Culture Independent Methods for Detection &

Enumeration of Gut Microflora

IntroductionLess 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

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 speciesBifidobacterium longumBifidobacterium catenulatumBifidobacterium adolescentis

2. Design of Hybridization ProbesSome probes --- Assessment of

Intestinal microbiota

Detection & quantificationFISH/dot blot hybridization

After amlification – amplicons are labelled

hyridized with samples Common microbes in fecal

samples

3. PCR-ELISACombination 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

4. Sequence Analysis of Randomly Amplified 16S rRNA Genes16S rRNA genes amplificationUniversal or group-specific

primersCloning and sequencing of

product DNAPredominant species –

ClostridiumIncreases with ageClostridium rRNA cluster XIVa –

decrease with age – decrease in Ruminococcus obeum

5. Temperature Gradient Gel Electrophoresis (TGGE)

16S rRNA genes amplificationUniversal 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

6. Denaturing High Performance Liquid Chromatography

PCR amplification of 16S rRNA

Seperation of amlified products – Denaturing HPLC

Seperated products – flourescent dyed

Detected – flourescent detecter

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

8. Oligonucleotide Arrays

Specie specific probes – detection of predominant human intestinal bacteria – fecal samples

Rapid & accurate – thousands of bacteria – simultaneous detection

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

10. FISHFISH with different group specific

probes90% fecal bacteria – detectedBacteroids, prevotella and

Clostridium – higher numberAssessment of changes in levels

of – predominant groups – consumption of probiotics

Effects of breast feeding – FISH – predominance of Bifidobacteria

11. Quantitative Real-Time PCR

Rapid, accurate Quantitative techniqueCharacterization and comparision

--- healthy and hospitalized subjects

Different assays – developed

SYBR Green dye – fecal Bifidobacterium, Desulfovibrio

5 nuclease assayTAQMAN probes –Bifidobacterium LactobacillusProbes labelled with flourescent

lanthanide

12. OmicsMetagenomics and

MetaproteomicsoMetagenomics – microbiota of large intestine Diversity of fecal microbiota –

crohan’s diseaseoMetaproteomics – Intestinal

microbiota in infants