Diagnostic Virology Flashcards
What is a serological test?
check blood/ antibodies present/ check viruses present
How were HTLV-1 infections originally detected?
HTLV-1 infections were originally detected using enzyme-linked immunosorbent assay (ELISA) assays.
In such an ELISA assay, viral antigens are detected with virus specific antibodies and the specificity of the assay can be further confirmed by Western-blot analysis.
A problem with the ELISA and Western-blot detection assay is that sometimes the results are inconclusive/indeterminate.
What method do we use now to detect HTLV-1 infections and what has been associated with a greater rate of trnasmission?
- HTLV-1 DNA in peripheral blood mononuclear cells (PBMCs)
- modified quantitative real time PCR (qRT-PCR)
Using this methods, proviral DNA can be detected in most HTLV-1 seropositive patients and its concentration is higher in patients with HAM than in asymptomatic carriers.
- Higher proviral load has been associated with a greater likelihood of transmission.
To detect proviral HTLV-1 DNA in patient samples, blood is taken from patients and PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCs) isolated from the whole blood sample. This is done by Ficoll gradient centrifugation, where red blood cells are separated from the plasma and PBMCs.
How is the qRT-PCR used to test the presence of the HTLV-1 virus?
blood is taken from patients and PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCs) isolated from the whole blood sample. This is done by Ficoll gradient centrifugation.
Next, the DNA is isolated from the PBMCs and used as template DNA in a PCR reaction.
In the first PCR reactions, primers designed to amplify the pol or tax gene of HTLV-I can be used. As part of our practicum, we will use primers to amplify the tax gene and we expect to see an approximately 300 bp DNA fragment if the patient is infected and no PCR product if the patient is not infected with HTLV-1.
As positive control for the PCR, DNA isolated from a cell line with integrated HTLV-1 DNA will be used and as negative control DNA isolated from PBMCs from an uninfected person will be used.
Within the HTLV-1 virus particle, in which form is the genetic material?
ssDNA
What type of cell does HTLV-1 preferentially infect?
T cells
How does HTLV-1 replicate?
HTLV-1 can integrate within the chromosome of infected cells and replicate as dsDNA as part of the host cell division process
What are 3 possible routes to become infected with HTLV-1 ?
Blood transfusion
From mother to infant by breastfeeding
Sexual Contact
What does HAM mean in severe form of HTLV-1 disease?
HTLV-1 associated myelopathy (myelopathy is injury to spinal cord)
What does ATLL mean in severe form of HTLV-1 disease?
Adult T-cell leukaemia/ lymphoma
What diseases can be caused by HTLV-1?
HTLV-1-associated myelopathy
Leukaemia
What are PCR methods used to detect HTLV-1 based on?
The presence of the tax gene
What is PCR?
polymerase chain reaction
A standard PCR reaction uses DNA as the starting material, with the aim of amplifying a specific region. A typical PCR reaction will have 30-40x cycles of the 3 main steps. The PCR product is visualised on an agarose gel using a stain that intercalates into the DNA and fluoresces under a specific light source.
What are the three steps of the western blot method
- Separation
- Transfer
- Staining
What does the western blot method look for?
Presence of antibodies specific to the virus
Describe the separation stage of the western blot procedure?
Viral proteins separated based on size on plyacrylamide protein gel
Which proteins will migrate fastest on the polyacrylamide gel of the western blot?
The smaller proteins
What happens to the western blot after the viral proteins are separated by size and have formed bands?
Incubated with human serum - if the human serum contains antibodies it will bind to the viral proteins
What happens in the staining phase of the western blot method?
Secondary antibodies which have an enzyme attached are added - they bind to the Fc region of the already bound antibodies
Then substrate is added which reacts with the enzyme to produce a signal
What must be detected for a positive result for HTLV-1?
MTA1
p53
p24
p19
gd21
- it means that antibodies against these proteins are present in the sample
What are the five components of PCR based analysis?
- Buffer
- Nucleotides - dNTPs (Deoxynucleoside triphosphates)
- DNA Polymerases
- Primers
- DNA Template
What is the purpose of the buffer in PCR?
Provides an appropriate pH for reactions to occur / polymerase to work
What are the three steps of PCR?
- Denature
- Anneal
- Elongate
What temperature does denaturation occur at?
90 degrees - breaks H bonds between double-stranded DNA
What must the primer have?
A free hydroxyl group
What is the temperature at which annealing takes place?
50 degrees
What temperature does elongation occur at?
73-75 degrees
Allows Polymerase to work
What DNA is used for PCR analysis?
PBMC - peripheral blood mononuclear cells = mixture of T cells B cell and NK cells
What type of genetic material can by amplified by a standard PCR
dsDNA
ssDNA
HTLV-1 has a single strand RNA genome, but a standard PCR can still be used for diagnosis. What is the reason for this?
During virus replication, viral RNA is reverse transcribed to ssDNA and converted to dsDNA that integrates into the host-cell genome. Viral infection can thus be determined by standard PCR using host cell DNA as template
What 5 key components must be included in a PCR mix?
DNA Template Primers Pair Taq Polymerase reaction Buffer Deoxynucleotides
How many grams of agarose do you need to make 50 ml of a 1% (weight/volume) agarose gel solution?
0.5
How do you estimate the size of your PCR product on the agarose gel?
Run a DNA ladder alongside your PCR reactions
What is the purpose of adding a loading buffer with dye to the PCR sample before loading on an agarose gel?
- Makes samples sink into wells
- Visualise how far the gel has migrated
- Visualise which wells contain samples
What is the typical number of repeat cycles for the three steps in a CR reaction?
30-40
What type of nucleic acid genetic material do SARS-CoV-2 virus particles contain?
ssRNA
What two types of primers are added to PCR and during what step?
During annealing step, forward and reverse primers are needed
What is assumed about the disease severity of HTLV-1?
Assumed that the number of T cells containing HTLV-1 correlate with the disease severity and also the likelihood of transmitting the virus
How long does the denaturation stage of PCR last?
1 minute
How long does the annealing stage of PCR last?
45 seconds
How long does the elongation stage of PCR last?
2 minutes
What is added during the elongation stage of PCR?
deoxynucleotides - dNTPs
What is added to Quantative Real Time PCR that is not found in in normal PCR?
TaqMan Probe
What extra information for qRT-PCR provide over normal PCR?
Provides information on the amount of viral DNA present in a sample and therefore helps predict the severity of disease
What does the TaqMan probe in qRT-PCR Bind to?
Binds to only the gene being amplified, in between the forward primer and reverse primer
What does the TaqMan Probe have attached to it?
A fluorophore and a quencher
What happens in qRT-PCR when the quencher and fluorophore get separated?
Probe degrades and becomes fluorescent
How does the fluorescence relate to the DNA amplified?
They are proportional - the more fluorescence produced, the more DNA that was amplified
How does qRT-PCR provide information on the quantity of viral load?
The fewer PCR cycles that are needed in order to reach the threshold of fluorescence (which is proportional to the amount of DNA amplified hence more viral load) indicates a higher viral load
What is the relationship between CT Values and tax gene copy number?
CT Values are linear with the Log10 of tax gene copy number
What is the purpose of adding a loading buffer with dye to the PCR sample before loading on an agarose gel?
- Makes samples sink into wells
- Visualise how far the gel has migrated
- Visualise which wells contain samples
What is the key function of Tax gene on HTLV-1?
viral transcription and oncogenesis
What is 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 nucleus and integrates into host genome
Viral genome can replicate as part of the host
chromosome
What are peripheral mononuclear cells (PMBCs) made of?
Mix of cells
Lymphocytes
T cells
B cells
NK cells
How does DNA gel electrophoresis work?
Separate DNA based on size
DNA is negatively charged and migrates towards the positive anode
Visualize DNA with intercalating DNA stain:
Ethidium bromide – UV light
Sybr Safe – blue light
Purpose of DNA loading dye:
Increase density/weight of the sample
See which well contains a sample
Indicate how far the DNA fragments have migrated during run
DNA marker/ladder:
Estimate the size of the PCR fragment
What is outline of how a PCR based analysis is usually performed?
- Set up a PCR reaction (10 min)
- Run PCR in thermocycler (2h)
- Prepare an agarose gel for analysis of PCR products (10 min + 30 min to solidify)
- Agarose gel electrophoresis to separate PCR product (20-30 min)
- Visualize and Image PCR products (10 min)
Interpret your PCR result and diagnose your patients
How does a PCR work?
1) Denature double stranded DNA template at high temperature – usually 94 C – 96 C
2) Anneal primers onto the DNA template by lowering the temperature – usually 45-65C
3) Extend the primers using a DNA-polymerase – to amplify the template – usually done at 72 C
Requirements for this step: A heat stable DNA polymerase which works in a specific reaction buffer and nucleotides dATP, dTTP, dGTP, dCTP
4) Steps 1 to 3 are repeated 30 to 35 times, this will amplify the DNA so that you obtain sufficient amounts for subsequent visualization
What is the purpose of a PCR?
The “Goal” of a PCR is to amplify a specific DNA fragment and the specificity comes from the primer set. The amplification of the DNA fragment will allow you to visualize this fragment afterwards.
What is being amplified when it comes to HTLV-1?
tax gene
Can you draw a typical qRT-PCR result curve and indicate some key parameters such as the cycle threshold (CT)?
Which way would the curve shift in a qRT-PCR reaction if you have more viral DNA in your sample and why?
Shift to the left- as threshold above the background would be reached earlier if more template DNA is present at the beginning of the reaction.
What is the advantage of using a qRT-PCR method compared to a standard PCR when diagnosing an infection?
It will not only give an indication if someone has an infection but also information on the amount of DNA of the infectious agent that is present
In the context of diagnosing HTLV-1 infection, why would it be important to know more about the amount of viral DNA present in the sample?
Knowing more about the amount of proviral DNA can give an indication of disease progressing. In the case of HTLV-1, the proviral DNA concentration is usually higher in patients with HAM than in asymptomatic carriers. Furthermore, higher proviral load has been associated with a greater likelihood of transmission.
CAn you use the isolated RNA material directly for a PCR based detection method, if not what do you need to add?
No, use reverse transcriptase to make a ssDNA template
