A Double Polymerase Chain Reaction Method for Detecting African Swine Fever and Swine Vesicular Disease Virus

Abstract

Purpose: To establish a double polymerase chain reaction (PCR) method for the simultaneous detection of African swine fever virus (ASFV) and swine vesicular disease virus (SVDV). Methods: By using reference sequences of ASFV and SVDV, this study synthesized parts of the genes connected to the 19-T vector which was inserted into competent DH5α cells to establish recombinant plasmids. Two specific primers of ASFV P72 proteins and SVDV genome were designed to amplify the two target genes. Two pairs of primers and two kinds of recombinant plasmids were added to one PCR reaction system to establish a double PCR assay for detection of the two diseases simultaneously. The double PCR conditions were optimized and the sensitivity and specificity of the assay determined. Results: The reaction was optimal with a final concentration of 0.36 μM for each primer, and a final annealing temperature of 55.5 oC. The lowest target gene copy number for detecting SVDV and ASFV was 7.6 × 102 and 1.5×105 copies/μL, respectively. The assay has a high level of specificity as only the recombinant plasmids of ASFV and SVDV were amplified and control plasmids for three other diseases - porcine circovirus (PCV), pseudorabies virus (PRV), and porcine parvovirus (PPV) - failed amplification. Conclusion: This study provides a rapid, sensitive and specific double PCR method for the simultaneous detection of ASFV and SVDV.