Carrier assembly and application thereof

Abstract

The invention provides a carrier assembly and application thereof, and belongs to the technical field of biology. Wherein the carrier assembly comprises a support layer, the support layer is arranged on the surface of the carrier, and the support layer comprises a plurality of separated areas which are not connected; and a plurality of monomer units, the plurality of monomer units are arranged in the separation area, and the monomer units comprise a target sequence. Wherein the target sequence is connected with the support layer. The vector assembly provided by the embodiment of the invention can be used for simultaneous capture, amplification and high-sensitivity detection of one or more types of nucleic acids.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, to a vector assembly and its application, especially to a vector assembly, a nucleic acid amplification method, a method for detecting the expression amount of multiple genes, and a nucleic acid sequencing method. BACKGROUND

[0002] In the past few decades, the research on nucleic acids (including DNA and RNA) has attracted extensive attention in the field of clinical transformation. This is mainly because DNA can guide the synthesis of functional proteins, so the variation of DNA sequence (such as insertion, deletion, single nucleotide polymorphism) plays a key role in the determination of cell function, indicating its important potential as a biomarker in disease diagnosis. In the field of clinical diagnosis and prevention of infectious diseases, there is an urgent need for rapid and accurate detection methods. Nucleic acid detection technology (NATs) has thus become a rapidly developing field.

[0003] In recent years, the rapid interpretation of human genome sequences has accelerated the progress of biomedical research, providing an effective means for discovering disease-related genes and cellular pathways. In particular, DNA microarray technology has significantly improved the sensitivity of chromosomal imbalance detection, having a positive impact on the diagnosis of childhood diseases such as major intellectual disabilities and birth defects.

[0004] Inspired by DNA replication and RNA transcription in cells, in vitro nucleic acid detection technologies have been widely developed to improve the specificity, sensitivity, and ease of operation of detection. However, existing in vitro nucleic acid detection technologies still have deficiencies in DNA molecule detection.

[0005] Therefore, the present application proposes a multi-dimensional nucleic acid detection method.

[0006] SUMMARY

[0007] The present application is based on the following findings of the inventors:

[0008] Currently, in vitro nucleic acid detection technologies are mainly divided into two categories: one is quantitative PCR technology (qPCR) based on nucleic acid pairing and replication amplification principles, and the other is microarray gene chip technology based on gene hybridization principles. However, the qPCR technology has limitations such as limited number of tests, complex design, and high demand for significant fluorescent probes; while the microarray gene chip technology faces challenges such as low capture rate, difficulty in accurate detection at low concentration, and complex production.

[0009] The present application aims to solve at least one of the related technical problems to some extent. To this end, the present application proposes a multi-dimensional nucleic acid detection method.

[0010] Specifically, the technical solution of the present application is as follows:

[0011] In a first aspect, the present application provides a carrier assembly. According to embodiments of the present application, the carrier assembly comprises a support layer disposed on a surface of the carrier, the support layer comprising a plurality of unconnected partition regions; and a plurality of monomer units disposed on the partition regions, the monomer units comprising a target sequence; wherein the target sequence is connected to the support layer. The carrier assembly according to embodiments of the present application can be used for simultaneous capture, amplification and detection of one or more types of nucleic acids.

[0012] According to embodiments of the present application, the carrier assembly can further comprise at least one of the following technical features:

[0013] According to embodiments of the present application, the target sequence is connected to the support layer by covalent bonding or non-covalent bonding. The non-covalent bonding is selected from at least one of van der Waals force, hydrogen bond, ionic interaction and hydrophobic interaction.

[0014] According to embodiments of the present application, the target sequence is connected to the support layer by covalent bonding. According to some specific embodiments of the present application, the 5' end of the target sequence is connected to the support layer by wherein n1 is an integer between 1 and 4; n2 is an integer between 1 and 3. Optionally, n1 is 1, 2, 3 or 4; n2 is 1, 2 or 3.

[0015] According to embodiments of the present application, the target sequences comprised in the plurality of monomer units are the same or different. The same target sequence can be used for capture, amplification and detection of the same type of nucleic acid; different target sequences can be used for capture, amplification and high-sensitivity detection of multiple different types of nucleic acids.

[0016] According to embodiments of the present application, the target sequence is selected from a primer sequence.

[0017] The present application does not make specific limitations on the support layer for fixing the target sequence. For example, the support layer can be selected from at least one of vesicles, colloids, biological membranes, droplets, silica, silica stone, glass, ceramic, silicon, quartz and plastic. In some preferred examples of the present application, the support layer is selected from silicon. The silicon includes silica.

[0018] According to embodiments of the present application, the distance between the same target sequences is 0.5-500 nm, which can be 1 nm, 10 nm, 20 nm, 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm or 500 nm. The arrangement of the target sequences based on the distance can ensure the effective performance of the PCR reaction, avoid competitive amplification or mutual interference between primers, thereby improving the amplification efficiency and product quality.

[0019] According to an embodiment of the present application, the dispersion degree of the aforementioned plurality of monomer units on the aforementioned partition region is 1-1000 μm, that is, the center distance of adjacent partition regions does not exceed 1000 μm, thereby ensuring effective performance of different types of nucleic acid amplification and avoiding mutual interference in the amplification process.

[0020] Illustratively, the aforementioned support layer surface has a predetermined plurality of partition regions, each of which indicates a plurality of target sequences (such as primer sequences) that are combined again. The same partition region can combine the same target sequence or different target sequences, and different partition regions can combine the same target sequence or different target sequences, based on which the aforementioned target sequence can be used to capture a nucleic acid template for amplification. For example, for qualitative or quantitative detection of the same type of nucleic acid, the same type of target sequence is arranged in different partition regions; if different types of nucleic acids are to be qualitatively or quantitatively detected, different target sequences are arranged in different partition regions. Based on the carrier assembly, simultaneous amplification of multiple nucleic acids can be achieved.

[0021] In a second aspect of the present application, a method for preparing the aforementioned carrier assembly is provided. According to an embodiment of the present application, the aforementioned method comprises: performing first contact treatment of a support layer connected with a maleimide and a target sequence, the 5' end of the target sequence being connected with a thiol group, and the target sequence being used for amplification of a nucleic acid to be detected. A stable thioether bond is formed through specific reaction between the thiol group of the target sequence and the maleimide, thereby preparing a carrier assembly with stable performance.

[0022] In the preparation process of the aforementioned carrier assembly, the target sequence can be flexibly designed based on experimental requirements.

[0023] According to an embodiment of the present application, the method for preparing the aforementioned carrier assembly can further comprise at least one of the following technical features:

[0024] According to an embodiment of the present application, the aforementioned support layer connected with a maleimide is obtained by: performing amination treatment on a support layer with a hydroxyl-modified surface; and performing second contact treatment of the support layer after amination treatment and 3-(maleimido)propionic acid N-hydroxysuccinimidyl ester, so as to obtain the aforementioned support layer connected with a maleimide. The aforementioned amination treatment is performed by performing third contact treatment of the support layer with a hydroxyl-modified surface and (3-aminopropyl)trimethoxysilane.

[0025] According to embodiments of the present application, the aforementioned third contact treatment is performed in a mixture of (3-aminopropyl)trimethoxysilane and anhydrous ethanol. The volume ratio of (3-aminopropyl)trimethoxysilane to anhydrous ethanol is 1:15. In some examples of the present application, the volume ratio of (3-aminopropyl)trimethoxysilane to anhydrous ethanol can also be selected as 1:14 or 1:16.

[0026] According to embodiments of the present application, the aforementioned third contact treatment time is 2 hours.

[0027] According to embodiments of the present application, the aforementioned support layer with hydroxyl modification is obtained by treating the support layer to be hydroxylated with hydrogen peroxide and concentrated sulfuric acid. In some specific embodiments of the present application, the aforementioned hydrogen peroxide is selected as a 30% hydrogen peroxide solution.

[0028] According to embodiments of the present application, the aforementioned support layer to be hydroxylated is treated with hydrogen peroxide and concentrated sulfuric acid for 30 min. Too long reaction time can cause side reactions, while too short reaction time can result in incomplete reaction. The inventors have verified through a large number of experiments that treating the support layer to be hydroxylated with hydrogen peroxide and concentrated sulfuric acid for 30 min can fully carry out the reaction, avoid side reactions, and make the dispersion of target sequences on the surface of the support layer and the distance between target sequences suitable for the simultaneous capture and amplification of nucleic acids.

[0029] According to embodiments of the present application, the aforementioned support layer is selected from at least one of vesicles, colloids, biological membranes, droplets, silica, silica stone, glass, ceramic, silicon, quartz, and plastic. According to some preferred embodiments of the present application, the aforementioned support layer is selected from silicon, preferably silicon dioxide.

[0030] Based on the aforementioned method for preparing a carrier assembly, in one specific example of the present application, the aforementioned carrier assembly is obtained as shown in Figure 3 .

[0031] In a third aspect of the present application, a nucleic acid amplification method is provided. According to embodiments of the present application, the aforementioned method comprises: performing a capture treatment on a nucleic acid to be amplified using the carrier assembly of any one of the first aspect, at least part of the sequence upstream of the 3' end of the aforementioned nucleic acid to be amplified is suitable for complementary pairing with at least part of the sequence upstream of the 3' end of the target sequence of the aforementioned carrier assembly; and performing an amplification reaction using the aforementioned target sequence as a primer and the aforementioned nucleic acid to be amplified as a template. The nucleic acid amplification method according to embodiments of the present application can achieve the simultaneous amplification and detection analysis of multiple nucleic acids.

[0032] According to embodiments of the present application, the aforementioned nucleic acid amplification method can further comprise at least one of the following technical features:

[0033] According to embodiments of the present application, the aforementioned amplification reaction is performed in an amplification reaction system, and the amplification reaction system further contains dNTPs.

[0034] According to embodiments of the present application, the aforementioned dNTPs are labeled with fluorescence. Based on the dNTPs labeled with fluorescence, the detection of nucleic acid sequences can be achieved on the carrier assembly.

[0035] According to some embodiments of the present application, the aforementioned amplification reaction system contains a plurality of fluorescent probes, and the aforementioned fluorescent probes are used to label target gene sequences. Through the labeling of different gene sequences by different fluorescent probes, the simultaneous detection of a plurality of nucleic acids in one reaction system can be achieved.

[0036] According to embodiments of the present application, the aforementioned nucleic acid amplification method further comprises: 1) denaturing the amplification reaction product; 2) the at least partial sequence upstream of the 3' end of the amplification single strand obtained after the denaturation is suitable for the second complementary pairing with the at least partial sequence upstream of the 3' end of the nearest adjacent target sequence; 3) performing a second amplification reaction with the nearest adjacent target sequence as a primer and the amplification single strand as a template. Through the cycle of steps 1) to 3), the amplification product of a predetermined number of nucleic acids to be amplified can be obtained.

[0037] Exemplarily, the above-mentioned method is used to amplify a single nucleic acid molecule. When amplifying a single nucleic acid molecule, the carrier assembly only contains one primer, and the sequence upstream of the 3' end of the primer is used to capture the sequence upstream of the 3' end of the nucleic acid to be amplified. After the primer on the carrier assembly captures the nucleic acid to be amplified, an extension reaction is performed in a reaction system containing dNTPs and DNA polymerase, with the nucleic acid to be amplified as a template, to form an amplification product Figure 1 ).

[0038] Then, through denaturation treatment (such as under the condition of 95°C), the amplification product is dissociated into two single-stranded DNAs. Under the annealing condition, the at least partial sequence upstream of the 3' end of the amplification single-stranded DNA is secondarily complementary paired with the at least partial sequence upstream of the 3' end of any of the primers in the vicinity. Then, a second extension reaction is performed in a reaction system containing dNTPs and DNA polymerase, with the amplification single-stranded DNA as a template. Through the cycle of the above-mentioned steps, a large amount of amplification product of the nucleic acid molecule can be obtained Figure 2 ), thereby realizing the efficient amplification of a single nucleic acid molecule.

[0039] In another example of the present application, a plurality of nucleic acid molecules are amplified by the above method. When a plurality of nucleic acid molecules are amplified simultaneously, the carrier assembly contains multiple types of target sequences (different target sequences are located in different compartments), and the same 3' end upstream sequence of the primers is used to capture the 3' end upstream sequence of the nucleic acid to be amplified. After the primers on the carrier assembly capture the nucleic acid to be amplified, an extension reaction is performed in a reaction system containing dNTP and DNA polymerase, using the nucleic acid to be amplified as a template, to form an amplification product.

[0040] Subsequently, the amplification products in different regions are all dissociated into two single-stranded DNAs by denaturation treatment (e.g., at 95°C). Under annealing conditions, at least part of the sequence upstream of the 3' end of each single-stranded DNA amplified in different regions is paired with at least part of the sequence upstream of the 3' end of any of the primers in the vicinity. Then, a second extension reaction is performed in a reaction system containing dNTP and DNA polymerase, using the single-stranded DNA as a template. By repeatedly performing the above steps, a large amount of amplification products of each nucleic acid molecule can be obtained, thereby achieving efficient amplification of multiple nucleic acid molecules simultaneously in one reaction. Figure 7 ).

[0041] In a fourth aspect of the present application, a method for detecting the expression amount of multiple genes is provided. According to an embodiment of the present application, the above method comprises: capturing and amplifying a plurality of genes to be detected by the method of the third aspect; detecting the fluorescence signal of the amplification product; and determining the expression amount of the plurality of genes to be detected based on the intensity and type of the fluorescence signal. Based on this method, the simultaneous detection of the expression amount of one or more genes in one reaction system can be achieved.

[0042] According to an embodiment of the present application, the above method for detecting the expression amount of multiple genes can further comprise at least one of the following technical features:

[0043] According to an embodiment of the present application, the above determination of the expression amount of the plurality of genes to be detected based on the intensity and type of the fluorescence signal is performed by: the carrier assembly comprises a plurality of non-connected compartments, and the plurality of non-connected compartments contain at least one primer, each compartment contains at least one primer, and the primer is used to amplify the gene to be detected, each gene to be detected corresponds to a fluorescence probe; and the expression amount of the plurality of genes to be detected is determined based on the positional relationship of the plurality of non-connected compartments and the type of the fluorescence signal.

[0044] In some examples of the present application, the aforementioned carrier assembly comprises N compartments, the N compartments comprise the same primer sequence, and one target gene is detected based on one fluorescent probe. Specifically, when detecting the expression level of one gene, the carrier assembly is mixed with amplification reagents containing templates, and one fluorescent dye is added to label the amplified DNA. Based on the brightness of the fluorescent signal, combined with a standard curve or a relative quantitative method, the expression level of one gene is quantitatively analyzed.

[0045] In some examples of the present application, the aforementioned carrier assembly comprises N compartments, the primer sequences of different compartments are different, and the primer sequences of each compartment are the same, and N target genes can be simultaneously detected based on one fluorescent probe. Specifically, the carrier assembly is mixed with amplification reagents containing templates, and one fluorescent dye is added to label the amplified DNA. Based on the brightness of the fluorescent signal of different compartments, combined with a standard curve or a relative quantitative method, the expression levels of N genes are quantitatively analyzed.

[0046] In some examples of the present application, the aforementioned carrier assembly comprises N compartments, the primer sequences of different compartments are different, and the primer sequences of each compartment are the same, and N target genes can be simultaneously detected based on one fluorescent probe. Specifically, the carrier assembly is mixed with amplification reagents containing templates, and one fluorescent dye is added to label the amplified DNA. Based on the brightness of the fluorescent signal of different compartments, combined with a standard curve or a relative quantitative method, the expression levels of N genes are quantitatively analyzed.

[0047] In some examples of the present application, the content of the target nucleic acid molecules in the sample solution can also be qualitatively or quantitatively detected by staining or electrochemical labeling methods.

[0048] In a fifth aspect of the present application, a genotyping method is provided. According to an embodiment of the present application, the aforementioned method comprises: detecting the expression levels of a plurality of target genes based on the method of the fourth aspect; and determining the genotype of the target genes based on the expression levels of the plurality of target genes. The method according to the embodiment of the present application can quickly and accurately detect the genotype of the sample.

[0049] For example, a SNP site in any genome is typed, assuming that the site has two alleles: A and B. The A gene primer is immobilized in the first compartment of the carrier assembly, and the B gene primer is immobilized in the second compartment. The carrier assembly is mixed with the amplification reagent containing the template, and 1 type of dye-labeled amplification DNA is added. If only the first compartment produces a fluorescence signal, the sample is homozygous, carrying the A allele; if only the second compartment produces a fluorescence signal, the sample is homozygous, carrying the B allele; if the first compartment and the second compartment both produce a fluorescence signal, the sample is heterozygous, carrying the A allele and the B allele.

[0050] In a sixth aspect of the present application, a nucleic acid sequencing method is provided. According to an embodiment of the present application, the aforementioned sequencing method comprises: amplifying the nucleic acid to be tested by the method of the third aspect of the present application; performing an adapter ligation on the amplification product; performing an adapter ligation on the amplification product; and performing a sequencing on the adapter-ligated product to obtain a sequencing result. Based on the method, efficient and accurate sequencing of the nucleic acid molecule to be tested can be achieved.

[0051] Advantages

[0052] The carrier assembly of the present application can be used for detecting target nucleic acid molecules in multiple dimensions. By immobilizing the same or different primer sequences in different compartments of the carrier assembly, the target nucleic acid molecules can be selectively captured, amplified, and detected. The method does not require the sample to be tested to be divided, and multiple target genes can be identified and qualitatively or quantitatively analyzed in one reaction system.

[0053] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0054] The above and / or additional aspects and advantages of embodiments of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0055] Figure 1 is a schematic diagram of surface DNA amplification of the carrier assembly provided by an embodiment of the present application;

[0056] Figure 2 is a schematic diagram of continuous surface DNA amplification of the carrier assembly provided by an embodiment of the present application;

[0057] Figure 3 is a schematic diagram of the synthesis route of the carrier assembly provided by an embodiment of the present application;

[0058] Figure 4is a schematic diagram of a verification result of primer connection on a surface of a carrier assembly provided by an embodiment of the present application.

[0059] Figure 5 is a schematic diagram of an amplification reaction result of a carrier assembly provided by an embodiment of the present application.

[0060] Figure 6 is a schematic diagram of a result of fluorescence intensity of experiments (groups) 1-6 provided by an embodiment of the present application.

[0061] Figure 7 is a schematic diagram of a carrier assembly amplification based on multiple primers provided by an embodiment of the present application. DETAILED DESCRIPTION

[0062] Embodiments of the present application are described in detail below. The present application can be implemented in many different forms and is not limited to the embodiments described herein. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application and cannot be understood as limiting the present application.

[0063] In this document, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. "A set" or "a plurality" refers to two or more.

[0064] In this document, "comprising" or "including" is an open expression that includes the stated elements or steps, but also includes additional elements or steps not expressly stated or shown.

[0065] The present application is illustrated below by way of examples, but this should not be understood as limiting the scope of the subject matter of the present application to the examples below. Any technology implemented based on the above description of the present application falls within the scope of the present application. The compounds or reagents used in the following examples can be obtained commercially or prepared by conventional methods known to those skilled in the art; the experimental instruments used can be obtained commercially.

[0066] Example 1: Preparation of a carrier assembly

[0067] In this embodiment, a silicon medium carrier layer is selected as a support layer for the preparation of a carrier assembly, and two different primer sequences (T1 and T2) are successfully connected on the surface of the carrier assembly.

[0068] 1. Hydroxylation of the surface of a silicon wafer

[0069] In the fume hood, the cut silicon wafer was placed in a beaker and washed with deionized water three times, the water was poured out, and 5 mL of 30% H2O2 solution was immediately added to the beaker with a pipette, then 15 mL of concentrated sulfuric acid was added with a pipette, and the mixture was slowly shaken and mixed uniformly, then left to stand for 30 min. After pouring off the reaction solution, it was washed with deionized water three times, and the hydroxylated silicon wafer was stored in a large amount of deionized water.

[0070] 2. Amino-functionalization of the silicon wafer surface

[0071] The silicon wafer treated in step 1 was washed with anhydrous ethanol three times, and after pouring off the washing solution, APTES:C2H5OH = 1:15 (v / v) mixed solution was quickly added, and shaken for 2 h. After the reaction was completed, the reaction solution was poured off and washed with anhydrous ethanol three times.

[0072] 3. Maleimide group on the surface of the silicon wafer

[0073] 1 mg of 3-maleimide propionic acid N-hydroxysuccinimide ester (BMPS) solid was dissolved in 40 μL of anhydrous ethanol, and after being blown and mixed uniformly, it was reserved. 360 μL of PBS buffer solution (pH = 8) was added to the BMPS mixture, and after being blown and mixed uniformly, 6 μL of anhydrous ethanol was used to wet the surface of the silicon wafer, and 100 μL of the BMPS solution was quickly added to the surface of the silicon wafer with a pipette gun, and the reaction was carried out at room temperature overnight. The silicon wafer was washed with PBS (pH = 8) buffer solution three times.

[0074] 4. Thiol-containing primer connection

[0075] The silicon wafer treated in step 3 was wetted with 6 μL of anhydrous ethanol, and 100 μL of 1 μm thiol primer mixture (which can match the new crown N gene template plasmid) was quickly added to the surface of the silicon wafer, and a control group of 100 μL of PBS (pH = 8) buffer solution was set up, and the reaction was carried out at room temperature overnight. After washing with PBS (pH = 7.2) buffer solution repeatedly, the silicon wafer was dried and reserved.

[0076] 5. Primer verification on the surface of the silicon wafer

[0077] A small amount of matched fluorescent marker 10 μm was added to each of the experimental group and the control group in step 4, incubated for 15 min, washed with TE buffer, and dried with nitrogen, and the difference in fluorescence intensity between the experimental group and the control group was observed under a fluorescence microscope.

[0078] The results are shown in Table 1 and Figure 4 The difference in light intensity between the experimental group and the control group was higher than 150, indicating that the thiol-containing primer was successfully introduced to the surface of the silicon wafer in the experimental group, and a higher fluorescence signal was generated.

[0079] Table 1

[0080]

[0081] Example 2: Verification of carrier assembly amplification reaction

[0082] This example is based on the carrier assembly prepared in Example 1 to carry out a primer amplification experiment.

[0083] The silicon wafer in Example 1 was placed in a PCR tube containing 30 μL of amplification reagent containing the new crown N gene template plasmid, and a certain amount of SYBR green was added for detecting whether double-stranded amplification occurred on the surface, and the surface amplification was carried out following the amplification procedure of 95°C for 1 min pre-denaturation, 40 cycles (95 degrees for 5 s, 60 degrees for 10 s). After the reaction, the silicon wafer was washed with ultrapure water and observed under a fluorescence microscope for the fluorescence intensity on the surface. At the same time, different plasmid control groups (no amplification plasmid, new crown N gene template plasmid, NADC-30 plasmid, new crown RNA) were set, and unmodified primer silicon wafer was used as a primer specificity control group (new crown N gene template plasmid, no amplification plasmid). The specific experimental scheme is as follows:

[0084] Experiment (group) 1: with primer, no plasmid;

[0085] Experiment (group) 2: with primer, with amplification plasmid;

[0086] Experiment (group) 3: with primer, with NADC-30 plasmid;

[0087] Experiment (group) 4: with primer, new crown RNA;

[0088] Experiment (group) 5: no primer, with amplification plasmid;

[0089] Experiment (group) 6: no primer, no plasmid.

[0090] The new crown N gene amplification primer and amplification sequence used in the above scheme are shown in Table 2.

[0091] The results are shown in Table 2. Figures 5-6

[0092] Table 2

[0093]

[0094] ​In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0095] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary, and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments without departing from the principles and spirits of the present application within the scope of the present application.

Claims

1. A carrier component, characterized in that, include: A support layer is disposed on the surface of the carrier, and the support layer includes several non-contiguous partitioned areas; as well as A plurality of single-unit cells are disposed in the partition region, and each single-unit cell includes a target sequence; The target sequence is connected to the support layer.

2. The carrier assembly according to claim 1, characterized in that, The connection is selected from covalent bonds or non-covalent bonds; Optionally, the non-covalent connection is selected from at least one of van der Waals forces, hydrogen bonds, interionic interactions, and hydrophobic interactions.

3. The carrier assembly according to claim 2, characterized in that, The connection is selected from covalent bonds; Optionally, the 5' end of the target sequence is... It is connected to the support layer, wherein n1 is an integer between 1 and 4; and n2 is an integer between 1 and 3.

4. The carrier assembly according to claim 3, characterized in that, The target sequences included in the plurality of individual units may be the same or different; Optionally, the target sequence is selected from primer sequences.

5. The carrier assembly according to claim 3, characterized in that, The support layer is selected from at least one of vesicles, colloids, biofilms, droplets, silica, silica, glass, ceramics, silicon, quartz and plastics, preferably silicon.

6. The carrier assembly according to claim 4, characterized in that, The distance between identical target sequences is 0.5-500 nm.

7. The carrier assembly according to claim 4, characterized in that, The dispersion of the several monomer units in the partition region is 1-1000 μm.

8. A method for nucleic acid amplification, characterized in that, include: The vector assembly according to any one of claims 1 to 7 is used to capture and process the nucleic acid to be amplified, wherein at least a portion of the sequence upstream of the 3' end of the nucleic acid to be amplified is adapted to be complementary to at least a portion of the sequence upstream of the 3' end of the target sequence of the vector assembly; The target sequence is used as a primer and the nucleic acid to be amplified is used as a template for the amplification reaction.

9. The method according to claim 8, characterized in that, The amplification reaction is carried out in an amplification reaction system, which further contains dNTPs; Optionally, the dNTPs are fluorescently labeled.

10. The method according to claim 9, characterized in that, The amplification reaction system contains a variety of fluorescent probes, which are used to label the target gene sequence.

11. The method according to claim 8, characterized in that, Further includes: 1) Denature the amplification reaction products; 2) At least a portion of the sequence upstream of the 3' end of the amplified first strand obtained after the denaturation treatment is adapted to perform a second complementary pairing with at least a portion of the sequence upstream of the 3' end of its nearest neighboring target sequence; 3) Using the nearest neighbor target sequence as a primer and the amplified first strand as a template, a second amplification reaction is performed.

12. The method according to claim 11, characterized in that, Repeat steps 1) to 3) to obtain a predetermined number of amplified nucleic acids.

13. A method for detecting the expression levels of multiple genes, characterized in that, include: The method described in any one of claims 8 to 12 is used to capture and amplify multiple genes to be tested; Fluorescence signal detection processing was performed on the amplification products; The expression levels of the multiple target genes are determined based on the intensity and type of fluorescence signals.

14. The method according to claim 13, characterized in that, The determination of the expression levels of the multiple target genes based on fluorescence signal intensity and type is performed in the following manner: The vector component includes several non-contiguous partitions, each partition containing at least one primer. The primers are used to amplify the gene to be tested, and each gene to be tested corresponds to a fluorescent probe. Based on the positional relationship of the several non-connected separators and the type of fluorescence signal, the expression levels of the multiple test genes are determined.

15. A genotyping method, characterized in that, include: The method described in claim 13 or 14 is used to detect the expression levels of multiple target genes; Based on the expression levels of the multiple test genes, the genotype of the test genes is determined.

16. A nucleic acid sequencing method, characterized in that, include: The nucleic acid to be tested is amplified using the method described in any one of claims 8 to 12; The amplified products were then subjected to adapter addition. as well as The adapter-treated products are then sequenced to obtain sequencing results.

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