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CYP2D6 gene mutation detection liquid-phase chip and detection method

A technology of CYP2D6C100T and CYP2D6G1846A, applied in CYP2D6 gene mutation detection liquid chip, medical and biological fields, can solve the problems of low sensitivity, poor repeatability of detection results, high price, etc., achieve high specificity and accuracy, and accurate detection results Reliability, effect of detection sensitivity improvement

Active Publication Date: 2012-07-18
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, domestic and foreign products for detecting CYP2D6 gene mutations, such as Amersham Bioscience (GE healthcare)'s CodeLink P450, Roche's AmpliChip CYP450 Test, etc., are mainly based on traditional solid-phase chips, which are expensive and have low sensitivity. poor reproducibility of results
Other PCR-based techniques for detecting gene mutations, such as direct sequencing and PCR-single-strand conformational polymorphism (SSCP) detection, have the disadvantages of low sensitivity, easy sample contamination, and high false positive rate.
However, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis technology and the allelic difference analysis method based on TaqMan technology can only detect one mutation at a time, which is time-consuming and laborious.

Method used

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  • CYP2D6 gene mutation detection liquid-phase chip and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 CYP2D6 gene mutation detection liquid chip mainly includes:

[0031] 1. ASPE Primers

[0032] The two normal genotypes of CYP2D6 gene are CYP2D6*1 and CYP2D6*2, and CYP2D6*2 is C2850T mutation. The five domestic common mutation genotypes are CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*10 and CYP2D6 gene duplication mutation. CYP2D6*3 is the A2549del deletion mutation, CYP2D6*4 is the common mutation of C100T and G1846A, CYP2D6*5 is the entire gene deletion mutation, CYP2D6*10 is the C100T mutation, and there are also duplication mutations of the entire gene. Specific primers were designed for the two normal genotypes CYP2D6*1 and CYP2D6*2 of the CYP2D6 gene and the gene duplication mutations of the five domestic common mutant genotypes CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*10 and CYP2D6 sequence. ASPE primers consist of "Tag + specific primer sequence". ASPE primer sequences are shown in the table below:

[0033] Table 1 ASPE primer sequence (Tag+ specific primer...

Embodiment 2

[0050] Example 2 Detection of Clinical Samples Using CYP2D6 Gene Mutation Detection Liquid Chip

[0051] The formula of described various solutions is as follows:

[0052] 50mM MES buffer (pH5.0) formula (250ml):

[0053] 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

[0054] 2×Tm hybridization buffer

[0055] 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

[0056] Store at 4°C after filtration.

[0057] ExoSAP-IT kit was purchased from US USB Company.

[0058] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[00...

Embodiment 3

[0124] Example 3 Detection of CYP2D6 C2850T, CYP2D6 Deletion Gene Mutation Detection Liquid Chip on Clinical Samples

[0125] Use CYP2D6 C2850T, CYP2D6 Deletion gene mutation detection liquid chip to detect serum samples 1-20, the synthesis of ASPE primers, Anti-tag sequence coated microspheres, amplification primers, detection methods, etc. are as described in Example 1 and Example 2 mentioned.

[0126] Table 6 Sample test results (MFI)

[0127] serial number

[0128] 4

[0129] Table 7 Analysis results of CYP2D6 C2850T and CYP2D6 Deletion gene mutations in samples

[0130] serial number

[0131] 8

[0132] It can be seen from the results of Example 2 and Example 3 that only CYP2D6 C2850T and CYP2D6 Deletion gene mutations in Example 3 are detected by the liquid chip, which is consistent with that of Example 2.

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Abstract

The invention provides a CYP2D6 gene mutation detection liquid-phase chip which comprises ASPE (Allele Specific Primer Extension) primers aiming at CYP2D6 C2850T and CYP2D6Deletion mutational sites, three microballoons respectively enveloped with a specific anti-tag sequence and amplification primers aiming at the CYP2D6C2850T and the CYP2D6 Deletion mutational sites. The CYP2D6 gene mutation detection liquid-phase chip can simultaneously detect aiming at the CYP2D6 C2850T and the CYP2D Deletion mutational sites and has excellent signal to noise ratio. The coincidence ratio with a sequencing method of the CYP2D6 gene mutation detection liquid-phase chip reaches up to 100 percent, and the CYP2D6 gene mutation detection liquid-phase chip has higher specificity and precision compared with intra-class correlation products.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a CYP2D6 gene mutation detection liquid chip and a detection method. Background technique [0002] Tamoxifen (TAM, tamoxifen, also known as tamoxifen) is a selective estrogen receptor modulator whose structure is similar to estrogen. After entering breast cancer cells, estrogen can bind to estrogen receptor (ER, estrogen receptor), promote the synthesis of DNA and m-RNA of tumor cells, and stimulate the growth of tumor cells. When TAM enters the cell, it can compete with estrogen to bind to the estrogen receptor, preventing the function of estrogen, thereby inhibiting the proliferation of breast cancer cells. TAM treatment belongs to one of the endocrine treatment of breast cancer, and another commonly used endocrine therapy drug is aromatase inhibitors (aromatase inhibitors). However, aromatase inhibitors are only suitable for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 许嘉森何嘉英曾涛李国强朱泽尧
Owner SUREXAM BIO TECH
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