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Immuno > Diagnostic Virology > Flashcards

Diagnostic Virology 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
Q

How many grams of agarose do you need to make 50 ml of a 1% (weight/volume) agarose gel solution?

A

0.5

26
Q

How do you estimate the size of your PCR product on the agarose gel?

A

Run a DNA ladder alongside your PCR reactions

27
Q

What is the purpose of adding a loading buffer with dye to the PCR sample before loading on an agarose gel?1.

A
  1. Makes samples sink into wells
  2. Visualise how far the gel has migrated
  3. Visualise which wells contain samples
28
Q

What is the typical number of repeat cycles for the three steps in a CR reaction?

A

30-40

29
Q

What type of nucleic acid genetic material do SARS-CoV-2 virus particles contain?

A

ssRNA

30
Q

What two types of primers are added to PCR and during what step?

A

During annealing step, forward and reverse primers are needed

31
Q

What is assumed about the disease severity of HTLV-1?

A

Assumed that the number of T cells containing HTLV-1 correlate with the disease severity and also the likelihood of transmitting the virus

32
Q

How long does the denaturation stage of PCR last?

A

1 minute

33
Q

How long does the annealing stage of PCR last?

A

45 seconds

34
Q

How long does the elongation stage of PCR last?

A

2 minutes

35
Q

What is added during the extension stage of PCR?

A

deoxynucleotides - dNTPs

36
Q

What is added to Quantative Real Time PCR that is not found in in normal PCR?

A

TaqMan Probe

37
Q

What extra information for qRT-PCR provide over normal PCR?

A

Provides information on the amount of viral DNA present in a sample and therefore helps predict the severity of disease

38
Q

What does the TaqMan probe in qRT-PCR Bind to?

A

Binds to only the gene being amplified, in between the forward primer and reverse primer

39
Q

What does the TaqMan Probe have attached to it?

A

A fluorophore and a quencher

40
Q

What happens in qRT-PCR when the quencher and fluorophore get separated?

A

Probe degrades and becomes fluorescent

41
Q

How does the fluorescence relate to the DNA amplified?

A

They are proportional - the more fluorescence produced, the more DNA that was amplified

42
Q

How does qRT-PCR provide information on the quantity of viral load?

A

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
Q

What is the relationship between CT Values and tax gene copy number?

A

CT Values are linear with the Log10 of tax gene copy number

44
Q

What is the purpose of adding a loading buffer with dye to the PCR sample before loading on an agarose gel?1.

A
  1. Makes samples sink into wells
  2. Visualise how far the gel has migrated
  3. Visualise which wells contain samples

Immuno (14 decks)

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