diagnostic virol Flashcards
what is HTLV -1
Human T-cell Leukaemia Virus Type I
Diseases caused by the virus
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)
transmission
Mother to Infant (breast feeding/during birth)
S contact
Blood (e.g. blood transfusion)
sharing needles
can symptoms, disease and disease severity vary between indivisuals
yes making diagnostic difficult
descirbe the structure of the virus
single stranded envelope
Genome
ssRNA
does it have reverse transcitepase
yes
what does the viral tax protien do
viral transcription process
affects cell progression and signillaing leading to oncogenisis
specifically found in htlv-1 so pcr targets the tax gene
what does it infect
infects preferentially T-cells / T-helper cells
Replication cycle
HTLV-1 enters T-cell
ssRNA released into host cell cytosol
ssRNA reverse transcribed (RT-enzyme) to ssDNA
ssDNA converted to dsDNA
dsDNA enters nucleus and integrates into host genome
viral genome can replicate as part of the host chromosome
Assumed that the number of T-cells containing HTLV-1 DNA correlates with
Disease severity
Likelihood of transmitting the virus
another way to transfer
some dna trancibed to rna
make viral protien
infect neiobirng cells
but not stable so doesn’t transmit via freee viral protiens person to person
when diagnosing
not only to important if they have the virus but also gain information about the actual viral load
aim of pcr
(amplifying) (specific) region. of dna
how is it viewed
visualised on an (agarose) gel using a stain that (intercalates) into the DNA and (fluoresces) under a specific light source.
3 steps of pcr
denature
primer anealing
extending dna
what temp is denature
95
temp for annealing
55
temp for extending
72
do people develop antibodies against the HTLV 1
yes
against different viral protiens
Western-blot method
blood taken to Method used to
assess if patients have
Antibodies specific to
HTLV-1 proteins
step 1
seperation
what happens in step 1
different viral protein will be separated based on the size
smaller protien move faster
what gel is used
ployacrylamide protein gel
step 2
trasnfer
what happens
protien trasnfered using electric transfer system onto a PVDF membrane and viral protines will stick
what it casue
image of gel with distcint viral protiens as distinct bands on membrane
BUT NOT VISIBLE
STEP 3
staining
they are immobilised
what happens
Incubate membrane with
human serum (primary AB)
AB bind to to htlv protien if they recognise
2: Wash membrane- remove all unbound antibodies
3: Incubate membrane with secondary AB linked to enzyme- recognise the fc region on promary AB, conjugated to an enzyme
4: Wash membrane- unbound secondary removed
5: Add substrate for enzyme linked to secondary AB
step 4
visualisation
what can happen
colour precipitate, luminicesnt signal
how are viral proteins obtained
in vitro grown viral cultures
is it a positive criteria in other countris
yes, react to only some viral protiens
in tropical countries
which specific gene is amplified for HTLV-1 pcr
tax gene
what extends the dna in pcr
dna polymerase
how many cyles of pcr
30- 40
how many primers are required for pcr of HTLV-1
2
forward and reverse
what does pcr have
DNA template
2: Primers
(forwards and reverse)
3: DNA polymerase
4: dNTPs
5: Reaction Buffer
3 steps sample prep
1 take blood
2 isolate PBMC ]
3 isolate DNA
which blood is used
Peripheral Blood
Peripheral blood
mononuclear cells(PBMCs)
what r PBMCs
are a mix of cells
Monocytes Lymphocytes T-cells B-cells NK-cells
what happens in step 2
centrigugation
seperated into plasma, PBMC, granulocytes and RBC fraction
which fraction do you use
PBMC
why is a special temp resistant DNA polymerase needed
PCR is perfomed at high temp
normally taq polymerase used
what else do you need to test for/set up
+ control and - control
what is + control
DNA sample known to contaon HTLV-1
- control
DNA sample free of HTLV-1
what happens when pcr is completed
anaylsed by DNA gel electropherisis (agrose)
how is it seperaetd
Separate DNA based on size
DNA is negatively charged and migrates towards the positive anode
what migrates faster
smaller fragments
Visualize DNA by adding intercalating DNA stain
yes
why do you need to add DNA loading dye
when loading dna sample
Increase density/weight of the sample
See which well contains a sample
Indicate how far the DNA fragments have migrated during run
how to determine actual size of pcr product
add DNA marker/ladder
what is it
contains differnet dna fragments of known size
what happens if the control results are not valid
assay is invalid
results can’t be used to diagnose patient
what can be amplified using PCR
dsDNA or ssDNA
deoxynucleotide or nucleotide used in pcr
deoxynucleotide
can pcr be used for bacteria
yes
what is (qRT-PCR
Quantitative Real Time PCR
diff between pcr and qRT-PCR
pcr provides yes or no amswer to infection
qRT PCR Provides information on amount of viral DNA
present in sample
It is quantitive
what can qRT PCR predict
Will help to predict severity of disease
Will help to predict transmission likelihood
function of qRT PCR
same as pcr for same gene
how do you anaylise the amount of pcr in qRT PCR
the amount of pcr is monitored throughout the cycle
how do you monitor
flouresent signal is proportional to amount of DNA
how do you if they are infected
If infected you pass the threshold level (CT)
if not you don’t
what are the 2 main methods for qRT PCR
Fluorescence dye-based method
(SYBR Green dye method)
(dye not flouresengt in solution but flourecsent when in binds to DNA)
DNA probe-based qRT-PCR method (TaqMan method)
diff between DNA probe-based qRT-PCR method (TaqMan method) and pcr
3rd probe added (oligo)
binds to specifc gene that will be apmlified
when does flouresence occur
when the fluerophore and quencher are no longer in close proximity
how are they seperated
when DNA polymerase binds to oligo
it will continue to sythesise the DNA
but degrade oligo that is bound to the fluerphore and quencher
- of DNA probe-based qRT-PCR method (TaqMan method)
expesnive
diff bin base pairs of pcr and qrt
pcr 300 bp
qrt 100 bp
how are CT and tax gene copy related
CT values are linear
with the
Log10 of tax gene copy number
Low HTLV-1 load (10)
High CT number (35)
High HTLV-1 load (107)
Low CT number (15)
What type of patient samples could you take to detect SARS-CoV-2 using a PCR-based method? (tick all that apply)
Stool
Nasal
Sputum
how is taqman probe
can’t be extended no free OH group
covalently bonded to reporter and quencher
r on 5’
q 3’
