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Kit and detection method for detecting BRAF gene mutation

The technology of a kit, PB-600-M-R2, is applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc. It can solve the problems of large demand for detection samples, low accuracy, and low detection throughput. , to achieve the effect of low demand for detection samples, high accuracy and high detection throughput

Pending Publication Date: 2020-07-17
天津普瑞赛斯分子诊断技术有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sequencing method has low sensitivity (only 20%), which leads to high false negatives, and there are many steps and complicated operations
Fluorescent quantitative PCR method is currently the mainstream detection method, with high sensitivity and simple operation, but there are also shortcomings in the large demand for detection samples, low detection throughput and low accuracy.

Method used

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  • Kit and detection method for detecting BRAF gene mutation
  • Kit and detection method for detecting BRAF gene mutation
  • Kit and detection method for detecting BRAF gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] In this example, the plasmid constructed by genetic engineering was used as the positive plasmid, and the wild-type human genomic DNA was used as the control. BRAF gene mutation and 60 cases of clinical samples were detected by Highly Multiplexed PCR (Highly Multiplexed PCR) and high-precision capillary electrophoresis technology of the present invention, and compared with fluorescent quantitative PCR detection. The specific steps and methods are as follows:

[0060] 1. Test sample processing and DNA extraction:

[0061] (1) Plasmid treatment and extraction: each plasmid was extracted using a plasmid extraction kit, and the specific extraction steps were operated according to the kit instructions. The extracted plasmid was dissolved in Tris-EDTA (10mmol / L, pH 8.0), the quality of the extraction was detected by Nanodrop, and its concentration was determined, and then the concentration of the plasmid was adjusted to different copy numbers with Tris-EDTA (10mmol / L, pH 8.0...

Embodiment 2

[0073] In this example, the plasmid constructed by genetic engineering was used as the positive plasmid, and the wild-type human genomic DNA was used as the control. The BRAF gene mutation and 60 cases of clinical samples were detected by using Highly Multiplexed PCR (Highly Multiplexed PCR) and high-precision capillary electrophoresis technology of the present invention. The specific steps and methods are as follows:

[0074] 1. Test sample processing and DNA extraction:

[0075] (1) Plasmid treatment and extraction: each plasmid was extracted using a plasmid extraction kit, and the specific extraction steps were operated according to the kit instructions. The extracted plasmid was dissolved in Tris-EDTA (10mmol / L, pH 8.0), the quality of the extraction was detected by Nanodrop, and its concentration was determined, and then the concentration of the plasmid was adjusted to different copy numbers with Tris-EDTA (10mmol / L, pH 8.0), and used as PCR Template, take 5 μL for PCR ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a kit and a detection method for detecting BRAF gene mutation. The kit for detecting the BRAF gene mutation comprises a BRAF reaction solution, a BRAF positive control and a BRAF negative control, wherein the BRAF reaction solution consists of primers and a PCR buffer solution, the BRAF positive control is plasmidDNA, and the BRAF negative control is wild-type human genome DNA. The method is high in accuracy, low in cost, good in specificity, low in requirement on a detection sample, high in detection flux, high in accuracy and simple in operation; and the detection speed is high, and the whole detection process can be completed only in 150 minutes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit and a detection method for detecting BRAF gene mutation. Background technique [0002] The vast majority of BRAF mutations are BRAF V600E mutations, which mainly occur in melanoma, colon cancer and thyroid cancer. In patients with colorectal cancer, the mutation rate of BRAF gene is 15%, while the mutation rate of BRAF is 80% in primary melanoma, 68% in metastatic melanoma, and up to 10% in benign moles. 82%. [0003] At present, BRAF gene detection has been listed as a routine examination before treatment for colorectal cancer patients in Europe and the United States. Vemurafenib, a new targeted drug developed for BRAF gene mutation, has a significant effect on patients with positive BRAF gene mutation. Human BRAF gene V600E mutation gene detection has very important guiding significance for guiding the treatment of patients with colon cancer and melanoma. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686
CPCC12Q1/6886C12Q1/686C12Q2600/156
Inventor 王晨光王海瑶纪东华孙翼辰党晓萌
Owner 天津普瑞赛斯分子诊断技术有限责任公司
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