The operating principle of PCR-ELISA 1. Use a special microplate as the assay plate, coat the microplate with avidin, then label the 3 ends of the probe with biotin, and then pass the biotin and coat in the microwell The avidin on the plate is connected, and the probe is fixed on the microwell plate to form a solid-phase capture system. Note: The 5 end of the probe and the 5 end of the target sequence to be tested should be complementary. 2, during amplification, The primers are labeled with antigen (biotin, digoxin, or luciferase, etc.), so that the amplified product will infiltrate the antigen. 3. The PCR amplified product hybridizes with the probe on the microplate to capture the target sequence to be tested As the amplification product has already infiltrated the antigen, after adding HRP-labeled antibody to the microwell, the enzyme-labeled antibody is immunocombined with the antigen of the target sequence, and finally the enzyme substrate is added for enzymatic color development, and accurate quantification is achieved by optical density measurement . PCR-ELISA has its unique advantages over PCR itself in virus typing, gene polymorphism analysis and cloning identification. In addition, the isotope labeling is not applied, thus avoiding the harm caused by radioactivity, and the entire experiment cycle only requires About 6h, much shorter experiment time than Southern blot hybridization, easy to operate , Can be used for the detection of a large number of specimens. Although compared with fluorescein stained gel electrophoresis, the specificity, sensitivity and accuracy of PCR-ELISA test results have been significantly improved, but ELISA is basically an open reaction system. Especially when washing the ELISA reaction plate, it is easy to cause pollution and cause false positives. Therefore, during the entire experiment of PCR-ELISA, strict aseptic operations must be carried out. The same is true for PCR products and probes. The hybridization conditions (dilution of PCR products, hybridization temperature and time) are strict. The synthesis and intersection of immunoenzyme technology and other technologies are the obvious characteristics of this development process. It is worth noting that electronic computer science technology and molecular biology The development of science and technology such as physics, chemistry, material science and mathematics will affect the development of immunoenzyme technology. In the future, immunoenzyme technology is very likely to be combined with electronic computer science and technology, in-depth tips on the regulation of organism quantity genes and physical chemistry special New materials form new infiltrations and integrations, enabling humans to study biological sciences and to detect and study pathogens, bacteria, viruses and related products Unprecedented depth and height.
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