How Does the SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit Work?
Due to its high sensitivity and specificity, reverse transcriptase-polymerase chain reaction (RT-PCR) testing has been widely recognized as the gold standard for detecting and confirming COVID-19 infections worldwide. Among the various forms of nucleic acid amplification tests (NAATs) used for diagnosing infections with SARS-CoV-2, real-time RT-PCR is one of the most reliable, convenient, and widely used methods. Following the removal of pandemic restrictions worldwide, there is a growing need for diagnostic solutions to address the challenges arising from the increasing circulation of other viruses alongside SARS-CoV-2. With the development of multiplex real-time RT-PCR assays, it is now easier than ever to differentiate between COVID-19 and other common respiratory infections with similar clinical presentations and seasonal patterns, such as influenza and respiratory syncytial virus (RSV). Read along to learn more about the SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit, its principle, optimal timing, and advantages.
What is the SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit?
The SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit is a multiplex RT-PCR assay intended to detect and differentiate infections with SARS-CoV-2. Although the principles of RT-PCR and PCR largely overlap, RT-PCR assays combine reverse transcription of RNA into DNA with the amplification of specific DNA targets via polymerase chain reaction (PCR) since the detection of RNA viruses such as SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) requires the conversion of their viral RNA into DNA. Therefore, the SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit allows for the conversion of short pieces of viral RNA for each of the given viruses and their replication through many cycles until the presence of the virus becomes detectable.
As a multiplex assay, the SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit targets viral genes specific to each of these viruses for the simultaneous detection of SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) within a single sample. Finally, as a real-time assay, it allows the amplification and detection processes to proceed simultaneously. This feature reduces the turn-around time of the results relative to regular PCR tests. You can find further details on the steps of diagnostic testing via the SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit below.
As illustrated by the figure above, the typical sample workflow for real-time RT-PCR includes sample collection, sample storage and/or transportation to a laboratory, specimen lysis, purification of viral RNA via nucleic acid extraction, and real-time RT-PCR ampliﬁcation, detection, and analysis. Following its collection and transportation to a laboratory setting, the sample is lysed with physical or chemical agents to provide access to the viral RNA. Next, the viral RNA is purified through nucleic acid extraction to remove potential contaminants that could interfere with the RT-PCR process. These steps can be performed manually or fully automated.
After specimen lysis and nucleic acid extraction are completed, RT-PCR can be initiated. The first step of RT-PCR involves the creation of a cDNA from the extracted viral RNA by an enzyme called reverse transcriptase. The second DNA strand is completed in accordance with the forward primer and cDNA. In turn, the resulting double-stranded DNA (dsDNA) is used as a template throughout the denaturation, annealing, and extension stages required for real-time PCR. The PCR stage of the process contains a ﬂuorescent probe or dye which generates a fluorescent signal if any of the genes targeted by the kit are amplified during cycling through the denaturation, annealing, and extension stages of real-time PCR. A real-time thermo-cycler then captures these signals. If the captured fluorescence crosses a defined threshold, the PCR cycle that it occurs is referred to as the threshold cycle (Ct). The threshold cycle value (Ct) assists in interpreting the test results as positive, negative, or indeterminate.
When should the SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit be used?
The real-time RT-PCR method typically allows for high overall accuracy levels throughout different stages of infection. However, the performance of real-time RT-PCR may depend on various factors such as sample type, the timing of sample collection, the targeted genes within the viral RNA, the operational parameters of the device, and the thresholds defined for the interpretation of results, along with the primers, probes, and reagents utilized in the different steps of the procedure. As the viral RNA tends to remain detectable for prolonged periods in the upper or lower respiratory specimen and its amplification boosts the sensitivity of the kit, the SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit offers a wide detection window for these viruses. However, the timing of sample collection can be optimized to minimize the risk of false-negatives further. According to the United Kingdom Health Security Agency, the level of SARS-CoV-2 viral RNA within samples is highest throughout the first week following the symptom onset. Repeat testing can also be considered after an initially negative result if there is a high level of clinical suspicion.
What are the advantages of the SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit?
The SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit is intended to assist medical professionals in detecting and differentiating infections with SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) in patients with clinical and epidemiological risk factors. Although there are slight differences among the signs and symptoms of COVID-19, influenza, and RSV which may help you determine which of these viruses you may have most likely been infected with, the clinical presentations and seasonal patterns of these infections often mimic each other. Thus, if you are experiencing symptoms common to these infections, such as fever, chills, cough, sneezing, sore throat, runny or congested nose, headache, fatigue, and myalgia, you can consider contacting your healthcare provider and taking a test.
As the viral genes targeted by the SARS-CoV-2, Flu & RSV Real-Time PCR Detection Kit remain detectable for extended periods in the collected specimen, the kit offers a much wider detection window compared to rapid antigen tests. Moreover, amplifying the targeted genes through many cycles afforded by the PCR portion of the process allows for a significantly higher level of sensitivity relative to methods based on antigen detection. Finally, the simultaneous proceeding of the amplification and detection processes shortens the turn-around time of results relative to regular PCR testing. This can help increase efficiency, especially for large-scale mass testing purposes.
Safiabadi Tali, S. H., LeBlanc, J. J., Sadiq, Z., Oyewunmi, O. D., Camargo, C., Nikpour, B., Armanfard, N., Sagan, S. M., & Jahanshahi-Anbuhi, S. (2021). Tools and Techniques for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) /COVID-19 Detection. Clinical Microbiology Reviews, 34(3). https://doi.org/10.1128/cmr.00228-20