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