CN114250276B - 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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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6851—Quantitative amplification
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention discloses a microRNA detection system and method based on an exponential amplification reaction and Argonaute nuclease. The invention provides a microRNA detection system based on an exponential amplification reaction and Argonaute nuclease, which comprises the following components: (a) The guide DNA is an amplification product obtained by amplifying the target microRNA by adopting an exponential amplification reaction system; (b) Argonaute nuclease; (c) And the detection probe is provided with a fluorescent group and a quenching group, and comprises a region complementary to an amplification product obtained by amplifying the target 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 multiplex 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 method based on an exponential amplification reaction and Argonaute nuclease.
Background
Mature microRNA (miRNA) is a single-stranded non-coding RNA molecule with the length of 18-28 bases, and plays an important role in regulating various vital activities such as immune system, growth and development of cells, proliferation and differentiation, apoptosis and the like. mirnas are receiving extensive attention from researchers in various fields of development, biology, aging and metabolism, disease pathology research, disease treatment, and the like. mirnas are transcribed from some DNA into RNA and processed, which can bind to mRNA, thereby inhibiting gene expression after transcription, and abnormal levels of mirnas are closely related to various diseases. For example, up-regulation of miRNA-21, miRNA-92a, miRNA-31 and miRNA-141 levels is an important factor (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):427-448.). causing cancers, the occurrence of one disease is often related to the deregulation of a plurality of miRNAs, and the multiplex detection of miRNAs has very important clinical significance for diagnosis and prognosis of diseases. mirnas are widely present in blood, and free microRNA markers of blood circulation have been used in liquid biopsies of tumors to aid in early diagnosis and screening of cancer (Journal of CLINICAL MEDICINE 4.10.10 (2015): 1890-1907). On the other hand, there are family members in which there is only a single or two nucleotide difference between the family members, i.e., single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP), such as let 7miRNA family, and there are 12 species in total in human-derived let 7miRNA family members (Trends in molecular medicine,2008,14 (9): 400-409.Endocrine-RELATED CANCER,2010,17 (1): F19-F36.). The accurate differentiation or typing of miRNA family members will drive basic research of mirnas, e.g. identify a certain key miRNA member causing a disease, and by typing analysis can help to diagnose certain disease types such as cancers (CLINICA CHIMICA ACTA (2012) 1092-1097.bioorg.med.chem.46 (2021) 116363), with important scientific research implications and clinical value.
The gold standard for miRNA detection at present is that the reverse transcription of reverse transcription polymerase chain reaction (reverse transcription-polymerase chain reaction,RT-PCR,Nature Reviews Genetics 13.5(2012):358-369.),miRNA mainly comprises two methods, namely a tail-added reverse transcription method and a stem-loop reverse transcription method, wherein the former method needs to design a primer of a stem-loop structure to prolong miRNA so as to obtain longer-chain cDNA, and the latter method comprises the steps of adding poly (A) at the tail to prolong the miRNA chain, and then reversely transcribing the miRNA into cDNA by using a designed universal primer template. 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 techniques such as rolling circle amplification (Rolling Circle Amplification, RCA), exponential amplification reactions (Exponential Amplification Reaction, EXPAR), double-strand specific nuclease signal amplification (Duplex-Specific Nuclease Signal Amplification, DSNSA) have been developed for detection of mirnas, and the lack of specificity and sensitivity of these methods currently limits their further popularization and application in practical 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 object, so that the detection efficiency is low, and the available clinical information is limited and insufficient for clinical diagnosis.
In addition, current methods of miRNA typing are very lacking, RCA, while suitable for miRNA typing analysis, is less sensitive and less specific to achieve single base resolution (ANALYTICA CHIMICA ACTA 1076 (2019) 138e 143). Traditional genotyping relies primarily on sequencing and allele-specific PCR and is not suitable for miRNA typing analysis. Sequencing needs to be carried out by a large instrument, and is mainly aimed at a longer target sequence, so that the cost is high and the period is long. Allele-specific PCR requires extensive primer design and screening, is complex to detect, and is very limited to detectable targets. In recent years CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology is widely studied in the field of nucleic acid detection, a CRISPR/Cas system is a programmable high-specificity, rapid and efficient nucleic acid shearing system, specific nucleic acid is accurately identified and sheared by Cas nuclease under the mediation of guide RNA, and various Cas enzymes have a bystander cutting function, and can cut other nucleic acid probes (ACS Nano 2021,15,7848-7859) which are dissociated in the system while shearing targets. The problems of several aspects exist in the concentration in the actual nucleic acid detection process, firstly, the concentration of the target substance is low, the target substance is unstable and is easy to degrade, the direct detection mode usually needs to use expensive equipment such as sequencing and the like, and special operation is difficult to be practically applied; secondly, the existing nucleic acid amplification technology has poor specificity accuracy and is easy to generate false positive; third, the current technology has limited detection efficiency, and can not realize multiple detection in a single reaction system, namely simultaneous detection of a plurality of targets. Although CRISPR/Cas combined with amplification techniques such as recombinase polymerase amplification (Recombinase Polymerase Amplification, RPA), loop-mediated isothermal amplification (Loop-Mediated Isothermal Amplification, LAMP) achieve high specificity, high sensitivity and even multiplex nucleic acid detection (Science, 2018,360,439-444; science,2018,360,436-439;Cell Discovery 2018,4,20), guide RNAs have sequence dependencies, which are more complex in design, and in addition, the cost of guide RNA synthesis and screening is higher, RNA is easier to degrade, multiplex detection requires Cas enzymes with multiple nucleic acid preferences, which are rare on the one hand, poor in practicality, and on the other hand, result in complex and costly detection systems.
Argonaute (Ago) is a programmable DNA endonuclease, ago requires only a 5' -phosphorylated single-stranded DNA (16-18 bases) as guide DNA (guide DNA, gDNA), targets to recognize specific nucleic acid sequences complementary to gDNA, and cleaves complementary DNA or RNA nucleic acid sequences at the 10 th-11 th base position of gDNA (Nature.507 (7491), 258-261.). The Ago enzyme has greater sequence-independent dependence than Cas nucleases, and is more versatile and viable in practical detection applications. In addition, gDNA is easy to design, low in cost and stable. The Ago enzyme has a precise recognition and cleavage function with single base resolution, wherein the Ago nuclease (Thermus thermophilus Ago, ttAgo) from thermophilic thermus has been proved to be used for detecting tumor gene mutation such as KRAS, EGFR, BRAF, the detection sensitivity is improved by 100 times compared with the traditional clamp PCR (clamp PCR) mediated by peptide Nucleic acid (Peptide Nucleic Acid, PNA) and heteronucleic acid (Xenonucleic Acid, XNA), and the single copy level is reached (Nucleic ACIDS RESEARCH 48.4.4 (2020): e19-e 19.). Recent studies have shown that the efficiency of cleavage of detection systems based on Ago nucleases (Pyrococcus furiosus Ago, pfAgo) from thermophilic archaea is equivalent or even better than CRISPR technology, with the shortest possible cleavage to be done and a strong detection signal to be generated in 3-5min (Nature communications 12.1.1 (2021): 1-9.).
Up to now, there has been no report of single or multiplex miRNA detection using Ago novel molecular enzyme technology and reports for miRNA typing analysis including SNPs and other base mutants. The existing miRNA detection method still has the following problems:
(1) The mirnas are various, low in abundance and short in fragment, so that the detection of the mirnas in biological samples is still a technical problem. The EXPAR technology can directly amplify miRNA for signal amplification and detection without a reverse transcription process, but has serious false positive phenomenon and limited sensitivity, and is difficult to realize application in clinical detection (ANGEWANDTE CHEMIE International Edition 49.32 (2010): 5498-5501). The EXPAR technology completes the detection of a certain miRNA in about 30min at a constant temperature of 55 ℃, and does not need a reverse transcription process, but has poor amplification specificity, is easy to generate false positive, and is difficult to distinguish family members (single base or multiple base differences) of the miRNA by the EXPAR. On the other hand, EXPAR cannot realize simultaneous detection of multiple targets in one reaction system.
(2) While the occurrence of diseases such as cancer is closely related to the deregulation of a plurality of miRNAs, most of the current methods only detect single miRNA targets at a time, the detection efficiency is low, and the clinical requirements are difficult to meet. The multiplex detection of miRNA can obtain more comprehensive detection result information, and is beneficial to improving the accuracy and efficiency of diagnosis and prognosis. There is currently a great lack of methods for highly specific, highly sensitive multiplex detection of mirnas. In addition, single/polynucleotide polymorphism exists among miRNA family members, but a specific member miRNA is often in 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 CRISPR/Cas technology can detect miRNA at cellular level, which is difficult to use for practical 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 sub-femtomole (sub-femtomolar) level, but still cannot achieve multiplex detection of the same system (chem.sci., 2020,11,7362). The CRISPR/Cas technology is used for typing analysis of miRNA, and has the advantages of high difficulty, high cost and no detection.
Disclosure of Invention
Problems to be solved by the invention
The invention provides a microRNA detection system and a microRNA detection method based on an exponential amplification reaction and Argonaute nuclease, which realize single-base specific and high-sensitivity miRNA detection, multiple miRNA detection and miRNA typing analysis so as to overcome the following problems and defects in the prior art:
1. The problem of nonspecific amplification of nucleic acid, and the defect of false positive detection;
2. The defect of insufficient clinical detection sensitivity;
3. A low-efficiency single detection mode; the miRNA detection system and the method provided by the invention realize the efficient, rapid and accurate detection of a plurality of miRNAs by one reaction system, and can be truly applied to clinical diagnosis;
4. the current mature and reliable miRNA typing analysis technology is still blank, and the invention also provides a high-efficiency, simple and accurate miRNA typing analysis method.
Solution for solving the problem
The invention develops an EXPAR and Argonaute nuclease-based miRNA detection system and a detection method, and realizes high-specificity and high-sensitivity miRNA detection, miRNA multiplex detection and miRNA typing analysis. The whole miRNA detection system related by the invention is divided into an EXPAR pre-amplification system and an Argonaute nuclease-based miRNA detection system. In the miRNA detection system based on the Argonaute nuclease, the Argonaute nuclease is endonuclease, has the specificity of single base resolution, is efficient and sensitive, is combined with the EXPAR pre-amplification system, and the amplicon generated by the miRNA through the EXPAR pre-amplification system is used as a gDNA triggering and mediating specific shearing detection probe of the Argonaute nuclease, so that a fluorescent signal is generated, and the high-specificity miRNA detection is realized; and secondly, specific detection probes are designed aiming at different miRNAs, so that the multiplex detection and typing analysis of the miRNAs can be realized in a tube.
The first aspect of the present invention provides a microRNA detection system based on an exponential amplification reaction and an Argonaute nuclease, which utilizes the efficient, specific and programmable nucleic acid cleavage capability of the Argonaute nuclease to identify and detect a target object, the microRNA detection system comprising:
(a) The guide DNA is an amplification product obtained by amplifying the target miRNA by adopting an exponential amplification reaction system;
(b) Argonaute nucleases;
(c) The detection probe is provided with a fluorescent group and a quenching group, and comprises a region complementary to an amplification product obtained by amplifying a target miRNA by using an exponential amplification reaction system.
In some specific 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 according to the sequence of the miRNA of interest. The amplified template comprises a3 'end sequence, a 5' end sequence and a nicking enzyme recognition sequence between the 3 'end sequence and the 5' end sequence, wherein the lengths of the 3 'end sequence and the 5' end sequence are the same and are 15-18 nucleotides. In some preferred embodiments, the 3' end of the amplification template further has an adenine base.
In some embodiments of the invention, the 3 'and 5' end sequences are identical and are complementary paired to the miRNA of interest. Since mirnas are typically 18-28 nucleotides in length, whereas the 3 'and 5' sequences of the present invention are 15-18 nucleotides in length, in some embodiments of the present invention, amplification template design may be based on a continuous sequence of 15-18 nucleotides in length in a miRNA. In other embodiments, the amplification template design may be 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 one base mismatch with the target miRNA such that the first base at the 5' end of the amplicon of the exponential amplification reaction is thymine.
In some preferred embodiments of the 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, 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,Reaction buffer, nt.bstnbi buffer and DEPC water.
In some more specific embodiments, the exponential amplification reaction system is amplified at a constant temperature of 50-60 ℃ for 10-30min; preferably, the amplification is carried out at a constant temperature of 50-60 ℃ for 15-20min; more preferably, the amplification is carried out at a constant temperature of 50-60℃for 20min. In other more specific embodiments, the exponential amplification reaction system is amplified at a constant temperature of 55℃for 10-30min; preferably, the amplification is carried out at a constant temperature of 55 ℃ for 15-20min; more preferably, the amplification is carried out at a constant temperature of 55℃for 20min.
In some more specific embodiments, in the detection probe, the fluorescent moiety and the quenching moiety 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 a hairpin structure or a linear structure. In some preferred embodiments, the detection probe is in a hairpin structure, the stem of the 5 '-end of the detection probe is provided with a fluorescent group, the stem of the 3' -end of the detection probe is provided with a quenching group, and the loop of the detection probe is a region complementary to an amplification product obtained by amplifying a target miRNA by using an exponential amplification reaction system.
In some more specific embodiments, the fluorophore is selected from FAM, HEX, CY, 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 Thermus thermophilus, pfAgo nuclease from archaea thermophilus Pyrococcus furiosus, and/or CbAgo nuclease from clostridium butyricum Clostridium butyricum.
In some preferred embodiments, the Argonaute nuclease is TtAgo nuclease from thermus thermophilus Thermus thermophilus. In these preferred embodiments, the microRNA detection system based on an exponential amplification reaction and Argonaute nuclease has a reaction temperature of 65-85℃and a reaction time of 5-40min; in some more preferred embodiments, the reaction time is from 10 to 30 minutes; more preferably 15-25min, 15-20min, and most preferably 15min; in still more preferred embodiments, the reaction temperature is 70-80 ℃, 75-80 ℃, most preferably 80 ℃.
In some preferred embodiments, the concentration of Argonaute nuclease in a microRNA detection system based on an exponential amplification reaction and Argonaute nuclease is 0.01-200nM; more preferably, the Argonaute nuclease has a concentration of 10-190nM, 20-180nM, 30-170nM, 40-160nM, 50-150nM, 60-140nM, 70-130nM, 80-120nM, 90-110nM or 90-100nM; most preferably, the concentration of Argonaute nuclease is 100nM.
In some specific embodiments, the microRNA detection system based on an exponential amplification reaction and Argonaute nuclease further comprises: (d) divalent metal ions.
In some more specific embodiments, the divalent metal ion is selected from Mn 2+、Mg2+、Co2+, and the like. In some more specific embodiments, the divalent metal ion is Mn 2+, such as MnCl 2. In these more specific embodiments, the concentration of MnCl 2 in the microRNA detection system based on an exponential amplification reaction and Argonaute nuclease is 0.01-1000 μm; preferably the concentration of MnCl 2 is 100-900. Mu.M; more preferably, the concentration of MnCl 2 is 200-800. Mu.M, 300-750. Mu.M, 400-750. Mu.M, 500-750. Mu.M, 600-750. Mu.M, 700-750. Mu.M; most preferably 750 μm.
In some specific embodiments, in a microRNA detection system based on an exponential amplification reaction and Argonaute nuclease, further comprising: (e) a reaction buffer. The reaction buffer is not particularly limited, and one skilled in the art can select according to the Argonaute nuclease used, for exampleReaction 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 templates are respectively designed for the at least two miRNAs; the detection probes are respectively designed for at least two miRNAs, and different detection probes have different fluorescent groups.
In some specific embodiments, microRNA detection systems based on exponential amplification reactions and Argonaute nucleases are used for single miRNA detection, multiplex miRNA detection, or miRNA typing analysis.
In some more specific embodiments, when microRNA detection systems based on exponential amplification reactions and Argonaute nucleases are 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 amplified simultaneously in one exponential amplification reaction system. Correspondingly, in a microRNA detection system, an amplified product triggers Argonaute nuclease to shear, and at the moment, detection probes which are specific to a plurality of target miRNAs and have different fluorescent groups are mixed in the microRNA detection system, and multiple miRNAs are detected through different fluorescent signals. For another example, in the case of performing miRNA typing analysis, pre-amplification is performed on a nucleic acid sample to be detected containing nucleic acid in an exponential amplification reaction system containing a plurality of amplification templates, after 100% complementary pairing of the amplification product and a corresponding detection probe, the Argonaute nuclease in the microRNA detection system is triggered and started to cleave the detection probe, and finally the miRNA typing analysis can be performed through the generated fluorescent signal.
The second aspect of the invention provides a microRNA detection kit, which comprises the microRNA detection system based on the exponential amplification reaction and Argonaute nuclease.
The third aspect of the invention provides a microRNA detection method, which detects microRNA by using the microRNA detection system of the first aspect of the invention, and the microRNA detection method comprises 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 miRNA in the nucleic acid sample to be detected through exponential amplification reaction to obtain an amplified product;
Step 2, providing or preparing a microRNA detection system, and adding the amplification product obtained in the step 1 into the microRNA detection system for reaction;
And step 3, obtaining a fluorescent signal in the reactant after the reaction of the microRNA detection system in the step 2, so as to realize the detection of miRNA.
In some specific embodiments, the test nucleic acid sample comprises nucleic acid from a biological sample selected from the group consisting of: blood, cells, serum, saliva, body fluids, plasma, urine, prostatic fluid, bronchial lavage, cerebrospinal fluid, gastric fluid, bile, lymph, peritoneal fluid, stool, and the like, or combinations thereof.
In some specific embodiments, a step of pretreatment of the biological sample, comprising extracting total micrornas in the biological sample, is further included prior to step 1. For example, a commercially available kit may be used to extract total mirnas in biological samples.
ADVANTAGEOUS EFFECTS OF INVENTION
The microRNA detection system and the detection method based on the exponential amplification reaction and the Argonaute nuclease provided by the invention are used for generating a detection miRNA signal by combining EXPAR pre-amplification and Argonaute nuclease specificity shearing. 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, miRNA is detected with high sensitivity, the minimum detection limit is 1 attomole per liter (aM), and the miRNA has good sensitivity in actual detection;
Thirdly, designing specific nucleic acid probes with different molecular markers aiming at different miRNA targets can realize multiple detection, provide comprehensive detection information for clinical diagnosis and improve diagnosis accuracy;
The Argonaute nuclease has the advantage of high specificity of single base resolution, solves the bottleneck of the current technology in the aspect of 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 an EXPAR and Argonaute nuclease-based miRNA detection system and a detection method provided in an embodiment of the present invention.
Fig. 2 is a schematic diagram of a miRNA detection system and a detection method for multiple miRNA detection or miRNA typing analysis based on EXPAR and Argonaute nucleases provided in the embodiments of the present invention.
FIG. 3 is a real-time fluorescence signal monitoring graph of an EXPAR amplification product of miRNA-21 detected by a miRNA detection system and a detection method based on EXPAR and Argonaute nucleases provided in the embodiment of the invention; among them, the EXPAR negative control is ultrapure water (labeled as water in the figure).
FIG. 4 is a diagram showing the detection of miRNA by high-specificity recognition at single-base resolution level based on EXPAR and Argonaute nucleases provided in the examples of the present invention; the EXPAR amplification product of the miRNA-21 was simulated by using miDNA-21 and single base mismatches of miDNA-21, miDNA-21 successfully triggered the initiation of the TtAgo shearing simulation probe, whereas the single base mismatches of miDNA-21 failed to initiate the TtAgo shearing simulation probe due to the single base mismatch with the simulation probe.
FIG. 5 is a graph depicting the sensitivity of detection of miRNA by the detection system and detection method based on EXPAR and Argonaute nucleases and comparing the sensitivity with conventional fluorescence EXPAR methods; taking miRNA-21 as an example, wherein A in FIG. 5 represents the signal intensity of an miRNA-21 target object with the detection concentration of 0-10nM based on an EXPAR and TtAgo system miRNA detection method; b in fig. 5 represents a standard curve of detection of miRNA-21 targets by a miRNA detection method based on the EXPAR and TtAgo systems; FIG. 5C shows a real-time fluorescence curve of a conventional fluorescence EXPAR method for detecting miRNA-21 target with a concentration of 0-100 nM; d in FIG. 5 represents a standard curve of the detection of miRNA-21 target by the conventional fluorescence EXPAR method.
FIG. 6 shows a multiplex detection of miRNAs based on EXPAR and Argonaute nucleases and methods provided in the examples of the present invention; taking miRNA-21, miRNA-92a, miRNA-31 and miRNA-141 as examples, firstly, simultaneously pre-amplifying the four target miRNAs in an EXPAR reaction system, and adding the obtained amplicons into a TtAgo nuclease-based miRNA detection system containing four detection probes; the fluorescent molecules marked by the fluorescent probes corresponding to the miRNA-21, the miRNA-92a, the miRNA-31 and the miRNA-141 are respectively: JOE, HEX, cy5 and ROX.
FIG. 7 shows a miRNA typing detection system and a detection method based on EXPAR and Argonaute nucleases provided in the examples of the present invention; let 7 family miRNA typing is exemplified. Fluorescent molecules marked by fluorescent probes corresponding to the Let 7a, the Let 7b and the Let 7i are respectively as follows: JOE, HEX, and ROX.
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention, in conjunction with the accompanying drawings. Specific materials and sources thereof used in embodiments of the present invention are provided below. It will be understood that these are merely exemplary and are not intended to limit the invention, as materials identical or similar to the type, model, quality, nature or function of the reagents and instruments described below may be used in the practice of the invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Definition of the definition
The terms "a" and "an" as used in this specification mean at least one unless explicitly indicated otherwise. In this specification, the use of the singular includes the plural unless specifically stated otherwise.
It should be understood that there is an implicit "about" prior to the temperatures, concentrations, times, etc. discussed in this specification so that minor and insubstantial deviations are within the scope of the teachings herein. Also, the use of "including," "comprising," "having," "containing" and "containing" is not intended to be limiting. It is to be understood that both the foregoing general description and the detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
As used herein, the term "or a combination thereof" refers to all permutations and combinations of items listed before the 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 BA, CA, CB, ACB, CBA, BCA, BAC or CAB if the order is important in a particular context. Continuing with this example, explicitly included are duplicate combinations comprising one or more entries or items, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Unless otherwise indicated as apparent from context, one of ordinary skill in the art will appreciate that there is generally no limit to the number of items or items in any combination.
The term "and/or" when used in connection with two or more selectable items is understood to mean any 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) joined by internucleotide phosphodiester linkages or internucleotide analogs, as well as associated counterions, such as H +、NH4+, trialkylammonium, tetraalkylammonium, mg 2+、Na+, and the like. The nucleic acid may be a polynucleotide or an oligonucleotide. The nucleic acid may consist entirely of deoxyribonucleotides, entirely of ribonucleotides, or may be a chimeric mixture thereof. Nucleotide monomer units can 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 monomer units, e.g., 5-40 to thousands of monomer 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 may be present on or in a biological sample.
The length of a nucleic acid can be expressed as a base, base pair (abbreviated "bp"), nucleotide/nucleotide residue (abbreviated "nt"), or kilobase ("kb") according to conventions used in the art. The terms "base", "nucleotide residue" may describe polynucleotides that are single-stranded or double-stranded, where the context permits. When this term is applied to a double stranded molecule, it is used to refer to the entire length and is understood to correspond to the term "base pair".
The term "base" refers to derivatives of purines and pyrimidines, and is a component of nucleic acids, nucleosides, and nucleotides. There are 5 bases: 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., fluorescent label, fluorophore) that can be used to detect or identify its corresponding target nucleotide or polynucleotide by hybridization to 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 that 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 the detected signal by fluorescence, chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Examples of labels include fluorophores, chromophores, radioactive atoms (particularly 32p and 125I), electron dense reagents, enzymes, and ligands with specific conjugates.
As used herein, the term "dNTP" refers to deoxynucleoside triphosphates. NTP refers to ribonucleoside triphosphates. Purine bases (Pu) include adenine (a), guanine (G) and derivatives and analogues thereof. Pyrimidine bases (Py) include cytosine (C), thymine (T), uracil (U) and derivatives and analogues thereof.
As used interchangeably herein, the terms "complementary," "complementary sequence," "complementary," and "complementarity" generally refer to a sequence that is fully complementary and hybridizable to a given sequence. The sequence that hybridizes to a given nucleic acid is referred to as the "complementary sequence" or "reverse complementary sequence" of a given molecule, provided that its base sequence on a given region is capable of binding complementarily 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 terms "amplification," "nucleic acid amplification," or "amplified" refer to the production of multiple copies of a nucleic acid template, or the production of 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 such as 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 products of an amplification reaction. The amplicon may be double-stranded or single-stranded, and may include separate constituent strands obtained by denaturing the double-stranded amplification product. In certain embodiments, the amplicon of one amplification cycle may be used as a template in a subsequent amplification cycle.
The term "exponential amplification reaction" or "EXPAR" is a technique for the efficient exponential amplification of short-chain nucleic acid sequences at constant temperature, using linear amplification to generate an oligonucleotide product as a novel primer that is bound to a template comprising two repeated sequences, and cleavage by a nicking enzyme to generate an oligonucleotide product of the same sequence, resulting in an exponential increase in the oligonucleotide product.
As used herein, the term "polymerase" generally refers to an enzyme (e.g., natural or synthetic) capable of catalyzing a polymerization reaction. Examples of polymerases can include nucleic acid polymerases (e.g., DNA polymerase or RNA polymerase), transcriptases, and ligases (liage). The polymerase may be a polymerase (polymerization enzyme). The term "DNA polymerase" generally refers to an enzyme capable of catalyzing the polymerization reaction 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). The nicking enzyme 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 may be a nicking enzyme (e.g., a restriction endonuclease, a nicking endonuclease, etc.). The nicking enzyme may bind to a nicking site of a double-stranded nucleic acid molecule to create a nick (or gap) 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
1. Design of amplification template and detection probe for EXPAR pre-amplification
1. The design principle of the amplification template for EXPAR pre-amplification is as follows:
1) The 3' end of the amplification template can be fixed to an adenine (A) base and has phosphorylation modification;
2) The "NNNNNNNNNNNNNNNN" on either side of the amplification template is a nucleic acid sequence complementary to the miRNA of interest, 15-18 nucleotides in length, e.g., the above-described exemplary "NNNNNNNNNNNNNNNN" is 16 nucleotides, which may also be 15, 17 or 18 nucleotides;
3) The middle ACTCAGACAA (SEQ ID NO: 1) of the amplified template is a fixed sequence, and is the recognition sequence of a nicking endonuclease (nicking endonuclease) Nt.BstNBI endonuclease in an 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 be mismatched with the sequence of the amplified template by 1 base;
5) The resulting amplicon is complementarily paired with 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. Universal template: 3'-P-ANNNNNNNNNNNNNNNNACTCAGACAANNNNNNNNNNNNNNNN-5'.
2. The detection probe design follows the principle:
The detection probe may be a hairpin or linear gene sequence, preferably a hairpin is selected. The 5 'end of the probe stem is modified with a signal molecule, such as JOE, HEX, cy, ROX and other signal groups, and the 3' end of the probe stem is modified with a quenching group, such as BHQ1, BHQ2 and the like. The detection probe ring part is combined with the target amplicon in a specific complementary pairing mode, and when the detection probe ring part is matched with the target amplicon in a 100% complementary mode, the TtAgo shearing detection probe is started to release and generate a fluorescent signal.
The multiplex detection targets and the targets of the typing analysis may be any miRNA, and in the following examples, the multiplex detection is exemplified by miRNA-21, miRNA-92a, miRNA-31 and miRNA-141. Genotyping analysis of mirnas including SNP and polybasic mutation typing of mirnas taking let 7 family (let 7a, let 7b, let 7c, let 7d and let 7 i) as an example, the corresponding EXPAR amplification templates and the corresponding detection probes were designed. The miRNA sequences used in the examples are specifically described in table 1:
Table 1: miRNA sequences used in the examples
| Nucleic acid name | 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) |
2. EXPAR pre-amplification system and Tttago nuclease-based miRNA detection system
EXPAR Pre-amplification System: in the examples, 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. The solution A comprises Nt.BstNBI buffer solution, an amplification template, dNTPs, an RNase inhibitor and a nucleic acid sample to be detected (which possibly contains target miRNA); the liquid B comprisesReaction buffer, nt.BstNBI, vent (exo-) DNA polymerase and DEPC water. According to the number of the nucleic acid samples to be detected in the solution A, the number of the amplification templates to be used is determined, and then the respective volumes of the solution A and the solution B can be adjusted. Preparing solution A and solution B respectively, mixing the prepared solution A and solution B, and immediately placing the mixed solution A and solution B at 55 ℃ for reaction. The EXPAR pre-amplification reaction temperature is in the range of 50-60℃and preferably 55 ℃. The EXPAR pre-amplification reaction time is 10-30min, preferably 20min.
The concentration of each component in the EXPAR pre-amplification system (solution A and 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(NH4)2SO4,2mM MgSO4,0.1％TritonX-100;Tris＝2- amino-2- (hydroxymethyl) -1, 3-propanediol (2-amino-2-hydroxymethylpropane-1, 3-diol), prepared by/>The stock solution of the reaction buffer is diluted and prepared in a pre-amplification system, and the stock solution of the reaction buffer is 0.5 XNt.BstNBI buffer (25 mM Tris-HCl, pH 7.9, 50mM NaCl,5mM MgCl 2 and 0.5mM Dithiothreitol (DTT) and is diluted and prepared in the pre-amplification system from the stock solution of the Nt.BstNBI buffer. Only a single corresponding amplification template is needed to be added for single miRNA detection, a plurality of corresponding amplification templates (0.1 mu M of each amplification template) are added for multiple miRNA detection, and the addition amount of each nucleic acid sample is 1-4 mu L.
TtAgo nuclease-based miRNA detection system: the miRNA detection system based on Tttago nuclease is 10 mu L and comprisesReaction buffer (20 mM Tris-HCl,10mM (NH 4)2SO4,10mM KCl,2mM MgSO4, 0.1%/>)X-100, pH 8.8), mnCl 2, detection probes (0.1. Mu.M), ttAgo nuclease (enzyme), ultrapure water and EXPAR amplicon. The EXPAR amplicon stock or 10-fold dilution was added to 1. Mu.L to a TtAgo nuclease-based miRNA detection system. The reaction temperature of the miRNA detection system based on Tttago nuclease ranges from 65 ℃ to 85 ℃, and the preferable reaction temperature is 80 ℃; the reaction time is 5-40min, preferably 15min. MnCl 2 concentration ranges from 0 to 1000. Mu.M, preferably 750. Mu.M; tttago enzyme concentration is 0-200nM, preferably 100nM. When detecting a plurality of targets, a plurality of corresponding detection probes are added, and each detection probe is 0.1 mu M.
The reagents and nucleic acid sequence sources used are shown in Table 2 below.
Table 2: information on reagents used in the examples
Examples
The invention provides a system and a method for detecting miRNA in a high-specificity, high-sensitivity and multiple modes. The method for detecting miRNA 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 treated, after a low-abundance nucleic acid sample is obtained through nucleic acid extraction, miRNA is specifically amplified through EXPAR, and then the amplified amplicon is added into a detection system containing TtAgo enzyme and a detection probe, and the amplicon is used as gDNA to trigger and mediate the TtAgo shearing detection probe, so that a detection signal is generated. The detection signals can be detected by real-time monitoring and terminal detection modes, including a real-time fluorescence PCR instrument, various fluorescence measuring instruments, a lateral flow immunochromatography test strip, a naked eye observation method and the like. The method has the advantages of simplicity, rapidness, low cost and the like, obtains the detection result about 30 minutes, has the sensitivity reaching aM, and can be well used for detecting clinical miRNA. The invention can be widely applied to disease diagnosis, clinical research and life science research, such as tumor liquid biopsy or early screening of cancers.
Specifically, the core of the invention is that a large amount of amplicons generated by amplifying miRNA by using EXPAR are utilized, tttago specifically recognizes and shears a detection probe under the mediation of the amplicons, and the simultaneous detection of multiple miRNAs by a one-pot method is realized. The principle is as in fig. 1, the details are as follows: each miRNA binds to a specifically designed and complementarily paired amplification template, which is amplified by EXPAR to yield a large amount of 5' phosphorylated single-stranded DNA (about 16 bases of amplicon). The generated nucleic acid specific amplicon is further used as a gDNA mediated TtAgo recognition and specific shearing detection probe to generate a fluorescent signal. Multiplex detection As shown in FIG. 2, the EXPAR system is added with multiple targets and corresponding amplification templates, the resulting amplicon is added to the TtAgo system containing multiple probes capable of specifically binding to the amplicon, and one-pot multiplex detection is achieved by the generation of different signals. The technical advantages are as follows: 1) Tttago has the specificity of single base resolution, and 100% of the amplicon sequence is matched with a detection probe to start shearing to generate a signal, so that the Tttago has extremely high detection specificity; 2) The detection system can be designed aiming at any miRNA target sequence, and the disorder sequence preference is achieved; 3) Multiple detection of a single TtAgo enzyme on a plurality of miRNAs in a single reaction system is realized; 4) Realizing 30min rapid detection and detection, and reaching aM sensitivity; 5) And various terminal detection modes can be flexibly combined.
The feasibility verification of the method is shown in figure 3, and the method is verified by firstly pre-amplifying miRNA-21 through EXPAR and then through a Tttago detection system. When the target is miRNA-21, the amplicon of the target successfully mediates the TtAgo shearing detection probe, and a fluorescent signal is generated quickly; the negative control (water) has no amplified product, no Tttago shearing detection probe can be started, and no fluorescence signal enhancement process is performed.
Example 1: high specificity detection capability of Tttago single base resolution
This example explores the shear specificity of TtAgo. First miDNA-21 and single base mismatched miDNA-21 (as shown in Table 2) were synthesized, as well as a mock probe (i.e., detection probe) as shown in Table 3.
Wherein miDNA-21 is 100% complementary paired with the mimetic, and a single base mismatch miDNA-21 has one base mismatch with the mimetic. The EXPAR pre-amplification products of miRNA-21 were simulated using miDNA-21, tttago nuclease-based detection systems comprising miDNA-21 (5. Mu.M) or single base mismatched miDNA-21 (5. Mu.M), a simulated probe (1. Mu.M), tttago (100 nM),Reaction buffer, mnCl 2 (750. Mu.M) and ultrapure water. The reaction temperature of a detection system based on Tttago nuclease is 80 ℃; the reaction time was 30min. In the Tttago nuclease-based detection system, the cleavage products (short-chain nucleic acids) of Tttago nuclease were confirmed by 15% urea-polyacrylamide gel electrophoresis under 130V for 80min.
As a result, as shown in FIG. 4, miDNA-21 was able to mediate the successful cleavage of the mimetic by Tttago nuclease, i.e., no mimetic bands around 30bp were shown, while the cleaved mimetic bands appeared below the miDNA-21 bands. However, the single-base mismatched miDNA-21 does not activate the Tttago nuclease cleavage mimetic probe, the mimetic probe band is still displayed at the position of about 30bp, and no trace of the cleaved nucleic acid product is below the single-base mismatched miDNA-21 band, which indicates that the Tttago nuclease-based detection system in the embodiment has high specificity of single base resolution. The single base specificity of the Tttago nuclease has great advantages, can solve the problems of nonspecific amplification and false positive caused by poor specificity of the traditional EXPAR, plays a great role in practical detection application, and can be used for accurate detection of miRNA 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 Tttago systems
This example explores the sensitivity of the methods for miRNA detection 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, 100pM, 1nM, 10 nM) were first pre-amplified based on the EXPAR pre-amplification system in the materials and methods section, with reaction conditions of 55 ℃ for 20min. Tttago nuclease-based detection system comprising pre-amplification product (1. Mu.L), detection probe (1. Mu.M), tttago (100 nM),Reaction buffer, mnCl 2 (750. Mu.M) and ultrapure water. The reaction temperature of a detection system based on Tttago nuclease is 80 ℃; the reaction time was 30min. The detection result is shown as A in FIG. 5, and the detection sensitivity reaches 1aM (10 -18 M) (B in FIG. 5).
In this embodiment, the sensitivity of the method for detecting miRNA based on the EXPAR and TtAgo nucleases is further compared with the sensitivity of the conventional fluorescent EXPAR detection miRNA, and the conventional fluorescent EXPAR system is as follows: the total volume of the solutions A and B was 10. Mu.L. Solution A consists of Nt.BstNBI buffer, amplification template, dNTPs, RNase inhibitor and a series of concentrations of miRNA-21 (0, 100fM, 1pM, 10pM, 100pM, 1nM and 10 nM); the solution B is prepared fromReaction buffer, nt.BstNBI, vent (exo-) DNA polymerase, SYBR Green and DEPC water. And (3) immediately placing the mixed solution A and solution B in a PCR instrument for reaction. The EXPAR reaction temperature was 55deg.C and the reaction time was 35min, and fluorescence was read every 30 seconds. The concentration of each component in the EXPAR system 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), SYBR Green (final concentration 5000-fold dilution of stock solution, soxhaust organism),/> Reaction buffer and 0.5 XNt.BstNBI buffer. The real-time fluorescence results of the conventional fluorescent EXPAR detection of miRNA-21 are shown as C in FIG. 5, and the amplification curve is very close to that of the negative control (water) (false positive amplification signal) at a concentration of 100fM, so that the detection sensitivity is 100fM (10 -13 M) (D in FIG. 5). In contrast, the sensitivity of the detection method based on EXPAR and Tttago nuclease miRNA is improved by several orders of magnitude compared with the original traditional EXPAR, and false positive signals are avoided.
Example 3: method for detecting miRNA based on EXPAR and Tttago nuclease to detect multiple miRNAs
This example explores the ability of the inventive methods for detecting mirnas based on EXPAR and TtAgo nucleases to detect multiple mirnas.
Taking four target substances miRNA of miRNA-21, miRNA-92a, miRNA-31 and miRNA-14 as examples, the detection process is divided into two steps, firstly, on the basis of an EXPAR pre-amplification system described in the materials and methods, four amplification templates corresponding to the miRNA and four corresponding target substances miRNA (serving as nucleic acid samples) are added into the EXPAR pre-amplification system, the concentration of the target substances miRNA is 100pM, and the four target substances miRNA are pre-amplified simultaneously in the same reaction tube, wherein the reaction condition is 55 ℃ for 20min. Secondly, a TtAgo nuclease-based miRNA detection system comprises detection probes corresponding to four amplicons and EXPAR multiplex amplicons, other components are consistent with the TtAgo system described in the materials and methods, the reaction condition is 80 ℃ for 15min, the reaction is completed in a PCR instrument, and fluorescent intensity values before and after the reaction are read by the PCR instrument for qualitative and quantitative detection.
The detection results of the method for detecting multiple miRNAs based on EXPAR and Tttago nucleases are shown in FIG. 6, the system can detect one miRNA of four targets through different fluorescent channels, and simultaneously detect two, three or four targets, in the embodiment, JOE signal represents miRNA-21, HEX signal represents miRNA-92a, cy5 signal represents miRNA-31 and ROX signal represents miRNA-141. The whole reaction comprises EXPAR pre-amplification and Tttago nuclease-based miRNA detection only needs 35 minutes, has simple and convenient reaction operation, and has great application prospect in the aspects of clinical diagnosis, prognosis, patient drug resistance and other monitoring.
Example 4: miRNA (micro ribonucleic acid) detection method based on EXPAR and Tttago nuclease for detecting miRNA typing
Targeting the let 7 family (let 7a, let 7b and let 7 i), the detection system and method of the present invention was further evaluated for miRNA family typing capability. Standard sequences of the three targets are respectively synthesized, and corresponding EXPAR amplification templates and three detection probes are synthesized. The FAM, HEX and ROX tagged sequence-specific detection genes are 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 with amplification in an EXPAR system comprising three templates at 55℃for 30 minutes. mu.L of EXPAR amplification product was loaded into a miRNA detection system (20. Mu.L) containing TtAgo nuclease of three detection probes and reacted at 80℃for 15 minutes. The results demonstrate that this example achieves accurate identification of the let 7 subtype by combining EXPAR pre-amplification with Ago detection (FIG. 7). Further proves that the method has wide application prospect.
Sequence listing
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Claims (13)
1. The microRNA detection system based on the exponential amplification reaction and Argonaute nuclease is characterized by comprising:
(a) The guide DNA is an amplification product obtained by amplifying a target microRNA by adopting an exponential amplification reaction system;
(b) Argonaute nucleases;
(c) The detection probe is provided with a fluorescent group and a quenching group, and comprises a region complementary to an amplification product obtained by amplifying a target microRNA by adopting an exponential amplification reaction system;
The exponential amplification reaction system comprises an amplification template;
The amplification template is designed according to the sequence of the target microRNA, and comprises a 3 'end sequence, a 5' end sequence and a nickase recognition sequence between the 3 'end sequence and the 5' end sequence, wherein the lengths of the 3 'end sequence and the 5' end sequence are the same and are 15-18 nucleotides;
The 3 'end sequence and the 5' end sequence are the same and are complementary matched with the target microRNA;
Or the first base at the 3' end of the 3' end sequence is mismatched with the target microRNA, so that the first base at the 5' end of the amplicon of the exponential amplification reaction system is thymine base;
the Argonaute nuclease is TtAgo nuclease from Thermus thermophilus (Thermus thermophilus);
the reaction temperature of the microRNA detection system based on the exponential amplification reaction and the Argonaute nuclease is 70-80 ℃;
The exponential amplification reaction system comprises an amplification template, DNA polymerase, nicking enzyme, dNTPs, RNase inhibitor and enzyme buffer solution;
In the detection probe, the fluorescent group and the quenching group are respectively and independently positioned at the 5 'end and the 3' end of the detection probe.
2. The microRNA detection system of claim 1, wherein the concentration of Argonaute nuclease in the microRNA detection system is 0.01-200nM.
3. The microRNA detection system based on an exponential amplification reaction and Argonaute nuclease as claimed in claim 1 wherein the 3' end of the amplified template further has an adenine base.
4. The microRNA detection system of claim 1, wherein the twelfth base at the 5' end of the amplicon of the exponential amplification reaction system is not an adenine base.
5. The microRNA detection system of claim 1, wherein the nicking enzyme is selected from the group consisting of restriction endonucleases and nicking endonucleases.
6. The microRNA detection system based on an exponential amplification reaction and Argonaute nuclease as claimed in claim 5 wherein the nicking enzyme is a restriction endonuclease.
7. The microRNA detection system of claim 1, wherein the detection probe is a hairpin structure or a linear structure based on an exponential amplification reaction and an Argonaute nuclease.
8. The microRNA detection system based on an exponential amplification reaction and Argonaute nuclease as claimed in claim 7 wherein the detection probe is of hairpin structure.
9. The microRNA detection system of claim 1, wherein the microRNA detection system based on an exponential amplification reaction and an Argonaute nuclease further comprises: (d) divalent metal ions;
And/or, (e) a reaction buffer.
10. A non-diagnostic microRNA detection method for detecting micrornas using the microRNA detection system of any one of claims 1-9, the non-diagnostic microRNA detection method comprising the steps of:
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 exponential amplification reaction to obtain an amplified product;
Step 2, providing or preparing a microRNA detection system, and adding the amplification product obtained in the step 1 into the microRNA detection system for reaction;
and step 3, obtaining a fluorescent signal in the reactant after the reaction of the microRNA detection system in the step 2, so as to realize the detection of microRNA.
11. The method of claim 10, wherein the sample of nucleic acid to be detected comprises nucleic acid from a biological sample selected from the group consisting of: blood, cells, serum, saliva, body fluids, plasma, urine, prostatic fluid, bronchial lavage, cerebrospinal fluid, gastric fluid, bile, lymph, peritoneal fluid, stool, or combinations thereof.
12. The method according to claim 11, further comprising a step of pretreatment of the biological sample before step 1, wherein the step of extracting total micrornas in the biological sample is performed.
13. The non-diagnostic microRNA detection method of claim 10, wherein the microRNA detection is selected from a single microRNA detection, a multiplex microRNA detection, a microRNA typing analysis, or a combination thereof.
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