ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Virology
Development and Validation of a Novel Triplex Droplet Digital PCR Assay for Simultaneous Detection of African Swine Fever Virus, Pseudorabies Virus, and Porcine Parvovirus
Provisionally accepted
Jiarong Yu1*
Lu Zhu2
Yuanyuan Zuo1
Shengbin Gao1Qinghua Wang1
Jiao Xu1Linlin Fang1Yumeng Liu1Shuang Liu1Xiaozhen Wang1Xiaohua Wang1Yingli Wang1Jingming Li1
Jingyue Bao1*Zhiliang Wang1*- 1China Animal Health and Epidemiology Center, Qingdao, China
- 2Qingdao Agricultural University, Qingdao, China
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Coinfection with multiple viruses significantly complicates swine disease management. DNA viruses including African swine fever virus (ASFV), pseudorabies virus (PRV), and porcine parvovirus (PPV) are causing reproductive failure in sows, with overlapping clinical presentations that challenge differential diagnosis. To address this, we developed a novel triplex droplet digital PCR (ddPCR) assay using three distinct fluorescent probe sets for simultaneous detection of these three viruses. The multiplex ddPCR demonstrated superior analytical performance with high specificity, sensitivity, and reproducibility. When compared to conventional quantitative PCR (qPCR), the ddPCR assay achieved limits of detection (LOD) of 0.08, 3.41, and 3.38 copies/μL for ASFV, PRV, and PPV respectively-representing approximately 10-fold lower LOD values than corresponding qPCR methods. Cross-reactivity tests confirmed absolute specificity against other important swine pathogens. Clinical validation using 217 field samples revealed marked diagnostic advantages: ddPCR detected positive cases in 137 samples (63.1%) versus 115 (53.0%) by qPCR. Species-specific positivity rates showed ddPCR improved detection by 5.99% (ASFV), 0.46% (PRV), and 3.69% (PPV) compared to qPCR. Concordance analysis demonstrated strong agreement between methods (94.01%-99.54%), with ddPCR showing superior sensitivity particularly for low-viral-load samples. This study establishes the first multiplex digital PCR platform for simultaneous differential diagnosis of ASFV, PRV, and PPV. The assay provides a robust tool for enhanced surveillance and control of these economically significant pathogens in swine populations.
Keywords: clinical diagnosis, multiplex droplet digital PCR, ASFV, prv, PPV
Received: 22 Sep 2025; Accepted: 03 Dec 2025.
Copyright: © 2025 Yu, Zhu, Zuo, Gao, Wang, Xu, Fang, Liu, Liu, Wang, Wang, Wang, Li, Bao and Wang. 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:
Jiarong Yu
Jingyue Bao
Zhiliang Wang
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