Download Detection, Quantification and Lab diagnosis Dr. Sobia Manzoor

Detection, Quantification and
Lab diagnosis
Dr. Sobia Manzoor
ASAB, LECTURE 4
Contents
• Rationale use of the laboratory
• Specimen processing
• Identification of viral components
Rationale use of the laboratory
Viral diseases are likely to take place. Even
chemotherapeutic agents are available. Advances in
virology paves the way to explore new viruses or drug
resistant viruses. For all this, we need to have a
system where these viruses can be isolated and
identified.
2. Management of the patient or prognosis is
influenced by the diagnosis.
1.
3. Infections may demand public health measures. E.g. blood
banks are routinely screened for HIV, HBV & HCV. Herpes
simplex is easily transmittable thorough sex workers. All
these information come through the effective use of lab
diagnosis.
4. Surveillance of viral infections help to monitor disease
prevalence, genotype dominance, history, significance as
well as evaluating immunization programs.
Specimen processing
 Collection of
viral sample is
a critical step
in diagnosis.
Identification of viral components
1.
Viral isolation
2. Antigen detection
1.
Immunoflourscence
2.
ELISA
3.
Electron microscopy
4.
serology
3. Nucleic acid detection
1. Viral isolation
 Advances in virology greatly relies on viral
propagation.
 Appropriate cell line, cytopathic effect (CPE) is
formed.
 Monkey kidney cell line, human fibroblast cell line etc.
Advantages
Disadvantage
Sensitive
Slow
Catch all
Labor intensive
Isolates
Multiple cell line required
Detect viable virus
Antigen detection
Immunoflourscence
 Rapid diagnostic tool.
 Initially undertaken with polyclonal anti-sera and then
with monoclonal antibody.
 The method makes use of a labeled Ab. Usually its
florescence.
Enzyme linked immunosorbant assay
(ELISA)
Serological tests
• Viruses generate an
immune response--important serological
target for diagnostics.
• Serological markers are:
IgG, IgM, IgE, etc.
Advantages of serological tests
1.
IgG----good indicator of prior infection
2. IgM---good indicator of recent infection
3. Automated
4. Rapid
5. Don’t need culturing
6. Can utilize non-invasive samples such as urine and
saliva.
Disadvantages of serological tests
1.
In certain cases, cross reactivity.
2. Insensitive against some congenital diseases such as
cytomegalovirus (CMV).
3. Not suitable for immunocompromised individuals.
Nucleic acid detection
PCR-Reagents
 DNA template
 Primer (F & R)
 MgCl2
 dNTP
 Taqpolymerase
 Nuclease free water
Generally, the volume of
the mixture is 20µl