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Infectious Disease Reports
Volume 17
Issue 4
10.3390/idr17040093
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Detection of Microorganisms Causing Human Respiratory Infection Using One-Tube Multiplex PCR

by
Isabela L. Lima
1,
Adriana F. Neves
2,
Robson J. Oliveira-Júnior
1,
Lorrayne C. M. G. Honório
1,
Vitória O. Arruda
2,
Juliana A. São Julião
3,
Luiz Ricardo Goulart Filho
1 and
Vivian Alonso-Goulart
1,*
1
Instituto de Biotecnologia, Universidade Federal de Uberlândia, Uberlândia 38405-302, Minas Gerais, Brazil
2
Instituto de Biotecnologia, Universidade Federal de Catalão, Catalão 75704-020, Goiás, Brazil
3
Technical Department, BioGenetics Tecnologia Molecular Ltda, Uberlândia 38400-446, Minas Gerais, Brazil
*
Author to whom correspondence should be addressed.
Infect. Dis. Rep. 2025, 17(4), 93; https://doi.org/10.3390/idr17040093 (registering DOI)
Submission received: 23 May 2025 / Revised: 22 July 2025 / Accepted: 24 July 2025 / Published: 4 August 2025
Review Reports Versions Notes

Abstract

Background/Objectives: Due to the significant overlap in symptoms between COVID-19 and other respiratory infections, a multiplex PCR-based platform was developed to simultaneously detect 22 respiratory pathogens. Target sequences were retrieved from the GenBank database and aligned using Clustal Omega 2.1 to identify conserved regions prioritized for primer design. Primers were designed using Primer Express® 3.0.1 and evaluated in Primer Explorer to ensure specificity and minimize secondary structures. A multiplex strategy organized primers into three groups, each labeled with distinct fluorophores (FAM, VIC, or NED), allowing for detection by conventional PCR or capillary electrophoresis (CE). Methods: After reverse transcription for RNA targets, amplification was performed in a single-tube reaction. A total of 340 clinical samples—nasopharyngeal and saliva swabs—were collected from patients, during the COVID-19 pandemic period. The automated analysis of electropherograms enabled precise pathogen identification. Results: Of the samples analyzed, 57.1% tested negative for all pathogens. SARS-CoV-2 was the most frequently detected pathogen (29%), followed by enterovirus (6.5%). Positive results were detected in both nasopharyngeal and saliva swabs, with SARS-CoV-2 predominating in saliva samples. Conclusion: This single-tube multiplex PCR-CE assay represents a cost-effective and robust approach for comprehensive respiratory pathogen detection. It enables rapid and simultaneous diagnosis, facilitating targeted treatment strategies and improved patient outcomes.
Keywords: diagnosis; respiratory syndromes; RT-PCR; SARS-CoV-2 diagnosis; respiratory syndromes; RT-PCR; SARS-CoV-2

Share and Cite

MDPI and ACS Style

Lima, I.L.; Neves, A.F.; Oliveira-Júnior, R.J.; Honório, L.C.M.G.; Arruda, V.O.; São Julião, J.A.; Goulart Filho, L.R.; Alonso-Goulart, V. Detection of Microorganisms Causing Human Respiratory Infection Using One-Tube Multiplex PCR. Infect. Dis. Rep. 2025, 17, 93. https://doi.org/10.3390/idr17040093

AMA Style

Lima IL, Neves AF, Oliveira-Júnior RJ, Honório LCMG, Arruda VO, São Julião JA, Goulart Filho LR, Alonso-Goulart V. Detection of Microorganisms Causing Human Respiratory Infection Using One-Tube Multiplex PCR. Infectious Disease Reports. 2025; 17(4):93. https://doi.org/10.3390/idr17040093

Chicago/Turabian Style

Lima, Isabela L., Adriana F. Neves, Robson J. Oliveira-Júnior, Lorrayne C. M. G. Honório, Vitória O. Arruda, Juliana A. São Julião, Luiz Ricardo Goulart Filho, and Vivian Alonso-Goulart. 2025. "Detection of Microorganisms Causing Human Respiratory Infection Using One-Tube Multiplex PCR" Infectious Disease Reports 17, no. 4: 93. https://doi.org/10.3390/idr17040093

APA Style

Lima, I. L., Neves, A. F., Oliveira-Júnior, R. J., Honório, L. C. M. G., Arruda, V. O., São Julião, J. A., Goulart Filho, L. R., & Alonso-Goulart, V. (2025). Detection of Microorganisms Causing Human Respiratory Infection Using One-Tube Multiplex PCR. Infectious Disease Reports, 17(4), 93. https://doi.org/10.3390/idr17040093

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