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Nucleic acid-based methods offer a variety of tools for the detection of parasites. This field of veterinary and medical sciences is rapidly evolving. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers. In this paper we present the usefulness of LAMP method in the diagnosis of babesiosis in various animal species. Babesia species-specific LAMP assay may have potential clinical application in the detection and differentiation of Babesia species, particularly in countries in which babesiosis is endemic. It can be assumed that in the near future this technique will be a routinely used diagnostic test in veterinary practices.
Duck plague (DP) caused by duck enteritis virus (DEV) is an acute infection of waterfowl. Apart from geese and ducks free-ranging swans are susceptible to infection. The exact epidemiological situation in relation to DEV infection in Eastern Europe is unknown. Therefore it was reasonable to conduct a study on DEV occurrence using the modern but simple loop-mediated isothermal amplification (LAMP). The use of LAMP facilitated DEV DNA detection in a time below 30 min. The method was also specific solely for DEV, while its detection limit was 1 pg of DNA. The aim of this study was the optimization of LAMP for detection of DEV infections among free-ranging water birds. The presence of DEV DNA was identified in 96 birds (72.7%), predominantly in wild ducks (Anas platyrhynchos) and mute swans (Cygnus olor). The results were recorded in visible light as a green color of reaction mixtures and their fluorescence under ultraviolet light. The presented study indicates the usefulness of the developed LAMP technique for DEV detection and the high percentage of free-ranging water birds infected with this virus. The study will be continued in the future for the determination of DEV occurrence in domestic geese and ducks.
Loop mediated isothermal amplification (LAMP) is an innovative technique of gene amplification and a simple diagnostic tool for the early detection and identification of diseases. The whole process of LAMP is easy and fast; amplification can be performed in less than one hour under isothermal conditions using a set of six specifically designed primers acting on eight different sequences of the target gene by incubation in a single tube. Products of amplification can be detected using agarose gel electrophoresis as well as monitoring without optical instruments or by a turbidimeter. The LAMP technique does not require a thermocycler and it can only be applied using a heating block or hot water bath. Given the benefits of rapid amplification, simplicity of the procedure and easy detection, as well as the lack of the need for specialized equipment or qualified staff, LAMP has potential application for clinical diagnosis and even in control of communicable diseases, contributing in this way to the development of personal medicine.
Listeria monocytogenes is an important foodborne pathogen that causes a disease known as listeriosis, which is especially dangerous for pregnant women. Infection with L. monocytogenes may also result in stillbirths, abortions and premature deliveries, as well as meningitis, septicaemia, encephalitis, and meningoencephalitis. Conventional detection methods of these bacteria are time-consuming; therefore, rapid alternative methods, including those based on molecular tests are needed. PCR is sensitive and specific; however, it requires the use of an agarose gel, which increases the time of analysis. A technique that allows the elimination of this step is real-time PCR, which enables the quantitative determination of L. monocytogenes in foods. A modification of PCR is multiplex PCR that allows detection of several genes at the same time and distinguishes between different species of microorganisms. Techniques such as RT-PCR or NASBA, where the target molecule is RNA, are used to detect viable cells and also allow quantitative analyses to be performed. Another rapid and specific method is LAMP, which can be performed in one hour in a water bath or heating block, without the use of a thermocycler. Biosensors and microarrays are examples of new technologies that due to the possibility to use anywhere and immediate interpretation of the results can be routinely used in the future for identification of L. monocytogenes in food.
Marek’s disease is a viral disease, a type of poultry cancer. It is caused by MDV serotype 1 (Marek’s disease virus, MDV) viruses, also referred to as Gallid herpesvirus 2 belonging to the family Herpesviridae. The complete, fully infectious virus particles are present in the feathers and the nodules months can survive in the environment for many months. The use of isothermal amplification methods of genetic material (Loop-Mediated Isothermal Amplification, LAMP) virus MDV enables rapid and precise identification of the virus in difficult diagnostic material, including dust. By optimizing the reaction conditions and using at least two pairs of primers which bind to specific sites in the genome of the virus, this technique has high sensitivity and specificity. The technique makes it possible to detect of single copies of the meq oncogene – a unique region of DNA of the first type of virus MDV.
Diagnostics of Mycoplasma synoviae (MS) is generally based on serological, culturing and molecular methods. Rapid diagnosis and identification of infections is very important in the poultry industry. The use of PCR or real-time PCR makes it possible to shorten the time for obtaining results from research and the effective detection of genetic material of MS. There are many variations of the PCR method, one of them is the LAMP method, which is rapid, very sensitive and does not require specialist equipment. This article reviews the molecular methods used for the diagnosis of Mycoplasma synoviae infection in poultry.
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