A lyophilization-compatible colorimetric LAMP assay for cold chain-free, extraction-free and rapid post-enrichment...

Developing reliable dry-format colorimetric LAMP assays remains challenging because most existing systems depend on pH-sensitive dyes e.g. Hydroxy Naphthol Blue and Phenol Red. These dyes lose performance after lyophilization or when exposed to extraction-free sample buffers, as they require low-buffer conditions to generate accurate color transitions. Lyophilization disrupts pH stability, and common lysis additives further interfere with signal clarity. Consequently, creating a robust, field-ready colorimetric LAMP platform has been constrained. Here, we developed a Mn-PAPS Colorimetric LAMP assay, a metal ion-based colorimetric system that is inherently pH-independent and fully compatible with freeze-drying. Using Escherichia coli as a model organism, this study demonstrates the feasibility, and diagnostic performance of the lyophilized colorimetric LAMP platform. The Mn-PAPS Colorimetric LAMP platform demonstrated sensitivity in a single-digit CFU range and maintained clear visual contrast with a high analytical accuracy across 69 spiked and unspiked reference samples. Incorporation of daughter primers reduced incubation time from 120 to 45 minutes. Two lyophilized formats demonstrated room-temperature stability, supporting field deployment. • Resolves the long-standing limitation of pH-sensitive colorimetric LAMP through a metal-ion dye system. • Demonstrates single-digit CFU sensitivity and high analytical accuracy using a micropipette-free workflow. • Provides stable performance under ambient conditions through lyophilization-compatible reagent formats.