CN115992206A - Argonaute-mediated one-pot method microRNA detection system and detection method - Google Patents
Argonaute-mediated one-pot method microRNA detection system and detection method Download PDFInfo
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Abstract
The invention discloses an Argonaute-mediated one-pot method microRNA detection system and a detection method. The invention provides a microRNA detection method, which comprises the steps of mixing a nucleic acid sample to be detected, an exponential amplification reaction system and a microRNA detection system to form a mixed reaction system, and reacting to obtain a fluorescent signal in a reactant so as to realize detection of microRNA; the microRNA detection system comprises a guide DNA, argonaute nuclease and a detection probe, and an amplification product obtained by amplifying the target microRNA by the exponential amplification reaction system is used as a guide DNA. The system and the method establish the one-pot detection of the integration of EXPAR amplification and Ago specific shearing for the first time, realize the simultaneous amplification, shearing and single or multiple target object detection in a single reaction system, avoid aerosol pollution, and have convenient operation and high efficiency.
Description
Technical Field
The invention relates to the technical field of microRNA detection, in particular to an Argonaute-mediated one-pot method microRNA detection system and a detection method; more particularly, it relates to a programmable Argonaute nucleic acid specific cleavage enzyme mediated one-pot microRNA detection/molecular diagnostic system and method.
Background
MicroRNA (miRNA) is a short-chain non-coding RNA which plays an important role in regulating gene expression by targeting mRNA and thus inhibiting gene expression after transcription (Cell, 2004,116 (2): 281-297). The increase or decrease of the miRNA abundance level is closely related to the occurrence of diseases and is used as one of important markers for diagnosing and monitoring the diseases. For example, studies over the past decades have shown that abnormal levels of miRNAs are closely related to the occurrence, progression, metastasis, etc. of tumors and are considered as important markers for tumor diagnosis and prognosis (Nature reviews cancer,2006,6 (4): 259-269.). Because of the complexity of the tumor itself, the sensitivity and specificity of tumor markers are poor, and the early and accurate diagnosis of the tumor is always a difficult problem for people at present. And miRNA stably exists in a plurality of body fluids, such as serum, saliva, urine and the like, can resist degradation of RNase, is stable at a certain pH value and temperature, and opens up a new idea for diagnosis of tumors. Detection of miRNA expression levels in circulating blood is promising as an optimal choice for early diagnosis of malignancy.
Nucleic acid detection techniques including reverse transcription-polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR), isothermal amplification and high throughput sequencing have all been developed and widely used in molecular medicine diagnostics, but suffer from deficiencies in sensitivity, specificity and ease of operation. In addition, because miRNA fragments are short, various types are high, sequence similarity is high, and the interference objects of complex biological samples are numerous, the target content is extremely small, and the traditional method has great limitation when being applied to miRNA detection. For example, PCR requires complex designs, reverse transcription and PCR processes, and has limited sensitivity and specificity. High throughput sequencing has the problems of long time consumption, high cost, complicated steps and the like. Currently, there is no satisfactory detection method for mirnas.
The CRISPR-Cas system is a mechanism formed by bacteria to defend against virus invasion, and programmable endonucleases (Cas enzymes) can specifically recognize targets and precisely cleave nucleic acid chains with high efficiency, and in recent years, the CRISPR-Cas system is rapidly becoming a high-efficiency tool in a series of fields such as genome editing, transcription regulation, nucleic acid detection and the like, and is considered as a powerful tool for improving the specificity and sensitivity of gene detection. CRISPR technology combines amplification methods such as PCR, recombinase polymerase amplification (recombinase polymerase amplification, RPA), loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP), rolling circle amplification (rolling circle amplification, RCA) and the like, and various targets can be detected by activating the trans-shearing activity of Cas enzyme through the amplicon of the target and amplifying the signal through the shearing probe, including pathogens, gene mutation and the like (ACS synthetic biology,2020,9 (6): 1226-1233.Biosensors and Bioelectronics,2020,165:112430). The main disadvantages of CRISPR are the following three points: 1. the pre-spacer adjacent motif (protospacer adjacent motif, PAM)/PFS (protospacer flanking sequence) sequence dependence of Cas enzymes limits its use in any target sequence; 2. cas enzyme is used for identifying a shearing target and simultaneously cutting a free single-chain fluorescent probe in a nonspecific trans-form, so that fluorescence is released to achieve the detection purpose, and multiple Cas enzymes are needed to be relied on for simultaneously detecting multiple targets, so that a multiple detection system is high in complexity, difficult to realize and high in cost; 3. cas enzymes rely on RNA as a guide, which is costly to synthesize and less stable. At present, the target miRNA can be detected by using the trans-cleavage mediated by Cas enzyme, but the detection can not be realized by a two-step method, namely, the nucleic acid amplification and the shearing signal release can not be completed in one step in a single reaction in a one-pot detection system, and multiple targets can not be synchronously and rapidly and effectively detected. Therefore, there is an urgent need in the art to develop a one-pot detection method with high sensitivity, good specificity and high throughput for the target miRNA.
Argonaute proteins (Ago proteins/Ago nucleases) are a large family of proteins, including prokaryotes Ago (pAgo) and eukaryotes Ago (eAgo) (Genome biology,2008,9 (2): 1-8.Nature Reviews Genetics,2013,14 (7): 447-459.). eAgo uses small RNA guides to find its mRNA targets, regulating gene expression and inhibiting mobile genetic elements in eukaryotes. pAgo exists in many bacterial and archaeal species, and unlike eukaryotic proteins, pAgo uses a small DNA guide to cleave DNA, a process known as DNA interference. This unique endonuclease activity guided by DNA/RNA molecules is of increasing interest, and Ago nucleases (or Argonaute nucleic acids, short Ago enzymes or Argonaute enzymes) have the precise recognition capability and high-efficiency cleavage capability of single-base resolution level by complementary pairing of guide DNA with target bases, and show great application prospects in gene editing and gene detection (Emerging Argonaute-based nucleic acid biosensors, trends in Biotechnology, 2022.). Ago (PfAgo) from archaea hyperthermophilum Pyrococcus furiosus can perform precise DNA cleavage guided by short-chain 5' -phosphorylated Single-stranded DNA (ssDNA) as guide DNA (guide DNA) at 95 ℃ (Nucleic acids research,2015,43 (10): 5120-5129). Thermophilic Ago from thermophilic thermus Thermus thermophilus (TtAgo) and methanococcus jannaschii Methanocaldococcus jannaschii (mjoago) showed similar endonuclease activity as PfAgo, with a difference in optimal cleavage temperature (Journal of Advanced Research,2020, 24:317-324.). In addition, double-stranded DNA (dsDNA) substrates can be cleaved by Ago after incubation at high temperature for long periods of time without guide, suggesting that ssDNA randomly generated by dsDNA instability may be used as a new guide and direct further DNA cleavage. Tttago has been successfully used for low abundance single base variation (single nucleotide variants, SNV) gene enrichment as a programmable endonuclease to increase SNV detection sensitivity (Nucleic acids research,2020,48 (4): e19-e 19.). The nucleic acid detection method of coupling Pfago and PCR reaction can realize detection of gene mutation with abundance of 0.01% and efficiency improved by more than 5500 times (Nucleic acids research,2021,49 (13): e75-e 75.). Clostridium perfringens Clostridium perfringens Argonaute (CpAgo) is a protein expressed by clostridium perfringens Clostridium perfringens, a human intestinal microorganism, and is found to have a cleavage activity in the temperature range of 4-60 ℃ by CpAgo, to cleave a single-stranded nucleic acid target under the mediation of gDNA, and to cleave a plasmid over a long period of time (Cell Discovery,2019,5 (1): 1-4.). Saline-alkali bacillus griseus Natronobacterium gregoryi Argonaute (NgAgo), clostridium butyricum Clostridium butyricum Argonaute (CbAgo) and Limnothrix rosea Argonaute (lraggo), which can perform DNA-mediated DNA cleavage in vitro at physiological temperatures. Typically, ago requires only a 5' -phosphorylated single-stranded DNA (typically 15-20 bases) as gDNA, targets a specific nucleic acid sequence that recognizes complementary pairs to the gDNA, and cleaves the complementary DNA or RNA nucleic acid sequence at the 10-11 base positions of the gDNA. Compared with Cas nuclease, ago enzyme has no PAM sequence dependence and has more universality in practical detection application. In addition, gDNA is easy to design, low in cost and stable. Therefore, the Argonaute enzyme has great advantages and application prospect in the aspect of nucleic acid detection.
To date, most nucleic acid detection methods involve cumbersome and time-consuming steps, such as RT-PCR for miRNA detection requiring two steps of reverse transcription and PCR, time-consuming and open-lid prone to aerosol contamination. CRISPR-Cas detection methods typically require 2-3 steps to complete: reverse transcription is performed firstly, then pre-amplification is performed, and finally Cas enzyme detection is performed; or pre-amplification followed by transcription and finally Cas enzyme detection. The adoption of a two-step or multi-step reaction system also easily causes inaccurate quantification, the operation steps can be greatly reduced by the highly integrated one-pot detection, the operation flow is simplified, the detection convenience is improved, the pushing to practical application is facilitated, the overall detection time is shortened, aerosol pollution caused by uncovering is avoided, and the performance advantages such as the accuracy of the detection method are improved. At present, a detection method integrating amplification and detection is lacking, and in particular, novel one-pot molecular diagnosis and miRNA detection application based on the mediation of programmable Argonaute nucleic acid specific shearing enzyme are not reported yet.
Disclosure of Invention
Problems to be solved by the invention
As described above, the miRNA fragments are short, the content is small, and the base difference between family members is small, so that accurate detection of miRNA has a certain difficulty and challenge in actual clinic, and still is a technical problem at present. There are two main methods for traditional PCR detection of mirnas: the tail addition method and the stem loop method have complex primer design and require reverse transcription steps, and are difficult to accurately quantify. Other isothermal amplification methods, such as Rolling Circle Amplification (RCA) and exponential amplification reaction (Exponential Amplification Reaction, EXPAR) techniques, suffer from non-specific amplification problems, are prone to false positives, have limited sensitivity, and are difficult to implement for clinical detection. The miRNA detection technology combined with CRISPR involves more steps, has a complex system and is not beneficial to practical application; in addition, cas enzymes used in CRISPR technology have PAM sequence dependence and poor universality, and the signal amplification mode of the Cas enzyme non-specific cleavage probe makes it difficult to realize multiplex detection in one reaction system. Research shows that the occurrence of tumors is closely related to the deregulation of a plurality of miRNAs, but the current method basically relies on a multi-step method to detect a single miRNA target, so that the detection efficiency is low, and the clinical requirements are difficult to meet. Therefore, development of a method capable of detecting miRNA in multiple ways is urgently needed, detection efficiency is improved in the method, detection operation flow is simplified, and detection time is shortened; in clinical diagnosis application, the method helps to obtain more comprehensive detection information so as to improve the accuracy and reliability of diagnosis and prognosis. There is currently a lack of a universal miRNA detection method that enables simple, rapid, low cost, multiplexed and one-pot detection.
Therefore, the invention provides an Argonaute-mediated one-pot method microRNA detection system and a detection method, which are used for detecting miRNA based on a novel programmable Argonaute (Ago) specific nucleic acid shearing enzyme-mediated one-pot method, and the amplicon generated by EXPAR is used as guide DNA (gDNA), so that Ago targeted identification and efficient shearing (multi-turn over) single-stranded fluorescent probes are mediated, and the one-step method, single-base specific, high-sensitivity and efficient and rapid detection of multiple miRNA in one reaction tube is realized. The method has a plurality of detection advantages, overcomes the problems and the defects existing in the prior art, and has wide application prospect in practice.
Solution for solving the problem
The invention develops a novel one-pot method miRNA detection system and method based on Ago endonuclease for the first time, realizes the integrated amplification and detection steps of a single reaction system, detects miRNA, and simultaneously has the remarkable advantages of high specificity, high sensitivity, rapidness and multiple detection. The system is based on a one-pot method, and is a mixed reaction system of an exponential amplification reaction system (or called EXPAR amplification system) and a microRNA detection system (or called Ago detection system) as a whole.
In the one-pot microRNA detection system and method provided by the invention, an EXPAR amplification template (template) and a detection probe (probe) are designed according to a miRNA sequence. Adding the nucleic acid sample to be detected into a mixed reaction system of an EXPAR amplification system and an Ago detection system, namely a one-pot detection system, wherein the reaction conditions are that the reaction is firstly carried out for 10-30min at 50-60 ℃ and then carried out for 10-30min at 30-50 ℃. The target miRNA in the nucleic acid sample to be detected is combined with an amplification template, extends under the action of Vent (exo-) DNA polymerase, and the nicking enzyme Nt.BstNBI recognizes a shearing site, shears an amplicon generated by the polymerase, and a large amount of amplicons generated by the amplification serve as specific gDNA triggering and mediated Ago specific recognition and shearing detection probes, and generate fluorescent signals so as to realize detection of miRNA.
When the miRNA is detected in multiple ways, a nucleic acid sample to be detected is added into a one-pot method mixed system containing a plurality of miRNA amplification templates and a plurality of corresponding probes, and the corresponding amplicons are generated by simultaneous amplification in a reaction system, and correspondingly, the amplicons trigger Ago to cut probes which are specific to the corresponding targets and are marked by different fluorescence, and finally, the multiple miRNA is detected through different fluorescence signal channels.
The first aspect of the invention provides a microRNA detection method, which comprises the following steps: mixing a nucleic acid sample to be detected, an exponential amplification reaction system and a microRNA detection system to form a mixed reaction system, and reacting to obtain a fluorescent signal in a reactant so as to realize detection of microRNA; the microRNA detection system comprises a guide DNA, argonaute nuclease and a detection probe, and an amplification product obtained by amplifying a target object microRNA by the exponential amplification reaction system is used as the guide DNA.
In some embodiments, the Argonaute nuclease is selected from one or more of a CpAgo nuclease from clostridium perfringens (Clostridium perfringens), an IbAgo nuclease from escherichia coli (Intestinibacter bartlettii), an NgAgo nuclease from saline-alkali bacillus griseus (Natronobacterium gregoryi).
In some preferred embodiments, the Argonaute nuclease is a CpAgo nuclease from clostridium perfringens (Clostridium perfringens).
In some specific embodiments, the Argonaute nuclease is present in the mixed reaction system in an amount of 0.01-200nM.
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 specific embodiments, the DNA polymerase is Vent (exo-) DNA polymerase or Bst DNA polymerase.
In some specific embodiments, the nicking enzyme is a restriction endonuclease or a nicking endonuclease.
In some preferred embodiments, the nicking enzyme is a restriction endonuclease.
In some more specific embodiments, the restriction endonuclease is nt.bstnbi.
In some embodiments, the enzyme buffer comprises
Reaction buffer and NEBuffer TM r 3.1.
In some embodiments, the detection probe carries a fluorescent group and a quenching group, and the detection probe includes a region complementary to an amplification product obtained by amplifying the target microRNA using an exponential amplification reaction system.
In some preferred 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 embodiments, the test nucleic acid sample comprises nucleic acid from a biological sample.
In some optional embodiments, the biological sample is 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.
In some embodiments, a mixed reaction system is formed, and the reaction conditions under which the reaction is performed are: reacting at 50-60deg.C for 10-30min, and then reacting at 30-50deg.C for 10-30min.
In some embodiments, the method further comprises a step of pretreatment of the biological sample prior to performing the microRNA detection method, comprising extracting total micrornas in the biological sample.
The second aspect of the invention provides a one-pot method microRNA detection system, which comprises a nucleic acid sample to be detected, an exponential amplification reaction system and a microRNA detection system; the microRNA detection system comprises a guide DNA, argonaute nuclease and a detection probe, and an amplification product obtained by amplifying a target object microRNA by the exponential amplification reaction system is used as the guide DNA; the Argonaute nuclease is selected from one or more of CpAgo nuclease from clostridium perfringens (Clostridium perfringens), ibAgo nuclease from Enterobacter butler (Intestinibacter bartlettii), ngAgo nuclease from saline-alkali bacillus griseus (Natronobacterium gregoryi).
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.
ADVANTAGEOUS EFFECTS OF INVENTION
The existing method for detecting miRNA based on nucleic acid amplification requires reverse transcription, amplification, detection and other steps to cover, aerosol pollution is easy to cause, the steps are tedious, and the time consumption is long. The system and the method provided by the invention establish the one-pot detection integrating EXPAR amplification and Ago specific shearing for the first time, combine the advantages of EXPAR rapid amplification and Ago specific shearing amplification detection signals, realize amplification and shearing at the same time in a single reaction system (one reaction tube), avoid aerosol pollution, and fully show the convenience of operation and the high efficiency of the method. The method has the following advantages in terms of detection performance:
the specificity is high, the accuracy is high, and the high-specificity detection of single base resolution is realized;
(II) broad detection concentrationThe miRNA is detected in a range with high sensitivity, and the minimum detection limit is 1zM (10 -21 Power mole per liter), has good sensitivity in actual detection;
the method can detect a plurality of target objects simultaneously by a one-pot method, the multiple detection capability is strong in application in practical application, the detection efficiency is improved, comprehensive detection information is provided for clinical diagnosis, and the diagnosis accuracy is improved;
The method provides a high-efficiency tool in miRNA typing analysis, and has great significance in the basic research fields such as medical research, life science field, clinical application and the like;
and fifth, the method is simple and quick, does not depend on large-scale expensive special equipment and special raw materials, has good feasibility in practical application, and has remarkable application prospect.
Drawings
Fig. 1 is a schematic diagram of a one-pot microRNA detection system and a detection method provided in an embodiment of the invention, which detect miRNA based on Ago one-pot method. The one-pot mixed reaction system comprises a miRNA amplification template, vent (exo-) DNA polymerase (shown as Vent DNA polymerase in the figure), nicking enzyme (endonuclease Nt.BstNBI), a detection probe (shown as a probe in the figure) and Argonaute nuclease (shown as Ago in the figure); dNTPs, mn not shown in the figure 2+ And an enzyme buffer.
Fig. 2 is a schematic diagram of a principle of performing miRNA multiplex detection based on an Ago one-pot method in a one-pot method microRNA detection system and a detection method provided by an embodiment of the invention.
FIG. 3, which comprises A and B, wherein A in FIG. 3 is the result of detecting the relative fluorescence intensity of miRNA-141 by a one-pot microRNA detection system, and the concentration range is 0-1nM; b in fig. 3 is the corresponding standard curve. The probe channel is ROX; the concentration takes log values as the abscissa and is expressed as Lg target concentration in the figure.
FIG. 4, which comprises A and B, wherein A in FIG. 4 is the result of detecting the relative fluorescence intensity of miRNA-21 by a one-pot microRNA detection system, and the concentration range is 0-1nM; b in fig. 4 is the corresponding standard curve. The probe channel is FAM; the concentration takes log values as the abscissa and is expressed as Lg target concentration in the figure.
FIG. 5, which comprises A and B, wherein A in FIG. 5 is the result of detecting the relative fluorescence intensity of miRNA-92a by a one-pot microRNA detection system, and the concentration range is 0-1nM; b in fig. 5 is the corresponding standard curve. The probe channel is HEX; the concentration takes log values as the abscissa and is expressed as Lg target concentration in the figure.
FIG. 6, which comprises A and B, wherein A in FIG. 6 is the result of detecting the relative fluorescence intensity of miRNA-31 by a one-pot microRNA detection system, and the concentration range is 0-1nM; b in fig. 6 is the corresponding standard curve. The probe channel is CY3; the concentration takes log values as the abscissa and is expressed as Lg target concentration in the figure.
Fig. 7 is a specific investigation of Ago one-pot detection of miRNA-141 by the one-pot microRNA detection system and the detection method provided by the embodiment of the invention. Wherein a in fig. 7 is a real-time fluorescence monitoring graph. B in fig. 7 is a histogram of relative fluorescence intensity values. The probe channel is ROX; the concentration takes log values as the abscissa and is expressed as Lg target concentration in the figure.
Fig. 8 is a schematic diagram of a one-pot microRNA detection system and a detection method for detecting multiple mirnas by one-pot method according to an embodiment of the present invention. Taking miRNA-141, miRNA-21, miRNA-92a and miRNA-31 as examples, an amplification template containing four targets and probes corresponding to the four targets in one reaction system are used for detecting one, two, three or four targets at the same time. The fluorescent molecules marked by the detection probes corresponding to the miRNA-141, the miRNA-21, the miRNA-92a and the miRNA-31 are respectively: ROX, FAM, HEX and CY3.
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. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, as materials that are the same as 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 present 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, and associated counter ions, such as H + 、NH4 + Trialkyl groupAmmonium, tetraalkylammonium, mg 2+ 、Na + Etc. 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 (enzymes). 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.
As described above, the current miRNA detection method has the defects of sensitivity, specificity, high efficiency, universality and other performances, is difficult to be truly applied to clinical detection, and in order to overcome the problems and realize accurate detection of miRNA, a novel one-pot molecular diagnosis method based on the mediation of programmable Argonaute (Ago) specific nucleic acid shearing enzyme is provided for detecting miRNA, an amplicon generated by EXPAR is used as guide DNA (gDNA), an Ago targeting recognition and high-efficiency shearing (multi-turn over) single-chain fluorescent probe is mediated, and a signal is generated by amplifying and shearing in one reaction tube by a one-step method, so that experimental steps are greatly simplified, aerosol pollution is avoided, detection efficiency is improved, detection speed is accelerated, and a powerful novel method is provided for practical application. In addition, the Ago enzyme has the specificity of single base resolution and high shearing efficiency, and can realize the miRNA detection with high specificity and high sensitivity. CRISPR requires multiple Cas endonuclease with different single-stranded nucleic acid preferences to realize multiple detection, the more the detection targets, the more complex the reaction system, the influence on detection efficiency and easy generation of strong background signals, but at present Cas enzyme has limited types and sequence dependence, and is difficult to realize multiple miRNA detection. In contrast, the Ago endonuclease mediated detection system can realize multiple detection by only a single Ago enzyme, is applicable to any target, can be programmed randomly and has wide universality.
Specifically, the invention provides a method for detecting miRNA in a high-specificity, high-sensitivity and one-pot manner. The invention realizes the integrated detection of EXPAR pre-amplification and Ago shearing amplification signals. The detection sample can be biological samples from different sources, such as tissues and serum, and after the nucleic acid extraction is carried out to obtain a low-abundance nucleic acid sample, the nucleic acid extraction sample is added into a one-pot detection system, and an amplicon generated by EXPAR is used as gDNA to trigger and mediate Ago shearing detection probes, 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 zM, 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.
The core of the invention is that EXPAR amplification and Ago shearing are combined into a one-pot method, and the multiple miRNAs of the one-pot method are realized by utilizing a large number of amplicon-mediated Ago shearing target specific probes generated by EXPAR amplification miRNA. The principle is as in fig. 1, the details are as follows: each miRNA binds to a specifically designed and complementarily paired amplification template, strand extension is performed by Vent DNA polymerase to generate nucleic acid strands complementary to the template, nicking enzyme nt.bstnbi recognizes the specific sites and cleaves the amplified strands, and the cleaved nucleic acid strands further bind to the amplification template to amplify and cleave, thus cycling repeatedly to generate 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 Ago recognition and specific shearing detection probe to generate a fluorescent signal. Multiplex detection As shown in figure 2, the EXPAR system is added into a sample to be detected, the corresponding amplification template and the probes of multiple targets, the generated amplicon mediates Ago to be specifically combined with the corresponding probes through base complementation pairing, and the multiplex detection of a one-pot method is realized through detecting different fluorescent signals.
The following describes the technical scheme of the invention in detail.
< microRNA detection method and one-pot method microRNA detection System >
In some aspects of the invention, a microRNA detection method is provided, comprising: mixing a nucleic acid sample to be detected, an exponential amplification reaction system and a microRNA detection system to form a mixed reaction system, and reacting to obtain a fluorescent signal in a reactant so as to realize detection of microRNA.
In other aspects of the invention, a one-pot method microRNA detection system is provided, which comprises the nucleic acid sample to be detected in the microRNA detection method, an exponential amplification reaction system and a microRNA detection system.
microRNA detection system
In the present invention, the microRNA detection system includes Argonaute nuclease and detection probe. In some specific embodiments, the microRNA detection system further comprises a guide DNA, wherein the guide DNA is an amplification product obtained by amplifying the target microRNA in the exponential amplification reaction system in the mixed reaction system. Because the one-pot method microRNA detection system and the microRNA detection method creatively adopt the scheme of the one-pot method, namely, all reagents are added before the reaction without adding the reagents for the second time, in the reaction process of the one-pot method microRNA detection system and the microRNA detection method, an amplification product obtained by amplifying a target object microRNA by the exponential amplification reaction system can be directly used as guide DNA for the microRNA detection system to carry out the reaction of microRNA detection.
In some specific embodiments, the Argonaute nuclease is selected from one or more of a CpAgo nuclease from clostridium perfringens (Clostridium perfringens), an IbAgo nuclease from escherichia coli (Intestinibacter bartlettii), a NgAgo nuclease from saline-alkali bacillus griseus (Natronobacterium gregoryi). In some preferred embodiments, the Argonaute nuclease is a CpAgo nuclease from clostridium perfringens (Clostridium perfringens).
The Argonaute nuclease, in particular CpAgo nuclease, is used, so that the microRNA detection system in the mixed reaction system is compatible with the reaction temperature and the reaction/enzyme buffer solution of the exponential amplification reaction system, the two reactions of amplification and detection (namely, one-pot method) can be realized by mixing the reaction raw materials once, and the operation flow is simplified. In addition, in the reaction system of the one-pot method, an amplification product obtained by amplifying the microRNA of the target object by the exponential amplification reaction system is directly used as guide DNA of Argonaute nuclease, so that the problem that aerosol pollution is easy to generate when the product is added into the microRNA detection system after the exponential amplification reaction is also avoided. Because of the compatibility of the reaction/enzyme buffer solution, the exponential amplification reaction product does not need to be secondarily added into the microRNA detection system, so that all reaction products can be used for detection reaction, thereby having higher sensitivity, and the nuclease has higher activity under the mixed reaction system.
In some more specific embodiments, the Argonaute nuclease is present in the mixed reaction system in an amount of 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 embodiments, the detection probe carries a fluorescent group and a quenching group, and the detection probe includes a region complementary to an amplification product obtained by amplifying the target microRNA using an exponential amplification reaction system.
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, 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 content of each detection probe in the mixed reaction system is 0.05 to 0.5. Mu.M, preferably 0.05 to 0.4. Mu.M, 0.05 to 0.3. Mu.M, 0.05 to 0.2. Mu.M, 0.05 to 0.1. Mu.M, 0.07 to 0.1. Mu.M, 0.09 to 0.1. Mu.M.
In some specific embodiments, the microRNA detection system further comprises a divalent metal ion.
In some more specific embodiments, the divalent metal ion is selected from Mn 2+ 、Mg 2+ 、Co 2+ Etc. In some more specific embodiments, the divalent metal ion is Mn 2+ For example MnCl 2 . In these more specific embodiments, mnCl is present in the mixed reaction system 2 The concentration of (2) is 0.01-1000 mu M; preferably MnCl 2 The concentration of (2) is 100-900 mu M; more preferably, mnCl 2 The concentration of (2) is 200-800. Mu.M, 300-750. Mu.MM, 400-750. Mu.M, 500-750. Mu.M, 600-750. Mu.M, 700-750. Mu.M; most preferably 750 μm.
No additional reaction buffer for Argonaute nuclease is needed in the microRNA detection system. Because the Argonaute nuclease selected by the invention and the constructed mixed reaction system are mutually matched with the DNA polymerase and the nicking enzyme reaction/enzyme buffer solution, the experimental raw materials are saved, the activities of various enzymes are reserved or improved, and the mutual influence is avoided.
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.
Exponential amplification reaction system
The exponential amplification reaction system provides guide DNA for Argonaute nuclease in the microRNA detection 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 according to the sequence of the miRNA of interest. The amplified 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 lengths of the 3 'end sequence and the 5' end sequence are the same and are 15-20 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 15-20 nucleotides in length, in some embodiments of the present invention, amplification template design may be based on a continuous sequence of 15-20 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 more specific embodiments, the amount of each amplification template in the mixed reaction system is 0.05 to 0.5. Mu.M, preferably 0.05 to 0.4. Mu.M, 0.05 to 0.3. Mu.M, 0.05 to 0.2. Mu.M, 0.05 to 0.1. Mu.M, 0.07 to 0.1. Mu.M, 0.09 to 0.1. Mu.M; exemplary, 0.1 μm.
In some specific embodiments, the DNA polymerase is Vent (exo-) DNA polymerase or Bst DNA polymerase. In some more specific embodiments, the DNA polymerase is present in an amount of 0.01 to 0.1U. Mu.L in the mixed reaction system -1 Preferably 0.01 to 0.09U. Mu.L -1 、0.01～0.08UμL -1 、0.01～0.07UμL -1 、0.01～0.06UμL -1 、0.02～0.06UμL -1 、0.03～0.06UμL -1 、0.04～0.06UμL -1 The method comprises the steps of carrying out a first treatment on the surface of the Exemplary, 0.04U μL -1 、0.05UμL -1 Or 0.06 U.mu.L -1 。
In some 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 more specific embodiments, the amount of nicking enzyme in the mixed reaction system is from 0.1 to 1U. Mu.L -1 Preferably 0.1 to 0.9U. Mu.L -1 、0.1～0.8UμL -1 、0.1～0.7UμL -1 、0.1～0.6UμL -1 、0.1～0.5UμL -1 、0.2～0.5UμL -1 、0.3～0.5UμL -1 The method comprises the steps of carrying out a first treatment on the surface of the Exemplary, 0.4U μL -1 、0.5UμL -1 。
In some preferred embodiments, the DNA polymerase is Vent (exo-) DNA polymerase and the endonuclease is Nt.BstNBI; in these preferred casesIn embodiments, the enzyme buffer may be
Reaction buffer and NEBuffer TM One or more of r 3.1; in these preferred embodiments, the exponential amplification reaction system comprises an amplification template, dNTPs, nt.BstNBI, vent (exo-) DNA polymerase, RNase inhibitor, (-) -A/D>
Reaction buffer and NEBuffer TM r3.1。
In some specific embodiments, the dNTP is present in the mixed reaction system in an amount of 100 to 500. Mu.M, preferably 100 to 400. Mu.M, 100 to 300. Mu.M, 200 to 300. Mu.M; exemplary are 200. Mu.M, 250. Mu.M, 300. Mu.M. In some specific embodiments, the RNase inhibitor is present in the mixed reaction system in an amount of 0.1 to 1.5U. Mu.L -1 Preferably 0.3 to 1.5U. Mu.L -1 、0.5～1.5UμL -1 、0.5～1.3UμL -1 、0.5～1UμL -1 、0.7～1UμL -1 The method comprises the steps of carrying out a first treatment on the surface of the Exemplary, 0.8U μL -1 、0.9UμL -1 。
Reaction conditions
In the one-pot method microRNA detection system and the microRNA detection method, a mixed reaction system is formed, and the reaction conditions for the reaction are as follows: reacting at 50-60deg.C for 10-30min, and then reacting at 30-50deg.C for 10-30min.
In some preferred embodiments, a mixed reaction system is formed, and the reaction conditions under which the reaction is carried out are: reacting for 15-25 min at 50-57 ℃, and then reacting for 10-20 min at 30-45 ℃; it is further preferable that the reaction is carried out at 53 to 57℃for 15 to 25 minutes, and then at 35 to 45℃for 10 to 20 minutes.
In some more preferred embodiments, a mixed reaction system is formed, and the reaction conditions under which the reaction is carried out are: the reaction was carried out at 55℃for 20min, followed by 37℃for 10min.
Nucleic acid sample to be tested
In the one-pot method microRNA detection system and the microRNA detection method of the invention, the nucleic acid sample to be detected comprises nucleic acid from a biological sample, and the biological sample is 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.
Other steps
In some specific embodiments, the method of microRNA detection of the present invention is preceded by a step of pretreatment of the biological sample comprising extracting total micrornas in the biological sample. For example, total micrornas in biological samples can be extracted using commercially available kits.
Applicable microRNA detection
In some specific embodiments, the one-pot microRNA detection system and microRNA detection method can be used for single miRNA detection, multiplex miRNA detection, or miRNA typing analysis.
In some more specific embodiments, when the one-pot microRNA detection system and microRNA detection method 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 a mixed reaction system containing a plurality of amplification templates for target miRNA amplification, and a plurality of target mirnas are amplified simultaneously. Meanwhile, in the mixed reaction system, the amplified product triggers the 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 mixed reaction 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 sample of nucleic acid to be detected containing nucleic acid in a mixed reaction system containing a plurality of amplification templates, after 100% complementary pairing of the amplification product and the corresponding detection probe, the Argonaute nuclease in the mixed reaction system is triggered and started to shear the detection probe, and finally the miRNA typing analysis can be performed through the generated fluorescent signal.
Examples
Example one, amplification template and detection Probe design
1. EXPAR amplification template design reference (Angewandte Chemie International Edition,2010,49 (32): 5498-5501.) the brief principle is as follows:
1) The first base at the 3' end of the template is a fixed A base and is modified by phosphorylation;
2) The repeated sequences at two ends of the template are complementarily paired with the miRNA of the target object, and the length of the repeated sequences is 15-20 nucleotides;
3) The middle ACTCAGACAA (SEQ ID NO: 1) of the template is a fixed sequence, and the repeated sequences at the two ends are connected, and the sequence is the recognition sequence of Nt.BstNBI nucleic acid shearing enzyme of incision endonuclease (nicking endonuclease) in EXPAR reaction;
4) The resulting amplicon is complementarily paired with the template repeat.
The universal template sequence is (taking 17 nucleotides as an example of the repetitive sequences at both ends of the template): 3'-P-ANNNNNNNNNNNNNNNNNACTCAGACAANNNNNNNNNNNNNNNNN-5' (wherein P stands for phosphorylation).
2. The detection probe design follows the principle: the detection probe may be a hairpin type or linear single-stranded DNA sequence, and hairpin type probes are preferably used. The 5 'end of the probe stem is modified with a signal molecule, such as FAM, HEX, ROX, cy5, and the 3' end of the probe stem is modified with a quenching group, such as BHQ1, BHQ2, etc. The probe loop sequence is designed to specifically complement and pair with the target amplicon, and when the probe loop is 100% complementary and paired with the target amplicon, the Ago shearing probe is activated and the fluorescent signal is released.
Four miRNAs are selected as targets for detection, namely miRNA-21, miRNA-92a, miRNA-31 and miRNA-141, and the corresponding EXPAR amplification templates and corresponding detection probes are designed (shown in table 1).
Table 1: miRNA sequence, EXPAR amplification template, and miRNA detection probe.
Wherein P in the sequence shown in Table 1 represents phosphorylation.
Ago-based one-pot detection system in second embodiment and microRNA detection method
The embodiment mainly establishes an Ago-based one-pot miRNA detection system in the microRNA detection method, namely a mixed reaction system of an exponential amplification reaction system and a microRNA detection system. The total volume of the system can be 10 or 20 mu L, and specifically comprises nicking enzyme Nt.BstNBI, vent (exo-) DNA polymerase, amplification template, dNTP, RNase inhibitor, 1×
Reaction buffer (20 mM Tris-HCl,10mM (NH) 4 ) 2 SO 4 ,10mM KCl,2mM MgSO 4 and 0.1％
X-100)，0.5×NEBuffer TM r3.1(50mM NaCl,25mM Tris-HCl,5mM MgCl 2 50. Mu.g/ml Recombinant Albumin), cpAgo nuclease, mnCl 2 And a detection probe.
The concentration of each component in the system is as follows: each detection probe (0.1. Mu.M), each amplification template (0.1. Mu.M), dNTPs (250. Mu.M), nicking enzyme Nt.BstNBI (0.4U. Mu.L) -1 ) Vent (exo-) DNA polymerase (0.05 U.mu.L) -1 ) RNase inhibitor (0.8U. Mu.L) -1 )。MnCl 2 750 μm; cpAgo nuclease 100nM.
The reaction conditions are divided into two stages, the first stage of the pre-amplification reaction, at a temperature in the range 50-60℃and preferably 55 ℃. The pre-amplification reaction time is 10-30min, preferably 20min. The second stage shear produces signal at a reaction temperature in the range of 30-50deg.C, preferably 37deg.C, for a reaction time of 10-30min, preferably 10min. The detection of a single miRNA only needs to add a single corresponding amplification template and probe, and the detection of a plurality of miRNAs adds a plurality of corresponding amplification templates and a plurality of target specific probes. The reaction is carried out in a PCR instrument, and a real-time fluorescence signal is acquired.
The reagents and nucleic acid sequence sources used are shown in Table 2 below.
Table 2: information on reagents used in the examples
Example 3, ago-based one-pot system detection miRNA sensitivity in microRNA detection method
In the embodiment, the sensitivity of detecting miRNA by using an Ago-based one-pot system in the microRNA detection method is explored. Taking miRNA-141, miRNA-21, miRNA-92a and miRNA-31 as examples, preparing a series of miRNA (0, 10zM, 100zM, 1aM, 10aM, 100aM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM and 10 nM) with concentration as a nucleic acid sample to be tested. The total volume of the one-pot system was 10. Mu.L, and 1. Mu.L of the nucleic acid sample to be tested containing the above configuration was selected, and the nicking enzyme Nt.BstNBI (0.4U. Mu.L) -1 ) Vent (exo-) DNA polymerase (0.05 U.mu.L) -1 ) Amplified template (0.1. Mu.M), dNTP (250. Mu.M), RNase inhibitor (0.8U. Mu.L) -1 )，1×
Reaction buffer (20 mM Tris-HCl,10mM (NH) 4 ) 2 SO 4 ,10mM KCl,2mM MgSO 4 and 0.1％/>
X-100)，0.5×NEBuffer TM r3.1(50mM NaCl,25mM Tris-HCl,5mM MgCl 2 ,50μg/ml Recombinant Albumin)、750μM MnCl 2 CpAgo nuclease 100nM and detection probe (0.1. Mu.M). Sequence specific probes for detecting miRNA-141, miRNA-21, miRNA-92a and miRNA-31 respectively label ROX, FAM, HEX, CY fluorescence.
The reaction conditions are divided into two stages, wherein the pre-amplification reaction temperature in the first stage is 55 ℃ and the pre-amplification reaction time is 20min. The second stage of shearing to generate signal has reaction temperature of 37 deg.c and reaction time of 10min. The reaction is carried out in a PCR instrument, and a real-time fluorescence signal is acquired by a fluorescence channel of a corresponding probe. The difference between the end fluorescent signal and the initial signal is used as the relative intensity value for quantitative analysis. The detection result of miRNA-141 is shown as A in FIG. 3, the fitting linear relation is shown as B in FIG. 3, and the detection sensitivity reaches 1zM. Similarly, the detection results of miRNA-21, miRNA-92a and miRNA-31 are respectively shown as A in FIG. 4, A in FIG. 5 and A in FIG. 6, the fitting linear relationship is respectively shown as B in FIG. 4, B in FIG. 5 and B in FIG. 6, and the detection sensitivity reaches 1zM. The method has high sensitivity and universality.
Example 4 Ago-based one-pot method system detection specificity investigation in microRNA detection method
The embodiment explores the specificity of one-pot system detection based on Ago in the microRNA detection method. miRNA-141 is used as a detection target, and other miRNAs are used as interferent controls, including miRNA-21, miRNA-92a, miRNA-31, miRNA-125b, miRNA-30a, miRNA-375, miRNA-100, let 7a, let 7b, let 7c, let 7i and water (blank control). All miRNA concentrations were 1pM.
The total volume of the one-pot system was selected to be 10. Mu.L, containing the nicking enzyme Nt.BstNBI (0.4U. Mu.L) -1 ) Vent (exo-) DNA polymerase (0.05 U.mu.L) -1 ) Amplified template (0.1. Mu.M), dNTP (250. Mu.M), RNase inhibitor (0.8U. Mu.L) -1 )，1×
Reaction buffer (20 mM Tris-HCl,10mM (NH) 4 ) 2 SO 4 ,10mM KCl,2mM MgSO 4 and 0.1％/>
X-100)，0.5×NEBuffer TM r3.1(50mM NaCl,25mM Tris-HCl,5mM MgCl 2 ,50μg/ml Recombinant Albumin)、750μM MnCl 2 CpAgo nuclease 100nM and detection probe (0.1. Mu.M) for detection of miRNA-141. The concentration of miRNA as a sample of nucleic acid to be measured in each reaction system was 1pM.
The reaction is carried out in two stages, wherein the temperature of the first stage of pre-amplification reaction is 55 ℃, and the time of the pre-amplification reaction is 20min. The second stage of shearing produces signal at 37 deg.c for 30min. The reaction is carried out in a PCR instrument, and a real-time fluorescence signal is acquired. The difference between the fluorescence signal and the initial signal at 30min of reaction was used as the relative intensity value for investigation of the specificity analysis. The specific real-time fluorescence results for miRNA-141 are shown as a in fig. 7, when the target miRNA-141 is present in the system, the fluorescence signal starts to be significantly enhanced in the second phase and reaches a maximum value rapidly into the plateau phase. And when other miRNAs are added into the system, extremely weak fluorescent signals are generated in the second stage. The relative fluorescence intensity value is further obtained, as shown in B in FIG. 7, the target miRNA-141 causes strong signal enhancement, and the signal change of other miRNAs is negligible, which indicates that the detection system of the Ago nuclease in the microRNA detection method provided by the invention has extremely high specificity, plays a significant role in practical detection application, and can be used for accurate detection of miRNA.
Example 5 Ago-based one-pot system detection of multiple miRNAs in microRNA detection methods
The embodiment explores the capability of the Ago system to detect multiple miRNAs in the microRNA detection method. Taking four targets of miRNA-141, miRNA-21, miRNA-92a and miRNA-31 as examples, the concentration of all miRNAs (serving as nucleic acid samples to be detected) is 1pM. The total volume of the one-pot system was selected to be 10. Mu.L, containing the nicking enzyme Nt.BstNBI (0.4U. Mu.L) -1 ) Vent (exo-) DNA polymerase (0.05 U.mu.L) -1 ) Four amplification templates (0.1. Mu.M each), dNTPs (250. Mu.M), RNase inhibitor (0.8U. Mu.L) -1 )，1×
Reaction buffer (20 mM Tris-HCl,10mM (NH) 4 ) 2 SO 4 ,10mM KCl,2mM MgSO 4 and 0.1％/>
X-100)，0.5×NEBuffer TM r3.1(50mM NaCl,25mM Tris-HCl,5mM MgCl 2 ,50μg/ml Recombinant Albumin)、750μM MnCl 2 100nM CpAgo nuclease and four probes (0.1. Mu.M each). The miRNA concentrations used as the nucleic acid samples to be tested were 1pM.
The reaction is carried out in two stages, wherein the temperature of the first stage of pre-amplification reaction is 55 ℃ and the time of the pre-amplification reaction is 20min. The second stage of shearing to generate signal has reaction temperature of 37 deg.c and reaction time of 10min. The reaction is carried out in a PCR instrument, and a real-time fluorescence signal is acquired. The fluorescence intensity values before and after the reaction are read by a PCR instrument for qualitative and quantitative detection. Wherein FAM signal represents miRNA-21, HEX signal represents miRNA-92a, CY3 signal represents miRNA-31, and ROX signal represents miRNA-141. The result of the Ago one-pot method for detecting miRNA is shown in figure 8, the system can detect one miRNA in four targets through different fluorescent channels, simultaneously detect two, three or four targets, has no obvious interference, only needs 30 minutes for the whole reaction time, has simple and convenient reaction operation, and has great application prospect in the aspects of monitoring of clinical diagnosis, prognosis, drug resistance of patients and the like.
To sum up, the advantages and features of the method of the invention are verified by the examples as follows:
1) Ago disorder array dependence, programmable targeting of arbitrary nucleic acid, strong universality;
2) Ago has single base specificity recognition capability, extremely strong detection specificity, avoids false positive signals, and is particularly advantageous for miRNAs with high similarity;
3) Ago is a multi-turn nucleic acid shearing enzyme, which has high shearing efficiency and quick reaction, and greatly shortens the reaction time;
4) The method integrates EXPAR amplification and Ago shearing, simplifies the whole reaction flow to one-pot one-step detection, avoids aerosol pollution, is simple and convenient, and is beneficial to practical application and popularization;
5) The method further realizes the efficient, rapid and accurate detection of the multiple miRNAs by the one-pot method, has great value in clinical diagnosis, provides a powerful tool for the analysis of miRNA expression profiles, and can serve various researches related to miRNAs.
Claims (11)
1. A microRNA detection method, comprising: mixing a nucleic acid sample to be detected, an exponential amplification reaction system and a microRNA detection system to form a mixed reaction system, and reacting to obtain a fluorescent signal in a reactant so as to realize detection of microRNA; wherein,
The microRNA detection system comprises a guide DNA, argonaute nuclease and a detection probe, and an amplification product obtained by amplifying a target microRNA by the exponential amplification reaction system is used as the guide DNA.
2. The microRNA detection method of claim 1, wherein the Argonaute nuclease is selected from one or more of a CpAgo nuclease from clostridium perfringens (Clostridium perfringens), an IbAgo nuclease from enterobacter butler (Intestinibacter bartlettii), a NgAgo nuclease from salinomyces griseus (Natronobacterium gregoryi);
preferably, the Argonaute nuclease is a CpAgo nuclease from clostridium perfringens (Clostridium perfringens).
3. The microRNA detection method of claim 1 or 2, wherein the Argonaute nuclease is contained in the mixed reaction system in an amount of 0.01-200nM.
4. The microRNA detection method as in any of claims 1-3, wherein the exponential amplification reaction system comprises an amplification template, a DNA polymerase, a nicking enzyme, dntps, an RNase inhibitor and an enzyme buffer.
5. The microRNA detection method as in claim 4, wherein the DNA polymerase is Vent (exo-) DNA polymerase or Bst DNA polymerase; and/or the number of the groups of groups,
The nicking enzyme is restriction endonuclease or nicking endonuclease;
preferably, the nicking enzyme is a restriction endonuclease;
more preferably, the restriction endonuclease is nt.bstnbi.
6. The microRNA detection method of claim 4 or 5, wherein the enzyme buffer comprises
Reaction buffer and NEBuffer TM r 3.1.
7. The microRNA detection method of any one of claims 1 to 6, wherein the detection probe has a fluorescent group and a quenching group, and the detection probe comprises a region complementary to an amplification product obtained by amplifying the target microRNA using an exponential amplification reaction system;
preferably, in the detection probe, the fluorescent group and the quenching group are each independently located at the 5 'end and the 3' end of the detection probe.
8. The microRNA detection method of any of claims 1-7, wherein the nucleic acid sample to be detected comprises nucleic acid from a biological sample;
optionally, the biological sample is 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.
9. The microRNA detection method of any of claims 1-8, wherein a mixed reaction system is formed, and the reaction conditions for performing the reaction are: reacting at 50-60deg.C for 10-30min, and then reacting at 30-50deg.C for 10-30min.
10. The microRNA detection method of any of claims 1-9, wherein the step of pre-treating the biological sample comprising extracting total micrornas in the biological sample is further included before performing the microRNA detection method.
11. The one-pot method microRNA detection system comprises a nucleic acid sample to be detected, an exponential amplification reaction system and a microRNA detection system;
the microRNA detection system comprises a guide DNA, argonaute nuclease and a detection probe, and an amplification product obtained by amplifying a target object microRNA by the exponential amplification reaction system is used as the guide DNA;
the Argonaute nuclease is selected from one or more of CpAgo nuclease from clostridium perfringens (Clostridium perfringens), ibAgo nuclease from Enterobacter butler (Intestinibacter bartlettii) and NgAgo nuclease from saline-alkali bacillus griseus (Natronobacterium gregoryi);
optionally, the exponential amplification reaction system comprises an amplification template, a DNA polymerase, a nicking enzyme, dntps, an RNase inhibitor, and an enzyme buffer.
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