Method for identifying high-risk individual of vte and kit thereof

Abstract

The present application relates to the technical field of molecular diagnosis and genotyping, and particularly relates to a VTE high-risk individual identification multiplex isothermal amplification detection method and a VTE high-risk individual identification multiplex isothermal amplification detection kit, a 4-7 SNP site detection panel containing SERPINC1 antithrombin deficiency variation, PROC protein C defect hotspot (p.Arg189Trp) and at least one SNP site in MTHFR, PAI-1, FGG and ABO is constructed; an allele-specific forward primer pair with a 3' end plus the second or third artificial mismatch double-mismatch anchoring structure, a universal reverse primer and a tetrahydrofuran site probe are designed for each SNP site; a risk stratification result is output by using an OR value weighted scoring model of a Chinese population cohort, the detection is rapid, single-tube multiplex, sample consumption is low, zero-instrument optional, and is suitable for rapid screening of genetic risk of venous thromboembolism in primary medical institutions of Chinese population.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics and genotyping technology, specifically to a method and kit for identifying high-risk individuals for venous thromboembolism (VTE) using multiple isothermal amplification (MIA) assays. These methods are applicable to rapid screening of genetic risk of venous thromboembolism in the Chinese population at the primary healthcare level, early warning of postoperative venous thromboembolism, and thrombosis risk assessment during pregnancy and postpartum. Background Technology

[0002] Venous thromboembolism (VTE) encompasses two clinical manifestations: deep vein thrombosis (DVT) and pulmonary embolism. It is the third leading cause of cardiovascular disease after myocardial infarction and stroke, affecting approximately 10 million people globally each year. In recent years, the incidence of VTE in China has been steadily rising, with approximately 600,000 new hospitalized cases annually. Early identification and prevention of high-risk individuals is urgently needed in primary healthcare institutions.

[0003] Venous thromboembolism (VTE) is a multifactorial disease resulting from the combined effects of genetic and environmental factors. Compared to European and American populations, the genetic background of VTE in the Chinese population shows significant differences. Factor VLeiden and prothrombin G20210A variants, common in European and American populations, are extremely rare in the Chinese population. However, SERPINC1 antithrombin deficiency variants and PROC protein C deficiency hotspot variants (such as p.Arg189Trp) are important genetic pathogenic factors for VTE in the Chinese population. Protein S deficiency is also a common cause of VTE in East Asian populations, but the distribution of PROS1 pathogenic variants is more dispersed in the Chinese population. Meanwhile, loci such as MTHFR C677T, MTHFR A1298C, PAI-1 4G / 5G, FGG rs2066865, and ABOrs8176719 show high frequency and independent risk effects in the Chinese population, constituting population markers for genetic susceptibility to VTE in this population.

[0004] Current genetic risk testing protocols for venous thromboembolism mainly rely on multiplex PCR combined with Sanger sequencing or high-throughput sequencing, requiring a thermal cycler and sequencer. A single test takes 12 to 48 hours from sample collection to result output, costing 3,000 to 5,000 RMB, making it difficult to popularize in primary healthcare institutions, emergency rooms, or community screening. Recombinase polymerase amplification, an isothermal technique that can complete nucleic acid exponential amplification within 30 minutes at a constant temperature of 37 °C to 42 °C, provides a foundational platform for point-of-care testing in molecular diagnostics. However, recombinase polymerase amplification technology has long faced a core bottleneck in multi-site simultaneous detection scenarios: multiple competitive inhibition. When different SNP sites coexist in the same reaction system, amplification efficiency varies significantly. The amplification products of high-efficiency sites rapidly occupy core system resources, leading to undetectable signals from low-efficiency sites. This makes it difficult for existing multiplex recombinase polymerase amplification protocols to exceed the upper limit of simultaneous detection of 3 to 4 sites.

[0005] Chinese patent application CN106367413A discloses a method for nucleic acid amplification and its application. This method uses multiplex recombinase polymerase amplification as a pre-amplification step in loop-mediated isothermal amplification to enrich the content of the target gene. It employs the outer primers F3 and B3 from the loop-mediated isothermal amplification primer pair as primers for recombinase polymerase amplification to reduce primer design difficulty and secondary structure interference. However, this approach positions multiplex recombinase polymerase amplification at the nucleic acid enrichment stage rather than directly performing allele-level genotyping. It does not directly address the competitive inhibition problem of simultaneous allele-level genotyping at multiple SNP sites during multiplex recombinase polymerase amplification. Specifically, the difference in amplification efficiency at different sites leads to excessively rapid accumulation of products at high-efficiency sites and insufficient signal at low-efficiency sites, making it difficult to achieve balanced amplification and simultaneous genotyping of multiple sites in a single-step multiplex reaction.

[0006] Studies using polygenic risk scoring models based on Chinese population cohorts have demonstrated that simplified panels with 10 to 44 SNP loci can effectively stratify the genetic risk of venous thromboembolism (VTE) in the Chinese population, with an area under the curve (AUC) of 0.71 to 0.74. However, these studies remain at the mathematical modeling level, relying on genome-wide typing chips or high-throughput sequencing platforms for initial input, without any engineering adaptation to point-of-care testing platforms. A significant technological translation gap exists between the actual pathogenic spectrum of VTE in the Chinese population and point-of-care testing platforms.

[0007] Therefore, there is an urgent need in this field for a detection method that can simultaneously resolve the following three contradictions: accurately identify multiple SNP loci corresponding to the true pathogenic spectrum of venous thromboembolism in the Chinese population; simultaneously complete allelic-level typing of multiple loci in a single-tube reaction while overcoming multiple competitive inhibitions of recombinase polymerase amplification; and output a stratified genetic risk index without the need for dedicated instruments to adapt to the real-time testing scenarios in primary healthcare. Summary of the Invention

[0008] The purpose of this invention is to provide a multiplex isothermal amplification detection method for identifying individuals at high risk of venous thromboembolism (VTE). This method involves constructing a core SNP detection panel for genetic susceptibility to VTE in the Chinese population, designing an allele-specific double mismatch primer architecture, employing a primer concentration non-uniform reverse gradient matching strategy to mitigate the competitive inhibition effect of multiplex recombinase polymerase amplification, and integrating multicolor fluorescence real-time monitoring with dual-mode reading of multi-label lateral flow chromatography test strips, as well as a weighted scoring model based on the OR value of the Chinese population cohort. This enables simultaneous allele-level genotyping and genetic risk stratification of multiple VTE risk SNP loci under single-tube multiplex reaction conditions. This shortens the time required for existing VTE gene detection, reduces reliance on thermal cyclers and sequencers, and meets the needs of rapid VTE screening in primary healthcare settings in the Chinese population.

[0009] To achieve the above-mentioned objectives, this invention provides a method for identifying high-risk individuals for VTE using multiple isothermal amplification detection, comprising the following steps:

[0010] Step S1: Construct a VTE genetic risk SNP detection panel. The SNP detection panel includes SNP loci from SERPINC1 and PROC corresponding to the genetic susceptibility of venous thromboembolism in the Chinese population, as well as at least one SNP locus from MTHFR, PAI-1, FGG, and ABO. The number of SNP loci contained in the SNP detection panel is 4 to 7.

[0011] Step S2 involves designing a pair of allele-specific forward primers, a universal reverse primer, and a probe containing a tetrahydrofuran-modified site for each SNP site in the SNP detection panel. The 3' terminal base of the allele-specific forward primer is complementary to the allele base of the SNP site, and the second-to-last or third-to-last position of the 3' terminal of the allele-specific forward primer contains an artificially mismatched base. The probe is an EXO probe corresponding to the multicolor fluorescence real-time monitoring mode or an NFO probe corresponding to the multi-tag sideflow chromatography test strip mode.

[0012] Step S3: The allele-specific forward primer, the universal reverse primer, and the probe are mixed in a single-tube reaction system. The single-tube reaction system also contains UvsX recombinase, gp32 single-stranded binding protein, and Bsu large-fragment polymerase. The allele-specific forward primer is set according to a non-uniform primer concentration ratio. This non-uniform primer concentration ratio is based on a reverse gradient matching according to the amplification efficiency of the flanking sequences of each SNP site, with primer concentrations lower for sites with high amplification efficiency than for sites with low amplification efficiency.

[0013] Step S4: The amplification reaction is carried out at a constant temperature of 37 °C to 42 °C for 20 to 40 minutes.

[0014] Step S5: The genotype of each SNP locus is obtained by reading the amplification products through at least one of the following methods: real-time monitoring of multicolor fluorescence and multi-label lateral flow chromatography test strips.

[0015] Step S6: Based on the genotype, a comprehensive genetic risk index is calculated using a weighted scoring model with the OR value of the cohort effect size of venous thromboembolism in the Chinese population as the weighting coefficient, and the risk stratification results are output.

[0016] This invention also discloses a VTE high-risk individual identification multiplex isothermal amplification detection kit, comprising all allele-specific forward primers, universal reverse primers, probes containing tetrahydrofuran-modified sites, UvsX recombinase, gp32 single-stranded binding protein, Bsu large fragment polymerase, reaction buffer, exonuclease III or endonuclease IV, and multi-tag sideflow chromatography test strips.

[0017] Compared with the prior art, the present invention has the following significant advantages:

[0018] First, representative SNPs in the pathogenesis spectrum of venous thromboembolism in the Chinese population, such as SERPINC1 antithrombin deficiency variants, PROC protein C deficiency hotspot variants (p.Arg189Trp), MTHFR, PAI-1, FGG, ABO pathways, and population effect sites, are integrated into a simplified panel of 4 to 7 SNP sites. The panel information is optimized for the main components of the genetic risk of venous thromboembolism in the Chinese population. Compared with the existing universal panels for Europe and the United States with Factor V Leiden and prothrombin G20210A as the core, the fit of the Chinese population is significantly improved. While greatly reducing the number of detection sites and costs, the ability to identify high-risk individuals in the Chinese population is retained.

[0019] Second, the 3' end base of the allele-specific forward primer is complementary to the allele base at the SNP site, and an artificial mismatch base is introduced at the second to third position from the end of the 3' end to form a double mismatch anchoring structure. Compared with primer design with only a single mismatch at the 3' end, the double mismatch synergistically increases the energy barrier for primer extension on the erroneous allele template, which helps to improve the allele discrimination ability in the recombinase-mediated isothermal amplification system and reduce the non-specific amplification of mismatch templates in multiplex reactions.

[0020] Third, the primer concentration non-uniformity reverse gradient matching strategy applies and standardizes known primer concentration optimization techniques in the field of multiplex PCR to the multiplex recombinase polymerase amplification system. It reduces primer concentration for sites with flanking sequences where the GC content is in the high-efficiency amplification range and increases primer concentration for sites where the GC content deviates from the high-efficiency range. This compensates for the differences in amplification efficiency at different sites from the primer supply side, which helps to alleviate the competitive inhibition effect between sites in multiplex recombinase polymerase amplification and improves the uniformity of amplification at multiple sites in a single tube reaction.

[0021] Fourth, structurally adapted probes were configured for both the real-time fluorescence monitoring and multi-tag lateral flow chromatography test strip reading modes. The real-time fluorescence monitoring mode uses an exo probe with fluorescent and quenching groups labeled on either side of the internal tetrahydrofuran site, which generates fluorescence upon cleavage by exonuclease III. The lateral flow chromatography mode uses an nfo probe with a 5' end labeled with a fluorescent or hapten tag, containing a tetrahydrofuran site internally, and a 3' end blocking structure, which is cleaved by endonuclease IV (Nfo). The probe's dependence on perfectly complementary double-strand cleavage, combined with the allele-specific double mismatch primers, provides additional specificity assurance at the amplification product level.

[0022] Fifth, the dual-mode reading architecture of multicolor fluorescence real-time monitoring and multi-label lateral flow chromatography test strips gives the present invention cross-scenario adaptability. The multicolor fluorescence mode is adapted to primary hospitals and emergency scenarios equipped with portable fluorescence detectors, while the multi-label lateral flow test strip mode does not require any instruments and is adapted to village clinics, remote areas and home screening scenarios.

[0023] Sixth, a weighted scoring model using the OR value of the cohort effect size of venous thromboembolism in the Chinese population as a weighting coefficient directly converts genotype into a comprehensive genetic risk index and outputs three risk stratification results of low, medium and high, completing the integrated transformation from genotype to clinical decision-making.

[0024] Seventh, the detection time is significantly shorter than the existing multiplex PCR combined with Sanger sequencing protocol. The isothermal amplification reaction is completed in 20 to 40 minutes at 37 °C to 42 °C. Combined with rapid sample pretreatment and real-time reading, the total time of the entire process is significantly lower than the 12 to 48 hours of the existing protocol. The equipment dependence is reduced from thermal cycler plus sequencer to portable constant temperature device or zero instrument (lateral flow test strip mode). Single tube multiplex reaction replaces multi-tube single reaction, and the sample volume is reduced to 5 μL to 10 μL of whole blood or an equivalent amount of saliva or buccal swab DNA extract. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the overall process of the VTE high-risk individual identification multiple isothermal amplification detection method of the present invention;

[0026] Figure 2This is a schematic diagram of the molecular design of the allele-specific forward primer of the present invention with a double mismatch anchoring structure at the 3' end of the second to last or third to last position;

[0027] Figure 3 This is a schematic diagram of the molecular-level configuration of the tetrahydrofuran cleavage sites and labeling groups of the exo probe and nfo probe of the present invention;

[0028] Figure 4 This is a schematic diagram showing the relationship between the primer concentration non-uniformity reverse gradient matching strategy of the present invention and the efficiency curve of UvsX recombinase.

[0029] Figure 5 This is a comparison of the kinetic curve of the single-tube multiplex recombinase polymerase amplification reaction of the present invention and the kinetic curve of the reaction with the same concentration ratio in the comparative example;

[0030] Figure 6 This is a schematic diagram of the dual-channel genotype determination of the exo probe in the multicolor fluorescence real-time monitoring mode of the present invention;

[0031] Figure 7 This is a schematic diagram of the structure of the multi-label side-flow chromatography test strip of the present invention and the spatial resolution reading of 7 pairs of parallel detection lines;

[0032] Figure 8 This is a schematic diagram of the genetic risk comprehensive index weighted scoring model and the output logic of low, medium and high risk stratification in this invention;

[0033] Figure 9 This is a bar chart showing the statistical relationship between the sensitivity and specificity of clinical sample detection in this invention. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention. Experimental methods not specifically described in the embodiments shall be carried out under conventional conditions or manufacturer's recommended conditions; reagents used, unless otherwise specified, are commercially available.

[0035] Example 1: Construction of a multi-site SNP panel for VTE in the Chinese population

[0036] This embodiment constructs a core detection panel containing 7 SNP loci based on evidence of genetic susceptibility to venous thromboembolism in the Chinese population. For example... Figure 1 As shown, the overall process of this invention includes a complete technical chain from whole blood sample collection, single-tube multiplex isothermal amplification, dual-mode result reading to the weighted score output of the genetic risk comprehensive index. The panel construction principle is to take the pathogenic variants of anticoagulation deficiency reported in the Chinese population as the high-effect core, supplemented by metabolic and fibrinolytic pathway regulatory sites, and matched with high-frequency population effect sites, so as to cover both high-effect factors and population effect factors of genetic risk of venous thromboembolism in the Chinese population with a relatively small number of sites.

[0037] The constructed panel for detecting seven SNP loci is as follows: The first locus is MTHFR rs1801133, which is the C→T mutation at position 677 of the MTHFR gene, corresponding to an amino acid change in Ala222Val. The T allele frequency in the Chinese population is 40% to 45%, and the TT homozygous genotype is associated with elevated plasma homocysteine ​​levels, with a reported relative risk of approximately 1.5 to 1.8 times. The second locus is MTHFR rs1801131, which is the A→C mutation at position 1298 of the MTHFR gene, corresponding to an amino acid change in Glu429Ala. The C allele frequency in the Chinese population is 18% to 22%. The third locus is PAI-1. rs1799889, or plasminogen activator inhibitor 1 (PAI-1) promoter region 4G / 5G insertion / deletion polymorphism, has a 4G allele frequency of 55% to 60% in the Chinese population. Homozygous 4G / 4G genotypes lead to upregulation of PAI-1 expression and downregulation of fibrinolytic function. The fourth locus is the SERPINC1 gene variant corresponding to an antithrombin deficiency reported in the Chinese population. Antithrombin is an important physiological anticoagulant protein, and its functional deficiency significantly increases the risk of venous thromboembolism. Although this type of variant has a low frequency in the general population, its effect size is large in carriers. The fifth locus is PROC... c.565C>T corresponds to the Arg189Trp variation in protein C (rs146922325), a confirmed protein C defect hotspot variant in the Chinese population, with an allele frequency of approximately 0.4%. Heterozygous carriers have reportedly a 5- to 7-fold increased risk of venous thromboembolism. The sixth locus, FGGrs2066865, is a fibrinogen γ chain gene variant that affects fibrinogen γ' isoform levels, with a C allele frequency of 28% to 32% in the Chinese population. The seventh locus, ABO rs8176719, is an ABO gene c.261delG variant that distinguishes between blood type O and non-O. In the Chinese population, non-O blood individuals have higher levels of plasma coagulation factor VIII and von Willebrand factor than those with blood type O, which is associated with a slightly increased risk of venous thromboembolism.

[0038] Of the seven SNP sites mentioned above, SERPINC1 antithrombin deficiency variant and PROC p.Arg189Trp are high-effect core sites, MTHFR rs1801133 and PAI-1 rs1799889 are pathway regulatory sites, and MTHFR rs1801131, FGG rs2066865 and ABO rs8176719 are population-effect sites. In some implementation scenarios, a simplified 4-site core version can be further implemented according to clinical needs, for example, retaining SERPINC1, PROC, MTHFR rs1801133, and PAI-1 as the simplest core panel. It should be noted that although protein S deficiency is an important cause of venous thromboembolism in East Asian populations, the pathogenic variants of PROS1 in the Chinese population are highly heterogeneous and lack a single high-frequency hotspot suitable for allele-specific typing. Therefore, this panel does not include a single PROS1 locus. The well-known PROS1 Tokushima variant (p.Lys196Glu) in East Asian populations has been reported to be missing in the Chinese population and is not applicable to the Chinese population panel.

[0039] Example 2: Design of allele-specific primers and probes

[0040] In this embodiment, for each of the seven SNP loci in the panel constructed in Example 1, a pair of allele-specific forward primers, a universal reverse primer, and a probe were designed. The allele-specific forward primers adopt a double mismatch anchoring structure with the second-to-last or third-to-last mismatched base at the 3' end. The 3' end base strictly corresponds to the allele base of the SNP locus to form the first anchoring mismatch, and an artificial mismatched base is introduced at the second-to-last or third-to-last position to form the second anchoring mismatch. The double mismatches synergistically increase the energy barrier for primer extension on the erroneous allele template.

[0041] The specific implementation of the double mismatch architecture is illustrated using the MTHFR rs1801133 site as an example. Figure 2As shown, the double mismatch anchoring architecture forms two recognition barriers at the 3' end of the primer. The wild-type allele at this site is C, and the mutant allele is T. The wild-type specific forward primer has a G base at the 3' end (complementary to wild-type C), and an artificial mismatch is introduced at the second-to-last position of the 3' end. When the wild-type primer binds to the wild-type template, the GC pair at the 3' end is perfect, and the second-to-last position forms a weak mismatch with the corresponding base in the template, allowing for extension. When the wild-type primer incorrectly binds to the mutant template, the GT mismatch at the 3' end, combined with the second-to-last mismatch, constitutes a double mismatch combination, resulting in stronger inhibition of primer extension on the mismatched template. The mutant-specific forward primer has an A base at the 3' end (complementary to mutant T), and an artificial mismatch is also introduced at the second-to-last position of the 3' end. Considering that the UvsX recombinase actively mediates primer-template pairing in the recombinase polymerase amplification system and has a different tolerance for single 3' end mismatches than conventional PCR, the double mismatch design aims to enhance the ability to distinguish alleles. The specific artificial mismatch base type needs to be determined after actual verification of primer-target sequences, and the preferred mismatch types are CT mismatch, GT mismatch, CA mismatch, etc., which have moderate intensity.

[0042] Each SNP site corresponds to a universal reverse primer, located within a conserved region of 80 to 150 bp downstream of the SNP site, with a length of 30 to 34 nt. The Tm value is uniformly designed within the range of 58 °C to 62 °C. The 3' end avoids any known polymorphic sites, and the 5' end is not modified with any molecular tag or fluorescent group. The use of universal reverse primers reduces the total number of primers in multiplex reactions. The 7-site panel has a total of 7 reverse primers, which is half the number of primers compared to the scheme of designing two reverse primers for each site independently, which helps to reduce the probability of primer cross-collision.

[0043] Each SNP site is configured with a corresponding probe according to the readout mode, with probe lengths ranging from 46 nt to 52 nt. The EXO probe corresponds to the multicolor fluorescence real-time monitoring mode, containing a tetrahydrofuran debasement mimicry site. Fluorescent groups (FAM, HEX, Cy5, ROX, etc.) and quencher groups (BHQ-1, BHQ-2, etc., selected according to spectral matching) are labeled on either side of the tetrahydrofuran site. A C3 spacer blocking group is labeled at the 3' end to prevent probe extension. The NFO probe corresponds to the multi-tag side-flow chromatography test strip mode, with a fluorescent group or hapten tag (such as FAM) labeled at the 5' end, containing a tetrahydrofuran debasement mimicry site, and a blocking group labeled at the 3' end. The NFO probe does not contain a quencher group.

[0044] The two probes have different working mechanisms. For example... Figure 3As shown, the exo probe is used for real-time fluorescence monitoring: after the probe hybridizes perfectly complementary with the amplification product, exonuclease III recognizes and cleaves the tetrahydrofuran site, causing spatial separation of the fluorescent group and the quenching group, releasing a fluorescence signal. If the amplification product fails to form or forms an imperfect double strand due to double mismatch blocking, cleavage does not occur, and no fluorescence signal is released. The nfo probe is used for lateral flow chromatography detection: after the probe hybridizes with the amplification product, endonuclease IV (Nfo) cleaves the tetrahydrofuran site, releasing the 3' blocking group, allowing the probe to be extended and integrated into the amplification product by Bsu polymerase. The opposing primer is simultaneously labeled with another molecular tag, ultimately forming a double-labeled amplification product for capture by the lateral flow test strip. The cleavage dependence of the two probes on perfectly complementary double strands, combined with the allele-specific double mismatch primers, constitutes an additional guarantee of specificity at the amplification product level. Even if the mismatch primers produce a small amount of non-specific amplification product, the probe will not produce an effective signal because it does not cleave the imperfectly complementary sequence.

[0045] For the lateral flow chromatography test strip reading mode, the wild-type specific forward primer for each SNP locus is labeled with one molecular tag at its 5' end, and the mutant specific forward primer is labeled with another molecular tag at its 5' end. A total of several different molecular tags are required for the 7-locus dual primers. Typical molecular tags include FITC, Biotin, DIG, Texas Red, Cy3, Alexa Fluor 647, His tag, and FLAG tag. A single molecular tag is uniformly labeled at the 5' end of the universal reverse primer for gold-labeled binding detection on the test strip.

[0046] The thermodynamic parameter verification of primer design specifically implemented the following seven quality gates: First, primer length was controlled between 30 and 35 nt to meet the optimal primer length requirements of the recombinase polymerase amplification system; second, primer Tm values ​​were designed within the range of 65 °C to 72 °C to ensure stable primer-template binding ability under isothermal conditions of 37 °C to 42 °C; third, primer GC content was controlled between 40% and 60% to avoid abnormal amplification efficiency; fourth, the number of GCs in the last 5 bases of the 3' end of the primer did not exceed 3 to prevent excessive stability leading to nonspecific extension; fifth, the primer's own secondary structure free energy ΔG was greater than -3 kcal / mol to prevent hairpin structures from inhibiting effective primer binding; sixth, the free energy ΔG for primer dimer formation between primer pairs was greater than -5 kcal / mol to eliminate significant primer dimer interference; and seventh, the primer pair's whole-genome BLAST alignment E value was greater than 1 × 10⁻⁶. -3 Under these conditions, no other significant matches were found besides the target site to rule out off-target amplification. The above seven thermodynamic parameters were validated using a combination of Primer3, OligoAnalyzer, and NCBI BLAST. If any parameter failed, the process returned to the primer design stage for re-optimization.

[0047] For the ABO rs8176719 site, since this site represents a single-base deletion of c.261delG rather than a typical point mutation, a special strategy is required for the 3' end design of wild-type and mutant primers. The 3' end of the wild-type primer is designed to span the sequence before and after the deletion site, while the 3' end of the mutant primer is designed to be the corresponding base of the continuous sequence after the deletion. Artificial mismatches at the second-to-last or third-to-last position are also introduced to enhance distinguishability. This design has been validated to provide allele distinguishability for ABO deletion variants equivalent to other point mutation sites, clearly differentiating heterozygous and homozygous genotypes.

[0048] Example 3: Reverse Gradient Matching Strategy for Primer Concentration Non-uniformity

[0049] This embodiment elucidates the design principle and specific parameter settings of the primer concentration non-uniformity reverse gradient matching strategy of the present invention. Primer concentration optimization to balance multiplex reactions is a known technique in the field of multiplex PCR. This invention applies it to the multiplex recombinase polymerase amplification system and regularizes it. The amplification efficiency of different SNP sites in multiplex amplification varies due to the combined influence of multiple factors such as flanking sequence GC content, local secondary structure, and primer-template pairing kinetics. In single-tube reactions with multiple sites coexisting, sites with higher amplification efficiency tend to accumulate products rapidly in the early stages of amplification and consume shared reaction resources, inhibiting product accumulation at sites with lower amplification efficiency. This strategy compensates for this efficiency difference through a concentration gradient on the primer supply side.

[0050] The inventors first analyzed the GC content of the ±100 bp flanking sequences of each SNP site in the 7-site panel constructed in Example 1. The analysis results showed that the GC content of the flanking sites of MTHFR rs1801133 and ABO rs8176719 was in the range of approximately 48% to 52%, which are high-efficiency amplification sites; the GC content of the flanking sites of MTHFR rs1801131, PAI-1 rs1799889, and FGG rs2066865 was in the range of approximately 42% to 47%, which are medium-efficiency amplification sites; and the GC content of the flanking sites of SERPINC1 antithrombin deficiency variant and PROC c.565C>T was in the range of approximately 37% to 40%, which are low-efficiency amplification sites.

[0051] Based on the above classification of amplification efficiency, primer concentration settings follow a reverse matching principle: primer concentrations are reduced for sites with high amplification efficiency and increased for sites with low amplification efficiency. Specific examples are as follows: the allele-specific forward primer concentration for MTHFR rs1801133 and ABOrs8176719 is set at approximately 80 nmol / L; the allele-specific forward primer concentration for MTHFR rs1801131, PAI-1 rs1799889, and FGG rs2066865 is set at approximately 150 nmol / L; and the allele-specific forward primer concentration for SERPINC1 and PROC is set at approximately 300 nmol / L. The universal reverse primer concentration is set in accordance with the corresponding forward primer concentration.

[0052] The working principle of primer concentration reverse gradient matching is as follows: Figure 4 As shown, the amplification efficiency exhibits a non-monotonic distribution with range-dependent flanking GC content, and the reverse gradient distribution of primer concentration forms a mirror-complementary relationship with the amplification efficiency distribution. For high-efficiency sites, reducing primer concentration slows down the amplification rate due to primer supply limitations, reducing the excessive consumption of shared reaction resources and reserving an amplification window for low-efficiency sites. For low-efficiency sites, increasing primer concentration increases the density of effective amplification events, compensating for the amplification rate. The synergy of these two approaches helps to reduce the differences in the amount of amplified products at each site and improves the comparability of the sensitivity of each channel in the downstream signal reading process.

[0053] The reverse matching rules for primer concentration gradients can be dynamically updated based on the addition of new sites or panel adjustments. For any newly included SNP site, the GC content of its ±100 bp flanking sequences is first calculated, and the corresponding primer concentration level is matched according to the interval in which the GC content falls. Then, fine-tuning is performed based on the actual amplification efficiency test results of the site.

[0054] The inventors further characterized the independent efficiency of singleton amplification reactions at each locus using quantitative fluorescence detection, serving as a reference for primer concentration reverse gradient matching rules. Specifically, singleton recombinase polymerase amplification reactions were performed using allele-specific primer pairs for each locus, along with universal reverse primers. Genomic DNA templates of the same concentration were added, and amplification was carried out isothermally at 37 °C. The relative order in which the amplification curves at each locus reached the threshold signal was recorded. Characterization results showed that MTHFR rs1801133 and ABO rs8176719 loci reached the threshold fastest, followed by MTHFR rs1801131, PAI-1, and FGG loci, while SERPINC1 and PROC loci were the slowest. The ranking of singleton amplification efficiency largely corresponded to the GC content classification flanking each locus, providing experimental evidence for primer concentration reverse gradient matching.

[0055] The effectiveness of primer concentration reverse gradient matching was evaluated using multiple reaction experiments. For example... Figure 5 As shown, when using a non-uniform primer concentration ratio, the fluorescence amplification curves of each site successively reach the threshold within similar time windows, and the curve family clusters well. However, when using an equal primer concentration ratio (see Comparative Example 1), the amplification curves of high-efficiency sites quickly enter the plateau phase, while the amplification curves of low-efficiency sites lag behind or even fail to reach the threshold, and the curve family exhibits obvious high- and low-efficiency differentiation. The results indicate that the primer concentration reverse gradient matching strategy helps to move the amplification kinetics of multiple sites from high- and low-efficiency differentiation towards a more balanced and synchronized state.

[0056] Example 4: Multiplex Recombinase Polymerase Amplification Reaction System and Amplification Kinetics

[0057] This embodiment constructs a reaction system for 7-site multiplex recombinase polymerase amplification and evaluates the reaction kinetics. The total volume of a single tube of the reaction system is 50 μL, and the composition includes: 1× reaction buffer (containing Tris-HCl buffer system, Mg...). 2+ The following ingredients were added: potassium acetate, polyethylene glycol, dithiothreitol, a dNTP mixture of approximately 200 μmol / L, an ATP regeneration system (creatine kinase and phosphocreatine), UvsX recombinase, UvsY recombinase loading factor, gp32 single-strand binding protein, and Bsu large fragment polymerase. Appropriate cleavage enzymes were added according to the readout mode: exonuclease III was added to cleave the exo probe in multicolor fluorescence real-time monitoring mode, and endonuclease IV (Nfo) was added to cleave the nfo probe in multi-tag lateral flow chromatography mode. Allele-specific forward primers corresponding to the 7 loci were added according to the non-homogenized ratio described in Example 3, universal reverse primers were added at the corresponding concentrations, and probes were added at appropriate concentrations for each locus.

[0058] The sample nucleic acid addition volume was 5 μL. Sample types included EDTA-anticoagulated whole blood lysis buffer, saliva DNA extraction buffer, and buccal swab DNA extraction buffer. Whole blood samples were rapidly processed using an alkaline lysis method. 5 μL of whole blood was added to 20 μL of lysis buffer (200 mmol / L potassium hydroxide, 10 mmol / L EDTA), vortexed, and denatured at 95 °C for 5 min. The mixture was then neutralized to pH 7.5-8.0 with 20 μL of neutralization buffer (Tris-HCl 900 mmol / L, pH 8.0) and used directly as a nucleic acid template in the reaction system. The concentration of genomic DNA template in whole blood samples was generally 10 ng / μL to 50 ng / μL, corresponding to approximately 3 × 10⁻⁶ genomic copy numbers per site. 3 Up to 1.5×10 4 copies / μL.

[0059] The reaction conditions were amplification at a constant temperature of 37 °C to 42 °C for 20 to 40 minutes. In multicolor fluorescence real-time monitoring mode, the signals of each fluorescence channel were recorded in real time during amplification. In multi-tag side-flow chromatography test strip mode, after amplification, an appropriate amount of amplification product was diluted in side-flow running buffer and added to the sample pad of the test strip; the detection result was read after several minutes. It should be noted that multiplexing recombinase polymerase amplification is challenging in practice; the more sites, the more complex the primer interactions. The reverse gradient primer concentration described in this embodiment is one measure to alleviate multi-site competitive inhibition. In actual implementation, it is preferable to first verify the system stability with a 4-site core panel and then gradually expand the number of sites.

[0060] Example 5 Genotype determination using a multicolor fluorescence real-time monitoring mode

[0061] In this embodiment, a portable multi-channel fluorescence detector was used to monitor the amplification products of the multicolor fluorescence real-time monitoring mode in Example 4 and determine the genotype. Two exo probes were configured for each SNP site. The first exo probe sequence was perfectly complementary to the wild-type allele amplification product and labeled the first fluorescence channel (e.g., the FAM channel corresponding to site 1 wild-type). The second exo probe sequence was perfectly complementary to the mutant allele amplification product and labeled the second fluorescence channel (e.g., the HEX channel corresponding to site 1 mutant).

[0062] The rules for genotype determination are as follows: Figure 6 As shown, the dual-channel fluorescence signal combination for each SNP locus is mapped to three genotype interpretation outputs. A signal above the threshold in the first fluorescence channel alone, with no signal in the second fluorescence channel, indicates a homozygous wild type (e.g., the CC genotype at the MTHFR rs1801133 locus); both the first and second fluorescence channels showing signals above the threshold simultaneously indicate a heterozygous type (CT genotype); and the second fluorescence channel showing a signal above the threshold alone, with no signal in the first fluorescence channel, indicates a homozygous mutant type (TT genotype). The threshold is set as the mean of the corresponding channel signal in the negative control reaction plus three standard deviations.

[0063] This embodiment tested samples from 50 patients with clinical venous thromboembolism and 50 healthy controls. Patient samples were obtained from a cohort of venous thromboembolism patients in the vascular surgery and respiratory departments of a tertiary-level hospital, diagnosed by ultrasound or CT pulmonary angiography. Healthy controls were obtained from routine physical examinations, excluding individuals with a family history of venous thromboembolism or previous thrombotic events. Each sample was tested in parallel using the method of this invention and traditional multiplex PCR combined with Sanger sequencing. The Sanger sequencing results were used as the gold standard to evaluate the performance of the method of this invention.

[0064] In clinical sample validation, the genotyping results of the method of this invention for each SNP locus on the panel showed good consistency with the Sanger sequencing gold standard. High consistency was observed in the genotyping of common polymorphic sites (MTHFR, PAI-1, FGG, ABO), and low-frequency, high-effect sites (SERPINC1 antithrombin deficiency variant, PROC p.Arg189Trp) could be accurately identified in carrier samples. This method is applicable to various sample types, including whole blood, saliva, and buccal swabs.

[0065] The actual interpretation process of the multicolor fluorescence real-time monitoring mode of this invention is illustrated using a typical sample detection result as an example. The fluorescence signal characteristics of a patient's sample with deep vein thrombosis in the lower extremities at 7 loci are as follows: Locus 1, MTHFR rs1801133, shows signals above the threshold in both the FAM wild-type channel and the HEX mutant channel, indicating a CT heterozygous genotype; Locus 2, MTHFR rs1801131, shows a signal in the FAM wild-type channel and no signal in the HEX mutant channel, indicating an AA homozygous wild-type genotype; Locus 4, SERPINC1, an antithrombin deficiency variant, shows signals in both the wild-type and mutant channels, indicating a heterozygous genotype, suggesting that the patient carries an antithrombin deficiency variant; Locus 5, PROC c.565C>T, shows a signal in the wild-type channel and no signal in the mutant channel, indicating a CC homozygous wild-type genotype; the remaining loci are interpreted according to the same rules. The genotype combination of this sample can be used to calculate the corresponding genetic risk comprehensive index and risk stratification results using the weighted scoring model in Example 7.

[0066] For the interpretation of fluorescence signals, this invention further provides a quantitative judgment rule based on the ratio of dual-channel signal intensity to reduce the subjectivity of manual interpretation. Specifically, the rule is as follows: if the ratio of the FAM channel endpoint signal intensity to the HEX channel endpoint signal intensity is greater than 10:1, it is determined to be homozygous wild-type; if the ratio is between 0.5:1 and 2:1, it is determined to be heterozygous; if the ratio is less than 1:10, it is determined to be homozygous mutant; if the ratio is between 2:1 and 10:1 or between 1:2 and 1:10, it is determined to be uncertain and requires re-detection. This quantitative judgment rule can be integrated into the embedded software of a portable fluorescence detector to achieve automated genotype output.

[0067] Example 6: Visual Reading of Multi-Label Sideflow Chromatography Test Strip Patterns

[0068] This embodiment uses a multi-label lateral flow chromatography test strip to visually read the amplification products of Example 4, without any instrument assistance. Figure 7As shown, the test strip structure, from top to bottom, consists of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The conjugate pad contains a detection probe with a gold nanoparticle-conjugated anti-universal reverse primer 5' molecular tag (e.g., a gold particle-anti-FAM antibody complex). Parallel detection lines are fixed on the nitrocellulose membrane. Each SNP site corresponds to two detection lines for wild-type and mutant types. Each detection line contains a specific antibody with a corresponding wild-type or mutant allele-specific forward primer 5' molecular tag. There are a total of 7 pairs (14 parallel detection lines) for 7 sites. An additional control line is included to indicate the effectiveness of the chromatographic flow path.

[0069] The test strip works as follows: After the reaction, 2 μL of the amplification product is diluted in 100 μL of side-flow running buffer and added to the sample pad. The amplification product carries a dual-tag label: a wild-type or mutant-specific forward primer 5' molecular tag and a universal reverse primer 5' molecular tag. As it flows through the binding pad, the universal reverse primer 5' end molecular tag binds to the gold particle-antibody complex and flows with the buffer to the nitrocellulose membrane. The amplification product carrying the wild-type forward primer tag is captured by the antibody immobilized on the corresponding detection line, forming a red band. The amplification product carrying the mutant forward primer tag is captured by the other detection line. When no amplification product passes through, the detection line remains uncolored; only the control line shows color.

[0070] The test strip reading rules are as follows: for each SNP locus, if only the wild-type test line shows color, it is determined to be homozygous wild-type; if both wild-type and mutant test lines show color simultaneously, it is determined to be heterozygous; if only the mutant test line shows color, it is determined to be homozygous mutant. Overall interpretation requires only visual observation and is completed within 5 minutes from amplification completion to result output. This embodiment also tested clinical patient samples and healthy control samples using the lateral flow test strip mode. The interpretation results showed good consistency with the multicolor fluorescence real-time monitoring mode, demonstrating the usability of the lateral flow mode as a zero-instrument reading solution.

[0071] The side-flow test strip mode is applicable to environments without fluorescent detectors, such as village clinics, community clinics, medical points in remote areas, and home screening. Its zero instrument dependence makes this invention have a significant accessibility advantage in the scenario of venous thromboembolism risk screening in primary healthcare.

[0072] Example 7: Weighted scoring and risk stratification based on the comprehensive genetic risk index

[0073] This embodiment is based on the genotype results of 7 SNP loci obtained in Example 5 or Example 6. It uses a weighted scoring model with the OR value of the cohort effect size of venous thromboembolism in the Chinese population as the weighting coefficient to calculate the comprehensive genetic risk index and output the risk stratification results.

[0074] The formula for calculating the weighting coefficient is: w i =ln(OR i), where OR i This represents the odds ratio of the risk genotype to the normal genotype reported for the i-th SNP locus in a Chinese cohort study of venous thromboembolism. Example weighting coefficients are given (specific values ​​should be based on the reports from the Chinese cohort studies used): MTHFR rs1801133 approximately 0.50, MTHFR rs1801131 approximately 0.34, PAI-1 rs1799889 approximately 0.59, SERPINC1 antithrombin deficiency variant approximately 1.6, PROCp.Arg189Trp approximately 1.8, FGG rs2066865 approximately 0.44, and ABO rs8176719 approximately 0.50. The weighting coefficients for the two high-effect loci, SERPINC1 and PROC, are significantly higher than those for pathway and population-effect loci, reflecting the dominant role of anticoagulation deficiency variants in risk scoring.

[0075] The genotype coding rule is: homozygous wild-type G i =0, heterozygous G i =1, homozygous mutant G i =2. The formula for calculating the Genetic Risk Composite Index, also known as the Polygenic Risk Score, is PRS = Σ(w i ×G i This means summing the product of the weight coefficients of each SNP site within the panel and the genotype coding.

[0076] Risk stratification criteria are based on percentile thresholds of the Chinese population cohort distribution: such as Figure 8 As shown, the PRS calculation results are mapped to low, medium, and high risk stratification output logic through three threshold divisions. Individuals with a PRS greater than the 90th percentile threshold corresponding to the Chinese population are classified as high-risk; those with a PRS between the 10th and 90th percentiles are classified as medium-risk; and those with a PRS below the 10th percentile are classified as low-risk. The specific PRS values ​​corresponding to each percentile threshold should be determined after verification in the target population cohort. The risk stratification results are output as suggestive information. This method is an in vitro detection method, and the detection object is an ex vivo nucleic acid sample; it does not include diagnostic or treatment steps for humans.

[0077] It should be noted that although high-effect loci such as SERPINC1 antithrombin deficiency variants and PROC p.Arg189Trp have large effect sizes, their allele frequencies in the general population are low (PROC p.Arg189Trp approximately 0.4%). Therefore, individuals with a high-risk genetic risk index are relatively few in the general population, mainly consisting of individuals carrying the aforementioned low-frequency, high-effect anticoagulation deficiency variants; while individuals carrying risk genotypes of common pathways and population effect loci mostly fall into the medium-risk range. The specific thresholds and stratification efficacy of this weighted scoring model should be determined after validation in the target population cohort.

[0078] The risk stratification results output by this invention are suggestive information for clinicians to consider in conjunction with the patient's clinical condition. This method is an in vitro detection method, testing isolated nucleic acid samples, and does not include diagnostic steps involving living human subjects, nor any treatment steps. Subsequent clinical management based on the risk stratification results should be independently determined by clinicians according to relevant clinical guidelines and is not within the scope of this method.

[0079] This invention's weighted scoring model is constructed based on real pathogenic loci in the Chinese population. Compared to models using common European and American loci (Factor V Leiden, Prothrombin G20210A), it exhibits higher detectability of risk genotypes in the Chinese population. This invention's panel model offers significant advantages over multi-locus high-throughput genotyping schemes in terms of detection time, cost, and equipment accessibility: isothermal amplification is completed in 20 to 40 minutes, it is portable or requires no instruments for reading, and it allows for multiple operations per tube, enabling genetic risk scoring to be implemented in point-of-care testing scenarios at the primary healthcare level—something that more complex high-locus panel models struggle to achieve.

[0080] Comparative Example 1: Control Experiment with Isoconcentrated Multiplex Recombinase Polymerase

[0081] The purpose of this comparative example is to verify the advantages of the non-uniform primer concentration reverse gradient matching strategy of the present invention over the equal concentration multiplex reaction strategy. The reaction system, enzyme system, and probe configuration of Comparative Example 1 are the same as those of Example 4, the only difference being that the concentrations of allele-specific forward primers for all loci are set to the same value.

[0082] The results of simultaneous multi-site amplification in Comparative Example 1 showed that the fluorescence channel signals corresponding to high-efficiency amplification sites (MTHFR rs1801133, ABOrs8176719) rapidly reached the plateau phase, while the fluorescence channel signals corresponding to low-efficiency amplification sites (SERPINC1 antithrombin deficiency variant, PROC) lagged significantly. Some low-efficiency sites failed to reach the effective threshold within the reaction time, leading to a decrease in the reliability of genotyping determination for these sites. In contrast, when using a reverse gradient primer concentration in the embodiments of this invention, the signal uniformity at each site was significantly improved.

[0083] Comparative Example 1 shows that in the amplification of multiplex recombinase polymerase at equal concentrations, high-efficiency sites rapidly accumulate products and consume shared reaction resources, while low-efficiency sites are difficult to amplify effectively due to resource competition, resulting in a severe imbalance of signals at multiple sites. The primer concentration non-uniformity reverse gradient matching strategy of this invention improves the balance of multi-site amplification through supply-side compensation, which helps to improve the feasibility of simultaneous detection of multiple sites.

[0084] Comparative Example 2: Control experiment with primers without artificially mismatched bases

[0085] The purpose of this comparative example is to verify the advantages of the double mismatch architecture of the allele-specific forward primers of the present invention, which features an artificial mismatched base at the second or third to last position of the 3' end, compared to traditional single mismatched primers at the 3' end. The reaction system, enzyme system, and primer concentration ratio of Comparative Example 2 are completely identical to those of Example 4. The only difference is that all allele-specific forward primers retain only the single mismatch design where the 3' end base is complementary to the SNP allele, and the artificial mismatched base at the second or third to last position of the 3' end is removed.

[0086] The results of Comparative Example 2 show that when only allele-specific primers with a single mismatch at the 3' end are used, both wild-type primers paired with homozygous mutant templates and mutant primers paired with homozygous wild-type templates may produce a certain proportion of non-specific amplification, increasing the risk of misclassification between adjacent genotypes (such as homozygous and heterozygous). The double mismatch anchoring architecture of this invention introduces stronger extension inhibition on the mismatched template, helping to reduce the proportion of non-specific amplification and improve the reliability of genotype determination. In recombinase-mediated isothermal amplification systems, since recombinase can promote primer-template pairing, the distinguishing ability of single mismatches is relatively limited; therefore, the gain of the double mismatch design is particularly significant in this system.

[0087] Comparative Example 2 results show that the single mismatch primer architecture has relatively limited allele differentiation ability under recombinase-mediated multiple isothermal amplification environment, and there is a risk of non-specific amplification. The double mismatch anchoring architecture of this invention increases the energy barrier of erroneous template extension by adding artificial mismatched bases at the second to third to last position at the 3' end, which helps to improve the reliability of genotype determination under multiple reaction conditions.

[0088] Comparative Example 3: Panel Control Experiment Common in Europe and America

[0089] The purpose of this comparative example is to verify the superior suitability of the SNP panel constructed for the Chinese population based on the actual pathogenic spectrum of the Chinese population compared to existing universal panels used in Europe and America. Comparative Example 3 uses the core sites Factor V Leiden (rs6025) and prothrombin G20210A (rs1799963) of the SNP panel for venous thromboembolism in the European and American population disclosed in existing US Patent US20190249253A1, combined with MTHFR rs1801133 and PAI-1 rs1799889 to construct a 4-site control panel. Other reaction conditions are the same as in Example 4.

[0090] The results of Comparative Study 3 showed that among the 50 Chinese patients with venous thromboembolism, there were 0 Factor V Leiden carriers and 0 prothrombin G20210A carriers, with a positive detection rate of 0%. Only the risk genotypes corresponding to the MTHFR rs1801133 and PAI-1 rs1799889 loci showed differences between the patient and control groups. However, the area under the curve of the risk scoring model constructed using only two points was only 0.55, which is close to the random prediction level and cannot be used for effective stratification of the genetic risk of venous thromboembolism in the Chinese population.

[0091] Comparative Example 3 confirmed that major pathogenic variants in European and American populations, such as Factor V Leiden and prothrombin G20210A, are extremely rare in the Chinese population, with allele frequencies of less than 0.1% and 0.05%, respectively. Directly using the commonly used European and American panels for genetic risk screening of venous thromboembolism in the Chinese population has no clinical value. This invention constructs a panel based on the actual pathogenic spectrum of the Chinese population, which includes SERPINC1 antithrombin deficiency variants, the PROC p.Arg189Trp core site, and MTHFR, PAI-1, FGG, ABO pathways, and population effect sites, achieving effective coverage of the genetic risk of venous thromboembolism in the Chinese population.

[0092] To systematically compare the technical features of the present invention with those of the comparative solutions, the key technical features of the panel of the present invention compared with those of Comparative Examples 1 to 3 are summarized in the table below; Meanwhile, as Figure 9 As shown, the differences in technical features between the panel of this invention and the three control groups in terms of multi-site synchronization capability, signal uniformity, and coverage of pathogenic sites in the Chinese population can be intuitively compared. It should be noted that the table below is a qualitative comparison of the technical features, and the specific performance indicators involved need to be further determined in actual sample verification.

[0093]

[0094] The following key results can be observed from the summary table of test data:

[0095] Firstly, regarding the ability to simultaneously amplify multiple sites. This invention improves the balance of multi-site amplification in a single tube by employing a reverse gradient matching strategy with non-uniform primer concentrations, which helps to simultaneously detect multiple sites in a single tube reaction. In Comparative Example 1, the high- and low-efficiency site amplification was severely unbalanced under the isoconcentration scheme, limiting the number of sites that could be detected simultaneously. Comparative Example 2, although capable of multi-site amplification, had limited allele discrimination ability. Comparative Example 3 showed insufficient coverage of pathogenic sites in the Chinese population by European and American panels.

[0096] Secondly, regarding allele differentiation and specificity assurance, this invention employs a multi-specificity assurance mechanism using double mismatch primers and probes for perfect complementary cleavage and dual-channel reading, which helps reduce non-specific amplification in multiplex reactions; in contrast, the allele differentiation capability of the single mismatch scheme in Comparative Example 2 is relatively limited.

[0097] Thirdly, regarding the detection of low-efficiency sites. This invention improves the amplification of low-efficiency sites (SERPINC1 antithrombin deficiency variant, PROC) by compensating for differences in amplification efficiency through a reverse primer concentration gradient; in contrast, the low-efficiency sites in the isoconcentration scheme of Comparative Example 1 are prone to insufficient amplification or even missed detection.

[0098] Fourth, regarding the coverage of pathogenic sites in the Chinese population. The panel of this invention focuses on high-effect variants reported in the Chinese population, such as SERPINC1 antithrombin deficiency variant and PROC p.Arg189Trp; in contrast, the core sites Factor V Leiden and prothrombin G20210A in the European and American panels are extremely rare in the Chinese population and are difficult to use for identifying the genetic risk of venous thromboembolism in the Chinese population.

[0099] Fifth, regarding panel simplification and translational value. This invention focuses on high-effect pathogenic variants and major pathways and population-effect sites in the Chinese population with fewer SNP sites. While retaining the ability to identify high-risk individuals in the Chinese population, it significantly reduces detection complexity and cost. Compared to multi-site risk scoring models that require high-throughput genotyping platforms, it is more suitable for translational application in primary healthcare point-of-care testing scenarios.

[0100] Sixth, regarding detection efficiency and cost. The isothermal amplification reaction of this invention is completed in 20 to 40 minutes. Combined with rapid sample pretreatment and immediate reading, the total time is significantly lower than the 12 to 48 hours of the existing multiplex PCR combined with Sanger sequencing protocol. The equipment dependence is reduced from a thermal cycler plus a sequencer to a portable constant temperature device or zero instrument (lateral flow test strip mode). Single-tube multiplex PCR replaces multi-tube single-tube PCR, and the sample volume is reduced to 5 μL to 10 μL of whole blood or an equivalent amount of saliva or buccal swab DNA extract.

[0101] Based on the above embodiments and comparative examples, the VTE high-risk individual identification multiplex isothermal amplification detection method provided by this invention, through the synergy of technical elements such as a simplified panel specific to the Chinese population, double mismatched allele-specific primers, primer concentration non-uniform reverse gradient matching, dual-mode reading of exo and nfo probes, and OR value weighted scoring, offers a comprehensive solution for the needs of accurate identification, rapid detection, multi-site synchronization, and equipment accessibility in the context of primary healthcare for venous thromboembolism in the Chinese population. Compared with existing technologies, it has application value in terms of clinical suitability, detection efficiency, and cost accessibility in the Chinese population. Further performance confirmation of this invention requires systematic validation with real clinical samples.

[0102] Those skilled in the art should understand that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the protection scope of the claims of the present invention.

Claims

1. A multiple isothermal amplification detection method for identifying high-risk individuals for VTE, characterized in that... Includes the following steps: S1, Construct a VTE genetic risk SNP detection panel. The SNP detection panel includes SNP loci corresponding to the genetic susceptibility to venous thromboembolism in the Chinese population (SERPINC1, PROC) and at least one SNP locus from MTHFR, PAI-1, FGG, and ABO. The number of SNP loci in the SNP detection panel is 4 to 7. S2, Design a pair of allele-specific forward primers, a universal reverse primer, and a probe containing a tetrahydrofuran-modified site for each SNP locus in the SNP detection panel. The gene-specific forward primer has a 3' terminal base complementary to the allele base at the SNP site, and contains an artificial mismatched base at the second-to-last or third-to-last position at the 3' end; S3, the allele-specific forward primer, the universal reverse primer, and the probe are mixed in a single-tube reaction system containing UvsX recombinase, gp32 single-stranded binding protein, and Bsu large fragment polymerase. The allele-specific forward primer concentration is matched in reverse gradient according to the amplification efficiency of the flanking sequences of each SNP site, with the primer concentration lower for sites with high amplification efficiency than for sites with low amplification efficiency; S4, amplification is performed at 37 °C to 42 °C for 20 to 40 min; S5, the amplification products are read using at least one method, such as real-time multicolor fluorescence monitoring and multi-label lateral flow chromatography test strips, to obtain the genotype; S6, based on the genotype, a genetic risk comprehensive index is calculated using a weighted scoring model with the OR value of the Chinese population venous thromboembolism cohort, and the risk stratification results are output.

2. The method for identifying high-risk individuals for VTE using multiple isothermal amplification detection according to claim 1, characterized in that, The SNP detection panel contains at least four SNP sites selected from MTHFR rs1801133, MTHFR rs1801131, PAI-1 rs1799889, SERPINC1 antithrombin deficiency-related variant site, PROC c.565C>T, FGG rs2066865, and ABO rs8176719, and contains at least one of SERPINC1 antithrombin deficiency-related variant site and PROC c.565C>T.

3. The method for identifying high-risk individuals for VTE using multiple isothermal amplification detection according to claim 1 or 2, characterized in that, The artificial mismatched base at the second or third position from the end of the 3' end of the allele-specific forward primer is selected from one of CT mismatch, GT mismatch, CA mismatch, or AC mismatch.

4. The method for identifying high-risk individuals for VTE using multiple isothermal amplification detection according to claim 3, characterized in that, The reverse gradient matching of primer concentrations is set according to the following rules: if the GC content within the 200 bp range flanking the SNP site falls within the range of 45% to 55%, the corresponding allele-specific forward primer concentration is set to 50 nmol / L to 100 nmol / L; if the flanking GC content falls within the range of 40% to 45% or 55% to 60%, the corresponding primer concentration is set to 120 nmol / L to 180 nmol / L; if the flanking GC content is below 40% or above 60%, the corresponding primer concentration is set to 200 nmol / L to 400 nmol / L.

5. The method for identifying high-risk individuals for VTE using multiple isothermal amplification detection according to claim 4, characterized in that, The probe containing the tetrahydrofuran-modified site is an exo probe or an nfo probe; the exo probe is labeled with a fluorescent group and a quenching group on both sides of the tetrahydrofuran-modified site and is cleaved by exonuclease III; the nfo probe is labeled with a fluorescent group or hapten tag at the 5' end and contains a blocking group at the 3' end and is cleaved by endonuclease IV; the fluorescent group is selected from one of FAM, HEX, Cy5 or ROX, and the probe length is 46 nt to 52 nt.

6. The method for identifying high-risk individuals for VTE using multiple isothermal amplification detection according to claim 5, characterized in that, When the multi-tag lateral flow chromatography test strip is used to read the amplification products, the 5' ends of the allele-specific forward primers are labeled with two different molecular tags selected from FITC, Biotin, DIG, Texas Red, Cy3, Alexa Fluor 647, His tag, or FLAG tag. Each SNP site in the detection area of ​​the multi-tag lateral flow chromatography test strip corresponds to two parallel detection lines for wild type and mutant type, and each parallel detection line is fixed with a corresponding molecular tag-specific antibody.

7. The method for identifying high-risk individuals for VTE using multiple isothermal amplification detection according to claim 6, characterized in that, The weighted scoring model weight coefficient w i The natural logarithm (ln(OR)) of the corresponding SNP locus was used. i The genetic risk index is expressed in the form of PRS = Σ(w i × G i ) calculate, where G i The genotype coding value for the i-th SNP locus is 0 for homozygous wild type, 1 for heterozygous type, and 2 for homozygous mutant type. The risk stratification result determination rule is that PRS greater than the 90th percentile threshold of the corresponding Chinese population distribution is determined as high risk, PRS between the 10th and 90th percentiles is determined as medium risk, and PRS lower than the 10th percentile is determined as low risk.

8. The method for identifying high-risk individuals for VTE using multiple isothermal amplification detection according to claim 7, characterized in that, When the multicolor fluorescence real-time monitoring is used to read the amplification products, two exo probes are configured for each SNP site. The first exo probe complements the wild-type allele amplification product and is labeled with the first fluorescence channel, and the second exo probe complements the mutant allele amplification product and is labeled with the second fluorescence channel. The genotype determination rule is that the presence of a signal in the first fluorescence channel alone indicates a homozygous wild-type, the presence of signals in both the first and second fluorescence channels simultaneously indicates a heterozygous type, and the presence of a signal in the second fluorescence channel alone indicates a homozygous mutant type.

9. The method for identifying high-risk individuals for VTE using multiple isothermal amplification detection according to claim 8, characterized in that, The amplification reaction sample is 5 μL to 10 μL of whole blood, saliva, or buccal swab DNA extract. The sample is treated with lysis buffer for no more than 5 min and then directly added to the single-tube reaction system.

10. A multiplex isothermal amplification assay kit for identifying high-risk individuals for VTE, characterized in that... It comprises all allele-specific forward primers, universal reverse primers, probes containing tetrahydrofuran-modified sites, UvsX recombinase, gp32 single-stranded binding protein, Bsu large fragment polymerase, reaction buffer, and exonuclease III or endonuclease IV for implementing the VTE high-risk individual identification multiple isothermal amplification detection method according to any one of claims 1 to 9, as well as multi-labeled lateral flow chromatography test strips.

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