Development and validation of a novel triplex droplet digital PCR assay for simultaneous detection of African swine fever virus, pseudorabies virus, and porcine parvovirus
Jiarong Yu, Lu Zhu, Yuanyuan Zuo, Shengbin Gao, Qinghua Wang, Jiao Xu, Linlin Fang, Yumeng Liu, Shuang Liu, Xiaozhen Wang, Xiaohua Wang, Yingli Wang, Jingming Li, Jingyue Bao, Zhiliang Wang
TL;DR
A new digital PCR test can detect three swine viruses at once, offering better accuracy and sensitivity than traditional methods.
Contribution
The first triplex droplet digital PCR assay for simultaneous detection of African swine fever virus, pseudorabies virus, and porcine parvovirus.
Findings
The triplex ddPCR assay achieved lower detection limits than conventional qPCR for all three viruses.
Clinical validation showed ddPCR detected 10-20% more positive cases than qPCR in field samples.
The assay demonstrated strong specificity and high concordance with existing methods.
Abstract
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…
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Taxonomy
TopicsAnimal Disease Management and Epidemiology · Animal Virus Infections Studies · Viral Infections and Immunology Research