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Kit for detecting KRAS gene mutation

A kit and multiple detection technology, applied in the fields of biotechnology and tumor diagnosis, can solve the problems of high price, low sensitivity, sequence splicing burden, etc.

Pending Publication Date: 2020-03-24
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] ①Sanger sequencing method: The detection of gene mutations requires the amplification, purification, sequencing, and sequence analysis of the samples to be tested. Detect mutations with an abundance of more than 20%, with many false negatives
[0009] ④High-throughput sequencing method: High-throughput sequencing method is expensive, and the read length is about 30-250bp, which is shorter than Sanger sequencing, which imposes a burden on sequence splicing. The test results still need to be verified by Sanger sequencing, and for the detection of exon and endon Insufficient accuracy of small fragment deletions containing subjunction regions
The high-throughput sequencing method is cumbersome to operate, which limits the sequencing speed

Method used

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  • Kit for detecting KRAS gene mutation
  • Kit for detecting KRAS gene mutation
  • Kit for detecting KRAS gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] The invention provides a method for detecting multiple gene mutations in lung cancer, primers, probes and a kit. The specific implementation steps are as follows:

[0112] (1) Extraction of nucleic acid templates from samples to be tested

[0113] Nucleic acid extraction or purification reagents from Sun Yat-Sen University Daan Gene Co., Ltd. (Tissue samples can use Yuesui Machinery Equipment No. 20170666; ascites and plasma samples can use Yuesui Machinery Equipment Number 20180628), and nucleic acid extraction is performed according to the kit instructions. The nucleic acid after tissue sample extraction is diluted to a concentration of 2-100ng / μL (the concentration of nucleic acid extracted from pleural fluid and plasma is not less than 2ng / μL), and the purity should meet A 260 / A 280 The ratio ranges between 1.7-2.3. The template can be used directly for subsequent experiments or stored at -80°C for future use, avoiding repeated freezing and thawing.

[0114] (2...

Embodiment 2

[0133] Embodiment 2 Sensitivity and accuracy detection

[0134] The sensitivity reference product with a nucleic acid concentration of 2ng / μL is composed of the detection gene locus plasmid DNA or cell line, and the wild-type cell line nucleic acid is mixed in a certain proportion, and the gene mutation rate of the mixture is 1%.

[0135] (1) Sample addition

[0136] Take 3 μL of DNase system and add it to each PCR reaction tube. Centrifuge briefly for 15s.

[0137] Take 5 μL negative quality control substance, sensitivity reference substance, and positive quality control substance respectively, and add them to the reaction tube in sequence according to the order in Table 5. Close the cap of the PCR reaction tube tightly, mix well, and centrifuge briefly for 10s.

[0138] For each sensitivity reference product, 5 repeated experiments.

[0139] (2) Real-time fluorescent PCR amplification:

[0140] Set the real-time fluorescent PCR amplification instrument to detect FAM sig...

Embodiment 3

[0148] Embodiment 3 clinical sample detection

[0149] Extraction of test sample nucleic acid:

[0150] (1) Extraction of nucleic acid templates from clinical samples to be tested

[0151] Collect 100 negative clinical samples, and randomly mix in one case each of G12S mutation, G12R mutation, G12C mutation, G12D mutation, G12V mutation, G12A mutation, G13C mutation, and G13D mutation confirmed by conventional methods. Nucleic acid extraction or purification reagents from Gene Co., Ltd. (Tissue samples can use Yuesui Machinery No. 20170666; ascites and plasma samples can use Yuesui Machinery No. 20180628). Nucleic acid extraction is carried out according to the kit instructions. The nucleic acid is diluted to a concentration of 2-100ng / μL (the concentration of nucleic acid extracted from pleural fluid and plasma is not less than 2ng / μL), and the purity should meet A 260 / A 280 The ratio ranges between 1.7-2.3. The template can be used directly for subsequent experiments or...

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Abstract

The invention provides a kit for detecting KRAS gene mutation. Specifically, the invention discloses a primer for multiplex detection of KRAS gene mutation, a probe, and a kit containing a primer andprobe mixed solution. According to the invention, 8 mutation types of the lung cancer KRAS gene can be simultaneously detected. The method and the kit of the invention have the advantages of simple detection process, multiple detectable mutation types, high sensitivity, small sample dependence and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology and tumor diagnosis, in particular, the invention relates to a kit and a method for multiple detection of KRAS gene mutation. Background technique [0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality, among which non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer patients, and the 5-year survival rate of advanced non-small cell lung cancer patients is about 15%. With the development of technology, more and more attention has been paid to gene mutations in tumors. Non-small cell lung cancer has a high degree of heterogeneity, resulting in extremely diverse clinical manifestations and therapeutic effects. [0003] The KRAS gene is located on chromosome 12 and is a member of the ras gene family. The KRAS gene is a downstream regulatory gene of the EGFR signal transduction pathway. When it is mutated, it can automatically activate the path...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2537/143C12Q2545/113C12Q2563/107
Inventor 蒋析文朱小亚邓洁黄志文钟灵秀
Owner DAAN GENE CO LTD
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