Polymerase Chain Reaction (PCR) rapidly creates millions of copies of a specific segment of genetic material, allowing for the detection of pathogens or genetic markers with high sensitivity. This amplification process is routinely used in clinical diagnostics, forensic science, and research laboratories worldwide. The reliability of this technique depends on rigorous quality assurance checks known as PCR controls. These controls are separate reactions run concurrently with experimental samples to confirm that the entire process, from reagents to equipment, is performing accurately. Including these checks ensures that any result is a true reflection of the sample and not an artifact of a flawed procedure.
External Controls: Ensuring Reagents and Cleanliness
External controls are reactions that operate independently of the sample material, validating the overall function of the laboratory environment and the chemical components used in the reaction. The Positive Control (PC) and the Negative Control, also known as the No Template Control (NTC), are the two primary checks in this category. The Positive Control contains a known quantity of the target nucleic acid sequence, guaranteeing that the reaction’s chemical components, such as the DNA polymerase enzyme and the primers, are active and functional. If the PC fails to produce the expected amplification signal, it indicates a systemic failure, such as degraded reagents or a thermal cycling machine malfunction. This failure suggests that any negative result obtained from the samples is likely a false negative, invalidating the run, which must then be repeated with fresh components.
Conversely, the Negative Control (NTC) is set up with all the same reagents, but purified water replaces the target nucleic acid template. This control confirms that the reaction mixture and the preparation environment are free of contaminating genetic material. If the NTC produces an amplification signal, it demonstrates that contamination has occurred, possibly from previously amplified products or impure reagents. A contaminated NTC means that any positive result from a sample could be a false positive, potentially leading to an incorrect conclusion. The presence of amplification in the NTC invalidates the entire run, requiring the laboratory to decontaminate the workspace and use a new batch of reagents.
Internal Controls: Monitoring Sample Integrity
While external controls monitor the reaction setup, Internal Controls (IC) assess the quality of the individual biological sample itself. A common challenge in testing real-world samples, such as blood, mucus, or swabs, is the presence of substances that can inhibit the PCR reaction. These inhibitors, including host DNA or residual chemicals from the nucleic acid extraction process, can prevent the target sequence from amplifying, even if it is present. The Internal Control is a non-target sequence of DNA or RNA purposefully added to every sample before the amplification process begins.
The IC is designed to amplify using a different set of primers than the main target, creating a distinct signal that confirms the reaction is successfully occurring within the tube. If the main target is negative, but the IC successfully amplifies, it confirms the sample was properly extracted and that no significant inhibitors are present. If the IC fails to amplify in a sample, it signals that the sample contains potent inhibitors or that the nucleic acid extraction was unsuccessful. The IC prevents a true positive sample from being incorrectly reported as negative due to poor sample quality, thereby avoiding an undetected false negative.
Interpreting Results: What Control Failure Indicates
Controls establish the fundamental validity of the entire test run and provide a framework for troubleshooting. If the Positive Control fails to amplify, the laboratory must assume that the reagents are degraded, the machine is not cycling correctly, or the setup was flawed. Since the system failed to detect a known positive, all negative results from the samples are unreliable. The entire batch must be re-run following a thorough check of the equipment and reagents, confirming a failure of the assay components themselves.
The failure of the Negative Control (NTC), indicated by an unexpected amplification signal, points directly to contamination. This contamination could be from carryover DNA, impure water, or contaminated reaction mix components. The laboratory must immediately discard all reagents, clean the workspace, and prepare a new run, as any positive result from a sample is suspect. NTC failure suggests the potential for false positive results across the entire batch, invalidating the data.
If external controls function as expected, but the Internal Control fails within a specific sample, the problem is localized to that tube. This failure indicates the sample likely contains high levels of PCR inhibitors, such as heme from blood or melanin, which prevent the polymerase enzyme from functioning. The result for that specific sample is reported as inconclusive, requiring the sample to be diluted and re-tested to reduce the concentration of inhibitory substances. Interpreting these control patterns ensures that diagnostic results are accurate, preventing false negatives and false positives.