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Genome-informed LAMP assays for specific detection of bacterial spot-causing bacteria, Xanthomonas euvesicatoria and X. vesicatoria

Mohammed Arif: University of Hawaii at Manoa

<div>Bacterial leaf spot (BLS) is an economically important bacterial disease of tomato and pepper caused by four species, <em>Xanthomonas euvesicatoria </em>(<em>Xe</em>), <em>X. vesicatoria</em> (<em>Xv</em>)<em>,</em> <em>X. gardneri </em>(<em>Xg</em>) and<em> X. perforans </em>(<em>Xp</em>)<em>.</em> Symptoms produced by all four species are nearly indistinguishable. Currently, no point-of-care diagnostics exist for BLS. In this research, we developed two LAMP assays to rapidly and accurately identify and differentiate <em>Xe</em> and <em>Xv</em> using a field-deployable portable BioRanger instrument. Genomes of <em>Xe, Xv, Xg, Xp</em> and other species of <em>Xanthomonas</em> were retrieved from NCBI GenBank genome database and were aligned and curated using Mauve and Geneious, respectively. Unique genes for <em>Xe</em> and <em>Xv</em> were selected for design of LAMP primers. Assays were tested for specificity against 74 strains of <em>Xanthomonas</em> species and other closely related species. No false negatives or false positives were detected. Detection limits of both assays were determined using 10-fold serially diluted purified genomic DNA of <em>Xe</em> and <em>Xv</em>; both assays detected down to 100 fg of genomic DNA. However, in spiked assays when 1 µl of host DNA was added to each serial dilution of target genomic DNA, sensitivity was reduced to 1000 fg. Assays specifically detected target pathogens in infected tomato plant samples. The developed assays have applications in accurate diagnostics at point-of-care, surveillance, disease management and epidemiological studies.</div>