TECHNOLOGY AND CODE article
Front. Vet. Sci.
Sec. Veterinary Clinical, Anatomical, and Comparative Pathology
This article is part of the Research TopicCutting-edge diagnostics in livestock disease managementView all 4 articles
A Duplex Real-time Fluorescent Quantitative PCR Method for Simultaneous and Rapid Detection of Actinobacillus pleuropneumoniae and Influenza A Virus
Provisionally accepted
Yu-Jie Ren1
Hua Lin2Dao-Jian Yu3
Jing Zhang4Fei Yang1Bai-Yi Zhang5Ying-Qi Chen4Hai-Xin Long6Li-Li Ren1*- 1Science and Technology Research Center of China Customs, Beijing, China
- 2Chengdu Customs Technical Center, Chengdu, Chengdu, China
- 3Animal and Plant Inspection and Quarantine Technology Center Shenzhen Customs District, Shenzhen, China
- 4Chengdu Customs Technical Center, Chengdu, China
- 5Science and Technology Research Center of China, Beijing, China
- 6Beijing Coyote Biotech Co., Ltd., Beijing, China
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Co-infections involving Actinobacillus pleuropneumoniae (APP) and Influenza A (H1N1) virus present a serious diagnostic challenge in swine respiratory disease, complicating effective outbreak management and control. This study reports the development and validation of a novel duplex TaqMan real-time PCR assay, optimized for the Coyote Flash10 portable automated detection system, for the simultaneous identification and quantification of both pathogens. The assay employs primers and probes specific to the apxIVA virulence gene of APP and the conserved matrix (M) gene of H1N1, with porcine RNase P as an internal control. Validation using recombinant plasmid standards demonstrated a sensitivity of 1 copy/µL for each target, with strong linear correlation (R² > 0.99). Calculated amplification efficiencies (APP: 121.6%; H1N1: 118.6%) were marginally above the typical 90–110% range. However, the assay exhibited robust and consistent quantification without evidence of reaction competition. In a simulated clinical matrix, detection limits corresponded to 10,000-fold and 100-fold dilutions for APP and H1N1, respectively. The method showed excellent repeatability (intra-assay CV < 3%) and high specificity, with no cross-reactivity against six other common porcine respiratory pathogens. This rapid, closed-tube assay provides a complete result within one hour and offers a practical, point-of-care-compatible solution for on-farm surveillance and the differential diagnosis of complex respiratory co-infections in swine populations. A key limitation of this study is that validation was performed primarily using simulated samples; future validation with authentic clinical samples will further confirm the method's clinical utility.
Keywords: Actinobacillus pleuropneumoniae, duplex real-time fluorescent quantitative PCR, Influenza A (H1N1) virus, Point-of-care testing, Swine respiratory disease
Received: 24 Dec 2025; Accepted: 11 Feb 2026.
Copyright: © 2026 Ren, Lin, Yu, Zhang, Yang, Zhang, Chen, Long and Ren. 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: Li-Li Ren
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