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 method of real-time fluorescence quantitative PCR

In Real-timePCR, there are two strategies for template quantification, relative quantification and absolute quantification.

Introduction to Traditional Quantitative PCR Methods

1) Internal reference method: add the quantified internal standard and primers to different PCR reaction tubes, and the internal standard is synthesized by genetic engineering method. The upstream primers are fluorescently labeled, and the downstream primers are not. At the same time as the template is amplified, the internal standard is also amplified. In the PCR product, due to the different lengths of the internal standard and the target template, the amplification products of the two can be separated by electrophoresis or high performance liquid phase, and their fluorescence intensities can be measured respectively, and the internal standard can be used as a control to quantify the template to be detected.

2) Competition method: select an exogenous competitive template containing a new endonuclease site produced by mutant clones. In the same reaction tube, the sample to be tested and the competitive template are simultaneously amplified with the same pair of primers (one of the primers is fluorescently labeled). After amplification, the PCR product is digested with endonuclease, and the product of the competitive template is digested into two fragments, while the template to be tested is not digested by enzyme. The two products can be separated by electrophoresis or high performance liquid phase, and the fluorescence intensity can be measured separately , based on the known template to infer the initial copy number of the unknown template.

3) PCR-ELISA method: using labeled primers such as digoxigenin or biotin, the amplified product is bound by a specific probe on the solid phase plate, and then anti-digoxigenin or biotin enzyme-labeled antibody-horseradish peroxidation is added. Substrate enzyme conjugate, the enzyme develops the color of the substrate. The conventional PCR-ELISA method is only a qualitative experiment. If an internal standard is added to make a standard curve, the purpose of quantitative detection can also be achieved.