Q56425223 Dotaz Zobrazit nápovědu
- MeSH
- kongresy jako téma MeSH
The subject of this review covers modern experimental procedures for chromosomal gene replacement in Escherichia coli and related bacteria, which enable the specific substitution of targeted genome sequences with copies of those carrying defined mutations. Two principal methods for gene replacement were established. The first "in-out" method is based on integration of plasmid into bacterial chromosome and subsequent resolving of the cointegrate. The "linear fragment" method (recombineering) is based on homologous recombination mediated by short homology arms at the ends of linear DNA molecule. Many new protocols and improvements in targeted gene replacement were introduced during the last 10 years. These methods are well suited for high-throughput functional gene studies and for many biotechnological applications.
- MeSH
- amikacin farmakokinetika terapeutické užití MeSH
- aminoglykosidy genetika MeSH
- Enterococcus faecalis enzymologie genetika metabolismus MeSH
- enzymy genetika MeSH
- finanční podpora výzkumu jako téma MeSH
- gentamiciny farmakokinetika terapeutické užití MeSH
- geny genetika MeSH
- infekce spojené se zdravotní péčí etiologie farmakoterapie MeSH
- léková rezistence genetika účinky léků MeSH
- Publikační typ
- srovnávací studie MeSH
AIMS: A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. METHODS AND RESULTS: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method. CONCLUSIONS: The developed real-time PCR is suitable for rapid quantification of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.
- MeSH
- deoxyribonukleasy metabolismus MeSH
- DNA bakterií analýza MeSH
- Escherichia coli genetika izolace a purifikace MeSH
- financování organizované MeSH
- kinetika MeSH
- polymerázová řetězová reakce metody MeSH
- potravinářská mikrobiologie MeSH
- proteiny vnější bakteriální membrány genetika MeSH
- proteiny z Escherichia coli genetika MeSH
