Laboratory diagnosis of viral infection Flashcards
Virus isolation/culture
3 types
_____
_____
_____
Cell culture
Egg inoculation
Animal inoculation
Egg inoculation
________ are among the most useful form of living animal tissue
Used for the ____ of viruses or _____ viruses
Used for _________ cultivation in the production of viral vaccines
Embryonated eggs
isolation; titrating
large quantity
Animal inoculation
In 1909, Landsteiner & Popper used ______ to isolate Polio virus.
_____,______,_______,_______ are now commonly used
monkeys
Rabbit, Guinea pig, mouse, rat
Earliest method for cultivation of viruses causing human disease is by ______
Animal inoculation
Animal inoculation
The growth of virus in inoculated animals may be indicated by _____,_____ or _______
death, disease or visible lesions.
Disadvantages of animal inoculation are that they may ______ with viral growth and the animals often _________
interfere
harbour latent viruses.
Chorioallantoic membrane inoculation is used for which viruses
Herpes simplex
Pox
Rous sarcoma
Amniotic inoculation is used for which viruses
Influenza
Mumps
Yolk sac inoculation is used for which viruses
Herpes simplex
Allantoic inoculation is used for which viruses
Influenza
Mumps
Newcastle disease virus
Avian adenovirus
Cell culture
Primary cell culture
It consists of ____ cells, freshly taken from body & cultured.
They are capable of _____ growth in culture & cannot be maintained in _____
Eg. Monkey, kidney, human embryonic kidney, human amnion & chick embryo cell culture.
normal; limited; serial culture.
Cell culture
Primary cell culture
They are useful for __________ and for ________
isolation of viruses & for vaccine production.
_______ is best cell culture systems available
Why?
Primary cell culture
since they support the widest range of viruses
Primary cell culture is quite cheap
In Primary cell culture it is often difficult to obtain a reliable supply
T/F
F, they are very expensive
T
Cell culture
Diploid cell culture
These are used to ensure ___________
After ____ serial passages, they undergo ________.
Eg. Human _______
a continuous supply of cell line.
50; senescence
fibroblasts
Diploid cell culture are useful in viral vaccine production.
T/F
T
Cell culture
Continuous cell culture
These are single type usually derived from _____ cells that are capable of __________________________________.
Eg. _____ for rabies vaccine, ___ , HEp-2, LLC-MK2
cancer
continuous serial cultivation indefinitely
Verocell; Hela
Continuous cell culture are not useful for vaccine production.
T/F
F
They are
________ cell culture is most easy to handle
Continuous
the range of viruses supported by continuous cell culture is limitless
T/F
F
often limited
Mention the types of cell cultures
Primary
Diploid
Continuous
Cell cultures
Growing virus may produce
- ________
- ________
Cytopathic Effect (CPE)
Haemadsorption
Cell cultures
Growing virus may produce
- Cytopathic Effect (CPE) - such as the ______ of cells or ______ formation, may be specific or non-specific.
- Haemadsorption - cells acquire the ability to _______________
ballooning; syncytia
stick to mammalian red blood cells
Cell cultures
Confirmation of the identity of the virus may be carried out using ______,__________, or __________ tests.
neutralization, haemadsorption-inhibition or immunofluorescence
Syncytium formation is seen in cell culture caused by ____ ,and _____ virus
RSV
measles
Problems with cell culture
_____ period (up to _____ ) required for result.
Often very poor ________ ,which depends on a large extent on the _________.
Susceptible to ___________.
Long; 4 weeks
sensitivity; condition of the specimen.
bacterial contamination
Problems with cell culture
Susceptible to ________ which may be present in the specimen.
Some viruses will not grow in cell culture e.g. ________,________,________,________
toxic substances
Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus.
Light microscopy
Detecting the ____________ in smear & tissue.
viral inclusion body
Light microscopy
Inclusion bodies are ________ of stainable substances, usually proteins.
Can either be :
_______ inclusions or ________ inclusions
dense aggregates
Intra nuclear
Intra cytoplasmic
Electron microscopy
Virus particles are detected and identified on the basis of ______
______ virus particles per ml required for visualization
______-______ magnification normally used.
morphology
10^6
50,000 - 60,000
Electron microscopy
Use Vesicle Fluid for ______ or ____
Skin scrapings for _____,_____,____
Faeces for ____,____,_______,______ ,______
HSV VZV
papillomavirus, orf molluscum contagiosum
Rotavirus, Adenovirus Norwalk like viruses Astrovirus, Calicivirus
Molecular methods
________
_________ reaction
_______ reaction
__________ based amplification
________ mediated amplification
Hybridisation
Polymerase chain
Ligase chain
Nucleic acid sequence
Transcription
Polymerase chain reaction or PCR
is a simple laboratory technique to obtain ______ of _____ fragments even from samples containing only ____ quantities of DNA or RNA.
multiple copies
specific DNA
minute
Essential components of PCR
______
________
________
______ cations
Deoxynucleoside triphosphates (dNTPs)
________ solution
Template DNA
Primers
Thermostable DNA polymerase enzyme
Divalent
Buffer
Essential components of PCR
Template DNA that contains the ______ of interest to be amplified during the PCR.
target sequence
Essential components of PCR
Primers- A pair of synthetic ________ (______ and ____ primers) that are complementary to the ___’ ends of each of the two strands of target DNA.
oligonucleotides
forward and reverse
3
Essential components of PCR
Thermostable DNA polymerase enzyme like ____ polymerase (originally isolated from ————————- ) is a vital ingredient of a PCR to _____ the _____________________ of DNA.
Taq
thermophilic bacterium Thermus aquaticus
catalyse; template-dependent synthesis
Essential components of PCR
Divalent cations, usually ____ are required in (minute or optimum?) concentration for the activity of most __________ as well as for several other steps in PCR.
Mg 2+
optimum
thermostable DNA polymerases
Essential components of PCR
Deoxynucleoside triphosphates (dNTPs)-
________ amounts of each dNTP (dATP, dCTP, dGTP, dTTP), which are ______ used by the DNA polymerase enzyme to synthesize a new strand of DNA.
Equimolar
building blocks
Essential components of PCR
Buffer solution to maintain ____________ for optimum activity and ______ of the _____
suitable ionic environment
stability
DNA polymerase.
STEPS IN PCR
______
————
________
Denaturation
Annealing
Extension
STEPS IN PCR
Denaturation: The first step of a PCR where the sample is ____ to _____ or _____ the two strands of the DNA (>_____oC).
heated
separate or denature
90
STEPS IN PCR
Annealing: the reaction temperature is ______ (usually ___-__ 0C below the Tm of _____ )to allow the ————- to bind to the single strands of the template DNA.
lowered
3-5; primer
oligonucleotide primers
STEPS IN PCR
Extension: the temperature is ______, typically to ____°C, allowing specific enzymes to synthesize a new DNA strand complementary to the DNA template.
Raised
72
STEPS IN PCR
Typically about ___-___ cycles of PCR are performed depending upon the _________ used, the __________ and the ______________ for post-PCR processing.
25 to 45; type of PCR ; amount of initial template DNA ; number of amplicon copies desired
STEPS IN PCR
The PCR is commonly performed in a reaction volume of ____ μl in small reaction tubes (___-___ ml volumes) in a _______ that heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction.
10–200; 0.2–0.5
thermocycler
Immunological assay
Antigen detection
•_________ assay (ELISA)
•_____________
•___________/_________ assay
Enzyme linked immunosorbent
Immunoflourescence
Immunochromatographic /lateral flow
Immunological assay
Antibody detection and quantification
•_____
•_____________ test
•__________ test
ELISA
Complement fixation
Immunoflourescence Rapid
Post PCR analysis
Agarose gel electrophoresis.
PCR products can be visualized by staining the gel with _______ such as ______ which binds to DNA and intercalates between the stacked bases.
Confirmation of size of the DNA product is done by ______________________
fluorescent dye
ethidium bromide
comparing the size with DNA ladder.
The appearance of discrete band of the correct size may be indicative of a successful PCR amplification.
T/F
T
POST PCR analysis
______ electrophoresis
______ the PCR product
__________ with a specific _______
Agarose gel
Sequencing
Hybridization; oligonucleotide probe
