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
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
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