4.11 Diagnostic Virology Flashcards
Within the HTLV-1 virus particle, in which form is the genetic material?
ssRNA
What type of cell does HTLV-1 preferentially infect?
T cells
What proteins does HTLV-1 produce?
Reverse transcriptase
Tax protein
What does the Tax protein cause?
Induces viral transcription and leads to oncogenesis
How does HTLV-1 replicate?
The ssRNA of HTLV-1 is reverse transcribed to dsDNA which integrates into the nucleus of the infected cell and replicates as a part of the host chormosome
What are 3 possible routes to become infected with HTLV-1 ?
Blood transfusion
From mother to infant by breast feeding
Sexual contact
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 does number of T cells with HTLV-1 DNA correlate with (2 things) and what is this important for?
Correlates with disease severity and likelihood of transmission
Helps gain information on viral load for diagnosing patients
Why is no free virus required for transmission?
Viral genome can be integrated into host genetic information
Thus infected T cells in blood are why HTLV-1 can be transmitted via blood transfusions
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
What happens in the separation stage of the western blot procedure?
Viral proteins separated based on size on polyacrylamide protein gel
Which proteins will migrate fastest on the polyacrylamide gel of the western blot?
The smaller proteins
What happens in the transfer stage of the western blot procedure?
Proteins transferred onto PVDF membrane and become immobilised in bands but aren’t visible yet
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 conjugated with an enzyme 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 is the output signal from the western blot method?
Luminescent signal detected with a special camera
Which (5) HTLV-1 proteins do patients need antibodies against to be classed as positive for the criteria we’re using?
Synthetic peptide MTA-1
Viral core proteins p53, p24, p19
Recombinant glycoprotein gd21
Why is PCR needed in addition to the western blot method?
Indeterminate serological HTLV-1 tests may occur where there are reactive patterns to some but not all of the HTLV-1 proteins
What are the five components of PCR based analysis?
- Buffer
- Nucleotides - dNTPs
- 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?
- Denaturation
- Annealing
- Extension
What temperature does denaturation occur at and why?
95 degrees – breaks H bonds between double-stranded DNA
What must the primer have?
A free hydroxyl group
What is the temperature at which annealing occurs and why?
50 degrees – allows primer to anneal with the complementary DNA sequence
What temperature does extension occur at and why?
72 degrees – allows polymerase to work
What DNA is used for PCR analysis?
DNA is isolated from peripheral blood mononuclear cells
This is a mixture of T cells, B cell and NK cells
What type of genetic material can by amplified by a standard PCR?
dsDNA and ssDNA
HTLV-1 has a single stranded 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 are the names of the primers used to amplify the HTLV-1 gene?
Forward – HL43
Reverse – HL44
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 by increasing mass
- 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
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 extension stage of PCR?
Deoxynucleotides – dNTPs
What is the enzyme commonly used as DNA polymerase in PCR and why?
Taq polymerase because it is heat resistant
How are PCR DNA samples analysed?
Gel electrophoresis where DNA is separated based on size
Since it is negatively charged, it moves towards the positive electrode
How do we visualise DNA in gel electrophoresis?
Intercalating DNA stain
Ethidium bromide for UV, sybr safe for blue light
What other samples must be prepared alongside the patient’s during PCR?
Positive control –DNA sample containing HTLV-1
Negative control – DNA sample free of HTLV-1
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 and likelihood of transmission
What does the TaqMan probe in qRT-PCR Bind to?
Binds to the gene being amplified in a region 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 during probe cleavage?
DNA polymerase separates the fluorophore and the quencher
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 viral load?
The fewer PCR cycles that are needed in order to reach the threshold of fluorescence (proportional to the amount of DNA amplified hence viral load) indicates a higher viral load
The cycle number is the CT value
What is the relationship between CT Values and tax gene copy number?
CT Values are linear with the Log10 of tax gene copy number
Which cells can be infected by SARS-CoV-2?
Nasopharyngeal, lung, intestinal
What type of patient samples could you take to detect SARS-CoV-2 using a PCR-based method?
Sputum, nasal, stool
What is the name of the key virus protein that SARS-CoV2 uses to bind to host cells? What is the name of the corresponding host cell receptor it binds to?
Spike protein
Angiotensin converting enzyme 2
What viral protein is often used for the development of a PCR-based detection for SARS-COV2 virus?
Nucleocapsid
