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Primer pair as well as probe and kit for detecting human BRAF gene mutation

A technology for detecting primers and primer pairs, which is used in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc. problem, to achieve the effect of high sensitivity and fast detection speed

Inactive Publication Date: 2014-09-03
HELIXGEN GUANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the labor intensity is slightly higher, professional technicians are required, and it is not suitable for high-throughput detection and large-scale clinical application
[0009] 3. SSCP, DHPLC, gene chip, liquid phase chip and other methods are complicated to operate and require high equipment. They are only used in laboratories of scientific research institutions and are not suitable for large-scale promotion and use.

Method used

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  • Primer pair as well as probe and kit for detecting human BRAF gene mutation
  • Primer pair as well as probe and kit for detecting human BRAF gene mutation
  • Primer pair as well as probe and kit for detecting human BRAF gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] This example illustrates the detection performance of the present invention by taking BRAF gene V600 wild-type clinical samples and BRAF gene V600 wild-type cell line DNA (enterprise standard product) mixed with mutant plasmids as examples.

[0067] Experiments use BRAF gene V600 wild-type clinical paraffin-embedded tissue samples to verify the specificity of the present invention; respectively verify the repeatability of the present invention with high mutation rate and low mutation rate enterprise standards, and verify the present invention with 1% standard products sensitivity.

[0068] Detection consists of the following steps:

[0069] 1. DNA extraction

[0070] For clinical paraffin-embedded tissue samples, about 4 paraffin sections with a thickness of 6 μm to 10 μm were taken, and genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit or a commercial DNA extraction kit of the same performance. The specific operation steps were according to the kit instru...

Embodiment 2

[0088] Clinical trials were carried out in three tertiary hospitals, and the current gold standard detection method for gene mutations—Sanger method gene sequencing was used as a control to carry out clinical trial research on the kit of the present invention (ie, the kit in Example 1).

[0089] Clinical trials have tested more than a thousand clinical specimens, and the tumor types include thyroid cancer, melanoma and colorectal cancer. The sensitivity (positive coincidence rate) of the kit of the present invention is 97.4%, and the specificity (negative coincidence rate) is 90.6%. The correct rate (total consistency rate) is 93.5%. The Kappa test showed that the consistency of the two methods was excellent. Based on the above analysis, the human BRAF gene mutation detection kit (fluorescent PCR method) of the present invention has a high consistency with the gold standard Sanger gene sequencing method in detecting whether there is a BRAF gene mutation in clinical paraffin-e...

Embodiment 3

[0091] One case of clinical paraffin-embedded thyroid cancer sample collected in January 2013 was used to detect BRAF gene mutation by using the present invention.

[0092] 1. DNA extraction

[0093] Take about 4 paraffin sections with a thickness of 6 μm to 10 μm, and use the QIAamp DNA FFPE Tissue Kit or a commercial DNA extraction kit of the same performance to extract genomic DNA, and the specific operation steps follow the kit instructions. Use a spectrophotometer to measure the concentration of the extracted DNA stock solution, it should be at least 10ng / μL, and the OD 260nm / OD 280nm The ratio should be in the range of 1.6-2.0. Dilute the DNA to 10ng / μL working solution for later use.

[0094] 2. Detection of BRAF gene mutation

[0095] Prepare the sample DNA prepared above for the following reaction system preparation:

[0096] a) Prepare a mixed solution (Mix), and prepare according to the ratio shown in the table below.

[0097]

Volume per reacti...

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Abstract

The invention discloses a primer pair and a probe for detecting a human BRAF gene mutation. The sequences of the primer pair and probe are shown as SEQ ID NO: 1 to SEQ ID NO: 7. The invention also discloses a kit for detecting human BRAF gene mutation and used for detecting T-A base mutation occurring on 1799-postion nucleotide of the BRAF gene exon 15 by a fluorescent PCR. The kit disclosed by the invention has the advantages of simple and quick operation, high specificity and low misdetection rate and is applicable to the auxiliary clinical diagnosis and typing of thyroid cancer, or provides a clinical reference for selecting tyrosine kinase inhibitors of melanoma and colorectal cancer and BRAF gene mutation targeted drugs.

Description

technical field [0001] The invention relates to a biological detection reagent, more specifically, the invention relates to a human BRAF gene mutation detection primer pair and probe and a detection kit using the primer pair and probe. Background technique [0002] BRAF gene, the full name of V-Raf oncogenic homologue B1, is a member of the RAF gene family, encodes serine / threonine protein kinase, is an effector of the oncogene RAS, and is also a MAPK class Linked signal transduction components, involved in multiple physiological processes, involved in the regulation of intracellular cell growth, differentiation and apoptosis. In a variety of human malignant tumors, the continuous activation caused by BRAF gene mutation can cause MEK / ERK signal transduction disorder, resulting in excessive cell proliferation and malignant transformation, such as melanoma, colorectal cancer, and thyroid cancer, all of which have different proportions of BRAF mutation. About 80-90% of BRAF g...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2535/137C12Q2545/101C12Q2531/113
Inventor 曾庆张颖芬张中满
Owner HELIXGEN GUANGZHOU
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