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Method for detecting copy number variation of human CYP2D6 gene

A gene copy number and copy number variation technology, applied in the field of molecular biology, can solve the problems of cumbersome procedures, low accuracy, time-consuming and laborious, etc., and achieve the effect of convenient and fast operation, simplified operation and guaranteed accuracy

Active Publication Date: 2021-07-30
上海康黎诊断技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for detecting the copy number variation of human CYP2D6 gene, so as to solve the problems of low accuracy, cumbersome procedures, and time-consuming and laborious problems in the prior art for detecting the copy number variation of human CYP2D6

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  • Method for detecting copy number variation of human CYP2D6 gene
  • Method for detecting copy number variation of human CYP2D6 gene
  • Method for detecting copy number variation of human CYP2D6 gene

Examples

Experimental program
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Embodiment 1

[0031] Embodiment 1 design primer probe combination 1

[0032] According to this preferred embodiment, the present invention uses the TaqMAN fluorescence quantitative method to specifically amplify the CYP2D6 exon 9 region, and simultaneously increases the double internal reference TERT and RPPH as parameters for evaluating the qualification of the sample, so as to avoid the risk of misjudgment due to sample quality problems. Realize simultaneous detection of 3 target genes (2D6+TERT+RPPH) in a single tube. The present invention designs primers and probes for human CYP2D6 gene, TERT internal reference gene, and RPPH internal reference gene respectively. The nucleotide sequences thereof are shown in Table 1 below, and the preferred concentration of each primer and probe is also shown.

[0033] Table 1 The sequence of the primer probe combination 1 designed for the detection of human CYP2D6 copy number

[0034] Primer name sequence Concentration nM CYP2D6-F (...

Embodiment 2

[0035] Example 2 A double internal reference detection kit 1 for detecting copy number variation of human CYP2D6

[0036] 1. Main components

[0037] According to this preferred embodiment, a kit for detecting copy number variation of human CYP2D6 is provided, and the main components of the kit are shown in Table 2 below.

[0038] Table 2 The main components of the kit

[0039]

[0040] 2. Components that must be detected but not included in the kit

[0041] 1.5ml centrifuge tube (for configuring PCR reaction solution and DNA extraction), 0.2mL PCR tube or 8-tube strip or 96-well plate, tip with filter plug (1mL, 200μL and 10μL), DNA extraction kit (recommended Qiamp DNA Blood Mini Kit, Enrich Swab / Blood Spot Extraction Kit).

[0042] 3. Applicable instruments

[0043] Applicable to the Real-time PCR amplification instrument of ABI7500 model.

[0044] 4. Sample requirements

[0045] 4.1 Applicable samples are EDTA anticoagulated whole blood, oral swabs and blood cards...

Embodiment 3

[0080] Embodiment 3 design primer probe combination 2

[0081] In order to verify that the detection accuracy of CYP2D6 can be improved even if different double internal references are selected, an internal reference gene different from that in Example 1 was selected in this example, and a primer-probe combination 2 was designed.

[0082] According to this preferred embodiment, the TaqMAN fluorescence quantitative method is used to specifically amplify the CYP2D6 exon 9 region, and the double internal reference ALB and β-globin gene are added as parameters for evaluating the qualification of the sample, so as to avoid the risk of misjudgment due to sample quality problems , realize simultaneous detection of 3 target genes (2D6+ALB+β-globin) in a single tube. In this example, primers and probes were respectively designed for human CYP2D6 gene, ALB internal reference gene, and β-globin internal reference gene. The nucleotide sequences thereof are shown in Table 6 below, and the ...

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Abstract

The invention provides a method for detecting copy number variation of a human CYP2D6 gene, the method comprises the following steps of: selecting two DNA sequences which have certain copy number and do not have copy number variation in a known genome of a sample to be detected as reference genes, calculating the difference Ct0 of Ct values of the two reference genes, and judging whether the degradation degree of the sample reaches the inaccurate detection degree or not; if the Ct0 reaches a threshold value which cannot ensure the accuracy of the detection result, prompting to re-sample for DNA extraction; and if the Ct0 is within a threshold range for ensuring the accuracy of the detection result, calculating the difference value between the Ct value of the CYP2D6 gene and the Ct value of one reference gene to obtain Ct1, and further judging the CYP2D6 copy number variation condition of the sample to be detected. According to the invention, the quality problem of the sample is eliminated, the accuracy of the CNV detection result is ensured, the operation is convenient and fast, the detection time is shortened, and the high cost caused by sequencing is saved.

Description

technical field [0001] The present invention relates to the field of molecular biology, and more specifically relates to a method for detecting variation in the copy number of human CYP2D6 gene. Background technique [0002] CYP2D6 is responsible for the metabolism of 20%-30% of human drugs. Many studies have shown that there is a very high correlation between CYP2D6 enzyme activity and the polymorphism of the gene, and the activity is rarely changed by induction. CYP2D6 copy number variation (CNV) is closely related to its activity. For example, the homozygous mutation of *5 is 0 copy, and the human CYP2D6 enzyme activity of this genotype is extremely low, which easily leads to the accumulation of drugs metabolized by CYP2D6, and is prone to adverse reactions. However, people who carry 3 copies of CYP2D6 (3CNV) and above have very high enzyme activity and a high elimination rate of drugs metabolized by it. It is often difficult for conventional doses of drugs to reach eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2545/101C12Q2537/16C12Q2563/107
Inventor 魏宁何炯张辉韩燕
Owner 上海康黎诊断技术有限公司
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