Kit for guiding 5-fluorouracil medication

A fluorouracil and kit technology, which is applied in the field of kits for guiding the medication of 5-fluorouracil, can solve the problems of being unsuitable for clinical application, changing the curative effect, toxic and side effects, and being expensive, achieving safe, reasonable and effective individualized drug delivery, and improving the availability Predictive and targeted effects

Inactive Publication Date: 2015-08-26
北京奥维森基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

5-FU has a relatively narrow safe therapeutic range, and the toxicity increases with the increase of the dose. Therefore, in the host or tumor tissue, slight changes in gene expression of genes encoding key metabolic enzymes or 5-FU action sites will Change efficacy and side effects
[0005] 5-FU is widely used in tumor patients, and the curative effect is significant, but sometimes serious side effects and even death occur
The method of routinely measuring dihydropyrimkline dehydrogenase (DPD) activity to predict the response to 5-FU is not suitable for clinical application or screening of large sample cases due to its high price, difficulty in implementation, and complicated operation

Method used

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  • Kit for guiding 5-fluorouracil medication
  • Kit for guiding 5-fluorouracil medication
  • Kit for guiding 5-fluorouracil medication

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the preparation of specific primer pair, positive control and negative control

[0020] 1. Preparation of specific primer pairs

[0021] The specific primer pair for detecting IVS14+1G>A point mutation of DPYD gene consists of F1 and R1.

[0022] F1 (sequence 1 of the sequence listing): 5'-TTCATCAGGACATTGTGACA-3';

[0023] R1 (SEQ ID NO: 2 of the Sequence Listing): 5'-AAGCAACTGGCAGATTCTT-3'.

[0024] The specific primer pair for detecting the 665C>T point mutation of the MTHFR gene consists of F2 and R2.

[0025] F2 (sequence 3 of the sequence listing): 5'-GGTCAGAAGCATATCAGTCA-3';

[0026] R2 (SEQ ID NO: 4 of the Sequence Listing): 5'-AGCGGAAGAATGTGTCAG-3'.

[0027] The specific primer pair used to detect the IVS14+1G>A point mutation of the DPYD gene is also called primer pair A (target sequence 299bp). The specific primer pair used to detect the 665C>T point mutation of the MTHFR gene is also called primer pair B (target sequence 318bp). Synthesize p...

Embodiment 2

[0033] Embodiment 2, specific sensitivity detection of specific primer pair

[0034] 1. Sensitivity detection of primers to formazan

[0035] Using the positive control plasmid A as a template, the primer pair A was used for PCR amplification.

[0036] The total volume of the PCR amplification system is 25 μl. The PCR amplification system contains 0.5μM F1, 0.5μM R1 and 1U hot-start DNA polymerase. The PCR amplification system contains 50ng / μl, 25ng / μl, 15ng / μl, 10ng / μl, 8ng / μl, 5ng / μl, 2.5ng / μl or 1ng / μl positive control plasmid A respectively.

[0037] The PCR amplification program is: 95°C for 5 minutes; 30 cycles of 95°C for 30 seconds, 53°C for 30 seconds, and 72°C for 30 seconds; 72°C for 10 minutes; 15°C, hold.

[0038] The PCR amplification products were subjected to 2% agarose gel electrophoresis, and the results were shown in figure 1 . figure 1 Among them, T1 to T8 successively represent the systems in which the concentration of positive control plasmid A is 50...

Embodiment 3

[0046] Embodiment 3, the application of specific primer pair

[0047] 1. Collect blood samples from 8 volunteers who gave informed consent, and extract genomic DNA.

[0048] 2. Use each genomic DNA extracted in step 1 as a template, and use primers to perform PCR amplification on A (in the initial PCR amplification system, the content of genomic DNA is 10 ng); set a positive control with positive control plasmid A instead of genomic DNA Treatment, set the negative control treatment with negative control instead of genomic DNA.

[0049] The total volume of the PCR amplification system is 25 μl, containing 0.5 μM F1, 0.5 μM R1 and 1 U hot-start DNA polymerase.

[0050] The PCR amplification program is: 95°C for 5 minutes; 30 cycles of 95°C for 30 seconds, 53°C for 30 seconds, and 72°C for 30 seconds; 72°C for 10 minutes; 15°C, hold.

[0051] 3. Use each genomic DNA extracted in step 1 as a template, and use primer pair B to carry out PCR amplification (in the initial PCR ampli...

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Abstract

The present invention discloses a kit for guiding 5-fluorouracil medication. The invention provides a primer group, which comprises a primer pair A and a primer pair B, wherein the primer pair A comprises single-stranded DNA molecules represented by the sequence 1 and single-stranded DNA molecules represented by the sequence 2, and the primer pair B comprises single-stranded DNA molecules represented by the sequence 3 and single-stranded DNA molecules represented by the sequence 4. The present invention further provides the kit for identifying the IVSl4+lG>A point mutation of gene DPYD and the 665C>T point mutation of gene MTHFR, wherein the kit comprises the primer group. According to the present invention, the kit can be used for detecting the two SNP sites associated with the 5-fluorouracil sensitivity so as to be used to clinically guide the individualized use of the 5-fluorouracil, and improve the targeting property and the predictability of the clinical treatment.

Description

technical field [0001] The invention relates to a kit for guiding the administration of 5-fluorouracil. Background technique [0002] 5-fluorouracil (5-fluorouracil, 5-FU) is a pyrimidine fluoride, which belongs to anti-metabolism and anti-tumor drugs. It has a strong killing effect on proliferative cells and is one of the main chemotherapy drugs in clinical practice. , liver cancer and other tumors are effective. 5-FU must be converted into corresponding nucleotides in the body to exert the effect of inhibiting tumors. 5-FU is first converted into 5-fluoro-2-deoxyuridine nucleotides in vivo, and inhibits DNA biosynthesis by inhibiting thymidine nucleotide synthetase (TS). In addition, it can also be incorporated into RNA, preventing uracil from incorporating into RNA and inhibiting RNA synthesis. After 5-FU metabolism, there are two main active products: (1) fluorouracil triphosphate (FUTP), which binds to RNA and interferes with its function; (2) through the action of u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 文洁
Owner 北京奥维森基因科技有限公司
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