Primers, probes, kit and method for detecting polymorphism of human CYP2C19 gene

A technology of CYP2C19 and gene polymorphism, applied in the field of genetic engineering, can solve the problems of inability to accurately locate the position of gene changes, inability to accurately detect gene polymorphisms in samples, and expensive chips, etc., to achieve accurate fluorescence typing analysis and reduce costs. Intuitive and convenient effect of bottom signal strength and result interpretation

Inactive Publication Date: 2017-05-31
SHANGHAI TISSUEBANK MEDICAL LAB CO LTD +3
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  • 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

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  • Primers, probes, kit and method for detecting polymorphism of human CYP2C19 gene
  • Primers, probes, kit and method for detecting polymorphism of human CYP2C19 gene
  • Primers, probes, kit and method for detecting polymorphism of human CYP2C19 gene

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 CYP2C19*2 genes

[0047] In this example, the H2228 cell line containing the CYP2C19*2-GG genotype, the HCT116 cell line containing the CYP2C19*2-GA genotype, and the B7-549 cell line containing the CYP2C19*2-AA genotype were verified by sequencing. The feasibility and specificity of the invented detection method.

[0048] The specific detection method is as follows:

[0049] Using the PCR reaction system and reaction conditions described in this manual, DNA from the H2228 cell line, DNA from the HCT116 cell line and DNA from the B7-549 cell line 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 H2228DNA sample well is CYP2C19*2-GG type, the HCT116DNA...

Embodiment 2

[0052] Example 2: Using human genomic DNA samples to detect CYP2C19*2 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, the human genomic DNA sample was used as a template for fluorescent PCR genotyping detection. The genotyping results of 63 representative human genomic DNA samples are shown in the appendix of the manual. Figure 2A As shown, where NTC is the negative control, except for the positive control, 44 samples were CYP2C19*2-GG homozygous, 15 samples were CYP2C19*2-GA heterozygous, and...

Embodiment 3

[0057] Example 3: Using human genomic DNA samples to detect CYP2C19*2 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 9 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 the negative control. In addition to the positive control, 6 samples we...

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Abstract

The invention belongs to the technical field of gene engineering and discloses a composition of primers and probes for detecting polymorphism of human CYP2C19 gene, a kit containing the composition of the primers and the probes, and a fluorescence PCR method for detecting the polymorphism of the human CYP2C19 gene by using the composition of the primers and the probes or the kit. Based on a TaqMan fluorescence PCR technology, the primers, the probes and the kit are simple and rapid, and are high in sensitivity; in addition, through reasonable collocation of the primers and the probes, the interaction between the primers, the interaction between the primers and the probes and the interaction between the probes can be effectively avoided; the detection errors are reduced. When the primers, the probes and the kit, provided by the invention, are used for detecting the polymorphism of the human CYP2C19 gene, the primers, the probes and the kit has the advantages that the sensitivity is high, the specificity is high, the operation is simple, rapid and safe, the result is simply and intuitively determined and read, and blood samples or dried blood spots samples on filter paper can be directly used as templates.

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 CYP2C19 gene polymorphisms. Background technique [0002] CYP2C19 is an important member of the second subfamily of CYP450 enzymes and an important drug-metabolizing enzyme in the human body. About 2% of clinical drugs are metabolized by it. At present, the US FDA has announced that there are 19 drugs that use CYP2C19 as a pharmacogenomic biomarker, including clopidogrel, S-mephenytoin, omeprazole, voriconazole, diazepam, and norazepam. Gene polymorphisms of CYP2C19 can lead to individual differences in enzyme activity, causing the population to appear as an extensive metabolizer (EM), an intermediate metabolizer (IM) and a poor metabolizer (PM). There are many polymorphic sites in the CYP2C19 gene. The allelic types carried by Orientals are CYP2C19*2, CYP2C19*3 and CYP2C19*17. It can cause the loss of the enzyme...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/106C12Q2600/156C12Q2563/107
Inventor 郑仲征安琳芮立尤开
Owner SHANGHAI TISSUEBANK MEDICAL LAB CO LTD
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