ThermiQuant(TM) MegaScan: High-throughput isothermal reactor with quantitative colorimetric readout for paper-based nucleic acid amplification tests

Isothermal nucleic acid amplification tests (NAATs), such as loop-mediated isothermal amplification (LAMP) implemented on microfluidic paper-based analytical devices ({micro}PADs), enable inexpensive and rapid ([≤]60 min) colorimetric molecular diagnostics; however, no existing instrument supports high-throughput (>100 reactions) quantitative analysis of colorimetric isothermal assays on paper substrates under controlled laboratory conditions. To address this gap, we developed ThermiQuant MegaScan, a scanner- and water-bath-based platform that accommodates a 160-reaction {micro}PAD cartridge, maintains uniform incubation at 65 {+/-} 0.5 {degrees}C, and enables real-time imaging every 30 s. We also developed accompanying software, Amplimetrics, for automated {micro}PAD detection and kinetic colorimetric analysis. Using paper-based colorimetric LAMP targeting the SARS-CoV-2 orf7ab region, the assay achieved a limit of detection (LoD) of 50 copies per reaction (6.7 copies/{micro}L) and a limit of quantification (LoQ) of 1,000 copies per reaction (133 copies/{micro}L) using purified synthetic DNA targets, and achieved 72% sensitivity and 100% specificity relative to digital PCR (dPCR) for diluted human nasopharyngeal (NP) swab virus samples. We further evaluated the effects of viral and universal transport media (VTM/UTM) on assay performance and found that linear calibration derived from synthetic targets do not reliably translate to clinical samples in these media. Together, these results establish ThermiQuant MegaScan as a laboratory-grade, high-throughput reference platform for standardized evaluation, optimization, and benchmarking of paper-based colorimetric nucleic acid amplification assays.