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Primers, probe, kit for detection of CHO nucleic acid residue and detection method

A detection method and kit technology, applied in the field of molecular biology, can solve the problems of DNA hybridization method such as long experimental period, inability to detect single-stranded DNA, and lack of sequence specificity, and achieve the significance of people's drug safety and detection accuracy and increased sensitivity without cross-reactive effects

Inactive Publication Date: 2019-02-01
苏州蝌蚪生物技术有限公司
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AI Technical Summary

Problems solved by technology

The DNA hybridization method represented by the digoxin-labeled dot hybridization method has a long experimental period, complex operation, many interference factors, poor repeatability, only semi-quantitative analysis, non-specific color development, and sometimes false positives
Fluorescent dye method detects all double-stranded DNA, cannot detect single-stranded DNA, and has no sequence specificity

Method used

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  • Primers, probe, kit for detection of CHO nucleic acid residue and detection method
  • Primers, probe, kit for detection of CHO nucleic acid residue and detection method
  • Primers, probe, kit for detection of CHO nucleic acid residue and detection method

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Embodiment 1

[0086] Example 1 is used for the design of primers and Taqman probes for real-time fluorescent quantitative PCR detection of Chinese hamster ovary cells (CHO)

[0087]Referring to the Chinese hamster genome sequence (Genome ID: 2791) included in GenBank, the conserved regions of Chinese hamster ribosomal 18s gene, ND1 gene, and ACTB gene were selected, and ABI PrimerExpress 3.0 real-time fluorescent quantitative PCR primer design software was used to design synthetic primers and Taqman probes. Needle. For the fluorescent labeling of the probe, FAM (5' end) was selected as the reporter luminescent group, and TAMRA (3' end) was used as the quenching group.

[0088] Typical primer pairs and probe sequences are listed in Table 1 below:

[0089]

[0090]

[0091] Such as Figure 1a-Figure 1d As shown, the 10-fold serial dilution of the CHO nucleic acid standard real-time fluorescence quantitative PCR amplification curve, the result shows that the CHO-18s1 primer pair and pr...

Embodiment 2

[0093] Real-time fluorescent quantitative PCR detection of embodiment 2 Chinese hamster ovary cells (CHO)

[0094] 1. CHO positive quality control DNA extraction

[0095] The following operations must be carried out in the P2 laboratory negative pressure biological safety cabinet in strict accordance with the requirements.

[0096] (1) Put 100 mg of Chinese hamster ovary cells under sterile conditions into a 1.5 mL clean centrifuge tube, add 500 μL of extraction buffer (50 mM Tris pH8.0, 25 mM EDTA, 100 mM NaCl, 1% Triton X-100) and 20 μL ( 20mg / mL) proteinase K, in a water bath at 56°C for 30min.

[0097] (2) After lysing in a water bath, add 10 μL of magnetic beads (40 mg / mL, shake well before use), then add 250 μL of 30% PEG & 2M NaCl mixture, and vortex to mix.

[0098] (3) Rotate and mix the centrifuge tube in a mixer for 10 minutes (speed: 0.83-0.85 rev / s).

[0099] (4) After mixing, fix the adsorption on the magnetic stand, and observe with the naked eye that the mag...

Embodiment 3

[0129] The real-time fluorescent quantitative PCR detection kit of embodiment 3 Chinese hamster ovary cells (CHO)

[0130] Primers CHO-18s-F (10 μM) 100 μL, CHO-18s-R (10 μM) 100 μL, Taqman probe (10 μM) 70 μL and Taqman Mix1 used for real-time fluorescent quantitative PCR detection of Chinese hamster ovary cell (CHO) nucleic acid .5mL, CHO DNA (30ng / μL) 50μL, and sterilized double distilled water 1.5mL are packaged together to obtain a real-time fluorescent quantitative PCR detection kit for Chinese hamster ovary cell (CHO) nucleic acid.

[0131] To sum up, with the above technical solution, the embodiment of the present invention can very accurately detect the residues of CHO nucleic acid in Chinese hamster-derived biological products, which is of great significance for controlling the quality of Chinese hamster-derived related biological products. People's drug safety is of great significance. The detection method and the kit of the invention can be used for the detection of...

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Abstract

The invention discloses primers, a probe and a kit for detection of CHO (Chinese hamster ovary) nucleic acid residue and a detection method. The primer pair for detection of CHO nucleic acid residue comprises: a first primer and a second primer, and the sequences of the primers are respectively shown as SEQ ID NO.1 and SEQ ID NO.2. The sequence of the probe is shown as SEQ ID NO.3. The kit includes the primer pair and the probe. The detection method of the CHO nucleic acid residue includes: providing a to-be-detected DNA sample, and mixing the to-be-detected DNA sample with conventional components of the kit for PCR amplification detection to form a mixed solution; then utilizing the primer pair and probe contained in the kit for PCR amplification on the mixed solution; and detecting the PCR amplification product, and judging the residue condition of CHO nucleic acid in the DNA sample. The detection method and the kit provided by the invention can accurately detect the residue condition of CHO nucleic acid in Chinese hamster-derived biological products, and have broad application prospects.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, more specifically, to nucleic acid molecular biology detection methods, in particular to primers, probes and corresponding kits for real-time fluorescent quantitative PCR detection of Chinese hamster ovary cells (CHO), And a real-time fluorescent quantitative PCR detection method for detecting CHO nucleic acid residues by using the kit. Background technique [0002] CHO cells are Chinese hamster ovary cells (Chinese hamster ovary), which are immortal and can be passed down for more than 100 generations. They are currently widely used in bioengineering. There are more than 30 genetic engineering products officially approved for human disease treatment or disease prevention around the world, among which the CHO expression system can accurately post-transcriptionally modify the expressed protein in terms of molecular structure, physical and chemical properties and biological functi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6876C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6876C12Q2531/113
Inventor 薛峰陈伟苏静周莉质陆寿祥吴城霖
Owner 苏州蝌蚪生物技术有限公司
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