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 --- culture independent techniques 16S rDNA used – specific primers and probes

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 of Intestinal microbiota Detection & quantification FISH/dot blot hybridization After amlification – amplicons are labelled hyridized with samples Common microbes in fecal samples

5. 3. PCR-ELISA Combination of PCR and ELISA Amplified DNA – Labelled with digoxigenin – hybridized with probe immobilized in microtiter plate wells. 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 c lamp – 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 products – flourescent dyed Detected – flourescent detecter

9. 7. Terminal Restriction Fragment Length Polymorphism 16S rRNA amplification – labelled and unlabelled primers – one end of product is labelled PCR product – digested with endonucleases Length of labeled terminal restriction fragments – capillary electrophoresis

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 SYBR Green dye – fecal Bifidobacterium , Desulfovibrio

14. 5 nuclease assay TAQMAN probes – Bifidobacterium Lactobacillus Probes labelled with flourescent lanthanide

15. 12. Omics Metagenomics and Metaproteomics Metagenomics – microbiota of large intestine Diversity of fecal microbiota – crohan’s disease Metaproteomics – Intestinal microbiota in infants