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Updated: Sep 27, 2025
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
Recombinase Polymerase Amplification Combined with Real-Time Fluorescent Probe for Mycoplasma pneumoniae Detection
Tingting Jiang1, Yacui Wang2, Weiwei Jiao1,2
1Baoding Key Laboratory for Precision Diagnosis and Treatment of Infectious Diseases in Children, Baoding Children's Hospital, Baoding 071051, China.
Abstract:
Mycoplasma pneumoniae (M. pneumoniae) is one of the major causes of community-acquired pneumonia, accounting for 20-40% of total cases. Rapid and accurate detection of M. pneumoniae is crucial for the diagnosis and rational selection of antibiotics. In this study, we set up a real-time recombinase polymerase amplification (RPA) assay to detect the conserved gene CARDS of M. pneumoniae. The amplification can be finished in 20 min at a wide temperature range from 37-41 °C. The limit of detection of RPA assay was 10 fg per microliter. Cross-reaction with commonly detected respiratory pathogens was not observed using RPA assay. Among clinical sputum samples, the detection rate of RPA assay and real-time PCR assay was 48.4% (92/190) and 46.3% (88/190), respectively (p = 0.68). Therefore, the RPA assay for M. pneumoniae detection is rapid and easy to use and may serve as a promising test for early diagnosis of M. pneumoniae infection.
Insights
A new real-time recombinase polymerase amplification (RPA) assay rapidly detects Mycoplasma pneumoniae (M. pneumoniae) in 20 minutes. This sensitive and specific test shows promise for early diagnosis of M. pneumoniae infections.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Respiratory Infections
Background:
- Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia.
- Accurate and rapid detection is essential for timely treatment and antibiotic selection.
Purpose of the Study:
- To develop and evaluate a real-time recombinase polymerase amplification (RPA) assay for Mycoplasma pneumoniae detection.
- To assess the assay's speed, sensitivity, specificity, and performance in clinical samples.
Main Methods:
- A real-time RPA assay targeting the conserved CARDS gene of M. pneumoniae was established.
- Assay performance was evaluated for amplification time, temperature range, limit of detection, and cross-reactivity.
- The assay was validated using clinical sputum samples and compared to real-time PCR.
Main Results:
- The RPA assay detected M. pneumoniae within 20 minutes across a wide temperature range (37-41 °C).
- The limit of detection was as low as 10 fg/µL, with no observed cross-reactivity with common respiratory pathogens.
- Detection rates in clinical samples were comparable to real-time PCR (48.4% vs. 46.3%, p=0.68).
Conclusions:
- The developed RPA assay is a rapid, sensitive, and specific method for detecting M. pneumoniae.
- Its ease of use and speed make it a promising tool for early diagnosis of M. pneumoniae infections.
- This assay can aid in the rational selection of antibiotics for pneumonia cases.
