Method and kit for sensitively detecting human EGFR (epidermal growth factor receptor) gene mutation on basis of Sanger sequencing

A sensitive detection and kit technology, applied in the field of gene mutation detection, can solve the problems of high detection cost of imported kits, restrictions on clinical promotion and application, and restrictions on clinical application

Active Publication Date: 2016-01-20
FUJIAN MEDICAL UNIV
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Problems solved by technology

The EGFR detection kit developed based on the Scorpions-ARMS method, such as the EGFR detection kit from Qiagen, Germany, can detect 29 common mutations of the EGFR gene at the same time, with high sensitivity (can detect mutations as low as 1%), simple operation, rapid detection, etc. Advantages; however, the detection cost of such imported kits is too high (3000-5000 yuan per specimen) which limits its clinical promotion and application
The Sanger sequencing method is the gold standard

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  • Method and kit for sensitively detecting human EGFR (epidermal growth factor receptor) gene mutation on basis of Sanger sequencing
  • Method and kit for sensitively detecting human EGFR (epidermal growth factor receptor) gene mutation on basis of Sanger sequencing
  • Method and kit for sensitively detecting human EGFR (epidermal growth factor receptor) gene mutation on basis of Sanger sequencing

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Embodiment Construction

[0052] 1. The method for sensitive detection of human EGFR gene mutation based on Sanger sequencing and the principle of the kit

[0053] The Sanger sequencing method is the gold standard for gene diversity / mutation detection. The target gene fragment is amplified by primer pairs targeting the conserved regions at both ends; If the fragment content is less than 10%, it is difficult to accurately identify the fragment by sequencing. Tumor is a somatic disease. Since the somatic mutation component only accounts for a part of clinical samples, and there may be a large number of wild-type genes in addition, conventional Sanger sequencing cannot guarantee the sensitivity of gene mutation detection in such samples. need.

[0054] Aiming at the deficiency of Sanger sequencing method in the detection of EGFR gene mutation, the present invention proposes a connection inhibition method to improve, figure 1 is the schematic diagram of its principle. The present invention takes 29 c...

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Abstract

The invention belongs to the field of gene mutation detection and particularly relates to a method and a kit for sensitively detecting human EGFR (epidermal growth factor receptor) gene mutation on the basis of Sanger sequencing. In order to overcome the defects of a conventional human EGFR gene mutation detection method and kit, the method and the kit for flexibly detecting the human EGFR gene mutation on the basis of the Sanger sequencing are provided; heatproof DNA ligase and a specific connecting primer system aiming at a mutation site are introduced into an EGFR gene segment amplification system, so that amplification of wild type EGFR gene segments in a sample is greatly inhibited, and a wild type sample can be directly determined on the basis of amplified segment bands; or the proportion of mutation segments in a final PCR (polymerase chain reaction) product of a sample with low mutation content is enabled to be higher than 50%, accordingly, the accurate identification of the Sanger sequencing on a heterozygous gene segment system is guaranteed; the method and the kit can detect the EGFR gene mutation with the content as low as 1% in the sample on the basis of the Sanger sequencing, the accuracy rate is very high, the detection cost is low, and new unknown mutation can be possibly detected.

Description

technical field [0001] The invention belongs to the field of gene mutation detection, in particular to a method and a kit for sensitively detecting human EGFR gene mutation by using Sanger sequencing. Background technique [0002] Human epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) is a transmembrane glycoprotein with tyrosine kinase activity encoded by the proto-oncogene C-erbB-1 (HER-1), widely expressed in most normal epithelial cells middle. However, in many solid tumors (such as non-small cell lung cancer), there is often high or abnormal expression of EGFR, which is related to the inhibition of tumor cell proliferation, angiogenesis, invasion, metastasis and apoptosis, so EGFR has become a relevant tumor. (especially non-small cell lung cancer) targeted therapy is an important target. EGFR-targeted drugs currently used in clinical practice include: EGFR tyrosine kinase inhibitors (TKIs, such as gefitinib (Iressa) and erlotinib (Tarceva), a...

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6886C12Q2600/156C12Q2600/158C12Q2535/101
Inventor 黄志清施纯玫郑建伟陈强翁芳霞王劲
Owner FUJIAN MEDICAL UNIV
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