Objective An urgently needed rapid and simple method for diagnosing the endemic rabbit hemorrhagic disease caused by RHdV-2 identified in China in 2020 was established.

Method Vector NTI Alignment was used to analyze the genomic sequences of the two serotypes of RHdV, Type 1 and Type 2. The specific and conserved fragment VP60 of RHdV-2 was obtained to design probes and specific primers for the recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay. Based on the RAA amplification products, a CRISPR-derived RNA was selected with the RAA-CRISPR/Cas13a(Cpf1)-LFD reaction conditions optimized and assay sensitivity and specificity evaluated. Subsequently, in comparison with the RAA previously established in our laboratory, the newly developed assay was used to detect clinical specimens.

Result A specific and conserved sequence in VP60 of RHdV-2 was found that led to synthesis of the primers for RAA amplification. Using the amplified product as the reaction template, positive sample could be best detected with crRNA1. By adding 10 min to the RAA reaction time, a clear positive result on the test strip was obtained. The sensitivity test using the recombinant plasmid as the template showed the lowest number of detectable plasmid copies to be 6.7×10¹ copies·μL⁻¹. The new assay was highly specific as it detected only RHdV-2 as positive. And it tested 10 positives on 31 clinical specimens with a detection rate of 30.3%.

Conclusion The RAA-CRISPR/Cas13a(Cpf1)-LFD method was rapid and simple to operate and highly sensitive and specific in detecting RHdV-2. It significantly improved the diagnostic method of the hemorrhagic disease in rabbits.