Nucleic Acidbased Methods in Microbiological Testing

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Nucleic Acid-based Methods in Microbiological
Testing
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What can a microbiologist extract from a
soil/water sample?

• Microbes (fungi, bacteria, viruses)
• DNA
• RNA
• Proteins/enzymes
• Other signature molecules (fatty acids,
  metabolism by-products, etc)

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Techniques for separation of chemicals
Size exclusion chromatography separates small
from big molecules
Ion exchange chromatography charged from
non-charged, anions from cations
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Isolation of nucleic acids from environmental
samples

• Nucleases are present in soil/water samples
• add EDTA to chelate cation cofactors
• Steps in nucleic acid extraction
• Concentrate dilute samples
• Lyse cells (lysozyme, detergents, solvents, glass
  beads or freezing/thawing)
• Isolation (buffer in pH 8), then wash with
  organic solvents.
• Precipitation lower pH, add alcohol.

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Gel electrophoresis
apparatus
sample data
Separates nucleic acids based on the size of the
charged particle
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Nucleic Acid TechniquesGene Probes
target
labeled probe
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Nucleic Acid TechniquesGene Probes
sample
extract DNA
denature DNA
DNA is fixed on a filter
label
gene probe
From Environmental Microbiology, Maier et al
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Nucleic Acid TechniquesGene Probes
Microarrays.
From Environmental Microbiology, Maier et al
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Nucleic Acid TechniquesGene Probes
DNA-probe
DNA-probe
From Environmental Microbiology, Maier et al
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FISHfluorescent in-situ hybridization

• Based on the ability of DNA to re-anneal
  (hybridize)
• A probe is an oligo-nucleotide that hybridizes to
  the 16s rRNA gene
• The principle is similar to other probes, only
  allows to visualize sample in situ (i.e. in place)

Hogart et al., 2000
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FISHfluorescent in-situ hybridization
http//www.bio.davidson.edu/Courses/Molbio/MolStud
ents/spring2003/Baxter/MolecularTool.html
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How to design a probe

• Identify a target sequence
• for FISH, a short probe (15-25 nt) will do
• search http//www.ncbi.nlm.nih.gov/entrez/query.fc
  gi?dbNucleotide
• Get sequence
• Design a probe

Target sequence
5 ..C A T G C G T T A A T A T T G C G 3
probe
3 G T A C G C A A T T A T 5
Sequence of nucleic acids are always written
5-gt3, so the sequence of your probe is
5 T A T T A A C G C A T G 3
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How to identify a target sequence

• 16s rRNA gene - a common target for bacterial
  detection
• 16s rRNA gene encodes ribosomal RNA
• highly conserved regions of the gene can be used
  to identify all bacteria
• some regions of the 16s rRNA gene are less
  conserved --gt can be used to identify specific
  clades of bacteria.
• Go to http//www.ncbi.nlm.nih.gov/entrez/query.fcg
  i?dbNucleotide
• Search for 16s rRNA
• gt400K sequences should pop up

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Nucleic Acid Based Techniques

• Gene Probes
• Advantages
• kits for fast DNA preparation from any sample in
  lt1hr
• sensitive. Specific for particular genes (even
  distantly related bacteria easily share
  virulence, antibiotic resistance genes)
• kits for hybridization also available (several
  hrs required)

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Nucleic Acid Based Techniques

• Gene Probes
• Advantages
• kits for fast DNA preparation from any sample in
  lt1hr
• sensitive. Specific for particular genes (even
  distantly related bacteria easily share
  virulence, antibiotic resistance genes)
• kits for hybridization also available (several
  hrs required)
• Disadvantages
• expensive
• specialized equipment for detection (5-30K)
• false positives, negatives possible
• some probes are radioactive

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Nucleic Acid TechniquesPolymerase Chain Reaction
One cycle
1) Heat samples to denature (separate strands)
DNA
DNA from the sample
2) Add primers, nucleotides nucleotides anneal
to the homologous region of DNA
3) Add DNA-dependent DNA polymerase Polymerase
syntesizes the new DNA chain in 5 -gt3 direction
Off to the next cycle
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How to design PCR primers

• need two primers, each 20-25 bp long
• to synthesize a probe, select a short sequence
  ( 100-500 bp)

3 CTATAATCTGG 5
lt-primer 1
5 GATATTAGACCATAGACATCAGACACATTAGGGAC 3 3
CTATAATCTGGTATCTGTAGTCTGT GTAATCCCTG 5
5 CATTAGGGAC 3
primer 2
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Nucleic Acid Based Techniques

• Detection of E. coli by PCR.
• Grow on selective medium
• Test putative positives by PCR
• primers for gadAB, stx genes
• EHEC are -typically- uidA-, lac-

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Nucleic Acid TechniquesMultiplex PCR
Allows to identify multiple sequences in
one reaction.
From Environmental Microbiology, Maier et al
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DNA fingerprinting

• Repetitive element-PCR
• rapid test to ID bacteria
• geographic differences may exist, databases
  required
• Ribotyping
• rRNA genes are amplified
• highly reproducible
• labor-intensive, reference database required

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DNA fingerprinting

• Repetitive element-PCR
• rapid test to ID bacteria
• geographic differences may exist, databases
  required
• Ribotyping
• rRNA genes are amplified
• highly reproducible
• labor-intensive, reference database required
• PFGE (Pulse Field Gel Electrophoresis)
• bacterial DNA is isolated, digested and separated
• extremely sensitive (/-)
• 50 public labs submit data on E.coli O157H7,
  Salmonella, Shigella, Listeria to a National
  database