Amplification primer set and probe, kit, and typing method for vitamin D and calcium metabolism related gene typing

Abstract

The present invention discloses an amplification primer set and a probe, a kit, and a typing method for vitamin D and calcium metabolism-related gene typing. The amplification primer set and the probe include: an ARMS primer set and a probe for CYP2R1-rs10741657 site: a CYP2R1-rs10741657 wild-type forward primer, a mutant forward primer, a common reverse primer, and a common probe; an ARMS primer set and a probe for GC-rs2282679 site: a GC-rs2282679 wild-type forward primer, a mutant forward primer, a common reverse primer, and a common probe; an ARMS primer set and a probe for VDR-rs2228570 site: a VDR-rs2228570 wild-type forward primer, a mutant forward primer, a common reverse primer, and a common probe, and the sequences thereof are shown in SEQ ID NOs: 01 to 12, respectively. It can accurately detect and analyze the polymorphisms of three gene loci: CYP2R1‑rs10741657, GC‑rs2282679 and VDR‑rs2228570, with high detection sensitivity, short detection time, low detection cost and simple operation.

Description

Technical Field

[0001] The present application belongs to the field of gene detection technology, and specifically relates to an amplification primer set and probe, a kit, and a typing method for typing genes related to vitamin D and calcium metabolism. Background Art

[0002] Vitamin D is a fat-soluble vitamin. It is a nutrient necessary for maintaining human life activities. Its main function is to regulate calcium and phosphorus metabolism, promote intestinal calcium and phosphorus absorption and bone calcification, and maintain the balance of blood calcium and blood phosphorus. In addition, vitamin D also has the function of regulating cell growth and differentiation and immune regulation. Calcium is also an essential nutrient for the human body. It is not only the main inorganic component of bones and teeth, but also an essential element for neurotransmitters, muscle contraction, blood coagulation, hormone release and milk secretion.

[0003] Lack of vitamin D and calcium can lead to rickets in infants and osteomalacia in adults. It can also affect the nervous system, such as intellectual retardation and crying in infants, insomnia and dreaminess in adults. Lack of vitamin D can also reduce the body's immunity. In addition, when vitamin D and calcium are not taken in enough, the incidence of diabetes and cardiovascular diseases will increase, and even malignant tumors may be caused. Therefore, conducting individual nutritional needs testing is essential for everyone's health.

[0004] Usually, the detection of individual nutritional needs is mainly through biochemical detection methods, but this method can only detect the current nutritional status of the individual and is easily affected by external factors. Nutritional metabolism gene testing is a normal test of nutritional metabolism ability at the genetic level, and a single test will benefit you for life.

[0005] The main genes related to vitamin D and calcium metabolism include CYP2R1, GC and VDR. The VDR gene encodes the vitamin D receptor (VDR). As a nucleophilic protein, VDR is a nuclear macromolecule that mediates the biological effects of 1,25(OH)D. It plays an important role in maintaining calcium-phosphorus metabolism, regulating cell proliferation and differentiation. The mutation of rs2228570 in the VDR gene reduces the receptor affinity, thereby reducing the binding of vitamin D derivatives to the receptor and hindering the intestinal absorption of calcium. The GC gene encodes vitamin D binding protein, also known as GC globulin, which can bind to various forms of vitamin D and transport vitamin D metabolites between the skin, liver and kidneys and transport them to target organs. The mutation of the rs2282679 site reduces the activity of GC globulin and affects the absorption of vitamin D and calcium. The protein encoded by CYP2R1 is a member of the cytochrome P450 superfamily enzyme, which can convert vitamin D into an active form to bind to the vitamin D receptor, thereby promoting calcium absorption. The mutation of the rs10741657 site will also affect the metabolism of vitamin D and calcium. Therefore, the detection of vitamin D and calcium metabolism gene polymorphisms is of great significance for understanding the nutritional needs of individuals.

[0006] At present, the main methods for detecting SNP polymorphisms include Sanger sequencing, gene chip method, PCR-RFLP, Taqman probe method, etc. For example, Chinese patent CN113604558A discloses a vitamin D receptor gene SNP site detection reagent, and uses Sanger sequencing to detect the polymorphism of VDR gene rs2228570. Chinese patent CN108893531A discloses a children's vitamin requirement gene detection kit, which uses the gene chip method to detect the polymorphism of CYP2R1 rs10741657; the cost of these two methods is relatively high, which is not conducive to market promotion. Chinese patent CN110358844A discloses a sports genetic marker site detection method and its product based on nucleic acid mass spectrometry typing technology, and uses nucleic acid mass spectrometry to detect the polymorphism of GC rs2282679, but the mass spectrometry method has high requirements for equipment. In addition, the PCR-RFLP method is complicated and cumbersome to operate, and has low sensitivity. The traditional Taqman probe method requires the design of different fluorescent probes according to different base mutations, which is troublesome and costly. Chinese patent CN113215227A discloses a primer and probe for vitamin D receptor gene typing detection, which uses the ARMS-PCR method to detect the polymorphism of four sites of the VDR gene. However, the patent does not cover comprehensive gene detection related to vitamin D and calcium metabolism. Summary of the invention

[0007] In view of this, on the one hand, some embodiments disclose amplification primer sets and probes for vitamin D and calcium metabolism related gene typing, including: ARMS primer sets and probes for CYP2R1-rs10741657 site, ARMS primer sets and probes for GC-rs2282679 site, and ARMS primer sets and probes for VDR-rs2228570 site; wherein,

[0008] The ARMS primer set and probe for the CYP2R1-rs10741657 locus include:

[0009] CYP2R1-rs10741657 wild-type forward primer, the sequence of which is shown in SEQ ID NO: 01;

[0010] CYP2R1-rs10741657 mutant forward primer, whose sequence is shown in SEQ ID NO: 02;

[0011] CYP2R1-rs10741657 common reverse primer, the sequence of which is shown in SEQ ID NO: 03;

[0012] CYP2R1-rs10741657 common probe, the sequence of which is shown in SEQ ID NO: 04;

[0013] The ARMS primer set and probe for GC-rs2282679 locus include:

[0014] GC-rs2282679 wild-type forward primer, the sequence of which is shown in SEQ ID NO: 05;

[0015] GC-rs2282679 mutant forward primer, the sequence of which is shown in SEQ ID NO: 06;

[0016] GC-rs2282679 common reverse primer, the sequence of which is shown in SEQ ID NO: 07;

[0017] GC-rs2282679 common probe, the sequence of which is shown in SEQ ID NO: 08;

[0018] The ARMS primer set and probe for VDR-rs2228570 locus include:

[0019] VDR-rs2228570 wild-type forward primer, the sequence of which is shown in SEQ ID NO: 09;

[0020] VDR-rs2228570 mutant forward primer, the sequence of which is shown in SEQ ID NO: 10;

[0021] VDR-rs2228570 common reverse primer, the sequence of which is shown in SEQ ID NO: 11;

[0022] The sequence of the VDR-rs2228570 common probe is shown in SEQ ID NO:12.

[0023] On the other hand, some embodiments disclose a kit for vitamin D and calcium metabolism related gene typing, including an amplification primer set and a probe for vitamin D and calcium metabolism related gene typing.

[0024] Furthermore, some embodiments disclose a kit for vitamin D and calcium metabolism related gene typing, which also includes a PCR amplification reagent.

[0025] The kit for vitamin D and calcium metabolism related gene typing disclosed in some embodiments includes an amplification reaction system comprising an amplification primer set, a probe and a PCR amplification reagent configured as follows:

[0026]

[0027] On the other hand, some embodiments disclose a method for typing genes related to vitamin D and calcium metabolism, comprising: amplifying genes related to vitamin D and calcium metabolism using amplification primers and probes for typing genes related to vitamin D and calcium metabolism.

[0028] Furthermore, some embodiments disclose a method for typing genes related to vitamin D and calcium metabolism, comprising: using an ARMS primer set and probe at the CYP2R1-rs10741657 site, an ARMS primer set and probe at the GC-rs2282679 site, and an ARMS primer set and probe at the VDR-rs2228570 site to amplify genes related to vitamin D and calcium metabolism, respectively.

[0029] In some embodiments, the typing method of vitamin D and calcium metabolism related genes disclosed in the present invention comprises the following amplification reaction system:

[0030]

[0031] Some embodiments disclose a method for typing genes related to vitamin D and calcium metabolism, comprising: amplifying genes related to vitamin D and calcium metabolism using a kit for typing genes related to vitamin D and calcium metabolism.

[0032] Some embodiments disclose a method for typing genes related to vitamin D and calcium metabolism, using the ARMS primer set and probe for the CYP2R1-rs10741657 site, the ARMS primer set and probe for the GC-rs2282679 site, and the ARMS primer set and probe for the VDR-rs2228570 site in the kit to amplify the vitamin D and calcium metabolism related genes respectively.

[0033] The amplification primer set and probe, kit, and typing method for vitamin D and calcium metabolism related gene typing disclosed in the embodiments of the present invention can accurately detect and analyze the polymorphisms of the three gene loci CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570, and more comprehensively include gene loci related to vitamin D and calcium metabolism, which is conducive to a comprehensive understanding of individual nutritional needs. The amplification primer set and probe have good amplification specificity, high detection sensitivity, short detection time, low detection cost, and simple operation method, and have good prospects for promotion and application in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 .Amplification results of samples with genotype AA at CYP2R1-rs10741657;

[0035] Figure 2 .Amplification results of samples with genotype GG at CYP2R1-rs10741657;

[0036] Figure 3 .Amplification results of samples with CYP2R1-rs10741657 locus genotype of AG;

[0037] Figure 4 .Amplification results of samples with GC-rs2282679 locus genotype of TT;

[0038] Figure 5 .Amplification results of samples with genotype GG at GC-rs2282679;

[0039] Figure 6 .Amplification results of samples with GC-rs2282679 locus genotype of TG;

[0040] Figure 7 .Amplification results of samples with AA genotype at VDR-rs2228570;

[0041] Figure 8 .Amplification results of samples with the VDR-rs2228570 locus genotype of GG;

[0042] Fig. 9.Amplification results of samples with AG genotype at VDR-rs2228570;

[0043] Fig.10 .The amplification results of samples with the genotype of CYP2R1-rs10741657 site being GG homozygous, the genotype of GC-rs2282679 site being TG heterozygous, and the genotype of VDR-rs2228570 being AG heterozygous. DETAILED DESCRIPTION

[0044] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0045] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0046] The terms "substantially" and "approximately" used herein are used to describe small fluctuations. For example, they can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%. The numerical data represented or presented in the range format herein are used only for convenience and brevity, and should therefore be flexibly interpreted as including not only the values ​​clearly listed as the limits of the range, but also all independent values ​​or sub-ranges contained in the range. For example, the numerical range of "1-5%" should be interpreted as including not only the clearly listed values ​​of 1% to 5%, but also the independent values ​​and sub-ranges within the range shown. Therefore, independent values ​​such as 2%, 3.5% and 4% and sub-ranges such as 1%-3%, 2%-4% and 3%-5% are included in this numerical range. This principle also applies to the range of only one numerical value. In addition, such an interpretation applies regardless of the width of the range or the characteristics described. In this article, the forward primer is represented by "F", also known as the upstream primer, and the reverse primer is represented by "R", also known as the downstream primer.

[0047] In this document, including in the claims, transitional words such as "comprises," "includes," "with," "having," "containing," "involving," "accommodating," etc. are understood to be open-ended, i.e., meaning "including but not limited to." Only the transitional words "consisting of" and "composed of" are closed transitional words.

[0048] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0049] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0050] In some embodiments, the amplification primer set and probe for vitamin D and calcium metabolism related gene typing include: an ARMS primer set and probe for CYP2R1-rs10741657 site, an ARMS primer set and probe for GC-rs2282679 site, and an ARMS primer set and probe for VDR-rs2228570 site; wherein,

[0051] The ARMS primer set and probe for the CYP2R1-rs10741657 locus include:

[0052] CYP2R1-rs10741657 wild-type forward primer, the sequence of which is shown in SEQ ID NO: 01;

[0053] CYP2R1-rs10741657 mutant forward primer, whose sequence is shown in SEQ ID NO: 02;

[0054] CYP2R1-rs10741657 common reverse primer, the sequence of which is shown in SEQ ID NO: 03;

[0055] CYP2R1-rs10741657 common probe, the sequence of which is shown in SEQ ID NO: 04;

[0056] The ARMS primer set and probe for GC-rs2282679 locus include:

[0057] GC-rs2282679 wild-type forward primer, the sequence of which is shown in SEQ ID NO: 05;

[0058] GC-rs2282679 mutant forward primer, the sequence of which is shown in SEQ ID NO: 06;

[0059] GC-rs2282679 common reverse primer, the sequence of which is shown in SEQ ID NO: 07;

[0060] GC-rs2282679 common probe, the sequence of which is shown in SEQ ID NO: 08;

[0061] The ARMS primer set and probe for VDR-rs2228570 locus include:

[0062] VDR-rs2228570 wild-type forward primer, the sequence of which is shown in SEQ ID NO: 09;

[0063] VDR-rs2228570 mutant forward primer, the sequence of which is shown in SEQ ID NO: 10;

[0064] VDR-rs2228570 common reverse primer, the sequence of which is shown in SEQ ID NO: 11;

[0065] The sequence of the VDR-rs2228570 common probe is shown in SEQ ID NO:12.

[0066] Based on the Amplification Refractory Mutation System (ARMS), the inventors designed ARMS primers based on the principle of mismatched base replacement. When using a general thermostable DNA polymerase (lacking exonucleolytic proofreading activity), the 3'-end mismatch of the PCR primer leads to a sharp reduction in the product. According to the known mutation site, the corresponding ARMS primers are designed, and the 3'-end bases are complementary to the template bases of the wild-type and mutant types, respectively. Fluorescent PCR is performed on the samples to be tested and the amplification curves are monitored to directly achieve the purpose of distinguishing wild-type and mutant genes.

[0067] The mutant and wild-type primers designed by the inventors based on the allele-specific amplification method improve the specificity of amplification; they are also highly sensitive, with a minimum of only 10 ng of DNA template required to complete typing of human DNA samples; the detection time is short, with typing of the sample to be tested completed within 1 hour; the cost is low, and the reagents and consumables used are more commonly used in clinical practice, with a low cost, which is convenient for clinical promotion.

[0068] In some embodiments, a kit for typing genes related to vitamin D and calcium metabolism comprises an amplification primer set and a probe for typing genes related to vitamin D and calcium metabolism.

[0069] Furthermore, the kit for typing genes related to vitamin D and calcium metabolism also includes PCR amplification reagents.

[0070] The kit for vitamin D and calcium metabolism related gene typing disclosed in some embodiments includes an amplification reaction system comprising an amplification primer set, a probe and a PCR amplification reagent configured as follows:

[0071]

[0072] In some embodiments, the method for typing genes related to vitamin D and calcium metabolism includes: using amplification primers and probes to amplify the genes related to vitamin D and calcium metabolism in the sample to be tested.

[0073] Furthermore, some embodiments disclose a method for typing genes related to vitamin D and calcium metabolism, comprising: using an ARMS primer set and probe at the CYP2R1-rs10741657 site, an ARMS primer set and probe at the GC-rs2282679 site, and an ARMS primer set and probe at the VDR-rs2228570 site to amplify the vitamin D and calcium metabolism related genes of the sample to be tested, respectively.

[0074] Some embodiments disclose a method for typing genes related to vitamin D and calcium metabolism, which comprises amplifying the vitamin D and calcium metabolism related genes of a sample to be tested using a kit for typing genes related to vitamin D and calcium metabolism.

[0075] Some embodiments disclose a method for typing genes related to vitamin D and calcium metabolism, which utilizes the ARMS primer set and probe at the CYP2R1-rs10741657 site, the ARMS primer set and probe at the GC-rs2282679 site, and the ARMS primer set and probe at the VDR-rs2228570 site in the kit to amplify the vitamin D and calcium metabolism related genes of the sample to be tested, respectively.

[0076] Generally, a kit for typing genes related to vitamin D and calcium metabolism includes an amplification primer set and a probe, as well as an amplification reagent used in conjunction with the amplification primer set. The amplification primer set, the probe and the amplification reagent constitute an amplification reaction system for performing an amplification reaction.

[0077] As an optional embodiment, the configuration of the amplification reaction system is listed in Table 1.

[0078] Table 1 Amplification reaction system configuration

[0079]

[0080] Generally, the configuration amplification system is divided into two independent amplification reaction systems, namely, a wild-type reaction system and a mutant-type reaction system. The amplification reaction systems in Table 1 are uniformly applicable to the wild-type reaction system and / or the mutant-type reaction system;

[0081] For example, for the wild-type reaction system, 2×Taq Pro HS U Probe Master Mix includes PCR buffer, dNTP, and thermostable Taq polymerase; Primer Forward Mix refers to an upstream primer mixture including three gene loci: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Primer Reverse Mix refers to a downstream primer mixture shared by the three gene loci: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Probe Mix refers to a mixture of fluorescent probes shared by the three gene loci: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570;

[0082] For the mutant reaction system, 2×Taq Pro HS U Probe Master Mix includes PCR buffer, dNTP, and thermostable Taq polymerase; Primer Forward Mix refers to a mutant upstream primer mixture including three gene loci: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Primer Reverse Mix refers to a downstream primer mixture shared by the three gene loci: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Probe Mix refers to a mixture of fluorescent probes shared by the three loci: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570.

[0083] Amplification test

[0084] In order to verify the genotyping effect of the designed amplification primer set and probe, the following experiments were performed:

[0085] (1) Sample processing and nucleic acid extraction

[0086] The DNA of the sample to be tested was extracted using a commercial DNA extraction kit, and the concentration and purity of the extracted DNA were measured using a spectrophotometer. When the OD260 / OD280 of the sample to be tested was 1.8-2.0, it was considered a qualified sample and subsequent experiments could be carried out.

[0087] (2) Configure the amplification reaction system

[0088] In two amplification tubes, a wild-type amplification reaction system and a mutant amplification reaction system were respectively configured. The configuration system refers to Table 1 of this article. The primers and probes used were the ARMS primer set and probe for the CYP2R1-rs10741657 site, the ARMS primer set and probe for the GC-rs2282679 site, and the ARMS primer set and probe for the VDR-rs2228570 site; wherein,

[0089] The ARMS primer set and probe for the CYP2R1-rs10741657 locus include: CYP2R1-rs10741657 wild-type forward primer CYP2R1_F1, CYP2R1-rs10741657 mutant forward primer CYP2R1_F2, CYP2R1-rs10741657 common reverse primer CYP2R1_R, CYP2R1-rs10741657 common probe CYP2R1_P, and their sequences and sequence numbers are shown in Table 2;

[0090] The ARMS primer set and probe for the GC-rs2282679 locus include: GC-rs2282679 wild-type forward primer GC_F1, GC-rs2282679 mutant forward primer GC_F2, GC-rs2282679 common reverse primer GC_R, GC-rs2282679 common probe GC_P, and their sequences and sequence numbers are shown in Table 2;

[0091] The ARMS primer set and probe for the VDR-rs2228570 locus include: VDR-rs2228570 wild-type forward primer VDR_F1, VDR-rs2228570 mutant forward primer VDR_F2, VDR-rs2228570 common reverse primer VDR_R, and VDR-rs2228570 common probe VDR_P, whose sequences and sequence numbers are listed in Table 2.

[0092] Table 2 Amplification primer sets and their sequence lists

[0093]

[0094] (3) Perform amplification reaction

[0095] The configured wild-type amplification reaction system and mutant amplification reaction system were amplified using qPCR instrument ABI 7500, and fluorescence signals were collected; the fluorescence detection channels were VIC, CY5 and ROX, among which VIC fluorescence reflected the amplification of CYP2R1-rs10741657 site, CY5 fluorescence reflected the amplification of GC-rs2282679 site, and ROX fluorescence reflected the amplification of VDR-rs2228570 site;

[0096] The amplification reaction program settings are listed in Table 3;

[0097] Table 3 qPCR reaction program settings

[0098]

[0099]

[0100] (4) Experimental results

[0101] According to the above amplification test method, amplification tests are performed on multiple samples to be tested. The experimental results of multiple samples to be tested are listed below, and the experimental results are explained in conjunction with the accompanying drawings;

[0102] Figure 1 In the sample shown, the VIC channel was amplified normally in the CYP2R1-rs10741657 wild-type reaction system, while the VIC channel was not amplified in the CYP2R1-rs10741657 mutant reaction system, indicating that the sample genotype was AA homozygous.

[0103] Figure 2 In the sample shown, the VIC channel was amplified normally in the CYP2R1-rs10741657 mutant reaction system, while the VIC channel was not amplified in the CYP2R1-rs10741657 wild-type reaction system, indicating that the sample genotype was GG homozygous;

[0104] Figure 3 In the sample shown, the VIC channel was amplified normally in both the CYP2R1-rs10741657 mutant and wild-type reaction systems, indicating that the sample genotype was AG heterozygous;

[0105] like Figure 4 In the sample shown, the CY5 channel was amplified normally in the GC-rs2282679 wild-type reaction system, but the CY5 channel was not amplified in the GC-rs2282679 mutant reaction system, indicating that the genotype of the sample was TT homozygous;

[0106] like Figure 5 In the sample shown, the CY5 channel was amplified normally in the GC-rs2282679 mutant reaction system, while the CY5 channel was not amplified in the GC-rs2282679 wild-type reaction system, indicating that the genotype of the sample was GG homozygous;

[0107] like Figure 6 In the sample shown, the CY5 channel was amplified normally in both the GC-rs2282679 mutant and wild-type reaction systems, indicating that the sample genotype was TG heterozygous;

[0108] like Figure 7In the sample shown, the ROX channel was amplified normally in the VDR-rs2228570 wild-type reaction system, but the ROX channel was not amplified in the VDR-rs2228570 mutant reaction system, indicating that the genotype of the sample was AA homozygous;

[0109] like Figure 8 In the sample shown, the ROX channel was amplified normally in the VDR-rs2228570 mutant reaction system, while the ROX channel was not amplified in the VDR-rs2228570 wild-type reaction system, indicating that the genotype of the sample was GG homozygous;

[0110] like Fig. 9 In the sample shown, the ROX channel in both the VDR-rs2228570 mutant and wild-type reaction systems was amplified normally, indicating that the genotype of the sample was AG heterozygous;

[0111] like Fig.10 In the samples shown, the VIC channel was amplified normally in the CYP2R1-rs10741657 mutant reaction system, while the VIC channel was not amplified in the CYP2R1-rs10741657 wild-type reaction system, indicating that the genotype of the rs10741657 site of the sample was GG homozygous; the CY5 channel was amplified normally in both the GC-rs2282679 mutant and wild-type reaction systems, indicating that the genotype of the GC-rs2282679 site of the sample was TG heterozygous; the ROX channel was amplified normally in both the VDR-rs2228570 mutant and wild-type reaction systems, indicating that the genotype of the VDR-rs2228570 site of the sample was AG heterozygous.

[0112] Clinical sample validation test

[0113] Twenty clinical samples were randomly selected from the sample library, and the CYP2R1-rs10741657 gene locus, GC-rs2282679 gene locus and VDR-rs2228570 gene locus of the 20 clinical samples were typed using the ARMS-PCR method and the first-generation sequencing method, respectively.

[0114] Steps:

[0115] (1) Randomly select 20 clinical samples, use a commercial DNA kit to extract the DNA of the samples to be tested, and measure its concentration and purity; when the OD260 / OD280 of the samples to be tested is 1.8-2.0, they are qualified samples and can be used for subsequent experiments;

[0116] (2) Configure the ARMS amplification reaction system

[0117] The amplification reaction system was configured with reference to Table 1, and the sample DNA to be tested, primers, fluorescent probes, PCR buffer, dNTPs and thermostable Taq enzyme were added respectively; the primers in this embodiment refer to the ARMS primer set and probe for the CYP2R1-rs10741657 site, the ARMS primer set and probe for the GC-rs2282679 site, and the ARMS primer set and probe for the VDR-rs2228570 site disclosed in the examples of the present application, and their sequences are listed in Table 2;

[0118] (3) Perform PCR amplification reaction

[0119] According to the qPCR reaction program settings listed in Table 3, the qPCR instrument ABI 7500 was used to amplify and collect fluorescence signals; the detection channels were VIC, CY5 and ROX, among which VIC fluorescence reflected the amplification of the CYP2R1-rs10741657 site, CY5 fluorescence reflected the amplification of the GC-rs2282679 site, and ROX fluorescence reflected the amplification of the VDR-rs2228570 site;

[0120] (4) Determine the genotype of the clinical sample based on the fluorescence signal after the PCR reaction;

[0121] (5) The 20 selected clinical samples were sequenced and typed using the first-generation sequencing method and compared with the ARMS-PCR typing results.

[0122] The results are shown in Table 4. It was found that the consistency between the ARMS-PCR and first-generation sequencing results reached 100%, which also verified the accuracy of this method.

[0123] Table 4 Comparison of ARMS-PCR and first-generation sequencing typing results of clinical samples

[0124]

[0125] Some embodiments disclose a method for typing genes related to vitamin D and calcium metabolism, comprising using a kit to amplify a sample to be tested, specifically comprising:

[0126] (1) Sample processing, extracting DNA from the sample to be tested;

[0127] (2) using the amplification primer set and probe for vitamin D and calcium metabolism related gene typing in the kit to configure the ARMS amplification reaction system, the amplification primer set and probe sequences are listed in Table 2, and the amplification reaction system is listed in Table 1;

[0128] (3) performing PCR amplification reaction and detecting fluorescence signals;

[0129] (4) Determine the genotype of the sample to be tested based on the fluorescent signal of the amplification reaction.

[0130] The typing method of vitamin D and calcium metabolism related genes disclosed in some embodiments comprises using amplification primer sets and probes for vitamin D and calcium metabolism related genes to amplify the genes of the sample to be tested, respectively, and specifically comprises:

[0131] (1) Sample processing, extracting DNA from the sample to be tested;

[0132] (2) Amplification primer sets and probes for vitamin D and calcium metabolism-related genes are used to configure an ARMS amplification reaction system. The amplification primer sets and probe sequences are listed in Table 2, and the amplification reaction system is listed in Table 1;

[0133] (3) performing PCR amplification reaction and detecting fluorescence signals;

[0134] (4) Determine the genotype of the sample to be tested based on the fluorescent signal of the amplification reaction.

[0135] The amplification primer set and probe, kit, and typing method for vitamin D and calcium metabolism-related gene typing disclosed in the embodiments of the present invention can accurately detect and analyze the polymorphisms of three gene loci CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570, and more comprehensively include gene loci related to vitamin D and calcium metabolism. The amplification primer set and probe have good amplification specificity, high detection sensitivity, short detection time, low detection cost, and simple operation method, which is conducive to promotion in this field.

[0136] The technical solutions disclosed in this application and the technical details disclosed in the embodiments are merely illustrative of the inventive concept of this application and do not constitute a limitation on the technical solutions of this application. Any conventional changes, replacements or combinations of the technical details disclosed in this application have the same inventive concept as this application and are within the protection scope of the claims of this application. SEQUENCE LISTING <110> Zhengzhou University Henan Shenyou Medical Laboratory Co., Ltd. <120> Amplification primer sets and probes, kits, and typing methods for vitamin D and calcium metabolism related gene typing Law <130> 20220415 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <400> 1 gggagatact ttagcacgca 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> 2 gggagatact ttagcagtcg 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 ctgtcagccc tggaagactc 20 <210> 4 <211> 27 <212> DNA <213> Artificial sequence <400> 4 tgacattgga gctgagatca actgcta 27 <210> 5 <211> twenty four <212> DNA <213> Artificial sequence <400> 5 ctaacaataa aaaatacctg gctt 24 <210> 6 <211> twenty four <212> DNA <213> Artificial sequence <400> 6 ctaataataa aaaatacctg gctg 24 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 ggagctggga ctacagttgc 20 <210> 8 <211> 26 <212> DNA <213> Artificial sequence <400> 8 taagagacag agatttgctg ggcatg 26 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <400> 9 cttgctgttc ttacaggaat 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <400> 10 cttgctgttc ttacaggaac 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <400> 11 cccttcatgg aaacaccttg 20 <210> 12 <211> 27 <212> DNA <213> Artificial sequence <400> 12 aggtcatagc attgaagtga aagccag 27

Claims

1. Amplification primer set and probe for typing genes related to vitamin D and calcium metabolism, characterized in that: include: ARMS primer set and probe for CYP2R1-rs10741657, ARMS primer set and probe for GC-rs2282679, and ARMS primer set and probe for VDR-rs2228570; among them, The ARMS primer set and probe for the CYP2R1-rs10741657 site include: CYP2R1-rs10741657 wild-type forward primer, the sequence of which is shown in SEQ ID NO: 01; CYP2R1-rs10741657 mutant forward primer, whose sequence is shown in SEQ ID NO: 02; CYP2R1-rs10741657 common reverse primer, the sequence of which is shown in SEQ ID NO: 03; CYP2R1-rs10741657 common probe, the sequence of which is shown in SEQ ID NO: 04; The ARMS primer set and probe for the GC-rs2282679 site include: GC-rs2282679 wild-type forward primer, the sequence of which is shown in SEQ ID NO: 05; GC-rs2282679 mutant forward primer, the sequence of which is shown in SEQ ID NO: 06; GC-rs2282679 common reverse primer, the sequence of which is shown in SEQ ID NO: 07; GC-rs2282679 common probe, the sequence of which is shown in SEQ ID NO: 08; The ARMS primer set and probe of the VDR-rs2228570 locus include: VDR-rs2228570 wild-type forward primer, the sequence of which is shown in SEQ ID NO: 09; VDR-rs2228570 mutant forward primer, the sequence of which is shown in SEQ ID NO: 10; VDR-rs2228570 common reverse primer, the sequence of which is shown in SEQ ID NO: 11; The sequence of the VDR-rs2228570 common probe is shown in SEQ ID NO:

12.

2. A kit for typing genes related to vitamin D and calcium metabolism, characterized in that: It comprises the amplification primer set and probe for vitamin D and calcium metabolism related gene typing as described in claim 1.

3. The kit for genotyping related to vitamin D and calcium metabolism according to claim 2, characterized in that: Also included are PCR amplification reagents.

4. The kit for genotyping related to vitamin D and calcium metabolism according to claim 3, characterized in that: The amplification reaction system including the amplification primer set, the probe and the PCR amplification reagent is configured as follows: The amplification reaction system is applicable to wild-type reaction system and mutant reaction system; For the wild-type reaction system, 2×Taq Pro HS U Probe Master Mix includes PCR buffer, dNTPs, and thermostable Taq polymerase; Primer Forward Mix is ​​a mixture of wild-type forward primers including the three sites of CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Primer Reverse Mix is ​​a mixture of common reverse primers for the three sites of CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Probe Mix is ​​a mixture of common probes for the three sites of CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; For the mutant reaction system, 2×Taq Pro HS U Probe Master Mix includes PCR buffer, dNTP, and thermostable Taq polymerase; Primer Forward Mix is ​​a mutant forward primer mixture including three sites: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Primer Reverse Mix is ​​a common reverse primer mixture including three sites: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Probe Mix is ​​a mixture of common probes for the three sites: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570.

5. A non-diagnostic typing method for vitamin D and calcium metabolism related genes, characterized in that: include: The amplification primer set and probe described in claim 1 are used to amplify genes related to vitamin D and calcium metabolism.

6. The non-diagnostic typing method for vitamin D and calcium metabolism related genes according to claim 5, characterized in that: include: The ARMS primer set and probe at the CYP2R1-rs10741657 site, the ARMS primer set and probe at the GC-rs2282679 site, and the ARMS primer set and probe at the VDR-rs2228570 site were used to amplify genes related to vitamin D and calcium metabolism, respectively.

7. The non-diagnostic typing method for vitamin D and calcium metabolism related genes according to claim 6, characterized in that: The amplification reaction system is: The amplification reaction system is applicable to wild-type reaction system and mutant reaction system; For the wild-type reaction system, 2×Taq Pro HS U Probe Master Mix includes PCR buffer, dNTPs, and thermostable Taq polymerase; Primer Forward Mix is ​​a mixture of wild-type forward primers including the three sites of CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Primer Reverse Mix is ​​a mixture of common reverse primers for the three sites of CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Probe Mix is ​​a mixture of common probes for the three sites of CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; For the mutant reaction system, 2×Taq Pro HS U Probe Master Mix includes PCR buffer, dNTP, and thermostable Taq polymerase; Primer Forward Mix is ​​a mutant forward primer mixture including three sites: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Primer Reverse Mix is ​​a common reverse primer mixture including three sites: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Probe Mix is ​​a mixture of common probes for the three sites: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570.

8. A non-diagnostic typing method for vitamin D and calcium metabolism related genes, characterized in that: include: The kit according to any one of claims 2 to 4 is used to amplify genes related to vitamin D and calcium metabolism.

9. The non-diagnostic typing method for vitamin D and calcium metabolism related genes according to claim 8, characterized in that: The ARMS primer set and probe at the CYP2R1-rs10741657 site, the ARMS primer set and probe at the GC-rs2282679 site, and the ARMS primer set and probe at the VDR-rs2228570 site were used to amplify genes related to vitamin D and calcium metabolism, respectively.

10. The non-diagnostic typing method for vitamin D and calcium metabolism related genes according to claim 9, characterized in that: The amplification reaction system is: The amplification reaction system is applicable to wild-type reaction system and mutant reaction system; For the wild-type reaction system, 2×Taq Pro HS U Probe Master Mix includes PCR buffer, dNTPs, and thermostable Taq polymerase; Primer Forward Mix is ​​a mixture of wild-type forward primers including the three sites of CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Primer Reverse Mix is ​​a mixture of common reverse primers for the three sites of CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Probe Mix is ​​a mixture of common probes for the three sites of CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; For the mutant reaction system, 2×Taq Pro HS U Probe Master Mix includes PCR buffer, dNTP, and thermostable Taq polymerase; Primer Forward Mix is ​​a mutant forward primer mixture including three sites: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Primer Reverse Mix is ​​a common reverse primer mixture including three sites: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570; Probe Mix is ​​a mixture of common probes for the three sites: CYP2R1-rs10741657, GC-rs2282679, and VDR-rs2228570.

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