The so-called real-time Q-PCR technology of real-time fluorescence quantitative PCR instrument  refers to the method of adding fluorescent genes to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and quantitatively analyzing the unknown template through the standard curve. In the development of real-time technology, two important discoveries played a key role: (1) In the early 1990s, the discovery of the 5' exonuclease activity of Taq DNA polymerase, which degrades specific fluorescent probes needle, thus making indirect detection of PCR products possible. (2) The use of fluorescent double-labeled probes enables real-time monitoring of the entire reaction process in a closed reaction tube. The combination of these two findings and the commercial development of the corresponding instruments and reagents has led to the use of real-time Q-PCR methods in research work.

The number of DNA copies generated during the PCR reaction increases exponentially. With the increase of the number of reaction cycles, the PCR reaction no longer generates templates in an exponential manner, thus entering a plateau phase. In traditional PCR, gel electrophoresis is often used to separate and use fluorescent staining to detect the amplification products of PCR reactions, so there is uncertainty in quantifying PCR products by this end-point method. In real-time Q-PCR, the entire PCR reaction amplification process is monitored in real time and the amplification-related fluorescence signals are continuously analyzed. As the reaction time progresses, the changes in the monitored fluorescence signals can be plotted as a line curve. In the early stage of the PCR reaction, the level of fluorescence produced cannot be clearly distinguished from the background, and then the fluorescence production enters the exponential phase, the linear phase and the plateau phase, so the amount of PCR product can be detected at a certain point in the exponential phase of the PCR reaction, and From this, the content of the template is deduced. In order to facilitate the comparison of the detected samples, in the exponential phase of the real-time Q-PCR reaction, a certain threshold value of the fluorescence signal needs to be set first. Generally, this threshold value (threshold) is the fluorescence signal of the first 15 cycles of the PCR reaction. The signal was taken as the fluorescence background signal (baseline), and the default setting of the fluorescence threshold was 10 times the standard deviation of the fluorescence signal from 3 to 15 cycles. It can be used to define the threshold cycle number (Ct) for a sample if a fluorescent signal above a threshold is detected to be considered a true signal. The meaning of the Ct value is: the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value. Studies have shown that there is a linear relationship between the Ct value of each template and the logarithm of the initial copy number of the template, and the higher the initial copy number, the smaller the Ct value. A standard curve can be made by using a standard with a known initial copy number, so as long as the Ct value of an unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve.