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Detection method for CYP2C9*2 gene polymorphism, as well as nucleic acid probe and kit for method

A technology for gene polymorphism and detection probes, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc.

Inactive Publication Date: 2015-04-29
FUWAI HOSPITAL OF CARDIOVASCULAR DESEASE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, CYP2C9 and VKORC1 gene detection has been commercialized, but domestic medical institutions can only use western commercial products for warfarin sensitive gene detection

Method used

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  • Detection method for CYP2C9*2 gene polymorphism, as well as nucleic acid probe and kit for method
  • Detection method for CYP2C9*2 gene polymorphism, as well as nucleic acid probe and kit for method
  • Detection method for CYP2C9*2 gene polymorphism, as well as nucleic acid probe and kit for method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] 1. Preparation of probes and primer pairs for CYP2C9*2 gene

[0108]Probes for CYP2C9*2 genes and primer pairs for CYP2C9*2 genes were prepared using conventional techniques.

[0109] The specific sequence is as follows:

[0110] Probe for CYP2C9*2 gene

[0111] *2 Mutant probe FAM-aacacAgtcctcaAtgctcc-BHQ2

[0112] *2 wild-type probe HEX-ttgaacacGgtcctcaatgctc-BHQ2

[0113] Primer pair for CYP2C9*2 gene

[0114] Forward primer sequence 3D1-S-1: AAGGCAGCGGGCTTCCTC

[0115] Reverse primer sequence 4D1-A-1: GCTGCGGAATTTTGGGATGG.

[0116] Second, configure the PCR reaction solution,

[0117] The serving size is as follows:

[0118] Commercially available 2×PCR reaction solution (including taq enzyme, UNG enzyme, dNTP, dUTP) 10 μL

[0119] Primer and probe mix 1 μL

[0120] Internal control probe mixture 1 μL

[0121] RNase-free water 7 μL

[0122]

[0123] Aliquot, 19 μl per tube into 0.2ml PCR reaction tubes / plates, and transfer to the sample processing area...

Embodiment 2

[0137] Steps one to four:

[0138] In the same method and steps as in Example 1, the DNA sample was replaced with a DNA sample expressing wild type CYP2C9*2, and 1 μl of the wild type DNA sample was added to 19 μl of the PCR reaction solution.

[0139] 5. Results analysis

[0140] CYP2C9*2 expresses wild type results in figure 2 shown in the table. These graphs are analysis graphs showing changes in fluorescence intensity over time, that is, changes in fluorescence intensity as the number of amplification cycles increases. Such as figure 2 As shown, the fluorescence curve indicates that the CYP2C9*2 gene in the sample is wild type.

Embodiment 3

[0142] one,

[0143] 5 mL of venous blood was collected from 3 subjects A, B and C, and anticoagulated with EDTA (obtained samples 1, 2 and 3).

[0144] Methods of Using the RelaxGene Blood Genomic DNA Extraction Kit

[0145] 1. Add 3 mL of EDTA anticoagulated venous blood, add 5 mL of Lysis Solution CL to lyse blood cells, transfer the solution to a clean and sterile 15 mL centrifuge tube, invert and mix well; centrifuge at 2,000×g for 5 minutes at room temperature, discard the supernatant, and keep the precipitate ;

[0146] 2. Add 5ml lysate CL to the centrifuge tube, shake vigorously until the precipitate is completely dissolved, and centrifuge at 2,000×g for 5 minutes at room temperature;

[0147] 3. Discard the supernatant, add 2.5ml proteinase K digestion solution, vortex and mix; 56°C water bath for 10 minutes.

[0148] 4. Add 2.5ml of isopropanol, shake the centrifuge tube gently, and pick out the filamentous genomic DNA.

[0149] 5. Wash DNA in 70% ethanol and dr...

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Abstract

The invention discloses a detecting probe for CYP2C9*2 gene polymorphism. The nucleotide sequences of the detecting probe are selected from a sequence 1 and a sequence 2; a fluorescent group and a quenching group are respectively arranged at the ends (5'and 3'); the peak values of the wavelengths of the florescent light emitted from the fluorescent group are at different positions. Preferably, the quenching group is selected from MGB and BHQ2; preferably, the fluorescent group is selected from FAM, VIC, JOE, HEX, CY3, NED, TAMRA, ROX, TEXAS RED, CY5 and the like. The invention further discloses a primer pair used for gene amplification; the primer pair comprises a forward primer and a reverse primer; the forward primer is an oligonucleotide which consists of base sequences of a sequence 3; the reverse primer is an oligonucleotide which consists of base sequences of a sequence 4. The invention further discloses a kit which contains the primers and the probe, and a detecting method for the CYP2C9*2 gene polymorphism.

Description

technical field [0001] The invention relates to a detection probe and amplification primer for CYP2C9*2 gene polymorphism, belonging to the field of biotechnology. Background technique [0002] Warfarin is an oral anticoagulant that has been widely used in the fields of thrombosis prevention in atrial fibrillation, stroke, venous thromboembolism, and antithrombotic therapy after mechanical valve implantation in recent decades. The drug is a racemic isomer, wherein the R-type enantiomer and the S-type enantiomer have different action strengths, and the pharmacological effect of the S-type is 3-6 times that of the R-type. The drug has strong water solubility, is rapidly absorbed through the gastrointestinal tract, and has a bioavailability close to 100%. The peak plasma concentration is reached 90 minutes after oral administration, and the half-life is 36 to 42 hours. Combined with plasma protein in blood circulation (binding rate 98% ~ 99%). The two enantiomers accumulated...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/106C12Q2600/156
Inventor 李一石刘红韩璐璐田蕾娄莹王巍许建屏
Owner FUWAI HOSPITAL OF CARDIOVASCULAR DESEASE CHINESE ACAD OF MEDICAL SCI
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