Kit and method for detecting linear RNA

Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a kit and a method for detecting linear RNA (Ribonucleic Acid). The invention provides a kit for detecting linear RNA (Ribonucleic Acid). The kit comprises a molecular beacon, a hairpin combination for a primer exchange reaction and a reagent for the primer exchange reaction, the hairpin combination comprises an identification hairpin and an extension hairpin. The invention develops a nucleic acid isothermal amplification method for detecting linear RNA (Ribonucleic Acid) based on primer exchange reaction, such as miRNA (Micro Ribonucleic Acid). Compared with the existing technology for detecting miRNA, the kit and the method provided by the invention have the advantages of no need of expensive instruments, simple experimental process, simple design of nucleic acid recognition elements, high sensitivity, extensibility of detection objects and the like, and are high in practical applicability.

Description

Technical Field

[0001] The present application relates to the field of nucleic acid detection technology, and in particular to a kit and method for detecting linear RNA. Background Art

[0002] MicroRNAs (miRNAs) are a class of highly conserved endogenous noncoding RNAs, approximately 18 to 22 nucleotides in length, that are ubiquitous in eukaryotes. miRNAs can bind to messenger RNA (mRNA) through complementary base pairing, thereby inhibiting protein synthesis. Consequently, miRNAs can regulate post-transcriptional gene expression and play a crucial role in biological growth, development, cell differentiation, and apoptosis. In recent years, a wealth of research has demonstrated that miRNAs are involved in cellular regulatory processes in tumor cells, with their aberrant expression being closely associated with tumor development and progression. Furthermore, some studies have shown that serum miRNAs possess remarkable stability and resistance to degradation, being insensitive to RNases and extreme physical and chemical factors, demonstrating essential characteristics for their potential as ideal disease biomarkers. However, miRNAs are small in size, have low abundance, and share highly homologous family sequences, making sensitive and reliable detection of miRNAs in clinical samples challenging.

[0003] Traditional miRNA detection methods include polymerase chain reaction (PCR), microarray chips, and RNA sequencing (RNA-Seq), which generally have high sensitivity. In recent years, a series of detection technologies based on isothermal nucleic acid amplification have been widely studied in the field of miRNA detection due to their advantages of high specificity, high yield, and high sensitivity. These include rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), exponential amplification reaction (EXPAR), hybridization chain reaction (HCR), and catalytic hairpin assembly (CHA).

[0004] PCR-based detection methods, such as qRT-PCR, are considered the gold standard for miRNA detection, with characteristics such as high sensitivity and high sequence specificity. However, primer design is a major challenge, and contamination and noise may be introduced during sample purification, reverse transcription, and fragment replication. Microarray chip methods can quickly detect multiple miRNAs simultaneously, but due to the similarity of miRNA homology family sequences, this method results in a high proportion of false positive results. RNA sequencing has the advantage of being non-targeted, but due to its complicated operation steps and data processing process, and its reliance on expensive instruments, it is not suitable for routine detection. However, isothermal nucleic acid amplification strategies also have their own inherent defects: rolling circle amplification (RCA) requires auxiliary enzyme-chain and product separation processes; loop-mediated isothermal amplification (LAMP) and exponential amplification reaction (EXPAR) require multiple enzymes and primers to maintain the amplification reaction; hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) are overly complex due to the hairpin structure design and unclear reaction mechanism, and their detection sensitivity and stability are limited in actual sample detection. Summary of the Invention

[0005] In light of this, the present invention has developed a nucleic acid isothermal amplification method for detecting linear RNA, such as miRNA, based on the primer exchange reaction (PER). Compared with existing miRNA detection technologies, the kit and method provided in this application do not require expensive instruments, have a simple experimental process, simple nucleic acid recognition element design, high sensitivity, and scalable detection targets, and have strong practical applicability.

[0006] In a first aspect, the present application provides a kit for detecting linear RNA, comprising:

[0007] Molecular beacons, hairpin assemblies for primer exchange reactions, and reagents for primer exchange reactions;

[0008] The hairpin combination comprises an identification hairpin and an extension hairpin;

[0009] The recognition hairpin contains, in sequence: a nucleotide sequence A constituting a first hairpin structure, a nucleotide sequence B that is reverse complementary to the target RNA, and a polyT sequence; or

[0010] The recognition hairpin contains, in order: a nucleotide sequence A constituting a first hairpin structure, a nucleotide sequence C reversely complementary to the target RNA, a nucleotide sequence D reversely complementary to a portion of the nucleotide sequence C, and a nucleotide sequence E reversely complementary to the target RNA;

[0011] The extended hairpin sequentially contains: a nucleotide sequence A constituting a first hairpin structure, a nucleotide sequence F that is reverse complementary to a portion of the nucleotide sequence A, and a polyT sequence;

[0012] The molecular beacon contains: a nucleotide sequence G constituting a second hairpin structure, and a fluorescent group and a quenching group coupled to both ends of the nucleotide sequence G, wherein the nucleotide sequence G contains in sequence: a nucleotide sequence H that is reverse complementary to a partial sequence of the nucleotide sequence A, and a nucleotide sequence I that is reverse complementary to a partial sequence of the nucleotide sequence H.

[0013] Specifically, the nucleotide sequence A constituting the first hairpin structure and the nucleotide sequence G constituting the second hairpin structure contain self-complementary segments and non-complementary segments, constituting the "stem" structure and "stem-loop" structure of the hairpin.

[0014] Specifically, the recognition hairpin can have two structures. The first recognition hairpin contains, in sequence: a nucleotide sequence A constituting a first hairpin structure, a nucleotide sequence B that is reverse complementary to the target RNA, and a polyT sequence. The recognition hairpin contains a nucleotide sequence (nucleotide sequence B) that is reverse complementary to the target RNA; the second recognition hairpin contains, in sequence: a nucleotide sequence A constituting a first hairpin structure, a nucleotide sequence C that is reverse complementary to the target RNA, a nucleotide sequence D that is reverse complementary to a portion of the nucleotide sequence C, and a nucleotide sequence E that is reverse complementary to the target RNA. The recognition hairpin contains two nucleotide sequences (nucleotide sequence C and nucleotide sequence E) that are reverse complementary to the target RNA, and the nucleotide sequence D of the second recognition hairpin can be complementary to part of the nucleotide sequence C to form a "stem-loop" structure.

[0015] More specifically, Figure 1 The schematic diagram of the principle and detection scheme of the PER-based amplification method for detecting miRNA in this application is shown in FIG. Figure 1As shown in Figure A, "Target Identification", the target microRNA specifically binds to the recognition sequence at the 3' end of the recognition hairpin (iHP-1) through base complementary pairing. Subsequently, under the catalysis of Bst DNA polymerase, a polymerase-mediated strand displacement reaction (PMSDR) is triggered, which extends a specific sequence (in Figure 1 Then, the extended product is released from the recognition hairpin through branch migration (BM), and the recognition hairpin can be recycled to bind to new target microRNA. Figure 1 As shown in Figure B, "PER Cycle and Signal Output," the product's b domain, with the assistance of an extended hairpin and Bst DNA polymerase, triggers the next round of PMSDR and BM, ultimately releasing a single strand containing two b units at its 3' end. Crucially, this single strand can further initiate PMSDR and BM, forming a self-perpetuating process known as the PER cycle. The resulting single-stranded PER product contains numerous repeating b domains, and these repeating units can serve as carriers for signal production. For example, a fluorescent molecular beacon (Flu-MB) can be designed with a partial sequence that complements the repeating units. Under annealing conditions, Flu-MB folds onto itself, placing the fluorescent group 6-carboxyfluorescein (6-FAM) modified at its 5' end close to the Dabcyl group at its 3' end. In the absence of target product, the fluorescence of the 6-FAM at the 5' end is quenched by the Dabcyl group, resulting in no signal generation. Therefore, the system with target microRNA will produce a highly enhanced fluorescence signal, thereby enabling sensitive detection of target microRNA. Figure 1As shown in Figure C, this application has designed a new recognition structure for a hairpin (iHP-2). Based on the target miRNA sequence, two complementary paired sequences (nucleotide sequence C and nucleotide sequence E, which are reverse complementary to the target RNA) are designed. Under annealing conditions, this free sequence forms an additional hairpin structure based on the original structure (nucleotide sequence D is partially complementary to nucleotide sequence C to form an additional hairpin structure). The target miRNA binds to most of the sequence on the hairpin structure through complementary base pairing, thereby destroying the hairpin structure and triggering the subsequent amplification reaction. In the absence of a specific target, this additional hairpin structure can prevent nonspecific amplification, thereby reducing background signal and improving detection sensitivity.

[0016] Specifically, the preparation method of the recognition hairpin and the extension hairpin includes: dissolving the single-stranded DNA of the hairpin combination in ultrapure water so that the final concentration of the DNA single strand is 100 μM. The dissolved recognition hairpin and extension hairpin are diluted to 10 μM, and are added to TE buffer (pH 8.0) with 10 mM magnesium sulfate (MgSO4) at a volume ratio of 1:1, so that the final concentration of the DNA single strand is 1 μM. The mixed solution is placed in a PCR instrument at 95°C for 10 minutes, then annealed and slowly cooled to room temperature to form recognition hairpins and extension hairpins.

[0017] In some embodiments, the nucleotide sequence A constituting the first hairpin structure comprises the nucleotide sequence shown in SEQ ID NO.1.

[0018] In some embodiments, the target RNA is miR-196a or its single- or double-base mismatched miRNA, miR-196b, or miR-21.

[0019] In some embodiments, when the target RNA is miR-196a, the nucleotide sequence B that is reverse complementary to the target RNA comprises the nucleotide sequence shown in SEQ ID NO.2;

[0020] When the target RNA is miR-196a, the nucleotide sequence C that is reverse complementary to the target RNA comprises the nucleotide sequence shown in SEQ ID NO.3, the nucleotide sequence D that is reverse complementary to a portion of the nucleotide sequence C comprises the nucleotide sequence shown in SEQ ID NO.4, and the nucleotide sequence E that is reverse complementary to the target RNA comprises the nucleotide sequence shown in SEQ ID NO.5;

[0021] In some embodiments, in the extended hairpin, the nucleotide sequence F that is reverse complementary to the partial sequence of the nucleotide sequence A comprises the nucleotide sequence shown in SEQ ID NO.6.

[0022] In some embodiments, in the molecular beacon, the nucleotide sequence H that is reverse complementary to the partial sequence of nucleotide sequence A comprises the nucleotide sequence shown in SEQ ID NO.7, and the nucleotide sequence I that is reverse complementary to the partial sequence of nucleotide sequence H comprises the nucleotide sequence shown in SEQ ID NO.8.

[0023] In some embodiments, the polyT sequence is a nucleotide sequence having more than four consecutive Ts. Preferably, the polyT sequence is four consecutive Ts, five consecutive Ts, six consecutive Ts, seven consecutive Ts, eight consecutive Ts, nine consecutive Ts or ten consecutive Ts.

[0024] In some embodiments, when the target RNA is miR-196a, the recognition hairpin comprises the nucleotide sequence shown in SEQ ID NO. 9 or 10; the extended hairpin comprises the nucleotide sequence shown in SEQ ID NO. 11; and in the molecular beacon, the nucleotide sequence G constituting the second hairpin structure comprises the nucleotide sequence shown in SEQ ID NO. 12;

[0025] When the target RNA is miR-196b, the recognition hairpin comprises the nucleotide sequence shown in SEQ ID NO.18; the extended hairpin comprises the nucleotide sequence shown in SEQ ID NO.11; and in the molecular beacon, the nucleotide sequence G constituting the second hairpin structure comprises the nucleotide sequence shown in SEQ ID NO.12;

[0026] When the target RNA is miR-21, the recognition hairpin comprises the nucleotide sequence shown in SEQ ID NO.19; the extended hairpin comprises the nucleotide sequence shown in SEQ ID NO.11; and in the molecular beacon, the nucleotide sequence G constituting the second hairpin structure comprises the nucleotide sequence shown in SEQ ID NO.12.

[0027] Specifically, the nucleotide sequence structures of SEQ ID NOs. 9-11 and 18-19 are as follows. The italicized portion is the self-complementary region of the hairpin, forming the "stem" structure of the hairpin. The bold portions are the specific sequences of miR-196a, iHP-196b, and iHP-21 that recognize the targets, respectively. The underlined bases correspond to the "stem-loop" structure of the hairpin:

[0028] SEQ ID NO.9:

[0029]

[0030] SEQ ID NO.10:

[0031]

[0032] SEQ ID NO.11:

[0033]

[0034] SEQ ID NO.18:

[0035]

[0036] SEQ ID NO.19:

[0037]

[0038] Specifically, the target miR-196a sequence (SEQ ID NO.13):

[0039] 5'-CAACAACAUUAACCACCCGA-3'.

[0040] In some embodiments, the fluorescent group and the quenching group are conventional groups used for fluorescent molecular beacons. The fluorescent group can be a fluorescent group 6-carboxyfluorescein (6-FAM), Cy2 or ATTO 495; the quenching group can be a Dabcyl group, a BHQ1 group or a TAMRA group.

[0041] Specifically, the molecular beacon of the present application can be a fluorescent molecular beacon (Flu-MB), whose sequence contains a self-complementary sequence and folds under annealing conditions. At this time, the fluorescent group 6-carboxyfluorescein (6-FAM) modified at its 5' end is close to the Dabcyl group at its 3' end. When the target product is not present, the fluorescence of the 6-FAM at the 5' end is quenched by the Dabcyl group, and no signal is generated.

[0042] More specifically, the nucleotide sequence of the nucleotide sequence G constituting the second hairpin structure is as follows, the italicized portion is the region complementary to the nucleotide sequence G itself, and the bold portion is Figure 1 The sequence of the repeating unit b is identified; the nucleotide sequence G constituting the second hairpin structure is: The repeating sequence unit b (5'-AATAAGAGAT-3') of Flu-MB is reverse-complementary to the repeating sequence unit (5'-ATCTCTTATT-3') of the PER product and then opens to generate a fluorescent signal.

[0043] In some embodiments, the reagents for the primer exchange reaction include magnesium ions, Bst DNA polymerase, TE buffer, Bst Reaction Buffer and dNTPs, wherein N is A, T and C.

[0044] Specifically, the reaction system for the primer exchange reaction includes: a recognition hairpin, an extension hairpin, TE buffer, MgSO4, Bst DNA polymerase, Bst Reaction Buffer, and dNTPs (N = A, T, C). In a typical amplification reaction, 2-3 reaction system amounts more than the original reaction system of TE buffer, MgSO4, Bst reaction buffer, clean.G, dNTPs (N = A, T, C), Bst DNA polymerase, recognition hairpin, and extension hairpin are added to the same centrifuge tube, mixed and centrifuged, and then divided into 0.2mL centrifuge tubes. The miRNA sample to be tested is also added, and the tube is placed in a PCR thermostat and incubated at 37°C for 2 hours; then heated to 80°C for 20 minutes to inactivate the Bst DNA polymerase to terminate the reaction, and finally cooled to 4°C to store the product; the reaction volume of the above method is 50μL, and the concentrations of the reaction components are:

[0045] Recognize hairpin iHP: 100nM;

[0046] Extended hairpin eHP: 100 nM;

[0047] TE buffer: 1×;

[0048] MgSO4: 5mM;

[0049] Bst DNA polymerase: 8 U;

[0050] Bst Reaction Buffer: 1×;

[0051] dNTP (N=A, T, C): 100nM.

[0052] Specifically, the preparation method of the molecular beacon includes: first, using ultrapure water to dissolve the nucleotide sequence G (Flu-MB) constituting the second hairpin structure in the DNA single-stranded structure state modified with 6-carboxyfluorescein (6-FAM) at the 5' end and Dabcyl at the 3' end, so that its final concentration is 100μM for storage. The dissolved Flu-MB is diluted to 6μM, and 6μM Flu-MB and 10mM MgSO4 are added to TE buffer (pH 8.0) in a volume ratio of 1:1, so that the final concentration of Flu-MB is 600nM. The mixed solution is placed in a PCR instrument and maintained at 95°C for 10 minutes, and then slowly cooled to room temperature to obtain the hairpin structure of Flu-MB. The fluorescence of 6-FAM in the hairpin structure state is quenched by Dabcyl.

[0053] Specifically, the linear RNA may be a functional RNA, such as microRNA (miRNA), long non-coding RNA (lncRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), etc.

[0054] The second aspect of the present application provides a method for detecting linear RNA, comprising

[0055] Step 1: mixing the sample to be tested with the hairpin combination for primer exchange reaction and the reagent for primer exchange reaction of the kit for constant temperature reaction, and then heating to terminate the primer exchange reaction;

[0056] Step 2, mixing the mixture obtained in step 1 with the molecular beacon of the kit;

[0057] Step 3: Collect the signal of the molecular beacon of the reactant in step 2 and obtain the detection result after analysis.

[0058] Specifically, the method comprises: mixing the sample to be tested, the recognition hairpin, the extension hairpin, TE buffer, MgSO4, Bst DNA polymerase, Bst Reaction Buffer and dNTP (N = A, T, C), and incubating at 37 ° C for 2 hours; then heating to 80 ° C for 20 minutes to inactivate the Bst DNA polymerase to terminate the reaction; then, transferring 45 μL of the reaction product to a black 96-well plate, and then adding 10 μL of annealed Flu-MB (600 nM), and incubating in the dark for 10 minutes, and Flu-MB binds to the repeat sequence unit ( Figure 1 b) complementation, and then opening to generate a fluorescent signal. Obtain fluorescence data using a microplate reader: Place the microplate reader in the microplate reader for detection, and record the fluorescence emission spectrum from 512 nm to 600 nm at an excitation wavelength of 494 nm. The resulting data is processed using software.

[0059] The present invention is based on the isothermal nucleic acid amplification technology of the primer exchange reaction (PER), and on this basis, a kit for detecting miRNA is constructed. The kit includes a molecular beacon, a hairpin combination for the primer exchange reaction, and reagents for the primer exchange reaction. By designing a recognition hairpin and an extension hairpin with unique hairpin structures, as well as a molecular beacon with a partial sequence that can complementarily pair with the recognition hairpin, the target miRNA specifically binds to the recognition sequence at the 3' end of the recognition hairpin through base complementary pairing. Subsequently, under the catalysis of Bst DNA polymerase, a polymerase-mediated strand displacement reaction (PMSDR) is triggered, extending a specific sequence from the target miRNA. The extended product is then released from the recognition hairpin through branch migration (BM), and the recognition hairpin can be recycled to bind to new target miRNAs. Next, the specific sequence of the product, assisted by the extension hairpin and Bst polymerase, triggers another round of PMSDR and BM, ultimately releasing a single strand containing two specific sequences at its 3' end. The key is that this single strand can further activate PMSDR and BM, forming a self-perpetuating process called PER cycle. The final synthesized single-stranded PER product contains a large number of repeated specific sequences, which can be used as carriers for signal output. Molecular beacons can complementarily pair with these specific sequences to release fluorescent signals. Therefore, the system with target miRNA will produce a highly enhanced fluorescent signal, thereby enabling sensitive detection of target miRNA. Furthermore, based on the target miRNA sequence, the recognition hairpin of the present application contains two sequences that complementarily pair with the target miRNA sequence. Under annealing conditions, the free sequence forms an additional hairpin structure on the recognition hairpin based on the original structure. The target miRNA binds to most of the sequences on the hairpin structure through complementary base pairing, thereby destroying the hairpin structure and triggering subsequent amplification reactions; in the absence of a specific target, this additional hairpin structure can avoid nonspecific amplification, thereby reducing the background signal and improving the sensitivity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0061] Figure 1 This is a schematic diagram of the principle and detection scheme of the PER-based amplification method for detecting miRNA in this application;

[0062] Figure 2 Agarose gel electrophoresis of the characterization hairpin combination and amplification product of Example 3 of the present application;

[0063] Figure 3Schematic diagram of the workflow of the fluorescent molecular beacon Flu-MB provided in Example 4 of the present application;

[0064] Figure 4 Fluorescence emission spectra of the target miR-196a detected by different components of the kit provided in Example 4 of the present application;

[0065] Figure 5 The sensitivity results of the kit provided in Example 5 of this application to miRNA;

[0066] Figure 6 The specificity results of the kit provided in Example 5 of the present application for different targets;

[0067] Figure 7 The expression levels and significant differences of the recognition hairpin iHP-196a, recognition hairpin iHP-196b, recognition hairpin iHP-21, extended hairpin eHP, and Flu-MB in the serum of gastric cancer patients and healthy non-tumor patients provided in Example 6 of the present application were detected;

[0068] Figure 8 This is the ROC curve analysis of a single miRNA and a combination of three miRNAs in serum in Example 6 of the present application;

[0069] Figure 9 The base pairing of the hairpin iHP-2 identified in Example 7 of the present application;

[0070] Figure 10 The fluorescence spectra of different recognition hairpins iHP (iHP-1 and iHP-2) for the same target provided in Example 7 of the present application, wherein NC is a negative control;

[0071] Figure 11 The specificity of the second recognition hairpin (iHP-2) provided in Example 7 of the present application for recognizing the target miR-196a and the mismatch sequence. DETAILED DESCRIPTION

[0072] The present application provides a kit and method for detecting linear RNA, which is used to address the technical defects of the RNA detection methods in the prior art, such as reliance on expensive instruments, complicated operation steps and data processing, and low detection sensitivity and stability.

[0073] The following is a clear and complete description of the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0074] definition

[0075] The term "Primer Exchange Reaction (PER)" refers to a novel signal amplification technology. The classic PER contains a single hairpin, which can bind a specific sequence to the sticky end, generating a long repeat sequence that can bind a large number of signal molecules and achieve signal amplification.

[0076] "Hairpin (HP)" refers to a single-stranded DNA that contains two complementary sequences and can self-assemble and pair under annealing conditions to form a "hairpin"-like structure.

[0077] "Identify hairpin (iHP)" refers to a specific hairpin used to recognize and extend the target miRNA, which is called an identification hairpin.

[0078] "Extended hairpin (eHP)" refers to a universal hairpin that extends a single strand of DNA based on the PER cycle (denoted as "PER Cycle") amplification strategy, also known as an extended hairpin.

[0079] "Fluorescent Molecular Beacon (Flu-MB)" refers to a single-stranded DNA with a fluorescent group and a quencher modified at each end. It is also designed to contain two complementary sequences, so it can form a "hairpin" structure under annealing conditions. At this time, the quencher will quench the fluorescence; its sequence is also complementary to the amplified product. When the product is present, its "hairpin" structure will be destroyed, and the fluorescent group will emit fluorescence.

[0080] Among them, the reagents used in the following examples are all commercially available or homemade.

[0081] Sequence Listing

[0082]

[0083]

[0084] Example 1 Formation of Identification Hairpin and Extension Hairpin

[0085] This example provides a synthesis method and sequence for recognizing hairpin iHP and extending hairpin eHP:

[0086] First, dissolve the synthesized single-stranded DNA (iHP-196a (iHP) and eHP) in the appropriate volume of ultrapure water according to the synthesis instructions, achieving a final DNA concentration of 100 μM. The dissolved iHP and eHP were diluted to 10 μM and added to TE buffer (pH 8.0) with 10 mM magnesium sulfate (MgSO4) at a 1:1 volume ratio, resulting in a final DNA concentration of 1 μM. The mixed solution was placed in a PCR instrument at 95°C for 10 minutes, followed by annealing and slow cooling to room temperature to obtain a hairpin structure.

[0087] The nucleic acid sequences that recognize the hairpin iHP and the extended hairpin eHP are as follows. The italicized portion is the region complementary to the hairpin itself, forming the "stem" structure of the hairpin. The bold portion is the specific sequence of the miRNA that recognizes the target. The underlined bases correspond to the "stem-loop" structure of the hairpin:

[0088] Identifying hairpins

[0089] Extension hairpin

[0090] Example 2 Nucleic Acid Amplification Method Based on Primer Exchange Reaction (PER)

[0091] This example provides a PER reaction based on the recognition of the hairpin iHP and the extended hairpin eHP. The reaction system includes: iHP-196a (iHP), eHP, TE buffer, MgSO4, Bst DNA polymerase, Bst Reaction Buffer, and dATP / dTTP / dCTP. In a typical amplification reaction, TE buffer, MgSO4, Bst reaction buffer, clean.G, dNTPs (N=A, T, C), Bst DNA polymerase, iHP, and eHP (2-3 reaction volumes more than the original reaction volume) are added to a single centrifuge tube. After mixing and centrifugation, the tubes are aliquoted into 0.2 mL centrifuge tubes. A specific miRNA sample is also added, and the tubes are placed in a PCR amplification thermostat and incubated at 37°C for 2 hours. The reaction is then terminated by heating to 80°C for 20 minutes to inactivate the Bst DNA polymerase. Finally, the product is cooled to 4°C for storage. The PER product is a long single-stranded DNA containing a repeating sequence unit (5'-ATCTCTTATT-3') that can be used for subsequent different detection methods, including but not limited to fluorescence detection and visualization detection.

[0092] The sequence of the target miRNA sample is:

[0093] 5'-CAACAACAUUAAACCACCCGA-3'

[0094] The reaction volume of the above method is 50 μL, and the concentrations of the reaction components are:

[0095] iHP-196a (iHP): 100 nM;

[0096] eHP: 100nM;

[0097] TE buffer: 1×;

[0098] MgSO4: 5mM;

[0099] Bst DNA polymerase: 8 U;

[0100] Bst Reaction Buffer: 1×;

[0101] dNTP (N=A, T, C): 100nM.

[0102] Example 3 Characterization of hairpin structure and amplification product by agarose gel electrophoresis

[0103] Preparation of electrophoresis buffer: Dilute 5×TBE to 1×TBE with pure water. Specifically, use a graduated cylinder to measure 100 mL of 5×TBE into a 500 mL volumetric flask, dilute to 500 mL with pure water, invert the flask several times to mix well, and transfer to a covered glass bottle for storage.

[0104] To prepare an agarose gel: First, use an electronic balance to weigh 1g of agarose and add it to 50mL of 1× TBE, shaking thoroughly. Heat in a microwave oven until boiling twice, ensuring the gel solution is clear and free of bubbles. Once the temperature drops to approximately 50°C, add 5μL of 10000× Gel-Red dye to the 50mL gel and gently shake to mix thoroughly. Seal the ends of the gel plate with tape, insert an appropriate comb, pour the gel into the gel plate, and cool at room temperature to solidify. Before electrophoresis, place the agarose gel plate in a refrigerator at 4°C for at least 30 minutes.

[0105] According to the method of Example 1, the recognition hairpin iHP-1 (SEQ ID NO.9) and the extended hairpin eHP (SEQ ID NO.11) were prepared, and the PER product of iHP-1 (SEQ ID NO.9) + eHP (SEQ ID NO.11) + Target miRNA was prepared using the PER method of Example 2. 10 μL of each of the samples after the final reaction (recognition hairpin iHP-1, extended hairpin eHP, PER product) was added to an appropriate amount of DNA loading buffer (added to the previously prepared 2% agarose gel for electrophoresis experiment, wherein the electrophoresis voltage was 130 V and the gel was placed in 1×TBE buffer for 35 minutes. After the electrophoresis was completed, the gel was immediately placed under a gel imaging system for imaging analysis. The results are shown in FIG. Figure 2 ( Figure 2 Column M is the maker, column 1 is the recognition hairpin iHP-1, column 2 is the extended hairpin eHP, and column 3 is the PER product of iHP-1+eHP+Target miRNA). Figure 2 It can be seen that Example 1-2 successfully prepared the recognition hairpin iHP-1, extended hairpin eHP and PER products.

[0106] Example 4 Preparation of Fluorescent Molecular Beacon Flu-MB (Flu-MB)

[0107] The workflow diagram of the fluorescent molecular beacon Flu-MB is as follows: Figure 3 The fluorescent molecular beacon Flu-MB is prepared as follows: First, a single-stranded DNA structure containing a second hairpin structure modified with 6-carboxyfluorescein (6-FAM) at the 5' end and Dabcyl at the 3' end (Flu-MB, SEQ ID NO. 12) is dissolved in ultrapure water to a final concentration of 100 μM for storage. The dissolved Flu-MB is diluted to 6 μM. 6 μM Flu-MB and 10 mM MgSO4 are added to TE buffer (pH 8.0) at a 1:1 volume ratio to achieve a final Flu-MB concentration of 600 nM. The mixed solution is placed in a PCR instrument at 95°C for 10 minutes, then slowly cooled to room temperature to obtain the Flu-MB hairpin structure.

[0108] The target miR-196a was detected using the kit of the present application: divided into four groups, ① target miR-196a (SEQ ID NO.13), recognition hairpin iHP (SEQ ID NO.9) and extended hairpin eHP (SEQ ID NO.11), ② target miR-196a (SEQ ID NO.13) and recognition hairpin iHP (SEQ ID NO.9), ③ target miR-196a (SEQ ID NO.13) and extended hairpin eHP (SEQ ID NO.11), ④ recognition hairpin iHP (SEQ ID NO.9) and extended hairpin eHP (SEQ ID NO.11), respectively, the reagents of the above four groups were mixed with TE buffer, MgSO4, Bst DNA polymerase, Bst Reaction Buffer and dNTP (N = A, T, C), incubated at 37 ° C for 2h; then heated to 80 ° C for 20min to allow Bst The DNA polymerase was inactivated to terminate the reaction; 45 μL of the PER product was then transferred to a black 96-well plate, followed by the addition of 10 μL of annealed Flu-MB (600 nM). The plate was incubated in the dark for 10 minutes. The repeating sequence unit b (5'-AATAAGAGAT-3') of Flu-MB complemented the repeating sequence unit (5'-ATCTCTTATT-3') of the PER product and then opened to generate a fluorescent signal. Fluorescence data was obtained using a microplate reader: the microplate was placed in a microplate reader for detection, and the fluorescence emission spectrum from 512 nm to 600 nm was recorded at an excitation wavelength of 494 nm. The results are shown in Figure 2. Figure 4 Each experiment was performed in parallel three times, and the data were processed by software. Figure 4 It can be seen that the fluorescent molecular beacon Flu-MB binds to the recognition hairpin iHP and the extended hairpin eHP in the detection reaction system.

[0109] Example 5 Characterization of the performance of the kit

[0110] Sensitivity and specificity of the detection kit for miRNA.

[0111] 1. Sensitivity: Based on the aforementioned nucleic acid amplification reaction steps, miRNA with a concentration gradient was added as a target into the reaction system to explore the sensitivity of the sensor. The specific experimental steps are as follows: first, prepare the target miR-196a (SEQ ID NO.13) with 7 concentration gradients (0.1 pM-100 nM) required for the experiment, and dilute them in sequence with a dilution factor of 10 to obtain the corresponding concentration of miRNA. The prepared concentration gradient miRNA is added as a target to the reaction system (containing recognition hairpin iHP (SEQ ID NO.9), extension hairpin eHP (SEQ ID NO.11), TE buffer, MgSO4, Bst DNA polymerase, Bst Reaction Buffer and dNTP (N=A, T, C)), and incubated at 37°C for 2 hours; then heat to 80°C and maintain for 20 minutes to inactivate Bst DNA polymerase to terminate the reaction; then transfer 45 μL of PER product to a black 96-well plate, followed by adding 10 μL of annealed Flu-MB (600 nM) and incubating in the dark for 10 minutes. Use a microplate reader to obtain fluorescence data: Place the microplate reader in the microplate reader for detection, and record the fluorescence emission spectrum from 512nm to 600nm at an excitation wavelength of 494nm. The results are as follows: Figure 5 shown. Figure 5 It can be seen that the detection reaction system in which the fluorescent molecular beacon Flu-MB binds to the recognition hairpin iHP and the extended hairpin eHP has a high sensitivity.

[0112] 2. In the specificity part, based on the aforementioned nucleic acid amplification step, miRNAs with single base mismatch (OM) or double base mismatch (DM) were designed as targets and added to the reaction system to explore the specificity of the sensor. The specific experimental steps are as follows: miRNAs of different sequences and miRNAs with single base mismatch or double base mismatch (target miR-196a, 196a-OM, 196a-DM, miR-196b and miR-21, respectively) were added to the reaction system (containing recognition hairpin iHP (SEQ ID NO.9), extension hairpin eHP (SEQ ID NO.11), TE buffer, MgSO4, Bst DNA polymerase, Bst Reaction Buffer and dNTP (N = A, T, C)), incubated at 37 ° C for 2h; then heated to 80 ° C for 20min to allow Bst The DNA polymerase was inactivated to terminate the reaction; 45 μL of the PER product was then transferred to a black 96-well plate, followed by the addition of 10 μL of annealed Flu-MB (600 nM). The plate was incubated in the dark for 10 minutes. The repeating sequence unit b (5'-AATAAGAGAT-3') of Flu-MB was reverse-complemented with the repeating sequence unit (5'-ATCTCTTATT-3') of the PER product, and then opened to generate a fluorescent signal. Fluorescence data was obtained using a microplate reader: the microplate was placed in a microplate reader for detection, and the fluorescence emission spectrum from 512 nm to 600 nm was recorded at an excitation wavelength of 494 nm. The results are shown in Figure 2. Figure 6 All experiments were repeated three times. Figure 6 It can be seen that the detection reaction system of the fluorescent molecular beacon Flu-MB and the recognition hairpin iHP and extended hairpin eHP is specific.

[0113] The recognition hairpin iHP used in this example is iHP-196a (SEQ ID NO. 9), extended hairpin eHP (SEQ ID NO. 11) and Flu-MB (SEQ ID NO. 12).

[0114] The miRNA names and sequences used in this example are as follows, with the bold parts indicating base mismatch sites:

[0115] miR-196a: 5'-CAACAACAUUAAACCCACCCGA-3' (SEQ ID NO. 13);

[0116] 196a-OM:

[0117] 196a-DM:

[0118] miR-196b: 5'-UAGGUAGUUUCCUGUUGUUGGG-3' (SEQ ID NO. 16);

[0119] miR-21: 5'-UAGCUUAUCAGACUGAUGUUGA-3' (SEQ ID NO. 17).

[0120] Example 6: Research on the use of the kit to differentiate between gastric cancer and control groups

[0121] In this example, 25 gastric cancer serum samples and 10 control serum samples were collected. The cases were mainly pathologically diagnosed cases collected from the hospital, and the controls were healthy non-tumor patients.

[0122] The recognition hairpin iHP-196a (SEQ ID NO. 9), extended hairpin eHP (SEQ ID NO. 11), and Flu-MB (SEQ ID NO. 12) used in this example, and the remaining hairpin sequences are as follows:

[0123] Identification hairpin iHP-196b (SEQ ID NO. 18): (The italicized part is the complementary region of the hairpin itself, which constitutes the "stem" structure of the hairpin. The bold part is the specific sequence of the miRNA that recognizes the target. The underlined bases correspond to the "stem-loop" structure of the hairpin).

[0124] Identification of hairpin iHP-21 (SEQ ID NO. 19): (The italicized part is the complementary region of the hairpin itself, which constitutes the "stem" structure of the hairpin. The bold part is the specific sequence of the miRNA that recognizes the target. The underlined bases correspond to the "stem-loop" structure of the hairpin).

[0125] In this example, the serum miRNAs detected in gastric cancer cases and the control group were miR-196a (SEQ ID NO. 13), miR-196b (SEQ ID NO. 16), and miR-21 (SEQ ID NO. 17).

[0126] The expression levels of serum miR-196a, miR-196b, and miR-21 in gastric cancer cases and controls were examined according to the method of Example 2. The method comprises: adding a PER reaction system (containing a recognition hairpin iHP, an extension hairpin eHP, TE buffer, MgSO4, Bst DNA polymerase, Bst Reaction Buffer, and dNTPs (N = A, T, C)) to the serum of gastric cancer cases and controls, wherein the recognition hairpin of the PER reaction system is a single hairpin of iHP-196a, iHP-196b, or iHP-21, incubating at 37°C for 2 hours; then heating to 80°C for 20 minutes to allow Bst The DNA polymerase was inactivated to terminate the reaction. 45 μL of the PER product was then transferred to a black 96-well plate, followed by the addition of 10 μL of annealed Flu-MB (600 nM). The plate was incubated in the dark for 10 minutes. The repeating sequence b unit of Flu-MB (5'-AATAAGAGAT-3') reverse-complemented with the repeating sequence unit of the PER product (5'-ATCTCTTATT-3'), which then opened up and generated a fluorescent signal. Fluorescence data was acquired using a microplate reader: the plate was placed in a microplate reader for detection, and the fluorescence emission spectrum was recorded from 512 nm to 600 nm at an excitation wavelength of 494 nm. The test samples were clinical serum samples from non-cancer controls (NC, n = 10) and gastric cancer patients (GC, n = 25) to quantify the expression levels of these three miRNAs (miR-196a, miR-196b and miR-21) in serum. The significant difference analysis of the detection results between cancer and control groups was performed using SPSS software; Lasso regression was used to define the serum signal (Serum-Sig) of each sample as a weighted linear combination of miR-196a, miR-196b and miR-21. The results are shown in Figure 2. Figure 7 As shown, the significant difference analysis showed that compared with the single miRNA markers (miR-21, P = 0.0873; miR-196a, P = 0.2293; miR-196b, P = 0.0014), the P value of the combination of these three miRNAs was less than 0.0001, indicating that there was a significant difference between the two groups based on the detection analysis of the combination of these three miRNAs.

[0127] At the same time, the above data were analyzed by ROC curve. The results are as follows Figure 8As shown, the diagnostic performance of these serum-miRNA and EV-Sigs was quantitatively evaluated by receiver operating characteristic (ROC) curve analysis. The area under the ROC curve for each individual miRNA marker was 0.676 (miR-21), 0.612 (miR-196a), and 0.876 (miR-196b), respectively. The combined serum-Sig of miR-196a, miR-196b, and miR-21 significantly improved diagnostic performance, with an AUC of 0.908.

[0128] Example 7 Performance Study of the Second Identification Hairpin (iHP-2)

[0129] This embodiment innovatively designs a recognition hairpin with an additional hairpin structure (called iHP-2), the base pairing of which is as follows: Figure 9 As shown in the figure, two complementary sequences (nucleotide sequence C and nucleotide sequence E) were designed based on the target miRNA sequence (miR-196a). Under annealing conditions, this free sequence forms an additional hairpin structure based on the original structure. The target miRNA binds to the majority of the sequence in this hairpin structure through complementary base pairing, thereby destroying the hairpin structure and triggering the subsequent amplification reaction. In the absence of a specific target, this additional hairpin structure can prevent nonspecific amplification, thereby reducing background signal and improving detection sensitivity.

[0130] The kit used in this example includes: extended hairpin eHP (SEQ ID NO.11), target miRNA (miR-196a, SEQ ID NO.13) and Flu-MB (SEQ ID NO.12). The recognition sequences of hairpin iHP are:

[0131] iHP-1 (SEQ ID NO. 9):

[0132]

[0133] iHP-2 (SEQ ID NO. 10):

[0134]

[0135] The miRNA names and sequences used in this example are as follows, with the bold parts indicating base mismatch sites:

[0136] miR-196a: 5'-CAACAACAUUAAACCCACCCGA-3' (SEQ ID NO. 13);

[0137] 196a-OM:

[0138] 196a-DM:

[0139] This example uses fluorescence spectroscopy to quantitatively analyze the ability of the iHP-2 recognition hairpin, which has an additional hairpin structure, to reduce background signal and noise. Based on the nucleic acid amplification steps described in Examples 2-5, equal concentrations and volumes of recognition hairpins with different sequences (iHP-1 and iHP-2) were used as controls, and equal amounts of target (miR-196a) were added to investigate the performance of the designed iHP-2 recognition hairpin. The specific experimental steps are as follows: First, the same concentrations of recognition hairpin iHP (100 nM, iHP-1 and iHP-2) required for the experiment were prepared, and then divided into four groups, namely ① recognition hairpin iHP-1 + target miRNA (miR-196a, SEQ ID NO.13) + Flu-MB (SEQ ID NO.12), ② recognition hairpin iHP-2 + target miRNA (miR-196a, SEQ ID NO.13) + Flu-MB (SEQ ID NO.12), ③ recognition hairpin iHP-1 + Flu-MB (SEQ ID NO.12), ④ recognition hairpin iHP-2 + Flu-MB (SEQ ID NO.12). The above four groups were Figure 10 They were labeled as iHP-1, iHP-2, NC-iHP-1 and NC-iHP-2, respectively. These four groups of components were then added to the PER reaction system (TE buffer, MgSO4, Bst DNA polymerase, Bst Reaction Buffer and dNTP (N=A, T, C)) and incubated at 37°C for 2 hours. The mixture was then heated to 80°C for 20 minutes to inactivate the Bst DNA polymerase and terminate the reaction. 45 μL of the PER product was then transferred to a black 96-well plate, followed by the addition of 10 μL of annealed Flu-MB (600 nM). The plates were incubated in the dark for 10 minutes. The repeating sequence unit b (5'-AATAAGAGAT-3') of Flu-MB was reversely complemented with the repeating sequence unit (5'-ATCTCTTATT-3') of the PER product and then opened to generate a fluorescent signal. Use a microplate reader to obtain fluorescence data: Place the microplate reader in the microplate reader for detection, and record the fluorescence emission spectrum from 512nm to 600nm at an excitation wavelength of 494nm. The results are as follows: Figure 10 As shown, Figure 10 The results show that the background signal of the reaction system with an additional hairpin structure for recognizing hairpin iHP-2 is significantly reduced.

[0140] This example is based on the aforementioned nucleic acid amplification step and uses the previously designed single-base mismatch (OM) or double-base mismatch (DM) miRNAs as targets (i.e., miR-196a, 196a-OM, and 196a-DM) added to the reaction system to explore the specificity of iHP-2. The specific experimental steps are as follows: miRNAs with different sequences (target miR-196a, 196a-OM and 196a-DM) were added to the reaction system (containing iHP-2 (SEQ ID NO.10), extended hairpin eHP (SEQ ID NO.11), TE buffer, MgSO4, Bst DNA polymerase, Bst Reaction Buffer and dNTP (N = A, T, C)), and incubated at 37 ° C for 2 hours; then heated to 80 ° C for 20 minutes to inactivate Bst DNA polymerase to terminate the reaction; then 45 μL of PER product was transferred to a black 96-well plate, and then 10 μL of annealed Flu-MB (600 nM) was added, and incubated in the dark for 10 minutes. The repeat sequence unit b (5'-AATAAGAGAT-3') of Flu-MB and the repeat sequence unit (5'-ATCTCTTATT-3') of the PER product were reversely complemented and opened to generate a fluorescent signal. Use a microplate reader to obtain fluorescence data: Place the microplate reader in the microplate reader for detection, and record the fluorescence emission spectrum from 512nm to 600nm at an excitation wavelength of 494nm. All experiments were repeated three times. The results are as follows Figure 11 shown. Figure 10 The results showed that the recognition hairpin iHP-2 with an additional hairpin structure was specific.

[0141] In summary, the key point of this application is an isothermal amplification method based on a primer exchange reaction, using different recognition hairpin structures (iHP-1 and iHP-2) as recognition elements, combined with an extended hairpin (eHP) to generate a single-stranded product with a repeating sequence unit. The eHP designed in this application has universal applicability. By designing the sticky end sequence at the 3' end of the iHP, it can achieve rapid detection and analysis of different miRNAs. Modifying the sticky end with different recognition elements can also expand the detection targets beyond miRNA. Furthermore, the PER product of this application has a repeating b sequence unit, based on which Flu-MB can be used to quantify the expression level of the target miRNA through fluorescence signal, achieving the purpose of simple and rapid diagnosis. Subsequent detection can be combined with various detection methods, including but not limited to the fluorescence detection method mentioned in this application. Furthermore, the iHP-2 designed in this application is innovative, utilizing an additional hairpin structure to ensure detection specificity and reduce nonspecific amplification. Subsequent modification of the iHP with antibodies, aptamers, and other structures can also expand the detection targets to molecules other than nucleic acids, including but not limited to proteins and lipids.

[0142] The above is only a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A kit for detecting linear RNA, characterized in that: include: Molecular beacons, hairpin assemblies for primer exchange reactions, and reagents for primer exchange reactions; The hairpin combination comprises an identification hairpin and an extension hairpin; The recognition hairpin contains, in sequence: a nucleotide sequence A constituting a first hairpin structure, a nucleotide sequence B that is reverse complementary to the target RNA, and a polyT sequence; or The recognition hairpin contains, in order: a nucleotide sequence A constituting a first hairpin structure, a nucleotide sequence C reversely complementary to the target RNA, a nucleotide sequence D reversely complementary to a portion of the nucleotide sequence C, and a nucleotide sequence E reversely complementary to the target RNA; The extended hairpin sequentially contains: a nucleotide sequence A constituting a first hairpin structure, a nucleotide sequence F that is reverse complementary to a portion of the nucleotide sequence A, and a polyT sequence; The molecular beacon contains: a nucleotide sequence G constituting a second hairpin structure, and a fluorescent group and a quenching group coupled to both ends of the nucleotide sequence G, wherein the nucleotide sequence G contains in sequence: a nucleotide sequence H that is reverse complementary to a partial sequence of the nucleotide sequence A, and a nucleotide sequence I that is reverse complementary to a partial sequence of the nucleotide sequence H.

2. The kit according to claim 1, wherein The nucleotide sequence A constituting the first hairpin structure comprises the nucleotide sequence shown in SEQ ID NO.

1.

3. The kit according to claim 1, wherein The target RNA is miR-196a or its single- or double-base mismatched miRNA, miR-196b or miR-21.

4. The kit according to claim 3, wherein When the target RNA is miR-196a, the nucleotide sequence B that is reverse complementary to the target RNA comprises the nucleotide sequence shown in SEQ ID NO.2; When the target RNA is miR-196a, the nucleotide sequence C that is reverse complementary to the target RNA comprises the nucleotide sequence shown in SEQ ID NO.3, the nucleotide sequence D that is reverse complementary to the partial sequence of nucleotide sequence C comprises the nucleotide sequence shown in SEQ ID NO.4, and the nucleotide sequence E that is reverse complementary to the target RNA comprises the nucleotide sequence shown in SEQ ID NO.

5.

5. The kit according to claim 1, wherein In the extended hairpin, the nucleotide sequence F that is reverse complementary to the partial sequence of the nucleotide sequence A comprises the nucleotide sequence shown in SEQ ID NO.

6.

6. The kit according to claim 1, wherein In the molecular beacon, the nucleotide sequence H that is reverse complementary to the partial sequence of nucleotide sequence A comprises the nucleotide sequence shown in SEQ ID NO.7, and the nucleotide sequence I that is reverse complementary to the partial sequence of nucleotide sequence H comprises the nucleotide sequence shown in SEQ ID NO.

8.

7. The kit according to claim 1, wherein The polyT sequence is a nucleotide sequence having more than four consecutive Ts.

8. The kit according to claim 3, wherein When the target RNA is miR-196a, the recognition hairpin comprises the nucleotide sequence shown in SEQ ID NO. 9 or 10; the extended hairpin comprises the nucleotide sequence shown in SEQ ID NO. 11; and in the molecular beacon, the nucleotide sequence G constituting the second hairpin structure comprises the nucleotide sequence shown in SEQ ID NO. 12; When the target RNA is miR-196b, the recognition hairpin comprises the nucleotide sequence shown in SEQ ID NO.18; the extended hairpin comprises the nucleotide sequence shown in SEQ ID NO.11; and in the molecular beacon, the nucleotide sequence G constituting the second hairpin structure comprises the nucleotide sequence shown in SEQ ID NO.12; When the target RNA is miR-21, the recognition hairpin comprises the nucleotide sequence shown in SEQ ID NO.19; the extended hairpin comprises the nucleotide sequence shown in SEQ ID NO.11; and in the molecular beacon, the nucleotide sequence G constituting the second hairpin structure comprises the nucleotide sequence shown in SEQ ID NO.

12.

9. The kit according to any one of claims 1 to 8, characterized in that The reagents for the primer exchange reaction include magnesium ions, Bst DNA polymerase, TE buffer, Bst Reaction Buffer and dNTPs, wherein the N is A, T and C.

10. A method for detecting linear RNA, characterized in that: Step 1: mixing a sample to be tested with a hairpin combination for primer exchange reaction and a reagent for primer exchange reaction of the kit according to any one of claims 1 to 9, performing a constant temperature reaction, and then heating to terminate the primer exchange reaction; Step 2, mixing the mixture obtained in step 1 with the molecular beacon of the kit according to any one of claims 1 to 9; Step 3: Collect the signal of the molecular beacon of the reactant in step 2 and obtain the detection result after analysis.

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