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Nucleic acid, kit and method for detecting polymorphism of A118G locus of OPRM1 gene of human beings

A site polymorphism and kit technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of cross-contamination of PCR products, limited development and application, and high detection costs, achieving Avoid non-specific amplification problems, reliable detection results, and overcome the effect of easy contamination

Active Publication Date: 2017-05-31
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the direct sequencing method is called the gold standard, its detection sensitivity is low, the operation process is complicated, the sample is easily contaminated, it takes a long time, and the detection cost is high
The gene chip method and the liquid phase chip method have the same complicated operation process, high detection cost, long detection cycle, and the accuracy of the results is not high, and sample contamination is prone to occur, resulting in false positive results
The PCR-RFLP method is complicated to operate, and when there are many samples, it is easy to cause cross-contamination of PCR products, and it is prone to insufficient or excessive enzyme digestion, resulting in false negative or false positive results, with low reliability
The above shortcomings limit the development and application of these four methods in actual clinical work, and cannot meet the needs of rapid clinical evaluation

Method used

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  • Nucleic acid, kit and method for detecting polymorphism of A118G locus of OPRM1 gene of human beings
  • Nucleic acid, kit and method for detecting polymorphism of A118G locus of OPRM1 gene of human beings
  • Nucleic acid, kit and method for detecting polymorphism of A118G locus of OPRM1 gene of human beings

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Design of primers, probes, and verification templates

[0056] The basic principle of ARMS-PCR amplification: During PCR amplification, the extension of the primer starts from its 3'end, and this extension requires that the base at the 3'end of the primer and the template must be completely paired. Only in this way can the primer Extension, amplification can proceed to obtain the expected amplification product. If the 3'end of the primer cannot be paired with the template, the extension of the primer is blocked and the corresponding amplification product cannot be obtained. Based on the above facts, 3 The primers containing mutant bases at the'end are used to detect whether there are corresponding mutation sites in the target DNA, which is 3'-specific PCR amplification. Amplification blocking mutation system and allele-specific PCR. This reaction system contains two PCR amplification reactions. There are two pairs of primers but their 3'ends are different. One is...

Embodiment 2

[0078] Example 2 Method for detecting human OPRM1 gene A118G locus polymorphism using real-time fluorescent PCR

[0079] The wild-type upstream primers, mutant upstream primers, common downstream primers and common detection probes screened and designed in Example 1 were synthesized. The 5'end of the detection probe was connected with a fluorescent group and the 3'end was connected with a quenching group. Among them, the fluorescent group can be any one of FAM, JOE, CY3, and HEX, and the quenching group can be any one of MGB, BHQ1, TAMRA, and BHQ2.

[0080] The method for detecting the A118G polymorphism of human OPRM1 gene by real-time fluorescent PCR is as follows:

[0081] (1) Extract DNA from the sample;

[0082] (2) Perform real-time fluorescent PCR amplification detection of the human OPRM1 gene A118G site polymorphism on the extracted sample DNA, using a wild-type reaction system and a mutant-type reaction system, wherein the wild-type primer pair in the wild-type system The n...

Embodiment 3

[0104] Example 3 Kit for detecting polymorphism of human OPRM1 gene A118G site

[0105] The real-time fluorescent PCR kit for detecting the polymorphism of human OPRM1 gene A118G site includes the following components:

[0106] Wild-type upstream primer: the nucleotide sequence is shown in SEQ ID No. 1;

[0107] Mutant upstream primer: the nucleotide sequence is shown in SEQ ID No. 2;

[0108] Common downstream primer: the nucleotide sequence is shown in SEQ ID No. 3

[0109] Common detection probe: the nucleotide sequence is shown in SEQ ID No. 4, the 5'end of the common detection probe is connected with a fluorescent group, and the 3'end is connected with a quenching group;

[0110] Wild-type positive control: contains the nucleotide sequence shown in SEQ ID No. 8;

[0111] Mutant positive control: contains the nucleotide sequence shown in SEQ ID No.9.

[0112] In order to avoid missed inspections and false inspections, it also includes: quality control primer pairs, quality control prob...

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Abstract

The invention discloses nucleic acid and a kit used for detecting polymorphism of A118G locus of OPRM1 gene of human beings, and establishes a method for detecting polymorphism of A118G locus of OPRM1 gene of human beings with strong specificity, high sensitivity, high accuracy and simple operation, and provides a guide scheme for individual dose administration of opioid, such as morphine, fentanyl, tramadol, oxycodone & aceta minophen and dolantin. The detection method provided in the invention adopts an operation in a completely-closed tube, the operation is simple, convenient and rapid, the detection result can be directly obtained by detecting the fluorescent signal value in the PCR process, and no PCR post-treatment or electrophoresis detection is needed, thereby overcoming the difficult problems of easy pollution of the traditional PCR technology, easy false positive rate, and capability of effectively avoiding non-specific amplification, and being applicable to detection of large-scale samples.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a nucleic acid, a kit and a method for detecting the polymorphism of the A118G site of the human OPRM1 gene. Background technique [0002] Cancer pain is the pain caused by cancer. It is related to real or possible tissue damage caused by tumor cells invading the body tissues. It is a common complication of cancer patients and seriously affects the quality of life and treatment effects of advanced cancer patients. . According to statistics from the World Health Organization (WHO), at least one-third of the diagnosed tumor patients have varying degrees of pain, and the number of advanced patients is as high as 60% to 90%. Therefore, cancer pain control and cancer early prevention, early diagnosis, and early treatment are listed as four key programs of WHO. [0003] Although there are various pharmacological and non-pharmacological treatments for cancer pain, opioid analgesics ar...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2561/113C12Q2535/137C12Q2563/107
Inventor 邹芳杨惠娟段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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