Garlanda 4 Flashcards
nucleic acid based methods detect
organism specific DNA or RNA
multiplex assays
where a single nucleic acid based test can detect more than 2 causative organisms
nucleic acid tests are quantitative or qualitative?
qualitative, but quantification methods exist for a limited number of infections (HIV, CMV, HTLV)
nucleic acid quantitative tests are useful for
diagnosis and monitoring response for treatment
tests for target amplification
PCR on DNA
RT-PCR on RNA and cDNA
process of PCR has 3 steps? What can you use to automate them?
denaturation, priming, extension
Use a thermocycler
Different kinds of amplification
- signal: e.g. branched DNA assays, hybrid capture (labelled nucleic acid probes)
- probe: ligase chain reaction (amplifies the nucleic acid used as the probe)
- post amplification analysis: sequencing of the amplified product, microarray analysis
Samples should be refrigerated and not frozen if labile viruses are suspected, these are
varicella zoster virus, influenza virus, HIV-2
unamplified testing occurs in which cases
where the sample has already been cultured, or the organism was present in high concentrations already
what are the 3 kinds of reporters for unamplified testing
- radioactive reporter
- biotin avidin reporter
- chemi-luminescent reporter
DNA microarrays
consist of molecules of immobilized ssDNA. Fluorescent labelled DNA washed over the array will adhere only to the locations where there are complementary DNA sequences
Uses of DNA microarrays
- monitoring gene expresssion
- diagnosis of infection
- ID of organisms in an environmental sample
Metagenomic sequencing
amplification is unbiased, so there are no primers for a specific infectious agent. The amplification is followed by NGS, then you compare between the genetic material found in sample with a large database of genomes of all pathogens
Chromatographic methods rely on
comparison of an organisms fatty acids to a database. They can be used to ID bacteria, mycobacteria, and fungi. Testing accuracy depends on the conditions used to culture the specimen and the quality of the database
mass spectrometry depends on
Id of specific macromolecules based on mass/charge ratio. This method is limited bc its not readily deployable
Automated blood culture depends on
change in color due to CO2 production
T/F chromatographic methods for pathogen identification are nucleic acid based
F
How to use chromatographic methods
microbial components or products are separated and ID by using high performance liquid chromatography (HPLC) or gas chromatography.
ID is done by comparison of organisms fatty acids to a database. Can ID an/aerobic, mycobacteris, fungi.
MALDI-TOF
is mass spectrometry
- results in <1 hr
- proteins are detected with a sensor to create a spectrum that represents the protein makeup of the whole sample. Used for CULTURED MICROORGANISMS
VITEK mass spectrometry works in minutes, and can provide single choice ID of species
The protein spectrum generated is
compared to spectra of a database.
The database is built to allow for
discrimination between species. This is
accomplished by including spectra for each
species generated from:
• An average of >14 isolates/species and
average of 36 spectra/species
• Geographical diversity of isolates
• Isolates of different sample origins (e.g.
blood, tissue, etc.)
• Isolates subcultured on different media
• Isolates with different incubation times
