Primer, kit and method for detecting CHO cell DNA residues

A kit and cell technology, applied in the field of quantitative detection of DNA content in CHO cells, can solve the problems of comparable specificity, not widely used, and low success rate, and achieve the effect of high specificity and good sensitivity

Inactive Publication Date: 2016-08-17
LIVZON MABPHARM
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

EvaGreen dye has no specificity for the binding of double-stranded DNA and cannot recognize DNA fragments from different sources. Although it is stronger than the original SYBR Green fluorescent dye in terms of false positives and anti-interference, its specificity cannot be compared with Taqman probes. technology comparable
[0008] Chinese patent applications with application numbers CN201110099204.1, CN201210475980.1 and CN201210161343.7 disclose methods for detecting DNA residues in CHO cells using Taqman probe technology real-time fluorescent PCR, wherein CN201210475980.1 uses multiplex PCR technology to detect, multiplex PCR technology Difficult and cumbersome operation, low success rate, complex data processing, many affected factors, not widely used

Method used

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  • Primer, kit and method for detecting CHO cell DNA residues
  • Primer, kit and method for detecting CHO cell DNA residues
  • Primer, kit and method for detecting CHO cell DNA residues

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Embodiment 1

[0050] Example 1: Methodological Confirmation

[0051] 1.1 Materials and reagents: 2×Q-PCR master mix is ​​a commercially available product, the ViiA 7 fluorescent quantitative PCR instrument is a product of Life Technologies, the DNA dilution is prepared by our laboratory using molecular biology grade reagents, and the genomic DNA extraction kit is a product of Life Technologies ; The IPC template in the IPC reagent is the PGEM-T Easy Vector series product of Promage Company, and the primers and corresponding probes involved in the experiment are synthesized by Life Technologies Company.

[0052] The primer sequences and probe sequences used for methodological confirmation are as follows:

[0053] Forward primer sequence: 5'-CTACCAGAGGTCCTGAGTTCAATT-3' (SEQ ID NO: 1)

[0054] Reverse primer sequence: 5'-GGGCAC CAGGTCTCATAACG-3' (SEQ ID NO:2)

[0055] The nucleotide sequence of the Taqman probe is:

[0056] Taqman probe: 5'-CCAGCAACCA CATGGTGGCT CAC-3' (SEQ ID NO: 3)

[...

Embodiment 2

[0093] The preparation of embodiment 2 kit

[0094] Prepare a kit comprising the following components:

[0095] DNA diluent (5mL / tube) 1 tube, Q-PCR master mix (1.5mL / tube) 1 tube, IPC reagent (250μl / tube) 1 tube, CHO cell DNA standard (40μl / tube) 1 tube, forward Primer (100 μl / tube) 1 tube, reverse primer (100 μl / tube) 1 tube, Taqman probe (50 μl / tube) 1 tube, pure water (1 mL / tube) 1 tube.

[0096] CHO cell DNA standard, the concentration is 3.0×10 7 fg / μl.

[0097] IPC reagent: Each 2.5 μl IPC reagent contains 1 μl 1fg / μl IPC template, 0.5 μl 10 μM IPC-F, 0.5 μl 10 μM IPC-R, 0.5 μl 5 μM IPC-Probe.

[0098] The forward primer, reverse primer, and Taqman probe are all 10 μM.

[0099] DNA diluent: use deionized water as solvent, Tris-HCl concentration is 10mM / L, EDTA concentration is 0.5mM / L, adjust pH to 8.0 with NaOH solution.

[0100] 2×Q-PCR master mix: Q-PCR master mix (2×).

[0101]

[0102]

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Abstract

The present invention provides a primer pair for detecting CHO cell DNA residues. The invention uses a nucleotide sequence with GenBank accession number J00052.1 for design, provides a Taqman probe based on the nucleotide sequence, and a corresponding detection method. The method is a real-time PCR method; and based on an obtained standard curve, the DNA amount in CHO cells in a sample to be tested is calculated. The detection method can be used to detect bioproteins using CHO cells as an expression cell line in genetic engineering, such as antibodies, therapeutic proteins and vaccines. The real-time PCR method for detecting DNA of CHO cells and a PCR kit based on the method achieve limit of quantitation as low as 0.1fg / mul for the detection of DNA residues in CHO cells, which shows that the method has good sensitivity and high specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a primer, a kit and a detection method for quantitatively detecting the DNA content of CHO cells (Chinese Hamster Ovary, Chinese hamster ovary cells). Background technique [0002] In modern times, a large number of biological proteins such as monoclonal antibody drugs, recombinant protein drugs, and vaccines have been widely used clinically. The host cells that produce these biological proteins are mainly bacteria, yeast cells, and mammalian cells. Thus, the DNA of these host cells will remain in the final purified protein product. Although the amount of residual DNA is very low, the residual DNA in protein products is potentially dangerous to the human body. It is generally believed that the host cell DNA may contain certain unknown DNA fragments, some DNA fragments that may integrate viruses may cause human infection, and some DNA fragments with cancerous genes may induce tumors. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 彭育才艾军文朱保国
Owner LIVZON MABPHARM
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