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Peer-reviewedCC BY 4.0PLOS One

Development of a multiplex real-time PCR assay with fluorescence probe-melting-curve analysis for one-tube detection of respiratory pathogens

  • Mingyu Ji1
  • Hongjiao Dong2
  • Yihui Xu3
  • Yunshan Wang4
  • Qianqian Zhao
4 affiliations
  1. 1Medical Integration and Practice Center, Shandong University, Jinan, Shandong, China
  2. 2Medical Research and Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
  3. 3Research and Development Department, GuoRan Gene Technology (Shandong) Company Limited, Jinan, Shandong, China
  4. 4Shandong LaiBo Biotechnology Company Limited, Jinan, Shandong, China
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Abstract

Pathogens causing respiratory infections have reemerged globally since the COVID-19 pandemic. Rapid and accurate identification of respiratory pathogens is critical for diagnosis and treatment. In this study, we developed and evaluated a multiplex real-time PCR assay with a fluorescence probe melting curve (mqPCR-PMC) assay to simultaneously detect six common pathogens, namely, influenza virus A and B (IFV-A and -B), respiratory syncytial virus (RSV), human rhinovirus (HRV), human adenovirus (HAdV) and Mycoplasma pneumoniae (MP). The primers and probes optimized in this assay exhibited a specificity of 100% with a panel of 57 pathogens. The detection limits of the assay ranged from 248 copies/ml to 394 copies/ml. Moreover, the coefficients of variation ranged from 0.11% to 0.30% for Tm and from 1.59% to 4.10% for Rm. The clinical accuracy of the assay was validated using 760 pharyngeal samples. Compared with a commercial kit based on conventional fluorescence RT-PCR, the assay showed more than 87% sensitivity and 99% specificity for each pathogen. The kappa values ranged from 0.927 to 1.000. Moreover, the assay was more precise for HRV and more sensitive on IFV-A, HAdV, MP and RSV. In summary, the mqPCR-PMC assay was demonstrated to be highly sensitive, highly specific and high throughput, and has potential for clinical use in respiratory infectious disease diagnosis.

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Cite

BibTeXRIS
BibTeX
@article{ji2026development,
  title = {{Development of a multiplex real-time PCR assay with fluorescence probe-melting-curve analysis for one-tube detection of respiratory pathogens}},
  author = {Mingyu Ji and Hongjiao Dong and Yihui Xu and Yunshan Wang and Qianqian Zhao},
  journal = {PLOS One},
  year = {2026},
  month = sep,
  doi = {10.1371/journal.pone.0357377},
  url = {https://doi.org/10.1371/journal.pone.0357377},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Development of a multiplex real-time PCR assay with fluorescence probe-melting-curve analysis for one-tube detection of respiratory pathogens
AU  - Mingyu Ji
AU  - Hongjiao Dong
AU  - Yihui Xu
AU  - Yunshan Wang
AU  - Qianqian Zhao
PY  - 2026
DA  - 2026/09/09
JO  - PLOS One
DO  - 10.1371/journal.pone.0357377
UR  - https://doi.org/10.1371/journal.pone.0357377
AB  - Pathogens causing respiratory infections have reemerged globally since the COVID-19 pandemic. Rapid and accurate identification of respiratory pathogens is critical for diagnosis and treatment. In this study, we developed and evaluated a multiplex real-time PCR assay with a fluorescence probe melting curve (mqPCR-PMC) assay to simultaneously detect six common pathogens, namely, influenza virus A and B (IFV-A and -B), respiratory syncytial virus (RSV), human rhinovirus (HRV), human adenovirus (HAdV) and Mycoplasma pneumoniae (MP). The primers and probes optimized in this assay exhibited a specificity of 100% with a panel of 57 pathogens. The detection limits of the assay ranged from 248 copies/ml to 394 copies/ml. Moreover, the coefficients of variation ranged from 0.11% to 0.30% for Tm and from 1.59% to 4.10% for Rm. The clinical accuracy of the assay was validated using 760 pharyngeal samples. Compared with a commercial kit based on conventional fluorescence RT-PCR, the assay showed more than 87% sensitivity and 99% specificity for each pathogen. The kappa values ranged from 0.927 to 1.000. Moreover, the assay was more precise for HRV and more sensitive on IFV-A, HAdV, MP and RSV. In summary, the mqPCR-PMC assay was demonstrated to be highly sensitive, highly specific and high throughput, and has potential for clinical use in respiratory infectious disease diagnosis.
SN  - 1932-6203
ER  -