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Lab Diagnosis of Viral Infections
Virus culture and isolation
-CPE
-Hemagglutination
-Plaque assay
-TCID50 assay
Detection of viral antibody
-Hemagglutination -inhibition test
-EIA/ELISA
Detection of viral antigen
Immunofluorescence
-EIA/ELISA
-Western Blot
Immunoprecipitation
Detection of viral genome
-(PCR)
-southern & northern blot
Lab Diagnosis of Viral Infections
Detect and identify the viruses
-Microscopy in Cell Culture
-Immunofluorescence (IF)
-Molecular Methods
Quantify the viruses
-measuring infectivity
-examine nucleic acid and protein
-counting
particles
• Provides viable isolate of the virus
• Detects multiple viruses
• Cell culture types:
• 1. Primary cells - Monkey Kidney
• 2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts
• 3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK
• Cell monolayers are then examined by lm for CPE
• (centrifugation enhanced shell vial tech)
• detection of virus infections by identifying virus antigens using marked Abs.
• +ves : very rapid
• -ves : very expensive d.t cost of Abs and variability may occur as a result of non specific binding or cross reaction of the Abs
• amplification techniques including (PCR), (NASBA) and (LMDA) are rapid detection and molecular identification method for most known human viruses.
• +ves: They are extremely sensitive and rapid.
• -ves: very expensive and is useless in case of viral mutation
Lab Diagnosis of Viral Infections
Detect and identify the viruses
-Microscopy in Cell Culture
-Immunofluorescence (IF)
-Molecular Methods
Quantify the viruses
-measuring infectivity
-examine nucleic acid and protein
-counting
particles
• What is a “plaque” ?
• +ves: inexpensive
• -ves: considered time consuming, laborious and relative error may occur
• What is TCID50 ?
• -ves: time consuming, laborious
• +ves: inexpensive and gives more accurate results than plaque assay
• IFA utilizes an antibody based staining methods to detect virally infected cells.
• +ves: more sensitive and faster than traditional plaque assays or TCID50
• -ves: may be quite expensive and variability may also be introduced
Lab Diagnosis of Viral Infections
Detect and identify the viruses
-Microscopy in Cell Culture
-Immunofluorescence (IF)
-Molecular Methods
Quantify the viruses
-measuring infectivity
-examine nucleic acid and protein
-counting
particles
• detection &Amplification of viral genome.
• Reverse transcriptase PCR (RT-PCR).
• “real-time” PCR.
• +ves: very rapid, very accurate
• -ves: expensive, requiers skilled operators
• it employs antigens or antibodies coupled to an easily-assayed enzyme.
• Types of ELISA
• +ves: a rapid, highly sensitive method
• -ves: expensive , variability may also occur
• (HA) is the most common indirect method to quantify amount of virus particles.
• +ves: fast, inexpensive with simple principle
• -ves: considered laborious
• For visualization of specific DNA, RNA and protein among thousands of contaminating molecules.
• Types of blotting
• 1 ) Southern blotting ( to detect DNA )
• 2 ) Northern blotting ( to detect RNA )
• 3 ) Western blotting ( to detect protein )
Lab Diagnosis of Viral Infections
Detect and identify the viruses
-Microscopy in Cell Culture
-Immunofluorescence (IF)
-Molecular Methods
Quantify the viruses
-measuring infectivity
-examine nucleic acid and protein
-counting
particles
• It uses fluorescent dyes such as (SYBR Green I) for the DNA of viruses.
• +ves: allows acquisition of statistically reliable data with little effort
• -ves: does not provide high sensitivity
• (Virus Counter® 2100) two dyes tech.
• For viruses that are very small to be seen directly under light microscope.
• +ves: it doesn’t require virus-specific reagents
• -ves: high instrument cost and amount of space and facilities required