PCR detection primers for seven different species of cells and detection method and use thereof

A technology for detecting primers and cells, applied in the field of cell detection, can solve the problems of high cost, narrow species range, time-consuming and labor-intensive, etc., and achieve high sensitivity, strong specificity, and broad application prospects

Inactive Publication Date: 2016-10-26
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used detection methods in the laboratory mainly include chromosome analysis, STR analysis, and isoenzyme map analysis. These methods are costly, time-consuming and laborious, and the range of species that can be identified is narrow.

Method used

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  • PCR detection primers for seven different species of cells and detection method and use thereof
  • PCR detection primers for seven different species of cells and detection method and use thereof
  • PCR detection primers for seven different species of cells and detection method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Primers designed for PCR detection of cells (MDBK, MDCK, BHK21, ST, SP20, 293, VERO) derived from seven different species

[0051] Mitochondrial DNA (mtDNA) is often used as the target gene in PCR detection, and the inventors analyzed and confirmed that the cytochrome b (Cytb) gene in mitochondrial DNA is more suitable as the target gene for species identification. This is because the full length of the Cytb gene is about 1100-1200bp, and there is no significant difference in sequence length between species, but there is a bias in nucleotide composition and there are differences between species. The coding sequence region of Cytb gene evolves slowly, is relatively conserved, and exists in all mammals.

[0052] Based on the above ideas, the inventor retrieved the nucleotide sequences of the cytochrome b (Cytb) gene of seven different species of cells (MDBK, MDCK, BHK21, ST, SP20, 293, VERO) on Genbank, and used bioinformatics The method compares the nucleotid...

Embodiment 2

[0060] Embodiment 2, establish the PCR detection method of the cell (MDBK, MDCK, BHK21, ST, SP20, 293, VERO) of seven kinds of different species sources

[0061] The present invention uses the primers in Example 1 to carry out PCR detection on seven different species-derived cells (MDBK, MDCK, BHK21, ST, SP20, 293, VERO), including the following steps:

[0062] 1) Extract the genomic DNA of the cells to be tested

[0063] Seven kinds of cells, MDBK, MDCK, BHK21, ST, SP20, 293 and VERO, were selected from the laboratory to extract genomic DNA. The specific steps are as follows:

[0064] (1) Take two tubes of cryopreserved MDBK, MDCK, BHK21, ST, SP20, 293 and VERO 7 kinds of cells, thaw the cryopreserved tubes in warm water at 37°C, and collect the cells in 1.5mL centrifuge tubes, 8000rmp Centrifuge for 10min, discard the supernatant, and resuspend the cells with 1mL PBS;

[0065] (2) Take 200 μL of the resuspended cells into a new 1.5 mL centrifuge tube, add 1 mL of DNAzol Ly...

Embodiment 3

[0072] Embodiment 3, the specific detection of primer and detection method of the present invention

[0073] Using the genomic DNA of cells from seven different species (MDBK, MDCK, BHK21, ST, SP20, 293, VERO) at a concentration of 10 ng / μL as templates, the seven templates were tested with the seven pairs of primers designed in Example 1, respectively. PCR amplification to detect the specificity of each pair of primers. The PCR reaction system and PCR reaction procedure were the same as those in Example 2. After the PCR reaction was completed, the PCR products were detected by 1.5% agarose gel electrophoresis, and the loading volume was 10 μL.

[0074] The result is as Figure 2-Figure 8 Shown:

[0075] figure 2 It is the specific detection result of the bovine-derived primer pair (C-F and C-R) on 7 kinds of cellular genomic DNA templates. The results show that the bovine-derived specific primers only amplify a 932bp fragment on the DNA template of bovine-derived MDBK cel...

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Abstract

The invention discloses PCR detection primers for seven different species of cells such as MDBK, MDCK, BHK21, ST, SP20, 293 and VERO commonly used in a laboratory and a detection method and use thereof. The detection primers comprise seven pairs of primers, each pair of the primers only produce specific amplification effects on a cytochrome b (Cytb) gene fragment of the corresponding specie of cells so that the primers can synchronously detect seven different species of cells, have high specificity, realize identification of specie sources (ie. cell source reliability) of cells commonly used in a laboratory, realize determination of cross contamination of different species of cells in cell culture and provides a scientific basis for cell culture and subsequent experiment smooth implementation. The PCR detection primers have the advantages of good specificity, high sensitivity, fastness and simpleness.

Description

technical field [0001] The invention belongs to the field of cell detection, in particular to PCR detection primers and detection methods for seven kinds of cells from different species (MDBK, MDCK, BHK21, ST, SP20, 293, VERO) commonly used in laboratories, and is applicable to the identification of cell sources and detection of cross-contamination between cells. Background technique [0002] Animal cells cultured in vitro are currently one of the most widely used and most important experimental tools in the fields of medicine and life sciences. To determine the reliability of the source of cells and whether there is cross-contamination of cells of different species during the culture process is crucial to the success of the experiment. important. At present, the commonly used detection methods in the laboratory mainly include chromosome analysis, STR analysis, and isoenzyme map analysis. These methods are costly, time-consuming and laborious, and the range of species that ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6888C12Q2600/16
Inventor 韩志玲郝鹏陈光达商晓桂闫聪陈君彦张贵刚范秀丽刘国英魏学峰
Owner JINYUBAOLING BIO PHARMA CO LTD
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