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A nucleic acid detection kit for quantifying trace residues in mouse-derived tissues

A technology for detection kits and kits, applied in the field of bioengineering, to achieve good sensitivity and good specificity

Active Publication Date: 2020-09-18
天津欧德莱生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no nucleic acid identification product of mouse-derived tissue. To meet the requirements of this detection parameter, from the beginning of research and development, we have quantitatively searched for and designed methods and products that can accurately determine the amount of nucleic acid in trace amounts of mouse-derived tissue.

Method used

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  • A nucleic acid detection kit for quantifying trace residues in mouse-derived tissues
  • A nucleic acid detection kit for quantifying trace residues in mouse-derived tissues
  • A nucleic acid detection kit for quantifying trace residues in mouse-derived tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Kit preparation

[0038] The preparation of the kit includes the following components:

[0039] Strain C57BL / 6J genomic DNA (30ng / ul) (40ul / tube) 1 tube, NS0 cell genomic DNA (30ng / ul) (40ul / tube) 1 tube, SP2 cell genomic DNA (30ng / ul) (40ul / tube) 1 tube, 1 bottle of DNA diluent (7ml / bottle), 1 tube of 2×qPCR master mix (750ul / tube), 1 tube of 10×primer-probe mixture (150ul / tube), ultrapure water (1.5ml / tube) 1 tube.

[0040] Among them, the 2×qPCR master mix is ​​a commercial reagent (Tiangen), the DNA diluent is prepared by the laboratory using molecular biology grade reagents, and the 10× primer-probe mixture: forward primer: reverse primer: Taqman probe according to Mix at a ratio of 1:1:0.2 to obtain a 10× primer-probe mixture. The primers and probes were synthesized and purified by Shanghai Sangong, and the sequences are as follows:

[0041] Forward primer sequence: ACAGGDTTTCTCTGTRTAG (Seq ID No:4)

[0042] Reverse primer sequence: CGGATYTCTGAGTTTGAG (Seq ID...

Embodiment 2

[0048] qPCR test method for genomic DNA of strain C57BL / 6J, NS0, SP2 cells

[0049] DNA extraction and calibration: Genomic DNA was extracted from strain C57BL / 6J tissue, NS0 and SP2 cells according to the instructions of Genomic DNA Extraction Kit (Tiangen), and then the concentration of genomic DNA was calibrated to 30ng / ul using Qubit (Invitrogen).

[0050] Take out the reagents required for the PCR reaction: 2×qPCR master mix, primer-probe mixture, quantitative standard, ultrapure water.

[0051] Determine the final number of reaction tubes according to the number of samples to prepare qPCRmix, as shown in Table 1 below.

[0052] After adding the samples, PCR amplification was performed on the ABI7500 real-time fluorescent quantitative PCR instrument (ABI). The reaction conditions are: 50 o C 2min, 95 o C pre-denaturation 10min; 95 o C 10sec, 55 o C 1min, 40 cycles.

Embodiment 3

[0054] Drawing of Standard Curve of Genomic DNA of NS0 Cells

[0055] The standard reference genome of the corresponding mouse-derived tissue or cell line is selected according to the actual detection requirements for trace residues of mouse-derived tissue. The purpose of this example is to quantify the residual DNA of NSO cells, and extract and calibrate the genomic DNA of NSO cells (30ng / ul) was serially diluted with DNA diluent to obtain ST1 (300pg / ul), ST2 (30pg / ul), ST3 (3pg / ul), ST4 (0.3pg / ul), ST5 (0.03pg / ul) , ST6 (0.003pg / ul). Carry out sample addition according to the sample addition system in Table 1, then run on the machine and react according to the reaction procedure. figure 1 It is a diagram of the detection results of the NSO cell DNA quantitative standard of the present invention; the Ct value is 10-40, the amplification curve has an obvious exponential growth period, and becomes S-shaped, which can be clearly determined as positive. Use the log value of eac...

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Abstract

The invention discloses a nucleic acid detection kit for quantifying trace residues of mouse source tissues. The kit can detect the DNA residues of the mouse source tissues or cells, and the minimum concentration is 0.1 fg / [mu]l; the kit has the high specificity while having the high sensitivity, and can be used for food pollution detection, environmental monitoring and precise quantification of the DNA residues of the mouse source cells in biological medicines such as monoclonal antibodies; and a reliable quality control evidence can be provided for production links of semi-finished productsand finished products of the biological medicines such as the monoclonal antibodies, and important guarantee is provided for safety production and use of vaccines.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a nucleic acid detection kit for quantifying trace residues in mouse source tissues. Background technique [0002] Real-time fluorescent PCR technology is a nucleic acid detection technology that has developed rapidly in recent years. A PCR amplification instrument with a nuclear power coupling device (CCD) is used to reflect the amplification of each cycle of PCR in real time by detecting the dynamic changes of fluorescent signals. increase level. The CCD can periodically emit excitation light of a specific wavelength according to a certain program, collect and detect fluorescent signals, and analyze and summarize them to the workstation through software to obtain the amplification curve. TaqMan PCR technology is a kind of real-time fluorescent PCR. Compared with the traditional PCR, it adds a probe whose two ends are respectively labeled with a fluorescent ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 常子嵩李婷陈涛孙明娣王博玮李英
Owner 天津欧德莱生物医药科技有限公司