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PSYCH > Diagnostic Virology COPY > Flashcards

Diagnostic Virology COPY Flashcards

1
Q

Within the HTLV-1 virus particle, in which form is the genetic material?

A

ssDNA

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2
Q

What type of cell does HTLV-1 preferentially infect?

A

T cells

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3
Q

How does HTLV-1 replicate?

A

HTLV-1 can integrate within the chromosome of infected cells and replicate as dsDNA as part of the host cell division process

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4
Q

What are 3 possible routes to become infected with HTLV-1 ?

A

Blood transfusion
From mother to infant by breast feeding
Sexual Contact

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5
Q

What diseases can be caused by HTLV-1?

A

HTLV-1-associated myelopathy

Leukaemia

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6
Q

What are PCR methods used o detect HTLV-1 based on?

A

The presence of the tax gene

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7
Q

What are the three steps of the western blot method

A
  1. Separation
  2. Transfer
  3. Staining
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8
Q

What does the western blot method look for?

A

Presence of antibodies specific to the virus

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9
Q

Describe the separation stage of the western blot procedure?

A

Viral proteins separated based on size on plyacrylamide protein gel

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10
Q

Which proteins will migrate fastest on the polyacrylamide gel of the western blot?

A

The smaller proteins

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11
Q

What happens to the western blot after the viral proteins are separated by size and have formed bands?

A

Incubated with human serum - if the human serum contains antibodies it will bind to the viral proteins

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12
Q

What happens in the staining phase of the western blot method?

A

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

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13
Q

What must be detected for a positive result for HTLV-1?

A 
MTA1
p53
p24
p19
gd21
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14
Q

What are the five components of PCR based analysis?

A
  1. Buffer
  2. Nucleotides - dNTPs
  3. DNA POlymerases
  4. Primers
  5. DNA Template
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15
Q

What is the purpose of the buffer in PCR?

A

Provides an appropriate pH for reactions to occur / polymerase to work

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16
Q

What are the three steps of PCR?

A
  1. Denature
  2. Anneal
  3. Elongate
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17
Q

What temperature does denaturation occur at?

A

90 degrees - breaks H bonds between double-stranded DNA

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18
Q

What must the primer have?

A

A free hydroxyl group

19
Q

What is the temperature at which annealing takes place?

A

50 degrees

20
Q

What temperature does elongation occu at?

A

73-75 degrees

Allows Polymerase to work

21
Q

What DNA is used for PCR analysis?

A

PBMC - peripheral blood mononuclear cells = mixture of T cells B cell and NK cells

22
Q

What type of genetic material can by amplified by a standard PCR

A

dsDNA

ssDNA

23
Q

HTLV-1 has a single strand RNA genome, but a standard PCR can still be used for diagnosis. What is the reason for this?

A

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

24
Q

What 5 key components must be included in a PCR

mix

A 
DNA Template
Primers Pair
Taq Polymerase
reaction Buffer
Deoxynucleotides
25
How many grams of agarose do you need to make 50 ml of a 1% (weight/volume) agarose gel solution?
0.5
26
How do you estimate the size of your PCR product on the agarose gel?
Run a DNA ladder alongside your PCR reactions
27
What is the purpose of adding a loading buffer with dye to the PCR sample before loading on an agarose gel?1.
1. Makes samples sink into wells 2. Visualise how far the gel has migrated 3. Visualise which wells contain samples
28
What is the typical number of repeat cycles for the three steps in a CR reaction?
30-40
29
What type of nucleic acid genetic material do SARS-CoV-2 virus particles contain?
ssRNA
30
What two types of primers are added to PCR and during what step?
During annealing step, forward and reverse primers are needed
31
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
32
How long does the denaturation stage of PCR last?
1 minute
33
How long does the annealing stage of PCR last?
45 seconds
34
How long does the elongation stage of PCR last?
2 minutes
35
What is added during the extension stage of PCR?
deoxynucleotides - dNTPs
36
What is added to Quantative Real Time PCR that is not found in in normal PCR?
TaqMan Probe
37
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
38
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
39
What does the TaqMan Probe have attached to it?
A fluorophore and a quencher
40
What happens in qRT-PCR when the quencher and fluorophore get separated?
Probe degrades and becomes fluorescent
41
How does the fluorescence relate to the DNA amplified?
They are proportional - the more fluorescence produced, the more DNA that was amplified
42
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
43
What is the relationship between CT Values and tax gene copy number?
CT Values are linear with the Log10 of tax gene copy number
44
What is the purpose of adding a loading buffer with dye to the PCR sample before loading on an agarose gel?1.
1. Makes samples sink into wells 2. Visualise how far the gel has migrated 3. Visualise which wells contain samples

PSYCH (78 decks)

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