CN114214402B

CN114214402B - Primer group, kit and detection method for detecting blood hypercoagulability or venous thrombosis risk gene polymorphism and application

Info

Publication number: CN114214402B
Application number: CN202111634960.XA
Authority: CN
Inventors: 黄新祥; 周冰钏
Original assignee: Jiangsu Innovo Biotechnology Co ltd
Current assignee: Jiangsu Innovo Biotechnology Co ltd
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2024-02-09
Anticipated expiration: 2041-12-29
Also published as: CN114214402A

Abstract

The invention relates to a primer group, a kit and a detection method for detecting blood hypercoagulability or venous thrombosis risk gene polymorphism and application thereof, belonging to the technical field of molecular biology. According to the invention, a specific primer is designed, a method and a risk evaluation system for detecting human venous thrombosis susceptibility based on Sanger sequencing are established, the primer can rapidly and qualitatively detect blood hypercoagulability or venous thrombosis risk gene polymorphism, the gene type of a site to be detected can be rapidly and sensitively judged through one-time PCR amplification directly and a sequencing result, and thus the blood hypercoagulability or venous thrombosis risk gene is accurately typed; the detection method of the invention has accurate and comprehensive detection results of blood hypercoagulability or venous thrombosis risk; the evaluation system provided by the invention is complete, can comprehensively detect the potential risk of the detected object, and avoids the defect that the detection of a single gene or a few combined genes is detected, other risk genes are ignored, and the potential risk of the detected object cannot be comprehensively reflected.

Description

Primer group, kit and detection method for detecting blood hypercoagulability or venous thrombosis risk gene polymorphism and application

Technical Field

The invention relates to a primer group, a kit and a detection method for detecting blood hypercoagulability or venous thrombosis risk gene polymorphism and application thereof, belonging to the technical field of molecular biology.

Background

The hypercoagulable state of blood is a state in which blood is more likely to coagulate than normal, and is due to activation of coagulation factors induced by a series of factors. Patients with high blood coagulation have a high risk of thrombosis, and should be actively prevented and anti-thrombus and anticoagulated treatments timely taken. Venous thrombosis is one of the leading diseases of death and disability worldwide, and recent studies have shown that the hazard of venous thrombosis in asian areas (particularly china) is becoming increasingly apparent. One of the factors of venous thrombosis includes genetic risk factors such as anti-protein S deficiency (type i), coagulation factor V Leiden, dyslipidemia and other coagulation related disorders.

The common examination methods for venous thrombosis are as follows: general examination, local examination, imaging examination, intravenous contrast, venous pressure measurement, etc. With the development of molecular biology, some thrombosponding genes become research hotspots.

PAI-1 protein is the main inhibitor of tissue plasminogen activator in blood plasma, and PAI-1 in normal plasma concentration regulates blood coagulation homeostasis by inhibiting the hydrolysis of plasma plasmin by tissue plasminogen activator. The 1G insertion or deletion polymorphism, namely PAI-1G/5G insertion or deletion polymorphism, exists upstream of transcription initiation and is expressed as 3 genotypes 4G/4G, 5G/5G and 4G/5G, and researches show that the risk of venous thrombosis is 6.35 times that of the 5G/5G type (normal fibrinolysis type) for the 4G/4G genotype; in the case of the 4G/5G genotype, the risk of developing venous thrombosis is 4.85 times that of the 5G/5G genotype. Patients at risk have reduced fibrinolytic capacity and an increased risk of thrombosis.

MTHFR, methylene tetrahydrofolate reductase, is a key enzyme in the homocysteine metabolic pathway, a metabolic enzyme that catalyzes the conversion of homocysteine to methionine. The MTHFR gene mutation has reduced metabolic activity, which results in a disturbed vasomotor factor balance, and Deep Vein Thrombosis (DVT), especially in children. More than ten polymorphisms exist in the MTHFR gene, most commonly the C677T polymorphism, followed by the A1298C polymorphism. Since the nucleotide C at the 677 site of MTHFR gene is mutated into T, the amino acid at the site is mutated from alanine (Ala) into valine (Val), thereby adding a new restriction enzyme site. Alanine is an important amino acid in the active center of MTHFR, and substitution of valine greatly reduces both enzyme activity and thermostability, resulting in increased risk of venous thrombosis, with TT being the least active of the 3 genotypes CC, CT and TT at this site, approximately 45% of the CC genotypes. Thus, the TT genotype polymorphism at position 677 of the MTHFR gene is associated with deep vein thrombosis, and the TT genotype polymorphism is 6 times higher than the risk of DVT in other genotypes. Similarly, mutation of base A at 1298 locus of MTHFR gene to C is 8.3 times higher risk of DVT in patients.

Factor V (factor V Leiden, F5) is an important cofactor in the clotting process, which activates prothrombin by factor Xa to form thrombin. Thrombin cleaves fibrinogen to form fibrin, which polymerizes to form a dense network that forms a blood clot. The 41721 base of the F5 gene is mutated, and the risk of developing venous thrombosis is 4.8 times and 22 times that of the GG genotype of the AG and AA genotypes of the F5 gene from G to A.

LINC01146 (long intergenic non-protein coding RNA 1146) is an RNA gene belonging to the lncRNA class. LINC01146 presence site: rs1887289, HIT1.TDAG8, also known as GPR65 (G-protein coupled receptor 65), is a protein-encoding gene. Diseases associated with GPR65 include gastric cancer and alapplication mink disease. Their associated pathways include the RET signaling pathway and the GPCR signaling pathway. TDAG8 presence site: rs3742704, HIT2.

HIT1 and HIT2 are both associated with a heparin-induced thrombocytopenia risk, HIT1 being TT-type, heparin-induced thrombocytopenia HIT being 16.83 times higher than normal; HIT2 is GG type, heparin-induced thrombocytopenia HIT risk 33.48 times higher than normal.

Therefore, the genes have non-negligible effect in the venous thrombosis process, and susceptible people or high risk groups can be screened in time through gene detection, so that the method has important significance in targeted prevention and treatment and reduction of blood hypercoagulability or venous thrombosis risk.

At present, various common methods for SNP detection, such as a PCR gene chip method, a PCR high-resolution fusion curve method, an allele-specific PCR method, a fluorescent quantitative PCR method, an in-situ hybridization method and the like, exist, but the existing detection method has the defects of poor specificity of the adopted primer, higher requirements on equipment, low sensitivity, unfavorable clinical popularization and the like. Therefore, the establishment of the method for detecting the blood hypercoagulability or venous thrombosis risk gene polymorphism is urgent, and the method is convenient to operate, low in cost, accurate and visual in result, easy to analyze, good in specificity and high in sensitivity.

Disclosure of Invention

The invention aims to overcome some technical problems in the prior art, and based on single nucleotide polymorphism sites on PAI-1, MTHFR, F5 and HIT genes, the invention improves a primer group, a kit and a detection method for detecting blood hypercoagulability or venous thrombosis risk gene polymorphism and application thereof.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the present invention first provides a primer set for detecting a blood hypercoagulability or venous thrombosis risk gene polymorphism, the primer comprising:

(1) Primers for detecting PAI-1 (4G/5G) polymorphism, including the primers shown in SEQ ID No.7 and SEQ ID No. 8;

(2) Primers for detecting MTHFR (677C > T) polymorphism include primers shown in SEQ ID.NO.9 and SEQ ID.NO. 10;

(3) Primers for detecting MTHFR (1298A > C) polymorphism include primers shown in SEQ ID.NO.11 and SEQ ID.NO. 12;

(4) Primers for detecting the F5 (41721G > A) polymorphism include the primers shown in SEQ ID.NO.13 and SEQ ID.NO. 14;

(5) Primers for detecting LINC01146 (C > T) HIT1 polymorphism include primers shown in SEQ ID.NO.15 and SEQ ID.NO. 16;

(6) Primers for detecting GPR65 (T > G) HIT2 polymorphism include the primers shown in SEQ ID No.17 and SEQ ID No. 18.

Wherein PAI-1, MTHFR, F5, HIT1 and HIT2 are respectively thrombus related proteins, and sites or forms with mutation are arranged in brackets, and the PAI-1 (4G/5G), MTHFR (677C > T), MTHFR (1298A > C), F5 (41721G > A), LINC01146 (C > T) HIT1 and GPR65 (T > G) HIT2 are abbreviated.

The invention also provides the application of the primer for detecting the blood hypercoagulability or venous thrombosis risk gene polymorphism in detecting the blood hypercoagulability or venous thrombosis risk gene polymorphism or the application of the primer for detecting the blood hypercoagulability or venous thrombosis risk gene polymorphism in preparing a product for detecting the blood hypercoagulability or venous thrombosis risk gene polymorphism.

The invention also provides a detection kit for detecting the blood hypercoagulability or venous thrombosis risk gene polymorphism, which comprises the primer for detecting the blood hypercoagulability or venous thrombosis risk gene polymorphism, PCR Mix and sterilizing deionized water.

In the kit, the PCR Mix is preferably 2 xTaq Master Mix, wherein the 2 xTaq Master Mix comprises Taq DNA Polymerase, dNTPs and an optimized buffer system;

the concentration of the primer was 10. Mu.M.

The invention also provides application of the detection kit in preparation of a product for detecting blood hypercoagulability or venous thrombosis risk gene polymorphism.

The invention also provides a method for detecting blood hypercoagulability or venous thrombosis risk gene polymorphism, which comprises the following steps:

(1) And extracting the genome DNA of the peripheral blood of the testee by using a genome DNA extraction kit to obtain a sample to be tested.

(2) The reaction container is respectively added with a sample to be detected and a detection reagent, and a negative control group and a positive control group are arranged at the same time, and target fragments are amplified by PCR reaction.

The detection reagent comprises a primer for detecting the risk gene polymorphism.

(3) Sequencing the amplified product, and judging according to the sequencing result.

Wherein, in the step (2), the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 1min, and cycling for 35 times; finally, the extension is carried out for 5min at 72 ℃.

In the step (2), the PCR Mix and the sterilized deionized water are also added in the PCR reaction.

In the step (2), the PCR reaction system is as follows: PCR Mix 12.5. Mu.L, amplification primer 10. Mu.M, sample genomic DNA to be tested 2-10ng, sterile deionized water make up to 25. Mu.L.

In step (3), according to the sequencing result, the peak of each color represents a base, green: a, black: g, red: t, blue: and C, directly reading each base on the target fragment, and comparing the base with a reference sequence in an NCBI database.

In the step (3), the judgment standard is a judgment system established by the invention, and the judgment system is as follows:

simultaneously or respectively detecting several genes, setting different weight scores according to genotypes of the genes, and adding the genotype scores into 5 risk grades: low risk, medium risk, higher risk, high risk, extremely high risk; wherein 0 to 5 are classified as low risk, 6 to 20 are classified as medium risk, 21 to 30 are classified as higher risk, 31 to 50 are classified as high risk, and more than 50 are classified as extremely high risk.

Further, the genotype correspondence scores of the respective genes were as follows:

site PAI-1 (4G/5G), dbSNP is called as rs1799762, if the genotype is 5G/5G, the score is 0 score, and the genotype is normal; if the genotype is 4G/5G and the score is 5, the method indicates that the fibrinolytic capacity of the detected object is weakened, and the risk of recurrent abortion and DVT is medium; if the genotype is 4G/4G and the score is 25 points, the method indicates that the fibrinolytic capacity of the detected object is weakened, and the risk of recurrent abortion and the risk of DVT are higher.

Site MTHFR (677C > T), dbSNP is called rs1801133, if the genotype is GG, the score is 0 score, and the genotype is normal; if the genotype is AG and the score is 5, the result shows that the folic acid metabolism absorption capacity of the detected object is weakened, which possibly leads to the rise of homocysteine in blood, and further leads to the risk of medium recurrent abortion and medium DVT; if the genotype is AA and the score is 15, the folic acid metabolism and absorption capacity of the detected object is extremely low, which possibly leads to the rise of homocysteine in blood, and further leads to higher recurrent abortion risk and higher DVT risk.

Site MTHFR (1298A > C), dbSNP is called rs1801131, if the genotype is TT, the score is 0 score, and the genotype is normal; if the genotype is TG and the score is 5 points, the result shows that the folic acid metabolism absorption capacity of the detected object is weakened, which possibly leads to the rise of homocysteine in blood, and then leads to the risk of medium recurrent abortion and medium DVT; if the genotype is GG and the score is 15 points, the folic acid metabolism and absorption capacity of the detected object is extremely low, which possibly leads to the rise of homocysteine in blood, and further leads to higher recurrent abortion risk and higher DVT risk.

Site F5 (41721G > A), dbSNP accession number rs6025, if genotype is CC, score 0 score, is normal; if the genotype is TC and the score is 5, the risk of moderate activated protein C resistance of the detected subject is indicated, and then the risk of moderate recurrent abortion and the risk of moderate DVT are caused; if the genotype is TT, the score is 25 points, which indicates that the detected object has higher risk of resisting the syndrome of activated protein C, and further causes high recurrent abortion risk and high DVT risk.

Site LINC01146 (C > T) HIT1, dbSNP is called as rs1887289, if the genotype is TT, the score is 0, and the genotype is normal; if the genotype is CT and the score is 5 points, the risk of heparin-induced thrombocytopenia in the detected subjects is indicated, and thrombus complications possibly occur; if the genotype is CC and the score is 10, the higher heparin-induced thrombocytopenia risk of the detected object is indicated, and thrombus complications can be possibly accompanied.

The locus GPR65 (T > G) HIT2, dbSNP is called as rs3742704, if the genotype is AA, the score is 0 score, and the genotype is normal; if the genotype is CA and the score is 5, the risk of heparin-induced thrombocytopenia in the detected subjects is indicated, and thrombus complications possibly occur; if the genotype is CC, the score is 10 points, and the higher heparin-induced thrombocytopenia risk is possibly accompanied by thrombosis complications.

The scoring system can also be presented by the table 1:

TABLE 1 evaluation System for blood hypercoagulability or venous thrombosis Risk

The beneficial effects of the invention are as follows:

(1) The primer provided by the invention has strong specificity and can amplify target fragments in a targeted manner.

(2) The kit can rapidly and qualitatively detect the blood hypercoagulability or venous thrombosis risk gene polymorphism, directly carry out one-time PCR amplification, and rapidly and sensitively judge the gene type of the locus to be detected through a sequencing result, thereby accurately typing the blood hypercoagulability or venous thrombosis risk gene.

(3) The detection method has accurate and comprehensive detection results of blood hypercoagulability or venous thrombosis risk, has low detection cost and can detect unknown mutation compared with methods such as fluorescent PCR and the like.

(4) The evaluation system provided by the invention is complete, has wide gene coverage, can comprehensively detect potential risks of the detected object, can more accurately reflect the accurate condition of the detected object compared with the detection of a single gene, and avoids the defects that the detection of a single gene or a few combination genes occurs, the detection of other risk genes is ignored, and the potential risks of the detected object cannot be comprehensively reflected.

Drawings

FIG. 1 shows the results of primer set-specific assays; lanes 1-6 are the specific amplification results for polymorphic sites PAI-1 (4G/5G), MTHFR (677C > T), MTHFR (1298A > C), F5 (41721G > A), LINC01146 (C > T) HIT1, GPR65 (T > G) HIT2, respectively, with M being Marker,100bp ladder.

FIG. 2 shows the results of 6 polymorphic site detection in healthy pregnant women.

FIG. 3 shows the results of 6 polymorphic site detection in pregnant women in a hypercoagulable state.

Detailed Description

In order to enable those skilled in the art to better understand the technical scheme of the present invention, the following detailed description of the preferred embodiments of the present invention is provided, but the following embodiments do not limit the scope of the present invention.

In the examples of the present invention, which are not described in detail, the procedures of PCR, cleavage, ligation, codon optimization, etc., which are all performed by conventional molecular biology experimental methods, are understood and easily accomplished by those skilled in the art based on the product specifications or the basic knowledge of the art, and thus will not be described in detail.

In the embodiment of the invention, the adopted nucleic acid extraction reagent is a blood genome DNA column type small-amount extraction kit, and CW2087 is selected as the reagent; the PCR Mix is 2 xTaq Master Mix, and the Novoxam P111 is selected; the primers involved were synthesized by Suzhou Hongsun biotechnology Co.

Example 1:

specific amplification primers were designed based on the polymorphic sites of the PAI-1, MTHFR, F5, TDAG8 (also known as GPR 65) genes in the dbSNP database, dbSNP accession number information, and the nucleotide sequences of the genes.

Wherein, the locus PAI-1 (4G/5G) has a dbSNP accession number of rs1799762; site MTHFR (677C > T), dbSNP accession number rs1801133; site MTHFR (1298A > C), dbSNP accession number rs1801131; site F5 (41721G > A), dbSNP accession number rs6025; site LINC01146 (C > T) HIT1, dbSNP accession number rs1887289; the locus GPR65 (T > G) HIT2, dbSNP accession number rs3742704.

The partially amplified nucleotide sequences of the genes are as follows:

PAI-1(4G/5G)：SEQ.ID.NO.1

MTHFR(677C>T)：SEQ.ID.NO.2

MTHFR(1298A>C)：SEQ.ID.NO.3

F5(41721G>A)：SEQ.ID.NO.4

LINC01146(C>T)HIT1：SEQ.ID.NO.5

GPR65(T>G)HIT2：SEQ.ID.NO.6

wherein, the underlined part in the sequence is the primer sequence position, the shaded and bolded position is the mutation site, wherein, 4 continuous G are in SEQ ID No.1, and 5 continuous G are possibly mutated; the G in SEQ ID No.2 may be mutated to A or C; t in SEQ ID No.3 may be mutated to G; the mutation of C in SEQ ID No.4 to A or T is possible; t in SEQ ID No.5 may be mutated to A or C; a in SEQ ID No.6 may be mutated to C.

The detailed information of the primer is as follows:

example 2:

according to example 1, a primer set for detecting a blood hypercoagulability or venous thrombosis risk gene polymorphism, said primer comprising:

In this embodiment, a detection kit is prepared for detecting a blood hypercoagulability or venous thrombosis risk gene polymorphism, and the kit comprises a primer for detecting the blood hypercoagulability or venous thrombosis risk gene polymorphism, a PCR Mix and sterilized deionized water.

In the kit, the primer for detecting the blood hypercoagulability or venous thrombosis risk gene polymorphism comprises the following components:

(1) Primers for detecting PAI-1 (4G/5G) polymorphism, including the primers shown in SEQ ID No.7 and SEQ ID No. 8; the primer is a primer aiming at the polymorphic locus of the PAI-1 gene rs1799762, and the size of an amplified product is 369bp.

(2) Primers for detecting MTHFR (677C > T) polymorphism include primers shown in SEQ ID.NO.9 and SEQ ID.NO. 10; the primer is a primer aiming at the rs1801133 polymorphic site of the MTHFR gene, and the size of an amplified product is 309bp.

(3) Primers for detecting MTHFR (1298A > C) polymorphism include primers shown in SEQ ID.NO.11 and SEQ ID.NO. 12; the primer is a primer aiming at the MTHFR gene rs1801131 polymorphic site, and the size of an amplified product is 377bp.

(4) Primers for detecting the F5 (41721G > A) polymorphism include the primers shown in SEQ ID.NO.13 and SEQ ID.NO. 14; the primer is a primer aiming at the rs6025 polymorphic site of the MTHFR gene, and the size of an amplified product is 332bp.

(5) Primers for detecting LINC01146 (C > T) HIT1 polymorphism include primers shown in SEQ ID.NO.15 and SEQ ID.NO. 16; the primer is a primer aiming at the polymorphic locus of the TDAG8 gene rs1887289, and the size of an amplified product is 317bp.

(6) Primers for detecting GPR65 (T > G) HIT2 polymorphism include the primers shown in SEQ ID No.17 and SEQ ID No. 18. The primer is a primer aiming at the polymorphic locus of the TDAG8 gene rs3742704, and the size of an amplified product is 321bp.

In the kit, the PCR Mix is preferably 2 xTaq Master Mix, wherein the 2 xTaq Master Mix comprises Taq DNA Polymerase, dNTPs and an optimized buffer system; the concentration of the primer was 10. Mu.M.

In the embodiment, the pregnant woman blood DNA is collected as a sample to be detected for amplification, and the specificity and accuracy of the primer are verified. Respectively preparing amplification systems for amplifying different bit sequences, wherein each amplification system is provided with 3 parallels.

Preparing a PCR system:

system composition	Additive amount
		2×Taq Master Mix	12.5μL
Amplification primers	Upstream and downstream primers were 10. Mu.M each
		Sample genomic DNA	10ng
Sterilized deionized water	Make up to 25. Mu.L

The amplification procedure was: 2min pre-denaturation at 96 ℃, 30s at 57 ℃, 2min at 72 ℃ and 35 cycles; extended 5min at 72℃and endless circulation at 4 ℃.

The specificity or effectiveness of the primers was verified by agarose gel electrophoresis of the amplified products. The amplification result proves that 3 parallels of each pair of primers can effectively amplify single fragments; and sequencing the PCR product, and performing blast verification on the whole sequence of the sequencing result, wherein the amplified fragment is the required target fragment, and the 6 pairs of primers are proved to be specific and specific. As shown in FIG. 1, each primer can amplify target fragment effectively, lanes 1-6 are specific amplification results of polymorphic sites PAI-1 (4G/5G), MTHFR (677C > T), MTHFR (1298A > C), F5 (41721G > A), LINC01146 (C > T) HIT1, GPR65 (T > G) HIT2, M is Marker,100bp ladder, and commercial supplier is well known as century, and product number CW0636S.

Example 3:

in the embodiment, genome fragments extracted from blood cells of healthy pregnant women are used as templates, primers designed for SNP loci related to the venous blood hypercoagulability are amplified, and a method for detecting human venous thrombosis susceptibility based on Sanger sequencing is established on the basis. Mainly comprises the following steps and reagents:

in this example, the primers and kits described in examples 1 and 2 were used for detection.

In the kit, the 2 xTaq Master Mix as an amplification reagent contains Taq DNA Polymerase, dNTPs and an optimized buffer system, and can be amplified only by adding primers and templates. The protective agent added into the amplification system can keep stable activity after repeated freeze thawing of the 2X Master Mix.

(1) Genomic DNA extraction: the 200 mu L EDTA anticoagulated blood is extracted by using a small-amount extraction kit of a genomic DNA column type of CW2087 blood, and the electrophoresis of the genomic DNA of each sample is not degraded and has good brightness, thus proving that the extraction effect is good; the extraction amount is l-2 mug, and the A260/A280 is between 1.8 and 2.0, which meets the amount and purity required by amplification.

(2) Specific fragment amplification:

the amplification system was formulated as follows:

For each sample, setting a total of 6 tube systems, and respectively adding 6 pairs of different amplification primers; amplifying on a gene amplification instrument under the following conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 1min, and cycling for 35 times; finally, the extension is carried out for 5min at 72 ℃.

(3) Sequencing of the target gene fragment:

all amplified products were labeled and were sequenced by HongXuehong Biotech Inc., and the sequencing peaks are shown in FIG. 2 (only one-way sequencing pattern was shown). The key sites and genotypes of the samples are indicated.

According to FIG. 2 and the judgment system of the invention, FIG. 2A shows the detection results of PAI-1 (4G/5G) and rs1799762 sites, wherein the genotypes of the sites are 5G/5G, and the scores are 0 points; FIG. 2B shows the detection results of MTHFR (677C > T) and rs1801133 locus, wherein the locus genotype is AG, heterozygous mutation occurs, and the score is 5 minutes; FIG. 2C shows the detection results of MTHFR (1298A > C) and rs1801131 locus, wherein the locus genotype is TT, no mutation occurs, and the score is 0 score; FIG. 2D shows the detection results of F5 (41321G > A) and rs6025 sites, wherein the genotype of the sites is CC, no mutation occurs, and the score is 0; FIG. 2E shows the detection results of LINC01146 (C > T) HIT1 and rs1887289 sites, wherein the genotype of the sites is CT, heterozygous mutation occurs, and the score is 5 points; FIG. 2F shows the results of GPR65 (T > G) HIT2 and rs3742704 locus, the genotype of the locus is AA, no mutation occurs, and the score is 0.

According to the judging system provided by the invention, the risk score of the detected target is 10, the risk has medium risk of blood hypercoagulability or venous thrombosis, the probability of thrombotic diseases of the object to be detected is high, and the related examination of the blood hypercoagulability state can be regularly carried out.

Example 4:

in the embodiment, genome fragments extracted from blood of pregnant women with thrombus hypercoagulability are taken as templates, primers designed for SNP loci related to venous blood hypercoagulability are amplified, and a method for detecting human venous thrombus susceptibility based on Sanger sequencing is established on the basis. Mainly comprises the following steps and reagents:

(2) Specific fragment amplification:

the amplification system was formulated as follows:

(3) Sequencing of the target gene fragment:

all amplified products were labeled and were sequenced by HongXuehong Biotech Inc., and the sequencing peaks are shown in FIG. 3 (only one-way sequencing pattern was shown). The key sites and genotypes of the samples are indicated.

According to FIG. 3 and the judgment system of the invention, FIG. 3A shows the detection results of PAI-1 (4G/5G) locus rs1799762, and the 159 th base begins to have two sets of different peak shapes overlapped together, which indicates that 4G/5G heterozygous mutation occurs at the locus, and the score is 5 minutes; FIG. 3B shows the detection results of MTHFR (677C > T) and rs1801133 locus, wherein the locus genotype is AA, homozygous mutation occurs, and the score is 15 minutes; FIG. 3C shows the detection results of MTHFR (1298A > C) and rs1801131 locus, wherein the locus genotype is TG, heterozygous mutation occurs, and the score is 5; FIG. 3D shows the detection results of F5 (41321G > A) and rs6025 sites, wherein the genotype of the sites is CC, no mutation occurs, and the score is 0; FIG. 3E shows the detection results of LINC01146 (C > T) HIT1 and rs1887289 sites, wherein the genotype of the sites is CC, homozygous mutation occurs, and the score is 10 points; FIG. 3F shows the results of GPR65 (T > G) HIT2 and rs3742704 locus, the genotype of the locus is AA, no mutation occurs, and the score is 0.

According to the judgment system of the invention, the risk score of the detected target is 35 points, and the risk of suffering from blood hypercoagulability or venous thrombosis is high. Detecting that the PAI-1 locus of a pregnant woman subject is 4G/5G type, the PAI-1 activity is increased to different degrees, and the probability of occurrence of thrombotic diseases is increased; PAl-1 is related to infiltration of trophoblast to intima and fibrin deposition in early gestation, and is prone to adverse events related to early gestation, such as recurrent abortion, stillbirth, premature birth, etc. Can be used for periodically checking the blood hypercoagulability, and measures are taken to prevent thrombus in early pregnancy.

The MTHFR gene encodes a key enzyme in the folate metabolic pathway that maintains normal levels of homocysteine in the body and is involved in DNA synthesis. Detecting heterozygous mutation of MTHFR gene 677C > T locus of pregnant woman object, which shows that the activity of methylene tetrahydrofolate reductase is slightly reduced, and proper amount of folic acid is required to be supplemented according to medical advice during pregnancy; if the mutation is homozygous, the activity of the methylene tetrahydrofolate reductase is reduced more, and the risk of recurrent abortion and the risk of thrombotic diseases are high, so that the folic acid needs to be supplemented in a large amount according to the order of doctors. The MTHFR gene 1298A > C site was judged as above.

In the embodiment, the F5 gene V factor locus of the detection object is not mutated, if the locus is mutated, activated Protein C Resistance (APCR) is easily caused, an APCR screening test is needed, and measures are taken to prevent thrombus in early pregnancy; if heterozygous, there is a moderate risk of thrombotic disease and recurrent abortion; if homozygous, there is a high risk.

LINC01146 (C > T) HIT1 and rs1887289, the heterozygous mutation is medium risk, and the thromboembolism of patients receiving heparin treatment or patients after HIT is obviously increased.

Sequence listing

<110> Jiangsu Yino biological technology Co.Ltd

<120> primer set, kit and detection method for detecting blood hypercoagulability or venous thrombosis risk gene polymorphism and application

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ttatgggctc tcctgggccc ctcacctgga tgggaaagat cccggggacg atggggcaag 240

tgatgcccat gtcggtgcat gccttcacaa agcggaagaa tgtgtcagcc tcaaagaaaa 300

gctgcgtgat gatgaaatcg gctcccgcag acaccttctc cttcaagtgc ttcaggtcag 360

cctcaaagct ccctgcttcg gggtggcctt tggggtaacc tgccaatagg gatgacagtc 420

aggagaggct ggcctccacc tgttcaaggc gagggatgaa gagaccacag ggactgggca 480

gagagagtcc tctgctttgg gggctctgat cttgcacctt ccaacagcac agcacagcct 540

gaactaaaac agtgagtggc tgggctcatg actgatatgc ttctgacccc agagatgact 600

ctcaggggag gtggctcaga gcccagttcc aagagtgggg gtggattgct ccaaactccc 660

<210> 3

<211> 720

<212> DNA

<213> human (homosapiens)

<400> 3

aggcaagtca cctgggagag acggtgagct ggctggggcg accatcaggt ttggcaccct 60

gagtccctct cacggccccc aacaaagacc cagcctgtct ttgcctccct aagcccttcc 120

aggtggaggt ctcccaactt acccttctcc ctttgccatg tccacagcat ggaggggagg 180

gcacaggatg gggaagtcac agccccgcag cctggcctgc agctggggtc aggccagggg 240

caggggatga accagggtcc ccactccagc atcactcact ttgtgaccat tccggtttgg 300

ttctcccgag aggtaaagaa cgaagacttc aaagacactt tcttcactgg tcagctcctc 360

cccccacatc ttcagcagct cctccttggg ggacttgctc ttcaggtaga agaggtagta 420

gtccttcagc tccccaaagg caggggaaga ggaattgccc ctggcagagg ggtgcccaga 480

ggtcagggca cactcctgac agagggcagt gccaccacat gcccaggagg ccattcctgt 540

aaattctgcc cctgactcct cccaggtcaa ccacaagcat gcaaacttct tctgccctcc 600

cgctcccaag aacaaagatg tatttgcaag gaaggtctgc aggccctcac cagcggccgt 660

tagggaactc gtcccactcc tgggtacggt agatgtaact ctttggtctg gaggcccaga 720

<210> 4

<211> 660

<212> DNA

<213> human (homosapiens)

<400> 4

aaaaggaaga aattaggaaa ggctaatcaa cttgctcaac acatccaata ccaacagacc 60

tggaatttga aactaagaca aaatatgtta tcacactcta gacttgcctt cggcagtgat 120

ggtactgata aaaatagaca agacaaaaaa aaaaaaagaa taaatgttat cacactggtg 180

ctaaaaagga ctacttgaca attactgttc tcttgaagga aatgccccat tatttagcca 240

ggagacctaa catgttctag ccagaagaaa ttctcagaat ttctgaaagg ttacttcaag 300

gacaaaatac ctgtattcct cgcctgtcca gggatctgct cttacagatt agaagtagtc 360

ctattagccc agaggcgatg tctctcatga tgtccacgtc actgtagtat ggtcttgtta 420

agcactgggc atcattttct gtgggttcat caaactctaa gatgttccac ttataagtat 480

aggtttcccc tggttgaact gctctgatca tggtgttgtt cctgcctgaa agaaaatata 540

ttcaaaattg ttttcatttg caaagttatt tcatgataat aaataaataa ataagctttc 600

gctggaacca attaatattg caaaaggaat tcttttattt ttattttttt taaattatac 660

<210> 5

<211> 550

<212> DNA

<213> human (homosapiens)

<400> 5

taatccagat cttagagtct tcccctactg catctctcta aagaagatta aagaaaaata 60

ttctgagaaa tgtagccaag aagtgaaaag aaaagcttat gcatctctac taaataggtc 120

aacagtagca acacaccacc ttggatatgc tatgtgccaa atactctgga gtccttgtct 180

gaaaagcttc aagaagagtg acataactgt taattttctc taccttagtg gtgtaacacc 240

aaaatgagaa tatccaccgt tctcattact gccacatttc cccaaatctc caaaagacat 300

ctaagtgtca tctggtccca cgatccctcc ctagacccac cacatttgct ctgagaggtc 360

ctctagtccc tactggaaca cagtcgggaa tgggaactca cggcctcccc aggcagcccc 420

tttcatcttt gcatggctca gagataaaat tattctgagc ctggatctca tgccctgtgg 480

tttctgctca tggttctgcc tttcgaaatt cagaataatt ttaatccatc ttttacatga 540

cagctgtcat 550

<210> 6

<211> 550

<212> DNA

<213> human (homosapiens)

<400> 6

tgctgtcatg ttgtgggaag atgaaacagt tgttgaatat tgcgatgccg aaaagtctaa 60

ttttacttta tgctatgaca aatacccttt agagaaatgg caaatcaacc tcaacttgtt 120

caggacgtgt acaggctatg caataccttt ggtcaccatc ctgatctgca accggaaagt 180

ctaccaagct gtgcggcaca ataaagccac ggaaaacaag gaaaagaaga gaatcataaa 240

actacttgtc agcatcacag ttacttttgt cttatgcttt actccctttc atgtgatgtt 300

gctgattcgc tgcattttag agcatgctgt gaacttcgaa gaccacagca attctgggaa 360

gcgaacttac acaatgtata gaatcacggt tgcattaaca agtttaaatt gtgttgctga 420

tccaattctg tactgttttg taaccgaaac aggaagatat gatatgtgga atatattaaa 480

attctgcact gggaggtgta atacatcaca aagacaaaga aaacgcatac tttctgtgtc 540

tacaaaagat 550

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gggcagctcg aagaagtga 19

<210> 8

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

ggaccacctc caggaaaga 19

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gagggagctt atgggctctc 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ctgcccagtc cctgtggtct 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

acaggatggg gaagtcacag 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ggagtcaggg gcagaattta 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

aaatgttatc acactggtgc 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ccaggggaaa cctatactta 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

aacagtagca acacaccacc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gccatgcaaa gatgaaaggg 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

atgcaatacc tttggtcacc 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

atcttcctgt ttcggttaca 20

Claims

1. A primer set for detecting a blood hypercoagulability or venous thrombosis risk gene polymorphism, said primer comprising:

2. Use of the primer set of claim 1 for preparing a product for detecting blood hypercoagulability or venous thrombosis risk gene polymorphism.

3. A test kit for detecting a blood hypercoagulability or venous thrombosis risk gene polymorphism, comprising the primer set for detecting a blood hypercoagulability or venous thrombosis risk gene polymorphism of claim 1.

4. A kit according to claim 3, further comprising other reagents required for a PCR reaction, including PCR mix or Taq DNA Polymerase, dntps.

5. A kit according to claim 3, wherein the concentration of primer in the kit is 10 μm.