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Tacrolimus metabolic gene detection kit and application method thereof

A tacrolimus and gene detection technology, applied in the field of gene detection kits related to tacrolimus metabolism, can solve the problems of difficult promotion and high detection cost

Active Publication Date: 2020-01-03
成都仕康美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In addition, in the prior art, sequencing or PCR-RFLP methods are mostly used for the detection of genotyping. Both methods require blood, tissue and other samples for detection, and the detection cost is high, so it is difficult to promote in clinical practice.

Method used

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  • Tacrolimus metabolic gene detection kit and application method thereof
  • Tacrolimus metabolic gene detection kit and application method thereof
  • Tacrolimus metabolic gene detection kit and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Preparation of Tacrolimus Metabolic Gene Detection Kit

[0054] A tacrolimus metabolism gene detection kit, comprising:

[0055] Primers and probes for detecting CYP3A5 6986A>G gene site mutation:

[0056] Upstream primer: 5'-AGTATTTTAAACATATAAAAC-3'

[0057] Downstream primer: 5'-TTCTAGTTCATTAGGGTGTGA-3'

[0058] Probe 1: 5'-TTCAATATCTCTTCC-3', labeled with FAM at the 5' end, connected with MGB at the 3' end

[0059] Probe 2: 5'-TTCAGTATCTCTTCC-3', labeled VIC at the 5' end, and connected to MGB at the 3' end

[0060] Primers and probes for detection of ABCB1 1236C>T gene site mutation:

[0061] Upstream primer: 5'-ACAGTGATAAATGATTAATCAA-3'

[0062] Downstream primer: 5'-ATGTGACTGCTGATCACCGCAG-3'

[0063] Probe 1: 5'-AGGGCCTGAACCTGA-3', FAM labeled at the 5' end, MGB at the 3' end

[0064] Probe 2: 5'-AGGGTCTGAACCTGA-3', 5' labeled VIC, 3' connected to MGB

[0065] Primers and probes for detecting POR 1508C>T gene site mutation:

[0066] Upstream pri...

Embodiment 2

[0081] Example 2 Detection of Tacrolimus Metabolism Type Using Tacrolimus Metabolism Gene Detection Kit

[0082] (1) Human (A, male, 34 years old) oral epithelial cells were collected, and total free DNA was extracted, and total free DNA was extracted using QIAamp Circulating Nucleic Acid Kit from QIAGEN, and the DNA concentration was adjusted to 10-40ng / μL;

[0083] (2) Determination of CYP3A5 6986A>G gene locus mutation typing: Add 0.5 μL of upstream primer (SEQ ID No.1), 0.5 μL of downstream primer (SEQ ID No.2), probe 1 (SEQ ID No. .3) 0.5 μL, probe 2 (SEQ IDNo.4) 0.5 μL, real-time qPCR MasterMix 10 μL and ultrapure water 8 μL, then add the DNA template prepared in step (1) to prepare a PCR reaction solution, and prepare the prepared The reaction solution was transferred to a 96-well plate for PCR reaction; after the reaction, the 96-well plate was transferred to a fluorescent quantitative PCR reader to read the results, and the CYP3A5 6986A>G gene site mutation of the tes...

Embodiment 3

[0087] Example 3 Detection of Tacrolimus Metabolism Type Using Tacrolimus Metabolism Gene Detection Kit

[0088] (1) Collect human (B, female, 86 years old) oral epithelial cells, and extract total free DNA: the method is the same as in Example 2;

[0089] (2) Determine the CYP3A5 6986A>G gene locus mutation type: the method is the same as in Example 2. After the reaction, transfer the 96-well plate to a fluorescent quantitative PCR reader to read the results. The CYP3A56986A>G gene locus Point mutation is AG type;

[0090] (3) Determine the ABCB1 1236C>T gene site mutation typing: the method is the same as in Example 2. After the reaction, transfer the 96-well plate to a fluorescent quantitative PCR reader to read the results, and the test subject ABCB11236C>T gene site Point mutation is CC typing;

[0091] (4) Determine the POR 1508C>T gene site mutation typing: the method is the same as in Example 2. After the reaction, transfer the 96-well plate to a fluorescent quantita...

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Abstract

The invention discloses a tacrolimus metabolic gene detection kit. The tacrolimus metabolic gene detection kit comprises a primer pair shown as SEQ ID No.1-2, a probe pair shown as SEQ ID No.3-4, a primer pair shown as SEQ ID No.5-6, a probe pair shown as SEQ ID No.7-8, a primer pair shown as SEQ ID No.9-10, and a probe pair shown as SEQ ID No.11-12, wherein the primer pair shown as SEQ ID No.1-2and the probe pair shown as SEQ ID No.3-4 are used for detecting CYP3A5 6986A>G gene locus mutation, the primer pair shown as SEQ ID No.5-6 and the probe pair shown as SEQ ID No.7-8 are used for detecting ABCB1 1236C>T gene locus mutation, and the primer pair shown as SEQ ID No.9-10 and the probe pair shown as SEQ ID No.11-12 are used for detecting POR 1508C>T gene locus mutation. The kit can accurately judge the tacrolimus metabolic type of a to-be-detected person, meanwhile the tacrolimus metabolic degree can further be judged by combining data, and the more reliable reference basis is provided for clinical safe and effective medication.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a gene detection kit for detection related to tacrolimus metabolism and an application method thereof. Background technique [0002] Drug metabolism refers to the biotransformation process of drugs in the body, and drug metabolism reaction refers to the biotransformation reactions of drugs in the body, and the products are metabolites. Studies have shown that drug metabolism in vivo is mainly controlled by metabolic enzymes. Oxidation and binding reactions in the process of drug metabolism, induction or inhibition of drug metabolism enzymes, drug metabolism species, organ tissue, gender differences, stereoselective differences in drug metabolism, and gene polymorphisms of enzymes involved in drug metabolism, etc. All of them are related to drug toxicity, which in turn affects the safety of clinical medication. Therefore, drug metabolism research can eff...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 魏亮
Owner 成都仕康美生物科技有限公司
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