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PoM > Immunology - Diagnostic Virology > Flashcards

Immunology - Diagnostic Virology Flashcards

1
Q

qRT - PCR meaning

A

quantitative real time PCR (DNA template) or quantitative reverse transcription PCR (RNA template, RT- = real time)

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2
Q

PCR stands for

A

Polymerase chain reaction

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3
Q

Standard PCR starting material

A

DNA

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4
Q

PCR aim

A

amplifying a specific region

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5
Q

PCR cycles

A

30-40x of 3 main steps

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6
Q

PCR product visualisation

A

On agarose gel using stain that intercalates into the DNA and fluorescent under a specific light source

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7
Q

PCR steps

A

1: denaturing dsDNA at 95ºC (1 minute)
2: primer annealing at 55ºC (45ºC, forward and reverse primers)
3: extending DNA strands at 72 ºC (2 minutes but length varies based on sample size, only dNTPs)

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8
Q

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

A

ssRNA

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9
Q

Which type of cell does HTLV-1 preferentially infect?

A

T cells/T-helper cells

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10
Q

HTLV-1 replication

A

Can integrate within the chromosome of infected cells and replicate as dsDNA as part of the host cell division process

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11
Q

Possible routes of HTLV-1 infection

A

Blood transfusion, from mother to infant by breast feeding, sexual contact

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12
Q

HAM in severe forms of HTLV-1 disease indicates…

A

HTLV-1-associated myelopathy

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13
Q

ATLL in severe forms of HTLV-1 disease indicates…

A

Adult T-cell leukaemia/lymphoma

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14
Q

HTLV-1 meaning

A

Human T-cell Leukaemia Virus Type I

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15
Q

Identification of HTLV-1

A

Mid-late 1970s Japan, associated with adult T-cell leukaemia (ATL) cases, first virus associated with human malignancy

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16
Q

Prevalence of HTLV-1

A

15-20 million ppl, worldwide distribution but more prevalent in some areas (Japan, Caribbean, Iran, Romania), approximately 5% will develop ATL

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17
Q

Diseases caused by HTLV-1

A

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 (HUS)

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18
Q

Type of virus (HTLV-1)

A

enveloped

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19
Q

Genetic info during HTLV-1 infection

A

DNA

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20
Q

Gag, Pro, Pol proteins (HTLV-1)

A

virion assembly and maturation

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21
Q

Env proteins (HTLV-1)

A

viral entry and transformation tropism

22
Q

p13 protein (HTLV-1)

A

viral persistence

23
Q

p13 protein (HTLV-1)

A

viral infectivity and persistence

24
Q

Rex protein (HTLV-1)

A

positive post-transcriptional regulation

25
Q

Tax protein (HTLV-1)

A

viral transcription and oncogenesis, instead of viral homologue of cellular proto-oncogene, tested for in several PCR-based methods because specific to HTLV-1

26
Q

p30 protein (HTLV-1)

A

transcriptional and post-transcriptional regulation

27
Q

Replication cycle (HTLV-1)

A
  • HTLV-1 enters T-cell
  • ssRNA released into host cell cytosol
  • ssRNA rveersse transcribed (RT-enzyme) to ssDNA
  • ssDNA converted to dsDNA
  • dsDNA enters nucleus and integrates into host genome
  • viral genome can replicate as part of host chromosome
28
Q

Dominant contributor to increase in number of HTLV-1 infected cells

A

clonal proliferation and host cell division rather than de novo infection of cells with free virus particles but some DNA can be transcribed to RNA and released

29
Q

Number of T-cells containing HTLV-1 DNA correlates with…

A

disease severity, cancer likelihood, likelihood of transmitting the virus

30
Q

Western-blot method

A

Used to assess if patients have antibodies specific to HTLV-1 proteins (in serum), steps:

1: separation (in polyacrylamide protein gel, smaller proteins migrate faster)
2: transfer (with electric system on PVDF membrane, protein show as bands, PVDF membrane and gel between filter paper and sponge with positive and negative plates)
3: staining (primary antibody binds to protein, , secondary antibody then binds alongside enzyme, converting substrate to signal)
4: visualization (output signal = color reaction, generation of precipitate, luminescent signal detected with special camera housed in Western-blot imager)

31
Q

Western-blot method - staining

A

1: incubate membrane with human serum (primary antibody (AB))
2: wash membrane
3: incubate membrane with secondary AB linked to enzyme
4: wash membrane
5: add substrate for enzyme linked to secondary AB

32
Q

Positive result

A
  • Synthetic pepride MTA-1
  • Viral core proteins p53, p24, p19
  • Recombinant glycoprotein gd21
33
Q

Inconclusive results

A

especially in tropical areas; need additional tests ie PCR

34
Q

PCR method - DNA polymerase

A

extends strand in 5’ to 3’ direction

35
Q

PCR method - Primers

A

forward: 5’ to 3’, reverse: 3’ to 5’ (but read 5’ to 3’)

36
Q

PCR method - components

A
  1. DNA template
  2. Primers (forward and reverse)
  3. DNA polymerase
  4. dNTPs
  5. Reaction buffer for proper DNA polymerase functioning
37
Q

PCR method - sample preparation

A

Peripheral blood used, peripheral blood mononuclear cells isolated (mix of monocytes, lymphocytes (T-cells, B-cells, NK-cells); with separation medium and CFG, sample separates into (from top) plasma, PBMC, granulocytes and RBCs), DNA isolated (cell lysis/protein degradation, capture and cleaning of DNA, elution of DNA)

38
Q

PCR - 50 μl reaction volumes

A

37.6 ddH2O (autoclaved)
5 10X reaction buffer
2 DNA template (positive control or negative control or patient’s)
1.5 each of primer 1 and 2
2 40 mM dNTPs (10 mM each nucleotide)
0.4 DNA polymerase (often Taq polymerase (derived from thermophilic bacterium Thermus aquaticus))

39
Q

DNA gel electrophoresis

A

Separates DNA based on size, DNA is negatively charged and migrates towrads the positive anode, visualize DNA with intercalating DNA stain (ethidium bromide (UV light), sybr safe (blue light)), purpose of DNA loading dye (increase density/weight of sample, see which wells contain a sample, indicate how far DNA fragments have migrated during run), DNA marker/ladder (estimate size of PCR fragment)

40
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)

41
Q

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

A

Visualise which wells contain samples, visualise how far the gel has migrated, makes samples sink into wells

42
Q

qRT-PCR

A

Provides information on amount of viral DNA present in sample, will help predict severity of disease and likelihood of transmission

43
Q

DNA probe-based qRT-PCR method (TaqMan method)

A
  1. polymerization and strand displacement
  2. probe cleavage (by DNA polymerase, release of reporter dye, fluorescence occurs when reporter and quencher dye are no longer in close proximity)
  3. completion of polymerisation
44
Q

TaqMan data reading

A

High HTLV-1 load = low cycle threshold number

45
Q

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

A

Nasopharyngeal, lung, intestinal

46
Q

What type of patients sample could you take to detect SARS-CoV-2 using a PCR method?

A

Stool, nasal, sputum

47
Q

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

A

ssRNA

48
Q

What is the infectious material in the case of SARS-CoV-2?

A

Free virus particles

49
Q

What is the most common infection route with SARS-CoV-2?

A

Droplets

50
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

51
Q

What is the name of the key SARS-CoV-2 protein that binds to host cells, and what receptor does it bind to?

A

Spike protein, angiotensin-converting enzyme (ACE2)

52
Q

Which protein is often used for the development of a PCR-based detection for SARS-CoV-2 virus?

A

Nucleocapsid

PoM (33 decks)

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