Polymerase Chain Reaction (PCR) as a Diagnostic Tool

What is PCR?

Polymerase Chain Reaction

• Laboratory technique for rapidly producing (amplifying) millions to billions of copies (amplicons) of a specific segment of DNA (target)

• A means to detect/isolate nucleic acid copies

What are the key components for a PCR protocol?

• Template DNA - e.g. blood, tissue, swabs

• Primers - short synthetic oligonucleotides (Forward and reverse)

• Taq Polymerase - DNA polymerase (Facilitates extension of the DNA strand)

• Deoxynucleotide triphosphates (dNTPs) - DNA building blocks (Making up the new copies of the new nucleic acid)

• Buffer (Mgz+) (Creates an effective environment where DNA polymerase can function)

Describe the principles of PCR.

• Target DNA is DENATURED to separate strands [95° C]

• Two primers are designed to amplify the target region of DNA

• Reaction is cooled to allow primers to ANNEAL to target [55 - 65° C]

• Primers then EXTENDED using Taq polymerase [72° c]

• This cycle is then repeated another 25-30 times

• Until sufficient material and copies are produced

What determines the area of DNA which is amplified?

The primers

How does reverse transcriptase PCR or RT-PCR occur?

Unlike original PCR, starts with an RNA template

• RNA must be converted to complementary DNA (cDNA) using enzyme reverse transcriptase, which produces the DNA copy from the RNA

• This can be done as 1 step, and then PCR performed, or done as one process, which is less susceptible to conatmination

How are PCR products detected in conventional PCR/RT-PCR?

• product at end of reaction is detected (qualitative, not quantitative re quantity of target nucleic acid)

• product can be recovered for further investigation/application (if required) e.g. gel/capillary electrophoresis

1. Agarose or polyacrylamide gel prepared

2. Load sample dye into well of gel

3. Pass current through gel - fragments of different sizes travel different distances

How are PCR products detected in real-time or quantitative PCR/RT-PCR?

• Correctly termed qPCR (quantitative PCR) or RT-qPCR (for RNA target), but you may see it mislabelled as RT-PCR which is more correctly used for reverse transcriptase PCR)

• Based on PCR principles

• Product is measured as the reaction progresses, in real time, with product quantification after each cycle

.. To enable detection, the amplified product is labelled with a fluorescent dye

• Dye is incorporated within amplicon, and machine reads the fluorescence

What are some examples of dyes used in real-time PCR?

• SYBR Green

• non-sequence-specific fluorescent dyes that intercalates with all dsDNA product

• Fluorescence proportional to all amplified product (specific and non- specific)

• TaqMan

• sequence-specific DNA probes attached to fluorochrome are added which bind through sequence complementarity to dsDNA product

• fluorescence proportional to specific product

Describe the qPCR amplification curve.

Exponential Phase

• Characteristics: During this phase, all reaction components (template, primers, DNA polymerase, dNTPs) are in excess, and the reaction efficiency is optimal and consistent. In an ideal reaction (100% efficiency), the amount of PCR product and the associated fluorescence signal approximately double with each cycle.

• Ct (threshold cycle): The cycle threshold (Ct) value is defined as the point at which the qPCR fluorescence amplification curve crosses a set threshold line, typically in the early exponential phase of the reaction

  • Low Ct - high amount of target in samples

  • High Ct - low amount of target in samples

Non-Exponential Phase

• Baseline Phase: This is the initial phase (typically cycles 3-15) where the fluorescence signal is low and largely indistinguishable from background noise. Very little product has accumulated, and changes in product quantity are undetectable.

• Threshold line - this line separates the non-exponential (baseline/background) phase from the exponential phase of the reaction's fluorescence output. The machine calculated level of fluorescence which is statistically higher than the baseline.

• Plateau Phase: In the final phase, the reaction stops or nearly stops, as one or more critical components (such as dNTPs, primers, or DNA polymerase enzyme activity) become limiting, and accumulated product may also inhibit the reaction. The amplification curve flattens out, and the amount of final product is no longer related to the initial amount of template. Traditional PCR takes its measurement at this endpoint, which is less reliable for quantification due to sample-specific variations in when the plateau is reached. 

What are some veterinary uses of PCR?

• Genetic screening

• Tumour diagnostics

• Detection of potential pathogens

• Monitoring progression of disease/infection

What are some examples of instances where PCR might be used in the context of genetic screening?

• Hypertrophic cardiomyopathy (MYBPC3 gene) - Maine Coon and Ragdoll

• Polycystic kidney disease (PKD1 gene) in Persian and Persian- related cats

• Progressive retinal atrophy in dogs - Multiple types and genes involved e.g. rcd4-PRA, NECAP1

What are some examples of instances where PCR might be used in the context of tumour or cancer diagnostics?

• PARR (PCR testing for Antigen Receptor Rearrangement) for lymphoma or lymphoid leukaemia

• Canine mast cell tumours (c-Kit gene mutation)

• Canine transitional cell and prostate carcinoma (BRAF mutation)

What is the role of a vet in helping to conduct PCR testing?

1. What sample do you take from animal?

2. How do you collect and handle the sample to send to the lab?

**The lab performs the test and sends a report with the result

• Ensure suitable sample is submitted in a suitable medium

• Ensure posted in appropriate packaging

• Laboratory will advise suitable collection sample/medium

3. How do you interpret the result and inform the client?

What are some things that might affect the PCR results under responsibility of a veterinarian?

1. Poor sample collection technique

2. Sample contamination

3. Sample degradation: RNA viruses (RNA degrades faster than DNA)

4. PCR inhibitory substances - Substances contained within the clinical sample that interfere with the PCR reaction: FALSE NEGATIVE

• E.g. (Lithium) heparin, fluorescein, cat litter, charcoal, Heme, bilirubin, bile salts (faeces).

What are some things that might affect the PCR results under responsibility of the laboratory.

False positive

• Laboratory contamination

• Non-specific primers: then some products may not be of desired sequence

False negative

• Non-specific primers: target sequence may not be amplified efficiently

• Primers may not bind to mutant strains

• Reaction failed

What are the advantages of PCR?

• Rapid (24-48hr report), sensitive, specific diagnosis

• Identify pathogens non-cultivable/slow to culture/dangerous to culture/overgrown by other agents

• Can be performed on formalin-fixed, paraffin-embedded tissues

• Identification of carriers + shedders

• Strain-specific identification

• Vaccine vs field infections (sequence differences)

• Quantitative: pathogen load

What are the disadvantages of PCR?

• No information on viability/infectivity

• No data on antimicrobial susceptibility (bacterial pathogens)

• Commensal or not clinically significant