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.

In the early stage of the PCR reaction, the level of fluorescence generated cannot be clearly distinguished from the background, and then the fluorescence generation enters the exponential phase, the linear phase and the final plateau phase, so the amount of PCR products can be detected at a certain point in the exponential phase of the PCR reaction. And from this to deduce the initial content of the template. 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 detected fluorescence signal above a threshold is 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.