Point-of-Care Radiation Biodosimetry via a Body-Heated Reverse Transcription-Recombinase Polymerase Amplification Coupled to a Paper-Based Vertical Flow Immunoassay

Abstract

Rapid biodosimetry at the point of care is critical for efficient triage and clinical management during mass-casualty radiological events. Here, we introduce a power-free biodosimetry platform integrating one-step reverse transcription recombinase polymerase amplification (RT-RPA) with vertical flow immunoassay (VFI) for the multiplex detection of two radiation-responsive transcripts (CDKN1A and DDB2) and housekeeping gene (MRPS5) from fingerstick blood. One-step RT-RPA showed optimal efficiency at 36 °C for 20 min, comparable to two-step RT-RPA and standard PCR. This temperature could be provided by the human body, where the axilla delivered a stable heat source (36.2 °C) with minimal interindividual variability across a range of environmental conditions (from 4 to 42 °C), supporting amplification comparable to thermocycler-based reactions. Amplified products were detected using a capillary-driven VFI platform. The assay demonstrated strong specificity, uniform signal distribution, and reproducibility with intra- and interassay coefficient of variation below 10% and 15%, respectively, across all three transcripts. When applied to ex vivo X-ray irradiated blood samples (1 and 2 Gy), a dose-dependent increase in CDKN1A and DDB2 signals was observed. Receiver operating characteristic analysis demonstrated that discrimination between sham- and 1 Gy-irradiated was not significant, whereas the assay distinguished sham- from 2 Gy-irradiated with area under the curve values of 0.82 (95% CI = [0.66–0.98]; p = 0.0052) and 0.89 (95% CI = [0.76–1.00]; p = 0.0006) for CDKN1A and DDB2, yielding sensitivity/specificity of 84.62%/69.23% and 92.31%/84.62%, respectively. These findings supported the feasibility of the body-heat one-step RT-RPA VFI as a rapid, equipment-free, field-deployable triage tool for large-scale radiation biodosimetry.