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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