Primer set for amplification of CYP2C19 gene, reagent for amplification of CYP2C19 gene comprising the same, and use of the same

Abstract

Disclosed is a primer set for amplifying a target region in CYP2C19 gene which contains a site to be detected by a gene amplification method, and which enables to amplify the region specifically. Two pairs of primer sets are used, each of which comprises a forward primer comprising a nucleotide sequence depicted in each of SEQ ID NOs:12 and 32 and a reverse primer comprising a nucleotide sequence depicted in each of SEQ ID NOs:22 and 48, respectively. These primer sets enable to amplify two regions each containing a site having each of two types of polymorphisms occurring in CYP2C19 gene (CYP2C19 gene*2, CYP2C19*3) simultaneously in a single reaction solution.

Description

technical field

[0001] 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

[0002] 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 persons (PMs) who have very low metabolism of a CYP2C19 substrate drug (antiepileptic agent), namely (S)-Mephenytoin (S-Mep) . CYP2C19*2 and CYP2C19*3 are known as important gene polymorphisms related to the PMs. The former is a mutation in which guanine (position 681) of exon 5 is changed to adenine, which causes a splicing defect and changes the translation initiation site of ...

Examples

Embodiment 1

[0146] 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

[0169] 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).

[...

technical field

[0001] 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

[0002] 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 persons (PMs) who have very low metabolism of a CYP2C19 substrate drug (antiepileptic agent), namely (S)-Mephenytoin (S-Mep) . CYP2C19*2 and CYP2C19*3 are known as important gene polymorphisms related to the PMs. The former is a mutation in which guanine (position 681) of exon 5 is changed to adenine, which causes a splicing defect and changes the translation initiation site of ...

Examples

Embodiment 1

[0146] 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

[0169] 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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