Diagnostic Virology Flashcards
What are the 4 stages in western blotting?
- Gel electrophoresis to separate the proteins according to size
- Transfer to a soluble medium
- Staining (basically same process as Elisa) but starts with initial incubation with human serum then followed by two sets of antibodies and a flurophore
- Visualisation
What are the proteins recognised by the HTLV proteins required for a positive test?
MTA-1 p53 P24 P19 gd21
What is the direction DNA always presented?
5” to 3” on the top
What direction does DNA polymerase work in?
5” to 3” direction (i.e. starts from the 3” end and adds in the 5” direction)
3 stages of PCR
Denaturation
Annealing using both forward and revers primers
Extension using DNTPs
Why does PCR work for detecting HTLV?
As the single strand RNA strand in the virus is integrated into the double stranded human genome
What gene is targeted in the PCR of HTLV?
The tax gene
What direction should the sequence of the primer be written down?
Both should be in the 5” to 3” direction, so the reverse complement regions should be written back to front
What are 5 key reagents in the PCR?
DNA template Forward and reverse primers DNA polymerase (Taq polymerase - temperature resistant) DNTPs Reaction buffer
How is the sample prepared for collecting detection of HTLV (PCR)?
Using peripheral blood Placed on separation medium Centrifuged to fractions Remove the PBMC section (peripheral blood mononuclear cells - lymphocytes and monocytes) Then isolate DNA from these cells
How many controls should be used?
2 one positive (has the HTLV) One negative (no HTLV)
How is PCR result analysed?
Using DNA gel electrophoresis
What direction does the DNA travel?
It is placed at the cathode and it travels to the positive anode (DNA is negatively charged)
What is difference between PCR and qPCR?
qPCR provide real time information about the amount of viral DNA present in a sample
so can help predict the severity of disease
Predict likelihood of transmission
