Manual PCR, also referred to as conventional PCR, is time consuming and requires manual manipulation. Each step of the protocol involves multiple transfers of samples, reagents, and consumables, i.e., pipette tips, tubes, gloves, etc. Once the PCR product is obtained, secondary analysis, such as gel electrophoresis, is needed for detection. Definitive identification often requires genomic sequence analysis or other alternative methods. Thus, conventional PCR is not ideal for rapid diagnostic use in most clinical laboratories.
Real-time PCR, also called quantitative PCR (qPCR), is an automated technique that allows for the detection and quantification of the target material. Results are generated at the same time that the assay is being run, hence the designation "real-time." The key feature in real-time PCR is that amplification of DNA is detected in “real-time” as the PCR is in progress. The assessment of the PCR progression is performed using a fluorescent reporter which measures the fluorescent signal to directly assess the number of amplified DNA molecules.
Although both conventional PCR and real-time PCR follow a similar procedure, there are many advantages to real-time PCR, including the following:
- The ability to monitor the progress of the PCR reaction (amplification) as it occurs in real-time.
- The ability to precisely measure the amount of amplified DNA product, or amplicon at each cycle, which allows highly accurate quantification of the amount of starting material in samples.
- Provides a dynamic range of detection (from as little as a single strand of target to millions of copies in a sample).
- Amplification and detection occur in a single tube, eliminating post-PCR manipulations.
