Culture Independent Methods
for Detection & Enumeration
of Gut Microflora
Introduction


Less than 25% of the intestinal –
cultivated



Many bacteria have not been cultured
yet



Molecular
bi...
1. Design of PCR Primers for
DNA Amplification
Specie or group specific primers – GIT
 rDNA specific primers --- rapid &
...
2. Design of Hybridization
Probes
Some

probes --- Assessment
Intestinal microbiota

of

Detection

& quantification
FI...
3. PCR-ELISA
 Combination

of PCR and ELISA

DNA – Labelled with
digoxigenin – hybridized with probe
immobilized in micro...
4. Sequence Analysis of
Randomly Amplified 16S rRNA
Genes

16S rRNA genes amplification
 Universal or group-specific prim...
5. Temperature Gradient Gel
Electrophoresis (TGGE)
16S rRNA genes amplification
 Universal or group-specific primers –
on...
6. Denaturing High Performance
Liquid Chromatography


PCR amplification of 16S rRNA



Seperation of amlified products ...
7. Terminal Restriction Fragment
Length Polymorphism


16S rRNA amplification – labelled and
unlabelled primers – one end...
8. Oligonucleotide Arrays


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

...
9. Relative Amount of Group or
Specie-rRNA


Quantitative study – quantification of
relative amount of each group with
re...
10. FISH
FISH with different group specific
probes
 90% fecal bacteria – detected
 Bacteroids, prevotella and Clostridiu...
11. Quantitative Real-Time
PCR
Rapid, accurate
 Quantitative technique
 Characterization and comparision --healthy and h...
5

nuclease assay
 TAQMAN probes –Bifidobacterium
Lactobacillus
 Probes labelled with flourescent
lanthanide
12. Omics
Metagenomics and Metaproteomics
o Metagenomics –
microbiota of large intestine
Diversity of fecal microbiota –...
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Culture independent methods for detection & enumeration of gut microflora

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Culture independent methods for detection & enumeration of gut microflora

  1. 1. Culture Independent Methods for Detection & Enumeration of Gut Microflora
  2. 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. 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. 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. 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. 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. 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. 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. 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. 10. 8. Oligonucleotide Arrays  Specie specific probes – detection of predominant human intestinal bacteria – fecal samples  Rapid & accurate – thousands of bacteria – simultaneous detection
  11. 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. 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. 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. 14. 5 nuclease assay  TAQMAN probes –Bifidobacterium Lactobacillus  Probes labelled with flourescent lanthanide
  15. 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 
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