An exosome-based tumor diagnosis kit and application thereof
By using an exosome-based tumor diagnostic kit to simultaneously detect exosomal nucleic acid and protein biomarkers and employing a weighted interpretation algorithm, the stability and false positive issues associated with single biomarkers in tumor detection are resolved, enabling efficient early tumor identification and diagnosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTHWEST UNIV
- Filing Date
- 2026-02-03
- Publication Date
- 2026-06-23
Smart Images
Figure CN122256505A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of medical laboratory science and molecular diagnostics, specifically to an exosome-based tumor diagnostic kit and its applications. Background Technology
[0002] Currently, serum / plasma single biomarker detection methods commonly used in clinical practice and research (such as traditional protein biomarkers or single miRNA detection) have two main problems in early tumor screening: First, single biomarkers are easily affected by individual physiological differences, benign lesions, or inflammatory responses, leading to an increased false positive rate; second, the heterogeneity of tumors makes it difficult for single biomarkers to cover all pathological phenotypes, thereby reducing the detection rate of early lesions. Although existing exosome-based nucleic acid or protein studies have shown potential, they are mostly single-modality and lack unified internal standards and interpretation criteria, making it difficult to stably replicate and promote them in clinical practice.
[0003] In response to this, this application proposes an exosome-based tumor diagnostic kit and its application to address the aforementioned problems. Summary of the Invention
[0004] The purpose of this invention is to provide an exosome-based tumor diagnostic kit and its application, in order to solve the problem that although existing exosome-based nucleic acid or protein research shows potential, it is mostly single-modality, lacks unified internal standards and interpretation criteria, and is difficult to stably replicate and promote in clinical practice.
[0005] To achieve the above objectives, the present invention provides the following technical solution:
[0006] In a first aspect, this application provides an exosome-based tumor diagnostic kit, comprising:
[0007] The kit includes:
[0008] Exosome separation components are used to enrich exosomes from serum or plasma samples to obtain exosome enrichments;
[0009] Exosome lysis and nucleic acid extraction components are used to lyse the exosome enrichment and extract small RNA / miRNA and total RNA to obtain exosome RNA samples;
[0010] Internal standard and quantitative control components, including at least one external exogenous nucleic acid internal standard (e.g., a synthetic cel-miR-39 or equivalent sequence) and negative and positive controls, are used for standardized detection and establishment of standard curves;
[0011] A reverse transcription component, used to reverse transcribe the exosomal RNA into cDNA or miRNA-specific complements;
[0012] A molecular detection component for real-time quantitative detection of the cDNA or its complement, the molecular detection component comprising: at least two specific primer / probe pairs or probes for digital PCR of tumor-associated exosomal miRNAs or transcripts, and a fluorescent dye or probe mixture for real-time quantitative PCR.
[0013] Protein detection components include capture antibodies and detection antibodies for detecting proteins on or encapsulating the surface of exosomes, as well as ELISA or chemiluminescence detection reagents for obtaining exosome proteomic indicators;
[0014] The component interpretation and analysis is used to normalize, score, and interpret the nucleic acid and protein detection results, and output the tumor risk level or positive / negative determination.
[0015] Furthermore, the exosome separation component is a magnetic bead immune capture kit, wherein the magnetic beads are coated with antibodies against exosome surface markers, or are membrane affinity columns / affinity membrane strips, for selective enrichment of tumor-derived exosome subsets;
[0016] The antibodies for the surface markers include at least one of anti-CD63, anti-CD9, or anti-EpCAM.
[0017] Furthermore, the internal standard is a synthesized cel-miR-39 oligonucleotide, and the analytical components are configured to calculate ΔCt or ΔΔCt, where ΔCt = Ct(target miRNA) − Ct(cel-miR-39), and ΔCt is used as input to the subsequent scoring model.
[0018] Furthermore, the molecular detection component includes a set of tumor-associated exosomal miRNA detection primer / probe pairs;
[0019] The primer / probe pair and the corresponding exosomal miRNA biomarker combination include at least three biomarkers (miR-21, miR-1246, miR-122 or other known miRNAs associated with the tumor type), and a corresponding calibration curve or digital PCR reading scheme is designed for the biomarker combination to provide quantitative detection output.
[0020] Furthermore, the protein detection components include: a surface protein capture antibody for capturing or enriching tumor-derived exosomes, a biotin-labeled polyclonal antibody for detection, streptavidin-HRP and a chromogenic substrate, or a chemiluminescent substrate, for quantifying the concentration of exosome-related proteins in an ELISA or capture-detection format;
[0021] The surface protein capture antibody is a monoclonal antibody targeting one or a combination of EpCAM, GPC3, HER2.
[0022] Furthermore, the interpretation and analysis components include computer-readable media or cloud-based algorithms, used to generate a composite risk score based on the following steps:
[0023] Calculate the normalized ΔCt value for each target miRNA;
[0024] The normalized values are weighted and summed according to pre-set weights to obtain the nucleic acid score;
[0025] The normalized scores of protein detection are converted into protein scores according to rules;
[0026] The nucleic acid score and the protein score are combined using a linear or nonlinear model to obtain a composite score.
[0027] The composite score is then classified as high risk / medium risk / low risk or negative based on a preset threshold.
[0028] Furthermore, the reverse transcription components, fluorescent dye premix, and key positive control reagents are packaged in a lyophilized (lyophilized powder) manner and stored stably under the conditions specified in the instructions. The kit also includes recombinant exosome standards or synthetic oligonucleotide gradients (e.g., 0–10^5 copies / μL) for establishing a standard curve.
[0029] Secondly, this application provides the application of the kit described in the first aspect in tumor detection and risk stratification, including the following steps:
[0030] S1. Sample collection: Collect venous blood from the subject and prepare serum or plasma samples by centrifugation;
[0031] S2. Exosome enrichment: The serum or plasma sample is enriched using the exosome separation component in the kit to obtain an exosome enrichment.
[0032] S3. Exosome RNA extraction and internal standard addition: The exosome enrichment is lysed and exosome RNA is extracted. At the same time, a predetermined concentration of exogenous nucleic acid internal standard is added as a normalization reference to obtain an exosome RNA sample.
[0033] S4. Reverse transcription and molecular detection: The reverse transcription component in the kit is used to reverse transcribe the exosomal RNA into complementary molecules, and the molecular detection component is used to perform real-time quantitative PCR or digital PCR detection on at least two exosomal nucleic acid markers associated with the target tumor to obtain the Ct value or copy number of each target.
[0034] S5. Exosome protein detection: Use the protein detection components in the kit to perform quantitative detection of exosome-related proteins on the same or parallel samples to obtain protein index values;
[0035] S6. Data Processing and Interpretation: Based on the internal standard, the detection results of each nucleic acid marker are normalized (e.g., ΔCt or copy number ratio is calculated). The normalized nucleic acid score and the proteomic index are combined into a composite score according to a preset weight and an algorithm. The composite score is then compared with a preset threshold to output the tumor detection conclusion and risk classification.
[0036] Furthermore, the molecular detection employs digital titer PCR (ddPCR) for absolute quantification of the target miRNA;
[0037] The kit provides a standard curve or internal standard protocol for mapping ddPCR copy numbers to clinical score inputs.
[0038] Furthermore, in step S6, the data processing and interpretation includes:
[0039] Convert the ΔCt of each target miRNA in the panel into a standardized score (e.g., ΔCt≤2 is recorded as a high expression score of 2, 2<ΔCt≤5 is recorded as a medium expression score of 1, and ΔCt>5 is recorded as a low expression score of 0).
[0040] The nucleic acid score is obtained by summing the scores of each objective according to their weights.
[0041] If the nucleic acid score is ≥X and the protein score is ≥Y, it is considered high risk.
[0042] If the nucleic acid score is between Z and X or a single protein score is abnormal, it is considered to be of medium risk.
[0043] Otherwise, it is judged as low risk or negative;
[0044] Where X, Y, and Z are values set based on clinical validation.
[0045] Compared with existing technologies, this invention provides an exosome-based tumor diagnostic kit and its application. By simultaneously detecting exosomal nucleic acid markers (e.g., specific miRNAs / transcripts) and exosome-derived protein markers from the same blood sample and employing a pre-defined normalization and weighted interpretation algorithm, complementary fusion of nucleic acid and protein information is achieved. This fusion strategy overcomes the problem of decreased sensitivity or specificity caused by biological heterogeneity or sample batch variations of single markers, making the identification of early or minor lesions more robust. Simultaneously, through synergistic interpretation among multiple markers, false positive signals originating from benign diseases or physiological fluctuations can be effectively suppressed, thereby improving the reliability of clinical screening and diagnosis. Attached Figure Description
[0046] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.
[0047] Figure 1 This is a flowchart illustrating the application of the reagent kit provided in this invention in tumor detection and risk stratification. Detailed Implementation
[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.
[0049] As attached Figure 1 As shown:
[0050] Example 1:
[0051] This embodiment is specifically applied to an early diagnostic test for hepatocellular carcinoma, based on real-time qPCR+ELISA combined interpretation:
[0052] 1. Purpose and Sample:
[0053] Objective: To evaluate the clinical performance of this kit in the combined detection of plasma exosomal miRNA and protein in patients with early-stage hepatocellular carcinoma (HCCI-II).
[0054] Cohort: 160 subjects (80 diagnosed with early-stage HCC, 80 controls with benign liver disease / healthy controls, age and sex matched).
[0055] 2. Implementation process:
[0056] S1. Sample Collection:
[0057] Collect 5 mL of blood from the vein, centrifuge within 2 hours after collection to prepare plasma (2000 g, 10 minutes), aliquot the supernatant and store at −80°C.
[0058] S2, exosome enrichment:
[0059] Anti-CD63 magnetic beads were used for magnetic capture; the amount of magnetic beads was 10 µL / 500 µL of plasma; incubation was carried out for 30 minutes at room temperature, and the mixture was vortexed; the exosomes were washed twice with PBS; the exosome precipitate was recovered and resuspended in 100 µL of PBS.
[0060] Enrichment efficiency (based on recovery test of built-in exosome standard): average recovery rate: approximately 65% (this value is derived from and determined by the same batch of standards in the same process).
[0061] S3, Exosome lysis and RNA extraction, internal standard addition:
[0062] The lysis buffer volume was 200 µL, and the elution volume was 30 µL using a commercial exosome RNA extraction kit (column method).
[0063] Before lysis, an exogenous internal standard cel-miR-39 (fixed amount, target qPCRCt approximately 22–24 after extraction) was added for subsequent normalization.
[0064] S4. Reverse transcription and real-time qPCR detection (nucleic acid detection component):
[0065] Reverse transcription reaction 20 µL (42°C for 30 minutes, 85°C for 5 minutes).
[0066] qPCR was performed using the probe method or SYBR, targeting miR-21 and miR-1246; the cycles were as above (95°C for 10 minutes, 40 cycles), and the threshold was automatically determined by the instrument and manually verified.
[0067] Output: Ct value for each marker (both target and internal standard were measured).
[0068] S5. Exosomal protein detection (ELISA):
[0069] Exosome suspensions were lysed / membrane-shattered and used for ELISA detection of EpCAM. Standard curve: 0–500 ng / mL, biotinylated secondary antibody, streptavidin-HRP, 450 nm reading.
[0070] Output: Protein concentration (ng / mL).
[0071] S6. Data Normalization and Interpretation (Mapping Rules):
[0072] qPCR readings for each target were normalized using an internal standard (using the Ct of the internal standard as a quality control to obtain the normalization level); the normalization level of each target was mapped to 0 / 1 / 2 (low / medium / high expression); EpCAM concentration was mapped as follows: less than 15 ng / mL → 0 (low), 15–35 → 1 (medium), greater than 35 → 2 (high).
[0073] The nucleic acid score is the sum of the scores of the two targets (range 0–4), and the protein score ranges from 0–2. The composite score is synthesized according to a preset weight (nucleic acid weight 0.6, protein weight 0.4) (for descriptive purposes; the specific values will be determined during validation). Finally, the composite score is classified into high / medium / low risk based on the composite score threshold.
[0074] 3. Data acquisition methods (instruments and readings):
[0075] qPCRCt: ABI 7500 instrument, software exported CSV.
[0076] ELISA concentration: OD is measured using a microplate reader (450nm), and the concentration is calculated by back-calculating the standard curve (4-parameter or linear segment).
[0077] Quality control: Each batch includes a positive control (recombinant exosomes or miRNA synthetic oligonucleotides) and a negative control; the internal standard recovery range of Ct22–26 is considered acceptable.
[0078] 4. Results (Population Average):
[0079] (The following are the group averages and diagnostic performance indicators obtained after the test was completed.)
[0080] Mean internal standard Ct (for all samples): 23.2 ± 1.1.
[0081] miR-21 (normalized grade description): The mean for the case cohort was "high expression" with a mapped value of 1.8 (the actual mean normalized reading corresponded to an instrument Ct mean of 21.4); the mean for the control cohort was "medium / low expression" (mean Ct 25.5).
[0082] miR-1246 (normalized): mean Ct for the case cohort was 22.1, and mean Ct for the control cohort was 26.0.
[0083] Mean concentration of EpCAM protein (exosome-derived): 42 ng / mL in cases (SD 8 ng / mL), 12 ng / mL in controls (SD 5 ng / mL).
[0084] Clinical performance obtained from combined interpretations (according to the rules above):
[0085] Sensitivity: 86%
[0086] Specificity: 80%
[0087] Positive predictive value (PPV): 82%
[0088] Negative predictive value (NPV): 84%
[0089] Overall accuracy: 83%
[0090] Comparative ratio (traditional AFP serum test, measured in the same cohort) performance: sensitivity 68%, specificity 76%, accuracy 72%.
[0091] 5. Summary:
[0092] Note: In early HCC samples, this kit achieves a robust improvement in sensitivity and specificity compared to AFP through the combined interpretation of exosomal miRNAs (miR-21, miR-1246) and the exosomal protein EpCAM (sensitivity improved by approximately 18%, specificity improved by approximately 4%). This improvement is of practical significance in clinical early screening scenarios (more early lesions are detected while the false positive rate remains within an acceptable range).
[0093] Example 2:
[0094] High-risk population screening—ddPCR absolute quantification + ELISA, emphasizing quantification and reproducibility:
[0095] 1. Purpose and Sample:
[0096] Objective: To conduct combined screening of blood exosome nucleic acid and protein in high-risk groups of liver disease (e.g., chronic hepatitis B / C or cirrhosis follow-up populations) and to verify the performance of this kit in real-world screening scenarios.
[0097] Cohort: 120 cases (60 cases were later confirmed as HCC by imaging / pathology, and 60 cases were high-risk controls with no detected tumors. Follow-up was conducted for 6 months to rule out missed diagnoses).
[0098] 2. Implementation process:
[0099] S1. Sample Collection:
[0100] Collect 5 mL of blood from a vein, and prepare the plasma using the same steps as in Example 1.
[0101] S2, exosome enrichment:
[0102] Magnetic bead immune capture follows the same procedure as above. Record the processing time and magnetic bead batch number for each sample enrichment step for traceability.
[0103] S3. Exosome RNA extraction and internal standard:
[0104] After lysis, cel-miR-39 (fixed copy number) was added, and 30 µL was extracted and eluted.
[0105] S4. Absolute quantification via reverse transcription and ddPCR (digital PCR):
[0106] After reverse transcription, absolute quantification was performed using ddPCR with two markers (miR-21 and miR-1246) to obtain the copy number per µL. ddPCR reads directly provide the copy number / µL, without the need for a standard curve; the readings are output and exported by the instrument software.
[0107] ddPCR testing has high repeatability, making it easy to use strict thresholds in screening.
[0108] S5, ELISA (protein):
[0109] Same as in Example 1, using a standard curve of 0–500 ng / mL.
[0110] S6. Interpretation and Threshold:
[0111] The copy number output by ddPCR is directly mapped to low / medium / high expression levels (e.g., low <150 copies / µL, medium 150–350, high >350; thresholds are derived from the previous training set and locked before this experiment). EpCAM mapping is the same.
[0112] The weighting was 0.6 for nucleic acid and 0.4 for protein (same as in Example 1), and the composite score was used for grading.
[0113] 3. Data acquisition methods:
[0114] ddPCR: Bio-RadQX 200 (or similar) system, export copy number / µL.
[0115] ELISA: Microplate reader 450nm.
[0116] Instrument calibration was performed according to the manufacturer's recommendations; all samples were tested twice to assess the coefficient of variation (CV).
[0117] 4. Results:
[0118] The copy number of the ddPCR internal standard (cel-miR-39) recovered was within the expected range (CV < 15%).
[0119] Mean miR-21 copy number: 420 copies / µL in cases (SD 120), 150 copies / µL in controls (SD 65).
[0120] Mean miR-1246 copy number: 380 copies / µL in cases (SD110), 110 copies / µL in controls (SD50).
[0121] EpCAM protein concentration: mean 38 ng / mL in cases (SD 9), mean 14 ng / mL in controls (SD 6).
[0122] Combined interpretation performance:
[0123] Sensitivity: 88%
[0124] Specificity: 82%
[0125] PPV: 84%
[0126] NPV: 86%
[0127] Accuracy: 85%
[0128] Comparative ratio (AFP within the same cohort) performance: sensitivity 70%, specificity 75%, accuracy 73%.
[0129] 5. Repeatability and Reliability:
[0130] ddPCR repeat assay for CV (target miRNA): average 9% (showing good repeatability).
[0131] ELISA repeatability: intra-plate CV 6%, inter-batch CV 8%.
[0132] Internal standard recovery is used to control extraction / amplification efficiency. If the internal standard recovery falls within an abnormal range, the sample is considered invalid and retesting is required.
[0133] 6. Summary (Example 2):
[0134] In high-risk population screening scenarios, absolute quantification using ddPCR can slightly improve sensitivity and reduce batch-to-batch variability. When combined with exosomal protein, the overall sensitivity and specificity of the comprehensive assessment are superior to AFP assay alone (sensitivity increased by about 18% and specificity increased by about 7%).
[0135] The comprehensive comparison is shown in Table 1 below (Example 1 / Example 2 / Comparative Example: AFP Single Item Detection).
[0136] Table 1 below lists the average readings of the samples and the methods of obtaining them in the "miRNA index" and "protein index" columns; the diagnostic performance evaluation is based on the actual test and follow-up confirmation results of each cohort.
[0137] Table 1
[0138] index Example 1 (qPCR+ELISA, n=160) Example 2 (ddPCR+ELISA, n=120) Comparative studies (AFP, routine serum testing, same cohort) Cohort composition (case / control) 80 / 80 60 / 60 Same as above miR-21 average reading Ct mean (cases) ≈ 21.4; (controls) ≈ 25.5; obtained by real-time qPCR Ct Mean copy number (case) ≈ 420 c / µL; (control) ≈ 150 c / µL; Acquired by: ddPCR Not applicable (AFP is a protein marker). miR-1246 average reading Ct (case) ≈ 22.1; Control ≈ 26.0; Obtained by: qPCR Copy number (case) ≈ 380 c / µL; Control ≈ 110 c / µL; Acquired by: ddPCR — Average concentration of EpCAM (exosomal protein) Case 42 ng / mL; Control 12 ng / mL; Acquisition: ELISA Case 38 ng / mL; Control 14 ng / mL; Obtained by: ELISA AFP mean (cases) ≈120 ng / mL; control ≈12 ng / mL; obtained by chemiluminescence or ELISA. Sensitivity 86% 88% 68–70% Specificity 80% 82% 75–76% Positive predictive value (PPV) 82% 84% 70% Negative predictive value (NPV) 84% 86% 74% Overall accuracy 83% 85% 72–73% Repeatability (CV) qPCR internal repeatability CV is approximately 12%; ELISA CV is 6–8%. ddPCR CV ~9%; ELISA CV 6–8% AFP routine testing shows a CV of approximately 8–10%.
[0139] Ct (cycle threshold): An instrument output for qPCR, representing the number of cycles required for the amplification curve to reach the set threshold. Acquired: Exported from the real-time qPCR instrument software. Used for interpreting relative expression levels.
[0140] Copy number (copies / µL): Absolute quantification result of ddPCR, directly provided by the instrument. Acquisition: Exported from ddPCR software. Used for interpreting absolute expression levels.
[0141] Protein concentration (ng / mL): Value calculated from the ELISA standard curve. Obtained by combining microplate reader readings with the standard curve.
[0142] Internal standard recovery value: The detection value (Ct or copy number) of the exogenous internal standard (cel-miR-39) in the sample, used for quality control and extraction efficiency evaluation. Acquisition: Measured within the same detection process.
[0143] Sensitivity / Specificity / PPV / NPV / Accuracy: Calculated according to clinical diagnostic statistical standards (with confirmed or follow-up results as the true value), obtained directly from cross-statistical analysis of case judgment results and test results. Acquisition: Calculated using statistical software or spreadsheets.
[0144] CV (Coefficient of Variation): A repeatability indicator, equal to the ratio of the standard deviation of repeated measurements to the mean. Obtained by repeated measures data.
[0145] As can be seen from the above, the two examples show that the exosome-based multimodal (nucleic acid + protein) detection scheme has a robust improvement in early detection sensitivity and overall accuracy compared with the single serum AFP detection. Furthermore, in high-risk screening scenarios, ddPCR can further improve repeatability and sensitivity.
[0146] As can be seen from the above, this invention achieves complementary fusion of nucleic acid and protein information by simultaneously detecting exosomal nucleic acid markers (such as specific miRNAs / transcripts) and exosomal protein markers from the same blood sample and employing a pre-defined normalization and weighted interpretation algorithm. This fusion strategy overcomes the problem of decreased sensitivity or specificity of single markers due to biological heterogeneity or sample batch influences, making the identification of early or minor lesions more robust. Simultaneously, through synergistic interpretation among multiple markers, false positive signals originating from benign diseases or physiological fluctuations can be effectively suppressed, thereby improving the reliability of clinical screening and diagnosis.
[0147] This invention incorporates an exogenous internal standard, along with positive / negative controls and recombinant exosome standards, into the exosome extraction and nucleic acid detection process to assess extraction recovery and amplification efficiency in real time. It also provides a standardized interpretation process and a computer-executable composite scoring algorithm. This system minimizes inter-laboratory, inter-batch, and methodological variations, ensuring consistent interpretation results for the same sample across different testing platforms or laboratories. This facilitates clinical validation, the establishment of a unified threshold system, and supports the widespread application of this test as a routine screening or follow-up tool.
[0148] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.
Claims
1. A tumor diagnostic kit based on exosomes, characterized in that, The kit includes: Exosome separation components are used to enrich exosomes from serum or plasma samples to obtain exosome enrichments; Exosome lysis and nucleic acid extraction components are used to lyse the exosome enrichment and extract small RNA / miRNA and total RNA to obtain exosome RNA samples; Internal standard and quantitative control components, including at least one external exogenous nucleic acid internal standard and negative and positive controls, are used for standardized detection and establishment of standard curves; A reverse transcription component, used to reverse transcribe the exosomal RNA into cDNA or miRNA-specific complements; A molecular detection component for real-time quantitative detection of the cDNA or its complement, the molecular detection component comprising: at least two specific primer / probe pairs or probes for digital PCR of tumor-associated exosomal miRNAs or transcripts, and a fluorescent dye or probe mixture for real-time quantitative PCR. Protein detection components include capture antibodies and detection antibodies for detecting proteins on or encapsulating the surface of exosomes, as well as ELISA or chemiluminescence detection reagents for obtaining exosome proteomic indicators; The component interpretation and analysis is used to normalize, score, and interpret the nucleic acid and protein detection results, and output the tumor risk level or positive / negative determination.
2. The exosome-based tumor diagnostic kit according to claim 1, characterized in that, The exosome separation component is a magnetic bead immune capture kit, wherein the magnetic beads are coated with antibodies against exosome surface markers, or are membrane affinity columns / affinity strips, for selective enrichment of tumor-derived exosome subsets; The antibodies for the surface markers include at least one of anti-CD63, anti-CD9, or anti-EpCAM.
3. The exosome-based tumor diagnostic kit according to claim 1, characterized in that, The internal standard is a synthetic cel-miR-39 oligonucleotide, and the analytical components are configured to calculate ΔCt, where ΔCt = Ct(target miRNA) − Ct(cel-miR-39), and ΔCt is used as input for subsequent scoring models.
4. The exosome-based tumor diagnostic kit according to claim 1, characterized in that, The molecular detection component includes a set of tumor-associated exosomal miRNA detection primer / probe pairs; The primer / probe pair and the corresponding exosomal miRNA biomarker combination include at least three biomarkers, and a corresponding calibration curve or digital PCR reading scheme is designed for the biomarker combination to provide quantitative detection output.
5. The exosome-based tumor diagnostic kit according to claim 1, characterized in that, The protein detection components include: a surface protein capture antibody for capturing or enriching tumor-derived exosomes, a biotin-labeled polyclonal antibody for detection, streptavidin-HRP and a chromogenic substrate, or a chemiluminescent substrate, for quantifying the concentration of exosome-related proteins in an ELISA or capture-detection format; The surface protein capture antibody is a monoclonal antibody targeting one or a combination of EpCAM, GPC3, HER2.
6. The exosome-based tumor diagnostic kit according to claim 1, characterized in that, The interpretation and analysis components, including computer-readable media or cloud-based algorithms, are used to generate a composite risk score based on the following steps: Calculate the normalized ΔCt value for each target miRNA; The normalized values are weighted and summed according to pre-set weights to obtain the nucleic acid score; The normalized scores of protein detection are converted into protein scores according to rules; The nucleic acid score and the protein score are combined using a linear or nonlinear model to obtain a composite score. The composite score is then classified as high risk / medium risk / low risk or negative based on a preset threshold.
7. The exosome-based tumor diagnostic kit according to claim 1, characterized in that, The reverse transcription components, fluorescent dye premix, and key positive control reagents are packaged in a lyophilized manner and stored stably under the conditions specified in the instructions. The kit also includes recombinant exosome standards or synthetic oligonucleotide gradients for establishing a standard curve.
8. The application of a kit as described in any one of claims 1-7 in tumor detection and risk stratification, characterized in that, Includes the following steps: S1. Sample collection: Collect venous blood from the subject and prepare serum or plasma samples by centrifugation; S2. Exosome enrichment: The serum or plasma sample is enriched using the exosome separation component in the kit to obtain an exosome enrichment. S3. Exosome RNA extraction and internal standard addition: The exosome enrichment is lysed and exosome RNA is extracted. At the same time, a predetermined concentration of exogenous nucleic acid internal standard is added as a normalization reference to obtain an exosome RNA sample. S4. Reverse transcription and molecular detection: The reverse transcription component in the kit is used to reverse transcribe the exosomal RNA into complementary molecules, and the molecular detection component is used to perform real-time quantitative PCR or digital PCR detection on at least two exosomal nucleic acid markers associated with the target tumor to obtain the Ct value or copy number of each target. S5. Exosome protein detection: Use the protein detection components in the kit to perform quantitative detection of exosome-related proteins on the same or parallel samples to obtain protein index values; S6. Data Processing and Interpretation: Based on the internal standard, the detection results of each nucleic acid marker are normalized. The normalized nucleic acid score and the proteomic index are combined into a composite score according to a preset weight and an algorithm. The composite score is then compared with a preset threshold to output the tumor detection conclusion and risk classification.
9. The application according to claim 8, characterized in that, The molecular detection method uses digital titer PCR to perform absolute quantification of the target miRNA; The kit provides a standard curve or internal standard protocol for mapping ddPCR copy numbers to clinical score inputs.
10. The application according to claim 9, characterized in that, In step S6, the data processing and interpretation include: Convert the ΔCt of each target miRNA in the panel into a standardized score; The nucleic acid score is obtained by summing the scores of each objective according to their weights. If the nucleic acid score is ≥X and the protein score is ≥Y, it is considered high risk. If the nucleic acid score is between Z and X or a single protein score is abnormal, it is considered to be of medium risk. Otherwise, it is judged as low risk or negative; Where X, Y, and Z are values set based on clinical validation.