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Kit for detecting EGFR gene hot spot mutation and detection method

A kit and reagent technology, applied in the field of kits for detection of EGFR gene hotspot mutations, can solve problems such as limiting the application of Sanger sequencing method, and achieve the effects of short operation time, simple operation, and improved sensitivity

Active Publication Date: 2014-01-01
武汉思泰得医学检验实验室有限公司
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Problems solved by technology

[0007] In tumor tissue, EGFR wild-type cells and mutant cells are mixed, and the sensitivity of Sanger sequencing method is only 10-20%, that is, when mutant cells account for 10-20% of the total detected cells, it can be detected, so it is extremely Limits the application of Sanger sequencing

Method used

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  • Kit for detecting EGFR gene hot spot mutation and detection method
  • Kit for detecting EGFR gene hot spot mutation and detection method
  • Kit for detecting EGFR gene hot spot mutation and detection method

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Embodiment 1

[0037] 1. Synthesis of PCR primer sequences, probe sequences and PNA sequences in the kit

[0038] 1.1 According to the requirements of EGFR gene hotspot mutation region and sequencing fragments (see Figure 1 ~ Figure 4 ), design the following four pairs of mutually independent primer sequences and probe sequences, and synthesize them artificially:

[0039] EGFR18F: TTGTCTCTGTGTTCTTGTCCC (SEQ. ID NO. 1)

[0040] EGFR18R:TTCCCAAACACTCAGTGAAAC (SEQ. ID NO. 2)

[0041] EGFR18P:FAM-CCCAGCTTGTGGAGCCTCTTACACCC-BHQ1 (SEQ. ID NO. 9)

[0042] EGFR19F: ACATTATCAGGCTTAGGTGCG (SEQ. ID NO. 3)

[0043] EGFR19R: CAGACAGTAGAAAAGGTGGGC (SEQ. ID NO. 4)

[0044] EGFR19P:FAM-CTCCACAGCCCCAGTGTCCCTCA-BHQ1 (SEQ. ID NO. 10)

[0045] EGFR20F: TCCCTGTGCTAGGTCTTTTG (SEQ. ID NO. 5)

[0046] EGFR20R: TCCCTTCCCTGATTACCTTT (SEQ. ID NO. 6)

[0047] EGFR20P:FAM-CGATCTGCACACACCAGTTGAGC-BHQ1 (SEQ. ID NO. 11)

[0048] EGFR21F: CTTTCATGCGCCAT (SEQ. ID NO. 7)

[0049] EGFR21R: GCCACCTCCTTACTTTGCCCTTTCT (...

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Abstract

Belonging to the field of biotechnology, the invention discloses a kit for detecting EGFR gene hot spot mutation and a detection method. The kit provided by the invention comprises a PNA reagent, and clamp technology is utilized for enclosing a wild type EGFR gene sequence, thereby raising sensitivity of a sanger sequencing method and detection rate of EGFR gene mutation. The detection method provided by the invention is simple and safe, does not require many expensive reagents, and is economical and efficient; meanwhile, the detection method has the advantages of short operation time and can be completed within 4-6 h.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for detecting EGFR gene hotspot mutations and a detection method thereof. Background technique [0002] EGFR (epidermal growth factor receptor), the epidermal growth factor receptor, is normally embedded in the cell membrane on the cell surface and is a membrane protein that plays an important role in the reproduction, growth, repair and survival of tumor cells. [0003] At present, some targeted drugs for clinical treatment of cancer such as monoclonal antibody drugs (Eribtux, Cectibix / Panitumumab), small molecule drugs (Iressa / Gefitinib) , Tarceva / Erlotinib), through blocking the activity of EGFR and inhibiting its phosphorylation and signal transduction, so as to play an anti-tumor effect, and can also increase the anti-tumor efficacy of chemotherapy and radiotherapy. [0004] Iressa and Tarceva are two tumor-targeted therapeutic drugs with high specificity deve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/106C12Q2600/156
Inventor 叶伦李雪梅付金玲陈刚
Owner 武汉思泰得医学检验实验室有限公司
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