Primer and probe compositions, kits and uses thereof for vte-associated genes

Abstract

The present application relates to a primer and probe composition for VTE related genes, a kit and its application, belonging to the technical field of VTE related gene typing, comprising a first primer pair and a first fluorescent probe for PROC gene rs1469223 site, a second primer pair and a second fluorescent probe for APOH gene rs52797880 site, a third primer pair and a third fluorescent probe for THBD gene rs16984852 site, and a fourth primer pair and a fourth fluorescent probe for SERPINC1 gene rs2227589 site; the primer and probe composition can realize the genotyping of multiple VTE gene sites in a single tube by relying on asymmetric PCR combined with probe melting curve technology, and the detection sensitivity, detection accuracy and typing specificity are all very excellent, which can meet the application requirements of large-scale genotyping screening of clinical venous thrombosis susceptible genes.

Description

Technical Field

[0001] This invention relates to the field of VTE-related gene typing technology, specifically to a primer and probe composition, kit, and application for VTE-related genes. Background Technology

[0002] Venous thromboembolism (VTE) includes deep vein thrombosis (DVT) and pulmonary embolism (PE). Deep vein thrombosis is a blood clot (thrombus) that forms in the deep veins, primarily in the legs, and symptoms include pain, tenderness, erythema, and swelling in the affected leg. Pulmonary embolism occurs when part or all of the thrombus breaks off (embolism) and travels upwards to the lungs, blocking the pulmonary artery. Pulmonary embolism has a high incidence, high disability rate, and high mortality rate. The risk of developing venous thromboembolism (VTE) is steadily increasing in cancer patients, up to nine times that of the general population. This risk varies between 0.5% and 20% in the first year, depending on cancer type, stage, treatment method, and non-cancer-related risk factors. The contribution of pre-thrombotic gene mutations to the development of venous thromboembolism has been well-established. For example, THBDc.-151G>T combined with PROC c.565C>T (which increases the risk of VTE by 6.06 times) and PROC c.574_576dup / del (which increases the risk of VTE by 6.06 times) can explain about 10% of VTE events in the Chinese population.

[0003] Data on pre-thrombotic genetic variations have been incorporated into a polygenic venous thromboembolism (VTE) risk score, which reflects an individual's genetic susceptibility to VTE in a weighted, continuous scoring system. Since these polygenic VTE risk scores do not change throughout cancer progression, they may help identify cancer patients at high risk of VTE, thus better guiding thrombosis prevention decisions. Recent research has confirmed this view, finding that polygenic risk scores can identify high-risk VTE patients independently of cancer type. Genetic testing of high-risk populations can aid in prevention efforts and provide a basis for personalized anticoagulation therapy.

[0004] For SNP detection, the mainstream methods currently include nucleic acid sequencing, amplification arrest mutation assay (ARMS-PCR), high-resolution melting curve assay (HRM), and melting curve probe assay. Among these, nucleic acid sequencing is the "gold standard" for confirming single-point mutations, but it has the limitation of a long detection cycle. While ARMS-PCR is fast, its single-tube multiplex detection capability is limited, and false positive control is highly dependent on strict primer design. High-resolution melting analysis (HRM) is a nucleic acid sequence variation detection technology based on real-time quantitative PCR that does not require specific probes. After PCR amplification, the amplicons are subjected to a fine gradient temperature increase. By observing the fluorescence changes of saturated DNA-binding dyes, differences in DNA double-strand unwinding behavior can be accurately distinguished, and sequence alterations such as base mutations, SNPs, insertions / deletions, and methylation can be identified. This method can significantly reduce aerosol contamination of amplification products through a fully enclosed reaction system. However, the underlying technology of this method is highly dependent on the temperature-dependent quenching characteristics of saturated dyes and the precision of the measurement and control instruments. For example, dye performance defects can directly inhibit PCR amplification efficiency and compress the dynamic range of fluorescence signals, reducing the system's detection sensitivity. Non-specific quenching of dyes, combined with instrument temperature and signal acquisition errors, can distort the melting curve profile and deviate from the calculated melting temperature value. This not only reduces the accuracy of the detection results but also smooths out the characteristic signal differences between different genotypes, severely weakening the specificity of variant typing. Consequently, the resolution of HRM is limited, and it is often unable to accurately distinguish specific variant types for sites with extremely small changes in Tm values. Melting curve probe assay (MCP) is a closed-tube genotyping technique mediated by sequence-specific probes. It first enriches single-stranded target nucleic acids through asymmetric PCR, then collects the fluorescent signal of probe-target hybridization and double-strand dissociation upon heating. The Tm difference, resulting from the difference in probe-template matching, distinguishes between wild-type and mutant genotypes. Genotyping is achieved by relying on site-specific fluorescent probe hybridization and dissociation signals, unaffected by amplicon GC distribution or non-specific amplification products. The detection results are almost independent of dye temperature quenching characteristics and instrument temperature control accuracy, exhibiting significantly better genotyping specificity and stability than HRM. In practice, the design quality of primers and probes directly determines the sensitivity, accuracy, and genotyping specificity of SNP detection using the melting curve probe assay. However, for SNP detection of VTE-related pathogenic genes, current technologies lack primer and probe combinations capable of high sensitivity, high accuracy, and high specificity for VTE-related genes, a problem that urgently needs to be addressed. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems, the first aspect of the present invention provides a primer and probe composition for VTE-related genes. This composition can achieve genotyping of multiple VTE gene loci in a single tube. It not only has the advantages of simple operation, short time consumption, and high detection efficiency, but also has excellent detection sensitivity, detection accuracy and genotyping specificity.

[0006] A primer and probe composition for VTE-related genes includes a first primer pair and a first fluorescent probe targeting the rs146922325 site of the PROC gene. The first primer pair includes a dominant primer and a dominant primer, the nucleotide sequence of which is shown in SEQ ID NO:1, the nucleotide sequence of which is shown in SEQ ID NO:2, and the nucleotide sequence of which is shown in SEQ ID NO:3. A second primer pair and a second fluorescent probe targeting the rs52797880 site of the APOH gene are also included. The second primer pair includes a dominant primer and a dominant primer, the nucleotide sequence of which is shown in SEQ ID NO:4, the nucleotide sequence of which is shown in SEQ ID NO:5, and the nucleotide sequence of which is shown in SEQ ID NO:6. A third primer pair and a third fluorescent probe targeting the rs16984852 site of the THBD gene are also included. The third primer pair includes a dominant primer and a dominant primer, the nucleotide sequence of which is shown in SEQ ID NO:7, and the nucleotide sequence of which is shown in SEQ ID NO:8. As shown in NO:8, the nucleotide sequence of the third fluorescent probe is shown in SEQ ID NO:9; and the fourth primer pair and the fourth fluorescent probe targeting the rs2227589 site of the SERPINC1 gene, the fourth primer pair including a dominant primer and a dominant primer, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:10, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:11, and the nucleotide sequence of the fourth fluorescent probe is shown in SEQ ID NO:12. The primer and probe composition provided in this protocol is suitable for detecting single nucleotide polymorphisms (SNPs) of pathogenic genes related to venous thromboembolism (VTE). Combined with the melting curve probe method, it has the advantages of simple operation, short detection cycle, and high detection efficiency. This primer and probe composition can not only simultaneously perform genotyping of the PROC gene rs146922325, APOH gene rs52797880, THBD gene rs16984852, and SERPINC1 gene rs2227589 in a single reaction tube, but also has excellent detection sensitivity, detection accuracy, and genotyping specificity, which can meet the application needs of clinical venous thrombosis susceptibility gene genotyping detection.

[0007] Furthermore, the protocol includes a fifth primer pair and a fifth fluorescent probe targeting the rs1799963 site of the F2 gene. The fifth primer pair comprises a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:13, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:14, and the nucleotide sequence of the fifth fluorescent probe is shown in SEQ ID NO:15. In this protocol, the addition of an asymmetric primer pair and matching fluorescent probe targeting the rs1799963 site of the F2 gene allows for compatibility with primers and probes from other sites within the same reaction system. This enables simultaneous genotyping of the rs1799963 site of the F2 gene alongside other VTE risk genes, effectively broadening the detection target coverage, resulting in clear genotyping signals and excellent genotype differentiation.

[0008] Furthermore, the protocol includes a sixth primer pair and a sixth fluorescent probe targeting the rs2036914 site of the F11 gene. The sixth primer pair comprises a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:16, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:17, and the nucleotide sequence of the sixth fluorescent probe is shown in SEQ ID NO:18. In this protocol, the addition of an asymmetric primer pair and matching fluorescent probe targeting the rs2036914 site of the F11 gene allows for compatibility with primers and probes from other sites within the same reaction system. This enables simultaneous genotyping of the rs2036914 site of the F11 gene alongside other VTE risk genes, effectively broadening the detection target coverage, resulting in clear genotyping signals and excellent genotype differentiation.

[0009] Furthermore, the protocol also includes a seventh primer pair and a seventh fluorescent probe targeting the rs2066865 site of the FGG gene. The seventh primer pair comprises a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:19, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:20, and the nucleotide sequence of the seventh fluorescent probe is shown in SEQ ID NO:21. In this protocol, the addition of an asymmetric primer pair and matching fluorescent probe targeting the rs2066865 site of the FGG gene allows for compatibility with primers and probes from other sites within the same reaction system. This enables simultaneous genotyping of the rs2066865 site of the FGG gene alongside other VTE risk genes, effectively broadening the detection target coverage, resulting in clear genotyping signals and excellent genotype differentiation.

[0010] Furthermore, the protocol includes an eighth primer pair and an eighth fluorescent probe targeting the MTHFR gene rs1801133 site. The eighth primer pair comprises a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:22, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:23, and the nucleotide sequence of the eighth fluorescent probe is shown in SEQ ID NO:24. In this protocol, the addition of an asymmetric primer pair and matching fluorescent probe targeting the MTHFR gene rs1801133 site allows for compatibility with primers and probes from other sites within the same reaction system. This enables simultaneous genotyping of the MTHFR gene rs1801133 site with other VTE risk genes, effectively broadening the detection target coverage, resulting in clear genotyping signals and excellent genotype differentiation.

[0011] In practice, placing multiple sets of asymmetric primers and probes in the same reaction system can easily lead to primer competition, amplification bias, non-specific amplification, and fluorescence channel crosstalk. To address this technical problem, the second aspect of this invention further divides the primer pairs and fluorescent probes targeting the eight sites into two reaction units: a first reaction unit and a second reaction unit. The first reaction unit includes primer pairs and fluorescent probes targeting the PROC gene rs146922325, SERPINC1 gene rs2227589, FGG gene rs2066865, and MTHFR gene rs1801133 sites. This first reaction unit is used to achieve single-tube quadruple detection. The second reaction unit includes primer pairs and fluorescent probes targeting the APOH gene rs52797880, THBD gene rs16984852, F2 gene rs1799963, and F11 gene rs2036914 sites. This second reaction unit is also used to achieve single-tube quadruple detection. This leads to a dual-tube, quadruple VTE-SNP genotyping technique. In existing technologies, when multiple sets of asymmetric primers and fluorescent probes are concentrated in a single PCR reaction system for simultaneous multi-site SNP genotyping, a series of technical challenges arise as the number of primers and probes increases. These challenges include primer-substrate competition, amplification efficiency imbalance, non-specific cross-binding amplification, fluorescence signal interference, and melting peak overlap / crosstalk. Furthermore, the asymmetric amplification system itself has concentration differences between dominant and suboptimal primers. When multiple asymmetric amplification systems coexist, amplification bias is further amplified, easily leading to suppressed amplification at some sites, loss of genotype signals, blurred distinction between heterozygous peaks, and disordered fluorescence channel signals. Ultimately, this results in a significant decrease in the accuracy of simultaneous multi-site genotyping, making stable and accurate simultaneous detection of the eight key VTE SNP sites impossible, severely limiting the clinical application of high-throughput VTE susceptibility genotyping. To address the core technical bottlenecks of the existing technologies, this invention innovatively proposes a dual-tube quadruple grouping and genotyping system. This system rationally groups and arranges the primer-probe combinations for eight VTE-related SNP sites, dividing them into two independent single-tube quadruple reaction units: the first reaction unit integrates primer-probe combinations for four sites (PROC, SERPINC1, FGG, and MTHFR), and the second reaction unit integrates primer-probe combinations for four sites (APOH, THBD, F2, and F11). Through precise site grouping and matching, this system fundamentally solves the technical defects caused by the coexistence of multiple asymmetric amplification systems.

[0012] Furthermore, preliminary experiments have shown that this invention, through specific grouping optimization, avoids malicious substrate competition and cross-specific amplification between primers at different sites, balances the amplification efficiency within each quadruple system, eliminates fluorescence channel crosstalk and melting peak overlap caused by multiple probe coexistence, and effectively corrects amplification bias caused by the coexistence of multiple sets of asymmetric primers. This ensures that all four sets of primers and probes in each reaction unit can efficiently and specifically amplify the target fragment. Ultimately, it achieves accurate genotyping of all eight VTE-susceptible SNP sites in a single run using only two reaction units per sample. Even in the complex single-tube quadruple system, it still ensures excellent melting peak separation, clear genotype signals, and no signal interference or genotyping errors at each site. Compared with existing methods such as single-tube typing of a small number of sites, multi-tube separate testing, or multi-site co-transfer disordered typing, this invention greatly simplifies the detection operation, reduces the detection time, reduces sample and reagent consumption, and increases the detection throughput. At the same time, it greatly improves the stability, specificity, and accuracy of multi-site simultaneous typing, overcoming the technical barriers of multiple asymmetric PCR typing that have long existed in this field.

[0013] Preferably, the molar ratio of the dominant primer to the suboptimal primer at each detection site is 100:1 to 1:1. This molar ratio effectively addresses the shortcomings of uncontrollable amplification bias, site amplification imbalance, and poor heterozygous peak resolution in multiple asymmetric systems. This ratio range is a suitable interval obtained through extensive gradient experiments, taking into account the characteristics of the dual-tube quadruple genotyping system. By controllably adjusting the amplification efficiency difference between upstream and downstream primers, the amplification bias can be flexibly optimized according to the sequence characteristics of different sites, effectively suppressing primer competition and non-specific amplification in multiple systems, stabilizing the melting peak position and peak shape discrimination, adapting to complex systems with four sets of primers and probes coexisting, and significantly improving the universality, stability, and accuracy of simultaneous multi-site genotyping.

[0014] Preferably, in a 1–100 μL reaction system, the final concentration of each dominant primer is 0.1–1 μM, the final concentration of each inferior primer is 0.01–0.5 μM, and the final concentration of each probe is 0.05–0.5 μM.

[0015] Multiplex asymmetric PCR genotyping systems commonly suffer from technical challenges such as intense amplification competition, severe amplification bias, increased nonspecific amplification, and significant fluorescence crosstalk. Conventional asymmetric primer ratios are only suitable for single-site detection, with parameters often based on broad empirical settings, making them unsuitable for the complex coexistence of four primers in a single tube. This easily leads to defects such as site amplification imbalance, large differences in signal intensity, blurred heterozygous double peaks, and overlapping melting peaks, resulting in poor stability and low accuracy in multiplex genotyping systems, hindering the simultaneous and accurate detection of multiple sites. To address these existing technical problems, this paper further refines the system by setting the concentration of the dominant primer stock solution for each site to 20 μM and the addition volume to 0.5 μL; the concentration of the suboptimal primer stock solution to 2 μM and the addition volume to 0.5 μL; and the probe stock solution to 20 μM and the addition volume to 0.2 μL. In a 20 μL system, the corresponding final concentrations are 0.5 μM, 0.05 μM, and 0.2 μM, respectively. The molar ratio of the dominant to the suboptimal primer is 10:1. This invention further defines precise primer and probe concentration ratios for a dual-tube quadruple genotyping system. In a 20 μL reaction system, the final concentrations of the dominant primer (0.5 μM), the suboptimal primer (0.05 μM), and the probe (0.2 μM) are strictly limited, maintaining a fixed molar ratio of 10:1 between the dominant and suboptimal primers. This precise ratio is not easily obtained through conventional experiments in this field; rather, it represents the optimal combination obtained by the inventors through multiple rounds of concentration gradient screening, amplification balance adjustments, and quadruple system compatibility verification. It is not a simple selection of conventional parameters. This specific 10:1 asymmetric molar ratio can precisely control the amplification efficiency at each site, controllably creating a moderate amplification bias, significantly widening the melting peak temperature difference between wild-type and mutant types, effectively improving the bimodal resolution of heterozygotes, and avoiding genotype ambiguity. Simultaneously, the standardized final concentrations of primers and probes can balance the amplification competitiveness of the four primer groups within the same quadruple system, significantly reducing substrate competition between primers, suppressing non-specific amplification, and effectively solving the problem of high-activity sites suppressing low-activity site amplification in multiplex systems. Matched probe concentrations reduce fluorescence background and channel crosstalk, ensuring signal stability and excellent discrimination. This parameter system synergistically enhances the aforementioned dual-tube quadruple site grouping structure, addressing the industry pain points of multiple asymmetric genotyping from both the reaction architecture and reaction parameters perspectives. This allows the single-tube quadruple system to maintain high specificity, high sensitivity, and high genotyping accuracy, significantly increasing detection throughput and reducing sample and reagent consumption. Compared to existing crude mixing schemes, this represents a significant technological advancement.

[0016] Preferably, the 5' end of the probe is connected to a corresponding fluorescent group, and the 3' end is connected to a quenching group.

[0017] A third aspect of this invention addresses the problem of providing a kit that is compatible with VTE-related gene loci and has excellent overall detection performance. Furthermore, it provides a kit for VTE-related genes, comprising the primer and probe composition, and reaction solutions 1 and 2 separated from each other. In this solution, by configuring the primer and probe composition in the kit, the kit is suitable for detecting single nucleotide polymorphisms (SNPs) of pathogenic genes related to venous thromboembolism (VTE); it can simultaneously complete the genotyping of multiple VTE-related gene SNP loci in a single tube, with high detection sensitivity, accurate genotyping, and strong specificity; the operation process is simple, the detection cycle is short, and genotype can be directly interpreted without additional sequencing verification. It can provide a standardized, high-throughput detection tool for clinical screening of susceptibility genes for venous thromboembolism, adapting to the needs of large-scale population VTE risk stratification and precision diagnosis and treatment.

[0018] Furthermore, it also includes 2×PCR Mix, negative control and / or positive control.

[0019] Furthermore, the use of the primer and probe composition described above in the preparation of reagents or products for SNP typing of VTE-related genes is provided.

[0020] Furthermore, the kit described herein is provided for use in the preparation of reagents or products for VTE-related gene SNP typing.

[0021] The use of a primer and probe composition as described in any one of claims 1-5 in the preparation of reagents or products for SNP typing of VTE-related genes.

[0022] Compared with existing technologies, the present invention provides a primer and probe composition, kit and its application for VTE-related genes. Relying on asymmetric PCR combined with melting curve probe technology, it can achieve genotyping of multiple VTE gene loci in a single tube. It not only has the advantages of simple operation, short time consumption and high detection efficiency, but also has excellent detection sensitivity, detection accuracy and genotyping specificity, which can fully meet the application needs of large-scale genotyping screening of venous thrombosis susceptibility genes in clinical practice. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1Melting curves for different genotypes of eight VTE-related genes are shown. A represents MTHFR (Tm value of 65.5℃ for CC genotype and 55.5℃ for TT genotype); B represents PROC (Tm value of 62℃ for CC genotype and 52.5℃ for TT genotype); C represents APOH (Tm value of 69.5℃ for CC genotype and 60.5℃ for TT genotype); and D represents THBD (Tm value of 69.5℃ for GG genotype and 60.5℃ for TT genotype). 57.5℃); E is F2 (Tm value of GG type melting peak is 63℃, Tm value of AA type melting peak is 52℃); F is SERPINC1 (Tm value of GG type melting peak is 61℃, Tm value of AA type melting peak is 51℃); G is F11 (Tm value of CC type melting peak is 60.5℃, Tm value of TT type melting peak is 50.5℃) and H is FGG (Tm value of GG type melting peak is 58℃, Tm value of AA type melting peak is 48℃).

[0025] Figure 2 The results of TaqMan probe assays for 8 VTE-related SNP sites are shown. A is MTHFR; B is PROC; C is APOH; D is THBD; E is F2; ​​F is SERPINC1; G is F11 and H is FGG.

[0026] Figure 3 Real-time fluorescence curves for the detection of VTE-related genes at gradient concentrations. A represents MTHFR; B represents PROC; C represents APOH; D represents THBD; E represents F2; ​​F represents SERPINC1; G represents F11; and H represents FGG.

[0027] Figure 4 The results represent the detection of the presence / absence combinations of four targets in the first reaction unit, where MTHFR, PROC, SERPINC1, and FGG correspond to the FAM, HEX, ROX, and Cy5 channels, respectively.

[0028] Figure 5 The results show the detection results of the presence / absence combinations of four targets in the second reaction unit, where THBD, APOH, F2 and F11 correspond to the FAM, HEX, ROX and Cy5 channels respectively.

[0029] Figure 6 The melting curve for the VTE-related genotyping of the first obtained clinical specimen is shown, with S1 representing the clinical genotype sample.

[0030] Figure 7 The melting curve for the VTE-related genotyping of the second clinical specimen obtained is shown, where S2 represents the clinical genotype sample.

[0031] Figure 8The melting curve for the VTE-related genotyping of the third clinical specimen obtained, where S3 represents the clinical genotype sample.

[0032] Figure 9 The melting curve for the VTE-related genotyping of the fourth clinical specimen obtained, where S4 represents the clinical genotype sample.

[0033] Figure 10 The melting curve for the VTE-related genotyping of the fifth clinical specimen obtained, where S5 represents the clinical genotype sample.

[0034] Figure 11 Melting curve for VTE-related genotyping of the 6th clinical specimen obtained, where S6 represents the clinical genotype sample.

[0035] Figure 12 The melting curve for the VTE-related genotyping of the 7th clinical specimen obtained, where S7 represents the clinical genotype sample. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0037] Example 1

[0038] 1. Design of primer and probe compositions for VTE-related genes

[0039] Using publicly available SNP information from the NCBI dbSNP database, primer and probe sequences were designed for SNP sites of eight VTE-related genes. These eight VTE-related genes are applicable to the genetic characteristics of Chinese individuals and are: PROC rs146922325, APOH rs52797880, THBD rs16984852, SERPINC1 rs2227589, F2 rs1799963, F11 rs2036914, FGG rs2066865, and MTHFR rs1801133. Primers were designed at both ends of the detected mutation sites, and probes covered the mutation sites.

[0040] This invention targets primer and probe sequences for eight VTE-susceptible SNP sites. The screening process involved multiple rounds of initial sequence screening, in vitro amplification verification, melting curve evaluation, and multiple system compatibility testing. After eliminating numerous candidate sequences and optimizing the systems, the optimal primer and probe combinations of SEQ ID NO:1–24 were finally obtained. The specific screening process is as follows:

[0041] Round 1: Batch candidate sequence generation and preliminary theoretical screening. For each target SNP site, 20-30 sets of upstream and downstream primers and matching probes of different lengths and offsets were designed around the conserved regions upstream and downstream of the mutation site, resulting in over two hundred candidate sequence combinations. Preliminary screening was conducted through secondary structure prediction, dimer analysis, and Tm value uniformity. Unqualified sequences with severe hairpin structures, cross-sequence complementary pairing, high-probability dimers between primers, and probe-primer mismatches were eliminated. Candidate sequences with relatively qualified theoretical parameters were retained for experimental validation.

[0042] Round Two: Screening and Elimination of Singleton Amplification Performance. Singleton PCR melting curve typing experiments were performed on each retained candidate sequence, with standardized amplification conditions and system concentrations. Experimental results showed that most sequences designed using conventional software had significant defects: some sequences exhibited poor amplification specificity, resulting in non-specific melting peaks; the melting temperature difference between wild-type and mutant products was too small in some sequences, leading to peak overlap and inability to distinguish between homozygous and heterozygous genotypes; some primers showed unbalanced amplification efficiency, resulting in heterozygous double peaks of varying strengths and unstable interpretation. This round eliminated defective sequences exhibiting non-specific amplification, insufficient melting peak discrimination, and unstable typing, retaining only high-quality sequences with accurate singleton typing, clean peak shapes, and stable baselines.

[0043] The third round: Melting peak temperature difference optimization screening. Based on the sequences retained from the second round, the differences in the Tm of the wild / mutant melting peaks of each candidate combination were further refined. Sequence combinations with large bimodal temperature differences, symmetrical peak shapes, no tailing, and no overlap were prioritized for screening. This completely avoids the problems of adjacent peak positions, easy cross-interference, and ambiguous heterozygosity interpretation that are common in conventional designs, ensuring that different genotypes have independent, clear, and stably distinguishable characteristic melting intervals.

[0044] Round Four: Dual System Compatibility Screening. Most sequences with excellent single-site performance cannot be directly adapted to multi-site coexistence systems. This invention constructs dual amplification systems by combining primers and probes from different sites in pairs, batch-verifying system compatibility, and focusing on eliminating issues such as primer cross-interference, probe competition, mutual amplification inhibition, melting peak shift, and increased background. Many conventional sequences exhibited problems such as decreased amplification efficiency, disappearance of heterozygous peaks, and resurgence of non-specific signals under dual coexistence conditions, and were all eliminated one by one. After multiple rounds of pairwise pairing and trial-and-error verification, the final set of SEQ ID NO:1–24 sequence combinations can maintain their respective amplification specificity in the dual composite system, with no mutual inhibition or cross-interference, stable melting peak positions, and excellent separation, fully meeting the system compatibility requirements for simultaneous multi-site genotyping.

[0045] After multiple rounds of progressive screening and trial-and-error optimization, this embodiment finally obtained a VTE gene primer-probe composition with excellent comprehensive performance. Compared with traditional sequences, it can basically eliminate primer dimers and non-specific amplification, has a large temperature difference between wild-type and mutant melting peaks with no peak overlap, clear and stable heterozygous genotyping, and is compatible with dual-multiplex amplification systems and has good multi-sequence coexistence compatibility. The primer and probe composition sequences are shown in Table 1 below.

[0046] Table 1. Primer and probe composition sequence listing

[0047] The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. After quantification, the primers and probes were stored at -20℃ protected from light.

[0048] 2. PCR amplification

[0049] Using the specific primer and probe combinations designed above, PCR amplification reactions were performed using the target gene plasmid standard as a template. The PCR amplification reaction system is shown in Table 2.

[0050] Table 2 PCR amplification reaction system

[0051] According to the above reaction system, the samples were added to eight-tube strips, with each target containing corresponding wild-type, mutant, and heterozygous samples. The specific amplification process was as follows: Asymmetric PCR amplification was performed by adding the sample to be tested, primer pairs, and fluorescent probes. Melting curve analysis was performed on the PCR products, and the genotype of the sample was determined by the presence or absence of a specific melting peak. In this embodiment, the conditions for the fluorescent PCR amplification reaction were: 95℃ (3-10 min), 95℃ (10-30 s) → 55-65℃ (30-60 s), 30-50 cycles. The melting curve analysis conditions were: 30℃ → 80℃. For example, in this embodiment, after mixing the reaction system, PCR amplification was performed under the following conditions: 95℃ (3 min) → 95℃ (10 s) → 60℃ (30 s), 45 cycles (using a BIO-RADCFX amplification instrument), melting curve conditions: 30℃ → 80℃, and the fluorescence signal was collected using the FAM, HEX, ROX, or Cy5 channels.

[0052] 3. Analysis of Test Results

[0053] The specific method for determining the genotyping results is as follows: In the sample detection melting curve, if the MTHFR gene shows two melting curve peaks, with one melting peak having a Tm value between 63-67℃ and the other melting peak having a Tm value between 53-57℃, the sample genotype is determined to be heterozygous; otherwise, it is homozygous.

[0054] If the PROC gene shows two melting curve peaks, with one melting peak having a Tm value between 60-64℃ and the other melting peak having a Tm value between 50-55℃, the sample genotype is determined to be heterozygous; otherwise, it is homozygous.

[0055] If the APOH gene shows two melting curve peaks, with one melting peak having a Tm value between 68-72℃ and the other melting peak having a Tm value between 58-62℃, the sample genotype is determined to be heterozygous; otherwise, it is homozygous.

[0056] If the THBD gene shows two melting curve peaks, with one melting peak having a Tm value between 68-72℃ and the other melting peak having a Tm value between 55-60℃, the sample genotype is determined to be heterozygous; otherwise, it is homozygous.

[0057] If SERPINC1 shows two melting curve peaks, with one melting peak having a Tm value between 59-63℃ and the other melting peak having a Tm value between 49-53℃, the sample genotype is determined to be heterozygous; otherwise, it is homozygous.

[0058] If the F2 gene shows two melting curve peaks, with one melting peak having a Tm value between 61-65℃ and the other melting peak having a Tm value between 50-54℃, the sample genotype is determined to be heterozygous; otherwise, it is homozygous.

[0059] If the F11 gene shows two melting curve peaks, with one melting peak having a Tm value between 58-63℃ and the other melting peak having a Tm value between 48-53℃, the sample genotype is determined to be heterozygous; otherwise, it is homozygous.

[0060] If the FGG gene shows two melting curve peaks, with one melting peak having a Tm value between 56-60℃ and the other melting peak having a Tm value between 46-50℃, the sample genotype is determined to be heterozygous; otherwise, it is homozygous.

[0061] The melting curves of the eight genes obtained in this embodiment are shown in the appendix. Figure 1 As shown, the Tm value of the MTHFR CC type melting peak is 65.5℃, the Tm value of the TT type melting peak is 55.5℃, and the CT type shows a distinguishable double peak; the Tm value of the PROC CC type melting peak is 62℃, the Tm value of the TT type melting peak is 52.5℃, and the CT type shows a distinguishable double peak; the Tm value of the APOH CC type melting peak is 69.5℃, the Tm value of the TT type melting peak is 60.5℃, and the CT type shows a distinguishable double peak; the Tm value of the THBD GG type melting peak is 69.5℃, the Tm value of the TT type melting peak is 57.5℃, and the GT type shows a distinguishable double peak; the Tm value of the F2 GG type melting peak is 63℃, the Tm value of the AA type melting peak is 52℃, and the GA type shows a distinguishable double peak; SERPINC1 The Tm value of the GG type melting peak is 61℃, the Tm value of the AA type melting peak is 51℃, and the GA type shows a distinguishable double peak; the Tm value of the F11 CC type melting peak is 60.5℃, the Tm value of the TT type melting peak is 50.5℃, and the CT type shows a distinguishable double peak; the Tm value of the FGG GG type melting peak is 58℃, the Tm value of the AA type melting peak is 48℃, and the GA type shows a distinguishable double peak.

[0062] This embodiment employs asymmetric PCR combined with melting curve probe method, utilizing a matching specific primer and probe composition to perform genotyping detection on wild-type, mutant, and heterozygous plasmid templates of eight VTE-related genes. Homozygous wild-type and homozygous mutants of each gene exhibit a single characteristic melting peak, while heterozygous samples show two well-separated sets of specific melting peaks simultaneously. The Tm intervals corresponding to each genotype are clearly defined, with no peak overlap or mutual interference. Based on the preset Tm range, accurate genotyping of eight VTE susceptibility gene SNP loci (MTHFR, PROC, APOH, THBD, F2, SERPINC1, F11, and FGG) can be achieved. These results confirm that the primer and probe composition provided by this invention has high specificity, the asymmetric amplification system produces clear melting curves, significant differences in melting temperatures between wild-type, mutant, and heterozygous individuals, effective differentiation of genotypes, and clear genotyping criteria. This enables reliable and accurate genotyping of eight key VTE gene loci, allowing for accurate determination of different genotypes based on melting curves.

[0063] Example 2

[0064] In this embodiment, to compare the allele discrimination effects of different SNP genotyping methods, the TaqMan probe method was used to perform parallel detection on wild-type, mutant, and heterozygous plasmid templates at the same gene locus. Using wild-type, mutant, and heterozygous plasmid standards for each gene locus as templates, real-time fluorescent PCR amplification using the primer and probe combination designed in Example 1 was performed. The reaction program was: 95℃ (3 min) → 95℃ (10 s) → 60℃ (30 s), 45 cycles (using a BIO-RAD CFX amplification instrument). The PCR amplification reaction system is shown in Table 3.

[0065] Table 3 PCR amplification reaction system

[0066] The results of the probe real-time fluorescence curve detection are as follows Figure 2 As shown, the allele signal separation at different gene loci varies. Some loci exhibit relatively clear genotype clusters, while the separation of the two allele signals at APOH and THBD loci is relatively insufficient, easily affecting genotype interpretation. In contrast, the asymmetric PCR combined with probe melting curve method used in this application can classify genotypes based on the Tm difference between perfectly matched and mismatched hybridization products. This allows different genotypes at APOH and THBD loci to exhibit identifiable characteristic melting peaks, thereby improving the accuracy and intuitiveness of genotype interpretation at these loci.

[0067] Example 3

[0068] In this embodiment, the plasmid standard of the target gene fragment was serially diluted 10-fold using TE buffer to prepare 10... 7 10 6 10 5 10 4 A series of templates with concentrations of 10³, 10², and 10¹ copies / μL were prepared, with dd H₂O (double-distilled water) as a negative control. The specific details are shown in Table 4.

[0069] Table 4 PCR amplification reaction system

[0070] Using the primer and probe composition designed in Example 1, the mixture was added to eight tubes according to the above reaction system. After mixing, PCR amplification was performed under the following conditions: 95℃ (3 min) → 95℃ (10 s) → 60℃ (30 s), 45 cycles (using a BIO-RAD CFX amplification instrument). The sensitivity of different genotypes was tested based on the real-time fluorescence curve.

[0071] The real-time fluorescence curve results are attached. Figure 3 As shown, in a 20 μL system, the primer and probe composition designed in Example 1 can detect 10 6 ~1 copy / μL of plasmid target carrying the MTHFR gene; capable of detecting 10 6 ~1 copy / μL of plasmid target carrying the PROC gene; capable of detecting 10 6 ~1 copy / μL of plasmid target carrying the APOH gene; capable of detecting 10 6 ~1 copy / μL of plasmid target carrying the THBD gene; capable of detecting 10 6 ~1 copy / μL of plasmid target carrying the F2 gene; capable of detecting 10 6 ~1 copy / μL of plasmid target carrying the SERPINC1 gene; capable of detecting 10 6 ~1 copy / μL of plasmid target carrying the F11 gene; capable of detecting 10 6 ~1 copy / μL of plasmid target carrying the FGG gene. It is evident that this system can reliably detect target templates as low as 1 copy for each gene locus, demonstrating a significant advantage over conventional primers and probes. This indicates that the system possesses excellent capture and recognition capabilities when processing trace samples, with an extremely low detection limit, proving that the aforementioned detection system has extremely high nucleic acid detection sensitivity.

[0072] Example 4

[0073] To verify the anti-interference ability, amplification balance, and genotyping specificity of the dual-tube quadruple grouping system, asymmetric primer ratio parameters, and multi-probe multi-channel coexistence scheme of this invention in complex multiplex reactions, this embodiment conducts multiplex simultaneous genotyping verification on plasmid standards with different combinations. For the eight VTE-SNP sites, the dual-reaction unit grouping strategy defined in this invention is adopted: the first reaction unit contains four primer-probe combinations: MTHFR, PROC, SERPINC1, and FGG; the second reaction unit contains four primer-probe combinations: THBD, APOH, F2, and F11. Both reaction units use the preferred asymmetric primer ratio system of this invention: 20 μM of dominant primer stock solution, 0.5 μL added; 2 μM of inferior primer stock solution, 0.5 μL added; and 20 μM of probe stock solution, 0.2 μL added, uniformly forming a stable 10:1 amplification molar ratio in a 20 μL system, matched for four-channel fluorescence detection: FAM, HEX, ROX, and Cy5.

[0074] The specific quadruple PCR reaction system is shown in Table 5. In Table 5, A, B, C, and D represent MTHFR, PROC, SERPINC1, and FGG in the first reaction unit, or THBD, APOH, F2, and F11 in the second reaction unit, respectively. The four sets of primers and probes coexist synchronously in the system, simulating complex detection scenarios such as simultaneous positive results at multiple sites, partial positive results, single positive results, and template-free results in clinical practice. To fully verify the anti-competition, anti-crosstalk, and anti-nonspecific amplification performance of the multiplex system, this embodiment sets 16 target combination modes for each quadruple reaction unit, covering all combinations of target-free, single-target, dual-target, triple-target, and quadruple-target all-positive conditions, maximizing the simulation of the coexistence of diverse genotypes in clinical samples.

[0075] Table 5 PCR amplification reaction system

[0076] After the reaction system was thoroughly mixed, the PCR amplification program used uniform asymmetric amplification conditions: 95℃ for 3 min; 95℃ for 10 s, 60℃ for 30 s, for 45 cycles of asymmetric PCR. After amplification, the temperature was increased from 30℃ to 80℃ for melting curve scanning. Simultaneously, the fluorescence melting signals of the four channels (FAM, HEX, ROX, and Cy5) were acquired, and the specificity of each melting peak, peak shape resolution, and channel interference were analyzed. The results for the first four-fold system are as follows: Figure 4 As shown, the results for the second quartet system are as follows: Figure 5As shown, the added target exhibits a characteristic peak in its corresponding channel, while the corresponding channel without the added target does not show a corresponding characteristic peak. When all four targets are present, all four channels maintain identifiable signals. The detection results indicate that this embodiment effectively overcomes the primer competition, amplification bias, non-specific amplification, and fluorescence crosstalk problems commonly found in multiple asymmetric systems through specific site grouping, precise asymmetric primer ratio control, and multi-channel probe matching design. All experimental groups only show characteristic melting peaks in the fluorescence channels with the added target; channels without targets show no extraneous peaks, no background interference, and no cross-signal leakage. Regardless of whether it is a single site, multiple sites coexisting, or all four sites being positive, the melting peak positions of each channel are stable, the peak shapes are clean, and the separation is good. There is no overlap or shift in the characteristic Tm intervals of different genotypes. The results confirm that the dual-tube quadruple grouping architecture combined with the optimized asymmetric amplification ratio in this embodiment can ensure that the four sets of asymmetric primers and probes do not inhibit or interfere with each other in the same system, and that the amplification is balanced and stable. This greatly improves the compatibility and reliability of multi-site synchronous typing and effectively solves the technical bottlenecks of existing multiple VTE-SNP typing systems, such as poor stability, easy interference, and difficulty in accurate quadruple synchronous typing.

[0077] Example 5

[0078] 1. Extraction of genomic DNA from clinical specimens

[0079] First, collect 7 whole blood samples, centrifuge at 3000 rpm and discard the supernatant plasma. Transfer 200 μL of the leukocyte layer to a flow cytometry tube. Add 3 volumes of erythrocyte lysis buffer, mix thoroughly by pipetting, and incubate at 4°C for 7 min. Centrifuge at 450 g for 5 min and discard the supernatant. Then, add 2 volumes of erythrocyte lysis buffer, mix thoroughly, and incubate at 4°C for 7 min. Centrifuge at 450 g for 5 min and discard the supernatant. Next, add 1 volume of erythrocyte lysis buffer, mix thoroughly, and incubate at 4°C for 7 min. Centrifuge at 450 g for 5 min and discard the supernatant. Finally, resuspend the cells in 300 μL of PBS buffer to obtain a cell suspension containing nuclei. Take 200 μL of the above-mentioned mixed cell suspension and extract genomic DNA using an automated nucleic acid extractor (e.g., Nathch CS) and its matching nucleic acid extraction reagent (S10015) to obtain a high-purity genomic DNA solution for subsequent detection.

[0080] 2. Quadruple amplification reaction

[0081] Take the above DNA solution and prepare the reaction solution according to the reaction system shown in Table 6. In Table 6, A, B, C, and D represent MTHFR, PROC, SERPINC1, and FGG in the first group of reaction units, or THBD, APOH, F2, and F11 in the second group of reaction units, respectively.

[0082] Table 6 PCR amplification reaction system

[0083] It is understood that each extracted target may contain corresponding wild-type, mutant, and heterozygous types. Amplification was performed according to the above reaction system: the above reaction solution was added to eight-tube strips, and plasmids carrying the corresponding homozygous genotypes (wild-type and mutant) were added as positive controls. After mixing the reaction system, PCR amplification was performed under the following conditions: 95℃ (3 min) → 95℃ (10 s) → 60℃ (30 s), 45 cycles (using a BIO-RAD CFX amplification instrument), melting curve conditions: 30℃ → 80℃, while simultaneously acquiring fluorescence signals from the FAM, HEX, ROX, and Cy5 channels.

[0084] 3. Results Analysis

[0085] Under the condition of amplification with a positive plasmid control, different genotypes were determined based on the melting curve. For example... Figures 3-9 The results showed that specific amplification occurred in each reaction tube containing the template, and the Tm values ​​of the PCR products of homozygotes and heterozygotes could be well distinguished.

[0086] Specifically, such as Figure 6 As shown, the genotypes of the first clinical specimen were MTHFR (CT), PROC (CC), APOH (TT), THBD (GG), F2 (GG), SERPINC1 (GA), F11 (CT), and FGG (GG).

[0087] like Figure 7 As shown, the genotypes of the second clinical specimen were MTHFR (CC), PROC (CC), APOH (TT), THBD (GG), F2 (GG), SERPINC1 (GG), F11 (CC), and FGG (GA).

[0088] like Figure 8 As shown, the genotypes of the third clinical specimen were MTHFR (CC), PROC (CT), APOH (TT), THBD (GG), F2 (GG), SERPINC1 (GA), F11 (CC), and FGG (GG).

[0089] like Figure 9 As shown, the genotypes of the fourth clinical specimen were MTHFR (CT), PROC (CC), APOH (TT), THBD (GG), F2 (GG), SERPINC1 (GG), F11 (CC), and FGG (GA).

[0090] like Figure 10As shown, the genotypes of the fifth clinical specimen were MTHFR (CC), PROC (CC), APOH (TT), THBD (GG), F2 (GG), SERPINC1 (GA), F11 (CC), and FGG (GA).

[0091] like Figure 11 As shown, the genotypes of the 6th clinical specimen were MTHFR (CT), PROC (CC), APOH (TT), THBD (GG), F2 (GG), SERPINC1 (GG), F11 (CT), and FGG (GG).

[0092] like Figure 12 As shown, the genotypes of the 7th clinical specimen were MTHFR (CT), PROC (CC), APOH (CT), THBD (GG), F2 (GG), SERPINC1 (GG), F11 (CT), and FGG (GA).

[0093] The detection results of the above gene samples were compared with the gold standard sequencing results. All sample genotyping results were completely consistent with the sequencing gold standard, with 100% detection accuracy, no missed detections, and no misjudgments. The separation effect of melting peaks of different genotypes was excellent, proving that the primer-probe combination can stably adapt to clinical specimens and achieve stable and accurate genotyping of VTE-related multi-gene SNP sites. The genotyping specificity and detection accuracy are excellent, which can meet the clinical needs of venous thrombosis susceptibility gene genotyping detection.

[0094] Example 6

[0095] This embodiment provides a thrombosis risk screening kit suitable for VTE screening in cancer patients. The kit includes the primer and probe composition in Example 1, which can simultaneously detect multiple VTE core susceptibility gene SNPs by single-tube multiplex fluorescent PCR, achieving high-throughput rapid typing detection of samples and meeting the needs of clinical VTE screening and precision diagnosis and treatment.

[0096] Furthermore, in a more refined embodiment, the kit also includes PCR buffer, hot-start Taq DNA polymerase, and dNTPs. The PCR buffer is typically an aqueous solution composed of a Tris buffer system, potassium ions, magnesium ions, etc., used to maintain a suitable pH and ionic environment for the PCR reaction. Additionally, the kit includes negative and positive controls; in specific implementations, the negative control can be water, and the positive control includes a plasmid carrying the corresponding genotype.

[0097] The kit provided in this embodiment was used to test 362 gene samples for validation. All samples were sequenced using the gold standard sequencing method. The following genotypes were detected: MTHFR CC (120 cases), CT (201 cases), TT (41 cases); PROC CC (300 cases), CT (50 cases), TT (12 cases); APOH TT (320 cases), CT (30 cases), CC (12 cases); THBD GG (357 cases), GA (5 cases), AA (0 cases); F2 GG (359 cases), GA (2 cases), AA (1 case); SERPINC1 GG (220 cases), GA (120 cases), AA (22 cases); F11 CC (194 cases), CT (138 cases), TT (30 cases); FGG... 132 cases were identified as GG genotype, 149 as GA genotype, and 81 as AA genotype. The results of this kit on 362 gene samples were compared with the gold standard sequencing results, showing complete agreement with no missed or incorrect detections, achieving 100% accuracy. The kit demonstrated 100% sensitivity for the target genotype and no cross-positive signals for non-target genotype samples, exhibiting 100% specificity. These results confirm that the kit is accurate in genotyping, free of false positives and false negatives, and exhibits stable and reliable performance, meeting the application needs for large-scale clinical screening and precision diagnosis of venous thrombosis susceptibility genes in cancer patients.

[0098] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A primer and probe composition for VTE-related genes, characterized in that, The invention includes a first primer pair and a first fluorescent probe targeting the rs146922325 site of the PROC gene. The first primer pair includes a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:1, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:2, and the nucleotide sequence of the first fluorescent probe is shown in SEQ ID NO:

3. A second primer pair and a second fluorescent probe targeting the rs52797880 site of the APOH gene. The second primer pair includes a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:4, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:5, and the nucleotide sequence of the second fluorescent probe is shown in SEQ ID NO:

6. A third primer pair and a third fluorescent probe targeting the rs16984852 site of the THBD gene, wherein the third primer pair comprises a dominant primer and a dominant primer, the nucleotide sequence of which is shown in SEQ ID NO:7, the nucleotide sequence of which is shown in SEQ ID NO:8, and the nucleotide sequence of the third fluorescent probe is shown in SEQ ID NO:9; and The fourth primer pair and fourth fluorescent probe are for the rs2227589 site of the SERPINC1 gene. The fourth primer pair includes a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:10, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:11, and the nucleotide sequence of the fourth fluorescent probe is shown in SEQ ID NO:

12.

2. The primer and probe composition for VTE-related genes according to claim 1, characterized in that, It also includes a fifth primer pair and a fifth fluorescent probe targeting the rs1799963 site of the F2 gene. The fifth primer pair includes a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:13, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:14, and the nucleotide sequence of the fifth fluorescent probe is shown in SEQ ID NO:

15. It also includes a sixth primer pair and a sixth fluorescent probe targeting the rs2036914 site of the F11 gene. The sixth primer pair includes a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:16, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:17, and the nucleotide sequence of the sixth fluorescent probe is shown in SEQ ID NO:

18.

3. The primer and probe composition for VTE-related genes according to claim 2, characterized in that, It also includes a seventh primer pair and a seventh fluorescent probe targeting the rs2066865 site of the FGG gene. The seventh primer pair includes a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:19, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:20, and the nucleotide sequence of the seventh fluorescent probe is shown in SEQ ID NO:

21. It also includes an eighth primer pair and an eighth fluorescent probe targeting the rs1801133 site of the MTHFR gene. The eighth primer pair includes a dominant primer and a dominant primer. The nucleotide sequence of the dominant primer is shown in SEQ ID NO:22, the nucleotide sequence of the dominant primer is shown in SEQ ID NO:23, and the nucleotide sequence of the eighth fluorescent probe is shown in SEQ ID NO:

24.

4. The primer and probe composition for VTE-related genes according to claim 4, characterized in that, The primer pairs and fluorescent probes targeting eight sites are divided into two reaction units, namely the first reaction unit and the second reaction unit. The first reaction unit includes primer pairs and fluorescent probes targeting the PROC gene rs146922325 site, the SERPINC1 gene rs2227589 site, the FGG gene rs2066865 site, and the MTHFR gene rs1801133 site. The first reaction unit is used to achieve quadruple detection in a single tube. The second reaction unit includes primer pairs and fluorescent probes targeting the APOH gene rs52797880 site, the THBD gene rs16984852 site, the F2 gene rs1799963 site, and the F11 gene rs2036914 site. The second reaction unit is used to achieve single-tube quadruple detection.

5. The primer and probe composition for VTE-related genes according to claim 4, characterized in that, The molar ratio of dominant primer to inferior primer for each detection site is 100:1 to 1:

1.

6. A kit for VTE-related genes, characterized in that, Includes the primer and probe composition according to any one of claims 1-5.

7. The kit for VTE-related genes according to claim 6, characterized in that, It also includes PCR buffer.

8. The use of a primer and probe composition as described in any one of claims 1-5 in the preparation of a reagent or product for SNP typing of VTE-related genes.

9. The use of a kit as described in any one of claims 6-7 in the preparation of an assessment reagent or product for assessing the risk of venous thromboembolism.

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