The principle of real-time fluorescent quantitative PCR (qPCR) technology is as follows:

Real-time fluorescent quantitative PCR is a technology that adds fluorescent groups to the PCR reaction system and realizes the quantification of the initial template nucleic acid through real-time detection and analysis of fluorescent signals. Its principle is mainly based on the following aspects:

Basis of polymerase chain reaction (PCR)

PCR is a technology for in vitro amplification of specific DNA fragments. Through multiple cycles of three steps of high-temperature denaturation, low-temperature annealing, and moderate-temperature extension, the target DNA fragment can be replicated in large quantities in a short time. In real-time fluorescent quantitative PCR, the same basic principle is followed. Using the enzymatic reaction of DNA polymerase and guided by a pair of specific primers, new DNA strands are synthesized on the basis of template DNA.

Principle of fluorescence signal generation

• Fluorescent dye method: Commonly used fluorescent dyes such as SYBR Green I can specifically bind to the minor groove of double-stranded DNA. In the annealing and extension stages of the PCR reaction, as new double-stranded DNA is continuously synthesized, SYBR Green I binds to double-stranded DNA, and the intensity of its fluorescence signal is proportional to the amount of double-stranded DNA. When the PCR reaction proceeds, the fluorescence signal will increase with the increase of double-stranded DNA, so that the accumulation of PCR products can be monitored in real time.
• Fluorescent probe method: Such as TaqMan probe, it is an oligonucleotide probe with a fluorescent reporter group labeled at the 5′ end and a fluorescent quenching group labeled at the 3′ end. In the free state, due to the close distance between the fluorescent reporter group and the fluorescent quenching group, the fluorescence signal of the fluorescent reporter group is absorbed by the quenching group and cannot emit fluorescence. When the PCR reaction proceeds to the extension stage, Taq DNA polymerase has 5’→3′ exonuclease activity and will gradually degrade the TaqMan probe specifically bound to the template from the 5′ end, separating the fluorescent reporter group and the fluorescent quenching group, and the fluorescent reporter group emits a fluorescence signal.

Quantitative principle

• Relationship between Ct value and initial template amount: In the real-time fluorescent quantitative PCR reaction, the fluorescence signal is detected once in each cycle. When the fluorescence signal reaches the set threshold, the corresponding cycle number is called the Ct value (Cycle threshold). There is a logarithmic linear relationship between the Ct value and the initial template amount. That is, the more the initial template amount, the smaller the Ct value; the less the initial template amount, the larger the Ct value. By making a standard curve with standard products of known initial template amounts, according to the Ct value of the sample, the amount of the initial template in the sample can be calculated on the standard curve.