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Method of preparing DNA probe, kit and application thereof

A technology of DNA probes and biological products, applied in the field of biomedicine, can solve the problems of different fragment sizes, many interference factors, and difficulties, and achieve the effects of high safety, high specificity, and improved specificity and accuracy

Inactive Publication Date: 2018-02-13
WUHAN OPTICS VALLEY HUMANWELL BIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the variety of exogenous DNA hosts, fragment sizes, and trace impurities, there are many interference factors in the determination, how to perform specific, sensitive, rapid and accurate detection is a difficult task

Method used

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  • Method of preparing DNA probe, kit and application thereof
  • Method of preparing DNA probe, kit and application thereof
  • Method of preparing DNA probe, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The preparation of embodiment 1 probe DNA

[0043] The inventor explored a variety of experimental conditions when preparing the probe DNA in the early stage, so as to break the Escherichia coli genomic DNA into fragments of appropriate size, so that the fragment size is suitable for DNA hybridization and probe labeling. Specifically, the inventors screened a large number of conditions for ultrasonic treatment of Escherichia coli genomic DNA, and finally screened out test conditions suitable for the preparation of probe DNA, which were successfully applied to the detection of host DNA content in recombinant biological products, ensuring the safety of clinical medication sex.

[0044] Condition 1:

[0045] (1) Dilute the genomic DNA standard (136 μg / ml) with purified water to 40 μg / ml, and perform ultrasonic treatment with an ultrasonic instrument. The ultrasonic parameters are shown in Table 4.

[0046] Table 4:

[0047] Genomic DNA concentration

Ultraso...

Embodiment 2

[0092] Example 2 Residual Detection of Recombinant Biological Product Host (Escherichia coli) DNA

[0093] The inventors used the DNA probes prepared under different conditions in Example 1 to detect and compare the amount of host DNA residues in recombinant biological products, in order to study the hybridization efficiency and detection effect of the DNA probes prepared under different conditions, in order to screen out the optimal The method is used for quality control of host DNA residues in recombinant biological products to ensure the safety of clinical medication.

[0094] Condition 1:

[0095] The inventor used the probe DNA prepared in Example 1 condition 1 to detect and analyze the residual amount of recombinant biological product host DNA (derived from the Roche kit), and the detection steps included denaturation and dilution of the test sample and positive control (positive Control gDNA is diluted to 2ug / ml, 0.2ug / ml, 0.02ug / ml with salmon sperm DNA diluent; the t...

Embodiment 3

[0110] Example 3 Comparative Study on Sample Diluents in Residual Detection of Recombinant Biological Products Host DNA

[0111] When the inventor was developing the method in the early stage, the positive control gDNA and the test product were diluted with purified water or TE buffer solution, resulting in distortion of the test results, and the hybridization spots were not in the same color system, which could not be judged, so it is not suitable for biological products. Quantitative determination. After screening and optimization in the later stage, the inventor found that the salmon sperm DNA diluent was used for dilution, and the probe DNA prepared under condition 3 or 4 of Example 1 was used for hybridization, and the test results were stable and reliable. The results of using different dilutions in the determination of the residual amount of biological product host DNA are as follows: Figure 9 shown. Figure 9 The results showed that when the positive control gDNA an...

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Abstract

The invention provides a method of preparing a DNA probe, a kit, a method of detecting content of bacillus coli DNA in a biological product, and a method of determining a production process of a biological product. The method of preparing the DNA probe comprises the steps of (1) ultrasonic crushing cell genomic DNA of bacillus coli, wherein in the ultrasonic crushing treatment, the concentration of the cell genomic DNA of bacillus coli is 20 mcg / ml; and (2) incubating the product of ultrasonic crushing and a labeling reagent so as to acquire the DNA probe, wherein the DNA probe carries the label. The DNA probe prepared by the method can detect the volume of residual bacillus coli DNA in biological products (comprising middle sample, semi-finished product and finished product) exclusively,flexibly, quickly and accurately.

Description

technical field [0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to a method for preparing a DNA probe, a kit and applications thereof. More specifically, the present invention relates to a method for preparing a DNA probe, a kit, and detection of Escherichia coli DNA in biological products. method of content and method of determining the manufacturing process of biological products. Background technique [0002] In order to ensure the product quality of therapeutic recombinant DNA drugs, both the European and American Food and Drug Administration and the State Food and Drug Administration have put forward clear requirements on the amount of host cell DNA residues in biological products. The amount of residual DNA in prokaryotic organisms should not be higher than 10ng / dose, and the amount of residual DNA in CHO cells should not be higher than 100pg / dose. However, due to the wide variety of exogenous DNA hosts, fr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/689C12Q1/6806
CPCC12N15/1003C12Q1/6806C12Q1/689
Inventor 许勇周昌茂王学海黄璐马铮马梵辛肖强刘哲
Owner WUHAN OPTICS VALLEY HUMANWELL BIO PHARMA
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