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CYP2D6 gene mutation detection primer and kit

A technology for detection kits and detection primers, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc. Low, high sensitivity, high specificity effect

Inactive Publication Date: 2019-01-11
江门市妇幼保健院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinically, patients with CYP2D6*10 / *10 genotype are not suitable for TAM treatment, or need a larger dose to achieve effective blood drug concentration, but this may cause more serious adverse reactions and increase the risk of patients using drugs. Therefore, determining the CYP2D6 genotype has guiding significance for the drug use of breast cancer patients

Method used

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  • CYP2D6 gene mutation detection primer and kit
  • CYP2D6 gene mutation detection primer and kit
  • CYP2D6 gene mutation detection primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] An embodiment of the CYP2D6 gene mutation detection kit of the present invention, comprising the upstream primer shown in SEQ ID No.1, the downstream primer shown in SEQ ID No.2 and the probe shown in SEQ ID No.3; The 5' end of the probe is connected with a fluorescent reporting group ROX, and the 3' end is connected with a fluorescent quenching group BHQ2.

[0038] SEQ ID No.1: 5'-TTGGTAGTGAGGCAGGTAT-3';

[0039] SEQ ID No.2: 5'-TGGTCGAAGCAGTATGGT-3';

[0040] SEQ ID No.3: 5'ROX-GCTGGGCTGCACGCTACCCACCAGGCCCCCTG

[0041] CCACTG-BHQ2 3'.

[0042] The primers were synthesized by a biological company and configured according to the instructions.

Embodiment 2

[0044] In this example, the kit described in Example 1 is used to detect the CYP2D6 gene.

[0045] 1. Reaction system configuration

[0046] Configure the reaction system according to the components and component final concentrations shown in Table 1:

[0047] Table 1 reaction system

[0048]

[0049] The DNA template is DNA extracted from peripheral blood cells of the patient.

[0050] 2. Reaction procedure

[0051]The PCR reaction program is shown in Table 2:

[0052] Table 2 Reaction program

[0053]

[0054]

[0055] 3. Judgment criteria for results

[0056] The kit of the invention can be used to detect wild type, heterozygous mutation and homozygous mutation of CYP2D6*10 (rs1065852 C100T) site. The criteria for judging the test results are as follows:

[0057] (1) CYP2D6 gene wild type: Ct value is less than 30; Tm value: 74.48±0.68°C, single peak;

[0058] (2) CYP2D6 gene heterozygous mutant: Ct value is less than 30; Tm value: (66.88±0.75°C) / (74.48±0.6...

Embodiment 3

[0066] In this example, the CYP2D6 gene of 15 clinical samples was detected. Take the peripheral blood sample of the subject to be tested, extract the DNA, use the kit described in Example 1, and perform detection according to the detection method described in Example 2, and at the same time perform the first-generation sequencing on the sample. The test results are shown in Table 3:

[0067] Table 3 Test results

[0068]

[0069]

[0070] The above results show that the kit of the present invention has good accuracy when used for CYP2D6 gene detection, and the detection result is completely consistent with the sequencing result.

[0071] Table 4 Mg 2+ Final concentration optimization reaction system

[0072]

[0073] The reaction procedure of table 5 reaction system optimization process

[0074]

[0075]

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Abstract

The invention discloses a CYP2D6 gene mutation detection primer, comprising an upstream primer as shown in SEQ ID No. 1, a downstream primer as shown in SEQ ID No. 2 and a probe as shown in SEQ ID No.3. The invention also discloses the use of the detection primer in preparing a CYP2D6 gene mutation detection reagent and a CYP2D6 gene mutation detection reagent kit. The detection primer provided by the invention can realize the simultaneous detection of wild type of CYP2D6*10 (rs1065852 C100T) site in the same tube PCR reaction, heterozygous mutation and homozygous mutation, and has the advantages of simple operation, high sensitivity, high specificity and low cost.

Description

technical field [0001] The invention belongs to the technical field of gene mutation detection, in particular to a CYP2D6 gene mutation detection primer and a kit. Background technique [0002] Cytochrome P450 (CYP450) enzyme belongs to monooxygenase, which is a metabolic enzyme of group B cytochrome superfamily gene with heme as the prosthetic group. It is named for its reduced state absorption peak at 450nm. It catalyzes many reactions related to drug metabolism and the synthesis of glutathione (Cho). [0003] CYP2D6 is a member of the CYP450 family. It is an important enzyme involved in the metabolism of exogenous substances in the body. It is mainly distributed in the liver, accounting for 4% of the total liver CYP450 enzymes, and involved in the metabolism of 25% of commonly used clinically. Drugs are the first drug-metabolizing enzymes found to have genetic polymorphisms. The CYP2D6 gene is a fully functional gene, and the gene encoding CYP2D6 is located at 22q13.2, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2563/107
Inventor 唐佳曾钦龙徐进美黄晖
Owner 江门市妇幼保健院
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