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.
Need Writing Help?
Get feedback on grammar, clarity, concision and logic instantly.Check your paper »
- Microbiology Review Paper 1.What are (a) the hypotheses tested and (b) the logically formulated rationale for each. Explain both LOGICALLY and in terms of the Biological Sciences. The hypothesis of this experiment is clearly stated and tested. They hypothesized that wounds within a hospital are the cause of the cross-contamination spread of methicillin-resistant Staphylococcus aureus (MRSA). Methicillin resistant S. aureus is one of the more dangerous pathogenic strains of S. aureus, because it is highly resistant to antimicrobials thereby limiting treatment.... [tags: Staphylococcus aureus, Antibiotic resistance]
1143 words (3.3 pages)
- The Impact of Ancient DNA Discovered in Siberia For many decades, there has been an overwhelming curiosity about what life was like before humans ruled the earth - back when life on land, off land, and even the very land itself was very different than it is today. Pieces of this puzzle have been discovered all over the world, ranging from perfectly preserved skeletons at the bottom of bodies of water to fragments discovered in vast deserts. Due to the conditions of the ancient finds, there is only so much that can be deciphered about the past, which leads to the creations of various theories of what life was like in different areas and why and when it changed.... [tags: Exploratory Essays Research Papers]
1417 words (4 pages)
- Bacterial Strains, Plasmids, and Standard Media Bacterial strain which was used as a host for recombinant plasmid propagation was E. coli DH5 α, whereas for subcloning and expression was B. subtilis DB104 ; Plasmid used in this experiment was pSKE194 an E.coli-Bacillus shuttle vector (both bacterial strain and plasmid were precious gift from Prof Meinhardt, Muenster University). For regenerating Bacillus protoplasts containing desired recombinant plasmid the 1.5% (w/v) agar containing regeneration medium DM3 was used (Chang and Cohen 1984), and for selection of positive Bacillus transformant, LB medium containing erythromycin 5 µg mL-1 were used.... [tags: Bacteria, Escherichia coli, Enzyme, DNA]
1076 words (3.1 pages)
- Worldwide, Staphylococcus species are notorious as human pathogens and are the focus of comprehensive studies among researchers in the scientific field. More than, 40 different Staphylococcus species cause diseases known to be clinically and economically significant (Lindsay 2008). Public health has become threatened by the antibiotic –resistance strains that passes on from staphylococcus species to humans (Lindsay 2008). Therefore, Lindsay believes that each species or their habitat might correlate with the type of infection produced (Lindsay 2008).... [tags: Staphylococcus aureus, Staphylococcus, Bacteria]
1005 words (2.9 pages)
- "Plasmids" Bacteria, yeast, and some higher eukaryotic cells contain molecules know as plasmids. "Plasmids are circular, double stranded, DNA molecules that are separate from a cell 's chromosomal DNA. " They have the ability to replicate themselves separately within the host cell. Some bacterial cells may contain hundreds of plasmid molecules that have replicated. Plasmids display either a parasitic or symbiotic relationship towards the host cell. An example of a symbiotic relationship is that a drug-resistant plasmid can help the bacterial host cell to survive against antibiotics.... [tags: DNA, Bacteria, Gene, Plasmid]
848 words (2.4 pages)
- Diagnosis Diagnosis The most appropriate test to detect Neisseria gonorrhoeae is the nucleic acid amplification test, also know as NAAT. NAAT consist of a urine sample, endocervical and vaginal swab for women, and urethral swab in men (CDC, 2015). Test detects the genetic material of the bacteria, in this case N. gonorrhoeae. Theoretically the test is able to detect a single copy of the nucleic acid of the bacteria (AACC, 2014). The test amplifies the number of copies of the genetic material in order for the bacteria to be identified.... [tags: DNA, DNA replication, Bacteria, Gonorrhea]
829 words (2.4 pages)
- DNA cloning and genetic engineering are a vital aspect of the field of genetics. DNA cloning provides the ability to isolate and replicate genes of interest and study their direct impact on organisms. Genetic engineering and transformation provide an insight on an issue of great caliber, the increase of antibiotic resistant bacteria. The extraction and inserting of genetic material are known as miniprep and transformation, respectively. A vector is essential in order to transform genetic material between bacteria.... [tags: DNA, Molecular biology, Bacteria]
1428 words (4.1 pages)
- Microbial world frequently face abrupt changes in the surrounding environment. For the survival in such a dynamic environment, it is advantageous to take decision of future action at the population level. Bacteria do so by a phenomenon called quorum sensing (QS), which is sensing the presence of other bacteria in surrounding environment, and taking the decision at certain threshold cell density. In QS active state, bacteria induces certain set of target genes at population level. QS among bacteria is mediated by autoinducers (AIs), whose concentration is a function of cell density, and above a certain concentration of cells AI mediate QS.... [tags: germs, strain, dna]
1361 words (3.9 pages)
- DNA Extraction In extracting chromatin from the cells of wheat germ there are seven steps to follow. The optimal cell to use would be the polyploidal eukaryotic. Eukaryotes have nucleus membrane-bound organelles, while prokaryotic does not. The polyploidal eukaryotic cell has DNA that is held in the nucleus while the prokaryote has DNA that floats freely around the cell. The DNA of eukaryotes is more complex and extensive than the other. Prokaryote is a bacterial cell that usually has DNA in one large strand and only has one chromosome while the eukaryotic cell has more than one chromosome and is considered to be a higher organism.... [tags: Process Biology Biological Essays]
585 words (1.7 pages)
- Extraction by Bacteria Leaching Copper and Gold can be extracted from their ores by using bacteria leaching. A long time ago, engineers investigating the possibility of reopening the Roman workings noticed a blue-green liquid running from the mountains of excavated rock. When the fluid ran across old iron implements, it left a brown film. When scraped off, the film was pure copper. In 1947, US microbiologists discovered that the transformation was in fact the work of microorganisms.... [tags: Papers]
860 words (2.5 pages)