Development of Heat-Dry RT-LAMP Bioassay for Rapid Latent Detection of Botrytis cinerea

The global food security crisis is emphasized by the alarming amount of food waste, where about one-third of the world’s food production, roughly 1.3 billion metric tons, is lost annually. Pathogens, such as Botrytis cinerea, contribute significantly to this loss by attacking stored agricultural produce. These attacks typically start when pathogens infiltrate small fruit wounds, remain dormant, and then switch to an aggressive necrotrophic stage upon ripening, causing significant postharvest food losses. In response to this challenge, this study presents an innovative application of Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP). This method is increasingly recognized for its simplicity and effectiveness, distinguishing itself from more complex molecular diagnostic techniques. This study focuses on developing a heat-dry RT-LAMP desiccation method designed to be simple, robust, rapid, sensitive, and specific in detecting Botrytis cinerea. This method lies in its utilization of a desiccation process, where heat is utilized to preserve crucial components such as primers and enzymes in the presence of trehalose. A 5% trehalose with an amplification time of 1 h and 40 min was optimal for the assay detection of latent Botrytis cinerea. This method exhibited a sensitivity of 10 femtograms and was tailored specifically to the Botrytis cinerea PLF marker. Validation was performed using RNA extracted from an infected tomato, establishing a detection threshold of 1 ng/µL, approximately 500 pg of synthesized DNA target marker. This discovery holds significant implications, suggesting the potential for developing dry RT-LAMP kits that are adaptable for both laboratory and field usage. Furthermore, this method shows promise as a diagnostic tool for other neglected pathogenic diseases, representing a substantial advancement in agricultural pathology and supporting endeavors to enhance food security.