RESUMO
Gut flora analysis is hampered by the complexity of the intestinal microbiota and by inherent limitations of culture-based approaches. Therefore, culture-independent molecular methods based upon 16S rRNA gene analysis were applied successfully for the analysis of complex microbial communities. However, generally accepted and validated profiling methods such as denaturing and temperature gradient gel electrophoresis (DGGE/TGGE) are still laborious and time consuming. Thus, we adapted the separation of amplified bacterial 16S rRNA gene fragments by denaturing high performance liquid chromatography (DHPLC) using the WAVE Microbial Analysis System as a rapid and convenient means to display complex intestinal bacterial communities and to monitor changes in the gut flora. The separation of 16S rRNA gene fragments amplified from reference strains representing main gut bacterial populations and from human stool samples revealed that DHPLC analysis effectively detects bacterial groups predominant in the human gut flora. The investigation of faecal samples from hospitalized patients before, during and after antibiotic therapy showed that PCR-based DHPLC can be used to monitor gut flora changes. Results from DHPLC analysis were comparable with DGGE profiles generated from the same samples, demonstrating that the adapted DHPLC protocol is well suited for the analysis of complex microbial communities.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fezes/microbiologia , Intestinos/microbiologia , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
A novel denaturing high-performance liquid chromatography (DHPLC)-based technique allows rapid high-resolution analysis of PCR products. We used this technique for unequivocal molecular identification of seven Candida species. We show the application of this PCR/DHPLC approach for direct detection and identification of yeast species from blood cultures and for detection of Candida colonization in the gastrointestinal tract of allogeneic transplant patients.
