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Fluorescence quantitative detection primers and probe for BRAF gene V600E mutation

A technology of DNA probes and probes, applied in the field of biomedicine, can solve the problems of unnecessary use, high sequencing cost, and long experiment cycle, and achieve the effects of reducing false positives, short experiment cycle, and low cost

Inactive Publication Date: 2017-08-25
MYGENOSTICS (CHONGQING) GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are unavoidable shortcomings such as high sequencing costs and long experimental cycles, which also lead to the fact that sequencing technology is generally not used for primary screening, but as a means of confirmation after primary screening

Method used

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  • Fluorescence quantitative detection primers and probe for BRAF gene V600E mutation
  • Fluorescence quantitative detection primers and probe for BRAF gene V600E mutation
  • Fluorescence quantitative detection primers and probe for BRAF gene V600E mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the design and selection of primers and probes for detecting BRAF gene V600E mutation

[0034] The design principles of the primers and probes used to detect the BRAF gene V600E mutation in the present invention are as follows: a specific fluorescent probe is added while adding a pair of primers during PCR amplification, and a reporter fluorescent group is marked at both ends of the probe. group and a quencher fluorophore. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme degrades the probe, so that the reporter fluorescent group and the quencher group The fluorescent group is separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, every time a DNA strand is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescent signal is completely synchronized with t...

Embodiment 2

[0047] Embodiment 2, the test of experimental reaction system and reaction condition

[0048] The DNA extracted from tissue samples and FFPE paraffin section samples (NGS was positive for BRAF V600E), as well as the fluorescence quantitative kit KAPA PROBE FAST qPCR Kits (KAPA BIOSYSTEMS, #KK4703) were used for the experiment. The size of the experimental system was determined according to the kit recommendation as 20 μl.

[0049] In the case of the same experimental environment and other conditions, the concentration of forward and reverse primers in the experimental system was optimized and tested, and finally it was determined that the final concentration of 500nM can achieve the best detection effect.

[0050] Under the same experimental environment and other conditions, the concentration of fluorescent probes in the experimental system was optimized and tested, and it was finally determined that the final concentration of 500nM can achieve the best detection effect.

[0...

Embodiment 3

[0056] Embodiment 3, determination of standard sample detection lower limit

[0057] For different types of standard samples (Horizin BRAF V600E cfDNA Reference StandardSet and Horizon BRAF V600E FFPE Reference Standard), use standard positive samples and negative samples to configure positive samples with different mutation frequencies, and then perform detection limit experiments. The specific experimental method steps are the same as in Example 2.

[0058] See the experimental results figure 1 , figure 2 with image 3 . It can be seen that the present invention can detect the lowest 50ng of DNA and the lowest mutation rate of 0.5%, and the final fluorescence curve Ct value is less than or equal to 36. It shows that the primers and probes provided by the present invention have good sensitivity.

[0059] Mykino (Chongqing) Gene Technology Co., Ltd.

[0060] Primers and probes for fluorescent quantitative detection of BRAF gene V600E mutation

[0061] GNCLN171097

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Abstract

The invention discloses fluorescence quantitative detection primers and a probe for BRAF gene V600E mutation. The fluorescence quantitative detection primers for BRAF gene V600E mutation are primer 1 and primer 2, the primer 1 is a single stranded DNA shown in SEQ ID NO. 1 in a sequence listing, and the primer 2 is a single stranded DNA shown in SEQ ID NO. 2 in the sequence listing; the probe is the single stranded DNA probe shown in SEQ ID NO. 3 in the sequence listing. For tissue sample DNA and FFPE paraffin sections, the fluorescence quantitative detection primers and the probe for BRAF gene V600E mutation can perform detection with a minimum 50 ng DNA, a minimum mutation rate of 0.5% and a final fluorescence curve Ct value being less than or equal to 36, indicating that the primer pair and the probe provided by the invention have good sensitivity. The fluorescence quantitative detection primers and the probe for BRAF gene V600E mutation are significant for screening BRAF gene V600E mutant-associated cancers.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a fluorescent quantitative detection primer and probe for BRAF gene V600E mutation. Background technique [0002] The BRAF gene is located on chromosome arm 7q34. It encodes B-raf, a serine-threonine kinase that is part of the Ras-Raf-Mek-Erk-MAPK (mitogen-activated protein kinase) signaling cascade. Activation of this pathway is involved in promoting the growth, proliferation and differentiation of cells. [0003] There are three functional RAF proteins in humans, ARAF, BRAF and CRAF. Among them, BRAF has the highest basal kinase activity and is the most potent activator of the MAPK pathway. [0004] The BRAF gene consists of 18 exons, and the most common activating mutation is found in exon 15 at nucleotide position 1799, involving the conversion of thymine to adenine. This resulted in the substitution of glutamic acid for valine at position 600, which was designated V600E. The mut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2563/107C12Q2545/114
Inventor 伍建姬晓雯冯越任爽华
Owner MYGENOSTICS (CHONGQING) GENE TECH CO LTD
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