diagnostic Virology Flashcards
- How is HTLV-1 transmitted?
- What type of genome is HTLV-1?
Mother to infant (breast feeding/during birth)
Sexual contact Blood (e.g. blood transfusion/sharing needles) ssRNA
- Is HTLV-1 enveloped?
- What does reverse transcriptase do?
Yes
Converts RNA to DNA
- What does HTLV-1 have instead of a viral homologue of a proto-oncogene?
- What is the function of this protein?
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
- Outline the replication cycle of HTLV-1?
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
- What type of cells do HTLV-1 preferentially infect?
- What does the number of T-cells containing HTLV-1 DNA correlate with?
T cells
Disease severity Likelihood of transmitting the virus
- What is taken from patients to detect whether they are infected or not?
- What can the Western-blot method be used for?
Blood sample
Used to assess if patients have antibodies specific to HTLV-1 proteins
- What is the 3rd step of the Western-blot method and outline the process?
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
- What are the antibodies produced against when there is a positive result for HTLV-1 protein detection by western-blot?
Synthetic peptide: MTA-1
Viral Core Proteins: p53 p24 p19 Recombinant glycoprotein: gd21
- What is the aim of PCR?
- What does DNA polymerase need to act on, on a strand of DNA?
To amplify a specific piece of DNA
A primer with a free 3' hydroxyl group
- Outline the steps of PCR (include temperatures)
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
- 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?
30-40 times
Forward primer, Reverse primer
- How does the size of the fragment affect the extension time?
Larger fragment = longer extension time
- What direction are these primers written in?
5’ to 3’
Forward already written like this Reverse has to be written backwards as originally it is 3' to 5'
- What are the 5 components of a PCR?
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
- How are the sample results analysed?
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
- What is the purpose of the DNA loading dye?
Increase density/weight of the sample to allow the samples to sink into wells
See which wells contain a sample Indicate how far DNA fragments have migrated during run
- What type of genetic material can by amplified by a standard PCR?
dsDNA
ssDNA
- What are each of the layers of the centrifuged sample show from top to bottom?
Plasma
PBMC Separation medium granulocytes RBCs
- How is a sample prepared for PCR to detect HTLV-1 Tax gene?
Take blood - peripheral blood to get Peripheral Blood Mononuclear Cells (PBMCs) which are a mix of lymphocytes and monocytes
Isolate PBMCs - blood layered on separation medium with a specific density and tube is subject to centrifugation step. Blood will be separated into PBMC and plasma to isolate the PBMCs Isolate DNA - Cell lysis and degradation of sample → Capture and cleaning of DNA → elution of DNA
- What information does qRT-PCR provide us with and how can we use it?
Amount of viral DNA present in sample
Helps to predict severity of disease Will help to predict transmission likelihood
- What are the 2 common methods of qRT-PCR?
- What is added in qRT-PCR TaqMan method that is not added in normal PCR?
Fluorescent dye-based method (SYBR Green dye method)
DNA probe-based method (TaqMan method)
Oligo probe with a fluorophore attached on 5' end and a quencher attached on the 3' end
- Why is no fluorescence detected at the start of qRT-PCR?
Because the quencher quenches the fluorescence so no fluorescence can be detected
- Explain the TaqMan method of qRT-PCR
Polymerisation & Strand Displacement - Oligo TaqMan probe binds to DNA template strand in a region between the forward and reverse primer
Probe Cleavage - As the DNA polymerase acts on the DNA strand it displaces the TaqMan probe which leads to the fluorophore breaking off from the probe, giving off fluorescence as the quencher and fluorophore are no longer in close proximity Completion of Polymerisation - The DNA polymerase acts further which also displaces the rest of the oligo probe and quencher
- What is the problem with qRT-PCR?
- What does CT value mean in qRT-PCR?
It is very expensive
Threshold value is cycle number at which fluorescence generated within a reaction crosses the fluorescence threshold (signal above background fluorescence)
- How can you identify a high number of viral DNA?
By looking at the amount of fluorescence detected
e.g Low CT value = High HTLV-1 load
- If you have viral RNA and you want to detect it, what process would you use?
RT-qRT-PCR (Reverse Transcription Quantitative Real Time PCR)
