keith (L24) Flashcards
The Purpose of Viral Diagnosis
For clinical management of patients
- Acute diagnosis (new infection)
- Management of chronic/persistent infections
For public health management
Screening the blood supply
Monitoring and planning health care provision
Requirements for Ideal Diagnostic Test
Specific & sensitive (minimal false-positives, false-negatives) Reproducible Cheap and robust High-throughput Quick to perform
different types of samples we can take
nasal swab urine saliva faeces bipsies blood CSF
non invasive methods are very easy and cheap
diagnostic tests from the liver to find virus - most accurate but too invasive
Diagnostic Methods
Direct detection:
- Testing for the presence of virus
- Testing for viral proteins (usually as antigens)
- Testing for viral nucleic acid
- May not be able to get the right samples for detection if virus is not systemic
Indirect detection:
- Testing for the presence of specific antibodies
- A positive result doesn’t necessarily indicate current infection as antibodies persist after infection is gone
detection of whole virus
Virus isolation (whole organisms, eggs, cells)
Cytopathology
Electron microscopy
Haemagglutination
process of haemagglutination (nonspecific)
sample and dilute it across the plate
add some rbc to each well
initially the rbcs are suspended up in the mixture
if there is no virus there, the heavy rbcs will drop to the bottom of the well
if there is virus present, it can hemagglutinate it and form the lattice which will float up
it is quantitative since we know the diluted concentrations
process of haemagglutination (specific)
maintain in a lab antibody samples
when a sample comes in and you suspect it might be due to virus A, you can take your reference serum to virus A,
make a dilution across the plate
add sample that contains the virus
if the virus is A, then the antibody will bind to it and stop it from agglutinating the rbds
this is an HA inhibition test - HA reaction was inhibited by antibody
if you get that outcome, then we know that the antibody choice matches the virus
tells us that patient is infected by that virus
only takes a few hours
Virus Detection: Antibody Neutralization Test
classic test for presence of virus - by testing it with neutralising antibodies
test for virus A by mixing the sample with antibodies for virus A - will destroy its infectivity, and we can detect if the infectivity remains or not
not done often anymore
DIAGRAM AND PROCESS IN L24 S11
Specific Antibody Detection
Protein immunoblotting
Standard enzyme-linked immunosorbent assay (ELISA)
Standard enzyme-linked immunosorbent assay (ELISA) PROCESS
- bind standard virus antigen to the well
- add test serum, specific ab binds if present
- add standard antibody that binds to the first ab and is linked to an enzyme
- add a substrate that the enzyme can turn into a coloured product
- read absorbance, colour = positive result
IgM capture ELISA PROCESS
- bind IgM specific ab to well
- add test serum, IgM binds
- add standard antigen to detect the presence of specific IgM
- add IgG specific for the standard antigen, with coupled enzyme
5/ add a substrate that the enzyme can turn into a coloured product - read the absorbance, colour = positive result
Viral Nucleic Acid Detection
- Slot (or dot)-blot nucleic acid hybridization ((in situ hybridisation)
- PCR: depends on primer hybridization for specificity (qualitative nucleic acid amplification by polymerase chain reaction (after RT for RNA detect)
- Quantitative nucleic acid amplification by real-time PCR (qPCR)
- Quantitative nucleic acid detection by branched (b)DNA assay (mimics ELISA for protein detection, but based on nucleic acid base-pairing)
Nucleic Acid Detection: ‘omics:
- DNA sequencing
- High density oligonucleotide arrays on microchips
- High throughput sequencing (virome analysis)
–> no one single test is efficient because of the inevitable tradeoff between sensitivity and specificity
2 types of herpes simplex virus and how to detect one from the other
type 1 infects the mouth
type 2 infects the geniitals
they are similar in nucleic acid sequences
you get absolute specific detection of type 1 infection with a particular reagent or a type 2 infection with a different ragent (no crossover between the 2)
base pairing is very specific detection of a type nucleic acid
we can use this by detecting viral dna in situ
only one type that this is relevant for - papilloma virus
process of nucleic acid detection by PCR
- denature and anneal primers
- extend primers by transcription
- denature and anneal primers
CYCLE
relatively qualitative, difficult to get quantitative information from it
but we just use it to get a +ve or -ve result
Quantitation using real-time PCR – Light Cycler reactions
Use very sensitive DNA-binding dye (SYBR green) to quantify DNA made in the PCR as it proceeds
- Needs Light-Cycler PCR machine
- SYBR green fluorescence when bound to dsDNA is»_space; than alone in solution or bound to ssDNA
Set threshold value in the exponential phase of PCR
Number of cycles required for product to cross threshold – known as Ct – is measure of input template amount
take template and every 10 fold dilution gives a right shift of the curve of 3.3 cycles in the point at which the curve crosses the threshold
NOTES AND DIAGRAMS IN L24 S22-23
Quantitative nucleic acid detection by branched (b)DNA assay
Mimics ELISA for protein detection, but based on nucleic acid base-pairing
like doing an ELISA on nucleic acid
configuration in a microwell with sequential addition of reagents, ending up in the detection of the presence of the viral nucleic acid in a patient’s sample
instead of using successive antibodies to do our detections, we use successive bits of nucleic acids
ending up with immobilisation of a yellow blob enzyme in the well (only ends up in the well if the specific viral nucleic acid was present and gives a colour reaction when we add the substrate)
reagents are different - because we are doing specificity of base pairing instead of antibody-antigen interactions
start with a microwell, and make it receptive to the binding of nucleic acids by hooking thiis pair of molecules
2 different molecules to make it commercially practical:
covalently couple the capture probe onto the well (this nucleic acid is nothing to do with virus, just a spike that is pre prepared to bind stuff to)
make it specific to the thing we are looking for, by base pairing to it the capture extender (another nucleic acid thing we can synthesise)
- it will base pair to the probe
- make it specific to what we want
- turn it into an assay for virus A to compare it against another virus B by chosing the right sequence to be able to base pair for what we are looking for
- now put in the patient’s sample
- if it does contain target dna, it will base pair with the capture molecule and will be immobilised in the well
- then we need to detect the ones that did get immobilised
- use probes that are specific to the thing we are looking for
- put the pair of them in, if they bind then these stalky bits will also be present and that will allow the detection molecule to bind
