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Kit, amplification method and detection method for detecting SNP sites related to tacrolimus and cyclosporine a personalized medicine

A technology of tacrolimus and kit, which is applied in the field of detection kits and amplification and detection of SNP sites related to individualized medicine of tacrolimus and cyclosporine A, can solve the problem of increased susceptibility to infection, grafts Rejection, transplanted diabetes and other problems, to avoid false positive results, reduce operation steps, and achieve good repeatability

Inactive Publication Date: 2011-12-14
UNION STEMCELL & GENE ENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The effective therapeutic range of tacrolimus and cyclosporine A is very narrow. Insufficient dosage or low blood concentration may lead to graft rejection, while excessive dosage will induce a series of adverse reactions, including nephrotoxicity, neurotoxicity, Transplant diabetes, increased susceptibility to infection, cancer, hypertension, and gastrointestinal disturbances

Method used

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  • Kit, amplification method and detection method for detecting SNP sites related to tacrolimus and cyclosporine a personalized medicine
  • Kit, amplification method and detection method for detecting SNP sites related to tacrolimus and cyclosporine a personalized medicine
  • Kit, amplification method and detection method for detecting SNP sites related to tacrolimus and cyclosporine a personalized medicine

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Step 1: Preparation of whole blood cell lysate

[0056] Take 300 μl of peripheral venous blood from the subject, add 700 μl of cell lysate, invert and mix 5 times, centrifuge at 12,000 rpm for 1 minute, discard the supernatant, and place the centrifuge tube upside down on clean absorbent paper for 2 minutes to ensure that the precipitate remains in the tube , add 300 μl double distilled water, vortex and shake to form a cell lysate suspension.

[0057] Step 2: PCR amplification reaction

[0058] 1. Configure the wild-type reaction system: Add 4.8 μl of cell lysis suspension, 5 μl of 2× wild-type amplification buffer and 0.2 μl of polymerase (2.5 U / μl) into the PCR tube to form an independent reaction system. Mix well and centrifuge briefly, see the table below for details:

[0059] 2× wild-type amplification buffer solution 5μl Cell Lysis Suspension 4.8μl Polymerase (2.5U / μl) 0.2μl

[0060] 2. Configure the mutant reaction system: add 4.8...

Embodiment 2

[0073] Step 1: Extraction and dilution of whole blood genomic DNA

[0074] Take 300 μl of peripheral venous blood from the subject, and extract whole blood genomic DNA according to the instructions of the whole blood genomic DNA extraction kit. Measure the concentration of DNA with a spectrophotometer and dilute to 15-20 ng / μl.

[0075] Step 2: PCR amplification reaction

[0076] 1. Configure the wild-type reaction system: add 4.8 μl of cell lysis suspension, 5 μl of 2× wild-type amplification buffer, and 0.2 μl of polymerase (2.5 U / μl) into the PCR tube to form an independent reaction system. Mix well after adding the sample. Evenly, briefly centrifuge, see the table below for details:

[0077] 2× wild-type amplification buffer solution 5μl Cell Lysis Suspension 4.8μl Polymerase (2.5U / μl) 0.2μl

[0078] 2. Configure the mutant reaction system: add 4.8 μl of cell lysis suspension, 5 μl of 2× mutant amplification buffer and 0.2 polymerase (2.5 U / ...

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Abstract

The invention discloses a detection kit and a detection method for detecting SNP sites related to individualized drug use of tacrolimus and cyclosporine A by using multiplex PCR technology combined with SNP sensitive molecular switch technology. The kit is used to type three SNP loci associated with the administration of tacrolimus and cyclosporine A, including: rs2242480 SNP locus on the CYP3A4 gene, rs776746 SNP locus on the CYP3A5 gene, and rs776746 SNP locus on the MDR1 gene rs1045642 SNP site. The kit contains wild-type 2× amplification buffer, mutant 2× amplification buffer, and polymerase. The two buffers contain sequence-specific primers and internal reference primers corresponding to the SNP wild-type and mutant phenotypes, which can The typing of the above three SNP sites was completed in two multiplex PCR reactions, so as to provide a molecular biological basis for the rational use of tacrolimus and cyclosporine A.

Description

technical field [0001] The invention relates to a kit for detecting SNP sites and a PCR amplification method thereof, in particular to a kit for detecting SNP sites related to individualized drug use of tacrolimus and cyclosporin A by using multiplex PCR combined with molecular switch technology and the kit and its method. Multiplex PCR amplification method and detection method. Background technique [0002] Tacrolimus, also known as FK506, is a fermentation product isolated from Streptomyces tsukubaensis, and its chemical structure is a 23-membered macrolide antibiotic. It is a powerful new immunosuppressant. It has a selective inhibitory effect on T cells, mainly inhibiting the release of IL-2, IL-3, IFN-γ by Th cells and inhibiting the expression of IL-2R by binding to intracellular immunophilin FK-binding protein (FKBP). In recent years, as a first-line drug for liver and kidney transplantation, it has been marketed in 14 countries including Japan and the United States...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 韩俊领杜宏伟周毓玲崔丽娟
Owner UNION STEMCELL & GENE ENG
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