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Primer group, kit and detection method for detecting polymorphism of risk genes of blood hypercoagulation or venous thrombosis and application

A gene polymorphism and venous thrombosis technology, applied in the field of molecular biology, can solve the problems of poor primer specificity, unfavorable clinical promotion, and low sensitivity, and achieve the effect of complete evaluation system, wide coverage of genes, and accurate detection results

Pending Publication Date: 2022-03-22
江苏意诺飞生物科技有限公司
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0010] At present, there are many commonly used methods for SNP detection, such as PCR gene chip method, PCR high-resolution fusion curve method, allele-specific PCR method, fluorescent quantitative PCR method, in situ hybridization, etc., but the current detection methods have limitations. The primers used have poor specificity, relatively high equipment requirements, low sensitivity or are not conducive to clinical promotion, etc.

Method used

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  • Primer group, kit and detection method for detecting polymorphism of risk genes of blood hypercoagulation or venous thrombosis and application
  • Primer group, kit and detection method for detecting polymorphism of risk genes of blood hypercoagulation or venous thrombosis and application
  • Primer group, kit and detection method for detecting polymorphism of risk genes of blood hypercoagulation or venous thrombosis and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] According to the polymorphic sites of PAI-1, MTHFR, F5, and TDAG8 (also called GPR65) genes in the dbSNP database, the dbSNP request number information, and the nucleotide sequences of each gene, design specific amplification primers.

[0060] Among them, the site PAI-1 (4G / 5G), the dbSNP access number is rs1799762; the site MTHFR (677C>T), the dbSNP access number is rs1801133; the site MTHFR (1298A>C), the dbSNP access number is rs1801131; For point F5 (41721G>A), the dbSNP accession number is rs6025; for the locus LINC01146(C>T)HIT1, the dbSNP accession number is rs1887289; for the locus GPR65(T>G)HIT2, the dbSNP accession number is rs3742704.

[0061] The partial amplified nucleotide sequence of each gene is as follows:

[0062] PAI-1 (4G / 5G): SEQ.ID.NO.1

[0063]

[0064] MTHFR(677C>T): SEQ.ID.NO.2

[0065]

[0066] MTHFR(1298A>C): SEQ.ID.NO.3

[0067]

[0068]

[0069] F5(41721G>A): SEQ.ID.NO.4

[0070]

[0071] LINC01146 (C>T) HIT1: SEQ.ID.NO.5 ...

Embodiment 2

[0080] According to embodiment 1, a primer set for detecting blood hypercoagulability or venous thrombosis risk gene polymorphism, the primers include:

[0081] (1) Primers for detecting PAI-1 (4G / 5G) polymorphisms, including primers shown in SEQ.ID.NO.7 and SEQ.ID.NO.8;

[0082] (2) primers for detecting MTHFR (677C>T) polymorphism, including primers shown in SEQ.ID.NO.9 and SEQ.ID.NO.10;

[0083] (3) primers for detecting MTHFR (1298A>C) polymorphism, including primers shown in SEQ.ID.NO.11 and SEQ.ID.NO.12;

[0084] (4) Primers for detecting F5 (41721G>A) polymorphism, including primers shown in SEQ.ID.NO.13 and SEQ.ID.NO.14;

[0085] (5) Primers for detecting LINC01146(C>T)HIT1 polymorphism, including primers shown in SEQ.ID.NO.15 and SEQ.ID.NO.16;

[0086] (6) Primers for detecting GPR65(T>G)HIT2 polymorphism, including primers shown in SEQ.ID.NO.17 and SEQ.ID.NO.18.

[0087] In this embodiment, a detection kit is prepared for detecting the polymorphism of blood hyperc...

Embodiment 3

[0102] In this example, using the genome fragments extracted from the blood cells of healthy pregnant women as templates, 6 pairs of primers designed for SNP sites related to venous blood hypercoagulability were amplified, and on this basis, a method for detecting human venous thrombosis susceptibility based on Sanger sequencing was established. . It mainly includes the following steps and reagents:

[0103] In this example, the primers and kits described in Examples 1 and 2 were used for detection.

[0104] In the kit, 2×Taq Master Mix as an amplification reagent contains Taq DNA Polymerase, dNTP and an optimized buffer system, which can be amplified only by adding primers and templates. The protective agent added to the amplification system makes the 2×Master Mix maintain stable activity after repeated freezing and thawing.

[0105] (1) Genomic DNA extraction: Use Kangwei CW2087 Blood Genomic DNA Column Small Extraction Kit to extract 200 μL EDTA anticoagulated blood. The ...

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Abstract

The invention relates to a blood hypercoagulation or venous thrombosis risk gene polymorphism detection primer group, a kit, a detection method and application, and belongs to the technical field of molecular biology. By designing the specific primer, a method and a risk evaluation system for detecting human venous thrombosis susceptibility based on Sanger sequencing are established, the primer can rapidly and qualitatively detect the blood hypercoagulation or venous thrombosis risk gene polymorphism, and the sensitivity of the human venous thrombosis risk gene can be greatly improved through one-time PCR amplification. The gene type of the to-be-detected site can be quickly and sensitively judged according to a sequencing result, so that the blood hypercoagulable or venous thrombosis risk gene can be accurately typed; the detection method provided by the invention is accurate and comprehensive in detection result of the blood hypercoagulation or venous thrombosis risk; the evaluation system is complete and can comprehensively detect the potential risk of the detected object, and the defect that the potential risk of the detected object cannot be comprehensively reflected due to the fact that other risk genes are ignored when a single gene or a small number of combined genes are detected is avoided.

Description

technical field [0001] The invention relates to a primer set, a kit, a detection method and an application for detecting a polymorphism of a blood hypercoagulability or venous thrombosis risk gene, and belongs to the technical field of molecular biology. Background technique [0002] The hypercoagulable state of blood refers to the state in which the blood is more likely to coagulate than normal, and is due to the activation of coagulation factors induced by a series of factors. Patients whose blood is in a hypercoagulable state have a high risk of thrombosis, and should be actively prevented, and antithrombotic and anticoagulant treatments should be taken in time. Venous thrombosis is one of the major diseases causing death and disability in the world. Recent studies have shown that the harm of venous thrombosis in Asia (especially China) is becoming more and more apparent. One of the factors for venous thrombosis includes genetic risk factors such as antiprotein S deficie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11C12Q1/6869
CPCC12Q1/6883C12Q1/6858C12Q1/6869C12Q2600/156C12Q2531/113C12Q2535/101
Inventor 黄新祥周冰钏
Owner 江苏意诺飞生物科技有限公司
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