Lecture 6: Diagnostics Flashcards
(41 cards)
viremia =
presence of virus in the blood.
what are the current techniques to diagnose a viral infection?
virus isolation: gold standard detection of viral antigens detection of viral nucleic acids detection of virus specific antibodies visualization and identification of viruses by electron microscopy
when does the max titer of a virus often occur?
it will coincide with the peak fever, this may precede the onset of other clinical signs
virus isolation
slow
agent independent
cytopathic effects are indicative of the virus involved - often indicative of the virus involved
provides inexhuastible amount of virus
what specific cell lines are known to grow many viruses?
embryonated hen’s eggs
suckling mice
what characteristic is often times sufficient in assigning a virus to the correct family?
morphology
viral diagnosis by EM
useful in detection of non-cultivable viruses.
agent independent
negative staining
thick-sectioning (most cells must contain virus, expensive takes days)
detection of viral antigen by immunofluorescence is used on what type of samples?
cryostat sections
lesion smears tissue cultures
what is IF not compatible with?
formalin fixation
direct IF uses what?
FITC-labeled specific viral antiserum
indirect IF uses what?
labeled anti-species antiserum after 1st antibody
increases sensitivity but also non-specific background
immunohistochemical (IHC) staining: what enzyme is usually used? what color does it become once it reacts with substrate?
horse radish peroxidase
brown
what are the advantages of IHC over IF:
needs only a light microscope
amplifies reaction product and color; gets stronger by increasing incubation time
what other information can you get from IHC that you dont get from other diagnostic techniques?
provides evidence of antigen localization inside cells
“point of care”: immunochromatography
migration of antibody-conjugate complexes thru a filter matrix or lateral flow matrix
- all controls are included in the membrane
- results are seen as colored spots
- rapid and simple assay: each test contains a positive and negative control
- sensitivity varies
detection of viral nucleic acids: what types of viruses can be detected
detects viruses that are non-cultivable
- allows detection of viruses that are not viable
- detects latent infections
- detects viruses that have been bound or complexed
- detects virus in formalin-fixed tissues
detection of nucleic acids: PCR
- is an in-vitro method for the enzymatic synthesis of specific DNA sequences
- it amplifies DNA
- primer annealing provides dsDNA for the polymerase to extend the DNA synthesis
- primers can also degenerate to increase the test sensitivity whenever there is variability
reverse transcription polymerase chain reaction (RT-PCR): for RNA viruses
what versions are there?
conventional two-tube RT-PCR
single-tube RT-PCR: all components for both reactions are contained in a single reaction tube
PCR usually consists of 3 steps:
- denaturation of dsDNA
- primer annealing
- extension
some PCR precautions:
- lab becomes contaminated with amplified DNA products as tubes are opened and false positives are obtained in subsequent PCRs
- nested PCR assays are contamination-prone
- real-time PCR or qPCR measures accumulation of DNA as it is amplified and has solved most of these contamination problems
deep sequencing
new viruses are discovered by random nucleic acid amplification and relatively low cost sequencing.
real-time polymerase chain reaction
once tubes are set, there are no longer opened - preventing cross-contamination.
- PCR product is measured by increases in fluorescence intensity generated by reporter molecules.
- this is more sensitive than conventional PCRs
examples of reporter molecules
DNA binding dyes like SYBR GREEN I, TaqMan probes and quenchers
advantages of PCR
- useful to detect virus that wont grow in culture: papillomavirus, norovirus, astrovirus, rotavirus
- can be used on any sample appropriate for virus isolation
- fast
- preferred for initial ID of dangerous viruses (rabies, hendra virus, nipah virus, influenza, ebola, etc)
