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

Abstract

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 × 101 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.

Keywords: African swine fever virus (ASFV); CP204 gene; genotype I; genotype II; recombinase polymerase amplification (RPA).

Figure 1

Analytical performance of the real-time RPA assay for ASFV. The synthesized DNA standards in concentrations ranging from 5 × 10⁶ to 5 × 10⁰ copies were amplified as templates for the real-time RPA assay. A representative of three experiments is shown.

Figure 2

Comparison of the real-time RPA assay and qPCR for ASFV isolates and clinical specimens. The time to detection of the real-time RPA assay and qPCR were measured using11 ASFV isolates and clinical specimens collected from pigs challenged with ASFV (a) Volgograd-v (genotype II) or (b) Congo-v (genotype I). The time to a positive result for the tested specimens is expressed in seconds.

Figure 3

Results of the real-time RPA assay with clinical specimens from pigs challenged with ASFV genotypes I and II. Comparison between the performance of real-time RPA and qPCR on ASFV DNA samples (a). Ct values for real-time RPA and qPCR are shown in (b,c), respectively. Large white piglets were challenged with 1 × 10³ TCID₅₀ of ASFV Volgograd-v (genotype II) or Congo-v (genotype I), and the blood samples were collected at the designated time point after infection. DNA was extracted from the collected blood samples and used as a template for the real-time RPA assay.