Kit for detecting thrombophilia related gene loci on MALDI-TOF (matrix-assisted laser desorption / ionization-time of flight) mass spectrum platform

Abstract

The invention belongs to the technical field of gene detection, discloses a kit for detecting thrombophiliosis related gene loci through an MALDI-TOF mass spectrum platform, and provides a group of multiple PCR amplification primers and single base extension primers for detecting the thrombophiliosis related gene loci through the MALDI-TOF mass spectrum platform and application of the multiple PCR amplification primers and the single base extension primers in the kit. The detected object comprises the genotypes of seven gene loci related to thrombophilia. The established detection kit has both flexibility and expandability, is simple to operate, high in throughput, automatic, high in throughput and low in cost, can effectively screen the problems of thrombophiliosis susceptibility and the like caused by genetic polymorphism in advance, and can help doctors to adjust a medication scheme so as to obtain an efficient, safe and economic optimal treatment effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene detection, and particularly relates to a kit for detecting thrombophilia related gene sites on a MALDI-TOF mass spectrometry platform. BACKGROUND

[0002] Thrombophilia refers to a pathological state of being prone to thrombosis and thromboembolism due to various genetic or acquired factors. The main clinical manifestations of thrombophilia are venous thromboembolism (VTE), such as deep vein thrombosis, pulmonary embolism, intracranial venous thrombosis, portal vein thrombosis, and mesenteric vein thrombosis. Some types of thrombophilia can manifest as young-onset acute coronary syndrome, ischemic stroke, and other arterial thrombotic events. The repeated occurrence of thrombotic events caused by thrombophilia significantly increases the morbidity and mortality of patients, seriously endangering human health. The Thrombosis and Hemostasis Group of the Chinese Society of Hematology published the “Guidelines for the Diagnosis and Prevention of Thrombophilia in China (2021 Edition)”, which clearly states that 60% of the differences in individual susceptibility to VTE are due to genetic factors. The guidelines point out that genetic thrombophilia is often caused by mutations in genes such as antithrombin (AT), protein C (PC), and other genes that lead to the loss of protein anticoagulant function, or mutations in genes such as coagulation factor V Leiden and prothrombin G20210A that lead to enhanced protein procoagulant function, ultimately causing thromboembolism. The guidelines point out that protein C deficiency, antithrombin deficiency, elevated FⅪ levels, F5 Leiden mutation, and F2 G20210A mutation are high-risk factors for VTE, and elevated PAI-1 levels are a medium-to-low-risk factor.

[0003] Protein C is involved in maintaining the dynamic homeostasis of coagulation and the balance of the body's anticoagulant and fibrinolytic system. Protein C c.565C>T (rs146922325) variation leads to anticoagulant protein deficiency, and the activity decreases to appear thrombophilia. F2 c.*97G>A (G20210A, rs1799963) mutation belongs to an autosomal dominant inheritance, and the coagulation function is activated due to abnormal mutation, and this mutation can increase the risk of thromboembolism by 3 times. PAI-1 inhibits plasminogen activation g.4332dupG (rs1799889) maintains PAI-1 at a normal level, and 4G / 5G or 4G / 4G genotype can cause elevated plasma PAI-1 concentration, promote atherosclerosis, reduce plasmin activity, increase platelet adhesion and aggregation function, and cause thrombosis. FXI is related to thrombin production and indirectly inhibits fibrinolysis, and F11 c.1481-188C>T (rs2289252), c.56-282T>C (rs2036914) variation increases the activity level of F11, promotes coagulation effect, and increases the risk of thrombosis. F5 gene encodes the 5th coagulation factor, and F5 c.1601G>A (F5 Leiden mutation, rs6025) can inhibit the effect of active protein C, cause excessive thrombin production, cause blood clotting too fast, and increase the risk of thrombosis. AT antithrombin inhibits thrombin activity and interferes with the early stage of coagulation cascade to regulate clot formation, and c.41+141G>A (rs2227589) variation leads to significant reduction of antithrombin and anti-FXa activity levels, leading to easy thrombophilia.

[0004] The polymorphisms of PAI-1 gene 4G / 5G site, F11 gene c.1481-188C>T site, F11 gene c.56-282T>C, F5 gene c.1601G>A site, F2 gene c.*97G>A site, PC gene c.565C>T site and AT gene c.41+141G>A site were qualitatively detected, and the correlation of the polymorphisms of the above sites with genetic thrombophilia was mainly from the reports of domestic and foreign literatures, and was supported by a large amount of clinical experimental data.

[0005] Currently, the most commonly used SNP detection means in clinic mainly include Sanger sequencing, fluorescent quantitative PCR, low-density gene chip and pyrosequencing, etc. These technologies greatly promote the application of gene detection in clinical medicine. However, with the deepening research on disease mechanism and treatment means, the demand for SNP detection in clinic is gradually transferred from a limited number of sites of single gene to multiple genes and multiple sites. The above-mentioned existing clinical commonly used gene detection technologies are not completely suitable for the detection requirement of multiple genes and multiple sites. In comparison, MALDI-TOF mass spectrometry utilizes multiplex PCR technology, i.e. one reaction tube can simultaneously detect multiple SNP sites (up to dozens of sites), which can greatly improve the detection efficiency of multiple genes and multiple sites and reduce the sample amount, meeting the new requirements of clinic. SUMMARY

[0006] To overcome the above technical problems, the present application provides a kit for detecting thrombophilia related gene sites by MALDI-TOF mass spectrometry platform.

[0007] The present application adopts the following technical solutions: A set of MALDI-TOF mass spectrometry platform detection of thrombophilia related gene sites of multiplex PCR amplification primer, nucleotide sequence as shown in SEQ ID NO: 1-14.

[0008] Multiplex PCR amplification primer detailed table 1: The present application also discloses a set of MALDI-TOF mass spectrometry platform detection of thrombophilia related gene sites of single base extension primer, including single base extension primer group, the nucleotide sequence of single base extension primer group is as shown in SEQ ID NO: 15-21.

[0009] Single base extension primer detailed table 2: The single base extension primer group carries out single base extension reaction in a system to detect 7 gene sites related to thrombophilia susceptibility risk.

[0010] The present application also discloses the application of one or several of the multiplex PCR amplification primer and / or one or several of the single base extension primer in the preparation of a kit for detecting the genotype of thrombophilia related gene sites.

[0011] The application also discloses a kit for detecting the genotype of a gene locus susceptible to thrombosis, comprising: a multiplex PCR reaction primer mixture containing nucleotide sequences as shown in SEQ ID NO: 1-14, a single base extension primer mixture containing nucleotide sequences as shown in SEQ ID NO: 15-21, and a multiplex PCR reaction mixture, a multiplex PCR enzyme, a SAP reaction mixture, a SAP enzyme mixed single base extension reaction mixture, a single base extension enzyme, desalting resin, and a mass spectrometry chip; The multiplex PCR reaction mixture comprises ribonucleotides, divalent magnesium ions, chloride ions, water, Tris-Hcl and potassium chloride, the multiplex PCR enzyme comprises polymerase and glycerol, the SAP reaction mixture comprises magnesium chloride buffer and Tris-Hcl, the SAP enzyme comprises shrimp alkaline phosphatase and glycerol, and the single base extension reaction mixture comprises ammonium sulfate, potassium chloride, Tris-Hcl and dideoxynucleotides, and the single base extension enzyme comprises glycerol and high-specificity polymerase.

[0012] The kit contains nucleotide sequences as shown in SEQ ID NO: 1-14 in the multiplex PCR reaction primer mixture, and the multiplex PCR amplification primers are all 0.2 μM. The kit contains nucleotide sequences as shown in SEQ ID NO: 15-22 in the single base extension primer mixture, wherein SEQ ID NO: 15 is 8.83 μM, SEQ ID NO: 16 is 12.76 μM, SEQ ID NO: 17 is 12.10 μM, SEQ ID NO: 18 is 11.02 μM, SEQ ID NO: 19 is 11.81 μM, SEQ ID NO: 20 is 9.97 μM, and SEQ ID NO: 21 is 9.14 μM.

[0013] Compared with the prior art, the application has the following beneficial effects: 1. The object detected by the application includes the genotypes of 7 gene loci related to thrombosis, and the detection kit established has flexibility and expandability, is simple to operate and high-throughput and automatic, is high-throughput and low-cost, and can effectively screen problems such as susceptibility to thrombosis caused by genetic polymorphism in advance.

[0014] 2. The primer set provided by the application does not need to add other markers such as fluorescein and biotin, only needs to synthesize ordinary primers, has low detection cost, and is suitable for large-scale population molecular screening and routine genetic diagnosis.

[0015] 3. The application realizes detection of thrombosis gene loci in the same kit, can effectively solve problems such as thrombosis caused by genetic polymorphism, and can help doctors adjust treatment plans to achieve efficient, safe and economical optimal treatment effects. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the electrophoresis result of VTE7 gene multiplex amplification; Figure 2 is the mass spectrum of sample detection result; Figure 3 is the mass spectrum result of PAI-1 gene g.4332dupG site of the sample detected by the method of Example 1; Figure 4 is the mass spectrum result of F2 gene c.*97G>A site of the sample detected by the method of Example 1; Figure 5 is the mass spectrum result of F5 gene c.1601G>A site of the sample detected by the method of Example 1; Figure 6 is the mass spectrum result of AT gene c.41+141G>A site of the sample detected by the method of Example 1; Figure 7 is the mass spectrum result of PC gene c.565C>T site of the sample detected by the method of Example 1; Figure 8 is the mass spectrum result of F11 gene c.1481-188C>T site of the sample detected by the method of Example 1; Figure 9 is the mass spectrum result of F11 gene c.56-282T>C site of the sample detected by the method of Example 1; Figure 10 is the heterozygous mass spectrum result of PAI-1 gene g.4332dupG site of the sample detected by the method of Example 1; Figure 11 is the 5G5G mass spectrum result of PAI-1 gene g.4332dupG site of the sample detected by the method of Example 1; Figure 12 is the heterozygous mass spectrum result of F11 gene c.56-282T>C site of the sample detected by the method of Example 1. DETAILED DESCRIPTION

[0017] The embodiments of the present application are described in detail below, the examples of the embodiments are shown in the accompanying drawings, and if not specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art, and the methods in the embodiments are the conventional methods in the art, unless otherwise specified. The embodiments described below with reference to the accompanying drawings are exemplary and are used to explain the present application, and cannot be understood as a limitation of the present application.

[0018] A kit for detecting a gene site related to thrombophilia by MALDI-TOF mass spectrometry platform I. Composition The nucleotide sequence of the multiplex PCR amplification primer is shown in SEQ ID NO: 1-14, the nucleotide sequence of the single base extension primer is shown in SEQ ID NO: 15-21, the negative control and the positive control, the multiplex PCR reaction reagent (including: multiplex PCR reaction mixture, multiplex PCR reaction enzyme mixture), dNTPs removal reaction reagent (including: SAP reaction mixture, SAP enzyme) and single base extension reaction reagent (including: extension reaction mixture, extension enzyme mixture), desalting resin, and mass spectrometry chip.

[0019] II. Method of use 1. Perform multiplex PCR reaction After extracting the total DNA of the sample to be tested, perform multiplex PCR reaction amplification.

[0020] According to the amount of specimen required for detection, first prepare the PCR reaction amplification primer mixture according to Table 4, shake well, centrifuge for a moment, then add the components in turn (except for template DNA) to the multiplex PCR reaction system, shake for 10 seconds, centrifuge for a moment, and then divide and store in the corresponding holes of the 8-hole row, finally add the template DNA, shake well, centrifuge for a moment, and then put into the PCR amplification instrument.

[0021] Table 4: Perform multiplex amplification according to the procedure in Table 5.

[0022] Table 5: 2. dNTPs removal reaction Prepare the dNTPs removal mixture reaction system according to Table 6, and then add it to the reaction tube, and perform the reaction according to the reaction procedure: 37℃ for 40min; 85℃ for 5min; 4℃ for storage.

[0023] Table 6: 3. Single base extension reaction Add the single base extension reaction system reagent to each reaction tube, and perform extension on the PCR instrument according to the reaction conditions. The extension reaction system is shown in Table 7.

[0024] Table 7: The reaction procedure is: 95℃ pre-denaturation for 30sec; [95℃ for 5sec, (48℃ for 5sec, 80℃ for 5sec, 5 cycles), 40 cycles]; 72℃ for 3min; 4℃ for storage.

[0025] The composition of the single base extension primer mixture is shown in Table 8.

[0026] Table 8: 4. Mass spectrometry detection After the reaction, centrifuge at 2000 rpm for a moment, add 14 μL of sterilized double distilled water to each well, shake well after mixing, centrifuge for a moment, pour the desalting resin into each reaction well, and desalt for 30 minutes. Mix the extension product and matrix liquid 1:1, spot 1 μL on the mass spectrometry chip, and perform mass spectrometry detection.

[0027] III. Result judgment (1) The time-of-flight mass spectrometry detection system collects and stores the pulse signals by the ion trap instrument, and performs mass spectrometry analysis. By comparing the signal intensity of A, T, G and C and the mass difference between adjacent signals (ddATP=297, ddCTP=273, ddGTP=313, ddTTP=342), the information of the template sequence can be obtained, thereby realizing the detection result of the gene site.

[0028] (2) When the result of the analyzed gene site is wild type or homozygous, a single arrow appears. If the result of the analyzed gene site is heterozygous, a double arrow appears.

[0029] The genotype results of each site are shown in Table 9 Table 9: Although the embodiments of the present application have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, replacements and variations can be made to the above embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A primer set for multiplex PCR amplification for detecting a gene locus related to thrombophilia by MALDI-TOF mass spectrometry platform, characterized in that, comprises at least one of the following primer pairs 1 to 7; The primer nucleotide sequences of primer pair 1 are shown as SEQ ID NO: 1-2; The primer nucleotide sequences of primer pair 2 are shown as SEQ ID NO: 3-4; The primer nucleotide sequences of primer pair 3 are shown as SEQ ID NO: 5-6; The primer nucleotide sequences of primer pair 4 are shown as SEQ ID NO: 7-8; The primer nucleotide sequences of primer pair 5 are shown as SEQ ID NO: 9-10; The primer nucleotide sequences of primer pair 6 are shown as SEQ ID NO: 11-12; The primer nucleotide sequences of primer pair 7 are shown as SEQ ID NO: 13-14.

2. A single base extension primer set for detecting a gene locus related to thrombophilia using a MALDI-TOF mass spectrometry platform, characterized by comprises at least one of the following 7 groups; The nucleotide sequence of group 1 is shown as SEQ ID NO: 15; The nucleotide sequence of group 2 is shown as SEQ ID NO: 16; The nucleotide sequence of group 3 is shown as SEQ ID NO: 17; The nucleotide sequence of group 4 is shown as SEQ ID NO: 18; The nucleotide sequence of group 5 is shown as SEQ ID NO: 19; The nucleotide sequence of group 6 is shown as SEQ ID NO: 20; The nucleotide sequence of group 7 is shown as SEQ ID NO:

21.

3. Use of the multiplex PCR amplification primer group of claim 1 and / or the single base extension primer group of claim 2 in the preparation of a kit for detecting the genotype of a gene site related to thrombophilia.

4. A kit for detecting the genotype of a gene locus that poses a susceptibility risk to thrombosis, characterized in that, comprises: a multiplex PCR reaction primer mixture containing multiplex PCR amplification primers with nucleotide sequences shown as SEQ ID NO: 1-14, a single base extension primer mixture containing single base extension primers with nucleotide sequences shown as SEQ ID NO: 15-21, and a multiplex PCR reaction mixture, a multiplex PCR enzyme, a SAP reaction mixture, a SAP enzyme mixture single base extension reaction mixture, a single base extension enzyme, a desalting resin, and a mass spectrometry chip.

5. The kit of claim 4, wherein The multiplex PCR reaction mixture comprises ribonucleotides, dipotassium magnesium ions, chloride ions, water, Tris-Hcl and potassium chloride, the multiplex PCR enzyme comprises polymerase and glycerol, the SAP reaction mixture comprises magnesium chloride buffer and Tris-Hcl, the SAP enzyme comprises shrimp alkaline phosphatase and glycerol, the single base extension reaction mixture comprises ammonium sulfate, potassium chloride, Tris-Hcl and dideoxynucleotides, and the single base extension enzyme comprises glycerol and high-specificity polymerase.

6. The kit of claim 4, wherein The kit contains nucleotide sequences as shown in SEQ ID NO: 1-14 in the multiplex PCR reaction primer mixture, and the multiplex PCR amplification primer is 0.2 μM; the kit contains nucleotide sequences as shown in SEQ ID NO: 15-22 in the single base extension primer mixture, wherein SEQ ID NO: 15 is 8.83 μM, SEQ ID NO: 16 is 12.76 μM, SEQ ID NO: 17 is 12.10 μM, SEQ ID NO: 18 is 11.02 μM, SEQ ID NO: 19 is 11.81 μM, SEQ ID NO: 20 is 9.97 μM, and SEQ ID NO: 21 is 9.14 μM.

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