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Detection kit for methotrexate metabolic markers and detection method and application of detection kit

A technology of metabolic markers and detection kits, applied in the field of gene detection, which can solve the problems of low specificity, easy contamination of amplified products, and difficulty in primer design

Pending Publication Date: 2021-10-22
湖南菲思特精准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the sequencing method and the chip method have cumbersome operation steps, long detection cycle, and the amplification product is prone to contamination; the high-resolution melting curve method has simple steps, low specificity, and high requirements for equipment; The specific amplification method uses ARMS primers for specific amplification, and the design of the primers is difficult to optimize, and the detection conditions are strict.
Taqman fluorescent probe method has high test cost, and the amplification throughput of multiple genes is not high

Method used

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  • Detection kit for methotrexate metabolic markers and detection method and application of detection kit
  • Detection kit for methotrexate metabolic markers and detection method and application of detection kit
  • Detection kit for methotrexate metabolic markers and detection method and application of detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, the preparation of kit

[0039] The kit of the present invention designs specific amplification primers and sequencing primers for ABCB1 (C3435T), MTHFR (C677T), and SLCO1B1 (c.1865+4846T>C), which are used for constant temperature amplification and pyrosequencing detection. The design of primers based on the recombinant enzyme polymerase amplification technology is one of the keys of the present invention. The primer design of this technology cannot be carried out by auxiliary software, and can only rely on manual design. In order to ensure the amplification speed and detection sensitivity, the primer length should be controlled at 30-35 bp. If the primer design is too short, non-specific amplification will easily increase and cause false positives. If the primer design is too long, it will easily lead to failure of amplification. Gene polymorphism sequence is subject to the public sequence in Genebank.

[0040] (1) The primer sequences of this embodimen...

Embodiment 2

[0052] Embodiment 2, pyrophosphate detection

[0053] The instrument that adopts among the present invention is as follows: thermostat;

[0054] Pyrosequencer: Wuhan First Biotechnology Co., Ltd.

[0055] (1) Reagent preparation (reagent preparation room)

[0056] Take out the reagent in advance, vortex reagent 1 for 15 seconds, and centrifuge at low speed for later use. Add 440ul of Reagent 1 directly to Reagent 2 (lyophilized), and vortex for 15 seconds to mix well. Determine the number of reactions N, N = number of samples to be tested (n) + number of quality control products (1) + blank control. It is recommended to conduct positive control and blank control analysis for each PCR experiment at the same time. Then the reaction solution was dispensed into PCR reaction tubes at 20 μL / tube.

[0057] (2) Sample testing (sample preparation room)

[0058] Add the sample DNA, positive control and blank control into the PCR reaction tube according to the amount of 5 μL, close...

Embodiment 3

[0087] Example 3. Correlation between genetic test results and methotrexate drug dosage and adverse reactions

[0088] Correspondence between gene loci and drugs

[0089]

[0090]

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PUM

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Abstract

The invention discloses a detection kit of methotrexate metabolism markers, and a detection method and application of the detection kit. The kit is used for detecting the gene polymorphism of the methotrexate metabolism markers ABCB1 (C3435T), MTHFR (C677T) and SLCO1B1 (c.1865+4846T>C). According to the invention, multiple RPA amplification and optimized pyrosequencing technologies are combined to detect the methotrexate drug dosage and the gene polymorphism related to adverse reaction prediction, the kit can be used for simultaneously detecting ABCB1 (C3435T), MTHFR (C677T) and SLCO1B1 (c.1865+4846T>C) gene polymorphism methotrexate drugs, and suggestions from the gene perspective are provided for clinical personalized medication.

Description

technical field [0001] The invention relates to a detection kit for methotrexate metabolic markers and a detection method and application thereof, belonging to the field of gene detection. Background technique [0002] Methotrexate (methotrexate, MTX) is a folic acid antagonist, through the binding of dihydrofolate reductase, tetrahydrofolate production barrier to interfere with DNA synthesis, thereby inhibiting tumor cell proliferation. HDMTX chemotherapy can cause a variety of toxic and side effects, mainly including bone marrow suppression, manifested as leukopenia, and whole blood suppression in severe cases. In addition to common nausea and vomiting, gastrointestinal reactions also have oral and perianal mucosal ulcers. Large doses or long-term application can damage liver and kidney function. Adverse reactions caused by chemotherapy drugs lead to the suspension or termination of chemotherapy, which affects the outcome of the disease. Pharmacogenomics studies have sho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6886C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2600/16C12Q2600/106C12Q2521/507C12Q2522/101C12Q2531/119C12Q2537/143C12Q2565/301
Inventor 孙悦刘丹
Owner 湖南菲思特精准医疗科技有限公司
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