阅读说明：本技术 用于检测基因多态性的引物组、试剂盒及方法 (Primer group, kit and method for detecting gene polymorphism ) 是由 冯薇 赵方圆 智慧芳 倪君君 于 2020-12-21 设计创作，主要内容包括：本发明提供了用于检测基因多态性的引物组、试剂盒及方法,该引物组包括：用于检测APOE基因、CYP4F2基因、GGCX基因、NPC1L1基因、NQO1基因和VKORC1基因的引物对。利用该引物组进行多重PCR检测时,通过一次PCR反应可以同时对上述至少一个基因的突变进行检测,使得基因检测通量提高。(The invention provides a primer group, a kit and a method for detecting gene polymorphism, wherein the primer group comprises: primer pairs for detecting APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene. When the primer group is used for multiple PCR detection, the mutation of at least one gene can be simultaneously detected through one PCR reaction, so that the gene detection flux is improved.)

1. A primer set for detecting gene polymorphism, comprising: at least one of the following primer pairs;

the primer pair is used for detecting 388T > C sites and 526C > T sites of APOE genes, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2;

the primer pair is used for detecting the rs3093168T > C locus of the CYP4F2 gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.3, the nucleotide sequence of a downstream primer is shown as SEQ ID No.4, and the nucleotide sequence of a sequencing primer is shown as SEQ ID No. 5;

the primer pair is used for detecting the site rs2108622G & gtA of the CYP4F2 gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.6, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 7;

the primer pair is used for detecting the INV2-1G > T site of the GGCX gene, wherein the nucleotide sequence of the upstream primer of the primer pair is shown as SEQ ID NO.8, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 9;

a primer pair for detecting Val255Met site of GGCX gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.10, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 11;

the primer pair is used for detecting a Phe299Ser site, a Ser300Phe site, a Gln374Ter site, a Leu394Arg site, an Arg476His \ Cys site, an Arg485Pro site, a Trp493Ser site, a Trp501Ser site, a Gly537Tyr site and a Gly558Arg site of the GGCX gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.12, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.13, and the nucleotide sequence of a sequencing primer is shown as SEQ ID NO. 14;

a primer pair used for the Leu216Ala locus of the NPC1L1 gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.15, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 16;

a primer pair for detecting Pro187Ser sites of NQO1 gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.17, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 18;

the primer pair is used for detecting rs9923231-1369G > A sites of VKORC1 genes, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.19, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 20;

the primer pair is used for detecting Arg98Trp site of VKORC1 gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.21, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 22.

2. A kit for detecting gene polymorphism, comprising: the primer set of claim 1.

3. The kit according to claim 2,

the kit also comprises: DNA polymerase, PCR buffer solution corresponding to the DNA polymerase, mixture of 4 dNTPs and ultrapure water.

4. The kit according to claim 3,

the dosage of the DNA polymerase is 0.5-5U, the final concentration of each dNTP is 50-500 mu M, and the final concentration of each primer in the primer group is 20-400 nM.

5. The kit according to claim 3 or 4,

the DNA polymerase includes: taq polymerase, KOD FX polymerase, Pfu polymerase or Phusion polymerase.

6. A method for detecting a gene polymorphism, comprising:

designing the primer set of claim 1, or removing the primer set in the kit of any one of claims 2 to 5;

extracting genome DNA from a sample to be detected as an amplification template;

preparing a multiple Polymerase Chain Reaction (PCR) reaction system containing the primer group and the amplification template;

performing multiple PCR amplification reaction on the multiple PCR reaction system to obtain a PCR product;

and determining the genotypes of the APOE gene, the CYP4F2 gene, the GGCX gene, the NPC1L1 gene, the NQO1 gene and the VKORC1 gene of the sample to be detected according to the PCR product.

7. The method of claim 6,

determining the genotypes of the APOE gene, the CYP4F2 gene, the GGCX gene, the NPC1L1 gene, the NQO1 gene and the VKORC1 gene of the sample to be detected according to the PCR product, wherein the genotypes of the APOE gene, the CYP4F2 gene, the GGCX gene, the NPC1L1 gene, the NQO1 gene and the V:

detecting the PCR product by electrophoresis to obtain the size of an amplified fragment of the PCR product;

and when the size of the amplified fragment of the PCR product is correct, carrying out nucleotide sequence determination on the PCR product to obtain the genotypes of the APOE gene, the CYP4F2 gene, the GGCX gene, the NPC1L1 gene, the NQO1 gene and the VKORC1 gene of the sample to be detected.

8. The method of claim 6,

the multiplex PCR reaction system comprises: DNA polymerase, PCR buffer solution corresponding to the DNA polymerase, mixture of 4 kinds of dNTP and ultrapure water;

wherein the dosage of the DNA polymerase comprises 0.5-5U, the final concentration of each dNTP is 50-500 mu M, and the final concentration of each primer in the primer group is 20-400 nM.

9. The method of claim 8,

the DNA polymerase includes: taq polymerase, KOD FX polymerase, Pfu polymerase or Phusion polymerase.

10. The method according to any one of claims 6 to 9,

the reaction conditions of the multiplex PCR reaction system are as follows: pre-denaturation is carried out for 60-600 s at the temperature of 94-98 ℃; denaturation at 94-98 ℃ for 5-60 s, annealing at 50-68 ℃ for 10-120 s, and extension at 68-72 ℃ for 30-360 s, wherein the denaturation, annealing and extension are circulated for 25-45 times; the final extension is 0-1800 s at 68-72 ℃.

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer group, a kit and a method for detecting gene polymorphism.

Background

Vitamin K is an essential fat-soluble vitamin for blood clot formation, and when the vitamin K is deficient, blood coagulation and bone metabolism abnormality of the body can be caused. The APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene are related to the metabolism of vitamin K.

At present, Polymerase Chain Reaction (PCR) detection tests can be designed for APOE genes, CYP4F2 genes, GGCX genes, NPC1L1 genes, NQO1 genes and VKORC1 genes, respectively, to detect gene mutation conditions.

However, since each PCR reaction is directed to only one exon/mutation site, the throughput of detecting gene polymorphism is low.

Disclosure of Invention

The invention provides a primer group, a kit and a method for detecting gene polymorphism, which can improve the detection flux of gene polymorphism.

The present invention provides a primer set for detecting gene polymorphism, comprising: at least one of the following primer pairs; wherein the content of the first and second substances,

the primer pair is used for detecting 388T > C sites and 526C > T sites of APOE genes, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2; when the 388T > C site, 526C > T site and the upstream and downstream genes of the APOE gene are amplified by the upstream primer SEQ ID NO.1 and the downstream primer SEQ ID NO.2, the fragment length of the corresponding amplification product is 490 bp.

The primer pair is used for detecting the rs3093168T > C locus of the CYP4F2 gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.3, the nucleotide sequence of a downstream primer is shown as SEQ ID No.4, and the nucleotide sequence of a sequencing primer is shown as SEQ ID No. 5; when the rs3093168T > C locus of the CYP4F2 gene and the genes at the upstream and downstream thereof are amplified by using an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.4, the length of a fragment of a corresponding amplification product is 581 bp.

The primer pair is used for detecting the site rs2108622G & gtA of the CYP4F2 gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.6, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 7; when the rs2108622G A locus of CYP4F2 gene and the upstream and downstream genes thereof are amplified by using the upstream primer SEQ ID NO.6 and the downstream primer SEQ ID NO.7, the length of the fragment of the corresponding amplification product is 904 bp.

The primer pair is used for detecting the INV2-1G > T site of the GGCX gene, wherein the nucleotide sequence of the upstream primer of the primer pair is shown as SEQ ID NO.8, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 9; when the upstream primer SEQ ID NO.8 and the downstream primer SEQ ID NO.9 are used for amplifying the INV2-1G > T site of the GGCX gene and the upstream and downstream genes thereof, the fragment length of the corresponding amplification product is 159 bp.

A primer pair for detecting Val255Met site of GGCX gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.10, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 11; when the upstream primer SEQ ID NO.10 and the downstream primer SEQ ID NO.11 are used for amplifying the Val255Met site of the GGCX gene and the upstream and downstream genes thereof, the segment length of the corresponding amplification product is 300 bp.

A primer pair for detecting and detecting a Phe299Ser site, a Ser300Phe site, a Gln374Ter site, a Leu394Arg site, an Arg476His \ Cys site, an Arg485Pro site, a Trp493Ser site, a Trp501Ser site, a Gly537Tyr site and a Gly558Arg site of a GGCX gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.12, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.13, and the nucleotide sequence of a sequencing primer is shown as SEQ ID NO. 14; when the upstream primer SEQ ID NO.12 and the downstream primer SEQ ID NO.13 are used for amplifying a Phe299Ser site, a Ser300Phe site, a Gln374Ter site, a Leu394Arg site, a Arg476His \ Cys site, a Arg485Pro site, a Trp493Ser site, a Trp501Ser site, a Gly537Tyr site, a Gly558Arg site and upstream and downstream genes thereof of the GGCX gene, the fragment length of a corresponding amplification product is 2368 bp.

A primer pair used for the Leu216Ala locus of the NPC1L1 gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.15, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 16; when the upstream primer SEQ ID NO.15 and the downstream primer SEQ ID NO.16 are used for amplifying the Leu216Ala locus of the NPC1L1 gene and the upstream and downstream genes thereof, the fragment length of the corresponding amplification product is 697 bp.

A primer pair for detecting Pro187Ser sites of NQO1 gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.17, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 18; when the Pro187Ser site of the NQO1 gene and the upstream and downstream genes thereof are amplified by using the upstream primer SEQ ID NO.17 and the downstream primer SEQ ID NO.18, the fragment length of the corresponding amplification product is 1126 bp.

The primer pair is used for detecting rs9923231-1369G > A sites of VKORC1 genes, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.19, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 20; when rs9923231-1369G > A locus of VKORC1 gene and upstream and downstream genes thereof are amplified by using an upstream primer SEQ ID NO.19 and a downstream primer SEQ ID NO.20, the length of a fragment of a corresponding amplification product is 404 bp.

The primer pair is used for detecting Arg98Trp site of VKORC1 gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.21, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 22. When the Arg98Trp site of VKORC1 gene and the upstream and downstream genes thereof are amplified by using the upstream primer SEQ ID NO.21 and the downstream primer SEQ ID NO.22, the fragment length of the corresponding amplification product is 222 bp.

The accuracy of polymorphism detection of APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene can be improved by the primer group consisting of the primer pair, and the detection flux can be improved to the maximum extent.

Multiplex PCR is a novel amplification technique developed on the basis of conventional PCR, i.e., two or more pairs of primers can be added into a reaction system to simultaneously amplify a plurality of nucleic acid fragments. The multiplex PCR has important application in microbe, genetic disease, tumor and pharmacogenomics.

Based on this, the present invention also provides a kit for detecting gene polymorphism, comprising: a primer set consisting of at least one primer pair as described above.

In an embodiment of the present invention, the kit further includes: DNA polymerase, PCR buffer solution corresponding to the DNA polymerase, mixture of 4 dNTPs and ultrapure water.

Wherein the dosage of the DNA polymerase comprises 0.5-5U, the final concentration of each dNTP is 50-500 mu M, and the final concentration of each primer in the primer group is 20-400 nM.

The DNA polymerase includes: taq polymerase, KOD FX polymerase, Pfu polymerase or Phusion polymerase. Wherein the concentration degree of the PCR buffer solution corresponding to the DNA polymerase includes 1X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X or 10X.

For the amount of the DNA polymerase, 0.5-5U is any value in the range of 0.5U to 5U, for example, 0.5U, 1U, 1.5U, 2U, 2.5U, 3U, 3.5U, 4U, 4.5U, and 5U.

For each dNTP final concentration, 50-500. mu.M refers to any value in the range of 50M to 500M, such as 50. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, 250. mu.M, 300. mu.M, 350. mu.M, 400. mu.M, 450. mu.M and 500. mu.M.

For the final concentration of each primer, 20-400 nM refers to any value in the range of 20nM to 400nM, e.g., 20nM, 50nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, and 400 nM.

Specifically, when the kit is manufactured, a reagent for extracting the DNA of a sample to be detected or a professional DNA extraction kit can be selectively configured according to actual needs; the DNA of the sample to be detected can be obtained more conveniently and rapidly, and the convenience and the rapidity of the detection finished product kit are enhanced. The sample to be tested can be any blood, cell, tissue or buccal swab sample containing DNA.

The present invention also provides a method for detecting gene polymorphism, comprising:

designing the primer group consisting of at least one primer pair, or transferring the primer group in the kit;

extracting genome DNA from a sample to be detected as an amplification template;

preparing a multiplex PCR reaction system comprising the primer group and the amplification template;

performing multiple PCR amplification reaction on the multiple PCR reaction system to obtain a PCR product;

and determining the genotypes of the APOE gene, the CYP4F2 gene, the GGCX gene, the NPC1L1 gene, the NQO1 gene and the VKORC1 gene of the sample to be detected according to the PCR product.

Specifically, when performing a PCR amplification reaction using a primer set in a kit, formulating a multiplex PCR reaction system including the primer set and the amplification template may include:

and preparing a PCR reaction system by using a primer group, DNA polymerase, a PCR buffer solution corresponding to the DNA polymerase, a mixture of 4 dNTPs, ultrapure water and an amplification template in the kit.

In an embodiment of the present invention, the determining the genotypes of the APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene, and VKORC1 gene of the test sample according to the PCR product includes:

detecting the PCR product by electrophoresis to obtain the size of an amplified fragment of the PCR product;

and when the size of the amplified fragment of the PCR product is correct, carrying out nucleotide sequence determination on the PCR product to obtain the genotypes of the APOE gene, the CYP4F2 gene, the GGCX gene, the NPC1L1 gene, the NQO1 gene and the VKORC1 gene of the sample to be detected.

Specifically, agarose gel electrophoresis or polyacrylamide gel electrophoresis can be used to resolve DNA fragments of different lengths.

For example, when KOD FX is used as the DNA polymerase and 2 × concentrated PCR buffer is used, the above PCR reaction system may be configured such that the following components are used: 0.5-2 μ l of DNA polymerase, 18-30 μ l of PCR buffer solution, 2-12 μ l of mixture of various dNTPs, 2-10 μ l of mixture of 10 primer pairs, 5-1000 ng of DNA, and a proper amount of ultrapure water for water supplement to 50 μ l. Other volume sizes configured in the same proportions may also be used.

Specifically, in an embodiment of the present invention, the reaction conditions of the PCR reaction system are as shown in table 1:

TABLE 1

Table 1 also shows that the storage conditions of the PCR products are 2-8 ℃.

For the pre-denaturation temperature and the denaturation temperature, 94-98 ℃ means any value in the range of 94 ℃ to 98 ℃, such as 94 ℃, 95 ℃, 96 ℃, 97 ℃ and 98 ℃.

For the pre-denaturation time, 60-600 s refers to any value in the range of 60s to 600s, such as 60s, 100s, 150s, 200s, 250s, 300s, 350s, 400s, 450s, 500s, 550s, and 600 s.

In terms of denaturation time, 5 to 60s means 5s is any value within the range of 60s, for example, 5s, 10s, 20s, 30s, 40s, 50s, 60 s.

The annealing temperature is 50 to 68 ℃ and means any value in the range of 68 ℃ at 50 ℃, for example, 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃, 66 ℃ and 68 ℃.

For the annealing time, 10 to 120s means any value in the range of 10s to 120s, for example, 10s, 15s, 20s, 30s, 40s, 50s, 60s, 70s, 80s, 90s, 100s, 110s, and 120 s.

For the temperature of the elongation and the temperature of the final elongation, 68 to 72 ℃ means any value in the range of 68 ℃ to 72 ℃, for example, 68 ℃, 69 ℃, 70 ℃ and 72 ℃.

For the extension time, 30-360 s refers to any value within the range of 30 s-360 s, such as 30s, 50s, 100s, 150s, 200s, 250s, 300s, and 360 s.

For the final extension time, 0 to 1800s means any value in the range of 0s to 1800s, for example, 0s, 10s, 50s, 100s, 200s, 300s, 400s, 500s, 600s, 700s, 800s, 900s, 1000s, 1100s, 1200s, 1300s, 1400s, 1500s, 1600s, 1700s, and 1800 s.

The method for detecting a gene polymorphism is a method not intended for diagnosis.

Specifically, the method for extracting genomic DNA from a sample to be tested comprises the following steps: extracting by hand or kit, and extracting the extracted DNA to obtain the genome DNA.

Compared with the prior art, the invention at least has the following beneficial effects:

(1) the interpretation of the amplification result is visual and accurate: the primers provided by the invention can ensure that the sizes of the amplification products of the primer pairs are fully distinguished, and nonspecific products or dimers cannot be generated due to interaction among the amplification fragments, so that the accuracy of the result is ensured.

(2) And (3) improving the detection flux: while the common PCR only aims at one primer pair to amplify to generate one nucleic acid fragment in each reaction, the multiplex PCR of the invention can simultaneously amplify at least two nucleic acid fragments, and can detect a plurality of hot spot mutation sites in APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene through a single-tube one-time reaction.

(3) The cost is reduced: the invention can reduce the PCR reaction system from a plurality of systems/procedures to one system/procedure, thereby reducing the use amount of reagents and consumables such as DNA polymerase, dNTP and the like and greatly reducing the detection cost.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 shows the result of agarose gel electrophoresis detection according to an embodiment of the present invention;

FIG. 2 is a partial nucleotide base sequence of the mutation site c.388T > C of the APOE gene in the PCR product sequence determination result provided by an embodiment of the present invention;

FIG. 3 is a partial nucleotide base sequence of the mutation site c.526C > T of the APOE gene in the PCR product sequence determination result provided by one embodiment of the present invention;

FIG. 4 is a partial nucleotide base sequence of the mutation site rs3093168T > C for CYP4F2 gene in the result of sequencing PCR products according to an embodiment of the present invention;

FIG. 5 is a partial nucleotide base sequence of the mutation site rs2108622G > A of the CYP4F2 gene in the result of sequencing the PCR product according to an embodiment of the invention;

FIG. 6 shows a partial nucleotide base sequence of AGGCX gene mutation site INV2-1G > T in the PCR product sequence determination results provided by an embodiment of the present invention;

FIG. 7 is a partial nucleotide base sequence of Val255Met for GGCX gene in the result of sequencing of PCR products according to an embodiment of the present invention;

FIG. 8 shows the partial nucleotide base sequence of Leu216Ala directed against NPC1L1 gene in the PCR product sequencing result provided by an embodiment of the present invention;

FIG. 9 is a partial nucleotide base sequence of Pro187Ser for NQO1 gene in a PCR product sequence assay provided by an embodiment of the present invention;

FIG. 10 shows the partial nucleotide base sequence of the mutation site rs9923231-1369G > A of VKORC1 gene in the PCR product sequence determination result provided by an embodiment of the present invention;

FIG. 11 shows the partial nucleotide base sequence of Arg98Trp for VKORC1 gene in the PCR product sequence determination results provided by an embodiment of the present invention.

Detailed Description

Specifically, the reagents used in the implementation of the invention are all commercial products, and the databases used in the implementation of the invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.

Once vitamin K is utilized by the body, it becomes inactive and must be reactivated to exert its biological effects. This vitamin K recovery process is referred to as the vitamin K cycle. The essential role of vitamin K is an essential cofactor for the formation of Gla proteins (coagulation factors, osteocalcin, etc.), and vitamin K circulation aids in the formation of Gla proteins.

The reasons for vitamin K deficiency are as follows: abnormality of intestinal flora; ② too little vitamin K intake; ③ abnormal absorption of vitamin K; and fourthly, abnormal circulation of the vitamin K. Therefore, by determining the content of the vitamin K in the body, not only the influence of the vitamin K in the food on the absorption and metabolism of the human body can be known, but also the difference of the sensitivity of the vitamin K in various foods to different human genes can be known.

Plasma vitamin K levels are related to APOE genotype: APOE2> APOE3> APOE4, demonstrating that vitamin K levels are regulated by different APOE genotypes. The result of this is an increased rate of metabolism of plasma chylomicron CM and very low density lipoprotein VLDL due to the relatively strong binding of APOE4 to the receptor. Vitamin K, which is highly lipophilic, is present in CM and is removed at the same rate as CM, thereby accelerating vitamin K metabolism, which in turn affects the carboxylation of osteocalcin, the concentration of uncarboxylated osteocalcin increases, calcium salt deposits and bone mineralization is hindered, so that osteogenesis is reduced, and bone density and bone strength are reduced. However, CM is finally metabolized by the liver and vitamin K bioavailability in APOE4(E3/4+ E4/4) for the treatment of blood clotting abnormalities is high.

The protein encoded by CYP4F2 has the function of oxidizing and hydrolyzing vitamin K, and the level of reduced vitamin K in the vitamin K circulation is reduced by hydroxylating the phenyl side chain of the vitamin K. rs3093168(T > C) and rs2108622(G > A) are mutated, resulting in reduced enzyme activity and dyssynthesis of blood coagulation factors.

Glutamyl carboxylase is encoded by the GGCX gene and is a VK hydroxylation-dependent enzyme. The vitamin K dependent proteins such as anticoagulation factors II, VII, IX, X, proteins C, S and Z can only play an anticoagulation role after the catalytic hydroxylation of GGCX. After mutation of Val255Met, Phe299Ser, Ser300Phe, Gln374Ter, Leu394Arg, Arg476His \ Cys, Arg485Pro, Trp493Ser, Trp501Ser, Gly537Tyr, Gly558Arg, INV2-1G > T, the ability of glutamyl carboxylase to hydroxylate vitamin K dependent protein is reduced, which results in vitamin K coagulation factor deficiency type 1 (VKCFD1) disease or pseudoxanthoma elastosis, and the level of coagulation factors partially returns to normal after taking VK.

The NPC1L1 protein is a cholesterol transport protein and plays an important role in intestinal VK absorption, and the intake of Leu216Ala is reduced after mutation, which causes the body to lack VK.

The NQO1 gene encodes quinone oxidoreductase, also known as DT-diaphorase (DT-diaphorase), which provides electrons from NADH or NAD (P) H to catalyze quinone compounds, reducing the various quinone compounds to hydroquinone compounds. The quinone (VK) form of vitamin K is converted to vitamin K hydroquinone (VKH 2). The Pro187Ser mutation results in reduced enzyme activity, normal wild-type enzyme activity, low to moderate heterozygosity enzyme activity, and inactivation of mutant enzymes, thereby interfering with VK cycling.

Vitamin K2, 3-epoxide reductase complex subunit 1(VKORC1) is an essential enzyme in the vitamin K cycle. VKORC1 catalyzes the reduction of vitamin K2, 3-epoxide (VK0) to the quinone (VK) form of vitamin K, which in turn is reduced to vitamin K hydroquinone (VKH 2). Insufficient VKORC1 enzyme activity leads to vitamin k dependent clotting factor deficiency, leading to bleeding. This phenotype is known as vitamin K coagulation factor deficiency type 2 (VKCFD 2). Arg98Trp homozygous form, the only mutation found so far that is associated with VKCFD 2. The VKORC1-1639G & gtA gene polymorphism is obviously related to the acute procoagulant effect of vitamin K, and the INR value of the international standardization ratio in a G allele carrier is reduced more rapidly.

Therefore, it is desirable to provide a primer set for detecting the mutation of the above-mentioned gene. The method specifically comprises the following steps:

example 1: design and Synthesis of primer set

Step 1.1: designing specific amplification upstream and downstream primers and corresponding sequencing primers based on an APOE gene, a CYP4F2 gene, a GGCX gene, an NPC1L1 gene, a NQO1 gene and a VKORC1 gene.

For the designed primers, Primer Quest and Primer Premier 5.0 are adopted to design the primers and analyze the mismatching of the dimer and the stem loop, the primers are designed at two ends containing the site to be detected, and the annealing temperatures of 10 pairs of primers are basically kept consistent.

The primer set provided in this example covers all hot-spot gene mutations of the APOE gene, CYP4F2 gene, GGCX gene, NPC1L1 gene, NQO1 gene and VKORC1 gene. Since the small sequence change can cause the primer amplification efficiency to be obviously reduced and the specificity to be poor, multiple PCR primer sets are respectively designed aiming at different sites/exons, and after the screening of a pre-experiment, the primer sets with the best amplification effect shown in the following table 2 are selected by integrating the fragment length and the site/exon inclusion conditions of products.

TABLE 2

Step 1.2: and (3) synthesizing the primer group designed in the step 1.1.

Example 2: extraction of DNA from a sample to be tested

Step 2.1: mouth-shed cells were collected with a mouth swab or fresh peripheral blood samples were collected with a blood collection tube.

Step 2.2: DNA was extracted from the specimen using a Tiangen buccal swab genomic DNA extraction kit (DP322) or a blood/cell/tissue genomic DNA extraction kit (DP304), and the concentration and purity of the DNA were determined using NP80-touch (IMPLEN, Germany), and the DNA whose test results were within a preset range was stored.

Example 3: preparing a PCR reaction system by using the primer group synthesized in the step 1.2 and the DNA preserved in the step 2.2

Step 3.1: and (3) taking the genome DNA stored in the step 2.2 as an amplification template, and adopting the primer group synthesized in the step 1.2 to prepare a multiple PCR reaction system.

In this example, a multiplex PCR amplification system was prepared by using DNA polymerase and buffer as basic raw materials in KOD FX enzyme system (cat. KFX-101) manufactured by Toyobo, Inc., and adjusting the primer concentration, dNTP concentration, buffer concentration and enzyme amount based on the amplification system in the enzyme system specification, and the specific composition of this reaction system is shown in Table 3 below.

TABLE 3

Reagent compositionIs divided into	Volume of
		2×PCR buffer for FX	25μl
2mM dNTP	12.5μl
		Primer Mix	8.25μl
KOD FX(1U/μl)	1.25μl
		DNA	2μl
Ultrapure water	1μl

It should be noted that, the proportional scaling up/down of the reaction system is within the protection scope of the embodiment of the present invention; the amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate proportion.

Step 3.2: the procedure of the PCR instrument was set according to the multiplex PCR reaction conditions shown in Table 4 below, and a multiplex PCR amplification reaction was performed on the multiplex PCR reaction system prepared in step 3.1 to obtain a PCR product.

TABLE 4

It is also shown in Table 4 that the PCR product after amplification can be stored at 4 ℃ until use.

Example 4: electrophoretic detection

Step 4.1: and (3) detecting the PCR product obtained in the step 3.2 by agarose gel electrophoresis to obtain the size of the PCR product fragment.

The detection results are shown in fig. 1, wherein FW, JYX, LS, PH, SLP, ZFY, and ZHF shown in fig. 1 are used to characterize different samples to be tested, the left-most column of fig. 1 shows a ruler bar for characterizing the length of a fragment, and the right-most column of fig. 1 shows the electrophoresis results of PCR products of the blank control group.

Referring to FIG. 1, according to the comparison of the position of the bright band of each product with the left-side scale bar, it can be identified which exon amplification product corresponds to the bright band of each product. For example, 10 bright bands from top to bottom are usually PCR amplification products corresponding to the gene segments of GGCX-3, NQO1, CYP4F2-2, NPC1L1, CYP4F2-1, APOE, VKORC1-1, GGCX-2, VKORC1-2 and GGCX-1, respectively.

According to the electrophoresis result of the blank group, environmental factors have no adverse effect on the electrophoresis detection result of the sample to be detected. According to the electrophoresis result of each sample to be detected, 10 bright bands respectively correspond to PCR amplification products of GGCX-3, NQO1, CYP4F2-2, NPC1L1, CYP4F2-1, APOE, VKORC1-1, GGCX-2, VKORC1-2 and GGCX-1 gene segments, and the number of the bright bands is consistent with theory; the 10 bright bands are clear and have obvious intervals, and different bright bands have no overlapping, no smear and the like, so that the bright band effect is good. Thus, it can be shown that when the PCR amplification primer set designed in step 1.1 is used for PCR amplification, only the expected target product is generated, but no other irrelevant product is generated, and the design of the primer set is reasonable.

Step 4.2: after the size of the PCR product fragment is verified to be correct, the sequence of the PCR product can be determined.

Example 5: sequence determination

Step 5.1: and 4.2, after the sizes of the PCR product fragments are verified to be correct, carrying out sequence determination on the PCR products obtained in the step 3.2 to obtain a sequencing result in a format of ab 1.

Step 5.2: the sequencing results obtained in step 5.1 were analyzed by Chromas sequence analysis software to obtain mutations in the genes GGCX-3, NQO1, CYP4F2-2, NPC1L1, CYP4F2-1, APOE, VKORC1-1, GGCX-2, VKORC1-2, and GGCX-1.

The partial sequencing results are shown in FIGS. 2 to 11.

FIG. 2 shows nucleotide base sequences at and upstream and downstream of the mutation site c.388T > C of the APOE gene. Referring to the box line part in FIG. 3, it can be seen that the site at position 388 is C, but not T, i.e., a gene mutation occurs at this site.

FIG. 3 shows the nucleotide base sequence at and upstream and downstream of the site c.526C > T of the mutation of the APOE gene. Referring to the box line portion in FIG. 4, it can be seen that the site at position 526 is T, but not C, i.e., a gene mutation occurs at this site.

FIG. 4 shows nucleotide base sequences at and upstream and downstream of the mutation site rs3093168T > C of the CYP4F2 gene. Referring to the box line portion in fig. 5, it can be seen that the site rs3093168T > C is C, but not T, i.e. a gene mutation occurs at the site.

FIG. 5 shows the nucleotide base sequence at the site rs2108622G > A of the mutation of CYP4F2 gene and upstream and downstream thereof. Referring to the box line part in fig. 6, it can be seen that the rs2108622G > site a is a, but not G, i.e. the gene mutation occurs at this site.

FIG. 6 shows nucleotide base sequences at and upstream and downstream of the mutation site INV2-1G > T of GGCX gene. Referring to the box line part in FIG. 7, it can be seen that the INV2-1G > T site is G, but not T, i.e., no gene mutation occurs at the site.

FIG. 7 shows a partial map of the sequencing peaks of the Val255Met wild homozygote of the GGCX gene. Referring to the line in FIG. 8, it can be seen that the amino acid at position 255 is Val (GTG) instead of Met (ATG), i.e., no genetic mutation has occurred at this amino acid.

FIG. 8 shows a partial map of the sequencing peaks of the Leu216Ala wild homozygote of the NPC1L1 gene. Referring to the box line portion of FIG. 9, it can be seen that the amino acid at position 216 is Leu (CTG) rather than Ala (GCN), i.e., no gene mutation has occurred at this amino acid.

Fig. 9 shows a partial map of the sequencing peaks of Pro187Ser mutant hybrids of NQO1 gene. Referring to the box line part in FIG. 10, it can be seen that the amino acid at position 187 is Ser (TCT), rather than Pro (CCT), at which the gene mutation occurs.

FIG. 10 shows the nucleotide base sequences at and upstream and downstream of the mutation site rs9923231-1369G > A of VKORC1 gene. Referring to the box line portion in FIG. 11, it can be seen that the site rs9923231-1369G > A is A, but not G, i.e., the gene mutation occurs at the site.

FIG. 11 shows a partial map of the sequencing peaks of wild homozygote Arg98Trp of VKORC1 gene. Referring to the box line portion of FIG. 11, it can be seen that the amino acid at position 98 is Arg (CGG) instead of Trp (TGG), i.e., no genetic mutation has occurred at this amino acid.

The missing figures in fig. 1 to 11 do not affect the technical content of the present solution.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.