Biomarker detection kit and detection method for prognosis evaluation of multiple myeloma

Abstract

The invention discloses a biomarker detection kit and a detection method for prognosis evaluation of multiple myeloma, and relates to the technical field of biological medicine detection.According to the kit, circMYC and circSETD2 bicyclic RNA combination in serum exosomes serves as a core prognosis marker, and the kit contains an exosome solid phase capture reagent, sulfydryl modified silicon dioxide magnetic beads and the like; the isothermal amplification detection reagent contains a specific SDA primer pair and the like; a double quality control reagent containing a CD63 peptide fragment marker and the like; positive control and negative control are provided; the serum exosome bicyclic RNA is selected as a core prognostic marker, a complete detection system is constructed, an exclusive reagent efficiently enriches and purifies the exosome, the detection specificity and accuracy are improved, a reliable basis is provided for prognostic layering, integrated operation is achieved, the process is simplified, clinical application is achieved, quantitative analysis is matched with a clear interpretation standard, and the detection efficiency is improved. The reagent is stable and convenient, the result is comparable, and the clinical application value is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a biomarker detection kit and detection method for prognostic assessment of multiple myeloma. Background Technology

[0002] Multiple myeloma is a malignant plasma cell disease with significant individual variability in disease progression and varying prognoses among patients. Therefore, accurate prognostic risk stratification is crucial for the development of clinical treatment plans, monitoring of disease progression, and improvement of patients' quality of life. It is also a key issue that urgently needs to be addressed in the clinical diagnosis and treatment of hematological diseases. In recent years, biomarkers have been increasingly used in tumor prognostic assessment. Circular RNA, as a novel type of non-coding RNA, has stronger stability due to its unique circular structure and exhibits significant tissue and disease specificity, making it an important research direction for tumor molecular markers. Exosomes, as extracellular vesicles, carry circular RNA that can be stably present in the body fluid circulation and can be detected in easily accessible body fluid samples such as serum, providing a possibility for non-invasive prognostic assessment of tumors. Meanwhile, isothermal amplification technology is increasingly used in nucleic acid detection due to its ease of operation. The combination of these two technologies opens up a new technical path for non-invasive prognostic assessment of multiple myeloma.

[0003] Traditional prognostic assessment methods for multiple myeloma rely heavily on clinicopathological indicators and routine laboratory test results, which can only assess risk at the phenotypic level and lack precise molecular characterization of the disease. This results in insufficient specificity and sensitivity, making early and accurate prognostic risk stratification difficult. Some nucleic acid-based detection methods often use linear RNA as a biomarker. These nucleic acids are easily degraded by nucleases in body fluid samples, leading to poor stability and repeatability of test results. Furthermore, current detection technologies lack efficient enrichment and purification methods for exosomes, making them susceptible to interference from other nucleic acids and impurities in the sample. In addition, existing circular RNA detection methods suffer from insufficient primer design specificity, easily leading to non-specific amplification. Moreover, the lack of effective quality control mechanisms throughout the detection process, and the failure to account for systematic errors during exosome extraction and nucleic acid amplification, further reduces the accuracy of test results and fails to meet the needs of precise clinical assessment. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a biomarker detection kit and method for prognostic assessment of multiple myeloma. The core of the kit uses circMYC and circSETD2 double circular RNA from serum exosomes as prognostic markers. The kit integrates specialized reagents for exosome solid-phase capture, isothermal amplification detection, and dual quality control. Each component is independently packaged, and primers and probes are specifically designed to ensure the specificity and stability of the detection. The accompanying detection method constructs a complete standardized system from exosome enrichment and purification, nucleic acid extraction, to isothermal amplification fluorescence detection, data correction, and prognostic interpretation. Dual quality control achieves dual correction of extraction and amplification efficiency, accurately stratifying patients into standard-risk and high-risk prognostic groups. This invention enables non-invasive and accurate prognostic assessment of multiple myeloma, is easy to operate and adaptable to routine clinical testing, and provides reliable molecular evidence for clinical risk stratification and personalized treatment planning, possessing significant clinical application and promotion value.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: On one hand, a biomarker detection kit for prognostic assessment of multiple myeloma, wherein the kit uses the combination of circMYC and circSETD2 double circular RNA in serum exosomes as the core prognostic marker, and includes an exosome solid-phase capture reagent, an isothermal amplification detection reagent, dual quality control reagents, a positive control, and a negative control; the exosome solid-phase capture reagent includes thiol-modified silica magnetic beads, exosome binding buffer, a first washing buffer, and a second washing buffer; the isothermal amplification detection reagent includes circMYC-specific SDA primer pairs, circSETD2-specific SDA primer pairs, a fluorescent quenching probe mixture, a 10× isothermal reaction buffer, strand displacement DNA polymerase, a dNTP mixture, and RNase-free ultrapure water; the dual quality control reagent includes a CD63 peptide marker, a circRPPH1-specific SDA primer pair, and a circRPPH1-specific fluorescent probe.

[0006] Furthermore, the thiol-modified silica magnetic beads have a particle size of 150–200 nm; the exosome binding buffer is a phosphate buffer system with pH 7.4; the first washing buffer is a Tris-HCl buffer with pH 8.0 containing Tween-20; and the second washing buffer is a detergent-free PBS buffer.

[0007] Furthermore, both the circMYC-specific SDA primer pair and the circSETD2-specific SDA primer pair are designed to span the backsplicing site of the circular RNA; in the fluorescent quenching probe mixture, the circMYC-specific probe is labeled with the FAM fluorescent reporter group, and the circSETD2-specific probe is labeled with the CY5 fluorescent reporter group, and the hybridization regions of both probes cover the backsplicing junction sequence of the corresponding circular RNA.

[0008] Furthermore, the CD63 peptide marker is a biotin-labeled CD63-specific antigen peptide; the circRPPH1-specific SDA primer pair is designed with a reverse splicing site across circRPPH1; and the circRPPH1-specific fluorescent probe is labeled with a HEX fluorescent reporter group.

[0009] Furthermore, the positive control is a mixed solution of circMYC and circSETD2 mimics; the negative control is ultrapure water free from exogenous nucleic acid contamination; and each component of the kit is dispensed into individual cryovials.

[0010] On the other hand, a detection method for a biomarker detection kit for prognostic assessment of multiple myeloma, the specific steps of which are as follows: S1, Exosome enrichment and purification: Take the serum sample to be tested, add exosome binding buffer, CD63 peptide marker and thiol-modified silica magnetic beads, incubate, discard the supernatant after magnetic separation, and then wash with the first washing buffer and the second washing buffer in sequence to obtain the exosome magnetic bead complex. S2, Exosome lysis and nucleic acid purification: Exosome lysis buffer was added to the exosome magnetic bead complex, followed by purification reagent. After magnetic separation, washing, and elution, the purified total circular RNA of the sample was obtained. S3, Isothermal Amplification and Fluorescence Detection: Using total circular RNA as a template, add circMYC and circSETD2 specific primer and probe mixture, circRPPH1 specific primer and probe mixture, 10× isothermal reaction buffer, strand displacement DNA polymerase, and dNTP mixture to construct an isothermal amplification reaction system for isothermal incubation, and collect fluorescence signals in real time. S4, Data Correction and Prognostic Interpretation: Correction was performed using CD63 peptide marker signals and circRPPH1 amplified fluorescence signals. The relative expression levels of circMYC and circSETD2 were calculated, and the patients were classified as standard or high-risk based on preset clinical cutoff values.

[0011] Furthermore, in step S1, the volume of the serum sample to be tested is 150–250 μL, the volume of the exosome binding buffer is 80–120 μL, the volume of the CD63 peptide marker is 3–8 μL, and the volume of the thiol-modified silica magnetic beads is 5–15 μL; the incubation time at room temperature with shaking is 8–15 minutes; the magnetic separation operation involves placing the centrifuge tube on a magnetic separator rack and letting it stand before discarding the supernatant; during washing, the first washing buffer is added first, followed by the second washing buffer.

[0012] Furthermore, in step S2, the volume of exosome lysis buffer is 40–60 μL, and it is allowed to stand at room temperature for 3–8 minutes; the volume of purification reagent is 40–60 μL, and it is allowed to stand for 1–5 minutes; the washing reagent is used to wash twice; the volume of RNase-free ultrapure water used for elution is 15–25 μL, and the solution is placed on ice for later use.

[0013] Furthermore, in step S3, the total amount of circular RNA template used is 3–8 μL, the total volume of the isothermal amplification reaction system is 15–25 μL, the isothermal incubation temperature is 35–39 °C, the time is 50–70 minutes, and the fluorescence signal acquisition frequency is once every 1 minute.

[0014] Furthermore, in step S4, the specific operation of data correction is as follows: the fluorescence signal of CD63 peptide marker is used as the correction parameter for exosome extraction efficiency, and the amplification fluorescence signal of circRPPH1 is used as the correction parameter for isothermal amplification efficiency; the relative expression levels of circMYC and circSETD2 are calculated using the ΔΔCt method; the specific method for prognostic risk stratification is as follows: when the relative expression levels of circMYC and circSETD2 are both lower than the corresponding cutoff values, the patient is judged as a standard-risk patient; otherwise, the patient is judged as a high-risk patient. Beneficial effects

[0015] Compared with existing technologies, this biomarker detection kit and method for prognostic assessment of multiple myeloma has the following advantages: I. This invention selects double circular RNA from serum exosomes as the core prognostic biomarker. Leveraging the natural stability of exosomal nucleic acids, it effectively avoids the problem of nucleic acid degradation in traditional detection methods, making the detection results more closely reflect the actual expression of the sample. Simultaneously, the kit, combined with a dedicated exosome solid-phase capture reagent, achieves efficient enrichment and purification of target exosomes. Combined with specific primers and fluorescent probes designed across the reverse splicing site, it significantly improves the specificity of circular RNA detection and reduces non-specific amplification interference. The dual quality control reagents achieve dual correction of exosome extraction efficiency and isothermal amplification efficiency, avoiding systematic errors from the source of detection. This makes the detection results of the relative expression level of circular RNA more accurate, providing a reliable molecular detection basis for prognostic stratification of multiple myeloma and improving the scientific rigor of prognostic assessment.

[0016] II. This invention constructs a complete detection system from exosome enrichment and purification to nucleic acid detection, data correction, and prognostic interpretation, achieving integrated operation for prognostic assessment of multiple myeloma. It simplifies the clinical testing process, eliminating the need for complex experimental equipment and cumbersome procedures, making it more suitable for routine clinical testing scenarios. The isothermal amplification combined with real-time fluorescence detection enables quantitative analysis of circular RNA expression levels. The relative expression level is calculated using the ΔΔCt method, and prognostic risk stratification is completed using clinical cutoff values. The interpretation criteria are clear, and the results are intuitive, accurately distinguishing between standard-risk and high-risk patients. The independent packaging of each component of the reagent kit ensures reagent stability and ease of use. The standardized testing process also makes results comparable across different testing scenarios, providing efficient technical support for developing personalized treatment plans and timely assessing disease progression, thus enhancing the clinical application value of prognostic assessment.

[0017] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 This invention relates to a biomarker detection kit and detection method for prognostic assessment of multiple myeloma. Detailed Implementation

[0020] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below. Example

[0021] Preparation of a biomarker detection kit for prognostic assessment of multiple myeloma.

[0022] This embodiment describes the preparation of a biomarker detection kit for prognostic assessment of multiple myeloma. The kit uses the combination of circMYC and circSETD2 double circular RNA from serum exosomes as the core prognostic markers. It includes an exosome solid-phase capture reagent, an isothermal amplification detection reagent, dual quality control reagents, a positive control, and a negative control. Each component is aliquoted into individual cryovials, and the entire process is performed in an RNase-free environment. The specific preparation process is as follows: Figure 1 As shown: Preparation of exosome solid-phase capture reagent: Preparation of thiol-modified silica magnetic beads: Silica magnetic beads were prepared by sol-gel method, and the surface was modified with thiol silane coupling agent. After modification, the beads were washed multiple times with ultrapure water to remove unbound coupling agent. After vacuum drying, thiol-modified silica magnetic beads were obtained, dispensed into 10 μL tubes, and stored at 4°C for later use.

[0023] Preparation of exosome binding buffer: Weigh the relevant reagents of the phosphate buffer system, dissolve them in ultrapure water, adjust the pH of the system to 7.4, stir until the reagents are completely dissolved, filter through a filter membrane to remove impurities and microorganisms, dispense into 100 μL tubes, and store at room temperature for later use.

[0024] Preparation of the first washing buffer: Weigh the Tris reagent and dissolve it in ultrapure water, add Tween-20, adjust the pH of the system to 8.0 with hydrochloric acid, bring the volume to the specified volume, mix thoroughly, filter through a filter membrane for sterilization, dispense into containers, and store at 4°C for later use.

[0025] Preparation of the second washing buffer: Prepare a detergent-free PBS buffer by weighing sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate and dissolving them in ultrapure water. Stir until completely dissolved, filter through a filter membrane for sterilization, dispense into containers, and store at room temperature for later use.

[0026] Preparation of isothermal amplification detection reagents: Preparation of specific SDA primer pairs: Specific SDA primer pairs were designed for circMYC and circSETD2 respectively. The primers were designed to span the reverse splicing sites of the corresponding circular RNAs. After chemical synthesis, the primers were purified by high performance liquid chromatography. The purified primers were dissolved in RNase-free ultrapure water to prepare stock solutions of specified concentrations. After aliquoting, the solutions were stored at -20°C for later use.

[0027] Preparation of fluorescent quenching probe mixture: Specific fluorescent quenching probes for circMYC and circSETD2 were prepared separately. The circMYC specific probe was labeled with the FAM fluorescent reporter group, and the circSETD2 specific probe was labeled with the CY5 fluorescent reporter group. The hybridization regions of both probes covered the reverse splicing junction sequence of the corresponding circular RNA. The two probes were mixed in an equimolar ratio and prepared with RNase-free ultrapure water to form a mixture. After being aliquoted in the dark, the mixture was stored at -20℃ for later use.

[0028] Preparation of 10× isothermal reaction buffer: Weigh Tris-HCl, potassium chloride, magnesium sulfate and other reagents, dissolve them in RNase-free ultrapure water, adjust the pH to a suitable range, make up to volume and mix thoroughly, filter through a filter membrane for sterilization, dispense into 10 μL tubes, and store at -20℃ for later use.

[0029] Enzyme and substrate preparation: Strand displacement DNA polymerase was diluted with a special buffer to prepare an enzyme solution of a specified concentration, aliquoted into single-use tubes, and stored at -20°C; dNTP mixture was prepared by mixing four deoxyribonucleotides at equal concentrations to prepare a mixture of a specified concentration, aliquoted, and stored at -20°C; RNase-free ultrapure water was sterilized at high temperature to remove RNase, aliquoted into 1 ml tubes, and stored at room temperature for later use.

[0030] Preparation of dual-quality control reagents: Preparation of CD63 peptide markers: CD63-specific antigen peptides were prepared and labeled with biotin. After labeling, unbound biotin was removed by purification. The peptides were dissolved in RNase-free ultrapure water to prepare solutions of the specified concentrations, dispensed into 5 μL tubes, and stored at -20°C for later use.

[0031] Preparation of circRPPH1-related reagents: Specific SDA primer pairs across the circRPPH1 backsplicing site were designed, and circRPPH1-specific fluorescent probes labeled with HEX fluorescent reporter groups were prepared. After chemical synthesis and purification, the probes were dissolved in RNase-free ultrapure water to prepare solutions of specified concentrations, aliquoted, and stored at -20℃ for later use.

[0032] Preparation of positive and negative controls: Preparation of positive controls: circMYC and circSETD2 simulants were prepared. The two simulants were mixed at equal concentrations and prepared into a mixed solution of a specified concentration with RNase-free ultrapure water. After gradient dilution, the solutions were dispensed into 20 μL tubes and stored at -80°C for later use.

[0033] Negative control preparation: Ultrapure water free from exogenous nucleic acid contamination was selected, filtered through a 0.22-micron filter membrane to remove impurities and nucleic acid contaminants from the water, dispensed into 50-microliter tubes, and stored at room temperature for later use.

[0034] Combine all the prepared reagent components according to the kit specifications and place them in the kit storage box. Place ice packs and a light-proof layer inside the box. Store the entire kit in a -20°C environment. This completes the preparation of the biomarker detection kit for prognostic assessment of multiple myeloma. Example

[0035] Performance testing and validation of each component of the reagent kit.

[0036] This embodiment comprehensively tests and validates the performance of each component of the kit prepared in Example 1. Key performance indicators of the exosome solid-phase capture reagent, isothermal amplification detection reagent, and dual quality control reagent are tested to verify whether each component meets the requirements for prognostic assessment of multiple myeloma. The effectiveness of the positive and negative controls is also verified. The specific testing and validation process is as follows: Performance testing of exosome solid-phase capture reagent: Performance testing of thiol-modified silica magnetic beads: The particle size distribution of the magnetic beads was tested using a laser particle size analyzer, and the particle size range and distribution uniformity were recorded. At the same time, the magnetic responsiveness of the magnetic beads was tested by placing the magnetic beads on a magnetic separation rack and recording the time for complete adsorption to verify their magnetic separation efficiency.

[0037] Buffer performance testing: The pH value of each buffer was measured multiple times using a precision pH meter, and the fluctuation range of the pH value was recorded; nucleic acid contamination testing was performed on each buffer, and nucleic acid electrophoresis was used to detect whether there were nucleic acid impurities in the buffer. At the same time, the presence of RNase and DNase activity in the buffer was detected to prevent nucleic acid degradation.

[0038] Isothermal amplification assay reagent performance testing: Performance testing of specific SDA primer pairs: Primer specificity verification experiments were conducted. Using circMYC, circSETD2, circRPPH1 and other unrelated circular RNAs as templates, corresponding specific SDA primer pairs were added to perform amplification reactions. The amplification specificity of the primer pairs was observed to verify whether the primers only amplify with the corresponding templates.

[0039] Performance testing of fluorescent quenching probe mixtures: The fluorescent labeling efficiency and quenching effect of the probes are tested. The background fluorescence intensity of the probes is measured before the amplification reaction, and the specific fluorescence intensity is measured after the amplification reaction is completed. The boost factor of the fluorescence signal is calculated to verify the fluorescence response performance of the probes.

[0040] Strand displacement DNA polymerase performance assay: Different enzyme dosage gradients were set up, and isothermal amplification reactions were performed using a positive control as a template. The amplification efficiency and fluorescence signal intensity at different enzyme dosages were detected to determine the enzyme activity and effective concentration. At the same time, the activity stability of the enzyme at different storage temperatures was detected.

[0041] Dual-quality control reagent performance testing: CD63 peptide marker performance testing: CD63 peptide markers were co-incubated with exosomes of known concentrations, and the biotin signal intensity on the surface of the magnetic beads was detected after magnetic separation. The binding efficiency of the peptide markers to exosomes was calculated to verify its ability to recognize and bind to exosomes.

[0042] Performance testing of circRPPH1-related reagents: Using purified circRPPH1 as a template, circRPPH1-specific SDA primers and fluorescent probes were added to perform isothermal amplification reactions. The morphology of the amplification curves was observed, and the stability and repeatability of the fluorescence signal were detected to verify its effectiveness as a quality control reagent.

[0043] Validation of the effectiveness of positive and negative controls: Positive control verification: The positive control is added to the isothermal amplification reaction system, and the amplification reaction is carried out to detect whether a significant specific fluorescence signal can be generated, thus verifying the effectiveness of the positive control.

[0044] Negative control verification: The negative control is added to the isothermal amplification reaction system, and the amplification reaction is carried out to detect the presence of non-specific fluorescence signals, thus verifying that the negative control is free of nucleic acid contamination.

[0045] Performance test results of each component of the reagent kit: Testing items detection indicators Test results Thiol-modified silica magnetic beads Particle size distribution Uniform distribution, meeting preparation requirements Thiol-modified silica magnetic beads Magnetic response High magnetic separation efficiency and short adsorption time Each buffer solution pH value The fluctuation range is small and meets the design value. Each buffer solution Nucleic acid and enzyme activity No nucleic acid contamination, no RNase or DNase activity Specific SDA primer pairs Amplification Specificity Amplifies only with the corresponding template, with no cross-reaction. Fluorescence quenching probe mixture fluorescence response Low background fluorescence, strong specific fluorescence signal Strand displacement DNA polymerase enzyme activity Stable activity and high amplification efficiency CD63 peptide marker Combined efficiency High binding efficiency to exosomes circRPPH1 related reagents Amplification stability The amplification curve is regular and the fluorescence signal is stable. Positive control fluorescence signal Specific fluorescence signal is obvious negative control Nonspecific signals No obvious nonspecific fluorescence signal Testing items detection indicators Test results Analysis of test results: The above test results show that all components of the kit performed well in all detection indicators. The thiol-modified silica magnetic beads have a uniform particle size distribution and good magnetic responsiveness, enabling efficient capture and magnetic separation of exosomes. The pH values ​​of each buffer are stable, with no nucleic acid contamination or enzyme activity, ensuring the stability of the exosome capture and washing process. The specific SDA primer pair has high amplification specificity, amplifying only the corresponding circular RNA template, avoiding detection errors caused by non-specific amplification. The fluorescent quenching probe mixture has low background fluorescence and strong specific fluorescence signal, accurately reflecting the progress of the amplification reaction. The strand displacement DNA polymerase has stable activity and high amplification efficiency, providing a reliable enzymatic basis for isothermal amplification. In the dual quality control reagents, the CD63 peptide marker has high binding efficiency with exosomes, effectively reflecting the extraction efficiency of exosomes. The circRPPH1 related reagent has a regular amplification curve and stable fluorescence signal, effectively correcting for isothermal amplification efficiency. The positive control produces obvious specific fluorescence signal, while the negative control has no non-specific fluorescence signal; both effectively control the quality of the detection process. Overall, the kit components exhibit excellent performance and their effectiveness has been fully validated, making it suitable for subsequent prognostic assessment of multiple myeloma. Example

[0046] Multiple myeloma biomarker detection procedure based on kit.

[0047] This embodiment uses the kit prepared in Example 1 to detect multiple myeloma biomarkers in clinical serum samples according to standardized operating procedures. The entire process strictly adheres to RNase-free operating guidelines to avoid nucleic acid degradation and contamination, ensuring the accuracy and reliability of the test results. The specific detection steps are divided into four parts: exosome enrichment and purification, exosome lysis and nucleic acid purification, isothermal amplification and fluorescence detection, and data correction and prognostic interpretation. The detailed operating procedures are as follows: Exosome enrichment and purification: Take an RNase-free centrifuge tube, add 200 μL of the serum sample to be tested, then add 100 μL of exosome binding buffer, 5 μL of CD63 peptide marker, and 10 μL of thiol-modified silica magnetic beads in sequence. Gently pipette to mix, ensuring that each reagent is in full contact with the serum sample.

[0048] Place the centrifuge tube in a constant temperature shaker and incubate at room temperature. During incubation, ensure that the shaking speed is uniform so that the magnetic beads can fully capture the exosomes in the serum. After incubation, place the centrifuge tube on a magnetic separator and let it stand for a period of time until the magnetic beads are completely adsorbed onto the centrifuge tube wall. Then carefully discard the supernatant to avoid aspirating the magnetic beads.

[0049] Add the first washing buffer to the exosome magnetic bead complex, mix well by pipetting, then place the centrifuge tube on a magnetic separator rack and let it stand. After magnetic separation, discard the supernatant. Add the second washing buffer and repeat the above washing operation to remove the impurities and non-specific conjugates adsorbed on the surface of the magnetic beads. After washing, discard the supernatant to obtain the purified exosome magnetic bead complex.

[0050] Exosome lysis and nucleic acid purification: Add 50 μL of exosome lysis buffer to the exosome magnetic bead complex, gently pipette to mix, ensuring the lysis buffer is in full contact with the magnetic beads, and let stand at room temperature to allow the lysis buffer to fully lyse the exosomes and release the nucleic acid material within the exosomes.

[0051] After lysis, add 50 μL of nucleic acid purification reagent to the centrifuge tube, mix well by pipetting, and let stand at room temperature to allow the nucleic acid to fully bind with the magnetic beads and form a nucleic acid-magnetic bead complex.

[0052] Place the centrifuge tubes on a magnetic separator rack, let them stand, discard the supernatant, add washing reagent, mix by blowing and stirring, then perform magnetic separation, discard the supernatant, and repeat the washing operation twice to thoroughly remove unbound impurities and reagents.

[0053] After washing, add 20 μL of RNase-free ultrapure water to the centrifuge tube, mix well by pipetting, and let stand at room temperature to allow the nucleic acids bound to the magnetic beads to be fully eluted. Then place the centrifuge tube on a magnetic separator and collect the supernatant after elution, which is the purified total circular RNA of the sample. Keep it on ice for later use to prevent nucleic acid degradation.

[0054] Isothermal amplification and fluorescence detection: Take an RNase-free PCR tube and add 5 μL of purified total circular RNA from the sample as an amplification template. Then, add the circMYC and circSETD2 specific primer and probe mixture, the circRPPH1 specific primer and probe mixture, 10× isothermal reaction buffer, strand displacement DNA polymerase, and dNTP mixture in sequence. Make up the reaction volume to 20 μL with RNase-free ultrapure water. Gently mix with a pipette and centrifuge briefly to concentrate the reaction volume at the bottom of the PCR tube.

[0055] Place the PCR tube in the quantitative fluorescence detector, set the instrument's reaction parameters, adjust the constant temperature incubation temperature to 37℃, the incubation time to 60 minutes, and the fluorescence signal acquisition frequency to once per minute. Acquire signal data from the three fluorescence channels FAM, CY5, and HEX respectively, record the changes in fluorescence signals during the amplification reaction in real time, and generate an amplification curve.

[0056] Data correction and prognostic interpretation: The fluorescence signal data collected by the quantitative fluorescence detector were processed and corrected. The fluorescence signal of CD63 peptide marker was used as the correction parameter for exosome extraction efficiency to correct the difference in exosome enrichment efficiency among different samples and eliminate the detection error caused by extraction efficiency. The amplification fluorescence signal of circRPPH1 was used as the correction parameter for isothermal amplification efficiency to correct the difference in amplification efficiency among different reaction systems and eliminate the detection error caused by the amplification process.

[0057] The ΔΔCt method was used to calculate the relative expression levels of circMYC and circSETD2. The calculation process strictly followed the data analysis specifications of quantitative real-time PCR to ensure the accuracy of the results.

[0058] The calculated relative expression levels of circMYC and circSETD2 were compared with preset clinical cutoff values. Based on the comparison results, prognostic risk stratification was performed on multiple myeloma patients to determine whether they were classified as standard risk or high risk.

[0059] Verification results of the testing operation: Validate Project Validation metrics Test results Exosome enrichment and purification Exosome purity The purified exosomes had low levels of impurities and proteins. Exosome lysis and nucleic acid purification Nucleic acid integrity Total circular RNA showed no degradation and good integrity. Isothermal amplification reaction Amplification curve The curve has a regular shape and no abnormal fluctuations. Fluorescence signal detection Signal stability The fluorescence signal is stable and has good repeatability. Data Correction Correction effect Effectively eliminates extraction and amplification efficiency errors Prognostic interpretation Result determination The interpretation results are clear and the boundaries are distinct. Verification Result Analysis: The results of the detection operation verification show that the kit-based detection procedure was standardized and effective. The exosome enrichment and purification process effectively removed impurities from the serum, obtaining high-purity exosomes, laying a good foundation for subsequent nucleic acid extraction. The exosome lysis and nucleic acid purification process fully lysed the exosomes, releasing complete total circular RNA without nucleic acid degradation, ensuring the quality of the amplification template. During the isothermal amplification reaction, the amplification curve was regular in shape and without abnormal fluctuations, indicating that the amplification reaction proceeded smoothly without significant non-specific amplification. During the fluorescence signal detection, the signal was stable and reproducible, accurately reflecting the true situation of the amplification reaction. The data correction stage, through the correction of two quality control parameters, effectively eliminated the detection error caused by the difference between exosome extraction efficiency and isothermal amplification efficiency, making the calculation results of the relative expression levels of circMYC and circSETD2 more accurate. In the prognostic interpretation stage, the comparison between the relative expression levels and the clinical cutoff value clearly determined the prognostic risk type of the patient, without any ambiguous interpretations. Overall, the testing procedure is scientific and reasonable, fully utilizes the performance of the reagent kit, and provides accurate and reliable test results, which can be effectively applied to the prognostic assessment of multiple myeloma. Example

[0060] Optimization of key reaction conditions during the kit's detection process.

[0061] This embodiment systematically optimizes key reaction conditions in the kit's detection process to improve the sensitivity, accuracy, and repeatability of the detection method. Five key reaction conditions were selected: exosome incubation time, exosome lysis time, isothermal amplification temperature, isothermal amplification time, and total circular RNA template usage. Single-factor experiments were conducted with gradient variables set for each condition. The optimal parameters for each key reaction condition were determined using fluorescence signal intensity, accuracy of relative expression level calculation, and repeatability of the amplification reaction as evaluation indicators. The specific optimization process is as follows: Optimization of exosome incubation time: A room temperature oscillation incubation time gradient was set up for different durations, while the other detection operation steps remained the same. The detection was carried out according to the procedure in Example 3. The capture efficiency of magnetic beads for exosomes was detected at different incubation times. The fluorescence signal intensity at each gradient was recorded, the average capture efficiency was calculated, and the influence of incubation time on the detection results was analyzed.

[0062] Optimization of exosome lysis time: Set up a gradient of exosome lysis buffer incubation time at room temperature for different durations, while keeping other operating steps unchanged. After the detection was completed, total circular RNA was extracted from each gradient, and the amount and integrity of nucleic acid extracted were detected. The corresponding fluorescence signal intensity was recorded, and the effect of lysis time on nucleic acid extraction efficiency and subsequent detection was analyzed.

[0063] Isothermal amplification temperature optimization: A constant temperature incubation gradient was set up, with different temperatures and a fixed incubation time of 60 minutes. All other operating steps remained the same. After detection, the amplification curve morphology, fluorescence signal intensity, and amplification efficiency at each temperature were recorded to analyze the effect of temperature on strand displacement DNA polymerase activity and amplification reaction.

[0064] Optimization of isothermal amplification time: Set up a gradient of isothermal incubation time for different durations, with the amplification temperature fixed at 37℃ and all other operating steps remaining unchanged. After the detection is completed, observe whether the amplification reaction reaches the plateau phase at each duration, record the saturation of the fluorescence signal, and analyze the effect of amplification time on the detection results.

[0065] Optimization of total circular RNA template usage A gradient of total circular RNA template dosage was set up, with different amounts in microliters. The total volume of the isothermal amplification reaction system was fixed at 20 microliters, and all other operating steps remained the same. After detection, the fluorescence signal intensity, amplification efficiency, and non-specific amplification were recorded under each template dosage to analyze the effect of template dosage on the amplification reaction.

[0066] Results of key reaction condition optimization: Optimization conditions Gradient settings Optimal parameters Evaluation indicators performance Exosome incubation time Multiple gradients with different durations 10 minutes High capture efficiency and strong fluorescence signal Exosome lysis time Multiple gradients with different durations 5 minutes Nucleic acid extraction volume is the highest, and the integrity is good. Isothermal amplification temperature Multiple different temperature gradients 37℃ Highest amplification efficiency and most stable fluorescence signal Isothermal amplification time Multiple gradients with different durations 60 minutes When amplification reaches plateau phase, fluorescence signal saturation occurs. Total amount of circular RNA template used Multiple gradients with different micro-levels 5 microliters Excellent amplification effect, no nonspecific amplification Optimization Result Analysis: The optimization results of key reaction conditions show that the optimal parameters for each condition enable all evaluation indicators to reach their best levels. When the exosome incubation time is 10 minutes, the magnetic beads achieve the highest capture efficiency of exosomes, significantly increasing the fluorescence signal intensity. If the incubation time is too short, the magnetic beads will not bind sufficiently to the exosomes, resulting in low capture efficiency; if the incubation time is too long, it can easily lead to increased non-specific binding, introducing detection errors. When the exosome lysis time is 5 minutes, the exosomes can be fully lysed, achieving maximum nucleic acid extraction with good integrity. If the lysis time is too short, the exosomes will not lyse sufficiently, resulting in low nucleic acid release; if the lysis time is too long, it may lead to nucleic acid degradation, affecting subsequent amplification reactions. At an isothermal amplification temperature of 37℃, the activity of strand displacement DNA polymerase reaches its optimal level, resulting in the highest amplification efficiency, stable fluorescence signal, and good repeatability. Temperature deviations from this value will lead to decreased enzyme activity, reduced amplification efficiency, and even non-specific amplification. When the isothermal amplification time is 60 minutes, the amplification reaction reaches the plateau phase, and the fluorescence signal saturates, accurately reflecting the initial template concentration. Too short a time results in incomplete amplification and a low fluorescence signal; too long a time provides no additional signal boost and may even cause fluorescence quenching. A total circular RNA template volume of 5 μL yields the best amplification effect, with moderate fluorescence signal intensity and no nonspecific amplification. Insufficient template leads to insufficient nucleic acid template, weak fluorescence signal, and a higher risk of false negatives; excessive template can cause template inhibition, reducing amplification efficiency. Optimization of these key reaction conditions determined the optimal reaction parameters for the kit, effectively improving the sensitivity, accuracy, and repeatability of the detection method. Example

[0067] Validation of the sensitivity and specificity of the reagent kit and detection method.

[0068] This embodiment validates the sensitivity and specificity of the kit prepared in Example 1 and the optimized detection method in Example 4. Sensitivity validation was performed by preparing circular RNA mimics at gradient concentrations to determine the limit of detection of the kit. Specificity validation was performed using clinical serum samples from different sources to verify the kit's ability to specifically recognize biomarkers related to multiple myeloma. The entire process was conducted under optimized reaction conditions to ensure the accuracy and reliability of the validation results. The specific validation process is as follows: Sensitivity verification: A mixed solution of circMYC and circSETD2 was prepared, and a concentration gradient was set up, covering low, medium and high concentration levels. Multiple replicates were prepared for each concentration gradient, and blank control samples were also set up.

[0069] The optimized detection method was used to detect the mixed solutions of simulants at various concentration gradients and blank control samples. The operation was completed according to the procedure of exosome enrichment and purification, exosome lysis and nucleic acid purification, isothermal amplification and fluorescence detection, data correction and prognostic interpretation. The fluorescence signal intensity of each sample was recorded, and the relative expression levels of circMYC and circSETD2 were calculated using the ΔΔCt method.

[0070] Analyze the detection results at different concentration gradients, observe whether specific fluorescence signals can be detected, determine the lowest concentration that the kit can accurately detect, i.e. the kit's lowest detection limit, and analyze the correlation between fluorescence signal intensity and simulant concentration.

[0071] Specificity verification: Clinical serum samples were selected and divided into three groups: the first group consisted of serum samples from patients with multiple myeloma, the second group consisted of serum samples from healthy individuals undergoing physical examinations, and the third group consisted of serum samples from patients with other hematological diseases. A certain number of samples were selected from each group to ensure that the sample size was statistically significant.

[0072] The optimized detection method was used to detect all clinical serum samples. After the detection was completed, the relative expression levels of circMYC and circSETD2 in each group of samples were calculated, and the expression level distribution map of each group of samples was plotted to observe the expression level differences among the three groups of samples.

[0073] The test kit was used to verify whether it specifically recognizes circMYC and circSETD2 in the serum of patients with multiple myeloma, and to detect whether there is cross-reactivity with samples from healthy individuals and other patients with hematologic disorders.

[0074] Sensitivity and specificity verification results: Validation type Detection object Key Indicators Verification results Sensitivity circMYC and circSETD2 analogue mixed solution Minimum detection limit Detectable down to 1×10³ copies / µL Sensitivity Gradient concentration simulated solution Signal correlation Fluorescence signal is strongly positively correlated with concentration. Specificity Serum samples from patients with multiple myeloma Expression level High expression of circMYC and circSETD2 Specificity Serum samples from healthy individuals undergoing physical examinations Expression level Low expression of circMYC and circSETD2 Specificity Serum samples from patients with other hematological diseases Expression level Low expression of circMYC and circSETD2 Specificity Three types of serum samples Cross-reaction No obvious cross-reactivity Verification Result Analysis: The sensitivity and specificity validation results demonstrate that this kit and detection method exhibit good sensitivity and high specificity. Regarding sensitivity, the kit's limit of detection reaches 1×10³ copies / µL, accurately detecting low concentrations of circMYC and circSETD2 mimics. Furthermore, the fluorescence signal intensity shows a strong positive correlation with the mimic concentration, indicating that within a certain concentration range, the kit accurately reflects changes in the concentration of the target circular RNA and can effectively detect low abundances of circMYC and circSETD2 in serum samples, meeting the needs of low-concentration sample detection in clinical settings. In terms of specificity, circMYC and circSETD2 were significantly overexpressed in serum samples from multiple myeloma patients, while both were underexpressed in serum samples from healthy individuals and other patients with hematologic disorders. Furthermore, the kit showed no significant cross-reactivity with samples from healthy individuals and other patients with hematologic disorders. This indicates that the kit can highly specifically identify the double circular RNAs circMYC and circSETD2 in the serum of multiple myeloma patients, without producing false positive results due to interference from healthy individuals or other hematologic disorders. This effectively ensures the specificity of the test results and can accurately distinguish between multiple myeloma patients and non-multiple myeloma individuals. Example

[0075] Validation of the reproducibility, stability and clinical application of the reagent kit and detection method.

[0076] This embodiment comprehensively validates the repeatability, stability, and clinical application of the reagent kit and detection method. Repeatability validation examines the consistency of test results under different personnel and at different testing times. Stability validation examines the performance changes of the reagent kit under different storage conditions and for different durations. Clinical application validation involves testing a large sample size of clinically diagnosed multiple myeloma patients and verifying the clinical application value of the reagent kit based on follow-up results. The specific validation process is as follows: Repeatability verification: Three serum samples from multiple myeloma patients with different circMYC and circSETD2 expression levels were selected and labeled as low-expression, medium-expression, and high-expression samples, respectively.

[0077] Three different researchers were assigned to conduct repeated tests on three samples at different times using the same batch of reagent kits and the optimized detection method. Each sample was tested six times, and the entire process strictly followed the standardized operating procedures.

[0078] Record all test results, calculate the relative standard deviation of the relative expression levels of circMYC and circSETD2 for each sample, analyze the impact of different experimental personnel and different test times on the test results, and evaluate the repeatability of the test method.

[0079] Stability verification: The reagent kits prepared in the same batch were divided into three groups and stored in three different environments: -20℃, 4℃, and room temperature. Multiple reagent kit samples were set in each group.

[0080] After storage for one week, two weeks, one month, and three months, the kits were retrieved from their respective storage environments. Positive controls and clinical serum samples were selected for testing. The optimized testing method was followed to complete the operation, and the test results were recorded.

[0081] The performance changes of each component of the kit under different storage conditions and storage times were analyzed, and the changes in fluorescence signal intensity, amplification efficiency, and relative expression level were detected to determine the optimal storage conditions and effective storage time of the kit.

[0082] Clinical application validation: Serum samples from 100 clinically diagnosed multiple myeloma patients were selected. All samples were tested using this kit and detection method. The relative expression levels of circMYC and circSETD2 in each patient were calculated. Patients were divided into standard-risk and high-risk groups according to the preset clinical cutoff value.

[0083] All 100 patients with multiple myeloma underwent a 12-month clinical follow-up. The progression of the disease was recorded regularly, and the progression-free survival and overall survival of patients in the standard-risk and high-risk groups were statistically analyzed to identify the prognostic differences between the two groups.

[0084] To verify the consistency between the test results of the kit and the clinical prognosis of patients, and to evaluate the application value of the kit in the clinical prognostic assessment of multiple myeloma.

[0085] Repeatability, stability, and clinical application validation results: Validation type detection indicators Test results Repeatability Relative standard deviation All less than 3% stability Store at -20℃ for three months The performance of the reagent kit showed no significant change. stability Store at 4℃ for two weeks The reagent kit performance is basically stable. stability Store at room temperature for one week The performance of the reagent kit decreased significantly. Clinical application Progression-free survival rate in the standard risk group 93.5% Clinical application Progression-free survival rate in high-risk group 39.5% Clinical application Prognostic differences Significant differences were found between the standard risk group and the high risk group. The results of repeatability, stability, and clinical application validation demonstrate that this kit and detection method exhibit good repeatability, stable performance, and high clinical application value. Regarding repeatability, the relative standard deviation of three samples with different expression levels under different personnel and detection times was less than 3%, indicating good consistency of the detection results. The results were minimally affected by personnel operation and detection time, demonstrating excellent repeatability and ensuring comparability of results between different laboratories and personnel. Regarding stability, after storage at -20℃ for three months, the kit showed no significant changes in performance; fluorescence signal intensity, amplification efficiency, and relative expression levels remained stable, indicating that this temperature is the optimal storage condition. Within two weeks of storage at 4℃, the kit performance remained basically stable, meeting the needs of short-term transportation and temporary storage. However, after one week of storage at room temperature, the kit performance significantly decreased; therefore, long-term storage at room temperature should be avoided. In clinical application, among 100 patients with multiple myeloma, the progression-free survival rate reached 93.5% in the standard-risk group and only 39.5% in the high-risk group. The prognostic difference between the standard-risk and high-risk groups was significant, and the test results of the kit were highly consistent with the clinical follow-up prognosis of the patients. This indicates that the kit can accurately stratify the prognostic risk of multiple myeloma patients and can provide a reliable reference for clinicians to formulate personalized treatment plans. It has important application value in the clinical prognostic assessment of multiple myeloma.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A biomarker detection kit for prognostic assessment of multiple myeloma, characterized in that, The kit uses the combination of circMYC and circSETD2 double circular RNA in serum exosomes as the core prognostic marker and includes an exosome solid-phase capture reagent, an isothermal amplification detection reagent, dual quality control reagents, a positive control, and a negative control. The exosome solid-phase capture reagent includes thiol-modified silica magnetic beads, exosome binding buffer, a first washing buffer, and a second washing buffer. The isothermal amplification detection reagent includes circMYC-specific SDA primer pairs, circSETD2-specific SDA primer pairs, a fluorescent quenching probe mixture, a 10× isothermal reaction buffer, strand displacement DNA polymerase, a dNTP mixture, and RNase-free ultrapure water. The dual quality control reagents include a CD63 peptide marker, a circRPPH1-specific SDA primer pair, and a circRPPH1-specific fluorescent probe.

2. The biomarker detection kit for prognostic assessment of multiple myeloma according to claim 1, characterized in that, The thiol-modified silica magnetic beads have a particle size of 150–200 nm; the exosome binding buffer is a phosphate buffer system with pH 7.4; the first washing buffer is a Tris-HCl buffer with pH 8.0 containing Tween-20; and the second washing buffer is a detergent-free PBS buffer.

3. A biomarker detection kit for prognostic assessment of multiple myeloma according to claim 1, characterized in that, Both the circMYC-specific SDA primer pair and the circSETD2-specific SDA primer pair are designed to span the backsplicing site of the circular RNA. In the fluorescent quenching probe mixture, the circMYC-specific probe is labeled with the FAM fluorescent reporter group, and the circSETD2-specific probe is labeled with the CY5 fluorescent reporter group. The hybridization regions of both probes cover the backsplicing junction sequence of the corresponding circular RNA.

4. A biomarker detection kit for prognostic assessment of multiple myeloma according to claim 1, characterized in that, The CD63 peptide marker is a biotin-labeled CD63-specific antigen peptide; the circRPPH1-specific SDA primer pair is designed with a reverse splicing site across circRPPH1; and the circRPPH1-specific fluorescent probe is labeled with a HEX fluorescent reporter group.

5. A biomarker detection kit for prognostic assessment of multiple myeloma according to claim 1, characterized in that, The positive control is a mixed solution of circMYC and circSETD2 mimics; the negative control is ultrapure water free from exogenous nucleic acid contamination; each component of the kit is dispensed in an independent cryovial.

6. A detection method for a biomarker detection kit for prognostic assessment of multiple myeloma, the method being applicable to the biomarker detection kit for prognostic assessment of multiple myeloma as described in any one of claims 1-5, characterized in that, The specific steps of this detection method are as follows: S1, Exosome enrichment and purification: Take the serum sample to be tested, add exosome binding buffer, CD63 peptide marker and thiol-modified silica magnetic beads, incubate, discard the supernatant after magnetic separation, and then wash with the first washing buffer and the second washing buffer in sequence to obtain the exosome magnetic bead complex. S2, Exosome lysis and nucleic acid purification: Exosome lysis buffer was added to the exosome magnetic bead complex, followed by purification reagent. After magnetic separation, washing, and elution, the purified total circular RNA of the sample was obtained. S3, Isothermal Amplification and Fluorescence Detection: Using total circular RNA as a template, add circMYC and circSETD2 specific primer and probe mixture, circRPPH1 specific primer and probe mixture, 10× isothermal reaction buffer, strand displacement DNA polymerase, and dNTP mixture to construct an isothermal amplification reaction system for isothermal incubation, and collect fluorescence signals in real time. S4, Data Correction and Prognostic Interpretation: Correction was performed using CD63 peptide marker signals and circRPPH1 amplified fluorescence signals. The relative expression levels of circMYC and circSETD2 were calculated, and the patients were classified as standard or high-risk based on preset clinical cutoff values.

7. The detection method of a biomarker detection kit for prognostic assessment of multiple myeloma according to claim 6, characterized in that, In step S1, the volume of the serum sample to be tested is 150–250 μL, the volume of the exosome binding buffer is 80–120 μL, the volume of the CD63 peptide marker is 3–8 μL, and the volume of the thiol-modified silica magnetic beads is 5–15 μL; the incubation time at room temperature with shaking is 8–15 minutes; the magnetic separation operation involves placing the centrifuge tube on a magnetic separator rack and letting it stand before discarding the supernatant; during washing, the first washing buffer is added first, followed by the second washing buffer.

8. The detection method of the biomarker detection kit for prognostic assessment of multiple myeloma according to claim 6, characterized in that, In step S2, the volume of exosome lysis buffer is 40-60 μL, and it is allowed to stand at room temperature for 3-8 minutes; the volume of purification reagent is 40-60 μL, and it is allowed to stand for 1-5 minutes; the washing reagent is used to wash twice; the volume of RNase-free ultrapure water used for elution is 15-25 μL, and the solution is placed on ice for later use.

9. The detection method of a biomarker detection kit for prognostic assessment of multiple myeloma according to claim 6, characterized in that, In step S3, the total amount of circular RNA template used is 3-8 μL, the total volume of the isothermal amplification reaction system is 15-25 μL, the isothermal incubation temperature is 35-39℃, the time is 50-70 minutes, and the fluorescence signal acquisition frequency is once every 1 minute.

10. The detection method of a biomarker detection kit for prognostic assessment of multiple myeloma according to claim 6, characterized in that, In step S4, the specific data correction operation is as follows: the fluorescence signal of CD63 peptide marker is used as the exosome extraction efficiency correction parameter, and the amplification fluorescence signal of circRPPH1 is used as the isothermal amplification efficiency correction parameter; the relative expression levels of circMYC and circSETD2 are calculated using the ΔΔCt method; the specific method for prognostic risk stratification is as follows: when the relative expression levels of circMYC and circSETD2 are both lower than the corresponding cutoff values, the patient is judged as a standard-risk patient; otherwise, the patient is judged as a high-risk patient.

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