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The aim of the study was to apply the real-time PCR method with the intercalating dye SYBR Green I for the detection of genes of Bacillus anthracis located on plasmids pXO1 and pXO2 and the specific chromosomal rpoB sequence. The research was conducted on one vaccinal and four field strains of Bacillus anthracis. The assessment of the specificity of the tests involved isolates of other species of the genus Bacillus as well as strains of six other species of microorganisms. The studies were conducted with the PCR QuantiTect kit (Qiagen) and primers specific for the gene pag coding PA protein, gene cap coding capsule, and primers for the amplification of the specific chromosomal sequence. PCR enabled the detection of all genes under examination by the observation of amplification curves. The specificity of real-time PCR was confirmed by estimating melting temperatures of PCR products. It was shown that the melting temperatures of amplicons obtained in the reaction were 78°C, 79°C and 76°C in cases of detecting the chromosomal rpoB sequence, pag gene, and cap-C gene, respectively. The sensitivity and linearity of the reactions were determined using a regression coefficient. A high regression coefficient of 0.99 was demonstrated for all the reactions. The real-time tests were highly sensitive and specific.
93 environmental samples were collected and tested using molecular biology techniques. The presence of chromosomal sequence Ba813 was detected in three soil samples (SS1, SS3, ZL2) and in three dental pulp samples (ZZ3, ZZ5, ZZ8). The presence of pag gene (210 bp) was observed in SS3 and ZL2 samples. There were no positive reactions with primers targeting the capsule encoding gene (777 bp). Multiplex PCR reaction enables identification of capC gene (264 bp) in five samples: SS3, ZL2, ZZ3, ZZ5, ZZ8. The presence of pag gene (SS3, ZL2) and cap gene (SS3, ZL2, ZZ5) were observed in standard PCR. Bacillus anthracis spores were detected by SMART test in three samples: SS3, ZL2 and ZZ5. These strains were non-hemolytic, sensitive to penicillin and gamma phage. The PCR positive for pag gene isolates were able to produce PA (protective antigen).
The aim of the study was to use selected genetic analysis to differentiate plasmid cured strains of Bacillus anthracis from transitional strains (Bacillus sp. Ba 813). Two different research techniques (macro restriction analysis and PCR) were used and they confirmed that the PCR technique facilitated detecting the presence of the Ba 813 chromosomal sequence in both strains (plasmid cured Bacillus anthracis strain and Bacillus sp. Ba 813). This result indicated that applying the PCR method to differentiate plasmid cured Bacillus anthracis strains from Bacillus sp. Ba 813 was not possible. The macro restriction method, however, facilitated a precise differentiation of plasmid cured Bacillus anthracis strains from transitional Bacillus sp. Ba 813.
Laseczka wąglika posiada szereg genów bezpośrednio lub pośrednio związanych ze zjadliwością, zlokalizowanych zarówno w plazmidach jak też w chromosomie. Na podstawie wyników PCR ze starterami swoistymi dla ośmiu genów (capA, capB, capC, pagA, lef, cya, gerXA i sap) kodujących potencjalne czynniki wirulencji określono wirulotypy szczepów Bacillus anthracis izolowanych na terenie kraju.
To date, antibiotics have been the primary drugs used in the treatment of anthrax infections. However, their effectiveness is questionable, especially during the phase of intensive toxin production in the course of infection, and the number of drug-resistant strains continues to rise. Successful treatment of anthrax infection is therefore becoming more difficult. The article discusses some of the new methods of inhibiting anthrax infections: the inhibition of spore germination and of the attachment of PA to the host cell receptor, the inhibition of the enzymatic process of cleaving PA into PA63 and PA20, and of PA63 oligomerization, endocytosis and translocation (their influence on the protection of macrophages against lysis was also discussed). In addition, the neutralization of B. anthracis LeTx and EdTx toxins was presented as another potential method of inhibiting anthrax infections.
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