Diagnostic virology/bacteriology

Question 1

Neutrophil key function

Answer 1

Phagocytosis of microbes

Question 2

Eosinophil key functions

Answer 2

Cytokines
Helminth destruction

Question 3

Basophil key functions

Answer 3

Cytokines
Helminth destruction

Question 4

Macrophage key functions

Answer 4

Phagocytosis of microbes/apoptotic cells
Cytokines

Question 5

What is the coagulase test testing for and what shows a pos/neg test?

Answer 5

Tests for the ability of the bacteria to produce coagulase, which cross-links fibrinogen to form clots on the bacterial surface

Positive-Clumps(coagulation of plasma)
Negative-No clumps

Question 6

What is the use of the catalase test in bacerial testing and what are the pos/neg results?

Answer 6

To see if the bacteria can produce catalase, an enzyme which catalyses the breakdown of H2O2

Positive-O2 bubbles
Neg-No bubbles

Question 7

What is the purpose of lactose fermentation in bacterial testing?

Answer 7

To see if the bacteria can ferment lactose

Question 8

Describe the outcomes of the bacterial haemolysis test

Answer 8

Alpha-Partial haemolysis(Opaque zone)
Beta-Complete haemolysis(Transparent greenish zone)
Gamma-No haemolysis :(

Question 9

Why investigate a bacteria for haemolysis?

Answer 9

To see its ability to produce haemolysins

Question 10

Answer 10

Gram pos cocci

Question 11

Answer 11

Gram pos cocci

Question 12

Answer 12

Gram neg bacilli

Question 13

Why do gram-neg bacteria turn pink under gram staining?

Answer 13

Alcohol washes out the crystal-violet/iodine as there is only a thin peptidoglycan layer.
-Safranin then stains the bacteria pink

Question 14

What is the purpose of alcohol in gram staining?

Answer 14

Dehydrates the peptidoglycan, making it tighter and better at retaining the stain in gram pos cells

Question 14

If you had viral RNA and wanted to detect the viral load present, what PCR type would you use?

Answer 14

RT-qRT-PCR(Reverse transcriptase quantitive real time polymerase chain reaction)
-Detects viral RNA so this would be used

Question 15

Branches of PCR

Answer 15

Question 16

What is qRT-PCR?

Answer 16

Quantitative reverse transcriptase PCR

Question 17

What is RT-PCR?

Answer 17

Reverse-transcriptase PCR, used for RNA viruses

Question 18

What is qPCR?

Answer 18

Quantitative PCR, used for measuring viral load

Question 19

What is the problem is qRT-PCR?

Answer 19

Spenny

Question 20

How is qPCR quantititative?

Answer 20

The volume of HTLV-1 positive cells dictates how quickly the CT is reached

Question 21

What does a positive and negative test look like on a qPCR diagram and how can you identify a high/low viral load using the CT level?

Answer 21

Question 22

How do you calculate transmittance?

Answer 22

Transmittance = Light passing through the sample/incident light

Question 23

How does qPCR differ from PCR?

Answer 23

Question 24

What type of genetic material can be amplified by standard PCR?

Answer 24

ssDNA dsDNA

Question 25

What is the advantage of qPCR over PCR?

Answer 25

qPCR(Quantitative real time PCR) can tell us how many cells from the sample contain the gene, rather than just yes/no on whether the sample has the gene -Used to predict the severity of the disease

Question 26

What must be prepared alongside a patients' sample of DNA testing for HLTV-1?

Answer 26

A positive and negative control

Question 27

What does the DNA marker/ladder do?

Answer 27

Is used to estimate the size of the DNA fragment-Use of the intercalating stain

Question 28

How are PCR DNA samples analysed and what is the purpose of DNA loading dye when loading it onto gel?

Answer 28

DNA gel electrophoresis -Use of an intercalating stain of ethidium bromide for UV light and Sybr Safe for blue light allowing the DNA fragments to be visualised Loading dye uses: Sample weight is increased Lets you see which cells contain a sample Indicates how the DNA fragments have moved during the electrophoresis run **Intercalating dye is different to loading dye** -Loading dye is used to give the DNA colour and density whereas ETBR is used to track the DNA under UV light

Question 29

What enzyme is commonly used as DNA polymerase and why?

Answer 29

Taq polymerase-Used because its' derived from thermophilic bacterium thermus aquaticus and has a high heat resistance, which is useful for PCR as it's done at high temperatures

Question 30

What are the 5 key components of PCR?

Answer 30

DNA template Primers-Binds to the tax gene to amplify the 300 bp region DNA polymerase dNTPs(deoxynucleotides) Reaction buffer-Provides appropriate salt+pH environment for polymerase to work

Question 31

What are the names of the primers used to amplify the HTLV-1 gene?

Answer 31

Forward-HL43 Reverse-HL44 -The region amplified is 300 base pairs of the tax gene

Question 32

How can PCR be done on HTLV-2 even though it's an RNA virus?

Answer 32

Part of its' replication cycle involves turning its' RNA into DNA -So standard PCR can be used to detect ssDNA or dsDNA of the virus that's been integrated

Question 33

Describe the 3 step process of PCR

Answer 33

Question 34

What does DNA polymerase need to act on DNA?

Answer 34

Primer with a '3 OH group

Question 35

Purpose of PCR and which direction does it act in?

Answer 35

Amplify a piece of DNA -Acts in '5 to '3 direction

Question 36

Which 5 HTLV-1 proteins do patients need antibodies against to return a positive test for the criteria we're using?

Answer 36

MTA-1 p53 p24 p19 gd21 -Issues arise when patients have antibodies against some, but not all HTLV-1 proteins

Question 37

Describe the sequence of the western-blot method

Answer 37

1. Separation-Different viral proteins are separated using gel electrophoresis in polyacrylamide gel. - 2. Transfer-Proteins are transferred using an electric transfer system onto PVDF membrane, immobilising distinct proteins which aren't visible yet - 3. Staining-Membrane is incubated with serum and any antibodies present will bind to the HTLV-1 proteins, the membrane is washed and incubated with a secondary enzyme conjugated antibody which binds to the Fc region on the primary antibody. The membrane is washed again and then a substrate is added to the membrane, causing a reaction with the conjugate enzyme(ELISA like test) - 4. Visualisation-Output of substrate-enzyme reaction can be a colour change or a precipitate produced(Most commonly a luminescent sigal that can be seen with a camera in a western-blot imager

Question 38

Why is no free virus needed for transmission?

Answer 38

Genetic material of the virus can be incorporated into host cells

Question 39

What does the western blot method assess within an HTLV-1 infection

Answer 39

Whether a patient has any specific HTLV-1 antibodies present in their serum.

Question 40

What does the number of T cells with HTLV-1 DNA correlate with and what is that important for?

Answer 40

1. Disease severity 2. Likelihood of transmitting the virus Important bc-Gaining info on viral load, useful for diagnosis

Question 41

Outline HTLV-1 replication

Answer 41

HTLV-1 enters T cell ssRNA released into cytosol ssRNA reverse transcribed into ssDNA ssDNA converted into dsDNA dsDNA enters nucleus and intergrated randomly into host genome Viral genome replicates alongside host genome, producing more viruses

Question 42

How does PCR detect HTLV1?

Answer 42

Detection of the tax gene

Question 43

What does the tax protein do?

Answer 43

Induces viral transcription, can affect the cell cycle process, leading to oncogenesis

Question 44

What proteins does HTLV-1 make?

Answer 44

Reverse transcriptase Viral tax protein

Question 45

Is HTLV-2 enveloped?

Answer 45

Yes

Question 46

What type of genome is HTLV-1?

Answer 46

ssRNA

Question 47

How is HTLV-1 transmitted?

Answer 47

Blood Sexual fluids Mother-infant

Question 48

What diseases does HTLV-1 cause?

Answer 48

ATL HAM(HLTV-1-associated myelopathy) HTLV-1-associated infectious dermatitis HTLV-1-associated uveitis(HUS)