Polymerase Chain Reaction (PCR) as a Diagnostic Tool

M.9, W.1, L.10

What is a polymerase chain reaction (PCR)?

lab technique to rapidly produce millions to billions of copies of DNA

What are the key components of PCR? (5)

1. Template DNA (blood, tissue, swabs)

2. Primers (short synthetic oligonucleotides)

3. Taq polymerase (DNA polymerase)

4. Deoxynucleotide triphosphates (DNA building blocks)

5. Buffer (Mg2+)

How does PCR work in simple terms?

• Target DNA denatured to separate strands (95°C)

• Cool reaction = primer can anneal to target (55 - 65°C)

• Primers then extended using Taq polymerase (72°C)

• Repeat 25-30 times

  • until you get sufficient material

What is reverse transcriptase PCR (RT-PCR)?

an RNA template that is converted to complementary DNA using enzyme reverse transcriptase

*convert RNA to DNA then continue using same steps as previous flashcard

List the 6 components for a RT-PCR?

1. Template RNA

2. Primers

3. Reverse transcriptase

4. Taq polymerase

5. Deoxynucleotide triphosphates

6. Buffer

How is the product of PCR detected?

Electrophoresis (lab technique that separates charged molecules based on size and electrical charge using electric field)

What is real-time PCR (qPCR)?

lab technique that uses fluorescent dye to measure product as reaction progresses in real time

List the two detection methods for a qPCR

1. SYBR Green

2. TaqMan

Describe what SYBR Green is

• non-sequence-specific

• flourescent dye that binds to dsDNA

• emits green fluorescent signal upon binding, allowing researchers to monitor amplification in real time

• specific and non-specific product is amplified (binds to any dsDNA)

Describe TaqMan qPCR

• sequence-specific

• DNA probes attached to fluorochrome - binds to target sequence

• only bind to specific target sequence

  • allows for precise, real-time monitoring of amplification

What is baseline in PCR?

Calculation of background fluorescence

What is the threshold line in PCR?

the level at which fluorescence is statistically higher than baseline

What is Ct in PCR?

the cycle number where the fluorescence signal crosses a predefined threshold

• value refers to # of cycles needed to replicate enough DNA/RNA to be detected

What does a low Ct mean?

High amount of target (DNA/RNA) in sample

What does a high Ct mean?

Low amounts of target (DNA/RNA) in sample

List 4 veterinary uses of PCR

1. genetic screening

2. tumor diagnostics

3. detection of potential pathogens

4. monitoring progression of disease/infection

What gene can PCR be used to detect hypertrophic cardiomyopathy in Maine Coons and Ragdolls?

MYBPC3 gene

What gene can PCR be used to detect polycystic kidney disease in Persian and Persian-related cats?

PKD1 gene

What geneS can PCR be used to detect progressive retinal atrophy in dogs?

Multiple types and genes:

• rcd4-PRA

• NECAP1

what kind of PCR test is used for diagnosing lymphoma and lymphoid leukemia

PARR - PCR testing for Antigen Receptor Rearrangement

What gene mutation can PCR be used to detect canine mast cell tumors?

C-kit gene mutation

What gene mutation can PCR be used to detect canine transitional cell and prostate carcinomas?

BRAF mutation

what can affect PCR results

1. Poor sample collection technique

2. sample contamination

3. sample degradation

4. PCR inhibitory substance (e.g. heparin, charcoal, bile salts)

What can cause a false positive PCR?

• Lab contamination

• non-specific primers

What can cause a false negative PCR?

• Non-specific primers

• mutant strains

• Failed reaction

list some advantages of PCR

• rapid

• specific diagnosis

• identifies carriers+shedders

• strain-specific identification

list some limitations about PCR

• Result Interpretation

  • no info on infectivity (if its intact or alive)

  • no data on antimicrobial susceptibility

  • is it clinically significant?