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Detection kit for venous thrombosis risk assessment and detection method and application of detection kit

A detection kit and venous thrombosis technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of long detection cycle, easy pollution of amplification products, and cumbersome operation steps

Pending Publication Date: 2021-12-07
菲思特(上海)生物科技有限公司
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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 for venous thrombosis risk assessment and detection method and application of detection kit
  • Detection kit for venous thrombosis risk assessment and detection method and application of detection kit
  • Detection kit for venous thrombosis risk assessment and detection method and application of detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the preparation of kit

[0044] The rapid reaction kit of the present invention designs specific amplification primers and sequencing primers for PAI-1 (4G / 5G) and F5 (G1691A) 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. Gene polymorphism sequence is subject to the public sequence in Genebank.

[0045] (1) The primer sequences of this embodiment are as follows:

[0046]

[0047]

[0048] (2) The detection kit of the present embodiment comprises the following components:

[0049]

[0050] (3) The single serving configuration system of the detection kit PCR reaction solution of the present embodiment is as follows:

[0051] The concentrations of the components in the PCR reaction solution are: PAI-1 (4G / 5G) pre-prim...

Embodiment 2

[0054] Embodiment 2, pyrophosphate detection

[0055] The instruments adopted in the present invention are as follows: amplification instrument, pyrosequencer (Wuhan First Biotechnology Co., Ltd.).

[0056] (1) Reagent preparation (reagent preparation room)

[0057] 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.

[0058] (2) Sample testing (sample preparation room)

[0059] 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 s...

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Abstract

The invention discloses a detection kit for venous thrombosis risk assessment and a detection method and application of the detection kit. Markers for detecting venous thrombosis metabolism are PAI-1 (4G / 5G) and F5 (G1691A), the kit designs a specific amplification primer and a sequencing primer aiming at the polymorphism of the PAI-1 (4G / 5G) and F5 (G1691A), the kit comprises the following components: an amplification reaction solution, a PAI-1 (4G / 5G) sequencing primer, an F5 (G1691A) sequencing primer and a positive control. According to the detection kit, blood direct expansion, rapid amplification and optimized pyrosequencing technologies are combined to detect the gene polymorphism related to the venous thrombosis risk assessment, and suggestions from the gene perspective are provided for clinical personalized medication for the venous thrombosis risk assessment.

Description

technical field [0001] The invention relates to a detection kit for risk assessment of venous thrombosis, a detection method and application thereof, and belongs to the field of gene detection. Background technique [0002] Venous thromboembolism (VTE) is a disease caused by multiple factors such as genetics, environment, and behavior. 50% to 60% of the occurrence of VTE can be attributed to genetic factors. There are large differences. Protein C deficiency, protein S deficiency, and antithrombin deficiency are the main types of genetic defects in Asian populations, mainly manifested as deep vein thrombosis (DVT) and pulmonary embolism (PE). With the attention of clinical work on this disease and the development of medical and health services, the incidence of venous thrombosis in Asia is increasing year by year. Mutations and gene polymorphisms play an important role in the occurrence of diseases. Genetic factors showed obvious regional and racial differences. [0003] 4...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6869C12Q1/686C12N15/11
CPCC12Q1/6883C12Q1/6869C12Q1/686C12Q2600/106C12Q2600/156C12Q2565/301C12Q2537/143
Inventor 陈立波刘丹孙悦
Owner 菲思特(上海)生物科技有限公司
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