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A duplex real-time fluorescent quantitative PCR method for simultaneous and rapid detection of Actinobacillus
Yu-Jie Ren1, Hua Lin2, Dao-Jian Yu3
1Science and Technology Research Center of China Customs, Beijing, China.
Abstract:
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 (R 2 > 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.
Insights
A new duplex real-time PCR assay simultaneously detects Actinobacillus pleuropneumoniae (APP) and influenza A (H1N1) virus in swine. This rapid, portable test aids in diagnosing complex respiratory co-infections for better disease management.
Area of Science:
- Veterinary Diagnostics
- Molecular Biology
- Animal Health
Background:
- Swine respiratory disease is often caused by co-infections, complicating diagnosis and control.
- Actinobacillus pleuropneumoniae (APP) and influenza A (H1N1) virus are common pathogens involved in swine respiratory disease.
- Accurate and rapid diagnostic tools are crucial for managing swine respiratory outbreaks.
Purpose of the Study:
- To develop and validate a novel duplex TaqMan real-time PCR assay for simultaneous detection of APP and H1N1.
- To optimize the assay for a portable automated detection system (Coyote Flash10).
- To provide a rapid, point-of-care diagnostic solution for swine respiratory co-infections.
Main Methods:
- Developed a duplex TaqMan real-time PCR assay targeting the apxIVA gene of APP and the M gene of H1N1.
- Utilized porcine RNase P as an internal control.
- Validated the assay using recombinant plasmid standards and simulated clinical matrices.
Main Results:
- Achieved high sensitivity (1 copy/μL) and linearity (R² > 0.99) for both targets.
- Demonstrated robust quantification and excellent repeatability (intra-assay CV <3%) and specificity.
- The assay provided results within one hour, with detection limits of 10,000-fold for APP and 100-fold for H1N1 in simulated samples.
Conclusions:
- The developed duplex real-time PCR assay is a rapid and specific tool for simultaneous detection of APP and H1N1 in swine.
- The portable nature of the assay makes it suitable for on-farm surveillance and differential diagnosis.
- Further validation with authentic clinical samples is recommended to confirm clinical utility.
