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Detection method of types of CYP2D6 gene

A detection method and genotyping technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of long detection cycle, high cost, and low efficiency of CYP2D6 genotyping detection

Inactive Publication Date: 2019-08-23
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection cycle of the next-generation sequencing method is long and the cost is high, resulting in low detection efficiency of CYP2D6 genotyping

Method used

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  • Detection method of types of CYP2D6 gene
  • Detection method of types of CYP2D6 gene
  • Detection method of types of CYP2D6 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0192] In Example 1, the detection method of CYP2D6 genotyping may include:

[0193] Step C1: pre-synthesizing specific amplification primers shown in SEQ ID NO.1-28 and single-base extension primers shown in SEQ ID NO.29-69.

[0194] Step C2: extract the genomic DNA of the sample to be tested from the sample to be tested, and use the genomic DNA of the sample to be tested as a DNA template, wherein the concentration of the DNA template is 50 ng / μl.

[0195] Among them, the genomic DNA of the sample to be tested is extracted from the fresh peripheral blood sample of the sample to be tested by using the blood / cell / tissue genomic DNA extraction kit (DP304), and the concentration is determined by NP80-touch at 5-1000ng / Genomic DNA within the range of μl, and the OD260 / OD280 ratio is within the range of 1.6-2.2.

[0196] Step C3a: Prepare 0.9 μl of distilled water, 25 mmol of MgCl 2 0.4μl, 0.1μl of 20mmol deoxyribonucleoside triphosphate mixture, 1μl of the specific amplificati...

Embodiment 2

[0209] In Example 2, the detection method of CYP2D6 genotyping may include:

[0210] Step D1: pre-synthesizing specific amplification primers shown in SEQ ID NO.1-28 and single-base extension primers shown in SEQ ID NO.29-69.

[0211] Step D2: extract the genomic DNA of the test sample from the test sample, and use the test sample genomic DNA as a DNA template, wherein the concentration of the DNA template is 5 ng / μl.

[0212] Among them, the genomic DNA of the sample to be tested is extracted from the oral exfoliated cells of the sample to be tested using the Tiangen Oral Swab Genomic DNA Extraction Kit (DP322), and the concentration is measured by NP80-touch in the range of 5 to 1000 ng / μl Genomic DNA with OD260 / OD280 ratio between 1.6 and 2.2.

[0213] Step D3a: Prepare 0.8 μl of distilled water, 25 mmol of MgCl 2 0.3 μl, 0.1 μl of 20 mmol deoxyribonucleoside triphosphate mixture, 0.8 μl of the specific amplification primer mixture shown in SEQ ID NO.1-14, 0.1 μl of TaqDN...

Embodiment 3

[0226] In embodiment 3, the detection method of CYP2D6 genotyping may include:

[0227] Step E1: pre-synthesizing specific amplification primers shown in SEQ ID NO.1-28 and single-base extension primers shown in SEQ ID NO.29-69.

[0228] Step E2: extract the genomic DNA of the sample to be tested from the sample to be tested, and use the genomic DNA of the sample to be tested as a DNA template, wherein the concentration of the DNA template is 119 ng / μl.

[0229] Among them, the genomic DNA of the sample to be tested is extracted from the tissue sample of the sample to be tested by using the blood / cell / tissue genomic DNA extraction kit (DP304), and the concentration is determined by NP80-touch within the range of 5-1000ng / μl , and the OD260 / OD280 ratio is within the range of 1.6 to 2.2.

[0230] Step E3a: Prepare 1 μl of distilled water, 25 mmol of MgCl 2 0.5 μl, 0.2 μl of 20 mmol deoxyribonucleoside triphosphate mixture, 1.2 μl of the specific amplification primer mixture show...

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Abstract

The invention provides a detection method of types of a CYP2D6 gene. The detection method comprises the steps of preparing a first polymerase chain reaction (PCR) amplification system comprising specific amplification primers shown in SEQ ID NO.1-14 and a second PCR amplification system comprising specific amplification primers shown in SEQ ID NO.15-28, carrying out amplification reactions in sequence, and then conducting digestive treatment; obtaining a first digestion reaction system and a second digestion reaction system; preparing a first single base extension reaction system comprising single base extension primers shown in SEQ ID NO.29-49 and a second single base extension reaction system comprising single base extension primers shown in SEQ ID NO.50-69; carrying out extension reactions on the first digestion reaction system joining in the first single base extension reaction system and the second digestion reaction system joining in the second single base extension reaction system, then conducting purification treatment, sequentially utilizing a detection instrument comprising a mass spectrometer for detection, and determining genotypes of SNP loci of the CYP2D6 gene of a to-be-detected sample. By means of the detection method, the detection efficiency of the types of the CYP2D6 gene can be improved.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a detection method for CYP2D6 genotyping. Background technique [0002] Cytochrome P450 (CYP450) is a gene family composed of heme-thiolate proteins, which can participate in the synthesis of sterol hormones and the metabolism of endogenous and exogenous substances in organisms. CYP2D6 gene is one of the members of CYP450 family, which is of great significance in genetic pharmacology. [0003] Currently, NGS is usually used to detect CYP2D6*2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *12, *13, *14, * 15, *16, *17, *19, *20, *21, *29, *33, *35, *36, *38, *40, *41, *42 genotypes. However, the detection cycle of the next-generation sequencing method is long and the cost is high, which leads to low detection efficiency of CYP2D6 genotyping. Contents of the invention [0004] The embodiment of the present invention provides a detection method of CYP2D6 genotyping, which can i...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858
Inventor 赵方圆翟瑞雪智慧芳倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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