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Gut microbiota
1. Akram Najafi, Bushehr University
of Medical Science, Iran
Gut Microbiota
Presented by:
Akram Najafi
2. Akram Najafi, Bushehr University
of Medical Science, Iran
What is Gut Microbiota?
• Skin, mouth, and gut act as host to an enormous variety
of microbes, bacterial, archaeal, fungal, and viral.
• The human gut microbiota is estimated to be composed
of approximately 1014 bacterial cells.
• Approximately 400-500 bacterial species
• Their total genome capacity is 150 times larger than the
human gene complement, with an estimated 3.3 million
microbial genes
4. Akram Najafi, Bushehr University
of Medical Science, Iran
• These microbes help us:
- To digest our food
- To harvest energy from the diet
- To stimulate of the proliferation of the intestinal epithelium
- To regulate of fat storage in the host
- To maintain our immune systems
• More recently, studies strongly suggest that dysbiosis
contributes to:
- IBS, intestinal cancers, obesity, type 1 diabetes…
13. Akram Najafi, Bushehr University
of Medical Science, Iran
Techniques used to characterize the gut microbiota:
• Culture
• qPCR (real time PCR)
• Fluorescence in situ hybridization (FISH)
• Denaturing gradient gel electrophoresis (DGGE)
• Terminal restriction fragment length polymorphism (T-RFLP)
• DNA micro-arrays
• Direct sequencing of 16S rRNA (Pyrosequencing)
14. Akram Najafi, Bushehr University
of Medical Science, Iran
Culture:
• Isolation of bacteria on selective media
• Advantages:
- Cheap, semi-quantitative
• Disadvantages:
- <30% of gut microbiota have been cultured to date
15. Akram Najafi, Bushehr University
of Medical Science, Iran
16 SrRNA is able to demonstrate the following:
• The microbial diversity of the gut microbiota
• Qualitative & quantitative information on bacterial species
• Changes in the gut microbiota in relation to disease
16. Akram Najafi, Bushehr University
of Medical Science, Iran
qPCR (real time PCR):
• Amplification and quantification of 16S rRNA. Reaction
mixture contains a compound that fluoresces when it binds
to double-stranded DNA.
• Advantages:
- Phylogenetic identification, quantitative, fast
• Disadvantages:
- PCR bias, unable to identify unknown species
17. Akram Najafi, Bushehr University
of Medical Science, Iran
Fluorescence in situ hybridization (FISH):
• Fluorescently labelled oligonucleotide probes hybridize
complementary target 16S rRNA sequences. When
hybridization occurs, fluorescence can be enumerated using
flow cytometry.
• Advantages: Phylogenetic identification, semi-
quantitative, no PCR bias, fast
• Disadvantages: Dependent on probe sequences, unable to
identify unknown species
19. Akram Najafi, Bushehr University
of Medical Science, Iran
DNA micro-arrays (DNA chip):
• Fluorescently labelled oligonucleotide probes hybridize with
complementary nucleotide sequences. Fluorescence detected
with a laser.
• Mainly used in studies to compare the microbiota between
different populations.
• Advantages: Phylogenetic identification, semi-
quantitative, fast
• Disadvantages: Cross hybridization, PCR bias, species
present in low levels can be difficult to detect.
21. Akram Najafi, Bushehr University
of Medical Science, Iran
Denaturing gradient gel electrophoresis (DGGE):
• Gel separation of 16S rRNA amplicons using denaturant/
temperature
• Advantages: Fast, semi-quantitative, bands can be
excised for further analysis
• Disadvantages: No phylogenetic identification, PCR bias
22. Akram Najafi, Bushehr University
of Medical Science, Iran
Terminal restriction fragment length polymorphism (T-RFLP):
• Fluorescently labelled primers are amplified and then
restriction enzymes are used to digest the 16S rRNA
amplicon.
• Advantages: Fast, semi-quantitative, cheap
• Disadvantages: No phylogenetic identification, PCR bias, low
resolution
23. Akram Najafi, Bushehr University
of Medical Science, Iran
Direct sequencing of 16S rRNA amplicons:
• Massive parallel sequencing of partial 16S rRNA amplicons
for example, 454 Pyrosequencing® (Roche Diagnostics
GMBH Ltd, Mannheim, Germany)
• Amplicon immobilized on beads, amplified by emulsion
PCR, addition of luciferase results in a chemoluminescent
signal
• Advantages: Phylogenetic
identification, quantitative, fast, identification of unknown
bacteria
• Disadvantages: PCR bias, expensive, laborious
24. Akram Najafi, Bushehr University
of Medical Science, Iran
Advantages:
• Pyrosequencing can sequence 500 million bases, at 99%
or better accuracy, in a single run.
• It represents an approximately 2,000-fold increase in
throughput over Sanger sequencing.
• As shorter sequences are read (approximately one half of
the read lengths generated in Sanger sequencing), thus
bacteria that are in low abundance can be detected.
• Phylogenetic
identification, quantitative, fast, identification of
unknown bacteria.
25. Akram Najafi, Bushehr University
of Medical Science, Iran
Sampling:
• The majority of published studies have actually been based
on results from stool samples rather than mucosal biopsy
samples or luminal content analysis.
• Stool samples are used as a proxy for the study of the gut
microbiota as these samples are easier to collect than biopsy
samples, especially in healthy volunteers.
• Questionnaire
27. Akram Najafi, Bushehr University
of Medical Science, Iran
• The DNA qualities and concentrations in the samples will
apply using the gel electrophoresis and spectrophotometer.
• Pyrosequencing of Barcoded 16S rRNA Gene Amplicons:
- PCR reactions will run in a thermal controller using the
following cycling parameters:
- 5 min of denaturation at 95C,
- 30 s at 95 C (denaturing),
- 30 s at 56 C (annealing),
- 90 s at 72 C (elongation),
- A final extension at 72C for 7 min.