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Bio 459 - Diseases > Midterm 2 - Notes 1 (Part 3) > Flashcards

Midterm 2 - Notes 1 (Part 3) Flashcards

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1
Q

What are 6 epidemiologic problems associated by molecular strain typing?

A
  1. Dynamics of disease transmission
  2. Risk determination in sporadic occurrence of disease
    - risk of getting the disease when there is an outbreak
  3. Stratifying data and refining study designs
  4. Distinguishing pathovars and non-pathovars
  5. Addressing nosocomial infections
    - eg) USA 100 and 300 staphococcus aureas
  6. Identifying genetic determinants of disease transmission
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2
Q

Pathovars

A

Bacterial strains with similar characteristics, that is differentiated at infra-subspecific level from other strains of the same species on the basis of distinctive pathogenicity to one more plant hosts
- can case a disease

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3
Q

Non-pathovars

A

Will not cause the disease

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4
Q

What is pla involved with?

A

The immune response from the host

  • typically found in the plague virus
  • found on the smallest plasmid
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5
Q

Simplicity

A

Molecular techniques may be similar to execute and simpler to train people to use

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6
Q

High throughput

A

Capacity of a test to process a large number of specimens simultaneously

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7
Q

Cost

A

Widespread use of molecular biology reagents have reduced costs in developing countries

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8
Q

Appropriateness

A

The capacity of a test to address epidemiologic problems not possible to address by conventional methods
- if conventional test can be used and there is no disadvantage to use it, then there is no need to use a molecular technique

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9
Q

Typeability

A

Ability of a technique to generate an unambiguous result for an isolate tested

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10
Q

Reproducibility

A

Ability of a test to produce identical results when a strain is tested repeatedly

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11
Q

Ease of interpretation

A

Information derived from molecular techniques has to serve as a stratum in epidemiological study

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12
Q

Ease of use

A

Same as simplicity

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13
Q

Stability

A

The character used for molecular typing is not subject to rapid evolution or last from host
- if you lose the plasmid it will not be stable

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14
Q

Epidemiologic concordance

A

Molecular typing compares favourably with a previously validated test

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15
Q

What is the validity of a test?

A

It is its ability to correctly predict or identify those who truly have the characteristics the test is trying to detect and exclude those who do not have the characteristics

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16
Q

What is the point for validating a disease?

A

Confirms methods for pathogens causing disease recognized to occur as an outbreak

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17
Q

What are the types of phenotypic strain typing? (4)

A
  1. Typing by growth and morphologic characteristics
  2. Typing based on biochemical characteristics
  3. Typing by serologic characteristics
  4. Typing by fundamental or physiologic characteristics
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18
Q

What is an example of typing by growth and morphological characteristics?

A

Colour and shape of colonies on agar plates

- S. aureus on Vogel Johnson agar are black colonies surrounded by a cleared yellow zone

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19
Q

What are 2 examples of typing based on biochemical characteristics?

A
  1. Targeting an enzyme associated with a disease
    - lactose fermenting E. coli cause diarrhea
    - targeting lactose fermentation for detection of infectious agents
  2. Mannitol fermentation for staphylococcus aureus
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20
Q

What is typing by serological characteristics based on?

A

Differences in antigenic determinants of infectious agents

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21
Q

What are 2 examples of serological characteristics?

A
  1. E. coli can be subtype by O polysaccharide and further sub-typed by flagellar protein H
    - hamburger disease (E. coli O157:H7) –> associated with cattle
  2. Influenza virus is typed by H and N antigenic proteins (H5N1)
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22
Q

What is an O polysaccharide important for?

A

Important to invade the immune system

- involved in the immune response from the host

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23
Q

What are 7 examples of typing by functional or physiological characteristics?

A
  1. Antimicrobial susceptibility
  2. Phage tagging
  3. Colicin (bacteriocin) tying
  4. Cell culture assay
  5. Survival characteristics (in vitro or in vivo)
  6. Toxigenicity bioassays
  7. Multilocus enzyme electrophoresis (MLEE)
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24
Q

What are 2 examples of antimicrobial susceptibility?

A
  1. Betalactamase (ampicillin)

2. Rifampin

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25
Q

What is colicin typing based on?

A

Susceptibility to bacteriocins: short peptides that inhibits other bacteria

26
Q

Bacteriocins

A

Disrupts the cell wall membranes of bacteria

27
Q

What is an example of cell culture assay?

A

Enteropathogenic E. coli can be differentiated from other E. coli by their ability to attach to HeLa cells and their effect

28
Q

What are survival characteristics based on?

A

Susceptibility to stress conditions

29
Q

How can shinga-like toxins or vertoxins E. coli be identified?

A

By their cytotoxic effects on vero cells

- african green monkey kidney cells

30
Q

What do toxin producing clostridium difficile cause?

A

Cytopathic effects on fibroblast cell monolayers

31
Q

MLEE

A

Multilocus enzyme electrophoresis

32
Q

What is MLEE based on?

A

Metabolic enzymes that are highly conserved and that may reflect differences visualized by migration of enzymes in a starch gel

33
Q

What does typing by ELISA do?

A

It detects the presence of an antibody of an antigen in the given sample
- enzyme linked immunosorbent

34
Q

What are the 5 phases of ELISA typing?

A
  1. Antibodies to virus bound to wells of microtiter plates
  2. Add patient sample (secretions, serum, etc) suspected of containing virus particles or virus antigens and wash wells with a buffer
  3. Add antivirus antibody containing conjugated enzyme
  4. Wash with buffer
  5. Add substrate for the enzyme and measure the amount of coloured products
35
Q

What are the typical results for ELISA?

A

You are looking for the coloured parts

  • quantitation = colour product produced is proportional to the amount of anigens
  • colour and antigens are positively correlated
36
Q

What is typing of western (immuno-) blotting used for?

A

Used to identify specific amino acid sequences in proteins by using antibodies

37
Q

What are the 5 phases of western blotting?

A
  1. Denature proteins by boiling detergent and subject to electrophoresis; proteins separated by molecular weight
  2. Blot the separated proteins from the gel to the membrane
  3. Treat membrane containing blotted proteins with antibodies; each antibody recognizes and binds to a specific protein
  4. Add marker to bind to antigen-antibody complexes, either radioactive staphylococcus protein A125I or antibody containing conjugated enzyme
  5. Expose to film (125I) or enzyme substrate and develop to reveal antibody labeled proteins
38
Q

What can western blotting be used to identify?

A

HIV

39
Q

What are the 3 types of genotypic strain typings?

A
  1. Nucleic acid extraction
  2. Analysis of extrachromosomal DNA elements
  3. Genome-based typing methods
40
Q

What are 5 types of genome based typing?

A
  1. Restriction endonuclease analysis
  2. Southern blot hybridization
  3. Pulse field gel electrophoresis (PFGE)
  4. Whole genome sequence comparison
  5. Microarray comparison
41
Q

Nucleic acid extraction

A

DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods

42
Q

What do you need in DNA isolation?

A

Mechanical destruction of the cell

43
Q

What are the 3 phases of nucleic acid extraction?

A
  1. Lysis
  2. Precipitation
  3. Purification
44
Q

What happens in the lysis phase of nucleic acid extraction?

A

The cell is broken by mechanical disruption to release DNA

45
Q

What happens in the precipitation phase of nucleic acid extraction? (3)

A
  1. DNA is freed from the nucleus and is mixed with mashed up bits from the cell
  2. They get separated
    - Na+ neutralizes negative change (making it more stable and less water soluble)
  3. Alcohol is added and causes the DNA to precipitate out because it is not soluble in alcohol
46
Q

What happens in the purification phase in nucleic acid extraction?

A

Rinsed with alcohol to get ride of any last debris

47
Q

What are the 4 phases of analysis of extrachromosomal DNA elements?

A
  1. 2 samples are cut by restriction endonuclease
  2. Then they are precipitation
  3. Centrifuged
  4. The remains are put in a gel electrophoresis and compared to each other
48
Q

What does restriction endonuclease analysis do?

A

The analysis of DNA molecules by the use of restriction enzymes
- DNA is cleaved with the enzymes and the fragments obtained are generally separated by gel electrophoresis

49
Q

What does gel electrophoresis do?

A

It is a method used to separate mixture of DNA, RNA or proteins according to their molecular size
- molecule are pushed by an electrical field through a gel that contains small pores

50
Q

What does gel electrophoresis create?

A

Bands that can be used to determine the contents and can compared them to other samples

51
Q

What is southern blot?

A

A procedure for identifying specific sequences of DNA in which fragments separated on a gel are transferred directly to a 2nd medium on which detection by hybridization may be carried out

52
Q

What does the southern blot need to be located on to be probed?

A

A membrane

53
Q

What is the southern blot a combination of?

A

Gel electrophoresis and hybridization

54
Q

What is pulse field gel electrophoresis?

A

A technique used for separation of large DNA molecules by applying to a gel matrix on an electric field that periodically changes direction

55
Q

Why does PFGE take longer than normal gel electrophoresis?

A

Because the of the size of the fragments and that the DNA do not move in a straight line

56
Q

What is whole genome sequence comparison?

A

Is the process of determining the complete DNA sequence of an organisms genome at a single time
- can be used to compare to other genome sequences

57
Q

What is microarray comparison?

A

It is a collection of microscopic DNA spots attached to a solid surface

58
Q

Why do we use microarrays? (2)

A
  1. To measure the expression levels of large numbers of genes simultaneously
  2. Can genotype multiple regions of a genome
59
Q

What do CMA chips do?

A

They use labels or probes that bond to specific chromosome regions in order to identify it and can determine the expression levels of the gene

60
Q

What are 2 PCR targets?

A
  1. Coding regions = genes

2. Non-coding regions = repetitive elements

61
Q

What do PCRs do?

A

They amplify the gene you are looking at

Bio 459 - Diseases (21 decks)

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