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Development of genome-informed diagnostics for detection of Pectobacterium species using recombinase polymerase amplification coupled with LFD

Firas Ahmed: University of Hawaii At Manoa

<div><em>Pectobacterium </em>species can be devastating pathogens that cause bacterial soft rot diseases of a number of agroeconomically important fruit and vegetable crops worldwide, including tomato and potato. A rapid, accurate, and simple method is required to detect bacterial infections for timely management of disease. Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for specific and sensitive detection of <em>Pectobacterium</em> directly from infected plant tissue without the need for DNA purification. The specificity of RPA was tested against 27 strains of different <em>Pectobacterium</em> species and 12 non-<em>Pectobacterium</em> sp. including <em>Dickeya</em> sp. No false positives or negatives were observed. RPA primers and probes were designed to target tomato and potato host genome regions to enhance the reliability of the assay. Assays specifically detected the target pathogen in infected host tissues. The detection limit for both sensitivity and spiked sensitivity assays was down to 10 fg. RPA showed no inhibitory effects when pathogen or host targets were directly detected in infected potato and tomato sap. This RPA method has applications in diagnostics at point-of-care, surveillance, biosecurity, disease management and epidemiological studies.</div>