Specific Detection of RHDV GI.1 and GI.2 by RT-LAMP-CRISPR/Cas12a Platform.

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.