Micro ribonucleic acid and alpha-fetoprotein combined detection kit

Abstract

This invention relates to the field of medical reagent kit technology, and discloses a combined detection kit for microRNA and alpha-fetoprotein, comprising reagent one and reagent two. Reagent one includes: reverse transcriptase mixture, reverse transcription buffer, dilution buffer, amplification detection solution 1, amplification detection solution 2, amplification detection solution 3, amplification detection solution 4, positive control, negative control, blank control, and a value card; reagent two includes: labeled antibody, coating antibody, calibrator 1, calibrator 2, calibrator dilution buffer, washing buffer, and substrate solution. This invention provides an integrated kit that combines the analysis of three specific microRNAs (miR-21, miR-125a, miR-150) in serum extracellular vesicles, a stable internal control (miR-16), and serum AFP concentration to generate a comprehensive score (E-value), significantly improving the sensitivity and specificity of HCC auxiliary diagnosis and solving the problem of poor diagnostic performance of single indicators.

Description

Technical Field

[0001] This invention relates to the field of medical reagent kit technology, specifically a combined detection kit for microRNA and alpha-fetoprotein. Background Technology

[0002] MicroRNAs have been extensively studied as tumor markers. In human serum, miR-21, miR-125a, and miR-150 are potential diagnostic markers for liver disease or liver cancer [1-3]. Extracellular vesicles are widely distributed in serum, and microRNAs are not degraded under the protection of the extracellular vesicle membrane [4]. The expression level of miR-16 in the total microRNA profile of serum exosomal tissues is intermediate, and it is stably expressed in healthy individuals, those with chronic hepatitis B, those with hepatitis B cirrhosis, and those with liver cancer, without changing due to disease status. Therefore, miR-16 can serve as a stable internal reference for the quantification of extracellular vesicle microRNAs in liver cancer patients. α1-AFP is a glycosylated albumin with a molecular weight of 69 kDa derived from the yolk sac, undifferentiated hepatocytes, and the fetal gastrointestinal tract [5]. AFP levels are elevated in 70-95% of patients with primary liver cancer. AFP levels are also elevated to some extent in metastatic liver cancer and extrahepatic tumors. AFP can be detected in the serum of patients with acute and chronic hepatitis and cirrhosis, generally at only (50~200) ng / mL, but in a few patients it can temporarily rise to over 400 ng / mL.56 A systematic comparison of the comprehensive changes in the expression levels of microRNAs in serum extracellular vesicles between patients with and without liver cancer revealed that the combined detection of alpha-fetoprotein with miR-21, miR-125a, and miR-150 in serum extracellular vesicles significantly improves the specificity of detection and has higher diagnostic value than traditional tumor markers.

[0003] Currently, alpha-fetoprotein (AFP), as a single serum biomarker for liver cancer, has limited sensitivity and specificity, exhibiting high false negative and false positive rates. Although some studies indicate that specific microRNAs have the potential to serve as novel biomarkers, the diagnostic value of single microRNAs is insufficient, and there is a lack of stable clinically applicable detection systems that effectively integrate multiple microRNAs with AFP. Existing technologies (such as qPCR or chemiluminescence alone) cannot provide a unified, multi-indicator combined solution, resulting in insufficient accuracy in the auxiliary diagnosis of primary hepatocellular carcinoma, especially early-stage liver cancer. Summary of the Invention

[0004] The purpose of this invention is to provide a combined detection kit for microRNA and alpha-fetoprotein to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a combined detection kit for microRNA and alpha-fetoprotein, comprising reagent one and reagent two; The reagent 1 includes: reverse transcriptase mixture, reverse transcription buffer, dilution solution, amplification detection solution 1, amplification detection solution 2, amplification detection solution 3, amplification detection solution 4, positive control, negative control, blank control, and value card; The second reagent includes: labeled antibody, coated antibody, calibrator 1, calibrator 2, calibrator diluent, washing solution, and substrate solution; In the first reagent, the reverse transcriptase mixture contains reverse transcriptase and PolyA polymerase, the reverse transcription buffer contains RT buffer, dNTPs, ATP and reverse transcription primers, the diluent is nuclease-free water, amplification detection solution 1 contains qPCR Premix, miR-21 primers and fluorescent probes, amplification detection solution 2 contains qPCR Premix, miR-125a primers and fluorescent probes, amplification detection solution 3 contains qPCR Premix, miR-150 primers and fluorescent probes, and amplification detection solution 4 contains qPCR Premix, miR-16 primers and fluorescent probes; In Reagent 2, the labeled antibody contains ALP enzyme-labeled mouse anti-human AFP monoclonal antibody, Tris buffer, and BSA; the coating antibody contains magnetic particle-coated mouse anti-human AFP monoclonal antibody, phosphate buffer, and BSA; calibrator 1 contains lyophilized recombinant antigen, BSA, trehalose, and mannitol; calibrator 2 contains lyophilized AFP recombinant antigen, BSA, trehalose, and mannitol; and the substrate solution contains a solution of 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1 and 2-dioxane disodium salts.

[0006] Preferably, in reagent one, the positive control is the cell culture supernatant containing extracellular vesicles of liver cancer cells, the negative control is the cell culture supernatant containing extracellular vesicles of normal cells, and the blank control is nuclease-free water.

[0007] Preferably, in the second reagent, the calibrator diluent is purified water and the washing solution is Tris buffer.

[0008] Preferably, the reagent one comprises: 58 μL / tube × 1 reverse transcriptase mixture, 320 μL / tube × 1 reverse transcription buffer, 1 mL / tube × 2 diluent, 1060 μL / tube × 1 amplification detection solution 1, 1060 μL / tube × 1 amplification detection solution 2, 1060 μL / tube × 1 amplification detection solution 3, 1060 μL / tube × 1 amplification detection solution 4, 1.2 mL / tube × 5 positive control, 1.2 mL / tube × 5 negative control, 1.2 mL / tube × 5 blank control, and 1 value card.

[0009] Preferably, in reagent two, there are 80 μL × 48 labeled antibody, 50 μL × 48 coated antibody, 0.5 mL × 2 calibrator 1, 0.5 mL × 2 calibrator 2, 1.0 mL × 2 calibrator diluent, 1.35 mL × 48 washing solution, and 0.2 mL × 48 substrate solution.

[0010] The method of using the combined detection kit for microRNA and alpha-fetoprotein is characterized by including the following steps: MicroRNA Detection Step 1. Sample Pretreatment Take 1 mL of serum sample and isolate extracellular vesicles from the serum; extract total RNA containing the target microRNA from the extracted extracellular vesicles; Step 2. Reverse transcription reaction Take out the reverse transcriptase mixture and reverse transcription buffer, equilibrate to room temperature and mix well. Aliquot the premixed solution into PCR tubes at 7 μL / tube and store at 2-8℃. Add 3 μL of extracted RNA template to each aliquoted premixed solution, mix and centrifuge. Place the PCR tubes into the PCR instrument. After the reaction is complete, obtain the cDNA product, mix and centrifuge for later use. Step 3. Real-time fluorescence quantification Take out amplification detection solutions 1, 2, 3, and 4, equilibrate to room temperature, and mix well. Aliquot each amplification detection solution into corresponding PCR tubes at 20 μL / tube and store at 2-8℃ in the dark. Take 40 μL of dilution buffer and add it to the cDNA product from step 2, mix well, and dilute. Take 5 μL of the diluted cDNA and add it to the PCR tubes containing the four different amplification detection solutions, mix well, and centrifuge. Place the PCR tubes into a real-time quantitative PCR instrument for reaction. Step 4. Result Reading After the reaction is complete, the instrument software automatically analyzes the curve and reads the Ct value of each target; Alpha-fetoprotein (AFP) testing Step 5. Add 500 μL of calibrator diluent to each of calibrator 1 and calibrator 2, and gently mix until completely dissolved; Step 6. Use the reconstituted calibrator 1 and calibrator 2 as standards, add 100 μL to each well, place the standards on the sample position of the instrument, start the calibration program, and the instrument will automatically generate or verify the standard curve for this test based on the concentration of the two standards and the corresponding luminescence value. Step 7. Place no less than 100 μL of serum sample, positive control, negative control and blank control into the sample position of the instrument for detection. The instrument will automatically calculate the concentration of AFP in each sample based on the luminescence intensity of the sample and the standard curve. Joint analysis and result determination Step 8. Input the four Ct values ​​obtained in Step 4 and the AFP concentration value obtained in Step 7 into the microRNA and alpha-fetoprotein combined analysis software to calculate the comprehensive score, i.e., the E value. An E value ≥ 0.50 is considered positive, and an E value < 0.50 is considered negative.

[0011] Compared with the prior art, the beneficial effects of the present invention are: In this invention, specific primers and probes are used to specifically identify and amplify target microRNAs (miR-21, miR-125a, miR-150) and an internal control microRNA (miR-16). Reverse transcriptase and polymerase are used to reverse transcribe the microRNAs into cDNA and perform PCR amplification. Buffer and dNTPs provide a suitable reaction environment and substrate. Antibody-coated magnetic microparticles capture AFP in the sample. ALP-labeled anti-AFP antibody binds to the captured AFP, forming a sandwich complex, and generates a signal through an enzymatic reaction. A fluorescence signal is generated under ALP catalysis, and its intensity is proportional to the AFP concentration. Calibrators are used to establish a standard curve and quantify the AFP concentration. By providing an integrated kit, three specific microRNAs (miR-21, miR-125a, miR-150) in serum extracellular vesicles, a stable internal control (miR-16), and serum AFP concentration are analyzed together to generate a comprehensive score (E-value), which significantly improves the sensitivity and specificity of HCC auxiliary diagnosis and solves the problem of poor diagnostic performance of single indicators. Detailed Implementation

[0012] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0013] Minimal RNA (miR-21, miR-125a, miR-150, miR-16) detection steps (PCR fluorescent probe method) Step 1: Sample Pretreatment Extracellular vesicle extraction: Take 1 mL of serum sample and use the matching extracellular vesicle extraction reagent according to its instructions to separate extracellular vesicles from the serum. microRNA extraction: Immediately use the accompanying nucleic acid extraction or purification reagent to extract total RNA (containing the target microRNA) from the extracted extracellular vesicles according to their instructions. The extracted RNA should be used immediately for the next reaction, or stored at -85°C to -75°C for no more than one month.

[0014] Step 2: Reverse transcription (RT) Reagent preparation (Reagent preparation area): Remove the reverse transcriptase mixture and reverse transcription buffer, equilibrate to room temperature, and mix well.

[0015] Prepare the reverse transcription premix according to the following system (N = number of samples + number of controls): Reverse transcriptase mixture: N × 1 μL Reverse transcription buffer: N × 6 μL Aliquot the premixed solution into PCR tubes at 7 μL / tube and store at 2-8℃ for later use.

[0016] Sample loading (sample processing area): Add 3 μL of extracted RNA template to each aliquot of premixed solution, mix well and centrifuge.

[0017] Reaction (Amplification Detection Region): Place the PCR tube into the PCR instrument and run the following program: 42℃, 60 minutes 85℃, 5 minutes 4℃, keep After the reaction was complete, the cDNA product was obtained, mixed and centrifuged for later use.

[0018] Step 3: Real-time quantitative PCR (qPCR) Reagent preparation (Reagent preparation area): Take out 4 tubes of amplification detection solution (corresponding to miR-21, miR-125a, miR-150, and miR-16 respectively), equilibrate to room temperature, and mix well.

[0019] Aliquot each amplification detection solution into the corresponding PCR tube at 20 μL / tube (each detection solution is aliquoted into N tubes) and store at 2-8℃ in the dark for later use.

[0020] Sample addition (specimen processing area): Add 40 μL of diluent (nuclease-free water) to the above cDNA product and mix well to dilute.

[0021] Take 5 μL of diluted cDNA and add it to PCR tubes containing four different amplification and detection solutions. (That is, each sample requires 4 PCR tubes to detect 4 targets respectively).

[0022] Mix well and centrifuge.

[0023] Amplification and Detection (Amplification and Detection Area): Place the PCR tube into the ABI 7500 Real-Time PCR instrument.

[0024] Run the following program: Phase 1: 95℃, 5 minutes, 1 cycle (pre-denaturation) Phase 2: 95℃ for 15 seconds → 60℃ for 32 seconds (collecting fluorescence from the FAM channel), for a total of 45 cycles. Fluorescence channel settings: Reporter Fluorescence: FAM Quenching fluorescence: NFQ-MGB Reference Dye: ROX Step 4: Result Reading After the reaction is complete, the instrument software automatically analyzes the curve.

[0025] Set the threshold to 0.15 and adjust the baseline as needed.

[0026] The software automatically provides the Ct value for each target (miR-21, miR-125a, miR-150, miR-16).

[0027] Quality control requirements: Blank control: Ct value > 39 or no value.

[0028] Positive / negative controls and all samples with Ct values ​​≤ 39.

[0029] The Ct values ​​for each target in the positive / negative control should be within the specified range (refer to the value card).

[0030] The amplification curve should have a typical logarithmic growth phase.

[0031] Alpha-fetoprotein (AFP) Detection Procedure (Magnetic Particle Chemiluminescence Immunoassay) This part was conducted in the immunology testing laboratory, using a fully automated chemiluminescence immunoassay analyzer to detect AFP in serum.

[0032] Step 5: Preparation of Instruments and Reagents Applicable instruments: Hotgen Biotech C2000, MQ60 smart, C3000 or C6000 fully automated chemiluminescence immunoassay analyzer.

[0033] Reagent equilibration: Remove the kit (Reagent II) from the refrigerator at 2-8°C and allow it to equilibrate to room temperature (10-30°C).

[0034] Calibrator reconstitution: Add 500 μL of calibrator diluent to each of the lyophilized calibrator 1 (CAL-1) and calibrator 2 (CAL-2), and gently mix until completely dissolved.

[0035] Step 6: Calibration (required when using a new batch of reagents) Enter the standard curve: Enter the standard curve data of the new batch of reagents into the instrument software (usually provided in the form of a QR code or data table).

[0036] Run calibration: Use the reconstituted CAL-1 and CAL-2 as standards, add 100 μL to each well, and it is recommended to perform replicates.

[0037] Place the standard on the sample position of the instrument and start the calibration program.

[0038] The instrument automatically performs the following steps: adding antibody-coated magnetic beads, enzyme-labeled antibodies, washing, adding luminescent substrate, and detecting the luminescent signal.

[0039] The instrument automatically generates or verifies the standard curve for this test based on the concentration and corresponding luminescence value of the two standards.

[0040] Frequency: After the initial calibration, recalibration is required at least once every 4 weeks.

[0041] Step 7: Sample Testing Sample loading: Place no less than 100 μL of serum sample, positive control, negative control and blank control into the sample slot of the instrument respectively.

[0042] Run the test: Select the "Sample Detection" program in the instrument software and start it.

[0043] Automated process: The instrument automatically completes the following reaction steps: Step A (Incubation): Take the sample, the magnetic bead suspension coated with antibody and the alkaline phosphatase (ALP) labeled antibody, mix and incubate to form a sandwich complex of "magnetic bead-antibody-antigen (AFP)-enzyme-labeled antibody".

[0044] Step B (Cleaning): Adsorb the magnetic bead complex under the action of a magnetic field, and clean to remove unbound free components.

[0045] Step C (Luminescence and Detection): Add the chemiluminescent substrate (AMPPD). The ALP catalyzes the decomposition of the substrate and the emission of light. The instrument measures the luminescence intensity (RLU).

[0046] Results calculation: The instrument automatically calculates the concentration of AFP in each sample (unit: ng / mL) based on the sample's luminescence intensity (RLU) and the standard curve.

[0047] Quality control requirements: Positive control AFP concentration ≥7 ng / mL.

[0048] The negative control AFP concentration was <7 ng / mL.

[0049] The blank control group had an AFP concentration of <0.8 ng / mL.

[0050] Step 8: Joint Analysis and Result Determination Data input: Input the four Ct values ​​obtained in step 4 and the AFP concentration value obtained in step 7 into the dedicated "MicroRNA and Alpha-fetoprotein Combined Analysis Software".

[0051] Software calculation: The software calculates the overall score (E-score) based on the built-in algorithm.

[0052] Result determination: If the E value is ≥ 0.50, it is considered positive.

[0053] If the E value is less than 0.50, it is considered negative.

[0054] Final report: This "positive" or "negative" result is an auxiliary diagnostic suggestion based on the combined analysis of multiple indicators, and is provided for clinicians' reference. It needs to be combined with other examinations such as imaging to make a comprehensive judgment.

[0055] The software for combined analysis of microRNA and alpha-fetoprotein has the following built-in algorithm: Overall risk score (E-score) = 1 / (1 + e^(-Z)) Where e is the natural constant, and Z is a linear combination, calculated as follows: Z = β0 + β1 × f(AFP) + β2 × g(miRNA Ct value) The following is a detailed breakdown of the meaning and calculation method of each part of the formula: Step 1: Data Preprocessing and Transformation Because AFP concentration and Ct value have different dimensions and their distribution may be skewed, they must be converted first.

[0056] 1. AFP concentration conversion: Usually, the logarithm is taken to approximate a normal distribution.

[0057] f(AFP) = ln(AFP concentration + 1) ("+1" is to avoid the fact that an AFP concentration of 0 has no mathematical meaning) 2. miRNA Ct value conversion: Ct values ​​need to be converted to relative expression levels first. Assume you are testing three miRNAs (e.g., miR-21, miR-122, miR-223).

[0058] First, calculate the ΔCt value of each miRNA relative to the internal reference gene (e.g., U6): ΔCt_miR-21 = Ct(miR-21) - Ct(U6) Then, convert to relative expression level (2^-ΔCt): g(miR-21) = -ΔCt_miR-21 Step 2: Determine the coefficients (β value) We obtained the following logistic regression results: • β0 (intercept) = -5.0 • β1 (AFP coefficient) = 0.8 • β2 (coefficient of miR-21) = 1.2 • β3 (coefficient of miR-122) = 0.9 • β4 (coefficient of miR-223) = -1.1 (the negative sign indicates that this indicator is a protective factor, and a high expression level means a low risk) Step 3: Calculate the Z value Substitute a patient's specific test value into the input. For example: · AFP = 25 ng / mL → f(AFP) = ln(25+1) ≈ 3.26 ·miR-21: ΔCt = 2.5 → g(miR-21) = -2.5 ·miR-122: ΔCt = 1.8 → g(miR-122) = -1.8 ·miR-223: ΔCt = 3.0 → g(miR-223) = -3.0 Therefore, Z = (-5.0) + 0.8×3.26 + 1.2×(-2.5) + 0.9×(-1.8) + (-1.1)×(-3.0) = -5.0 + 2.61 - 3.0 - 1.62 + 3.3 = -3.71 Step 4: Calculate the final comprehensive risk score Substitute the Z value into the core formula: Overall risk score = 1 / (1 + e^(-(-3.71))) = 1 / (1 + e^(3.71)) ≈ 1 / (1 + 40.9) ≈ 0.024 This result (0.024) is a probability value between 0 and 1, which can be interpreted as the probability that the sample is positive (such as having liver cancer) is approximately 2.4%.

[0059] In a study involving 884 participants conducted at 5 clinical trial sites, this kit was used to detect the virus, with clinical diagnostic criteria as the gold standard.

[0060] Clinical sensitivity: 94.27% Clinical specificity: 91.61% Precision: Intra-batch CV for Reagent I < 5.0%; Intra-batch CV for Reagent II ≤ 8.0%.

[0061] Detection limits: 20 copies / μL for Reagent I; 0.8 ng / mL for Reagent II.

[0062] Linearity: For reagent II, r ≥ 0.9900 in the range of [0.8, 1200] ng / mL.

[0063] Interference resistance: The test results are unaffected by the presence of interfering substances such as bilirubin, hemoglobin, triglycerides, rheumatoid factor, and commonly used drugs at concentrations listed in the instructions.

[0064] Configuration method of this application: Preparation of Reagent I: Prepare a mixture containing reverse transcriptase, reverse transcription buffer, and four separate amplification detection solutions (for miR-21, miR-125a, miR-150, and miR-16, respectively). Weigh each liquid component according to the formula, dissolve it in nuclease-free water, adjust the pH, and dispense.

[0065] Preparation of Reagent II: Prepare antibody-coated magnetic microparticle suspensions, ALP-labeled antibody solutions, AFP calibrator solutions of different concentrations, and chemiluminescent substrate solutions. The calibrators are usually lyophilized and should be reconstituted with calibrator diluent before use.

[0066] Process parameters: The preparation process must be carried out in a sterile, nuclease-free environment; enzymes and antibodies are usually operated in a cold environment of 2-8℃; the freeze-drying process has specific temperature, vacuum degree and time parameters.

[0067] In this application, specific primers and probes are used to specifically recognize and amplify target microRNAs (miR-21, miR-125a, miR-150) and internal control microRNAs (miR-16). Reverse transcriptase and polymerase are used to reverse transcribe the microRNAs into cDNA and perform PCR amplification. Buffer and dNTPs provide a suitable reaction environment and substrate. Antibody-coated magnetic microparticles capture AFP in the sample. ALP-labeled anti-AFP antibodies bind to the captured AFP, forming a sandwich complex, and generate a signal through an enzymatic reaction. The chemiluminescent substrate generates a fluorescent signal under ALP catalysis, the intensity of which is proportional to the AFP concentration. Calibrators are used to establish a standard curve and quantify the AFP concentration.

Claims

1. A combined detection kit for microRNA and alpha-fetoprotein, characterized in that, Including Reagent 1 and Reagent 2; The reagent 1 includes: reverse transcriptase mixture, reverse transcription buffer, dilution solution, amplification detection solution 1, amplification detection solution 2, amplification detection solution 3, amplification detection solution 4, positive control, negative control, blank control, and value card; The second reagent includes: labeled antibody, coated antibody, calibrator 1, calibrator 2, calibrator diluent, washing solution, and substrate solution; In the first reagent, the reverse transcriptase mixture contains reverse transcriptase and PolyA polymerase, the reverse transcription buffer contains RT-Buffer, dNTPs, ATP and reverse transcription primers, the diluent is nuclease-free water, amplification detection solution 1 contains qPCR Premix, miR-21 primers and fluorescent probes, amplification detection solution 2 contains qPCR Premix, miR-125a primers and fluorescent probes, amplification detection solution 3 contains qPCR Premix, miR-150 primers and fluorescent probes, and amplification detection solution 4 contains qPCR Premix, miR-16 primers and fluorescent probes; In Reagent 2, the labeled antibody contains ALP enzyme-labeled mouse anti-human AFP monoclonal antibody, Tris buffer, and BSA; the coating antibody contains magnetic particle-coated mouse anti-human AFP monoclonal antibody, phosphate buffer, and BSA; calibrator 1 contains lyophilized recombinant antigen, BSA, trehalose, and mannitol; calibrator 2 contains lyophilized AFP recombinant antigen, BSA, trehalose, and mannitol; and the substrate solution contains a solution of 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1 and 2-dioxane disodium salts.

2. The combined detection kit for microRNA and alpha-fetoprotein according to claim 1, characterized in that, In the first reagent, the positive control is the cell culture supernatant containing extracellular vesicles of liver cancer cells, the negative control is the cell culture supernatant containing extracellular vesicles of normal cells, and the blank control is water without nuclease.

3. The combined detection kit for microRNA and alpha-fetoprotein according to claim 1, characterized in that, In the second reagent, the calibrator diluent is purified water, and the washing solution is Tris buffer.

4. The combined detection kit for microRNA and alpha-fetoprotein according to claim 1, characterized in that, The reagent 1 contains: 58 μL / tube × 1 reverse transcriptase mixture, 320 μL / tube × 1 reverse transcription buffer, 1 mL / tube × 2 dilution buffer, 1060 μL / tube × 1 amplification detection solution 1, 1060 μL / tube × 1 amplification detection solution 2, 1060 μL / tube × 1 amplification detection solution 3, 1060 μL / tube × 1 amplification detection solution 4, 1.2 mL / tube × 5 positive control, 1.2 mL / tube × 5 negative control, 1.2 mL / tube × 5 blank control, and 1 value card.

5. The combined detection kit for microRNA and alpha-fetoprotein according to claim 1, characterized in that, The reagent 2 contains 80 μL × 48 labeled antibody, 50 μL × 48 coated antibody, 0.5 mL × 2 calibrator 1, 0.5 mL × 2 calibrator 2, 1.0 mL × 2 calibrator diluent, 1.35 mL × 48 washing solution, and 0.2 mL × 48 substrate solution.

6. The method of using the microRNA and alpha-fetoprotein combined detection kit according to claim 1, characterized in that, Includes the following steps: MicroRNA Detection Step 1. Sample Pretreatment Take 1 mL of serum sample and isolate extracellular vesicles from the serum; extract total RNA containing the target microRNA from the extracted extracellular vesicles; Step 2. Reverse transcription reaction Take out the reverse transcriptase mixture and reverse transcription buffer, equilibrate to room temperature and mix well. Aliquot the premixed solution into PCR tubes at 7 μL / tube and store at 2-8℃. Add 3 μL of extracted RNA template to each aliquoted premixed solution, mix and centrifuge. Place the PCR tubes into the PCR instrument. After the reaction is complete, obtain the cDNA product, mix and centrifuge for later use. Step 3. Real-time fluorescence quantification Take out amplification detection solutions 1, 2, 3, and 4, equilibrate to room temperature, and mix well. Aliquot each amplification detection solution into corresponding PCR tubes at 20 μL / tube and store at 2-8℃ in the dark. Take 40 μL of dilution buffer and add it to the cDNA product from step 2, mix well, and dilute. Take 5 μL of the diluted cDNA and add it to the PCR tubes containing the four different amplification detection solutions, mix well, and centrifuge. Place the PCR tubes into a real-time quantitative PCR instrument for reaction. Step 4. Result Reading After the reaction is complete, the instrument software automatically analyzes the curve and reads the Ct value of each target; Alpha-fetoprotein (AFP) testing Step 5. Add 500 μL of calibrator diluent to each of calibrator 1 and calibrator 2, and gently mix until completely dissolved; Step 6. Use the reconstituted calibrator 1 and calibrator 2 as standards, add 100 μL to each well, place the standards on the sample position of the instrument, start the calibration program, and the instrument will automatically generate or verify the standard curve for this test based on the concentration of the two standards and the corresponding luminescence value. Step 7. Place no less than 100 μL of serum sample, positive control, negative control and blank control into the sample position of the instrument for detection. The instrument will automatically calculate the concentration of AFP in each sample based on the luminescence intensity of the sample and the standard curve. Joint analysis and result determination Step 8. Input the four Ct values ​​obtained in Step 4 and the AFP concentration value obtained in Step 7 into the microRNA and alpha-fetoprotein combined analysis software to calculate the comprehensive score, i.e., the E value. An E value ≥ 0.50 is considered positive, and an E value < 0.50 is considered negative.