Integration of paper-based DNA extraction with digitized image analysis for colorimetric LAMP-based V....

Vibrio parahaemolyticus (V. parahaemolyticus) contamination in seafood is a major contributor to foodborne illness and poses significant public health risks, particularly in regions where seafood is consumed raw or minimally cooked. Although colorimetric loop-mediated isothermal amplification (LAMP) has emerged as a promising diagnostic approach, its practicality is often limited by complex DNA extraction procedures and the lack of a standardized digital analysis method, as results are typically interpreted solely by eye based on color change, which prevents objective and quantitative comparison. In this study, we developed a paper-based DNA extraction method combined with a digital image analysis-assisted colorimetric LAMP assay to enable rapid and digitized detection of V. parahaemolyticus. The paper-based extraction required no bulky equipment and was completed within 20 min, followed by successful DNA amplification by colorimetric LAMP in 25 min. The assay demonstrated high specificity, effectively distinguishing V. parahaemolyticus from other bacteria. Colorimetric results were captured using a smartphone and processed in ImageJ, where multiple color formulas were compared. Green-channel normalization provided the highest contrast between positive and negative samples (ΔI = 0.0602), and threshold-coupled ratio analysis further enhanced contrast to 0.288. The effect of different ROI shapes (Tube-shaped ROI, Rectangular ROI, and Paper-shaped ROI) was compared yielding ΔI values of 0.018, 0.013, and 0.029, respectively. The sensitivity for V. parahaemolyticus detection was as low as 10² CFU mL^-1. The optimized workflow reduces subjective bias, provides a portable, low-cost, and field-ready diagnostic strategy for seafood safety monitoring.