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Abstract:
Background: The analysis of microbial communities is of importance in many cases of life sciences and bioengineering. Traditional techniques of investigations like culture or cloning methods are characterized by many disadvantages. They are by and large unable to give a total qualitative and quantitative view of the total amount of microorganisms themselves, their interactions among each other and with their environment. Method: We generated species specfic and fluorescently labeled 16S rRNA gene fragments by the T-RFLP technique. For this purpose bacterial DNA of clinically important microorganisms of different genome sizes (P. aeruginosa, Campylobacter jejuni, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Burkholderia cepacia, Mycobacterium avium, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans) were amplified by PCR using FAM-labeled 8-27f primer and unlabeled 1507-1492r primer (E. coli 16S rRNA);, using 8-27f and 1507-1492r. The amplfied DNA was digested with the restriction enzyme Hha I. For the separation of these fragments we validated a T-RFLP technique to an automated high throughput multi-capillary electrophoresis device (ABI 3100 Genetic Analysis?) with regard to a precise qualitative and quantitative characterization of microbial communities. We constructed a single stranded ROX labeled sizing standard up to for accurate sizing up to 780bp. The differences from the nominal size was less than half a base pair. Additionally we generated an internal quantification standard and investigated the minimal detectable concentration. The standard error was less than 3
