Development of a Real-Time Recombinase Polymerase Amplification Assay for the Rapid Detection of African Swine Fever Virus Genotype I and II

African swine fever (ASF) is a contagious viral disease in pigs and wild boars which poses a major threat to the pig industry. Rapid and accurate diagnosis is necessary to control ASF. Hence, we developed a rapid diagnostic method using a recombinase polymerase amplification (RPA) assay targeting the conserved sequences of CP204L (p30) thatcan rapidly detect ASF virus (ASFV) genotype strains I and II. The lower detection limit of the real-time RPA assay was 5 x 10(1) copies per reaction. The real-time RPA assay effectively detected ASFV isolates and clinical specimens belonging to ASFV genotypes I and II. The sensitivity and specificity of the assay were 96.8% (95% confidence interval (CI): 83.3-99.9) and 100% (95% CI: 88.4-100.0), respectively. The agreement between the real-time RPA assay and a reference commercial real-time quantitative polymerase chain reaction (qPCR) was 100%. The real-time RPA assay had a detection time of 6.0 min (95% CI: 5.7-6.2), which was significantly shorter than that of qPCR (49 min; 95% CI: 47.4-50.6; p < 0.001). Thus, the developed real-time RPA assay is a rapid and accurate diagnostic tool for detecting ASFV genotypes I and II.