Diagnostic Virology

Question 1

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

Answer 1

ssRNA

Question 2

What type of cell does HTLV-1 preferentially infect?

Answer 2

T cells

Question 3

How does HTLV-1 replicate?

Answer 3

HTLV-1 can integrate within the chromosome of infected cells and replicate as dsDNA as part of the host cell division process

Question 4

What are 3 possible routes to become infected with HTLV-1 ?

Answer 4

Blood transfusion
From mother to infant by breast feeding
Sexual Contact

Question 5

What diseases can be caused by HTLV-1?

Answer 5

HTLV-1-associated myelopathy
Leukaemia

Question 6

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

Answer 6

The presence of the tax gene

Question 7

What are the three steps of the western blot method

Answer 7

1. Separation
2. Transfer
3. Staining

Question 8

What does the western blot method look for?

Answer 8

Presence of antibodies specific to the virus

Question 9

Describe the separation stage of the western blot procedure?

Answer 9

Viral proteins separated based on size on polyacrylamide protein gel

Question 10

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

Answer 10

The smaller proteins

Question 11

What happens to the western blot after the viral proteins are separated by size and have formed bands?

Answer 11

Incubated with human serum - if the human serum contains antibodies it will bind to the viral proteins

Question 12

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

Answer 12

Secondary antibodies which have an enzyme attached 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

Question 13

What must be detected for a positive result for HTLV-1?

Answer 13

MTA1
p53
p24
p19
gd21

Question 14

What is the purpose of the buffer in PCR?

Answer 14

Provides an appropriate pH for reactions to occur / polymerase to work

Question 15

What are the three steps of PCR?

Answer 15

1. Denature
2. Anneal
3. Elongate

Question 16

What temperature does denaturation occur at?

Answer 16

94 degrees - breaks H bonds between double-stranded DNA

Question 17

What must the primer have?

Answer 17

A free hydroxyl group

Question 18

What is the temperature at which annealing takes place?

Answer 18

54 degrees

Question 19

What temperature does elongation occur at?

Answer 19

72 degrees
Allows Polymerase to work

Question 20

What DNA is used for PCR analysis?

Answer 20

PBMC - peripheral blood mononuclear cells = mixture of lymphocytes and monocytes

Question 21

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

Answer 21

dsDNA
ssDNA

Question 22

HTLV-1 has a single strand RNA genome, but a standard PCR can still be used for diagnosis. What is the reason for this?

Answer 22

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

Question 23

What 5 key components must be included in a PCR
mix

Answer 23

DNA Template
Primers Pair
Taq Polymerase
reaction Buffer
Deoxynucleotides

Question 24

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

Answer 24

0.5

Question 25

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

Answer 25

Run a DNA ladder alongside your PCR reactions

Question 26

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

Answer 26

1. Makes samples sink into wells
2. Visualise how far the gel has migrated
3. Visualise which wells contain samples

Question 27

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

Answer 27

30-40

Question 28

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

Answer 28

ssRNA

Question 29

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

Answer 29

During annealing step, forward and reverse primers are needed

Question 30

What is assumed about the disease severity of HTLV-1?

Answer 30

Assumed that the number of T cells containing HTLV-1 correlate with the disease severity and also the likelihood of transmitting the virus

Question 31

How long does the denaturation stage of PCR last?

Answer 31

1 minute

Question 32

How long does the annealing stage of PCR last?

Answer 32

45 seconds

Question 33

How long does the elongation stage of PCR last?

Answer 33

2 minutes

Question 34

What is added during the extension stage of PCR?

Answer 34

deoxynucleotides - dNTPs

Question 35

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

Answer 35

TaqMan Probe

Question 36

What extra information for qRT-PCR provide over normal PCR?

Answer 36

Provides information on the amount of viral DNA present in a sample and therefore helps predict the severity of disease

Question 37

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

Answer 37

Binds to only the gene being amplified, in between the forward primer and reverse primer

Question 38

What does the TaqMan Probe have attached to it?

Answer 38

A fluorophore and a quencher

Question 39

What happens in qRT-PCR when the quencher and fluorophore get separated?

Answer 39

Probe degrades and becomes fluorescent

Question 40

How does the fluorescence relate to the DNA amplified?

Answer 40

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

Question 41

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

Answer 41

The fewer PCR cycles that are needed in order to reach the threshold of fluorescence (which is proportional to the amount of DNA amplified hence more viral load) indicates a higher viral load

Question 42

What is the relationship between CT Values and tax gene copy number?

Answer 42

CT Values are linear with the Log10 of tax gene copy number