Nucleic acid detection method based on DNA nanometer machine, kit and biosensor

Abstract

The invention discloses a nucleic acid detection method based on a DNA nano machine and application of the nucleic acid detection method in a kit or a biosensor. The detection method comprises the following steps: designing a padlock probe and a DNAzyme chain according to a to-be-detected target gene sequence, correspondingly designing a primer, a protection chain and a substrate chain, realizing amplification of the target gene sequence through a NickRCA technology, constructing a DNA nano machine by using DNAzyme, combining an amplification product with the DNA nano machine to change the conformation of DNAzyme, and detecting the target gene sequence under the action of metal ions. The method comprises the following steps: activating enzyme activity to cut a substrate chain to realize directional amplification of a signal, adding a nano marker into a reaction system as a signal reference substance, and detecting by using a sensor to realize qualitative and / or quantitative analysis of a to-be-detected target gene. The system and the method have the advantages of integration, portability, low cost and the like, can be suitable for completing on-site rapid instant detection in an environment with limited conditions, and are of great significance in disease prevention and control.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology detection, and in particular relates to a nucleic acid detection method, a kit and a biosensor based on a DNA nanomachine. Background Art

[0002] Malaria is a vector-borne infectious disease caused by protozoan parasites of the genus Plasmodium. It is transmitted through the bites of infected female Anopheles mosquitoes and is listed as one of the world's three major health threats, along with tuberculosis and AIDS. The WHO's "World Malaria Report 2021" shows that in 2021, there were approximately 247 million new cases of malaria worldwide and approximately 619,000 deaths. In 2021, the WHO announced that China had obtained malaria-free certification, which means that China has eliminated the occurrence of local malaria. However, with the globalization of the economy and the increase in international cooperation, the number of people entering and leaving the country has increased, the number of imported malaria cases has remained high, and deaths have occurred from time to time. The situation of malaria prevention and control is still not optimistic. The clinical diagnosis of malaria infection usually adopts the peripheral blood smear staining microscopy method, which is the gold standard for positive diagnosis of malarial parasite infection. However, the best testing period for this method is when the patient has a high fever, and it requires high experience and professionalism of the test personnel. It is easy to miss the test and produce false negatives in the early infection and when the number of malarial parasites in the blood is small; immunological test methods have disadvantages such as high false positive rate and limited detection window period; the use of polymerase chain reaction to detect malaria requires precise temperature control, and has certain requirements for detection equipment and laboratory hardware. Since malarial infection usually occurs in economically backward environments, limited sanitary conditions and scarce medical resources, the development of high-sensitivity molecular diagnostic tools that are portable, accurate, cost-effective and compatible with mobile power supplies is of great clinical significance for the early detection and standardized treatment of malaria.

[0003] Nucleic acid detection remains an important detection strategy for diagnosing diseases. Ultrasensitive detection of specific DNA or RNA sequences is of great significance for genetic research, diagnosis and personalized medicine. Among them, PCR technology is still the most popular amplification technology for amplifying specific DNA fragments and detecting low-abundance nucleic acids. However, PCR requires precise control of temperature cycles to successfully amplify DNA, and the resulting instrument limitations have hindered its wider application. At the same time, various isothermal amplification technologies are rapidly developing and applying. Among them, the rolling circle amplification (RCA) method proposed in the 1990s is an isothermal enzymatic nucleic acid amplification method with the advantages of simplicity, versatility and robustness. Nicking-enhanced RCA (NickRCA) is a process in which a restriction site is introduced during rolling circle amplification to create a nick on the product chain and serve as the starting site for the polymerase. In this process, multiple polymerases work simultaneously on the same template, using the chain displacement properties of the polymerase to displace the old chain and perform a new round of amplification to obtain a periodically repeated product with a product length that is (n-1) times the template length (n∈Z+, and n>=2).

[0004] Functional nucleic acid is a DNA or RNA molecule isolated from a DNA library using in vitro screening technology. It is a nucleic acid sequence with specific recognition of target substances and catalytic function. Its types include aptamers, deoxyribonucleases (DNAzymes), G-quadruplexes and other DNA assemblies. Among them, DNAzyme is a single-chain DNA enzyme with catalytic activity. Compared with protein enzymes, DNAzyme has the advantages of simple structure, stable activity and low synthesis cost. It has been widely used in biosensors, bioimaging, environmental monitoring, nanomachines and other fields. DNAzyme with RNA cleavage activity has high catalytic efficiency and strong specific recognition ability under the action of metal ions. Its structure includes a highly conserved catalytic core and two sequence-editable binding arms. It specifically recognizes the substrate chain through the two binding arms and catalyzes the cleavage of the ribonucleotide phosphodiester bond in the middle of the substrate chain. DNA nanomachine is a molecular machine that integrates specific functions of functional nucleic acids (aptamers, DNAzymes, etc.), relies on programmed sequence-specific interactions of DNA chains, and performs tasks according to molecular or environmental signals.

[0005] Medical testing technology in the traditional sense refers to the use of centralized clinical laboratories with automated technology to perform large-scale tests at low cost. In decentralized and resource-limited environments, traditional analytical methods are limited. Therefore, point-of-care testing (POCT) has emerged as an alternative form of medical testing and is one of the main directions for the development of medical testing in the future. It is expected to achieve major breakthroughs in accessibility, speed, and accuracy of results, and does not require complex laboratory infrastructure and specialized laboratory personnel. However, POCT also has various requirements such as ease of use, short detection time, high sensitivity, and low cost. In most cases, biosensors made of single functional materials are difficult to meet. This has also promoted the rapid development of current in vitro diagnostics (IVD) technology and nanotechnology. For example, magnetic nanoparticles (MNPs) have both magnetic particle and nanoparticle characteristics and can generate magnetic responses in a magnetic field. MNPs have excellent operability, biocompatibility, signal contrast (common biological samples have no magnetic signal interference, and MNPs can be used as markers or contrast agents to obtain excellent signal-to-noise ratios), penetration, and stability. The magnetic properties of MNPs make them very easy to enrich and separate, and they can be directed to move or rotate under the guidance of an external magnetic field. Super-parallel MNPs can only be magnetized when subjected to an external magnetic field, and the magnetization axis is in the same direction as the magnetic field. In the absence of a magnetic field, no residual magnetization is retained. The physicochemical stability of super-parallel MNPs does not affect biological interactions, making in vitro and in vivo detection and magnetic manipulation possible. Functionalized super-parallel MNPs are widely used in biomedical applications. Summary of the invention

[0006] After in-depth research, the inventors realized the feasibility of isothermal amplification technology combined with DNA nanomachines such as deoxyribozymes (DNAzymes) and nanotechnology in the field of nucleic acid detection. By utilizing the low background noise advantage of nanotechnology such as magnetic nanoparticles (MNPs), DNAzymes are connected to MNPs for assembly. Through isothermal amplification reactions, directional conduction and amplification of detection signals can be achieved. Combined with magnetic biosensors, rapid real-time detection of pathogens can be completed under limited conditions.

[0007] One of the purposes of the present invention is to provide a nucleic acid detection method based on DNA nanomachines, which can be used for on-site rapid and instant detection in an environment with backward economy, limited sanitary conditions and scarce medical resources; the second / third purpose of the present invention is to provide a kit / biosensor using the nucleic acid detection method, which has the advantages of integration and portability; the fourth / fifth / sixth purpose of the present invention is to provide a kit / biosensor for detecting malarial parasites and a method of using the same, which has a wide range of applications and is of great significance for malaria prevention and control.

[0008] The above purpose is achieved through the following technical solutions:

[0009] A nucleic acid detection method based on a DNA nanomachine comprises the following steps:

[0010] S1, screening of target genes to be tested;

[0011] S2, designing a padlock probe and a DNAzyme chain according to the target gene sequence to be tested, and correspondingly designing primers, protection chains and substrate chains; wherein the padlock probe has a phosphorylated 5' end, and is provided with a target gene recognition region, a primer recognition region and an endonuclease recognition region, the target gene recognition region is provided at both ends of the padlock probe, and is complementary to the target gene sequence to be tested, and the endonuclease cleavage site of the endonuclease recognition region is methylated; the protection chain has the same sequence as a partial region of the padlock probe, and has a chemically closed 3' end; the DNAzyme chain is complementary to a substrate chain with a single RNA site, and the sequence of a partial region of the DNAzyme chain is the same as the sequence of the target gene recognition region in the padlock probe; the DNAzyme chain and the substrate chain have biotinylated 3' ends;

[0012] S3, mixing the sample solution with the padlock probe, primers, and DNA ligase to perform a ligase reaction to obtain a ligase reaction product;

[0013] S4, preparing a nanocomplex system solution; the nanocomplex is generated by combining a DNAzyme chain, a substrate chain, and a nano-label;

[0014] S5, taking the ligase reaction product and the nanocomplex system solution, adding a metal ion solution, a DNA polymerase, an endonuclease, T4 polynucleotide kinase and a protective chain, mixing, and performing a nicking-enhanced rolling circle amplification reaction, a DNAzyme endonuclease reaction, and a chain displacement reaction to obtain a final reaction system;

[0015] S6, using a sensor to detect the final reaction system to achieve qualitative analysis and / or quantitative analysis of the target gene to be detected.

[0016] In the above technical scheme, the target gene sequence is amplified by NickRCA, and a nanomachine is constructed using DNAzyme. The amplified product binds to it to change the DNAzyme conformation. Under the action of metal ions, the enzyme activity is activated to cut the substrate chain to achieve directional amplification of the signal. Specifically, the target gene sequence and the two ends of the padlock probe match each other. After the two are hybridized, a ligase reaction is carried out under the action of a ligase to form a circular template; a methylated enzyme cleavage site (AGCT) is introduced into the padlock probe. After the primer binds to the padlock probe, isothermal nucleic acid amplification NickRCA is initiated under the action of a polymerase and an endonuclease to obtain a single-stranded amplification product; in this process, a protective chain is added to assist the endonuclease to fully cut and generate more short-chain products while binding to the 3' end of the amplified product to prevent the single-stranded amplification product from being corrected and chopped by the exonuclease activity of the added DNA polymerase; the ends of the DNAzyme chain and the substrate chain are biotinylated and combined with a nano-marker to form a nano-complex. Since the sequence of some regions of the DNAzyme chain is the same as that of the padlock probe, The amplification product is complementary to the nanocomplex, mediating its conformational change. Under the action of metal ions, the enzyme activity is stimulated, the specific site in the substrate chain is recognized, RNA cutting is performed, and the substrate chain is divided into two parts, generating 2'(3')-cyclic phosphate and 5'-hydroxyl terminal cutting products. The 5'-hydroxyl terminal cutting product carrying the nano marker is separated from the complex and free in the system. The 2'(3')-cyclic phosphate cutting product generates a 3'hydroxyl product under the action of T4 polynucleotide kinase, and then undergoes a chain displacement reaction under the correction and extension of DNA polymerase. The amplification product is released and re-enters the system for recycling. After the reaction is terminated, the sensor is used to detect the required parameter indicators of the marker in the system to achieve qualitative and quantitative analysis of the target gene in the test solution.

[0017] Furthermore, in step S4, the molar ratio of the DNAzyme chain to the substrate chain in the nanocomplex is (1.5-1):1, preferably 1:1.

[0018] Furthermore, in step S4, the nano-marker includes one or more of magnetic nanoparticles, gold nanoparticles, and fluorescent groups.

[0019] Most DNAzymes use metal ions as coenzymes and rely on metal ions to stimulate their activity and produce catalytic reactions.

[0020] Furthermore, in step S4, the DNAzyme is Mg 2+ Dependent DNAzyme, the metal ion solution is Mg 2+ Solution.

[0021] Preferably, the DNAzyme is 8-17 DNAzyme.

[0022] Furthermore, in step S6, the sensor includes one or more of a photomagnetic endpoint phase difference detection system, an electron paramagnetic resonance spectrometer, an ultraviolet-visible spectrophotometer, and a fluorescence microscope.

[0023] Furthermore, the DNA polymerase is phi29 DNA polymerase; the endonuclease is a restriction endonuclease or a nicking endonuclease.

[0024] A kit comprises reaction solution a, reaction solution b and reaction solution c; wherein the reaction solution a comprises a padlock probe, a primer and a DNA ligase; the reaction solution b comprises a nanocomplex formed by combining a DNAzyme chain, a substrate chain and a nano-marker; the reaction solution c comprises T4 polynucleotide kinase, DNA polymerase, an endonuclease, a protective chain and a metal ion solution; the padlock probe and the DNAzyme chain are designed according to the target gene sequence to be detected, and the primer, the protective chain and the substrate chain are designed accordingly; the padlock probe has a phosphorylated 5' end, and is provided with a primer recognition region, an endonuclease recognition region, and target gene recognition regions complementary to the target gene sequence to be detected are provided at both ends, and the endonuclease cleavage site of the endonuclease recognition region is methylated; the protective chain has the same sequence as a partial region of the padlock probe and has a chemically closed 3' end; the DNAzyme chain is complementary paired with a substrate chain with a single RNA site, and the sequence of the partial region of the DNAzyme chain is the same as the sequence of the target gene recognition region in the padlock probe; the DNAzyme chain and the substrate chain have biotinylated 3' ends.

[0025] Furthermore, the above-mentioned kit is used to detect Plasmodium, wherein the sequence of the padlock probe is shown in SEQ ID NO:1, the sequence of the primer is shown in SEQ ID NO:3, the sequence of the protection chain is shown in SEQ ID NO:4, the sequence of the DNAzyme chain is shown in SEQ ID NO:5, and the sequence of the substrate chain is shown in SEQ ID NO:6.

[0026] A biosensor comprises the above-mentioned kit.

[0027] A biosensor for detecting malarial parasites comprises the above-mentioned reagent kit for detecting malarial parasites.

[0028] A method for detecting Plasmodium using the above-mentioned kit or biosensor for detecting Plasmodium comprises the following steps:

[0029] S1, mixing a test solution containing the target gene with reaction solution a, performing a ligase reaction, and obtaining a ligase reaction product;

[0030] S2, taking the ligase reaction product and mixing it with the reaction solution b and the reaction solution c, performing nicking-enhanced rolling circle amplification reaction, DNAzyme endonuclease reaction, and strand displacement reaction to obtain a final reaction system;

[0031] S3, using a sensor to detect the final reaction system to achieve qualitative and / or quantitative analysis of the target gene to be detected;

[0032] Wherein, the sequence of the target gene is shown as SEQ ID NO:2.

[0033] Furthermore, in a specific embodiment of the present invention, the nano-marker is a magnetic nanoparticle (MNP); and the sensor is a photomagnetic endpoint phase difference sensor.

[0034] Its mechanism of action is to use the optical occlusion changes caused by the periodic rotation of MNPs under the action of an external alternating magnetic field to detect the substance to be tested that is bound to the MNPs. The presence of the target molecules causes the MNPs to combine together to form aggregates of different sizes and shapes. The concentration of the target molecules is further determined based on the physical properties of the aggregates and their rotational dynamics in response to an external oscillating magnetic field.

[0035] The main reason for the periodic rotation of MNP is Brownian relaxation. During optical magnetic detection, since the applied magnetic field is a sinusoidal magnetic field, when the magnetic field exists, the MNP or MNP conjugate is magnetized, and the MNP magnetization axis points to the direction of the magnetic field; when the magnetic field disappears, the magnetization disappears, and the MNP or MNP conjugate resumes random orientation. The movement process of MNP from magnetization to magnetization disappearance is called Brownian relaxation, and the time it takes is called Brownian relaxation time, which is proportional to the hydrodynamic volume of MNP. Therefore, the substance bound to MNP is detected, and the presence of target gene molecules causes MNP to aggregate. The quantification of target gene molecules is further achieved through Brownian relaxation affected by the change in the hydrodynamic volume of MNP. In one embodiment of the present invention, a DNAzyme chain modified with MNPs is hybridized with a substrate chain modified with MNPs. Due to the principle of minimum energy, the MNPs spontaneously aggregate to form an assembly, the overall hydrodynamic volume of the MNPs increases, and the Brownian relaxation time increases. When the target gene is present, the ligase reaction and the NickRCA reaction are triggered to amplify the target gene signal, and a nanocomplex (MNP) mediated by the NickRCA product and combined with MNPs is obtained. D&S ), changing the conformation of the complex, in Mg 2+ The RNA cleavage activity of the DNAzyme chain is activated under the assistance, the substrate chain is cut, and the end modified with MNP is free in the system, and the hydrodynamic volume is reduced. In this process, MNP is used as a marker, and the signal change is measured according to the change in the hydrodynamic volume of MNP before and after the reaction for qualitative and quantitative analysis, and the detection limit can reach the fM level.

[0036] Existing laboratory malaria diagnosis is mainly divided into three categories: pathogen detection, immunological detection and molecular biological detection. However, due to the widespread spread of malaria, which often occurs in economically backward and sanitary conditions, existing laboratory detection methods are difficult to meet local display requirements on a large scale. Compared with the existing technology, the nucleic acid detection method and application based on DNA nanomachines proposed in the present invention have the following beneficial effects:

[0037] (1) The detection method provided by the present invention has low sample pretreatment requirements during the detection process (human tissue samples are non-magnetic) and can be used for detection in almost any body fluid and sample, eliminating potential sample loss and speeding up the analysis.

[0038] (2) The kit or biosensor provided by the present invention can be used independently or as an auxiliary detection of RT-PCR technology, which greatly reduces the detection cost during a major epidemic outbreak and alleviates the financial burden of underdeveloped regions.

[0039] (3) The detection method, kit or biosensor provided by the present invention combines the nicking rolling circle amplification technology (NickRCA) with the RNA cleavage activity of DNAzyme for the first time to detect the conserved region sequence of Plasmodium, which greatly improves the efficiency of DNAzyme. The selected target gene sequence is widely present in various Plasmodium species, which increases the scope of application of malaria detection. Compared with RT-PCR and other existing malaria detection methods, this method has high integration, low cost, short time consumption, relatively low technical requirements for operators, strong specificity, high accuracy, and can be applied to on-site detection under limited environmental conditions.

[0040] (4) Construct a nanomachine with DNAzyme as the core, use an optomagnetic sensing mode that can be adapted to a mobile power source and use MNP as a marker to detect Plasmodium pathogens. This is suitable for malaria-prone areas with poor medical conditions and limited sanitary environment, greatly reducing the detection cost and achieving rapid, real-time, and sensitive POC detection of malaria.

[0041] (5) Relying on the molecular mechanism contained in the detection method or kit or biosensor provided by the present invention, the biosensor system is constructed by replacing the target gene and its related sequence target gene binding region. It is expected that rapid real-time detection of multiple pathogens can be completed under limited conditions, which is of great significance for the prevention and control of widely spread dangerous pathogens.

[0042] Instruction Manual

[0043] Figure 1 Schematic diagram of a magnetic biosensor based on DNA nanomachines in one embodiment.

[0044] Figure 2 This is a diagram of the molecular amplification electrophoresis verification results in an embodiment.

[0045] Figure 3 This is a diagram showing the electrophoresis verification results of the combination product of NickRCA product and DNA nanomachine in one embodiment.

[0046] Figure 4 This is a diagram showing the hydrodynamic volume results of the relevant MNP assembly structure detected using dynamic light scattering in one embodiment.

[0047] Figure 5 Atomic force microscopy verification result of the MNP assembly structure in one embodiment

[0048] Figure 6 This is a diagram showing the detection results based on the optical magnetic endpoint phase difference sensor in one embodiment. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with the accompanying drawings and examples so that those skilled in the art can implement it with reference to the text of the specification. Unless otherwise defined, all technical and scientific terms used in the examples have the same meanings as those commonly understood by those skilled in the art of the present invention. Unless otherwise specified, the methods used are all conventional methods in the art; the medicaments used can be prepared by commercial routes or known public methods.

[0050] Taking the detection of Plasmodium pathogen as an example, the present invention is further described in detail.

[0051] Example 1 Sequence Design in Biosensor for Detecting Plasmodium

[0052] 1.1 Screening of target gene sequences

[0053] The detection site of Plasmodium pathogen nucleic acid is generally located on 18S rRNA, but because the mitochondrial genome has a higher copy number than 18S rRNA, the detection target was screened and compared with 151 existing Plasmodium mitochondrial gene (mtDNA) sequences, and a conservation analysis was performed to obtain a highly conservative target gene sequence containing 36nt. The sequence information is shown in Table 1.

[0054] 1.2 Design of padlock probe

[0055] The total length of the padlock probe sequence is 78nt. Since the double-stranded DNA torque is about 10.44bp / turn, in order to reduce the difficulty of strand displacement of the polymerase during amplification, the length of the padlock probe should be an integer multiple of the unnatural DNA torque. The padlock probe has 18nt at both ends that complement the target sequence, and a restriction endonuclease site is introduced therein. The cleaved bases are modified with methylation to prevent themselves from being cut, and the 5' end of the padlock probe needs to be phosphorylated to meet the working conditions of the ligase. The sequence is calculated by the online nucleic acid secondary structure prediction tool NUPACK software to calculate the binding force between the padlock probe and the target gene sequence and predict the secondary structure of the padlock probe. Under the premise of ensuring the binding force, the stability of irrelevant secondary structures (such as intramolecular hairpin structures and intermolecular dimers) is minimized to improve the amplification efficiency. The sequence information of the padlock probe is shown in Table 1.

[0056] 1.3 Primer design

[0057] The primer sequence was designed according to the padlock probe sequence (see Table 1). The primer sequence matched the padlock probe sequence and was distinguished from the target gene sequence and the protective strand.

[0058] 1.4 Design of protection chain (RO)

[0059] The sequence information of the protection strand (RO) is shown in Table 1. The protection strand is 25 nt in length, completely complementary to the NickRCA product, and chemically blocked at the 3' end. The main functions of RO are twofold: RO introduces a restriction enzyme site near the 5' end to recognize the RCA product, and the 3' end of the NickRCA product generated after nicking matches RO, so it will not be digested by the polymerase.

[0060] 1.5 Design of DNA nanomachines

[0061] The DNA nanomachine integrates the DNAzyme chain and the corresponding substrate chain, specifically:

[0062] (1) DNAzyme chain (hereinafter referred to as "D chain") was verified by electrophoresis and 8-17 DNAzyme with RNA cleavage activity was selected. The designed D chain is 130 nt in length. From both ends to the middle of the D chain, it consists of: substrate chain binding arm region, enzyme active center, padlock probe same region and random sequence. The 3' end of the D chain is modified with biotin. The sequence information is shown in Table 1.

[0063] (2) The two ends of the designed substrate chain (hereinafter referred to as "S chain") match the D chain, and a cleavage site matching the D chain is provided in the middle region. The 3' end of the S chain is modified with biotin. The sequence of the S chain refers to the 8-17 DNAzyme binding chain in the literature. The sequence information is shown in Table 1.

[0064] Table 1 DNA sequences involved

[0065]

[0066]

[0067] The padlock probe in Table 1 contains a methylated C (bold and underlined); the substrate strand contains a cleavage site a (rA, bold and underlined).

[0068] The 8-17 DNAzymes in Table 1 were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the remaining sequences were sent to Sangon Biotechnology (Shanghai) Co., Ltd. for synthesis.

[0069] Example 2 Electrophoresis verification of molecular products

[0070] The nucleic acid molecules involved in this example are as described in Example 1. Agarose electrophoresis is used to verify each reaction product and to verify the generation of the expected nucleic acid fragment. It should be noted that the target gene-containing solution in this example is a standard solution, which can directly trigger rolling circle amplification without adding primers. The following are the main reagents and equipment involved:

[0071] (1) Reagents: TE buffer (Beijing Solebow Technology, T1120); 10×TBE buffer (Beijing Prilai Gene Technology Co., Ltd., B1111-500 mL); TAE buffer (Thermo Fisher, B1110); Tris-HCl (Beijing Prilai Gene Technology Co., Ltd., B1011), 1 M; zinc sulfate heptahydrate (Shanghai Hushi, 7446-20-0); magnesium sulfate (New England BioLabs, M0374S), 100 mM; bovine serum albumin (BSA) (Beijing Solebow Technology, 109Z054); agarose gel (Burgos, Spain, BY-R0100); SYBR Gold (Thermo Fisher, S11494); 6×Loading dye (Hunan Aikerui Biotechnology Co., Ltd., AG11902); DNA Marker (Thermo Fisher, SM0371); GL DNAMarker5000 (Hunan Aikerui Bioengineering Co., Ltd., AG11906); dNTP mixture (Beijing Solebow Technology, PC2200), 10 mM; DNA ligase supporting reagents: 10×Ampligase DNA ligase buffer, Ampligase DNA ligase (5 U / μL, Biosearch technologiesr, A32750); DNA polymerase supporting reagents: 10×polymerase buffer, phi29DNA polymerase (10 U / μL, Thermo Fisher, EP0094); DNA kinase supporting reagents: 10×polynucleotide kinase buffer, T4 polynucleotide kinase (New England BioLabs, M0201V), 10 U / μL; AluI restriction endonuclease (NewEngland BioLabs, R0137S), 10 U / μL.

[0072] Among the above molecular reagents, phi29 DNA polymerase is a polymerase with high processivity (above 70kb) and can achieve efficient isothermal DNA amplification. It has strong strand displacement activity and is a common tool enzyme for RCA reaction. phi29 DNA polymerase also has 3'→5' exonuclease (correction reading) activity that preferentially acts on single-stranded DNA or RNA; AluI restriction endonuclease recognizes specific sites in the double strand for cutting.

[0073] (2) Equipment: Gel imaging system (Guangzhou BIO-OI Biotechnology Co., Ltd., OI100); metal bath thermostat (Jiangsu Wuxi Nice Biotechnology Co., Ltd., HS11000); electrophoresis apparatus (Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd., JY600C).

[0074] 2.1 Electrophoresis to verify the molecular amplification product:

[0075] The migration rate of circular DNA structures in agarose gel is slower than that of linear DNA structures of the same length, so electrophoresis can be used to qualitatively verify whether the padlock probe is connected into a circle by the ligase. The rolling circle amplification (RCA) product has a complex structure and a large molecular weight, and its migration rate in agarose gel is extremely low. It is used to qualitatively verify whether the reaction amplification and hybridization reaction are designed successfully.

[0076] (1) Ligase reaction (20 nM): Mix 5 μL of ligase buffer (10×), 1 μL of padlock probe (1 μM), 3 μL of target gene (1 μM), 33.5 μL of water, 5 μL of bovine serum albumin (2 mg / mL) and 2.5 μL of Ampligase DNA ligase (5 U / μL) and react at 50°C for 15 min.

[0077] (2) RCA reaction: 6 μL of ligase reaction product (1 nM), 3 μL of polymerase buffer (10×), 3 μL of bovine serum albumin (2 mg / mL), 1 μL of deoxyribonucleotide dNTP (10 mM), 16 μL of water, and 1 μL of phi29 DNA polymerase (10 U / μL) were mixed and reacted at 37°C for 60 min.

[0078] (3) NickRCA reaction: 6 μL of ligase reaction product (1 nM), 3 μL of polymerase buffer (10×), 3 μL of bovine serum albumin (2 mg / mL), 1 μL of deoxynucleotide dNTP (10 mM), 15 μL of water, 1 μL of phi29 DNA polymerase (10 U / μL), and 1 μL of AluI restriction endonuclease (10 U / μL) were mixed and reacted at 37°C for 60 min.

[0079] (4) NickRCA and protection chain reaction: 6 μL of ligase reaction product (1 nM), 3 μL of polymerase buffer (10×), 3 μL of bovine serum albumin (2 mg / mL), 1 μL of deoxynucleotide dNTP (10 mM), 9 μL of water, and 1 μL of phi29 DNA polymerase (10 U / μL) were mixed, and 1 μL of AluI restriction endonuclease (10 U / μL) and 6 μL of protection chain (20 μM) were added, and the reaction was carried out at 37°C for 60 min.

[0080] (5) Electrophoresis verification: Agarose gel electrophoresis (2.5%, 1×TAE buffer), take 15 μL of the sample to be analyzed, mix it with 1 μL SYBR Gold and 3 μL loading buffer, perform gel electrophoresis at room temperature (100V, 45 minutes) and take an image. The sample to be analyzed includes:

[0081] (a) Lane 1: 4 μM target gene chain;

[0082] (b) Lane 2: 5 μM padlock probe strand;

[0083] (c) Lane 3: 250 nM of the above ligase reaction product;

[0084] (d) Lane 4: RCA reaction product initiated by 200 pM ligase reaction product;

[0085] (e) Lane 5: NickRCA reaction product initiated by 200 pM ligase reaction product;

[0086] (f) Lane 6: NickRCA reaction product assisted by protective strands initiated by 200 pM ligase reaction product;

[0087] (g) Lane M: Maker.

[0088] The electrophoresis results are shown in Figure 2 , analysis shows that except for the shortest chain in lane 3 which is the excess target chain, the other bands are all above the padlock probe chain, proving that the ligase reaction proceeds normally; in lane 4, an extra-long RCA product is produced; in lane 5, a smear-like product appears, but the long-chain product is more than the short-chain product, and the NickRCA product is generated; in lane 6, a smear-like product appears, and compared with lane 5, more obviously shorter products are produced, proving that the protection chain can assist in cutting to generate more short-chain products.

[0089] 2.2 Electrophoresis verification of the binding reaction between NickRCA products and DNA nanomachines

[0090] As mentioned above, the 8-17 type DNAzyme chain (D chain) and the corresponding substrate chain (S chain) were selected. The D chain has the same region as the padlock probe, so that it can match the NickRCA product. The D&S complex of the D chain and the S chain was prepared and added to the same reaction system as the NickRCA product. After the D&S complex binds to the NickRCA product, the D chain catalytic active center is formed. 2+ Under the action of the D chain, after cutting the RNA site in the S chain, a cleavage product with 2'(3')-cyclic phosphate and 5'-hydroxyl termini is produced. According to literature research, T4 polynucleotide kinase can convert 2'(3')-cyclic phosphate into 3' hydroxyl. The phi29 DNA polymerase has a 3'-5' exonuclease (correction reading) activity that preferentially acts on single-stranded DNA or RNA. Therefore, the phi 29 DNA polymerase preferentially recognizes the mismatched bases converted to 3' hydroxyl by T4 polynucleotide kinase, and extends after correction to displace the NickRCA chain bound to the D chain.

[0091] The electrophoresis verification experimental plan is as follows:

[0092] (1) Ligase reaction (20 nM): Mix 10 μL ligase buffer (10×), 2 μL padlock probe (1 μM), 6 μL target gene (1 μM), 67 μL water, 10 μL bovine serum albumin (2 mg / mL) and 5 μL Ampligase DNA ligase (5 U / μL) and react at 50°C for 15 min.

[0093] (2) Synthesis of D&S complex: 18 μL D chain (20 μM), 12 μL S chain (20 μM), and 18 μL water were mixed and reacted at 90°C for 10 min to open the secondary structure of the DNA chain. The temperature was then lowered by 10°C every 10 min until it reached 37°C to allow the two to fully combine and form a D&S complex. The ratio of D chain to S chain was 3:2 to ensure that there was no free S chain in the system.

[0094] (3) NickRCA reaction

[0095] System 1: Mix 1.2 μL of ligase reaction product (5 nM), 3 μL of polymerase buffer (10×), 3 μL of bovine serum albumin (2 mg / mL), 2 μL of deoxynucleotide dNTP (10 mM), 10.8 μL of water, and 2 μL of phi29 DNA polymerase (10 U / μL), add 2 μL of AluI restriction endonuclease (10 U / mL) and 6 μL of protective chain (20 μM), and react at 37°C for 120 minutes. Take out 15 μL of the mixed solution and add 6 μL of MgSO4 (50 mM).

[0096] System 2: Mix 3 μL of ligase reaction product (5 nM), 7.5 μL of polymerase buffer (10×), 7.5 μL of bovine serum albumin (2 mg / mL), 5 μL of deoxynucleotide dNTP (10 mM), 27 μL of water, and 5 μL of phi29 DNA polymerase (10 U / μL), add 5 μL of AluI restriction endonuclease (10 U / mL) and 15 μL of protective chain (20 μM), and react at 37°C for 120 minutes. Take out 15 μL of the mixed solution for later use.

[0097] (4) NickRCA and D&S complex strand displacement reaction (two-step enzyme inactivation)

[0098] System 3: The remaining mixed solution of System 2 was reacted at 80°C for 20 minutes to inactivate phi 29 DNA polymerase and AluI restriction endonuclease, and then 24 μL of MgSO4 (50 mM) and 12 μL of D&S complex (5 μM) were added and mixed; three portions of 24 μL of the mixed solution (3.A, 3.B, 3.C) were separated from System 3, wherein:

[0099] System 3.A: Add 6 μL of water to system 3.A and react at 37°C for 120 minutes;

[0100] System 3.B: Add 3 μL T4 polynucleotide kinase buffer (10×) and 1 μL water to system 3.B, react at 37°C for 60 minutes, then add 1 μL phi29 DNA polymerase (10 U / μL) and 1 μL T4 polynucleotide kinase (10 U / μL), mix thoroughly and react at 37°C for another 60 minutes;

[0101] System 3.C: Add 3 μL T4 polynucleotide kinase buffer (10×), 1 μL water, 1 μL phi29 DNA polymerase (10 U / μL), and 1 μL T4 polynucleotide kinase (10 U / μL) to system 3.C and react at 37°C for 120 minutes.

[0102] (5) NickRCA and D&S complex strand displacement reaction (two-step method without inactivating the enzyme)

[0103] System 4: Add 6 μL of MgSO4 (50 mM) to 15 μL of the mixed solution taken out from the above system 2, mix, add 3 μL of D&S complex (5 μM), react at 37°C for 60 minutes, then add 1 μL of water, 3 μL of T4 polynucleotide kinase buffer (10×), and 1 μL T4 polynucleotide kinase (10 U / μL), mix thoroughly, and react at 37°C for 60 minutes.

[0104] (6) Chain displacement reaction of NickRCA and D&S complex (one-step method)

[0105] System 5: 3 μL of ligase reaction product (5 nM), 7.5 μL of polymerase buffer (10×), 7.5 μL of bovine serum albumin (2 mg / mL), 5 μL of deoxynucleotide dNTP (10 mM), 27 μL of water, and 5 μL of phi29 DNA polymerase (10 U / μL) were mixed, and 5 μL of AluI restriction endonuclease (10 U / μL), 15 μL of protective chain (20 μM), 9.5 μL of MgSO4 (50 mM), and 7.5 μL of D&S complex (5 μM) were added and mixed thoroughly. 26 μL was taken out and added to 3 μL of T4 polynucleotide kinase buffer (10×) and 1 μL of T4 polynucleotide kinase (10 U / μL), mixed thoroughly, and reacted at 37°C for 120 minutes.

[0106] (7) Electrophoresis verification: Agarose gel electrophoresis (2.5%, 1×TBE buffer), take 15 μL of the sample to be analyzed, mix it with 1 μL SYBR Gold and 3 μL loading buffer, perform gel electrophoresis at room temperature (100V, 45 minutes) and take an image. The sample to be analyzed includes:

[0107] (a) Maker, placed in lane M;

[0108] (b) Lane 1: 1 μM S chain;

[0109] (c) Lane 2: 1 μM D chain;

[0110] (d) Lane 3: 1 μM of the above D&S complex;

[0111] (e) Lane 4: product of system 1 primed by 200 pM target gene.

[0112] (f) Lane 5: System 3.A product triggered by 200 pM target gene;

[0113] (g) Lane 6: System 3.B product triggered by 200 pM target gene;

[0114] (h) Lane 7: System 3.C product triggered by 200 pM target gene;

[0115] (i) Lane 8: product of system 4 primed by 200 pM target gene.

[0116] (j) Lane 9: product of system 5 primed by 200 pM target gene.

[0117] The electrophoresis verification results are shown in Figure 3 . Analysis showed that the product in lane 3 was the 3:2 combination product of lanes 1 and 2; a smear-like product appeared in lane 4, proving that the NickRCA reaction was successful; lane 5 was the combination product of the NickRCA reaction product and the D&S complex. Compared with the product band in lane 4, the product disappeared at the corresponding position in this lane (position a in lane 4) and a new band appeared (position b), thus proving that the D&S complex was combined with the NickRCA product; in lane 6, phi29 was added to the product in lane 5. DNA polymerase and T4 polynucleotide kinase can cut the S chain, extend it after correction, and the corresponding band in lane 5 is divided into two in lane 6 (c position), which are the chain displacement product D+D' and the combination product of NickRCA product and D&S complex, proving that DNAzyme works normally and chain displacement reaction occurs; lane 7 is the same as lane 6; lane 8 is a two-step enzyme that is not inactivated, and the restriction endonuclease in NickRCA is always working, so there are fewer super-long chain products in the hole, and the D+D' band appears in the second lane, proving that DNAzyme works normally and chain displacement reaction occurs; lane 9 is a one-step reaction, and D+D' appears, proving that DNAzyme works normally and chain displacement reaction occurs.

[0118] Example 3 MNP assembly (M D&S ) Preparation and characterization

[0119] In this embodiment, magnetic nanoparticles (BNF-Starch-1mL (Micromod, 10-19-102)) were selected as nano-markers; nucleic acid sequences were used as scaffolds for MNPs, and MNP assemblies were formed through nucleic acid sequences. As mentioned above, the D chain and the S chain in the DNA nanomachine were each modified with biotin at their 3' ends, so that the MNPs were modified on the D chain and the S chain respectively, and the D&S complex was formed through the complementary base pairing of the two chains, and the MNP assembly was further formed. According to the calculation in the MNP product manual, the MNPs were modified with a streptavidin load of 0.2 times, that is, 40 nucleic acid chains were modified on each MNP.

[0120] 3.1M D&S Preparation

[0121] The specific experimental steps are as follows:

[0122] (1) System 1: Take 20 μL of MNP (10 mg / mL) and perform magnetic separation to elute free streptavidin;

[0123] (2) System 2: MNP combined with D chain (M D 18 μL D chain (0.2 μM) and 22 μL Tris-HCl buffer (50 mM, PH = 8.0) were mixed, heated in a metal bath at 60°C for 10 minutes to open the secondary structure of the DNA chain, and then cooled rapidly for 5 minutes. 10 μL of system 1 solution was added and reacted at 37°C for 30 minutes. The streptavidin on the MNP specifically bound to the biotin modified on the D chain. Finally, Tris-HCl buffer was added to 100 μL to obtain MNP. D System solution.

[0124] (3) System 3: MNP combined with S chain (M S 18 μL S chain (0.2 μM) and 22 μL water were mixed and heated at 60°C for 10 minutes in a metal bath to open the secondary structure of the DNA chain. The temperature was quickly cooled for 5 minutes. 10 μL of the system-1 solution was added and reacted at 37°C for 30 minutes. The streptavidin on the MNP specifically bound to the biotin modified on the S chain. Finally, Tris-HCl buffer was added to 100 μL to obtain MNP. S System solution.

[0125] (3) M D System solution and M S The system solution was mixed, frozen at -20°C for 60 minutes, thawed at room temperature (25°C) for 10 minutes, and then incubated at 25°C for 60 minutes on a rotating magnetic plate. D With M S Fully combined to form MNP assembly M D&S , stored at 4℃ for 48 hours before use.

[0126] 3.2M D&S Characterization

[0127] Dynamic light scattering (DLS) and atomic force microscopy (AFM) were used for verification.

[0128] (1) DLS verification

[0129] The BeNano 90 nanoparticle size analyzer (Bettersize Instruments, Dandong, China) was used to verify the hydrodynamic volume of the MNP assembly before and after the reaction, including M D , 3.6pM target gene triggers amplification reaction and decomposes the structure of MNP and M D&S , related parameters: laser light source wavelength 671nm, detection angle 90°, detection temperature: 25℃.

[0130] Test results such as Figure 4 Shown: The formed M D&S There is a small part that is not aggregated, and the fluid dynamics volume of the aggregated part increases to about 1000nm; after the 3.6pM target triggers the amplification reaction and decomposes the structure, the fluid dynamics volume of the MNP decreases to about 254nm.

[0131] (2) Atomic force microscopy verification

[0132] Testing instrument: Asylum Research MFP-3D BIO biological atomic force microscope (Oxford Instruments plc, A-bingdon); Parameters: Probe: AC160TSR3 silicon probe, aluminum reflective coating, tip radius: 7nm, scanning area: 5×5μm 2 The resolution was 256 × 256, and the scanning speed was about 1 Hz. AFM images were processed using Gwyddion 2.43 software.

[0133] like Figure 5 As shown: (a) M D (b) M D&S , (c) The free magnetic beads in the system after the amplification reaction decomposition. As can be seen from the figure, (b) MNPs are aggregated in large quantities, M D&S Assembly formation; (c) 3.6pM target triggers amplification reaction to M D&S The assembly structure opens.

[0134] Example 4: Optical and magnetic endpoint phase difference detection verification

[0135] As described above, the selected malarial conserved region was used as the target gene, and after rolling circle amplification, it was combined with the M prepared in Example 3. D&S The assembly reacts, and the optical magnetic endpoint phase difference sensor is used to verify the sensitivity of the molecular amplification system and to perform qualitative and quantitative analysis of the amplification products. The specific experimental steps are as follows:

[0136] (1) Ligase reaction (20 nM): Mix 10 μL ligase buffer (10×), 2 μL padlock probe (1 μM), 6 μL target gene (1 μM), 67 μL water, 10 μL bovine serum albumin (2 mg / mL) and 5 μL Ampligase DNA ligase (5 U / μL) and react at 50°C for 15 min.

[0137] (2) Prepare M as in step 3.1 above. D&S Assembly.

[0138] (3) Amplification reaction and M D&S Solution: 30 μL ligase reaction product (0.5 fM-1000 fM), 6 μL polymerase buffer (10×), 6 μL bovine serum albumin (2 mg / mL), 3 μL deoxynucleotide dNTP (10 mM), 3 μL MgSO4 (100 mM), 3 μL M D&S The assembly (5 μM), 1 μL phi29 DNA polymerase (10 U / μL), 1 μL AluI restriction endonuclease (10 U / μL), 3 μL T4 polynucleotide kinase buffer (10×), 1 μL T4 polynucleotide kinase (10 U / μL), and 6 μL protective chain (20 μM) were fully mixed and incubated at 37°C with a rotating magnetic for 75 min.

[0139] (4) Target gene detection analysis: The sensitivity of the molecular amplification system was verified using an optical magnetic endpoint phase difference sensor, where the optical magnetic detection parameters were: 1 mT magnetic field, 450 nm wavelength light source, and detection at room temperature. The results were as follows: Figure 6 As shown, the test involves 10 groups of samples, including a blank control group, different target genomes with concentrations of 0.5fM-1000fM (respectively 0.5fM, 1fM, 3.2fM, 10fM, 32fM, 50fM, 100fM, 320fM, 1000fM), and the 10 groups of samples differ only in the concentration of the ligase reaction product, and the relative phase difference of the optical and magnetic signals is the difference between the average of the 0.5Hz-10.7Hz phase signal and the blank. The sensitivity of this molecular amplification system can reach the fM level.

[0140] In the above examples, the qualitative verification of the amplified product was verified by electrophoresis; the quantitative verification was performed by calculating the M of the target triggered by 0.5fM-1000fM concentration. D&SAfter the structure is solved, the phase average of the optical and magnetic detection phase (0.5Hz-10.7Hz) caused by the fluid dynamics volume is quantified.

[0141] Further explanation: the concentrations of enzymes, probes, primers, and protective chains involved in the above embodiments are all used in excess to ensure maximum reaction efficiency. If the concentration is low, the reaction efficiency will be reduced. The reaction time of each step is the shortest time to obtain the best signal, which is determined by the reaction efficiency. If the reaction efficiency is low, the same reaction result can generally be achieved by extending the reaction time. The reaction temperature of each step depends on the optimal working temperature of the enzyme, which is generally 37°C.

[0142] The technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above-described embodiments only express several implementation methods of the present invention, but cannot be understood as limiting the scope of the patent of the present invention. The scope of the present invention is defined by the attached claims rather than the above description. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, other implementations and modifications can be easily realized according to the contents disclosed in this patent document. Therefore, all equivalent implementations and changes that do not deviate from the present invention and fall within the meaning and scope of the equivalent elements of the claims fall within the protection scope of the present invention.

Claims

1. A nucleic acid detection method based on DNA nanomachines, characterized in that: The following steps are involved: S1, screening of target genes to be tested; S2, designing a padlock probe and a DNAzyme chain according to the target gene sequence to be tested, and correspondingly designing primers, protection chains and substrate chains; wherein the padlock probe has a phosphorylated 5' end, and is provided with a target gene recognition region, a primer recognition region and an endonuclease recognition region, the target gene recognition region is provided at both ends of the padlock probe, and is complementary to the target gene sequence to be tested, and the endonuclease cleavage site of the endonuclease recognition region is methylated; the protection chain has the same sequence as a partial region of the padlock probe, and has a chemically closed 3' end; the DNAzyme chain is complementary to a substrate chain with a single RNA site, and the sequence of a partial region of the DNAzyme chain is the same as the sequence of the target gene recognition region in the padlock probe; the DNAzyme chain and the substrate chain have biotinylated 3' ends; S3, mixing the sample solution with the padlock probe, primers, and DNA ligase to perform a ligase reaction to obtain a ligase reaction product; S4, preparing a nanocomplex system solution; the nanocomplex is generated by combining a DNAzyme chain, a substrate chain, and a nano-label; S5, taking the ligase reaction product and the nanocomplex system solution, adding metal ion solution, DNA polymerase, endonuclease, T4 polynucleotide kinase and protective chain, mixing, and performing nicking-enhanced rolling circle amplification reaction, DNAzyme endonuclease reaction, and strand displacement reaction to obtain a final reaction system; S6, using a sensor to detect the final reaction system to achieve qualitative analysis and / or quantitative analysis of the target gene to be detected.

2. The detection method according to claim 1, characterized in that: In step S4, the molar ratio of the DNAzyme chain to the substrate chain in the nanocomplex is (1.5-1):1, preferably 1:

1.

3. The detection method according to claim 1, characterized in that: In step S4, the nano-markers include one or more of magnetic nanoparticles, gold nanoparticles, and fluorescent groups.

4. The detection method according to any one of claims 1 to 3, characterized in that: In step S6, the sensor includes one or more of a photomagnetic endpoint phase difference detection system, an electron paramagnetic resonance spectrometer, an ultraviolet-visible spectrophotometer, and a fluorescence microscope.

5. A kit, characterized in that: comprising reaction liquid a, reaction liquid b, and reaction liquid c; The reaction solution a includes a padlock probe, a primer, and a DNA ligase; the reaction solution b includes a nanocomplex formed by combining a DNAzyme chain, a substrate chain, and a nano-label; the reaction solution c includes T4 polynucleotide kinase, a DNA polymerase, an endonuclease, a protective chain, and a metal ion solution; A padlock probe and a DNAzyme chain are designed according to the target gene sequence to be detected, and primers, protection chains and substrate chains are designed accordingly; the padlock probe has a phosphorylated 5' end, and is provided with a primer recognition region, an endonuclease recognition region, and target gene recognition regions complementary to the target gene sequence to be detected at both ends, and the endonuclease cleavage site of the endonuclease recognition region is methylated; the protection chain has the same sequence as a partial region of the padlock probe and has a chemically closed 3' end; the DNAzyme chain is complementary to a substrate chain with a single RNA site, and the sequence of the partial region of the DNAzyme chain is the same as the sequence of the target gene recognition region in the padlock probe; the DNAzyme chain and the substrate chain have biotinylated 3' ends.

6. The kit according to claim 5, characterized in that The sequence of the padlock probe is shown in SEQ ID NO: 1, the sequence of the primer is shown in SEQ ID NO: 3, the sequence of the protection chain is shown in SEQ ID NO: 4, the sequence of the DNAzyme chain is shown in SEQ ID NO: 5, the sequence of the substrate chain is shown in SEQ ID NO: 6, and the kit is used to detect Plasmodium.

7. A biosensor, characterized in that: Comprising the kit as claimed in claim 5.

8. A biosensor for detecting malarial parasites, characterized in that: Comprising the kit as claimed in claim 6.

9. A method for detecting malarial parasites using the kit according to claim 6 or the biosensor according to claim 8, characterized in that: The following steps are involved: S1, mixing a test solution containing the target gene with a reaction solution a to perform a ligase reaction to obtain a ligase reaction product; S2, taking the ligase reaction product and mixing it with the reaction solution b and the reaction solution c, performing nicking-enhanced rolling circle amplification reaction, DNAzyme endonuclease reaction, and strand displacement reaction to obtain a final reaction system; S3, using a sensor to detect the final reaction system to achieve qualitative and / or quantitative analysis of the target gene to be detected; Wherein, the sequence of the target gene is shown as SEQ ID NO:

2.

10. The method according to claim 9, characterized in that The nano marker is a magnetic nanoparticle; and the sensor is a photomagnetic endpoint phase difference sensor.

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