MiRNA biomarkers, compositions, kits, uses and methods thereof

Abstract

The invention discloses a miRNA biomarker, a composition, a kit, application and a method thereof. The invention provides a group of miRNA biomarkers capable of being used for early screening of cerebral apoplexy, and the diagnosis accuracy of early cerebral apoplexy is improved. The method comprises the following steps: firstly, screening a set of miRNA molecular markers by adopting a plasma miRNA high-throughput sequencing technology through clinical queue research; based on a miRNA molecular marker, a fluorescent quantitative PCR technology without reverse transcription is developed, and high-sensitivity detection of miRNA can be completed within 1 hour and 30 minutes. Based on fluorescent quantitative results of the four miRNA molecular markers, a model is constructed to predict the cerebral apoplexy, the AUC of the model is equal to 0.957, the diagnosis sensitivity is 86.8%, and the specificity is 98.2%.

Description

Technical Field

[0001] This article generally relates to the field of biotechnology, and in particular to a miRNA biomarker, composition, kit, its uses and methods for early screening of stroke. Background Technology

[0002] Stroke is a devastating neurological disease characterized by high mortality and disability rates. In my country, stroke is the leading cause of death among residents, bringing immense suffering and economic burden to families, and creating a significant socioeconomic burden.

[0003] Regarding current early screening technologies for stroke, firstly, diagnosis mainly relies on imaging techniques such as CT / MRI, but this has limitations such as high equipment requirements, high cost, post-onset diagnosis, and inability to dynamically monitor disease progression; secondly, a combination method using homocysteine ​​(enzymatic method) and glycated hemoglobin immunoscattering rate method is used, which, although low in cost, has a clinical diagnostic sensitivity of only 40%-60% and a specificity of only about 70%; thirdly, RT-qPCR technology is used, but the detection time is long and the cost is high. Therefore, there is an urgent need to develop humoral biomarkers with high specificity and sensitivity to solve this problem. Summary of the Invention

[0004] This application provides a miRNA biomarker for early screening of stroke, wherein the miRNA biomarker is selected from two or more of the following: hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p and hsa-miR-26a-5p.

[0005] On the other hand, this application also provides the use of the miRNA biomarkers described herein in the preparation of kits for early screening of stroke.

[0006] On the other hand, this application also provides a composition for detecting the miRNA biomarkers described herein for early stroke screening, comprising: First capture probe, second capture probe, forward primer, reverse primer and third fluorescent probe; The first capture probe is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 5-8; the second capture probe is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 9-12; the forward primer is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of SEQ ID NO: 13; the reverse primer is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of SEQ ID NO: 14; and the third fluorescent probe is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 15-18.

[0007] In another aspect, this application also provides a kit comprising the composition described herein for detecting miRNA biomarkers for early screening of stroke.

[0008] On the other hand, this application also provides the use of the composition or kit containing the miRNA biomarker described herein for detecting miRNA biomarkers for early screening of stroke in detecting miRNA biomarkers, wherein the miRNA biomarker is selected from two or more of the following: hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p and hsa-miR-26a-5p.

[0009] On the other hand, this application also provides the use of the compositions described herein for detecting miRNA biomarkers for early stroke screening or kits containing such biomarkers in early stroke screening.

[0010] On the other hand, this application also provides a method for detecting the content of miRNA biomarkers in a sample, wherein the miRNA biomarkers are selected from two or more of the following: hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p, and hsa-miR-26a-5p; the method comprises: using the composition described herein for detecting miRNA biomarkers for early screening of stroke or a kit containing the composition described herein, as well as performing the following operations: 1) Collect RNA from the sample; 2) Anneal and hybridize the RNA obtained in step 1) with the first and second capture probes, and ligate the hybridization products with DNA ligase; 3) Perform qPCR on the product from step 2) using forward primers, reverse primers, and a third fluorescent probe. Determine the content of miRNA in the sample based on the qPCR results.

[0011] On the other hand, this application also provides a composition for detecting miRNA, comprising: First capture probe, second capture probe, forward primer, reverse primer and third fluorescent probe; The first capture probe contains a first universal sequence and a sequence complementary to the latter half (5' to 3') of the miRNA, from 5' to 3' respectively. The second capture probe contains a sequence complementary to the first half (5' to 3') of the miRNA and a second universal sequence, from 5' to 3' respectively. The second capture probe 5' is phosphorylated. The forward primer is identical to a portion of the first universal sequence, and the reverse primer is inversely complementary to a portion of the second universal sequence. The third fluorescent probe is modified with fluorescent reporter groups and corresponding fluorescent quenching groups at both ends; the fluorescent reporter groups are FAM, VIC, HEX or ROX, the fluorescent quenching groups are BHQ2, BHQ1 or BHQ3, and the 3' end of the third fluorescent probe also contains MGB.

[0012] On the other hand, this application also provides a kit for detecting miRNA, wherein the kit contains the composition for detecting miRNA described herein.

[0013] On the other hand, this application also provides the use of the compositions described herein for detecting miRNAs or kits containing the same in detecting miRNAs.

[0014] On the other hand, this application also provides a method for detecting the content of miRNA in a sample, comprising: Perform the following procedures using the composition for detecting miRNAs described herein or a kit containing such composition: 1) Collect RNA from the sample; 2) Anneal and hybridize the RNA obtained in step 1) with the first and second capture probes, and ligate the hybridization products with DNA ligase; 3) Perform qPCR on the product from step 2) using forward primers, reverse primers, and a third fluorescent probe. Determine the content of miRNA in the sample based on the qPCR results.

[0015] The diagnosis of stroke mainly relies on imaging techniques such as CT / MRI, but these techniques have limitations such as high equipment requirements, high cost, and inability to make early predictions.

[0016] The purpose of this application is to provide a set of microRNA (miRNA) biomarkers for early stroke screening, thereby improving the diagnostic accuracy of early stroke. Firstly, a set of miRNA molecular markers was screened using plasma miRNA high-throughput sequencing technology through a clinical cohort study. Then, based on these miRNA molecular markers, a reverse transcription-free quantitative real-time PCR technique was developed, enabling highly sensitive miRNA detection within 1 hour and 30 minutes. Based on the quantitative real-time results of four miRNA molecular markers, a model was constructed to predict stroke. The model had an AUC of 0.957, a diagnostic sensitivity of 86.8%, and a specificity of 98.2%.

[0017] The beneficial technical effects of this application include: 1. miRNA molecular markers are derived from plasma, making sampling convenient and suitable for large-scale screening. They can also be extended to scenarios such as postoperative recurrence monitoring and efficacy evaluation. 2. NRT-qPCR technology has a miRNA detection sensitivity of up to 60 copies per microliter and can effectively identify single bases with high specificity; 3. By combining NRT-qPCR with machine learning, a diagnostic model for stroke was constructed, achieving a sensitivity of 86.8% and a specificity of 98.2%. 4. The diagnosis time for stroke can be shortened to 1.5 hours.

[0018] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and the accompanying drawings. Overview of the attached figures The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0019] Figure 1 This is a schematic flowchart illustrating the implementation scheme of this application.

[0020] Figure 2 This is a library peak diagram from Embodiment 1 of this application.

[0021] Figure 3 This is a graph showing the results of miRNA expression levels in Example 2 of this application.

[0022] Figure 4 This is a graph showing the results of miRNA expression levels in Example 2 of this application.

[0023] Figure 5 This is a diagram showing the functional enrichment results in Embodiment 2 of this application.

[0024] Figure 6 This is a diagram showing the functional enrichment results in Embodiment 2 of this application.

[0025] Figure 7 This is a schematic diagram of the clamping structure formed in Embodiment 3 of this application.

[0026] Figure 8 This is a graph showing the detection results of hsa-miR-146a-5p in Example 4 of this application.

[0027] Figure 9 This is a graph showing the detection results of hsa-miR-26a-5p in Example 4 of this application.

[0028] Figure 10 This is a graph showing the detection results of hsa-miR-126-3p in Example 4 of this application.

[0029] Figure 11 This is a graph showing the detection results of hsa-miR-199a-5p in Example 4 of this application.

[0030] Figure 12 This is a graph showing the results of the standard curve in Example 4 of this application.

[0031] Figure 13 This is a diagram of the hybridization complementarity structure in Example 5 of this application.

[0032] Figure 14 This is a graph showing the detection results of miRNA in Example 5 of this application.

[0033] Figure 15 This is the ROC curve diagram in Embodiment 6 of this application.

[0034] Figure 16 This is a bar chart showing AUC, sensitivity, specificity, and accuracy in Example 6 of this application.

[0035] Detailed Explanation Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood that this is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0036] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0037] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0038] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0039] miRNAs are small RNA molecules of about 22 nt that regulate posttranscriptional gene expression through complementary binding to target transcripts. Studies have shown that dysregulation of circulating miRNAs is associated with cardiovascular risk factors, atherosclerosis and coronary heart disease, and also plays a role in the pathophysiology of stroke.

[0040] In this application, we used high-throughput miRNA sequencing on stroke clinical cohort samples to screen differentially expressed miRNAs between healthy controls and the disease group. We then performed functional analysis on these differentially expressed miRNAs to explore the association between the screened miRNAs and cellular signaling pathways in stroke, identifying miRNA molecular markers for stroke diagnosis. Machine learning was then used to construct a model for stroke prediction, and its sensitivity and specificity were evaluated. Furthermore, this application developed a unique reverse transcription-free real-time quantitative PCR (NRT-qPCR) technology. After performance testing and validation using standards, the technology was tested with 100 clinical cohort samples, including 69 healthy controls and 31 stroke patients. The differential miRNA expression characteristics (ΔCT) between healthy individuals and patients were identified, and a common classifier algorithm was used to construct a model to evaluate its sensitivity and specificity, enabling rapid diagnosis of stroke. The overall technical route is described in [link to technical details]. Figure 1 .

[0041] In this application, differentially expressed miRNAs were screened using a stroke clinical cohort, addressing the lack of molecular biomarkers for early stroke diagnosis; miRNA molecular biomarkers were integrated and modeled using machine learning, addressing the problem of insufficient accuracy in early stroke diagnosis; NRT-qPCR technology was developed, addressing the issues of insufficient sensitivity and specificity in miRNA detection; and NRT-qPCR technology was used to screen miRNAs with differential ΔCT values, addressing the problems of long diagnostic time, cumbersome experimental procedures, and high costs associated with miRNA diagnosis.

[0042] In one aspect, this application provides a miRNA biomarker for early screening of stroke, wherein the miRNA biomarker includes: hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p and hsa-miR-26a-5p.

[0043] In some implementations, the miRNA biomarkers consist of hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p, and hsa-miR-26a-5p.

[0044] In some embodiments, the nucleotide sequence of hsa-miR-146a-5p is shown in SEQ ID NO:1, the nucleotide sequence of hsa-miR-126-3p is shown in SEQ ID NO:2, the nucleotide sequence of hsa-miR-199a-5p is shown in SEQ ID NO:3, and the nucleotide sequence of hsa-miR-26a-5p is shown in SEQ ID NO:4.

[0045] On the other hand, this application also provides the use of the miRNA biomarkers described herein in the preparation of kits for early screening of stroke.

[0046] On the other hand, this application also provides a composition for detecting the miRNA biomarkers described herein for early stroke screening, comprising: First capture probe, second capture probe, forward primer, reverse primer and third fluorescent probe; The first capture probe is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 5-8; the second capture probe is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 9-12; the forward primer is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of SEQ ID NO: 13; the reverse primer is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of SEQ ID NO: 14; and the third fluorescent probe is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 15-18.

[0047] In some embodiments, the first capture probe is selected from one or more sequences shown in SEQ ID NO: 5-8; the second capture probe is selected from one or more sequences shown in SEQ ID NO: 9-12; the forward primer is the sequence shown in SEQ ID NO: 13; the reverse primer is the sequence shown in SEQ ID NO: 14; and the third fluorescent probe is selected from one or more sequences shown in SEQ ID NO: 15-18.

[0048] In some embodiments, the first capture probe is the sequence shown in SEQ ID NO: 5-8; the second capture probe is the sequence shown in SEQ ID NO: 9-12; the forward primer is the sequence shown in SEQ ID NO: 13; the reverse primer is the sequence shown in SEQ ID NO: 14; and the third fluorescent probe is the sequence shown in SEQ ID NO: 15-18.

[0049] In some implementations, primer and probe sequences are provided as shown in the table below: In all respects disclosed elsewhere in this document, primers may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Primers may contain one or more non-natural nucleotides. Non-natural nucleotides may be, for example, deoxyinosine. Primers may be forward primers. Primers may be reverse primers. Primer length may be from about 5 to about 50 nucleotides. Primer length may be at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 or more base pairs. Primer length may be at most 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 5 nucleotides. The primer length can be approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 base pairs.

[0050] A primer pair may consist of a forward primer (upstream primer) and a reverse primer (downstream primer). The forward primer may be configured to hybridize with a first region (e.g., the 3' end) of the nucleic acid sequence, while the reverse primer may be configured to hybridize with a second region (e.g., the 5' end) of the nucleic acid sequence, thereby being configured to amplify the nucleic acid sequence under conditions sufficient for nucleic acid amplification. Different primer pairs may be configured to amplify different target nucleic acid sequences.

[0051] In this paper, “capture probe” refers to the probe sequence used to capture miRNA, including a first capture probe and a second capture probe.

[0052] In this document, the probe may be a nucleic acid (e.g., DNA, RNA, etc.). The probe may contain a region complementary to a region of the target nucleic acid. The concentration of the probe may be in excess relative to other components in the detection mixture. The probe may be a signal-generating nucleic acid probe. A signal-generating nucleic acid probe may contain the characteristics of a probe as described herein, and the characteristics of generating a signal upon application of a stimulus, modification of the probe, or hydrolysis of the probe. For example, the probe may be hydrolyzed by the exonuclease activity of a polymerase to generate a signal. The signal-generating nucleic acid probe may generate any signal as described elsewhere herein. For example, the signal-generating probe may generate a fluorescent signal. The signal-generating probe may contain a signal tag. The signal tag may generate a signal. The signal tag may generate a signal in response to a reaction or stimulus. For example, the signal tag may generate a signal upon degradation by exonuclease activity.

[0053] In some implementations, a signal can be generated simultaneously with hybridization of the probe to a nucleic acid region. For example, a probe (e.g., a molecular beacon) can generate a signal (e.g., a fluorescent signal) after hybridization with a nucleic acid. In some cases, a signal can be generated after the probe has hybridized with a nucleic acid region, or after the probe has been degraded by a nuclease. Where the probe contains a signal tag, the probe can be degraded when it binds to a region of a primer, thereby generating a signal. For example, a probe (e.g., a probe) can generate a signal after hybridization with a nucleic acid and subsequent degradation by a polymerase (e.g., during amplification such as PCR amplification). The probe can be degraded by the exonuclease activity of a nuclease.

[0054] In some implementations, the probe sequence is modified with fluorescent reporter groups and corresponding fluorescence quenchers at both ends. The probe, modified with both fluorescent reporter and fluorescence quencher groups, is used in qPCR to display the relative amount of synthesized DNA product. In the absence of amplification, the fluorescent reporter and fluorescence quencher groups at both ends of the probe sequence are attached together, and the fluorescence of the reporter group is quenched by the fluorescence quencher group, resulting in no fluorescence emission. When PCR synthesis begins, the probe sequence complementaryly pairs with the target fragment in the genomic DNA and anneals together. If the target microorganism (i.e., the target fragment) is present in the sample, the probe sequence can bind to the target fragment. Once the PCR reaction begins, if the PCR reaction occurs, the Taq enzyme dissociates the probe sequence bound to the target fragment, thereby separating the fluorescent reporter group from the fluorescence quencher group. At this point, the fluorescence emitted by the fluorescent reporter group can be detected. As the PCR reaction proceeds, the more DNA amplification product is synthesized, the more fluorescent reporter groups are dissociated. By detecting the fluorescence intensity, the amount of DNA amplification product can be accurately quantified.

[0055] In some embodiments, the third fluorescent probe is modified at both ends with a fluorescent reporter group and a corresponding fluorescent quencher group; the fluorescent reporter group is FAM, VIC, HEX or ROX, and the fluorescent quencher group is BHQ2, BHQ1 or BHQ3.

[0056] In some implementations, the 3' end of the third fluorescent probe also contains an MGB.

[0057] In another aspect, this application also provides a kit comprising the composition described herein for detecting miRNA biomarkers for early screening of stroke.

[0058] In some implementations, the kit may also contain one or more of the following: DNA polymerase, DNA ligase, miRNA standard, dNTP, RNase, metal cation, protein protectant, yeast RNA, and buffer.

[0059] In some implementations, the DNA ligase is Splint R ligase.

[0060] The kit may contain one or more nucleases. The nuclease may be a nucleic acid polymerase. The nucleic acid polymerase may be a deoxyribonucleic acid polymerase (DNase). The DNase may be Taq polymerase or a variant thereof. The nuclease may be a ribonucleic acid polymerase (RNase). The RNase may be RNase III. The nuclease may be an endonuclease. The endonuclease may be endonuclease I. Endonuclease I may be T7 endonuclease I. The nuclease may be able to degrade nucleic acids containing non-natural nucleotides. The nuclease may be endonuclease V, such as E. coli endonuclease V. The nuclease may be a polymerase (e.g., DNA polymerase). The polymerase may be Taq polymerase or a variant thereof. Non-restricted examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, PlatinµMTaq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragments, and their variants, modified products, and derivatives. For a given hot-start polymerase, a denaturation step of 2 to 10 minutes at 94–95°C may be required, which may vary the thermal profile depending on the polymerase. Nucleases can degrade probes under appropriate conditions. For example, a nuclease can be a polymerase and has exonuclease activity, degrading the probe to generate a detectable signal. Under appropriate conditions, the nuclease may be able to release a quencher from the probe. The kit may include instructions on the use of any of the aforementioned substances described herein.

[0061] On the other hand, this application also provides the use of the composition or kit containing the miRNA biomarker described herein for detecting miRNA biomarkers for early screening of stroke in detecting miRNA biomarkers, wherein the miRNA biomarker is selected from two or more of the following: hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p and hsa-miR-26a-5p.

[0062] On the other hand, this application also provides the use of the compositions described herein for detecting miRNA biomarkers for early stroke screening or kits containing such biomarkers in early stroke screening.

[0063] On the other hand, this application also provides a method for detecting the content of miRNA biomarkers in a sample, wherein the miRNA biomarkers are selected from two or more of the following: hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p, and hsa-miR-26a-5p; the method comprises: using the composition described herein for detecting miRNA biomarkers for early screening of stroke or a kit containing the composition described herein, as well as performing the following operations: 1) Collect RNA from the sample; 2) Anneal and hybridize the RNA obtained in step 1) with the first and second capture probes, and ligate the hybridization products with DNA ligase; 3) Perform qPCR on the product from step 2) using forward primers, reverse primers, and a third fluorescent probe. Determine the content of miRNA in the sample based on the qPCR results.

[0064] In some embodiments, the sample is blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, tears, or a tissue biopsy sample. Preferably, the sample is blood.

[0065] On the other hand, this application also provides a composition for detecting miRNA, comprising: First capture probe, second capture probe, forward primer, reverse primer and third fluorescent probe; The first capture probe contains a first universal sequence and a sequence complementary to the latter half (5' to 3') of the miRNA, from 5' to 3' respectively. The second capture probe contains a sequence complementary to the first half (5' to 3') of the miRNA and a second universal sequence, from 5' to 3' respectively. The second capture probe 5' is phosphorylated. The forward primer is identical to a portion of the first universal sequence, and the reverse primer is inversely complementary to a portion of the second universal sequence. The third fluorescent probe is modified with fluorescent reporter groups and corresponding fluorescent quenching groups at both ends; the fluorescent reporter groups are FAM, VIC, HEX or ROX, the fluorescent quenching groups are BHQ2, BHQ1 or BHQ3, and the 3' end of the third fluorescent probe also contains MGB.

[0066] On the other hand, this application also provides a kit for detecting miRNA, wherein the kit contains the composition for detecting miRNA described herein.

[0067] In some implementations, the kit may also contain one or more of the following: DNA polymerase, DNA ligase, miRNA standard, dNTP, RNase, metal cation, protein protectant, yeast RNA, and buffer.

[0068] In some implementations, the DNA ligase is Splint R ligase.

[0069] On the other hand, this application also provides the use of the compositions described herein for detecting miRNAs or kits containing the same in detecting miRNAs.

[0070] On the other hand, this application also provides a method for detecting the content of miRNA in a sample, comprising: Perform the following procedures using the composition for detecting miRNAs described herein or a kit containing such composition: 1) Collect RNA from the sample; 2) Anneal and hybridize the RNA obtained in step 1) with the first and second capture probes, and ligate the hybridization products with DNA ligase; 3) Perform qPCR on the product from step 2) using forward primers, reverse primers, and a third fluorescent probe. Determine the content of miRNA in the sample based on the qPCR results.

[0071] In this paper, the method for detecting the content of miRNA in a sample described herein is also known as the NRT-qPCR (Non-reverse transcription qPCR) method.

[0072] In some embodiments, the sample is blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, tears, or a tissue biopsy sample. Preferably, the sample is blood.

[0073] This application describes several embodiments, but these descriptions are exemplary and not limiting, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature or element of any embodiment may be used in combination with, or may replace, any feature or element of any other embodiment.

[0074] This application includes and contemplates combinations of features and elements known to those skilled in the art. The embodiments, features, and elements disclosed in this application can also be combined with any conventional features or elements to form unique inventive solutions. Any feature or element of any embodiment can also be combined with features or elements from other inventive solutions to form another unique inventive solution. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0075] Furthermore, in describing representative embodiments, the specification may have presented methods and / or processes as a specific sequence of steps. However, the method or process should not be limited to the specific order of steps described herein, to the extent that it does not depend on such a specific order. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to the steps performed in the written order, and those skilled in the art will readily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0076] Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. Unless otherwise defined or stated, all technical and scientific terms used in this application have the same meaning as those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this application.

[0077] Example Example 1. Sample Collection and miRNA Sequencing 1. Clinical Sample Collection The clinical research in this application was conducted in collaboration with the First Affiliated Hospital of Chongqing Medical University. The scientific review approval document is KX2025-KYC0420-02; the ethics review approval document is 2025-734-01.

[0078] 1) The samples were obtained from patients with hemorrhagic stroke and healthy individuals, totaling 102 cases, including 40 patients and 64 healthy individuals. 8 mL of whole blood was collected from each patient and stored in the PAXgene® Blood ccfDNA Tube (brand: QIAGEN; catalog number: 768115). After blood collection, the samples were stored at room temperature (18-25℃), and plasma extraction must be performed within a maximum of 72 hours.

[0079] 2) For the first centrifugation, use a balanced centrifuge at 2000 × g for 15 min at room temperature (15-25℃).

[0080] 3) Use a pipette to transfer the plasma into a 15ml conical centrifuge tube, making sure not to touch the white blood cell layer and the red blood cell portion.

[0081] 4) For the second centrifugation, use a balanced centrifuge at room temperature, 2000 × g, for 10 min. Use a pipette to transfer the plasma into the bottom centrifuge tube of a 15 ml conical tube, ensuring that no residual blood cell particles are disturbed. Store at -80℃ (approximately 4 mL of clean plasma should be obtained, and risk control should be carried out based on the color to determine whether there is hemolysis).

[0082] 2. miRNAs were extracted from plasma samples using the QIAGEN-miRNeasy SerµM-Plasma Kit (brand: QIAGEN; catalog number 217184).

[0083] 3. miRNA library construction 1) Prepare the reaction system on ice according to the reagents and volumes listed in the table below: Note: Reagents 3' Adapter II, 3' Adapter Ligation Buffer II, and 3' Adapter Ligation Enzymes II were purchased from Aibotek (catalog number: RK20312), with 3' Adapter II diluted 20 times.

[0084] 2) Mix the prepared reagent system by pipetting, centrifuge briefly to the bottom of the tube, and immediately place it in a PCR instrument for the following reaction procedure: 3) After the reaction is complete, remove the product and place it on ice. Prepare the reaction system according to the following table: Note: The Adapter Depletion Mix was purchased from Aibotek (item number: RK20312). 4) Mix the prepared reagent system by pipetting, centrifuge briefly to the bottom of the tube, and immediately place it in a PCR instrument for the following reaction procedure: 5) Connect the 5' connector, dissolve and mix the 5' Adapter (after denaturation at 80℃) and 5' Adapter Ligation Buffer on ice, then briefly centrifuge to collect the solution to the bottom of the tube. Prepare the reaction system according to the table below: Note: The reagents 5' Adapter, 5' Adapter Ligation Buffer, and 5' Adapter Ligation Enzymes were purchased from Aibotek (item number: RK20312). 6) Mix the prepared reagent system by pipetting, centrifuge briefly to the bottom of the tube, and immediately place it in a PCR instrument for the following reaction procedure: 7) Prepare the reaction system according to the following table: Note: 10 × RT Mix and HiScript II Enzyme Mix were purchased from Nanjing Novizan Pharmaceutical Co., Ltd. (item number: MR101). 8) Gently mix by pipetting, centrifuge briefly, and place in a PCR instrument to perform the following reaction procedure: 9) Prepare the reaction system according to the following table: Note: The 2X LA PCR Mix, sRNA Universal Primer, and Index primer were purchased from Ibotek (catalog number: RK20312). 10) Gently mix using a pipette, centrifuge briefly, and place in a PCR instrument to perform the following reaction procedure: 11) Magnetic bead purification: Add 90µL of Novizan purification magnetic beads (catalog number: N411-01) to the product from the previous step, mix thoroughly, and let stand at room temperature for 5 min. Place on a magnetic rack for about 5 min to allow the magnetic beads to be completely adsorbed and the solution to become clear. Carefully remove the supernatant. Add 200µL of freshly prepared 80% ethanol for rinsing, incubate at room temperature for 30-60 s, carefully remove the supernatant, and repeat once. After the magnetic beads are dry, add 26µL of ultrapure water for elution, let stand at room temperature for 3 min, place on a magnetic rack, and when the solution becomes clear, aspirate 25µL of the supernatant for use.

[0085] 4. Sequencing: Dilute the library to 1 ng / μL, and take 1 μL for sequencing using an Agilent 4200 Tapestation System (Agilent Technologies, USA). The library peak shape is normal (see...). Figure 2 Additionally, 1 µL was used for qPCR detection. The library was diluted to the required level (2 nmol) and sequenced using PE150 sequencing on the Illumina Novaseq sequencing platform. Each sample yielded 3 G of data.

[0086] All samples yielded more than 3g, and Q30 was greater than 80%, meeting the criteria for proceeding to the next step of analysis.

[0087] Example 2. miRNA analysis and signaling pathway analysis 1. Differential miRNA analysis 1) Raw Data Quality Control and Adapter Filtering: The raw data undergoes quality assessment and processing, primarily removing adapter sequences, eliminating low-quality bases (Phred score < 20), and filtering out sequences that are too short (< 18 nt) or too long (> 28 nt) to ensure that only high-quality miRNA sequences are used in subsequent analyses. After processing, clean data (Clean FASTQ) is generated for subsequent analysis.

[0088] 2) Sequence overlap: The sequencing strategy used in this study was PE150. R1 and R2 are essentially sequenced from both ends of the same miRNA molecule, and are reverse complementary sequences. R1 and R2 are then overlapped and spliced. This overlap is equivalent to the same miRNA molecule being sequenced twice. By comparing these two independent sequencing results, random sequencing errors that may occur in a single read can be effectively corrected, thereby obtaining a base sequence with extremely high confidence.

[0089] 3) Reference sequence alignment: The overlapping file obtained in the previous step is aligned with the mature human miRNA reference sequence. The alignment allows for a maximum of one base mismatch to account for both biological variations of miRNA and sequencing errors.

[0090] 4) Expression quantification: The number of sequences aligned to each known mature miRNA is counted to obtain the accurate raw expression count for each mature miRNA. Finally, a raw expression count matrix is ​​generated, where the rows are miRNA names, the columns are sample names, and the values ​​are the expression count values ​​of the corresponding miRNA in each sample.

[0091] 5) Differential miRNA analysis and candidate biomarker screening: The original count matrices of all samples were normalized to correct for differences in sequencing depth between samples. Subsequently, a generalized linear model based on a negative binomial distribution was used to fit the data, and the Wald test was used to accurately calculate the expression difference between the patient group and the healthy control group. Strict screening criteria were used (corrected p-value padj < 0.05 and absolute value of the fold change |Log2(FoldChange)| > 1). Finally, four statistically significant miRNAs were identified, and these four differentially expressed miRNAs could be effectively clustered, with significant differences in expression levels (see...). Figures 3-4 Therefore, these miRNAs will be used as candidate biomarkers for constructing diagnostic models. The biomarker information is shown in the table below: Note: miRNA ID: Unique standard nomenclature for mature miRNAs baseMean: The normalized average expression abundance of this miRNA across all samples. Log2 Fold Change: The logarithm of the fold change in expression level between the disease group and the healthy control group (base 2); negative values ​​indicate downregulation. lfcSE is a measure of the uncertainty of the Log2 Fold Change estimate; a smaller value indicates a more accurate estimate. stat: The Wald test statistic used to test the significance of differences, derived by dividing Log2 Fold Change by its standard error. pvalue: The original p-value of the statistical test. padj: The p-value after multiple test correction (padj) 2. Differential miRNA target gene analysis and signaling pathway analysis 1) Target Gene Prediction: Considering the high false positive rate of a single prediction database, the best practice for obtaining reliable results is to use multiple databases for prediction. All the significantly differentially expressed miRNAs identified above were used for target gene prediction, employing multiple databases and integrating their prediction results. To improve prediction accuracy and reduce the false positive rate, genes jointly predicted by two databases were selected as high-confidence candidate target genes. The specific implementation steps are as follows: a. Predict each differentially expressed miRNA using multiple databases; b. Select the top m genes from each database prediction, ranked by score; c. Integrate target genes from multiple databases, retaining only target genes that are predicted in two or more databases.

[0092] In this case study, taking hsa-miR-199-5p as an example, with an m value of 150, the following high-confidence candidate target genes were identified: ALCAM, ANK2, ANO1, AP1S2, AP3B1, ARF6, ARMCX2, C21orf91, C4orf46, CALB2, CAPZA1, CCDC43, CCNDBP1, CCN G1, CLDN14, CMTR2, CPEB2, CSNK1A1, CTNNA1, CXCL11, DCTN2, DDHD2, DLX3, DNAJC14, DOK6, DYRK1B, EIF4E3, EMB, ENPEP, FAM19A5, FBN1, FBXL3, FKBP7, FOXN2, FOXP1, FOXP4, FRMD6, FSTL1, FYTTD1, GABRB2, GALNT3, GO T1, GPR137C, ID4, IPO4, ITGA6, ITM2B, KCNJ2, KCNMB2, KLF5, LDLRAP1, LIN28B, LYVE1, MAEA, MAGT1, MDGA2, MTHFD2, MTMR2, MYOCD, NID2, NR5A2, NXPE3, ONECUT1, ONECUT2, PCGF5, PCGF6, PDK4, PHLDB2, PI4K2A, POU2F 1. POU2F2, PRDM1, PRDM6, PRRX1, PTAR1, PXDN, PYGO2, RAB34, RANBP17, RBMS3, RHOQ, RNF150, SDC2, SEC23IP , SFXN2, SGMS2, ​​SHC1, SIX5, SLC10A3, SLC31A2, SLC39A9, SLC50A1, SLC6A2, SLC8A1, SMARCD2, SNX25, SOCS5 , SPTLC2, STK3, SYAP1, TESK2, TGFBI, TNC, TRIM71, TRPM7, UBE3C, UHMK1, VAV3, VGLL4, WASF2, YBX3, ZBTB20.

[0093] 2) Enrichment and pathway analyses were performed on this high-confidence target gene list. Entries with a p-value < 0.05 were considered statistically significant. The functional enrichment results are as follows: Figure 5-6 As shown.

[0094] By grouping the dataset (stroke group and healthy control group), four differentially expressed miRNAs were screened. Further analysis of the target genes and related enriched signaling pathways of the differentially expressed miRNAs revealed that the KEGG pathway was associated with the glutamatergic synaptic pathway. Abnormalities in the glutamatergic synaptic pathway are a core pathological hub in stroke, and abnormal signal transduction pathways become key mechanisms leading to neuronal "poisoning" and death. At the same time, GO analysis showed that the target genes were associated with vascular transport pathways. Abnormal vascular transport leads to the disruption of the blood-brain barrier, abnormally inducing cerebral hemorrhage. Therefore, the biological significance of the association between these four miRNAs and stroke was derived.

[0095] Example 3. Construction of the capture probe, primers, and detection probe (MGB probe) 1. Based on the sequences of hsa-miR-146a-5p, hsa-miR-26a-5p, hsa-miR-126-3p, and hsa-miR-199a-5p, two capture probes were designed for each. The miRNA and capture probe sequences are shown in the table below: 2. After capture probe 1 and capture probe 2 hybridize and anneal the miRNA, a sandwich structure is formed (e.g., Figure 7 The sequence is shown in the table below: 3. Based on the annealing product sequence, amplification primers and MGB probes were designed, as shown in the table below: 4. miRNA, probe and primer were synthesized at Shanghai Sangon Biotech. miRNA was dissolved and diluted to 5µM, probe was dissolved and diluted to 5µM, and primer was dissolved and diluted to 10µM.

[0096] Example 4. Sensitivity Test 1. Preparation of Standards: Four miRNA molecular markers screened by miRNA sequencing can be synthesized artificially to prepare sensitivity test standards. The sequences were synthesized by Shanghai Sangon Biotech, and the miRNAs are single-stranded, with a synthesis amount of 5 nmol.

[0097] 2. Standard Dilution: Dissolve the synthesized miRNA in 50 µL of enzyme-free water. Define the dissolved sample as miRNA-D1. Determine the concentration using the Qubit™ microRNA Quantitative Kit (Thermo Fisher Scientific, catalog number: Q32880) and dilute to 7.06 ng / µL. Calculate the molar concentration (1 µM) using the formula [Concentration (µM) = Concentration (ng / µL) / Number of nucleotides / 0.321]. Define this concentration as miRNA-D2 and perform serial dilutions as shown in the table below: Note: D2 was serially diluted 10-fold to D12, and the copy number was estimated to be approximately 60 based on Avogadro's constant. 3. Prepare the reagent reaction system according to the following table: Note: 10× PBCV buffer was purchased from Yisheng Biotechnology (catalog number: 14962ES80), and yeast RNA was purchased from Thermo Fisher Scientific (catalog number: AM7120G). 4. Prepare the following reaction system immediately: Note: PBCV buffer was purchased from Yisheng Biotechnology (item number: 14962ES80), and DTT was purchased from Shanghai Sangon Biotech (item number: A620058). 5. Mix thoroughly, place in a PCR instrument, and proceed with the following reaction program: 6. After the reaction in step 5 is complete, immediately prepare the following reaction system: Note: The 2x AceQ Universal U+ Probe Master Mix was purchased from Novizan (item number: Q513-02). 7. Gently pipette or vortex to mix, and briefly centrifuge to bring the reaction solution to the bottom of the tube. Place the tube in a qPCR instrument and perform the following reaction: Sensitivity tests were performed on four miRNAs: hsa-miR-146a-5p, hsa-miR-26a-5p, hsa-miR-126-3p, and hsa-miR-199a-5p. Artificially synthesized standards were diluted to 60 copies / µL. NRT-qPCR was performed on samples from days D6 to D12 of the miRNA count. The results showed that NRT-qPCR could effectively detect miRNAs up to 60 copies / µL. Figures 8-11And by selecting the standard products D6-D10 of has-miR-199a-5p, a standard curve can be prepared, and R can be obtained. 2 =0.99, amplification efficiency of approximately 95.4% ( Figure 12 The overall testing time is approximately 1.5 hours. Detailed CT values ​​are shown in the table below. Example 5. Specificity Test 1. Preparation of Standards: Using the miRNA122 sequence as a reference, homologous sequences with one differential base were chemically synthesized at different positions at the 3' end. Corresponding capture probes and fluorescent probes were designed. The sequences were synthesized by Shanghai Sangon Biotech Co., Ltd., and the sequence information is shown in the table below: 2. Dilute the above standard to 100 pM, the hybridization complementary structure is as follows. Figure 13 As shown, subsequent experiments were conducted according to steps 3-8 of Example 4.

[0098] Using the miRNA122 sequence as a reference, sensitivity tests involving single-base differential sequences at different positions effectively identified the target miRNA, such as... Figure 14 Detailed CT values ​​are shown in the table below. Example 6. Clinical Sample Validation 1. Clinical sample collection: 100 plasma samples were collected, including 69 healthy individuals and 31 stroke patients. The plasma volume was 1 mL. The specific collection and miRNA extraction protocols were the same as in Example 1.

[0099] 2. NRT-qPCR detection: Take 5µL of miRNA sample and prepare the reaction system according to the table below: 3. Subsequent steps are consistent with steps 3-7 of Example 4. 4. Diagnostic Model Construction and Performance Evaluation: After all clinical samples underwent NRT-qPCR testing for hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p, and hsa-miR-26a-5p, the dataset of 100 samples was split into training and testing sets in a 7:3 ratio. In the training set, ΔCT values ​​were used as features, and a predictive model was constructed using a multivariate logistic regression algorithm. The model performance was evaluated using the receiver operating characteristic (ROC) curve.

[0100] Furthermore, NRT-qPCR detection of four miRNA biomarkers was performed on 100 plasma samples (69 healthy individuals and 31 stroke patients), with hsa-miR-16-5p as the internal control. The ΔCT value for each sample was obtained, and a model was constructed. The area under the curve (AUC) for stroke diagnosis reached 0.957. At the optimal cutoff point, the model's diagnostic sensitivity was 86.8%, and its specificity was 98.2%. Figures 15-16 As shown.

[0101] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A miRNA biomarker for early screening of stroke, wherein, The miRNA biomarkers are selected from two or more of the following: hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p, and hsa-miR-26a-5p.

2. The miRNA biomarker according to claim 1, wherein, The miRNA biomarkers consist of hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p, and hsa-miR-26a-5p.

3. The miRNA biomarker according to claim 1 or 2, wherein, The nucleotide sequence of hsa-miR-146a-5p is shown in SEQ ID NO:1, the nucleotide sequence of hsa-miR-126-3p is shown in SEQ ID NO:2, the nucleotide sequence of hsa-miR-199a-5p is shown in SEQ ID NO:3, and the nucleotide sequence of hsa-miR-26a-5p is shown in SEQ ID NO:

4.

4. Use of the miRNA biomarker according to any one of claims 1-3 in the preparation of a kit for early screening of stroke.

5. A composition for detecting the miRNA biomarker according to any one of claims 1-3, comprising: First capture probe, second capture probe, forward primer, reverse primer and third fluorescent probe; The first capture probe is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 5-8; the second capture probe is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 9-12; the forward primer is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of SEQ ID NO: 13; the reverse primer is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of SEQ ID NO: 14; and the third fluorescent probe is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 15-18.

6. The composition according to claim 5, wherein, The first capture probe is selected from one or more sequences shown in SEQ ID NO: 5-8; the second capture probe is selected from one or more sequences shown in SEQ ID NO: 9-12; the forward primer is the sequence shown in SEQ ID NO: 13; the reverse primer is the sequence shown in SEQ ID NO: 14; and the third fluorescent probe is selected from one or more sequences shown in SEQ ID NO: 15-18.

7. The composition according to claim 6, wherein, The first capture probe is the sequence shown in SEQ ID NO: 5-8; the second capture probe is the sequence shown in SEQ ID NO: 9-12; the forward primer is the sequence shown in SEQ ID NO: 13; the reverse primer is the sequence shown in SEQ ID NO: 14; and the third fluorescent probe is the sequence shown in SEQ ID NO: 15-18.

8. A reagent kit, wherein, The kit comprises the composition of any one of claims 5-7.

9. The kit according to claim 8, wherein, The kit also contains one or more of the following: DNA polymerase, DNA ligase, miRNA standard, dNTP, RNase, metal cation, protein protectant, yeast RNA, and buffer; preferably, the DNA ligase is Splint R ligase.

10. Use of the composition of any one of claims 5-7 or the kit of claim 8 or 9 in the detection of miRNA biomarkers, wherein, The miRNA biomarker is selected from two or more of the following: hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p, and hsa-miR-26a-5p.

11. Use of the composition of any one of claims 5-7 or the kit of claim 8 or 9 in early screening for stroke.

12. A method for detecting the content of miRNA biomarkers in a sample, wherein, The miRNA biomarker is selected from two or more of the following: hsa-miR-146a-5p, hsa-miR-126-3p, hsa-miR-199a-5p, and hsa-miR-26a-5p; the method comprises: performing the following operations using the composition of any one of claims 5-7 or the kit of any one of claims 8 or 9: 1) Collect the RNA from the sample; 2) Anneal and hybridize the RNA obtained in step 1) with the first capture probe and the second capture probe, and ligate the hybridization products using DNA ligase; 3) Perform qPCR on the product from step 2) using the forward primer, the reverse primer, and the third fluorescent probe. Determine the content of the miRNA in the sample based on the qPCR results.

13. A composition for detecting miRNA, comprising: First capture probe, second capture probe, forward primer, reverse primer and third fluorescent probe; The first capture probe contains, from 5' to 3', a first universal sequence and a sequence complementary to the latter half (5' to 3') of the miRNA, and the second capture probe contains, from 5' to 3', a sequence complementary to the first half (5' to 3') of the miRNA and a second universal sequence, and the second capture probe 5' is phosphorylated. The forward primer is identical to a portion of the first universal sequence, and the reverse primer is inversely complementary to a portion of the second universal sequence; The third fluorescent probe is modified with a fluorescent reporter group and a fluorescent quencher group corresponding to the fluorescent reporter group at both ends; the fluorescent reporter group is FAM, VIC, HEX or ROX, the fluorescent quencher group is BHQ2, BHQ1 or BHQ3, and the 3' end of the third fluorescent probe also contains MGB.

14. A kit for detecting miRNA, wherein, The kit comprises the composition of claim 13.

15. Use of the composition of claim 13 or the kit of claim 14 in the detection of miRNA.

16. A method for detecting the content of miRNA in a sample, comprising: Perform the following operations using the composition of claim 13 or the kit of claim 14: 1) Collect the RNA from the sample; 2) Anneal and hybridize the RNA obtained in step 1) with the first capture probe and the second capture probe, and ligate the hybridization products using DNA ligase; 3) Perform qPCR on the product from step 2) using the forward primer, the reverse primer, and the third fluorescent probe. Determine the content of the miRNA in the sample based on the qPCR results.

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