Probe, primer and kit for detecting gene polymorphism of tacrolimus individualized medication

A technology of gene polymorphism and tacrolimus is applied in the field of probes, primers and kits for detecting gene polymorphism of tacrolimus individualized medicine, which can solve the problem of high cost, inappropriate detection, time-consuming and labor-intensive, etc. problem, to achieve the effect of simple operation, clear genotyping and high sensitivity

Pending Publication Date: 2021-02-02
上海利康精准医疗技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the commonly used mutation detection method is direct DNA sequencing method. PCR products can be directly analyzed by DNA sequence, which can clarify the mutation site, but it has the disadvantages of time-consuming, laborious, expensive, and not suitable for detection of a large number of samples.

Method used

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  • Probe, primer and kit for detecting gene polymorphism of tacrolimus individualized medication
  • Probe, primer and kit for detecting gene polymorphism of tacrolimus individualized medication
  • Probe, primer and kit for detecting gene polymorphism of tacrolimus individualized medication

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: probe and primer design

[0028] The present invention designs probe and primer sequences for the CYP3A5 677A>G SNP site. The specific principle is to use the conformational change of the fluorescent probe and the target sequence after hybridization to release the fluorescent dye, and judge the genotyping results according to the peak diagrams and Tm values ​​at different temperatures after hybridization. In the absence of target DNA, the fluorophore and quencher can be stably combined together, and no fluorescent signal can be detected; when there is target DNA, the structure of the fluorescently labeled probe is destroyed, and the fluorophore and quencher The extinguishing groups are separated from each other, and the fluorescent signal can be detected.

[0029] Design the probe and primers so that when the template does not exist at the annealing temperature, the probe is in a stem-loop state, including the loop sequence and the reverse-complementary s...

Embodiment 2

[0036] Embodiment 2: Detect different genotype standard products

[0037] 1. Use the plasmid CYP3A5 6986 to construct and prepare wild-type standard plasmids and mutant standard plasmids containing the rs776746 site of the target gene (the source of the plasmid, and the synthesis of the plasmid containing the target gene were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. .The accuracy of the sequence was determined by sanger sequencing. The genotype of the wild-type standard plasmid rs776746 was AA;

[0038] 2. Using the probes and primers in Example 1.

[0039] 3. PCR reaction system:

[0040] 1) Add 7.5ul of PCR Mix, 0.5uM of primer 1 solution and 0.5uM of primer 2 solution, and 0.1uM of probe into each PCR reaction well, and then add the wild-type standard plasmid into 3 different PCR reaction wells respectively 2ul each of DNA, mutant standard plasmid DNA, mixed DNA (wild type standard plasmid and mutant standard plasmid are mixed at a ratio of 1:1), make up ...

Embodiment 3

[0043] Embodiment 3: detection method performance analysis experiment

[0044] 1. Precision experiment

[0045] Take a copy of wild-type standard plasmid DNA and mixed-type DNA (the wild-type standard plasmid and mutant standard plasmid are mixed at a ratio of 1:1), and test 3 times a day for 5 consecutive days. The method in example 2, see the result figure 2 . It shows that the fluorescent PCR amplification reaction of the present invention has good repeatability (the coincidence rate is greater than 95%, and the variation coefficient CV of the AG value of the detection result is less than 5%).

[0046] 2. Conformity rate experiment

[0047] Select 20 cases of DNA samples from healthy volunteers in Shanghai, apply the method in Example 2 to detect the rs776746 site, and use the Sanger sequencing method to verify at the same time, compare the consistency of the detection results of the two methods, and the results show that the method in Example 2 can be classified The c...

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Abstract

The invention provides a probe for detecting gene polymorphism of tacrolimus individualized medication. The sequence of the probe is shown as SEQ ID NO.: 1, or the two ends of the sequence of SEQ ID NO.: 1 are modified by groups. The invention also provides a primer for detecting gene polymorphism of tacrolimus individualized medication. The sequences of the primer are shown as SEQ ID NO.: 2 and SEQ ID NO.: 3. The probe and primer for gene polymorphism of tacrolimus individualized medication have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and thelike when being used for detecting the gene polymorphism of tacrolimus individualized medication.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to probes, primers and kits for detecting gene polymorphisms of tacrolimus individualized medicine. Background technique [0002] Tacrolimus (Tacrolimus, FK506) is a macrolide immunosuppressant, which is widely used clinically in the immunosuppressive treatment of liver, kidney, heart, lung, pancreas and other organ transplant patients. Its main adverse reactions include secondary Infection, nephrotoxicity, neurotoxicity, gastrointestinal reactions, metabolic disorders, lymphoproliferative diseases and tumors, etc. Low blood drug concentration of tacrolimus in organ transplant patients can lead to acute rejection and decreased drug sensitivity; high blood drug concentration is prone to nephrotoxicity, neurotoxicity, diabetes, hyperlipidemia, hypertension and gastrointestinal disorders. Adverse reactions such as tract disorders. Lead to the occurrence of tacrolimus side effects. C...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6883C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 卞雪莲潘峰毛丹丹张奕
Owner 上海利康精准医疗技术有限公司
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