Essay on Bacteria strains and DNA extraction

Essay on Bacteria strains and DNA extraction

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Materials and method
Bacteria strains and DNA extraction
A collection of standard bacterial strains containing E. amylovora strains and several species of bacteria confirmed by Biochemical, Carbohydrates and Virulence tests for identification of E. amylovora isolates (data not shown) were exploited to estimate the specificity test (table 1). Furthermore, in order to assess the performance of two PCR methods and LAMP assay, about 208 symptomatic plant samples, were used. This collection was obtained from various plant tissues (e.g., flowers, shoots, leaves, fruits, and limbs) belonging to apple, pear and quince cultivars of different regions of Iran, during spring and summer of 2009 and 2010. For preparing samples, the same method of (Gorris et al. 1996) was used: 100 micro liters of the each dilution and other standard bacterial routinely cultivated on Luria-Bertani agar (LB) or LB agar medium and incubated at 28 °C for 48h. In the following, total genomic DNA of each strain was isolated by lysis of bacterial pellets from 1 ml of broth culture, incubated overnight in DNA extraction buffer, purified with phenol–chloroform–isoamyl alcohol (25:24:1) and precipitated with isopropyl alcohol (Llop et al. 1999; Schaad et al. 2001). At last, each strain DNA was eluted in 100 µl of elution buffer and stored at −20 ◦C prior to further assessment. whenever pure bacterial cultures were used, the optical density of bacterial culture was measured by a spectrophotometer at 600 nm (2×107 CFU/ml), and one μl of each dilution of bacterial suspension was directly added to the LAMP and PCR reaction mix. In contrast, for infected plant samples, were used Plant Mini Kit (Qiagen). DNA isolation was performed according to the manufacturer’s protocol fo...

... middle of paper ...

... clearly specific-specific for E. amylovora.

Comparison of LAMP with Conventional and Nested PCR assays
To assess the ability of the LAMP and other PCR methods for detecting E. amylovora in naturally infected plant material (described previously), first of all LAMP method was tested with infected plant material. Among three methods, LAMP assay showed the highest power of detecting the pathogen in all symptomatic samples (Table 3). Unlike single PCR and nested PCR, in LAMP method there is no need to electrophoresis and time detection is reduced to 45 min. The existence of the pathogen in the positive samples was confirmed by its isolation in culture medium. These remarkable results demonstrated that, in one hand, the method has higher specificity and sensitivity than single PCR and on the other, it is slightly better than nested-PCR in a single closed tube.

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