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Kit for detecting mutation of locus L858R of EGFR gene

A site mutation and kit technology, applied in the field of nucleic acid detection, can solve problems such as low sensitivity and difficult DNA content, achieve high sensitivity, optimize the reaction system, and reduce human interference

Inactive Publication Date: 2020-05-29
宁波胤瑞生物医学仪器有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing reagents for detecting EGFR gene mutations based on digital PCR are relatively small, and there are still some shortcomings, including the difficulty of effectively detecting samples with extremely low DNA content, and low sensitivity, etc.

Method used

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  • Kit for detecting mutation of locus L858R of EGFR gene
  • Kit for detecting mutation of locus L858R of EGFR gene
  • Kit for detecting mutation of locus L858R of EGFR gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Design and synthesis of primer pairs and probes for detection of EGFR gene L858R site mutation

[0055] According to the 858th mutation of exon 21 of EGFR (i.e. L858R mutation, c2573T>G), design and synthesize the primer pair A and fluorescent probe B for detecting the L858R site mutation of the EGFR gene based on digital PCR technology; the specific sequence as follows:

[0056] The primer pair A includes an upstream primer and a downstream primer;

[0057] The nucleotide sequence of the upstream primer is: 5'-CACCGCAGCATGTCAAGATCA-3';

[0058] The nucleotide sequence of the downstream primer is: 5'-CTTTGCCTCCTTCTGCATGGTAT-3'

[0059] Fluorescent probe B includes mutant fluorescent probes and wild-type fluorescent probes;

[0060] The nucleotide sequence of the mutant fluorescent probe is: 5'-TTGGGCGGGCCAAAC-3';

[0061] The nucleotide sequence of the wild-type fluorescent probe is: 5'-TTGGGCTGGCCAAAC-3';

[0062] Both the 7th base at the 5' end of the ...

Embodiment 2

[0064] Example 2: A kit for detecting the L858R site mutation of the EGFR gene

[0065] The kit provided by the present invention includes: primer pair A provided in Example 1, fluorescent probe B, positive quality control substance, negative quality control substance and reaction premix.

[0066] The preparation method of the positive quality control product is as follows: artificially synthesize two kinds of DNA fragments with a length of 200 bp containing the wild-type and mutant-type mutation sites respectively, and load them into the plasmid vector pET-23d(+) (Promega). Use Qubit 3.0 for quantification, calculate the copy number concentration of the two types of plasmids, and mix the two plasmids (containing 1% L858R mutant DNA) according to the copy number ratio mutant type: wild type = 1:100, and then beat the plasmid mixture by ultrasound Break into fragments of about 180bp, quantify to 10ng / μL, and use it as a positive quality control.

[0067] The negative quality c...

Embodiment 3

[0070] Embodiment 3: the method for detection EGFR gene L858R site mutation

[0071] Using the kit described in Example 2, set up a PCR reaction system according to Table 1, and set up a PCR reaction system according to ddH 2 O. The order of the reaction master mix, probe, primer, and template DNA, add the above samples into the PCR tube according to the reaction system in Table 1, mix the mixed system for 15 seconds with a gentle vortex, and collect the solution by short-term centrifugation to the bottom of the test tube. Load the prepared reaction system onto the PCR chip to form a micro-reaction unit. Put the chip into a digital PCR instrument, perform PCR reaction according to the PCR reaction conditions in Table 2, and select FAM and CY3 as the channels for fluorescence detection.

[0072] Table 1: Reaction System

[0073]

[0074] Table 2: PCR reaction conditions

[0075]

[0076]

[0077] After the amplification is completed, the effective fluorescent posit...

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Abstract

The invention discloses a kit for detecting mutation of a locus L858R of an EGFR gene and relates to the technical field of nucleic acid detection. The kit comprises a primer pair for detecting the mutation of the locus L858R, a mutant type fluorescent probe, a wild type fluorescent probe, a positive quality control, a negative quality control and a reaction premix. According to the kit, in view of the locus L858R of the EGFR gene, primers of specific sequences and locked nucleic acid probes are designed, a reaction system is optimized, high-sensitivity detection on the mutation of the locus L858R of the EGFR gene is achieved by using a digital PCR platform, and mutations of 1: 3,000 of low-concentration DNA samples can be effectively distinguished; and operations are simple, results are stable, the accuracy is high, the data amplification effect is good, and thus, the kit can be extensively applied to early screening of EGFR mutant types of non-small cell lung cancer and drug resistance monitoring of a therapy process.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection, in particular to a kit for detecting the L858R site mutation of the EGFR gene. Background technique [0002] In recent years, the number of new cancer cases per year has reached 4.29 million, among which lung cancer ranks first in morbidity and mortality, accounting for 13% and 18% of new cases of malignant tumors, although the diagnostic methods and treatment methods of lung cancer are constantly improving. , but the mortality rate of lung cancer has not been effectively controlled. Among all lung cancers, non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for more than 80%. The most common pathogenic mutation in Chinese patients with NSCLC comes from the epidermal growth factor receptor gene (EGFR). ). [0003] Epidermal growth factor receptor (EGFR) is a transmembrane protein widely distributed on the cell membranes of various tissues in the human body, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q2600/156C12Q2600/106C12Q2531/113C12Q2563/107
Inventor 刘一博金鑫浩任鲁风张未来于军
Owner 宁波胤瑞生物医学仪器有限责任公司
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