Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

POM > Diagnostic Virology > Flashcards

Diagnostic Virology Flashcards

1
Q

What is HTLV-1?

A 
Human T-Cell Leukaemia Virus type 1
Causes:
adult T cell leukaemia (ATL)
adult T cell leukaemia/lymphoma (ATLL)
HTLV-1 associated myelopathy (HAM)
Tropical spastic paraparesis (TSP)
HTLV-1 associated infectious dermatitis
HTLV-1 associated uveitis (HAU)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

How is HTLV-1 transmitted?

A

Mother to infant (breastfeeding/ Birth)
Sexual contact
Blood

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

How is HTLV-1 structured?

A

single strand enveloped RNA virus
HTLV-1 genetic information presented as double stranded DNA during infection
Virus encodes reverse transcriptase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is the tax gene?

A

Viral tax protein is a transactivator protein- induces transcription of viral mRNA and has a role in causing cancer
Found only in HTLV-1
Several PCR methods are designed to detect the tax gene of HTLV-1

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What types of cells does HTLV-1 infect and how does it infect them?

A

Infects T cells/ T helper cells
Once its infected cells its ssRNA will reach the cytoplasm
There reverse transcriptase will convert ssRNA to ssDNA which in converted to dsDNA
dsDNA will enter nucleus and integrate into the host’s genome allowing cell to replicate the viral genome

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is the western blot method?

A

If someone has developed antibodies to HTLV-1 these can be detected using the western blot method

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How is the western blot method carried out?

A
  1. Seperation
    - different viral proteins derived from in vitro viral cultures will be separated based on their size using polyacrylamide gel electrophoresis
    - smaller protein will migrate further
  2. Transfer
    - proteins transferred using electric transfer system onto PVDF membrane
    - viral proteins stick to membrane
    - different viral proteins present on distinct bands on the membrane- bands aren’t visible
  3. Staining
    - membrane incubated with protein serum
    - if antibodies recognise HTLV-1 protein they’ll bind to it
    - membrane washed to get rid of unbound antibodies and incubated with secondary antibody (conjugated to enzyme) which recognises Fc region of antibody
    - membrane washed again
    - substrate which will be recognised by enzyme added, enzyme will react to give a signal once bound to substrate
  4. Visualisation
    - membrane observed
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What does PCR stand for?

A

Polymerase chain reaction

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Whats the aim of PCR?

A

To amplify a specific piece of DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

How does PCR work?

A
  1. Denaturation
    - sample is heated to 95 degrees C to denature double strand DNA to produce single strand DNA
  2. Annealing
    - sample is cooled to 50-60 degrees. This allows primmer to anneal to DNA with complimentary sequence
    - For a standard PCR you need 2 primers- forward and reverse primer
  3. Extension
    - sample heated to 70-72 degrees- thermostable DNA polymerase extends DNA to produce second DNA strand
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How many times os this PCR cycle repeated?

A

30-40 times- machine takes about 2 hours

The DNA produced is separated on an agarose gel and stained and visulaised

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What primer is used to amplify the HTLV-1 tax gene?

A

HTLV-1 genome can be retrieved from data base
Forward primer is HL43 and has same sequence as top strand
Reverse primer is HL44 and has same sequence as bottom strand
The region amplifies is a 300bp region of the tax gene
Primers are written from 5’ to 3’

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are the components of PCR?

A
  1. DNA template
  2. Primers
  3. DNA polymerase
  4. dNTPs- building blocks of DNA
  5. Reaction buffer- appropriate salt and pH
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How is a patient sample for PCR prepared?

A

Blood taken and peripheral blood mononuclear cells (PBMCs) are isolated:
PBMCs is mix of lymphocytes
Blood is layered on separation medium and tube is centrifuged
Blood separated into plasma and PBMC fraction
PBMC fraction is taken and DNA is extracted from cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What other samples should be made alongside the patient sample?

A

Control positive: DNA sample with HTLV-1 virus

Control negative: DNA sample without HTLV-1 virus

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How are fragments analysed?

A

Using agarose electrophoresis
In electrophoresis:
-DNA is negatively charged and migrates towards positive anode- small fragments migrate further
-stain is added for visualisation of DNA

17
Q

What stains are commonly used for agarose electrophoresis?

A

Ethidium bromide- need UV light- toxic

Sybr safe- needs blue light

18
Q

What may be needed to load DNA onto agarose gel?

A

May need a DNA loading dye
This makes PCR solution heavier so it sinks to bottom of wells
It also has a colour dye allowing you to see it and estimate how far DNA has migrated on gel

19
Q

How do you determine size of PCR product?

A

You will need to run a DNA marker with samples

A DNA marker is a sample which has DNA fragments of known size

20
Q

What information can a qPCR give us?

A

Amount of viral DNA in a sample
Will help predict the severity of a disease and transmission likelihood
Can tell us how many cells in a patient sample have HTLV-1 in their DNA

21
Q

What are 2 common methods used for qPCR?

A

Florescence dye-based method (SYBR green dye)- fluoresce dye is added to reaction mixture- dye become fluorescent once bound to DNA

DNA probe based method (TaqMan method)

22
Q

How does qPCR work?

A

Increase in fluorescence is measured over time and in real time
Fluorescent signal is proportional to amount of viral DNA in sample
In an infected patient sample fluoresce will rise until to gets to a cycle threshold- this threshold isn’t reached in a negative sample

23
Q

Hw does the DNA probe qRT-PCR method work?

A

A third or oligo probe is added to mixture which binds to specific gene that’ll be amplified
Oligo binds to region between DNA region thats amplified by forward and reverse primers
Oligo contain flurophere and quencher molecule at 5’ and 3’ end- when the oligo is intact these molecules are close together
Flouresence of sample increases when flurophere and quencher molecule are separated
In PCR when DNA polymerase extends it’ll reach the oligo and degrade it causing flurophere and quencher molecule to be separated

24
Q

How is the TaqMan probe designed to detect HTLV-1 ?

A

TaqMan probe was designed to anneal similar to the forward primer
5” end of oligo is attached to flurophere (FAM)
3’ end of oligo is attached to quencher (BHQ1)

25
Q

How is data from the q-PCR used to workout how much tax gene is present?

A

A logarithmic graph of cycle threshold against gene number (X axis- log10 ) is produced
Once a standard curve is produced you can estimate the number of tax genes

POM (48 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions