A nucleic acid complex for detecting a single nucleotide variation and a method for detecting the same

Abstract

The present application relates to the technical field of bioinformatics, and particularly relates to a nucleic acid complex for detecting single nucleotide variation and a detection method.The nucleic acid complex comprises: a single-stranded nucleic acid, on which n detection units are connected in series; each detection unit comprises a respective standing sequence and a binding sequence; the detection site of the detection unit is located at the adjacent position of the standing sequence in the direction of the binding sequence, and the length of the standing sequence is 5-7 bp; n-1 FP-DNAs, each of which is combined with part or all of the binding sequence of a detection unit and part or all of the standing sequence of the adjacent detection unit; and a detectable signal part combined with part or all of the binding sequence of the terminal detection unit of the single-stranded nucleic acid.The present application provides a nucleic acid complex detection system for detecting single nucleotide variation and a detection method, which can realize the simultaneous detection of multiple SNVs sites, has high accuracy and sensitivity, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of bioinformatics technology, and in particular to a nucleic acid complex for detecting single nucleotide variants and a detection method thereof. Background Technology

[0002] Single nucleotide variants (SNVs) refer to substitutions, insertions, or deletions of a single base in the genome. They are widely present in human genes and are closely related to various genetic diseases, tumor susceptibility, and drug metabolism. Therefore, accurate and rapid detection of SNVs is of great significance for disease diagnosis, precision medicine, and personalized treatment.

[0003] Existing SNV detection technologies mainly include PCR and its extension methods, DNA sequencing, CRISPR / Cas system detection, and DNA microarrays. PCR methods, such as qPCR or ARMS-PCR, achieve amplification and detection through specific primers, are simple to operate, and have high sensitivity. However, they are prone to non-specific amplification in multi-site joint detection and rely on thermal cycling equipment, making it difficult to meet the needs of rapid, isothermal detection. DNA sequencing technologies, including high-throughput sequencing (NGS) and Sanger sequencing, can provide comprehensive sequence information and are suitable for in-depth analysis, but they are costly, complex to operate, and have long detection cycles, making them unsuitable for rapid on-site diagnosis. CRISPR / Cas systems utilize the sequence recognition characteristics of Cas proteins combined with signal amplification strategies to achieve SNV detection, possessing certain specificity and sensitivity. However, the system design is complex, with strict requirements for target sequence design, and multi-site joint detection still faces technical challenges. DNA microarrays achieve parallel detection of multiple sites through oligonucleotide probes, suitable for large-scale screening, but the preparation process is complex and costly, the detection sensitivity for low-abundance mutations is insufficient, and rapid, isothermal detection is difficult to achieve. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a method for detecting single nucleotide variants in nucleic acid complexes.

[0005] In a first aspect, the present invention provides a nucleic acid complex, comprising: A single-stranded nucleic acid, wherein n detection units are tandemly connected on the single-stranded nucleic acid, where n is an integer greater than 1; each detection unit includes its own base sequence and binding sequence; The detection site of the detection unit is located at an adjacent position of the footing sequence in the direction of the binding sequence, and the length of the footing sequence is 5~7 bp; n-1 FP-DNAs, each FP-DNA being bound to a partial or complete binding sequence of a detection unit and a partial or complete binding sequence of its adjacent detection units; The detectable signal portion binds to part or all of the binding sequence of the single-stranded nucleic acid terminal detection unit.

[0006] This invention, based on the fundamental principle of strand displacement reactions, ingeniously constructs a logic gate system for the simultaneous detection of multiple target sequences. When the anchoring sequence of the initiating detection unit is identified, strand displacement occurs, FP-DNA is expelled from the single-stranded nucleic acid, and the anchoring sites of adjacent detection units are exposed, providing an anchoring site for the triggering strand of another target sequence. Based on this, multiple detection units can be sequentially connected in series. When the anchoring site of the outermost detection unit is exposed, the triggering strand of the last target fragment binds and triggers a strand displacement reaction, releasing the detectable signal portion and triggering subsequent signal detection reactions. When a preset signal is detected, it indicates that these target sequences are present simultaneously, thereby achieving the detection of multiple target sequences.

[0007] The method for designing the target sequence and binding sequence provided by this invention can ensure the accuracy of detection, guaranteeing both the binding efficiency of the target sequence trigger strand and the normal occurrence of the strand displacement reaction (successfully extruding FR-DNA). This method can effectively ensure the specificity and sensitivity of the detection results, while suppressing false positives caused by single-point mismatches.

[0008] When the base sequence is 5~7bp, it has better chain substitution reaction efficiency and reaction stability, while when it is preferably 6bp, it performs best in terms of chain hybridization efficiency, reaction stability and system balance.

[0009] Furthermore, the FP-DNA can also bind to a portion of the base sequence of the corresponding detection unit, but not all of it, provided that the base can still stably undergo a strand displacement reaction.

[0010] Furthermore, the detectable signal portion is: a signal nucleic acid chain, or a nucleic acid chain-signal generating molecule; Preferably, the recognition sequence of the signal nucleic acid chain includes the following nucleotide sequence: GACGTGACT (nucleotide sequence as shown in SEQ ID NO.15).

[0011] The detectable signal portion in the method provided by this invention can employ various signal molecules in the art, such as a CHA amplification reaction triggered by a signal probe. Alternatively, it can be in the form of a nucleic acid chain-signal generating molecule, where the signal generating molecule can be a fluorescent group-quencher luminescence system (based on the FRET effect), an enzyme-catalyst substrate luminescence system (e.g., an HRP enzyme + TMB substrate colorimetric system), etc. Preferably, the CHA amplification reaction is triggered by a signal probe, using the aforementioned signal nucleic acid chain as the signal probe.

[0012] Furthermore, n can be 2, 3, or 4.

[0013] The present invention preferably uses 2-4 detection units. With this number of detection units, the length of the single-stranded nucleic acid will not be too long, and a high strand substitution reaction efficiency can be maintained.

[0014] As a preferred embodiment, if Alzheimer's disease RS7412 and RS429358 are detected simultaneously, a nucleic acid complex as shown in SEQ ID NO.1-SEQ ID NO.3 is provided, with SEQ ID NO.1 as the single-stranded nucleic acid, wherein TTTTTTTTTTT is used for ligation with the solid-phase carrier, Toholds1 is CAGGCA, and Toholds2 is ACTTCA. SEQ ID NO.2 is used as FP-DNA, and SEQ ID NO.3 is used as the detectable signal portion (for CHA amplification reaction).

[0015] As a preferred embodiment, if both RS94704480 and RS1564282 of Parkinson's syndrome are detected simultaneously, a nucleic acid complex as described in SEQ ID NO. 8-10 is provided, with SEQ ID NO. 8 as the single-stranded nucleic acid, Toholds 1 as TTAGTC, and Toholds 2 as TCTCTA. SEQ ID NO. 9 is the FP-DNA, and SEQ ID NO. 10 is the detectable signal portion (for CHA amplification reaction).

[0016] As a preferred embodiment, the present invention provides a method for preparing the aforementioned nucleic acid complex, wherein the reaction system comprises: The single-stranded nucleic acid, the FP-DNA, the detectable signal portion, SMB, PBST, and TBE.

[0017] The reaction procedure includes: 92~98℃, 3~10min; The temperature drops to 50-60℃ at a rate of 0.5-1.5℃ / min. 50~60℃, 3~10min; The temperature drops to 20-30℃ at a rate of 0.5-1.5℃ / min. 20~30℃, 3~10min; The temperature drops to 3~8℃ at a rate of 0.5~1.5℃ / min.

[0018] In a second aspect, the present invention provides a nucleic acid detection composition comprising: a solid-phase carrier, and a nucleic acid complex as described above immobilized on the solid-phase carrier.

[0019] Furthermore, the solid support is selected from magnetic beads, polystyrene microspheres, nanoparticles, test strips, planar supports, container supports, or electrodes; Preferably, the nucleic acid complex is immobilized on the solid support using a biotin-streptavidin system.

[0020] This invention further employs a solid-phase carrier enrichment logic gate structure, which can also provide anchor points in subsequent centrifugation separation, reducing the probability of false positives and non-specific reactions during the screening process.

[0021] The present invention preferably uses magnetic beads, and the logic gate system is connected by magnetic beads-biotin-streptavidin-flexible linkers.

[0022] Furthermore, it also includes: The signal amplification system corresponding to the detectable signal portion; Preferably, the signal amplification system includes one or more of the following: a catalytic hairpin assembly reaction system, a hybridization chain reaction system, a rolling circle amplification system, a CRISPR-Cas signal amplification system, a chain displacement amplification system based on biological enzymes, an exponential amplification reaction system, or a deoxyribozyme catalytic amplification system; More preferably, the signal amplification system includes a hairpin structure sequence for the CHA reaction; More preferably, the hairpin structure sequence comprises nucleotide sequences as shown in SEQ ID NO.15 and SEQ ID NO.16.

[0023] This invention further employs a CHA cyclic amplification system for signal amplification. While the basic reaction principle of the CHA system used in this study still belongs to the traditional catalytic hairpin assembly (CHA) mechanism, optimizations were made to the hairpin structure stability, chain substitution kinetics, and background suppression capability to meet the requirements of single nucleotide variant detection. The optimizations mainly included parameters such as the stem-loop lengths of the H1 and H2 hairpins, the Toehold length of the region binding to the SP chain, the Toehold binding length between H1 and H2, and the length of the self-locking double arms. Furthermore, NUPACK was used to simulate and analyze the thermodynamic stability and chain substitution efficiency under different structural conditions. Through these optimizations, this invention reduces system background leakage while improving the reaction efficiency after target chain triggering, thereby enhancing the specificity, stability, and versatility of the system.

[0024] The CHA signal amplification system is not a conventional amplification unit independent of the logic gate module, but rather forms a kinetic coupling and structural synergy with the preceding DNA logic gate. The logic gate output strand also serves as the specific initiation strand for the CHA reaction, and its release concentration determines the initiation efficiency of the CHA cycle. Compared to conventional signal amplification strategies such as HCR, RCA, or PCR, the CHA system used in this invention has higher compatibility with DNA logic gates in terms of strand substitution mechanism, isothermal reaction conditions, and dynamic hairpin structure. It can improve the sensitivity and specificity of single nucleotide variant detection while maintaining low background leakage, and can form a more stable synergistic amplification effect.

[0025] Thirdly, the present invention provides a kit comprising the aforementioned nucleic acid complex or the aforementioned nucleic acid detection composition.

[0026] Fourthly, the present invention provides the application of the aforementioned nucleic acid complex, or the aforementioned nucleic acid detection composition, or the aforementioned kit in the detection of target sequences; Preferably, the target sequence includes any one of the following: single nucleotide variant nucleotide sequences, insertion or deletion mutant nucleic acid sequences, gene fusion characteristic nucleic acid sequences, pathogen characteristic nucleic acid sequences, or microRNAs; More preferably, the target sequence is a plurality of single nucleotide variant nucleotide sequences.

[0027] The method provided by this invention is applicable to a variety of target sequences, as long as they can bind to the site and trigger subsequent chain displacement reactions.

[0028] Fifthly, the present invention provides a method for detecting single nucleotide variant nucleotide sequences, comprising: The aforementioned nucleic acid complex, or the aforementioned nucleic acid detection composition, or the aforementioned kit is used to detect the sample to be tested; The polymorphism of the single nucleotide variant is determined based on the signal detection results of the detectable signal portion.

[0029] Furthermore, the detection conditions include: Under conditions of 25~37℃, the test was conducted for 30-40 minutes.

[0030] The present invention has the following beneficial effects: This invention provides a nucleic acid complex detection system and corresponding detection method for detecting single nucleotide variants. It adopts a modular design to connect multiple detection units in series and constructs logic gates based on the basic principle of strand substitution reaction, which can simultaneously detect multiple SNVs.

[0031] The method provided by this invention can be flexibly extended to different SNVs or to add new detection functions. It is easy to operate and has advantages such as high specificity, high sensitivity, and universal detection of multiple sites, and has important application value. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 This is a schematic diagram of the logic gate construction provided in an embodiment of the present invention.

[0034] Figure 2 This is a reaction flow diagram provided by the present invention.

[0035] Figure 3 The results are from room temperature logic gate PAGE electrophoresis provided in this embodiment of the invention.

[0036] Figure 4 This is the result of PAGE electrophoresis of logic gates after freezing, provided in an embodiment of the present invention.

[0037] Figure 5 The results are fluorescence recordings from a room-temperature logic gate qPCR instrument provided in this embodiment of the invention.

[0038] Figure 6 The results are fluorescence recordings from a frozen logic gate qPCR instrument provided in this embodiment of the invention.

[0039] Figure 7 These are the experimental amplification electrophoresis results of room-temperature AD logic gates and PD logic gates provided in the embodiments of the present invention; where A is the AD logic gate and B is the PD logic gate.

[0040] Figure 8 These are the experimental amplification electrophoresis results of AD and PD logic gates after being kept at room temperature and frozen, as provided in the embodiments of the present invention; where A is the AD logic gate and B is the PD logic gate. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0042] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0043] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0044] Example 1 This embodiment provides a method for detecting SNVs, including the following process: 1. Nucleic acid sequence.

[0045] Design and synthesize functional nucleic acid sequences as needed, including: Table 1. Nucleic acid sequences suitable for logic gate construction and experiments in Alzheimer's disease.

[0046] Table 2. Nucleic acid sequences suitable for logic gate construction and experiments in Parkinson's disease.

[0047] Table 3. Sequence design suitable for catalytic hairpin assembly reactions

[0048] The nucleic acids required for the reaction are dissolved in ddH2O / TBE buffer and stored at 5°C for later use. For long-term storage, they can be frozen at -20°C. 5mM KCl, 12.5mM MgCl2 and other ionic components can be selectively added as needed to maintain stability.

[0049] 2. Experimental materials.

[0050] In the experiment, the present invention uses the aforementioned MT (mutant gene) and WT (wild-type gene) fragments to define the mutant gene as "1" and the wild-type gene as "0" to simulate the "1 / 0" signal input mode of the electronic logic gate; (1,1) is the double mutant gene experimental group; since there are two judgment points in the CP chain, the (1,0) and (0,1) experimental groups are set according to the order of appearance of the mutant gene in the order of 5'→3'; (0,0) is the double wild-type gene experimental group; A represents the example disease Alzheimer's disease, and P represents the example disease Parkinson's syndrome.

[0051] 3. Experimental procedure.

[0052] 2μL CP chain (10μM), 1μL FP chain (10μM), 1μL SP chain (10μM), SMB 2μL, 1×PBST 98μL, 50μL 1×TBE.

[0053] Magnetic beads and CP chains can be pre-mixed and fixed at room temperature, then washed with PBST before use. Magnetic beads are primarily used to enrich logic gate structures and also provide anchor points for subsequent centrifugation.

[0054] The reaction procedure is as follows: 2 μL CP (10 μM), 1 μL FP (10 μM), 1 μL SP (10 μM), 2 μL SMB, 98 μL 1×PBST, 50 μL 1×TBE; reaction conditions: 95℃, 5 min; 95-55℃, 1℃ / min, 40 min; 55℃, 5 min; 55℃-25℃, 1℃ / min; 25℃, 5 min; 25-5℃, 1℃ / min, 20 min; can be stored for a long time at 5℃.

[0055] The reaction is carried out in a PCR instrument.

[0056] The results are as follows Figure 1 The logic gate detection system shown has the following detection principle: Figure 2 As shown.

[0057] The logic gate system after the reaction was subjected to PAGE electrophoresis, and the results are as follows: Figure 3 and Figure 4 As shown in the results above, this invention successfully constructed a logic gate detection system based on chain substitution reaction.

[0058] (2) Detection method.

[0059] The experimental materials were divided into four groups, with the mutant gene sequence as "1" and the wild-type gene sequence as "0". Wild-type (1 μL, 1 μM) and / or mutant gene (1 μL, 1 μM) were added to the solution for constructing complete logic gates in the manner of (0,0)(0,1)(1,0)(1,1).

[0060] Add the logic gate detection system (10 μL, 1 μM) obtained in step (1) and perform the screening reaction (PCR instrument). The reaction conditions are: 37℃, 30 min.

[0061] The CHA reaction system was added, consisting of 10 μL H1 (1 μM), 10 μL H2 (1 μM), and 1 μL LSP. The reaction conditions were: 37 °C for 60 min.

[0062] Characterization was performed using real-time fluorescence detection results.

[0063] The logic gate detection system was then replaced with 200nM and 500nM gates for repeated experiments.

[0064] (3) The present invention further freezes the logic gate detection system at -20°C for 14 days. After freezing, it is thawed at room temperature and the previous detection process is repeated.

[0065] (4) Test results.

[0066] The results are as follows Figure 5 and Figure 6 As shown, from Figure 5 and Figure 6 As can be seen, in group (1,1), the fluorescence signal typically rises rapidly within cycles 12-15, significantly earlier than in other input combinations. The system is only fully activated and enters the efficient amplification phase when both SNV mutation sites are present simultaneously; the deletion of either site fails to trigger sufficient SP release, thus maintaining a low background signal. Furthermore, superior detection results were achieved under different concentration conditions (minimum 200 nM) and after cryopreservation. This demonstrates that the detection system provided by this invention has high detection accuracy and stability.

[0067] Figure 7 and Figure 8 The results show that the CHA reaction is as expected. The system exhibits significant and rapid fluorescence enhancement only when the target SNVs are perfectly matched. Conversely, the signal is insignificant or increases slowly under single-site mismatch or complete non-match conditions. This verifies the high specificity, high sensitivity, and versatility of this system in the detection of multi-site SNVs. Furthermore, it maintains excellent detection efficiency even after cryopreservation, indicating its superior stability.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nucleic acid complex, characterized in that, include: A single-stranded nucleic acid, wherein n detection units are tandemly connected on the single-stranded nucleic acid, where n is an integer greater than 1; each detection unit includes its own base sequence and binding sequence; The detection site of the detection unit is located at an adjacent position of the footing sequence in the direction of the binding sequence, and the length of the footing sequence is 5~7 bp; n-1 FP-DNAs, each FP-DNA being bound to a partial or complete binding sequence of a detection unit and a partial or complete binding sequence of its adjacent detection units; The detectable signal portion binds to part or all of the binding sequence of the single-stranded nucleic acid terminal detection unit.

2. The nucleic acid complex according to claim 1, characterized in that, The detectable signal component is: a signal nucleic acid chain, or a nucleic acid chain-signal generating molecule; Preferably, the recognition sequence of the signal nucleic acid chain includes the following nucleotide sequence: GACGTGACT.

3. The nucleic acid complex according to claim 1 or 2, characterized in that, n is 2, 3, or 4.

4. A nucleic acid detection composition, characterized in that, include: A solid support, and a nucleic acid complex as described in any one of claims 1-3 immobilized on the solid support.

5. The nucleic acid detection composition according to claim 4, characterized in that, The solid support is selected from magnetic beads, polystyrene microspheres, nanoparticles, test strips, planar supports, container supports, or electrodes; Preferably, the nucleic acid complex is immobilized on the solid support using a biotin-streptavidin system.

6. The nucleic acid detection composition according to claim 4 or 5, characterized in that, Also includes: The signal amplification system corresponding to the detectable signal portion; Preferably, the signal amplification system includes one or more of the following: a catalytic hairpin assembly reaction system, a hybridization chain reaction system, a rolling circle amplification system, a CRISPR-Cas signal amplification system, a chain displacement amplification system based on biological enzymes, an exponential amplification reaction system, or a deoxyribozyme catalytic amplification system; More preferably, the signal amplification system includes a hairpin structure sequence for the CHA reaction; More preferably, the hairpin structure sequence comprises the nucleotide sequences shown in SEQ ID NO.15 and SEQ ID NO.

16.

7. A reagent kit, characterized in that, Includes the nucleic acid complex according to claim 1 or 2, or the nucleic acid detection composition according to any one of claims 4-6.

8. The use of the nucleic acid complex according to claim 1 or 2, or the nucleic acid detection composition according to any one of claims 4-6, or the kit according to claim 7 in detecting target sequences; Preferably, the target sequence includes: Any one of the following: single nucleotide variant nucleotide sequence, insertion or deletion mutant nucleic acid sequence, gene fusion characteristic nucleic acid sequence, pathogen characteristic nucleic acid sequence, or microRNA; More preferably, the target sequence is a plurality of single nucleotide variant nucleotide sequences.

9. A method for detecting single nucleotide variant nucleotide sequences, characterized in that, include: The nucleic acid complex of claim 1 or 2, or the nucleic acid detection composition of any one of claims 4-6, or the kit of claim 7, is used to detect the sample to be tested; The polymorphism of the single nucleotide variant is determined based on the signal detection results of the detectable signal portion.

10. The method according to claim 9, characterized in that, The detection conditions include: Under conditions of 25~37℃, the test was conducted for 30-40 minutes.

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