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Updated: Sep 21, 2025
Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip
Published on: March 29, 2024
Development and evaluation of a multiplex quantitative polymerase chain reaction assay for detecting bacteria
Xianxia Zhuo1, Jiankang Zhao2, Lei Wang3
1Department of Pulmonary and Critical Care Medicine, Capital Medical University, Beijing, China; Laboratory of Clinical Microbiology and Infectious Diseases, Department of Pulmonary and Critical Care Medicine, China-Japan Friendship Hospital, Beijing, China; Department of Pulmonary and Critical Care Medicine, Centre for Respiratory Diseases, China-Japan Friendship Hospital, Beijing, China; Institute of Respiratory Medicine, Chinese Academy of Medical Science; National Clinical Research Center of Respiratory Diseases, Beijing, China.
Objectives:
This study aimed to establish a multiplex quantitative polymerase chain reaction (MQ-PCR) assay for 12 bacterial pathogens found in lower respiratory tract infection (LRTI) and to evaluate its performance in a cohort of 211 patients with LRTI.
Methods:
The study was divided into two stages: a pilot study to establish the methodology and a clinical validation study to evaluate its performance. In the pilot study, we established the MQ-PCR and analyzed its performance regarding limits of detection, reproducibility, specificity, and efficiency. In the clinical validation study, we obtained 211 sputum and/or bronchoalveolar lavage fluid (BALF) samples and detected pathogens by MQ-PCR. The MQ-PCR time was 3 h from sample collection to complete pathogen detection.
Results:
The limit of detection was 1000 copies/ml, and the maximum efficiency was >95%. When cutoffs of ≥105 copies/ml in sputum and ≥104 copies/ml in BALF were applied, the sensitivity, specificity, and positive and negative predictive values of the MQ-PCR were 77% (95% confidence interval [CI] 67-88%), 94% (95% CI 93-95%), 25% (95% CI 19-31%), and 99% (95% CI 99-100%), respectively.
Conclusions:
This study demonstrates that the new MQ-PCR assay is time-saving, more effective and sensitive, and brings us closer to mainstream adoption of quantitative molecular detection of bacteria.
