Rapid reaction kit for predicting dosage of traserine, and detection method and application thereof

A dose prediction, rapid response technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of high test cost, difficult primer design, and low throughput of multiple gene amplification. question

Pending Publication Date: 2021-10-19
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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  • Rapid reaction kit for predicting dosage of traserine, and detection method and application thereof
  • Rapid reaction kit for predicting dosage of traserine, and detection method and application thereof
  • Rapid reaction kit for predicting dosage of traserine, and detection method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the preparation of kit

[0046] The rapid reaction kit of the present invention designs specific amplification primers and sequencing primers for CYP2C19 (681G>A) and CYP2C19 (636G>A) for amplification and pyrosequencing detection. Designing primers based on rapid amplification technology is one of the keys of the present invention. In order to ensure the amplification speed and detection sensitivity, the amplification length should be controlled at 60-120bp as much as possible. Gene polymorphism sequence is subject to the public sequence in Genebank.

[0047] (1) The primer sequences of this embodiment are as follows:

[0048]

[0049] (2) The detection kit of the present embodiment comprises the following components:

[0050]

[0051]

[0052] (3) The single serving configuration system of the detection kit PCR reaction solution of the present embodiment is as follows:

[0053] The final concentrations of each component of the PCR reaction so...

Embodiment 2

[0057] Embodiment 2, pyrophosphate detection

[0058] The instruments adopted in the present invention are as follows: amplification instrument, pyrosequencer (Wuhan First Biotechnology Co., Ltd.).

[0059] (1) Reagent preparation (reagent preparation room)

[0060] Take out the reagents in advance, vortex the PCR reaction solution for 15 seconds, and centrifuge at low speed for later use. 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 16 μL / tube.

[0061] (2) Sample testing (sample preparation room)

[0062] Add EDTA anticoagulated whole blood, positive control and blank control into the PCR reaction tube according to the sample volume of 4 μL, close the tube cap tightly, centrifuge at low speed for 15 seco...

Embodiment 3

[0089] Example 3, Amplification efficiency under different EDTA anticoagulated whole blood volumes

[0090] Blood Direct PCR Master Mix (2×), containing blood resistant HemoTaq TM DNA polymerase and anti-inhibitor, showing super resistance to various PCR inhibitors such as heme in whole blood. EDTA anticoagulated whole blood was added to account for 5%, 10%, 20%, 30%, 40%, and 50% of the volume of the PCR reaction solution, respectively. Tested for maximum spiked whole blood sample volume. For test results, see figure 1 , the test showed that 20% volume EDTA anticoagulated whole blood had no effect on the amplification efficiency.

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Abstract

The invention discloses a rapid reaction kit for predicting the dosage of traserine, and a detection method and application thereof. The detection kit is used for detecting the gene polymorphism of two genes of traserine metabolic markers CYP2C19 681G>A and CYP2C19 636G>A. The kit comprises a CYP2C19 681G>A amplification primer, a CYP2C19 681G>A sequencing primer, a CYP2C19 636G>A amplification primer, a CYP2C19 636G>A sequencing primer and a positive control. According to the present invention, the multiple RPA amplification and the optimized pyrosequencing technology are combined to detect the gene polymorphism related to the traserine drug treatment effect prediction, and the kit can simultaneously detect the CYP2C19 (681G>A) gene polymorphism and the CYP2C19 (636G>A) gene polymorphism so as to provide the gene perspective suggestion for the clinical personalized medication.

Description

technical field [0001] The invention relates to a rapid response kit for dose prediction of Traserin, a detection method and application thereof, and belongs to the field of gene detection. Background technique [0002] Traserline is a selective serotonin reuptake inhibitor (SSRIs) that increases serotonergic activity by reducing presynaptic serotonin reuptake. Traserline is a common antidepressant used in pediatric patients for various indications. Traserline is metabolized primarily by the cytochrome P450 2C19 (CYP2C19) enzyme, with contributions from other enzymes (eg, cytochrome P450 2D6) to a lesser extent. Genetic variation in CYP2C19 leads to individual differences in CYP2C19 function. PM individuals may experience toxic side effects when using conventional doses of drugs, and the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines recommend considering reducing the initial sertraline dose by 50% in PM individuals. EM and intermediate metabolizers...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2565/301
Inventor 张玉曾芳周涛龚卫静
Owner 湖南菲思特精准医疗科技有限公司
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