Development of a triplex real-time PCR assay for detection and differentiation of gene-deleted and wild-type African swine fever virus.

Abstract African swine fever (ASF) is an infectious disease of domestic and wild pigs, caused by ASF virus (ASFV). In this study, a triplex real-time PCR assay was developed to detect and differentiate the gene-deleted and wild-type ASFV strains. Three pairs of primers and probes were designed to target the conserved region of B646 L gene (p72), MGF_360-14 L gene (located in the middle of MGF360-505R gene) and CD2v gene, respectively. Gene-deleted (with MGF360-505R and / or CD2v genes deletion) and wild-type ASFV strains were detected specifically and simultaneously by the assay developed without cross-reactions with other nucleic acids of PCV-2, CSFV, PRRSV, FMDV or SVA. The detection limits of the triplex rPCR were 7.9 copies, 9.7 copies, and 9.6 copies of standard plasmid DNA containing B646 L gene, MGF_360-14 L gene and CD2v gene, respectively. A total of 1215 field samples were tested in parallel by the triplex rPCR and real-time PCR recommended by OIE, and the B646 L gene detection results were completely consistent between these two assays. The triplex rPCR assay was successfully developed to identify pigs infected with wild-type ASFV strains or immunized with the ASFV gene-deleted vaccine.