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Kit for detecting EGFR gene mutation and detection method thereof

A kit and base technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of easy contamination, positive results can not confirm the specific unknown, etc., to achieve strong specificity, test personnel and environment. No harm, good safety effect

Active Publication Date: 2015-07-08
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can detect both known and unknown mutation sites, and the sensitivity is about 5%. However, the reaction tube needs to be opened during the detection process, which is easy to cause pollution, and the positive result cannot confirm the specific unknown, and finally needs to be confirmed by sequencing

Method used

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  • Kit for detecting EGFR gene mutation and detection method thereof
  • Kit for detecting EGFR gene mutation and detection method thereof
  • Kit for detecting EGFR gene mutation and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The EGFR gene negative quality control product is human serum genomic DNA without EGFR gene mutation, and then diluted to 10 3 copy number. The EGFR gene positive quality control product is connected to the pUC57-Amp vector by the mutation sequence of 29 mutation sites corresponding to the 4 exons of the EGFR gene, EGFR18, EGFR19, EGFR20 and EGFR21. After transformation into Escherichia coli, it is extracted and purified, and finally diluted into Concentration is 10 3 Copy number / μl. The mutation site information comes from the Cosmic database, which are G719A, G719S, G719C, E746_A750del(1), E746_A750del(2), L747_P753>S, E746_T751>I, E746_T751del, E746_T751>A, E746_S752>A, E746_S752 E746_S752>D、L747_A750>P、L747_T751>Q、L747_E749del、L747_T751del、L747_S752del、L747_A750>P、L747_P753>Q、L747_T751>S、L747_T751del、L747_T751>P、T790M、S768I、H773_V774insH、D770_N771insG、V769_D770insASV、L858R、L861Q, Among them, the deletion mutation site on EGFR19 was named 19-del; the insertion mut...

Embodiment 2

[0080] Accurately dilute the 29 mutation-positive quality control products and Control quality control products, and uniformly dilute to 10 3 Copy number, the dilution solution is 10ng / μl wild-type DNA, and then the positive quality control product and the Control quality control product are respectively 0:100, 0.01:99.99, 0.5:99.5 according to the ratio of the positive quality control product and the Control quality control product , 5:95 to prepare sensitivity reference products. Considering that there may be differences caused by adding samples or personal factors during the experiment, we choose to repeat the measurement 3 times, and compare and analyze the mixed sample with 0% each time to test its sensitivity, and analyze the corresponding sensitivity according to the average value in the results interval. According to the results of the minimum detection limit experiment, no amplification occurred in the blank samples of all sites, and the experiment is credible. When...

Embodiment 3

[0086] Collect 100 lung cancer patient tissue samples and matched preoperative plasma samples. The samples are all collected from the First Affiliated Hospital of Zhejiang University School of Medicine. Immediately after sample collection, store in a -80°C refrigerator. The DNA in the tissue samples and the free DNA (cf-DNA) in the plasma were extracted using Qiagen kits, and subjected to agarose gel electrophoresis and concentration determination.

[0087] Then, the method of Example 1 was used to detect mutation sites in the tissue samples and plasma samples of 15 lung cancer patients, and the tissue samples were sequenced at the same time, and the type of mutation was determined according to the sequencing results. The results are shown in Table 9.

[0088] Table 9, adopt the method of the present invention to detect lung cancer patient result

[0089]

[0090] The results show that the coincidence rate of mutations in the plasma samples detected by the method of the pr...

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Abstract

The invention discloses a kit for detecting EGFR gene mutation and a detection method thereof. The kit comprises a specific probe modified with LNA locked nucleic acid. The specific probe aims at an EGFR gene mutation site. The specific probe can be combined with a wild type DNA and can detect a DNA sample contained with 0.01 percent of the EGFR gene mutation. The detecting method has the advantages that the specificity is strong, the sensitivity is high, pollution is small, the operation is simple and quick, security is high and the like. The detection method is especially suitable for detecting the gene mutation from body fluid contained with low-content mutation like blood plasma, urine and saliva and is suitable for conducting early screening and diagnosis on lung cancer, and guidance is provided for individualized medication.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for detecting EGFR gene mutation, which relates to a method for using the kit to detect EGFR gene mutation in non-disease diagnosis. Background technique [0002] Epidermal growth factor receptor (EGFR) is the expression product of the proto-oncogene C-erbB-1 (HER-1), located on the cell membrane, and is a transmembrane receptor tyrosine kinase. EGFR mainly transmits signals to the nucleus through two pathways, one is Ras→Raf→MAPK pathway; the other is PI3K→PKC→IKK pathway. When the signal is transmitted to the nucleus, it will cause an increase in the transcription level of genes in the nucleus, resulting in cell proliferation and transformation. Abnormality of EGFR signaling is the cause of many tumors. [0003] At present, targeted therapy has become an important means of clinical treatment of non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC). Ire...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王弢李宗飞孙爱娟李杰张丽
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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