RT-RPA combined with CRISPR/Cas12a one-tube dual mode for detection of Akabane virus

Akabane virus (AKAV) is an arbovirus, transmitted through the bite of blood-sucking insects on susceptible ruminants, belonging to the order Bunyavirales , family Peribunyaviridae , genus Orthobunyavirus . The virus induces central nervous system abnormalities in developing fetuses, leading to arthrogryposis-hydrocephalus (A-H) syndrome, which poses a major threat to animal husbandry. Currently, there is a lack of effective treatment for Akabane disease. Therefore, rapid and accurate testing is essential for the prevention and control of Akabane disease. In this study, we selected a highly conserved S gene fragment for detection by sequence comparison and established the first single-tube assay for AKAV detection by combining RT-RPA with CRISPR/Cas12a. Meanwhile, the reaction conditions of the single-tube method were optimized, and the results were interpreted using a multifunctional enzyme marker and the lateral flow strip. When applied to 30 clinical samples, this CRISPR-Cas12a-based assay demonstrated a positivity rate of 20%, and the concordance of the results with those of RT-qPCR detection was 100%, indicating that the method has good detection ability and consistency.The method can complete the detection of AKAV within 50 min, with a limit of detection of 1 copy/μL and good specificity with no cross-reactivity observed with other related viruses. These characteristics enable the rapid and visualized detection of AKAV.