CN117551759A - Detection primer probe group and kit for detecting FCER2 gene polymorphism - Google Patents

Abstract

The application discloses a detect primer probe group and kit for detecting FCER2 gene polymorphism, detect primer probe group including forward primer and reverse primer of targeted FCER2 gene: the wild type probe and the mutant type probe of the targeted FCER2 gene are characterized in that a first fluorescent group is modified at the 5 'end of the wild type probe, a first fluorescence quenching group is modified at the 3' end of the wild type probe, a second fluorescent group is modified at the 5 'end of the wild type probe, and a second fluorescence quenching group is modified at the 3' end of the wild type probe. Specific primers and probes are constructed aiming at the rs28364072 locus of the FCER2 gene, and the qPCR method is adopted, so that the specificity is high, the detection cost is low, the experimental method is stable, the FCER2 gene polymorphism can be rapidly detected, and the treatment and prevention of bronchial asthma are facilitated. Meanwhile, by adopting a double-probe design and matching with a primer with high specificity, the accuracy and stability of a detection result can be greatly improved.

Description

Detection primer probe group and kit for detecting FCER2 gene polymorphism

Technical Field

The application belongs to the technical field of genetic engineering, and in particular relates to a detection primer probe group and a kit for detecting FCER2 gene polymorphism.

Background

Bronchial asthma (bronchial asthma) is a heterogeneous disease characterized by chronic inflammation of the airways involving various cells (e.g. eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components.

The inhaled hormone is the most effective drug for inhibiting airway mucosa inflammation, can quickly and directly reach local inflammation parts after being inhaled, directly inhibit inflammatory cells related to asthma, reduce inflammatory mediators released by the inflammatory cells, alleviate mucosa edema, inhibit airway mucosa gland hypersecretion, increase mucus clearance and reduce tracheal hyperreactivity. Inhaled glucocorticoids (ICS) are the most effective first-line drug for asthma treatment, and studies have shown that the mutation sites of several genes on the glucocorticoid pathway, including the cci1, CRHR1, NR3C1, FCER2, STIP1, DUSP1 genes, ADCY9, TBX21, NK2R genes, and also the drug metabolizing enzymes CYP3A4, asthma susceptibility gene ORMDL3, are involved in the clinical phenotype of asthma and the response of ICS in asthma treatment. Wherein the Glucocorticoid Receptor (GR) gene (NR 3C 1) is located on chromosome 5q31-32, and variation of the NR3C1 gene affects GR conformation and activity, and indirectly alters patient response to ICS by altering inflammatory mechanisms of asthma by encoding T-bet transcription factors and helping to enhance TH1 lymphocyte populations, and IgE receptor II (FCER 2).

The FCER2 (Fc epsilonreceptor II) gene encodes a protein that is a B cell specific antigen and is a low affinity receptor for IgE. It plays an important role in B cell growth and differentiation and in regulating IgE production. When the FCER2 gene is subject to single nucleotide variation, there is a risk of severe exacerbation of asthma even if ICS is used by the patient, so that the patient needs to increase daily ICS dose. Therefore, the mutation status of the FCER2 gene of a patient is clarified through gene detection, which is very important for the treatment and prevention of bronchial asthma, and no kit for detecting FCER2 gene polymorphism exists at present.

Disclosure of Invention

The invention aims to provide a detection primer probe group and a kit for detecting FCER2 gene polymorphism, which are used for solving the technical problems that in the prior art, the kit for detecting FCER2 gene polymorphism is not available, the site mutation condition of the FCER2 gene of a patient is difficult to be clarified, and the treatment and the prevention of bronchial asthma are not facilitated.

In order to achieve the above purpose, a technical scheme adopted in the application is as follows:

provided is a detection primer probe set for detecting FCER2 gene polymorphism, comprising:

forward primer and reverse primer of the rs28364072 locus of the targeted FCER2 gene, wherein the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2.

In one or more embodiments, further comprising: wild type probes and mutant probes targeting the rs28364072 locus of the FCER2 gene;

the sequence of the wild type probe is shown as SEQ ID NO.3, a first fluorescent group is modified at the 5 'end of the wild type probe, and a first fluorescence quenching group is modified at the 3' end of the wild type probe;

the sequence of the mutant probe is shown as SEQ ID NO.4, a second fluorescent group is modified at the 5 'end of the mutant probe, and a second fluorescence quenching group is modified at the 3' end of the mutant probe.

In one or more embodiments, the first fluorescent group is FAM, the second fluorescent group is VIC, and the first and second fluorescence quenching groups are BHQ1.

In order to achieve the above purpose, a technical scheme adopted in the application is as follows:

there is provided a kit for detecting FCER2 gene polymorphism, comprising:

a forward primer and a reverse primer of the rs28364072 locus of the targeted FCER2 gene, wherein the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2;

PCR reaction reagent.

In one or more embodiments, the PCR reaction reagents include 2 x Phanta Max Master Mix;

the reaction system of the kit is configured to:

2X Phanta Max Master Mix uL, 10pmol/uL forward primer 2uL, 10pmol/uL reverse primer 2uL, 1uL of DNA to be detected, and 20uL of sterile water without enzyme.

In order to achieve the above purpose, a technical scheme adopted in the application is as follows:

there is provided a kit for detecting FCER2 gene polymorphism, comprising:

a forward primer and a reverse primer of the rs28364072 locus of the targeted FCER2 gene, wherein the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2;

a wild type probe and a mutant type probe of an rs28364072 locus of the targeted FCER2 gene, wherein the sequence of the wild type probe is shown as SEQ ID NO.3, a first fluorescent group is modified at the 5 'end of the wild type probe, a first fluorescence quenching group is modified at the 3' end of the wild type probe, the sequence of the mutant type probe is shown as SEQ ID NO.4, a second fluorescent group is modified at the 5 'end of the mutant type probe, and a second fluorescence quenching group is modified at the 3' end of the mutant type probe;

qPCR reaction reagent.

In one or more embodiments, the qPCR reaction reagents include 2 x AceQ qPCR Probe Master Mix;

the reaction system of the kit is configured to:

2X AceQ qPCRProbe MasterMix uL, 8pmol/uL forward primer 0.4uL, 8pmol/uL reverse primer 0.4uL, 5pmol/uL wild-type probe 0.4uL, 5pmol/uL mutant probe 0.4uL, 5uL of DNA to be detected, and 3.4uL of sterile, enzyme-free water.

In one or more embodiments, the first fluorescent group is FAM, the second fluorescent group is VIC, and the first and second fluorescence quenching groups are BHQ1.

The beneficial effect of this application is, in contrast to prior art:

specific primers and probes are constructed aiming at the rs28364072 locus of the FCER2 gene, and the qPCR method is adopted, so that the specificity is high, the detection cost is low, the experimental method is stable, the FCER2 gene polymorphism can be rapidly detected, and the treatment and prevention of bronchial asthma are facilitated. Meanwhile, by adopting a double-probe design and matching with a primer with high specificity, the accuracy and stability of a detection result can be greatly improved.

Detailed Description

In order to better understand the technical solutions in the present application, the following description will clearly and completely describe the technical solutions in the embodiments of the present application in conjunction with the embodiments of the present application, and it is obvious that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.

The applicant provides a kit for detecting a single nucleotide polymorphism of the FCER2 gene, wherein the single nucleotide polymorphism (single nucleotide polymorphism, SNP) mainly refers to a DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. It is the most common one of the human heritable variants, accounting for over 90% of all known polymorphisms.

Specifically, the kit of the present application includes a forward primer and a reverse primer targeting the rs28364072 locus of the FCER2 gene.

In one embodiment, the forward and reverse primer sequences are as follows:

forward primer: CGAGAGGTTGGGTAGAGT (SEQ ID NO. 1);

reverse primer: CTGTGACGACATGGAAGG (SEQ ID NO. 2).

To further enable fluorescent quantitative analysis, in one embodiment, the kit may further include a wild-type probe and a mutant probe targeting the rs28364072 locus of the FCER2 gene.

Specifically, the sequences of the wild-type probe and the mutant probe may be as follows:

wild type probe: FAM-TGCCCACCACCTCTGCAGAGCCCCAGC (SEQ id No. 3) -BHQ1;

mutant probes: VIC-AGAGCCCCGGCCCACCTGCTCCTCCGG (SEQ ID NO. 4) -BHQ1.

In other embodiments, the 5' ends of the wild type probe and the mutant type probe can be modified with other fluorescent groups, and the 3' ends of the wild type probe and the mutant type probe can be modified with other fluorescent quenching groups, so that the 5' ends of the wild type probe and the mutant type probe are modified with different fluorescent groups.

In one embodiment, a kit may include: the forward primer, the reverse primer and the PCR reaction reagent.

Wherein the PCR reagent may be a PCR pre-mix solution, such as 2X Phanta Max Master Mix; of course, in other embodiments, the PCR reagents may also include PCR buffers, dNTPs, DNA polymerases, and the like.

In another embodiment, a kit may include: the forward primer, reverse primer, wild type probe, mutant probe and qPCR reaction reagent.

Wherein the qPCR reaction reagent may comprise a qPCR pre-mix solution, such as 2 x AceQ qPCRProbe Master Mix; of course, in other embodiments, qPCR reagents may also include PCR buffers, dntps, DNA polymerase, and the like.

The following describes the advantageous effects of the technical scheme of the present application in further detail in conjunction with specific examples.

Example 1:

whole blood samples of 5 volunteers were taken, and 200uL of the whole blood sample of each volunteer was used to extract genomic DNA using the Norvezan whole blood extraction kit. The extracted genomic DNA was quantified using a qubit kit and the DNA extraction concentration was recorded.

The reaction system was configured according to the following table:

of these, 2× Phanta Max MasterMix is a PCR mix reagent of novinay.

PCR amplification was performed based on the above reaction system, and the PCR reaction procedure was as follows:

after the running procedure is finished, 5ul of PCR products are taken for gel electrophoresis detection, and whether the band is single is checked.

The detection shows that the bands of each sample are single, and the target fragment is between 100 and 200 bp. The remaining PCR products were then subjected to a first generation sequencing validation. Comparing the sequence of the first generation sequencing result with the original target sequence, and confirming whether the sequence is consistent with the original target sequence or not to obtain the following data:

of the 5 samples, 3 samples were AA homozygotes and 2 samples were AG heterozygotes.

The embodiment carries out PCR amplification based on the specific primers, and the genotype of the sample is confirmed by comparing sequences through first-generation sequencing, so that the specificity is high, the detection cost is low and the experimental method is stable.

Example 2:

oral epithelial cell samples of 30 volunteers were collected, the swab with the oral epithelial cells was placed into a centrifuge tube containing 1ml of 1xPBS buffer, the tube was thoroughly shaken, transiently centrifuged, the swab was removed, and the tube was placed into a centrifuge and centrifuged at 4000rpm for 10 minutes. The supernatant was discarded, the cell pellet in the centrifuge tube was retained, and genomic DNA of the cell pellet was extracted using a cell extraction kit for novzan.

The reaction system was configured according to the following table:

component (A)	Volume (ul)
		2×AceQqPCRProbeMasterMix	10
Forward primer (8 pmol/ul)	0.4
		Reverse primer (8 pmol/ul)	0.4
Wild type probe (5 pmol/ul)	0.4
		Mutant probe (5 pmol/ul)	0.4
DNA	5
		Asepsis water without enzyme	3.4
Total volume of	20

Of these, 2× AceQ qPCRProbe MasterMix is a qPCR reagent of novinay.

PCR amplification was performed based on the above reaction system, and the PCR reaction procedure was as follows:

and after the running program is finished, performing fluorescence analysis on the result, and judging the result according to the amplification curve.

Specifically, the result determination criteria are as follows:

FAM with amplification curve	VIC has an amplification curve	FAM and VIC all have curves
			AA	GG	AG

The result determination was performed on 30 samples according to the above-described determination criteria, and the results were as follows:

genotype of the type	AA	GG	AG
				Quantity of	20	2	8

In the embodiment, a qPCR method is adopted, so that the specificity is high, the detection cost is low, and the experimental method is stable. Meanwhile, by adopting a double-probe design and matching with a primer with high specificity, the accuracy and stability of a detection result can be greatly improved. Compared with the gene chip and the second generation sequencing, the method has the advantages of short time consumption, simple steps and easy operation. The content is clear, the understanding is easy, and the result interpretation is simpler.

It will be evident to those skilled in the art that the present application is not limited to the details of the foregoing illustrative embodiments, and that the present application may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims (8)

1. A detection primer probe set for detecting FCER2 gene polymorphism, comprising:

forward primer and reverse primer of the rs28364072 locus of the targeted FCER2 gene, wherein the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2.

2. The set of detection primer probes of claim 1, further comprising: wild type probes and mutant probes targeting the rs28364072 locus of the FCER2 gene;

the sequence of the wild type probe is shown as SEQ ID NO.3, a first fluorescent group is modified at the 5 'end of the wild type probe, and a first fluorescence quenching group is modified at the 3' end of the wild type probe;

the sequence of the mutant probe is shown as SEQ ID NO.4, a second fluorescent group is modified at the 5 'end of the mutant probe, and a second fluorescence quenching group is modified at the 3' end of the mutant probe.

3. The set of detection primer probes of claim 2, wherein the first fluorophore is FAM, the second fluorophore is VIC, and the first and second fluorescence quenching groups are BHQ1.

4. A kit for detecting FCER2 gene polymorphism, comprising:

a forward primer and a reverse primer of the rs28364072 locus of the targeted FCER2 gene, wherein the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2;

PCR reaction reagent.

5. The kit of claim 4, wherein the PCR reaction reagents comprise 2 x PhantaMax MasterMix;

the reaction system of the kit is configured to:

2X Phanta Max Master Mix uL, 10pmol/uL forward primer 2uL, 10pmol/uL reverse primer 2uL, 1uL of DNA to be detected, and 20uL of sterile water without enzyme.

6. A kit for detecting FCER2 gene polymorphism, comprising:

a forward primer and a reverse primer of the rs28364072 locus of the targeted FCER2 gene, wherein the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2;

a wild type probe and a mutant type probe of an rs28364072 locus of the targeted FCER2 gene, wherein the sequence of the wild type probe is shown as SEQ ID NO.3, a first fluorescent group is modified at the 5 'end of the wild type probe, a first fluorescence quenching group is modified at the 3' end of the wild type probe, the sequence of the mutant type probe is shown as SEQ ID NO.4, a second fluorescent group is modified at the 5 'end of the mutant type probe, and a second fluorescence quenching group is modified at the 3' end of the mutant type probe;

qPCR reaction reagent.

7. The kit of claim 6, wherein the qPCR reaction reagents comprise 2 x AceQ qPCR Probe MasterMix;

the reaction system of the kit is configured to:

2X AceQ qPCRProbe MasterMix uL, 8pmol/uL forward primer 0.4uL, 8pmol/uL reverse primer 0.4uL, 5pmol/uL wild-type probe 0.4uL, 5pmol/uL mutant probe 0.4uL, 5uL of DNA to be detected, and 3.4uL of sterile, enzyme-free water.

8. The kit of claim 6 or 7, wherein the first fluorescent moiety is FAM, the second fluorescent moiety is VIC, and the first and second fluorescence quenching moieties are BHQ1.