Droplet PCR (Polymerase Chain Reaction) kit for detecting mutation of human KRAS gene

A kit and human detection technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of difficult acquisition of tissue samples, and achieve dynamic tracking, convenient and accurate analysis of experimental data The effect of sensitivity

Inactive Publication Date: 2017-02-22
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

At present, the detection of K-ras gene mutation status generally uses tissue, but in clinical application, tissue samples are not easy to obtain, for example, for patients in the advanced stage, elderly patients, patients with importa

Method used

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  • Droplet PCR (Polymerase Chain Reaction) kit for detecting mutation of human KRAS gene
  • Droplet PCR (Polymerase Chain Reaction) kit for detecting mutation of human KRAS gene
  • Droplet PCR (Polymerase Chain Reaction) kit for detecting mutation of human KRAS gene

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0094] Example 1

[0095] A droplet PCR kit for detecting human KRAS gene mutation, its composition is shown in Table 7:

[0096] Table 7:

[0097]

[0098] The packaging and contents of each component of the droplet PCR kit in this example are shown in Table 8:

[0099] Table 8:

[0100]

[0101]

Example Embodiment

[0102] Example 2: Linear detection of samples of the kit of the application

[0103] 1. Prepare line quality control products

[0104] The K-ras gene G12D mutant plasmid DNA (SEQ ID NO: 12) and the K-ras negative HT-1080 cell line genomic DNA were mixed in proportion, the total genomic DNA concentration was 5000 copies / μl, the mutation ratio was 27%, the above sample As the sample line quality control product L1 of the kit of Example 1 of this application, the L1 sample was serially diluted with 5000 copies / μl of K-ras negative HT-1080 cell line genomic DNA in a 3-fold gradient to obtain the line properties. Control products L2, L3 and L4 are made into linear control products. The details are shown in Table 9:

[0105] Table 9:

[0106]

[0107] 2. Preparation of amplification reagents: Take out the KRAS (exon18) ddPCR reaction solution from the kit, melt and mix at room temperature, centrifuge at 2000 rpm for 10 seconds, and prepare the PCR master mix for each test as follows: 4μl ...

Example Embodiment

[0125] Example 3: Accuracy detection of the kit of this application

[0126] 1. Prepare accurate property control products

[0127] Select the qualified K-ras-positive cell line genomic DNA and mutant plasmid DNA, and mix them in proportion with the K-ras-negative HT-1080 cell line genomic DNA to form the accurate property control product of the kit of Example 1 of the application. The details are shown in Table 11:

[0128] Table 11:

[0129]

[0130]

[0131] 2. Other steps are the same as in embodiment 2;

[0132] The test results obtained are shown in Table 12:

[0133] Table 12:

[0134]

[0135] It can be seen from the experimental results in Table 12 that the positive coincidence rate of the test results for each accurate property control substance in the corresponding reaction solution is 100%.

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Abstract

The application relates to the field of the detection of gene mutation, and particularly speaking, relates to a droplet PCR (Polymerase Chain Reaction) kit for detecting the mutation of a human KRAS gene. The kit comprises a nucleic acid amplification reagent and a reference substance, wherein KRASddPCR reaction liquid contains primers and a probe which are used for detecting the mutation of No. 12 and No. 13 codon. A digital PCR technique is adopted for the kit provided by the application; quantitative detection is carried out on the mutation states of the No. 12 and No. 13 codon of a K-ras gene of a human being; the droplet PCR kit can be used for detecting the mutation states of the K-ras genes in free DNA (Deoxyribonucleic Acid) in paraffin-embedded pathological section tissues or the peripheral blood of patients suffered from colorectal cancer, lung cancer and the like; a reference is provided for a clinical doctor to select a tumor-targeted medicine for treatment and monitoring; the droplet PCR kit has absolute quantification, 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 KRAS 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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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2600/166C12Q2545/101C12Q2563/107C12Q2545/113C12Q2535/131C12Q2531/113
Inventor 陶慧卿李云航谢立群陈嘉铮孙彦波童光铨徐任高峰英杨建清
Owner 上海睿昂基因科技股份有限公司
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