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.