Full text
| |
| Author(s): |
Roberta Novaes Amorim Almeida
Total Authors: 1
|
| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2009-04-14 |
| Examining board members: |
Rene Peter Schneider;
Irma Nelly Gutierrez Rivera;
Beny Spira
|
| Advisor: | Rene Peter Schneider |
| Abstract | |
A method based on extraction of rRNA, followed by RT-PCR of 16S rRNA, cloning and ARDRA was optimized and validated for identification of bacteria active in biofilms. The method was first tested with artificial three-membered consortia. Cloning and RT did not lead to significant changes in the composition of the artificial consortia, but a reduction in cycle number in the PCR reaction from 30 to 10 was necessary for limiting the distortion in the proportion of amplicons relative to that of the templates. Analysis of real biofilms revealed that clones from active organisms occurred in frequencies >2% in the clone library, but reproducibility of analysis was unsatisfactory, probably due to factors such as the spatial heterogeneity of colonization of biofilms by microbes, PCR bias and more than one ARDRA clone per organism. Storage of biofilm samples at -20 °C for 2 months did not lead to important changes in composition. Very different clone profiles were obtained in the analysis of the same biofilm sample with the optimized method and with a kit (Mo Bio) for extraction of RNA. (AU) | |