Culture independent methods for detection & enumeration of gut microflora
Download as PPTX, PDF6 likes5,761 views
This document discusses various culture-independent methods for detecting and enumerating gut microflora, including: 1) Design of PCR primers and hybridization probes to target specific species or groups; 2) PCR-ELISA to combine PCR amplification with ELISA detection; 3) Sequence analysis of randomly amplified 16S rRNA genes; 4) Temperature gradient gel electrophoresis (TGGE) and denaturing high performance liquid chromatography (DHPLC) to separate amplified DNA fragments by size; 5) Terminal restriction fragment length polymorphism (T-RFLP) to generate terminal restriction fragments for profiling; 6) Oligonucleotide arrays and quantitative real-time PCR for rapid, accurate detection of thousands of bacteria; 7) Fluorescence in
Culture independent methods for detection & enumeration of gut microflora
- 1. Culture Independent Methods for Detection & Enumeration of Gut Microflora
- 2. Introduction Less than 25% of the intestinal – cultivated Many bacteria have not been cultured yet Molecular biology --independent techniques 16S rDNA used – specific primers and probes culture
- 3. 1. Design of PCR Primers for DNA Amplification Specie or group specific primers – GIT rDNA specific primers --- rapid & specific detection Matsuki – Bifidobacteria in fecal samples Common species Bifidobacterium longum Bifidobacterium catenulatum Bifidobacterium adolescentis
- 4. 2. Design of Hybridization Probes Some probes --- Assessment Intestinal microbiota of Detection & quantification FISH/dot blot hybridization amlification – amplicons are labelled hyridized with samples Common microbes in fecal samples After
- 5. 3. PCR-ELISA Combination of PCR and ELISA DNA – Labelled with digoxigenin – hybridized with probe immobilized in microtiter plate wells. Amplified Presence of hybridized DNA digoxigenin-targeted antibodies Analysis of Bifidobacterium species –
- 6. 4. Sequence Analysis of Randomly Amplified 16S rRNA Genes 16S rRNA genes amplification Universal or group-specific primers Cloning and sequencing of product DNA Predominant species – Clostridium Increases with age Clostridium rRNA cluster XIVa – decrease with age – decrease in Ruminococcus obeum
- 7. 5. Temperature Gradient Gel Electrophoresis (TGGE) 16S rRNA genes amplification Universal or group-specific primers – one has GC clamp – attached to 5 end of DNA – avoid complete dissociation of two DNA Amplified products – seperated – TGGE Predominant bands – sequenced – identity of most abundant microorganisms
- 8. 6. Denaturing High Performance Liquid Chromatography PCR amplification of 16S rRNA Seperation of amlified products – Denaturing HPLC Seperated dyed Detected – flourescent detecter products – flourescent
- 9. 7. Terminal Restriction Fragment Length Polymorphism 16S rRNA amplification – labelled and unlabelled primers – one end of product is labelled PCR product endonucleases Length of labeled terminal restriction fragments – capillary electrophoresis – digested with
- 10. 8. Oligonucleotide Arrays Specie specific probes – detection of predominant human intestinal bacteria – fecal samples Rapid & accurate – thousands of bacteria – simultaneous detection
- 11. 9. Relative Amount of Group or Specie-rRNA Quantitative study – quantification of relative amount of each group with regard to total 16S rRNA in sample – specific probes 6 bacterial groups – 70% of total fecal RNA Bacteroids, Prevotella – 37% of 16S rRNA
- 12. 10. FISH FISH with different group specific probes 90% fecal bacteria – detected Bacteroids, prevotella and Clostridium – higher number Assessment of changes in levels of – predominant groups – consumption of probiotics Effects of breast feeding – FISH – predominance of Bifidobacteria
- 13. 11. Quantitative Real-Time PCR Rapid, accurate Quantitative technique Characterization and comparision --healthy and hospitalized subjects Different assays – developed Green dye – fecal Bifidobacterium, Desulfovibrio SYBR
- 14. 5 nuclease assay TAQMAN probes –Bifidobacterium Lactobacillus Probes labelled with flourescent lanthanide
- 15. 12. Omics Metagenomics and Metaproteomics o Metagenomics – microbiota of large intestine Diversity of fecal microbiota –crohan’s disease o Metaproteomics – Intestinal microbiota in infants