Mirna detection method capable of distinguishing mutations

Abstract

The present invention provides a miRNA quantitative detection method capable of distinguishing mutations. More specifically, the present invention provides a primer and a probe which are sensitive to miRNA mutations, and provides a miRNA quantitative detection method capable of distinguishable mutations based on the primer and probe.

Description

A method for detecting mutant miRNA distinguishable from each other

[0001] This application claims the benefit of Chinese application No. 202411105961.9, filed on August 13, 2024, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of molecular biology detection, in particular, the present application relates to a method for quantitatively detecting mutant miRNA distinguishable from each other. More particularly, the present application provides primers and probes sensitive to miRNA mutation, and provides a method for quantitatively detecting mutant miRNA distinguishable from each other based on the primers and probes. BACKGROUND

[0003] MicroRNA (miRNA) is a class of single-stranded non-coding RNA with about 19-25 nucleotides, which is an important post-transcriptional regulatory factor of gene expression in human cells (Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004 Jan 23; 116(2): 281-97. doi: 10.1016 / s0092-8674(04)00045-5.; Shi R, Chiang VL. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004 Jul; 5(7): 522-31. doi: 10.1038 / nrg1379.). miRNA not only plays an important role in cell proliferation, apoptosis, differentiation and other processes, but also participates in the occurrence and development of various diseases including cardiovascular diseases and cancers (Berindan-Neagoe I, et al. MicroRNAome genome: a treasure for cancer diagnosis and therapy. CA Cancer J Clin. 2014 Sep-Oct; 64(5): 311-36. doi: 10.3322 / caac.21244.). In addition, it has been found that there are circulating miRNAs in blood which are very stable, and the expression profiles of circulating miRNAs in the blood of healthy people and patients are different (Chen X, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 2008 Oct; 18(10): 997-1006. doi: 10.1038 / cr.2008.282.; Mitchell PS, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008 Jul 29; 105(30): 10513-8. doi: 10.1073 / pnas.0804549105.), indicating that miRNA has broad prospects as a potential non-invasive biomarker for early diagnosis and prognosis evaluation of human cancers and other major diseases.

[0004] Traditional miRNA detection techniques such as Northern blotting, in situ hybridization, gene chip, etc. have poor amplification sensitivity for low-abundance miRNAs. Real-time quantitative PCR (RT-qPCR)-based detection techniques have always been considered one of the most sensitive miRNA detection methods. However, conventional RT-qPCR techniques are difficult to detect miRNAs with a length of about 19-25 nucleotides. In order to overcome this problem, researchers have developed poly-A tailing method, stem-loop primer reverse transcription method, etc. These methods increase the length of reverse transcription products to facilitate qPCR primer design. Shi et al. developed a poly(A) tailing method that uses poly(A) polymerase to add a poly(A) tail to the 3' end of miRNA, and then uses an oligo(dT) sequence-containing primer for reverse transcription (Shi R, Chiang VL. Facile means for quantifying microRNA expression by real-time PCR. Biotechniques. 2005 Oct; 39(4): 519-25. doi: 10.2144 / 000112010.). Due to the universality of the reverse transcription primer, the poly(A) tailing method has the advantage of low detection cost, but also reduces the specificity and sensitivity of the detection. Chen et al. developed a stem-loop primer method in which the reverse transcription primer contains a stem-loop structure at the 5' end and 6 specific nucleotides at the 3' end that pair with the 3' end of miRNA, allowing specific reverse transcription reaction (Chen C, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005 Nov 27; 33(20): e179. doi: 10.1093 / nar / gni178.). The stem-loop primer method is usually superior to the poly(A) tailing method in specificity and sensitivity. However, the nucleotide sequence of the probe in the stem-loop primer method overlaps with the nucleotide sequence of the reverse transcription primer, which contributes little to the specificity in real-time PCR. The specificity of real-time PCR is only achieved by the forward PCR primer, which is not enough to distinguish many miRNAs of the same family, similar miRNAs, and RNAs.In addition, some studies have reported that stem-loop primer system is prone to produce non-specific amplification when detecting no-template negative control (Varkonyi-Gasic E, et al. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods. 2007 Oct 12; 3: 12. doi: 10.1186 / 1746-4811-3-12.; Jung U, et al. A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA. RNA. 2013 Dec; 19(12): 1864-73. doi: 10.1261 / rna.040501.113.), which is likely to affect the accuracy of miRNA quantitative detection and reduce the detection sensitivity.

[0005] MiRNA biomarkers are disease markers with potential clinical diagnostic and prognostic value. However, to date, most of the published research results of miRNA as diagnostic and prognostic biomarkers have not been clinically applied. The main reason may be that the current miRNA quantitative detection method has some limitations, and therefore a new high-performance miRNA quantitative detection method is needed to promote the clinical transformation of miRNA biomarkers. SUMMARY

[0006] The present application provides a method for detecting mature miRNA in a sample, which has high sensitivity, good specificity, high quantitative accuracy, and can distinguish mutations, and can be widely used for expression analysis and clinical detection of mature miRNA.

[0007] Detection method

[0008] Therefore, in one aspect, the present application provides a method for detecting the presence and / or content of a 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, and 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 3' end of the reverse transcription primer comprises a capture sequence complementary to the first region of the target miRNA and can initiate an extension reaction;

[0011] (2) contacting the first primer, the second primer, and the fluorescent probe with the product obtained in the previous step under conditions permitting nucleic acid amplification, and performing a fluorescent quantitative PCR reaction;

[0012] wherein the first primer comprises a sequence complementary to a sequence complementary to the second region of the target miRNA (As sequence) at its 3' end, and is capable of initiating an extension reaction;

[0013] the second primer is capable of annealing to a sequence complementary to the reverse transcription primer, and is capable of initiating an extension reaction;

[0014] the fluorescent probe comprises (i) a sequence complementary to the third region of the target miRNA (Bs1 sequence), or (ii) a sequence complementary to a sequence complementary to the third region of the target miRNA (Bs2 sequence);

[0015] and the second region of the target miRNA is located upstream of the first region, and the second region and the first region comprise a spacer region therebetween;

[0016] the third region of the target miRNA comprises a spacer region between the first region and the second region of the target miRNA;

[0017] (3) assessing the presence and / or the amount of the target miRNA in the sample to be tested based on the fluorescent signal of the fluorescent quantitative PCR reaction in step (2).

[0018] In certain embodiments, in step (1), the reverse transcription primer is contacted with the sample to be tested at an annealing temperature (e.g., 12-20°C, 12-18°C, 12-16°C, 14-20°C, 14-18°C, 14-16°C, 16-20°C, 16-18°C, 16°C) permitting annealing of the reverse transcription primer to the target miRNA, and a reverse transcription reaction is performed under conditions permitting nucleic acid polymerization.

[0019] 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.

[0020] In certain embodiments, the sample to be tested is suspected of containing the target miRNA.

[0021] In certain embodiments, the third region overlaps the second region by 0-3 (e.g., 0, 1, 2, 3) nucleotide residues.

[0022] In certain embodiments, the capture sequence has a length of 4-9 nt.

[0023] In certain embodiments, the As sequence is 8-16 nt in length.

[0024] In certain embodiments, the fluorescent probe is 8-25 nt in length.

[0025] In certain embodiments, the spacer region of the target miRNA between the first region and the second region is 1-8 nt in length (e.g., the 3' end of the second region of the target miRNA is spaced 1-8 nucleotide residues from the 5' end of the first region).

[0026] In certain embodiments, the fluorescent probe comprises a sequence (Bs1 sequence) that is complementary to the third region of the target miRNA.

[0027] In certain embodiments, the fluorescent probe comprises the Bs1 sequence at its 3' end.

[0028] In certain embodiments, the Bs1 sequence is 8-25 nt, 8-20 nt, 10-25 nt, 10-20 nt, 8-18 nt, 10-18 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt in length.

[0029] In certain embodiments, the Bs1 sequence is fully complementary to the sequence of the third region of the target miRNA.

[0030] As will be readily understood by one skilled in the art, as used herein, "fully complementary" means that each nucleic acid base shown within a sequence of one nucleic acid molecule is capable of base pairing with each nucleic acid base shown within a corresponding segment of a sequence of a second nucleic acid molecule. Thus, in certain embodiments, each nucleic acid base of the Bs1 sequence comprised by the fluorescent probe is capable of forming a base pair with a nucleic acid base of a corresponding segment of a complementary strand of a reverse transcription product of the target miRNA.

[0031] In certain embodiments, the fluorescent probe further comprises, at its 5' end, a sequence or a partial sequence of the sequence of the reverse transcription primer that is upstream of and adjacent to the capture sequence.

[0032] In certain embodiments, the fluorescent probe comprises a sequence (Bs2 sequence) that is complementary to a sequence that is complementary to the third region of the target miRNA.

[0033] In certain embodiments, the fluorescent probe comprises the Bs2 sequence at its 5' end.

[0034] In some embodiments, the Bs2 sequence is 8-25 nt, 8-20 nt, 10-25 nt, 10-20 nt, 8-18 nt, 10-18 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt in length.

[0035] In some embodiments, the Bs2 sequence is fully complementary to the complement of the third region of the target miRNA.

[0036] In some embodiments, each nucleic acid base of the Bs2 sequence comprised by the fluorescent probe is capable of forming base pairing with a nucleic acid base of the corresponding segment in the reverse transcription product of the target miRNA.

[0037] In some embodiments, the fluorescent probe further comprises, at its 3' end, a sequence or a partial sequence of the sequence of the reverse transcription primer upstream of and adjacent to the capture sequence.

[0038] In some embodiments, the method comprises one or more features selected from the following:

[0039] (1) the first region is located at the 3' end of the target miRNA;

[0040] (2) the second region is located at the 5' end of the target miRNA;

[0041] (3) the 3' end of the second region of the target miRNA is spaced apart from the 5' end of the first region by 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide residues;

[0042] (4) the third region comprises the first region;

[0043] (5) the Tm value of the capture sequence is 12-20 °C (e.g., 12-18 °C, 12-16 °C, 14-20 °C, 14-18 °C, 14-16 °C, 16-20 °C, 16-18 °C, 16 °C);

[0044] (6) the capture sequence is located at the 3' end of the reverse transcription primer;

[0045] (7) the length of the capture sequence is 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, or 9 nt;

[0046] (8) the As sequence is located at the 3' end of the first primer;

[0047] (9) the As sequence is 8-16 nt, 8-14 nt, 8-12 nt, 11-16 nt, 11-14 nt, 11-12 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, or 16 nt in length;

[0048] (10) the As sequence has a Tm value of 20-55°C (e.g., 20-40°C, 20-30°C, 25-55°C, 25-40°C, 25-30°C, 30-55°C, 30-40°C);

[0049] (11) the fluorescent probe has a Tm value of 32-60°C (e.g., 32-55°C, 32-50°C, 40-60°C, 40-55°C, 40-50°C, 45-60°C, 45-55°C, 45-50°C);

[0050] (12) the fluorescent probe is 8-25 nt, 8-20 nt, 10-25 nt, 10-20 nt, 8-18 nt, 10-18 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt in length;

[0051] (13) the target miRNA is a mature miRNA;

[0052] (14) the reverse transcription primer comprises modified nucleotides (e.g., locked nucleic acids); preferably, the capture sequence of the reverse transcription primer comprises one or more modified nucleotides (e.g., locked nucleic acids);

[0053] (15) the first primer comprises modified nucleotides (e.g., locked nucleic acids); preferably, the As sequence of the first primer comprises one or more modified nucleotides (e.g., locked nucleic acids).

[0054] In certain embodiments, the capture sequence has a Tm value of 0-20°C (e.g., 0-18°C, 0-16°C, 4-18°C, 4-16°C, 8-18°C, 8-16°C, 12-18°C, 12-16°C, 14-20°C, 14-18°C, 14-16°C, 16-20°C, 16-18°C, 16°C).

[0055] In certain embodiments, the As sequence has a Tm value of 2-60°C (e.g., 2-55°C, 2-50°C, 2-40°C, 2-30°C, 10-60°C, 10-55°C, 10-50°C, 10-40°C, 10-30°C, 20-60°C, 20-55°C, 20-50°C, 20-40°C, 20-30°C, 25-60°C, 25-55°C, 25-50°C, 25-40°C, 25-30°C, 30-60°C, 30-55°C, 30-40°C).

[0056] In certain embodiments, the As sequence has a Tm value of 45-60°C (e.g., 45-58°C, 45-55°C, 50-60°C, 50-58°C, 50-55°C, 52-60°C, 52-58°C, 52-55°C, 55°C).

[0057] In certain embodiments, the reverse transcription primer does not comprise a nucleotide modification that blocks DNA polymerase extension.

[0058] In certain embodiments, the reverse transcription primer is 34-70 nt in length.

[0059] In certain embodiments, the first primer is 18-25 nt in length.

[0060] In certain embodiments, the second primer is 18-25 nt in length.

[0061] In certain embodiments, the fluorescent probe is modified with a fluorescent group and a fluorescence quenching group.

[0062] In certain embodiments, when the fluorescent probe is not bound to the amplification product of the target miRNA, the fluorescent group and the fluorescence quenching group can achieve fluorescence resonance energy transfer, so that the fluorescence signal of the fluorescent group is quenched completely or partially; and when the fluorescent probe is bound to the amplification product of the target miRNA, the fluorescent group and the fluorescence quenching group cannot achieve fluorescence resonance energy transfer, so that the fluorescent group produces a corresponding fluorescence signal.

[0063] In certain embodiments, when the fluorescent probe is not bound to the amplification product of the target miRNA, the fluorescent group and the fluorescence quenching group cannot achieve fluorescence resonance energy transfer, so that the fluorescent group produces a corresponding fluorescence signal; and when the fluorescent probe is bound to the amplification product of the target miRNA, the fluorescent group and the fluorescence quenching group can achieve fluorescence resonance energy transfer, so that the fluorescence signal of the fluorescent group is quenched completely or partially.

[0064] 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.

[0065] In certain embodiments, 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 fluorescent group and 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] In certain embodiments, in step (1), the annealing temperature of the reverse transcription reaction is set at 12-20°C (e.g., 12-18°C, 12-16°C, 14-20°C, 14-18°C, 14-16°C, 16-20°C, 16-18°C, 16°C).

[0068] In certain embodiments, in step (1), the annealing temperature of the reverse transcription reaction is set at 16°C.

[0069] In certain embodiments, in step (2), the annealing temperature of the fluorescent quantitative PCR reaction is set at 45-60°C (e.g., 45-58°C, 45-55°C, 50-60°C, 50-58°C, 50-55°C, 52-60°C, 52-58°C, 52-55°C, 47°C, 49°C, 55°C).

[0070] In certain embodiments, in step (2), the annealing temperature of the fluorescent quantitative PCR reaction is set at 55°C.

[0071] In certain embodiments, in step (2), the annealing temperature of the fluorescent quantitative PCR reaction is set at 47°C.

[0072] In certain embodiments, in step (2), the annealing temperature of the real-time PCR reaction is set at 49°C.

[0073] In certain embodiments, in step (2), the annealing temperature of the real-time PCR reaction is set at the same temperature as the extension temperature.

[0074] In certain embodiments, the reverse transcription primer does not comprise a stem-loop structure.

[0075] In certain embodiments, the reverse transcription primer further comprises a universal sequence at its 5' end.

[0076] In certain embodiments, the second primer is capable of annealing to the complement of the universal sequence.

[0077] In certain embodiments, the reverse transcription primer comprises a stem-loop structure.

[0078] In certain embodiments, the stem-loop structure is located upstream of the capture sequence.

[0079] In certain embodiments, the second primer is capable of annealing to the complement of the loop region sequence of the reverse transcription primer.

[0080] In certain embodiments, in step (3), the method further comprises comparing the fluorescence signal of the real-time PCR reaction in step (2) with the fluorescence signal corresponding to the target miRNA standard, to assess the presence and / or the amount of the target miRNA in the sample to be tested.

[0081] 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.

[0082] In certain embodiments, in step (3), the method assesses the presence and / or the amount of the target miRNA in the sample to be tested by comparing the Ct value of the real-time PCR reaction in step (2) with the Ct value corresponding to the target miRNA standard.

[0083] It is readily understood by one skilled in the art that the Ct value corresponding to the target miRNA standard should be obtained in the same manner as the Ct value of the sample to be tested. That is, the reverse transcription primer, the first primer, the second primer and the fluorescent probe for the target miRNA provided herein should be used, and the same processing steps should be followed, to obtain the Ct value of the target miRNA standard.

[0084] In some embodiments, the sample to be tested is suspected to contain a miRNA similar to the target miRNA, which has one or several nucleotide residue substitutions, deletions or additions (e.g., 1, 2 or 3 nucleotide residue substitutions, deletions or additions) compared to the target miRNA; the method further comprises:

[0085] Ct value 1 is obtained by detecting the sample to be tested using the system corresponding to the similar miRNA, and Ct value 2 is obtained by detecting the sample to be tested using the system corresponding to the target miRNA, and the difference between Ct value 1 and Ct value 2, i.e., sample ΔCt, is calculated; the similar miRNA standard and the target miRNA standard with known content are mixed in different proportions (e.g., 10000:0 to 0:10000), and the corresponding Ct values are obtained by detecting using the system corresponding to the similar miRNA and the system corresponding to the target miRNA, respectively, and the difference between the Ct values, i.e., standard ΔCt, is calculated; sample ΔCt is compared with multiple groups of standard ΔCt to evaluate whether the similar miRNA exists in the sample to be tested.

[0086] In some embodiments, after determining whether the similar miRNA exists, the corresponding Ct values of the target miRNA standard series after dilution are further obtained and a standard curve is established, and the corresponding Ct values of the similar miRNA standard series after dilution are further obtained and a standard curve is established; the Ct value of the sample to be tested is substituted into the target miRNA standard curve or the similar miRNA standard curve to calculate the content of the target miRNA or the similar miRNA in the sample to be tested.

[0087] As understood by those skilled in the art, the system corresponding to the similar miRNA means that, based on the sequence of the similar miRNA, reverse transcription primers, first primers, second primers and fluorescent probes specific to the sequence of the similar miRNA are designed according to the method of the present application, and a detection system is constructed from the designed primers and probes, and the detection system detects samples (e.g., samples to be tested or standards) according to the method of the present application.

[0088] As understood by those skilled in the art, the system corresponding to the target miRNA means that, based on the sequence of the target miRNA, reverse transcription primers, first primers, second primers and fluorescent probes specific to the sequence of the target miRNA are designed according to the method of the present application, and a detection system is constructed from the designed primers and probes, and the detection system detects samples (e.g., samples to be tested or standards) according to the method of the present application.

[0089] In some embodiments, the target miRNA standard is a sample (e.g., a purified sample) containing a known concentration or copy number of the target miRNA.

[0090] In some embodiments, the mimic miRNA standard is a sample (e.g., a purified sample) comprising a known concentration or copy number of the mimic miRNA.

[0091] It is easy for one skilled in the art to understand that the Ct value corresponding to the target miRNA standard and the Ct value corresponding to the mimic miRNA standard should be obtained in the same way as the Ct value of the sample to be tested. That is, the reverse transcription primer, the first primer, the second primer and the fluorescent probe provided in the present application for the target miRNA should be used, and the same processing steps should be adopted to obtain the Ct value of the target miRNA standard and the Ct value corresponding to the mimic miRNA standard.

[0092] In some embodiments, the mimic miRNA has one or several nucleotide residue substitutions, deletions or additions (e.g., 1, 2 or 3 nucleotide residue substitutions, deletions or additions) at positions corresponding to the first region and / or the second region of the target miRNA compared to the target miRNA.

[0093] In some embodiments, the method further comprises, after step (2), collecting the melting curve of the reaction product of step (2) using the fluorescent probe to obtain the melting curve and / or the melting temperature of the reaction product of step (2).

[0094] In some embodiments, the method further comprises comparing the melting curve and / or the melting temperature obtained with the melting curve and / or the melting temperature corresponding to the target miRNA standard.

[0095] In some embodiments, the target miRNA standard is a purified sample of the target miRNA (e.g., a purified sample comprising a known concentration or copy number of the target miRNA).

[0096] In some embodiments, when the melting curve and / or the melting temperature of the reaction product of step (2) obtained is different from the melting curve and / or the melting temperature corresponding to the target miRNA standard, it indicates that there is a mimic oligonucleotide (e.g., mimic miRNA) of the target miRNA in the sample to be tested, wherein the mimic oligonucleotide (e.g., mimic miRNA) has one or several nucleotide residue substitutions, deletions or additions (e.g., 1, 2 or 3 nucleotide residue substitutions, deletions or additions) compared to the target miRNA.

[0097] The skilled person in the art would readily understand that the way of obtaining the melting curve and / or the melting temperature of the target miRNA standard should be the same as that of the sample to be tested. That is, the reverse transcription primer, the first primer, the second primer and the fluorescent probe provided in the present application for the target miRNA should be used and the same processing steps should be adopted to obtain the melting curve and / or the melting temperature of the target miRNA standard.

[0098] In certain embodiments, when the obtained reaction product of step (2) comprises a single melting temperature, and the melting temperature is the same as that of the target miRNA standard, it indicates that the sample to be tested comprises the target miRNA.

[0099] In certain embodiments, when the obtained reaction product of step (2) comprises multiple (e.g., two or more) melting temperatures, and the melting temperature is lower than that of the target miRNA standard, it indicates that one or more of the similar oligonucleotides (e.g., similar miRNAs) are present in the sample to be tested.

[0100] In certain embodiments, the Tm value of the complementary pairing between the similar oligonucleotides (e.g., similar miRNAs) and the reverse transcription primer is lower than the annealing temperature of the reverse transcription in step (1).

[0101] In certain embodiments, the Tm value of the complementary pairing between the complementary sequence of the similar oligonucleotides (e.g., similar miRNAs) and the first primer is lower than the annealing temperature of the fluorescent quantitative PCR in step (2).

[0102] In certain embodiments, the Tm value of the complementary pairing between the similar oligonucleotides (e.g., similar miRNAs) or its complementary sequence and the fluorescent probe is lower than the annealing temperature of the fluorescent quantitative PCR in step (2).

[0103] In certain embodiments, compared with the target miRNA, the similar oligonucleotides (e.g., similar miRNAs) have one or several nucleotide residue substitutions, deletions or additions (e.g., 1, 2 or 3 nucleotide residue substitutions, deletions or additions) at the position corresponding to the interval region between the first region and the second region in the target miRNA.

[0104] In certain embodiments, the analogous miRNA and the target miRNA are derived from the same primary miRNA (pri-miRNA) or the same pre-miRNA (pre-miRNA). In certain embodiments, the analogous miRNA and the target miRNA are derived from the same family. In certain embodiments, the analogous miRNA and the target miRNA differ only by individual nucleotide residues (e.g., 1, 2, or 3 nucleotide residues are substituted, deleted, or added) and the analogous miRNA and the target miRNA each have a different origin (e.g., derived from different families, derived from different pri-miRNAs, or derived from different pre-miRNAs).

[0105] 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.

[0106] As will be readily appreciated by one skilled in the art, the methods of the present application are not limited to the detection of a particular species of miRNA, and can be used to detect any species of miRNA (e.g., any species of miRNA of which the sequence is known). For example, for any miRNA of a given sequence, a reverse transcription primer, a first primer, a second primer, and a fluorescent probe can be designed and provided for the miRNA based on the methods of the present application, and the miRNA can be specifically detected by the methods of the present application.

[0107] In certain embodiments, the target miRNA is selected from the group consisting of hcmv-miR-UL22A-5p, ebv-miR-BART3-3p, hcmv-miR-US25-2-3p, hcmv-miR-US4-3p, hcmv-miR-US25-1-5p, ebv-miR-BART2-5p, ebv-miR-BART2-3p, ebv-miR-BART5-5p, ebv-miR-BART9-5p, ebv-miR-BART10-3p, ebv-miR-BHRF1-3, and any combination thereof.

[0108] In certain embodiments, the target miRNA is hcmv-miR-UL22A-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 2, the first primer has a sequence as set forth in SEQ ID NO: 10, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 22.

[0109] In certain embodiments, the target miRNA is ebv-miR-BART3-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 5, the first primer has a sequence as set forth in SEQ ID NO: 16, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 26.

[0110] In certain embodiments, the target miRNA is hcmv-miR-US25-2-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 114, the first primer has a sequence as set forth in SEQ ID NO: 149, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 193.

[0111] In certain embodiments, the target miRNA is hcmv-miR-US4-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 118, the first primer has a sequence as set forth in SEQ ID NO: 155, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 199.

[0112] In certain embodiments, the target miRNA is hcmv-miR-US25-1-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 124, the first primer has a sequence as set forth in SEQ ID NO: 159, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 203.

[0113] In certain embodiments, the target miRNA is ebv-miR-BART2-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 128, the first primer has a sequence as set forth in SEQ ID NO: 162 or 221, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 206. In certain embodiments, the target miRNA is ebv-miR-BART2-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 128, the first primer has a sequence as set forth in SEQ ID NO: 221, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 206.

[0114] In certain embodiments, the target miRNA is ebv-miR-BART2-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 131, the first primer has a sequence as set forth in SEQ ID NO: 169, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 209.

[0115] In certain embodiments, the target miRNA is ebv-miR-BART5-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 135, the first primer has a sequence as set forth in SEQ ID NO: 175, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 211.

[0116] In certain embodiments, the target miRNA is ebv-miR-BART9-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 141, the first primer has a sequence as set forth in SEQ ID NO: 179, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 213.

[0117] In certain embodiments, the target miRNA is ebv-miR-BART10-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 144, the first primer has a sequence as set forth in SEQ ID NO: 183, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 215.

[0118] In certain embodiments, the target miRNA is ebv-miR-BHRF1-3, and the reverse transcription primer has a sequence as set forth in any one of SEQ ID NOs: 217-129, the first primer has a sequence as set forth in SEQ ID NO: 220, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 223. In certain embodiments, the target miRNA is ebv-miR-BHRF1-3, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 218 or 219, the first primer has a sequence as set forth in SEQ ID NO: 220, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 223.

[0119] In certain embodiments, the target miRNA is hcmv-miR-UL22A-5p, and the reverse transcription primer has the sequence of SEQ ID NO: 2, the first primer has the sequence of SEQ ID NO: 10, the second primer has the sequence of SEQ ID NO: 18, and the fluorescent probe has the sequence of SEQ ID NO: 22.

[0120] In certain embodiments, the target miRNA is ebv-miR-BART3-3p, and the reverse transcription primer has the sequence of SEQ ID NO: 5, the first primer has the sequence of SEQ ID NO: 16, the second primer has the sequence of SEQ ID NO: 19, and the fluorescent probe has the sequence of SEQ ID NO: 26.

[0121] In certain embodiments, the target miRNA is hcmv-miR-US25-2-3p, and the reverse transcription primer has the sequence of SEQ ID NO: 114, the first primer has the sequence of SEQ ID NO: 149, the second primer has the sequence of SEQ ID NO: 185, and the fluorescent probe has the sequence of SEQ ID NO: 193.

[0122] In certain embodiments, the target miRNA is hcmv-miR-US4-3p, and the reverse transcription primer has the sequence of SEQ ID NO: 118, the first primer has the sequence of SEQ ID NO: 155, the second primer has the sequence of SEQ ID NO: 186, and the fluorescent probe has the sequence of SEQ ID NO: 199.

[0123] In certain embodiments, the target miRNA is hcmv-miR-US25-1-5p, and the reverse transcription primer has the sequence of SEQ ID NO: 124, the first primer has the sequence of SEQ ID NO: 159, the second primer has the sequence of SEQ ID NO: 187, and the fluorescent probe has the sequence of SEQ ID NO: 203.

[0124] In certain embodiments, the target miRNA is ebv-miR-BART2-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 128, the first primer has a sequence as set forth in SEQ ID NO: 162 or 221, the second primer has a sequence as set forth in SEQ ID NO: 188, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 206. In certain embodiments, the target miRNA is ebv-miR-BART2-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 128, the first primer has a sequence as set forth in SEQ ID NO: 221, the second primer has a sequence as set forth in SEQ ID NO: 188, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 206.

[0125] In certain embodiments, the target miRNA is ebv-miR-BART2-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 131, the first primer has a sequence as set forth in SEQ ID NO: 169, the second primer has a sequence as set forth in SEQ ID NO: 189, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 209.

[0126] In certain embodiments, the target miRNA is ebv-miR-BART5-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 135, the first primer has a sequence as set forth in SEQ ID NO: 175, the second primer has a sequence as set forth in SEQ ID NO: 190, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 211.

[0127] In certain embodiments, the target miRNA is ebv-miR-BART9-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 141, the first primer has a sequence as set forth in SEQ ID NO: 179, the second primer has a sequence as set forth in SEQ ID NO: 191, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 213.

[0128] In certain embodiments, the target miRNA is ebv-miR-BART10-3p, the reverse transcription primer has a sequence as set forth in SEQ ID NO: 144, the first primer has a sequence as set forth in SEQ ID NO: 183, the second primer has a sequence as set forth in SEQ ID NO: 192, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 215.

[0129] In certain embodiments, the target miRNA is ebv-miR-BHRF1-3, the reverse transcription primer has a sequence as set forth in any one of SEQ ID NOs: 217-129, the first primer has a sequence as set forth in SEQ ID NO: 220, the second primer has a sequence as set forth in SEQ ID NO: 222, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 223. In certain embodiments, the target miRNA is ebv-miR-BHRF1-3, the reverse transcription primer has a sequence as set forth in SEQ ID NO: 218 or 219, the first primer has a sequence as set forth in SEQ ID NO: 220, the second primer has a sequence as set forth in SEQ ID NO: 222, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 223.

[0130] In certain embodiments, the method is for diagnostic purposes.

[0131] In certain embodiments, the method is for non-diagnostic purposes.

[0132] Kit

[0133] In another aspect, the present application also provides a kit comprising a reverse transcription primer, a first primer and a second primer and a fluorescent probe for detecting a target miRNA;

[0134] wherein the reverse transcription primer comprises a capture sequence complementary to a first region of the target miRNA at its 3' end and is capable of initiating an extension reaction;

[0135] the first primer comprises a sequence (As sequence) complementary to a sequence complementary to a second region of the target miRNA at its 3' end and is capable of initiating an extension reaction;

[0136] the second primer is capable of annealing to a sequence complementary to the reverse transcription primer and is capable of initiating an extension reaction;

[0137] The fluorescent probe comprises: (i) a sequence complementary to the third region of the target miRNA (a Bs1 sequence), or (ii) a sequence complementary to a sequence complementary to the third region of the target miRNA (a Bs2 sequence);

[0138] The second region of the target miRNA is located upstream of the first region, and the second region and the first region comprise a spacer region therebetween.

[0139] The third region of the target miRNA comprises a spacer region between the first region and the second region of the target miRNA.

[0140] In certain embodiments, the third region overlaps the second region by 0-3 (e.g., 0, 1, 2, 3) nucleotide residues.

[0141] In certain embodiments, the capture sequence is 4-9 nt in length.

[0142] In certain embodiments, the As sequence is 8-16 nt in length.

[0143] In certain embodiments, the fluorescent probe is 8-25 nt in length.

[0144] In certain embodiments, the spacer region between the first region and the second region of the target miRNA is 1-8 nt in length (e.g., the 3’ end of the second region of the target miRNA is spaced 1-8 nucleotide residues from the 5’ end of the first region).

[0145] In certain embodiments, the fluorescent probe comprises a sequence complementary to the third region of the target miRNA (a Bs1 sequence).

[0146] In certain embodiments, the fluorescent probe comprises the Bs1 sequence at its 3’ end.

[0147] In certain embodiments, the Bs1 sequence is 8-25 nt, 8-20 nt, 10-25 nt, 10-20 nt, 8-18 nt, 10-18 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt in length.

[0148] In certain embodiments, the Bs1 sequence is fully complementary to the sequence of the third region of the target miRNA.

[0149] In some embodiments, each nucleic acid base of the Bs1 sequence comprised by the fluorescent probe is capable of base pairing with a nucleic acid base of a corresponding segment of a complementary strand of the reverse transcription product of the target miRNA.

[0150] In some embodiments, the fluorescent probe further comprises, at its 5' end, a sequence or a partial sequence thereof of the sequence of the reverse transcription primer located upstream of and adjacent to the capture sequence.

[0151] In some embodiments, the fluorescent probe comprises a sequence complementary to a complementary sequence of the third region of the target miRNA (Bs2 sequence).

[0152] In some embodiments, the fluorescent probe comprises, at its 5' end, the Bs2 sequence.

[0153] In some embodiments, the Bs2 sequence has a length of 8-25 nt, 8-20 nt, 10-25 nt, 10-20 nt, 8-18 nt, 10-18 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt.

[0154] In some embodiments, the Bs2 sequence is fully complementary to a complementary sequence of the third region of the target miRNA.

[0155] In some embodiments, each nucleic acid base of the Bs2 sequence comprised by the fluorescent probe is capable of base pairing with a nucleic acid base of a corresponding segment of the reverse transcription product of the target miRNA.

[0156] In some embodiments, the fluorescent probe further comprises, at its 3' end, a sequence complementary to a sequence or a partial sequence thereof of the sequence of the reverse transcription primer located upstream of and adjacent to the capture sequence.

[0157] In some embodiments, the kit comprises one or more features selected from the following:

[0158] (1) the first region is located at the 3' end of the target miRNA;

[0159] (2) the second region is located at the 5' end of the target miRNA;

[0160] (3) the 3' end of the second region of the target miRNA is separated from the 5' end of the first region by 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide residues;

[0161] (4) the third region comprises the first region;

[0162] (5) the Tm value of the capture sequence is 12-20°C (e.g., 12-18°C, 12-16°C, 14-20°C, 14-18°C, 14-16°C, 16-20°C, 16-18°C, 16°C);

[0163] (6) the capture sequence is located at the 3' end of the reverse transcription primer;

[0164] (7) the length of the capture sequence is 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, or 9 nt;

[0165] (8) the As sequence is located at the 3' end of the first primer;

[0166] (9) the length of the As sequence is 8-16 nt, 8-14 nt, 8-12 nt, 11-16 nt, 11-14 nt, 11-12 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, or 16 nt;

[0167] (10) the Tm value of the As sequence is 20-55°C (e.g., 20-40°C, 20-30°C, 25-55°C, 25-40°C, 25-30°C, 30-55°C, 30-40°C);

[0168] (11) the Tm value of the fluorescent probe is 32-60°C (e.g., 32-55°C, 32-50°C, 40-60°C, 40-55°C, 40-50°C, 45-60°C, 45-55°C, 45-50°C);

[0169] (12) the length of the fluorescent probe is 8-25 nt, 8-20 nt, 10-25 nt, 10-20 nt, 8-18 nt, 10-18 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt;

[0170] (13) the target miRNA is a mature miRNA;

[0171] (14) the reverse transcription primer comprises modified nucleotides (e.g., locked nucleic acids); preferably, the capture sequence of the reverse transcription primer comprises one or more modified nucleotides (e.g., locked nucleic acids);

[0172] (15) the first primer comprises modified nucleotides (e.g., locked nucleic acids); preferably, the As sequence of the first primer comprises one or more modified nucleotides (e.g., locked nucleic acids).

[0173] In certain embodiments, the Tm value of the capture sequence is 0-20°C (e.g., 0-18°C, 0-16°C, 4-18°C, 4-16°C, 8-18°C, 8-16°C, 12-18°C, 12-16°C, 14-20°C, 14-18°C, 14-16°C, 16-20°C, 16-18°C, 16°C).

[0174] In certain embodiments, the Tm value of the As sequence is 2-60°C (e.g., 2-55°C, 2-50°C, 2-40°C, 2-30°C, 10-60°C, 10-55°C, 10-50°C, 10-40°C, 10-30°C, 20-60°C, 20-55°C, 20-50°C, 20-40°C, 20-30°C, 25-60°C, 25-55°C, 25-50°C, 25-40°C, 25-30°C, 30-60°C, 30-55°C, 30-40°C).

[0175] In certain embodiments, the Tm value of the As sequence is 45-60°C (e.g., 45-58°C, 45-55°C, 50-60°C, 50-58°C, 50-55°C, 52-60°C, 52-58°C, 52-55°C, 55°C).

[0176] In certain embodiments, the reverse transcription primer does not comprise a nucleotide modification that blocks DNA polymerase extension.

[0177] In certain embodiments, the reverse transcription primer is 34-70 nt in length.

[0178] In certain embodiments, the first primer is 18-25 nt in length.

[0179] In certain embodiments, the second primer is 18-25 nt in length.

[0180] In certain embodiments, the fluorescent probe is modified with a fluorescent group and a fluorescence quenching group.

[0181] In certain embodiments, when the fluorescent probe is not bound to the amplification product of the target miRNA, the fluorescent group and the fluorescence quenching group can achieve fluorescence resonance energy transfer, so that the fluorescence signal of the fluorescent group is quenched completely or partially; and when the fluorescent probe is bound to the amplification product of the target miRNA, the fluorescent group and the fluorescence quenching group cannot achieve fluorescence resonance energy transfer, so that the fluorescent group produces a corresponding fluorescence signal.

[0182] In certain embodiments, the fluorescent probe, when not bound to the amplification product of the target miRNA, the fluorescent group and the fluorescent quencher group are unable to achieve fluorescence resonance energy transfer, thereby causing the fluorescent group to generate a corresponding fluorescent signal; and, when the fluorescent probe binds to the amplification product of the target miRNA, the fluorescent group and the fluorescent quencher group are able to achieve fluorescence resonance energy transfer, thereby causing the fluorescent group to generate a corresponding fluorescent signal to be completely or partially quenched.

[0183] 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.

[0184] In certain embodiments, the fluorescent quencher group is selected from the group consisting of BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, and any combination thereof.

[0185] In certain embodiments, the combination of the fluorescent group and the fluorescent quencher 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.

[0186] In certain embodiments, the reverse transcription primer does not comprise a stem loop structure.

[0187] In certain embodiments, the reverse transcription primer further comprises a universal sequence at its 5' end.

[0188] In certain embodiments, the second primer is capable of annealing to the complement of the universal sequence.

[0189] In certain embodiments, the reverse transcription primer comprises a stem loop structure.

[0190] In certain embodiments, the stem loop structure is located upstream of the capture sequence.

[0191] In certain embodiments, the second primer is capable of annealing to a sequence complementary to the loop region sequence of the reverse transcription primer.

[0192] Without being limited by theory, the kit provided by the present application can be used to detect a target miRNA of any origin. For example, the target miRNA can be derived from a body fluid sample, a cell sample, an extracted sample, or any combination thereof.

[0193] As will be readily appreciated by one skilled in the art, the kit of the present application 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, the kit based on the present application can provide a reverse transcription primer, a first primer, a second primer, and a fluorescent probe for the miRNA.

[0194] In certain embodiments, the target miRNA is selected from the group consisting of hcmv-miR-UL22A-5p, ebv-miR-BART3-3p, hcmv-miR-US25-2-3p, hcmv-miR-US4-3p, hcmv-miR-US25-1-5p, ebv-miR-BART2-5p, ebv-miR-BART2-3p, ebv-miR-BART5-5p, ebv-miR-BART9-5p, ebv-miR-BART10-3p, ebv-miR-BHRF1-3, and any combination thereof.

[0195] In certain embodiments, the target miRNA is hcmv-miR-UL22A-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 2, the first primer has a sequence as set forth in SEQ ID NO: 10, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 22.

[0196] In certain embodiments, the target miRNA is ebv-miR-BART3-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 5, the first primer has a sequence as set forth in SEQ ID NO: 16, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 26.

[0197] In certain embodiments, the target miRNA is hcmv-miR-US25-2-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 114, the first primer has a sequence as set forth in SEQ ID NO: 149, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 193.

[0198] In certain embodiments, the target miRNA is hcmv-miR-US4-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 118, the first primer has a sequence as set forth in SEQ ID NO: 155, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 199.

[0199] In certain embodiments, the target miRNA is hcmv-miR-US25-1-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 124, the first primer has a sequence as set forth in SEQ ID NO: 159, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 203.

[0200] In certain embodiments, the target miRNA is ebv-miR-BART2-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 128, the first primer has a sequence as set forth in SEQ ID NO: 162 or 221, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 206. In certain embodiments, the target miRNA is ebv-miR-BART2-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 128, the first primer has a sequence as set forth in SEQ ID NO: 221, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 206.

[0201] In certain embodiments, the target miRNA is ebv-miR-BART2-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 131, the first primer has a sequence as set forth in SEQ ID NO: 169, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 209.

[0202] In certain embodiments, the target miRNA is ebv-miR-BART5-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 135, the first primer has a sequence as set forth in SEQ ID NO: 175, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 211.

[0203] In certain embodiments, the target miRNA is ebv-miR-BART9-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 141, the first primer has a sequence as set forth in SEQ ID NO: 179, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 213.

[0204] In certain embodiments, the target miRNA is ebv-miR-BART10-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 144, the first primer has a sequence as set forth in SEQ ID NO: 183, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 215.

[0205] In certain embodiments, the target miRNA is ebv-miR-BHRF1-3, and the reverse transcription primer has a sequence as set forth in any one of SEQ ID NOs: 217-129, the first primer has a sequence as set forth in SEQ ID NO: 220, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 223. In certain embodiments, the target miRNA is ebv-miR-BHRF1-3, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 218 or 219, the first primer has a sequence as set forth in SEQ ID NO: 220, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 223.

[0206] In certain embodiments, the target miRNA is hcmv-miR-UL22A-5p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 2, the first primer has a sequence as set forth in SEQ ID NO: 10, the second primer has a sequence as set forth in SEQ ID NO: 18, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 22.

[0207] In certain embodiments, the target miRNA is ebv-miR-BART3-3p, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 5, the first primer has a sequence as set forth in SEQ ID NO: 16, the second primer has a sequence as set forth in SEQ ID NO: 19, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 26.

[0208] In certain embodiments, the target miRNA is hcmv-miR-US25-2-3p, and the reverse transcription primer has the sequence of SEQ ID NO: 114, the first primer has the sequence of SEQ ID NO: 149, the second primer has the sequence of SEQ ID NO: 185, and the fluorescent probe has the sequence of SEQ ID NO: 193.

[0209] In certain embodiments, the target miRNA is hcmv-miR-US4-3p, and the reverse transcription primer has the sequence of SEQ ID NO: 118, the first primer has the sequence of SEQ ID NO: 155, the second primer has the sequence of SEQ ID NO: 186, and the fluorescent probe has the sequence of SEQ ID NO: 199.

[0210] In certain embodiments, the target miRNA is hcmv-miR-US25-1-5p, and the reverse transcription primer has the sequence of SEQ ID NO: 124, the first primer has the sequence of SEQ ID NO: 159, the second primer has the sequence of SEQ ID NO: 187, and the fluorescent probe has the sequence of SEQ ID NO: 203.

[0211] In certain embodiments, the target miRNA is ebv-miR-BART2-5p, and the reverse transcription primer has the sequence of SEQ ID NO: 128, the first primer has the sequence of SEQ ID NO: 162 or 221, the second primer has the sequence of SEQ ID NO: 188, and the fluorescent probe has the sequence of SEQ ID NO: 206. In certain embodiments, the target miRNA is ebv-miR-BART2-5p, and the reverse transcription primer has the sequence of SEQ ID NO: 128, the first primer has the sequence of SEQ ID NO: 221, the second primer has the sequence of SEQ ID NO: 188, and the fluorescent probe has the sequence of SEQ ID NO: 206.

[0212] In certain embodiments, the target miRNA is ebv-miR-BART2-3p, and the reverse transcription primer has the sequence of SEQ ID NO: 131, the first primer has the sequence of SEQ ID NO: 169, the second primer has the sequence of SEQ ID NO: 189, and the fluorescent probe has the sequence of SEQ ID NO: 209.

[0213] In certain embodiments, the target miRNA is ebv-miR-BART5-5p, and the reverse transcription primer has the sequence of SEQ ID NO: 135, the first primer has the sequence of SEQ ID NO: 175, the second primer has the sequence of SEQ ID NO: 190, and the fluorescent probe has the sequence of SEQ ID NO: 211.

[0214] In certain embodiments, the target miRNA is ebv-miR-BART9-5p, and the reverse transcription primer has the sequence of SEQ ID NO: 141, the first primer has the sequence of SEQ ID NO: 179, the second primer has the sequence of SEQ ID NO: 191, and the fluorescent probe has the sequence of SEQ ID NO: 213.

[0215] In certain embodiments, the target miRNA is ebv-miR-BART10-3p, and the reverse transcription primer has the sequence of SEQ ID NO: 144, the first primer has the sequence of SEQ ID NO: 183, the second primer has the sequence of SEQ ID NO: 192, and the fluorescent probe has the sequence of SEQ ID NO: 215.

[0216] In some embodiments, the target miRNA is ebv-miR-BHRF1-3, and the reverse transcription primer has a sequence as set forth in any one of SEQ ID NOs: 217-129, the first primer has a sequence as set forth in SEQ ID NO: 220, the second primer has a sequence as set forth in SEQ ID NO: 222, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 223. In some embodiments, the target miRNA is ebv-miR-BHRF1-3, and the reverse transcription primer has a sequence as set forth in SEQ ID NO: 218 or 219, the first primer has a sequence as set forth in SEQ ID NO: 220, the second primer has a sequence as set forth in SEQ ID NO: 222, and the fluorescent probe has a sequence as set forth in SEQ ID NO: 223.

[0217] In another aspect, the present application also provides a method for designing a miRNA detection primer, comprising the following steps:

[0218] S1: providing a target miRNA sequence;

[0219] S2: designing a reverse transcription primer sequence, a first primer sequence, a second primer sequence, and a fluorescent probe sequence according to the target miRNA sequence;

[0220] wherein the reverse transcription primer, the first primer, the second primer, and the fluorescent probe are as defined above.

[0221] In some embodiments, the target miRNA is a mature miRNA.

[0222] In another aspect, the present application also provides a computer readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the steps of the method for designing a miRNA detection primer as described above.

[0223] In another aspect, the present application also provides an electronic device, comprising: a computer readable storage medium as described above; and

[0224] one or more processors configured to execute the program in the computer readable storage medium.

[0225] In another aspect, the present application also provides the use of the kit, the method for designing a miRNA detection primer, the computer readable storage medium, or the electronic device as described above in detecting a miRNA.

[0226] In some embodiments, the target miRNA is a mature miRNA.

[0227] In another aspect, the present application also provides use of the kit as described above in the preparation of a detection reagent for detecting miRNA.

[0228] In certain embodiments, the target miRNA is a mature miRNA.

[0229] Definitions of terms

[0230] In the present application, unless otherwise indicated, 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 viral, biochemical, immunological laboratory procedures described herein are described in the manner considered to be conventional by those of ordinary skill in the corresponding fields. In addition, the following terms are defined and explained as follows in order to better define the present application.

[0231] When the terms "for example," "for instance," "such as," "including," "containing," or "comprising" or variations thereof are used herein, these terms are not to be interpreted in an exclusionary sense, but rather in an illustrative sense.

[0232] Unless otherwise indicated herein, the singular forms "a," "an," and "the" and the like are to be construed as covering both the singular and the plural unless the context otherwise permits or requires.

[0233] As used herein, the term "upstream" is used to describe the relative position of two nucleic acid sequences (or two nucleic acid molecules) and has the meaning generally understood by those of ordinary skill in the art. For example, the expression "a 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 forward 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."

[0234] For a variety of reasons, the nucleic acids or polynucleotides of the present application (e.g., "reverse transcription primer," "first primer," "second primer," "fluorescent probe," 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.

[0235] 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. For purposes 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 pre-determined 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.

[0236] As used herein, the terms "complementary" or "complementarity" are used in reference to the sequence of nucleotides linked in accordance with the base-pairing rules. For example, the sequence "A-G-T" is complementary to the sequence "3'-U-C-5'." Complementarity can be "partial," in which only some of the nucleic acid bases match according to the base-pairing rules. Or, there can be "complete" or "total" complementarity between the nucleic acids.

[0237] As used herein, the term "mature miRNA" has the meaning commonly understood by those skilled in the art, and refers to a class of small, non-coding RNAs, typically consisting of about 21-25 nucleotides, that are generally involved in the regulation of gene expression within cells. A mature miRNA present within 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.

[0238] Advantages of the invention

[0239] (1) The method of the present application has good specificity of the detection system itself, and there is no non-specific amplification when detecting a template-free negative water sample. Compared with the current commonly used stem-loop primer miRNA detection method, the present method has higher specificity of the detection probe. At the same time, a spacer sequence is designed between the 3' end of the reverse transcription primer and the 3' end of the first amplification primer, which effectively ensures the specificity of the detection system itself.

[0240] (2) The method of the present application has high sensitivity, and the detection range can span multiple concentration gradients, and has high accuracy and precision for quantitative detection of miRNA.

[0241] (3) The method has good quantitative accuracy, and the quantitative result is not easily affected by mutant target miRNA. By specific primer sequences of reverse transcription oligonucleotide and target miRNA 3' end complementary pairing, specific sequences of first amplification primer 3' and target miRNA reverse complementary sequence complementary pairing, specific sequences of detection probe 3' end and 5' spacer sequence of 3' extension region in reverse transcription product complementary pairing, full-length coverage of target miRNA to be detected is realized. When the mutant target miRNA exists, the Tm value of the mutant miRNA is lower than the target annealing temperature, so that the reverse transcription, amplification or detection efficiency is reduced, and therefore the detection signal of the mutant target miRNA and the detection signal of the non-mutant target miRNA can be effectively distinguished. When the target miRNA is detected, the influence of the mutant target detection signal can be minimized, so that the target miRNA can be accurately quantified.

[0242] (4) The method can well distinguish a few or single base mismatches, and by analyzing the melting curve of the amplification product, it can be judged whether the mutant exists in the sample and the proportion of the mutant. Therefore, compared with the prior art, the method has enhanced detection specificity for target miRNA in the presence of miRNA mutant or similar sequence in the sample.

[0243] (5) The method can be used for determination of expression amount of heterologous miRNA in different species, and the specificity of determination of heterologous miRNA in host nucleic acid background is good.

[0244] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not limited to the scope of the present application. According to the following detailed description of the preferred embodiments and the drawings, the various purposes and advantages of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0245] Figure 1 is a schematic diagram of the method of the present application.

[0246] Figures 2A to 2C are design rules and verification results of primers and probes in the method of the present application.

[0247] Figure 3 is a result diagram of analyzing the specificity of the method of the present application based on real-time fluorescent quantitative PCR.

[0248] Figure 4 is a result diagram of analyzing the sensitivity of the method of the present application based on real-time fluorescent quantitative PCR.

[0249] Figure 5 is a result diagram of distinguishing different miRNA mutants using the method of the present application.

[0250] Figure 6: Resulting graph of differentiating different mutants by melting curve using the method of the present application.

[0251] Figure 7: Resulting graph of detecting heterogenous miRNA in healthy mice using the method of the present application.

[0252] Figure 8: Resulting graph of detection performance of the method of the present application for clinical samples of hematopoietic stem cell transplantation.

[0253] Figure 9: Resulting graph of detection performance of the method of the present application for clinical samples of nasopharyngeal carcinoma.

[0254] Figure 10: Resulting graph of improving detection performance by Lock Nucleic Acid modification of primer probe nucleotide sequence in the method of the present application. DETAILED DESCRIPTION

[0255] 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 present application).

[0256] 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, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples describe the present application in terms of preferred embodiments, and that the present application is not intended to be limited to the preferred embodiments described in the examples.

[0257] Unless otherwise indicated, the various materials used in the following examples were obtained from commercial suppliers and used as received, unless otherwise indicated. The primers and probes were synthesized by Shanghai Generay Biotech Co., Ltd.

[0258] Example 1: Design rules and verification results of primers and probes in the present application

[0259] The EBV miRNA or CMV miRNA sequences detected in the present application are from miRBase, and different reverse transcription primers, first amplification primers, second amplification primers and detection probes are designed according to the respective miRNA sequences. The primer and probe sequences of EBV miRNA or CMV miRNA are shown in Table 1. On the basis that the designed primers and probes can stably complementarily pair with the template, the 3' end of the reverse transcription primer, the 3' end of the first amplification primer and the 5' end of the detection probe are further designed to be truncated by a single nucleotide at the complementary region, and the amplification or detection efficiency of a series of primers and probes with different lengths is compared.

[0260] The cDNA was generated by reverse transcription reaction using the reverse transcription primer in Table 1 as a template with the synthesized miRNA, and the reverse transcription primer was stem loop primer or straight chain primer. In this embodiment, stem loop primer was used. The reverse transcription reaction system was as follows:

[0261] The reverse transcription instrument was C1000 TM Thermal Cycler, and the reaction condition was 16°C for 30 minutes, 42°C for 30 minutes, and 95°C for 5 minutes. Two repeated holes were set for each reverse transcription reaction.

[0262] The cDNA generated by reverse transcription was used as a template for PCR amplification, and Real-time PCR quantitative detection was performed using the first amplification primer, the second amplification primer and the detection probe in Table 1. The reaction system of Real-time PCR was as follows:

[0263] The Real-time PCR instrument was CFX96 TM Real-Time System, and the reaction condition was pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 10 seconds, annealing and extension at 55°C for 30 seconds, and 45 cycles. Two repeated holes were set for each PCR reaction. The data analysis used GraphPad Prism 8 software. The final result was expressed as Ct average value + SD (standard deviation).

[0264] The detection results are shown in FIGS. 2A-2C. From the Ct values of the detection, the primer probe shown in the boxes in FIGS. 2A-2C can maintain high detection efficiency as the primer probe with longer sequence for the same concentration of template, but the detection efficiency is obviously decreased after the primer is further truncated by 1 nt. In another aspect, the truncation of 1 nt can be considered as a deletion of 1 nt mutation, which indicates that the primer probe shown in the boxes in FIGS. 2A-2C (i.e., RT-2 (SEQ ID NO: 2), F-2 (SEQ ID NO: 10), P-3 (SEQ ID NO: 22) for detection of hcmv-miR-UL22A-5p; RT-1 (SEQ ID NO: 5), F-5 (SEQ ID NO: 16), P-3 (SEQ ID NO: 26) for detection of ebv-miR-BART3-3p; RT-4 (SEQ ID NO: 114), F-3 (SEQ ID NO: 149), P-1 (SEQ ID NO: 193) for detection of hcmv-miR-US25-2-3p; RT-2 (SEQ ID NO: 118), F-4 (SEQ ID NO: 155), P-4 (SEQ ID NO: 199) for detection of hcmv-miR-US4-3p; RT-4 (SEQ ID NO: 124), F-3 (SEQ ID NO: 159), P-3 (SEQ ID NO: 203) for detection of hcmv-miR-US25-1-5p; RT-3 (SEQ ID NO: 128), F-2 (SEQ ID NO: 162), P-1 (SEQ ID NO: 206) for detection of ebv-miR-BART2-5p; RT-1 (SEQ ID NO: 131), F-4 (SEQ ID NO: 169), P-1 (SEQ ID NO: 209) for detection of ebv-miR-BART2-3p; RT-1 (SEQ ID NO: 135), F-3 (SEQ ID NO: 175), P-1 (SEQ ID NO: 211) for detection of ebv-miR-BART5-5p; RT-3 (SEQ ID NO: 141), F-3 (SEQ ID NO: 179), P-1 (SEQ ID NO: 213) for detection of ebv-miR-BART9-5p; RT-2 (SEQ ID NO: 144), F-3 (SEQ ID NO: 183), P-1 (SEQ ID NO: 215) for detection of ebv-miR-BART10-3p) can well distinguish the deletion mutation.

[0265] Example 2: Specificity verification of the real-time fluorescent quantitative PCR detection system of the method of the application

[0266] In embodiment 1, primers and probes capable of distinguishing deletion mutations were obtained for each target. This embodiment takes target hcmv-miR-UL22A-5p and ebv-miR-BART3-3p as an example, and evaluates the specificity of the detection system composed of these primers and probes (i.e., for hcmv-miR-UL22A-5p, the reverse transcription primer used is shown as SEQ ID NO: 2, the first amplification primer used is shown as SEQ ID NO: 10, the second amplification primer used is shown as SEQ ID NO: 18, and the probe used is shown as SEQ ID NO: 22; for ebv-miR-BART3-3p, the reverse transcription primer used is shown as SEQ ID NO: 5, the first amplification primer used is shown as SEQ ID NO: 16, the second amplification primer used is shown as SEQ ID NO: 19, and the probe used is shown as SEQ ID NO: 26), and simultaneously selects the widely used ABI As a reference method, 32 no-template negative water samples and 1 positive control sample are set up for each system of the method of the present application, and 12 no-template negative water samples and 1 positive control sample are set up for each system of the reference method.

[0267] The reverse transcription reaction system and procedure and the Real-time PCR reaction system and procedure of the method of the present application are the same as in embodiment 1.

[0268] The reverse transcription of the reference method uses commercial reverse transcription reagents and RT primers, and the reaction system is as follows:

[0269] The reverse transcription instrument is C1000 TM Thermal Cycler, and the reaction conditions are: 16℃ for 30 minutes, 42℃ for 30 minutes, and 85℃ for 5 minutes.

[0270] For the reference method, the cDNA generated by the above reverse transcription is used as the template for PCR amplification, and commercial ABI MicroRNA Assays and primers and probes are used for Real-time PCR quantitative detection, and the Real-time PCR reaction system is as follows:

[0271] The Real-time PCR instrument is 7500 Real Time PCR System, and the reaction conditions are: pre-denaturation at 95℃ for 10 minutes, denaturation at 95℃ for 15 seconds, annealing and extension at 60℃ for 60 seconds, for 45 cycles.

[0272] The detection results are shown in Figure 3. The results show that in the detection system designed by the method of the application for two miRNA targets of EBV miRNA or CMV miRNA, the positive control is normally amplified, and no non-specific amplification occurs in the 32-hole template-free negative water samples; while in the reference method, the positive control of the two miRNA targets is normally amplified, but non-specific amplification occurs under the condition of using fewer 12-hole negative water samples, in which 4 holes of the CMV miRNA target and 2 holes of the EBV miRNA target show non-specific amplification. It is shown that the real-time fluorescent quantitative PCR detection system of the method of the application has good specificity, which is superior to the widely used commercial reference method.

[0273] Example 3: Sensitivity verification of the real-time fluorescent quantitative PCR detection system of the method of the application

[0274] The primers and probes obtained in Example 1 for the distinguishable deletion mutations of the target miRNAs hcmv-miR-UL22A-5p and ebv-miR-BART3-3p (i.e. for hcmv-miR-UL22A-5p, the reverse transcription primer used is shown in SEQ ID NO: 2, the first amplification primer used is shown in SEQ ID NO: 10, the second amplification primer used is shown in SEQ ID NO: 18, and the probe used is shown in SEQ ID NO: 22; for ebv-miR-BART3-3p, the reverse transcription primer used is shown in SEQ ID NO: 5, the first amplification primer used is shown in SEQ ID NO: 16, the second amplification primer used is shown in SEQ ID NO: 19, and the probe used is shown in SEQ ID NO: 26), this example is for sensitivity evaluation of the system composed of primers and probes, and the widely used commercial ABI TaqMan® MicroRNA Assays is selected as a reference method to detect the standard samples of the two targets of EBV miRNA and CMV miRNA with 10-fold gradient dilution. The standard samples of the two targets of EBV miRNA and CMV miRNA are diluted from 6x10 4 (copies / μL) to 6x10 0 (copies / μL) for detection by the method of the application and the reference method.

[0275] The reverse transcription reaction system and procedure and the Real-time PCR reaction system and procedure of the method of the application are the same as those in Example 1.

[0276] The reverse transcription reaction system and procedure and the Real-time PCR reaction system and procedure of the reference method are the same as those in Example 2.

[0277] The results show (Figure 4) that for the hcmv-miR-UL22A-5p target, the sensitivity of the real-time fluorescent quantitative PCR of the method of the application is 10 times higher than that of the reference method, with a sensitivity of 6 x 10 1 (copies / μL); for the ebv-miR-BART3-3p target, the sensitivity of the real-time fluorescent quantitative PCR of the method of the application is consistent with that of the reference method, with a sensitivity of 6 x 10 2 (copies / μL).

[0278] Example 4: Verification of the discrimination ability of the real-time fluorescent quantitative PCR detection system of the method of the application for different miRNA mutants

[0279] In order to verify whether the real-time fluorescent quantitative PCR detection system of the method of the application can discriminate different site substitution mutations in addition to deletion mutations, the standard miRNA sequence was aligned with the human genome and transcriptome sequences on NCBI, and the base mutation information of different sites of the miRNA was obtained according to the alignment results, and at the same time, the miRNA mutants were obtained by artificially performing single base substitution mutation on the standard miRNA sequence according to this information (Table 2). According to the different positions of base matching of the miRNA with the RT primer, the first amplification primer and the detection probe, the mutants were divided into 3 categories, and the primer probes corresponding to the mutants were designed at the same time (Table 3) as controls. The primer probes capable of discriminating deletion mutations obtained in Example 1 (experimental group) and the primer probes corresponding to the mutants provided in Table 3 (control group) were used to detect all miRNA mutants at the same concentration.

[0280] In the experimental group, for the detection of hcmv-miR-UL22A-5p mutants, the reverse transcription primer used is shown as SEQ ID NO: 2, the first amplification primer used is shown as SEQ ID NO: 10, the second amplification primer used is shown as SEQ ID NO: 18, and the probe used is shown as SEQ ID NO: 22; for the detection of ebv-miR-BART3-3p mutants, the reverse transcription primer used is shown as SEQ ID NO: 5, the first amplification primer used is shown as SEQ ID NO: 16, the second amplification primer used is shown as SEQ ID NO: 19, and the probe used is shown as SEQ ID NO: 26.

[0281] For detection of the mutant hcmv-miR-UL22A-5p-mutant 1 to hcmv-miR-UL22A-5p-mutant 15 in the control group, the second amplification primer used is shown as SEQ ID NO: 18, wherein (i) for detection of the mutant hcmv-miR-UL22A-5p-mutant 1 to hcmv-miR-UL22A-5p-mutant 8, the reverse transcription primer and the probe are shown as SEQ ID NO: 2 and 22, and the first amplification primer used is shown as SEQ ID NO: 65-72, respectively; (ii) for detection of the mutant hcmv-miR-UL22A-5p-mutant 9 to hcmv-miR-UL22A-5p-mutant 12, the reverse transcription primer and the first amplification primer are shown as SEQ ID NO: 2 and 10, respectively, and the probe used is shown as SEQ ID NO: 73-76, respectively; (iii) for detection of the mutant hcmv-miR-UL22A-5p-mutant 13 to hcmv-miR-UL22A-5p-mutant 15, the first amplification primer and the probe are shown as SEQ ID NO: 10 and 22, respectively, and the reverse transcription primer used is shown as SEQ ID NO: 77-79, respectively.

[0282] For detection of the mutant ebv-miR-BART3-3p-mutant 1 to ebv-miR-BART3-3p-mutant 11 and ebv-miR-BART3-3p-mutant 14 to ebv-miR-BART3-3p-mutant 24 in the control group, the second amplification primer used is shown as SEQ ID NO: 19, wherein (i) for detection of the mutant ebv-miR-BART3-3p-mutant 1 to ebv-miR-BART3-3p-mutant 11, the reverse transcription primer and the probe are shown as SEQ ID NO: 5 and 26, respectively, and the first amplification primer used is shown as SEQ ID NO: 80-90, respectively; (ii) for detection of the mutant ebv-miR-BART3-3p-mutant 14 to ebv-miR-BART3-3p-mutant 19, the reverse transcription primer and the first amplification primer are shown as SEQ ID NO: 5 and 16, respectively, and the probe used is shown as SEQ ID NO: 91-96, respectively; (iii) for detection of the mutant ebv-miR-BART3-3p-mutant 20 to ebv-miR-BART3-3p-mutant 24, the first amplification primer and the probe are shown as SEQ ID NO: 16 and 26, respectively, and the reverse transcription primer used is shown as SEQ ID NO: 97-101, respectively.

[0283] The reverse transcription reaction system and procedure and the Real-time PCR reaction system and procedure of the method of the application are the same as those in Example 1.

[0284] The detection results are shown in Figure 5. The results show that for all single-base miRNA substitution mutants, the detection system using the method of the present application (corresponding to Figure 5, mutant sensitive type) can effectively distinguish substitution mutations at different sites compared with the control detection system (corresponding to Figure 5, non-sensitive type). In particular, the detection probe can achieve a discrimination degree of no amplification signal for single-base substitution mutant miRNA. It is shown that the real-time fluorescent quantitative PCR detection system of the method of the present application has good discrimination ability for different miRNA mutants.

[0285] Table 2, single-base mutant miRNA mutants in the present application

[0286] Example 5: Verification of the method of the present application for distinguishing different mutants by melting curve

[0287] After verification in Example 4, different miRNA mutants can be distinguished by PCR system. This example verifies whether melting curve can also be used to distinguish different mutants. In actual application, if one method has poor discrimination effect, the other method can also be used as a supplement. Standard cDNA, deletion mutant cDNA and substitution mutant cDNA (Table 4) are designed for hcmv-miR-UL22A-5p and ebv-miR-BART3-3p targets, and the primers and probes obtained in Example 1 capable of distinguishing deletion mutants are used for amplification (i.e. for hcmv-miR-UL22A-5p, the reverse transcription primer used is shown in SEQ ID NO: 2, the first amplification primer used is shown in SEQ ID NO: 10, the second amplification primer used is shown in SEQ ID NO: 18, and the probe used is shown in SEQ ID NO: 22; for ebv-miR-BART3-3p, the reverse transcription primer used is shown in SEQ ID NO: 5, the first amplification primer used is shown in SEQ ID NO: 16, the second amplification primer used is shown in SEQ ID NO: 19, and the probe used is shown in SEQ ID NO: 26), and the amplification product is subjected to melting curve analysis according to the following procedure

[0288] 95°C for 1 minute, 30°C-85°C for melting curve collection, collection every 0.5°C increase, collection time 5s. The results show that the deletion mutant cDNA and the substitution mutant cDNA can be distinguished from the standard cDNA, and their melting temperatures Tm and melting curves have obvious differences (Figure 6).

[0289] Table 4 Standard cDNA, deletion mutant cDNA and substitution mutant cDNA designed for hcmv-miR-UL22A-5p and ebv-miR-BART3-3p targets in the present application

[0290] Example 6: Specificity evaluation of the real-time fluorescent quantitative PCR detection system of the method of the present application in plasma samples of healthy mice

[0291] In order to further verify the specificity of the method of the present application, the plasma of healthy background mice was collected, total RNA was isolated, and the real-time fluorescent quantitative PCR detection system of the method of the present application was used for detection.

[0292] The plasma collection process was as follows: blood was collected in a blood collection tube containing EDTA anticoagulant, centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant was the plasma. The plasma sample was stored at -80°C before use.

[0293] Qiagen miRNeasy Serum / Plasma Advanced Kit was used for miRNA extraction, 200 μL of plasma sample was taken, and the manufacturer's instructions were followed for extraction. Finally, 50 μL of nuclease-free water was used for elution to obtain pure plasma total miRNA, which was stored at -80°C for standby.

[0294] The reverse transcription reaction system and procedure and the Real-time PCR reaction system and procedure of the method of the present application were the same as in Example 1.

[0295] The reverse transcription reaction system and procedure and the Real-time PCR reaction system and procedure of the reference method were the same as in Example 2.

[0296] The results (Figure 7) showed that for the hcmv-miR-UL22A-5p target, 19 out of 20 healthy background mouse plasma total miRNA samples detected using the method of the present application did not produce non-specific amplification, and only one produced a weak non-specific signal; for the ebv-miR-BART3-3p target, all healthy background mouse plasma total miRNA samples detected using the method of the present application did not produce non-specific amplification. However, the hcmv-miR-UL22A-5p and ebv-miR-BART3-3p detection of the reference method produced very strong non-specific amplification, indicating that the specificity of the real-time fluorescent quantitative PCR detection system of the method of the present application was good and superior to the existing widely used commercial reference method.

[0297] Example 7: Performance verification of the real-time fluorescent quantitative PCR detection system of the method of the present application for detection of clinical specimens of hematopoietic stem cell transplantation

[0298] To evaluate the clinical application potential of the method of the present application, 12 plasma samples positive for CMV-DNA from different transplant patients and 12 plasma samples from different healthy people (CMV-DNA negative) were collected for preliminary verification.

[0299] Plasma separation and miRNA extraction were the same as in Example 5.

[0300] The reverse transcription reaction system and procedure and the Real-time PCR reaction system and procedure of the method of the present application were the same as in Example 1.

[0301] The reverse transcription reaction system and procedure and the Real-time PCR reaction system and procedure of the reference method were the same as in Example 2.

[0302] The results showed (Figure 8) that 10 out of 12 plasma samples from transplant patients positive for CMV-DNA were detected positive for hcmv-miR-UL22A-5p using the method of the present application, and 1 out of 12 plasma samples from healthy people was detected positive; however, all the 12 plasma samples from healthy people were detected positive using the reference method. Under the condition that the sensitivity of the detection system of the method of the present application was higher than that of the reference method and that the plasma samples from healthy people were negative for CMV-DNA detected by the method of the present application, it could be inferred that the positive signals in the plasma samples from healthy people detected by the reference method were false positives. In conclusion, the method of the present application was more specific than the reference method in detecting healthy people, indicating that the method of the present application was superior to the existing widely used real-time fluorescent quantitative PCR method for clinical specimen detection, and had a high clinical application potential.

[0303] Example 8: Performance verification of the real-time fluorescent quantitative PCR detection system of the method of the present application for nasopharyngeal carcinoma clinical specimen detection

[0304] To further evaluate the clinical application potential of the method of the present application, 12 plasma samples from different nasopharyngeal carcinoma patients and 12 plasma samples from different healthy people (EBV-DNA negative) were collected for preliminary verification.

[0305] Plasma separation and miRNA extraction were the same as in Example 5.

[0306] The reverse transcription reaction system and procedure and the Real-time PCR reaction system and procedure of the method of the present application were the same as in Example 1.

[0307] The reverse transcription reaction system and procedure and the Real-time PCR reaction system and procedure of the reference method were the same as in Example 2.

[0308] The results show (Figure 9) that 9 out of 12 nasopharyngeal carcinoma patient plasma samples were detected positive for ebv-miR-BART3-3p using the method of the present application, while no positive signal was detected in 12 healthy human plasma samples; however, 12 healthy human plasma samples were detected positive using the reference method; under the condition that the sensitivity of the detection system of the method of the present application is consistent with the reference method and the healthy human plasma samples are negative for EBV-DNA detected by the method of the present application, it can be inferred that the positive signals in the healthy human plasma samples detected by the reference method are false positives. In summary, the method of the present application is more specific than the reference method in detecting healthy humans, indicating that the method of the present application is superior to the existing widely used real-time fluorescent quantitative PCR method for clinical specimen detection, and has high clinical application potential.

[0309] Example 9: Performance verification of the method of the present application for detecting target miRNA after nucleotide sequence LNA modification

[0310] In some cases, the nucleotide amplification sequence designed according to the target miRNA in the present study not only has mutation recognition ability, but also further LNA modification on the nucleotide sequence can improve the amplification efficiency of the target miRNA. The results show (Figure 10) that the use of modified nucleotide sequence, i.e. LNA-modified nucleotide sequence on the reverse transcription primer capture sequence (i.e. the sequence that anneals to the target miRNA) and the first amplification primer As sequence (i.e. the sequence that anneals to the reverse transcription product of the target miRNA) (Table 5) for target miRNA amplification detection shows that LNA modification can reduce the detection Ct value, i.e. can improve the amplification efficiency of the target miRNA.

Claims

A method for detecting the presence and / or the amount of a target microRNA (miRNA) in a test sample, comprising the following steps: (1) providing a reverse transcription primer and a test sample, contacting the reverse transcription primer with the test sample under conditions allowing reverse transcription, and performing a reverse transcription reaction; wherein the 3' end of the reverse transcription primer comprises a capture sequence complementary to a first region of the target miRNA and is capable of initiating an extension reaction; (2) contacting a first primer, a second primer, and a fluorescent probe with the product obtained in the previous step under conditions allowing nucleic acid amplification, and performing a fluorescent quantitative PCR reaction; wherein the 3' end of the first primer comprises a sequence (As sequence) complementary to a sequence complementary to a second region of the target miRNA and is capable of initiating an extension reaction; the second primer is capable of annealing to a sequence complementary to the reverse transcription primer and is capable of initiating an extension reaction; the fluorescent probe comprises (i) a sequence (Bs1 sequence) complementary to a third region of the target miRNA, or (ii) a sequence (Bs2 sequence) complementary to a sequence complementary to a third region of the target miRNA; and the second region of the target miRNA is located upstream of the first region, and the first region and the second region comprise a spacer region therebetween; the third region of the target miRNA comprises a spacer region between the first region and the second region of the target miRNA; (3) assessing the presence and / or the amount of the target miRNA in the test sample based on the fluorescent signal of the fluorescent quantitative PCR reaction in step (2). The method of claim 1, having one or more features selected from the following: (i) the third region overlaps the second region by 0-3 (e.g., 0, 1, 2, 3) nucleotide residues; (ii) the capture sequence has a length of 4-9 nt; (iii) the As sequence has a length of 8-16 nt; (iv) the fluorescent probe has a length of 8-25 nt; (v) the spacer region between the first region and the second region of the target miRNA has a length of 1-8 nt (e.g., the 3' end of the second region of the target miRNA is spaced apart from the 5' end of the first region by 1-8 nucleotide residues). The method of claim 1 or 2, wherein, the fluorescent probe comprises a sequence (Bs1 sequence) complementary to the third region of the target miRNA; preferably, the fluorescent probe comprises the Bs1 sequence at its 3' end; preferably, the Bs1 sequence is fully complementary to the sequence of the third region of the target miRNA; preferably, the fluorescent probe further comprises, at its 5' end, a sequence or a partial sequence of the sequence of the reverse transcription primer upstream of and adjacent to the capture sequence. The method of claim 1 or 2, wherein, the fluorescent probe comprises a sequence (Bs2 sequence) complementary to a sequence complementary to the third region of the target miRNA; preferably, the fluorescent probe comprises the Bs2 sequence at its 5' end; Preferably, the Bs2 sequence is fully complementary to the complement of the third region of the target miRNA; Preferably, the fluorescent probe further comprises at its 3' end the complement of the sequence or partial sequence of the reverse transcription primer which is upstream of and adjacent to the capture sequence. The method of any one of claims 1-4, which has one or more features selected from the group consisting of: (1) the first region is located at the 3' end of the target miRNA; (2) the second region is located at the 5' end of the target miRNA; (3) the 3' end of the second region of the target miRNA is spaced apart from the 5' end of the first region by 1, 2, 3, 4, 5, 6, 7 or 8 nucleotide residues; (4) the third region comprises the first region; (5) the capture sequence is located at the 3' end of the reverse transcription primer; (6) the capture sequence has a length of 4 nt, 5 nt, 6 nt, 7 nt, 8 nt or 9 nt; (7) the As sequence is located at the 3' end of the first primer; (8) the As sequence has a length of 8-16 nt, 8-14 nt, 8-12 nt, 11-16 nt, 11-14 nt, 11-12 nt, 8 nt, 11 nt or 12 nt; (9) the fluorescent probe has a Tm value of 32-60°C (e.g., 32-55°C, 32-50°C, 40-60°C, 40-55°C, 40-50°C, 45-60°C, 45-55°C, 45-50°C); (10) the fluorescent probe has a length of 8-25 nt, 8-20 nt, 10-25 nt, 10-20 nt, 8-18 nt, 10-18 nt, 8 nt, 11 nt, 12 nt, 13 nt, 14 nt, 17 nt or 18 nt; (11) the target miRNA is a mature miRNA; (12) the reverse transcription primer comprises modified nucleotides (e.g., locked nucleic acids); preferably, the capture sequence of the reverse transcription primer comprises one or more modified nucleotides (e.g., locked nucleic acids); (13) the first primer comprises modified nucleotides (e.g., locked nucleic acids); preferably, the As sequence of the first primer comprises one or more modified nucleotides (e.g., locked nucleic acids). The method of any one of claims 1-5, wherein in step (1), the annealing temperature of the reverse transcription reaction is set to be 12-20°C (e.g., 12-18°C, 12-16°C, 14-20°C, 14-18°C, 14-16°C, 16-20°C, 16-18°C, 16°C). The method of any one of claims 1-6, wherein in step (2), the annealing temperature of the fluorescent quantitative PCR reaction is set to be 45-60°C (e.g., 45-58°C, 45-55°C, 50-60°C, 50-58°C, 50-55°C, 52-60°C, 52-58°C, 52-55°C, 55°C); Preferably, in step (2), the annealing temperature and the extension temperature of the fluorescent quantitative PCR reaction are set to be the same. The method of any one of claims 1-7, wherein, The reverse transcription primer does not comprise a stem-loop structure; Preferably, the reverse transcription primer further comprises a universal sequence at its 5' end; Preferably, the second primer is capable of annealing to the complement of the universal sequence. The method of any one of claims 1-7, wherein, The reverse transcription primer comprises a stem-loop structure; Preferably, the stem-loop structure is located upstream of the capture sequence; Preferably, the second primer is capable of annealing to the complement of the loop region sequence of the reverse transcription primer. The method of any one of claims 1-9, wherein in step (3), the method further comprises comparing the fluorescence signal of the real-time PCR reaction in step (2) with the fluorescence signal corresponding to the standard miRNA to evaluate the presence and / or the amount of the target miRNA in the sample to be tested. Preferably, in step (3), the method compares the Ct value of the real-time PCR reaction in step (2) with the Ct value corresponding to the standard miRNA to evaluate the presence and / or the amount of the target miRNA in the sample to be tested. The method of any one of claims 1-10, wherein, The method further comprises, after step (2), acquiring the melting curve of the reaction product of step (2) using the fluorescent probe to obtain the melting curve and / or the melting temperature of the reaction product of step (2); Preferably, the method further comprises comparing the acquired melting curve and / or melting temperature with the melting curve and / or melting temperature corresponding to the standard miRNA; Preferably, when the acquired melting curve and / or melting temperature of the reaction product of step (2) is different from the melting curve and / or melting temperature corresponding to the standard miRNA, it indicates that there is a similar oligonucleotide (e.g., a similar miRNA) to the target miRNA in the sample to be tested, wherein the similar oligonucleotide (e.g., a similar miRNA) has one or several nucleotide residue substitutions, deletions or additions (e.g., 1, 2 or 3 nucleotide residue substitutions, deletions or additions) compared to the target miRNA. A kit comprising a reverse transcription primer, a first primer and a second primer and a fluorescent probe for detecting a target miRNA; wherein, The reverse transcription primer comprises a capture sequence complementary to the first region of the target miRNA and capable of initiating an extension reaction; The first primer comprises a sequence (As sequence) complementary to the complement of the second region of the target miRNA and capable of initiating an extension reaction; The second primer is capable of annealing to the complement of the reverse transcription primer and capable of initiating an extension reaction; The fluorescent probe comprises: (i) a sequence (Bs1 sequence) complementary to the third region of the target miRNA, or (ii) a sequence (Bs2 sequence) complementary to the complement of the third region of the target miRNA; The second region of the target miRNA is located upstream of the first region, and the second region and the first region comprise a spacer region therebetween; The third region of the target miRNA comprises the spacer region between the first region and the second region of the target miRNA. The kit of claim 12, which has one or more of the following features selected from: (i) the third region overlaps the second region by 0-3 (e.g., 0, 1, 2, 3) nucleotide residues; (ii) the length of the capture sequence is 4-9 nt; (iii) the length of the As sequence is 8-16 nt; (iv) the length of the fluorescent probe is 8-25 nt; (v) the length of the spacer region between the first region and the second region in the target miRNA is 1-8 nt (e.g., the 3’ end of the second region of the target miRNA is spaced apart from the 5’ end of the first region by 1-8 nucleotide residues). The kit of claim 12 or 13, wherein, the fluorescent probe comprises a sequence complementary to the sequence of the third region of the target miRNA (Bs1 sequence); preferably, the fluorescent probe comprises the Bs1 sequence at its 3’ end; preferably, the Bs1 sequence is fully complementary to the sequence of the third region of the target miRNA; preferably, the fluorescent probe further comprises at its 5’ end a sequence or a partial sequence of the sequence of the reverse transcription primer upstream of and adjacent to the capture sequence. The kit of claim 12 or 13, wherein, the fluorescent probe comprises a sequence complementary to the sequence complementary to the third region of the target miRNA (Bs2 sequence); preferably, the fluorescent probe comprises the Bs2 sequence at its 5’ end; preferably, the Bs2 sequence is fully complementary to the sequence complementary to the third region of the target miRNA; preferably, the fluorescent probe further comprises at its 3’ end a sequence complementary to the sequence of the reverse transcription primer upstream of and adjacent to the capture sequence. The kit of any one of claims 12-15, which has one or more of the following features selected from: (1) the first region is located at the 3’ end of the target miRNA; (2) the second region is located at the 5’ end of the target miRNA; (3) the 3’ end of the second region of the target miRNA is spaced apart from the 5’ end of the first region by 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide residues; (4) the third region comprises the first region; (5) the capture sequence is located at the 3’ end of the reverse transcription primer; (6) the length of the capture sequence is 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, or 9 nt; (7) the As sequence is located at the 3’ end of the first primer; (8) the length of the As sequence is 8-16 nt, 8-14 nt, 8-12 nt, 11-16 nt, 11-14 nt, 11-12 nt, 8 nt, 11 nt, or 12 nt; (9) the Tm value of the fluorescent probe is 32-60°C (e.g., 32-55°C, 32-50°C, 40-60°C, 40-55°C, 40-50°C, 45-60°C, 45-55°C, 45-50°C); (10) the length of the fluorescent probe is 8-25 nt, 8-20 nt, 10-25 nt, 10-20 nt, 8-18 nt, 10-18 nt, 8 nt, 11 nt, 12 nt, 13 nt, 14 nt, 17 nt or 18 nt; (11) the target miRNA is a mature miRNA; (12) the reverse transcription primer comprises modified nucleotides (e.g. locked nucleic acids); preferably, the capture sequence of the reverse transcription primer comprises one or more modified nucleotides (e.g. locked nucleic acids); (13) the first primer comprises modified nucleotides (e.g. locked nucleic acids); preferably, the As sequence of the first primer comprises one or more modified nucleotides (e.g. locked nucleic acids). The kit of any one of claims 12-16, wherein, the reverse transcription primer does not comprise a stem-loop structure; preferably, the reverse transcription primer further comprises a universal sequence at its 5' end; preferably, the second primer is capable of annealing to the complement of the universal sequence. The kit of any one of claims 12-16, wherein, the reverse transcription primer comprises a stem-loop structure; preferably, the stem-loop structure is located upstream of the capture sequence; preferably, the second primer is capable of annealing to the complement of the loop sequence of the reverse transcription primer. A method for designing a miRNA detection primer, comprising the following steps: S1: providing a target miRNA sequence; S2: designing a reverse transcription primer sequence, a first primer sequence, a second primer sequence and a fluorescent probe sequence according to the target miRNA sequence; wherein the reverse transcription primer, the first primer, the second primer and the fluorescent probe are as defined in any one of claims 1-11. 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 19. An electronic device comprising: The computer readable storage medium of claim 20; and one or more processors for executing the program in the computer readable storage medium. The kit of any one of claims 12-18, the method of claim 19, the computer readable storage medium of claim 20 or the electronic device of claim 21, for use in detecting a miRNA. The kit of any one of claims 12-18, for use in the preparation of a detection reagent for detecting a miRNA.

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