Introduction Flashcards
Define Molecular Diagnostics
A quantitative or qualitative measure of DNA/RNA or proteins
Which diagnostic testing methods rely on direct examination?
Electron microscopy, genome identification, antigen detection, bacterial culture
What are 3 ways to detect organisms?
Direct visualization (light or electron microscopy), detecting protein (ELISA, IFA, IHC), or detecting DNA or RNA (PCR, NGS)
How does an ELISA test work?
There is an antibody on the plate. If antigen is in the sample it will bind to the antibody resulting in a color change
How does an immunofluorescent antibody test (IFA) work?
The antibody has a fluorescent tag, and exists within the cell culture or tissues. A specific antibody is added and if the viral agent is present in the culture/tissue it will fluoresce under a fluorescent microscope
*requires high viral titer
Describe immunohistochemistry
It requires the use of paraffin embedded tissues mounted on glass slides. Sections are incubated with specific antibodies tagged with a substrate. If antigen is present in the tissue, the substrate will give a color reaction
What does RT-PCR require?
- DNA primers and probes that are specific to the target DNA
- Enough good quality target DNA or RNA to amplify
- Validated test methods
- Well maintained equipment
- Procedures for limiting cross contamination
How does PCR work?
A probe is programmed to recognize a certain strand of DNA in the sample. If such DNA is present, the quencher on the probe will be cleaved and the fluorophore on the probe will fluoresce. The more DNA in the sample that matches with the sequence the probe is targeting, the higher amount of the DNA is present in the sample
What does it mean if the threshold cycle is reached sooner for PCR?
There is a higher viral load in the sample
What is the unique feature of in-situ hybridization that other modalities do not allow you to do?
It allows you to visualize the virus in the tissue at the site of infection–> determination of which virus is responsible for pathologic lesions
Which modality is preferred for detection of unknown viruses?
Next generation sequencing
Describe how immunodiffusion works to detect antibodies?
Antigen is loaded at the center of the gel, test serum is loaded on the periphery, then a charge is added and antibodies move toward the antigen causing precipitation of a band
What modality can be used to determine if antibodies are from a vaccine vs from infection?
Virus neutralization- indicates the antibody was produced by infection if you get a positive
What does a 4 fold increase in antibody titer indicate?
Acute infection
What are some reasons that a test may not give the expected result?
Technical problem, early infection, false positive test, sequestering in tissues
