Polymerase chain reaction (PCR) involves the sequential amplification of target sequences of DNA by repeated cycles of oligonucleotide primer-driven DNA synthesis.
The technique has revolutionized the investigation and diagnosis of health and disease at the molecular level.
Uses
Alone
PCR has numerous applications in clinical veterinary medicine.
Already, there is wide availability of PCR detection of infectious agents from a range of body fluid or tissue samples.
PCR detection is highly sensitive and can detect minute quantities of microbial DNA.
The technique is highly specific for the target DNA and avoids the complications of cross-reactions that cloud interpretation of serological diagnostic methods.
PCR provides evidence of infection , whereas seropositivity only indicates exposure to an infectious agent.
PCR can be performed rapidly (within one day) and thus has advantages over traditional culture-based techniques.
The technique of ´real-time´ PCR is quantitative, and therefore provides an indication of infectious load. This is valuable in determining the severity of infection, and in monitoring recovery or response to infection.
PCR has been applied to the detection of abnormal genes in animals with inherited genetic defects or neoplasia.
The technique of reverse-transcriptase PCR enables detection and quantification (by real-time RT-PCR) of mRNA transcripts from specific genes.
This method has been used to quantify mRNA encoding numerous biological molecules, eg cytokines, chemokines, inflammatory mediators, in samples from veterinary species, where this is not currently possible by traditional means due to the lack of appropriate immunological reagents. For example, semi-quantification of cytokine mRNA has allowed characterization of the tissue immune responses occurring in canine inflammatory bowel disease and atopic dermatitis.
PCR has potential uses in gene therapy, eg feline hyperthyroidism (Blackwood & Argyle, JSAP 2002), plasmid DNA vaccination, paternity cases.
The most widespread sample is whole EDTA anticoagulated blood, but PCR may be performed on any appropriate body fluid, on aspirates, eg bone marrow, splenic), or on tissue biopsies.
Quantity of test material
The nature of PCR is that very small quantities of starting DNA template are sufficient to perform the test.
A 1ml blood sample is generally adequate for most applications.
A tissue biopsy the size of a skin punch would be adequate for PCR analysis.
Quality control
Precautions
It is vital that samples for PCR analysis are collected in a sterile fashion to avoid contamination with extraneous DNA, eg from the human sample collector, from environmental organisms.
Blood should be taken from appropriately prepared venipuncture sites.
Timing of test
Freshly collected samples are optimal for PCR purposes, but this does depend on the nature of the test.
Blood samples for detection of an infectious agent may be sent via normal postal routes, however tissue samples for RT-PCR often require immediate processing which restricts some PCR methodologies to a research setting.
Sample requirements should be discussed with the laboratory.
Sample storage
Samples may be stored for future PCR analysis.
Whole EDTA blood samples may be frozen (at -20°C or -70°C) for later testing.
Optimally, the genetic material (DNA or RNA) will be extracted from the sample, and the nucleic acid stored frozen (usually frozen in ethanol).
Fresh tissue samples must be snap-frozen in liquid nitrogen and then stored at -70°C for subsequent testing. Alternatively, tissue samples may be formalin-fixed (as for standard histopathology) for some PCR applications.
The extraction of nucleic acid can subsequently be made from the fixed tissue ' even after it has been embedded in paraffin-wax blocks. This technique has enabled retrospective studies using decades-old material.
The exquisite sensitivity of PCR has even enabled the methodology to be applied to samples of bone marrow or dental pulp extracted from archeological remains several thousands of years old.
Sample transport
The testing laboratory will advise on optimum shipping conditions.
For most blood-based PCR assays, EDTA blood must be received by the laboratory within 24 hours.
Many laboratories now offer PCR testing, and availability will likely increase in the near future.
PCR testing requires particular laboratory design to avoid problems with contamination, so larger, high-throughput laboratories are more likely to establish PCR testing.
The sample is first processed using commercially available extraction kits to obtain a starting preparation of DNA or RNA.
In RT-PCR, the RNA is reverse transcribed to DNA.
The key elements of the PCR reaction are the substrate DNA (that is, the sample being analyzed), 'primers', a DNA polymerase enzyme and nucleotides. These are all mixed in optimum proportions within a single vessel (PCR tube or plate).
Primers are short sequences of oligonucleotides that have been specifically designed to target a complementary sequence within the region of DNA that is the target of the reaction, eg within the microbial gene.
Two sets of primers are included, one that binds a 5'and one a 3' region of the target DNA.
In the PCR reaction, there are alternate cycles of heating and cooling.
The double-stranded DNA helix is first heated to a temperature whereby the two DNA strands separate to single strands.
The reaction vessel is then cooled, so that the primers affix to the target areas within each of the two DNA strands.
The heat stable DNA polymerase enzyme then extends the primers by the sequential addition of nucleotides using the single-stranded DNA as a template.
This proceeds to a predetermined length of primer extension.
As this process has essentially re-created double-stranded DNA, the next stage of the reaction again heats the vessel to dissociate these newly formed strands.
These then act as templates for another round of primer binding and de-novo DNA synthesis.
In the second stage of the reaction there are four single stranded DNA templates.
This cycle of heating and cooling is repeated many times over (often in the order of 35-40 cycles), such that there is an exponential increase in the amount of the target DNA sequence relative to the non-target areas of DNA.
This large quantity of target DNA can be identified by electrophoretically separating the contents of the sample vessel on a gel to reveal a single band of amplified product of the predicted molecular mass relative to DNA standards present in the gel.
This standard PCR methodology provides a means of simply detecting the presence of the target DNA within a sample. Although various means have been applied to attempt to quantify the amount of DNA produced in one reaction versus another (ie between two different starting blood samples), this method is generally considered to be non-quantitative.
A relatively new development has been the ability to more accurately quantify the DNA amplified during this reaction by real-time PCR. This method utilizes a more sophisticated piece of equipment which has the ability to detect fluorescence within the PCR reaction vessel.
In real-time PCR, as the amount of DNA product increases throughout the cycling, so there is an increase in the emission of fluorescence which is monitored throughout the assay (in 'real-time').
The fluorescence comes from the addition into the reaction of either a 'reporter probe' or an 'intercalating dye' which selectively binds to double stranded DNA, so fluorescence increases with each generation of double stranded DNA.
The amount of target DNA in the original sample is related to the point at which the fluorescence value crosses a threshold detection value.
Control
The individual testing laboratory should ensure that appropriate controls are in place and that the PCR methodology employed is able to specifically detect the intended target.
This may be confirmed by subsequent cloning and sequence analysis of the amplified genetic material.
Availability
The individual testing laboratory should ensure that appropriate controls are in place and that the PCR methodology employed is able to specifically detect the intended target. This may be confirmed by subsequent cloning and sequence analysis of the amplified genetic material.
Validity
Sensitivity
PCR is a highly sensitive test that can detect minute quantities of target sequence.
Specificity
PCR is a highly specific test that can unambiguously identify specific nucleic acid sequences.
In the case of identification of microbes, the specificity of the PCR is related to the design of the primers used, eg primers may be used to detect sequence that is common to many microbes, sequence that is common to a Genus, or sequence that is specific to a species.
Predictive value
In the case of infectious disease, PCR may be positive before an animal seroconverts or before organisms become detected in blood by visual observation.
By contrast, an animal may be seropositive, indicating the presence of specific antibody as evidence of exposure to an infectious agent, but PCR negative, if the organism has been successfully eliminated.
Technique (intrinsic) limitations
PCR will detect minute quantities of target DNA, eg in the investigation of an infectious disease, PCR technology will allow the detection of very low levels of the infectious agent’s DNA or RNA.
However, it is important to appreciate that PCR will not discriminate between active clinical infection, and a chronic subclinical carrier state.
Furthermore, for some diseases, the nature of the sample is important, eg samples for PCR prepared from bone marrow are more likely to yield a positive result than samples prepared from a blood sample for detection of Leishmania.
Technician (extrinsic) limitations
PCR generally provides a 'yes or no' answer, ie a sample will be positive or negative for the presence of a particular target DNA.
Quantitative, real-time PCR will enumerate the level of target DNA (often as a 'copy number') and the laboratory should indicate the significance of this.
Willoughby K (2003) The ABC of PCR.In Practice25 (3), 140-145.
Tasker S, Helps C R, Belford C R, Birtles R J, Day M J, Sparkes A H, Gruffydd-Jones T J & Harbour D A (2001) 16s rDNA comparison demonstrates near identity between a United Kingdom Haemobartonella felisstrain and the American Californian strain. Vet Microbiol81 , 73-78.
Zarlenga D S & Higgins J (2001) PCR as a diagnostic and quantitative technique in veterinary parasitology. Vet Parasitol101 , 215-230.
German A J, Helps C R, Hall E J & Day M J (2000) Cytokine mRNA expression in mucosal biopsies from German shepherd dogs with small intestinal enteropathies. Digestive Diseases and Sciences 45 , 7-17.
Harley R, Helps C R, Harbour D A, Gruffydd-Jones T J & Day M J (1999) Analysis of intralesional cytokine mRNA expression in feline chronic gingivostomatitis. Clinical and Diagnostic Laboratory Immunology 6 , 471-478.
Vernau W & Moore P F (1999) An immunophenotypic study of canine leukaemias and preliminary assessment of clonality by polymerase chain reaction. Vet Immunol Immunopathol69 , 145-164.
Vetstream contributor(s)
Professor Michael J Day BSc BVMS PhD FASM DipECVP MRCPath FRCVS , Division of Veterinary Pathology, Infection and Immunity, Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU, UK.
Dr Helen R Milner BVSc(Dist) PhD CertVR MRCVS , PO Box 8730, Riccarton, Christchurch, New Zealand.
Organization(s)
Acarus Laboratory, Department of Clinical Veterinary Science, University of Bristol.
