Mirna detection method and kit

Abstract

Provided is a miRNA detection method. More specifically, provided is a method for detecting miRNA on the basis of a stem-loop target miRNA-related reverse transcription primer, and provided are a reverse transcription primer and amplification primers for implementing the method, and a method for designing miRNA detection primers.

Description

A method and kit for detecting miRNA

[0001] This application claims the benefit of Chinese application No. 202411108567.0, filed on August 13, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of molecular biology detection, in particular, the present application relates to a method for detecting miRNA. More specifically, the present application provides a method for detecting miRNA based on a stem region target miRNA related reverse transcription primer, and provides a reverse transcription primer for implementing the method and an amplification primer, and a method for designing miRNA detection primers. BACKGROUND

[0003] MicroRNA (miRNA) is a short chain (about 22 nucleotides) non-coding RNA, widely exists in animals, plants, nematodes and other eukaryotes, is an important post-transcriptional regulatory factor of gene expression, usually combined with messenger RNA to mediate direct cleavage or indirect inhibition of translation (Bartel, D. P. (2004). "MicroRNAs: genomics, biogenesis, mechanism, and function." Cell 116(2): 281-297; He, L. and G. J. Hannon (2004). "MicroRNAs: small RNAs with a big role in gene regulation." Nat Rev Genet 5(7): 522-531). Studies have shown that miRNA plays an important role in cell differentiation, proliferation, apoptosis, individual growth and development, and organ formation. There is a fine regulation mechanism of miRNA expression in vivo, which follows strict spatiotemporal specificity. In addition, studies have found that there are circulating miRNAs in blood and they are very stable (Chen, X., et al (2008). "Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases." Cell Res 18(10): 997-1006), more importantly, the abnormal expression of circulating miRNA is closely related to the occurrence and development of many diseases (Cortez, M. A., et al (2011). "MicroRNAs in body fluids--the mix of hormones and biomarkers." Nat Rev Clin Oncol 8(8): 467-477; Kawaguchi, T., et al (2016). "Circulating MicroRNAs: A Next-Generation Clinical Biomarker for Digestive System Cancers." Int J Mol Sci 17(9): 1459), suggesting that miRNA can be used as a liquid biopsy biomarker for early diagnosis and prognosis evaluation of major diseases such as cancer.

[0004] For a long time, reverse transcription real-time quantitative PCR (RT-qPCR) based detection technique has been considered as the most sensitive and effective method for quantifying RNA species. However, due to the short sequence of miRNA, traditional quantitative detection scheme designed for long chain RNA is not applicable. The RT-qPCR detection method based on Taqman probe developed by Chen et al (Chen, C., et al (2005). "Real-time quantification of microRNAs by stem-loop RT-PCR." Nucleic Acids Res 33(20): e179) uses miRNA specific reverse transcription stem-loop primer to enhance the thermal stability of miRNA and DNA (reverse transcription stem-loop primer) hybrid double-stranded, so that the reverse transcription has high sensitivity; at the same time, the space constraint produced by the stem-loop primer makes the reverse transcription have high specificity, which is conducive to the distinction between mature miRNA and miRNA precursor. However, due to the additional probe hydrolysis step, the compatibility of the RT-qPCR detection method based on Taqman probe with rapid thermal cycling scheme for detecting miRNA needs to be further optimized. In addition, the design of specific TaqMan probe for each miRNA is not only too high in cost but also technically challenging and faces practical application difficulties. In order to solve this problem, although there have been studies by using universal or common reverse PCR primers and / or fluorescent probes to amplify and detect multiple miRNAs (Varkonyi-Gasic, E., et al (2007). "Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs." Plant Methods 3: 12; Yang, H., et al (2009). "A novel real-time polymerase chain reaction method for high throughput quantification of small regulatory RNAs." Plant Biotechnol J 7(7): 621-630). This strategy avoids the complexity of designing different specific probes for different miRNAs and reduces the detection cost, but the specificity of real-time PCR of this strategy is only achieved by forward PCR primer, which is not enough to distinguish many kinds of homologous miRNAs.Wan et al. used a new method of deoxyuridine-embedded oligonucleotide and hemi-nested primer to quantitatively detect miRNA (Wan, G., et al (2010). "High-performance quantification of mature microRNAs by real-time RT-PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers." RNA 16(7): 1436-1445), by reducing / eliminating the interference of RT primers on PCR amplification, three-primer specific amplification and combination with dye detection, not only improved the specificity of miRNA detection, but also greatly reduced the cost of multiple miRNA detection. However, dye detection cannot distinguish between non-specific fluorescent signals caused by primer dimers during amplification and specific fluorescent signals of target amplification in nature. Non-specific signals may reduce the accuracy of miRNA quantification, thereby masking the specific fluorescent signal of low concentration template or making the quantitative detection result deviate from the true level, especially when multiple miRNA detection is performed, the number of nucleotide sequences in the reaction system increases, and the probability of generating dimers also increases.

[0005] miRNA is a promising liquid biopsy biomarker for early diagnosis and prognosis of major diseases such as cancer. However, the current miRNA detection has some problems mentioned above, which may be one of the reasons that limit its clinical application. Therefore, it is necessary to develop a better miRNA detection method that can solve these problems and pave the way for the clinical application of miRNA detection. SUMMARY

[0006] The present application provides a new method for detecting mature miRNA in a biological sample, which has good specificity and high sensitivity and can be widely used for quantitative detection of mature miRNA.

[0007] Detection method

[0008] Therefore, in a first aspect, the present application provides a method for detecting the presence and / or content of target miRNA (MicroRNA) in a sample to be tested, comprising the following steps:

[0009] (1) providing a reverse transcription primer and a sample to be tested, contacting the reverse transcription primer with the sample to be tested under conditions allowing reverse transcription, and performing a reverse transcription reaction;

[0010] wherein the 5' end of the reverse transcription primer comprises a stem-loop structure, the loop region of the stem-loop structure comprises a tag sequence (Ts sequence), and the stem region of the stem-loop structure comprises a local double-stranded structure formed by annealing of a stem sequence (Ss sequence) and its complementary sequence (SsC sequence), wherein the Ss sequence is downstream of the SsC sequence;

[0011] the 3' end of the reverse transcription primer comprises a capture sequence (Cs sequence) capable of annealing to the target miRNA and initiating an extension reaction;

[0012] (2) contacting the first primer and the second primer with the product obtained in the previous step under conditions allowing nucleic acid amplification, and performing a nucleic acid amplification reaction;

[0013] wherein the first primer comprises or consists of a first region and a second region from 5' to 3' direction; the first region comprises the Ts sequence, and the second region is capable of annealing to the cDNA sequence of the target miRNA and initiating an extension reaction;

[0014] the second primer comprises or consists of a first region, a second region and a third region from 5' to 3' direction; the first region comprises the Ts sequence, the second region comprises the Ss sequence, and the third region is capable of annealing to the complementary sequence of the cDNA sequence of the target miRNA and initiating an extension reaction;

[0015] (3) assessing the presence and / or the amount of the target miRNA in the sample to be tested according to the presence and / or the amount of the nucleic acid amplification product of step (2).

[0016] In certain embodiments, the sample to be tested contains nucleic acids. In certain embodiments, in step (1), the reverse transcription primer is contacted with the nucleic acids in the sample to be tested.

[0017] In certain embodiments, the sample to be tested is suspected to contain the target miRNA.

[0018] It is readily understood by those skilled in the art that the Ss sequence and the SsC sequence can have complete complementarity (i.e., each nucleic acid base in the Ss sequence can form Watson-Crick base pairing with a nucleic acid base in the SsC sequence), or partial complementarity, as long as the Ss sequence and the SsC sequence can anneal to each other to form an intra-chain local double-stranded structure.

[0019] In certain embodiments, the first region of the first primer comprises the Ts sequence and additional nucleotide sequences, or the first region of the first primer consists of the Ts sequence.

[0020] In certain embodiments, the first region of the second primer comprises the Ts sequence and additional nucleotide sequences, or the first region of the second primer consists of the Ts sequence.

[0021] In certain embodiments, the second region of the second primer comprises the Ss sequence and additional nucleotide sequences, or the second region of the second primer consists of the Ss sequence.

[0022] In certain embodiments, the second region of the first primer is capable of annealing to the 3' end (e.g., 3' terminal end) of the cDNA sequence of the target miRNA.

[0023] In certain embodiments, the second region of the first primer is complementary to the 3' end (e.g., 3' terminal end) sequence of the cDNA sequence of the target miRNA.

[0024] In certain embodiments, the Ss sequence comprises the sequence of the second region of the first primer or a partial sequence thereof (e.g., a 5' terminal partial sequence of the second region of the first primer), and the Ss sequence has one or more (e.g., 1-4, 1, 2, 3, or 4) substitution mutations of nucleotide residues compared to the sequence of the second region of the first primer or the partial sequence thereof (e.g., the 5' terminal partial sequence of the second region of the first primer).

[0025] In certain embodiments, the Ss sequence comprises a DNA sequence identical to the 5' end (e.g., 5' terminal end) sequence of the target miRNA, and the Ss sequence has one or more (e.g., 1-4, 1, 2, 3, or 4) substitution mutations of nucleotide residues compared to the DNA sequence.

[0026] As readily understood by one skilled in the art, the DNA sequence identical to the 5' end sequence of the target miRNA means a DNA sequence formed by replacing ribonucleotide residues in the 5' end sequence of the target miRNA with corresponding deoxyribonucleotide residues and replacing uracil ribonucleotide residues therein with thymine deoxyribonucleotide residues.

[0027] In certain embodiments, the Ss sequence comprised by the second primer has substitution mutations as defined above.

[0028] In certain embodiments, the second primer comprises a segment I consisting of an upstream sequence of the nucleotide residue containing the substitution mutation closest to the 5' terminal end of the second primer, a segment II consisting of a downstream sequence of the nucleotide residue containing the substitution mutation closest to the 3' terminal end of the second primer; and the second primer has one or more of the features selected from the following:

[0029] (1) the Tm value of the segment II is greater than or equal to 30°C;

[0030] (2) the Tm value of the segment II differs from the Tm value of the segment I by no more than 10°C;

[0031] (3) the Tm value of the segment II is greater than or equal to the Tm value of the segment I.

[0032] In certain embodiments, the loop region of the reverse transcription primer further comprises a universal sequence (Us sequence).

[0033] In certain embodiments, the Us sequence is located upstream of the Ts sequence.

[0034] In certain embodiments, the Us sequence has a length of 0-6 nt (e.g., 1-6 nt, 2-6 nt, 3-6 nt, 4-6 nt, 1-5 nt, 2-5 nt, 3-5 nt, 4-5 nt, 4 nt).

[0035] In certain embodiments, the third region of the second primer is capable of annealing to the 3’ end of the complement of the cDNA sequence of the target miRNA.

[0036] In certain embodiments, the third region of the second primer comprises a sequence complementary to the 3’ end (e.g., 3’ terminal) sequence of the target miRNA.

[0037] In certain embodiments, the third region of the second primer comprises the Cs sequence of the reverse transcription primer or a partial sequence thereof.

[0038] In certain embodiments, the method possesses one or more of the features selected from the group consisting of:

[0039] (1) the Ts sequence has a length of 3-15 nt (e.g., 4-15 nt, 5-15 nt, 3-12 nt, 4-12 nt, 5-12 nt, 3-10 nt, 4-10 nt, 5-10 nt, 5 nt, 10 nt);

[0040] (2) the Ss sequence has a length of 6-15 nt (e.g., 8-15 nt, 6-12 nt, 8-12 nt, 6-9 nt, 8 nt);

[0041] (3) the Cs sequence of the reverse transcription primer is capable of annealing to the 3’ end (e.g., 3’ terminal) of the target miRNA; for example, the Cs sequence of the reverse transcription primer is complementary to the 3’ end (e.g., 3’ terminal) sequence of the target miRNA;

[0042] (4) the length of the Cs sequence of the reverse transcription primer is 4-10 nt (e.g., 5-10 nt, 6-10 nt, 7-10 nt, 4-8 nt, 5-8 nt, 6-8 nt, 7-8 nt, 7 nt);

[0043] (5) the length of the second region of the first primer is 10-30 nt (e.g., 12-30 nt, 15-30 nt, 10-25 nt, 12-25 nt, 15-25 nt, 10-20 nt, 12-20 nt, 15-20 nt);

[0044] (6) the length of the third region of the second primer is 4-10 nt (e.g., 5-10 nt, 6-10 nt, 7-10 nt, 4-8 nt, 5-8 nt, 6-8 nt, 7-8 nt, 7 nt);

[0045] (7) the target miRNA is a mature miRNA.

[0046] In some embodiments, the target miRNA exists in a single-stranded form.

[0047] In some embodiments, the method is used to detect the presence and / or the amount of one or more target miRNAs in a test sample.

[0048] In some embodiments, the method is used to detect the presence and / or the amount of a plurality of target miRNAs in a test sample;

[0049] wherein, in step (1), a plurality of reverse transcription primers respectively for different kinds of target miRNAs are provided, and the plurality of reverse transcription primers are contacted with nucleic acids in the test sample under conditions allowing reverse transcription, and a reverse transcription reaction is performed; and,

[0050] In step (2), a plurality of first primers and a plurality of second primers respectively for different kinds of target miRNAs are provided;

[0051] In step (3), the first primer and the second primer for a specific kind of target miRNA are contacted with the product obtained in the previous step under conditions allowing nucleic acid amplification, and a nucleic acid amplification reaction is performed in different reaction systems.

[0052] In some embodiments, in step (3), the presence and / or the amount of the nucleic acid amplification product in each reaction system in step (2) is used to evaluate the presence and / or the amount of each of the plurality of target miRNAs in the test sample.

[0053] In some embodiments, in step (2), the nucleic acid amplification reaction is a fluorescent quantitative PCR or an isothermal amplification.

[0054] In certain embodiments, in step (3), the presence and / or the amount of the target miRNA in the sample to be tested is determined by the presence and / or the amount of the amplification product of the fluorescent quantitative PCR or isothermal amplification.

[0055] In certain embodiments, in step (3), the method further comprises comparing the amount of the amplification product of the target miRNA standard with the amount of the amplification product of the sample to be tested in step (2) to evaluate the presence and / or the amount of the target miRNA in the sample to be tested.

[0056] In certain embodiments, the target miRNA standard is a sample (e.g., a purified sample) comprising the target miRNA at a known concentration or copy number.

[0057] In certain embodiments, the first primer is modified with a first group, and the second primer is modified with a second group;

[0058] wherein the first group is a first fluorescent group, and the second group is a second fluorescent group; or, the first group is a fluorescent group, and the second group is a fluorescent quenching group; or, the first group is a fluorescent quenching group, and the second group is a fluorescent group;

[0059] and, the first group and the second group are capable of achieving fluorescence resonance energy transfer during amplification.

[0060] In certain embodiments, the first group and the second group in the same amplification product molecule are capable of achieving fluorescence resonance energy transfer.

[0061] In certain embodiments, the first group is located 6-9 (e.g., 6, 7, 8, 9) nucleotide residues away from the 3’ end of the first primer; and / or, the second group is located 6-9 (e.g., 6, 7, 8, 9) nucleotide residues away from the 3’ end of the second primer.

[0062] In certain embodiments, the first group is a first fluorescent group, and the second group is a second fluorescent group, and the combination of the first fluorescent group and the second fluorescent group is selected from:

[0063] TAMRA and FAM, CY3 and CY5, FAM and Dabcyl, FAM and Light Cycler Red 610, FAM and Light Cycler Red 640, FAM and Light Cycler Red 670, FAM and Light Cycler Red 705, FAM and TAMRA, CY5 and CY3, Dabcyl and FAM, Light Cycler Red 610 and FAM, Light Cycler Red 640 and FAM, Light Cycler Red 670 and FAM, Light Cycler Red 705 and FAM.

[0064] In certain embodiments, the first group is a fluorescent group and the second group is a fluorescent quenching group; or, the first group is a fluorescent quenching group and the second group is a fluorescent group.

[0065] In certain embodiments, the fluorescent group is selected from the group consisting of FAM, TAMRA, HEX, ROX, CY3, CY5, TET, VIC, NED, JOE, Texas Red, CY5.5, and any combination thereof; and the fluorescent quenching group is selected from the group consisting of BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, and any combination thereof.

[0066] In certain embodiments, the combination of the fluorescent group and the fluorescent quenching group is selected from the group consisting of: FAM and BHQ1, TET and BHQ1, JOE and BHQ1, VIC and BHQ1, HEX and BHQ1, CY3 and BHQ2, NED and BHQ2, TAMRA and BHQ2, ROX and BHQ2, Texas Red and BHQ2, CY5 and BHQ2 / BHQ3, CY5.5 and BHQ2 / BHQ3, FAM and Dabcyl, TET and Dabcyl, JOE and Dabcyl, VIC and Dabcyl, HEX and Dabcyl, FAM and Eclipse, TAMRA and Eclipse, ROX and Eclipse, JOE and Eclipse, FAM and MGB, TET and MGB, JOE and MGB, VIC and MGB, HEX and MGB.

[0067] Without being limited by theory, the methods provided herein can be used to detect a target miRNA of any origin. For example, the target miRNA can be derived from a bodily fluid sample, a cell sample, an extracted sample, or any combination thereof. For example, in step (1) of the method, the sample to be tested is a cell sample, the method further comprises lysing the cells to release nucleic acid molecules in the cells, thereby contacting the nucleic acid molecules in the sample to be tested with the reverse transcription primer.

[0068] As will be readily understood by those skilled in the art, the methods of the present aspect are not limited to detection of a specific kind of miRNA, and can be used to detect any kind of miRNA (e.g., any kind of miRNA whose sequence is known). For example, for any miRNA having a given sequence, reverse transcription primers and first and second primers directed to the miRNA can be designed and provided based on the methods of the present aspect, and specific detection of the miRNA can be achieved by the methods of the present aspect.

[0069] In certain embodiments, the target miRNA is selected from the group consisting of hsa-miR-92a-3p, hsa-miR-25-3p, and any combination thereof.

[0070] In certain embodiments, the target miRNA is hsa-miR-92a-3p, and the first primer has a sequence as set forth in SEQ ID NO: 37, and the second primer has a sequence as set forth in any one of SEQ ID NOs: 39-41, 43.

[0071] In certain embodiments, the target miRNA is hsa-miR-92a-3p, and the first primer has a sequence as set forth in SEQ ID NO: 81, and the second primer has a sequence as set forth in SEQ ID NO: 80.

[0072] In certain embodiments, the target miRNA is hsa-miR-92a-3p, and the reverse transcription primer has a sequence as set forth in any one of SEQ ID NOs: 76-79 (e.g., the reverse transcription primer has a sequence as set forth in any one of SEQ ID NOs: 77-79; e.g., the reverse transcription primer has a sequence as set forth in SEQ ID NO: 78), the first primer has a sequence as set forth in SEQ ID NO: 81, and / or the second primer has a sequence as set forth in SEQ ID NO: 80.

[0073] In certain embodiments, the target miRNA is hsa-miR-25-3p, and the first primer has a sequence as set forth in SEQ ID NO: 45, and the second primer has a sequence as set forth in any one of SEQ ID NOs: 48-49, 51-52, 54.

[0074] In certain embodiments, the method is for diagnostic purposes.

[0075] In certain embodiments, the method is for non-diagnostic purposes.

[0076] Kit

[0077] In a second aspect, the present application provides a kit comprising a reverse transcription primer for detecting a target miRNA, and a first primer and a second primer;

[0078] wherein the 5' end of the reverse transcription primer comprises a stem-loop structure, the loop region of the stem-loop structure comprises a tag sequence (Ts sequence), and the stem region of the stem-loop structure comprises a local double-stranded structure formed by annealing of a stem sequence (Ss sequence) and a complementary sequence (SsC sequence) thereof, wherein the Ss sequence is downstream of the SsC sequence;

[0079] the 3' end of the reverse transcription primer comprises a capture sequence (Cs sequence) capable of annealing to the target miRNA and initiating an extension reaction;

[0080] the first primer comprises or consists of, from 5' to 3' direction, a first region and a second region; the first region comprises the Ts sequence, and the second region is capable of annealing to the cDNA sequence of the target miRNA and initiating an extension reaction;

[0081] the second primer comprises or consists of, from 5' to 3' direction, a first region, a second region and a third region; the first region comprises the Ts sequence, the second region comprises the Ss sequence, and the third region is capable of annealing to the complement of the cDNA sequence of the target miRNA and initiating an extension reaction.

[0082] As will be readily understood by those skilled in the art, the Ss sequence and the SsC sequence can have complete complementarity (i.e., each nucleic acid base in the Ss sequence can form Watson-Crick base pairing with a nucleic acid base in the SsC sequence), or partial complementarity, as long as the Ss sequence and the SsC sequence can anneal to each other to form an intrachain local double-stranded structure.

[0083] In certain embodiments, the first region of the first primer comprises the Ts sequence and an additional nucleotide sequence, or the first region of the first primer consists of the Ts sequence.

[0084] In certain embodiments, the first region of the second primer comprises the Ts sequence and an additional nucleotide sequence, or the first region of the second primer consists of the Ts sequence.

[0085] In certain embodiments, the second region of the second primer comprises the Ss sequence and an additional nucleotide sequence, or the second region of the second primer consists of the Ss sequence.

[0086] In certain embodiments, the second region of the first primer is capable of annealing to the 3' end (e.g., 3' terminal end) of the cDNA sequence of the target miRNA.

[0087] In certain embodiments, the second region of the first primer is complementary to the 3' end (e.g., 3' terminal end) sequence of the cDNA sequence of the target miRNA.

[0088] In certain embodiments, the Ss sequence comprises the sequence of the second region of the first primer or a partial sequence thereof (e.g., a 5' terminal partial sequence of the second region of the first primer), and the Ss sequence has one or more (e.g., 1-4, 1, 2, 3, or 4) substitution mutations of nucleotide residues compared to the sequence of the second region of the first primer or the partial sequence thereof (e.g., the 5' terminal partial sequence of the second region of the first primer).

[0089] In certain embodiments, the Ss sequence comprises the same DNA sequence as the 5' end (e.g., 5' terminal end) sequence of the target miRNA, and the Ss sequence has one or more (e.g., 1-4, 1, 2, 3, or 4) substitution mutations of nucleotide residues compared to the DNA sequence.

[0090] As will be readily understood by one skilled in the art, the DNA sequence identical to the 5' end sequence of the target miRNA means a DNA sequence formed by replacing ribonucleotide residues in the 5' end sequence of the target miRNA with corresponding deoxyribonucleotide residues and replacing uracil ribonucleotide residues therein with thymine deoxyribonucleotide residues.

[0091] In certain embodiments, the Ss sequence of the second primer has substitution mutations as defined above.

[0092] In certain embodiments, the second primer comprises a segment I consisting of an upstream sequence of the nucleotide residue having the substitution mutation closest to the 5' terminal end of the second primer and a segment II consisting of a downstream sequence of the nucleotide residue having the substitution mutation closest to the 3' terminal end of the second primer; and the second primer has one or more of the following features selected from the group consisting of:

[0093] (1) the Tm value of the segment II is greater than or equal to 30°C;

[0094] (2) the Tm value of the segment II differs from the Tm value of the segment I by no more than 10°C;

[0095] (3) the Tm value of the segment II is greater than or equal to the Tm value of the segment I.

[0096] In some embodiments, the loop region of the reverse transcription primer further comprises a universal sequence (Us sequence).

[0097] In some embodiments, the Us sequence is located upstream of the Ts sequence.

[0098] In some embodiments, the Us sequence has a length of 0-6 nt (e.g., 1-6 nt, 2-6 nt, 3-6 nt, 4-6 nt, 1-5 nt, 2-5 nt, 3-5 nt, 4-5 nt, 4 nt).

[0099] In some embodiments, the third region of the second primer is capable of annealing to the 3’ end of the complementary sequence of the cDNA sequence of the target miRNA.

[0100] In some embodiments, the third region of the second primer comprises a sequence complementary to the 3’ end (e.g., 3’ terminal end) sequence of the target miRNA.

[0101] In some embodiments, the third region of the second primer comprises the Cs sequence of the reverse transcription primer or a partial sequence thereof.

[0102] In some embodiments, the kit comprises one or more features selected from the following:

[0103] (1) the Ts sequence has a length of 3-15 nt (e.g., 4-15 nt, 5-15 nt, 3-12 nt, 4-12 nt, 5-12 nt, 3-10 nt, 4-10 nt, 5-10 nt, 5 nt, 10 nt);

[0104] (2) the Ss sequence has a length of 6-15 nt (e.g., 8-15 nt, 6-12 nt, 8-12 nt, 6-9 nt, 8 nt);

[0105] (3) the Cs sequence of the reverse transcription primer is capable of annealing to the 3’ end (e.g., 3’ terminal end) of the target miRNA; for example, the Cs sequence of the reverse transcription primer is complementary to the 3’ end (e.g., 3’ terminal end) sequence of the target miRNA;

[0106] (4) the Cs sequence of the reverse transcription primer has a length of 4-10 nt (e.g., 5-10 nt, 6-10 nt, 7-10 nt, 4-8 nt, 5-8 nt, 6-8 nt, 7-8 nt, 7 nt);

[0107] (5) the second region of the first primer has a length of 10-30 nt (e.g., 12-30 nt, 15-30 nt, 10-25 nt, 12-25 nt, 15-25 nt, 10-20 nt, 12-20 nt, 15-20 nt);

[0108] (6) the third region of the second primer has a length of 4-10 nt (e.g., 5-10 nt, 6-10 nt, 7-10 nt, 4-8 nt, 5-8 nt, 6-8 nt, 7-8 nt, 7 nt);

[0109] (7) the target miRNA is a mature miRNA.

[0110] In certain embodiments, the target miRNA exists in a single-stranded form.

[0111] In certain embodiments, the kit comprises the reverse transcription primer(s) for one or more target miRNAs, the first primer, and the second primer.

[0112] In certain embodiments, the first primer is modified with a first group, and the second primer is modified with a second group;

[0113] wherein the first group is a first fluorescent group, and the second group is a second fluorescent group; or, the first group is a fluorescent group, and the second group is a fluorescent quenching group; or, the first group is a fluorescent quenching group, and the second group is a fluorescent group;

[0114] and, the first group and the second group are capable of achieving fluorescence resonance energy transfer during amplification.

[0115] In certain embodiments, the first group and the second group in the same amplification product molecule are capable of achieving fluorescence resonance energy transfer.

[0116] In certain embodiments, the first group is located 6-9 (e.g., 6, 7, 8, 9) nucleotide residues away from the 3’ end of the first primer; and / or, the second group is located 6-9 (e.g., 6, 7, 8, 9) nucleotide residues away from the 3’ end of the second primer.

[0117] In certain embodiments, the first group is a first fluorescent group, and the second group is a second fluorescent group, and the combination of the first fluorescent group and the second fluorescent group is selected from:

[0118] TAMRA and FAM, CY3 and CY5, FAM and Dabcyl, FAM and Light Cycler Red 610, FAM and Light Cycler Red 640, FAM and Light Cycler Red 670, FAM and Light Cycler Red 705, FAM and TAMRA, CY5 and CY3, Dabcyl and FAM, Light Cycler Red 610 and FAM, Light Cycler Red 640 and FAM, Light Cycler Red 670 and FAM, Light Cycler Red 705 and FAM.

[0119] In certain embodiments, the first group is a fluorescent group and the second group is a fluorescent quenching group; or, the first group is a fluorescent quenching group and the second group is a fluorescent group.

[0120] and the fluorescent group is selected from the group consisting of FAM, TAMRA, HEX, ROX, CY3, CY5, TET, VIC, NED, JOE, Texas Red, CY5.5, and any combination thereof; and the fluorescent quenching group is selected from the group consisting of BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, and any combination thereof.

[0121] In certain embodiments, the combination of the fluorescent group and the fluorescent quenching group is selected from the group consisting of: FAM and BHQ1, TET and BHQ1, JOE and BHQ1, VIC and BHQ1, HEX and BHQ1, CY3 and BHQ2, NED and BHQ2, TAMRA and BHQ2, ROX and BHQ2, Texas Red and BHQ2, CY5 and BHQ2 / BHQ3, CY5.5 and BHQ2 / BHQ3, FAM and Dabcyl, TET and Dabcyl, JOE and Dabcyl, VIC and Dabcyl, HEX and Dabcyl, FAM and Eclipse, TAMRA and Eclipse, ROX and Eclipse, JOE and Eclipse, FAM and MGB, TET and MGB, JOE and MGB, VIC and MGB, HEX and MGB.

[0122] Without being limited by theory, the kits provided herein can be used to detect target miRNAs of any origin. For example, the target miRNAs can be derived from a bodily fluid sample, a cell sample, an extracted sample, or any combination thereof.

[0123] As will be readily appreciated by those skilled in the art, the kits of the present application can be used to detect any species of miRNA (e.g., any species of miRNA for which the sequence is known). For example, for any miRNA having a given sequence, the kits of the present application can provide a reverse transcription primer and a first primer and a second primer directed to the miRNA.

[0124] In certain embodiments, the target miRNA is selected from the group consisting of hsa-miR-92a-3p, hsa-miR-25-3p, and any combination thereof.

[0125] In certain embodiments, the target miRNA is hsa-miR-92a-3p, and the first primer has a sequence as set forth in SEQ ID NO: 37, and the second primer has a sequence as set forth in any one of SEQ ID NOs: 39-41, 43.

[0126] In certain embodiments, the target miRNA is hsa-miR-92a-3p, and the first primer has a sequence as set forth in SEQ ID NO: 81, and the second primer has a sequence as set forth in SEQ ID NO: 80.

[0127] In certain embodiments, the target miRNA is hsa-miR-92a-3p, and the reverse transcription primer has a sequence as set forth in any one of SEQ ID NOs: 76-79 (e.g., the reverse transcription primer has a sequence as set forth in any one of SEQ ID NOs: 77-79; e.g., the reverse transcription primer has a sequence as set forth in SEQ ID NO: 78), the first primer has a sequence as set forth in SEQ ID NO: 81, and / or the second primer has a sequence as set forth in SEQ ID NO: 80.

[0128] In certain embodiments, the target miRNA is hsa-miR-25-3p, and the first primer has a sequence as set forth in SEQ ID NO: 45, and the second primer has a sequence as set forth in any one of SEQ ID NOs: 48-49, 51-52, 54.

[0129] Primer design method

[0130] In a third aspect, the present application provides a method for designing a miRNA detection primer, comprising the following steps:

[0131] S1 : providing a target miRNA sequence;

[0132] S2: designing a reverse transcription primer, a first primer, and / or a second primer according to the target miRNA sequence;

[0133] wherein the reverse transcription primer, the first primer and the second primer are as defined above.

[0134] In some embodiments, the method is a method for designing miRNA detection primer sequences.

[0135] In a fourth aspect, the present application provides a computer readable storage medium, having stored thereon a computer program, which, when executed by a processor, implements the steps of the method of the third aspect.

[0136] In a fifth aspect, the present application provides an electronic device, comprising: the computer readable storage medium of the fourth aspect; and

[0137] one or more processors for executing the program in the computer readable storage medium.

[0138] Use

[0139] In a sixth aspect, the present application provides the use of the kit of the second aspect, the method of the third aspect, the computer readable storage medium of the fourth aspect, or the electronic device of the fifth aspect, in detecting miRNA.

[0140] In a seventh aspect, the present application provides the use of the kit of the second aspect in the preparation of a detection reagent for detecting miRNA.

[0141] Definitions of terms

[0142] In the present application, unless otherwise indicated or apparent from context, the scientific and technical terms used herein have the meanings that would be generally understood by one of ordinary skill in the art. Also, the virology, biochemistry, immunology laboratory procedures steps used herein are conventional procedures well known in the art. In addition, for better understanding of the present application, the definitions and explanations of relevant terms are provided as follows.

[0143] When the terms "for example", "for instance", "such as", "including", "containing" or variations thereof are used in this document, these terms are not to be interpreted in an exclusive sense, but are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components.

[0144] Unless otherwise indicated or apparent from context, the terms "a" and "an" and "the" and similar referents in the context of describing the application (especially in the context of the following claims) are to be interpreted in an inclusive rather than an exclusive sense.

[0145] As used herein, the term "upstream" is used to describe the relative positional relationship of two nucleic acid sequences (or two nucleic acid molecules) and has the meaning generally understood by those skilled in the art. For example, the expression "one nucleic acid sequence is upstream of another nucleic acid sequence" means that, when arranged in the 5' to 3' direction, the former is located at a more anterior position (i.e., a position closer to the 5' end) than the latter. As used herein, the term "downstream" has the opposite meaning of "upstream."

[0146] For a variety of reasons, the nucleic acids or polynucleotides of the present application (e.g., "reverse transcription primers," "first primers," "second primers," etc.) can include one or more modified nucleic acid bases, sugar moieties, or internucleoside linkages. For example, some reasons for using nucleic acids or polynucleotides that include modified bases, sugar moieties, or internucleoside linkages include, but are not limited to: (1) alteration of Tm; (2) alteration of susceptibility of the polynucleotide to one or more nucleases; (3) provision of a moiety for attachment of a label; (4) provision of a label or label quencher; or (5) provision of a moiety for attachment of another molecule in solution or bound to a surface.

[0147] As used herein, the terms "anneal," "annealing," "annealed," "hybridize," or "hybridization," and the like refer to the formation of a complex between nucleotide sequences having sufficient complementarity to form a complex via Watson-Crick base pairing. In the context of the present application, nucleic acid sequences that are "complementary to" or "complementary with" each other or that "hybridize" or "anneal" to each other are capable of forming or form a "hybrid" or "complex" that is sufficiently stable to serve a predetermined purpose. It is not required that every nucleic acid base within a sequence exhibited by one nucleic acid molecule be capable of base pairing or pairing or complexing with every nucleic acid base within a sequence exhibited by a second nucleic acid molecule in order for the two nucleic acid molecules or the respective sequences exhibited thereby to be "complementary to" or "anneal" or "hybridize" to each other. The terms "complementary" or "complementarity" are used as described herein when referring to sequences of nucleotides in connection with base pairing rules. For example, the sequence 5'-A-G-T-3' is complementary to the sequence 3'-T-C-A-5'. The complementarity can be "partial," in which only some of the nucleic acid bases match according to the base pairing rules. Alternatively, the complementarity can be "total" or "complete," in which all of the nucleic acid bases match according to the base pairing rules.

[0148] As used herein, the term "Fluorescence Resonance Energy Transfer" (FRET) has the meaning commonly understood by those skilled in the art, which is a process by which energy is transferred between two molecules through a non-radiative mechanism. This term is often used to describe a specific type of interaction that occurs between a fluorescent molecule (Donor) and another molecule that is capable of accepting energy (Acceptor). Donor: Usually a fluorescent molecule that, upon absorbing light energy, will be in an excited state and is able to release energy in the form of light. Acceptor: A molecule that is capable of accepting energy from the donor, usually another fluorescent molecule or a non-fluorescent molecule with an absorption spectrum that overlaps with the emission spectrum of the donor (e.g., a fluorescence-quenching group).

[0149] As used herein, the term "mature miRNA" has the meaning commonly understood by those skilled in the art, which is a class of small non-coding RNAs, usually consisting of about 21-25 nucleotides, generally involved in the regulation of gene expression in cells. The mature miRNA present in an organism or derived from a biological sample is generally generated from a longer primary miRNA (pri-miRNA) or precursor miRNA (pre-miRNA) through a multi-step process.

[0150] Advantages of the invention

[0151] (1) The detection system of the method of the present invention has good specificity itself, and there is no non-specific amplification when detecting a template-free negative water sample. On the one hand, in the method of the present invention, when the first amplification primer and the second amplification primer or the first amplification primer and the RT primer are non-specifically complementary to each other, a thermally stable stem-loop structure dimer amplicon can be generated, which can hinder the further amplification and accumulation of the dimer in the subsequent amplification process. On the other hand, compared with the dye method of miRNA detection, in some embodiments, the method of the present invention outputs fluorescence signal in the form of fluorescence resonance energy transfer when the first amplification primer and the second amplification primer specifically amplify the target miRNA, avoiding the influence of the accumulation of non-specific amplification or dimer fluorescence signal in the dye method on the detection.

[0152] (2) The method of the present invention has high sensitivity, and the detection range can span multiple concentration gradients, with high accuracy and precision for quantitative detection of miRNA.

[0153] (3) The method of the present invention has good quantitative accuracy, and the quantitative detection results are not easily affected by non-specific amplification products or dimers compared with the dye method.

[0154] (4) The method of the present application can be adapted to commercial fluorescent quantitative PCR instruments, and is compatible with rapid thermal cycling scheme for detecting miRNA.

[0155] Embodiments of the present application will now be described in detail, with reference to the accompanying drawings and examples, but as the skilled person will appreciate, the following drawings and examples are provided by way of illustration of the present application and are not limiting of the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to the skilled person, in light of the following detailed description of the drawings and preferred embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0156] Figure 1: Schematic diagram of the method of the present application.

[0157] Figure 2: Primer design rules and verification results of the method of the present application.

[0158] Figure 3: Design and application results of dimer self-limiting primers of the method of the present application.

[0159] Figure 4: Design and verification results of reverse transcription primers of the method of the present application.

[0160] Figure 5: FRET detection feasibility verification results of the method of the present application.

[0161] Figure 6: FRET detection feasibility verification results of the method of the present application.

[0162] Figure 7: Specificity verification results of real-time fluorescent quantitative PCR of the method of the present application.

[0163] Figure 8: Sensitivity verification results of real-time fluorescent quantitative PCR of the method of the present application. DETAILED DESCRIPTION

[0164] The present application will now be described with reference to the following examples, which are intended to illustrate the present application (but not to limit the scope of the present application).

[0165] Unless otherwise indicated, the molecular biology and immunological techniques utilized in the present application are performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995. The examples are intended to illustrate the present application, and are not intended to limit the scope of the present application as claimed.

[0166] Unless otherwise specified, the various raw materials used in the following examples are sourced from market sales, and the methods used are conventional methods, in which the primers and probes are synthesized by Shanghai Generay Biotech Co., Ltd.

[0167] Example 1: Primer design principles and verification results of the method of the present invention

[0168] Dimer is a common non-specific amplification product in dye-based PCR that affects detection performance. Avoiding the generation of dimers due to non-specific complementary pairing between primers is crucial for improving miRNA detection specificity. This invention designs three primer combinations. By progressively changing the 3' end sequence of the second amplification primer, increasing the difference between the 3' end sequence of the second amplification primer and the first amplification primer, and pairing different second amplification primers with the first amplification primer (Table 1), the specificity of different combinations is verified using a dye-based method. Eight template-free negative water samples are used for each combination. The Real-time PCR reaction system is as follows:

[0169] The real-time PCR instrument used was a CFX96. TM Real-Time System, reaction conditions: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 10 seconds, annealing extension at 55°C for 45 seconds, 45 cycles.

[0170] The results showed that when the 5' ends of the first and second amplification primers were mostly identical (i.e., the number of bases differing at the 3' ends of the first and second amplification primers was small), no non-specific amplification signal was generated (Figure 2). This indicates that primer combinations under these conditions are less likely to generate dimers, or dimers may have been generated in the initial stage of PCR, but because the generated dimers are stem-loop structures with higher Tm values, they limit the further amplification of dimers as templates in subsequent PCR reactions. Based on the pattern presented in the experimental results in Figure 2, to avoid dimer accumulation and ensure the sensitivity and specificity of miRNA detection, miRNA detection primers should be designed with the fewest possible identical base sequences at the 5' ends. Such primer combinations are called dimer self-restricting primer pairs.

[0171] Example 2: Design and application results of the self-restricting primers of the present invention

[0172] In order to verify the design rule of the dimer self-limiting primer obtained in Embodiment 1, the design rule of the primer was applied to the detection of two human miRNAs, hsa-miR-92a-3p and hsa-miR-25-3p. In order to verify the design rule, the relevant cDNA templates were synthesized according to the sequences of the human miRNAs, and the dimer self-limiting primers (Table 2) were designed. The dimer self-limiting primers comprise a first amplification primer and a second amplification primer with the same 5' end sequence. The 3' end sequence of the first amplification primer is complementary to the 5' sequence of the miRNA, and the 3' end sequence of the second amplification primer is complementary to the 3' sequence of the miRNA.

[0173] The Real-time PCR reaction system and procedure for the dye method detection were the same as those in Embodiment 1.

[0174] The results show that the hsa-miR-92a-3p-first amplification primer / hsa-miR-92a-3p-second amplification primer-1 and the hsa-miR-25-3p-first amplification primer / hsa-miR-25-3p-second amplification primer-1 have low amplification efficiency or no effective amplification for the positive template (6 x 10 3 copies / μL) (Figure 3). Further, the second amplification primer was designed by mutating a single or two bases in the second amplification primer, and the primer combination was obtained, which has high amplification efficiency for the positive template (6 x 10 3 copies / μL) (Figure 3) and no non-specific amplification for the negative water sample without a template (Figure 3). As shown in Figure 3, the first part of the second amplification primer shown in Figure 3 is composed of the upstream sequence of the nucleotide residue containing the mutant base closest to the 5' end of the second amplification primer, and the second part of the second amplification primer shown in Figure 3 is composed of the downstream sequence of the nucleotide residue containing the mutant base closest to the 3' end of the second amplification primer. It should be noted that the meanings of the "first part" and the "second part" in this embodiment are different from those defined in Figure 1. Further analysis of the detection results shows that the Tm value of the sequence of the first part (the first part shown in Figure 3) of the second amplification primer with better detection performance is preferably not more than 10°C compared with the Tm value of the sequence of the second part (the second part shown in Figure 3). On the basis of good specificity of the detection system, the Tm value of the sequence of the first part (the first part shown in Figure 3) is preferably designed to be less than or equal to the Tm value of the sequence of the second part (the second part shown in Figure 3), and the Tm value of the sequence of the second part (the second part shown in Figure 3) is preferably designed to be greater than or equal to 30°C (Figure 3).

[0175] Table 2, the dimer self-limiting primers preliminarily designed for the cDNA templates related to hsa-miR-92a-3p and hsa-miR-25-3p in the present application

[0176] Example 3: Design and verification results of reverse transcription primers of the method of the present application

[0177] After obtaining the design rules of the dimer self-limiting primers in Examples 1 and 2, the design rules of the reverse transcription primers were further explored. The designed RT primers included loop regions with or without Tag sequences, and two types of loop region base sequence lengths (Table 3). hsa-miR-92a-3p was used as a model miRNA to synthesize standard products, and 6x10 4 copies / μL concentration of miRNA was selected as a positive template, and 11 wells of negative control without template were set up to verify the detection performance of the designed 9 RT primers.

[0178] The reverse transcription primers in Table 3 were used for reverse transcription to generate cDNA, and the reverse transcription reaction system was as follows:

[0179] The reverse transcription instrument was C1000 TM Thermal Cycler, and the reaction conditions were 16℃ for 30 minutes, 42℃ for 30 minutes, and 95℃ for 5 minutes.

[0180] The results showed that the RT primers without Tag sequences would produce strong non-specific amplification (Figure 4). In addition, the loop region sequence of the RT primer should additionally include artificial base sequences except for the Tag sequence, but the length should not exceed 6 bases (Figure 4).

[0181] Table 3, RT primers and PCR amplification primers designed for hsa-miR-92a-3p target in the present application

[0182] Example 4: Feasibility verification of fluorescence resonance energy transfer detection scheme of the method of the present application

[0183] In Examples 1-3, the design rules of the dimer self-limiting primers and the RT primers were obtained, and in this example, the design rules based on the fluorescence resonance energy transfer system were explored. In order to verify the feasibility of the fluorescence resonance energy transfer detection method, the first amplification primer and the second amplification primer containing different fluorescent groups or quenching groups in the middle were designed for randomly generated miRNA sequences (Table 4).

[0184] The reaction system of Real-time PCR was as follows:

[0185] The Real-time PCR instrument was CFX96TM Real-Time System, the reaction conditions are: pre-denaturation 95 °C for 5 minutes, denaturation 95 °C for 10 seconds, annealing extension 55 °C for 45 seconds, 45 cycles.

[0186] The results show that, when detecting cDNA template, the fluorescence modification combination of primer pairs includes the combination of fluorescent group ROX and quenching group BHQ2 (1) and the combination of fluorescent group FAM and fluorescent group TAMRA (2), both of which can monitor the change of fluorescence signal in the amplification process, generating inverted or positive S-shaped amplification curve (Figure 5). Further analysis found that too long spatial distance can limit the realization of fluorescence resonance energy transfer, and the distance of 6-9 bases between fluorescent group or quenching group and the 3' end of primer is conducive to fluorescence resonance energy transfer. On the basis of the first randomly generated miRNA target verifying the feasibility of the fluorescence resonance energy transfer detection scheme, the feasibility of fluorescence resonance energy transfer is also proved on the other two cDNA targets (Figure 6).

[0187] Table 4, primers and templates for verifying the feasibility of the fluorescence resonance energy transfer detection method in the application

[0188] Example 5: Specificity verification of real-time fluorescent quantitative PCR of the method of the application

[0189] On the basis of the system design rules of the method of the application obtained in Examples 1-4, the specificity of real-time fluorescent quantitative PCR of the method of the application is verified in this example. Considering that multiple reverse transcription is more likely to interfere with the PCR detection system than single reverse transcription. In order to verify the specificity of the detection system of the method of the application based on multiple reverse transcription, 11 non-target related reverse transcription primers (Table 5) are additionally introduced on the basis of single reverse transcription for the target, simulating multiple reverse transcription reaction, and PCR is detected by using dye method and the method of the application respectively, and the primer sequences used by the two are the same (Table 6).

[0190] The Real-time PCR reaction system and procedure of the dye method detection are the same as those in Example 1.

[0191] The Real-time PCR reaction system and procedure of the method of the application detection are the same as those in Example 4.

[0192] The results show that under the condition of multiple reverse transcription, 12 non-specific amplifications are produced in 21 template-free negative water samples detected by the dye method, while no non-specific amplification is produced in 21 template-free negative water samples detected by the method of the application (Figure 7). It is illustrated that even under the condition of interference of multiple RT primers unrelated to the target to be detected, the specificity of the detection system of the method of the application is still superior to that of the dye method.

[0193] Table 5, Non-target related reverse transcription primers used for specificity verification of real-time fluorescent quantitative PCR of the method of the present application

[0194] Table 6, Primers and templates used for specificity and sensitivity verification of real-time fluorescent quantitative PCR of the method of the present application

[0195] Example 6: Sensitivity verification of real-time fluorescent quantitative PCR of the method of the present application

[0196] Based on the system design rules of the method of the present application obtained in Examples 1-4, this example verifies the sensitivity of real-time fluorescent quantitative PCR of the method of the present application. The cDNA template is serially diluted by 10 times, and PCR is detected by using the dye method and the method of the present application, respectively, and the primer sequences used by both are the same (Table 6).

[0197] The real-time PCR reaction system and procedure of the dye method detection are the same as in Example 1.

[0198] The real-time PCR reaction system and procedure of the method of the present application detection are the same as in Example 4.

[0199] The results (Figure 8) show that the sensitivity of the method of the present application and the control dye method detection can both reach 6 x 10 1 copies / μL, but 1 of the 12 wells of the dye method detection has a non-specific amplification signal, while the method of the present application has no non-specific amplification signal, indicating that the sensitivity of the method of the present application is comparable to or better than the dye method (depending on the severity of the non-specific amplification reaction of the dye method) and the specificity is better than the dye method.

[0200] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details in light of the overall teachings of the disclosure, and such modifications and changes are intended to be within the scope of the present application. The entire disclosure of the present application is given by the claims and any equivalents thereof.

Claims

1. A method for detecting the presence and / or the amount of a target miRNA (MicroRNA) in a sample to be tested, comprising the following steps: (1) providing a reverse transcription primer and a sample to be tested, contacting the reverse transcription primer with the sample to be tested under conditions allowing reverse transcription, and performing a reverse transcription reaction; wherein the 5' end of the reverse transcription primer comprises a stem-loop structure, the loop region of the stem-loop structure comprises a tag sequence (Ts sequence), and the stem region of the stem-loop structure comprises a local double-stranded structure formed by annealing of a stem sequence (Ss sequence) and its complementary sequence (SsC sequence), wherein the Ss sequence is downstream of the SsC sequence; the 3' end of the reverse transcription primer comprises a capture sequence (Cs sequence) capable of annealing to the target miRNA and initiating an extension reaction; (2) contacting a first primer and a second primer with the product obtained in the previous step under conditions allowing nucleic acid amplification, and performing a nucleic acid amplification reaction; wherein the first primer comprises or consists of, from 5' to 3' direction, a first region and a second region; the first region comprises the Ts sequence, and the second region is capable of annealing to the cDNA sequence of the target miRNA and initiating an extension reaction; the second primer comprises or consists of, from 5' to 3' direction, a first region, a second region and a third region; the first region comprises the Ts sequence, the second region comprises the Ss sequence, and the third region is capable of annealing to the complement of the cDNA sequence of the target miRNA and initiating an extension reaction; (3) evaluating the presence and / or the amount of the target miRNA in the sample to be tested according to the presence and / or the amount of the nucleic acid amplification product in step (2).

2. The method of claim 1, wherein, the second region of the first primer is capable of annealing to the 3' end (e.g., 3' terminal end) of the cDNA sequence of the target miRNA; preferably, the second region of the first primer is complementary to the sequence of the 3' end (e.g., 3' terminal end) of the cDNA sequence of the target miRNA.

3. The method of claim 1 or 2, wherein, the Ss sequence comprises the sequence of the second region of the first primer or a partial sequence thereof (e.g., a 5' end partial sequence of the second region of the first primer), and has one or more (e.g., 1-4, 1, 2, 3 or 4) substitution mutations of nucleotide residues compared to the sequence of the second region of the first primer or a partial sequence thereof (e.g., a 5' end partial sequence of the second region of the first primer).

4. The method of claim 1 or 2, wherein, the Ss sequence comprises a DNA sequence identical to the sequence of the 5' end (e.g., 5' terminal end) of the target miRNA, and has one or more (e.g., 1-4, 1, 2, 3 or 4) substitution mutations of nucleotide residues compared to the DNA sequence.

5. The method of claim 3 or 4, wherein, the Ss sequence comprised by the second primer has substitution mutations as defined in claim 3 or 4; Preferably, in the second primer, segment I of the second primer consists of the sequence upstream of the nucleotide residue containing the substitution mutation nearest to the 5' end of the second primer, segment II of the second primer consists of the sequence downstream of the nucleotide residue containing the substitution mutation nearest to the 3' end of the second primer; and the second primer possesses one or more of the following features: (1) the Tm value of segment II is greater than or equal to 30°C; (2) the Tm value of segment II differs from the Tm value of segment I by no more than 10°C; (3) the Tm value of segment II is greater than or equal to the Tm value of segment I.

6. The method of any one of claims 1-5, wherein, The loop region of the reverse transcription primer further comprises a universal sequence (Us sequence); Preferably, the Us sequence is located upstream of the Ts sequence; Preferably, the Us sequence has a length of 0-6 nt (e.g., 1-6 nt, 2-6 nt, 3-6 nt, 4-6 nt, 1-5 nt, 2-5 nt, 3-5 nt, 4-5 nt, 4 nt).

7. The method of any one of claims 1-6, wherein, The third region of the second primer is capable of annealing to the 3' end of the complement of the cDNA sequence of the target miRNA; Preferably, the third region of the second primer comprises a sequence complementary to the 3' end (e.g., 3' terminal) sequence of the target miRNA; Preferably, the third region of the second primer comprises the Cs sequence of the reverse transcription primer or a partial sequence thereof.

8. The method of any one of claims 1-7, possessing one or more of the following features: (1) the Ts sequence has a length of 3-15 nt (e.g., 4-15 nt, 5-15 nt, 3-12 nt, 4-12 nt, 5-12 nt, 3-10 nt, 4-10 nt, 5-10 nt, 5 nt, 10 nt); (2) the Ss sequence has a length of 6-15 nt (e.g., 8-15 nt, 6-12 nt, 8-12 nt, 6-9 nt, 8 nt); (3) the Cs sequence of the reverse transcription primer is capable of annealing to the 3' end (e.g., 3' terminal) of the target miRNA; for example, the Cs sequence of the reverse transcription primer is complementary to the 3' end (e.g., 3' terminal) sequence of the target miRNA; (4) the Cs sequence of the reverse transcription primer has a length of 4-10 nt (e.g., 5-10 nt, 6-10 nt, 7-10 nt, 4-8 nt, 5-8 nt, 6-8 nt, 7-8 nt, 7 nt); (5) the second region of the first primer has a length of 10-30 nt (e.g., 12-30 nt, 15-30 nt, 10-25 nt, 12-25 nt, 15-25 nt, 10-20 nt, 12-20 nt, 15-20 nt); (6) the third region of the second primer has a length of 4-10 nt (e.g., 5-10 nt, 6-10 nt, 7-10 nt, 4-8 nt, 5-8 nt, 6-8 nt, 7-8 nt, 7 nt); (7) the target miRNA is a mature miRNA.

9. The method of any one of claims 1-8, wherein, The method is used for detecting the presence and / or content of one or more target miRNAs in a sample to be tested.

10. The method of any one of claims 1-9, wherein, The method is used for detecting the presence and / or content of multiple target miRNAs in a sample to be tested; In step (1) of the method, a plurality of reverse transcription primers respectively for different target miRNAs are provided, and the plurality of reverse transcription primers are contacted with nucleic acids in the sample to be tested under conditions allowing reverse transcription, and a reverse transcription reaction is performed; and, In step (2), a plurality of first primers and a plurality of second primers respectively for different target miRNAs are provided; In step (3), the presence and / or content of the nucleic acid amplification product in each reaction system of step (2) is determined, and the presence and / or content of each of the plurality of target miRNAs in the sample to be tested is evaluated. In step (2), the nucleic acid amplification reaction is fluorescence quantitative PCR or isothermal amplification.

11. The method of any one of claims 1-10, wherein, In step (3), the presence and / or content of the nucleic acid amplification product of the fluorescence quantitative PCR or isothermal amplification is determined to determine the presence and / or content of the target miRNA in the sample to be tested. The first primer is modified with a first group, and the second primer is modified with a second group; 12. The method of claim 11, wherein, The first group is a first fluorescent group, and the second group is a second fluorescent group; or the first group is a fluorescent group, and the second group is a fluorescent quenching group; or the first group is a fluorescent quenching group, and the second group is a fluorescent group; The first group and the second group can achieve fluorescence resonance energy transfer during amplification. The first group is 6-9 (e.g., 6, 7, 8, 9) nucleotide residues away from the 3' end of the first primer; and / or the second group is 6-9 (e.g., 6, 7, 8, 9) nucleotide residues away from the 3' end of the second primer.

13. The method of claim 12, wherein, The first group is a first fluorescent group, and the second group is a second fluorescent group, and the combination of the first fluorescent group and the second fluorescent group is selected from:

14. The method of claim 12 or 13, wherein, TAMRA and FAM, CY3 and CY5, FAM and Dabcyl, FAM and Light Cycler Red 610, FAM and Light Cycler Red 640, FAM and Light Cycler Red 670, FAM and Light Cycler Red 705, FAM and TAMRA, CY5 and CY3, Dabcyl and FAM, Light Cycler Red 610 and FAM, Light Cycler Red 640 and FAM, Light Cycler Red 670 and FAM, Light Cycler Red 705 and FAM. ​ 15. The method of claim 12 or 13, wherein, the first group is a fluorescent group and the second group is a fluorescent quencher group; or the first group is a fluorescent quencher group and the second group is a fluorescent group; and the fluorescent group is selected from FAM, TAMRA, HEX, ROX, CY3, CY5, TET, VIC, NED, JOE, Texas Red, CY5.5 and any combination thereof; and the fluorescent quencher group is selected from BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB and any combination thereof.

16. A kit comprising a reverse transcription primer for detecting a target miRNA, and a first primer and a second primer; wherein the 5' end of the reverse transcription primer comprises a stem-loop structure, the loop region of the stem-loop structure comprises a tag sequence (Ts sequence), and the stem region of the stem-loop structure comprises a local double-stranded structure formed by annealing of a stem sequence (Ss sequence) and a complementary sequence (SsC sequence) thereof, wherein the Ss sequence is downstream of the SsC sequence; the 3' end of the reverse transcription primer comprises a capture sequence (Cs sequence) capable of annealing to the target miRNA and initiating an extension reaction; the first primer comprises or consists of, from 5' to 3' direction, a first region and a second region; the first region comprises the Ts sequence, and the second region is capable of annealing to the cDNA sequence of the target miRNA and initiating an extension reaction; the second primer comprises or consists of, from 5' to 3' direction, a first region, a second region and a third region; the first region comprises the Ts sequence, the second region comprises the Ss sequence, and the third region is capable of annealing to the complement of the cDNA sequence of the target miRNA and initiating an extension reaction.

17. The kit of claim 16, wherein, the second region of the first primer is capable of annealing to the 3' end (e.g., 3' terminal end) of the cDNA sequence of the target miRNA; Preferably, the second region of the first primer is complementary to the sequence of the 3' end (e.g., 3' terminal end) of the cDNA sequence of the target miRNA.

18. The kit of claim 16 or 17, wherein, the Ss sequence comprises the sequence of the second region of the first primer or a partial sequence thereof (e.g., a 5' end partial sequence of the second region of the first primer), and has one or more (e.g., 1-4, 1, 2, 3 or 4) substitution mutations of nucleotide residues compared to the sequence of the second region of the first primer or the partial sequence thereof (e.g., the 5' end partial sequence of the second region of the first primer).

19. The kit of claim 16 or 17, wherein, the Ss sequence comprises a DNA sequence identical to the 5' end (e.g., 5' terminal end) sequence of the target miRNA, and has one or more (e.g., 1-4, 1, 2, 3 or 4) substitution mutations of nucleotide residues compared to the DNA sequence.

20. The method of claim 18 or 19, wherein, the Ss sequence comprised by the second primer has substitution mutations as defined in claim 18 or 19; Preferably, in the second primer, segment I of the second primer consists of the sequence upstream of the nucleotide residue containing the substitution mutation nearest to the 5' end of the second primer, segment II of the second primer consists of the sequence downstream of the nucleotide residue containing the substitution mutation nearest to the 3' end of the second primer; and the second primer possesses one or more features selected from the following: (1) the Tm value of the segment II is greater than or equal to 30°C; (2) the Tm value of the segment II differs from the Tm value of the segment I by no more than 10°C; (3) the Tm value of the segment II is greater than or equal to the Tm value of the segment I.

21. The kit of any one of claims 16-20, wherein, the loop region of the reverse transcription primer further comprises a universal sequence (Us sequence); Preferably, the Us sequence is located upstream of the Ts sequence; Preferably, the Us sequence has a length of 0-6 nt (e.g., 1-6 nt, 2-6 nt, 3-6 nt, 4-6 nt, 1-5 nt, 2-5 nt, 3-5 nt, 4-5 nt, 4 nt).

22. The kit of any one of claims 16-21, wherein, the third region of the second primer is capable of annealing to the 3' end of the complement sequence of the cDNA sequence of the target miRNA; Preferably, the third region of the second primer comprises a sequence complementary to the 3' end (e.g., 3' terminal) sequence of the target miRNA; Preferably, the third region of the second primer comprises the Cs sequence of the reverse transcription primer or a partial sequence thereof.

23. The kit of any one of claims 16-22, which possesses one or more features selected from the following: (1) the Ts sequence has a length of 3-15 nt (e.g., 4-15 nt, 5-15 nt, 3-12 nt, 4-12 nt, 5-12 nt, 3-10 nt, 4-10 nt, 5-10 nt, 5 nt, 10 nt); (2) the Ss sequence has a length of 6-15 nt (e.g., 8-15 nt, 6-12 nt, 8-12 nt, 6-9 nt, 8 nt); (3) the Cs sequence of the reverse transcription primer is capable of annealing to the 3' end (e.g., 3' terminal) of the target miRNA; for example, the Cs sequence of the reverse transcription primer is complementary to the 3' end (e.g., 3' terminal) sequence of the target miRNA; (4) the Cs sequence of the reverse transcription primer has a length of 4-10 nt (e.g., 5-10 nt, 6-10 nt, 7-10 nt, 4-8 nt, 5-8 nt, 6-8 nt, 7-8 nt, 7 nt); (5) the second region of the first primer has a length of 10-30 nt (e.g., 12-30 nt, 15-30 nt, 10-25 nt, 12-25 nt, 15-25 nt, 10-20 nt, 12-20 nt, 15-20 nt); (6) the third region of the second primer has a length of 4-10 nt (e.g., 5-10 nt, 6-10 nt, 7-10 nt, 4-8 nt, 5-8 nt, 6-8 nt, 7-8 nt, 7 nt); (7) the target miRNA is a mature miRNA.

24. The kit of any one of claims 16-23, wherein, The kit comprises the reverse transcription primer, the first primer and the second primer for one or more target miRNAs.

25. The kit of any one of claims 16-24, wherein, The first primer is modified with a first group, and the second primer is modified with a second group; The first group is a first fluorescent group, and the second group is a second fluorescent group; or the first group is a fluorescent group, and the second group is a fluorescent quenching group; or the first group is a fluorescent quenching group, and the second group is a fluorescent group; The first group and the second group can realize fluorescence resonance energy transfer during amplification.

26. The kit of claim 25, wherein, The first group is 6-9 (e.g., 6, 7, 8, 9) nucleotide residues away from the 3' end of the first primer; and / or the second group is 6-9 (e.g., 6, 7, 8, 9) nucleotide residues away from the 3' end of the second primer.

27. The kit of claim 25 or 26, wherein, The first group is a first fluorescent group, and the second group is a second fluorescent group, and the combination of the first fluorescent group and the second fluorescent group is selected from: TAMRA and FAM, CY3 and CY5, FAM and Dabcyl, FAM and Light Cycler Red 610, FAM and Light Cycler Red 640, FAM and Light Cycler Red 670, FAM and Light Cycler Red 705, FAM and TAMRA, CY5 and CY3, Dabcyl and FAM, Light Cycler Red 610 and FAM, Light Cycler Red 640 and FAM, Light Cycler Red 670 and FAM, Light Cycler Red 705 and FAM.

28. The kit of claim 25 or 26, wherein, The first group is a fluorescent group, and the second group is a fluorescent quenching group; or the first group is a fluorescent quenching group, and the second group is a fluorescent group; The fluorescent group is selected from FAM, TAMRA, HEX, ROX, CY3, CY5, TET, VIC, NED, JOE, Texas Red, CY5.5 and any combination thereof; and the fluorescent quenching group is selected from BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB and any combination thereof.

29. A method for designing miRNA detection primers, comprising the following steps: S1: providing a target miRNA sequence; S2: designing a reverse transcription primer, a first primer and / or a second primer according to the target miRNA sequence; The reverse transcription primer, the first primer and the second primer are as defined in any one of claims 1-15.

30. A computer readable storage medium having stored thereon a computer program which, when executed by a processor, implements the steps of the method of claim 29.

31. An electronic device comprising: The computer readable storage medium of claim 30; and one or more processors to execute a program in the computer readable storage medium.

32. Use of the kit of any one of claims 16-28, the method of claim 29, the computer readable storage medium of claim 30, or the electronic device of claim 31, in detecting a miRNA.

33. Use of the kit of any one of claims 16-28 in the manufacture of a detection reagent for detecting a miRNA.

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