Primer pair for cyp2c19 gene amplification, reagent for cyp2c19 gene amplification containing same and use thereof

A technology of CYP2C19 and primer pairs, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of a large amount of labor, reduced reliability of analysis results, and impractical analysis, so as to reduce effort and cost, omitting pretreatment, and shortening the effect of the amplification reaction

Inactive Publication Date: 2011-12-14
ARKRAY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, when the genes encoding other isozymes are also amplified, for example, it becomes the analysis result of the specific polymorphism (CYP2C19*2 or CYP2C19*3) analysis of the CYP2C19 gene (Non-Patent Document 1 and Non-Patent Document 2) Causes of reduced reliability
In addition, like this, since a large amount of labor is required to analyze a single sample, there is also a problem that it is not practical to analyze a large number of samples.

Method used

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  • Primer pair for cyp2c19 gene amplification, reagent for cyp2c19 gene amplification containing same and use thereof
  • Primer pair for cyp2c19 gene amplification, reagent for cyp2c19 gene amplification containing same and use thereof
  • Primer pair for cyp2c19 gene amplification, reagent for cyp2c19 gene amplification containing same and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0147] Blood was collected from 4 subjects using lithium heparin blood collection tubes (samples 1 to 4). 10 μL of the obtained blood was mixed with 90 μL of distilled water, and 10 μL of the mixed solution was mixed with 90 μL of distilled water. 10 μL of this mixed solution was added to 40 μL of a PCR reaction solution having the following composition, and PCR was performed using a thermal cycler. The conditions of PCR were as follows: after treatment at 95°C for 60 seconds, 1 cycle at 95°C for 1 second and 10 seconds at 54°C was repeated for 50 cycles, and then at 95°C for 1 second, and then Treatment at 40°C for 60 seconds. Next, the above-mentioned PCR reaction solution was heated from 40° C. to 95° C. at a rate of temperature increase of 1° C. / 3 seconds, and changes in fluorescence intensity over time were measured. The measurement wavelengths are 450-480 nm (detection of the fluorescent dye Pacific Blue), and 515-555 nm (detection of the fluorescent dye BODIPY FL). N...

Embodiment 2

[0170] Blood was collected from two subjects using EDTA blood collection tubes (Samples 1-2). 10 μL of the obtained blood was mixed with 70 μL of the following diluent A, and 10 μL of the mixed solution was mixed with 70 μL of the following diluent B. 10 μL of this mixture was heat-treated at 95° C. for 5 minutes, then added to 46 μL of a PCR reaction solution having the following composition, and PCR was performed using a thermal cycler. The conditions of PCR are as follows: after treating at 95°C for 60 seconds, repeating 50 cycles of 1 second at 95°C and 15 seconds at 64°C, and then treating at 95°C for 1 second, Treat at 40°C for 60 seconds. Next, the above-mentioned PCR reaction solution was heated from 40° C. to 75° C. at a rate of temperature increase of 1° C. / 3 seconds, and changes in fluorescence intensity over time were measured. The measurement wavelength is 515-555nm (detection of fluorescent dye BODIPY FL) and 585-700nm (detection of fluorescent dye TAMRA).

[...

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Abstract

The present invention provides a primer pair for amplifying a target region containing a detection target site in the CYP2C19 gene using a gene amplification method, which can specifically amplify the region. Two primer pairs each including a forward primer containing the base sequences of SEQ ID NO: 12 and SEQ ID NO: 32 and a reverse primer containing the base sequences of SEQ ID NO: 22 and SEQ ID NO: 48 were used. By using this primer pair, two regions of the CYP2C19 gene containing the occurrence sites of two polymorphisms (CYP2C19 gene*2, CYP2C19*3) can be amplified simultaneously in the same reaction solution.

Description

[0001] This application is a divisional application, the international application number of the original application is PCT / JP2007 / 073205, the Chinese national application number is 2007800301350, the application date is November 30, 2007, and the invention name is "primer pair for CYP2C19 gene amplification, Reagent for CYP2C19 gene amplification containing it and its use". technical field [0002] The present invention relates to a pair of primers for amplifying CYP2C19 gene, a reagent for amplifying CYP2C19 gene containing the primer pair and use thereof. Background technique [0003] Cytochrome P450 is an enzyme classified into a superfamily, and there are multiple subfamilies (for example, CYP1A, CYP1B, CYP2C, CYP2D, CYP2E, CYP3A, etc.). Among them, CYP2C19, which is an isozyme of the human CYP2C subfamily, is known as an enzyme involved in drug metabolism. Furthermore, mutations in the gene encoding CYP2C19 (CYP2C19 gene) have been demonstrated by enzyme-deficient pe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156C12Q1/6876C12N15/09C12P19/34C12Q1/6844C12Q2527/107
Inventor 间岛智史吉永由纪
Owner ARKRAY INC
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