Accuracy of SARS-CoV-2 testing is critical when determining if someone is infected and needs to be quarantined and/or treated for a coronavirus infection. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays are the tool of choice for determining if someone has an active viral shedding of SARS-CoV-2. RT-PCR assays reverse transcribe the viral RNA into DNA for amplification and subsequent identification of target regions. PCR is extremely sensitive and only trace amounts of the template DNA or RNA are necessary for identification. This sensitivity makes the assay ideal for identifying the presence of this specific coronavirus in a sample. When used for pathogen detection, RT-PCR assays require the use of appropriate controls. These aid in the interpretation of results by identifying contamination during processing, inhibition of the reverse transcription and amplification reactions, or even if the pre-PCR step of extraction was successful or not
Negative Controls – Preventing False Positives
This same sensitivity also makes PCR assays very sensitive to contamination and can easily deliver false positive results unless an appropriate negative control is used in the assay. Care must be taken to avoid contamination of reagents with genetic material from samples, kit controls, the environment, or amplicons from previous reactions. Additionally, to prevent the reporting of false positives, negative controls are run during each experiment to ensure contamination is identified if it does occur. The negative control is expected to result in no amplification of the target regions. Due to the sensitivity of the primer/probe sets for RT-PCR, if amplicons were made and signal is shown for the SARS-CoV-2 target genes, then contamination of the PCR experiment with foreign DNA has occurred. If the negative control does not yield any signal for the target regions, then there is added confidence in not reporting false positives.
Positive Controls – Preventing False Negatives
Preventing false negatives is imperative to slowing down the spread of SARS-CoV-2. False negatives can occur if the reverse transcription and/or PCR reactions are not functioning properly. It is critical to include an appropriate positive control in every run of a RT-PCR assay to identify possible false negative samples. A positive control is expected to have amplification of the assay specific SARS-CoV-2 target regions. The resulting signaling show that the reagents are working properly. If something was inhibiting the reaction, then the positive control would not be able to make amplicons. Likewise, if the reagents for the reaction were not made or mixed properly, the positive control would also not work as expected. If the positive control works, then samples that come up negative are expected to be negative instead of falsely negative from inhibition or incorrect set-up. Thus, this control adds additional confidence to the results of the run.
Internal controls – Preventing False Negatives
An additional potential source of false negatives could stem from insufficient sample collection or sample extraction. To mitigate this, an internal control can be used. There are two different approaches in RT-PCR assay design for internal controls: endogenous and exogenous.
Endogenous internal controls leverage genetic knowledge of the samples. This type of internal control uses housekeeping genes to report the presence of genetic material from the sample. This guards against false negatives by showing that there is indeed sample DNA present and that the collection, extraction and amplification steps were all successful. This control type is not placed in a designated well but instead is present in every sample well.
Exogenous internal control systems are a bit more complex. They involve adding an outside source of encapsulated RNA to each sample before extraction. When the internal control target region is amplified and measured, it shows two things. One, the extraction method worked. Two, the reverse transcription worked. In these cases, it adds additional confidence that the likewise encapsulated SARS-CoV-2 was also successfully extracted, and that its genetic material in the form of RNA was also properly transcribed if present. Thus, when the internal controls are successful and present, any samples that are negative are believed to be truly negative. However, if the internal control is not present in a reaction without SARS-CoV-2 as well, then that sample cannot confidently be called negative and must be retested with an additional attempt at extraction or even collection.
The use of positive, negative, and internal controls is needed to ensure the accuracy of SARS-CoV-2 testing using RT-PCR assays by identifying contamination, inhibition of the reverse transcription and amplification reactions, and failure of nucleic acid extraction.
What to do with Inconclusive Samples
Read our blog post, How to Handle Inconclusive Samples with SARS-COV-2 Real-time PCR Tests, to learn how to access internal, positive and negative controls and what to do if you obtain inconclusive results.