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Published on: January 17, 2014
A Nanoconfined, Hydrogel-Partitioned LAMP Platform for Rapid, Extraction-Free Quantification of Mycoplasma pneumoniae
Xiaoyang Jin1,2, Yuxuan Li3,4, Yajun Guo2
1The School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325027, China.
Abstract:
Accurate quantification of Mycoplasma pneumoniae (M. pneumoniae) is important because pathogen load is associated with disease severity and treatment outcomes. However, quantitative polymerase chain reaction (qPCR) requires DNA extraction and standard-curve calibration, whereas digital polymerase chain reaction (dPCR) remains costly and less accessible. Here, we developed gLAMP-X, an extraction-free hydrogel-limited amplification (gLAMP) platform for rapid quantification of M. pneumoniae from clinical throat swabs. The "X" component denotes extraction-free lysis that disrupts the lipid bilayer of M. pneumoniae while preserving nucleic acid integrity. In gLAMP-X, a polyethylene glycol (PEG)-based nanoporous hydrogel partitions loop-mediated isothermal amplification (LAMP) into isolated nanoscopic microreactors, converting homogeneous amplification into a digital-like system in which each event forms a discrete fluorescent cluster for end point counting. Under optimized conditions, gLAMP-X achieved a limit of detection of 1.7 copies/μL within 20 min. In 90 pediatric throat swabs, gLAMP-X showed 100% sensitivity, 98.1% specificity, and strong quantitative agreement with qPCR.
Insights
We developed gLAMP-X, a rapid, extraction-free method for quantifying Mycoplasma pneumoniae (M. pneumoniae). This innovative technique offers accurate pathogen detection from throat swabs in just 20 minutes.
Area of Science:
- Molecular Biology
- Diagnostic Technologies
- Microbiology
Background:
- Accurate quantification of Mycoplasma pneumoniae (M. pneumoniae) is crucial for assessing disease severity and treatment effectiveness.
- Current methods like quantitative polymerase chain reaction (qPCR) require DNA extraction and calibration, while digital polymerase chain reaction (dPCR) is expensive and less accessible.
Purpose of the Study:
- To develop a rapid, extraction-free, and accessible platform for accurate M. pneumoniae quantification.
- To introduce gLAMP-X, a novel hydrogel-limited amplification (gLAMP) system for direct detection from clinical samples.
Main Methods:
- Developed gLAMP-X, an extraction-free lysis and hydrogel-partitioned loop-mediated isothermal amplification (LAMP) platform.
- Utilized a polyethylene glycol (PEG)-based nanoporous hydrogel to create nanoscopic microreactors for digital-like counting.
- Optimized conditions for rapid amplification and endpoint fluorescent cluster detection.
Main Results:
- gLAMP-X achieved a limit of detection of 1.7 copies/μL within 20 minutes.
- Demonstrated 100% sensitivity and 98.1% specificity in analyzing 90 pediatric throat swabs.
- Showed strong quantitative agreement with established qPCR methods.
Conclusions:
- gLAMP-X provides a fast, sensitive, and specific method for M. pneumoniae quantification directly from clinical throat swabs.
- The extraction-free and accessible nature of gLAMP-X addresses limitations of current diagnostic techniques.
- This platform holds potential for improved point-of-care diagnostics and disease management.
