Laboratory Flashcards
Examples of direct exam
LAVE light microscopy Ag detection Viral genome detection Electron microscopy
Diagnostic methods in virology
Direct
Indirect
Serology
Examples of indirect examination (3)
Cell culture
Eggs
Animals
Growing virus may produce?
Cytopathic effect
Hemadaorption
Termed as ballooning of cells or syncytia formation, may be non specific or specific
Cytopathic effect
Examples that give CPE
Ballooning: Enterovirus 71 HsV Syncytia: RSV Measles
Cells acquire the ability to stick to mammalian RBCs
Hemadsorption
In cell cultures, confirmation pf the identity of the virus may be acrried out using..
Neutralization
Hemadsorption-inhibition
IF tests
3 types of cell culture
Primary
Semi continuous
Continuous
Human embryonic kidney? Monkey kidney? HeLa? Vero? Skin fibroblast?
Semi Primary Cont Cont Semi
Most easy to handle but the range pf viruses suported is often limited
Continuous
Readily isolated by cell culture (9)
RICE PA MCH!!
Rsv InfluenZa Cmv Echo ParainfluenZa Adeno Mumps Cox b Hsv
Rapid culture how many days?
2-4 days
Explain CMV Deaff test
Cell grown in cover slips; spun for 1 hr; incubated for 2-4days; examined by IF
Cannot be grown in cell culture
Diarrheal viruses
Parvo
HPV
hepB
How many virus particles to be visualized in EM
10^6
Which specimens may virus be detects by EM?
Feces
Vesicle fluid
Skin scrapings
Feces?
Diarrheal: rota, adeno, calici, norwalk, astro
The specificity and sensitivity of em may be enhanced by
Immune electron microscopy
2 variants of immune em
Classic
Solid phase
Sample is treated with specific antisera before being put up for em
Classical
Grid is coated with antisera
Solid phase
Problems with em
Expensive
Experience
Low sensitivity
Detection of rising titers bet acute and convalescent stages or igM detection
Serology
Newer serology techniques?
Radio immunoassay
Enzyme linked immunosorbent assay
Particle agglutination
Western blot
Criteria for diagnosing primary infection (4)
4 fold increase in igG
Presence of igm
Seroconversion
Single high titer of igG
Criteria for reinfection?
Absence of slight increase in IgM
Viruses that produce clinical disease before appearance of antibodies
Respiratory
Diarrheal virus
Virus which produce clinical disease months or years after seroconversion
Hiv and rabies
Parts of a western blot
Lane 1 and 2
Sample a,b,c
Lane 1?
Positive control
Lane 2?
Negative control
Sample c?
Positive
Consists of 2 rows in which 4 fold increase is aignificant and indicates recent infection
Complement fixation
Used mainly for hsv and vzv encephalitis
Csf antibodies
Indicative of meningitis
Csf/serum > 1/100
Rapid diagnosis in blood
Cmv (pp65 antigenemia test)
Problems of IF
Reduced sensitivity and specificity compared to cell culture;
Expensive in terms of lab time
Nethods based on detection of viral genome
Molecular method
Examples of classical molecular method
Dot blot
Southern blot
In situ hybrid
Temps in pcr?
DenaturAtion - 94c
Annealing - 50
Extension - 72
Cycles of pcr?
25-40
Identificatin of pcr product by?
AGE
HYBRIDIZATION
rest enzyme analysis
Dna sequencing
In PCR, Each cycle _____ the copy number of target
Doubles
Linear amplification what method?
Branched dna
Promising technology that detects large number of virus, pathogenic potential, drug sensitivity at the same time
DNA chip
Techniques with target amplification?
Pcr
Nasba
Tma
Techniques that have Signal ampli?
Lcr
Qb replicase
Monoclonal antibody labeled
Dfa (30-45)
