Diagnostics: PCR Flashcards
Define assay
Refers to a test
What is a benchmark?
Measure of the true disease status of an animal
Define test sensitivity
Availability of test to identify the presence of a disease or illness correctly
Define test specificity
Ability of a test to correctly exclude individuals who don’t have a given disease or disorder
Why do you want a test to have high sensitivity and specificity?
For accuracy e.g. prevent unnecessary culling
When was PCR developed?
1980s
What are the three part processes of PCR?
- DNA extraction
- Polymerase Chain Reaction
- Gel electrophoresis
What happens during the polymerase chain reaction?
DNA is amplified and creates numerous copies
What happens during gel electrophoresis?
Visualisation
What are the components of PCR?
- DNA template
- DNA polymerase
- Primers
- Nucleotides
What are primers?
Single-stranded DNA of short pieces added to help polymerase generate and amplify DNA
What is used during the DNA polymerase stage?
- Taq DNA polymerase (thermis aquaticus)
- Pfu DNA polymerase (Pyrococcus furiosus)
What nucleotides are used during PCR?
-DNTPS
- Deoxynucleotide triphosphates
What are the 4 steps of PCR?
Template DNA
1. Denaturation- double to single strand process
2. Annealing
3. Extension- synthesis of new DNA
True or false: the shorter the sample of DNA, the more it will travel
True
What does gel electrophoresis compare to?
Known values
Why do we use primers during the gel electrophoresis stage?
Gives us the sensitivity for the wanted DNA
What is reverse transcriptase PCR used for?
Detects viruses using viral RNA
Why is nested PCR used?
Reduces non-specific binding in products
Why is multiplex PCR used?
Amplifies multiple targets in a single target and is good for identifying unknown diseases
Why is quantitative PCR used?
Very quick process which doesn’t require gel electrophoresis and easy to interpret
Why would arbitrary primed PCR be used?
For internal control
What can reverse Transcriptase PCR be used to identify e.g. disease?
- Feline Coronavirus
- Parasites e.g. Brugia malayi in mosquitoes
What are the advantages of Nested PCR
- Reduced non-specific binding sites
- Increases sensitivity and specificity
What are the negatives of nested PCR?
- Needs 2 primers
- Potential for carryover contamination
- Expensive
What are the characteristics of quantitative PCR?
- Set time for reaction
- Fluorescence measured
- Intensity directly related to quantity of DNA present in sample
- Compared to standard and blank
What does RAPD stand for?
Randomly Amplified Polymorphic DNA
What are the characteristics of RAPD?
- Sample DNA mixed with random primers
- Primers bind with “random loci”
- Creates genomic fingerprint
What are the applications of PCR?
- Identification and characterisation of infectious agents
- Investigation of strain relatedness of pathogen of interest
- Cloning genes
- PCR sequencing
What is a negative to PCR in relation to detection of infection?
Detects both live and dead disease in DNA- no way of knowing if infection is alive or dead
What are veterinary applications of PCR?
- Gene expression analysis
- Viral load
- Detection of genetically modified organisms
- SNP genptyping and allelic discrimination
What are advantages to PCR?
- Simple to use and understand
- Highly sensitive
- Produce millions of copies of a single product
- Can be used for gene expression analysis
What are disadvantages to PCR?
- Contamination fairly easy
- Primers need to be designated
- Primers can anneal to undesired sequences
- Incorrect nucleotides
What is Cryptosporidium?
- A zoonotic parasite
- Resistant to chlorine
- Causes watery diarrhoea
- Protozoa
What is the causative agent of foot and mouth disease?
RNA virus
How can foot and mouth disease be diagnosed?
- Rapid diagnostic assays recommended- ELISA ia not sensitive enough
- Rapid Reverse transcriptase PCR
- Portable smartcycler & Bioseeq platforms
What is Canine leishmaniasis?
- Protozoan and zoonotic
- Multiple pathogenic mechanisms
- Variable clinical presentations
- High seroprevalence in subclinical dogs
- Test positive with conventional PCR
- qPCR
Give an example of some of the hosts for canine distemper virus
- Domestic dogs
- African wild dogs
- Japanese monkeys
What are the clinical signs of canine distemper virus?
- Transient fever
- Immunosuppression
- Loss of appetite
- Slight depression
- Tonsillitis
- Ocular and nasal discharge
How can Canine distemper virus be detected?
- Using RT-PCR: sensitive and specific method to ID this virus
- Hemagglutinin gene amplified and sequenced: genetic variability of the local strains
What is the treatment for canine distemper virus?
- Live attenuated vaccine developed in 1950’s
- CDV in vaccinated animals have been reported throughout the world
