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Cyp2c9, cyp2c19 gene mutation detection liquid chip and its detection method

A technology of CYP2C19 and detection solution, which is applied in the fields of medicine and biology, can solve the problems that the detection flux cannot meet the clinical needs, the repeatability of the detection results is poor, and the price of solid-phase chips is expensive, so as to achieve accurate and reliable detection results and improve detection accuracy The effect of increasing the rate and fluorescence signal value

Active Publication Date: 2011-12-28
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Several PCR-based technologies for detecting CYP2C9 and CYP2C19 gene SNPs have been established, such as direct sequencing, semi-quantitative PCR technology, and PCR-single-strand conformation polymorphism (SSCP) detection. The above technologies have low sensitivity and sample Disadvantages such as easy pollution and high false positive rate, ordinary PCR method and fluorescent quantitative PCR cannot meet the clinical needs due to the limitation of detection throughput
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; traditional solid-phase chips Expensive, low sensitivity and poor reproducibility of test results

Method used

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  • Cyp2c9, cyp2c19 gene mutation detection liquid chip and its detection method
  • Cyp2c9, cyp2c19 gene mutation detection liquid chip and its detection method
  • Cyp2c9, cyp2c19 gene mutation detection liquid chip and its detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 CYP2C9 and CYP2C19 gene mutation detection liquid phase chip kit mainly includes:

[0032] 1. ASPE Primers

[0033] Specific primer sequences were designed for each of the two common mutant alleles of CYP2C9 and CYP2C19 genes. ASPE primers are composed of Tag+ specific sequences. The principle of specific sequence design is: the 3' end of the specific sequence is the mutation site; the specific primers used to detect the wild type and mutant type of a gene mutation site should come from DNA The same chain; the annealing temperature of the specific primer should be between 51°C and 56°C; the length of the primer is generally between 18-22bp. ASPE primer sequences are shown in the table below:

[0034] Table 1 ASPE primer sequence

[0035]

[0036]

[0037]

[0038]

[0039] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere (as shown in Table 2), and the 3' end ...

Embodiment 2

[0052] Example 2 Detection of Clinical Samples Using CYP2C9 and CYP2C19 Gene Mutation Detection Liquid Chip

[0053] The formula of described various solutions is as follows:

[0054] 50mM MES buffer (pH5.0) formula (250ml):

[0055] 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

[0056] 2×Tm hybridization buffer

[0057] 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

[0058] Store at 4°C after filtration.

[0059] ExoSAP-IT kit was purchased from US USB Company.

[0060] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co.,...

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PUM

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Abstract

The invention discloses a CYP2C9, CYP2C19 gene mutation detection liquid chip and a detection method thereof. The liquid chip includes: microspheres respectively coated with specific anti-tag sequences, and the anti-tag sequences are selected from SEQ ID NO .9 to the sequence in SEQ ID NO.16; wild-type and mutant ASPE primers, the wild-type and mutant specific sequences are respectively selected from: SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO. 3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and / or SEQ ID NO.7 and SEQ ID NO.8; primers. The fluorescence signal value detected by the liquid phase chip is greatly improved, thereby further improving the detection sensitivity, enhancing the signal-to-noise ratio, and making the detection result more accurate and reliable.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to medicine and biotechnology, in particular to a liquid phase chip for detecting mutations of genes CYP2C9 and CYP2C19 related to metabolism of phenytoin sodium and a detection method thereof. Background technique [0002] Phenytoin is an antiepileptic drug and an antiarrhythmic drug. It is commonly used in the prevention of epilepsy after craniocerebral surgery, and in the treatment of epilepsy, especially grand mal seizures. This drug was first used in the early 20th century and has been used as a first-line drug for the treatment of epilepsy for a long time. Although its curative effect is good, it has many dose-related adverse reactions, and there are great individual differences. If the dosage of the patient is insufficient, the drug cannot achieve the expected curative effect; and if the dosage is slightly excessive, it will cause serious toxic and side effects to the nervous sy...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 许嘉森何嘉英郭元杰
Owner SUREXAM BIO TECH