Collection and Preparation of Clinical Specimens Flashcards
You want to collect a specimen in the _____ phase of disease
Acute, before antibody formation
- can not detect a virus if it is bound to an antibody
What are examples of specimens commonly used?
- blood
- nasal swabs
- feces
- urine
- pus
- vesicular fluid
- skin lesions
- spinal fluid
- biopsy and necropsy tissue
It is important to collect specimens immediately _______
After death
- autolytic changes are detrimental to viruses
Specimens should be taken with ______ instruments, using ______ technique
Sterile; aseptic
What 3 methods are used for isolation of viruses?
- embryonated eggs (most common)
- cell cultures
- live animals
Fluid specimens
Must be bacteria free
- inoculated directly, or after dilution
Solid tissue preparation
10-20% solution in 0.15M PBS, pH 7.2
- centrifuged at 2,000 xg for 10 minutes to remove tissue and cellular debris
- bacteria must be eliminated prior to inoculation using 10-15% ether for 1-2 hours
Antibiotics
Penicillin at 1,000 U/ml +/- 100 ug/ml streptomycin
- used to eliminate contaminating bacteria
Sedimentation
Centrifugation at 40,000 xg for 1 hr will concentrate most viruses
- pellet will be resuspended in a small volume of PBS prior to filtration and inoculation
_____ filters will trap most bacteria
- 22 micron
- does not trap myoplasma!! (lack a cell wall)
Storage
Fresh tissues are frozen to -60 C after collection
- specimens may be placed at -20 C until dry ice is obtained
- vials must be air tight due to CO2 gaseous phase of dry ice that will lower pH and inactivate certain viruses
Are live animals used for isolation of viruses?
NO
- specifically not used in diagnostics
- exception: rabies virus in neonatal mice
What are the 2 methods for detection of viruses in clinical specimens?
- direct
- indirect
Direct method
The virus itself, or component antigens or viral molecules, are detected
Indirect methods
Serologic evidence, in the form of antibodies, is taken as a sign of viral infection
Are all test methods applicable to all viruses?
No, some test methods work well for some viruses, but not others
- due to variations between individual viruses and standardizes results among different labs for consistency
T/F: Only avian viruses will replicate in embryonated chicken eggs
False!
- cells and extra-embryonic membranes of chickens lack a high degree of specialization
Why are chicken embryos used for viral isolation?
- availability
- economy
- convenient size
- relative freedom from letent infection and extraneous contamination
- lack of production of antibodies against viral inoculum
Incubation process
Preliminary incubation at 100.4-102.2F with incubation of incoulated embryos at 98.9-99.5 F (can stay at this temp throughout the entire period)
- lower temps may be required under certain cirumstances
When are eggs obtained?
Get eggs at 9 days old, and they will hatch at 21 days
Yolk sac inoculation
Done on 5-7 day old eggs
- used with smaller viruses, that will invade the embryo and multiply in the body tissues
- 0.2-1.0 ml volume
Chorioallantoic cavity inoculation
Used for viruses that cause respiratory infections and encephalomyelitis viruses that readily multiply in the entodermal cells of the chorioallantoic sac wall
- 8-11 day old eggs
- 0.1-0.2 ml volume
Chorioallantoic membrane inoculation
Largest, cleanest, easiest to harvest
- 10-12 day old eggs
- 0.1-0.5 ml volume
Amniotic sac inoculation
Surrounds the embryo itself, is used primarily for isolation of influenza viruses from throat washings
- 7-15 day old eggs
- 0.1-0.2 ml volume
Intravenous inoculation
Not used often
- 10-15 day old eggs
- used for adaptation and replication of foot and mouth disease
- 0.02 - 0.05 ml volume
Intracerebral inoculations
Done on 8-14 day old eggs
- rabies and herpes simplex cultivated by this route
- 0.01 - 0.02 ml volume
What are factors influencing the growth of viruses in chicken embryos?
- age of the embryo
- route of inoculation
- concentration of virus and volume of inoculum
- temperature of incubation
- time of incubation following inoculation
Methods determining replication of a virus in embryos
- sampling of the virus in extraembryonic fluids and membranes
- pathologic alterations
- serologic tests
- hemagglutination
- antigenicity
- immunogenecity
_____ are the most widely used method for isolation of viruses from clinical material
Cell cultures
- denotes growing of cells in vitro
- use from homologous species (canine cells to propagate canine distemper)
Primary cell culture
Tissue culture started from material taken directly from an animal
- preferred due to increased susceptibility to viruses
- can become immortalized and are then referred to as cell lines, capable of indefinite subculturing
Established cell lines have advantage in _____ and ______
Consistency and uniformity
What allows for differentiation and preservation of architecture and function?
Growth of whole organs, or parts of an organ
- difficult
At ____ cuts, the cell starts to mutate and adapt to the culture
40
________ inoculation in mice is used to isolate neurotropic viruses
Intracerebral
Electron microscopy
Use negative contrast transmission to morphologically characterize viral particles (direct method)
- commonly used with enteric viruses
- examination of excretions, secretions, solid tissues
What is the preferred concentration of viral particles for electron microscopy?
10^8/ml permits rapid detection
- 10^6/ml is good, 10^5/ml is barely detectable
Knowledge of ____ and _____ is necessary to identify viruses with electron microscopy
- viral sizes
- morphology
Direct fluorescent antibody test
Quick, easy, need a special microscope to view results
- use a specimen from the host that contains the virus and attach commercially fluorescent labelled antibodies to the virus
- used to track multiple viruses within a host at a single time
- doe not work with all viruses
Indirect fluorescent antibody test
Specimen contains the virus and an unlabelled antibody (made from the host) and the commercial fluorescent labelled antiglobulin attaches to the antigen-antibody complex
- all antibodies are of known specificity and concentration
Enzyme linked immunosorbent assay
Used for detection of virus or viral antigen, that involves virus capture by specific antibody attached (absorbed or covalently bound) to a solid substrate
- captured antigen is detected by an enzyme labeled antiviral secondary antibody
- final washing step reveals result based on a visible color change following the addition of a substrate
What is the enzyme used in ELISA?
Usually horesradish peroxidase or alkaline phosphatase
ELISA can be designed to detect _____ or _____
Antigen (direct) or antibody (indirect)
Radioimmunoassay
Not commonly used
- similar to ELISA, but indicator system is isotope labeled antibody instead of enzyme labeled antibody
PCR
Common for labs, not clinic use
- nucleic amplification assays are universally applicable with the appropriate primers
PCR primer
Short, oligonucleotide sequence that attaches to a segment flanking the gene targeted for amplification
- after attachment, primers are extended into target gene region by the enzymatic addition of new nucleotides, added sequentially to the primer
What gene regions do primers attach to?
One serves as the upstream (forward) primer, the other as the downstream (reverse) primer
- bracket the target gene so that both opposing DNA strands are amplified = numerous double stranded copies
How are the double stranded copies turned into single strands?
Double strands are dissociated (melted) into single strands by increasing the reaction temperature
- temp is then decreased to reintroduce the primers
- cycle will repeat
General cycle of PCR
Dissociation –> annealing –> polymerase activity
- cycle will repeat 30-40 times
Taq polymerase
Heat-stable DNA polymerase commercially purified from the thermophilic organism Thermus aquaticus
- most expensive commercial ingredient
- high active temperature of this enzyme shortens temperature swings between strand “melting” and polymerase activity = quicker cycling
- extend primers between 2 fixed points on a strand of DNA
Thermocycler
Takes reaction mixture to 94 C for 60 sec to allow for melting –> 55 C for 60 sec to permit primer attachment –> 72 C for 120 sec to allow for polymerase addition of nucleotides to the primer
- final cycle of 72 C for 300 sec allows completion of new strands
What is the final step of PCR?
Detection and identification of amplified DNA
- PCR product, or amplicon
- done with agarose-gel electrophoresis, or gene probes
Gene probes
Species-specific oligonucleotide segments that incorporate markers to facilitate detection
- binding of probe to target gene demonstrates sequence homology, establishing identity
______ is critical to successful use of PCR
Primer selection
Restriction fragment length polymorphism
Electrophoretically analyze PCR products
- used to compare related organisms or characterize
Nested primers
Increase sensitivity and specificity in the detection of organisms
RT-PCR
Frequently applied in the detection and analysis of certain viruses, many of which utilize RNA in their genetic code
- requires an extra cycle to precede PCR to allow RT to convert viral RNA into DNA
PCR can be applied to _____ or _______
Viral DNA or RNA
