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Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae
Published on: February 24, 2023
Establishment and Application of a Multiple Cross Displacement Amplification Coupled With Nanoparticle-Based Lateral
Yacui Wang1, Yi Wang1, Shuting Quan1
1Key Laboratory of Major Diseases in Children, Ministry of Education, National Key Discipline of Pediatrics (Capital Medical University), National Clinical Research Center for Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, Beijing, China.
Abstract:
Mycoplasma pneumoniae (M. pneumoniae) is responsible for pneumonia, and is a causative agent of other respiratory tract infections (e.g., bronchiolitis and tracheobronchitis). Herein, we established and applied a multiple cross displacement amplification (MCDA) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (MCDA-LFB) for rapid, simple, and reliable detection of target pathogen. A set of 10 primers was designed based on M. pneumoniae-specific P1 gene, and optimal reaction conditions were found to be 30 min at 65°C. The detection results were visually reported using a biosensor within 2 min. The M. pneumoniae-MCDA-LFB method specifically detected only M. pneumoniae templates, and no cross-reactivity was generated from non-M. pneumoniae isolates. The analytical sensitivity for this assay was 50 fg of genomic templates in the pure cultures, as obtained from colorimetric indicator and real-time turbidimeter analysis. The assay was applied to 197 oropharyngeal swab samples collected from children highly suspected of M. pneumoniae infection, and compared to culture-based method and real-time PCR assay. The detection rates of M. pneumoniae using a culture-based method, real-time PCR assay, and MCDA-LFB assay were 8.1%, 33.0%, and 52.3%, respectively, which indicated that the MCDA-LFB assay was superior to the culture-based method and real-time PCR method for detection of target agent. Using this protocol, 25 min for rapid template extraction followed by MCDA reaction (30 min) combined with LFB detection (2 min) resulted in a total assay time of ~60 min. In conclusion, the MCDA-LFB assay established in this report was a simple, rapid, sensitive, and reliable assay to detect M. pneumoniae strains, and can be used as a potential diagnostic tool for M. pneumoniae in basic and clinical laboratories.
Insights
A new multiple cross displacement amplification (MCDA) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (MCDA-LFB) offers rapid and reliable detection of Mycoplasma pneumoniae (M. pneumoniae). This diagnostic tool shows superior sensitivity and specificity for M. pneumoniae detection in clinical samples.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Biosensor Technology
Background:
- Mycoplasma pneumoniae (M. pneumoniae) is a primary cause of bacterial pneumonia and other respiratory infections in children.
- Current diagnostic methods for M. pneumoniae, such as culture-based methods and real-time PCR, have limitations in terms of speed, sensitivity, or complexity.
- There is a need for rapid, sensitive, and reliable diagnostic tools for M. pneumoniae detection in clinical settings.
Purpose of the Study:
- To develop and validate a novel assay for the rapid, simple, and reliable detection of Mycoplasma pneumoniae.
- To evaluate the analytical sensitivity, specificity, and clinical performance of the developed assay.
- To compare the performance of the new assay with existing diagnostic methods for M. pneumoniae.
Main Methods:
- Development of a multiple cross displacement amplification (MCDA) assay using a set of 10 primers targeting the M. pneumoniae P1 gene.
- Coupling the MCDA assay with a nanoparticle-based lateral flow biosensor (LFB) for visual detection.
- Optimization of reaction conditions (30 min at 65°C) and assessment of analytical sensitivity (50 fg genomic templates).
- Validation using 197 oropharyngeal swab samples from children suspected of M. pneumoniae infection, compared to culture and real-time PCR.
Main Results:
- The developed MCDA-LFB assay demonstrated high specificity, with no cross-reactivity observed with non-M. pneumoniae isolates.
- Analytical sensitivity was determined to be 50 fg of genomic templates.
- In clinical samples, the MCDA-LFB assay detected M. pneumoniae in 52.3% of cases, significantly higher than culture (8.1%) and real-time PCR (33.0%).
- The total assay time, including sample preparation, was approximately 60 minutes.
Conclusions:
- The established MCDA-LFB assay is a simple, rapid, sensitive, and reliable method for detecting Mycoplasma pneumoniae strains.
- This assay shows superior performance compared to traditional culture-based and real-time PCR methods for M. pneumoniae detection.
- The MCDA-LFB assay holds potential as a valuable diagnostic tool for M. pneumoniae in both basic research and clinical laboratories.
