Ago-DNA polymerase synchronous double-cycle amplification system and application thereof

Abstract

This invention discloses an Ago-DNA polymerase simultaneous dual-cycle amplification system and its applications. The Ago-DNA polymerase simultaneous dual-cycle amplification system comprises: Ago enzyme, DNA polymerase, amplification template, dNTPs, MnCl2, and gDNA; the sequences of the amplification template and gDNA are obtained using the design method of this invention. This system can be applied to amplify and / or detect microRNA. This system enables integrated simultaneous dual-cycle amplification, simplifying the operation steps, reducing reaction complexity, avoiding aerosol contamination, reducing costs, and eliminating the requirement for specific sequences by restriction enzymes such as Cas and Nt.BstNBI. The amplification and / or detection methods using this system are highly versatile, accurate, and efficient, breaking through the bottlenecks of current molecular diagnostic technologies and holding promise for widespread application.

Description

Technical Field

[0001] This invention relates to the field of mRNA detection, and in particular to an Ago-DNA polymerase synchronous dual-cycle amplification system and its applications. Background Technology

[0002] Over the past four decades, nucleic acid detection technologies have developed rapidly, including nucleic acid hybridization, polymerase chain reaction (PCR), isothermal amplification, sequencing, and CRISPR-Cas-based molecular detection technologies. These technologies, targeting nucleic acid molecules, offer high accuracy and specificity and are widely used in clinical medical diagnosis, food safety monitoring, environmental monitoring, and quality control in agriculture and animal husbandry. Nucleic acid hybridization is the commonly used gold standard, but its detection sensitivity is relatively poor, and the process involves many steps and is time-consuming. PCR technology is currently the mainstream molecular diagnostic method, offering low cost. However, its limitations include reliance on strict procedures and partitioning facilities, and a limited detection limit for low-concentration samples. For example, the optimal sensitivity of PCR for single-base mutations (SNPs) is around 1%. Isothermal amplification methods such as EXPAR (Exponential Isothermal Amplification), LAMP (loop-mediated isothermal amplification), and RPA (recombinase polymerase amplification) avoid the need for precise temperature control during heating and cooling, offering rapid reactions suitable for on-site, point-of-care testing. However, their high amplification efficiency often leads to non-specific amplification signals, affecting detection accuracy. Sequencing technologies offer high throughput but are expensive, with complex testing procedures and data analysis, and are time-consuming, making them unsuitable for large-scale, grassroots testing needs. In recent years, the CRISPR-Cas (clustered regularly spaced short palindromic repeats / CRISPR-related proteins) system, especially Cas12 and Cas13 proteins, has been widely used to establish molecular diagnostic platforms. The Cas / sgRNA binary complex is activated upon recognizing the target sequence, initiating Cas's trans-cleavage activity, resulting in non-specific cleavage of free single-stranded (ss) DNA or ssRNA probes. The CRISPR-Cas system, due to the highly efficient cleavage activity of the Cas enzyme through multiple cycles, has greatly improved the sensitivity of molecular detection. Furthermore, RNA-mediated targeted recognition and cleavage significantly enhance detection specificity, demonstrating outstanding advantages in the field of nucleic acid detection. Combining the CRISPR-Cas system with amplification methods such as PCR, RPA, and LAMP, the amplicon of the target analyte activates the trans-cleavage activity of the Cas enzyme, cleaving the probe to amplify the signal, further improving the sensitivity and specificity of current nucleic acid detection technologies and promoting the development of molecular diagnostics. Currently, the CRISPR-Cas system faces many challenges due to its fundamental detection principle and the essential characteristics of the Cas enzyme. For example, non-specific cleavage caused by off-target effects of the Cas enzyme fundamentally affects the detection limit of the entire system.The sequence dependence of Cas enzymes, with Cas9 / 12 and Cas13 having PAM and PFS sequence requirements for target genes respectively, limits their universality for any gene and target site. The CRISPR-Cas system detects targets by recognizing and simultaneously initiating trans-cleavage, indirectly and non-specifically cleaving free single-stranded probes to release a signal. This necessitates the use of multiplexed Cas enzymes with different trans-cleavage sequence dependencies for simultaneous detection of multiple targets. These orthogonal reaction Cas enzymes are few in number, making screening difficult, and the multiple enzyme detection systems lead to high complexity and cost. Furthermore, the CRISPR-Cas system relies on RNA as the guide strand, which is approximately 50 to 100 nucleotides in length. RNA has poor stability and high synthesis costs, hindering its widespread practical application.

[0003] Argonaute (Ago) proteins are widely found in organisms, including bacteria, archaea, and almost all plants and animals. Ago is a highly conserved class of proteins, which can be divided into two main categories: eukaryotic Argonaute (eAgo) expressed in eukaryotes and prokaryotic Argonaute (pAgo) expressed in prokaryotes (Genomebiology, 2008, 9(2): 1-8. Nature Reviews Genetics, 2013, 14(7): 447-459.). Currently, eAgo proteins are mainly classified into three categories: Ago-like, PIWI-like, and WAGO. They are important proteins for regulating cell function and are widely involved in physiological processes such as cell growth, development, apoptosis, and carcinogenesis, as well as protecting cells from viral infections (Nature Structural & Molecular Biology, 2014, 21(9): 743-753; Nature, 2006, 442(7099): 199-202.). pAgos are very diverse, with 1711 species discovered as of 2020. Dozens of pAgos derived from bacteria and archaea have been expressed and studied (Nature, 2020, 587(7835): 632-637.). Similar to Cas enzymes, pAgo is a type of programmable endonuclease mediated by nucleic acids. Under the guidance of 5' phosphorylated or 5' hydroxylated ssDNA (guide DNA, gDNA) or ssRNA (guide RNA, gRNA), it can precisely recognize target sequences complementary to its guide strand bases and cleave the target at specific sites, distinguishing differences in individual nucleotides. Increasing research data confirms that pAgo participates in cellular defense, protecting bacteria and archaea from invasion by foreign viral nucleic acids, etc. (Science, 2018, 359(6379): eaar4120; Nature, 2021, 600(7887): 116-120.). pAgo's programmable targeting via gDNA, without being limited to target DNA with specific sequences such as PAM, coupled with the lower cost and greater stability of gDNA synthesis, and pAgo's multi-turnover enzymatic activity and single-base resolution specificity, make it a promising candidate for in vitro detection, molecular imaging, and genome editing.

[0004] Currently, detection methods based on CRISPR-Cas and Ago typically require two steps: pre-amplification followed by signal conversion and amplification using Cas enzymes or Ago cleavage probes. To simplify the process, shorten overall detection time, improve convenience, and avoid aerosol contamination from opening the container, thereby enhancing accuracy, several one-step detection methods based on CRISPR-Cas and Ago have been developed to promote the practical application of this technology. However, current one-step methods based on CRISPR-Cas and Ago suffer from complex components, affecting detection performance, and the multi-enzyme system is expensive. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a template and gDNA design method for use in the Ago-DNA polymerase synchronous dual-cycle amplification system.

[0006] The second objective of this invention is to provide the above-mentioned Ago-DNA polymerase synchronous dual-cycle amplification system.

[0007] A third objective of this invention is to provide the application of the above-described Ago-DNA polymerase synchronous dual-cycle amplification system.

[0008] The objective of this invention is achieved through the following technical solution: A template and gDNA design method, comprising the following steps: S1: Obtain the microRNA sequence; S2: Replace U with T in the microRNA sequence to obtain the amplicon sequence; S3: Replace A with T, C with G, T with A, and G with C in the amplicon sequence to obtain the complementary amplicon sequence; S4: Extract 16-20 consecutive bases from the complementary sequence of the amplicon; and record the sequence of all 16-20 bases. S5: Determine whether the first base of each of the recorded 16-20 base sequences is T; if not, execute S502 to replace the first base with T, and then execute S503 to generate a gDNA sequence after the replacement; if yes, directly execute S503 to generate a gDNA sequence from the 16-20 base sequence; the generated gDNA sequence can be one or more. S6: Summarize S5 to obtain the gDNA sequence; S7: Output all gDNA sequences; The method further includes the following steps: S3: Obtain the complementary sequence of the amplicon; S302: Reverse the order of the amplicon complementary sequences to obtain the amplicon inverse complementary sequences; connect the two amplicon inverse complementary sequences to obtain the template sequence; S303: Output template sequence.

[0009] Furthermore, the method further includes the following steps: S602: Determine whether the twelfth base of each gDNA sequence is G or C, and calculate the GC percentage in each sequence; S603: Outputs the results of each gDNA sequence, the determination of whether the twelfth base of the sequence is G or C, and the GC percentage of the sequence.

[0010] Further, the microRNA sequence described in step S1 is a sequence of 17–25 bp; even further, a sequence of 20–25 bp; and still even further, a sequence of 22–23 bp.

[0011] A computer-readable storage medium storing a computer program that, when executed by a processor, implements the steps of the method described above.

[0012] A computer program product includes a computer program that, when executed by a processor, implements the steps of the method described above.

[0013] A simultaneous dual-cycle amplification system using Ago-DNA polymerase includes: Ago enzyme, DNA polymerase, amplification template, dNTPs, MnCl2, RNase inhibitor, and gDNA; Furthermore, the amplification template and gDNA sequence are designed based on the desired amplification sequence using the above method; even further, when the Ago is TtAgo, the gDNA sequence is preferably selected from gDNA sequences whose twelfth base is G or C, and whose GC percentage is 20-60%.

[0014] Furthermore, the total volume of the Ago-DNA polymerase synchronous dual-cycle amplification system is 10–50 μL.

[0015] Furthermore, the DNA polymerase is Vent (exo-) DNA polymerase.

[0016] Furthermore, the Ago includes at least one of CpAgo, PfAgo, and TtAgo.

[0017] Further, the Ago-DNA polymerase synchronous dual-cycle amplification system comprises: 0–100 nM Ago, 0.05 U / μL DNA polymerase, 100–600 nM amplification template, 250 μM dNTPs, 0–1000 μM MnCl2, 0.5–1 U / μL RNase inhibitor, and 25–500 nM gDNA; even further, the Ago-DNA polymerase synchronous dual-cycle amplification system comprises: 25 nM Ago, 0.05 U / μL DNA polymerase, 200 nM amplification template, 250 μM dNTPs, 500 μM MnCl2, 0.8 U / μL RNase inhibitor, and 100 nM gDNA.

[0018] Furthermore, the Ago-DNA polymerase synchronous dual-cycle amplification system also includes: 1×thermoPol reaction buffer and 0.5×NEBuffer r3.1.

[0019] Furthermore, the 1×thermoPol reaction buffer comprises 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4 and 0.1% Triton® X-100.

[0020] Furthermore, the 0.5×NEBuffer r3.1 comprises 50 mM NaCl, 25 mM Tris-HCl, 5 mM MgCl2, and 50 µg / ml Recombinant Albumin.

[0021] The above-mentioned Ago-DNA polymerase synchronous dual-cycle amplification system is applied in the amplification and / or detection of microRNA.

[0022] The above-mentioned Ago-DNA polymerase synchronous dual-cycle amplification system is used in the preparation of products that amplify microRNA and / or products that detect microRNA.

[0023] A microRNA detection system includes the above-mentioned Ago-DNA polymerase synchronous dual-cycle amplification system and fluorescent reagent.

[0024] Furthermore, the fluorescent reagent is a fluorescent probe or a fluorescent dye; even further, the fluorescent dye includes 1×SYBR Green I.

[0025] The above-mentioned microRNA detection system is used in the detection of microRNA.

[0026] The above-mentioned microRNA detection system is used in the preparation of products for detecting microRNA.

[0027] A method for amplifying and / or detecting microRNA includes the following steps: (1) Add the sample to be amplified and / or detected to the above-mentioned Ago-DNA polymerase synchronous double-cycle amplification system; (2) Annealing: react at 50-60℃ for 10-60s; denaturation: react at 75-95℃ for 10-60s. (3) Repeat step (2) 60 to 90 times.

[0028] Further, step (2) is annealing: react at 55℃ for 30s, and denaturation: react at 80℃ for 30s.

[0029] Further, step (3) is to repeat step (2) 80 times.

[0030] A method for detecting microRNA includes the following steps: (1) Add the sample to be tested to the above microRNA detection system; (2) Annealing: react at 50-60℃ for 10-60s; denaturation: react at 75-95℃ for 10-60s. (3) Repeat step (2) 60 to 90 times.

[0031] Further, step (2) is annealing: react at 51℃ for 30s, and denaturation: react at 80℃ for 30s.

[0032] Further, step (3) is to repeat step (2) 80 times.

[0033] The present invention has the following advantages and effects compared with the prior art: This invention discloses a template and gDNA design method, and the template and gDNA obtained by this method are suitable for the Ago-DNA polymerase synchronous dual-cycle amplification system of this invention.

[0034] The Ago-DNA polymerase simultaneous dual-cycle amplification system of this invention involves the target miRNA binding to a designed amplification template, which extends under the action of Vent (exo-) DNA polymerase. The Ago enzyme cleaves the extended strand under the mediation of gDNA, thereby generating an amplicon. The amplicon then binds to the template strand again for amplification cycle (single cycle). Alternatively, the amplified strand can also undergo multiple amplification reactions due to the design of the repetitive sequence template (dual cycle). Further molecular beacons or fluorescent dyes can be designed to convert the amplification signal of the target into a fluorescent signal.

[0035] Unlike existing EXPAR methods, this system utilizes Ago to target and cleave the amplification strand under gDNA-mediated restriction, generating amplicons without relying on the site-specific restriction enzyme Nt. BstNBI to recognize and cleave 5'-GAGTCNNNN. Furthermore, the generated amplicons can both bind to the template to initiate new amplification and undergo multiple amplification. This method leverages the high specificity of Ago's single-base differentiation to achieve precise recognition and cleavage, solving the false-positive problem of EXPAR. Simultaneously, this system requires only DNA polymerase and Ago, eliminating the need for additional restriction endonucleases, thus reducing system complexity and cost.

[0036] This reaction achieves simultaneous amplification and cleavage in a single reaction. By designing repetitive sequence templates, a signal amplification strategy for simultaneous dual-cycle amplification is proposed, effectively improving detection sensitivity. Based on the precise recognition capability and programmability of Ago, this method possesses the advantages of high specificity in distinguishing single bases and arbitrary targeting. Furthermore, compared to existing Ago-based detection technologies, this system achieves integrated simultaneous dual-cycle amplification, simplifying operation steps, reducing reaction complexity, avoiding aerosol contamination, and reducing costs. The programmable targeting capability of Ago eliminates the requirement for specific sequences by endonucleases such as Cas and Nt. BstNBI, making this method highly versatile. Therefore, this amplification method is highly versatile, precise, and efficient, breaking through the bottlenecks of current molecular diagnostic technologies and holding promise for widespread application. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the template and gDNA design method used in the Ago-DNA polymerase simultaneous dual-cycle amplification system provided in the examples. Figure 2 This is a schematic diagram illustrating the principle of the synchronous dual-cycle amplification system based on Ago-DNA polymerase in this invention.

[0038] Figure 3 These are fluorescence amplification curves under different systems and the corresponding 15% polyacrylamide gel electrophoresis results of the amplification products.

[0039] Figure 4 This is a graph showing the sequencing results of microRNAs amplified using a simultaneous dual-cycle amplification system with Ago-DNA polymerase.

[0040] Figure 5The graph shows the analysis results of detecting different concentrations of microRNA-21 using an Ago-DNA polymerase simultaneous dual-cycle amplification system. A represents the polyacrylamide gel electrophoresis results of the amplified products, with target concentrations in lanes 1-5 being 50.0 nM, 25.0 nM, 12.5 nM, 10.0 nM, and 0 (Blank), respectively. B is the real-time fluorescence amplification curve, with target concentrations corresponding to the curves from left to right being 50.0 nM, 25.0 nM, 12.5 nM, 10.0 nM, and 0 (Blank), respectively. C is the corresponding standard curve.

[0041] Figure 6 The graphs show the analytical results of detecting different concentrations of microRNA-31, microRNA-106a, and microRNA-141 using an Ago-DNA polymerase synchronous double-cycle amplification system. Figure A shows the fluorescence intensity of microRNA-31, Figure B shows the fluorescence intensity of microRNA-106a, Figure C shows the fluorescence intensity of microRNA-141, Figure E shows the relative fluorescence intensity of microRNA-31, Figure F shows the relative fluorescence intensity of microRNA-106a, and Figure G shows the relative fluorescence intensity of microRNA-141.

[0042] Figure 7 The figures show the results of repeatability and specific cross-validation experiments of microRNA detected using the Ago-DNA polymerase synchronous dual-cycle amplification system; where A represents the results of the repeatability experiment and B represents the results of the specific cross-validation experiment. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents and raw materials used in the following embodiments are commercially available, and the experimental methods used in the embodiments are conventional methods in the art.

[0044] The reagents used in the experiments in the following examples are as follows: 10×NEBuffer™ r3.1 buffer (New England Biolabs), dNTP (TaKaRa Biotechnology Co. Ltd.), 10×ThermoPol reaction buffer (New England Biolabs), Vent (exo-) DNA polymerase (New England Biolabs), RNase inhibitor (TaKaRa Biotechnology Co. Ltd.), ultrapure water (Shanghai Sangon Biotech), TtAgo (New England Biolabs), MnCl2 (Aladdin), target, amplification template (Shanghai Sangon Biotech), and SYBR GREEN Ⅰ (Beijing Solarbio).

[0045] Figure 1 This is a schematic diagram of a template and gDNA design method used in a synchronous dual-cycle amplification system for Ago-DNA polymerase provided in an embodiment of the present invention. Specifically, the method includes the following steps: S1: Obtain the microRNA sequence to be detected; in some embodiments, the microRNA sequence to be detected is a sequence of 17-25 bp; preferably a sequence of 20-25 bp; more preferably a sequence of 22-23 bp; S2: Replace U with T in the microRNA sequence to obtain the amplicon sequence; S3: Replace A with T, C with G, T with A, and G with C in the amplicon sequence to obtain the complementary amplicon sequence; S4: Extract 16-20 consecutive bases from the complementary sequence of the amplicon; and record the sequence of all 16-20 bases; the recorded sequence of 16-20 bases can be one or more. S5: Determine whether the first base of each of the recorded 16-20 base sequences is T; if not, execute S502 to replace the first base with T, and then execute S503 to generate a gDNA sequence after the replacement; if yes, directly execute S503 to generate a gDNA sequence from the 16-20 base sequence; the generated gDNA sequence can be one or more. S6: Summarize S5 to obtain the gDNA sequence; S7: Output all gDNA sequences; The method further includes the following steps: S3: Obtain the complementary sequence of the amplicon; S302: Reverse the order of the amplicon complementary sequences to obtain the amplicon inverse complementary sequences; connect the two amplicon inverse complementary sequences to obtain the template sequence; S303: Output template sequence.

[0046] In some embodiments, the method further includes the following steps: S602: Determine whether the twelfth base of each gDNA sequence is G or C, and calculate the GC percentage in each sequence; S603: Outputs the results of each gDNA sequence, the determination of whether the twelfth base of the sequence is G or C, and the GC percentage of the sequence.

[0047] This invention also provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the steps of the above-described method.

[0048] This invention also provides a computer program product, including a computer program that, when executed by a processor, implements the steps of the above-described method.

[0049] Taking microRNA-21, microRNA-31, microRNA-141, and microRNA-106a as examples, templates and gDNA sequences were obtained using the above methods; some of the results are shown in Table 1.

[0050] Table 1

[0051] The Ago-DNA polymerase synchronous dual-cycle amplification system includes: Ago enzyme, DNA polymerase, amplification template, dNTPs, MnCl2, RNase inhibitor, and gDNA; the sequences of the gDNA and amplification template are obtained by the above method.

[0052] In some embodiments, the total volume of the Ago-DNA polymerase synchronous dual-cycle amplification system is 10–50 μL.

[0053] In some embodiments, the DNA polymerase is Vent (exo-) DNA polymerase.

[0054] In some embodiments, the Ago includes at least one of CpAgo, PfAgo, and TtAgo.

[0055] In some embodiments, the Ago is TtAgo, and the gDNA sequence is preferably selected from gDNA sequences whose twelfth base is G or C and whose GC percentage is 20-60%.

[0056] In some embodiments, the Ago-DNA polymerase synchronous dual-cycle amplification system comprises: 0–100 nM Ago, 0.05 U / μL DNA polymerase, 100–600 nM amplification template, 250 μM dNTPs, 0–1000 μM MnCl2, 0.5–1 U / μL RNase inhibitor, and 25–500 nM gDNA.

[0057] Preferably, in some embodiments, the Ago-DNA polymerase synchronous dual-cycle amplification system comprises: 25 nM Ago, 0.05 U / μL DNA polymerase, 200 nM amplification template, 250 μM dNTPs, 500 μM MnCl2, 0.8 U / μL RNase inhibitor, and 100 nM gDNA.

[0058] The Ago-DNA polymerase simultaneous dual-cycle amplification system further includes: 1×thermoPol reaction buffer, 0.5×NEBuffer r3.1, and fluorescent reagent. The 1×thermoPol reaction buffer comprises 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM M KCl, 2 mM MgSO4 and 0.1% Triton® X-100.

[0059] The 0.5×NEBuffer r3.1 contains 50 mM NaCl, 25 mM Tris-HCl, 5 mM MgCl2 and 50 µg / ml Recombinant Albumin.

[0060] The fluorescent reagent is a fluorescent probe or a fluorescent dye. Preferably, the fluorescent dye includes 1×SYBRGreen I.

[0061] The method for detecting microRNA using the above-described Ago-DNA polymerase simultaneous dual-cycle amplification system includes the following steps: (1) Add the sample to be tested to the Ago-DNA polymerase synchronous double-cycle amplification system, adding 1 μL of the sample to be tested for every 10 μL of system; (2) Annealing: react at 50-60℃ for 10-60s; denaturation: react at 75-95℃ for 10-60s. (3) Repeat step (2) 60 to 90 times.

[0062] Preferably, in some embodiments, step (2) is annealing: reacting at 51°C for 30 s, and denaturation: reacting at 80°C for 30 s.

[0063] Preferably, in some embodiments, step (3) is to repeat step (2) 80 times.

[0064] The target miRNA binds to the amplification template and extends under the action of Vent (exo-) DNA polymerase. Ago enzyme cleaves the extended strand under the mediation of gDNA, thereby generating an amplicon. The amplicon binds to the template strand again to complete the amplification cycle (single cycle). Alternatively, due to the design of the repetitive sequence template, the amplified strand can also undergo a multiple amplification reaction (double cycle). The schematic diagram of the amplification principle is shown below. Figure 2 As shown, the amplified signal is converted into a fluorescent signal by the addition of a fluorescent reagent, thereby enabling the detection of microRNA.

[0065] Example 1 Taking microRNA-21 as an example; the sequence of microRNA-21 is: 5'-UAGCUUAUCAGACUGAUGUUGA-3'; Using the template and gDNA design methods employed in the aforementioned Ago-DNA polymerase simultaneous dual-cycle amplification system, the following was obtained: microRNA-21 template sequence: 5'-CAACATCAGTCTGATAAACAGACTCATCAACATCAGTCTGATAA-3'; microRNA-21g DNA sequence: 5'-TATCAGACTGATGTTG-3'.

[0066] The above-mentioned microRNA-21 sequence, microRNA-21 template, and microRNA-21 gDNA were synthesized by Shanghai Sangon Biotech.

[0067] Eight experimental groups were set up for subsequent detection reactions in the amplification system. The components of the experimental groups are shown in Table 2. The Ago-DNA polymerase synchronous double-cycle amplification system of Group 1 included: 25 nM TtAgo, 0.05 U / μL Vent(exo-) DNA polymerase, 200 nM microRNA-21 template, 250 μM dNTPs, 500 μM MnCl2, 0.8 U / μL RNase inhibitor, 100 nM gDNA, 1×thermoPol reaction buffer, and 0.5×NEBuffer r3.1 and 1×SYBR Green I.

[0068] The 1×thermoPol reaction buffer comprises 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM M KCl, 2 mM MgSO4 and 0.1% Triton® X-100.

[0069] The 0.5×NEBuffer r3.1 contains 50 mM NaCl, 25 mM Tris-HCl, 5 mM MgCl2 and 50 µg / ml Recombinant Albumin.

[0070] The total volume of the system is 10 μL.

[0071] In Table 2, "+" indicates that the component is the same as in group 1; "-" indicates that the component is missing in the group; group 7 indicates that it contains only the template and 1×SYBR Green I, and group 8 indicates that it contains only gDNA and 1×SYBR Green I.

[0072] The method for detecting microRNA-21 using the above amplification system includes the following steps: (1) Add 1 μL of microRNA-21 to the above amplification system; the final target concentration is 50 nM.

[0073] (2) Annealing: react at 51℃ for 30s; denaturation: react at 80℃ for 30s. (3) Repeat step (2) 80 times.

[0074] Group 2 does not perform step (1).

[0075] Table 2

[0076] The results are as follows Figure 3As shown, when using the amplification system of Group 1 to detect microRNA-21, a strong fluorescent signal was generated as the reaction proceeded. However, no signal was generated in the Group 2 system when the target was absent. Similarly, when any of the above components were missing, the signal was extremely weak or even absent. These results demonstrate the feasibility of the simultaneous dual-cycle amplification of the target proposed in this patent. Furthermore, gel electrophoresis experiments were performed on these reaction products. The miR-21 amplification products were analyzed using 15% polyacrylamide gel electrophoresis (PAGE). First, the miR-21 amplification products were mixed with loading buffer at a 1:1 volume ratio. The mixture was then loaded onto a PAGE gel and electrophoretically separated at 130V for 90 minutes. The size of the amplified products was confirmed using DNA molecular weight standards of 20-100 bp. Next, the PAGE gel was stained for 10 minutes and analyzed using a Bio-Red imaging system. The gel bands containing the target amplification products were excised and purified using the UNIQ-10 Spin Column DNA Gel Extraction Kit. Finally, the purified amplification products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing analysis. The amplification products of this reaction showed ladder-like bands, which not only verified the feasibility of the amplification but also proved the existence of the polymerization reaction, thus confirming the feasibility of the simultaneous dual-cycle amplification design.

[0077] The sequencing results of the microRNA-21 amplification products were also completely consistent with the theoretical repeat amplification sequence (results are as follows). Figure 4 (As shown).

[0078] Example 2 The sequence of microRNA-21: 5'-UAGCUUAUCAGACUGAUGUUGA-3'; microRNA-21 template sequence: 5'-CAACATCAGTCTGATAAACAGACTCATCAACATCAGTCTGATAA-3'; microRNA-21g DNA sequence: 5'-TATCAGACTGATGTTG-3'.

[0079] The above-mentioned microRNA-21 sequence, microRNA-21 template, and microRNA-21 gDNA were synthesized by Shanghai Sangon Biotech.

[0080] The Ago-DNA polymerase simultaneous dual-cycle amplification system includes: 25 nM TtAgo, 0.05 U / μL DNA polymerase, 0.8 U / μL RNase inhibitor, 200 nM microRNA-21 template, 250 μM dNTPs, 500 μM MnCl2, 100 nM gDNA, 1×thermoPol reaction buffer, 0.5×NEBuffer r3.1, and 1×SYBR Green I. The total system volume is 10 μL.

[0081] The method for detecting microRNA-21 using the above amplification system includes the following steps: (1) Add the microRNA-21 sequence to the above amplification system so that the concentrations of microRNA-21 in the system are 50.0 nM, 25.0 nM, 12.5 nM, 10.0 nM and 0, respectively.

[0082] (2) Annealing: react at 51℃ for 30s; denaturation: react at 80℃ for 30s. (3) Repeat step (2) 90 times.

[0083] Test results as follows Figure 5 As shown, within the range of 10 nM to 50 nM, the fluorescence intensity of the target significantly increased with prolonged amplification time, while the negative control did not produce a fluorescence signal. PAGE gel analysis of the microRNA-21 amplification products further confirmed that this method can successfully amplify and detect microRNAs.

[0084] Example 3 To further verify the universality of this method, the template, gDNA, and microRNA in the system were replaced according to the sequence in Table 1 using the same method as in Example 2, and different concentrations of miR-31, miRNA-106a, and miRNA-141 were detected.

[0085] Test results as follows Figure 6 As shown, within the range of 10 nM to 50 nM, the fluorescence intensity of the target increased significantly with increasing amplification time, while the negative control did not produce a fluorescence signal. These results indicate that this method has the potential to become a powerful auxiliary tool for the analysis of microRNAs.

[0086] Example 4 Method stability and specificity study This embodiment investigated the stability and specificity of the Ago-Vent DNA polymerase-mediated simultaneous dual-cycle amplification system. Using four targets—miRNA-141, miRNA-21, miRNA-106a, and miRNA-31—as examples, the methods of Examples 2 and 3 were followed. In step (1) of the detection method, the final concentrations of miRNA-21, miRNA-31, miRNA-141, and miRNA-106a were 25 nM and 10 nM, respectively. The stability test was repeated six times. The results are as follows: Figure 7 As shown in A, this detection method has excellent reproducibility and can provide reliable results in practical applications.

[0087] In clinical diagnosis, highly specific detection methods are crucial for obtaining accurate results because clinical samples contain many potential sources of interference. To test the specificity of this method, cross-experiments were performed using miRNA-21, miRNA-31, miRNA-106a, and miRNA-141. Following the methods in Examples 2 and 3, four detection systems were established using templates for miRNA-21, miRNA-31, miRNA-141, and miRNA-106a individually. The results were tested by adding one of the targets—miRNA-21, miRNA-31, miRNA-141, or miRNA-106a—to systems containing the miRNA-21 template. The final concentration of miRNA added to each reaction system was 25 nM. The samples, templates, and gDNA used in the specificity experiments are shown in Table 3, and the results are as follows: Figure 7 As shown in B, the system exhibits no obvious cross-reactivity, demonstrating high specificity and the ability to effectively distinguish different miRNAs.

[0088] Table 3

[0089] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A template and gDNA design method, characterized in that, Includes the following steps: S1: Obtain the microRNA sequence; S2: Replace U with T in the microRNA sequence to obtain the amplicon sequence; S3: Replace A with T, C with G, T with A, and G with C in the amplicon sequence to obtain the complementary amplicon sequence; S4: Extract 16-20 consecutive bases from the complementary sequence of the amplicon; and record the sequence of all 16-20 bases. S5: Determine whether the first base of each of the 16-20 base sequences is T; if not, execute S502 to replace the first base with T, and then execute S503 to generate a gDNA sequence after the replacement; if yes, directly execute S503 to generate a gDNA sequence from the 16-20 base sequence. S6: Summarize S5 to obtain the gDNA sequence; S7: Output all gDNA sequences; The method further includes the following steps: S3: Obtain the complementary sequence of the amplicon; S302: Reverse the order of the amplicon complementary sequences to obtain the amplicon inverse complementary sequences; connect the two amplicon inverse complementary sequences to obtain the template sequence; S303: Output template sequence.

2. The method according to claim 1, characterized in that, The microRNA sequence described in step S1 is a sequence of 17–25 bp.

3. A computer-readable storage medium, characterized in that, It stores a computer program that, when executed by a processor, implements the steps of the method described above.

4. A computer program product, characterized in that, This includes a computer program that, when executed by a processor, implements the steps of the method described above.

5. An Ago-DNA polymerase synchronized dual-cycle amplification system, characterized in that, The Ago-DNA polymerase synchronous dual-cycle amplification system comprises: Ago enzyme, DNA polymerase, amplification template, dNTP, MnCl2, RNase inhibitor, and gDNA; the sequences of the amplification template and gDNA are designed by the method described in any one of claims 1 to 4.

6. The system according to claim 5, characterized in that, The total volume of the Ago-DNA polymerase synchronous dual-cycle amplification system is 10–50 μL; The DNA polymerase mentioned is Vent (exo-) DNA polymerase; The Ago includes at least one of CpAgo, PfAgo, and TtAgo; The Ago-DNA polymerase synchronous dual-cycle amplification system comprises: 0–100 nM Ago, 0.05 U / μL DNA polymerase, 100–600 nM amplification template, 250 μM dNTPs, and 0–1000 μM MnCl2.

7. The system according to claim 5, characterized in that, The Ago-DNA polymerase synchronous dual-cycle amplification system further includes: 1×thermoPol reaction buffer and 0.5×NEBuffer r3.1; The 1×thermoPol reaction buffer comprises 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4 and 0.1% Triton® X-100; The 0.5×NEBuffer r3.1 contains 50 mM NaCl, 25 mM Tris-HCl, 5 mM MgCl2 and 50 µg / ml Recombinant Albumin.

8. A microRNA detection system, characterized by, It includes the Ago-DNA polymerase synchronous dual-cycle amplification system as described in claims 5 to 7 and a fluorescent reagent; the fluorescent reagent is a fluorescent probe or a fluorescent dye.

9. The application of the Ago-DNA polymerase synchronous dual-cycle amplification system according to claims 5 to 7 or the microRNA detection system according to claim 8 in amplifying and / or detecting microRNA.

10. A method for amplifying and / or detecting microRNA, comprising the following steps: (1) Add the sample to be tested to the Ago-DNA polymerase synchronous dual-cycle amplification system as described in claims 5 to 7; (2) Annealing: react at 50-60℃ for 10-60s; denaturation: react at 75-95℃ for 10-60s. (3) Repeat step (2) 60 to 90 times.

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