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The aim of this study was to use the real time polymerase chain reaction in the detection of Babesia canis subclinical infestations in dogs and to compare the different DNA isolation methods on PCR sensitivity. The study included 6 dogs with suspected subclinical babesiosis. DNA for real time polymerase chain reaction were isolated by the phenol method as well as by Micro AX Gravity (A & A Biotechnology, Gdynia, Poland) and Blood mini (A & A Biotechnology, Gdynia, Poland) commercial kits. In the blood of all six specimens PCR demonstrated the presence of Babesia canis DNA. The most efficient proved to be a reaction to which the genetic material was isolated by the phenol method. The amount of total DNA obtained in this way, determined spectrometrically, ranged 43.7-54.3 ng/µl. Ct value in real-time PCR for DNA samples isolated in this manner was the lowest in comparison with other isolation methods, and averaged 22.5. Similar results were obtained when DNA was isolated from the blood with the Micro AX Gravity kit, while the least efficient was the Blood Mini Kit (amount of total DNA, depending on the sample was 14.0-25.1 ng/µl, amplification in real time occurred the slowest - average Ct value = 28). Readable sequences were obtained for all PCR products where DNA was isolated using the phenol method or by Micro AX Gravity. In the case of PCR products where DNA was isolated by the Blood Mini Kit, readable sequences were obtained only for 3 out of 6 tested samples. All sequences received in our study of the 18S RNA gene fragment showed a high 99.9-100% homology with the sequence of Babesia canis EU622792 These results confirm the usefulness of the real time PCR in the diagnosis of subclinical canine babesiosis and indicate the need for choosing such a DNA isolation method for this reaction that will guarantee the highest efficiency of amplification.
Bordetella bronchiseptica is a widespread Gram-negative pathogen occurring in different mammal species. It is known to play a role in the etiology of infectious atrophic rhinitis of swine, canine kennel cough, respiratory syndromes of cats, rabbits and guinea pigs, and sporadic human cases have also been reported. The aim of this article is to present the occurrence of infections caused by these bacteria in domestic cats with respiratory symptoms, as well as to conduct a molecular analysis of the flaA gene B. bronchiseptica for the purpose of ascertaining whether cats become infected with one or more bacteria strains. B. bronchiseptica was isolated from the respiratory system of 16 out of 35 domestic cats with symptoms of respiratory tract infections. Polymorphism analysis of polymerase chain reaction products of B. bronchiseptica flaA was performed to reveal the possible differences in nucleotide sequences of the flagellin gene. The phylogenetic analysis of nucleotide sequences obtained during PCR indicated that the isolates of bacteria from our own studies are characterised by 100% homology of the analysed fragment of the flaA gene, which suggests maintenance of a single genotype of these microorganisms in the cat population. Moreover, the bacteria revealed full homology with reference strain B. bronchiseptica ATCC 4617, and 99.4% homology with strain B. parapertussis ATCC 15311. This indicates that the PCR optimised for the Bordetella spp. flaA gene, combined with sequencing of amplicons obtained in PCR, is an effective diagnostic method allowing differentiation of Bordetella spp. type microorganisms.
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