Real-time fluorescence quantitative PCR technology effectively solves the limitation of traditional quantitative only end-point detection, realizes the detection of the intensity of the fluorescent signal once in each cycle, and records it in the computer software, through the calculation of the Ct value of each sample, Quantitative results were obtained according to a standard curve. Therefore, real-time PCR without internal standard is based on two foundations:

1) The reproducibility of the Ct value The PCR cycle has just entered the true exponential amplification phase (logarithmic phase) when it reaches the cycle number where the Ct value is located. At this time, the small error has not been amplified, so the reproducibility of the Ct value is good. That is, the same template is amplified at different times or amplified in different tubes at the same time, and the obtained Ct value is constant.

2) Linear relationship between Ct value and starting template Since there is a linear relationship between the Ct value and the logarithm of the starting template, the standard curve can be used to quantitatively determine the unknown sample. Therefore, real-time fluorescence quantitative PCR is a quantitative method using an external standard curve. Methods.

Compared with the internal standard method, the quantitative method of the external standard curve is an accurate and reliable scientific method. Real-time fluorescence quantitative PCR using external standard curve is a quantitative method with accurate quantification and good reproducibility so far. It has been recognized all over the world and is widely used in gene expression research, transgenic research, drug efficacy assessment, pathogen detection and many other fields.

Quantitative Real-time PCR (Quantitative Real-time PCR) is a method of measuring the total amount of products after each polymerase chain reaction (PCR) cycle with fluorescent chemicals in a DNA amplification reaction. A method for quantitative analysis of specific DNA sequences in a sample to be tested by an internal or external reference method. ·

Real-time PCR is the real-time detection of the PCR process through the fluorescent signal during the PCR amplification process. Since there is a linear relationship between the Ct value of the template and the initial copy number of the template in the exponential period of PCR amplification, it becomes the basis for quantification.