Garlanda 4

Question 1

nucleic acid based methods detect

Answer 1

organism specific DNA or RNA

Question 2

multiplex assays

Answer 2

where a single nucleic acid based test can detect more than 2 causative organisms

Question 3

nucleic acid tests are quantitative or qualitative?

Answer 3

qualitative, but quantification methods exist for a limited number of infections (HIV, CMV, HTLV)

Question 4

nucleic acid quantitative tests are useful for

Answer 4

diagnosis and monitoring response for treatment

Question 5

tests for target amplification

Answer 5

PCR on DNA

RT-PCR on RNA and cDNA

Question 6

process of PCR has 3 steps? What can you use to automate them?

Answer 6

denaturation, priming, extension

Use a thermocycler

Question 7

Different kinds of amplification

Answer 7

• 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

Question 8

Samples should be refrigerated and not frozen if labile viruses are suspected, these are

Answer 8

varicella zoster virus, influenza virus, HIV-2

Question 9

unamplified testing occurs in which cases

Answer 9

where the sample has already been cultured, or the organism was present in high concentrations already

Question 10

what are the 3 kinds of reporters for unamplified testing

Answer 10

• radioactive reporter
• biotin avidin reporter
• chemi-luminescent reporter

Question 11

DNA microarrays

Answer 11

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

Question 12

Uses of DNA microarrays

Answer 12

• monitoring gene expresssion
• diagnosis of infection
• ID of organisms in an environmental sample

Question 13

Metagenomic sequencing

Answer 13

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

Question 14

Chromatographic methods rely on

Answer 14

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

Question 15

mass spectrometry depends on

Answer 15

Id of specific macromolecules based on mass/charge ratio. This method is limited bc its not readily deployable

Question 16

Automated blood culture depends on

Answer 16

change in color due to CO2 production

Question 17

T/F chromatographic methods for pathogen identification are nucleic acid based

Answer 17

F

Question 18

How to use chromatographic methods

Answer 18

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.

Question 19

MALDI-TOF

Answer 19

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

Question 20

VITEK mass spectrometry works in minutes, and can provide single choice ID of species

Answer 20

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