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Updated: Sep 6, 2025
High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR
Published on: November 28, 2016
Development of a three-panel multiplex real-time PCR assay for simultaneous detection of nine canine respiratory
Junsheng Dong1, Wai Ning Tiffany Tsui2, Xue Leng3
1Kansas State Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, KS 66506, United States; Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu, China.
Abstract:
Infectious respiratory disease is one of the most common diseases in dogs worldwide. Several bacterial and viral pathogens can serve as causative agents of canine infectious respiratory disease (CIRD), including Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica, canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CHV-1), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine influenza virus (CIA) and canine respiratory coronavirus (CRCoV). Since these organisms cause similar clinical symptoms, disease diagnosis based on symptoms alone can be difficult. Therefore, a quick and accurate test is necessary to rapidly identify the presence and relative concentrations of causative CIRD agents. In this study, a multiplex real-time PCR panel assay was developed and composed of three subpanels for detection of the aforementioned pathogens. Correlation coefficients (R2) were >0.993 for all singleplex and multiplex real-time PCR assays with the exception of one that was 0.988; PCR amplification efficiencies (E) were between 92.1% and 107.8% for plasmid DNA, and 90.6-103.9% for RNA templates. In comparing singular and multiplex PCR assays, the three multiplex reactions generated similar R2 and E values to those by corresponding singular reactions, suggesting that multiplexing did not interfere with the detection sensitivities. The limit of detection (LOD) of the multiplex real-time PCR for DNA templates was 5, 2, 3, 1, 1, 1, 4, 24 and 10 copies per microliter for M. cynos, M. canis, B. brochiseptica, CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively; and 3, 2, 6, 17, 4 and 8 copies per microliter for CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively, when RNA templates were used for the four RNA viruses. No cross-detection was observed among the nine pathogens. For the 740 clinical samples tested, the newly designed PCR assay showed higher diagnostic sensitivity compared to an older panel assay; pathogen identities from selected samples positive by the new assay but undetected by the older assay were confirmed by Sanger sequencing. Our data showed that the new assay has higher diagnostic sensitivity while maintaining the assay's specificity, as compared to the older version of the panel assay.
Insights
A new multiplex real-time PCR assay accurately detects multiple canine infectious respiratory disease (CIRD) pathogens. This advanced diagnostic tool offers higher sensitivity and specificity than older methods for faster, more reliable canine disease identification.
Area of Science:
- Veterinary Diagnostics
- Molecular Biology
- Canine Infectious Diseases
Background:
- Canine infectious respiratory disease (CIRD) is a prevalent global issue in dogs.
- Multiple bacterial and viral agents cause CIRD, presenting similar clinical signs, complicating diagnosis.
- Accurate and rapid diagnostic tests are crucial for effective CIRD management.
Purpose of the Study:
- To develop and validate a multiplex real-time PCR panel assay for simultaneous detection of nine common CIRD pathogens.
- To evaluate the diagnostic sensitivity and specificity of the new assay compared to existing methods.
Main Methods:
- Development of a multiplex real-time PCR assay with three subpanels targeting nine CIRD pathogens (Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica, canine adenovirus type 2, canine herpesvirus 1, canine parainfluenza virus, canine distemper virus, canine influenza virus, and canine respiratory coronavirus).
- Assessed assay performance using correlation coefficients (R²), amplification efficiencies (E), and limit of detection (LOD) with plasmid and RNA templates.
- Validated the assay on 740 clinical samples, comparing results with an older panel assay and confirming positive samples via Sanger sequencing.
Main Results:
- The multiplex real-time PCR assay demonstrated high correlation coefficients (R² > 0.988) and amplification efficiencies (90.6–107.8%) comparable to singleplex assays.
- The assay achieved low limits of detection, ranging from 1 to 24 copies/µL for DNA and RNA targets, with no observed cross-reactivity.
- In clinical samples, the new assay exhibited significantly higher diagnostic sensitivity than the older panel assay while maintaining specificity.
Conclusions:
- The developed multiplex real-time PCR assay is a sensitive, specific, and efficient tool for diagnosing canine infectious respiratory disease.
- This assay facilitates rapid and accurate identification of multiple CIRD pathogens, improving diagnostic capabilities for canine respiratory illnesses.
- The enhanced diagnostic performance supports better clinical decision-making and disease control strategies in veterinary medicine.
