阅读说明：本技术 一种功能核酸磁生物传感器 (Functional nucleic acid magnetic biosensor ) 是由 许文涛 李舒婷 黄昆仑 朱龙佼 程楠 于 2021-02-08 设计创作，主要内容包括：本发明提供一种功能核酸磁生物传感器,包括：(1)BS-PCR扩增体系,(2)功能核酸磁层析分析体系。本发明通过巧妙地设计引物,使得在双链靶标存在下可对靶标进行超快扩增,并使扩增产物两端各带一个单链寡核苷酸。进一步利用构建的侧流磁层析试纸进行磁信号输出和分析,突破了目前功能核酸检测试纸信噪比高,信号分子不稳定的瓶颈,解决了传统PCR反应速度慢,产物难于可视化的难题,实现了对双链靶标的快速、高灵敏、可视化检测。不仅如此,本发明提供的磁传感器具有出色的长期重复磁信号读取的稳定性。(The invention provides a functional nucleic acid magnetic biosensor, which comprises: (1) a BS-PCR amplification system, and (2) a functional nucleic acid magnetic chromatography analysis system. The invention can carry out ultrafast amplification on the target in the presence of the double-stranded target by skillfully designing the primer, and two ends of the amplified product are respectively provided with a single-stranded oligonucleotide. The constructed lateral flow magnetic chromatography test paper is further utilized to output and analyze magnetic signals, the bottlenecks of high signal-to-noise ratio and unstable signal molecules of the existing functional nucleic acid detection test paper are broken through, the problems that the traditional PCR reaction speed is low and the product is difficult to visualize are solved, and the quick, high-sensitivity and visual detection of the double-chain target is realized. Furthermore, the magnetic sensor provided by the invention has excellent stability of repeated magnetic signal reading for a long time.)

1. A functional nucleic acid magnetic biosensor, comprising: (1) isolating an overspeed polymerase chain reaction BS-PCR amplification system, (2) a functional nucleic acid magnetic chromatography analysis system;

the sensor is used for performing functional nucleic acid magnetic chromatography analysis on products obtained after a sample to be detected sequentially reacts through the BS-PCR amplification system;

wherein the BS-PCR amplification system comprises: a forward partition adaptor primer and a reverse partition adaptor primer;

the functional nucleic acid magnetic chromatography analysis system comprises: chromatography buffer solution, magnetic signal probes, functional nucleic acid detection test paper and a functional nucleic acid magnetic chromatography detector.

2. The sensor of claim 1,

the forward blocking adaptor primer comprises: a general adaptor sequence A, a partition and a specific primer sequence B;

the reverse blocking adaptor primer comprises: a universal linker sequence A ', a partition and a specific primer sequence B';

the partition is positioned between the universal adaptor sequence and the specific primer sequence;

the nucleotide sequence of the specific primer sequence B, B 'is located at the 3' end of the forward block adapter primer and the reverse block adapter primer;

the spacers include structures that inhibit polymerase binding and/or structures that inhibit new strand extension during in vitro nucleic acid amplification reactions.

3. The sensor according to claim 1 or 2, wherein the concentration of the forward blocking adaptor primer is 1-5 μ M and the concentration of the reverse blocking adaptor primer is 1-5 μ M in the BS-PCR amplification system.

4. The sensor of claim 3, wherein the reaction process of the BS-PCR comprises: 95-98 ℃ for 0-10 s; at 55-65 deg.c for 25-40 cycles and 0-10 sec.

5. The sensor according to claim 4, wherein in the BS-PCR amplification system, the concentration of the forward blocking adaptor primer is 2 μ M, and the concentration of the reverse blocking adaptor primer is 2 μ M; the BS-PCR reaction process comprises the following steps: 96 ℃ for 4 s; 30 cycles at 60 ℃ for 6 s.

6. The sensor of claim 5, further comprising at least one of the following 1) -6):

1) the partition comprises a compound having a long chain structure;

2) the forward blocking adaptor primer comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 through the partition to obtain a primer;

3) the reverse blocking adaptor primer comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6 through the partition to obtain a primer;

4) universal linker sequence A, A 'is located 5' to the primer sequence;

5) the forward blocking adaptor primer comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO:1 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO:1 with the same function are connected through a partition to obtain a primer;

6) the reverse blocking adaptor primer comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 2 is substituted and/or deleted and/or added by one or more nucleotides and has a nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 2 with the same function through a partition.

7. The sensor according to claim 1, wherein the magnetic signal probe is a magnetic nanoparticle with a surface modified with a single-stranded nucleic acid sequence C;

the detection line of the functional nucleic acid detection test paper is modified with a single-stranded nucleic acid sequence D, and the quality control line is modified with a single-stranded nucleic acid sequence D';

the single-stranded nucleic acid sequence D is partially or completely complementary paired with the universal joint sequence A;

the single-stranded nucleic acid sequence D 'is partially or completely identical to the universal linker sequence A';

the single-stranded nucleic acid sequence D 'and the universal joint sequence A' are respectively complementary and paired with part or all of the single-stranded nucleic acid sequence C modified on the surface of the magnetic nano-particle.

8. The sensor of claim 1 or 7, wherein the functional nucleic acid magnetic tomography analysis process comprises: mixing 2-3 muL of a product obtained by BS-PCR reaction with 80-120 muL of chromatography buffer solution to form a sample to be detected, loading 1-3 muL of a magnetic signal probe on a bonding pad of functional nucleic acid detection test paper, inserting the detection test paper into the sample to be detected, changing the detection line of the test paper from colorless to brown after 3-10 minutes, observing with naked eyes, taking out the test paper, and placing the test paper into a functional nucleic acid magnetic chromatography detector to measure the strength of a magnetic signal.

9. The sensor of claim 8, further comprising at least one of the following 1) -4):

1) the magnetic signal probe includes: and (3) mixing the amino acid sequence shown in SEQ ID NO: 7 through interaction to modify the surface of the magnetic nano-particles to obtain a probe;

2) the functional nucleic acid detection test paper comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 8 at a detection line through interaction modification, and the nucleotide sequence shown in SEQ ID NO: 9, modifying the nucleotide sequence shown in the figure at the position of the quality control line through interaction to obtain detection test paper;

3) the magnetic signal probe includes: and (3) mixing the amino acid sequence shown in SEQ ID NO: 7 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 7, modifying the nucleotide sequence with the same function on the surface of the magnetic nano-particle through interaction to obtain a probe;

4) the functional nucleic acid detection test paper comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 8 is substituted and/or deleted and/or added by one or more nucleotides and has a nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 8 in the sequence table, modifying the nucleotide sequence with the same function at a detection line, and carrying out the following steps of: 9 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 9 has the same function, and is modified at the position of the quality control line through interaction.

10. Use of a sensor according to any one of claims 1 to 9 for detecting a double stranded nucleic acid target.

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a functional nucleic acid magnetic biosensor of a double-stranded nucleic acid target sequence.

Background

The PCR technology was developed in 1983 and is a typical nucleic acid temperature-variable amplification technology. Minute amounts of nucleic acid fragments can be increased millions of times in vitro. Since PCR has high sensitivity and specificity and a simple operation process, it has been applied to various fields such as food safety detection and supervision. However, PCR is not suitable for rapid on-site screening; and the amplification product is double-stranded, so that visual analysis is not easy to perform. Although many efforts have been made to reduce the time required for PCR amplification, there is still a need for improved speed, and simpler, and long-term stable signal readout for rapid field screening.

The side flow biosensor has the advantages of high detection precision, convenience, rapidness and the like. This is a visual analysis method developed for rapid screening. Compared with the immunochromatographic test paper based on the antibody, the lateral flow biosensor based on the functional nucleic acid has the advantages of wider target range, higher stability and lower cost. However, there are several factors that limit the development of functional nucleic acid strip technology. Firstly, the visualization and high-sensitivity detection of double-stranded DNA amplicons are difficult to realize simultaneously based on the traditional signal amplification method; secondly, due to the inherent characteristics of the signal molecules, the biological sample and the environment, the elimination of background interference is limited; the third is the stability of the signal molecule. Therefore, it is necessary to develop a novel functional nucleic acid test strip platform, which not only realizes visualization of double-stranded DNA amplicons, but also eliminates background interference and improves signal reading stability.

Magnetic materials on the nanometer scale have their own advantages. Firstly, the magnetic signal reading can resist background interference, and an actual biological sample has no magnetic background, so that high-sensitivity measurement can be realized without further processing the sample; secondly, the magnetic nanoparticles can provide qualitative visual output according to the aggregation-induced brown color thereof, and meanwhile, accurate magnetic signal intensity analysis is allowed to be carried out through the superparamagnetic signal response of the magnetic nanoparticles; thirdly, the magnetic signal has higher stability and longer decay period, which is helpful for establishing a stable screening platform. There are few reports on a functional nucleic acid lateral flow biosensor that uses magnetic nanoparticles as probes and outputs magnetic signals.

Disclosure of Invention

The invention aims to provide a functional nucleic acid magnetic biosensor.

Another purpose of the invention is to provide a method for rapidly and sensitively detecting a double-stranded nucleic acid target based on a partition overspeed PCR and functional nucleic acid magnetic chromatography test paper.

In order to realize the purpose of the invention, the inventor designs a general partition joint primer pair to perform partition overspeed Polymerase Chain Reaction (BS-PCR) according to a double-stranded nucleic acid target, and a Reaction product is analyzed by using newly constructed functional nucleic acid magnetic chromatography test paper, so that the novel functional nucleic acid magnetic biosensor based on the BS-PCR and the functional nucleic acid magnetic chromatography test paper is constructed.

In a first aspect, the present invention provides a functional nucleic acid magnetic biosensor comprising: (1) a BS-PCR amplification system, and (2) a functional nucleic acid magnetic chromatography analysis system.

The sensor detection system is used for performing functional nucleic acid magnetic chromatography analysis on products obtained after a sample to be detected sequentially reacts through the BS-PCR amplification system;

wherein the BS-PCR amplification system comprises: a forward partition adaptor primer and a reverse partition adaptor primer;

the functional nucleic acid magnetic chromatography analysis system comprises: chromatography buffer solution, magnetic signal probes, functional nucleic acid detection test paper and a functional nucleic acid magnetic chromatography detector.

Wherein the forward blocking adaptor primer comprises: a general adaptor sequence A, a partition and a specific primer sequence B;

the reverse blocking adaptor primer comprises: a universal linker sequence A ', a partition and a specific primer sequence B';

the partition is positioned between the universal adaptor sequence and the specific primer sequence;

the nucleotide sequence of the specific primer sequence B, B 'is located at the 3' end of the forward block adapter primer and the reverse block adapter primer;

the spacers include structures that inhibit polymerase binding and/or structures that inhibit new strand extension during in vitro nucleic acid amplification reactions.

The nucleotide sequence of the specific primer sequence B, B' specifically comprises a primer sequence designed according to the characteristic sequence of the double-stranded target fragment to be detected; the characteristic sequence comprises a characteristic sequence defined in the prior art or common general knowledge; the design includes the design methods described in the prior art or the common general knowledge.

Specifically, the sensor further comprises at least one of the following 1) to 2):

1) the concentration of the forward primer and the reverse primer in the reaction system of the BS-PCR reaction is more than 10 times of the concentration of the common PCR; specifically, 25 times;

2) the process of the BS-PCR reaction comprises the following steps: 95-98 ℃ for 0-10 s; at 55-65 deg.C for 25-40 cycles in 0-10 s; more specifically, the process of the BS-PCR reaction comprises: 96 ℃ for 4 s; 30 cycles at 60 ℃ for 6 s.

Specifically, the sensor further comprises at least one of the following 1) to 6):

1) the partition comprises a compound having a long chain structure;

2) the forward blocking adaptor primer comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 through the partition to obtain a primer;

3) the reverse blocking adaptor primer comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6 through the partition to obtain a primer;

4) universal linker sequence A, A 'is located 5' to the primer sequence;

5) the forward blocking adaptor primer comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO:1 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO:1 with the same function are connected through a partition to obtain a primer;

6) the reverse blocking adaptor primer comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 2 is substituted and/or deleted and/or added by one or more nucleotides and has a nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 2 with the same function through a partition.

More specifically, the partition is polyhexamethylene glycol.

Wherein the magnetic signal probe is a magnetic nanoparticle with a single-stranded nucleic acid sequence C modified on the surface.

The detection line of the functional nucleic acid detection test paper is modified with a single-stranded nucleic acid sequence D, and the quality control line is modified with a single-stranded nucleic acid sequence D'.

The single-stranded nucleic acid sequence D is partially or completely complementary paired with the universal joint sequence A;

the single-stranded nucleic acid sequence D 'is partially or completely identical to the universal linker sequence A';

the single-stranded nucleic acid sequence D 'and the universal joint sequence A' are respectively complementary and paired with part or all of the single-stranded nucleic acid sequence C modified on the surface of the magnetic nano-particle.

The A, A ', B, B ', C, D, D ' are used only to distinguish between different sequences and not for ordering.

The complementation includes complementation or reverse complementation defined by the prior art or the common general knowledge and/or complementation or reverse complementation according to the complementation principle defined by the prior art or the common general knowledge.

The test paper comprises test paper prepared by the prior art or common knowledge; the preparation method comprises the preparation method described in the prior art or common general knowledge.

Specifically, the functional nucleic acid magnetic chromatography analysis process comprises the following steps: mixing 2-3 muL of a product obtained by BS-PCR reaction with 80-120 muL of chromatography buffer solution to form a sample to be detected, loading 1-3 muL of a magnetic signal probe on a bonding pad of functional nucleic acid detection test paper, inserting the detection test paper into the sample to be detected, changing the detection line of the test paper from colorless to brown after 3-10 minutes, observing with naked eyes, taking out the test paper, and placing the test paper into a functional nucleic acid magnetic chromatography detector to measure the strength of a magnetic signal. Still more specifically, the functional nucleic acid magnetic chromatography analysis process comprises: mixing 2.5 mu L of a product obtained by BS-PCR reaction with 100 mu L of chromatography buffer solution to form a sample to be detected, loading 2 mu L of a magnetic signal probe on a bonding pad of the functional nucleic acid detection test paper, inserting the detection test paper into the sample to be detected, changing the color of a detection line of the test paper from colorless to brown after 5 minutes, observing by naked eyes, taking out the test paper, and placing the test paper into a functional nucleic acid magnetic chromatography detector to determine the strength of the magnetic signal.

Specifically, the sensor further comprises at least one of the following 1) to 4):

1) the magnetic signal probe includes: and (3) mixing the amino acid sequence shown in SEQ ID NO: 7 through interaction to modify the surface of the magnetic nano-particles to obtain a probe;

2) the functional nucleic acid detection test paper comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 8 at a detection line through interaction modification, and the nucleotide sequence shown in SEQ ID NO: 9, modifying the nucleotide sequence shown in the figure at the position of the quality control line through interaction to obtain detection test paper;

3) the magnetic signal probe includes: and (3) mixing the amino acid sequence shown in SEQ ID NO: 7 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 7, modifying the nucleotide sequence with the same function on the surface of the magnetic nano-particle through interaction to obtain a probe;

4) the functional nucleic acid detection test paper comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 8 is substituted and/or deleted and/or added by one or more nucleotides and has a nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 8 in the sequence table, modifying the nucleotide sequence with the same function at a detection line, and carrying out the following steps of: 9 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 9 has the same function, and is modified at the position of the quality control line through interaction.

The invention also provides the use of the aforementioned sensor for detecting double-stranded nucleic acid targets, which detection can be manifested as a qualitative detection.

In a second aspect, the present invention provides a method for qualitatively detecting a double-stranded nucleic acid target by using the sensor, comprising the following steps:

s1, adding a sample to be detected into the BS-PCR amplification system, and performing partition overspeed polymerase chain reaction to obtain a BS-PCR amplification product;

s2, performing magnetic chromatography analysis on the BS-PCR amplification product by using the functional nucleic acid magnetic chromatography analysis system.

S1 is specifically as follows: adding a sample to be detected into a BS-PCR amplification system, wherein the concentration of a forward partition joint primer in the system is 1-5 mu M, and the concentration of a reverse partition joint primer in the system is 1-5 mu M, and according to the program: performing 25-40 cycles of amplification at 95-98 deg.C for 0-10s, 55-65 deg.C for 0-10s to obtain BS-PCR amplification product.

The method also comprises the following specific steps: adding a sample to be detected into a BS-PCR amplification system, wherein the concentration of a forward partition joint primer in the system is 2 mu M, and the concentration of a reverse partition joint primer in the system is 2 mu M, and according to the program: performing 30 cycles of amplification at 96 ℃, 4s, 60 ℃ and 6s to obtain a BS-PCR amplification product.

S2 is specifically as follows: mixing 2-3 muL of a product obtained by BS-PCR reaction with 80-120 muL of chromatography buffer solution to form a sample to be detected, loading 1-3 muL of a magnetic signal probe on a bonding pad of functional nucleic acid detection test paper, inserting the detection test paper into the sample to be detected, changing the detection line of the test paper from colorless to brown after 3-10 minutes, observing with naked eyes, taking out the test paper, and placing the test paper into a functional nucleic acid magnetic chromatography detector to measure the strength of a magnetic signal.

And specifically, mixing 2.5 muL of a product obtained by BS-PCR reaction with 100 muL of chromatography buffer solution to form a sample to be detected, loading 2 muL of a magnetic signal probe onto a bonding pad of the functional nucleic acid detection test paper, inserting the detection test paper into the sample to be detected, changing the color of a detection line of the test paper from colorless to brown after 5 minutes, observing with naked eyes, taking out the test paper, and placing the test paper into a functional nucleic acid magnetic chromatography detector to measure the strength of the magnetic signal.

The invention provides a method for quickly and sensitively detecting a double-stranded nucleic acid target based on a partition overspeed PCR and functional nucleic acid magnetic chromatography test paper, which comprises the following steps:

firstly, designing a forward partition adaptor primer and a reverse partition adaptor primer, carrying out BS-PCR amplification on a sample containing a double-stranded nucleic acid target, wherein two ends of an amplified product are respectively provided with a section of universal adaptor sequence A and a section of universal adaptor sequence A'; modifying a single-stranded nucleic acid sequence C partially or completely complementary to a universal joint sequence A ' in a reverse partition joint primer on the surface of a magnetic nanoparticle to prepare a magnetic signal probe, modifying a single-stranded nucleic acid sequence D partially or completely complementary to the universal joint sequence A in the forward partition joint primer on a detection line of a test paper nitrocellulose membrane, modifying a single-stranded nucleic acid sequence D ' partially or completely identical to the universal joint sequence A ' in the reverse partition joint primer on a quality control line to prepare a functional nucleic acid detection test paper, and loading 1-3 mu L of the magnetic signal probe on a binding pad of the detection test paper; mixing 2-3 mu L of a product obtained by BS-PCR reaction with 80-120 mu L of chromatography buffer solution to form a sample to be detected, inserting the detection test paper loaded with the magnetic signal probe into the sample to be detected, changing the detection line of the test paper from colorless to brown after 3-10 minutes, and observing with naked eyes to further finish the quick visual detection of the double-stranded target. Or the test paper is taken out and put into a functional nucleic acid magnetic chromatography detector to measure the intensity of the magnetic signal for analysis.

The 5 'end of the forward partition joint primer is provided with a universal joint sequence A partially or completely complementary with a single-stranded nucleic acid sequence D on a detection line of the functional nucleic acid detection test paper, and the 3' end of the forward partition joint primer contains a specific primer sequence B capable of being combined with a target; the 5 'end of the reverse blocking joint primer is provided with a universal joint sequence A' partially or completely complementary with a single-stranded nucleic acid sequence C on the surface of the magnetic signal probe, and the 3 'end of the reverse blocking joint primer is provided with a specific primer sequence B' capable of being combined with a target; at least one partition is arranged between the 5 'end universal joint sequence and the 3' end specific primer sequence, and the partition can inhibit the combination of polymerase and/or can inhibit the extension of a new chain in the in vitro nucleic acid amplification reaction process.

Preferably, the partition is polyhexamethylene glycol.

The functional nucleic acid detection test paper is characterized in that a single-stranded nucleic acid sequence D at a detection line is partially or completely complementary to a universal joint sequence A, and a single-stranded nucleic acid sequence D 'at a quality control line is partially or completely complementary to a magnetic signal probe surface single-stranded nucleic acid sequence C'.

Preferably, in the method, the base sequences of the forward blocking adapter primer, the reverse blocking adapter primer, the single-stranded nucleic acid on the surface of the magnetic signal probe, and the single-stranded nucleic acid at the detection line and the quality control line of the functional nucleic acid detection test paper used in the detection of the double-stranded target are as follows:

forward blocking adaptor primer: 5 '-TTGGTCGTGGTGGTGGTTT-polyhexamethylene glycol-ACAGCCACCACTTCTCCTTG-3'

Reverse blocking adaptor primer: 5 '-CCTTCCCTCTTCCCCCC-polyhexamethylene glycol-CGGAAATGAAAGAAGGCTACCG-3'

Single-stranded nucleic acid on the surface of magnetic signaling probe: 5'-GGGGGGAAGAGGGAAGGTTTTT-3'

Single-stranded nucleic acid at detection line: 5'-AAACCACCACCACGACCAATTTTT-3'

Single-stranded nucleic acid at the control line: 5'-CCTTCCCTCTTCCCCCCTTTTT-3'

Wherein ttttttt denotes a spacer sequence.

The invention also provides a detection kit matched with the method, and the kit at least comprises the following components: a forward partition joint primer, a reverse partition joint primer, a chromatography buffer solution, a magnetic signal probe, functional nucleic acid detection test paper and the like.

The detection and analysis principle of the kit of the invention is as follows: firstly, designing a forward partition adaptor primer and a reverse partition adaptor primer, carrying out BS-PCR amplification on a sample containing a double-stranded nucleic acid target, wherein the partition in the primers prevents extension of polymerase, so that two ends of an amplified product are respectively provided with a single-stranded nucleic acid sequence; loading a magnetic signal probe on a combination pad of the functional nucleic acid detection test paper; mixing a product obtained by the BS-PCR reaction with a chromatography buffer solution to form a sample to be detected, inserting the detection test paper loaded with the magnetic signal probe into the sample to be detected, and changing the color of a detection line of the observation test paper from colorless to brown so as to complete the quick visual detection of the double-chain target. Or the test paper is taken out and put into a functional nucleic acid magnetic chromatography detector to measure the intensity of the magnetic signal for analysis.

The specific detection method comprises the following steps:

1) BS-PCR reaction: preparing a forward block adaptor primer, a reverse block adaptor primer, a double-stranded target, DNA polymerase, dNTP, reaction buffer and ddH₂A PCR reaction system consisting of O is used for carrying out BS-PCR amplification reaction;

2) functional nucleic acid magnetic chromatography analysis reaction: mixing 2-3 muL of a product obtained by BS-PCR reaction with 80-120 muL of chromatography buffer solution to form a sample to be detected, loading 1-3 muL of a magnetic signal probe on a bonding pad of functional nucleic acid detection test paper, inserting the detection test paper into the sample to be detected, changing the detection line of the test paper from colorless to brown after 3-10 minutes, observing with naked eyes, taking out the test paper, and placing the test paper into a functional nucleic acid magnetic chromatography detector to measure the strength of a magnetic signal.

Wherein, the formula of the chromatography buffer solution in the step 2) is as follows: 4 XSSC, 2% BSA, 0.05% Tween-20, 10 mM Tris-HCl and 0.002% TrtioX-100, pH 7.4.

Preferably, the BS-PCR reaction system in step 1) is as follows:

the PCR reaction procedure was as follows: 95-98 ℃ for 0-10 s; at 55-65 deg.c for 25-40 cycles and 0-10 sec.

The visual detection of the double-chain target can be realized by observing the existence of brown color at the detection line of the test paper by naked eyes; and (3) putting the test paper into a functional nucleic acid magnetic chromatography detector to measure the strength of the magnetic signal at the detection line for accurate analysis.

The detection sensitivity of the double-stranded target of the method is single copy.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention establishes a functional nucleic acid magnetic biosensor for the rapid and sensitive detection of double-stranded targets. Designing a partition joint primer to partition overspeed polymerase chain reaction of the double-stranded target, analyzing a reaction product by using newly constructed functional nucleic acid magnetic chromatography test paper, constructing a novel functional nucleic acid magnetic biosensor based on BS-PCR and the functional nucleic acid magnetic chromatography test paper, and providing a novel quick, sensitive and visual double-stranded target detection method. The detection time of the sample is greatly shortened, and the detection sensitivity is as low as single copy. The invention successfully breaks through the bottlenecks of high signal-to-noise ratio and unstable signal molecules of the current functional nucleic acid detection test paper, solves the problems of low reaction speed and difficult visualization of products of the traditional PCR, realizes the visualization and high-sensitivity detection of the double-stranded DNA amplicon by using the functional nucleic acid detection test paper, and has important practical significance for the real-time and rapid detection on site.

The method constructs a partition adaptor primer pair, carries out ultrafast amplification on the double-stranded target, reduces the time consumption of the traditional PCR process of about 3 hours to 5 minutes, and obviously reduces the time consumption of PCR reaction.

And (II) the use of the partition in the primer hinders the extension of polymerase, realizes the conversion of single-double strands of the PCR product and obtains the PCR product with single-stranded nucleic acid sequences at two ends.

Thirdly, functional nucleic acid magnetic chromatography detection test paper is constructed for the first time, magnetic signals are used as signal output, color change generated by aggregation induction of magnetic nanoparticles is used for visual qualitative analysis, and accurate analysis is carried out by measuring the magnetic field intensity by utilizing the superparamagnetic signal response of the magnetic nanoparticles to the magnetic field; the problems of background signal interference and signal molecule instability of the current functional nucleic acid detection test paper are solved.

And (IV) analyzing the BS-PCR amplification product by using the constructed functional nucleic acid magnetic chromatography test paper, solving the problem that the traditional PCR product is difficult to detect visually, and realizing the rapid, visual and high-sensitivity detection of the double-stranded DNA amplicon.

Drawings

FIG. 1 is a graph showing the results of the isolation of products of the ultrafast PCR reaction and analysis using functional nucleic acid magnetic chromatography detection paper in example 1 of the present invention; in FIG. 1 (a), lane 1: no target-forward and reverse blocking joint primer pair; lane 2: double-stranded target-common primer pair; lane 3: double-stranded target-forward partition adaptor primer-common reverse primer; lane 4: double-stranded target-common forward primer-reverse blocking adaptor primer; lane 5: double-stranded target-forward and reverse blocking adaptor primer pairs; 1-5 in FIG. 1 (b) corresponds to 1 to 5 in (a).

FIG. 2 shows the color intensity of the detection line of the functional nucleic acid detecting strip according to the present invention, which varies with the number of the double-stranded targets in example 1; wherein, the number of targets from 1 to 6 is respectively: 2X 10⁴、2×10³、2×10²、2×10¹、2×10⁰And 1X 10⁰Copy, 7 is blank.

FIG. 3 shows the variation of the difference between the magnetic signal intensity of the test group and the blank control at the detection line of the test paper according to the logarithm of the number of targets in example 1 of the present invention.

FIG. 4 shows the results of the specific detection of the biosensor in example 2 of the present invention.

FIG. 5 shows the verification result of the magnetic signal output stability of the biosensor in example 3 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

In the invention, the formula of the buffer solution for dissolving the magnetic signal probe comprises the following components: 20nM Na₃PO₄·12H₂O, 5% BSA, 0.25% Tween-20, 10% sucrose;

the formula of the buffer solution for magnetic chromatography is as follows: 4 XSSC, 2% BSA, 0.05% Tween-20, 10 mM Tris-HCl and 0.002% TrtioX-100, pH 7.4.

EXAMPLE 1 establishment of a functional nucleic acid magnetic biosensor

1. Experimental Material

10 XPCR buffer, EB nucleic acid dye, nucleic acid molecular weight standard 2000 DNA marker, dNTP, rTaq DNA polymerase, 20 XSSC, Bovine Serum Albumin (BSA), Tween-20, Tris (Tris), Triton X-100, MES, EDC, NHS, sucrose, etc. were purchased from Sigma company, USA. The experimental water was obtained from a Milli-Q pure water system.

The sequence design is as follows (SEQ ID NOS: 1-2, 7-9):

note: TTTTT represents a spacer sequence so as not to affect the function of the modified single-stranded nucleic acid;

the single-stranded nucleic acid on the surface of the magnetic signal probe is complementarily paired with the 5' end of the reverse partition joint primer and the single-stranded nucleic acid at the quality control line;

the 5' end of the forward blocking adapter primer is complementarily paired with the single-stranded nucleic acid at the detection line.

2. Construction and validation of magnetic signal probes

Preparing 50 mg/mL EDC and NHS solutions with 25 mM MES buffer (pH = 6); taking carboxyl modified magnetic nano ferroferric oxide, washing the magnetic nano ferroferric oxide by MES buffer solution for multiple times, and then suspending the magnetic nano ferroferric oxide; adding the same amount of EDC and NHS to the heavy suspension respectively, and activating at 37 ℃ for about 1 h; then washing with MES buffer solution for many times and then resuspending; adding a certain amount of amino modified single-stranded nucleic acid, and carrying out rotary incubation for about 1 h at room temperature. And washing for multiple times by using MES buffer solution to remove residual uncoupled nucleic acid, re-suspending the coupled magnetic nanoparticles by using a dissolving buffer solution, completing the preparation of the magnetic signal probe, and storing at 4 ℃ for later use.

And performing Zeta potential and dynamic light scattering characterization on the prepared magnetic signal probe by using a Zetasizer Nano ZS analyzer. The successful preparation of the magnetic signal probe is proved by the reduction of the Zeta potential and the increase of the particle size after the surface of the magnetic nano-particle is modified with the single-stranded nucleic acid.

3. Establishment and verification of functional nucleic acid magnetic biosensor

The double-chain target partition overspeed PCR system is as follows:

a20. mu.L reaction system was prepared on ice and quickly placed in an ultra-fast PCR reaction apparatus for temperature control. Ultra-speed PCR reaction procedure: 96 ℃ for 4s, 60 ℃ for 6s, 30 cycles, for a total of 5 min.

Completing the process of the overspeed PCR reaction, and verifying the amplification effect of the overspeed PCR reaction system by using 2% agarose gel electrophoresis, wherein the reaction conditions are as follows: 120V 0.5h, photographing system: molecular Imager Gel Doc XR (Bio-Rad).

Mixing 2.5 mu L of a product obtained by PCR reaction with 100 mu L of chromatography buffer solution to form a sample to be detected, loading 2 mu L of a magnetic signal probe on a bonding pad of a functional nucleic acid detection test paper, inserting the detection test paper into the sample to be detected, and observing a detection line of the test paper by naked eyes to turn from colorless to brown after 5 minutes.

Experimental results show that the successful design of the partition joint primer can perform overspeed PCR amplification, and the amplification product can be subjected to functional nucleic acid magnetic chromatography visualization analysis (figure 1).

4. Ultrasensitive visual rapid detection of double-chain target

According to the above-mentioned optimized system, respectively adding different quantities of 2X 10⁴ copies、2×10³ copies、2×10²copies、2×10¹ copies、2×10⁰copies and 1X 10⁰copy genome sample is subjected to partition overspeed PCR amplification, amplification products are subjected to magnetic chromatography analysis by using functional nucleic acid detection test paper, and brown with different depths, which can be seen by naked eyes, appears at the test line of the test paper (figure 2).

The test paper is put into a functional nucleic acid magnetic chromatography detector to measure the magnetic signal intensity at the detection line, and a curve (figure 3) is drawn according to the change of the difference value of the magnetic signal intensity of the experimental group and the blank control along with the logarithm of the number of the genome.

When the number of the genome is as low as a single copy, a macroscopic brown band also appears at the detection line of the test paper, the intensity of the magnetic signal is higher, and the difference with a blank control is obvious, so that the detection sensitivity of the MON810 is as low as a single copy.

Example 2 investigation of the specificity of the sensor

Biosensors constructed as in example 1, 2X 10 each²Transgenic maize MON810 genome of copies, 2X 10⁴The genome of the transgenic corn MIR604 and GA21, the transgenic soybean MON87705 and MON87769 and the transgenic rape RF3 of copies are added into the system for detection, and the result shows that the established magnetic biosensor has better specificity (figure 4).

Example 3 magnetic Signal stability verification

The 2-fold gradient diluted magnetic signal probe is used for performing functional nucleic acid magnetic chromatography analysis, the obtained test paper is stored at room temperature for one month, then the functional nucleic acid magnetic chromatography detector is used for measuring the magnetic signal intensity at the detection line again, and the magnetic signal intensity is compared with the measurement result before one month (figure 5).

The test paper is stored for one month, the strength of the measured magnetic signal is not obviously changed compared with that before one month, and the magnetic signal has excellent stability as a signal output.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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