A triplex PCR method to detect gene insertion for the distinction of
African swine fever virus wild-type versus deletion strains
Abstract
To date, there is no effective vaccine or antiviral therapy available to
prevent or treat African swine fever virus (ASFV) infections. ASFV gene
deletion strains have been proposed as promising anti-ASFV vaccine
candidates. In recent years, most ASFV gene deletion strains worldwide
have been recombinant strains expressing EGFP or mCherry
as markers. However, a method for their wide range detection is still
lacking. Therefore, in this study, a new triplex real-time PCR (RT-PCR)
method was established for the broad and accurate differentiation of
ASFV wild-type vs. gene deletion strains. We designed three pairs
of primers and probes to target B646L, EGFP, and
mCherry, and RT-PCR was used to detect these three genes
simultaneously. The detection method prevented non-specific
amplification of porcine reproductive and respiratory syndrome virus,
porcine epidemic diarrhea virus, circovirus type 2, pseudorabies virus,
and classical swine fever virus genes. The minimum copy number of
standard plasmid DNA detected using triplex RT-PCR was 9.49, 30.20, and
9.60 copies for B646L, EGFP, and mCherry,
respectively. Importantly, of the 1646 samples analyzed in this study,
67 were positive for ASFV, all corresponding to the wild-type virus.
Overall, our data show that the triplex RT-PCR method established in
this study can specifically identify both ASFV wild-type and gene
deletion strains.