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Primer group, kit and method for detecting c-kit gene mutation

A detection method and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve difficult detection, unknown and other problems, achieve high accuracy, good specificity, and improve The effect of sensitivity

Inactive Publication Date: 2018-08-07
GUANGZHOU KINGMED DIAGNOSTICS GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection methods of c-kit gene mutation mainly include quantitative PCR, Sanger sequencing and DHPLC, etc. Sanger sequencing method is considered as the gold standard for detecting gene mutation, but its detection sensitivity is about 15-20%. The locus is heterozygous, and when the content of one allele is less than 15-20%, it is difficult to detect it by Sanger sequencing
However, the mutation rate of c-kit gene in tumor cells is unknown, and the mutation rate of some exons is even lower than 10%.

Method used

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  • Primer group, kit and method for detecting c-kit gene mutation
  • Primer group, kit and method for detecting c-kit gene mutation
  • Primer group, kit and method for detecting c-kit gene mutation

Examples

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Embodiment 1

[0036] An embodiment of the primer set used for detecting c-kit gene mutation of the present invention, the primer set used for detecting c-kit gene mutation described in this example is shown in Table 2.

[0037] Table 2 Primer Sequence

[0038]

[0039]

[0040] Note: A in the primers represents the first-round amplification primer of nest-PCR, and B represents the nest primer in nest-PCR.

[0041] Using the primers in Table 1 can specifically amplify the corresponding exon fragments. Further, the primer set described in this embodiment for detecting c-kit gene mutation also includes the following sequencing primers: TGTAAAACGACGGCCAGT (upstream primer, shown in SEQ ID NO: 17) and CAGGAAACAGCTATGACC (downstream primer, shown in SEQ ID NO: 18) Show).

Embodiment 2

[0043] An embodiment of the test kit for detecting c-kit gene mutation of the present invention, the test kit for detecting c-kit gene mutation described in this embodiment includes the c-kit gene mutation described in embodiment 1 Primer set, DNA extraction reagent, DNA polymerase, buffer solution, MgCl 2 and agarose gel; wherein, the DNA extraction reagent comes from the FFPE tissue DNA extraction kit; the preparation method of the agarose gel is: take a sufficient amount of agarose, put it into a 200mL conical flask, add a certain amount of TAE Buffer solution (1×), heat in a microwave oven (SMC, J180) at medium-high heat for 3 minutes or boil until the solution is clear, cool to about 65°C at room temperature, pour into another dedicated 100mL conical flask, add acridine orange 3~ 5 μL, pour the gel, insert the tooth comb, and cool at room temperature for 10-15 minutes to obtain the agarose gel; the preparation method of the 50×TAE buffer solution used is: mix 242g Tris, 5...

Embodiment 3

[0045] An embodiment of the detection method of the c-kit gene mutation of the present invention, the detection method of the c-kit gene mutation described in this embodiment adopts the kit described in Example 2, and the specific method steps are as follows:

[0046] 1. DNA extraction: use FFPE tissue DNA extraction kit to extract the DNA in the sample to be tested;

[0047] 2. Use the primer set in Table 2 as a primer to take out Q5 TM Hot Boot Ultra Fidelity 2 x Master Mix, ddH 2 0, after melting at room temperature, briefly centrifuge to prepare a PCR amplification system as shown in Table 3 (the main purpose of adding DMSO in the amplification system is to increase specific amplification; the template in Table 3 is added in step 3); After a brief centrifugation, aliquot 23.0 μL to each PCR reaction tube;

[0048] Table 3 PCR reaction system

[0049]

[0050] 3. Add 2.0 μL (6.25-200ng) of template to the PCR reaction tube, cover the cap, shake and mix well, and perfo...

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Abstract

The invention relates to a primer group, a kit and a method for detecting c-kit gene mutation and belongs to the technical field of biological detection. The primer group comprises a PCR (polymerase chain reaction) amplification primer pair for detecting c-kit gene exon 9, a PCR amplification primer pair for detecting c-kit gene exon 11, a PCR amplification primer pair for detecting c-kit gene exon 13 and a PCR amplification primer pair for detecting c-kit gene exon 17. The novel primer group for detecting c-kit gene mutation can be used for detecting mutation of the c-kit gene exon 9, the exon 11, the exon 13 and the exon 17. The primer group is high in specificity, accuracy and sensitivity in c-kit gene mutation detection and can accurately detect samples with low mutation rate.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer set, a kit and a method for detecting c-kit gene mutation. Background technique [0002] The c-kit gene is located on human chromosome 4q11-q12 and belongs to class III tyrosine kinase receptors. Studies have shown that after c-kit binds to its ligand stem cell factor, dimerization and autophosphorylation occur, thereby activating downstream signaling pathways, mediating cell growth, proliferation, and inhibiting cell apoptosis. Mutations in the c-kit gene will lead to continuous activation of the c-Kit receptor, leading to uncontrolled cell proliferation and inhibition of apoptosis. [0003] Gastrointestinal stromal tumor (GIST) is a relatively common mesenchymal tumor of the digestive tract. Studies have shown that most GISTs are caused by mutations in the c-Kit gene, and the mutation rate of the c-kit gene in GIST is about 90%. c-Kit gene mu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 于世辉胡昌明黄晓强周丹燕毛琳琳徐艳艳海洋周杏子刘晶星赵薇薇赵强袁海明陈白雪喻长顺
Owner GUANGZHOU KINGMED DIAGNOSTICS GRP CO LTD
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