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POM > Diagnostic virology > Flashcards

Diagnostic virology Flashcards

1
Q

What is HTLV-1 ?

A

Human T-cell leukaemia virus type I - enveloped virus - ssRNA genome

Has viral proteins encoded on its RNA molecule

Genetic info will be present during infection as double stranded DNA

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2
Q

How is HTLV-1 spread? What cells does it preferentially invade?

A

Spread from cell to cell contact
Mother to infant - breast feeding /during birth

sexual contact

Blood transfusion

Infects T cells/ T helper cells

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3
Q

What are the key functions of HTLV-1?

A

virus assembly and maturation
Viral entry
Viral persistence
Viral infectivity and persistence

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4
Q

What diseases can be caused by HTV1?

A 
Adult T-cell leukaemia (ATL)
Adult T-cell leukaemia lymphoma (ATLL)
HTLV-1 associated myelopathy 
tropical spastic paraparesis 
HTLV-1 associated infectious dermatitis 
associated uveitis
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5
Q

What is the transforming entity of HTLV-1 that causes cancer?
What else does this gene do?

A

Viral tax protein - Viral transcription and oncogenesis

Affects cell cycle progression and signalling processes in the cell, both of which lead to oncogenesis

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6
Q

What is the replication cycle of HTLV-1?

A

HTLV-1 enters T-cell and releases ssRNA into the host cell cytosol
ssRNA reverse transcribed (RT-enzyme) to ssDNA
ssDNA converted to dsDNA which enters nucleus and integrates into host genome where it replicates with host DNA

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7
Q

Why is it important to determine viral load of a virus (ie number of T-cells containing HTLV-1 DNA)?

A

Correlates with…
disease severity
Likelihood of transmitting virus
Likelihood of cancer

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8
Q

What does the western blot method test for? What proteins need to be present for a positive result?

A

Assesses if patients have antibodies to specific HTLV-1 proteins

MTA-1 -synthetic peptide

P53, P24, P19 - viral core protein

gd21 - recombinant glycoprotein

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9
Q

What are the steps to a western blot?

A
  1. Viral proteins from cultures will be separated based on size on polycrylamide protein gel
    Smaller proteins migrate more than bigger
  2. Transferred using electric transfer system onto a PVDF membrane and the proteins will create an image - NOT VISIBLE
  3. staining -
    - membrane is incubated with the human serum (primary AB)
    - membrane washed to remove unbound AB
    - Incubate membrane with a second antibody thats linked to an enzyme
    - membrane washed to remove unbound AB
    - Substrate for enzyme linked to second antibody added to generate an output signal that can be seen
  4. Visualisation
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10
Q

Explain the three steps of the PCR reaction?

A

Step 1 - Denaturation at 94 degrees for one minute
heating produces single stranded DNA

Step 2- annealing at 54 degrees
allows primers to anneal to DNA with complimentary sequence

Step 3 - extension 72 degrees
Heated to optimal temp for thermostable DNA polymerase to extend DNA using nucleotides and produce a second strande

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11
Q

What are the key reagents used in PCR?

A

DNA template

Primers (forwards and reverse)

DNA polymerase - heat resistant

dNTPs - nucleotides

Reaction buffer - maintains optimum pH for polymerase

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12
Q

How is a sample prepared for pcr?

A

Blood collected

peripheral blood mononuclear cells (PBMCs) are isolated through centrifugation

Dna is isolated - by cell lysis, capture and cleaning of DNA and then elution

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13
Q

What is the purpose of DNA gel electrophoresis?

A

Separate DNA based on size

DNA is negatively charged so migrates towards the positive anode

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14
Q

What is the primer designed to amplify in HTLV-1?

A

Tax gene

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15
Q

What DNA loading dyes are used in gel electrophoreies and why?

A

To visualise DNA
ethidium bromide - UV light
Sybr safe - blue light

Purpose
increases weight of sample
See which well contains a sample
Indicate how far the fragments have migrated

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16
Q

What is a DNA ladder and what is its purpose?

A

Contains diff fragments with known size

Estimates size of PCR fragment

17
Q

What information does qRT-PCR give and why is this useful?

A

Provides information on amount of viral DNA present in sample

Will help to predict severity of disease
Will help to predict transmission of likelihood

18
Q

Name the two common qRT-PCR methods

A

Fluorescence dye-based method

DNA probe based method (TaqMan method)

19
Q

Explain the fluorescence dye-based method for qRT-PCR (SYBR green dye method)

A

Fluorescent dye added to mixture, non fluorescent in solution but when bound to DNA is fluorescent
increase in fluorescent signal is proportion to DNA in sample and is measure over time

20
Q

When is the DNA probe based method (TaqMan method) used?

A

oligo increases specificity to assay thus often used for diagnostic purposes

more expensive

21
Q

Explain the DNA probe based method (TaqMan method)

A

In addition to the two primers a third oligo probe is added

This binds to a region between the gene being amplified and contains a fluorophore and quencher

As the fluorophore and quencher become physically separated the fluorescence increases

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

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

A

Run a DNA ladder alongside your PCR reactions

25
Q
  1. What type of nucleic acid genetic material do SARS-CoV-2 virus particles contain?
A

ssRNA

26
Q

Once you have collected the patient sample what type of material would you isolate from it suitable for use in a SARS-CoV-2 PCR?

A

RNA

POM (42 decks)

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