Dtc detection kit based on double target joint and its risk assessment application

Abstract

The application is suitable for the field of in vitro diagnosis technology, and provides a DTC detection kit based on double-target joint and risk assessment application thereof, wherein the DTC detection kit is an ACTG2 and CKMT1A target joint detection kit, and contains detection reagents for specifically recognizing ACTG2 and CKMT1A. The application combines ACTG2 reflecting cytoskeleton remodeling and invasion and metastasis with CKMT1A reflecting energy metabolism reprogramming and dedifferentiation, fundamentally avoids the problem of false negative caused by traditional TgAb interference, breaks through the time lag of imageology which can only find macroscopic lesions, and realizes early, non-invasive, high-sensitivity and high-specificity evaluation of the risk of DTC distant metastasis and RAIR evolution.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, and particularly relates to a DTC detection kit based on dual-target combination and its risk assessment application. Background Technology

[0002] Currently, clinical monitoring of recurrence and metastasis after surgery and radioactive iodine (131I) treatment for differentiated thyroid cancer (DTC) mainly relies on the following two types of technologies, products, and methods:

[0003] 1. Serum Stimulated Thyroglobulin (ps-Tg) Detection Kit:

[0004] Product Structure: Existing ps-Tg detection kits are mainly based on enzyme-linked immunosorbent assay (ELISA) or chemiluminescent immunoassay (CLIA) platforms using a sandwich assay. Their core structural components include: a microplate or magnetic microparticles as a solid-phase support, an anti-thyroglobulin (Tg) capture antibody coated on the support, the Tg antigen in the serum sample to be tested, and an anti-Tg detection antibody conjugated with a signaling molecule (such as horseradish peroxidase HRP or acridinium ester).

[0005] Method and Procedure: Peripheral blood was drawn from patients after discontinuing thyroid hormone or receiving recombinant human thyroid-stimulating hormone (rhTSH) injection; the separated serum sample was added to the reagent kit reaction system, where the Tg antigen in the sample bound to the capture antibody and the free detection antibody on the solid phase, forming a sandwich immune complex of "capture antibody-Tg antigen-detection antibody"; after washing to remove non-specific free substances, substrate solution was added for color development or luminescence excitation; finally, the light signal intensity was read using an ELISA reader or chemiluminescence analyzer, and the quantitative concentration of Tg in the serum was calculated based on the standard curve.

[0006] 2. Traditional medical imaging detection systems:

[0007] Products and Processes: Primarily relies on large-scale radiological or nuclear medicine imaging equipment such as CT, 131I whole-body imaging (WBS), and 18F-FDG PET / CT. The core process involves: introducing a specific radionuclide tracer into the patient's body; after a certain period of in vivo metabolism and specific distribution, external detectors (such as PET detector rings or gamma cameras) capture the radiation emitted by the tissue; subsequently, a computer system performs three-dimensional image reconstruction, identifying metastatic lesions based on differences in anatomical density or metabolic activity between lesions and normal tissues.

[0008] Although the aforementioned products and methods constitute the current standard clinical procedure, the following structural and methodological deficiencies remain when assessing the risk of distant metastasis and radioactive iodine refractory (RAIR) progression in DTC:

[0009] 1. The inherent susceptibility to interference in the detection principle leads to an extremely high false negative rate: Current mainstream serum biomarkers (ps-Tg) are highly susceptible to interference from anti-thyroglobulin antibodies (TgAb) produced by the patient's own immune system when using the double-antibody sandwich assay. TgAb blocks the antigenic epitopes of Tg, creating a steric hindrance effect that prevents the binding of the detection antibody, resulting in a measured ps-Tg concentration far lower than the true level. This inherent flaw in the principle means that approximately 10%-25% of DTC patients with positive TgAb cannot obtain accurate metastasis risk assessment using conventional kits.

[0010] 2. Single tissue-specific markers lack tumor progression specificity: ps-Tg is only a tissue-specific marker of thyroid follicular cells, not a tumor-specific marker. Its serum concentration is also directly affected by the volume of normal residual thyroid tissue and the level of thyroid-stimulating hormone (TSH). Current technology cannot accurately distinguish whether an increase in ps-Tg is caused by the proliferation of residual normal tissue or by distant metastasis or highly invasive clonal evolution.

[0011] 3. Existing imaging and pathological diagnostic methods suffer from significant time lag: Traditional imaging equipment (such as ultrasound, CT, PET / CT, and 131I WBS) that relies on gross anatomy or metabolic imaging is limited by the physical resolution of the detectors and can only identify metastases that have already formed macroscopic lesions (usually on the millimeter to centimeter scale). For very early-stage patients in the micrometastasis stage, or those who have just undergone metabolic reprogramming leading to a decline in iodine uptake but whose morphology has not yet changed, existing imaging technologies have blind spots and cannot achieve early molecular warnings.

[0012] 4. Single-target detection cannot cover the multidimensional and complex mechanisms of DTC evolution into RAIR: The metastasis of DTC to distant sites and the malignant evolution of RAIR-DTC are essentially dual biological events involving cytoskeleton remodeling (leading to invasion and metastasis) and energy metabolism reprogramming (leading to changes in the local microenvironment and inhibition of NIS expression) in tumor cells. Existing single-target gene or protein detection kits cannot fully map this multidimensional and complex heterogeneity, leading to missed diagnoses or misdiagnoses of high-risk patients.

[0013] In response to the clinical challenges in existing DTC follow-up and prognostic assessment methods, there is an urgent need in this field to develop a highly sensitive, highly specific, and interference-resistant DTC detection kit and its risk assessment method. Summary of the Invention

[0014] The purpose of this invention is to provide a DTC detection kit based on dual-target combination and its risk assessment application, aiming to solve the problems mentioned in the background art.

[0015] To achieve the above objectives, the present invention provides the following technical solution:

[0016] The application of a biomarker combination in the preparation of a DTC detection kit, wherein the biomarker combination comprises ACTG2 and CKMT1A; the biomarker combination is used to assess the risk of distant metastasis and / or RAIR evolution of DTC.

[0017] Another objective of this invention is to provide a dual-target combined DTC detection kit, wherein the DTC detection kit is a combined detection kit for ACTG2 and CKMT1A targets, containing detection reagents that specifically identify ACTG2 and CKMT1A; the DTC detection kit is used to assess the risk of distant metastasis and / or RAIR evolution of DTC.

[0018] Furthermore, the DTC detection kit specifically includes a solid-phase carrier and detection reagents; the solid-phase carrier is coated with anti-ACTG2 capture antibody and anti-CKMT1A capture antibody; the detection reagents include an enzyme-labeled conjugate, ACTG2 standard, CKMT1A standard, and auxiliary reagents for color development and / or quantification; the enzyme-labeled conjugate includes enzyme-labeled anti-ACTG2 detection antibody and enzyme-labeled anti-CKMT1A detection antibody.

[0019] Furthermore, the solid-phase carrier is an enzyme-labeled plate or magnetic microparticles.

[0020] Furthermore, the enzyme is horseradish peroxidase or alkaline phosphatase.

[0021] Another object of the present invention is to provide a risk assessment application for the above-mentioned DTC detection kit, wherein the risk assessment method includes the following steps:

[0022] Obtain ACTG2 and CKMT1A detection values;

[0023] Based on the prefitted standard curve, the absolute concentrations of ACTG2 and CKMT1A were calculated according to the ACTG2 and CKMT1A detection values.

[0024] The absolute concentrations of ACTG2 and CKMT1A were substituted into a pre-defined multivariate risk assessment model to calculate the risk index.

[0025] The risk index is compared with a preset clinical cutoff value to determine the risk level of distant metastasis of DTC and / or the risk level of RAIR evolution.

[0026] Another objective of this invention is to provide a DTC risk assessment system based on dual-target combination, specifically including:

[0027] The above-mentioned DTC detection kit;

[0028] The signal detection module is used to acquire the ACTG2 detection value and the CKMT1A detection value;

[0029] The data processing module is used to calculate the absolute concentrations of ACTG2 and CKMT1A based on the prefitted standard curve and the ACTG2 and CKMT1A detection values.

[0030] The risk assessment module is used to substitute the absolute concentrations of ACTG2 and CKMT1A into a preset multivariate risk assessment model to calculate the risk index.

[0031] The result determination module is used to compare the risk index with a preset clinical cutoff value to determine the risk level of distant metastasis of DTC and / or the risk level of RAIR evolution.

[0032] Furthermore, the multivariate risk assessment model is a logistic regression assessment model, and its expression is:

[0033] ;

[0034] In the formula, P is the risk index; β0 is the intercept of the constant term; β1 and β2 are the partial regression coefficients of ACTG2 and CKMT1A, respectively; X1 and X2 are the absolute concentrations of ACTG2 and CKMT1A, respectively.

[0035] This invention fundamentally avoids the false negative problem caused by traditional TgAb interference by combining ACTG2, which reflects cytoskeleton remodeling and invasion and metastasis, and CKMT1A, which reflects energy metabolism reprogramming and dedifferentiation. At the same time, it overcomes the time lag of imaging, which can only detect macroscopic lesions, and realizes early, non-invasive, highly sensitive and highly specific assessment of the risk of distant metastasis of DTC and RAIR evolution. Attached Figure Description

[0036] Figure 1 This is a flowchart illustrating the DTC risk assessment method provided in an embodiment of the present invention.

[0037] Figure 2 This is a schematic diagram of the structure of the DTC risk assessment system provided in an embodiment of the present invention.

[0038] Figure 3 This is a schematic diagram illustrating the working principle of the DTC detection kit provided in this embodiment of the invention. Detailed Implementation

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] To address the technical problems existing in current techniques for assessing the risk of distant metastasis of DTCs and the evolution of RAIR, this invention aims to achieve the following technical objectives through a novel combination of biomarkers and a standardized detection process:

[0041] 1. This invention provides a novel, interference-resistant, and highly accurate serological detection tool: abandoning the traditional detection framework of thyroid-specific protein (Tg), it instead targets and detects tumor cell-derived cytoskeletal protein γ2-actin (ACTG2) and mitochondrial metabolic enzyme creatine kinase mitochondrial 1A (CKMT1A). This biomarker combination is completely unaffected by the binding interference and steric hindrance of serum TgAb in the immunological detection reaction, fundamentally solving the core problem of high false negative rates in existing technologies.

[0042] 2. Achieve non-invasive, early molecular early warning of distant metastasis of DTC and RAIR evolution: Overcome the time lag of traditional imaging examinations, by quantitatively detecting the trace concentration changes of ACTG2 and CKMT1A in peripheral blood, it can provide early molecular-level risk signals in the stage of tumor micrometastasis or early dedifferentiation (i.e. before the formation of morphological gross lesions), thus significantly advancing the monitoring window.

[0043] 3. Constructing a multidimensional joint prediction model to significantly improve predictive efficacy (AUC): By jointly detecting key molecules reflecting the two core mechanisms driving the evolution of RAIR-DTC, namely "invasive metastasis / epithelial-mesenchymal transition (ACTG2)" and "energy metabolism reprogramming (CKMT1A)," the limitations of single-marker detection are overcome. The risk assessment model established using dual targets significantly improves the sensitivity, specificity, and overall diagnostic efficacy for predicting DTC metastasis risk.

[0044] 4. Easy to translate into clinical applications and promote on a large scale: By combining biomarkers, the results can be transformed into standardized test kits that can be run on routine hospital laboratory equipment (such as enzyme-linked immunosorbent assay (ELISA) readers and fully automated chemiluminescence analyzers). The process is simple, the cost is controllable, and the reproducibility is good, filling the technological gap in the field of precise risk stratification of thyroid cancer in clinical practice.

[0045] Specifically, in one embodiment of the present invention, a combination of biomarkers is provided for use in the preparation of a DTC detection kit, wherein the combination of biomarkers includes ACTG2 and CKMT1A; the combination of biomarkers is used to assess the risk of distant metastasis of DTC and / or RAIR evolution.

[0046] In another embodiment of the present invention, a dual-target combined DTC detection kit is provided, wherein the DTC detection kit is a combined detection kit for ACTG2 and CKMT1A targets, containing detection reagents (such as probes, antibodies, ligands, etc.) that specifically recognize ACTG2 and CKMT1A; the DTC detection kit is used to assess the risk of distant DTC metastasis and / or RAIR evolution. It should be noted that the DTC detection kit may also exist in the form of a microarray chip; the present invention does not limit the physical product form of the DTC detection kit.

[0047] Specifically, the DTC detection kit includes a solid-phase carrier and detection reagents; the solid-phase carrier is coated with anti-ACTG2 capture antibody and anti-CKMT1A capture antibody; the detection reagents include an enzyme-labeled conjugate, ACTG2 standard, CKMT1A standard, and auxiliary reagents for colorimetric development and / or quantification; the enzyme-labeled conjugate includes enzyme-labeled anti-ACTG2 detection antibody and enzyme-labeled anti-CKMT1A detection antibody. Preferably, the solid-phase carrier is an ELISA plate or magnetic microparticles; the enzyme is horseradish peroxidase (HRP) or alkaline phosphatase. It should be noted that the enzyme label can also be replaced with a luminescent group or a fluorescein label, and is not limited thereto.

[0048] like Figure 1 As shown, in another embodiment of the present invention, a DTC risk assessment method based on the above-described DTC detection kit is also provided, comprising the following steps:

[0049] S1. Obtain the ACTG2 detection value and CKMT1A detection value;

[0050] S2. Based on the prefitted standard curve, the absolute concentrations of ACTG2 and CKMT1A are calculated according to the ACTG2 and CKMT1A detection values.

[0051] S3. Substitute the absolute concentrations of ACTG2 and CKMT1A into the preset multivariate risk assessment model to calculate the risk index;

[0052] S4. Compare the risk index with the preset clinical cutoff value to determine the risk level of distant metastasis of DTC and / or the risk level of RAIR evolution.

[0053] like Figure 2As shown, in another embodiment of the present invention, a DTC risk assessment system based on dual-target combination is also provided, specifically including:

[0054] The sample collection module is used to collect peripheral venous blood from the subject and obtain serum samples by centrifugation;

[0055] The above-mentioned DTC detection kit is used to detect ACTG2 and CKMT1A in serum samples based on a double antibody sandwich method;

[0056] The signal detection module is used to acquire ACTG2 detection values ​​and CKMT1A detection values; the detection values ​​are specifically optical density (OD) values.

[0057] The data processing module is used to calculate the absolute concentrations of ACTG2 and CKMT1A based on the prefitted standard curve and the ACTG2 and CKMT1A detection values.

[0058] The risk assessment module is used to substitute the absolute concentrations of ACTG2 and CKMT1A into a preset multivariate risk assessment model to calculate the risk index.

[0059] The result determination module is used to compare the risk index with a preset clinical cutoff value to determine the risk level of distant metastasis of DTC and / or the risk level of RAIR evolution.

[0060] The multivariate risk assessment model is a logistic regression assessment model, and its expression is:

[0061] ;

[0062] In the formula, P is the risk index; β0 is the intercept of the constant term; β1 and β2 are the partial regression coefficients of ACTG2 and CKMT1A, respectively; X1 and X2 are the absolute concentrations of ACTG2 and CKMT1A or their normalized values, respectively.

[0063] In this embodiment of the invention, ACTG2, which reflects cytoskeleton remodeling and invasion / metastasis, and CKMT1A, which reflects energy metabolism reprogramming and dedifferentiation, two independent yet synergistic key proteins leading to RAIR-DTC evolution, are combined for the first time. This two-dimensional mapping of "cytoskeleton + metabolism" is the biological basis for improving detection specificity and sensitivity (AUC). Moreover, this method completely avoids the binding site of the patient's endogenous TgAb, thoroughly eliminating the steric hindrance effect from a physical / chemical structural perspective, thus solving the problem of false negatives in up to 10%-25% of patients. In addition, this embodiment of the invention uses a multivariate risk assessment model that combines statistical algorithms such as logistic regression to transform two independent biochemical concentration indicators into a risk index that directly guides clinical decision-making. This transforms "lagging imaging diagnosis" that relies on macroscopic anatomical changes into "early molecular warning" based on peripheral blood microprotein concentrations, thereby achieving non-invasive screening at the micrometastasis stage.

[0064] It should be noted that, unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, all reagents and materials used are commercially available.

[0065] Example 1: This example provides a dual-target combined DTC detection kit, wherein the DTC detection kit is a combined ACTG2 and CKMT1A target detection kit, containing detection reagents (such as probes, antibodies, ligands, etc.) that specifically recognize ACTG2 and CKMT1A, and can be used to quantitatively detect the concentrations of ACTG2 and CKMT1A in serum samples. Specifically, the DTC detection kit includes a solid-phase support and detection reagents.

[0066] The solid-phase support was a 96-well polystyrene microplate. The microplate was physically divided into regions A and B. The inner surface of the wells in region A was pre-coated with a specific monoclonal capture antibody against human ACTG2, and the inner surface of the wells in region B was pre-coated with a specific monoclonal capture antibody against human CKMT1A. The coating concentration of both was 2-5 μg / mL.

[0067] The detection reagents include enzyme-labeled conjugates, standards, and auxiliary reagents for colorimetric development and / or quantification. The standards include lyophilized recombinant human ACTG2 and recombinant human CKMT1A standards, as well as standard reconstitution dilution buffer, used to prepare a standard curve with 6-8 gradient reference points for fitting the standard curve. The enzyme-labeled conjugates include HRP-covalently labeled anti-human ACTG2 and anti-human CKMT1A detection antibody solutions. The antigenic epitopes recognized by the detection antibodies are completely different from those of the capture antibodies to avoid competition. Auxiliary reagents include sample diluent (containing animal serum blocking HAMA interference), 20× concentrated wash buffer (PBS containing 0.05% Tween-20), colorimetric solution (3,3',5,5'-tetramethylbenzidine, TMB substrate solution), and reaction stop solution (2M H2SO4).

[0068] Example 2: As Figure 3 As shown, based on the principle of double-antibody sandwich ELISA, this embodiment provides a detection method for the above-mentioned DTC detection kit, specifically including the following steps:

[0069] 1. Sample pretreatment: Collect peripheral venous blood from the subject and centrifuge to obtain serum samples. Dilute the serum with the sample diluent provided in the kit at a ratio of 1:5 or 1:10.

[0070] 2. Specific Capture and Primary Antibody Incubation: Diluted serum samples and standard solutions of various gradients were added to the corresponding wells of the solid-phase carrier, 100 μL per well. The plates were covered with a sealing film and incubated at 37°C for 60 minutes. During this process, free ACTG2 and CKMT1A antigens in the samples were anchored by the specific capture antibodies at the bottom of the wells.

[0071] 3. First wash: Discard the liquid in the wells, wash the plate 4-5 times with the prepared 1× washing buffer, 300 μL per well each time, let stand for 1 minute, and then blot dry. This step aims to thoroughly remove unbound contaminating proteins and interfering antibodies such as TgAb that may be present in the serum.

[0072] 4. Detecting antibody binding: Add 100 μL of the corresponding HRP-labeled detection antibody solution to the microwells in regions A and B respectively, and incubate at 37°C in the dark for 30 minutes to form a sandwich-space complex structure of "coated antibody-target antigen-HRP detection antibody".

[0073] 5. Second round of washing: Same as step 3, wash away unbound free enzyme-labeled antibody to ensure that the subsequent colorimetric signal comes only from the specifically bound complex.

[0074] 6. Substrate chromogenic reaction: add 100 μL of TMB chromogenic solution to each well, and react for 15 minutes at room temperature (20-25°C) protected from light. HRP catalyzes the conversion of colorless TMB into a blue product.

[0075] 7. Termination of reaction and optical reading: quickly add 50 μL of stop solution to each well, and the solution changes from blue to yellow. Within 15 minutes after adding the stop solution, use a microplate reader to measure the OD value of each micro-well at a main wavelength of 450 nm (reference wavelength 630 nm), to obtain the ACTG2 detection value and CKMT1A detection value respectively.

[0076] Example 3: This example provides a DTC risk assessment method. After obtaining the ACTG2 detection value and CKMT1A detection value obtained in the above Example 2, the system will complete the conversion from chemical signals to clinical risk indexes through the following logic:

[0077] 1. Concentration calculation: the system takes the concentration of the standard substance as the abscissa and the corresponding OD value as the ordinate, and adopts four-parameter logistic regression (4-PL) or cubic spline interpolation to fit the standard curve. Substitute the OD values of the sample wells (that is, the ACTG2 detection value and the CKMT1A detection value) into the curve equation, and calculate the absolute concentrations (ng / mL) of ACTG2 (set as variable X1) and CKMT1A (set as variable X2) in the serum of the subject, respectively.

[0078] 2. Risk index calculation: input the obtained concentration variables X1 and X2 into the Logistic regression evaluation model pre-trained through large-sample clinical cohorts, as shown in the following formula:

[0079] ;

[0080] Wherein, β1 and β2 need to be calibrated according to actual large-sample clinical data, for example, β1=0.85, β2=0.62.

[0081] 3. Risk level output: the system compares the calculated risk index P value (between 0 and 1) with the preset clinical cutoff value (Cut-off threshold, such as 0.55) built in the kit. If P≥Cut-off, the system outputs "high metastasis / RAIR risk", indicating that the tumor may have undergone significant cytoskeletal remodeling and metabolic reprogramming; if P<Cut-off, the system outputs "low metastasis / RAIR risk", indicating routine follow-up.

[0082] In summary, compared with existing DTC diagnosis and treatment monitoring methods, the technical solution provided by the embodiment of the present invention has significant technical effects in the following four dimensions:

[0083] I. Eliminating endogenous antibody interference at the molecular structure level, significantly reducing false negative rate: This invention replaces the underlying target by selecting a combination of tumor cell-derived ACTG2 and CKMT1A proteins as the detection target. In in vitro immunological reactions, this combination completely avoids the binding site of TgAb in terms of both physical and chemical structure, eliminating antibody competition and cross-interference at the mechanistic level. This allows the kit provided by this invention to still provide accurate quantitative results even in DTC patients with high TgAb titers, filling a gap in existing technologies.

[0084] II. Breaking the Limits of Imaging Physical Resolution for Early Molecular Warning of Risk: This invention, through the detection of trace target proteins released from peripheral blood via blood circulation (liquid biopsy technology), can capture abnormal fluctuations in concentration at very early stages, such as when tumor cells have just entered the bloodstream after epithelial-mesenchymal transition (EMT) and formed micrometastases, or when glycolysis reprogramming has just begun to inhibit sodium / iodine cotransporter (NIS) expression. This technique significantly advances the time window for clinically detecting the malignant progression of DTCs, making early intervention possible.

[0085] III. Constructing a dual-dimensional "cytoskeleton-metabolism" model to significantly improve overall diagnostic efficacy: This invention creatively combines the target ACTG2, which characterizes "cytoskeleton remodeling and invasion metastasis," with the target CKMT1A, which characterizes "energy metabolism reprogramming and microenvironment alteration." Compared to existing single-target detection, the multivariate risk assessment model (Logistic regression algorithm) constructed in this invention achieves a larger area under the curve (AUC) in receiver operating characteristic (ROC) analysis. This dual-dimensional mapping mechanism effectively reduces the rate of missed diagnoses and false diagnoses, significantly improving the sensitivity and specificity of risk prediction.

[0086] IV. High-Level Standardization and Clinical Deployment Value: While achieving precise multi-target detection, this invention does not rely on costly or cumbersome mass spectrometry or high-throughput sequencing technologies. The kit provided in this invention utilizes a mature and standardized dual-antibody sandwich enzyme-linked immunosorbent assay (ELISA) platform. This technology has low hardware requirements, is perfectly compatible with existing standard ELISA readers and automated plate washers in laboratory departments of medical institutions at all levels, and features small batch-to-batch variation, stable reaction system, and controllable cost per test, making it highly feasible for large-scale mass production and clinical translation.

[0087] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. The application of a combination of biomarkers in the preparation of a DTC detection kit, characterized in that, The biomarker combination includes ACTG2 and CKMT1A; the biomarker combination is used to assess the risk of distant metastasis of DTC and / or RAIR evolution.

2. A DTC detection kit based on dual-target combination, characterized in that, The DTC detection kit is a combined detection kit for ACTG2 and CKMT1A targets, containing detection reagents that specifically identify ACTG2 and CKMT1A; the DTC detection kit is used to assess the risk of distant metastasis and / or RAIR evolution of DTC.

3. The DTC detection kit based on dual-target combination according to claim 2, characterized in that, The DTC detection kit specifically includes a solid-phase carrier and detection reagents; the solid-phase carrier is coated with anti-ACTG2 capture antibody and anti-CKMT1A capture antibody; the detection reagents include an enzyme-labeled conjugate, ACTG2 standard, CKMT1A standard, and auxiliary reagents for color development and / or quantification; the enzyme-labeled conjugate includes enzyme-labeled anti-ACTG2 detection antibody and enzyme-labeled anti-CKMT1A detection antibody.

4. The DTC detection kit based on dual-target combination according to claim 3, characterized in that, The solid support is an enzyme-labeled plate or magnetic microparticles.

5. The DTC detection kit based on dual-target combination according to claim 3, characterized in that, The enzyme is horseradish peroxidase or alkaline phosphatase.

6. A DTC risk assessment system based on dual-target combination, characterized in that, include: The DTC detection kit according to any one of claims 2-5; The signal detection module is used to acquire the ACTG2 detection value and the CKMT1A detection value; The data processing module is used to calculate the absolute concentrations of ACTG2 and CKMT1A based on the prefitted standard curve and the ACTG2 and CKMT1A detection values. The risk assessment module is used to substitute the absolute concentrations of ACTG2 and CKMT1A into a preset multivariate risk assessment model to calculate the risk index. The result determination module is used to compare the risk index with a preset clinical cutoff value to determine the risk level of distant metastasis of DTC and / or the risk level of RAIR evolution.

7. The DTC risk assessment system based on dual-target combination according to claim 6, characterized in that, The multivariate risk assessment model is a logistic regression assessment model, and its expression is: ； In the formula, P is the risk index; β0 is the intercept of the constant term; β1 and β2 are the partial regression coefficients of ACTG2 and CKMT1A, respectively; X1 and X2 are the absolute concentrations of ACTG2 and CKMT1A, respectively.

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