Jane E. Sykes, BVSc(Hons), PhD, DACVIM
School of Veterinary Medicine, University of California-Davis, Davis, CA, USA
The polymerase chain reaction (PCR) is a widely used molecular biologic tool that allows specific amplification of certain DNA sequences such that they can be detected using the naked eye, making it possible to detect DNA from a variety of different organisms, including uncultivable organisms and criminal suspects. The full potential of PCR was not realized until the spring of 1983, when Kary Mullis, an American biochemist from North Carolina, conceived the idea of using a pair of primers to bracket a desired DNA sequence and copying it with a DNA polymerase. In 1986, he started using the DNA polymerase of a bacterium that is adapted to live in hot springs, Thermophilus aquaticus (Taq). The polymerase of this organism is a heat-resistant polymerase that only needed to be added once. The resulting assay revolutionized molecular biology, genetics, medicine, and forensics.
In order to design a basic, conventional PCR assay, the exact DNA sequence of the two regions that flank a given region of interest in DNA must be known. Two primers, roughly 20 nucleotides long, are designed which match the sequence on the organism's DNA you are interested in detecting. The next step is to make up a master mix, a solution containing both primers, some Gs, Ts, As, and Cs for making new DNA, and the DNA polymerase enzyme. The DNA is then extracted from a clinical specimen - a skin biopsy, for example. The master mix is aliquoted, and the extracted DNA is added to the tube. The next step is performed in a thermocycler, which is programmed to heat and cool the tubes repeatedly, such that the DNA can be amplified. Initially, the DNA is heated, which causes the paired strands of DNA to denature and allow the primers to bind. Next, the tubes are cooled to a specific annealing temperature so that the primers bind to their respective sequences. The tubes are then heated to 72 degrees, which is the temperature at which the DNA polymerase extends the template DNA, using the primers as starting points. The cycle is then repeated 30–50 times, which results in logarithmic accumulation of PCR product.
Finally, the PCR product must be identified. The most basic way of achieving this is to run the sample on a gel. The longer the PCR product, the slower it moves through the gel, and by running a series of molecular weight markers alongside the sample of interest, the size of the PCR product can be determined after visualizing it using ultraviolet transillumination. The identity of the band can be confirmed by sequencing the resulting product or performing DNA hybridization.
This method is used when trying to detect RNA. After extraction, the enzyme reverse transcriptase is added to the RNA, together with As, Ts, Gs, and Cs to make DNA, and either a primer to initiate reverse transcription, 'random hexamers,' which are a mixture of 6-nucleotide primers representing all possible combinations of As, Ts, Cs, and Gs. The reverse transcriptase then makes a DNA copy of the RNA (cDNA), which can then be used in either conventional or real-time PCR.
Nested PCR involves subjecting a small amount of the amplified PCR product to a second round of conventional PCR using another set of internal primers. This increases the specificity of the PCR assay, but also greatly increases the sensitivity - so much so that false positives due to contamination can be extremely problematic with this method.
Multiplex PCR assays use multiple sets of primers in the same reaction tube to detect multiple organisms that may be present concurrently. The potential downside of multiplex reactions is that if one organism is present in relative abundance, amplification of this organism can consume reagents, reducing the sensitivity of the assay(s).
Conventional Versus Real-Time PCR
1. Conventional PCR. Conventional PCR involves running the assays described above, with a PCR product analysis that involves running the resultant sample on a gel and visualizing it using UV transillumination. Their use for diagnosis has largely become obsolete, because of the problems of PCR product carryover and contamination.
2. Real-time PCR. Real-time PCR assays rely on detection of a fluorescent signal as PCR product accumulates. The amount of fluorescence is proportional to the number of copies of template DNA in the sample, so the assay can be quantitative. The data are stored in an attached computer. Usually, the result is reported as a CT (cycle threshold) value, which is dependent on the number of cycles in the assay (usually < 40). The tube does not have to be opened to measure accumulation of PCR product over time, greatly reducing the chance of contamination. There are 2 major types of real-time PCR assays:
a. SYBR Green Real-time PCR assays. These assays use a double-stranded DNA binding dye that fluoresces in the presence of double-stranded DNA.
b. Fluorescent Probe Real-time PCR assays. These use one or more specific DNA probes, in addition to the 2 primers. The probe(s) are tagged with a dye that emits fluorescent light. Three types of probes may be used:
i. TaqMan (5' nuclease) probes. These probes are tagged with a fluorescent reporter dye at one end and a quencher dye at the other end. Normally, the close proximity of the quencher dye to the reporter dye prevents the reporter dye from emitting fluorescence. When the Taq copies the section of DNA between the two primers, its inherent exonuclease activity causes the polymerase enzyme to chop the probe into pieces. This separates the reporter from the quencher dye, such that the reporter dye emits fluorescence. Also, the probe adds specificity to the assay - in essence, you are including a hybridization step. Usually real-time PCR assays are run in plate format; the samples are added to a plastic plate with wells on it.
ii. Molecular beacons. This is a probe with fluorescent reporter and quencher dyes at each end, but when not annealed to the target sequence, it forms a hairpin loop that brings each dye in close proximity and stops them from emitting fluorescence. When the probe anneals, the dyes are separated and the fluorophore emits fluorescence.
iii. FRET hybridization probes. Assays using FRET hybridization probes utilize 2 probes that are expected to lie next to one another on the target sequence. One of the probes is tagged at the 3' end with a green fluorescent dye, the other at the 5' end with a red fluorescent dye. When the 2 dyes come together during annealing, the green fluorescent dye transfers its energy to the red probe, causing it to emit fluorescence, which can then be detected. Currently, there are very few reports of the use of this technology in veterinary patients.
Testing Platforms. Conventional PCR assays are performed in a desktop block thermocycler, which uses a metal block to heat and cool the tubes. Real-time PCR assays require more specialized instrumentation because of the fluorescence detection and analysis steps. Instruments vary in the number of samples they can analyze in one run, the method used for heating and cooling the tubes, and the time required for each run. In recent years, rapid thermocyclers have become available in several different formats. Roche has marketed the LightCycler, a special carousel thermocycler that rapidly heats and cools the samples, which are placed in specially designed glass capillaries. As a result, the assay is completed in 45 minutes, instead of the usual 4–5 hours.
PCR Assay Validation
For every PCR assay, the following factors must be determined as part of the validation process:
The sensitivity is the number of copies of the target DNA/RNA sequence the assay is capable of detecting. This requires serial dilutions of known concentrations of the template DNA to be tested using the PCR assay of interest. Sensitivity can also be defined in terms of whether all possible strains of a target organism can be detected. If the assay is very sequence-specific, for organisms with variable target sequences (such as RNA pathogens), only some strains may be detected. As part of the validation process, the assay should be used to detect as many strains as possible.
An assay has poor specificity if it generates a large number of false positives relative to a gold standard assay. A PCR assay has poor specificity if false positives occur as a result of contamination, or because of loading error. Specificity may also be poor if the primers amplify products from unrelated organisms. Specificity can be tested by searching the nucleotide database to ensure your primers do not match sequences other than those of the organism you are interested in. Specificity must also be tested as part of the validation process by showing that the PCR assay truly fails to amplify DNA from organisms that are closely related to the target organism, as well as DNA from the host species.
Reproducibility is determined for real-time PCR assays by determining whether the CT value varies significantly for the same sample when tested multiple times. Usually intra-assay (within the same run) and interassay (within different runs) variations are measured.
A real-time PCR assay that has an efficiency of 100% doubles the number of copies of template in every cycle. Efficiencies below 90% are unacceptable for quantitative assays, and ideally they should have efficiencies > 95%. The efficiency is determined by testing serial dilutions of a target DNA standard and by creating a chart of copy number versus CT value. Ideally, this is compared to a similar chart using dilutions of the standard DNA spiked into a clinical sample, to show that inhibitors within the clinical sample do not affect efficiency.
Problems and Pitfalls of PCR
False negatives can occur because of:
Insufficient sample size.
Sample degradation during handling or storage. This is particularly a problem with RNA.
Inhibition. A variety of organic and inorganic substances can inhibit PCR, including hemoglobin, polysaccharides, EDTA, and urea. It is possible to control for inhibition and sample degradation by concurrently testing the extracted sample for the presence of a housekeeping gene. Examples include GAPDH and 18S DNA. If the test for the housekeeping gene is negative, you know either the DNA degraded, or inhibitors were present in the sample.
Assay reagent failure.
False positives can occur for several reasons:
Contamination - this has the potential to occur both before and after sample collection and is most likely to be a problem when conventional PCR assays are used. Contamination can be minimized by careful sample collection, extraction, strict separation of work areas, use of closed tube post-PCR analysis, and routine monitoring. Additional systems are available to prevent reamplification of PCR products, such as the AMPErase UNG system. This system involves adding deoxyuridine residues instead of thymidine residues as reagents for PCR. Thus, resultant PCR products contain Us instead of Ts. At the start of PCR, the enzyme AMPErase is added to the mixture, and this destroys any DNA containing Us. When the tube is heated to initiate denaturation of DNA, the AMPErase enzyme is inactivated.
Factors influencing a clinician's interpretation of a PCR test result include:
1. The assay. It is important to know whether a laboratory is running conventional versus real-time PCR, because conventional PCR is more prone to contamination and false positives due to nonspecific PCR products. It is important to find out whether the laboratory is including appropriate controls for PCR inhibition. Both positive and negative controls should be included to ensure that the assay is working properly and that contamination or inhibition has not occurred.
2. The disease. If a PCR 'panel' is performed by the laboratory, a positive result may be obtained for an organism you were not expecting to be present. As with the result of any laboratory test, the result needs to be interpreted in light of the animal's clinical signs and the pathogenesis of the infection you are testing for. An understanding of the pathogenesis of infectious diseases and organism shedding patterns is crucial for interpreting positive PCR results. If the pathogenesis of a disease is chronic or largely immune-mediated, the organism you are testing for may be in such low concentrations it may be undetectable using the assay. If subclinical carrier states are common, a positive PCR result may not be relevant to the animal's disease. If the assay detects organisms that an animal has recently received a modified live vaccination for, a positive result may reflect the presence of replicating vaccine virus. PCR detects both viable and non-viable organisms, so bacterial PCR assays may be positive even after antimicrobials have been administered.
3. The specimen. The specimen type can be important for interpreting PCR results. The organism you are testing for may not be present in the specimen you have collected (i.e., other specimen types may have been more appropriate). The specimen may not have been collected properly. Shipping conditions can also affect the chance of receiving a positive result.
When collecting specimens for PCR, it is important to be aware that contamination can occur outside the PCR laboratory as well as within the PCR laboratory. Collecting specimens for PCR should be performed aseptically, and disposable instruments and gloves should be used (e.g., disposable scalpel blades, punch biopsy instruments). If possible, before collecting the specimen, the laboratory should be contacted to determine the optimal method of collection. If the laboratory is not available, the following are general guidelines. If testing for a DNA pathogen, either fresh or frozen tissue should be submitted. A small amount of saline can be added, but excessive dilution should be avoided as it reduces the sensitivity of the assay. For blood samples, EDTA is usually fine. Usually, overnight shipping of specimens on ice (DNA) or dry ice (RNA) is required. If specimens are to be stored before shipping, they should be frozen immediately after collection (preferably at or below -70 degrees Celsius). Tissues stored in formalin, especially for prolonged periods, are suboptimal for PCR because the formalin crosslinks the DNA in the specimen. Tissues fixed briefly in formalin, then paraffin-embedded are preferable to tissues stored in formalin, because the formalin is removed during the embedding process.