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Nucleic Acid-based Methods in Microbiological Testing

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Nucleic Acid-based Methods in Microbiological Testing

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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. FISH fluorescent 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. FISH fluorescent 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”

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