Real-time fluorescence quantitative PCR instrument is an instrument used for fluorescence measurement.

This device consists of a fluorescence quantification system and a computer, used to monitor the fluorescence of the cyclic process. Computers connected to real-time devices collect fluorescence data. The data is displayed in the form of charts through real-time analysis software equipped. The raw data is plotted as a graph of fluorescence intensity relative to the number of cycles. After collecting the raw data, analysis can begin. The software of real-time devices can normalize the collected data to compensate for differences in background fluorescence. After normalization, the threshold level can be set, which is the analysis of fluorescence number

The number of cycles that a sample undergoes to reach the threshold level is called the Ct value (the number of cycles at the limit point). The domain value should be set to maximize the amplification efficiency of the exponential period, in order to obtain the most accurate and reproducible data. If there are also standard samples labeled with corresponding concentrations being amplified simultaneously, linear regression analysis will generate a standard curve that can be used to calculate the concentration of unknown samples.

The so-called real-time qPCR technology refers to the method of adding fluorescent genes to the PCR reaction system, accumulating fluorescent signals to monitor the entire PCR process in real time, and finally quantitatively analyzing unknown templates through standard curves. In the development of real-time technology, two important discoveries have played a crucial role: (1) in the early 1990s, the discovery of the 5′ exonuclease activity of TaqDNA polymerase, which can degrade specific fluorescent probes, thus making indirect detection of PCR products possible. (2) Subsequently, 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 discoveries and the commercial development of corresponding instruments and reagents have led to the application of real-time qPCR methods in research work.

The DNA copy number generated during the PCR reaction increases exponentially. As the number of reaction cycles increases, the PCR reaction no longer generates templates exponentially and enters the plateau phase. In traditional PCR, gel electrophoresis is often used to separate and detect the final amplification product of PCR reaction with fluorescence staining, so this endpoint method is unreliable in quantifying PCR products. In real-time qPCR, the entire PCR amplification process was monitored in real-time and the fluorescence signals related to amplification were continuously analyzed. As the reaction time progressed, the changes in the monitored fluorescence signals could be plotted as a curve. In the early stage of PCR reaction, the level of fluorescence generated cannot be clearly distinguished from the background, and then the generation of fluorescence enters the exponential phase, linear phase, and final plateau phase. Therefore, the amount of PCR product can be detected at a certain point in the exponential phase of PCR reaction, and the initial content of the template can be inferred from this. In order to facilitate comparison of the detected samples, a certain threshold for fluorescence signal needs to be set during the exponential period of real-time qPCR reaction. Generally, this threshold is based on the fluorescence signal of the first 15 cycles of PCR reaction as the fluorescence background signal. The default setting for fluorescence threshold is 10 times the standard deviation of the fluorescence signal of 3-15 cycles. If a fluorescence signal exceeding the threshold is detected, it is considered a true signal and can be used to define the threshold cycle number (Ct) of the sample. The meaning of Ct value is the number of cycles experienced by the fluorescence signal in each reaction tube when it reaches the set threshold. Research has shown that there is a linear relationship between the Ct value of each template and the logarithm of the initial copy number of that template, with the Ct value decreasing as the initial copy number increases. A standard curve can be plotted using a standard sample with a known initial copy number, so as long as the Ct value of the unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve.