diagnostic virol Flashcards by J A

what is HTLV -1

Human T-cell Leukaemia Virus Type I

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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)

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transmission

Mother to Infant (breast feeding/during birth)
S contact
Blood (e.g. blood transfusion)
sharing needles

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can symptoms, disease and disease severity vary between indivisuals

yes making diagnostic difficult

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descirbe the structure of the virus

single stranded envelope

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Genome

ssRNA

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does it have reverse transcitepase

yes

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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

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what does it infect

infects preferentially T-cells / T-helper cells

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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

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Assumed that the number of T-cells containing HTLV-1 DNA correlates with

Disease severity

Likelihood of transmitting the virus

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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

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when diagnosing

not only to important if they have the virus but also gain information about the actual viral load

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aim of pcr

(amplifying) (specific) region. of dna

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how is it viewed

visualised on an (agarose) gel using a stain that (intercalates) into the DNA and (fluoresces) under a specific light source.

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3 steps of pcr

denature
primer anealing
extending dna

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what temp is denature

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temp for annealing

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temp for extending

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do people develop antibodies against the HTLV 1

yes

against different viral protiens

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Western-blot method

blood taken to Method used to
assess if patients have
Antibodies specific to
HTLV-1 proteins

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step 1

seperation

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what happens in step 1

different viral protein will be separated based on the size

smaller protien move faster

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what gel is used

ployacrylamide protein gel

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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'

diagnostic virol Flashcards

(74 cards)