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Method and kit for determining the content of Escherichia coli dna in biological products

A technology of Escherichia coli and biological products, which is applied in the field of biomedicine to achieve the effects of short time consumption, easy operation and improved accuracy

Active Publication Date: 2020-07-21
WUHAN OPTICS VALLEY HUMANWELL BIO PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, how to quickly and accurately detect residual DNA in biological products still needs further research and development

Method used

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  • Method and kit for determining the content of Escherichia coli dna in biological products
  • Method and kit for determining the content of Escherichia coli dna in biological products
  • Method and kit for determining the content of Escherichia coli dna in biological products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] In this example, the inventor introduces the materials and instruments used in the method for detecting the DNA content of Escherichia coli in biological products.

[0032] The materials used in the experiment are shown in Table 1:

[0033] Table 1

[0034]

[0035]

[0036] The instruments used in the experiment are shown in Table 2:

[0037] Table 2

[0038] Names of instruments and utensils Model / Specification Fluorescent quantitative PCR instrument StepOnePlus

Embodiment 2

[0039] Embodiment 2 prepares Escherichia coli (E.coli) quantitative standard substance

[0040] In the present embodiment, the inventor introduces Escherichia coli (E.coli) quantitative standard (genomic DNA) in detail Standard product) preparation process:

[0041] a. Take out the E.coli cultured overnight in the nutrient broth (37°C, 200rpm), and observe the growth of the bacteria;

[0042] b. Aspirate the bacterial liquid, 1ml / tube, and centrifuge at 12000rpm for 1min to enrich the bacterial cells;

[0043] c. Discard the supernatant, add 200ul GA buffer (from TIANGEN kit, Cat#DP302-02) to each tube, mix by pipetting, vortex for a few seconds, and resuspend the bacteria thoroughly;

[0044] d. Add 4ul RNase (RNAase) to each tube, place at room temperature for 5min, then add 20ul proteinase K (Proteinase K) to each tube, mix well; add 200ul GB buffer (from TIANGEN kit, Cat#DP302-02) (A large amount of white flocculent precipitates are produced), mix well, and mix ...

Embodiment 3

[0057] Example 3 Real-time fluorescent quantitative PCR technology (QPCR) detects E.coli host DNA residues

[0058] In this example, the inventors have introduced in detail the process and conditions for using QPCR to detect the DNA content of the E.coli host:

[0059] 1) The E.coli genomic DNA (gDNA) mother solution (136ng / microliter) extracted and prepared in Example 2 was sequentially serially diluted to 20ng / microliter, 2ng / microliter, 200pg / microliter, 20pg / microliter , 2pg / microliter, 200fg / microliter, 20fg / microliter, 2fg / microliter to draw the standard curve of CT-DNA amount.

[0060] 2) Sterilized ultrapure water (NTC): Freeze at -20°C after aliquoting and take as needed.

[0061] 3) Preparation of the reaction system, prepare the system (microliter) according to the reaction quantity, and the reaction system is as shown in Table 4:

[0062] Table 4

[0063] Water 8.5 10uM forward primer 1 10uM reverse primer 1 2X SYBR mix ...

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Abstract

A method for determining the escherichia coli DNA content of a biological product is provided. The method includes subjecting the biological product to real-time fluorescence quantitative PCR detection by utilizing an escherichia coli specific primer group to determine the escherichia coli DNA content of the biological product. By utilizing the method in an embodiment, the escherichia coli DNA content of the biological product can be quantificationally analyzed, operation is simple and convenient, time consumed is short, sensitivity is high, and the lowest detection limit of the escherichia coli DNA content of the biological product can be 2.0 fg / microlitre. The method can provide reliable quality control bases for production of biotech medicines adopting escherichia coli as a host, and provides important supports for development and safe production of recombinant biotech medicines.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method and a kit for determining the DNA content of Escherichia coli in 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 residual amount of prokaryotic DNA in biological products should not be higher than 10ng / dose, and the residual DNA amount of Chinese hamster ovary cells (CHO cells) should not be higher than 100pg / dose. [0003] However, how to quickly and accurately detect residual DNA in biological products still needs further research and development. Contents of the invention [0004] The present invention aims to solve one of the technical problems in the related art at least to a certain ext...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/10C12N15/11C12R1/19
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2563/107C12Q2561/113C12Q2545/113
Inventor 王学海周昌茂许勇马铮黄璐马梵辛肖强刘哲
Owner WUHAN OPTICS VALLEY HUMANWELL BIO PHARMA
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