• Polymerase chain reaction (PCR) involves the sequential amplification of target sequences of DNA by repeated cycles of oligonucleotide primer-driven DNA synthesis.
• It is traditionally qualitative but is often now more quantitative which has revolutionized the investigation and diagnosis of health and disease at the molecular level.

Uses

Indications for use

• PCR has numerous applications in clinical veterinary medicine, in part as it can be applied for numerous infectious agents on many sample types and potentially in the same test, not just on blood or serum samples.

Infectious

• PCR detection is highly sensitive and can detect minute quantities of RNA or DNA (viral, bacterial, fungal, protozoal, parasitic). It is therefore at risk of significant contamination – from the environment, sampler, processing and testing. Specific sterile well taken samples with a ‘closed’ system of extraction and amplification are ideal.
• The technique should be highly specific for the target DNA and avoid cross-reactions with other similar but unwanted material. This aspect relies on primers designed well to only detect the nucleic acid in question but still also cover different strains of virus. Primers then are often designed to target at least two regions, one that is conserved for that particular agent (as compared to a closely related species), with another that allows for strain variation (eg Calicivirus Feline calicivirus , parainfluenza). In this way the test reliably picks up natural variation in the target agent and can be designed to detect a Genus or particular species (eg Mycoplasma haemofelis cf. Mycoplasma haemominutum Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis'​) as required.
• PCR provides evidence of actual infectious agent, as compared to serology which uses the host’s antibody response to indicate exposure to that agent. PCR is similar to culture in this aspect but does not replace it. A positive PCR amplification does not indicate viable infectious agent. It is typically faster than culture but can be batch tested.
• Similarly, if slow growing or difficult to culture in vitro, PCR facilitates detection of such agents (eg Mycoplasma). It can be applied to available samples where organisms will not be viable, such as slides and frozen or EDTA samples.
• The newer technique of real-time PCR (qPCR) is quantitative, and therefore provides an indication of how much infectious agent was present (‘the infectious load’) in that sample at that time. This is valuable in determining the significance of a positive result and when monitoring disease or response to therapy.
• The technique of reverse-transcriptase PCR (RT-PCR) enables detection and quantification of RNA viruses or gene expression. Here the RNA is first converted into DNA before being amplified.
• When applied to tissue samples where an expected and appropriate inflammatory response has been seen on cytopathology or histopathology, qPCR testing is at its most powerful, as the context and significance as well as the amount of infectious agent are known. For example, a pleural effusion with a proteinaceous inflammatory pattern (eg A:G ratio <0.5) and associated biochemistry with hematology that yields a low Feline Coronaviral Ct value (say, Ct=18) would be typical of wet FIP as the pattern and response are consistent with the disease and large amounts of viral material have been found directly in association with that inflammation/pathology. Conversely, finding similar amounts of coronaviral material enterically in a fecal sample would not be consistent with clinical FIP Feline infectious peritonitis.
• PCR results should always be interpreted in the context of the case, sample tested, expected pathology and history, as well as in communication or discussion with the cytopathologist or histopathologist to accurately diagnose or help exclude infectious differentials.

Genetic

• PCR has now been widely applied in the detection of abnormal genes from animals with inherited genetic defects where it is designed to target a specific mutation in a gene sequence that is associated with a disease (eg Myotonia congenita Myotonia congenita).
• It can be used to detect virulence genes too (eg Clostridial enterotoxin gene).
• PCR can also be used to detect gene rearrangements in cell receptors, specifically T and B lymphocytes (PARR).

PARR

• PCR for Antigen Receptor Rearrangements. This is an ancillary tool in the diagnosis of lymphoproliferative disease Lymphoproliferative disease. Each T and B cell has a unique rearrangement of their V(D)J regions in their receptors (TCR and BCR respectively). This involves the deletion and addition DNA nucleotides within varying regions, resulting in varying lengths of DNA.
• Primers are specifically designed to bind both conserved regions and then amplify across the rearranged variable regions of each B and T cell V(D)J segment. 
• These amplicons vary in length and so can be separated by size in capillary electrophoresis.
• In health and when responding to infection, even though certain lymphocytes are selected for as the immune response mounts, the rearrangements remain variable, so their sizes differ to result in a smear when separated – polyclonal.
• When lymphocytes transform neoplastically, a unique rearrangement will come to dominate so when amplified, only a single band or peak will be seen corresponding to the length or size that rearranged amplicon – monoclonal.
• In lymphoma Lymphoma, we then expect a specific banding pattern to be produced, supporting neoplastic transformation. This can be applied to different tissues to stage the disease potentially and may in time allow for detection of minimal residual disease when monitoring response to chemotherapy.
• PARR must be applied in the correct context, either cytologically or histologically to avoid mis-diagnosis. 
  • Not all rearrangements are detected, resulting in a ‘false negative’.
  • Some lymphocytes clones rearrange both their T and B cell genes even though of one lineage/origin, ie a B cell lymphoma may show specific banding in both the TCR and BCR genes.
  • Myeloid leukemias Myeloid leukemia have been shown to rearrange their TCR and/or BCR genes – ‘a false positive’.
  • Over time, some chronic infections continue to select for more avid lymphocyte rearrangements to control an infection (eg Ehrlichia Ehrlichiosis). This results in a more restricted lymphocyte population and proliferation so banding can be seen as fewer variants persists – again a false positive (‘pseudoclonal’).
  • The clonal expansion will have to be detected relative to surrounding non lymphoid tissue or other non-neoplastic lymphocytes and inflammatory cells potentially.
• When applied in conjunction with morphology, grading and staging via cytopathology or histopathology, PARR complements the confident diagnosis of lymphoma to allow appropriate therapy.
• PARR is not phenotypic, so immunocytochemistry, immunohistochemistry Immunohistochemistry (IHC) and/or flow cytometric immunophenotyping Flow cytometry (immunophenotyping) can be required to classify a lymphoma fully (eg WHO), providing both prognostic and therapeutic information too.

General Principles

• It is important the primer sequence is designed to produce only one amplicon of known size.
• The amplicons can then be analysed and seen via traditional gel electrophoresis at a specific end point time. Or they can be visualized quantitatively - real time PCR (qPCR).
• In gel electrophoresis, the DNA amplicons migrate differing distances based upon their size, so when compared to reference size bands and known controls, a positive result is confirmed by seeing a band of a certain size.
• In qPCR the production of amplicons is monitored in a number of ways: non-specifically using a double stranded binding fluorescent dye to the DNA (eg SYBR Green I); specifically using a sequence specific DNA probe that fluoresces only when bound to the target amplicon.
• Often the thermocycler is linked to fluorescent monitoring system and analytical software intrinsically within one bench top platform.
• If the probe uses different dyes, more than one target can be detected in the same reaction using different primer sets – multiplex reaction.
• This multiplicity is very useful to allow inclusion of inherent controls within the process. This ensures successful extraction, sample addition and absence of inhibitors that would prevent amplification or allow a ‘false negative’ due to no extracted material or sample inhibition.
• Controls vary depending on the reaction and sample type in question.
  • Many use the host’s own DNA which ensures not only successful extraction and amplification but also a diagnostic sample in terms of cellularity, so that a negative or not detected result can be believed.
  • RNA targets (eg calicivirus) use a spiked synthetic RNA control designed not to cross react with the target sequence but ensure successful reverse transcription too.
  • Some samples, such as feces, often contain inhibitors and are very variably constituted with the amount of host DNA (eg shed epithelial cells) varying too. A synthetic DNA control is then also added to exclude sample inhibitors.