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Method for detecting CHO cell DNA

A cell and detection sensitivity technology, which is applied in the field of CHO cell DNA detection, can solve the problems of sample pretreatment, different primer and probe designs, and no unified standard substance, etc.

Active Publication Date: 2013-05-01
HUZHOU SHENKE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still has problems such as sample pretreatment, different primer and probe designs in each laboratory, and no unified standard substance, etc. The above problems still need to be further studied and solved.

Method used

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  • Method for detecting CHO cell DNA
  • Method for detecting CHO cell DNA
  • Method for detecting CHO cell DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Embodiment 1. evaluates the performance of the primer pair shown in SEQ ID NO:3 and SEQ ID NO:4

[0137] According to the above "Materials and methods used in the present invention", the primer pair shown in SEQ ID NO:3 and SEQ ID NO:4 was used for PCR detection. Experimental results such as figure 1 and figure 2 shown. in figure 1 is the reference product amplification curve, and the amplification curve shows an obvious exponential growth period. figure 2 is the reference standard curve. Depend on figure 2 It can be seen that when the concentration of the reference substance is 1000pg / μl, 100pg / μl, 10pg / μl, 1pg / μl, 100fg / μl, 10fg / μl, 1fg / μl and 0.1fg / μl, the slope of the standard curve drawn is -3.39 , the correlation coefficient (R2)=0.999, and the amplification efficiency was 97.23%. The standard curve has good linearity, and the detection sensitivity can reach 0.1fg / μl.

Embodiment 2

[0138] Example 2. Evaluation of the specificity of the primer pair shown in SEQ ID NO:3 and SEQ ID NO:4

[0139] In order to evaluate the specificity of the primer pair shown in SEQ ID NO:3 and SEQ ID NO:4, human and Escherichia coli contamination DNA common in production and mouse and rat genes with high homology to hamster cells were selected as system interference experiment. Specifically:

[0140] 1. Human and Escherichia coli contamination DNA system interference experiment

[0141] Add the same concentration of 10pg / μl CHO gene and human gene (human liver cancer cell SK-HEP-1 comes from the cell bank of the Type Culture Preservation Committee of the Chinese Academy of Sciences; extract with takara gene extraction kit) and the same concentration of 10pg in the detection system / μl CHO gene and Escherichia coli gene (E.coil DH5α strain from the Institute of Microbiology, Chinese Academy of Sciences, No. 1.1595, extracted with takara gene extraction reagent) were used as ...

Embodiment 3

[0147] Example 3. Evaluation of the performance of the primer pair shown in SEQ ID NO:5 and SEQ ID NO:6

[0148] According to the method described in Example 1, the performance of the primer pair shown in SEQ ID NO:5 and SEQ ID NO:6 was evaluated.

[0149] Experimental results such as Figure 7 and Figure 8 shown. in Figure 7 is the reference product amplification curve, and the amplification curve shows an obvious exponential growth period. Figure 8 is the reference standard curve. Depend on Figure 8 It can be seen that when the concentration of the reference substance is 1000pg / μl, 100pg / μl, 10pg / μl, 1pg / μl, 0.1pg / μl and 0.01pg / μl, the slope of the standard curve drawn is -3.33, and the correlation coefficient (R2)= 0.997, the amplification efficiency was 99.66%. The standard curve has good linearity, and the detection sensitivity can reach 10fg / μl.

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Abstract

The invention provides a primer pair used for detecting the CHO cell genome DNA, a kit containing the primer pair and a method for detecting the CHO cell genome DNA by using the primer pair, and the primer pair specifically combines to the sections shown in a SEQ ID NO: 1 and a SEQ ID NO: 2 on the CHO cell genome DNA. The PCR detection method which uses the primer pair has the advantages of simple and fast operation, and high sensitivity; can distinguish the interference DNA of escherichia coli or human, even mice or large mice which have homologous cell height with hamster.

Description

technical field [0001] The invention relates to the field of biological detection. Specifically, the present invention relates to a detection method of CHO cell DNA. Background technique [0002] In modern times, biological recombinant products have been widely used in the field of medicine and health, playing an increasingly important role. These biological products include recombinant protein drugs, gene recombinant vaccines, biological antigen antibodies and various cytokines, etc. The application of recombinant biological products is closely related to the cause of human health, and there are extremely strict requirements for its quality control and safety testing in the world. [0003] The vast majority of recombinant biological proteins are produced by large-scale genetic engineering host cells, and the complex non-target products in the cells are the main source of impurities in the final product, which directly affects the safety of biological products. The residu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/12C12Q1/68
CPCC12Q1/68C12Q1/6881C12Q2600/16
Inventor 孙玮洁杨志行吴婉欣文明
Owner HUZHOU SHENKE BIOTECHNOLOGY CO LTD
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