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BDS3 BAMS > Diagnostic and typing methods > Flashcards

Diagnostic and typing methods Flashcards

1
Q

What are the main bacteria associated with periodontal disease?

A
  • Porphyromonas gingivalis
  • Actinobacillus actinomycetemcomitans
  • Prevotella intermedia
  • Bacteroides forsythus
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2
Q

What are the main bacteria associated with dental caries?

A
  • Streptococcus mutans
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3
Q

What are the main bacteria associated with root canal infections?

A
  • Porphyromonas endodontalis
  • Fusobacterium nucleatum
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4
Q

What are the two bacterial detection methods?

A
  • Microbiological culture
  • Molecular biological
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5
Q

What is the microbiological culture method?

A
  • Vortex mix sample for 30 seconds to get homogenous suspension of bacteria
  • Serial dilute samples in FAB (fastidious anaerobe broth) to 10-^6
  • Spiral plate to agar media using either;
    a - Fastidious anaerobe agar (FAA) + 7.5% defibrinated horse blood
    b - same as a but supplemented with vancomycin for gram-negative anaerobes
  • Incubate anaerobically for 10 days
  • Obtain total bacterial counts
  • Can then sub culture specific bacteria on separate agar medium
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6
Q

What are the two molecular biological methods?

A
  • Use DNA probes
  • Polymerase chain reaction (PCR)
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7
Q

Why might we want to use vancomycin supplement when looking for oral disease?

A
  • Vancomycin is a selective agent for gram-negative anaerobes
  • Many oral disease are caused by gram- negative anaerobes so look for these spdecifically
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8
Q

Why must we conduct serial dilutions in microbiological culture methods?

A
  • If not then would be so many bacteria that would not be able to discern individual colonies
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9
Q

What colour do genera porphyromonas and prevotella bacteria appear on culture medium?

A
  • Appear black pigmented
  • Associated with periodontal disease
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10
Q

What are the 3 ways we can biochemically identify the isolated bacteria?

A
  • Anaerobes noted by their sensitivity to metronidazole disc (5ug/disc)
  • Gram stain
  • Rapid API 32 A: compare enzymatic activities , sugar fermentation of those bacteria to those held on central database
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11
Q

What is the outcome of gram staining?

A

Gram-positive bacteria = violet colour due to thick peptidoglycan layer in cell wall and retains the gram stain

Gram-negative bacteria = won’t stain as only thin peptidoglycan layer in cell wall

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12
Q

What does it mean if we see a zone of clearing in agar due to metronidazole disc?

A
  • Growing a gram-negative anaerobe
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13
Q

What are the advantages of microbiological culture methods?

A
  • Yields bacterial isolates
  • Can use these for future testing and to study e.g. for antibiotic sensitivities
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14
Q

What are the disadvantages to microbiological culture methods?

A
  • Requires viable cells (most die off and aren’t detected)
  • Insensitive (only show if 10^5-10^6 cells)
  • Only small numbers of samples analysed at once
  • Inconclusive results
  • Labour-intensive
  • Expensive process
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15
Q

What are DNA probes?

A
  • Segments of DNA that have been labelled with chemoluminescent, fluorescent or radioactive agents
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16
Q

What are some types of DNA probes?

A
  • Whole genomic (entire genome)
  • Cloned gene
  • Oligonucleotide (20-50 bases)
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17
Q

How sensitive are DNA probes compared to culture?

A
  • 10^3 cells required to detect species so more sensitive
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18
Q

How is the DNA probe prepared?

A
  • Probe is DNA double stranded molecule
  • Need to pull apart so label can be attached
  • Heat is used to denature the DNA and expose bases on both strands
  • One strand then labelled with either chemiluminescent, radioactive or fluorescent label
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19
Q

How is the sample prepared ready for the DNA probe?

A
  • Extract double stranded DNA from sample
  • Denature the DNA to single strands using heat
20
Q

What is a hybridisation reaction in DNA probe method?

A
  • Mix the single stranded sample and DNA probes
  • Probe binds to its complimentary sequence of DNA within sample if that bacterial species is present
  • Remove any non-binded DNA
  • Leaves use with labelled DNA probe identified within sample
21
Q

How are genomic probes made?

A
  • Extract and purify DNA from bacterial species you wish to investigate
  • DNA cut into smaller fragments and labelled
22
Q

What are some disadvantages of genomic/cloned gene probes?

A
  • Used back in 80’s when we didn’t have genetic info available for different bacterial species
  • Extremely non specific
  • Lots of cross-reactivity between whole genomic probes which made them unreliable
23
Q

How are cloned gene probes made?

A
  • A gene of interest would be cloned into E.coli
  • Cloned fragment isolated, purified and label attached
  • Gene is specific to bacterial species you are looking for (more specific than genomic)
24
Q

How do oligonucleotides probes work?

A
  • Very small in size
  • Target 16S ribosomal RNA gene (which all bacteria possess)
  • Possible to synthesise species specific probes that target one or more hypervariable regions
  • Synthesised single stranded oligonucleotides labelled and used as probe
  • Best DNA probe due to specificty
25
Q

Why is the 16S ribosomal RNA gene ideal for probes?

A
  • All bacteria possess it
  • Approx 1500 base pairs in length
  • Possesses nice hypervariable regions (V1 to V9) that contain DNA sequences that provide specific signature for each bacterial species
  • Conserved regions give a very broad range and useful as probes or PCR primers
26
Q

Why is Polymerase chain reaction good for detecting bacteria in clinical specimens?

A
  • Highly specific
  • Highly sensitive
  • Can be used to directly detect bacteria in clinical specimens
27
Q

What is required for PCR to work?

A
  • Double stranded DNA template from sample
  • Primers specififc to particular gene e.g. 16S ribosomal RNA of gene wanting to detect
  • Deoxynucleoside trisphospates
  • Enzyme Taq DNA polymerase which catalyses synthesis of new DNA strands
28
Q

How does PCR work?

A
  • Double stranded DNA from sample heat denatures at 94C (aka denaturation step)
  • PCR primers hybridise target sequences on each DNA strand (aka primer annealing step)
  • Taq DNA polymerase synthesises opposite strand incorporating dNTPs (aka primer extension step)
  • After each cycle of this the DNA is doubled
  • Cycle repeated up to 35 cycles
29
Q

What different types of primers are there?

A
  • General bacterial primers
  • Group-specific primers
  • Species-specific primers (either single primer pair or more than one primer pair)
  • All usually target 16S ribosomal RNA gene
30
Q

What does lanes with no PCR product mean and large amount of PCR?

A

No PCR = no bacteria you’re interested in
Large amount PCR = Greater the amount of bacteria interested in

31
Q

What are the advantages of DNA probes and PCR over microbiological culture?

A
  • Less time-consuming than culture methods
  • Very sensitive (DNA probes, 103 cells; PCR, 10 cells)
  • Can directly detect bacterial DNA within clinical samples
  • Do not require viable cells, samples do not have to be analysed immediately
  • Can detect uncultivable species
32
Q

What are the disadvantages of DNA probes and PCR over microbiological culture?

A
  • May detect dead cells
  • Detect only pre-selected species
33
Q

What is PCR-RFLP?

A
  • PCR - restriction fragment length polymorphisms
  • Identification of bacterial isolates
  • Digests PCR-amplified bacterial 16s rRNA gene with restriction enzymes
  • Yields specific patterns (fingerprints) for individual species
34
Q

What are the benefits of PCR-RFLP?

A
  • Rapid
  • Cheaper
  • More specific alternative to biochemical tests for identifying bacterial isolates from clinical samples
35
Q

Why is it important to subtype bacteria?

A
  • Track rotes of transmission during disease outbreaks
  • Study pathogenicity of specific strains
36
Q

What is subtyping?

A
  • Sufficient genetic diversity exists to allow identification of different clones (strains) among isolates of same species
37
Q

What are the traditional methods for subtyping bacteria?

A
  • Serotyping
  • Biotyping
38
Q

What are some problems with serotyping and biotyping?

A
  • Limited discriminatory capacity
  • Organism-specific methods
  • Specialised reagents required that are costly
39
Q

What is an alternative to traditional subtyping methods?

A
  • Molecular (genetic) typing methods
40
Q

What is restriction enzyme analysis (REA)?

A
  • Type of molecular typing method
  • Digest whole genomic DNA with restriction enzymes
  • But too many DNA fragments obtained so makes interpretation difficult
41
Q

What is gene probe typing?

A
  • Reduces number of DNA fragments generated by REA using suitable gene probe
42
Q

What is ribotyping?

A
  • Type of gene probe typing
  • Use E.coli rRNA operon as DNA probe following REA
    -Variation in number and size of fragments in bacterial DNA digest are complimentary with rRNA
43
Q

What is the procedure for any gene probe typing method?

A
  • Genomic DNA extracted from bacterial isolate or clinical sample
  • Digested with one or more restriction enzymes to give many DNA fragments
  • DNA fragments separated into distinct bands by agarose gel electrophoresis
  • Smaller ones migrate quicker
  • DNA bands transferred to nylon membrane
  • Hybridisation to labelled DNA probe
  • Probe is visualised and DNA fragments compared
44
Q

What is 16S-23S intergenic spacer region (IGS)?

A
  • Simple typing method is PCR-RFLP analysis of 16S-23S IGS
  • Very variable sequence
  • Amplify region by PCR using consensus primers
  • Digest PCR product with restriction enzymes to obtains stain-specific DNA finger prints
45
Q

What is DNA sequencing?

A
  • Sequences all the bases in stretch of DNA and detects even single base changes between different bacterial strains of same species
  • Best typing method
46
Q

How can we detect uncultivable and novel bacteria using DNA-based methods?

A
  • Genomic DNA extracted from sample
  • PCR amplifies 16S rRNA gene from bacterial DNA in clinical specimen
  • 16S rRNA genes cloned
  • 50 clones randomly chosen and sequenced
  • Clone sequences analysed to determine bacterial identity (BLAST)

BDS3 BAMS (15 decks)

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