Kit and method for detecting KRAS genetic mutation

A kit and gene technology, applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve the problems of complex operation, difficult clinical diagnosis products, low sensitivity, etc., and achieve detection sensitivity and specificity High, avoid cross-contamination, low reagent cost effect

Active Publication Date: 2012-01-04
HELIXGEN GUANGZHOU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sequencing is currently considered the gold standard method for detecting gene mutations because it can read the change of each base in DNA. However, due to its low sensitivity, the false negative rate is high when detecting clinical tumor tissue samples with high heterogeneity. At the same time, Sequencing steps are cumbersome, time-consuming, and have high requirements for equipment and operators, making it difficult to form clinical diagnostic products with standardized operations
Methods such as SSCP, DHPLC, gene chips, and liquid phase chips are currently only used in laboratories o

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  • Kit and method for detecting KRAS genetic mutation
  • Kit and method for detecting KRAS genetic mutation
  • Kit and method for detecting KRAS genetic mutation

Examples

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Example Embodiment

[0027] Example 1. Detection of the G12D (GGT>GAT) mutation at codon 12 of the KRAS gene in clinically collected colon cancer tumor tissue samples

[0028] In this embodiment, the method of the present invention is used to detect whether the gene of colon cancer tumor tissue sample is mutated at codon 12. The experimental plan is as follows:

[0029]1. Sample processing and genomic DNA extraction: accept colon cancer tumor tissue samples from clinical surgical resection, cut the tumor tissue into small pieces, and perform genomic DNA extraction according to the instructions of the QIAamp? DNA Mini Kit (Qiajie Biotechnology (Shanghai) Co., Ltd.) extract. Adjust the DNA concentration to 4 ng / μL and store at -20°C for later use.

[0030] 2. Specific amplification of sample DNA KRAS gene (LightCycler 480 real-time fluorescent quantitative PCR instrument of Roche Diagnostic Products (Shanghai) Co., Ltd.):

[0031] Preparation of reaction system:

[0032] ingredient...

Example Embodiment

[0043] Example 2. Detection of KRAS gene mutation at codon 13 G13D (GGC>GAC) in paraffin tissue samples of rectal cancer collected clinically

[0044] In this embodiment, the method of the present invention is used to detect whether the gene of the rectal cancer tumor tissue sample is mutated at codon 13. The experimental plan is as follows:

[0045] 1. Sample processing and genomic DNA extraction: accept paraffin tissue samples of rectal cancer from the clinic, cut the samples into 5 μm, and perform genomic DNA extraction according to the instructions of the QIAamp? DNA FFPE Tissue Kit (Qiagen Biotechnology (Shanghai) Co., Ltd.) . Adjust the DNA concentration to 2 ng / μl and store at -20°C for later use.

[0046] 2. Specific amplification of sample DNA KRAS gene (LightCycler 480 real-time fluorescent quantitative PCR instrument of Roche Diagnostic Products (Shanghai) Co., Ltd.):

[0047] Preparation of reaction system:

[0048] ingredients volume 2...

Example Embodiment

[0059] Example 3. Detection of KRAS gene mutation at codon 12 G12C (GGT>TGT) in clinically collected non-small cell lung cancer tumor tissue samples

[0060] In this embodiment, the method of the present invention is used to detect whether a mutation occurs at codon 13 in a tumor tissue sample of non-small cell lung cancer. The experimental plan is as follows:

[0061] 1. Sample processing and genomic DNA extraction: accept paraffin tissue samples from clinical non-small cell lung cancer, cut the samples into 5 μm, and perform genome analysis according to the instructions of QIAamp? DNA FFPE Tissue Kit (Qiaagen Biotechnology (Shanghai) Co., Ltd.) DNA extraction. Adjust the DNA concentration to 5 ng / μL and store at -20°C for later use.

[0062] 2. Specific amplification of sample DNA KRAS gene (LightCycler 480 real-time fluorescent quantitative PCR instrument of Roche Diagnostic Products (Shanghai) Co., Ltd.):

[0063] Preparation of reaction system:

[0064] ...

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Abstract

The invention provides a kit for detecting the mutation of codon 12 and/or codon 13 of a second extron of a KRAS gene. The kit comprises detection reaction liquid and a prime pair which is shown as SEQ ID NO. 1 and SEQ ID NO. 2. The invention also provides a method for detecting the mutation of the codon 12 and/or the codon 13 of the second extron of the KRAS gene. The method comprises the following steps of: (a) extracting deoxyribonucleic acid (DNA) of a genome of a sample to be detected; (b) performing polymerase chain reaction (PCR) amplification on the extracted DNA of the genome, wherein the prime pair shown as SEQ ID NO. 1 and SEQ ID NO. 2 is used during the amplification; and (c) performing high-resolution melting (HRM) analysis on an amplification product. The method is simple, efficient and quick, is low in cost and is suitable to be applied to clinical units.

Description

technical field [0001] The present invention relates to a test kit for detecting KRAS gene mutations and a detection method thereof, more specifically, the present invention relates to a test kit for detecting KRAS gene exon 2 codon 12 and / or codon 13 mutations and its detection method. Background technique [0002] Gene-guided individualized therapy is a major trend in clinical medicine and drug development in recent years. KRAS gene mutation is currently a relatively certain predictor of epidermal growth factor receptor (EGFR)-targeted drugs. Studies have shown that the status of KRAS gene mutation in tumor patients is closely related to the efficacy of EGFR-targeted drugs in the treatment of colon, rectal cancer, and non-small cell lung cancer. When the patient's tumor has KRAS gene mutation, the efficacy of targeted drugs is not good. KRAS gene status should be detected before treatment with EGFR-targeted drugs in patients with colorectal cancer and non-small cell lung...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 曾庆邹鸿志吴岚晓张颖芬
Owner HELIXGEN GUANGZHOU
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