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Kit for detecting mutation of V600E locus of BRAF (v-taf mourine sarcoma viral oncogene homolog B1) gene

A site mutation and kit technology, which is applied in DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem of low sensitivity of detection kits, achieve detection system optimization, reduce false positive reactions, highly specific effect

Pending Publication Date: 2020-06-19
宁波胤瑞生物医学仪器有限责任公司
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  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology describes an improved way to test if someone's cancer cells have certain genes that are associated with other types of malignancy or risk factors such as smoking habits. It involves designing primers and probings specifically designed against these targets while also utilizing a specialized DigitalPCR (Digital Polymerase Chain Reaction) process. By doing this, it allows researchers to accurately determine whether they may develop any inherited diseases like breast cancer caused by changes made during cell division.

Problems solved by technology

This patents discuss how molecular diagnostic tests are currently available but they all suffer from limitations like lacked precision and speed compared to other types of therapies (such as radiotherapy). There is also concern about potential side effects caused by chemopreventative agents given at high dosage levels. Overall, these technical problem addressed in this patented text relating to improving the performance of current molecence assays against advanced stages of breast cancer and metastatic castrate tamponade failure prediction.

Method used

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  • Kit for detecting mutation of V600E locus of BRAF (v-taf mourine sarcoma viral oncogene homolog B1) gene
  • Kit for detecting mutation of V600E locus of BRAF (v-taf mourine sarcoma viral oncogene homolog B1) gene
  • Kit for detecting mutation of V600E locus of BRAF (v-taf mourine sarcoma viral oncogene homolog B1) gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 design and synthesis are used to detect the probe of BRAF gene V600E site mutation, primer

[0029] According to the V600E site mutation on exon 15 of the BRAF gene, the locked nucleic acid probe and primer pair for detecting the V600E site mutation of the BRAF gene based on digital PCR technology of the present invention are designed and synthesized.

[0030] The nucleotide sequences of the locked nucleic acid probes include wild-type fluorescent probes and mutant fluorescent probes.

[0031] Wherein, the nucleotide sequence of the wild-type fluorescent probe is 5'-CATCGAGATTTCACTGTAGC-3', as shown in the sequence table SEQ ID No.1; the 4th position of the 5' end of the wild-type fluorescent probe and 13 bases to lock nucleic acid modified bases; the 5' end of the wild-type fluorescent probe is connected with a fluorescent reporter group FAM, and the 3' end is connected with a quenching group BHQ1.

[0032] Wherein, the nucleotide sequence of the mutant f...

Embodiment 2

[0037] Embodiment 2 detects the method for BRAF gene V600E site mutation

[0038] Include the following steps:

[0039] (1) extract the genomic DNA of the sample to be tested;

[0040](2) Preparation of reaction master mix: PCR Mix composed of dNTP, magnesium ions and hot-start Taq enzyme was purchased from New England Biolabs in the United States, and pyrophosphatase was added at a final concentration of 1 U according to the amount required for the reaction. The final concentration was 0.1% Triton-X-100 and BSA with a final concentration of 5 μg / μL were mixed to obtain a reaction premix; pyrophosphatase was purchased from New England Biolabs in the United States, Triton-X-100 and BSA were purchased from Sigma-Aldrich Shanghai Trading Co., Ltd. (Sigma);

[0041] (3) According to the 20 μL reaction system described in Table 1, add each component in turn to prepare the sample to be tested, and load the sample on the digital PCR chip to form a micro reaction unit. Put the chip...

Embodiment 3

[0046] The detection of embodiment 3 mutation standard substance

[0047] The positive quality control used in the present invention is a standard plasmid solution containing the V600E site mutant BRAF gene; the negative quality control is a plasmid containing the corresponding wild-type gene sequence.

[0048] The preparation method is as follows: separately synthesize the BRAF gene sequence and the wild-type gene sequence DNA containing the V600E site mutation, and respectively insert them into the plasmid vector pET-23d(+) (purchased from Promega). Quantification was performed using Qubit3.0 to calculate the copy number concentration of the two types of plasmids. According to the V600E site mutant genomic DNA: the wild-type genomic DNA ratio is 1:10, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000 and 1:3000 for mixing, and then The resulting mixed plasmids were fragmented into approximately 180 bp of fragmented DNA.

[0049] The 8 mutation standard products obtained according ...

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Abstract

The invention provides a kit for detecting mutation of a V600E locus of a BRAF (v-taf mourine sarcoma viral oncogene homolog B1) gene and belongs to the technical field of biologics. The kit compriseslocked nucleic acid probes and a primer pair, wherein the locked nucleic acid probes are used for detecting mutation of the V600E locus of the BRAF gene; the primer pair is used for detecting mutation of the V600E locus of the BRAF gene; the nucleotide sequences of the locked nucleic acid probe comprise a sequence shown in a sequence table SEQ ID No.1 of a wild florescent probe and a sequence shown a sequence table SEQ ID No.2 of a mutant fluorescent probe; and the nucleotide sequences of the primer pair are sequence in SEQ ID No.3 and SEQ ID No.4. The kit further comprises a reaction premixing liquid, a positive standard product and a negative standard product. The kit provided by the invention is capable of detecting mutation of the V600E locus of the BRAF gene, the sample demand amountis small, the sensitivity and the specificity are high, and the detection efficiency is also improved to a certain extent.

Description

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Claims

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Application Information

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Owner 宁波胤瑞生物医学仪器有限责任公司
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