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Method for quickly and quantitatively detecting mammalian cell deoxyribose nucleic acid (DNA)

A quantitative detection method, mammalian technology, applied in the field of quantitative detection of new genomic DNA, can solve the problems of increased cost, decreased specificity, increased probe usage, etc.

Inactive Publication Date: 2011-11-09
BEIJING XINYI YUANCHENG BIOLOGICAL TECH
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Problems solved by technology

[0003] The currently used detection method is random primers, and DNA polymerase synthesizes random probes. In production practice, we found that when the hybridization temperature increases, that is, the specificity increases, the sensitivity of this probe detection method decreases, and the application purpose is often not achieved. The detection limit is at the nanogram level, so we have to increase the amount of probes used and reduce the hybridization temperature. As a result, the sensitivity is improved, but the specificity is reduced, and the cost is increased; on the other hand, it takes 2.5 days from the synthesis of probes to the completion of hybridization and color development , too long

Method used

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  • Method for quickly and quantitatively detecting mammalian cell deoxyribose nucleic acid (DNA)
  • Method for quickly and quantitatively detecting mammalian cell deoxyribose nucleic acid (DNA)
  • Method for quickly and quantitatively detecting mammalian cell deoxyribose nucleic acid (DNA)

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example 3

[0028] Example 3 Add 1 μl of the reaction solution to the microwell with 45 μl of hybridization solution, place it at 37°C for 10 minutes, then wash the plate three times with 37°C PBS / T, add 50 μl (1×10 5 U / L) anti-digoxigenin peroxidase enzyme conjugate, placed at 37°C for 10min, then discarded the liquid in the well, washed the plate 3 times with PBS / T, then added 1 drop of TMB solution and 1 drop of 0.3 ml / L H 2 o 2 , place it in the dark for 10min, add 0.5mol / L H 2 SO 4 Terminate the color reaction, measure A 450nm .

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Abstract

The invention relates to a method for quantitatively detecting mammalian cell deoxyribose nucleic acid (DNA), which is used for detecting host cell DNA remained in a biological product which takes mammalian cells as matrixes. Multicopy genes in a mammalian cell genome are used as detection tag sequence templates, DNA polymerase, a molecular marker primer and molecular marker DNA are synthesized into a detection purpose sequence, and the detection purpose sequence is quantitatively analyzed. Compared with a molecular hybrid method, the method has the characteristics of short detection time (a detection process can be finished within two hours), stable detection results and high sensitivity.

Description

technical field [0001] The invention relates to a novel genome DNA quantitative detection method and kit, belonging to the field of biotechnology. Background of the invention [0002] With the development of biotechnology and the improvement of health levels, the demand for various viral vaccines and therapeutic antibodies has increased. Among them, various products produced by mammalian cell culture have gradually increased. Due to various cell lines such as VERO, CHO, MDCK, PER.C6, etc. have concerns about tumorigenesis in high-generation culture, so according to the requirements of the Pharmacopoeia of the People's Republic of China (2005 Edition), there are specific regulations on the residual amount of host cell DNA for products produced by various genera of such cells For example, each dose of purified rabies vaccine does not exceed 100pg, so it is necessary to detect the DNA content of host cells in the process of production, research and development, and quality insp...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 不公告发明人
Owner BEIJING XINYI YUANCHENG BIOLOGICAL TECH
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