The detection principle of the fluorescence quantitative PCR instrument is mainly based on the combination of PCR technology and fluorescence signal detection, and the details are as follows:

Basis of PCR Technology

PCR is a technique for amplifying DNA in vitro. Through the cycles of three steps, namely high-temperature denaturation, low-temperature annealing, and appropriate-temperature extension, the target DNA fragments can be amplified in large quantities in a short period of time. In fluorescence quantitative PCR, this serves as the basis for the quantitative analysis of the target DNA.

Generation and Detection of Fluorescence Signals

• Fluorescence Dye Method: Commonly used fluorescent dyes, such as SYBR Green I, can specifically bind to the minor groove of double-stranded DNA. During the PCR reaction process, as double-stranded DNA is synthesized, a large amount of SYBR Green I dye binds to the newly synthesized double-stranded DNA, thereby generating fluorescence signals. Moreover, the intensity of the fluorescence signal is proportional to the content of double-stranded DNA. The instrument can reflect the amplification status of DNA in the PCR reaction by detecting the intensity of the fluorescence signal.
• Probe Method: Specific fluorescent probes are used, with a fluorescent reporter group (R) labeled at the 5′ end and a fluorescent quencher group (Q) labeled at the 3′ end. In the PCR reaction system, the probe specifically hybridizes with specific regions of the template DNA. When the probe is intact, since the reporter group and the quencher group are very close to each other, the fluorescence emitted by the reporter group is absorbed or suppressed by the quencher group, and no fluorescence signal can be detected. During the PCR extension stage, the 5’→3′ exonuclease activity of Taq polymerase hydrolyzes the probe, separating the reporter group from the quencher group, and then the reporter group will emit fluorescence. The intensity of the fluorescence signal is proportional to the quantity of PCR products.

Principle of Quantitative Analysis

• Standard Curve Method: First, a series of gradient dilutions are made for the known concentration standards, and then fluorescence quantitative PCR reactions are carried out simultaneously for the standard samples and the samples to be tested. Taking the logarithm of the initial copy number of the standard samples as the abscissa and the corresponding Ct value (the number of PCR cycles when the fluorescence signal reaches the set threshold) as the ordinate, a standard curve is drawn. Under the same conditions, the samples to be tested are detected to obtain their Ct values, and then the initial copy number of the target DNA in the samples to be tested is calculated according to the standard curve.
• Absolute Quantification: By comparing the fluorescence signal intensities of the samples and the known concentration standards, the copy number or concentration of the target DNA in the samples is directly calculated without relying on the standard curve. This method requires that the instrument can accurately measure the absolute intensity of the fluorescence signal, and has relatively high requirements for the consistency and stability of the reaction system.
• Relative Quantification: In relative quantification, an internal reference gene is usually selected as a control. The expression level of the internal reference gene is relatively stable in different samples. By comparing the difference in Ct values between the target gene and the internal reference gene, the relative expression level of the target gene in different samples is calculated, so as to analyze the expression changes of the target gene under different conditions.

The fluorescence quantitative PCR instrument combines PCR amplification with fluorescence signal detection based on the above principles, realizing rapid and accurate quantitative analysis of DNA templates and playing an important role in multiple fields.