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POM 4 Immunology > 4.11 Diagnostic Virology > Flashcards

4.11 Diagnostic Virology Flashcards

1
Q

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

A

ssRNA

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2
Q

What type of cell does HTLV-1 preferentially infect?

A

T cells

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3
Q

What proteins does HTLV-1 produce?

A

Reverse transcriptase
Tax protein

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4
Q

What does the Tax protein cause?

A

Induces viral transcription and leads to oncogenesis

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5
Q

How does HTLV-1 replicate?

A

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

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6
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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7
Q

What diseases can be caused by HTLV-1?

A

HTLV-1-associated myelopathy
Leukaemia

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8
Q

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

A

The presence of the tax gene

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9
Q

What does number of T cells with HTLV-1 DNA correlate with (2 things) and what is this important for?

A

Correlates with disease severity and likelihood of transmission

Helps gain information on viral load for diagnosing patients

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10
Q

Why is no free virus required for transmission?

A

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

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11
Q

What are the three steps of the western blot method?

A
  1. Separation
  2. Transfer
  3. Staining
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12
Q

What does the western blot method look for?

A

Presence of antibodies specific to the virus

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13
Q

What happens in the separation stage of the western blot procedure?

A

Viral proteins separated based on size on polyacrylamide protein gel

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14
Q

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

A

The smaller proteins

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15
Q

What happens in the transfer stage of the western blot procedure?

A

Proteins transferred onto PVDF membrane and become immobilised in bands but aren’t visible yet

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16
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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17
Q

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

A

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

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18
Q

What is the output signal from the western blot method?

A

Luminescent signal detected with a special camera

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19
Q

Which (5) HTLV-1 proteins do patients need antibodies against to be classed as positive for the criteria we’re using?

A

Synthetic peptide MTA-1
Viral core proteins p53, p24, p19
Recombinant glycoprotein gd21

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20
Q

Why is PCR needed in addition to the western blot method?

A

Indeterminate serological HTLV-1 tests may occur where there are reactive patterns to some but not all of the HTLV-1 proteins

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21
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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22
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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23
Q

What are the three steps of PCR?

A
  1. Denaturation
  2. Annealing
  3. Extension
24
Q

What temperature does denaturation occur at and why?

A

95 degrees – breaks H bonds between double-stranded DNA

25
Q

What must the primer have?

A

A free hydroxyl group

26
Q

What is the temperature at which annealing occurs and why?

A

50 degrees – allows primer to anneal with the complementary DNA sequence

27
Q

What temperature does extension occur at and why?

A

72 degrees – allows polymerase to work

28
Q

What DNA is used for PCR analysis?

A

DNA is isolated from peripheral blood mononuclear cells

This is a mixture of T cells, B cell and NK cells

29
Q

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

A

dsDNA and ssDNA

30
Q

HTLV-1 has a single stranded 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

31
Q

What are the names of the primers used to amplify the HTLV-1 gene?

A

Forward – HL43
Reverse – HL44

32
Q

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

A

0.5

33
Q

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

A

Run a DNA ladder alongside your PCR reactions

34
Q

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

A
  1. Makes samples sink into wells by increasing mass
  2. Visualise how far the gel has migrated
  3. Visualise which wells contain samples
35
Q

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

A

30-40

36
Q

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

A

ssRNA

37
Q

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

A

During annealing step, forward and reverse primers are needed

38
Q

How long does the denaturation stage of PCR last?

A

1 minute

39
Q

How long does the annealing stage of PCR last?

A

45 seconds

40
Q

How long does the elongation stage of PCR last?

A

2 minutes

41
Q

What is added during the extension stage of PCR?

A

Deoxynucleotides – dNTPs

42
Q

What is the enzyme commonly used as DNA polymerase in PCR and why?

A

Taq polymerase because it is heat resistant

43
Q

How are PCR DNA samples analysed?

A

Gel electrophoresis where DNA is separated based on size

Since it is negatively charged, it moves towards the positive electrode

44
Q

How do we visualise DNA in gel electrophoresis?

A

Intercalating DNA stain

Ethidium bromide for UV, sybr safe for blue light

45
Q

What other samples must be prepared alongside the patient’s during PCR?

A

Positive control –DNA sample containing HTLV-1
Negative control – DNA sample free of HTLV-1

46
Q

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

A

TaqMan Probe

47
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 and likelihood of transmission

48
Q

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

A

Binds to the gene being amplified in a region between the forward primer and reverse primer

49
Q

What does the TaqMan Probe have attached to it?

A

A fluorophore and a quencher

50
Q

What happens in qRT-PCR during probe cleavage?

A

DNA polymerase separates the fluorophore and the quencher

Probe degrades and becomes fluorescent

51
Q

How does the fluorescence relate to the DNA amplified?

A

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

52
Q

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

A

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

53
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

54
Q

Which cells can be infected by SARS-CoV-2?

A

Nasopharyngeal, lung, intestinal

55
Q

What type of patient samples could you take to detect SARS-CoV-2 using a PCR-based method?

A

Sputum, nasal, stool

56
Q

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?

A

Spike protein

Angiotensin converting enzyme 2

57
Q

What viral protein is often used for the development of a PCR-based detection for SARS-COV2 virus?

A

Nucleocapsid

POM 4 Immunology (14 decks)

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