Diagnostic Virology Flashcards
HTLV-1 replication
- HTLV-1 enters T-cell
- ssRNA released into host cell cytosol
- ssRNA reverse transcribed to ssDNA
- ssDNA → dsDNA
- dsDNA enters nucleus and is integrated into host cell DNA by integrase
- Viral genome replicates as part of the host cell chromosome
How is HTLV-1 transmitted
Breast feeding
Sexual contact
Blood to blood contact
How is HTLV-1 detected
Blood taken from patients and peripheral blood mononuclear cells are isolated by centrifugation
Dna used as template to amplify tax gene
PCR
Has 30-40 cycles
Visualized on agarose gel using stain that intercalates into DNA and fluoresces under specific loght
Wester blot method
1)Separation- viral proteins separated depending on size with smaller proteins migrating first in ppolyacrymid gel using gel electrophoresis
2)Transfer- proteins are transferred using an electric transfer system onto PVDF membrane. Different viral proteins will create different bands
3)Staining- membrane incubated with human serum then washed and incubated with secondary ab which will recognize the Fc region of the first antibody .wash again and add substrate for enzyme linked to secondary AB
4)visualization-
Positive test
Shows bands at MTA-1,P53,P24,P19 and gd21
What are HL43 and HL44
Primers specific to tax gene
HL43 is our forward primer which will have same sequence as top strand and vice versa
Quantitative real time PCR
detects amount of viral DNA in sample
Fluorescent based qRT-PCR
Dna binding dye can fluoresce when bound to DNA
Eg SYBR green 8
DNA probe based method
Fluorescent probe binds to DNA sequence within amplified region
Primer contains fluorescent probe and quencher in close proximity
Quencher prevents generation of Floresce
Elongation of primer causes degradation of oligo probe so quencher is no longer in close proximity to fluorophore causing fluorescent signals
Oligoprobe has fluorophore attached to 5’ end and quencher on 3’ end
Quencher quenchesbthe fluorescent at the start
Taq man method of qRT-PCR
Polymerisation & Strand Displacement - Oligo TaqMan probe binds to DNA template strand in a region between the forward and reverse primer
Probe Cleavage - As the DNA polymerase acts on the DNA strand it displaces the TaqMan probe which leads to the fluorophore breaking off from the probe, giving off fluorescence as the quencher and fluorophore are no longer in close proximity
Completion of Polymerisation - The DNA polymerase acts further which also displaces the rest of the oligo probe and quencher
Stuns used to see dna on gel electrophoresis
Ethidium bromide then use uv light (toxic)
SYBR safe then blue light
