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Detection kit for cis-platinum metabolism marker and detection method and application thereof

A technology of metabolic markers and detection kits, applied in the field of gene detection, can solve the problems of long detection period, strict detection conditions, and complicated operation steps.

Inactive Publication Date: 2021-10-01
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 products are prone to contamination; the high-resolution melting curve method has low specificity and high requirements for equipment; allele-specific amplification The augmentation method uses ARMS primers for specific amplification, and its primer design 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 for cis-platinum metabolism marker and detection method and application thereof
  • Detection kit for cis-platinum metabolism marker and detection method and application thereof
  • Detection kit for cis-platinum metabolism marker and detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the preparation of kit

[0038] The kit of the present invention designs specific amplification primers and sequencing primers for TPMT*3C (A719G) and XPC (C2815A), 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.

[0039] (1) The primer sequences of this embodiment are as follows:

[0040]

...

Embodiment 2

[0050] Embodiment 2, pyrophosphate detection

[0051] The instruments adopted in the present invention are as follows: thermostat, pyrosequencer (Wuhan First Biotechnology Co., Ltd.).

[0052] (1) Reagent preparation (reagent preparation room)

[0053] 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.

[0054] (2) Sample testing (sample preparation room)

[0055] 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

[0080] Example 3. Correlation between genetic test results and cisplatin adverse reaction prediction

[0081] Correspondence between gene loci and drugs

[0082]

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Abstract

The invention discloses a detection kit for a cis-platinum metabolism marker and a detection method and application thereof. The kit is used for detecting gene polymorphism of two genes of the cis-platinum metabolism marker, namely TPMT*3C A719G and XPC C2815A. The kit comprises the following components: a TPMT*3C A719G amplification primer, a TPMT*3C A719G sequencing primer, an XPC C2815A amplification primer, an XPC C2815A sequencing primer and a positive control. According to the invention, multiplex RPA amplification and an optimized pyrosequencing technology are combined to detect the gene polymorphism related to the cisplatin adverse reaction prediction, and the kit can simultaneously detect cisplatin drugs with TPMT*3C (A719G) and XPC (C2815A) gene polymorphism so as to provide the suggestion for clinical personalized medication in the gene perspective.

Description

technical field [0001] The invention relates to a detection kit for cisplatin metabolic markers and a detection method and application thereof, belonging to the field of gene detection. Background technique [0002] Cisplatin has the characteristics of broad anti-cancer spectrum, strong effect, synergistic effect with many anti-tumor drugs, and no cross-drug resistance. It is one of the most commonly used drugs in current combination chemotherapy. Similar to bifunctional alkylating agents, it can inhibit the replication process of DNA. Cisplatin is widely used in the treatment of a variety of solid tumors, and ototoxicity is one of its main adverse reactions. The incidence of cisplatin-induced ototoxicity in children was as high as 61%, with bilateral hearing loss in most cases and often irreversible hearing loss. There are large individual differences in the clinical efficacy and adverse drug reactions of cisplatin drugs, so how to use thiopurine drugs for treatment reaso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6886C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2600/16C12Q2600/166C12Q2600/172C12Q2531/119C12Q2537/143C12Q2535/122C12Q2565/301C12Q2537/1376C12Q2521/507C12Q2522/101C12Q2545/113
Inventor 刘丹易倩春
Owner 湖南菲思特精准医疗科技有限公司