Primers, probes, kit and method for testing human VKORC1 and CYP2C9 gene polymorphisms

A gene polymorphism and kit technology, applied in the field of genetic engineering, can solve the problems of inability to accurately locate gene changes, expensive chips, inability to accurately detect gene polymorphisms in samples, etc., and achieve intuitive and convenient result interpretation and fluorescence analysis. Accurate analysis and the effect of reducing background signal strength

Inactive Publication Date: 2017-05-24
SHENZHEN TISSUEBANK PRECISION MEDICINE CO LTD +3
View PDF6 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the sequencing method is the "gold standard" for gene polymorphism detection, it requires a long detection time, complicated operation, and low sensitivity; the PCR-gene chip method has high operational requirements, and hybridization and washing must be protected from light operation, and the chip is expensive; the PCR-melting curve method is non-specific and cannot precisely locate the position of the gene change, and the difference in Tm caused by a single base difference is not very obvious, which usually results in inconspicuous peaks on the melting curve Therefore, there are certain difficulties in the clinical promotion of these three methods
The traditional fl...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primers, probes, kit and method for testing human VKORC1 and CYP2C9 gene polymorphisms
  • Primers, probes, kit and method for testing human VKORC1 and CYP2C9 gene polymorphisms
  • Primers, probes, kit and method for testing human VKORC1 and CYP2C9 gene polymorphisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Verification of the specificity of the detection system of the present invention using cell line DNA samples containing polymorphic sites of VKORC1 genes

[0047] In this example, the Kasumi-1 cell line containing the VKORC1-TT genotype, the HCT116 cell line containing the VKORC1-TC genotype, and the H2228 cell line containing the VKORC1-CC genotype were determined by sequencing to verify the feasibility of the detection method of the present invention and specificity.

[0048] The specific detection method is as follows:

[0049] Using the PCR reaction system and reaction conditions described in this specification, the Kasumi-1 cell line DNA, the HCT116 cell line DNA and the H2228 cell line DNA were used as templates, and each DNA sample was spotted once for detection.

[0050] The genotyping results of the test are shown in the attached instructions figure 1 shown. Among them, NTC is the negative control, the Kasumi-1DNA sample well is VKORC1-TT type, th...

Embodiment 2

[0052] Example 2: Using human genomic DNA samples to detect VKORC1 gene polymorphisms to verify the repeatability of the detection system of the present invention

[0053] In this example, human genomic DNA samples were used as templates for fluorescent PCR genotyping detection, and the detection results were directly compared with the "gold standard" sequencing results to verify the repeatability and accuracy of the detection system of the present invention.

[0054] The specific detection method is as follows:

[0055] Using the PCR reaction system and reaction conditions described in this manual, human genomic DNA samples were used as templates for fluorescent PCR genotyping detection. The genotyping results of 82 representative human genomic DNA samples are shown in the appendix of the manual. Figure 2A shown. Among them, NTC was the negative control. Except for the positive control, 68 samples were VKORC1-TT homozygous and 14 samples were VKORC1-TC heterozygous. Among ...

Embodiment 3

[0057] Example 3: Using human genomic DNA samples to detect VKORC1 gene polymorphisms to verify the sensitivity of the detection system of the present invention

[0058] In this embodiment, the sensitivity of the detection system of the present invention is also verified by using the human genome DNA sample as a template.

[0059] The specific detection method is as follows:

[0060] Using the PCR reaction system and reaction conditions described in this manual, select 7 cases of human genomic DNA samples as templates for fluorescent PCR genotyping detection, and set the detection limit groups of each DNA template as follows: 0.05ng; 0.1ng; 0.5ng ;1ng;10ng;50ng;100ng. The results show that clear genotyping results can still be obtained under the condition that the amount of DNA template is only 1ng. The representative genotyping results are shown in the attached instructions. image 3 As shown, NTC is a negative control. In addition to the positive control, 5 cases were VKOR...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a primer and probe combination for testing human VKORC1 and CYP2C9 gene polymorphisms, a kit containing the primer and probe combination and a fluorescent PCR (polymerase chain reaction) method for testing human VKORC1 and CYP2C9 gene polymorphisms by adopting the primer and probe combination. The invention, which is based on the TaqMan fluorescent PCR technique, is simple, efficient and highly sensitive. In addition, by reasonably matching primers with probes, the interaction between the primers, the interaction between the primers and the probes and the interaction between the probes are effectively prevented, thus reducing testing errors. When used for testing human VKORC1 and CYP2C9 gene polymorphisms, the primers, the probes and the kit which are provided by the invention have the advantages of high sensitivity, high specificity, easiness, quickness and safety in operation, simple and visual result judgment and the like, and can directly use a blood sample or a dried blood spot sample as a template.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, probes, kits and methods for detecting human VKORC1 and CYP2C9 gene polymorphisms. Background technique [0002] Warfarin is a commonly used anticoagulant drug in clinical practice, and it is the first-line drug for diseases such as deep vein thrombosis, atrial fibrillation, heart valve replacement and pulmonary embolism. However, the therapeutic window of warfarin is very narrow: if the dose is small, it cannot prevent the formation of thrombus; At present, more than 30 genes related to warfarin pharmacokinetics and pharmacodynamics are known, among which vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P450 2C9 (CytochromeP4502C9 , CYP2C9) gene polymorphism is the main factor influencing the individual dose difference of warfarin. [0003] There are wild-type CYP2C9*1 and mutant CYP2C9*2~CYP2C9*13 in the CYP2C9 gene, among which the m...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2561/101C12Q2563/107
Inventor 郑仲征安琳芮立尤开
Owner SHENZHEN TISSUEBANK PRECISION MEDICINE CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap