Reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR:
The standard method for reverse transcription-polymerase chain reaction (RT-PCR) uses RNA as the nucleic acid starting material, which serves as the template for the synthesis of complementary DNA (cDNA) using the enzyme RNA-dependent DNA polymerase. The newly synthesized cDNA is then amplified and detected using the polymerase chain reaction (PCR). Originally, the RT-PCR technique used radioactive isotope markers to detect specific genetic regions, but the technique was subsequently refined by replacing the isotopic labeling with specific markers, such as fluorescent dyes. This replacement in the technique allows for detecting and quantifying the patient’s viral copy in real time and is termed real-time RT-PCR. Real-time RT-PCR may also be written as RT-qPCR.
Since all coronaviruses have an RNA genome, most of the FDA-approved COVID-19 diagnostic molecular tests employ the real-time RT-PCR method using RNA as the starting material for in vitro nucleic acid amplification. The isolated RNA with cDNA is then amplified using primers and fluorescently labeled probes specific to the region of SARS-CoV-2.
