Equis ISSN 2398-2977

Polymerase chain reaction (PCR)

Contributor(s): Alastair Foote, Vetstream Ltd

Overview

  • 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.

Sampling

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Tests

Methodologies

  • Many laboratories now offer PCR testing.
  • PCR testing requires particular laboratory design to aviod 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.
  • The key elements of the PCR reaction are the substrate DNA/RNA (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 35-45 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.
  • Real-time PCR 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. This is commonly reported as the Ct value.
  • In real-time PCR lower Ct values indicate a lower number of amplification cycles required for detection and higher amounts of the detected DNA/RNA in the starting material.

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.

Validity

Sensitivity

  • PCR is a highly sensitive test that can detect minute quantities of target sequence.
  • Primers may not cover all possible viral/bacterial strains and care should be taken in selecting a test that is fit for purpose.

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. At a basic level, positive PCR results confirm the presence of the pathogen's genetic material, not that disease is caused by the pathogen.

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.
  • PCR may also remain positive after clinical recovery. Some virus DNA/RNA may persist in the body for long periods after recovery (persistence or latency).
  • Although detection is specific, the presence of the DNA/RNA does not always prove that a particular organism is causing disease. Results must be interpreted in the context of the clinical signs, eg some horses will excrete Lawsonia intracellulariswithout clinical signs of intestinal disease; clinically normal horses can carry latent EHV-1. 
  • 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.

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 agents DNA or RNA.
  • Some viruses show substantial variability in their genetic code and therefore PCR may not be capable of detecting all variants.
  • A sample may not contain detectable genetic material even if disease was caused by the organism being tested.
  • A positive test result indicates DNA/RNA of the detected organism in that sample, a negative result the failure to detect in that sample. The significance of these results must be interpreted by the clinician in the light of the clinical signs and history.
  • The nature of the sample is important in some diseases, eg samples for PCR prepared from a buffy coat blood sample are more likely to detect EHV-1 in case of neurological EHV infection during the viraemic phase than a CSF sample (although both can give positive results).

Further Reading

Publications

Refereed papers

  • Recent references fromPubMed.
  • Haralambus R, Burgstaller J, Klukowska-Rötzler J, Steinborn R, Buchinger S, Gerber V & Brandt S (2010)Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease. Equine Vet J42(4), 327-331PubMed.
  • Ousey J C, Palmer L, Cash R S, Grimes K J, Fletcher A P, Barrelet A, Foote A K, Manning F M & Ricketts S W (2009) An investigation into the suitability of a commercial real-time PCR assay to screen for Taylorella equigenitalisin routine prebreeding equine genital swabs. Equine Vet J41(9), 878-882PubMed.
  • Pusterla N, Jackson R, Wilson R, Collier J, Mapes S and Gebhart C (2009)Temporal detection of Lawsonia intracellularisusing serology and real-time PCR in Thoroughbred horses residing on a farm endemic for equine proliferative enteropathy.  Vet Microbiol136, 173-176PubMed.
  • Pusterla N, Madigan J E & Leutenegger C M (2006)Real-time polymerase chain reaction: a novel molecular diagnostic tool for equine infectious diseases.  J Vet Intern Med20(1), 3-12PubMed.
  • Willoughby K (2003)The ABC of PCR. In Pract25(3), 140-145.

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