Utilization of a New Hundred-Genomes Pipeline to Design a Rapid Duplex LAMP Detection Assay for Xanthomas euvesicatoria and X. vesicatoria in Tomato

Abstract

Xanthomonas euvesicatoria (Xe) and X. vesicatoria (Xv) are two economically important causal agents of bacterial spot (BS) of tomato and pepper. Management of BS in the field requires rapid and accurate detection. This work therefore aimed to develop a pipeline to design a simple, fast, and reliable assay for the detection of Xe and Xv by Loop-Mediated Isothermal Amplification (LAMP). A total of 109 publicly available whole genomic sequences of 24 different species of bacterial pathogens were used to design primers that would amplify the DNA of the two target species. Laboratory testing of the assay was performed on pure bacterial cultures and artificially infected plants, and amplification was conducted with both a sophisticated laboratory instrument and a simple mobile platform. The testing of the assay confirmed its specificity with a sensitivity reaching 1 pg μL−1 for both pathogens with an assay duration of 40 minutes on a mobile detection platform. Our diagnostics development pipeline enables the easy and fast design of a reliable detection assay in the genomics age. By validating the pipeline with Xe and Xv pathogens, we have simultaneously developed an assay with high specificity, sensitivity, and speed, which will allow it to be deployed, contributing to successful management of BS.