Development of a rapid detection method for Erwinia amylovora by loop-mediated isothermal amplification (LAMP)

Detection of plasmid pEA29 DNA of Erwinia amylovora by a loop-mediated isothermal amplification protocol (LAMP) was evaluated in laboratory assays. LAMP amplifies target DNA rapidly (I hour), isothermally (65 degrees C) and with high-specificity based on four primers designed to recognize six independent sequences of target DNA. A positive reaction results in a cloudy white precipitate of magnesium pyrophosphate in the PCR tube. With whole cell suspensions, our LAMP protocol had a detection limit of 25 colony forming units, which was similar to the sensitivity of nested PCR. The LAMP primers did not react with suspensions of Pantoea agglomerans, Pseudomonas fluorescens and Pseudomonas syringae. In experiments with apple, 0, 10, 100, or 1000 flowers collected from orchards were added to 5 liters of distilled water containing 0, 500 or 5000 CFU/ml of E. amylovora. Flower washes were sampled directly, or concentrated by filtration or centrifugation prior to the LAMP assay. The LAMP assay detected E. amylovora in 96% of samples with the pathogen and had no reaction with non-pathogen amended controls. The number of flowers in the suspension had no effect on LAMP results. Concentrating the wash suspension or extracting DNA improved the incidence of detection. A similar experiment with pear flowers resulted in 96 and 8% positive detection with LAMP for suspensions with and without the pathogen, respectively. Indigenous bacteria were isolated from flower suspensions at densities of 1.2 x 10(4) CFU/ml for apple and 3.9 x 10(4) CFU/ml for pear. LAMP represents a sensitive, low cost method for detection of E. amylovora. A LAMP assay can be subjected to quantitative monitoring of DNA amplification, and may be adapted for use in the field. When coupled with disease forecasting models, the ability to rapidly detect epiphytic populations of E amylovora with LAMP could improve fire blight management.