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Updated: Jun 29, 2026
High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR
Published on: November 28, 2016
Advancing multiplex diagnostics: A novel framework for simultaneous detection of diverse pathogens using respiratory
M Caputo1, S Ginart2, L Garrigos3
1Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Genética, Cátedra de Genética Forense, Centro de Referencia en Identificación Humana y Servicio de Huellas Digitales Genéticas, Junín 956, C1113AAD Buenos Aires, Argentina; CONICET - Consejo Nacional de Investigaciones Científicas y Técnicas, C1033AAJ Buenos Aires, Argentina..
This study introduces a rapid, cost-effective diagnostic method using qPCR and HRM analysis to simultaneously detect three common respiratory viruses: influenza A (H1N1), respiratory syncytial virus (RSV), and SARS-CoV-2. The assay accurately differentiates these pathogens in a single reaction, aiding differential diagnosis.
Area of Science:
- Molecular Diagnostics
- Virology
- Infectious Diseases
Background:
- Many infectious diseases present similar symptoms, complicating diagnosis and treatment.
- A single test capable of identifying multiple pathogens could significantly improve diagnostic resolution.
- Respiratory infections, in particular, are frequently caused by a variety of viral, bacterial, or fungal agents, necessitating rapid differentiation.
Purpose of the Study:
- To develop a rapid, specific, and cost-effective diagnostic method for the differential detection of common respiratory RNA viruses.
- To focus on a multiplex assay for the simultaneous identification of influenza A (H1N1), respiratory syncytial virus (RSV), and SARS-CoV-2.
- To establish a single-tube reaction system for improved diagnostic efficiency.
Main Methods:
- Development of a multiplex real-time quantitative PCR (qPCR) assay utilizing SYTO 9 fluorescent dye and pathogen-specific primers.
- Coupling qPCR with high-resolution melt (HRM) analysis for post-amplification discrimination of amplified products.
- Testing of samples with known concentrations (10^1-10^3 copies/μL) and preliminary validation with clinical samples against a commercial kit.
Main Results:
- The multiplex qPCR-HRM assay successfully generated distinct melt curves and peaks for SARS-CoV-2, influenza A (H1N1), and RSV.
- Mean melting temperatures (Tm) were distinct for each virus: 83.64 ± 0.08 °C (SARS-CoV-2), 80.45 ± 0.07 °C (influenza H1N1), and 75.99 ± 0.07 °C (RSV).
- The system demonstrated 100% accuracy in differentiating the three viruses and showed strong agreement with a commercial kit in clinical sample validation (κ = 0.87 for SARS-CoV-2, κ = 1.00 for RSV).
Conclusions:
- The developed multiplex qPCR-HRM method offers a rapid, specific, and cost-effective solution for the differential diagnosis of common respiratory RNA viruses.
- This single-tube assay provides a valuable alternative for clinical laboratories seeking efficient pathogen identification.
- The method's high accuracy and agreement with existing commercial tests support its potential for widespread adoption in respiratory diagnostics.
