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Animal Health and Disease > Diagnostics: PCR > Flashcards

Diagnostics: PCR Flashcards

1
Q

Define assay

A

Refers to a test

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2
Q

What is a benchmark?

A

Measure of the true disease status of an animal

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3
Q

Define test sensitivity

A

Availability of test to identify the presence of a disease or illness correctly

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4
Q

Define test specificity

A

Ability of a test to correctly exclude individuals who don’t have a given disease or disorder

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5
Q

Why do you want a test to have high sensitivity and specificity?

A

For accuracy e.g. prevent unnecessary culling

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6
Q

When was PCR developed?

A

1980s

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7
Q

What are the three part processes of PCR?

A
  1. DNA extraction
  2. Polymerase Chain Reaction
  3. Gel electrophoresis
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8
Q

What happens during the polymerase chain reaction?

A

DNA is amplified and creates numerous copies

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9
Q

What happens during gel electrophoresis?

A

Visualisation

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10
Q

What are the components of PCR?

A
  • DNA template
  • DNA polymerase
  • Primers
  • Nucleotides
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11
Q

What are primers?

A

Single-stranded DNA of short pieces added to help polymerase generate and amplify DNA

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12
Q

What is used during the DNA polymerase stage?

A
  • Taq DNA polymerase (thermis aquaticus)
  • Pfu DNA polymerase (Pyrococcus furiosus)
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13
Q

What nucleotides are used during PCR?

A

-DNTPS
- Deoxynucleotide triphosphates

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14
Q

What are the 4 steps of PCR?

A

Template DNA
1. Denaturation- double to single strand process
2. Annealing
3. Extension- synthesis of new DNA

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15
Q

True or false: the shorter the sample of DNA, the more it will travel

A

True

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16
Q

What does gel electrophoresis compare to?

A

Known values

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17
Q

Why do we use primers during the gel electrophoresis stage?

A

Gives us the sensitivity for the wanted DNA

18
Q

What is reverse transcriptase PCR used for?

A

Detects viruses using viral RNA

19
Q

Why is nested PCR used?

A

Reduces non-specific binding in products

20
Q

Why is multiplex PCR used?

A

Amplifies multiple targets in a single target and is good for identifying unknown diseases

21
Q

Why is quantitative PCR used?

A

Very quick process which doesn’t require gel electrophoresis and easy to interpret

22
Q

Why would arbitrary primed PCR be used?

A

For internal control

23
Q

What can reverse Transcriptase PCR be used to identify e.g. disease?

A
  • Feline Coronavirus
  • Parasites e.g. Brugia malayi in mosquitoes
24
Q

What are the advantages of Nested PCR

A
  • Reduced non-specific binding sites
  • Increases sensitivity and specificity
25
Q

What are the negatives of nested PCR?

A
  • Needs 2 primers
  • Potential for carryover contamination
  • Expensive
26
Q

What are the characteristics of quantitative PCR?

A
  • Set time for reaction
  • Fluorescence measured
  • Intensity directly related to quantity of DNA present in sample
  • Compared to standard and blank
27
Q

What does RAPD stand for?

A

Randomly Amplified Polymorphic DNA

28
Q

What are the characteristics of RAPD?

A
  • Sample DNA mixed with random primers
  • Primers bind with “random loci”
  • Creates genomic fingerprint
29
Q

What are the applications of PCR?

A
  • Identification and characterisation of infectious agents
  • Investigation of strain relatedness of pathogen of interest
  • Cloning genes
  • PCR sequencing
30
Q

What is a negative to PCR in relation to detection of infection?

A

Detects both live and dead disease in DNA- no way of knowing if infection is alive or dead

31
Q

What are veterinary applications of PCR?

A
  • Gene expression analysis
  • Viral load
  • Detection of genetically modified organisms
  • SNP genptyping and allelic discrimination
32
Q

What are advantages to PCR?

A
  • Simple to use and understand
  • Highly sensitive
  • Produce millions of copies of a single product
  • Can be used for gene expression analysis
33
Q

What are disadvantages to PCR?

A
  • Contamination fairly easy
  • Primers need to be designated
  • Primers can anneal to undesired sequences
  • Incorrect nucleotides
34
Q

What is Cryptosporidium?

A
  • A zoonotic parasite
  • Resistant to chlorine
  • Causes watery diarrhoea
  • Protozoa
35
Q

What is the causative agent of foot and mouth disease?

A

RNA virus

36
Q

How can foot and mouth disease be diagnosed?

A
  • Rapid diagnostic assays recommended- ELISA ia not sensitive enough
  • Rapid Reverse transcriptase PCR
  • Portable smartcycler & Bioseeq platforms
37
Q

What is Canine leishmaniasis?

A
  • Protozoan and zoonotic
  • Multiple pathogenic mechanisms
  • Variable clinical presentations
  • High seroprevalence in subclinical dogs
  • Test positive with conventional PCR
  • qPCR
38
Q

Give an example of some of the hosts for canine distemper virus

A
  • Domestic dogs
  • African wild dogs
  • Japanese monkeys
39
Q

What are the clinical signs of canine distemper virus?

A
  • Transient fever
  • Immunosuppression
  • Loss of appetite
  • Slight depression
  • Tonsillitis
  • Ocular and nasal discharge
40
Q

How can Canine distemper virus be detected?

A
  • Using RT-PCR: sensitive and specific method to ID this virus
  • Hemagglutinin gene amplified and sequenced: genetic variability of the local strains
41
Q

What is the treatment for canine distemper virus?

A
  • Live attenuated vaccine developed in 1950’s
  • CDV in vaccinated animals have been reported throughout the world

Animal Health and Disease (6 decks)

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