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1. Nucleic Acid-based Methods in Microbiological Testing
2. 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)
3. Techniques for separation of chemicals
4. 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.
5. Gel electrophoresis
6. Nucleic Acid Techniques:Gene Probes
7. Nucleic Acid Techniques:Gene Probes
8. Nucleic Acid Techniques:Gene Probes
9. Nucleic Acid Techniques:Gene Probes
10. 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)
11. FISHfluorescent in-situ hybridization
12. 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.fcgi?db=Nucleotide
Get sequence
Design a probe
13. 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 --> can be used to identify specific clades of bacteria.
Go to http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide
Search for: 16s rRNA
>400K sequences should pop up
14. Nucleic Acid Based Techniques Gene Probes
Advantages:
kits for fast DNA preparation from any sample in <1hr
sensitive. Specific for particular genes (even distantly related bacteria easily “share” virulence, antibiotic resistance genes)
kits for hybridization also available (several hrs required)
15. Nucleic Acid Based Techniques Gene Probes
Advantages:
kits for fast DNA preparation from any sample in <1hr
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
16. Nucleic Acid Techniques:Polymerase Chain Reaction
17. How to design PCR primers need two primers, each 20-25 bp long
to synthesize a probe, select a short sequence (~ 100-500 bp)
18. 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-
19.
Nucleic Acid Techniques:Multiplex PCR
20. 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
21. 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 O157:H7, Salmonella, Shigella, Listeria to a National database DNA “fingerprinting”