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Method for improving diagnosis efficiency of BRAF gene V600E mutation

A gene and mutation site technology, applied in the field of improving the diagnostic efficiency of the BRAF gene V600E mutation, can solve the problems of difficult and effective detection, PCR cannot be extended, and the proportion of tumor mutations cannot be given

Active Publication Date: 2019-01-04
GENOSABER BIOTECH CO LTD +1
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AI Technical Summary

Problems solved by technology

If the mismatch is located 3' of the primer, the PCR will not extend
The ARMS-PCR method can achieve a sensitivity of 1%, which can still meet the detection requirements for tumor tissue samples with a high mutation rate, but for tissue samples with low mutation content, especially liquid biopsy samples that are easy to obtain, such as blood, urine and Saliva samples, difficult to detect effectively
In addition, studies have shown that the proportion of sensitive gene mutations is closely related to the efficacy of targeted drugs, while sequencing and ARMS-PCR methods are both qualitative tests, which can only provide information on the presence or absence of gene mutations, but cannot give the proportion of tumor mutations. Insufficient to judge whether weak mutation positive patients can benefit from targeted therapy

Method used

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  • Method for improving diagnosis efficiency of BRAF gene V600E mutation
  • Method for improving diagnosis efficiency of BRAF gene V600E mutation
  • Method for improving diagnosis efficiency of BRAF gene V600E mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1, the selection of primer

[0076] The present inventor designs the mutation site to be detected on the front primer or the back primer, and the primer (containing a base complementary to the mutation site) comprising the detection site is a specific primer, and its 3' terminal base is compatible with the mutation site. The V600E site mutant is completely complementary, which is the key to distinguish the mutant from the wild-type gene. In this example, the ability of specific primers of different lengths to distinguish mutant genes from wild genes was investigated.

[0077]The V600E site is a total of 20 copies of the mutant plasmid pMUT-V600E, which is incorporated into 2.0×10 4 In the corresponding copy of the wild-type plasmid pWT-V600E, real-time fluorescent PCR detection was performed to calculate the difference between the two Ct values, ΔCt=Ct wild type-Ct mutant type.

[0078] The specific primers of different lengths used in this example, and the...

Embodiment 2

[0094] Embodiment 2, the effect of competitive Block oligonucleotide

[0095] Considering that tissue and blood samples contain a large amount of wild-type DNA in addition to mutant DNA, in order to further improve the specificity of detection, the inventors conducted analysis, comparison and combination experiments to verify the characteristics of the BRAF gene V600E mutation site and adjacent sequences. , to find a suitable competitive Block oligonucleotide. Block oligonucleotides are completely complementary to wild-type genes and partially complementary to mutant genes. In the presence of wild-type genes, Block oligonucleotides can block wild-type genes and prevent wild-type genes from being amplified. false positive.

[0096] A total of 20 copies of the V600E site as a mutant plasmid (pMUT-V600E) were incorporated into 2.0×10 4 In the corresponding wild-type plasmid (pWT-V600E) of the copy, real-time fluorescent PCR detection was performed, and the difference between th...

Embodiment 3

[0105] Embodiment 3, sensitivity

[0106] A total of 20 copies of the V600E site as a mutant plasmid (pMUT-V600E) were incorporated into 2.0×10 4 In the corresponding wild-type plasmid (pWT-V600E) of the copy, the incorporation ratio is shown in Table 9, and real-time fluorescent PCR detection was performed to investigate the sensitivity of the mutation detection system.

[0107] The specific primers, Block oligonucleotides, amplification primers on the other side, probe sequences, amplification reaction system and reaction procedures used in this example are the same as those in Example 2. The results are shown in Table 9.

[0108] Table 9. Sensitivity analysis results

[0109]

[0110] The above results show that, using the mutation detection system of the present invention, when the mutation ratio of the V600E site is 0.01%, there is still a good degree of discrimination from the wild-type plasmid.

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Abstract

The invention relates to a method for improving the diagnosis efficiency of BRAF gene V600E mutation. The inventor optimizes a detection reagent for detecting the BRAF gene V600E-site mutation, and onthis basis, a kit for detecting the BRAF gene V600E-site mutation, application and a detection method are provided.

Description

technical field [0001] The invention belongs to the field of diagnosis, and more specifically, the invention relates to a method for improving the diagnosis efficiency of BRAF gene V600E mutation. Background technique [0002] The BRAF gene, also known as the B1 gene of the carcinogenic homologue of the murine sarcoma filter virus, is one of the most important proto-oncogenes in humans, mainly occurring in melanoma, colon cancer and thyroid cancer. [0003] The vast majority of BRAF gene mutations are concentrated in the 1799th base of exon 15 (T>A), resulting in a change in the encoded amino acid (V600E). This mutation leads to continuous activation of the downstream MEK-ERK signaling pathway, driving the growth, proliferation, invasion and metastasis of tumor cells. The detection of BRAF gene V600E mutation can be used to guide the use of BRAF kinase inhibitors in melanoma patients and the targeted therapy of EGFR antibody in colorectal cancer, and can also be used as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/156C12Q2600/118
Inventor 杨国华吕娟焦晔
Owner GENOSABER BIOTECH CO LTD
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