Midterm 2 - Notes 1 (Part 3) Flashcards
What are 6 epidemiologic problems associated by molecular strain typing?
- Dynamics of disease transmission
- Risk determination in sporadic occurrence of disease
- risk of getting the disease when there is an outbreak - Stratifying data and refining study designs
- Distinguishing pathovars and non-pathovars
- Addressing nosocomial infections
- eg) USA 100 and 300 staphococcus aureas - Identifying genetic determinants of disease transmission
Pathovars
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
Non-pathovars
Will not cause the disease
What is pla involved with?
The immune response from the host
- typically found in the plague virus
- found on the smallest plasmid
Simplicity
Molecular techniques may be similar to execute and simpler to train people to use
High throughput
Capacity of a test to process a large number of specimens simultaneously
Cost
Widespread use of molecular biology reagents have reduced costs in developing countries
Appropriateness
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
Typeability
Ability of a technique to generate an unambiguous result for an isolate tested
Reproducibility
Ability of a test to produce identical results when a strain is tested repeatedly
Ease of interpretation
Information derived from molecular techniques has to serve as a stratum in epidemiological study
Ease of use
Same as simplicity
Stability
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
Epidemiologic concordance
Molecular typing compares favourably with a previously validated test
What is the validity of a test?
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
What is the point for validating a disease?
Confirms methods for pathogens causing disease recognized to occur as an outbreak
What are the types of phenotypic strain typing? (4)
- Typing by growth and morphologic characteristics
- Typing based on biochemical characteristics
- Typing by serologic characteristics
- Typing by fundamental or physiologic characteristics
What is an example of typing by growth and morphological characteristics?
Colour and shape of colonies on agar plates
- S. aureus on Vogel Johnson agar are black colonies surrounded by a cleared yellow zone
What are 2 examples of typing based on biochemical characteristics?
- Targeting an enzyme associated with a disease
- lactose fermenting E. coli cause diarrhea
- targeting lactose fermentation for detection of infectious agents - Mannitol fermentation for staphylococcus aureus
What is typing by serological characteristics based on?
Differences in antigenic determinants of infectious agents
What are 2 examples of serological characteristics?
- 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 - Influenza virus is typed by H and N antigenic proteins (H5N1)
What is an O polysaccharide important for?
Important to invade the immune system
- involved in the immune response from the host
What are 7 examples of typing by functional or physiological characteristics?
- Antimicrobial susceptibility
- Phage tagging
- Colicin (bacteriocin) tying
- Cell culture assay
- Survival characteristics (in vitro or in vivo)
- Toxigenicity bioassays
- Multilocus enzyme electrophoresis (MLEE)
What are 2 examples of antimicrobial susceptibility?
- Betalactamase (ampicillin)
2. Rifampin
What is colicin typing based on?
Susceptibility to bacteriocins: short peptides that inhibits other bacteria
Bacteriocins
Disrupts the cell wall membranes of bacteria
What is an example of cell culture assay?
Enteropathogenic E. coli can be differentiated from other E. coli by their ability to attach to HeLa cells and their effect
What are survival characteristics based on?
Susceptibility to stress conditions
How can shinga-like toxins or vertoxins E. coli be identified?
By their cytotoxic effects on vero cells
- african green monkey kidney cells
What do toxin producing clostridium difficile cause?
Cytopathic effects on fibroblast cell monolayers
MLEE
Multilocus enzyme electrophoresis
What is MLEE based on?
Metabolic enzymes that are highly conserved and that may reflect differences visualized by migration of enzymes in a starch gel
What does typing by ELISA do?
It detects the presence of an antibody of an antigen in the given sample
- enzyme linked immunosorbent
What are the 5 phases of ELISA typing?
- Antibodies to virus bound to wells of microtiter plates
- Add patient sample (secretions, serum, etc) suspected of containing virus particles or virus antigens and wash wells with a buffer
- Add antivirus antibody containing conjugated enzyme
- Wash with buffer
- Add substrate for the enzyme and measure the amount of coloured products
What are the typical results for ELISA?
You are looking for the coloured parts
- quantitation = colour product produced is proportional to the amount of anigens
- colour and antigens are positively correlated
What is typing of western (immuno-) blotting used for?
Used to identify specific amino acid sequences in proteins by using antibodies
What are the 5 phases of western blotting?
- Denature proteins by boiling detergent and subject to electrophoresis; proteins separated by molecular weight
- Blot the separated proteins from the gel to the membrane
- Treat membrane containing blotted proteins with antibodies; each antibody recognizes and binds to a specific protein
- Add marker to bind to antigen-antibody complexes, either radioactive staphylococcus protein A125I or antibody containing conjugated enzyme
- Expose to film (125I) or enzyme substrate and develop to reveal antibody labeled proteins
What can western blotting be used to identify?
HIV
What are the 3 types of genotypic strain typings?
- Nucleic acid extraction
- Analysis of extrachromosomal DNA elements
- Genome-based typing methods
What are 5 types of genome based typing?
- Restriction endonuclease analysis
- Southern blot hybridization
- Pulse field gel electrophoresis (PFGE)
- Whole genome sequence comparison
- Microarray comparison
Nucleic acid extraction
DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods
What do you need in DNA isolation?
Mechanical destruction of the cell
What are the 3 phases of nucleic acid extraction?
- Lysis
- Precipitation
- Purification
What happens in the lysis phase of nucleic acid extraction?
The cell is broken by mechanical disruption to release DNA
What happens in the precipitation phase of nucleic acid extraction? (3)
- DNA is freed from the nucleus and is mixed with mashed up bits from the cell
- They get separated
- Na+ neutralizes negative change (making it more stable and less water soluble) - Alcohol is added and causes the DNA to precipitate out because it is not soluble in alcohol
What happens in the purification phase in nucleic acid extraction?
Rinsed with alcohol to get ride of any last debris
What are the 4 phases of analysis of extrachromosomal DNA elements?
- 2 samples are cut by restriction endonuclease
- Then they are precipitation
- Centrifuged
- The remains are put in a gel electrophoresis and compared to each other
What does restriction endonuclease analysis do?
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
What does gel electrophoresis do?
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
What does gel electrophoresis create?
Bands that can be used to determine the contents and can compared them to other samples
What is southern blot?
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
What does the southern blot need to be located on to be probed?
A membrane
What is the southern blot a combination of?
Gel electrophoresis and hybridization
What is pulse field gel electrophoresis?
A technique used for separation of large DNA molecules by applying to a gel matrix on an electric field that periodically changes direction
Why does PFGE take longer than normal gel electrophoresis?
Because the of the size of the fragments and that the DNA do not move in a straight line
What is whole genome sequence comparison?
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
What is microarray comparison?
It is a collection of microscopic DNA spots attached to a solid surface
Why do we use microarrays? (2)
- To measure the expression levels of large numbers of genes simultaneously
- Can genotype multiple regions of a genome
What do CMA chips do?
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
What are 2 PCR targets?
- Coding regions = genes
2. Non-coding regions = repetitive elements
What do PCRs do?
They amplify the gene you are looking at
