Application of portable real-time recombinase-aided amplification (rt-RAA) assay in the clinical diagnosis of ASFV and prospective DIVA diagnosis

African swine fever, a serious infectious disease, has been found in many countries around the world over the last nearly 100 years, and causes untold damage to the economy wherever it occurs. Diagnosis is currently performed by real-time PCR, which is highly sensitive but can only be carried out in a diagnostic laboratory environment with sophisticated equipment and expertise. A sensitive, rapid diagnostic method that can be implemented in agricultural settings is thus urgently needed for the detection and control of African swine fever virus (ASFV) infection. In this study, we developed an isothermal amplification technology to achieve molecular diagnosis of ASFV in clinical samples, using recombinase-aided amplification (RAA) assay combined with a portable instrument. This assay method avoids the limitations of traditional real-time PCR and offers detection times within 20 min, enabling detection of as few as 10 copies of ASFV DNA molecules per reaction without cross-reaction with other common swine viruses. We evaluated clinical performance using 200 clinical blood samples. The coincidence rate of the detection results between rt-RAA and RT-qPCR was 96.94% positive, 100% negative, and 97.50% total. We have also developed an rt-RAA system for the detection of ASFV targeting the EP402R gene, with detection of as few as 10 copies of DNA per reaction; this offers the possibility of DIVA (differentiating infected from vaccinated animals) diagnosis, because CD2V gene-deleted ASFV could soon be approved to be the leading candidate for live attenuated vaccine in China. The rt-RAA assay is a reliable, rapid, highly sensitive method, and it offers a reasonable alternative to RT-qPCR for point-of-care detection of ASFV. Key points • The RT-RAA assay can detect as few as 10 copies of ASFV genome per reaction within 20 min. • The rt-RAA assay system targeting different genes can achieve differentiating infected from vaccinated diagnosis.