Related Experiment Video
Updated: May 9, 2025
Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip
Published on: March 29, 2024
Development and performance evaluation of a multiplex fluorescent PCR method for the detection of fastidious
Yijun Zhu1, Jingchao Shi1, Shihang Zhang2
1Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine (Jinhua Municipal Central Hospital), Jinhua, Zhejiang, China.
Introduction:
This study was performed to develop a multiplex fluorescent PCR method for the concurrent detection of six fastidious bacteria associated with respiratory tract infections. These bacteria include Streptococcus pneumoniae（SPN）, Bordetella pertussis (BP), Neisseria meningitidis (NM), Legionella pneumophila (LP), Moraxella catarrhalis (MC), and Haemophilus influenzae (HI).
Methodology:
A multiplex fluorescent PCR test using SYBR Green as a DNA dye was developed and optimised. Clinical samples from 296 children with respiratory tract infections were then tested using the proposed method to assess its applicability for detecting the six pathogens.
Results:
The SYBR Green-based multiplex fluorescent PCR method was successfully employed for the simultaneous identification of five pathogens through the analysis of melting curves and determination of the melting temperatures (Tm) values. However, the method exhibited limitations in distinguishing HI, necessitating separate detection using singleplex fluorescent PCR for this pathogen. In the methodological evaluation involving 296 clinical specimens, the multiplex fluorescent PCR successfully detected SPN, MC, NM, BP, and LP with sensitivities of 97.3%, 96.3%, 85.7%, 95.7%, and 80%, and areas under the ROC curve (AUC) of 0.977, 0.978, 0.927, 0.976 and 0.900, respectively.
Conclusions:
Compared to conventional bacterial culture methods, the multiplex fluorescent PCR method with SYBR Green as the DNA dye is a sensitive and cost-effective approach for the simultaneous and rapid identification of five fastidious bacteria, making it a valuable alternative for clinical diagnostic laboratories.
Insights
A new multiplex fluorescent PCR method accurately detects five key bacteria causing respiratory infections. This SYBR Green-based approach offers a sensitive, cost-effective alternative to traditional culture methods for rapid diagnosis.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Respiratory tract infections (RTIs) are often caused by fastidious bacteria.
- Accurate and rapid detection of these pathogens is crucial for effective treatment.
- Current diagnostic methods can be time-consuming and lack sensitivity.
Purpose of the Study:
- To develop a multiplex fluorescent PCR assay for simultaneous detection of six common RTI pathogens.
- To evaluate the performance of this assay using clinical samples.
Main Methods:
- Development and optimization of a multiplex fluorescent PCR assay utilizing SYBR Green dye.
- Testing of 296 clinical samples from children with RTIs.
- Analysis of melting curves and melting temperatures (Tm) for pathogen identification.
Main Results:
- The multiplex assay successfully identified five pathogens: Streptococcus pneumoniae (SPN), Moraxella catarrhalis (MC), Neisseria meningitidis (NM), Bordetella pertussis (BP), and Legionella pneumophila (LP).
- High sensitivities were observed for SPN (97.3%), MC (96.3%), NM (85.7%), BP (95.7%), and LP (80%).
- Haemophilus influenzae (HI) required separate singleplex PCR detection due to limitations in multiplex differentiation.
Conclusions:
- The SYBR Green-based multiplex fluorescent PCR is a sensitive and cost-effective method for rapid, simultaneous detection of five key RTI bacteria.
- This assay presents a valuable alternative to conventional bacterial culture for clinical diagnostic laboratories.
- Further optimization may be needed to include HI in the multiplex panel.
