Kit for detecting DNA residues of CHO cell and using method thereof

A kit and cell technology, applied in the field of quantitative detection of DNA residues in CHO cells, can solve the problems of low sensitivity, high price and high implementation cost, and achieve the effect of good sensitivity

Active Publication Date: 2011-09-14
SHANGHAI HENLIUS BIOTECH INC
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  • Abstract
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AI Technical Summary

Problems solved by technology

SYBR Green is the most commonly used fuel, but because of its strong affinity with DNA, it usually has an inhibitory effect on PCR reactions; Taqman probes have high sensitivity and strong specificity, but they are expensive. Orifice plate assays cost >$3,000, greatly limiting their use
[0005] The fluoresc

Method used

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  • Kit for detecting DNA residues of CHO cell and using method thereof
  • Kit for detecting DNA residues of CHO cell and using method thereof
  • Kit for detecting DNA residues of CHO cell and using method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Design of primers and preparation of reference materials for the detection of DNA residues in CHO cells by fluorescent quantitative PCR

[0042] 1. Materials

[0043] DNTPs, Taq DNA polymerase, fluorescent dyes and PCR buffer were purchased from BioRad in the United States. The 7500Fast fluorescent quantitative PCR instrument was a product of Applied Biosystems in the United States. The DNA dilution was prepared with analytical reagents. The genomic DNA extraction kit was from Bio- Tek products.

[0044] 2. Design and synthesis of primers and probes

[0045] Using the Genebank accession sequence (Genebank accession number EF540878.1) as a template, use the Primer Primer 5.0 software (PremierBiosoft, USA) to design PCR primers, and select the best combination from them through preliminary experiments:

[0046] CHO-F: 5'-CTGGCAGACATTCTAAATATC-3' (SEQ ID NO: 1),

[0047] CHO-R: 5'-TAGCAGACACTGTTGTAGAG-3' (SEQ ID NO: 2).

[0048] Primers were synthesized by Shanghai San...

Embodiment 2

[0056] Example 2: Application and Specificity Analysis of Detection of CHO Cell Residual DNA by Fluorescent Quantitative PCR

[0057] 1. Prepare a kit comprising the following components:

[0058] DNA diluent (1ml / tube) 1 tube, PCR amplification reaction solution (1ml / tube) 1 tube, negative quality control (150μl / tube) 2 tubes, positive quality control (150μl / tube) 2 tubes, quantitative reference Product (50μl / tube) 1 tube.

[0059] The positive quality control product is CHO cell genomic DNA, the concentration is 3.0×10 5 fg / ul.

[0060] The negative control substance is pure water.

[0061] Quantitative reference product is CHO cell genomic DNA, the concentration is 3.0×10 7 fg / ul.

[0062] DNA diluent (choose from any of the following):

[0063] Formula 1: use deionized water as a solvent, the concentration of Tris-HCl is 5.0 mM / L, the concentration of EDTA is 1.0 mM / L, and the pH is adjusted to 8.5 with NaOH solution.

[0064] Formula 2: use deionized water as solve...

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Abstract

The invention relates to a kit for detecting deoxyribose nucleic acid (DNA) residues of a Chinese hamster ovary (CHO) cell and a using method thereof. The kit comprises DNA extracting solution, polymerase chain reaction (PCR) amplification reaction liquid, a DNA quantitative reference product of a CHO cell genome, a negative quality control product, a positive quality control product and DNA diluent. In the kit, EvaGreen is used as a fluorescent dye, and the DNA of the CHO cell genome is detected by a real-time quantitative PCR technology; and products such as a treatment protein medicament, a recombinant vaccine, a monoclonal antibody and the like from the CHO cell can be accurately and quantitatively detected.

Description

technical field [0001] The invention relates to a kit for quantitatively detecting the residue of CHO cell DNA (namely, Chinese hamster ovary cell genome DNA) and its use. Background technique [0002] The preparation of therapeutic recombinant protein drugs through mammalian cell culture expression has become the mainstream technology in the field of biopharmaceuticals. Currently, 70% of the protein drugs that have been marketed and are undergoing clinical trials come from mammalian cells, among which CHO cells are the most Commonly used cell lines. CHO cells are derived from Chinese hamster ovary cells. At present, it is the preferred system for recombinant protein production; because it has many advantages compared with other expression systems: 1) It has accurate post-transcriptional modification function, and the expressed drug protein can pass through CHO cells undergo glycosylation modification, and its products are closest to natural protein molecules in terms of mo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 郎国竣郭新军姜伟东刘世高马辰张二辉
Owner SHANGHAI HENLIUS BIOTECH INC
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