Detection kit of paclitaxel metabolism marker and detection method and application thereof

A technology for detecting kits and metabolic markers, which is applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc. It can solve the problems of long detection cycle, strict detection conditions, and cumbersome operation steps.

Pending Publication Date: 2021-11-02
湖南菲思特精准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the sequencing method and the chip method have cumbersome operation steps, long detection cycle, and the amplification product is prone to contamination; the high-resolution melting curve method has simple steps, low specificity, and high requirements for equipment; The specific amplification method uses ARMS primers for specific amplification, and the design of the primers is difficult to optimize, and the detection conditions are strict.
Taqman fluorescent probe method has high test cost, and the amplification throughput of multiple genes is not high

Method used

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  • Detection kit of paclitaxel metabolism marker and detection method and application thereof
  • Detection kit of paclitaxel metabolism marker and detection method and application thereof
  • Detection kit of paclitaxel metabolism marker and detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, the preparation of kit

[0039] The kit of the present invention designs specific amplification primers and sequencing primers for CYP1B1 (432C / G) and ABCB1 (C3435T), and is used for constant temperature amplification and pyrosequencing detection. The design of primers based on the recombinant enzyme polymerase amplification technology is one of the keys of the present invention. The primer design of this technology cannot be carried out by auxiliary software, and can only rely on manual design. In order to ensure the amplification speed and detection sensitivity, the primer length should be controlled at 30-35 bp. If the primer design is too short, non-specific amplification will easily increase and cause false positives. If the primer design is too long, it will easily lead to failure of amplification. Gene polymorphism sequence is subject to the public sequence in Genebank.

[0040] (1) The primer sequences of this embodiment are as follows:

[0041] ...

Embodiment 2

[0051] Embodiment 2, pyrophosphate detection

[0052] The instruments adopted in the present invention are as follows: thermostat, pyrosequencer (Wuhan First Biotechnology Co., Ltd.).

[0053] (1) Reagent preparation (reagent preparation room)

[0054] Take out the reagent in advance, vortex reagent 1 for 15 seconds, and centrifuge at low speed for later use. Add 440ul of Reagent 1 directly to Reagent 2 (lyophilized), and vortex for 15 seconds to mix well. Determine the number of reactions N, N = number of samples to be tested (n) + number of quality control products (1) + blank control. It is recommended to conduct positive control and blank control analysis for each PCR experiment at the same time. Then the reaction solution was dispensed into PCR reaction tubes at 20 μL / tube.

[0055] (2) Sample testing (sample preparation room)

[0056] Add the sample DNA, positive control and blank control into the PCR reaction tube according to the amount of 5 μL, close the cap tigh...

Embodiment 3

[0081] Example 3. Correlation between genetic testing results and paclitaxel curative effect and adverse reactions

[0082] Correspondence between gene loci and drugs

[0083]

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Abstract

The invention discloses a detection kit of paclitaxel metabolism markers as well as a detection method and application of the detection kit. The detection kit is used for detecting gene polymorphism of two genes CYP1B1 432C/G and ABCB1C3435T of the paclitaxel metabolism markers, and the kit comprises the following components: a CYP1B1 432C/G amplification primer, a CYP1B1 432C/G sequencing primer, an ABCB1C3435T amplification primer, an ABCB1C3435T sequencing primer and a positive control. According to the present invention, the multiple RPA amplification and the optimized pyrosequencing technology are adopted as the combination to detect the gene polymorphism related to the prediction of the treatment effect and the adverse reaction of the paclitaxel, and the kit can simultaneously detect the CYP1B1 (432C/G) and the ABCB1 (C3435T) gene polymorphism paclitaxel so as to provide the gene perspective suggestion for the clinical personalized medication.

Description

technical field [0001] The invention relates to a paclitaxel metabolic marker detection kit, a detection method and an application thereof, and belongs to the field of gene detection. Background technique [0002] Paclitaxel, a natural anticancer drug, as a diterpene alkaloid compound with anticancer activity, has been widely used clinically in the treatment of breast cancer, ovarian cancer, some head and neck cancers and lung cancer. Microtubules, a component of eukaryotic cells, are formed by dimers of microtubules composed of two similar polypeptide (a and p) subunits. Under normal conditions, there is a dynamic equilibrium between microtubules and tubulin dimers. Paclitaxel can lose this dynamic balance between the two, induce and promote tubulin polymerization, prevent depolymerization, and stabilize microtubules. Paclitaxel inhibits the normal dynamic regeneration of the microtubule network necessary for mitosis, prevents the formation of the normal mitotic spindle, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6844C12Q1/6869C12N15/11
CPCC12Q1/6886C12Q1/6844C12Q1/6869C12Q2600/156C12Q2600/106C12Q2521/507C12Q2522/101C12Q2537/143C12Q2565/301
Inventor 刘丹易倩春
Owner 湖南菲思特精准医疗科技有限公司
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