A method for detecting contents of miR-205 and TCF21 in exosomes based on liquid phase chip, application and kit

Abstract

The application provides a method for detecting the contents of miR-205 and TCF21 in exosomes based on a liquid phase chip, an application and a kit; the method comprises the following steps: S1: obtaining an ex vivo plasma sample and extracting exosome precipitates from the ex vivo plasma sample; S2: extracting total RNA from the exosome precipitates of step S1, using a primer group to perform multiplex PCR amplification on the total RNA, and obtaining a multiplex PCR amplification product; wherein the targets of the multiplex PCR amplification include miR-205, TCF21 and ACTB; S3: performing a hybridization reaction on the multiplex PCR amplification product of step S2 and a multiplex fluorescent coding microsphere coupled with a specific probe, and obtaining a hybridization product; S4: detecting the fluorescence intensity of the hybridization product and calculating the contents of miR-205 and TCF21 in the ex vivo plasma sample. The method can realize high-sensitivity and high-specificity detection of miR-205 and TCF21 in exosomes.

Description

Technical Field

[0001] This application belongs to the field of biomedical detection, specifically relating to a method, application, and kit for detecting the content of miR-205 and TCF21 in exosomes based on liquid phase chip. Background Technology

[0002] Ovarian cancer is one of the most common types of gynecological malignancies. Although its incidence is relatively low, due to the lack of specific symptoms in the early stages and limited screening effectiveness, approximately 60% to 70% of patients are diagnosed at an advanced stage, resulting in poor treatment outcomes. Currently, clinical diagnosis mainly relies on serological markers and imaging examinations; however, these methods have limitations such as insufficient sensitivity, poor specificity, and high invasiveness.

[0003] Exosomes are extracellular vesicles with a diameter of approximately 30-150 nm, containing various cellular components such as DNA, RNA, and proteins, and are considered an ideal source of biomarkers for liquid biopsies. In recent years, microRNAs carried by exosomes have been increasingly explored as potential biomarkers for cancer diagnosis and prognostic assessment due to their regulatory roles in key biological processes such as cell differentiation, proliferation, and apoptosis. Previous studies have also confirmed that the expression levels of miR-205 and TCF21 in plasma exosomes can serve as valuable tumor biomarkers for improving the accuracy of ovarian cancer diagnosis.

[0004] However, traditional exosome isolation techniques, such as ultracentrifugation and polymer precipitation, generally suffer from problems such as cumbersome operation, long processing time, and low purity. Existing detection methods, such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), are complex in procedure and have limited detection efficiency. Multiplex polymerase chain reaction (MLCR) technology faces challenges in result interpretation, such as difficulty in distinguishing electrophoretic bands and inconsistent amplification efficiencies of different targets. Although quantitative real-time polymerase chain reaction (qPCR) has advantages such as speed, closed-tube operation, and low risk of contamination, it is difficult to achieve simultaneous detection of multiple targets due to limitations in the number of fluorescence channels and instrument performance.

[0005] Therefore, there is an urgent need to establish a simple, rapid and accurate analytical method to achieve quantitative detection of miR-205 and TCF21 in exosomes, thereby providing new ideas for the early diagnosis of ovarian cancer. Summary of the Invention

[0006] In view of the shortcomings of the above-mentioned related technologies, the purpose of this application is to provide a method, application and kit for detecting the content of miR-205 and TCF21 in exosomes based on liquid phase chip. This method can achieve high sensitivity and high specificity detection of miR-205 and TCF21 in exosomes, improve the sensitivity and specificity of diagnosis, and provide a new idea for the precision treatment of ovarian cancer.

[0007] To achieve the above and other related objectives, a first aspect of this application provides a method for detecting the levels of miR-205 and TCF21 in exosomes based on a liquid phase chip, comprising the following steps: S1: Obtain an isolated plasma sample and extract precipitate exosomes from the isolated plasma sample; S2: Extract total RNA from the precipitate exosomes of step S1, and perform multiplex PCR amplification on the total RNA using a primer set to obtain multiplex PCR amplification products; wherein, the targets of the multiplex PCR amplification include miR-205, TCF21 and ACTB. S3: The multiplex PCR amplification product obtained in step S2 is hybridized with multiplex fluorescently encoded microspheres coupled with specific probes to obtain hybridization products; S4: Detect the fluorescence intensity of the hybridization product and calculate the content of miR-205 and TCF21 in the isolated plasma sample.

[0008] In some specific embodiments, the process of extracting exosomes in step S1 includes: The isolated plasma sample was placed in a centrifuge tube, an anticoagulant was added, the mixture was stirred evenly, and centrifuged to obtain the supernatant. The supernatant and ethanol were mixed and allowed to stand to obtain a mixture; the mixture was then centrifuged to obtain precipitate exosomes.

[0009] In some specific embodiments, in step S2, total RNA is extracted from the exosomes using an exosome RNA extraction kit.

[0010] In some specific embodiments, in step S2, the primer set satisfies at least one of the following conditions: (1) The sequences of the primer set are shown in SEQ ID NO:1 to SEQ ID NO:6; The primer set includes an upstream primer for miR-205, a downstream primer for miR-205, an upstream primer for TCF21, a downstream primer for TCF21, an upstream primer for ACTB, and a downstream primer for ACTB. The upstream primer sequence of miR-205 is shown in SEQ ID NO:1, and the downstream primer sequence of miR-205 is shown in SEQ ID NO:2; The upstream primer sequence of TCF21 is shown in SEQ ID NO:3, and the downstream primer sequence of TCF21 is shown in SEQ ID NO:4; The upstream primer sequence for ACTB is shown in SEQ ID NO:5, and the downstream primer sequence for ACTB is shown in SEQ ID NO:6; (2) When performing the multiplex PCR amplification, the concentrations of the miR-205 upstream primer, the TCF21 upstream primer, and the ACTB upstream primer are all 0.15 µM, and the concentrations of the miR-205 downstream primer, the TCF21 downstream primer, and the ACTB downstream primer are all 0.15 µM, and the 5' end of the upstream primer is labeled with the cy5 fluorescent group. (3) The design of the primer set must meet at least one of the following conditions: i) Design specific primers based on the target sequence. Primer length should be 18-25 bp. ii) The difference between the upstream and downstream primers should not exceed 5 bp; iii) The G+C content of primers should be controlled between 40% and 60%; iv) Select an amplified fragment size of 200-300bp.

[0011] In some specific embodiments, the conditions for the multiplex PCR amplification in step S2 are as follows: Pre-denaturation at 90-96℃ for 5-8 min, followed by denaturation at 90-96℃ for 20-40 s, annealing at 60-65℃ for 20-40 s, extension at 70-75℃ for 20-40 s, for a total of 30 cycles, and finally extension at 70-75℃ for 5-8 min.

[0012] For example, in step S2, the sequences of the primer set are shown in SEQ ID NO:1 to SEQ ID NO:6; The primer set includes miR-205 upstream primer, miR-205 downstream primer, TCF21 upstream primer, TCF21 downstream primer, ACTB upstream primer, and ACTB downstream primer; The upstream primer sequence of miR-205 is shown in SEQ ID NO:1, and the downstream primer sequence of miR-205 is shown in SEQ ID NO:2; The upstream primer sequence of TCF21 is shown in SEQ ID NO:3, and the downstream primer sequence of TCF21 is shown in SEQ ID NO:4; The upstream primer sequence for ACTB is shown in SEQ ID NO:5, and the downstream primer sequence for ACTB is shown in SEQ ID NO:6.

[0013] For example, in step S2, when performing the multiplex PCR amplification, the concentrations of the miR-205 upstream primer, the TCF21 upstream primer, and the ACTB upstream primer are all 0.15 µM, and the concentrations of the miR-205 downstream primer, the TCF21 downstream primer, and the ACTB downstream primer are all 0.15 µM, and the 5' end of the upstream primer is labeled with the cy5 fluorescent group. For example, in step S2, the primer set design must meet at least one of the following conditions: i) Design specific primers based on the target sequence. Primer length should be 18-25 bp. ii) The difference between the upstream and downstream primers should not exceed 5 bp; iii) The G+C content of primers should be controlled between 40% and 60%; iv) Select an amplified fragment size of 200-300bp.

[0014] In some specific embodiments, the conditions for the multiplex PCR amplification in step S2 are as follows: Pre-denaturation at 90-96℃ for 5-8 min, followed by denaturation at 90-96℃ for 20-40 s, annealing at 60-65℃ for 20-40 s, extension at 70-75℃ for 20-40 s, for a total of 30 cycles, and finally extension at 70-75℃ for 5-8 min.

[0015] In some specific implementations, step S3 satisfies at least one of the following conditions: (1) The sequences of the specific probes are shown in SEQ ID NO:7 to SEQ ID NO:9; The miR-205 probe sequence is shown in SEQ ID NO:7; The TCF21 probe sequence is shown in SEQ ID NO:8; The ACTB probe sequence is shown in SEQ ID NO:9; (2) The conditions for the hybridization reaction are: reacting at 45℃-55℃ for 30-60 min; Preferably, the hybridization reaction is performed as follows: 5 µL of multiplex PCR amplification product, 5 µL of multiplex fluorescently encoded microspheres coupled with specific probes are mixed with 90 µL of hybridization solution and placed in a hybridization incubator at 45℃-55℃ for 30-60 min at 4500 rpm.

[0016] For example, in step S3, the sequence of the specific probe is shown in SEQ ID NO:7 to SEQ ID NO:9; The miR-205 probe sequence is shown in SEQ ID NO:7; The TCF21 probe sequence is shown in SEQ ID NO:8; The ACTB probe sequence is shown in SEQ ID NO:9.

[0017] For example, in step S3, the hybridization reaction conditions are: reacting at 45℃-55℃ for 30-60 minutes.

[0018] For example, in step S3, the hybridization reaction satisfies the following: take 5µL of multiplex PCR amplification product, 5µL of multiplex fluorescently encoded microspheres coupled with specific probes, and mix with 90µL of hybridization solution, place in a hybridization chamber at 45℃-55℃, and react at 4500rpm for 30-60min.

[0019] In some specific implementations, step S4 satisfies at least one of the following conditions: (1) The fluorescence intensity of the hybridization product was detected in a flow cytometry array analyzer; (2) Calculate the content of miR-205 and TCF21 in the isolated plasma sample according to the standard curve.

[0020] For example, in step S4, the fluorescence intensity of the hybridization product is detected in a flow cytometry array analyzer.

[0021] For example, in step S4, the levels of miR-205 and TCF21 in the isolated plasma sample are calculated based on the standard curve.

[0022] In some specific embodiments, the limit of detection of the method is 1000 copies / mL.

[0023] A second aspect of this application provides the use of the method described in the first aspect in the preparation of a kit for the diagnosis of early ovarian cancer.

[0024] A third aspect of this application provides a kit for detecting the levels of miR-205 and TCF21 in isolated plasma exosomes, comprising: Primer set for multiplex PCR amplification, the primer set containing primer pairs against miR-205, TCF21 and ACTB; Multiple fluorescently encoded microspheres coupled with specific probes, which can hybridize with the amplification products of miR-205, TCF21 and ACTB, respectively; Reagents used to detect the fluorescence intensity of hybridization products; Using the kit described above, the levels of miR-205 and TCF21 in exoplasma from isolated plasma were detected according to the method described in the first aspect.

[0025] The beneficial effects of this application are: 1. This application uses multiplex fluorescently encoded microspheres as detection carriers. Through the coupling of specific probes with target gene sequences, it achieves high sensitivity and high specificity detection of miR-205 and TCF21 in exosomes, thus overcoming the limitations of traditional methods (such as ultracentrifugation and polymer precipitation) such as cumbersome operation, long time consumption, and insufficient purity. Moreover, the multiplex fluorescently encoded microspheres of this application have the ability to detect multiple target substances simultaneously. This method can realize the simultaneous detection of multiple targets of miR-205 and TCF21, thus overcoming the shortcomings of existing quantitative real-time PCR technology, which is difficult to achieve simultaneous detection of multiple targets due to the limitations of fluorescence types and instruments.

[0026] 2. This application utilizes multiplex PCR amplification technology for gene amplification, combines it with a flow cytometry fluorescence array analyzer to detect fluorescence intensity, and calculates product concentration by constructing a standard curve, thereby realizing the quantification of miR-205 and TCF21 in a single tube sample, effectively avoiding the problems of complicated steps and low detection efficiency of methods such as qRT-PCR and ELISA.

[0027] 3. This application significantly improves the accuracy and reliability of the detection results by constructing a standard curve and directly measuring the fluorescence intensity, thereby solving the technical problems of difficulty in electrophoretic differentiation and uneven PCR efficiency in multiplex PCR technology.

[0028] 4. This application uses exosomes as biomarkers, combined with liquid biopsy technology, to achieve early diagnosis of ovarian cancer, significantly improving the sensitivity and specificity of diagnosis, thus providing a new strategy for the precision treatment of ovarian cancer.

[0029] Other features and advantages of this application will be described in detail in the following detailed description section. Attached Figure Description

[0030] Figure 1 This is a graph showing the test results of the sensitivity of the method described in this application; Figures 2a to 2b The miR-205 and TCF21 detection standard curves established for the methods described in this application; Figure 3 This is a graph showing the test results for the precision of the method described in this application. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0032] Terminology Explanation: Precipitated exosomes refer to exosomes obtained from isolated plasma samples in the form of precipitates through methods such as centrifugation. In this application, precipitated exosomes are used as the detection target. Enriching exosomes reduces interference from other impurities in the plasma, providing a pure sample for subsequent extraction of total RNA and detection of miR-205 and TCF21.

[0033] Multiplex fluorescently encoded microspheres coupled with specific probes are microsphere carriers whose surfaces are coupled with specific probes and which distinguish different targets through different fluorescent codes (such as differences in fluorescence intensity or wavelength). In this application, multiplex fluorescently encoded microspheres coupled with specific probes hybridize with multiplex PCR amplification products in a liquid environment, and recognize different targets (miR-205, TCF21, ACTB) through fluorescent coding, thereby achieving simultaneous detection of multiple targets.

[0034] Cy5 fluorescent group, a common fluorescent dye group, has high fluorescence intensity and stability. In this application, the Cy5 fluorescent group is used to label the 5' end of the upstream primer of the primer set, so that the multiplex PCR amplification product carries a fluorescent signal, which facilitates the detection of fluorescence intensity by flow cytometry after the hybridization reaction, and realizes quantitative analysis of the target.

[0035] ACTB, or β-actin gene, is a housekeeping gene widely expressed in eukaryotes, and its expression level is relatively stable. In this application, ACTB is used as an internal reference gene, co-amplified with miR-205 and TCF21 in multiplex PCR amplification to correct for systematic errors such as differences in sample RNA extraction and fluctuations in PCR reaction efficiency, ensuring the accuracy of miR-205 and TCF21 content detection results.

[0036] The first aspect of this application provides a method for detecting the levels of miR-205 and TCF21 in exosomes based on a liquid phase chip, comprising the following steps: S1: Obtain an isolated plasma sample and extract precipitate exosomes from the isolated plasma sample; S2: Extract total RNA from the precipitate exosomes of step S1, and perform multiplex PCR amplification on the total RNA using a primer set to obtain multiplex PCR amplification products; wherein, the targets of the multiplex PCR amplification include miR-205, TCF21 and ACTB. S3: The multiplex PCR amplification product obtained in step S2 is hybridized with multiplex fluorescently encoded microspheres coupled with specific probes to obtain hybridization products; S4: Detect the fluorescence intensity of the hybridization product and calculate the content of miR-205 and TCF21 in the isolated plasma sample.

[0037] The above-described technical solution extracts exosomes from plasma precipitates, enriching target molecules and reducing interference from plasma impurities, providing a pure sample basis for subsequent detection. Next, total RNA is extracted and multiplex PCR is used to amplify miR-205, TCF21, and the internal control ACTB. This achieves simultaneous amplification of multiple targets, improving efficiency, while the internal control corrects for experimental errors, ensuring quantitative accuracy. The amplified products are then hybridized with fluorescently encoded microspheres containing specific probes. Leveraging the high specificity and multi-channel detection advantages of liquid chromatography chips, target sequences are easily identified. Finally, fluorescence intensity is measured to calculate the content, achieving quantitative analysis of the two targets. Therefore, the method in this application balances sample purification, simultaneous multi-target detection, and result accuracy, enabling efficient and accurate detection of miR-205 and TCF21 content in exosomes, providing reliable technical support for the diagnosis of related diseases or molecular marker research.

[0038] In some embodiments, the process of extracting exosomes in step S1 includes: The isolated plasma sample was placed in a centrifuge tube, an anticoagulant was added, the mixture was stirred evenly, and centrifuged to obtain the supernatant. The supernatant and ethanol were mixed and allowed to stand to obtain a mixture; the mixture was then centrifuged to obtain precipitate exosomes.

[0039] Through the above technical solution, in step S1, adding an anticoagulant prevents plasma coagulation, ensuring the smooth progress of subsequent centrifugation. Centrifugation removes large particulate impurities such as cell debris, and RNA is extracted into the supernatant, thus initially purifying the sample. Next, the supernatant is mixed with ethanol and allowed to stand. The property of ethanol to change the polarity of the solution promotes the aggregation and precipitation of exosomes. Further centrifugation further enriches the exosomes in the precipitate. This exosome extraction process ensures both the recovery rate of exosomes and effectively reduces interference from impurities, providing a pure and high-quality sample basis for subsequent RNA extraction and the detection of miR-205 and TCF21 levels.

[0040] In some embodiments, the anticoagulant is EDTA-2Na. EDTA-2Na is chosen as the anticoagulant because it can block the coagulation reaction by chelating calcium ions in the blood, preventing plasma sample coagulation, facilitating subsequent centrifugation to separate the supernatant, avoiding mixing of exosomes with coagulation components, improving the purity and recovery rate of exosome extraction, and providing a stable and reliable sample basis for subsequent total RNA extraction and the detection of miR-205 and TCF21.

[0041] In some embodiments, in step S2, total RNA is extracted from the exosomes using an exosomal RNA extraction kit. By using the exosomal RNA extraction kit, the exosomal membrane structure can be specifically lysed, efficiently enriching trace amounts of total RNA, while simultaneously protecting RNA integrity and reducing degradation through system optimization.

[0042] In some embodiments, in step S2, the primer set satisfies at least one of the following conditions: (1) The sequences of the primer set are shown in SEQ ID NO:1 to SEQ ID NO:6; The primer set includes miR-205 upstream primer, miR-205 downstream primer, TCF21 upstream primer, TCF21 downstream primer, ACTB upstream primer, and ACTB downstream primer; The upstream primer sequence of miR-205 is shown in SEQ ID NO:1, and the downstream primer sequence of miR-205 is shown in SEQ ID NO:2; The upstream primer sequence of TCF21 is shown in SEQ ID NO:3, and the downstream primer sequence of TCF21 is shown in SEQ ID NO:4; The upstream primer sequence for ACTB is shown in SEQ ID NO:5, and the downstream primer sequence for ACTB is shown in SEQ ID NO:6; (2) When performing the multiplex PCR amplification, the concentration of the primer set is: (3) The design of the primer set must meet at least one of the following conditions: i) Design specific primers based on the target sequence. Primer length should be 18-25 bp. ii) The difference between the upstream and downstream primers should not exceed 5 bp; iii) The G+C content of primers should be controlled between 40% and 60%; iv) Select an amplified fragment size of 200-300bp.

[0043] Through the above embodiments, targeted primer sequences (SEQ ID NO: 1-6) are selected to target miR-205, TCF21, and the internal control ACTB to ensure amplification specificity and avoid interference from non-specific products. The primer set design requirements (length 18-25bp, upstream and downstream length difference ≤5bp, GC content 40%-60%, amplified fragment 200-300bp) enable each primer to amplify synergistically under the same reaction conditions, balancing efficiency and specificity, ensuring stable product quality, providing a high-quality template for subsequent hybridization detection, and supporting the accuracy of early ovarian cancer diagnosis.

[0044] In some embodiments, in step S2, the sequence of the primer set is as shown in SEQ ID NO:1 to SEQ ID NO:6; The primer set includes miR-205 upstream primer, miR-205 downstream primer, TCF21 upstream primer, TCF21 downstream primer, ACTB upstream primer, and ACTB downstream primer; The upstream primer sequence of miR-205 is shown in SEQ ID NO:1, and the downstream primer sequence of miR-205 is shown in SEQ ID NO:2; The upstream primer sequence of TCF21 is shown in SEQ ID NO:3, and the downstream primer sequence of TCF21 is shown in SEQ ID NO:4; The upstream primer sequence for ACTB is shown in SEQ ID NO:5, and the downstream primer sequence for ACTB is shown in SEQ ID NO:6; Specifically, SEQ ID NO:1 is 5'-GATCCTCAGACAATCCATGTGCT-3'; SEQ ID NO:2 is 5'-TGTCAGCTCCATGCCTCCTGAA-3'; SEQ ID NO:3 is: 5'-GCAGATCCTGGCTAACGACAAAT-3'; SEQ ID NO:4 is: 5'-CCACTTCTTTCAGGTCACTCTCGG-3'; SEQ ID NO:5 is: 5'-CCCTGGAGAAGAGCTACGAG-3'; SEQ ID NO:6 is: 5'-GATGCCACAGGACTCCATGC-3'.

[0045] Using the above technical solution, the primer set sequence is designed to target the specific regions of miR-205, TCF21, and the internal control ACTB, ensuring that each primer can bind to the target template and avoiding non-specific amplification. Specifically, the primer sequences for miR-205 and TCF21 match their conserved fragments, guaranteeing amplification efficiency; the primer for the internal control ACTB is used to correct for systematic errors such as differences in RNA extraction volume and fluctuations in PCR reaction efficiency during the experiment. This design achieves specific detection of the target gene and improves the reliability of the results through the introduction of the internal control, providing accurate and stable amplification products for subsequent hybridization and quantitative analysis, ensuring the reproducibility and accuracy of experimental data.

[0046] In some embodiments, during step S2, when performing the multiplex PCR amplification, the concentrations of the miR-205 upstream primer, the TCF21 upstream primer, and the ACTB upstream primer are all 0.15 µM, the concentrations of the miR-205 downstream primer, the TCF21 downstream primer, and the ACTB downstream primer are all 0.15 µM, and the 5' end of the upstream primer is labeled with the cy5 fluorescent group. In some embodiments, in step S2, the primer set is required to meet at least one of the following conditions: i) Design specific primers based on the target sequence. Primer length should be 18-25 bp. ii) The difference between the upstream and downstream primers should not exceed 5 bp; iii) The G+C content of primers should be controlled between 40% and 60%; iv) Select an amplified fragment size of 200-300bp.

[0047] Through the above technical solutions, the primer set design achieves a balance between specificity and binding efficiency at a length of 18-25 bp, avoiding non-specific binding due to excessively short primers or reduced amplification speed due to excessively long primers; a length difference of ≤5 bp between upstream and downstream primers ensures similar annealing temperatures, suitable for multiplex PCR to simultaneously amplify multiple targets (such as miR-205, TCF21, and the internal control ACTB); a G+C content of 40%-60% maintains primer structural stability and ensures efficient annealing; and an amplification fragment of 200-300 bp balances amplification efficiency with the convenience of subsequent detection. These conditions work together to optimize PCR amplification, improve detection accuracy and stability, reduce non-specific amplification, ensure target gene amplification, and provide a reliable template for subsequent hybridization and quantitative analysis.

[0048] In some embodiments, the conditions for the multiplex PCR amplification in step S2 are as follows: Pre-denaturation at 90-96℃ for 5-8 min, followed by denaturation at 90-96℃ for 20-40 s, annealing at 60-65℃ for 20-40 s, extension at 70-75℃ for 20-40 s, for a total of 30 cycles, and finally extension at 70-75℃ for 5-8 min.

[0049] The above technical solution involves pre-denaturation at 90-96℃ for 5-8 minutes to completely denature and unwind the template DNA while fully activating the thermostable DNA polymerase. In the subsequent denaturation-annealing-extension cycle, high-temperature denaturation ensures complete template unwinding, annealing at 60-65℃ optimizes primer binding and avoids non-specific amplification, and extension at 70-75℃ utilizes the enzyme's optimal temperature for efficient DNA synthesis. Thirty cycles control the product yield to meet detection requirements without generating excessive byproducts. The final extension ensures the complete synthesis of all fragments. This multiplex PCR amplification balances specificity and efficiency, providing a reliable amplification template for subsequent experiments, balancing amplification efficiency and specificity, and ensuring high-quality generation of the target product.

[0050] In some embodiments, step S3 satisfies at least one of the following conditions: (1) The sequences of the specific probes are shown in SEQ ID NO:7 to SEQ ID NO:9; The miR-205 probe sequence is shown in SEQ ID NO:7; The TCF21 probe sequence is shown in SEQ ID NO:8; The ACTB probe sequence is shown in SEQ ID NO:9; Specifically, SEQ ID NO:7 is 5'-CGGTGGAATGAAGGACAAGAGA-3'; SEQ ID NO:8 is 5'-CATAAAGGGCCACGTCAGGTTG-3'; SEQ ID NO:9 is 5'-CAGGACTCCATGCCCAGGAAGGA-3'.

[0051] (2) The conditions for the hybridization reaction are: react at 45℃~55℃ for 30-60 min.

[0052] Through the above embodiments, multiplex fluorescently encoded microspheres can simultaneously capture multiple target molecules, improving detection throughput. The specific probe sequences (SEQ ID NO: 7-9) are designed for miR-205, TCF21, and the internal control ACTB, ensuring specific binding to the amplification products and avoiding cross-reactions. The hybridization temperature of 45-55℃ and the reaction time of 30-60 min balance hybridization efficiency and specificity, allowing the probe to bind stably to the target sequences. These three elements work synergistically to achieve simultaneous detection of multiple indicators while ensuring accurate and reliable results, providing a solid foundation for subsequent quantitative analysis.

[0053] In some embodiments, in step S3, the sequence of the specific probe is shown as SEQ ID NO:7 to SEQ ID NO:9; The miR-205 probe sequence is shown in SEQ ID NO:7; The TCF21 probe sequence is shown in SEQ ID NO:8; The ACTB probe sequence is shown in SEQ ID NO:9; Specifically, SEQ ID NO:7 is 5'-CGGTGGAATGAAGGACAAGAGA-3'; SEQ ID NO:8 is 5'-CATAAAGGGCCACGTCAGGTTG-3'; SEQ ID NO:9 is 5'-CAGGACTCCATGCCCAGGAAGGA-3'.

[0054] In the above embodiments, by designing miR-205, TCF21, and ACTB probes, they can specifically bind to the target nucleic acid sequences, ensuring high detection specificity and effectively avoiding false positive results caused by non-specific hybridization. Secondly, different probes target different molecules, enabling simultaneous detection of multiple targets. The detection of miR-205, TCF21, and the internal reference gene ACTB can be completed simultaneously in a single tube reaction, significantly improving detection efficiency and reducing sample volume and detection time. Furthermore, the introduction of the ACTB internal reference probe allows for quality control of sample processing and detection procedures, correcting operational differences between different samples, ensuring the accuracy and reliability of detection results, and providing a solid data foundation for subsequent disease diagnosis and analysis based on the detection results.

[0055] In some embodiments, in step S3, the hybridization reaction is performed at 45°C to 55°C for 30-60 minutes. Performing the hybridization reaction under these conditions ensures specificity while improving detection efficiency.

[0056] Specifically, the hybridization reaction is performed as follows: 5 µL of multiplex PCR amplification product, 5 µL of multiplex fluorescently encoded microspheres coupled with specific probes, and 90 µL of hybridization solution are mixed and placed in a hybridization incubator at 45℃-55℃ for 30-60 min at 4500 rpm. This facilitates specific hybridization, and by combining multiplex fluorescently encoded microspheres, multiple targets can be detected simultaneously, enabling accurate and rapid multiplex analysis.

[0057] This application does not impose any particular restrictions on the preparation process of multiple fluorescently encoded microspheres coupled with specific probes, as long as the purpose of this application can be achieved. For example, the preparation process of multiple fluorescently encoded microspheres coupled with specific probes includes: first preparing host spheres and guest spheres, combining the host and guest spheres and linking them together with chemical bonds to obtain multiple fluorescently encoded microspheres; then activating the carboxyl groups on the surface of the multiple fluorescently encoded microspheres with EDC and NHS, coupling streptavidin, adding biotin-modified specific probes, reacting, and washing to remove free probes to obtain the microspheres. The specific probe sequences are shown in SEQ ID NO:7 to SEQ ID NO:9.

[0058] Specifically, the preparation process of multiplex fluorescently encoded microspheres coupled with specific probes includes the following steps: (1) Preparation of multiple fluorescently encoded microspheres This process involves separately encoding micron-sized host spheres and nano-sized guest spheres before assembling them. Specifically, the host spheres are prepared by using polystyrene microspheres as templates, sequentially loading magnetic particles and different predetermined amounts of red fluorescent quantum dots (605 nm) onto their surfaces, followed by polymer surface coating and modification to achieve host spheres with varying fluorescence intensities. The guest spheres are prepared by adding different predetermined amounts of green fluorescent dye to the Stöber reaction, followed by polymer coating and modification to achieve guest spheres with varying fluorescence intensities. Finally, the aforementioned host spheres and guest spheres with different fluorescence intensities are combined and linked by chemical bonds to achieve the preparation of multi-host-guest encoded microspheres.

[0059] (2) Coupling reaction The carboxyl groups on the surface of the encoded microspheres were activated using EDC and NHS. A 0.5 mg / ml streptavidin (SA) solution was added, and the mixture was rotated and mixed at 37 °C for 2 hours to obtain "SA-encoded microspheres". A specific probe modified with biotin was added to the "SA-encoded microspheres", and the mixture was reacted at room temperature for 20 minutes to bind through "SA-Biotin". Unbound free probes were washed away to obtain multiple fluorescently encoded microspheres coupled with specific probes.

[0060] In some embodiments, step S4 satisfies at least one of the following conditions: (1) The fluorescence intensity of the hybridization product was detected in a flow cytometry array analyzer; (2) Calculate the content of miR-205 and TCF21 in the isolated plasma sample according to the standard curve.

[0061] In the above embodiments, a flow cytometry array analyzer is used to detect the fluorescence intensity of the hybridization product, which can efficiently capture specific signals and ensure the sensitivity and specificity of the detection. The content is calculated using a standard curve, converting the fluorescence signal into a quantifiable molecular concentration value, ensuring the accuracy and repeatability of the results. The combination of these two methods solves the reliability problem of signal detection and achieves the transformation from qualitative to quantitative analysis, providing data support for the early diagnosis of ovarian cancer and assisting clinicians in making accurate judgments.

[0062] In some embodiments, the method has a limit of detection of 1000 copies / mL, which can capture low concentrations of miR-205 and TCF21 in plasma exosomes, meeting the need for detection of trace biomarkers in early ovarian cancer diagnosis.

[0063] A second aspect of this application provides the use of the method described in the first aspect in the preparation of a kit for the diagnosis of early ovarian cancer.

[0064] A third aspect of this application provides a kit for detecting the levels of miR-205 and TCF21 in isolated plasma exosomes, comprising: Primer set for multiplex PCR amplification, the primer set containing primer pairs against miR-205, TCF21 and ACTB; Multiple fluorescently encoded microspheres coupled with specific probes, which can hybridize with the amplification products of miR-205, TCF21 and ACTB, respectively; Reagents used to detect the fluorescence intensity of hybridization products; Using the kit described above, the levels of miR-205 and TCF21 in exoplasma from isolated plasma were detected according to the method described in the first aspect.

[0065] The following examples and comparative examples illustrate the implementation of this application in more detail. Those skilled in the art will understand that the preparation processes described in this application are merely examples, and any other suitable preparation processes are within the scope of this application. Various tests and evaluations were performed according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" are quality bases.

[0066] Example 1 The preparation process of multiplex fluorescently encoded microspheres coupled with specific probes includes the following steps: <Preparation of Multiple Fluorescent Encoded Microspheres> Preparation of the main sphere 1) Template ball selection: Polystyrene microspheres with a diameter of 5μm (Sigma-Aldrich, catalog number PS0500) were used as templates. Before use, they were ultrasonically cleaned with ethanol 3 times (power 300W, 5 minutes each time) and vacuum dried (60℃, 2 hours). 2) Magnetic particle loading: 10 mg template spheres were dispersed in 10 mL of deionized water, and 2 mL of Fe3O4 nanoparticle dispersion (concentration 5 mg / mL, particle size 10 nm, Aladdin reagent) was added. The mixture was reacted in a constant temperature shaker (THZ-300, speed 200 rpm) at 37 °C for 12 hours. After magnetic separation (magnetic field strength 0.5 T), the mixture was washed 3 times with PBS buffer (pH 7.4). 3) Red fluorescent quantum dot modification: The magnetically modified microspheres were resuspended in 5 mL of MES buffer (0.1 M, pH 6.0), and red fluorescent quantum dots (605 nm emission wavelength, Invitrogen, catalog number Q10121MP) at gradient concentrations of 0.1 μM, 0.3 μM, and 0.5 μM were added respectively. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added to a final concentration of 50 mM. The reaction was carried out at room temperature for 2 hours, and the mixture was collected by centrifugation (4000 × g, 10 min). 4) Polymer coating: The above microspheres were dispersed in 10 mL of ethanol / water mixture (volume ratio 3:1), 0.5 mL of 3-aminopropyltriethoxysilane (APTES) and 1 mL of 2% polyethylene glycol (PEG, molecular weight 5000) solution were added, and the mixture was refluxed at 50 °C for 4 hours. After washing, the surface-aminated host spheres were obtained (fluorescence intensity gradient was set to 3 levels).

[0067] Preparation of the object sphere: 1) Construction of the Stöber reaction system: 20 mL of anhydrous ethanol, 5 mL of deionized water and 0.5 mL of ammonia (28%, Sinopharm Group) were added to a 50 mL three-necked flask. Green fluorescent dye (FITC, Sigma-Aldrich) with gradient concentrations of 0.2 μM, 0.4 μM and 0.6 μM was added under magnetic stirring (500 rpm). 2) Synthesis of silica spheres: 2 mL of tetraethyl orthosilicate (TEOS) was added dropwise and reacted at 30 °C for 6 hours to generate green fluorescent silica spheres with a diameter of 500 nm. The spheres were collected by centrifugation (8000 × g, 15 min). 3) Surface carboxylation modification: Disperse the silica spheres in 10 mL of DMF solution, add 1 mL of methacrylic anhydride, react at 60 °C for 8 hours, dialyze (molecular weight cutoff 10 kDa) for 48 hours, and freeze dry to obtain surface carboxylated guest spheres (fluorescence intensity gradient set at 3 levels). Host and guest ball assembly 1 mg of host sphere and 1 mg of guest sphere were mixed in 5 mL of MES buffer (0.1 M, pH 6.0), and EDC (50 mM) and N-hydroxysuccinimide (NHS, 25 mM) were added. The mixture was reacted at room temperature for 2 hours to achieve covalent linkage between host and guest spheres via amide bonds, thus constructing a microsphere library with 3×3=9 re-encoded combinations. Fluorescent encoding was verified using flow cytometry (BD FACSCalibur).

[0068] <Coupling Reaction> The carboxyl groups on the surface of the microspheres are activated using EDC and NHS. The specific steps are as follows: 1) Activation reaction: Take 100 μL of coded microsphere suspension (concentration 10 mg / mL), wash twice with MES buffer (0.1 M, pH 6.0), resuspend in 500 μL MES buffer, add EDC (final concentration 50 mM) and NHS (final concentration 25 mM), and react at 37℃ in a shaker (speed 200 rpm) for 30 minutes; 2) SA Coupling: Add 100 μL of streptavidin (SA) solution (0.5 mg / mL, dissolved in PBS buffer pH 7.4, Sigma-Aldrich product number S4762), mix by rotating at 37°C (200 rpm) for 2 hours, separate magnetically (0.5T magnetic field), wash 3 times with PBST buffer (containing 0.05% Tween-20), and resuspend in 200 μL of PBS buffer to obtain "SA-encoded microspheres"; 3) Probe coupling: Add 50 μL of biotin-modified specific probe (concentration 10 μM, specific probe sequence as shown in SEQ ID NO:7 to SEQ ID NO:9) to “SA-encoded microspheres”, react at room temperature (25℃) in the dark for 20 minutes, and gently mix once every 5 minutes during the reaction. 4) Purification: Wash with PBST buffer (3 times) and PBS buffer (2 times) in sequence, centrifuge at 4000×g for 5 minutes each time, and finally resuspend in 100μL of PBS buffer containing 0.1% BSA. Store at 4℃ in the dark. The probe coupling efficiency (>90%) was detected by fluorescence microscopy (Olympus BX53) and flow cytometry.

[0069] Example 2 A method for detecting miR-205 and TCF21 levels in exosomes based on a liquid phase chip includes the following steps: S1. Extracting exosomes from plasma samples: S101. Place 5 mL of plasma sample into a 15 mL centrifuge tube, add 100 μL of 0.1 M EDTA-2 Na anticoagulant, and mix gently. S102. Perform a preliminary centrifugation for 15 minutes at room temperature (25℃) to obtain the supernatant; S103. Transfer the supernatant from step S102 to a new 15mL centrifuge tube, add 0.5mL of ethanol, mix gently, and let stand for 30 minutes to obtain the mixture. S104. Centrifuge the mixture from step S103 at 4°C for 15 minutes at high speed; discard the supernatant and retain a precipitate of about 100 μL to obtain the precipitate exosomes.

[0070] S2. Extract total RNA from exosomes and perform multiplex PCR amplification: S201. Total RNA was extracted from the precipitate exosomes in step S104 using the QIAGEN Exosome RNA Extraction Kit “Total Exosome RNA and Protein Isolation Kit”. The procedure was performed according to the kit instructions. S205, perform multiplex PCR amplification, the primer set includes specific primer pairs for miR-205, TCF21 and ACTB genes.

[0071] The specific primer sequences are as follows: miR-205 primers: 5'-GATCCTCAGACAATCCATGTGCT-3' (upstream primer) and 5'-TGTCAGCTCCATGCCTCCTGAA-3' (downstream primer); TCF21 primers: 5'-GCAGATCCTGGCTAACGACAAAT-3' (upstream primer) and 5'-CCACTTCTTTCAGGTCACTCTCGG-3' (downstream primer); ACTB primers: 5'-CCCTGGAGAAGAGCTACGAG-3' (upstream primer) and 5'-GATGCCACAGGACTCCATGC-3' (downstream primer).

[0072] In S205, the PCR reaction conditions were: 95℃ pre-denaturation for 5 minutes, followed by 95℃ denaturation for 30 seconds, 62℃ annealing for 30 seconds, 72℃ extension for 30 seconds, for a total of 30 cycles, and finally 72℃ extension for 5 minutes.

[0073] S3. Hybridize the amplification products with multiplex fluorescently encoded microspheres: S301. Mix the PCR amplification product with the multiplex fluorescently encoded microspheres with the specific probe conjugated in Example 1 in hybridization buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 0.1% SDS) and react at 50°C for 30 minutes to obtain the hybridization product.

[0074] The specific probe sequence is as follows: miR-205 probe: 5'-CGGTGGAATGAAGGACAAGAGA-3'; TCF21 probe: 5'-CATAAAGGGCCACGTCAGGTTG-3'; ACTB probe: 5'-CAGGACTCCATGCCCAGGAAGGA-3'.

[0075] S302. Wash the hybridization product twice with PBST buffer on a magnetic plate to obtain the hybridization product to be tested. S4. Detect the fluorescence intensity of the hybridization product to be tested using a flow cytometry array analyzer and calculate the product concentration: S401. Prepare standard curves based on miR-205 and TCF21 standard substances; S402. The fluorescence intensity of the hybridization product to be tested is detected by a flow cytometry array analyzer. The fluorescence intensity of the test samples and standard substances was measured using a flow cytometry fluorescence array analyzer. S403. Calculate the concentrations of miR-205 and TCF21 in the sample based on the standard curve and fluorescence intensity. Furthermore, the established method for detecting miR-205 and TCF21 content in exosomes based on liquid phase chip was evaluated methodologically.

[0076] 1. Sensitivity: miR-205 and TCF21 plasmids were prepared as detection samples to evaluate the limit of detection of the above method. The results are as follows: Figure 1 As shown, both miR-205 and TCF21 plasmids can be detected at concentrations as low as 1000 copies / mL, indicating that the detection limit for miR-205 and TCF21 reaches 1000 copies / mL.

[0077]

[0078] Figure 1 2. Linear: The linearity of the established detection method was evaluated using serially diluted miR-205 and TCF21 plasmids, and the results are as follows: Figure 2a , Figure 2b As shown, the linear regression equation for miR-205 is obtained as y = 120700In(x) - 873389, with a linear range of 10. 3 ~10 7 The correlation coefficient R = 0.98; the linear regression equation of TCF21 is y = 94271In(x) - 703372, with a linear range of 10. 3 ~10 8 The correlation coefficient R = 0.98.

[0079]

[0080]

[0081] Figure 2 3. Precision: The precision of the established methodology was assessed using miR-205 and TCF21 plasmids. Each sample was tested in 10 replicates, and the mean, standard deviation, and coefficient of variation (CV%) were calculated based on fluorescence intensity. Results are as follows: Figure 3 As shown, the CVs of miR-205 and TCF21 were 4.36% and 6.78%, respectively, indicating good reproducibility.

[0082] The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the principles of this application should be included within the protection scope of this application.

Claims

1. A method for detecting the content of miR-205 and TCF21 in exosomes based on a liquid phase chip, characterized in that, Includes the following steps: S1: Obtain an isolated plasma sample and extract precipitate exosomes from the isolated plasma sample; S2: Extract total RNA from the precipitate exosomes of step S1, and perform multiplex PCR amplification on the total RNA using a primer set to obtain multiplex PCR amplification products; wherein, the targets of the multiplex PCR amplification include miR-205, TCF21 and ACTB. S3: The multiplex PCR amplification product obtained in step S2 is hybridized with multiplex fluorescently encoded microspheres coupled with specific probes to obtain hybridization products; S4: Detect the fluorescence intensity of the hybridization product and calculate the content of miR-205 and TCF21 in the isolated plasma sample.

2. The method according to claim 1, characterized in that, The process of extracting exosomes in step S1 includes: The isolated plasma sample was placed in a centrifuge tube, an anticoagulant was added, the mixture was stirred evenly, and centrifuged to obtain the supernatant. The supernatant and ethanol were mixed and allowed to stand to obtain a mixture; the mixture was then centrifuged to obtain precipitate exosomes.

3. The method according to claim 1, characterized in that, In step S2, total RNA is extracted from the exosomes using an exosome RNA extraction kit.

4. The method according to claim 1, characterized in that, In step S2, the primer set satisfies at least one of the following conditions: (1) The sequences of the primer set are shown in SEQ ID NO:1 to SEQ ID NO:6; The primer set includes miR-205 upstream primer, miR-205 downstream primer, TCF21 upstream primer, TCF21 downstream primer, ACTB upstream primer, and ACTB downstream primer; The upstream primer sequence of miR-205 is shown in SEQ ID NO:1, and the downstream primer sequence of miR-205 is shown in SEQ ID NO:2; The upstream primer sequence of TCF21 is shown in SEQ ID NO:3, and the downstream primer sequence of TCF21 is shown in SEQ ID NO:4; The upstream primer sequence for ACTB is shown in SEQ ID NO:5, and the downstream primer sequence for ACTB is shown in SEQ ID NO:6; (2) When performing the multiplex PCR amplification, the concentrations of the miR-205 upstream primer, the TCF21 upstream primer, and the ACTB upstream primer are all 0.15 µM, and the concentrations of the miR-205 downstream primer, the TCF21 downstream primer, and the ACTB downstream primer are all 0.15 µM, and the 5' end of the upstream primer is labeled with the cy5 fluorescent group; (3) The design of the primer set must meet at least one of the following conditions: i) Design specific primers based on the target sequence. Primer length should be 18-25 bp. ii) The difference between the upstream and downstream primers should not exceed 5 bp; iii) The G+C content of primers should be controlled between 40% and 60%; iv) Select an amplified fragment size of 200-300bp.

5. The method according to claim 1, characterized in that, In step S2, the conditions for the multiplex PCR amplification are as follows: Pre-denaturation at 90-96℃ for 5-8 min, followed by denaturation at 90-96℃ for 20-40 s, annealing at 60-65℃ for 20-40 s, extension at 70-75℃ for 20-40 s, for a total of 30 cycles, and finally extension at 70-75℃ for 5-8 min.

6. The method according to claim 1, characterized in that, Step S3 satisfies at least one of the following conditions: (1) The sequences of the specific probes are shown in SEQ ID NO:7 to SEQ ID NO:9; The miR-205 probe sequence is shown in SEQ ID NO:7; The TCF21 probe sequence is shown in SEQ ID NO:8; The ACTB probe sequence is shown in SEQ ID NO:9; (2) The conditions for the hybridization reaction are: reacting at 45℃-55℃ for 30-60 min; Preferably, the hybridization reaction is performed as follows: 5 µL of multiplex PCR amplification product, 5 µL of multiplex fluorescently encoded microspheres coupled with specific probes are mixed with 90 µL of hybridization solution and placed in a hybridization incubator at 45℃-55℃ for 30-60 min at 4500 rpm.

7. The method according to claim 1, characterized in that, Step S4 satisfies at least one of the following conditions: (1) The fluorescence intensity of the hybridization product was detected in a flow cytometry array analyzer; (2) Calculate the content of miR-205 and TCF21 in the isolated plasma sample according to the standard curve.

8. The method according to any one of claims 1 to 7, characterized in that, The detection limit of the method is 1000 copies / mL.

9. Use of the method according to any one of claims 1 to 8 in the preparation of a kit for the diagnosis of early ovarian cancer.

10. A kit for detecting the levels of miR-205 and TCF21 in isolated plasma exosomes, characterized in that, include: Primer set for multiplex PCR amplification, the primer set containing primer pairs against miR-205, TCF21 and ACTB; Multiple fluorescently encoded microspheres coupled with specific probes, which can hybridize with the amplification products of miR-205, TCF21 and ACTB, respectively; Reagents used to detect the fluorescence intensity of hybridization products; Using the kit described above, the levels of miR-205 and TCF21 in isolated plasma exosomes were detected according to the method described in any one of claims 1 to 8.

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