Establishment and application of a rapid and visual detection method for Mycoplasma pneumoniae using enzymatic
Yue Song1, Yanfeng Lin2, Yingjia Xu2
1School of Public Health, Nanjing Medical University, Nanjing 211100, China.
Abstract:
Mycoplasma pneumoniae (MP) is a key pathogen of respiratory tract infections, and the development of rapid, sensitive, and simple point-of-care testing (POCT) methods is of great clinical significance. In this study, enzymatic recombinase amplification (ERA) was combined with a colloidal gold-based lateral flow dipstick (LFD) to establish a novel MP detection method. Specific primers targeting the P1 protein gene were designed, with the downstream primer and probe labeled with biotin and 6-carboxyfluorescein (FAM) at their 5' ends, respectively, and the primer pair combinations, reaction temperature, and time were optimized to construct the ERA-LFD method. The detection performance for different MP strains was evaluated using reference samples and compared with a commercial fluorescent quantitative polymerase chain reaction (qPCR) kit. Subsequently, specificity, sensitivity, and clinical applicability were assessed using clinical specimens. The results showed that the target gene fragment of MP could be successfully amplified at 37 °C within 15 min, and the ERA amplification products could be visualized on the colloidal gold LFD by the naked eye. The limit of detection (LoD) of the assay was as low as 10 copies/μL, which was 1,000-fold more sensitive than conventional PCR, with no cross-reactivity to nucleic acids from other common respiratory pathogens. Evaluation of 158 clinical samples using qPCR as the gold standard demonstrated that the method achieved 98.0% sensitivity, 100.0% specificity, 98.7% overall agreement, and a Kappa value of 0.973. Collectively, this study developed a simple, rapid, and sensitive MP detection method that is suitable for both routine clinical applications and POCT scenarios.
Insights
A new enzymatic recombinase amplification (ERA) combined with lateral flow dipstick (LFD) offers rapid, sensitive detection of Mycoplasma pneumoniae (MP). This point-of-care test is highly accurate for respiratory infections.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Biotechnology
Background:
- Mycoplasma pneumoniae (MP) is a significant cause of respiratory tract infections.
- There is a clinical need for rapid, sensitive, and simple point-of-care testing (POCT) methods for MP detection.
Purpose of the Study:
- To develop and validate a novel MP detection method combining enzymatic recombinase amplification (ERA) with a colloidal gold-based lateral flow dipstick (LFD).
- To assess the sensitivity, specificity, and clinical applicability of the developed ERA-LFD assay for MP detection.
Main Methods:
- Designed specific primers targeting the MP P1 protein gene.
- Optimized ERA reaction conditions (temperature, time) and primer/probe labeling (biotin, FAM).
- Evaluated performance using reference samples and clinical specimens, comparing with commercial qPCR.
Main Results:
- MP target gene amplification achieved within 15 min at 37°C, visualized by naked eye on LFD.
- Limit of detection (LoD) of 10 copies/μL, 1,000-fold more sensitive than conventional PCR.
- Clinical evaluation showed 98.0% sensitivity, 100.0% specificity, and 98.7% overall agreement with qPCR in 158 samples.
Conclusions:
- Developed a simple, rapid, and highly sensitive MP detection method using ERA-LFD.
- The assay is suitable for routine clinical applications and point-of-care testing (POCT).
- This method provides a valuable tool for diagnosing MP infections effectively.
