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Kit for detecting DNA residues of CHO cell and using method thereof
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A kit and cell technology, applied in the field of quantitative detection of CHO cell DNA residues, can solve the problems of low sensitivity, high implementation cost, limited use, etc., and achieve the effect of good sensitivity
Active Publication Date: 2013-07-17
SHANGHAI HENLIUS BIOTECH INC
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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 fluorescent PCR method of the present invention overcomes the shortcomings of the above-mentioned existing quantitative PCR detection methods such as low sensitivity and high implementation costs, and provides a simple, cheap, and high-accuracy quantitative fluorescent PCR detection method and a kit for the method
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Embodiment 1
[0041] Design of primers and preparation of reference materials for the detection of DNA residues in CHO cells by fluorescence quantitative PCR
[0042] 1. Materials
[0043] DNTPs, Taq DNA polymerase, fluorescent dyes and PCR buffers were purchased from BioRad, USA. 7500Fast fluorescence quantitative PCR instrument was produced by Applied Biosystems, USA. The DNA dilution was prepared with analytical reagents, and 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 Primer Primer 5.0 software (PremierBiosoft, USA) to design PCR primers, and select the best combination through pre-experiment:
[0046] CHO-F: 5'-CTGGCAGACATCTAAAATATC-3' (SEQ ID NO: 1),
[0047] CHO-R: 5'-TAGCAGACACTGTTGTAGAG-3' (SEQ ID NO: 2).
[0048] Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.
[0049] 3. T...
Embodiment 2
[0056] Example 2: Fluorescence quantitative PCR method for detection of residual DNA in CHO cells and its specificity analysis
[0057] 1. Prepare a kit comprising the following components:
[0058] 1 tube of DNA dilution solution (1ml / tube), 1 tube of PCR amplification reaction solution (1ml / tube), 2 tubes of negative quality control substance (150μl / tube), 2 tubes of positive quality control substance (150μl / tube), quantitative reference product (50μl / tube) 1 tube.
[0059] The positive control substance was CHO cell genomic DNA at a concentration of 3.0×10 5 fg / ul.
[0060] The negative control substance was pure water.
[0061] The quantitative reference material is CHO cell genomic DNA at a concentration of 3.0×10 7 fg / ul.
[0062] DNA Diluent (choose from any of the following):
[0063] Formulation 1: Deionized water is used as the 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 solu...
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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 CHO cell DNA (ie, Chinese hamster ovary cell genomic DNA) residues and its use. Background technique [0002] The preparation of therapeutic recombinant protein drugs through mammalian cell culture and expression has become the mainstream technology in the field of biopharmaceuticals. Among the protein drugs that have been marketed and are undergoing clinical trials, 70% come from mammalian cells, of which CHO cells are the most commonly used cell lines. CHO cells are derived from Chinese hamster ovary cells. Currently, it is the preferred system for recombinant protein production; compared with other expression systems, it has many advantages: 1) It has accurate post-transcriptional modification functions, and the expressed drug proteins can pass through CHO cells undergo glycosylation modification, and their products are closest to natural protein molecules in terms of molecular structure, ...
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