Trace DNA extraction method of biological product

Abstract

A trace DNA extraction method of a biological product comprises the following four steps: step 1, protein digestion; step 2, DNA binding; step 3, DNA washing; and step 4, DNA elution. After a protease K digestion process is conventionally operated, protein residues in a sample are removed by adopting a protein solubilizing agent, so that PCR inhibition caused by the residual protein is further reduced. DNA and magnetic beads are combined in a rotary uniform mixing mode, the uniform mixing is 360-degree up-down reversed rotary uniform mixing, no precipitate is generated, and the combination efficiency of the magnetic beads and the DNA can be improved. After a sample is washed with conventional ethanol, washing of a washing solution B without ethanol is added. The washing solution B can dissolve some PCR inhibition components which cannot be dissolved by ethanol, so that the purity of DNA can be improved, the PCR inhibition phenomenon is reduced, meanwhile, the PCR inhibition phenomenon cannot be generated by residues of the washing liquid B, the washing solution B can immediately enter a DNA elution step after being washed, and an ethanol drying step is not needed.

Description

technical field [0001] The invention relates to the technical field of DNA extraction methods for biological products, in particular to a method for extracting trace DNA from biological products. Background technique [0002] At present, DNA (deoxyribonucleic acid) extraction of biological products (such as monoclonal antibody drugs and virus extracts for CAR-T cell therapy) is mainly based on superparamagnetic magnetic beads with high binding force (magnetic beads are superparamagnetic After the surface of the nanoparticles is improved and modified, the superparamagnetic silica nano-magnetic beads prepared) are used as the basis to realize high-speed, harmless extraction and purification of DNA. During extraction, the magnetic beads can specifically recognize and efficiently bind DNA molecules through the silica microscopic interface in the environment of a chaotropic salt (such as guanidinium salt) buffer, and then under the action of an external magnetic field, the magnet...

AI Technical Summary

Problems solved by technology

[0003] In practice, due to the different purification processes, components and protein concentrations of various biological products, some components (such as residual proteins, residual components of chemical reagents, alcohols, sugars and residual cytoplasm, etc.) will be co-purified during extraction, so The extraction of DNA solution often has the phenomenon of low recovery rate of DNA extraction and PCR inhibition

The result of low recovery rate of DNA extraction and PCR inhibition will cause the Ct value (Cycle Threshold) of fluorescent quantitative PCR reaction (polymerase chain reaction), that is, the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value.

) hysteresis or no amplification signal at all, and the result of data interpretation is that the amount of DNA is reduced or there is no DNA, but this is not consistent with the actual situation. In this way, such as protein drugs expressed by cells, protein vaccines or cell therapy The DNA content of the host cell in the virus purification solution exceeds the standard, but the test result shows that the DNA content of the host cell is within the normal range (the product is actually unqualified, and the test result shows that it is qualified, that is, a false negative)

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