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A primer, probe, locked nucleic acid probe, kit and detection method for detecting c-kit gene mutation

A technology of locked nucleic acid probes and kits, used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as low sensitivity, achieve high sensitivity, improve sensitivity, and good recognition ability Effect

Active Publication Date: 2016-09-21
WUHAN BIOTECH GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem of low sensitivity of the method for detecting C-kit gene mutation in the prior art, the embodiment of the present invention provides a primer, probe, locked nucleic acid probe, kit and kit for detecting C-kit gene mutation. Detection method

Method used

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  • A primer, probe, locked nucleic acid probe, kit and detection method for detecting c-kit gene mutation
  • A primer, probe, locked nucleic acid probe, kit and detection method for detecting c-kit gene mutation
  • A primer, probe, locked nucleic acid probe, kit and detection method for detecting c-kit gene mutation

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Embodiment 1

[0052] The embodiment of the present invention provides a kind of primer and probe for detecting C-kit gene mutation, the primer and probe for detecting C-kit gene mutation include the No. 9 exon for detecting C-kit gene Primers and probes for exon 9 of C-kit gene, primers and probes for exon 11 of C-kit gene for detection of exon 11 of C-kit gene, for detection of C-kit Primers and probes of the No. 13 exon of the C-kit gene of the No. 13 exon of the gene, and primers and probes of the No. 17 exon of the C-kit gene used to detect the No. 17 exon of the C-kit gene probe, where

[0053] The forward primer of exon 9 of the C-kit gene is shown in SEQ ID NO.1 in the sequence listing;

[0054] The reverse primer of exon 9 of the C-kit gene is shown in SEQ ID NO.2 in the sequence listing;

[0055] The probe of exon 9 of the C-kit gene is shown in SEQ ID NO.3 in the sequence listing;

[0056] The forward primer of exon 11 of the C-kit gene is shown in SEQ ID NO.4 in the sequence l...

Embodiment 2

[0068] The embodiment of the present invention provides a locked nucleic acid probe for detecting C-kit gene mutation, the 3' end of the locked nucleic acid probe is connected with a PO4 group, and the locked nucleic acid probe includes:

[0069] Locked nucleic acid probe of C-kit gene No. 9 exon mutation type 502-503ins: 5'-GTTAA+AA+TA+G+GCA-3'-PO4 (shown in SEQ ID NO.13 in the sequence listing) ;

[0070] Locked nucleic acid probe of C-kit gene No. 11 exon mutation type 557-558del: 5'-AACC+TT+C+CA+C+TG-3'-PO4 (SEQ ID NO.14 in the sequence listing Show);

[0071] A locked nucleic acid probe whose mutation type is K642E in exon 13 of the C-kit gene: 5'-GGAC+T+TT+GA+GTTC-3'-PO4 (shown in SEQ ID NO.15 in the sequence listing);

[0072] Locked nucleic acid probe of C-kit gene No. 13 exon mutation type V654A: 5'-GATTC+A+CA+AT+ATT-3'-PO4 (shown in SEQ ID NO.16 in the sequence listing);

[0073] Locked nucleic acid probe of C-kit gene No. 17 exon mutation type D816V: 5'-CTTG+A+TG...

Embodiment 3

[0077] The embodiment of the present invention provides a kit for detecting C-kit gene mutation, the kit includes: the primers and probes provided in the first embodiment of the present invention, the locked nucleic acid probe provided in the second embodiment of the present invention, and the PCR reaction Solution, positive quality control, negative quality control, sequencing reaction solution, digestive enzyme system and sequencing PCR product purification reagents.

[0078] Specifically, in the kit, 0.75 μl each of 10 μmol / L forward primers, 0.75 μl each of 10 μmol / L reverse primers, 0.5 μl each of 10 μmol / L probes, and 0.5 μl each of 10 μmol / L locked nucleic acid probes.

[0079] Specifically, the PCR reaction solution includes: containing Mg 2+ 5μl of 5×PCR buffer, 1.5μl of 2.5mmol / L dNTPs, 0.2μl of 5U / μl Taq enzyme and 2μl of 20ng / μl template DNA.

[0080] Specifically, the digestive enzyme system includes alkaline phosphatase and exonuclease I, and the enzyme activity...

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Abstract

The invention discloses a primer, a probe, a locked nucleic acid probe, a kit and a detection method for detecting C-kit gene mutation, belonging to the technical field of biology. The primer and probe for detecting C-kit gene mutation comprise at least one of a primer and probe of a C-kit gene No.9 exon for detecting a No.9 exon of a C-kit gene, a primer and probe of a C-kit gene No.11 exon for detecting a No.11 exon of the C-kit gene, a primer and probe of a C-kit gene No.13 exon for detecting a No.13 exon of the C-kit gene, and a primer and probe of a C-kit gene No.17 exon for detecting a No.17 exon of the C-kit gene. The kit comprises the primer and the probe. The primer and probe can be used for highly-sensitively detecting whether the C-kit gene has mutation, meanwhile, the sensitivity of detecting the C-kit gene mutation can be greatly improved by adopting the locked nucleic acid probe, and whether the C-kit gene is mutated can be accurately detected by adopting the kit.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer, a probe, a locked nucleic acid probe, a kit and a detection method for detecting C-kit gene mutation. Background technique [0002] The proto-oncogene C-kit is located on human chromosome 4q11-12, and the C-kit gene contains 21 exons and 20 introns. The mutation of C-kit gene can stimulate the continuous proliferation of tumor cells and the uncontrolled anti-apoptotic signal. The mutation of C-kit gene contributes to the malignant growth of tumor. The mutation of C-kit gene has many forms, such as base pair loss , point mutations, and occasionally insertional mutations. The mutation rate of C-kit gene in gastrointestinal stromal tumor (GIST) is high, and the mutation site is mainly concentrated between 550Lys-256Val amino acid residues, and occurs in the extracellular region (exon 9 mutation is about 5 % to 10%), adjacent membrane regions (exon 11 mutations are about 57% ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6806C12Q2525/113C12Q2531/113C12Q2563/107
Inventor 林佳李品霍然唐景峰
Owner WUHAN BIOTECH GENE ENG
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