A loop-mediated isothermal amplification with bacterial enrichment assay for detection of Xanthomonas fragariae in strawberry
H. WANG (1), W. Turechek (2). (1) Univ of Florida, Ft Pierce, FL, U.S.A.; (2) ARS-USDA, Fort Pierce, FL, U.S.A.
We developed a rapid, sensitive and cost-effective loop-mediated isothermal amplification (LAMP) assay for detection of <i>Xanthomonas fragariae</i>, the cause of angular leaf spot of strawberry. <i>X. fragariae</i> is difficult to detect when present at low densities, thus a bacterial enrichment procedure was developed to precede the assay that consists of an overnight incubation step followed by a quick sample concentration and a boiling step to extract DNA. Extracted DNA was amplified by LAMP and qPCR using a related set of primers for comparison. Results showed that the LAMP primers amplified <i>X. fragariae</i> at 65 C within 30 min in reactions containing fluorescent dye for real-time detection or hydroxy naphthol blue dye for end-product detection. Primer specificity was determined by negative amplification with different pathogens and saprophytes isolated from strawberry and other <i>Xanthomonas</i> species. The LAMP detection limit was approx. 4 cells per reaction for pure bacteria culture and 3 x 102 cfu/ml for leaf and petiole samples. LAMP provided comparable detection sensitivity as qPCR, but was much faster. In addition, the whole assay required only basic instruments and reagents, thus making it a simple and cost effective procedure for identification of <i>X. fragariae</i> in strawberry leaf and petiole tissue.
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