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Detection kit for human BRAF gene V600E mutation

A detection kit and kit technology, which is applied in the field of kits for human BRAF gene V600E mutation detection, can solve the problems of unsatisfactory detection results of gene polymorphisms, achieve authentic and credible results, simple and objective interpretation methods, and facilitate analysis Effect

Inactive Publication Date: 2015-11-11
WUHAN YZY MEDICAL SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a detection kit for the V600E mutation of the human BRAF gene, so as to solve the problem that the detection effect of the gene polymorphism is not good when the sample size is small in the existing gene polymorphism detection technology. ideal question

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  • Detection kit for human BRAF gene V600E mutation
  • Detection kit for human BRAF gene V600E mutation
  • Detection kit for human BRAF gene V600E mutation

Examples

Experimental program
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Embodiment 1

[0058] The human BRAF gene V600E mutation detection kit is prepared, which specifically includes the following steps:

[0059] 1. Synthetic primers and probe sequences

[0060] Synthetic primer sequence SEQIDNO:1, SEQIDNO:2, internal standard primer sequence SEQIDNO:6 and SEQIDNO:7; synthetic specific probe sequence SEQIDNO:3 and SEQIDNO:5, and label the FAM fluorescent group at the 5' end, 3 The 'end is marked with quenching group NFQ and MGB modification group, the internal standard probe sequence SEQIDNO:8 is synthesized, and the 5' end is labeled with VIC fluorescent group, and the 3' end is marked with quenching group NFQ and MGB modification group group.

[0061] The above primer sequences were respectively prepared into 100 μM mother solution for storage, and the above probe sequences were respectively prepared into 100 μM mother solution for storage.

[0062] 2. Synthetic Locked Nucleic Acid Sequence

[0063] Synthesize the locked nucleic acid primer sequence SEQIDN...

Embodiment 2

[0071] The BRAF gene V600E mutation detection kit prepared in Example 1 was used to detect the samples to be tested.

[0072] In this embodiment, 50 cases of paraffin-embedded tissue sections of thyroid cancer patients diagnosed by clinical pathology were collected, and genomic DNA was extracted therefrom, and the BRAF gene V600E mutation detection kit obtained in Example 1 was used to detect whether there was BRAF gene in the samples to be tested. The V600E mutation is verified by the traditional sequencing method at the same time. The specific operation steps are:

[0073] 1. Extraction of Genomic DNA from Samples

[0074]Genomic DNA was extracted from the tissue samples of the lung cancer patients above using a DNA extraction kit (QIA & DNAFF PET issue Kit, Cat No. 56404). The DNA extraction method was carried out according to the instruction manual, and the operation steps were briefly described as follows: Place the clinically collected paraffin-embedded tissue (≤25mg) i...

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Abstract

The invention provides a detection kit and a detection method for human BRAF gene V600E mutation. The detection kit comprises a first reagent pack and a second reagent pack, wherein each of the first reagent pack and the second reagent pack comprises PCR buffer solution, dNTP, MgCl2, a specific probe, a locked nucleic acid (LNA) primer, an interior label system, HotStart Taq enzyme and UNG enzyme; the first reagent pack comprises a primer pair SEQ ID NO:1 and SEQ ID NO:2, and a probe SEQ ID NO:3; the second reagent pack comprises a primer pair SEQ ID NO:1 and SEQ ID NO:4, and a probe SEQ ID NO:5; and the SEQ ID NO:4 is LNA primer. The detection kit and the detection method can be used for detecting human BRAF gene V600E mutation, and has the advantages of strong specificity, high sensitivity, simplicity and rapidness in operation, high flux, safety, objective result determination and the like.

Description

technical field [0001] The invention relates to a kit for detecting the V600E mutation of the human BRAF gene, in particular to a kit for rapidly and effectively detecting the V600E mutation of the BRAF gene in a small amount of samples. Background technique [0002] The BRAF gene, whose full name is v-Raf mouse sarcoma virus proto-oncogene homolog B1 (v-RafmurinesarcomaviraloncogenehomologB1), was discovered and cloned by IKawa et al. Like ARAF and CRAF, it belongs to the RAF gene family. It is located on human chromosome 7q34, with a size of about 190kb. Its functional coding region consists of 2510 base pairs, and it encodes the serine-threonine protein kinase in the MAPK pathway. This enzyme converts signals from RAS is transduced to MEK1 / 2 to participate in the regulation of various biological events in cells. [0003] BRAF mutations occur in nearly 8% of human tumors, mainly in colorectal cancer, melanoma, and papillary thyroid cancer. According to statistics, about ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q1/6851C12Q1/6883C12Q2600/156C12Q2600/166
Inventor 周鹏飞蔡从利张喆叶婷张浩
Owner WUHAN YZY MEDICAL SCI & TECH
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