Confidence in Your PCR Results

PCR-based pathogen detection requires the use of appropriate controls. These aid in result interpretation by identifying adverse factors such as contamination, inhibition of the amplification reaction, or problems during nucleic acid extraction. For example, ruling out the possibility that your reaction has been contaminated leading to a false positive result requires the use of adequate negative controls. Alternatively, ensuring that your test would have detected the pathogen had it been present in the sample (i.e., reducing false negative results) requires the use of appropriate positive controls.

Negative controls are well established in detection workflows. Including multiple negative controls in an assay to rule out contamination is the basis for a valid positive result. The importance of controlling for false negative results in highly sensitive techniques such as PCR is recognized to a lesser extent. Several factors can generate a false negative result, such as errors in sample extraction or thermocycler malfunction. Assay failure due to PCR or RT-PCR inhibition is the most common cause. The most practical approach to control for the presence of inhibitors is to include an Internal Positive Control, or Internal Control (IC). This IC is simultaneously extracted and amplified (or only amplified) in the same tube with the pathogen target, and should always be combined with an external positive control to prove the functionality of the reaction mix for amplification of the pathogen target. This combination rules out inhibition, among other malfunctions, and confirms that a negative result is truly negative.

Not all internal controls are the same, and each IC concept has value for specific applications. Endogenous ICs occur naturally in test specimens, such as a sequence of the host genome (e.g., β-actin) or from normal microflora genomes (e.g., 16s). Exogenous ICs, on the other hand, are spiked into samples either during nucleic acid extraction or before PCR amplification.

Exogenous ICs can be homologous, where an artificial template is constructed with the same primer binding sites as the targeted pathogen sequence. Although the same primer set is used for target and IC, the sequence differs, enabling differentiation of pathogen and IC amplicons with different probes. Heterologous ICs, on the other hand, are designed with their own primers and probe.

Endogenous and exogenous homologous ICs carry the risk of impairing detection sensitivity for the pathogen target due to competition for reaction components. For example, a high starting amount of an endogenous IC template can impair assay sensitivity. This high starting amount can result from variations in the sample type or sampling technique. In the case of RNA applications, the high starting amount can also be due to enhanced expression levels of the IC due to disease-related cellular pathology. In the case of exogenous homologous ICs, using the same primers to amplify both target and IC leads to primer competition. Additionally, both endogenous and homologous ICs involve tedious IC design, and their use is restricted to a few applications or even individual assays.

In the context of process safety and workflow simplification, exogenous heterologous ICs are the most informative and flexible. The amount of IC template spiked into a sample is defined and consistent, and unrestricted design options enable optimization of IC properties. Only heterologous ICs allow for a design and setup that prevents competition for PCR components, and heterologous ICs are suited as universal controls, thereby making their implementation in new assays easy.

Features of internal controls

Feature	Exogenous homologous	Exogenous heterologous	Endogenous
Universal use in multiple assays		✓
Serves as control for purification procedure	✓	✓	✓
Differentiates purification errors from amplification errors	✓	✓
Template quantities are defined and consistent	✓	✓
Non-competitive internal control design		✓	✓

QIAGEN has created a robust set of internal controls that allow accurate detection of PCR inhibition and failure of extraction. These exogenous heterologous internal controls carry a unique artificial sequence that is not present in any biological sample material, and is detected using primers and probes that differ from those specific to the target pathogen. The IC DNA or RNA template is spiked into the sample during PCR or RT-PCR setup to ensure that the amplification process took place without problems. The IC template can also be spiked into the sample prior to the extraction process to additionally control for extraction failure. In both cases, the Internal Control RNA template additionally controls for successful reverse transcription.

• Because the QIAGEN Internal Control does not occur naturally in the test specimen, but is rather added to the sample, the amount of internal control template is defined and consistent, independent of sample type or sampling technique.
• The internal control is detected with primers and probes distinct from those used for the target pathogen, thereby preventing primer competition during the amplification reaction. Additionally, the non-competitive design of the IC assay prevents competition for other reaction components and assay pre-optimization with QuantiFast Pathogen Master Mix ensures that the sensitivity of pathogen detection is not impaired by duplexing with the IC (see figures High linearity and precision of singleplex and duplex detection and No loss of sensitivity when including the Internal Control).
• QIAGEN Internal Controls behave predictably in the presence of PCR inhibitors. A shift in the IC signal or failure of IC amplification give a clear indication of PCR inhibition or other errors, allowing the correct interpretation of negative results (see figure Correct interpretation of negative results)

QIAGEN Internal Controls are included in QuantiFast Pathogen +IC Kits.* The unique artificial sequence of the IC is detected with a dual-labeled probe assay using a MAX labeled probe (detectable in the same channel as HEX, JOE or VIC). The internal controls of these kits come in two variants to accommodate your specific needs: Internal Control DNA for co-detection with viral and bacterial DNA, and Internal Control RNA for co-detection with viral RNA. Highly efficient, QuantiFast Pathogen +IC Kits perform comparably to the well-known QuantiTect Virus Kit. Furthermore, they show lower threshold cycle values (C_T) and higher efficiency and linearity than high-quality kits from other suppliers (see figures No loss of sensitivity when including the Internal Control and Superior performance of the QuantiFast Pathogen +IC RT-PCR Kit compared to an alternative kit).

Test QuantiFast Pathogen +IC Kits and experience the certainty of knowing that you have reliable results.

* The lower concentrated IC for use as an amplification control is supplied with the kits. The higher concentrated IC for control of extraction and amplification must be ordered separately.