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Rapid detection kit for K-ras gene mutation

A detection kit and rapid technology, applied in the field of molecular biology, can solve the problems of EGFR-TKI curative effect differences, unnecessary, slow detection process, etc., and achieve the effect of rapid detection and determination of K-ras mutation and fast detection speed

Inactive Publication Date: 2019-07-12
邓定平
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Three EGFR-TKIs (gefitinib / Iressa, erlotinib / Tarceva, and Camistat) are currently the main drugs for molecularly targeted therapy in NSCLC, but in different patients EGFR- Efficacy of TKIs varies widely
However, since K-ras contains multiple sensitive mutation sites, the above-mentioned fluorescent PCR method has the problem that it needs to be divided into multiple PCR tubes for detection. Currently, fluorescent PCR detection kits on the market generally use 7 PCR tubes for detection , the analysis process is cumbersome and unnecessary, and the whole detection process is slow, it takes at least 1 day to get the result

Method used

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  • Rapid detection kit for K-ras gene mutation
  • Rapid detection kit for K-ras gene mutation
  • Rapid detection kit for K-ras gene mutation

Examples

Experimental program
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Effect test

preparation example Construction

[0066] Preparation of wild-type and mutant plasmids

[0067] Design a pair of PCR primers at the upstream and downstream of the K-ras mutation site to be detected. The length of the amplified fragment can be within the range of 500bp. Using gDNA as a template, the conventional PCR method is used to amplify this fragment. The fragment is cloned into the sequenced plasmid by means of AT cloning, usually using Invitrogen’s PCR2.1 Topo T vector kit, and the operation is carried out according to the instructions. T vector kits from other manufacturers can also be used, or even the T vector kit prepared by ourselves can be used. Vector plasmid. The positive clone obtained was verified by sequencing to prove the correctness of the sequence, and then the Stratagene company’s Quickchange kit was used to design another genotype primer corresponding to the site, and the positive clone of the mutant type was obtained by the Quickchange method, and the operation According to the instruc...

Embodiment 1

[0073] Example 1 Detection of mutant and wild-type plasmids

[0074] The mutant plasmids and wild-type plasmids of 10 kinds of mutations were used for fluorescent PCR amplification, and the plasmid copy number was 1.0×10 5 , calculate the difference between the two CT, △CT=CT 野生型 – CT 突变型 , the larger the difference, the better the discrimination effect of the kit. The reaction conditions are shown in Table 5.

[0075] Table 6

[0076]

[0077] The results show that the amplification process of the mutant and wild type plasmids has a larger △CT and has a better discrimination effect.

Embodiment 2

[0078] Example 2 The effect of blocking primers

[0079] Using the plasmid and fluorescent PCR method in Example 1, but without adding blocking primers, 10 kinds of mutated mutant plasmids and wild-type plasmids were used for fluorescent PCR amplification, and the plasmid copy number was 1.0×10 5 , calculate the difference between the two CT, △CT=CT 野生型 – CT 突变型 , the larger the difference, the better the discrimination effect of the kit. The reaction conditions are shown in Table 5.

[0080] Table 7

[0081]

[0082] The results show that the addition of blocking primers can effectively prevent the amplification of the wild-type template, improve ΔCT, improve the specificity of the detection kit, and improve the resolution effect.

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Abstract

The invention relates to a rapid detection kit for K-ras gene mutation, and belongs to the technical field of molecular biology. The kit consists of an ARMS upstream primer, a downstream primer, a trigger primer and a fluorescent probe for detection of 10 sensitive mutation sites. The K-ras gene detection kit can detect 10 major mutations simultaneously under a single-tube condition. With design of the trigger primer, a reaction system generates fluorescence when any kind of mutation exists, and the effect of rapid detection and determination of K-Ras mutation can be achieved. The kit has theadvantages of fast detection speed and the sensitivity degree reaching 0.001%.

Description

technical field [0001] The invention relates to a rapid detection kit for K-ras gene mutation, which belongs to the technical field of molecular biology. Background technique [0002] KRAS is a key downstream regulator in the EGFR (epidermal growth factor receptor) signaling pathway, involved in the regulation of cell growth, and plays an important role in the process of carcinogenesis. The KRAS gene product participates in the kinase signaling pathway that controls gene transcription, thereby regulating cell growth and differentiation. Alterations in the KRAS gene cause changes in the KRAS protein, as a result of which the KRAS protein no longer has the ability to cleave GTP molecules and release GTP molecules. Such a change causes this signal conduction path to always be in an "on" state. This "on" signal leads to cell growth and proliferation. Thus overexpression and amplification of KRAS leads to sustained cell proliferation, which is a critical step in tumorigenesi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 不公告发明人
Owner 邓定平
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