Nucleic acid detection method based on ternary DNA coding library

Abstract

The invention discloses a nucleic acid detection method based on a ternary DNA coding library, and belongs to the technical field of in-vitro nucleic acid detection. The method aims at rapidly judging whether a specific gene fragment exists in a solution or not through a coding library. A certain number of nucleic acid structures are arranged in a coding region on a carrier, and the sizes and the number of the nucleic acid structures in different regions determine the electric signal intensity of nanopores, so that a multi-stage electric signal is formed, and a multi-system coding library is created. And a probe and a marker nucleic acid structure are arranged in the carrier sensing region and are used for detecting a specific gene segment. According to the method, a database is established based on a DNA data coding structure, and the method is an emerging rapid detection mode and has the characteristics of high sensitivity and high throughput.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro nucleic acid detection, and in particular relates to a nucleic acid detection method based on a ternary DNA encoding library. Background Art

[0002] Nucleic acid testing is crucial for clinical diagnosis and genotyping, as reflected in its high sensitivity and specificity, which can accurately identify the virus in the early stages of infection, providing opportunities for early intervention. In addition, it provides important data for epidemiological research, helps understand infection rates and transmission routes, and plays a key role in monitoring virus mutations, providing a scientific basis for the formulation of public health policies.

[0003] Early nucleic acid detection widely used technologies such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) and DNA microarray detection. PCR is a molecular biology technology used to amplify specific DNA fragments. It is based on the principle of semi-conservative replication of DNA. It cycles through three basic steps in vitro: high-temperature denaturation (unwinding the DNA double strands into single strands), low-temperature annealing (primers bind to single-stranded DNA templates) and thermostatic extension (synthesizing new DNA chains with primers as the starting point under the action of DNA polymerase), so that the target DNA fragment increases exponentially. The operation process is relatively complicated, requiring professional instruments and equipment (such as PCR instruments) and technicians, and the detection time is long, usually several hours. It is easy to be contaminated. Once exogenous nucleic acids contaminate the sample or reaction system, false positive results may occur. DNA microarray detection technology has high requirements, and the chip production and detection process are complicated. The cost is high, including the cost of chip production and the cost of detection equipment. Data interpretation is also relatively complicated and requires professional bioinformatics knowledge. Although the existing methods have high specificity and sensitivity, they take a long time to detect, are expensive, require additional modifications, and require cumbersome sample processing, resulting in poor immediacy. In addition, the scope of application of early methods is limited, usually only specific pathogens can be detected, and they lack the ability to detect multiple targets simultaneously. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a nucleic acid detection method based on a ternary DNA encoding library; the DNA encoding library prepared by the present invention makes full use of the characteristics of DNA molecules, and encodes the corresponding DNA vectors according to the detection requirements of different pathogens. By analyzing the characteristic current peak signals generated by each DNA vector in the DNA encoding library in the nanopore, the type of pathogen detected can be effectively distinguished. This method not only provides a high-density information storage method, but also supports high-throughput detection, significantly improving the efficiency and accuracy of pathogen identification. Due to its unique encoding mechanism, the DNA encoding library can process a large number of samples at the same time, meeting the needs of large-scale screening.

[0005] The first object of the present invention is to provide a rapid detection method based on a ternary DNA encoding library.

[0006] The second object of the present invention is to propose a ternary coding structure, which increases the data density compared with the classic binary coding and can encode the mark 3 3 =27 different DNA vectors.

[0007] The third object of the present invention is to propose a detection method using a DNA encoding library, which has the characteristics of high throughput and high specificity.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical scheme is adopted:

[0009] A DNA coding structure based on ternary system, the DNA carrier includes a coding region and a sensor, the coding region includes two flag positions and three coding positions, and the three coding positions are located between the two flag positions;

[0010] A group of DNA dumbbell sequences from SEQ ID NO: 1 to SEQ ID NO: 11 are used to construct the first marker in the coding region of the DNA vector, and the nucleic acid sequences are shown in Table 1:

[0011] Table 1

[0012]

[0013] A group of DNA dumbbell sequences from SEQ ID NO: 12 to SEQ ID NO: 22 are used to construct a second marker in the coding region of a DNA vector, and the nucleic acid sequences are shown in Table 2:

[0014] Table 2

[0015]

[0016] A set of DNA dumbbell sequences from SEQ ID NO:23 to SEQ ID NO:29 are used to form the first coding position "1" in the ternary coding at the coding position of the DNA vector, and the sequence is shown in Table 3:

[0017] Table 3

[0018]

[0019] A set of DNA dumbbell sequences from SEQ ID NO:30 to SEQ ID NO:36 are used to form the "1" of the second coding position in the ternary coding at the coding position of the DNA vector, and the sequence is shown in Table 4:

[0020] Table 4

[0021]

[0022] A set of DNA dumbbell sequences from SEQ ID NO:37 to SEQ ID NO:43 are used to form the third coding position "1" in the ternary coding at the coding position of the DNA vector, and the sequence is shown in Table 5:

[0023] Table 5

[0024]

[0025] A group of DNA sequences from SEQ ID NO:44 to SEQ ID NO:53 are respectively combined with the WI(0) sequence of SEQ ID NO:54 to form 10 DNA three-way structure sequences, which are used to form the first coding position "2" in the ternary coding at the coding position of the DNA vector. The nucleic acid sequence is shown in Table 6:

[0026] Table 6

[0027]

[0028] A group of DNA sequences from SEQ ID NO:55 to SEQ ID NO:64 are respectively combined with the WII(0) sequence of SEQ ID NO:65 to form 10 DNA three-way structure sequences, which are used to form the second coding position "2" in the ternary coding at the DNA vector coding position. The nucleic acid sequence is shown in Table 7:

[0029] Table 7

[0030]

[0031] A group of DNA sequences from SEQ ID NO:66 to SEQ ID NO:75 are respectively combined with the WIII(0) sequence of SEQ ID NO:76 to form 10 DNA three-way structure sequences, which are used to form the third coding position "2" in the ternary coding at the DNA vector coding position. The nucleic acid sequence is shown in Table 8:

[0032] Table 8

[0033]

[0034] If the coding position of the DNA vector does not form a sequence of "1" in the ternary code and "2" in the ternary code, then the coding position is "0" in the ternary code.

[0035] The sensing area includes at least one sensing unit, and the sensing unit includes corresponding probe positions and label positions; the probe is used to capture target DNA;

[0036] The nucleotide sequences of SEQ ID NO:77 and SEQ ID NO:78 are used to form a probe position on a DNA structure, wherein "TTCCAGTGTG GCTGGTCATC" in SEQ ID NO:78 is used to capture the target DNA, and "GTAGCACCAT TACCATTAGC" is a part of SEQ ID NO:195, which is complementary to the DNA template strand; SEQ ID NO:77 is the remaining part of SEQ ID NO:195 used for complementing, and the corresponding label position is formed by SEQ ID NO:83-SEQ ID NO:88, constituting a sensing unit;

[0037] The nucleotide sequences of SEQ ID NO:79 and SEQ ID NO:80 form a probe site on the DNA structure, wherein "GTATTCCTCC AGATCTCTAC" in SEQ ID NO:80 is used to capture the target DNA, and "ACCGATATAT TCGGTCGCTG" is a part of SEQ ID NO:225, which is complementary to the DNA template strand; SEQ ID NO:79 is the remaining part of SEQ ID NO:225 used for filling the site, and the corresponding label site is formed by SEQ ID NO:89-SEQ ID NO:93, forming a sensing unit;

[0038] The nucleotide sequences of SEQ ID NO:81 and SEQ ID NO:82 form a probe position on the DNA structure, wherein "GCTCTGACGA ACGCTACAGG" in SEQID NO:82 is used to capture the target DNA, and "GGTCAGGATT AGAGAGTACC" is a part of SEQID NO:255, which is complementary to the DNA template chain; SEQ ID NO:81 is the remaining part of SEQ ID NO:255 used for filling the position, and the corresponding marking position is formed by SEQ ID NO:94-SEQ ID NO:98, constituting a sensing unit.

[0039] The DNA sequences of SEQ ID NO:77 to SEQ ID NO:82 are used to form probes at three sensing units in the sensing region of the DNA carrier, respectively. The nucleic acid sequences are shown in Table 9:

[0040] Table 9

[0041]

[0042] A set of DNA dumbbell sequences of SEQ ID NO:83 to SEQ ID NO:88 is used to form a marker at the first sensing unit of the DNA carrier sensing region, and the nucleic acid sequence is shown in Table 10:

[0043] Table 10

[0044]

[0045] A set of DNA dumbbell sequences of SEQ ID NO:89-SEQ ID NO:93 is used to form a marker at the second sensing unit of the DNA carrier sensing region, and the nucleic acid sequence is shown in Table 11:

[0046] Table 11

[0047]

[0048] A set of DNA dumbbell sequences of SEQ ID NO:94 to SEQ ID NO:98 is used to form a marker at the third sensing unit of the DNA carrier sensing region, and the nucleic acid sequence is shown in Table 12:

[0049] Table 12

[0050]

[0051] A ternary DNA coding library, each coding position in the coding structure has three options of "0", "1" and "2", and the coding library has 27 codes: 000, 001, 002, 010, 011, 012, 020, 021, 022, 100, 101, 102, 110, 111, 112, 120, 121, 122, 200, 201, 202, 210, 211, 212, 220, 221, 222.

[0052] The present invention provides a scheme of a DNA vector as follows:

[0053] The coding area uses ternary coding to represent 3 3 = 27 different detection sequences. The DNA vector is divided into a coding region and a sensor region:

[0054] The two ends of the coding region are each composed of 11 DNA dumbbell structures. These special DNA structures can generate current peaks when passing through the nanopore, thereby determining the location of the coding region.

[0055] The coding region includes two flag bits and three coding bits. Six DNA dumbbell structures are attached to a coding bit, representing "1" in the ternary code, which will generate a current peak when passing through the nanopore; while 10 DNA three-way structures are attached, representing "2" in the ternary code, which will generate a larger current peak when passing through the nanopore. At the coding bit, the part without the aforementioned structure to form the code "1" and code "2" is regarded as "0" in the ternary code.

[0056] The sensing area on the carrier includes a probe site and a label site. The probe site is connected to a probe sequence, which can specifically identify and capture the target nucleic acid, allowing it to bind to the carrier and be retained. There is also a DNA dumbbell structure on the label site next to the probe sequence as a marker. This DNA dumbbell structure can be used to mark the specific location of the target nucleic acid on the carrier, providing a basis for subsequent detection and identification.

[0057] A DNA vector, comprising the coding region and the sensing region according to claim 1, wherein the sensing region comprises a probe site and a labeling site; the probe is used to capture target DNA, and the label is used to label different detection sites.

[0058] A method for preparing a DNA vector comprises the following steps:

[0059] (1) Take the M13mp18 circular DNA solution, add buffer and a single-stranded DNA solution that is partially complementary to it; raise the temperature to 65°C and then decrease to room temperature at a rate of 1-3°C / min;

[0060] (2) Use 1 μL BamHI-HF enzyme and 1 μL EcoRI-HF enzyme to digest the mixed solution;

[0061] (3) Purify the DNA using the DNA CleanUp Kit to obtain the cut M13mp18 single-stranded DNA;

[0062] (4) Using the linear M13mp18 single-stranded DNA as the basic template, there are 190 complete complementary short chains (Nicholas A. W. Bell and Ulrich F. Keyser; NATURE NANOTECHNOLOGY, VOL 11, JULY 2016; nucleotide sequence table in the supporting information of Digitally encoded DNA nanostructures for multiplexed, single-molecule proteins ensing with nanopores), the complementary short chains remaining after the functional site template (i.e., the complementary short chains remaining after removing the short chains at the functional site position from the 190 short chains) are mixed with the short chains of the functional site, and 500 μL of 100 mM Tris buffer and 25 μL of 50 mM MgCl2 solution are added to prepare a mixed solution; the temperature is raised to 70°C, and then cooled to room temperature at a rate of 1-3°C / min to obtain a linear double-stranded DNA vector containing the coding region and the sensing region;

[0063] The short chain of functional bits is a short chain of flag bits, probe bits, marker bits, and coding bits.

[0064] An application of a DNA vector, wherein the DNA vector is applied to the detection of a target nucleic acid.

[0065] An application of a DNA vector, the detection method comprises the following steps:

[0066] (1) Mix the target nucleic acid to be detected with a linear double-stranded DNA vector containing a coding region and a sensing region; the target nucleic acid will be recognized and captured by the corresponding complementary probe on the vector;

[0067] (2) Injecting LiCl solution into the nanopore chip, extracting the LiCl solution and injecting it into the DNA carrier after capturing the target nucleic acid and ensuring that there are no bubbles remaining in the chip;

[0068] (3) The pore-crossing events are collected by a patch clamp device. By applying voltage to the chip, the DNA molecules pass through the nanopore, causing current changes. The detection of the target nucleic acid is confirmed by identifying the current peak.

[0069] The present invention provides a method for establishing a coding library for detection based on ternary coding, the steps are as follows:

[0070] (1) Perform nucleic acid pretreatment on the sample to be tested;

[0071] (2) Select relevant probes for the gene to be detected; one end of the probe sequence can be synthesized into the probe site, and the other end can be paired with the gene to be detected.

[0072] (3) Determine a set of codes according to the coding rules of the coding library;

[0073] (4) Synthesizing DNA vectors according to the coding scheme;

[0074] (5) Loading the sample and hybridizing the DNA carrier with the sample to be tested;

[0075] (6) Fabrication of nanopore chips;

[0076] (7) By applying voltage to the chip, the DNA molecules pass through the nanopore, causing a change in the current peak. The detection sample code is confirmed by identifying the current peak.

[0077] In the above steps, by applying voltage on the chip, the DNA molecules pass through the nanopore, causing current changes. The peak height and integrated area of ​​the current peak with a coding value of 2 are significantly higher than the peak height and integrated area of ​​the current peak with a coding value of 1, and can be accurately distinguished by the set threshold.

[0078] Furthermore, by applying voltage on the chip, the DNA molecule passes through the nanopore, causing a change in current. When the coding value is 0 and the DNA molecule passes through the nanopore, no change in current occurs.

[0079] The present invention also provides the application of the above sequence group or the method of establishing a coding library for detection: using ternary coding to prepare an identification probe library of the gene to be detected and detecting the target nucleic acid with high throughput and high specificity.

[0080] The beneficial effects of the present invention are:

[0081] The encoding library increases data density and can encode tags 3 3 = 27 different DNA vectors. A multi-level coding architecture and sensing structure are constructed into a DNA coding library, and the DNA coding is read by a DNA nanopore chip, and then the DNA vector to be detected is distinguished by the coding. Based on the research of technical methods, the present invention is committed to constructing a high-throughput, high-recognition, sensitive, low-cost and rapid nucleic acid detection method.

[0082] By setting a certain number of nucleic acid structures in the coding region on the carrier, the size and number of nucleic acid structures in different locations determine the strength of the nanopore electrical signal, thereby forming a multi-level electrical signal, and then creating a multi-binary coding library. Probes and marker nucleic acid structures are set in the sensing area of ​​the carrier to detect specific gene fragments. This method is based on the DNA data coding structure for library construction and is an emerging rapid detection method with the characteristics of high sensitivity and high throughput. Multi-binary DNA coding is a technology that uses the characteristics of DNA molecules to encode information into DNA nanostructures of different sizes at different locations; multi-binary DNA coding has a high information density and can store more data in the same DNA length. This makes multi-binary DNA coding have the potential for wide application in fields such as data storage and biosensor detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 This is a schematic diagram of different DNA structures in DNA vectors.

[0084] Figure 2 It is a schematic diagram of the self-assembly of a single-stranded DNA template, complementary short-stranded DNA and its functional DNA structure to form a DNA vector.

[0085] Figure 3 It is a schematic diagram of the coding region of a DNA vector.

[0086] Figure 4 Schematic diagram of the sensing region of the DNA vector.

[0087] Figure 5 This is a physical picture of the nanopore chip before sample addition.

[0088] Figure 6 The graph shows the characteristic current peaks generated by 27 encoding carriers passing through the nanopore chip.

[0089] Figure 7 The graph shows the characteristic current peaks generated by the DNA carrier C unit coded as 012 before and after capturing the target DNA through the nanopore chip. DETAILED DESCRIPTION

[0090] A DNA coding structure based on ternary system, the coding structure includes a coding region and a sensing region, the coding region includes two flag bits and three coding bits, the three coding bits are located between the two flag bits; the three coding bits are respectively a first coding bit, a second coding bit and a third coding bit; each coding bit can encode a value of 0, 1, 2, so as to be flexibly combined;

[0091] The nucleotide sequences of SEQ ID NO:1 to SEQ ID NO:11 are used to construct the first marker in the coding region of the DNA vector;

[0092] A group of DNA dumbbell sequences from SEQ ID NO:12 to SEQ ID NO:22 are used to construct a second marker in the coding region of a DNA vector;

[0093] A set of DNA dumbbell sequences of SEQ ID NO:23 to SEQ ID NO:29 are used to form the first coding position "1" in the ternary coding at the coding position of the DNA vector;

[0094] A set of DNA dumbbell sequences of SEQ ID NO:30 to SEQ ID NO:36 are used to form "1" of the second coding position in the ternary coding at the coding position of the DNA vector;

[0095] A set of DNA dumbbell sequences of SEQ ID NO:37 to SEQ ID NO:43 are used to form a "1" of the third coding position in a ternary code at the coding position of a DNA vector;

[0096] A group of DNA sequences from SEQ ID NO:44 to SEQ ID NO:53 are respectively combined with the WI(0) sequence of SEQ ID NO:54 to form 10 DNA three-way structure sequences, which are used to form the first coding position "2" in the ternary code at the coding position of the DNA vector;

[0097] A group of DNA sequences from SEQ ID NO:55 to SEQ ID NO:64 are respectively combined with the WII(0) sequence of SEQ ID NO:65 to form 10 DNA three-way structure sequences, which are used to form the second coding position "2" in the ternary code at the coding position of the DNA vector;

[0098] A group of DNA sequences from SEQ ID NO:66 to SEQ ID NO:75 are respectively combined with the WIII(0) sequence of SEQ ID NO:76 to form 10 DNA three-way structure sequences, which are used to form the third coding position "2" in the ternary code at the coding position of the DNA vector;

[0099] If the coding position of the DNA vector is in a simple double-stranded state and does not form a sequence of "1" in the ternary code or "2" in the ternary code, then the coding position is "0" in the ternary code.

[0100] In a specific implementation, each coding bit has three options: "0", "1" and "2", so the coding structure includes 27 codes: 000, 001, 002, 010, 011, 012, 020, 021, 022, 100, 101, 102, 110, 111, 112, 120, 121, 122, 200, 201, 202, 210, 211, 212, 220, 221, 222.

[0101] The sensing area includes at least one sensing unit, and the sensing unit includes corresponding probe positions and label positions; the probe is used to capture the target DNA.

[0102] In the first sensing unit, the nucleotide sequences of SEQ ID NO:77 and SEQ ID NO:78 are used to form a probe site on the DNA structure, and the nucleotide sequences of SEQ ID NO:83 to SEQ ID NO:88 form a marker site on the DNA structure;

[0103] In the second sensing unit, the nucleotide sequences of SEQ ID NO:79 and SEQ ID NO:80 are used to form a probe site on the DNA structure, and the nucleotide sequences of SEQ ID NO:89 to SEQ ID NO:93 form a marker site on the DNA structure;

[0104] In the third sensing unit, the nucleotide sequences of SEQ ID NO:81 and SEQ ID NO:82 are used to form a probe site on the DNA structure, and the nucleotide sequences of SEQ ID NO:94 to SEQ ID NO:98 form a marker site on the DNA structure;

[0105] In a specific embodiment, three target nucleic acids can be detected on each encoded DNA vector, so 81 types of DNA to be detected can be detected simultaneously; the DNA code is read by a DNA nanopore chip, and then the DNA vectors to be detected are distinguished by the coding and sensing units.

[0106] The present invention provides a detection method based on ternary DNA coding, and an example is as follows:

[0107] Example 1

[0108] Synthesize a DNA vector with the coding region coded as "012" and the sensing region detecting genes "A", "B", and "C":

[0109] (1) Take 44 μL of M13mp18 circular DNA solution, add 8 μL of 10x shearing buffer and 2 μL of Oligo solution, and then add appropriate amount of deionized water to make 80 μL of solution. Then, quickly raise the temperature to 65°C and slowly cool it down to 25°C at a rate of 1°C / min.

[0110] (2) Use 1 μL BamHI-HF enzyme and 1 μL EcoRI-HF enzyme to digest the mixed solution at 37°C for 1 hour. Use DNA CleanUp Kit to purify the DNA to obtain the cut M13mp18 single-stranded DNA.

[0111] (3) Using the linear M13mp18 single-stranded DNA as the basic template, the complementary short chains remaining from the reserved functional site template (i.e., the complementary short chains remaining after removing the short chains at the functional site position from the 190 short chains) are mixed with the short chains at the functional site mentioned above. (Schematic diagram as shown Figure 1 2.9 μL of 10 mM Tris-HCL buffer and 1 μL of 10 mM MgCl2 solution were added to prepare a 40 μL mixed solution with a final carrier concentration of 0.25 nM. The temperature was quickly raised to 75°C, and then slowly cooled to 25°C at a rate of 1°C / min to obtain a double-stranded DNA vector coded as "012" with a sensing area for detecting three different genes "A", "B" and "C".

[0112] Using this method, a DNA vector with the coding region coded as "012" and the sensing region detecting "A, B, C" genes was synthesized; the schematic diagram of the synthesis process is shown in Figure 2 shown. Figure 3 The ternary coding region of this example is presented. The coding region uses 6 DNA dumbbell structures and 10 DNA three-way structures to represent the codes "1" and "2" respectively. The 11 DNA dumbbell structures represented by two groups of gray blocks are used as reference points to mark the coding region. Figure 4 The sensing area of ​​this embodiment is shown. The sensing area is composed of three sensing units A, B, and C, each of which is composed of a probe and five DNA dumbbell structures. The five DNA dumbbell structures are used to mark different detection sites, while the probes are used to capture target DNA.

[0113] The above preparation method can be used to prepare other vectors with different codes or different probes. Each coding vector needs to be independently assembled due to the different short chain mixture combinations.

[0114] Example 2

[0115] Results collection for 27 coding vectors

[0116] The 27 encoding carrier mixtures were diluted into 4 M Li Cl buffer to a final carrier concentration of 0.25 nM and introduced into the tip side of the glass nanopore. Electrodes were placed on the opposite side of the nanopore and a 600 m V potential was applied to promote the transfer of negatively charged carrier molecules through the nanopore to generate characterization; characteristic current peak results were obtained after the carrier passed through the pore. The current signal was recorded using an Axopatch 200B patch clamp amplifier (Axon Instruments) at a sampling rate of 1 MHz and filtered by an external 50 kHz low-pass Bessel filter (900CT model, inverter). The signal was digitized using a 16-bit resolution data acquisition card (PCIe-6251 or PCIe-6351, National Instruments). Current analysis was performed using customized LabVIEW software and Python programs. Figure 6 The characteristic current peak results of 27 types of coding carriers after passing through the hole are shown as a standard diagram for subsequent detection comparison. Figure 5 This is a physical picture of the nanopore chip before sample addition.

[0117] Example 3

[0118] In the following experiments, the encoding DNA vector prepared in Example 2 was used, and the generated encoding signal was detected.

[0119] (1) First, order artificially synthesized dry powder of gene fragments that simulate real pathogens.

[0120] (2) Dissolve the simulated target sequence dry powder in nuclease-free water to prepare a 100 μM storage solution and store it at -20°C.

[0121] (3) The vector assembled in Example 1 was centrifuged and washed using a 100 kDa ultrafiltration membrane to remove excess unhybridized short chains, and the purified product was quantified and frozen for later use.

[0122] (4) The target nucleic acid and the DNA vector were incubated in TM buffer (10 mM Tris-HCl, 10 mM MgCl2, pH 8.0) at room temperature for 10 min. The target nucleic acid was recognized and captured by the corresponding complementary probe on the vector.

[0123] (5) The mixture was diluted into 4 M LiCl buffer to a final carrier concentration of 0.25 nM and introduced into the tip of the glass nanopore. Electrodes were placed on both sides of the nanopore and a 600 mV voltage was applied to promote the negatively charged carrier molecules to pass through the nanopore, generating a characteristic electrical signal.

[0124] (6) Using a patch clamp amplifier to collect the electrical signal of the through-hole, and obtaining the characteristic electrical signal of the carrier after the target nucleic acid is captured and passed through the hole; Figure 7 As shown, compared with the standard graph coded as "012" obtained in Example 3, the marker position C changes from a single peak to a double peak after capturing the target nucleic acid, proving that the DNA vector detects the target nucleic acid C.

Claims

1. A ternary-based DNA coding structure, characterized in that: The coding structure includes a coding area and a sensing area. The coding area includes two flag bits and three coding bits. The three coding bits are located between the two flag bits. The three coding bits are respectively a first coding bit, a second coding bit and a third coding bit. The value of each coding bit is selected from 0, 1 and 2. The nucleotide sequences of SEQ ID NO:1 to SEQ ID NO:11 are used to construct the first marker in the coding region of the DNA vector; A group of DNA dumbbell sequences from SEQ ID NO:12 to SEQ ID NO:22 are used to construct a second marker in the coding region of a DNA vector; A set of DNA dumbbell sequences of SEQ ID NO:23 to SEQ ID NO:29 are used to form the first coding position "1" in the ternary coding at the coding position of the DNA vector; A set of DNA dumbbell sequences of SEQ ID NO:30 to SEQ ID NO:36 are used to form "1" of the second coding position in the ternary coding at the coding position of the DNA vector; A set of DNA dumbbell sequences of SEQ ID NO:37 to SEQ ID NO:43 are used to form "1" of the third coding position in the ternary coding at the coding position of the DNA vector; A group of DNA sequences from SEQ ID NO:44 to SEQ ID NO:53 are respectively combined with the WI(0) sequence of SEQ ID NO:54 to form 10 DNA three-way structure sequences, which are used to form the first coding position "2" in the ternary code at the coding position of the DNA vector; A group of DNA sequences from SEQ ID NO:55 to SEQ ID NO:64 are respectively combined with the WII(0) sequence of SEQ ID NO:65 to form 10 DNA three-way structure sequences, which are used to form the second coding position "2" in the ternary code at the coding position of the DNA vector; A group of DNA sequences from SEQ ID NO:66 to SEQ ID NO:75 are respectively combined with the WIII(0) sequence of SEQ ID NO:76 to form 10 DNA three-way structure sequences, which are used to form the third coding position "2" in the ternary coding at the coding position of the DNA vector; If the coding position of the DNA vector is in a simple double-stranded state and does not form a sequence of "1" in the ternary code or "2" in the ternary code, then the coding position is "0" in the ternary code.

2. A ternary-based DNA coding structure according to claim 1, characterized in that: The sensing area includes at least one sensing unit, and the sensing unit includes corresponding probe positions and label positions; the probe is used to capture target DNA; The nucleotide sequence of SEQ ID NO:77-SEQ ID NO:78 is used to form a probe site on the DNA structure, and the corresponding label site is formed by SEQ ID NO:83-SEQ ID NO:88, constituting a sensing unit; The nucleotide sequence of SEQ ID NO:79-SEQ ID NO:80 is used to form a probe site on the DNA structure, and the corresponding label site is formed by SEQ ID NO:89-SEQ ID NO:93, constituting a sensing unit; The nucleotide sequence of SEQ ID NO:81-SEQ ID NO:82 is used to form a probe site on the DNA structure, and the corresponding label site is formed by SEQ ID NO:94-SEQ ID NO:98, forming a sensing unit.

3. A ternary-based DNA encoding library, characterized in that: The coding library comprises the DNA coding structure of claim 1 or 2, and the coding library comprises 27 codes: 000, 001, 002, 010, 011, 012, 020, 021, 022, 100, 101, 102, 110, 111, 112, 120, 121, 122, 200, 201, 202, 210, 211, 212, 220, 221, and 222.

4. A DNA vector, characterized in that: The DNA vector is a DNA vector comprising the coding structure according to claim 1 or 2.

5. The method for preparing a DNA vector according to claim 4, characterized in that: The following steps are involved: (1) Take the M13mp18 circular DNA solution, add buffer and oligo solution; raise the temperature to 50-70°C, and then reduce it to room temperature at a rate of 1-3°C / min; (2) Using BamHI-HF enzyme and EcoRI-HF enzyme to digest the mixed solution; (3) Purify the DNA using the DNA CleanUp Kit to obtain the cut M13mp18 single-stranded DNA; (4) Using the linear M13mp18 single-stranded DNA as the basic template, the template complementary short chain with the reserved functional site and the short chain of the functional site are mixed, and a buffer solution and MgCl2 solution are added to prepare a mixed solution; the temperature is raised to 60-80°C, and then cooled to room temperature at a rate of 1-3°C / min to obtain a linear double-stranded DNA vector containing the coding region and the sensing region; The short chain of functional bits is a short chain of flag bits, probe bits, marker bits, and coding bits.

6. The use of a DNA vector for non-disease diagnosis and treatment purposes as claimed in claim 4, characterized in that: The DNA vector is used for detecting target nucleic acid.

7. The use of a DNA vector for non-disease diagnosis and treatment purposes according to claim 6, characterized in that: The detection method includes the following steps: (1) The target nucleic acid to be detected is mixed with a linear double-stranded DNA vector containing a coding region and a sensing region; the target nucleic acid is recognized and captured by the corresponding complementary probe on the vector; (2) Inject 4M LiCl solution into the nanopore chip, and inject the DNA carrier diluted with 4M LiCl solution into the middle sample pool after capturing the target nucleic acid, and ensure that there are no bubbles in the chip; (3) The pore-passing event is collected by a patch clamp device to obtain the characteristic electrical signal of the carrier after the target nucleic acid is captured and passed through the pore. The detection of the target nucleic acid is confirmed by identifying the electrical signal.

8. The use of a DNA vector for non-disease diagnosis and treatment purposes according to claim 7, characterized in that: In step (3), a voltage is applied to the chip to allow the DNA molecule to pass through the nanopore, causing a current change, and the peak height and integrated area of ​​the current peak with a coding value of 2 are higher than the peak height and integrated area of ​​the current peak with a coding value of 1.

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