A Nanoconfined, Hydrogel-Partitioned LAMP Platform for Rapid, Extraction-Free Quantification of Mycoplasma pneumoniae.

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.