Real-Time Reverse Transcription Multienzyme Isothermal Rapid Amplification for Rapid Detection of African Horse Sickness Virus

African horse sickness (AHS) is an acute infectious disease of equids caused by the AHS virus (AHSV), which can cause up to 90% mortality in naive horses. Reliable and rapid diagnosis is crucial for the surveillance and control of AHSV. As one of the AHSV detection methods recommended by World Organization for Animal Health (WOAH), the RT-qPCR assay has the drawbacks such as complex operation, expensive instruments, and long detecting time, which limit its application in simple laboratories or outdoors. In this study, a real-time reverse transcription multienzyme isothermal rapid amplification (RT-MIRA) assay was established to detect AHSV. Primers and exo-probes were designed, synthesized, and screened based on the conserved regions of the AHSV Seg-7 gene. A series of experiments were conducted to evaluate the performances of the established real-time RT-MIRA for detecting AHSV. The valid testing results showed that this method was highly specific for the detection of AHSV, without exhibiting any cross-reactivity towards other equine viruses or other Orbivirus; its limit of detection (LOD) was 10 copies/μL, which was consistent with that of RT-qPCR, meaning it had good sensitivity for detecting AHSV. Furthermore, the real-time RT-MIRA for AHSV performed good repeatability, and its standard curve exhibited good linearity with a correlation coefficient of R² = 0.9898, which indicated that the established method could be used for the quantitative detection of ASHV. As no AHS infection cases have been reported in China, 120 simulated clinical samples were tested by the real-time RT-MIRA and RT-qPCR for AHSV, which results showed there was a significant correlation between the two assays, with a κ value of 0.966 and an R² value of 0.9576. Parallel detection of 396 equine blood samples and 1760 Culicoides by this method and the RT-qPCR showed that all samples were negative for AHSV. Furthermore, the results of the real-time RT-MIRA could be judged by naked eyes under a portable equipment with blue light (480 nm). In conclusion, the real-time RT-MIRA for AHSV was specific and sensitive and had the advantages of convenient operation, visualization, no need for special equipment, and could be a reliable tool for rapid screening and detection of AHSV in field or border ports.