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diagnostic virol Flashcards

1
Q

what is HTLV -1

A

Human T-cell Leukaemia Virus Type I

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2
Q

Diseases caused by the virus

A 
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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3
Q

transmission

A

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

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4
Q

can symptoms, disease and disease severity vary between indivisuals

A

yes making diagnostic difficult

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5
Q

descirbe the structure of the virus

A

single stranded envelope

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6
Q

Genome

A

ssRNA

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7
Q

does it have reverse transcitepase

A

yes

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8
Q

what does the viral tax protien do

A

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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9
Q

what does it infect

A

infects preferentially T-cells / T-helper cells

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10
Q

Replication cycle

A

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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11
Q

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

A

Disease severity

Likelihood of transmitting the virus

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12
Q

another way to transfer

A

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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13
Q

when diagnosing

A

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

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14
Q

aim of pcr

A

(amplifying) (specific) region. of dna

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15
Q

how is it viewed

A

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

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16
Q

3 steps of pcr

A

denature
primer anealing
extending dna

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17
Q

what temp is denature

A

95

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18
Q

temp for annealing

A

55

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19
Q

temp for extending

A

72

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20
Q

do people develop antibodies against the HTLV 1

A

yes

against different viral protiens

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21
Q

Western-blot method

A

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

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22
Q

step 1

A

seperation

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23
Q

what happens in step 1

A

different viral protein will be separated based on the size

smaller protien move faster

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24
Q

what gel is used

A

ployacrylamide protein gel

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25
Q

step 2

A

trasnfer

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26
Q

what happens

A

protien trasnfered using electric transfer system onto a PVDF membrane and viral protines will stick

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27
Q

what it casue

A

image of gel with distcint viral protiens as distinct bands on membrane
BUT NOT VISIBLE

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28
Q

STEP 3

A

staining

they are immobilised

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29
Q

what happens

A

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

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30
Q

step 4

A

visualisation

31
Q

what can happen

A

colour precipitate, luminicesnt signal

32
Q

how are viral proteins obtained

A

in vitro grown viral cultures

33
Q

is it a positive criteria in other countris

A

yes, react to only some viral protiens

in tropical countries

34
Q

which specific gene is amplified for HTLV-1 pcr

A

tax gene

35
Q

what extends the dna in pcr

A

dna polymerase

36
Q

how many cyles of pcr

A

30- 40

37
Q

how many primers are required for pcr of HTLV-1

A

2

forward and reverse

38
Q

what does pcr have

A

DNA template

2: Primers
(forwards and reverse)

3: DNA polymerase
4: dNTPs
5: Reaction Buffer

39
Q

3 steps sample prep

A

1 take blood
2 isolate PBMC ]
3 isolate DNA

40
Q

which blood is used

A

Peripheral Blood

Peripheral blood
mononuclear cells(PBMCs)

41
Q

what r PBMCs

A

are a mix of cells

Monocytes
Lymphocytes 
	T-cells
	B-cells
	NK-cells
42
Q

what happens in step 2

A

centrigugation

seperated into plasma, PBMC, granulocytes and RBC fraction

43
Q

which fraction do you use

A

PBMC

44
Q

why is a special temp resistant DNA polymerase needed

A

PCR is perfomed at high temp

normally taq polymerase used

45
Q

what else do you need to test for/set up

A

+ control and - control

46
Q

what is + control

A

DNA sample known to contaon HTLV-1

47
Q
  • control
A

DNA sample free of HTLV-1

48
Q

what happens when pcr is completed

A

anaylsed by DNA gel electropherisis (agrose)

49
Q

how is it seperaetd

A

Separate DNA based on size

DNA is negatively charged and migrates towards the positive anode

50
Q

what migrates faster

A

smaller fragments

51
Q

Visualize DNA by adding intercalating DNA stain

A

yes

52
Q

why do you need to add DNA loading dye

when loading dna sample

A

Increase density/weight of the sample
See which well contains a sample
Indicate how far the DNA fragments have migrated during run

53
Q

how to determine actual size of pcr product

A

add DNA marker/ladder

54
Q

what is it

A

contains differnet dna fragments of known size

55
Q

what happens if the control results are not valid

A

assay is invalid

results can’t be used to diagnose patient

56
Q

what can be amplified using PCR

A

dsDNA or ssDNA

57
Q

deoxynucleotide or nucleotide used in pcr

A

deoxynucleotide

58
Q

can pcr be used for bacteria

A

yes

59
Q

what is (qRT-PCR

A

Quantitative Real Time PCR

60
Q

diff between pcr and qRT-PCR

A

pcr provides yes or no amswer to infection
qRT PCR Provides information on amount of viral DNA
present in sample
It is quantitive

61
Q

what can qRT PCR predict

A

Will help to predict severity of disease

Will help to predict transmission likelihood

62
Q

function of qRT PCR

A

same as pcr for same gene

63
Q

how do you anaylise the amount of pcr in qRT PCR

A

the amount of pcr is monitored throughout the cycle

64
Q

how do you monitor

A

flouresent signal is proportional to amount of DNA

65
Q

how do you if they are infected

A

If infected you pass the threshold level (CT)

if not you don’t

66
Q

what are the 2 main methods for qRT PCR

A

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)

67
Q

diff between DNA probe-based qRT-PCR method (TaqMan method) and pcr

A

3rd probe added (oligo)

binds to specifc gene that will be apmlified

68
Q

when does flouresence occur

A

when the fluerophore and quencher are no longer in close proximity

69
Q

how are they seperated

A

when DNA polymerase binds to oligo
it will continue to sythesise the DNA
but degrade oligo that is bound to the fluerphore and quencher

70
Q
  • of DNA probe-based qRT-PCR method (TaqMan method)
A

expesnive

71
Q

diff bin base pairs of pcr and qrt

A

pcr 300 bp

qrt 100 bp

72
Q

how are CT and tax gene copy related

A

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)

73
Q

What type of patient samples could you take to detect SARS-CoV-2 using a PCR-based method? (tick all that apply)

A

Stool
Nasal
Sputum

74
Q

how is taqman probe

A

can’t be extended no free OH group
covalently bonded to reporter and quencher
r on 5’
q 3’

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