Diagnostic virology/bacteriology Flashcards
Neutrophil key function
Phagocytosis of microbes
Eosinophil key functions
Cytokines
Helminth destruction
Basophil key functions
Cytokines
Helminth destruction
Macrophage key functions
Phagocytosis of microbes/apoptotic cells
Cytokines
What is the coagulase test testing for and what shows a pos/neg test?
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
What is the use of the catalase test in bacerial testing and what are the pos/neg results?
To see if the bacteria can produce catalase, an enzyme which catalyses the breakdown of H2O2
Positive-O2 bubbles
Neg-No bubbles
What is the purpose of lactose fermentation in bacterial testing?
To see if the bacteria can ferment lactose
Describe the outcomes of the bacterial haemolysis test
Alpha-Partial haemolysis(Opaque zone)
Beta-Complete haemolysis(Transparent greenish zone)
Gamma-No haemolysis :(
Why investigate a bacteria for haemolysis?
To see its ability to produce haemolysins
Gram pos cocci
Gram pos cocci
Gram neg bacilli
Why do gram-neg bacteria turn pink under gram staining?
Alcohol washes out the crystal-violet/iodine as there is only a thin peptidoglycan layer.
-Safranin then stains the bacteria pink
What is the purpose of alcohol in gram staining?
Dehydrates the peptidoglycan, making it tighter and better at retaining the stain in gram pos cells
If you had viral RNA and wanted to detect the viral load present, what PCR type would you use?
RT-qRT-PCR(Reverse transcriptase quantitive real time polymerase chain reaction)
-Detects viral RNA so this would be used
Branches of PCR
What is qRT-PCR?
Quantitative reverse transcriptase PCR
What is RT-PCR?
Reverse-transcriptase PCR, used for RNA viruses
What is qPCR?
Quantitative PCR, used for measuring viral load
What is the problem is qRT-PCR?
Spenny
How is qPCR quantititative?
The volume of HTLV-1 positive cells dictates how quickly the CT is reached
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?
How do you calculate transmittance?
Transmittance = Light passing through the sample/incident light
How does qPCR differ from PCR?
What type of genetic material can be amplified by standard PCR?
ssDNA
dsDNA
What is the advantage of qPCR over PCR?
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
What must be prepared alongside a patients’ sample of DNA testing for HLTV-1?
A positive and negative control
What does the DNA marker/ladder do?
Is used to estimate the size of the DNA fragment-Use of the intercalating stain
How are PCR DNA samples analysed and what is the purpose of DNA loading dye when loading it onto gel?
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
What enzyme is commonly used as DNA polymerase and why?
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
What are the 5 key components of PCR?
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
What are the names of the primers used to amplify the HTLV-1 gene?
Forward-HL43
Reverse-HL44
-The region amplified is 300 base pairs of the tax gene
How can PCR be done on HTLV-2 even though it’s an RNA virus?
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
Describe the 3 step process of PCR
What does DNA polymerase need to act on DNA?
Primer with a ‘3 OH group
Purpose of PCR and which direction does it act in?
Amplify a piece of DNA
-Acts in ‘5 to ‘3 direction
Which 5 HTLV-1 proteins do patients need antibodies against to return a positive test for the criteria we’re using?
MTA-1
p53
p24
p19
gd21
-Issues arise when patients have antibodies against some, but not all HTLV-1 proteins
Describe the sequence of the western-blot method
- ## Separation-Different viral proteins are separated using gel electrophoresis in polyacrylamide gel.
- ## Transfer-Proteins are transferred using an electric transfer system onto PVDF membrane, immobilising distinct proteins which aren’t visible yet
- ## 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)
- 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
Why is no free virus needed for transmission?
Genetic material of the virus can be incorporated into host cells
What does the western blot method assess within an HTLV-1 infection
Whether a patient has any specific HTLV-1 antibodies present in their serum.
What does the number of T cells with HTLV-1 DNA correlate with and what is that important for?
- Disease severity
- Likelihood of transmitting the virus
Important bc-Gaining info on viral load, useful for diagnosis
Outline HTLV-1 replication
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
How does PCR detect HTLV1?
Detection of the tax gene
What does the tax protein do?
Induces viral transcription, can affect the cell cycle process, leading to oncogenesis
What proteins does HTLV-1 make?
Reverse transcriptase
Viral tax protein
Is HTLV-2 enveloped?
Yes
What type of genome is HTLV-1?
ssRNA
How is HTLV-1 transmitted?
Blood
Sexual fluids
Mother-infant
What diseases does HTLV-1 cause?
ATL
HAM(HLTV-1-associated myelopathy)
HTLV-1-associated infectious dermatitis
HTLV-1-associated uveitis(HUS)
