Identification of bacteria with

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2003-09-29
Identification of bacteria with 16S rRNA
sequencing
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2003-09-29
Identification of bacteria
Patient sample (blood, urin, faeces)
Primary culture
- Selective growth media - Antibiotics
Secondary culture
Identification?
Biochemical typing
API strip
Identification
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2003-09-29
Why sequencing?
Cultivation of bacteria fails - cultivation
methods are lacking - the bacteria are to few
to be cultivated - the bacteria are
inactive Biochemical identification fails -
methods are missing - the bacteria do not
perform biochemically correct
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2003-09-29
Identification Flowchart
Patient sample (blood, urin, faeces)
Primary culture fails
Sequencing
Secondary culture
Identification?
Biochemical typing
Identification fails
Sequencing
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2003-09-29
The Molecular Clock
Criteria -Randomly occuring mutations at
neutral sites -Constant mutation rate The
degree of sequence similarity reflects
phylogenetic distance, i.e. the more similar the
sequences- the more closely related the
species Ribosomal genes - contain regions
that are conserved among all self replicating
organisms, together with highly variable
regions -Are large enough to contain
adequate amount of information
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2003-09-29
Evolutionary tree of the domain Bacteria
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2003-09-29
The bacterial 16S rRNA molecule
Part of the ribosomal small subunit together with
a set of ribosomal proteins 16S the
sedimentation coefficient in ultra
centrifugation, measured in Svedberg rRNA
ribosomal RNA 1500 bases long RNA molecule
Conserved regions primer design Variable
regions identification
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2003-09-29
Historical highlights of the bacterial 16S rRNA
molecule

• Woese Fox 1977 Archaea, Bacteria and
  Eucaryotes could be classified acording to their
  phylogeny
• Classification of bacteria with phylogenetic
  trees
• Detection of non-cultivatible bacteria. 99 can
  not be cultivated!
• Wilson et al. 1991 Identification of the
  etiologic agent of Whipples disease

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2003-09-29
Sequencing of the 16S rRNA gene

• DNA extraction 1 h (more for gram positives)
• PCR 2,5 h
• Cycle sequencing 3 h
• Sequence analysis 1,5 h
• Editing of raw data 20 min
• Search in database 10 min lt 9 h

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2003-09-29
Conclusions
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2003-09-29
Lab. Groups
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2003-09-29
Wet-Lab content

• DNA preparation
• PCR with primers directed against 16S rDNA
• Analysis of PCR product on agarose gel
• Purification of PCR product
• Cyclic Sequencing reaction (PCR)
• Automated sequencing
• Identification of organism by database searches

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2003-09-29
PCR with primers directed against 16S rDNA

• Each PCR reaction containing
• Master Mix (dNTP, MgCl buffer)
• TaqDNApolymerase
• Forward Reverse Primers
• DNA Template
• Water to adjust the reaction volume

The samples are put in a Thermocycler 95C,
5min. Initial denaturation 95C,
45s. Denaturation of DNA 50C, 45s. Annealing of
Primers 72C, 1min. Elongation of new DNA 72C,
10min. Final elongation
X 35
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2003-09-29
Purification of PCR product

• Commercial kits (e.i QIAqiuck Spin) are used to
  remove the excess of primers and nucleotides from
  the PCR reaction
• The principle
• Binding of DNA to a silica filter at a suitable
  pH, adjusted with a buffer solution
• Washing the DNA with a wash buffer
• Elute the DNA by changing the pH with another
  buffer solution

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2003-09-29
Cyclic Sequencing reaction (PCR)
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2003-09-29
Identification of organism by database searches
BLAST www.ncbi.nlm.nih.gov
BLAST- search at the National Center for
Biotechnology Information (NCBI) site by using
the retrieved nucleotide sequence (or translated
amino acid sequence) in FASTA- format gtSeqname
Nucleotide sequence........
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2003-09-29
Computer sessions
http//www.kvir.uu.se/molbiokurs/molbiokurs_uu.htm
l
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2003-09-29
Demonstration of some useful techniques in
molecular biology

• Real- time PCR
• Automated Capillary Sequenator
• Amersham Pharmacia Biotech
• Gyros