PCR micro droplet kit for examining human EGFR gene mutation

A kit and human detection technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of wrongly killing normal cells and lack of specificity, and achieve high sensitivity, high accuracy, Operation without invasive effect

Inactive Publication Date: 2017-01-11
上海睿昂基因科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In tumor treatment, traditional chemotherapy mainly targets the DNA of cells, which often lacks specificity. While killing tumor cells, it also "wrongly kills" many normal cells.

Method used

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  • PCR micro droplet kit for examining human EGFR gene mutation
  • PCR micro droplet kit for examining human EGFR gene mutation
  • PCR micro droplet kit for examining human EGFR gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] A microdroplet PCR kit for detecting human EGFR gene mutation, its composition is as shown in Table 7:

[0110] Table 7:

[0111]

[0112]

[0113] The packaging and content of each component of the droplet PCR kit are shown in Table 8:

[0114] Table 8:

[0115]

[0116]

Embodiment 2

[0117] Example 2: Sample linearity detection of the kit of the present application

[0118] 1. Preparation of linear quality control products for EGFR (exon18) ddPCR reaction solution

[0119] Select EGFR gene mutant plasmid DNA and EGFR-negative HT-1080 cell line genomic DNA, mix in a certain proportion, the total genomic DNA concentration is 5000 copies / μl, and the mutation ratio is 27%. Quality control product L1, and then use 5000 copies / μl of EGFR-negative HT-1080 cell line genomic DNA to serially dilute the L1 sample with a 3-fold gradient to obtain linear quality control products L2, L3 and L4, respectively, to make a linear quality control products, as shown in Table 9.

[0120] Table 9:

[0121]

[0122] 2. Preparation of amplification reagents: Take out the EGFR (exon18) ddPCR reaction solution from the kit, melt and mix at room temperature, centrifuge at 2000rpm for 10s, and prepare the PCR master mix for each test: 4μl ddPCR+10μl ddPCR MIX3. A good PCR master...

Embodiment 3

[0142] Example 3: Detection of the accuracy of the kit of the present application

[0143] 1. Preparation of accurate quality control products

[0144] Select qualified EGFR-positive cell line genomic DNA and mutant plasmid DNA, and mix with EGFR-negative HT-1080 cell line genomic DNA in proportion to form the accurate quality control product of the kit of Example 1 of the present application. Among them, the DNA of the cell line was a commercially available product and was verified to be correct by sequencing. Specifically as shown in Table 11:

[0145] Table 11:

[0146]

[0147] 2, other steps are with embodiment 2;

[0148] The obtained experimental results are shown in Table 12:

[0149] Table 12:

[0150]

[0151]

[0152] From the experimental results in Table 12, it can be seen that each accurate quality control product in this application is in the corresponding reaction solution, and the positive coincidence rate of the test results is 100%.

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Abstract

The application relates to the examining field of gene mutation and particularly relates to a PCR micro droplet kit for examining human EGFR gene mutation. The kit includes a nucleic acid amplification reagent; a reference substance and micro droplet PCR generated oil. The nucleic acid amplification reagent includes a primer and a probe for examining the No. 18 extron mutant, a primer and a probe for examining the No. 19 extron mutant, a primer and a probe for examining the No. 20 extron mutant, and a primer and a probe for examining the No. 21 extron mutant. The present application can be used for examining the seven species of the No. 18, 19, 20, and 21 extron mutants for the EGFR genes in the DNAs extracted from the paraffin-embedded pathology tissue slices and the peripheral blood dissociate DNAs. The present application provides a reference for the patients to choose the tumor molecularly targeted drug remedy and monitor. The present invention has absolute ration, high specificity, accuracy and high sensitivity.

Description

technical field [0001] The application relates to the field of gene mutation detection, in particular to a microdroplet PCR kit for detecting human EGFR gene mutation. Background technique [0002] In tumor treatment, traditional chemotherapy mainly targets the DNA of cells, which often lacks specificity. While killing tumor cells, it also "wrongly kills" many normal cells. Targeted therapy is completely different from previous chemotherapy. There are many mechanisms involved in the pathogenesis of tumors, and these mechanisms are often unique to tumor cells. Targeted therapy is aimed at a specific signal transduction pathway or a certain pathogenesis in the pathogenesis of tumors, using monoclonal antibodies and small molecular substances to interrupt it, so as to achieve the purpose of treating tumors, but has a slight effect on normal cells. Targeted drugs intervene in the gene signaling pathways that control the growth and proliferation of tumor cells by binding to the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/106C12Q2600/118C12Q2600/136C12Q2600/156C12Q2600/166C12Q2545/101C12Q2563/107C12Q2545/113C12Q2535/131C12Q2531/113C12Q2563/159
Inventor 陶慧卿李云航谢立群陈嘉铮孙彦波童光铨徐任陈伟赵丹
Owner 上海睿昂基因科技股份有限公司
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