ORIGINAL RESEARCH article
Front. Vet. Sci.
Sec. Veterinary Infectious Diseases
Droplet digital PCR versus real-time PCR for quantitative analysis of the African Swine virus
Provisionally accepted
Silvia Dei Giudici*
Piero Bonelli
Maria Giovanna TiloccaSimona CauPier Paolo AngioiAnna Maria Sechi
Bruna Vodret
Annalisa Oggiano- Istituto Zooprofilattico Sperimentale della Sardegna G Pegreffi, Sassari, Italy
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The African swine fever (ASF) is a contagious viral disease of significant socio-economic and animal health impact with a high mortality rate and a cause of significant economic losses in many countries. ASF diagnosis is commonly diagnosed performed using molecular techniques. The World Organization of Animal Health (WOAH) has recommended two highly sensitive and specific quantitative real time PCR procedures (P1 qPCR and P2 qPCR) for the reliable and rapid detection of the ASF virus (ASFV). The present work aimed to standardizse two third-generation PCR technologies by using the same WOAH-recommended primer/probe sets. For this purpose, we developed two droplet digital PCR protocols ddPCRs and compared their analytical performances to the above-mentioned qPCRs. This validation involved testing serially diluted plasmid containing the vp72 gene sequence of the ASFV virus as the template. Clinical positive and negative samples were also analysed to validatecompare both PCR procedures. The ddPCR assays demonstrated excellent linearity (R²=0.999) across a dynamic range from 10⁴ to 1 copies/µLµL. The limits of detections (LOD) were 3.48 and 2.80 copies/µLµL and the limit of quantifications (LOQs) were ranging from 25 and 20 copies/µLµL for the P1 and the P2 procedures, respectively. The LOD values were comparable to those of qPCRs assays. The analysis of the clinical samples evidenced a strong agreement between the qPCR and the ddPCR, with bias values below 0.20, as determined by Bland-Altman analysis. The results of this study indicated that the ddPCR method can be adapted to existing validated qPCR protocols, enabling the quantification of low viral titer samples with similar analytical sensitivity. Moreover, the use of ddPCR allows for the absolute quantification without the need of a calibration curve, providing a reliable tool for ASFV diagnosis and outbreak management. It could also support global efforts to control the spread of ASFV. Further validation in diverse matrices (e.g., feed and environmental swabs) could expand its applicability in the One Health frameworks.
Keywords: digital PCR, African Swine Fever Virus, quantitative analysis, diagnosis, Real time PCR (qPCR)
Received: 12 Sep 2025; Accepted: 13 Nov 2025.
Copyright: © 2025 Dei Giudici, Bonelli, Tilocca, Cau, Angioi, Sechi, Vodret and Oggiano. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Silvia Dei Giudici, silvia.deigiudici@izs-sardegna.it
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