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Poster: Diseases of Plants: Disease Detection & Diagnosis


Development of a multiplex real-time PCR for the improved detection of citrus canker-causing Xanthomonas
V. MAVRODIEVA (1), G. Santillana (1), G. Dennis (2) (1) USDA APHIS PPQ S&T Beltsville Laboratory, U.S.A.; (2) USDA APHIS PPQ S&T, U.S.A.

To improve on the current assay for the detection of all citrus canker causing Xanthomonas which targets the pthA gene, a multiplex assay was developed to incorporate the 16S rRNA gene as a second target. Two new pthA amplisets designed to have better sensitivity than the current one, and two new 16S amplisets were evaluated individually, and in multiplex format. The PCR product of each ampliset was sequenced and verified to be the correct targets. Primers and probes interaction was determined and optimal concentrations selected followed by dNTP and MgCl2 concentrations optimization for the different ampliset combinations. In silico and experimental specificity analyses were performed. All amplisets were found to have a 100% match with the target organism. The pth-targeted amplisets showed cross-reactivity to some non-citrus Xanthomonas but not to other citrus-infecting genera. The method being developed targets symptomatic citrus therefore cross-reactions of the primers with Xanthomonas spp. of other crops should not interfere because most xanthomonads exhibit highly restricted host range (Delcourt et al., 2013). One of the 16S amplisets showed insufficient specificity thus was excluded from further optimization studies. Initial comparability studies suggest that the new pthA amplisets (used in multiplex with 16S rRNA gene) may be more sensitive than the currently used uniplex (pthA), having lower Ct values for the sets of archived diagnostic samples tested.