Hydrogel RT-LAMP Enables Rapid, Quantitative Assessment of Mycobacterium tuberculosis Immune Responses

Tuberculosis (TB) remains a global health challenge,
requiring
rapid and accessible diagnostics to guide preventive therapy. C-X-C motif chemokine ligand 10 (CXCL10) mRNA is a promising biomarker of TB infection, yet current assays
rely on long stimulation times and complex instrumentation. Here,
we present a hydrogel-based reverse transcription loop-mediated isothermal
amplification (HRT-LAMP) assay that partitions amplification reactions
within a porous hydrogel matrix, enabling digital-like quantification
of CXCL10 mRNA using only a heater and smartphone
imaging. A hand-held 3D-printed imaging device was developed to capture
fluorescence signals, which were subsequently quantified by a convolutional
neural network (CNN). The assay demonstrated a detection limit of
10 copies per reaction with excellent linearity (101–105 copies/test, R2 = 0.998), supporting
a dynamic range up to 105 copies/test via high-resolution
microscopy and 10 to 104 copies/test using the portable
smartphone platform. In 79 samples used to establish diagnostic thresholds,
HRT-LAMP reached 93.6% sensitivity and 100% specificity. In an independent
validation cohort of 91 samples, it achieved 91.2% agreement with
clinical diagnosis with sensitivity 92.3%, specificity 89.7%, κ
= 0.82, and area under the curve (AUC) = 0.918, outperforming interferon-γ
release assays (IGRA) used in clinics. Moreover, accurate results
were maintained with as little as 50 μL of blood (95.7% agreement).
HRT-LAMP thus enables rapid (<4 h), sensitive, and field-deployable
quantification of CXCL10 mRNA responses, offering
a practical platform for TB diagnosis in resource-limited and pediatric
settings.