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Human phlebothrombosis risk gene F5 and PAI-1 polymorphism detection kit as well as preparation method and application thereof

A detection kit and venous thrombosis technology, applied in the field of biomedical clinical detection, can solve the problems of low detection trace sensitivity, unfavorable clinical promotion, and low sensitivity, improve detection efficiency, avoid false negative and false positive problems, and ensure The effect of accuracy

Active Publication Date: 2018-12-21
WUHAN HEALTHCHART BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most common method is the sequencing method, which is low in cost, but takes a long time and is not sensitive; the high-resolution melting curve method requires relatively high equipment requirements, which is not conducive to clinical promotion; the traditional fluorescent PCR method is widely used in clinical practice. However, the detection sensitivity is not high

Method used

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  • Human phlebothrombosis risk gene F5 and PAI-1 polymorphism detection kit as well as preparation method and application thereof
  • Human phlebothrombosis risk gene F5 and PAI-1 polymorphism detection kit as well as preparation method and application thereof
  • Human phlebothrombosis risk gene F5 and PAI-1 polymorphism detection kit as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A human venous thrombosis risk gene F5 and PAI-1 polymorphism detection kit, including detection reagent 1, detection reagent 2, positive control substance and negative control substance; the detection reagent 1 is aimed at the rs6025 polymorphism site of F5 gene , comprising: a forward primer whose sequence is shown in SEQ ID NO.1, a reverse primer whose sequence is shown in SEQ ID NO.2, a sequence shown in SEQ ID NO.3 and identification of a VIC fluorescent group at the 5' end Primer, sequence shown in SEQ ID NO.4 and 5' end carrying FAM fluorescent group identification primer, sequence shown in SEQ ID NO.5 and 3' end carrying BHQ quenching group PNA, sequence as SEQ ID NO .6 and the 3' end carries the PNA of the BHQ quenching group, the internal reference forward primer whose sequence is shown in SEQ ID NO.7, the internal reference reverse primer whose sequence is shown in SEQ ID NO.8, and the sequence such as SEQ ID NO.8 Internal reference identification primer show...

Embodiment 2

[0074] A human venous thrombosis risk gene F5 and PAI-1 polymorphism detection kit, including detection reagent 1, detection reagent 2, positive control and negative control; the components of the detection reagent 1 and detection reagent 2 are as follows in Table 1 , the composition of the kit is shown in Table 2:

[0075] Table 1 detection reagent composition

[0076]

[0077]

[0078] Table 2 Kit Composition

[0079]

Embodiment 3

[0081] The minimum detection limit of the sample detected using the kit prepared in Example 2 specifically includes the following steps:

[0082] (1) Use Tiangen Biochemical Biotechnology Company Whole Blood DNA Extraction Kit to extract 5 human peripheral blood genomic DNAs. The specific operation steps are strictly in accordance with the kit instructions to obtain human genomic DNA samples to be tested, and dilute the samples to 2ng / μL , 0.5ng / μL, 0.1ng / μL, 0.05ng / μL.

[0083] (2) Take PCR eight-tubes, add 20 μL of gradient dilution samples to each well; add 30 μL of well-mixed F5 detection reagent or 30 μL of PAI-1 detection reagent to each well; shake and centrifuge the eight-tubes to mix The reaction solution;

[0084] (3) Put the eight tubes into the ABI 7500 fluorescent PCR instrument for amplification detection. The PCR reaction conditions are shown in Table 3:

[0085] Table 3 PCR reaction program

[0086]

[0087] (4) Analysis results:

[0088] Analyzing the t...

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Abstract

The invention eblogns to the technical field of biology and specifically relates to a human phlebothrombosis risk gene F5 and PAI-1 polymorphism detection kit as well as a preparation method and application thereof. The kit is prepared from a detection reagent, a positive control product and a negative control product, wherein the detection reagent is used for amplifying gene F5 and PAI-1 polymorphism related to the human phlebothrombosis risk, wherein the detection reagent applied to the F5 gene polymorphism is prepared from a primer sequence, a PNA sequence and PCR reaction liquid; the primer sequence is used for amplifying an rs6025 site of F5 gene; the detection reagent applied to the PAI-1 gene polymorphism is prepared from a primer sequence, a PNA sequence and PCR reaction liquid; the primer sequence is used for amplifying an rs17999762 site of the PAI-1 gene. The kit disclosed by the invention is used for detecting the human aspirin resistance gene polymorphism and has the advantages of high flexibility, high specificity, convenience in operation, reliable results and the like; furthermore, detection can be finished within 1 hour, and result reading is simple and objective.

Description

technical field [0001] The invention belongs to the technical field of biomedical clinical detection, and in particular relates to a human venous thrombosis risk gene F5 and PAI-1 polymorphism detection kit and its preparation method and application. Background technique [0002] The formation of thrombus is the body's normal protective response to vessel wall damage. When the procoagulant stimulus exceeds the body's natural protection ability to inhibit thrombus formation, the thrombus grows excessively beyond the physiological range and causes damage to the body. The formation of blood clots (thrombosis) in veins is venous thrombosis, and the risk factors for venous thrombosis are mainly divided into two categories: one is acquired risk factors; the other is genetic risk factors, such as anti-protein S deficiency (type I) , coagulation factor ⅤLeiden (FVL), dysfibrinogenemia and other coagulation-related diseases. Among them, FVL is the most common genetic risk factor for...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6883C12Q1/6858
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2600/166C12Q2563/107C12Q2535/137C12Q2545/113C12Q2525/107
Inventor 马晓晶李倩李雪梅叶伦程弘夏陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH
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