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

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
