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.
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.