diagnostic Virology Flashcards
1
Q
- How is HTLV-1 transmitted?
- What type of genome is HTLV-1?
A
Mother to infant (breast feeding/during birth)
Sexual contact Blood (e.g. blood transfusion/sharing needles) ssRNA
2
Q
- Is HTLV-1 enveloped?
- What does reverse transcriptase do?
A
Yes
Converts RNA to DNA
3
Q
- What does HTLV-1 have instead of a viral homologue of a proto-oncogene?
- What is the function of this protein?
A
Viral Tax protein
Important function in viral transcription process as well as several other different activities in the host cells It can affect cell-cycle progression and signalling processes in cells which can lead to oncogenesis
4
Q
- Outline the replication cycle of HTLV-1?
A
HTLV-1 enters T cell
ssRNA released into host cell cytosol ssRNA reverse transcribed (RT-enzyme) to ssDNA ssDNA converted to dsDNA dsDNA enters the nucleus and integrate into host genome viral genome can replicate as part of the host chromosome
5
Q
- What type of cells do HTLV-1 preferentially infect?
- What does the number of T-cells containing HTLV-1 DNA correlate with?
A
T cells
Disease severity Likelihood of transmitting the virus
6
Q
- What is taken from patients to detect whether they are infected or not?
- What can the Western-blot method be used for?
A
Blood sample
Used to assess if patients have antibodies specific to HTLV-1 proteins
7
Q
- What is the 3rd step of the Western-blot method and outline the process?
A
Staining:
1. Incubate membrane with human serum (primary AB) 2. Wash membrane 3. Incubate membrane with secondary AB linked to enzyme 4. Wash membrane 5. Add substrate for enzyme linked to secondary AB
8
Q
- What are the antibodies produced against when there is a positive result for HTLV-1 protein detection by western-blot?
A
Synthetic peptide: MTA-1
Viral Core Proteins: p53 p24 p19 Recombinant glycoprotein: gd21
9
Q
- What is the aim of PCR?
- What does DNA polymerase need to act on, on a strand of DNA?
A
To amplify a specific piece of DNA
A primer with a free 3' hydroxyl group
10
Q
- Outline the steps of PCR (include temperatures)
A
Denaturing dsDNA (94 degrees C)
Primer annealing (54 degrees C) allowing primers to anneal to single strands Extending DNA strands at optimal temperature (72 degrees C) for thermostable DNA polymerase to extend DNA and produce second strand
11
Q
- What is the typical number of repeat cycles for the three steps in a PCR reaction?
- For standard PCR what are the different primers required?
A
30-40 times
Forward primer, Reverse primer
12
Q
- How does the size of the fragment affect the extension time?
A
Larger fragment = longer extension time
13
Q
- What direction are these primers written in?
A
5’ to 3’
Forward already written like this Reverse has to be written backwards as originally it is 3' to 5'
14
Q
- What are the 5 components of a PCR?
A
DNA template
Primers DNA polymerase dNTPs - building blocks of DNA and needed for polymerase to copy DNA strands Reaction buffers - provides appropriate salt and pH environment for polymerase enzyme to function
15
Q
- How are the sample results analysed?
A
DNA Gel electrophoresis
DNA is -vely charged and migrates towards positive anode Smaller fragments migrate faster Visualise DNA with intercalating DNA stain (Ethidium bromide - UV light) Add loading dye Add DNA marker - estimates size of fragment
