Diagnostic Virology Flashcards
(42 cards)
Within the HTLV-1 virus particle, in which form is the genetic material?
ssRNA
What type of cell does HTLV-1 preferentially infect?
T cells
How does HTLV-1 replicate?
HTLV-1 can integrate within the chromosome of infected cells and replicate as dsDNA as part of the host cell division process
What are 3 possible routes to become infected with HTLV-1 ?
Blood transfusion
From mother to infant by breast feeding
Sexual Contact
What diseases can be caused by HTLV-1?
HTLV-1-associated myelopathy
Leukaemia
What are PCR methods used to detect HTLV-1 based on?
The presence of the tax gene
What are the three steps of the western blot method
- Separation
- Transfer
- Staining
What does the western blot method look for?
Presence of antibodies specific to the virus
Describe the separation stage of the western blot procedure?
Viral proteins separated based on size on polyacrylamide protein gel
Which proteins will migrate fastest on the polyacrylamide gel of the western blot?
The smaller proteins
What happens to the western blot after the viral proteins are separated by size and have formed bands?
Incubated with human serum - if the human serum contains antibodies it will bind to the viral proteins
What happens in the staining phase of the western blot method?
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
What must be detected for a positive result for HTLV-1?
MTA1
p53
p24
p19
gd21
What is the purpose of the buffer in PCR?
Provides an appropriate pH for reactions to occur / polymerase to work
What are the three steps of PCR?
- Denature
- Anneal
- Elongate
What temperature does denaturation occur at?
94 degrees - breaks H bonds between double-stranded DNA
What must the primer have?
A free hydroxyl group
What is the temperature at which annealing takes place?
54 degrees
What temperature does elongation occur at?
72 degrees
Allows Polymerase to work
What DNA is used for PCR analysis?
PBMC - peripheral blood mononuclear cells = mixture of lymphocytes and monocytes
What type of genetic material can by amplified by a standard PCR
dsDNA
ssDNA
HTLV-1 has a single strand RNA genome, but a standard PCR can still be used for diagnosis. What is the reason for this?
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
What 5 key components must be included in a PCR
mix
DNA Template
Primers Pair
Taq Polymerase
reaction Buffer
Deoxynucleotides
How many grams of agarose do you need to make 50 ml of a 1% (weight/volume) agarose gel solution?
0.5
