Brainscape's Knowledge GenomeTM

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


See full index

PoM (My deck) > Immuno - Diagnostic Virology > Flashcards

Immuno - Diagnostic Virology Flashcards

1
Q

What does HTLV-I stand for?

A

Human T-cell leukaemia virus type I

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

What family is the virus part of?

A

Delta retrovirus family

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

What is HTLV-I the causative agent of?

A

Tropical Spastic Paraparesis (TSP)
HTLV-I associated myelopathy (HAM)
adult T-cell leukaemia/lymphoma (ATLL)

other inflammatory diseases such as:

  • HTLV-I associated infectious dermatitis
  • HTLV-I-associated uveitis (HAU)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What kind of gene does the virus not harbour?

A

a viral homologue of a cellular proto-oncogene

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

What protein is the transforming entity ascribed to?

A

Viral Tax protein

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

What is the Tax protein and what does it affect?

A

It is a nuclear phosphoprotein and it can affect cell-cycle progression, cyclic AMP and nuclear factor kB (NF-kB)

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

Modes of Infection with HTLV-I virus

A

Cell to cell contact primarily:

  • Mother-infant (mainly through breast feeding)
  • Sexual contact
  • Parenteral transmission (other than the digestive system)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the dominant contributor to increase in number of HTLV-I?

A

Not de novo infection of cells but rather clonal proliferation

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

Describe the HTLV-I genetic material?

A

Monopartite, linear, dimeric, ssRNA(+) with a 5’-cap and a poly-A tail

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

Function of the 5’ cap

A

Added to the first nucleotide during transcription and it’s a modified Guanine nucleotide. It protects the transcript from being broken down and it also facilitates the attachment of the mRNA to the ribosome.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Role of the poly-A tail

A

A chain of adenine nucleotides added to mRNA in order to increase its stability and prevent it from being broken down.

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

What sample is needed for the detection of HTLV-I in patients?

A

Blood from which peripheral blood mononuclear cells (PBMCs) are isolated using Ficoll gradient centrifugation.

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

What is the first step of the PCR

A

Primers are designed to amplify the pol or tax gene of HTLV-I

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What does a PCR tube contain?

A

DNA template containing target, primers, free deoxynucleotides and DNA polymerase

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

Describe the steps of heating and cooling of the mixture

A
  1. Heated to 95 degrees to separate the DNA strands
  2. Cooled to 55 degrees so primers anneal to ssDNA
  3. Heated back up to 72 degrees so DNA polymerase starts to synthesise new strands of DNA starting from primers
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

what are the Tax and Rex proteins in HTLV-I used for?

A

Rex - positive post-transcriptional regulation

Tax - Viral transcription and oncogenesis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What does the number of infected cells correlate to?

A

Disease severity

Likelihood of transmission

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What is the separation method behind the Western blot?

A

Separated based on size on polyacrylamide protein gel so the smaller the DNA chain is the further it will go

PVDF membrane used

19
Q

How do you transfer the bands onto a PVDF membrane from the gel in Western blot?

A

Using electricity

20
Q

What is a positive HTLV-I test?

A

Bands for:

  • synthetic peptide - MTA-1
  • viral core proteins - p53, p24, p19
  • recombinant glycoprotein - gd21

Needs to have all of them otherwise the sample is inconclusive and requires further PCR analyses

21
Q

What key feature must the primer include?

A

A free 3’ hydroxyl group because the DNA polymerase extends strand in 5’ to 3’ direction

22
Q

What needs to be adjusted if the DNA fragment is larger/

A

Incubation time with temperature changes

23
Q

How are primer sequences written and what needs to be done to transform the reverse primer?

A

From 5’ to 3’ end so the reverse primer sequence needs to be written in reverse

24
Q

What sort of genetic material does PCR require

A

dsDNA or ssDNA

25
Q

What buffer is needed in the PCR

A

Salt buffer and pH for enzyme function

26
Q

What are PBMCs a mix of?

A

Monocytes

Lymphocytes - T, B and NK-cells

27
Q

What is used to separate the PBMCs from the blood plasma?

A

Separation medium with specific cell density which separates plasma on top then PBMCs and underneath it Granulocytes and finally RBCs

28
Q

What DNA polymerase is used in PCR and why?

A

Taq polymerase from thermophillic bacterium Thermus aquaticus bcs it’s resistant to the high temperatures

29
Q

Which electrode does DNA migrate to and why?

A

DNA migrates to positive anode because it’s negatively charged

30
Q

What stains are used to visualize DNA?

A

Ethidium bromide - UV light (toxic so not used anymore)

Sybr Safe - blue light

31
Q

What is the purpose of DNA loading dye?

A

Increase density/weight of sample so DNA sinks to bottom of wells properly
See which well contains a sample
Indicate how far the DNA fragments have migrated during run e.g. bromophenol blue migrates similar to 300 b.p. DNA fragment

32
Q

What else is essential in gel electrophoresis?

A

DNA marker/ladder

33
Q

What else is essential to be set up when performing the Gel electrophoresis?

A

Negative and Positive control samples

34
Q

What voltages is the electrophoresis run at?

A

80-100 so the gel doesn’t melt

35
Q

What determines the size of the pores on the agarose gel?

A

Its concentration which is usually 0.7% and 2.0%

the shorter the DNA fragment the higher the concentration needs to be

36
Q

What buffer is used for longer electrophoresis runs?

A

TBE buffer

NB. fill gel electrophoresis chamber with the same buffer used to prepare gel

37
Q

How do you increase the resolution of the DNA bands?

A

Wider bands (wider teeth ot thinner) or increasing length of running time

38
Q

Difference between PCR and qRT-PCR

A

The latter gives information on amount of viral DNA present in a sample which helps predict severity of disease and likelihood of transmission

39
Q

What happens during qRT-PCR?

A

Instead of doing gel electrophoresis at the end of the 30-40 cycle reaction the readings are shown throughout

Using fluorescent SYBR Green dye method or DNA probe-based method (TaqMan method)

40
Q

What is the TaqMan method?

A

Third oligoprobe added to the mix with a fluorophore molecule at the 5’ end and a quencher at the 3’ end

It sits between the forward and reverse primers and fluorescence occurs when reporter and quencher dye are no longer in close proximity (v. low at the beginning)

41
Q

Nomenclature of the reporter and quencher dyes

A

reporter - FAM (6-carboxyfluorescein)

quencher - BHQ1 (black hole quencher 1)

42
Q

What cells can be infected with SARS-Cov-2

A

intestinal, lung, nasopharengeal

43
Q

What type of samples do you take to detect COVID using PCR?

A

stool, nasal, sputum

PoM (My deck) (2 decks)

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