Materials and methodsBacterial strains and DNA extractionA collection of standard bacterial strains containing strains of E. amylovora and several species of bacteria confirmed by biochemical, carbohydrate and virulence tests for the identification of E. amylovora (data not shown) were used to estimate the specificity test (Table 1). Furthermore, in order to evaluate the performance of two PCR methods and the LAMP assay, approximately 208 symptomatic plant samples were used. This collection was obtained from various plant tissues (e.g. flowers, shoots, leaves, fruits and branches) belonging to cultivars of apple, pear and quince trees from different regions of Iran, in spring and summer. summer 2009 and 2010. To prepare the samples, the same method (Gorris et al. 1996) was used: 100 microliters of each dilution and other standard bacteria routinely cultured on Luria-Bertani (LB) agar or agar medium LB and incubated at 28 °C for 48 h. In the following, the total genomic DNA of each strain was isolated by lysis of bacterial pellets from 1 ml of culture broth, incubated overnight in DNA extraction buffer, purified with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated with isopropyl alcohol. alcohol (Llop et al. 1999; Schaad et al. 2001). Finally, DNA from each strain was eluted into 100 µl of elution buffer and stored at -20 ◦C before further evaluation. whenever pure bacterial cultures were used, the optical density of the bacterial culture was measured by a spectrophotometer at 600 nm (2 × 107 CFU/ml) and one µl of each bacterial suspension dilution was directly added to the LAMP reaction mixture and PCR. On the other hand, for infected plant samples, we used the Plant Mini Kit (Qiagen). DNA isolation was performed according to the manufacturer's protocol in the middle of the document......clearly specific to E. amylovora. Comparison of LAMP with Conventional and Nested PCR Assays To evaluate the ability of LAMP and other PCR methods to detect E. amylovora in naturally infected plant material (previously described), the first LAMP method was tested with infected plant material. Among the three methods, the LAMP assay showed the greatest power to detect the pathogen in all symptomatic samples (Table 3). Unlike single PCR and nested PCR, in the LAMP method, no electrophoresis is required and the detection time is reduced to 45 minutes. The existence of the pathogen in the positive samples was confirmed by its isolation in culture medium. These remarkable results demonstrated that, on the one hand, the method has higher specificity and sensitivity than single PCR and, on the other hand, it is slightly better than nested PCR in a single closed tube..