阅读说明：本技术 基于探针法实现维D受体Fok1位点准确分型的引物组合物、试剂、检测方法和系统 (Primer composition, reagent, detection method and system for realizing accurate typing of vitamin D receptor Fok1 locus based on probe method ) 是由 周梅华 龚强 余艳 李慧源 葛虎 陈鹏 代冰 于 2020-04-23 设计创作，主要内容包括：本发明提供一种基于探针法实现维D受体Fok1位点准确分型的引物组合物、试剂、检测方法和系统。所述基于探针法实现维D受体Fok1位点准确分型的引物组合物为根据维生素D受体基因Fok1(rs2228570)SNP位点多态性设计的引物和探针；基于探针法实现维D受体Fok1位点准确分型的试剂、检测方法和系统分别为采用了所述基于探针法实现维D受体Fok1位点准确分型的引物组合物的试剂、检测方法和系统。本发明提供的所述基于探针法实现维D受体Fok1位点准确分型的引物组合物解决了现有技术的Fok1位点多态性检测存在设备昂贵、气溶胶污染、步骤繁琐等技术问题。(The invention provides a primer composition, a reagent, a detection method and a system for realizing accurate typing of a vitamin D receptor Fok1 locus based on a probe method. The primer composition for realizing accurate typing of the vitamin D receptor Fok1 site based on the probe method is a primer and a probe designed according to the vitamin D receptor gene Fok1(rs2228570) SNP site polymorphism; the reagent, the detection method and the system for realizing the accurate typing of the vitamin D receptor Fok1 locus based on the probe method are respectively the reagent, the detection method and the system which adopt the primer composition for realizing the accurate typing of the vitamin D receptor Fok1 locus based on the probe method. The primer composition for realizing accurate typing of the vitamin D receptor Fok1 locus based on the probe method solves the technical problems of expensive equipment, aerosol pollution, complicated steps and the like in Fok1 locus polymorphism detection in the prior art.)

1. A primer composition for realizing accurate typing of a vitamin D receptor Fok1 site based on a probe method comprises a primer and a probe which are designed according to the polymorphism of a vitamin D receptor gene Fok1(rs2228570) SNP site, and is characterized in that the sequence is as follows:

forward amplification primer for VDR gene Fok1(rs 2228570):

CTGGCCCTGGCACTGACTCTGGCTCT；

reverse amplification primer of VDR gene Fok1(rs 2228570):

GGTCAAAGTCTCCAGGGTCAGG；

VDR gene Fok1(rs2228570) genotype T probe:

5’-CTTACAGGGATGGAG-3’；

VDR gene Fok1(rs2228570) genotype C probe:

5’-CTTACAGGGACGGAG-3’。

2. the primer composition for realizing accurate typing of the Fok1 locus of the vitamin D receptor based on the probe method according to claim 1, wherein the primer composition comprises the following components in percentage by weight: the fluorescent group at the 5 'end of the genotype T probe is VIC, and the quenching groups at the 3' end of the genotype T probe are Quencher and MGB.

3. The primer composition for realizing accurate typing of the Fok1 locus of the vitamin D receptor based on the probe method according to claim 1, wherein the primer composition comprises the following components in percentage by weight: the fluorescent group at the 5 'end of the genotype C probe is FAM, and the quenching group at the 3' end of the genotype C probe is Quencher and MGB.

4. The primer composition for realizing accurate typing of the vitamin D receptor Fok1 site based on the probe method according to any one of claims 1 to 3, wherein the primer and the probe exist in the form of a mixed solution, and the ratio is as follows: (the forward amplification primer: the reverse amplification primer: the genotype T probe: the genotype C probe: water) ═ 10:10:4:4: 192).

5. A reagent for realizing accurate typing of a vitamin D receptor Fok1 site based on a probe method is characterized in that: comprises the primer composition for realizing accurate typing of the Fok1 site of the dimension D receptor based on the probe method as claimed in any one of claims 1 to 4, and reagents required by a Q-PCR reaction.

6. A detection method for realizing accurate typing of a dimension D receptor Fok1 site based on a probe method is characterized by comprising the following steps: the method comprises the following steps:

the method comprises the following steps: extracting to obtain DNA of a sample to be detected;

step two: carrying out reaction on the DNA to be detected, the primer composition for realizing accurate typing of the Fok1 locus of the vitamin D receptor based on the probe method as claimed in any one of claims 1 to 4 and Q-PCR reaction reagent according to the steps of Q-PCR reaction.

7. A system for realizing accurate typing of a dimension D receptor Fok1 site based on a probe method is characterized by comprising the following steps:

the extraction module is used for extracting and obtaining DNA of a sample to be detected;

a detection module, which is used for carrying out reaction on the DNA of the sample to be detected, the primer composition for realizing accurate typing of the vitamin D receptor Fok1 locus based on the probe method as claimed in any one of claims 1 to 4 and a Q-PCR reaction reagent according to the steps of Q-PCR reaction.

Technical Field

The invention relates to the field of gene detection, in particular to a primer composition, a reagent, a detection method and a system for realizing accurate typing of a vitamin D receptor Fok1 locus based on a probe method.

Background

The vitamin D receptor gene (VDR) receptor is positioned at 12q13.11, has a total length of 63.5kb and 11 exons, has an mRNA length of 4,775nt, encodes a Vitamin D Receptor (VDR) protein consisting of 428 amino acid residues, is a nuclear hormone receptor of vitamin D3, and is also a second receptor of cholic acid. VDR belongs to a transcriptional regulator, is a member of the steroid hormone/thyroid hormone receptor superfamily, and functions as a ligand-induced replication factor. The regulation of gene expression by the protein is mainly realized through a series of metabolic reaction pathways, including an immune reaction pathway and a tumor activation pathway.

A plurality of domestic and foreign researches prove that the VDR gene and metal ions such as calcium are absorbed and metabolized in vivo, so that the VDR gene has obvious relevance to diseases such as fracture, osteoporosis, vitamin D dependent rickets II and the like. Fok1(rs2228570), the only known single nucleotide polymorphism of vitamin D receptor that can change protein structure, has population diversity. The genotype of the site is related to the transcriptional activation of human gingival fibroblasts and periodontal ligament cells, coronary artery diseases, type II diabetes, milk protein allergy of children, tuberculosis susceptibility, idiopathic low citric acid uropathy of white Yunnan people, hyperlipidemia of Chinese Han people and the like. Fok1(rs2228570) is located at position 47879112 of chromosome 12, and has three genotypes of TT, CT and CC.

Currently, commonly used methods for detecting the polymorphism of Fok1(rs2228570) include:

1. after PCR-based amplification and single base extension, SNP genotyping was performed using either MassARRAY or a generation sequencer. The existing equipment is expensive; after PCR amplification and single base extension, one round of enzyme digestion is needed, the steps are complicated, and the investment of manpower and reagent consumables is large; the PCR amplification product needs to be uncapped, so that the indoor aerosol pollution is easily caused, and the accuracy of the result is influenced.

2. Two forward primers and one reverse primer are utilized, after PCR amplification, product bands distinguished according to the sizes of fragments are seen under ultraviolet light through gel electrophoresis, and the genotype of a target SNP locus is judged according to the existence of the bands. The same problem of aerosol contamination exists; in the step of result judgment, gel electrophoresis is needed, and various complicated steps such as glue preparation, sample application, glue running, imaging and the like are involved, so that errors are easy to occur, and the labor investment is large; and the result is presented in the form of pictures, and the interpretation process depends on subjective factors, so that errors are easy to occur.

Disclosure of Invention

In order to solve the technical problems of expensive equipment, aerosol pollution, complicated steps and the like existing in the Fok1 locus polymorphism detection in the prior art, the invention provides the primer composition for realizing accurate typing of the vitamin D receptor Fok1 locus based on a probe method.

A primer composition for realizing accurate typing of a vitamin D receptor Fok1 site based on a probe method comprises a primer and a probe which are designed according to the polymorphism of a vitamin D receptor gene Fok1(rs2228570) SNP site, and the sequence is as follows:

forward amplification primer for VDR gene Fok1(rs 2228570):

CTGGCCCTGGCACTGACTCTGGCTCT；

reverse amplification primer of VDR gene Fok1(rs 2228570):

GGTCAAAGTCTCCAGGGTCAGG；

VDR gene Fok1(rs2228570) genotype T probe:

5’-CTTACAGGGATGGAG-3’；

VDR gene Fok1(rs2228570) genotype C probe:

5’-CTTACAGGGACGGAG-3’。

the fluorescent group at the 5 'end of the genotype T probe is VIC, and the quenching groups at the 3' end of the genotype T probe are Quencher and MGB. The fluorescent group at the 5 'end of the genotype C probe is FAM, and the quenching group at the 3' end of the genotype C probe is Quencher and MGB.

The primer and the probe exist in the form of mixed solution, and the proportion is as follows: (the forward amplification primer: the reverse amplification primer: the genotype T probe: the genotype C probe: water) ═ 10:10:4:4: 192).

A reagent for realizing accurate typing of a vitamin D receptor Fok1 site based on a probe method comprises a primer composition for realizing accurate typing of a vitamin D receptor Fok1 site based on the probe method and reagents required for performing a Q-PCR reaction.

A detection method for realizing accurate typing of a dimension D receptor Fok1 locus based on a probe method comprises the following steps:

the method comprises the following steps: extracting to obtain DNA of a sample to be detected;

step two: and (3) carrying out reaction on the DNA of the sample to be detected and the reagent for realizing accurate typing of the Fok1 locus of the dimensional D receptor based on the probe method according to the step operation of Q-PCR reaction.

A system for realizing accurate typing of a dimension D receptor Fok1 site based on a probe method comprises the following steps:

the extraction module is used for extracting and obtaining DNA of a sample to be detected;

and the detection module is used for carrying out reaction on the DNA of the sample to be detected and the reagent for realizing accurate typing of the Fok1 locus of the vitamin D receptor based on the probe method according to the step operation of Q-PCR reaction.

Compared with the prior art, the primer composition for realizing accurate typing of the vitamin D receptor Fok1 site by adopting the probe-based method is designed according to the polymorphism of the SNP site of the vitamin D receptor gene Fok1(rs2228570), the primer probe composition of the sequences is used for adjusting the GC content of the primer to be 60 +/-2% according to the rules of AT interchange and GC interchange under the condition of non-specific amplification, the adjusted primer Tm peak value is single, and the primer sequence is modified to form the primer and the probe. The detection can be completed by one-step Q-PCR, and the method has the advantages that:

1. simple and efficient: performing Q-PCR reaction once, wherein the operation is simple, and the time is 2-3 hours; 2. the popularization is strong, the related main equipment is a real-time fluorescence quantitative PCR instrument, the price is relatively low, and most of mechanisms can be equipped; 3. the result is accurate, and the result is presented in a digital form, is not influenced by subjective judgment, and is objective and real; 4. the compatibility is good, the result can be directly accessed into a corresponding table for fast interpretation, and the sample processing capability is strong; 5. the pollution risk is small, cover opening is not needed after PCR is finished, aerosol cannot be generated, and false positive cannot be caused.

Since the Quencher is a quenching group, when the Quencher and the fluorophore exist on one DNA strand at the same time, the fluorophore cannot emit fluorescence. Thus, normally, the device cannot detect a fluorescent signal. When the Q-PCR reaction is carried out, the probe is specifically combined to the SNP site, the DNA polymerase contained in the reaction liquid cuts the probe from the target fragment into the reaction liquid, and the fluorescent gene on the corresponding probe is separated from the quenching group Quencher and captured by equipment, so that the specific genotype of the target SNP is detected.

Drawings

FIG. 1 is a graph showing Melt cut obtained by detecting the primer composition for realizing accurate typing of a vitamin D receptor Fok1 locus by using a probe method;

FIG. 2 is a diagram showing the effect of site polymorphism typing obtained by detecting the primer composition for realizing accurate typing of the vitamin D receptor Fok1 site based on the probe method provided by the invention;

FIG. 3 is a tabular form diagram of FIG. 2;

FIG. 4 is a graph showing Melt cut results from prior art testing;

FIG. 5 is a graph showing the effect of typing of a site polymorphism detected by a conventional technique.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

The primer composition for realizing accurate typing of the Fok1 locus of the vitamin D receptor based on a probe method comprises the following steps:

forward amplification primer for VDR gene Fok1(rs 2228570):

CTGGCCCTGGCACTGACTCTGGCTCT (corresponding to SEQ No.1 of the sequence Listing);

reverse amplification primer of VDR gene Fok1(rs 2228570):

GGTCAAAGTCTCCAGGGTCAGG (corresponding to SEQ No.2 of the sequence Listing);

VDR gene Fok1(rs2228570) genotype T probe:

5'-CTTACAGGGATGGAG-3' (corresponding to SEQ No.3 of the sequence Listing);

VDR gene Fok1(rs2228570) genotype C probe:

5'-CTTACAGGGACGGAG-3' (corresponding to SEQ No.4 of the sequence Listing).

Wherein, the fluorescent group at the 5 'end of the genotype T probe is VIC, and the quenching group at the 3' end is Quencher and MGB. The fluorescent group at the 5 'end of the genotype C probe is FAM, and the quenching group at the 3' end of the genotype C probe is Quencher and MGB.

The reagent for realizing accurate typing of the vitamin D receptor Fok1 site based on the probe method comprises a primer composition for realizing accurate typing of the vitamin D receptor Fok1 site based on the probe method and a reagent required by Q-PCR, and specific components of the primer composition and the reagent are detailed in a detection method below.

The detection method for realizing accurate typing of the Fok1 locus of the dimensional D receptor based on a probe method comprises the following steps:

1. and (3) extracting DNA. 94 portions of DNA (S1 to S94) were extracted using the Crude DNA Extraction Kit (for Blood) (Vazyme) according to the procedure described in the specification, and stored at-20 ℃ for use.

2. A Q-PCR amplification system was prepared. SNP typing reaction is adopted in a reagent of ChamQ Geno-SNP Probe MasterMix (Vazyme), which is as follows:

reagent	Volume (μ L)
		TaqProbe 2X Q-PCR Mastermix	10.0
Primer&Probe Mix	2.0
		nuclease free water	3.0
Template	5.0
		Total volume	20

Wherein, the Primer & Probe Mix is a Primer composition for realizing accurate typing of the vitamin D receptor Fok1 site based on the Probe method. The components were mixed with double distilled water at a concentration of 100pmol/ul in a ratio of 10:10:4:4:192 (water) (see below).

Primer probe	Concentration of	Volume (μ L)
			Fok1(rs2228570) forward primer	100pmol/ul	5.0ul
Fok1(rs2228570) reverse primer	100pmol/ul	5.0ul
			Fok1(rs2228570) genotype T probe	100pmol/ul	2.0ul
Fok1(rs2228570) genotype C probe	100pmol/ul	2.0ul
			ddH2O		96.0ul

3. And (5) Q-PCR amplification. The following procedure was followed using an Applied Biosystems QuantStudio 5 apparatus:

4. and outputting the result. As shown in FIGS. 1 to 3, the results are the Melt cut display chart, the effect chart of the site polymorphism typing and the tabular form thereof, respectively.

In FIG. 2, the upper left part (blue) represents homozygous TT type, the middle part (green) represents heterozygous CT type, and the lower right part (red) represents homozygous CC type.

Please refer to FIG. 4 and FIG. 5, which are the Melt cut display graph and the polymorphism typing effect graph, respectively, obtained by the detection in the prior art. Compared with the prior art, the primer composition for realizing accurate typing of the vitamin D receptor Fok1 locus based on the probe method is adopted for detection, and the obtained peak image is single and has good specificity. The success rate of successful typing also increased from about 89% to nearly 100%.

A system for realizing accurate typing of a dimension D receptor Fok1 site based on a probe method comprises the following steps:

the extraction module is used for extracting and obtaining DNA of a sample to be detected;

the detection module is used for enabling the DNA of the sample to be detected and the reagent for realizing accurate typing of the Fok1 locus of the dimension D receptor based on the probe method to react according to the step operation of Q-PCR reaction and outputting a reaction result;

and an apparatus capable of automatically performing Q-PCR operation. The detection module detects the DNA of the sample to be detected according to the detection method for realizing accurate typing of the Fok1 locus of the vitamin D receptor based on the probe method by relying on the equipment; and the results are displayed as visual data.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Sequence listing

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