CN114250276A - MicroRNA detection system and method based on exponential amplification reaction and Argonaute nuclease - Google Patents
MicroRNA detection system and method based on exponential amplification reaction and Argonaute nuclease Download PDFInfo
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- CN114250276A CN114250276A CN202111522346.4A CN202111522346A CN114250276A CN 114250276 A CN114250276 A CN 114250276A CN 202111522346 A CN202111522346 A CN 202111522346A CN 114250276 A CN114250276 A CN 114250276A
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Abstract
The invention discloses a microRNA detection system and a method based on exponential amplification reaction and Argonaute nuclease. The invention provides a microRNA detection system based on exponential amplification reaction and Argonaute nuclease, which comprises: (a) the guide DNA is an amplification product obtained by amplifying a target object microRNA by adopting an exponential amplification reaction system; (b) argonaute nuclease; (c) the detection probe is provided with a fluorescent group and a quenching group, and comprises a region which is complementary with an amplification product obtained by amplifying the target object microRNA by adopting an exponential amplification reaction system. The microRNA detection system provided by the invention realizes single-base specific and high-sensitivity miRNA detection, and multiple miRNA detection and miRNA typing analysis.
Description
Technical Field
The invention relates to the technical field of microRNA detection, in particular to a microRNA detection system and a method based on exponential amplification reaction and Argonaute nuclease.
Background
Mature microRNA (miRNA) is a single-stranded non-coding RNA molecule with the length of 18-28 basic groups, and plays an important role in regulating various life activities of an immune system, growth and development of cells, proliferation and differentiation, apoptosis and the like. mirnas are receiving extensive attention from researchers in various fields such as developmental science, biology, aging and metabolism, disease pathology research, disease treatment, and the like. mirnas are transcribed from some DNAs into RNAs and processed, and bind to mrnas, thereby inhibiting the expression of transcribed genes, and abnormal levels of mirnas are closely related to various diseases. For example, upregulation of the levels of miRNA-21, miRNA-92a, miRNA-31 and miRNA-141 is an important factor in causing cancer (Biomedical reports 5.4(2016): 395-402; Journal of cancer research and clinical oncology 139.2(2013):223-229.Biomedicine & Pharmacotherapy 108(2018):1162-1169.Cellular Physiology and Biochemistry 38.2(2016): 427-. The occurrence of a disease is often related to the dysregulation of multiple miRNAs, and the multiplex detection of miRNAs has very important clinical significance for the diagnosis and prognosis of diseases. miRNA are widely present in blood, free microRNA markers of blood circulation have been used for liquid biopsy of tumors, contributing to early diagnosis and screening of cancer (Journal of clinical medicine 4.10(2015): 1890-1907). On the other hand, some miRNAs have family members, and the family members have only Single Nucleotide or two-Nucleotide difference, namely Single Nucleotide Polymorphism (SNP), such as let 7miRNA family, and 12 kinds of human-derived let 7miRNA family members (Trends in molecular mechanism, 2008,14(9):400-409. endogrine-related cancer,2010,17(1): F19-F36). Accurate differentiation or typing of miRNA family members will drive basic research of mirnas, e.g. identifying a certain key miRNA member causing disease, and by typing analysis can help to diagnose certain disease types such as cancer (Clinica Chimica Acta 413(2012) 1092-1097. bioorg. med. chem.46(2021)116363), with important scientific research significance and clinical value.
The current gold standard for miRNA detection is reverse transcription-polymerase chain reaction (RT-PCR, Nature Reviews Genetics 13.5(2012): 358-. And finally, designing a specific miRNA primer, and performing fluorescent quantitative PCR determination by a dye method. However, the current RT-PCR detection process of microRNA is complicated, time-consuming, high in cost and deficient in sensitivity. Nucleic acid Amplification technologies such as Rolling Circle Amplification (RCA), Exponential Amplification Reaction (EXPAR), double-strand Specific Nuclease Signal Amplification (DSNSA) and the like are developed for detection of miRNA, and at present, these methods have insufficient specificity and sensitivity, and limit further popularization and application thereof in actual clinical detection (chem. soc. rev.,2021,50, 4141-4161). On the other hand, each detection system of the methods can only detect a single miRNA target, so that the detection efficiency is low, and the available clinical information is limited and is not enough for clinical diagnosis.
In addition, the current miRNA typing analysis method is very lacking, and although RCA is suitable for miRNA typing analysis, its sensitivity is low and specificity is not sufficient to reach the resolution of a single base (analytical Chimica Acta 1076(2019)138e 143). Traditional genotyping relies mainly on sequencing and allele-specific PCR and is not suitable for miRNA typing analysis. Sequencing needs a large-scale instrument, mainly aims at a long target sequence, and has high cost and long period. Allele-specific PCR requires a large number of primer designs and screens, detection is complex, and detectable targets are very limited. CRISPR (clustered Regularly interleaved Short Palindromic repeats) technology is widely researched and used in the field of nucleic acid detection in recent years, a CRISPR/Cas system is a programmable high-specificity, rapid and high-efficiency nucleic acid cleavage system, a Cas nuclease accurately recognizes and cleaves specific nucleic acid under the mediation of guide RNA, and a plurality of Cas enzymes have the function of side-cut and can cleave other nucleic acid probes (ACS Nano 2021,15, 7848) -7859) dissociating in the system while cleaving a target. The problems in several aspects exist in the actual nucleic acid detection process, firstly, the target substance is low in concentration, unstable and easy to degrade, and the direct detection mode usually needs expensive equipment such as sequencing and the like, and is difficult to be applied practically; secondly, the specificity and accuracy of the existing nucleic acid amplification technology are poor, and false positive is easy to generate; thirdly, the detection efficiency of the current technology is limited, and multiple detections cannot be realized in a single reaction system, namely the simultaneous detection of a plurality of target objects. Although CRISPR/Cas combines with Amplification technology such as Recombinase Polymerase Amplification (RPA) and Loop-Mediated Isothermal Amplification (LAMP) to realize high specificity, high sensitivity and even multiple nucleic acid detection (Science,2018,360, 439-444; Science,2018,360, 436-439; Cell Discovery 2018,4,20), guide RNA has sequence dependence, the design is complex, the synthesis and screening costs of guide RNA are high, RNA is easy to degrade, multiple detection needs the aid of multiple nucleic acid-preferential Cas enzymes, and on one hand, the nucleic acid-specific enzymes are rare and have poor practicability, and on the other hand, the detection system is complex and has high cost.
Argonaute (Ago) is a programmable DNA endonuclease that requires only a stretch of 5' -phosphorylated single-stranded DNA (16-18 bases) as guide DNA (gDNA), targets to recognize a specific nucleic acid sequence complementary to gDNA, and cleaves a complementary DNA or RNA nucleic acid sequence at the 10 th-11 th base position of gDNA (Nature.507(7491), 258-. Compared with Cas nuclease, Ago enzyme has no sequence dependence, and has higher universality and feasibility in actual detection application. In addition, gDNA is easy to design, inexpensive and stable. The Ago enzyme has the function of accurately identifying and cutting with single base resolution, wherein Ago nuclease (Ttago) from Thermus thermophilus has been proved to be used for detecting tumor gene mutation such as KRAS, EGFR, BRAF and the like, and the detection sensitivity is improved by 100 times compared with the traditional clamped PCR (loop PCR) mediated by Peptide Nucleic Acid (PNA) and ribonucleic Acid (Xenucleic Acid) to reach the single copy level (Nucleic Acid research 48.4(2020): e19-e 19). Recent studies have shown that the cleavage efficiency of detection systems based on Ago nuclease (Pfago) derived from thermophilic archaea is equivalent to or even superior to that of CRISPR technique, and cleavage can be completed at least 3-5 min and strong detection signals can be generated (Nature communications 12.1(2021): 1-9.).
To date, there have been no reports of single or multiple miRNA detection using Ago novel molecular enzyme technology and for miRNA typing analysis including SNPs and other base mutants. The existing miRNA detection method still has the following problems:
(1) since mirnas are various in types, low in abundance and short in fragment, detection of mirnas in biological samples is still a technical problem. The EXPAR technology does not need a reverse transcription process, can directly amplify miRNA for signal amplification and detection, but has serious false positive phenomenon and limited sensitivity, and is difficult to realize application in clinical detection (Angewandte chemical International Edition 49.32(2010):5498- > 5501). The EXPAR technology completes the detection of a certain miRNA in about 30min at the constant temperature of 55 ℃, does not need the reverse transcription process, but has poor amplification specificity and is easy to generate false positive, and the EXPAR is difficult to distinguish the family members (single base or multi-base difference) of the miRNA. On the other hand, EXPAR cannot achieve simultaneous detection of multiple targets in one reaction system.
(2) The occurrence of a disease such as cancer is closely related to the disorder of multiple miRNAs, and most of the current methods only detect a single miRNA target at one time, have low detection efficiency and are difficult to meet the clinical requirements. The multiple detection of miRNA can obtain more comprehensive detection result information, which is helpful to improve the accuracy and efficiency of diagnosis and prognosis. Currently, a method for multiplex detection of miRNA with high specificity and high sensitivity is very lacking. In addition, single/polynucleotide polymorphism exists among miRNA family members, and miRNA of a specific member often has close relation with certain diseases, and the current technology cannot realize simple, rapid, low-cost, high-sensitivity and high-specificity miRNA typing analysis.
(3) The sensitivity of miRNA detection by the CRISPR/Cas technology can reach the cellular level, and the miRNA detection is difficult to be used for the actual clinical blood sample detection (anal. chem.2019,91, 5278-5285). CRISPR/Cas technology coupled with Catalytic Hairpin Assembly (CHA) nucleic acid amplification can detect micrornas at the sub-femtolor level, but still cannot achieve multiplex detection of the same system (chem.sci.,2020,11, 7362). The CRISPR/Cas technology for miRNA typing analysis needs multiple Cass, is difficult and high in cost, and does not have detection advantages.
Disclosure of Invention
Problems to be solved by the invention
The invention provides a microRNA detection system and a method based on exponential amplification reaction and Argonaute nuclease, which realize single-base-specificity and high-sensitivity miRNA detection, multiple miRNA detection and miRNA typing analysis and overcome the following problems and defects in the prior art:
1. the problem of non-specific amplification of nucleic acids, the disadvantage of detecting false positives;
2. the disadvantage of insufficient sensitivity of clinical detection;
3. a low efficiency single detection mode; the miRNA detection system and the method provided by the invention realize that one reaction system can efficiently, rapidly and accurately detect multiple miRNAs, and can be really applied to clinical diagnosis;
4. the current mature and reliable miRNA typing analysis technology is still a blank, and the invention also provides an efficient, simple and accurate miRNA typing analysis method.
Means for solving the problems
The invention develops an miRNA detection system and a detection method based on EXPAR and Argonaute nuclease, and realizes high specificity and high sensitivity miRNA detection, miRNA multiple detection and miRNA typing analysis. The miRNA detection system provided by the invention is integrally divided into an EXPAR pre-amplification system and an miRNA detection system based on Argonaute nuclease. In the miRNA detection system based on the Argonaute nuclease, the Argonaute nuclease is an endonuclease, has the specificity of single base resolution, is efficient and sensitive, is combined with an EXPAR pre-amplification system, and takes an amplicon generated by the miRNA through the EXPAR pre-amplification system as a gDNA triggering and mediating specific shearing detection probe of the Argonaute nuclease, so that a fluorescent signal is generated and high-specificity miRNA detection is realized; secondly, specific detection probes are designed aiming at different miRNAs, so that multiple detection and typing analysis of the miRNAs can be realized in one tube.
The invention provides a microRNA detection system based on exponential amplification reaction and Argonaute nuclease, which utilizes the efficient, specific and programmable nucleic acid shearing capability of the Argonaute nuclease to identify and detect a target object, and the microRNA detection system comprises:
(a) the guide DNA is an amplification product obtained by amplifying a target miRNA by adopting an exponential amplification reaction system;
(b) argonaute nuclease;
(c) the detection probe is provided with a fluorescent group and a quenching group, and comprises a region complementary with an amplification product obtained by amplifying a target miRNA by using an exponential amplification reaction system.
In some embodiments, the exponential amplification reaction system comprises an amplification template, a DNA polymerase, a nicking enzyme, dntps, an RNase inhibitor, and an enzyme buffer.
In some more specific embodiments, the amplification template is an amplification template designed based on the sequence of the target miRNA. The amplification template comprises a 3 'end sequence, a 5' end sequence and a nicking enzyme recognition sequence between the 3 'end sequence and the 5' end sequence, wherein the 3 'end sequence and the 5' end sequence have the same length and are 15-18 nucleotides. In some preferred embodiments, the amplification template further comprises an adenine base at the 3' end.
In some embodiments of the invention, the 3 'and 5' end sequences are identical and are complementary paired to the target miRNA. Since mirnas are typically 18-28 nucleotides in length, while the 3 'and 5' end sequences of the present invention are 15-18 nucleotides in length, in some embodiments of the present invention, amplification template design may be performed based on a contiguous stretch of 15-18 nucleotides in length in a miRNA. In other embodiments, amplification template design may be performed based on the full-length sequence of the miRNA.
In some embodiments of the invention, the first base at the 3 ' end of the 3 ' end sequence has a base mismatch with the target miRNA such that the first base at the 5 ' end of the amplicon of the exponential amplification reaction system is thymine.
In some preferred embodiments of the present invention, the twelfth base at the 5' end of the amplicon of the exponential amplification reaction system is not an adenine base.
In some more specific embodiments, the DNA polymerase is Vent (exo-) DNA polymerase or Bst DNA polymerase.
In some more specific embodiments, the nicking enzyme is a (restriction) endonuclease or a nicking endonuclease; preferably, the nicking enzyme is a restriction endonuclease, and more preferably, the restriction endonuclease is nt.
In some preferred embodiments, the DNA polymerase is Vent (exo-) DNA polymerase and the endonuclease is nt.bstnbi; in these preferred embodiments, the exponential amplification reaction system comprises an amplification template, dNTPs, Nt.BstNBI, Vent (exo-) DNA polymerase, RNase inhibitor, and the like,
Reaction buffer, nt.
In some more specific embodiments, the exponential amplification reaction system is amplified at a constant temperature of 50-60 ℃ for 10-30 min; preferably, the constant temperature amplification is carried out for 15-20min at 50-60 ℃; more preferably, the amplification is carried out at a constant temperature of 50-60 ℃ for 20 min. In other more specific embodiments, the exponential amplification reaction system is amplified at a constant temperature of 55 ℃ for 10-30 min; preferably, the constant temperature amplification is carried out for 15-20min at 55 ℃; more preferably, the isothermal amplification is carried out at 55 ℃ for 20 min.
In some more specific embodiments, in the detection probe, the fluorophore and the quencher are each independently located at the 5 'end and the 3' end of the detection probe.
In some more specific embodiments, the detection probe is in a hairpin configuration or a linear configuration. In some preferred embodiments, the detection probe is a hairpin-type structure, the stem of the 5 'end of the detection probe has a fluorescent group, the stem of the 3' end of the detection probe has a quencher group, and the loop of the detection probe is a region complementary to an amplification product obtained by amplifying a target miRNA using an exponential amplification reaction system.
In some more specific embodiments, the fluorophore is selected from FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or a combination thereof; the quenching group is selected from BHQ1, BHQ2, TAMRA, DABCYL, DDQ or a combination thereof.
In some more specific embodiments, the Argonaute nuclease is selected from any one of TtAgo nuclease from Thermus thermophilus, PfAgo nuclease from Pyrococcus thermophilus, and/or CbAgo nuclease from Clostridium butyricum.
In some preferred embodiments, the Argonaute nuclease is TtAgo nuclease from Thermus thermophilus. In the preferred embodiments, the reaction temperature of the microRNA detection system based on the exponential amplification reaction and the Argonaute nuclease is 65-85 ℃, and the reaction time is 5-40 min; in some more preferred embodiments, the reaction time is from 10 to 30 min; more preferably 15-25min, 15-20min, most preferably 15 min; in yet other more preferred embodiments, the reaction temperature is 70-80 ℃, 75-80 ℃, and most preferably 80 ℃.
In some preferred embodiments, the concentration of the Argonaute nuclease is 0.01 to 200nM in a microRNA detection system based on an exponential amplification reaction and Argonaute nuclease; more preferably, the concentration of Argonaute nuclease is 10-190nM, 20-180nM, 30-170nM, 40-160nM, 50-150nM, 60-140nM, 70-130nM, 80-120nM, 90-110nM or 90-100 nM; most preferably, the concentration of Argonaute nuclease is 100 nM.
In some specific embodiments, the microRNA detection system based on exponential amplification reaction and Argonaute nuclease further comprises: (d) a divalent metal ion.
In some more specific embodiments, the divalent metal ion is selected from Mn2+、Mg2+、Co2+And the like. In some more specific embodiments, the divalent metal ion is Mn2+E.g. MnCl2. In these more specific embodiments, MnCl is in a microRNA detection system based on an exponential amplification reaction and Argonaute nuclease2The concentration of (A) is 0.01-1000. mu.M; preferably MnCl2The concentration of (D) is 100-900 mu M; more preferably, MnCl2The concentration of the compound is 200-800 μ M, 300-750 μ M, 400-750 μ M, 500-750 μ M, 600-750 μ M, 700-750 μ M; most preferably 750. mu.M.
In some specific embodiments, the method further comprises the following steps in a microRNA detection system based on exponential amplification reaction and Argonaute nuclease: (e) reaction buffer. As the reaction buffer, the present invention is not particularly limited, and those skilled in the art can select the reaction buffer according to the Argonaute nuclease to be used, for example
Reaction buffer.
In some specific embodiments, the target miRNA is one miRNA or at least two mirnas. When the target miRNA is at least two miRNAs, correspondingly, the amplification template is an amplification template designed for the at least two miRNAs respectively; the detection probes are respectively designed aiming at least two miRNAs, and different detection probes have different fluorescent groups.
In some specific embodiments, the exponential amplification reaction and Argonaute nuclease based microRNA detection system is used for single miRNA detection, multiplex miRNA detection, or miRNA typing analysis.
In some more specific embodiments, when the microRNA detection system based on exponential amplification reaction and Argonaute nuclease is used for multiplex miRNA detection or miRNA typing analysis, the different detection probes have different fluorophores.
For example, in the case of multiplex miRNA detection, a nucleic acid sample to be detected is mixed into an exponential amplification reaction system containing a plurality of amplification templates for target miRNA amplification, and a plurality of target mirnas are simultaneously amplified in one exponential amplification reaction system. Correspondingly, in a microRNA detection system, the amplification product triggers the shearing of the Argonaute nuclease, and at the moment, detection probes which are specific to multiple target miRNA and have different fluorescent groups are mixed in the microRNA detection system, and multiple miRNA are detected through different fluorescent signals. For another example, when miRNA typing analysis is performed, pre-amplification is performed on a nucleic acid sample to be detected containing nucleic acid in an exponential amplification reaction system containing multiple amplification templates, after an amplification product is combined with a corresponding detection probe in a 100% complementary pairing manner, an Argonaute nuclease shearing detection probe in a microRNA detection system is triggered and started, and finally, miRNA typing analysis can be performed through a generated fluorescent signal.
The invention provides a microRNA detection kit in a second aspect, which comprises the microRNA detection system based on exponential amplification reaction and Argonaute nuclease in the first aspect.
The third aspect of the present invention provides a method for detecting microRNA, wherein the method for detecting microRNA uses the microRNA detection system of the first aspect of the present invention, and the method for detecting microRNA comprises the following steps:
step 2, providing or preparing a microRNA detection system, adding the amplification product obtained in the step 1 into the microRNA detection system, and reacting;
and 3, obtaining a fluorescent signal in the reactant after the reaction of the microRNA detection system in the step 2, and realizing the detection of miRNA.
In some embodiments, the test nucleic acid sample comprises nucleic acids from a biological sample selected from the group consisting of: blood, cells, serum, saliva, body fluids, plasma, urine, prostate fluid, bronchial lavage, cerebrospinal fluid, gastric fluid, bile, lymph fluid, peritoneal fluid, stool, and the like, or combinations thereof.
In some specific embodiments, step 1 is preceded by a step of pre-treatment of the biological sample, which comprises extracting total microRNA from the biological sample. For example, commercially available kits can be used to extract total mirnas in a biological sample.
ADVANTAGEOUS EFFECTS OF INVENTION
The microRNA detection system and the detection method based on the exponential amplification reaction and the Argonaute nuclease generate a miRNA detection signal by combining EXPAR pre-amplification and specific shearing of the Argonaute nuclease. The microRNA detection system and the detection method have the following technical effects:
the problem of traditional EXPAR false positive is solved, and high specificity detection with single base resolution is realized;
secondly, high-sensitivity miRNA detection is realized, the lowest detection limit is 1 attomole per liter (aM), and the miRNA detection kit has good sensitivity in actual detection;
thirdly, specific nucleic acid probes of different molecular markers are designed aiming at different miRNA targets, so that multiple detections can be realized, comprehensive detection information is provided for clinical diagnosis, and the diagnosis accuracy is improved;
the Argonaute nuclease has the advantage of high specificity of single base resolution, solves the bottleneck of the prior art in miRNA typing analysis, and has great significance in medical research, life science field, agriculture and clinical application.
Drawings
Fig. 1 is a schematic diagram of the principles of an miRNA detection system and a miRNA detection method based on EXPAR and Argonaute nuclease provided in the embodiment of the present invention.
Fig. 2 is a schematic diagram of the principle of multiple miRNA detection or miRNA typing analysis performed by the miRNA detection system and the miRNA detection method based on EXPAR and Argonaute nuclease provided in the embodiment of the present invention.
FIG. 3 is a real-time fluorescence signal monitoring chart of the miRNA detection system and the detection method based on the EXPAR and the Argonaute nuclease for detecting the EXPAR amplification product of miRNA-21 provided by the embodiment of the invention; the EXPAR negative control was ultrapure water (labeled as water in the figure).
FIG. 4 is a diagram for verifying miRNA detection with single base resolution and high specificity identification provided in the embodiments of the present invention based on miRNA detection system and method of EXPAR and Argonaute nuclease; by using the miDNA-21 and the single-base mismatched miDNA-21 to simulate the EXPAR amplification product of miRNA-21, the miDNA-21 successfully triggers and starts the TtAgo shearing simulation probe, and the single-base mismatched miDNA-21 cannot start the TtAgo shearing simulation probe due to the single-base mismatch with the simulation probe.
FIG. 5 is a graph illustrating the sensitivity of miRNA detection by the miRNA detection system and method based on EXPAR and Argonaute nuclease, compared with the conventional fluorescent EXPAR method; taking miRNA-21 as an example, wherein A in FIG. 5 represents the signal intensity of the miRNA-21 target with the detection concentration of 0-10nM by the miRNA detection method based on the EXPAR and TtAgo systems; b in FIG. 5 shows a standard curve for miRNA-21 target detection based on the miRNA detection method of the EXPAR and TtAgo systems; c in FIG. 5 represents the real-time fluorescence curve of miRNA-21 target with the detection concentration of 0-100nM by the traditional fluorescence EXPAR method; d in FIG. 5 represents a standard curve for detecting miRNA-21 target by the traditional fluorescence EXPAR method.
FIG. 6 shows the miRNA multiplex detection based on the miRNA detection system and the detection method of EXPAR and Argonaute nuclease provided in the embodiments of the present invention; taking miRNA-21, miRNA-92a, miRNA-31 and miRNA-141 as examples, firstly pre-amplifying the four target miRNA in an EXPAR reaction system simultaneously, and adding the obtained amplicon into a miRNA detection system containing four detection probes and based on TtAgo nuclease; the fluorescent molecules marked by the fluorescent probes for detecting the miRNA-21, the miRNA-92a, the miRNA-31 and the miRNA-141 are respectively as follows: JOE, HEX, Cy5, and ROX.
FIG. 7 shows miRNA typing detection based on the miRNA detection system and detection method of EXPAR and Argonaute nuclease provided in the examples of the present invention; miRNA typing of let 7 family is exemplified. The fluorescent molecules marked by the fluorescent probes corresponding to Let 7a, Let 7b and Let 7i are respectively as follows: JOE, HEX, and ROX.
Detailed Description
The invention is further illustrated by the following examples, but not by way of limitation, in connection with the accompanying drawings. The following provides specific materials and sources thereof used in embodiments of the present invention. However, it should be understood that these are exemplary only and not intended to limit the invention, and that materials of the same or similar type, quality, nature or function as the following reagents and instruments may be used in the practice of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Definition of
As used in this specification, the terms "a", "an" and "the" mean at least one unless explicitly stated otherwise. In this specification, the use of the singular includes the plural unless explicitly stated otherwise.
It is to be understood that there is an implied "about" preceding the temperatures, concentrations, times, etc. discussed in this specification in order to slight and insubstantial deviations within the scope of the teachings herein. Also, the use of "including," "comprising," "having," "containing," "owning," "with," and "containing" is not intended to be limiting. It is to be understood that both the foregoing general description and the foregoing detailed description are exemplary and explanatory only and are not restrictive of the invention.
As used herein, the term "or combinations thereof refers to all permutations and combinations of the items listed prior to that term. For example, "A, B, C or a combination thereof" is intended to include at least one of a, B, C, AB, AC, BC, or ABC, and if the order is important in a particular context, BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with the example, explicitly included are combinations that contain one or more repetitions of the entry or item, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. Unless otherwise apparent from the context, one of ordinary skill in the art will appreciate that there is generally no limitation on the number of items or terms in any combination.
The term "and/or" when used to connect two or more selectable items should be understood to mean either one of the selectable items or any two or more of the selectable items.
As used herein, the term "nucleic acid" means single-and double-stranded polymers of nucleotide monomers, including 2' -Deoxyribonucleotides (DNA) and Ribonucleotides (RNA) linked by internucleotide phosphodiester linkages or internucleotide analogs, and associated counterions, e.g., H+、NH4+Trialkyl ammonium, tetraalkyl ammonium, Mg2+、Na+And the like. The nucleic acid may be a polynucleotide or an oligonucleotide. The nucleic acid may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or may be a chimeric mixture thereof. The nucleotide monomer units may include any of the nucleotides described herein, including but not limited to naturally occurring nucleotides and nucleotide analogs. Nucleic acids typically range in size from a few monomeric units, e.g., 5-40 to several thousand monomeric nucleotide units. Nucleic acids include, but are not limited to, genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, fragmented nucleic acids, nucleic acids obtained from subcellular organelles such as mitochondria or chloroplasts, and nucleic acids obtained from microorganisms or DNA or RNA viruses that can be present on or in a biological sample.
According to the customary terminology in the art, the length of a nucleic acid can be expressed as bases, base pairs (abbreviated "bp"), nucleotides/nucleotide residues (abbreviated "nt"), or kilobases ("kb"). The terms "base", "nucleotide residue" may describe a polynucleotide, either single-stranded or double-stranded, where the context permits. When the term is applied to double-stranded molecules, it is used to refer to the entire length and should be understood as equivalent to the term "base pair".
The term "base" refers to purine and pyrimidine derivatives, which are components of nucleic acids, nucleosides, nucleotides. The total number of bases is 5: cytosine (abbreviated as C), guanine (G), adenine (a), thymine (T, DNA specific) and uracil (U, RNA specific).
The terms "microRNA", "miRNA" and "miR" are synonymous and refer to a collection of non-coding single-stranded RNA molecules of about 18-28 nucleotides in length that regulate gene expression. Mirnas are found in a wide range of organisms and have been shown to play a role in development, homeostasis and disease etiology.
As used herein, the term "probe" generally refers to a nucleotide or polynucleotide labeled with a label (e.g., a fluorescent label, a fluorophore) that can be used to detect or identify its corresponding target nucleotide or polynucleotide by hybridization with the corresponding target sequence in a hybridization reaction.
The term "label" refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) signal, which can be attached to a nucleic acid or protein by covalent or non-covalent interactions (e.g., by ionic or hydrogen bonding, or by immobilization, adsorption, etc.). Labels typically provide a signal for detection by fluorescence, chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-ray diffraction or absorption, magnetism, enzyme activity, and the like. Examples of labels include fluorophores, chromophores, radioactive atoms (especially 32p and 125I), electron-dense reagents, enzymes and ligands with specific binders.
As used herein, the term "dNTP" refers to a deoxynucleoside triphosphate. NTP refers to ribonucleoside triphosphates. Purine bases (Pu) include adenine (a), guanine (G), and derivatives and analogs thereof. Pyrimidine bases (Py) include cytosine (C), thymine (T), uracil (U), and derivatives and analogs thereof.
As used interchangeably herein, the terms "complementary," "complementary sequence," "complementary," and "complementarity" generally refer to sequences that are fully complementary to, and can hybridize to, a given sequence. The sequence that hybridizes to a given nucleic acid is referred to as the "complement" or "reverse complement" of a given molecule, provided that its base sequence on a given region is capable of complementarily binding to the base sequence of its binding partner, such that, for example, A-T, A-U, G-C and G-U base pairs are formed.
As used herein, the term "amplification", "nucleic acid amplification" or "amplified" refers to the generation of multiple copies of a nucleic acid template, or multiple copies of a nucleic acid sequence complementary to a nucleic acid template. The term (including the term "polymerization") may also refer to extending a nucleic acid template (e.g., by polymerization). The amplification reaction may be a polymerase mediated extension reaction, for example, a Polymerase Chain Reaction (PCR). However, any known amplification reaction may be suitable for the uses described herein.
As used herein, the terms "amplicon" and "amplification product" generally refer to the product of an amplification reaction. Amplicons may be double-stranded or single-stranded, and may include separate constituent strands obtained by denaturing double-stranded amplification products. In certain embodiments, amplicons of one amplification cycle may be used as templates in subsequent amplification cycles.
The term "exponential amplification reaction" or "EXPAR" is a technical method for amplifying a short-chain nucleic acid sequence exponentially with high efficiency under a constant temperature condition, in the method, an oligonucleotide product generated by linear amplification is used as a new primer to be combined on a template containing two sections of repeated sequences, and after the incision enzyme is cut by the incision enzyme, an oligonucleotide product with the same sequence is generated, so that a chain reaction is caused, and the oligonucleotide product grows exponentially.
As used herein, the term "polymerase" generally refers to an enzyme (e.g., natural or synthetic) that is capable of catalyzing a polymerization reaction. Examples of polymerases can include nucleic acid polymerases (e.g., DNA polymerases or RNA polymerases), transcriptases, and ligases (liages). The polymerase may be a polymerization enzyme. The term "DNA polymerase" generally refers to an enzyme capable of catalyzing the polymerization of DNA.
As used herein, the term "nicking enzyme" generally refers to a molecule (e.g., an enzyme) that cleaves one strand of a double-stranded nucleic acid molecule (i.e., "nicks" a double-stranded molecule). A nickase may be a nuclease that cleaves only a single DNA strand either because of its natural function or because it has been engineered (e.g., modified by mutation and/or deletion of one or more nucleotides) to cleave only a single DNA strand. The nicking enzyme can be an enzyme that generates nicks (e.g., a restriction endonuclease, a nicking endonuclease, etc.). Nicking enzymes can bind to a nicking site of a double-stranded nucleic acid molecule to create a nick (or nick) in one strand of the double-stranded nucleic acid molecule. The incision may be made within the incision site. Alternatively, the incision may be made near the incision site.
Materials and methods
First, EXPAR pre-amplified amplification template and detection probe design
1. The amplification template for EXPAR pre-amplification is designed according to the following principle:
1) the 3' end of the amplification template can be fixed to an adenine (A) base and has phosphorylation modification;
2) the amplification template is flanked on both sides by "NNNNNNNNNNNNNNNN" nucleic acid sequences that are complementary paired to the target miRNA and are 15-18 nucleotides in length, e.g., the exemplary "NNNNNNNNNNNNNNNN" described above is 16 nucleotides, which can also be 15, 17, or 18 nucleotides;
3) the middle ACTCAGACAA (SEQ ID NO:1) of the amplification template is a fixed sequence and is a recognition sequence of nicking endonuclease (nicking endonuclease) Nt.BstNBI endonuclease in the EXPAR pre-amplification reaction;
4) in order to ensure that the first base at the 5' end of the amplicon is T, the sequence of the target miRNA can have 1 base mismatch with the sequence of the amplification template;
5) the resulting amplicon is complementary paired to the "NNNNNNNNNNNNNNNN" sequence, and the first base at the 5' end of the amplicon is typically T; the twelfth base from the 5' end is avoided as A. A general template: 3 '-P-ANNNNNNNNNNNNNNNNACTCAGACAANNNNNNNNNNNNNNNN-5'.
2. The detection probe design follows the principle:
the detection probe may be a hairpin or a linear gene sequence, preferably, a hairpin. The 5 'end of the probe stem is modified with signal molecules, such as signal groups of JOE, HEX, Cy5, ROX and the like, and the 3' end of the probe stem is modified with quenching groups, such as BHQ1, BHQ2 and the like. The detection probe ring part is specifically and complementarily paired with the target object amplicon, and when the detection probe ring part is 100% complementarily paired with the target object amplicon, the TtAgo shearing detection probe is started to release and generate a fluorescent signal.
The target for multiplex detection and the target for typing analysis may be any miRNA, and in the following examples, miRNA-21, miRNA-92a, miRNA-31 and miRNA-141 are used as the multiplex detection. Typing analysis of miRNA includes SNP and multi-base mutation typing of miRNA, corresponding EXPAR amplification templates and corresponding detection probes are designed by taking let 7 families (let 7a, let 7b, let 7c, let 7d and let 7i) as examples. miRNA sequences used in the examples are specifically seen in table 1:
table 1: miRNA sequences used in the examples
| Name of nucleic acid | Sequence (5 '-3') |
| microRNA-21 | UAGCUUAUCAGACUGAUGUUGA(SEQ ID NO:2) |
| microRNA-92a | UAUUGCACUUGUCCCGGCCUGU(SEQ ID NO:3) |
| microRNA-31 | AGGCAAGAUGCUGGCAUAGCU(SEQ ID NO:4) |
| microRNA-141 | UAACACUGUCUGGUAAAGAUGG(SEQ ID NO:5) |
| let 7a | UGAGGUAGUAGGUUGUAUAGUU(SEQ ID NO:6) |
| let 7b | UGAGGUAGUAGGUUGUGUGGUU(SEQ ID NO:7) |
| let 7c | UGAGGUAGUAGGUUGUAUGGUU(SEQ ID NO:8) |
| let 7d | AGAGGUAGUAGGUUGCAUAGUU(SEQ ID NO:9) |
| let 7i | UGAGGUAGUAGUUUGUGCUGUU(SEQ ID NO:10) |
Second, EXPAR pre-amplification system and miRNA detection system based on TtAgo nuclease
EXPAR preamplification system: in the embodiment, an EXPAR pre-amplification miRNA system is mainly established. The EXPAR system mainly comprises: the total volume of the solution A and the solution B can be 10 or 20 mu L. BstNBI buffer solution, an amplification template, dNTP, RNase inhibitor and a nucleic acid sample to be detected (which may contain a target miRNA); the liquid B comprises
Reaction buffer, Nt.BstNBI, Vent (exo-) DNA polymerase and DEPC water. And determining the number of amplification templates to be used according to the number of the nucleic acid samples to be detected in the solution A, and further adjusting the volumes of the solution A and the solution B. Respectively preparing solution A and solution B, mixing the prepared solution A and solution B, and immediately reacting at 55 ℃. The temperature range of the EXPAR pre-amplification reaction is 50-60 ℃, and the preferred temperature range is 55 ℃. The EXPAR pre-amplification reaction time is 10-30min, preferably 20 min.
The concentration of each component in the EXPAR pre-amplification system (solution A + solution B) is as follows: amplification template (0.1. mu.M), dNTP (250. mu.M), Nt.BstNBI (0.4U. mu.L)-1) Vent (exo-) DNA polymerase (0.05U. mu.L)-1) RNase inhibitor (0.8U. mu.L)-1)、
Reaction buffer (20mM Tris-HCl, pH8.8,10mM KCl,10mM (NH)4)2SO4,2mM MgSO40.1% TritonX-100; tris ═ 2-amino-2- (hydroxymethyl) -1, 3-propanediol (2-amino-2-hydroxypropanediol-1, 3-diol) prepared from
Diluted reaction buffer stock solution in a pre-amplification system) and 0.5 XNt.BstNBI buffer solution (25mM Tris-HCl, pH 7.9, 50mM NaCl, 5mM MgCl)2And 0.5mM Dithiothreitol (DTT) prepared by diluting stock solution of Nt.BstNBI buffer solution in a pre-amplification system. A single miRNA detection only needs to add a single corresponding amplification template, a plurality of miRNA detections are added with a plurality of corresponding amplification templates (each amplification template is 0.1 mu M), and the addition amount of each nucleic acid sample is 1-4 mu L.
TtAgo nuclease-based miRNA detection system: TtAgo nuclease-based miRNA detection lines are 10 μ L, including
Reaction buffer (20mM Tris-HCl,10mM (NH)4)2SO4,10mM KCl,2mM MgSO4,0.1％
X-100,pH 8.8)、MnCl2Detection probes (0.1. mu.M), TtAgo nuclease (enzyme), ultrapure water and EXPAR amplicons. The EXPAR amplicon stock solution or 10-fold dilution plus 1. mu.L is added into a miRNA detection system based on TtAgo nuclease. The reaction temperature of the miRNA detection system based on the TtAgo nuclease is 65-85 ℃, preferably 80 ℃; the reaction time is 5-40min, preferably 15 min. MnCl2The concentration ranges from 0 to 1000. mu.M, preferably 750. mu.M; the concentration of the TtAgo enzyme is 0-200nM, preferably 100 nM. When a plurality of targets are detected, a plurality of corresponding detection probes are added, and each detection probe is 0.1 mu M.
The reagents and sources of nucleic acid sequences used are shown in Table 2 below.
Table 2: reagent information used in the examples
Examples
The invention provides a system and a method for multiple detection of miRNA with high specificity and high sensitivity. The miRNA detection method integrally comprises two steps of EXPAR pre-amplification and miRNA detection based on Argonaute nuclease. Firstly, biological samples such as tissues and serum from different sources are processed, a low-abundance nucleic acid sample is obtained through nucleic acid extraction, miRNA is specifically amplified through EXPAR, an amplicon obtained through amplification is added into a detection system containing TtAgo enzyme and a detection probe, and the amplicon is used as gDNA to trigger and mediate TtAgo shearing of the detection probe, so that a detection signal is generated. The detection signal can be detected by a real-time monitoring and terminal detection mode, and comprises a real-time fluorescence PCR instrument, various fluorescence detectors, a lateral flow immunochromatography test strip, a macroscopic observation method and the like. The method has the advantages of simplicity, rapidness, low cost and the like, the detection result is obtained in about 30min, the sensitivity reaches aM, and the method can be well used for detecting clinical miRNA. The invention can be widely applied to disease diagnosis, clinical research, life science research, such as tumor liquid biopsy or cancer early screening.
Specifically, the core of the invention is that a large amount of amplicons generated by EXPAR amplification miRNA are utilized, TtAgo specifically recognizes and cuts the detection probe under the mediation of the amplicons, and the simultaneous detection of multiple miRNA in one-pot method is realized. The principle is as shown in FIG. 1, and the details are as follows: each miRNA binds to a specifically designed and complementary paired amplification template, and undergoes amplification by EXPAR to produce a large amount of 5' -phosphorylated single-stranded DNA (around 16 bases, amplicon). The generated nucleic acid specific amplicon is further used as a gDNA mediated TtAgo recognition and specific shearing detection probe to generate a fluorescence signal. And (3) performing multiplex detection as shown in FIG. 2, adding a plurality of targets and corresponding amplification templates into an EXPAR system, adding the generated amplicons into a TtAgo system containing a plurality of probes capable of specifically binding the amplicons, and realizing one-pot multiplex detection by generating different signals. The technical advantages are that: 1) TtAgo has the specificity of single base resolution, and 100% of detection probes matched with the amplicon sequence start shearing to generate signals, so the detection specificity is extremely high; 2) the detection system can be designed aiming at any miRNA target sequence and has no sequence preference; 3) realizing multiple detection of a plurality of miRNAs by a single TtAgo enzyme in a single reaction system; 4) the rapid detection is realized within 30min, and the sensitivity reaches aM; 5) and various terminal detection modes can be flexibly combined.
The feasibility verification of the method is shown in figure 3, and is firstly verified by EXPAR pre-amplification miRNA-21 and then by a TtAgo detection system. When the target is miRNA-21, the amplicon successfully mediates the TtAgo shearing detection probe to quickly generate a fluorescence signal; and the negative control (water) has no amplification product, can not start the TtAgo shearing detection probe, and has no fluorescent signal enhancement process.
Example 1: high specificity detection capability of TtAgo single base resolution
This example explores the shear specificity of TtAgo. First, miDNA-21 and single base mismatched miDNA-21 (shown in Table 2), and a mock probe (i.e., a detection probe) were synthesized, as shown in Table 3.
Wherein the miDNA-21 is 100 percent complementary matched with the analogue probe, and the single base mismatching miDNA-21 has one base mismatching with the analogue probe. The miDNA-21 is used for simulating an EXPAR pre-amplification product of miRNA-21, a detection system based on the TtAgo nuclease comprises the miDNA-21(5 mu M) or the miDNA-21 with single base mismatch (5 mu M), a simulation probe (1 mu M), the TtAgo (100nM),
reaction buffer, MnCl2(750. mu.M) and ultrapure water. The reaction temperature of a detection system based on the TtAgo nuclease is 80 ℃; the reaction time was 30 min. In a detection system based on the TtAgo nuclease, a cleavage product (short-chain nucleic acid) of the TtAgo nuclease is verified by 15% urea-polyacrylamide gel electrophoresis under the electrophoresis condition of 130V for 80 min.
As a result, as shown in FIG. 4, miDNA-21 was able to mediate successful cleavage of the mock probe by TtAgo nuclease, i.e., no mock probe band was shown near 30bp, whereas a cleaved mock probe band appeared below the miDNA-21 band. The single-base mismatched miDNA-21 does not have the TtAgo nuclease cleavage simulation probe, the simulation probe band is still shown at the position of about 30bp, and the trace of the nucleic acid product which is cleaved is not left below the single-base mismatched miDNA-21 band, which shows that the detection system based on the TtAgo nuclease in the embodiment has high specificity with single-base resolution. The single base specificity of the TtAgo nuclease has great advantages, can solve the problems of nonspecific amplification and false positive caused by poor specificity of the traditional EXPAR, plays an important role in practical detection application, and can be used for accurate miRNA detection and typing among family members.
Table 3: MiDNA-21 and single base mismatched miDNA-21
Example 2: sensitivity of miRNA detection method based on EXPAR and TtAgo system
This example explores the sensitivity of miRNA detection methods based on EXPAR and TtAgo nucleases.
Taking miRNA-21 as an example, a series of concentrations of miRNA (as nucleic acid samples) (0, 1aM, 10aM, 100aM, 1fM, 10fM, 100fM, 1pM, 10pM, 1nM, 10nM) were first pre-amplified based on the EXPAR pre-amplification system in the materials and methods section, with reaction conditions of 55 ℃ and 20 min. TtAgo nuclease-based detection systems comprise a pre-amplification product (1. mu.L), a detection probe (1. mu.M), TtAgo (100nM),
reaction buffer, MnCl2(750. mu.M) and ultrapure water. The reaction temperature of a detection system based on the TtAgo nuclease is 80 ℃; the reaction time was 30 min. The detection result is shown as A in FIG. 5, and the detection sensitivity reaches 1aM (10)-18M) (B in fig. 5).
In this example, the sensitivity of the miRNA detection method based on EXPAR and TtAgo nuclease was further compared with the sensitivity of the conventional fluorescent EXPAR detection miRNA, and the conventional fluorescent EXPAR system was as follows: the total volume of solution A and solution B was 10. mu.L. The solution A comprises Nt.BstNBI buffer solution, amplification template, dNTP,RNase inhibitor and a series of miRNA-21(0, 100fM, 1pM, 10pM, 100pM, 1nM and 10nM) concentrations; solution B is prepared from
Reaction buffer, Nt.BstNBI, Vent (exo-) DNA polymerase, SYBR Green, and DEPC water. And mixing the solution A and the solution B, and immediately placing the mixture in a PCR instrument for reaction. The EXPAR reaction temperature was 55 ℃ and the reaction time was 35min, and the fluorescence was read every 30 seconds. The concentrations of the components in the EXPAR system are as follows: amplification template (0.1. mu.M), dNTP (250. mu.M), Nt.BstNBI (0.4U. mu.L)-1) Vent (exo-) DNA polymerase (0.05U. mu.L)-1) RNase inhibitor (0.8U. mu.L)-1) SYBR Green (final concentration 5000-fold dilution of stock solution, Solibao organism),
reaction buffer and 0.5 xnt. The real-time fluorescence result of the traditional fluorescent EXPAR detection miRNA-21 is shown as C in figure 5, and when the concentration is 100fM, the amplification curve is very close to that of a negative control (water) (false positive amplification signal), so the detection sensitivity is 100fM (10)-13M) (D in fig. 5). In contrast, the sensitivity of the miRNA detection method based on the EXPAR and the TtAgo nuclease, which is provided by the invention, is improved by several orders of magnitude compared with the original traditional EXPAR, and false positive signals are avoided.
Example 3: miRNA detection method based on EXPAR and TtAgo nuclease for detecting multiple miRNA
This example explores the ability of the miRNA detection methods based on the EXPAR and TtAgo nucleases of the present invention to detect multiple mirnas.
Taking four target objects of miRNA-21, miRNA-92a, miRNA-31 and miRNA-14 as examples, the detection process is divided into two steps, firstly, based on the EXPAR pre-amplification system described in the material and method, adding amplification templates corresponding to the four miRNA and four corresponding target objects of miRNA (as nucleic acid samples) into the EXPAR pre-amplification system, wherein the concentration of the target objects of miRNA is 100pM, pre-amplifying the four target objects of miRNA simultaneously in the same reaction tube, and the reaction conditions are 55 ℃ and 20 min. And secondly, the miRNA detection system based on the TtAgo nuclease comprises detection probes corresponding to the four amplicons and an EXPAR multiple amplicon, other components are consistent with the TtAgo system in the material and method, the reaction conditions are 80 ℃ and 15min, the reaction is finished in a PCR instrument, and the fluorescence intensity values before and after the reaction are read by the PCR instrument for qualitative and quantitative detection.
The detection result of detecting multiple miRNAs based on the miRNA method of EXPAR and TtAgo nuclease is shown in FIG. 6, the system can detect one miRNA of four targets through different fluorescence channels, and simultaneously detect two, three or four targets, in the embodiment, a JOE signal represents miRNA-21, a HEX signal represents miRNA-92a, a Cy5 signal represents miRNA-31, and a ROX signal represents miRNA-141. The time of the whole reaction including EXPAR pre-amplification and miRNA detection based on TtAgo nuclease only needs 35min, the reaction operation is simple and convenient, and the method has great application prospect in the aspects of clinical diagnosis, prognosis, patient drug resistance and the like.
Example 4: miRNA typing detection method based on EXPAR and TtAgo nuclease
The detection system and the method are further evaluated for miRNA family typing capability by taking let 7 families (let 7a, let 7b and let 7i) as targets. The standard sequences of the three targets are respectively synthesized, and the corresponding EXPAR amplification template and the three detection probes are simultaneously synthesized. Sequence-specific detection genes for FAM, HEX and ROX markers were used to distinguish between let 7a, let 7b and let 7i, respectively. First, each target was formulated in a series of different concentrations and reacted and amplified in an EXPAR system containing three templates at 55 ℃ for 30 minutes. 2 μ L of the EXPAR amplification product was loaded into a miRNA detection system (20 μ L) containing TtAgo nuclease with three detection probes and reacted at 80 ℃ for 15 minutes. The results show that this example achieved accurate identification of let 7 subtypes by combining EXPAR preamplification and Ago detection (fig. 7). Further proves that the method has wide application prospect.
Sequence listing
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Claims (14)
1. A microRNA detection system based on exponential amplification reaction and Argonaute nuclease is characterized by comprising:
(a) the guide DNA is an amplification product obtained by amplifying a target object microRNA by adopting an exponential amplification reaction system;
(b) argonaute nuclease;
(c) the detection probe is provided with a fluorescent group and a quenching group, and comprises a region complementary with an amplification product obtained by amplifying a target object microRNA by using an exponential amplification reaction system.
2. The system for detecting microRNA based on exponential amplification reaction and Argonaute nuclease according to claim 1, wherein the Argonaute nuclease is selected from any one of Tabout nuclease from Thermus thermophilus (Thermus thermophilus), Pfago nuclease from Thermus archaea (Pyrococcus furiosus) and/or Cbago nuclease from Clostridium butyricum (Clostridium butyricum);
preferably, the Argonaute nuclease is TtAgo nuclease from Thermus thermophilus.
3. The system for detecting microRNA based on exponential amplification reaction and Argonaute nuclease according to claim 1 or 2, wherein the concentration of the Argonaute nuclease in the system for detecting microRNA based on exponential amplification reaction and Argonaute nuclease is 0.01-200 nM.
4. The exponential amplification reaction and Argonaute nuclease-based microRNA assay system according to any of claims 1-3, wherein the exponential amplification reaction system comprises an amplification template, a DNA polymerase, a nickase, dNTPs, an RNase inhibitor and an enzyme buffer.
5. The system for detecting microRNA according to claim 4, wherein the amplification template is designed according to the sequence of the microRNA of the target, and comprises a 3 'end sequence, a 5' end sequence, and a nicking enzyme recognition sequence between the 3 'end sequence and the 5' end sequence, wherein the 3 'end sequence and the 5' end sequence have the same length and are 15-18 nucleotides;
preferably, the 3' end of the amplification template further has an adenine base.
6. The system for detecting microRNA based on exponential amplification reaction and Argonaute nuclease according to claim 5, wherein the 3 'terminal sequence and the 5' terminal sequence are the same and are complementary and paired with the microRNA of the target;
or, the first base at the 3 ' end of the 3 ' end sequence has one base mismatch with the target microRNA, so that the first base at the 5 ' end of the amplicon of the exponential amplification reaction system is a thymine base;
preferably, the twelfth base at the 5' end of the amplicon in the exponential amplification reaction system is not an adenine base.
7. The system for detecting microRNA based on an exponential amplification reaction and an Argonaute nuclease according to any of claims 4-6, wherein the nicking enzyme is selected from a restriction endonuclease or a nicking endonuclease;
preferably, the nicking enzyme is a restriction endonuclease.
8. The exponential amplification reaction and Argonaute nuclease-based microRNA detection system according to any of claims 1 to 7, wherein in the detection probe, the fluorescent group and the quencher group are independently located at the 5 'end and the 3' end of the detection probe;
optionally, the detection probe is in a hairpin structure or a linear structure, preferably in a hairpin structure.
9. The exponential amplification reaction and Argonaute nuclease-based microRNA assay system according to any one of claims 1-8, wherein the exponential amplification reaction and Argonaute nuclease-based microRNA assay system further comprises: (d) a divalent metal ion;
and/or, (e) a reaction buffer.
10. A microRNA detection kit comprising the exponential amplification reaction and Argonaute nuclease-based microRNA detection system of any one of claims 1-9.
11. The microRNA assay kit according to claim 10, wherein the microRNA assay is selected from the group consisting of a single microRNA assay, a multiplex microRNA assay, a microRNA typing assay, or a combination thereof.
12. A method for detecting microRNA by using the microRNA detection system of any one of claims 1 to 9, the method comprising the following steps:
step 1, providing or preparing an exponential amplification reaction system, adding a nucleic acid sample to be detected into the exponential amplification reaction system, and amplifying a target object microRNA in the nucleic acid sample to be detected through the exponential amplification reaction to obtain an amplification product;
step 2, providing or preparing a microRNA detection system, adding the amplification product obtained in the step 1 into the microRNA detection system, and reacting;
and 3, obtaining a fluorescent signal in the reactant after the reaction of the microRNA detection system in the step 2, and realizing the detection of the microRNA.
13. The method for detecting microRNA according to claim 12, wherein the nucleic acid sample to be detected comprises nucleic acid from a biological sample selected from the group consisting of: blood, cells, serum, saliva, body fluid, plasma, urine, prostate fluid, bronchial lavage fluid, cerebrospinal fluid, gastric fluid, bile, lymph fluid, peritoneal fluid, stool, or combinations thereof.
14. The method for detecting microRNA according to claim 13, further comprising a step of pre-treating the biological sample before step 1, wherein the step comprises extracting total microRNA from the biological sample.
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