Comparative evaluation of real-time PCR and droplet digital PCR targeting the B646L (p72) and CP204L (p30) genes for early detection of African swine fever virus.

This study compared the analytical performance, diagnostic accuracy, and early detection capability of four nucleic acid amplification assays for African swine fever virus (ASFV), including two real-time PCR (qPCR) assays and two droplet digital PCR (ddPCR) assays targeting the B646L (p72) and CP204L (p30) genes. Analytical performance was evaluated using plasmid standards, whereas diagnostic performance was assessed using 133 samples comprising 73 specimens from experimentally infected pigs and 60 ASFV-negative controls. Both qPCR assays exhibited excellent linearity (R² > 0.99) and acceptable amplification efficiencies. Among the four assays, p72-ddPCR achieved the highest detection rate (84.9%), diagnostic sensitivity (92.31%), and area under the receiver operating characteristic curve (AUC = 0.940) using p72-qPCR as the reference assay. McNemar's test showed no significant difference between p30-qPCR and the reference assay (p > 0.05), whereas both ddPCR assays differed significantly (p < 0.05). Bland-Altman and Spearman analyses demonstrated systematic differences but very strong correlations between qPCR and ddPCR. Longitudinal blood sample analysis showed that p72-ddPCR detected the greatest number of ASFV-positive samples at most post-infection time points, whereas p30-qPCR identified more positive samples than p72-qPCR at 3 days post-infection. These findings indicate that ASFV detection performance is influenced by both the amplification platform and target gene. Overall, B646L-targeted ddPCR provided superior sensitivity for detecting low-level ASFV DNA, whereas CP204L-targeted qPCR showed promise for improving early detection of viremia following experimental infection. The complementary strengths of these assays may facilitate more sensitive laboratory diagnosis and earlier ASFV detection.