Wu Mengting, Chen Mengmeng, Qiu Rulong, Ge Lei, Fan Zhiyu, Hu Bo, Wei Houjun, Li Yiming, Wang Fang, Song Yanhua
Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, China.
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China.
Transbound Emerg Dis. 2024 Nov 19;2024:3881457. doi: 10.1155/tbed/3881457. eCollection 2024.
Rabbit hemorrhagic disease is a highly contagious and acute fatal disease caused by rabbit hemorrhagic disease virus (RHDV). The first outbreak of RHDV2 in 2020 has posed a serious threat to the rabbit breeding industry in China. An effective and specific detection strategy for RHDV GI.1 (RHDV1) and GI.2 (RHDV2) is urgently needed. In this study, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-CRISPR/Cas12a-based dual readout portable detection platform. The platform showed excellent specificity to identify RHDV1 and RHDV2 strains and no cross-reaction with other prevalent pathogens of rabbit. The detection limit for RHDV1 and RHDV2 by RT-LAMP-CRISPR/Cas12a could reach 10 copies/μl of the VP60 gene per reaction. Furthermore, 74 clinical samples were detected for both RHDV1 and RHDV2. RT-LAMP-CRISPR/Cas12a-based dual readout portable detection platform showed 25.68% (19/74) RHDV1-positive samples, 43.24% (32/74) RHDV2-positive samples, and 8.11% (6/74) RHDV1/RHDV2 double positive samples, respectively. The coincidence rates of detection RHDV1 and RHDV2 between RT-LAMP-CRISPR/Cas12a and quantitative real-time-polymerase chain reaction (qPCR) were both 97.30%. RT-LAMP-CRISPR/Cas12a showed higher sensitivity and detection rate compared with qPCR. Moreover, the results were visible to the naked eye within 1.5 h combined with lateral flow strips (LFSs) and visual fluorescence. The RT-LAMP-CRISPR/Cas12a portable platform has the advantages of high sensitivity, specificity, fast, low equipment requirements, which can be used in clinical practice in rural areas and resource-limited settings.
兔出血性疾病是由兔出血性疾病病毒（RHDV）引起的一种高度传染性的急性致命疾病。2020年RHDV2的首次爆发对我国养兔业构成了严重威胁。迫切需要一种针对RHDV GI.1（RHDV1）和GI.2（RHDV2）的有效且特异的检测策略。在本研究中，我们建立了一种基于逆转录环介导等温扩增（RT-LAMP）-CRISPR/Cas12a的双读数便携式检测平台。该平台在鉴定RHDV1和RHDV2毒株方面表现出优异的特异性，且与兔的其他常见病原体无交叉反应。RT-LAMP-CRISPR/Cas12a对RHDV1和RHDV2的检测限可达每个反应10拷贝/μl的VP60基因。此外，对74份临床样本进行了RHDV1和RHDV2的检测。基于RT-LAMP-CRISPR/Cas12a的双读数便携式检测平台分别显示出25.68%（19/74）的RHDV1阳性样本、43.24%（32/74）的RHDV2阳性样本和8.11%（6/74）的RHDV1/RHDV2双阳性样本。RT-LAMP-CRISPR/Cas12a与定量实时聚合酶链反应（qPCR）检测RHDV1和RHDV2的符合率均为97.30%。与qPCR相比，RT-LAMP-CRISPR/Cas12a表现出更高的灵敏度和检测率。此外，结合侧向流动条（LFS）和视觉荧光，结果在1.5小时内肉眼可见。RT-LAMP-CRISPR/Cas12a便携式平台具有灵敏度高、特异性强、快速、设备要求低等优点，可用于农村地区和资源有限环境的临床实践。
