A real-time fluorescence quantitative PCR instrument , consisting of a fluorescence quantitative system and a computer, is used to monitor the fluorescence during the cycle. A computer connected to a real-time device collects fluorescence data. The data is displayed in the form of graphs through the developed real-time analysis software. Raw data are plotted as fluorescence intensity versus cycle number. Analysis can begin after raw data has been collected. The software of the real-time device normalizes the collected data to compensate for differences in background fluorescence. After normalization, a threshold level can be set, which is the level at which the fluorescence data is analyzed.

The number of cycles that the sample takes to reach the threshold level is called the Ct value (cycles to the limit point). The threshold should be set so that the amplification efficiency of the exponential phase is large, so that accurate and reproducible data can be obtained. If a standard labeled with the corresponding concentration is also amplified, the linear regression analysis will generate a standard curve that can be used to calculate the concentration of the unknown sample.

The so-called real-time Q-PCR technology 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 can degrade 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.