Diagnostics: PCR

Question 1

Define assay

Answer 1

Refers to a test

Question 2

What is a benchmark?

Answer 2

Measure of the true disease status of an animal

Question 3

Define test sensitivity

Answer 3

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

Question 4

Define test specificity

Answer 4

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

Question 5

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

Answer 5

For accuracy e.g. prevent unnecessary culling

Question 6

When was PCR developed?

Answer 6

1980s

Question 7

What are the three part processes of PCR?

Answer 7

1. DNA extraction
2. Polymerase Chain Reaction
3. Gel electrophoresis

Question 8

What happens during the polymerase chain reaction?

Answer 8

DNA is amplified and creates numerous copies

Question 9

What happens during gel electrophoresis?

Answer 9

Visualisation

Question 10

What are the components of PCR?

Answer 10

• DNA template
• DNA polymerase
• Primers
• Nucleotides

Question 11

What are primers?

Answer 11

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

Question 12

What is used during the DNA polymerase stage?

Answer 12

• Taq DNA polymerase (thermis aquaticus)
• Pfu DNA polymerase (Pyrococcus furiosus)

Question 13

What nucleotides are used during PCR?

Answer 13

-DNTPS
- Deoxynucleotide triphosphates

Question 14

What are the 4 steps of PCR?

Answer 14

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

Question 15

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

Answer 15

True

Question 16

What does gel electrophoresis compare to?

Answer 16

Known values

Question 17

Why do we use primers during the gel electrophoresis stage?

Answer 17

Gives us the sensitivity for the wanted DNA

Question 18

What is reverse transcriptase PCR used for?

Answer 18

Detects viruses using viral RNA

Question 19

Why is nested PCR used?

Answer 19

Reduces non-specific binding in products

Question 20

Why is multiplex PCR used?

Answer 20

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

Question 21

Why is quantitative PCR used?

Answer 21

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

Question 22

Why would arbitrary primed PCR be used?

Answer 22

For internal control

Question 23

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

Answer 23

• Feline Coronavirus
• Parasites e.g. Brugia malayi in mosquitoes

Question 24

What are the advantages of Nested PCR

Answer 24

• Reduced non-specific binding sites
• Increases sensitivity and specificity

Question 25

What are the negatives of nested PCR?

Answer 25

- Needs 2 primers - Potential for carryover contamination - Expensive

Question 26

What are the characteristics of quantitative PCR?

Answer 26

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

Question 27

What does RAPD stand for?

Answer 27

Randomly Amplified Polymorphic DNA

Question 28

What are the characteristics of RAPD?

Answer 28

- Sample DNA mixed with random primers - Primers bind with "random loci" - Creates genomic fingerprint

Question 29

What are the applications of PCR?

Answer 29

- Identification and characterisation of infectious agents - Investigation of strain relatedness of pathogen of interest - Cloning genes - PCR sequencing

Question 30

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

Answer 30

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

Question 31

What are veterinary applications of PCR?

Answer 31

- Gene expression analysis - Viral load - Detection of genetically modified organisms - SNP genptyping and allelic discrimination

Question 32

What are advantages to PCR?

Answer 32

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

Question 33

What are disadvantages to PCR?

Answer 33

- Contamination fairly easy - Primers need to be designated - Primers can anneal to undesired sequences - Incorrect nucleotides

Question 34

What is Cryptosporidium?

Answer 34

- A zoonotic parasite - Resistant to chlorine - Causes watery diarrhoea - Protozoa

Question 35

What is the causative agent of foot and mouth disease?

Answer 35

RNA virus

Question 36

How can foot and mouth disease be diagnosed?

Answer 36

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

Question 37

What is Canine leishmaniasis?

Answer 37

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

Question 38

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

Answer 38

- Domestic dogs - African wild dogs - Japanese monkeys

Question 39

What are the clinical signs of canine distemper virus?

Answer 39

- Transient fever - Immunosuppression - Loss of appetite - Slight depression - Tonsillitis - Ocular and nasal discharge

Question 40

How can Canine distemper virus be detected?

Answer 40

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

Question 41

What is the treatment for canine distemper virus?

Answer 41

- Live attenuated vaccine developed in 1950's - CDV in vaccinated animals have been reported throughout the world