How does Real-Time RT-PCR Work?
Sample Collection: To start the diagnostic test, a trained healthcare worker will use a swab to collect nasopharyngeal specimens from the patient’s
nasopharynx. The sample is then placed into a sterile tube containing viral transport media to keep the virus viable
Sample Preparation: Once the specimens arrive at the laboratory, researchers will use available commercial purification kits to extract RNA from the sample. Next, the RNA sample is added into one reaction mixture containing all the ingredients required to complete the diagnostic test, also known as “one-step RT-PCR”. The ingredients inside this mixture includes DNA polymerase, reverse transcriptase, DNA building blocks, and specific fluorophore probes and primers that recognize SARS-CoV-2.
- Step 1 – Denaturation/Separation: To begin, it is important to remember that DNA is a double-stranded structure. Thus, the next step is to unwind the DNA molecule into separate DNA stands. This is accomplished by heating the DNA to high temperatures (> 90°C) for amount 10 min.
- Step 2 – Primer Annealing: Next, is the addition of short fragments of DNA, called primers. Primers are designed with high specificity and will attach to specific targets within cDNA of the SARS-CoV-2 RNA virus. The specific lower temperature is needed for primer annealing too. In general, there 7 common gene targets used for testing COVID-19; each gene target is essential to the virus’ replication or structure(4). Those essential gene targets include RNA-dependent polymerase (RdRP), ORF1ab (SARS-CoV-2’s conserved open reading frame), S gene (spike protein), N gene (nucleocapsid protein), E gene (envelope; virus’ outer shell).
- Step 3 – Primer Extension/Elongation: Since DNA is a double stranded structure, there are two primers in this reaction mix, each one is designed to target one of the two DNA strands. Once the primers attach to their target DNAs, they will direct the DNA polyermase on where to begin and finish amplification on the DNA segment. This step results in an identical DNA copy of the target DNA.
And Repeat: Real-Time PCR will repeat the cycle multiple times (usually for 40 cycles). Every time RT-PCR completes a cycle, it will double the target DNA. Additionally, there are also fluorescent probes that bind specifically to the DNA targets, downstream of each primer. Every time DNA polyermase amplifies the DNA target, it will activate the probe to release a fluorescence signal. Thus, as the amount of target DNA increases, the fluorescence intensity will also increase.
