALV的传播至关重要。在本研究中，基于禽白血病病毒相对保守的序列设计了带有T7启动子的特异性CRISPR RNA（crRNA）和重组酶辅助扩增（RAA）引物。当crRNA识别靶序列时，Cas13a蛋白被激活以切割报告探针，然后使用侧向流动试纸条（LFD）读取检测结果。构建了RAA-CRISPR/Cas13a-LFD反应体系。对RAA扩增时间、Cas13a蛋白浓度、crRNA浓度和CRISPR反应时间进行了优化，以评估该体系的特异性、灵敏度和重复性。最后，将RAA-CRISPR/Cas13a-LFD方法与聚合酶链反应（PCR）-琼脂糖电泳法和qPCR法用于临床样本检测，并评估RAA-CRISPR/Cas13a-LFD方法的可靠性。结果表明，RAA-CRISPR/Cas13a-LFD方法可在37°C下40分钟有效扩增靶基因，检测结果可通过LFD目视观察确定。该方法具有良好的特异性，与马立克氏病病毒（MDV）、禽腺病毒（FAdV）、传染性法氏囊病病毒（IBDV）、新城疫病毒（NDV）、传染性喉气管炎病毒（ILTV）和传染性支气管炎病毒（IBV）无交叉反应。该方法的最低检测限为10拷贝/μL，具有良好的重复性和稳定性。临床检测符合率分别达到97.69%和99.23%。综上所述，本研究建立了一种简单、高效、准确且可视化的ALV检测方法，可用于禽白血病（AL）的预防和快速临床诊断。
