P-131 - Systematic identification of dye-additive-primer interactions influencing loop-mediated isothermal amplification for plant pathogen detection

Loop-mediated isothermal amplification (LAMP) is ideal for rapid in-field pathogen detection due to its speed and simplicity. However, false positives and inconsistent readouts, influenced by detection chemistry, reaction conditions, and primer efficiency, continue to provide challenges. Optimizing these parameters is critical for reliable LAMP diagnostics for biosecurity applications. This study experimentally evaluated interactions among three reaction additives, nine widely used detection dyes (pH-sensitive, colourimetric, and fluorescence-based dyes), and eight primer sets (two from literature, six newly designed), under various conditions, with and without loop primers. Phytophthora agathidicida and Phytophthora cinnamomi, primary pathogens causing kauri dieback, served as model pathogens. Certain dyes were found to initially show no or mainly false-positive results in standard LAMP reactions but became effective when paired with specific additives. By screening dye-additive combinations, reliable amplification signals could be produced even with less efficient primers or without loop primers. Our results provide valuable insights into the complex interactions between dyes, additives, and primer design, refining LAMP assay conditions and reducing false positives. The optimized assay supports the development of LAMP-based in-field diagnostics for plant pathogens, with direct applications in managing kauri dieback and strengthening biosecurity in New Zealand’s forests.