Objective Establishing a highly sensitive real-time fluorescence
quantitative PCR (qPCR) method for universal testing of epidemic African swine
fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design
primer probes covering 24 p72 genotypes. The optimal amount of
dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various
sensitivity and limit of detection (LOD) tests were performed, and clinical
samples from China and imported goods were tested. Results The optimal
primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal
for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p =
0.369). The method was employed to test 142 clinically suspected samples, of
which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the
positive testing rate for ASFV was higher than for the standard qPCR method
recommended by the Office International Des Epizooties (OIE), and for the
commercially available kit. Thus, our method is superior for testing weakly
positive samples with low virus titre, and epidemic strains present in imported
goods. Conclusion Our method could be employed for universal testing of
epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV
strains/endemic strains, reducing falsenegatives, and benefitting early diagnosis.
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