Chapter 20- Lab analysis of the immune response Flashcards
Why take the time to identify an infectious agent? (4)
- Many bacteria are resistant to certain antibiotics
- Antibiotic resistant bacteria and viruses are spreading across the world- you can prevent the spread if you know the pathogen
- Specific pathogens are associated with secondary disease complications
- Tracking the spread of disease can lead to its source
Classification
Placing organisms in groups of related species. Lists of characteristics of known organism
Identification
Matching characteristics of an “unknown” organism to lists of known organisms. Clinical lab identification is an example
Problem solving algorithms to identify bacteria
This is a step by step problem solving procedure. Lab technicians will often inoculate several tests at once to speed up inoculation- to eliminate certain pathogens or to make sure that other pathogens are present. They will interpret the results in a set sequence- if one test is positive, another test must also be positive.
Classic methodology of identifying bacteria (4)
- Isolate bacterium from patient
- Pure culture- cell and colony morphology on an agar plate. This is using Koch’s postulates
- Gram stain is always necessary
- Biochemical pathways/properties
Biochemical testing
Checks the metabolism of the organism, and whether the organism has certain enzymes present. You can check cell properties through gram stain or in pure culture, but not with biochemical testing
Strip testing
Strip testing can be used to check for changes in pH and for other tests mentioned in the dichotomous key. The strip is inoculated with a specific bacteria and monitored for a change in color
Taxonomic key
Dichotomous key that has paired statements in the form of “either-or”. Followed by statements to go to another pair of statements- kind of like a flow chart. For example, if you know that bacteria is gram negative, you do a glucose test- if negative, it’s probably pseudomonas, if positive, you do a lactose test
Genetic homology
GC content of the pathogen’s chromosomal DNA. Animal cells have very low GC content, while the GC content of bacteria is much higher
What does it mean if an organism has a high GC content?
Higher GC content means that the organism is more adaptable- fungi and protozoa also have a high GC content so they can adapt when the environment changes
Restriction fragment length polymorphism (RFLP) analysis
Compares the DNA banding patterns of different DNA samples after digestion using restriction enzymes. Restriction enzyme recognition sites are short and are found throughout the genome. Therefore, differences in DNA sequences in an individual’s genome leads to differences in the distribution of restriction enzyme recognition sites. This is visualized through the banding pattern of gel electrophoresis. It can be used to diagnose heritable diseases or establish paternity
Polymerase chain reaction (PCR)
Amplifies the number of copies of a specific DNA sequence for further analysis. The double stranded template DNA is denatured at a high temperature to separate the strands. The temperature is lowered so DNA primers that are complementary to the end of the target sequence bind to the template strands. The temperature is then raised again so that nucleotides can be added to the primer using the single stranded target as a template. Multiple sets of DNA primers can identify individual genes from a pathogen for more specific typing
Disadvantage of PCR
PCR may not be able to detect a pathogen if the amount in the sample is too low- organisms from the microbiota are also present in the sample, making it difficult
Quantitative reverse transcriptase PCR (qRT-PCR)
Used for obtaining DNA copies of a specific mRNA molecule. Uses a reverse transcriptase enzyme to convert mRNA molecules into cDNA. That cDNA is then used as a template for traditional PCR amplification. RT-PCR can detect whether a specific gene has been expressed in a sample. The quantitative method uses fluorescence probes
Why are fluorescent probes used in qRT-PCR?
To monitor the increase in a double stranded template during a PCR reaction as it occurs. The kinetics data can be used to quantify the amount of the original target sequence, or how much mRNA was present originally. Can be used to determine the number of DNA copies or organisms in a sample (like HIV viral load
Meaning of curves produced by qRT-PCR
Earlier CT (cycle time) means the lowest amount of fluorescence the machine could detect was detected sooner. Lower amounts of amplification means that it takes longer for fluorescence to be detected
Enzyme-linked immunosorbent assay (ELISA) (3 types)
Direct, indirect, sandwich
Direct ELISA
Antigens are immobilized in the well of the plate. An antibody that is specific for a particular antigen is combined with an enzyme that is added to each well. The antibody will bind if the antigen is present. The presence of the enzyme causes a substrate to convert to a colored end product if the antigen is present
Sandwich ELISA
Uses antibodies to quantify a specific antigen in a solution. Adds a primary antibody to the wells of the plate. The primary antibody captures the antigen, and a secondary antibody is added- it is a polyclonal antibody that is combined with an enzyme. The enzyme converts the substrate into a colored end product. Called a sandwich because the antigen is between 2 antibodies
Indirect ELISA
Used to quantify antigen-specific antibodies against a pathogen instead of antigens. Attaches the known antigen to the wells. Patient serum is added, and if antibodies (primary antibodies) are present, they will bind to the antigen. The secondary antibody with its conjugated enzyme is directed against the primary antibody. This secondary antibody allows us to quantify how much antigen specific antibody is present in the patient’s serum by the intensity of the color produced by the reaction
Antigen capture ELISA
Detects antigens in a patient sample or serum antibodies. Uses an antigen or antibody which will bind to the antigen/antibody in the patient sample (if present) and cause a color change
Fluorescent antibody staining
Antibody specific to particular pathogen types is tagged with a fluorescent tag. If the organism of interest is present, the antibody will bind to it and fluoresce
Phage typing
Receptor sites for bacteriophages are highly specific. Certain strains of a bacterium species are attacked only by particular types of phages. You can therefore use a bacteriophage to determine the bacterial species
Fatty acid sequencing
Identification of the fatty acid composition of the outer membrane and cytoplasmic membrane. Each microorganism has a different fatty acid phenotype- different patterns of fatty acids. Uses FAME- fatty acid methyl ester
Point of care laboratory tests
Used directly at the site of patient care. The result is obtained quickly in order to make appropriate treatment decisions. Good POC tests have high specificity and high sensitivity. Strep rapid tests are an example
Sensitivity
Refers to how small of a sample a test can detect. Less sensitivity would require more antigens to get a positive result
Specificity
Refers to how well a test can distinguish positives and negatives.
How do POC tests work?
When the sample is placed on a cassette, the antibodies will bind to it. The test has a test line and control line- antibodies bind to the control line, but the test line being present indicates the test is positive. Newer tests do mini PCR and are able to determine which antibiotics the organism may be resistant to based on its genetics
