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A Loop-Mediated Isothermal Amplification Assay and Sample Preparation Procedure for Sensitive Detection of Xanthomonas fragariae in Strawberry

Hehe Wang, William W. Turechek
Plos One 2016 v.11 no.1 pp. 1-21
DNA, Xanthomonas fragariae, bacterial diseases of plants, boiling, cost effectiveness, detection limit, disease control, leaf spot, loop-mediated isothermal amplification, microbial culture, microbial detection, nursery stock, petioles, plant pathogenic bacteria, plant tissues, quantitative polymerase chain reaction, rapid methods, sampling, strawberries
Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infections are common and contribute to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay with a bacterial enrichment procedure as a more efficient method for detection of asymptomatic infections of X. fragariae. The enrichment procedure calls for an overnight incubation of strawberry tissues with phosphate-buffered saline (PBS), followed by a quick sample concentration and a boiling step to extract DNA for amplification. The bacterial enrichment step allows sampling of a larger amount of plant tissue, hence increasing the detection rate in real-life samples. The detection limit of the LAMP assay was approximately 2000 CFU/mL for pure bacteria culture and 300 CFU/mL for strawberry leaf and petiole samples processed through enrichment assay. LAMP provided a 2—3 fold lower detection limit than the standard qPCR assay but was much faster, less expensive, and more user-friendly. Therefore, LAMP with bacterial enrichment assay will serve as a rapid, sensitive and cost-effective tool for detecting asymptomatic infections of X. fragariae in strawberry nursery stock and contribute to improved disease management in the industry.