OPRM1 (Opiate Receptor Mu 1) gene SNP (Single Nucleotide Polymorphism) detection specific primer, liquid-phase chip and detection method

A detection solution and specificity technology, which is applied in the field of molecular biology, can solve the problems that the detection flux cannot meet the clinical needs, the false positive rate is high, and the sample is easy to be polluted, so as to achieve accurate and reliable detection results, simple steps, and improved sensitivity Effect

Inactive Publication Date: 2012-08-22
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] At present, there are almost no products on the market to detect OPRM1 gene polymorphisms at home and abroad. Most of the reports are still in the experimental research stage and have not yet been commercialized. The existing detection technologies are mainly based on PCR technology, such as direct sequencing and real-time fluorescence quantification. PCR method, etc. These technologies have the disadvantages of easy sample contamination and high false positive rate. At the same time, due to the limitation of detection throughput, they cannot meet the clinical needs.
In addition, allelic difference analysis and denaturing high-performance liquid chromatography based on TaqMan technology can also be used for the detection of OPRM1 polymorphisms, but they can only detect one mutation at a time, which is time-consuming and laborious

Method used

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  • OPRM1 (Opiate Receptor Mu 1) gene SNP (Single Nucleotide Polymorphism) detection specific primer, liquid-phase chip and detection method
  • OPRM1 (Opiate Receptor Mu 1) gene SNP (Single Nucleotide Polymorphism) detection specific primer, liquid-phase chip and detection method
  • OPRM1 (Opiate Receptor Mu 1) gene SNP (Single Nucleotide Polymorphism) detection specific primer, liquid-phase chip and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 OPRM1 gene SNP detection liquid chip mainly includes:

[0030] 1. ASPE Primers

[0031] Specific primer sequences were designed for four common SNP sites of OPRM1: A118G, IVS2+G31A, IVS2+G691C and IVS3+A6151G. ASPE primers consist of "Tag + specific primer sequence". ASPE primer sequences are shown in the table below:

[0032] Table 1 ASPE primer sequence (Tag+ specific primer sequence)

[0033]

[0034]

[0035] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a wild-type or mutant specific primer sequence (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0036] 2. Microspheres coated with anti-tag sequences

[0037]According to the designed ASPE-specific primer...

Embodiment 2

[0049] Example 2 Detection of samples using the OPRM1 gene SNP detection liquid chip described in Example 1

[0050] The formula of described various solutions is as follows:

[0051] 50mM MES buffer (pH5.0) formula (250ml):

[0052] Reagent

source

Final concentration

Dosage per 250ml

MES(2[N-Morpholino]

ethanesulfonic acid)

Sigma M-2933

0.05M

2.44g

5M NaOH

Fisher SS256-500

---

5 drops

[0053] 2×Tm hybridization buffer

[0054] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5M NaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0055] Store at 4°C after filtration.

[0056] ExoSAP-IT kit was purchased from US USB Company.

[0057] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co.,...

Embodiment 3

[0116] Example 3 Detection of OPRM1 gene SNP detection gene mutation by liquid chip with different ASPE primers

[0117] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0118] Taking the detection liquid chip of the A118G site mutation of the OPRM1 gene as an example, the specific primer sequence at the 3' end of the ASPE primer was designed for the wild type and mutant type of A118G, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1 - 6 of SEQ ID NO.8, correspondingly, the anti-tag sequences coated on the microspheres and complementary to the corresponding tag sequences are selected from SEQ ID NO.17-SEQ ID NO.24. The specific design is shown in the following table (Table 6). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0119] Table 6 Design of liquid phase chip preparati...

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PUM

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Abstract

The invention discloses an OPRM1 (Opiate Receptor Mu 1) gene SNP (Single Nucleotide Polymorphism) detection liquid-phase chip which mainly comprises ASPE (Allele Specific Primer Extension) primer pairs respectively designed for SNP sites of an OPRM1 gene, microballoons and an amplification primer, wherein each ASPE primer comprises a tag sequence at a 5' end and a specific primer sequence at a 3'end aiming at an SNP site of a target gene; the microballoons are respectively enveloped with a specific anti-tag sequence; and the amplification primer is used for amplifying a target sequence of the OPRM1 gene with A118G, IVS2+G31A, IVS2+G691C and / or IVS3+A615G SNP sites. The invention also provides a detection method using the liquid-phase chip and an SNP detected specific primer of the OPRM1 gene. The coincidence ratio of the detection method with a sequencing method reaches up to 100 percent, and the prepared OPRM1 gene SNP detection liquid-phase chip has excellent signal to noise ratio. Moreover, a designed probe and the anti-tag sequence basically have no cross reaction, and the parallel detection of a plurality of SNP sites can be realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to OPRM1 gene SNP detection specific primers, a liquid phase chip and a detection method. technical background [0002] The mu opioid receptor (MOR) is the main site of action for opioids to exert analgesic effects. Opioids can achieve analgesic effects by increasing the pain threshold. How many opioids are currently used in the treatment of cancer pain? The common ones are morphine, heroin, fentanyl, pethidine, codeine, and oxycodone. The gene encoding the mu opioid receptor (OPRM1) is a leading candidate gene that affects the pharmacodynamic response to opioids. Opioids are very effective in the treatment of cancer pain. The pain treatment of most advanced cancer patients will eventually transition to the third step, that is, strong opioid treatment. However, it is found in clinical work that the effective analgesic dose varies g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森朱泽尧
Owner SUREXAM BIO TECH
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