Real-time PCR is a modification technique from the conventional PCR which was a time-consuming technique. Real-time PCR allows monitoring of PCR progress in actual time. Note that real-time PCR is not the same as RT-PCR, in which an enzymatic reaction step with Reverse Transcriptase is used to transcribe the RNA genome to cDNA amplification.
In real-time PCR, also known as quantitative PCR, amplification detection occurs simultaneously in the same reaction tube. Amplification occurs in the presence of a reporter. A probe specific to a target is bound to a fluorescent component. The signal intensity is directly proportional to the amount of amplified nucleic acid.
- Amplification and simultaneous quantitation of the target DNA is done in the same PCR instrument.
- There are several commercially available fluorescence-detecting thermocyclers.
- Fluorescent dyes specifically label DNA of interest.
- Amount of fluorescence generated is proportional to the quantity of DNA present.
Applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, species identification, detection of allergens, and much more.
33. Horvath, A. D. "Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis." Genes (BaseI). 2022 Dec 16; 13(12): 2387.4. "Real-Time PCR." Clinical Laboratory Science. Available at: https://clinicalsci.info/real-time-pcr/.
