ABSTRACT

Rapid and accurate detection of Salmonella is essential for effective clinical management. Conventional culture methods are time-consuming, and existing molecular or colorimetric assays often suffer from complex instrumentation requirements or ambiguous visual interpretation, particularly when analyzing complex matrices such as stool. To address these challenges, we developed an instrument-free, dual-indicator colorimetric loopmediated isothermal amplification (LAMP) assay targeting the Salmonella invA gene. The system combines Phenol Red and Hydroxy Naphthol Blue (HNB), yielding a sharp and distinct color change. Key parameters (including Mg2+ and SYTO 9 dye concentrations, reaction temperature, and primer ratios) were systematically optimized. A rapid, purification-free nucleic acid release protocol using polyvinylpyrrolidone (PVP) was incorporated to reduce the effects of amplification inhibitors in stool samples. The resulting assay was thoroughly evaluated for specificity, sensitivity, precision, and reagent stability. The dual-indicator system generated a clear visual transition from light purple (negative) and dark grey (positive). It showed 100% specificity against 12 non- Salmonella strains and achieved a limit of detection of 3.0 × 10^3 CFU/mL in pure culture, translating to 3.0 × 10^4 CFU/mL (~1.1 × 10^3 CFU/g of stool) in spiked stool within 30 min. High precision (coefficient of variation ≤4.29%) was maintained, and reagents remained functional after 6 freeze-thaw cycles, 72 h at 10–30 ◦C, or 14 days of refrigeration. This instrument-free, dual-indicator LAMP system offers a rapid, visually unambiguous, and robust solution for direct Salmonella detection in stool, effectively overcoming previous limitations related to interpretation ambiguity and matrix interferences. It is therefore well-suited for point-of-care diagnostics, especially in resource-limited settings.