Nucleic acid detection method

Abstract

The invention discloses a method for nucleic acid detection, which comprises the following steps: permeabilizing a fixed biological sample, so that a detection probe can enter the biological sample to be specifically combined with a target sequence of target nucleic acid to be detected; capturing the detection probe released from the biological sample by using a solid phase carrier; after multiple rounds of enzymatic chemical reactions, the captured detection probe modifies the sequenced linker, and finally, the nucleic acid of the biological sample is determined through sequencing and biosignal analysis. As long as a target sequence of mRNA exists, the detection probe can be combined with the target sequence to form a complex. After the detection probe is released, the solid-phase carrier not captures mRNA but captures the detection probe, so that the problem of low binding efficiency of the targeting probe caused by degradation of mRNA in a fixed biological sample can be avoided.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nucleic acid detection method. Background Art

[0002] Single-cell sequencing technology plays a crucial role in clinical medicine, enabling studies of tumor heterogeneity, the clonal development and evolution of cancer cells, the invasion of early-stage cancers, the mutation rate and types of cancer cells, tracking cancer cell metastasis and spread, revealing the tumor microenvironment, and understanding the evolution of cancer cell resistance during treatment. Currently, the most commonly used single-cell sequencing technology measures transcriptome expression levels within individual cells. This technology uses poly T sequences on encoded microspheres to capture polyadenylated mRNA (containing poly A sequences) expressed by eukaryotic cells, followed by enrichment and library construction for sequencing analysis. However, this method is unable to measure mRNA without poly A sequences. Furthermore, due to the unique nature of clinical samples, specimens removed from organisms often cannot be immediately sent to the laboratory for testing; they must be paraffin-embedded for pathological examination or stored in medical archives for several years. These situations lead to severe mRNA degradation and loss of poly A sequences. However, the poly T-based capture of mRNA poly A sequences requires that the mRNA contain a complete poly A sequence. If this sequence is missing, the mRNA cannot be captured and cannot be sequenced using these methods.

[0003] In the prior art, Chinese patent CN114774527A proposes a single-cell transcriptome determination method based on random primers: first, the random primer enters the cell nucleus and binds to the RNA, then reverse transcription is performed in situ in the cell nucleus, and a capture linker is added to the end of the obtained cDNA chain, and finally the cDNA in the single cell nucleus is captured, labeled, amplified, enriched and sequenced. However, since random primers are not specific, they will not only bind to the target RNA to be tested, but also to single-stranded DNA and exogenous nucleic acids in the cell nucleus, which ultimately leads to low qualified effective reads in the sequencing data and low reads matching rate with transcriptome gene comparisons. Patent CN117015617A designs targeted binding probes for known target nucleic acids to be tested, such as RNA, and uses the targeted probes to specifically bind to cellular RNA, and then captures and analyzes the targeted probes, thereby achieving the purpose of single-cell transcriptome analysis. Based on this method, 10x Genomics launched the Single Cell Flex Gene Expression Kit, which can specifically detect the target nucleic acid to be tested. However, this method does not consider the effect of cell fixation on the binding of the targeted probe to the target nucleic acid to be tested, resulting in low efficiency of targeted probe binding; single-cell capture and probe capture and extension are achieved based on droplet microfluidics technology, and multiple rounds of post-reaction cleaning cannot be performed, resulting in high background contamination and low reaction efficiency; when testing more fragile cells or cell nuclei, the high droplet generation rate causes damage to the cells or cell nuclei, reducing the quality of data results; expensive equipment is required to generate droplets, which increases the cost of sample testing. Summary of the Invention

[0004] In order to solve the problem of inaccurate nucleic acid detection results of fixed biological samples, the present invention proposes a nucleic acid detection method.

[0005] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0006] A nucleic acid detection method comprising the following steps:

[0007] Step 1) permeabilizing the biological sample to be tested; the biological sample is a biological sample that has been fixed;

[0008] Step 2) reacting the biological sample to be detected after the permeabilization treatment in step 1) with a detection probe, so that the detection probe specifically binds to the nucleic acid to be detected in the biological sample to form a nucleic acid-probe complex; the detection probe includes a sequencing adapter sequence, a target capture sequence and an immobilized nucleotide sequence, and the target capture sequence is used to specifically bind to the target sequence of the target nucleic acid to be detected in the nucleic acid to be detected;

[0009] Step 3) releasing the probe in the nucleic acid-probe conjugate from the biological sample and separating it from the nucleic acid to be detected; binding the released probe to a solid support; the solid support comprising a substrate having a coding sequence attached to its surface, the coding sequence comprising a PCR primer sequence and a probe capture sequence; the probe capture sequence being adapted to bind to the fixed nucleotide sequence;

[0010] Step 4) performing a DNA polymerase reaction on the solid phase to obtain a DNA polymerase reaction product, and sequencing the DNA polymerase reaction product to obtain a detection result of the nucleic acid to be detected.

[0011] Optionally, the biological sample to be detected includes at least one of tissue organs, cells, cell clusters, and cell nuclei.

[0012] Optionally, the step 1) further comprises performing a de-crosslinking treatment on the biological sample to be detected;

[0013] The decrosslinking treatment includes physical decrosslinking treatment or chemical decrosslinking treatment; the physical treatment includes at least one of thermal decomposition, photodecomposition, and sonication decomposition;

[0014] The chemical cross-linking treatment is to treat the fixed biological sample with a cross-linking reagent; the cross-linking reagent includes at least one of DTT, TCEP, Tween-20, Triton X-100, SDS, PBS, TE, citrate, and biological enzymes;

[0015] Preferably, before the de-crosslinking treatment, the paraffin on the surface of the fixed biological sample is removed.

[0016] Optionally, the permeabilization treatment in step 1) includes physical permeabilization treatment or chemical permeabilization treatment; the physical permeabilization treatment includes at least one of heating, light irradiation, and sonication;

[0017] The chemical permeabilization treatment is to treat the biological sample to be detected with a permeabilization reagent; the permeabilization reagent includes at least one of Tween-20, Triton X-100, SDS, PBS, TE, citrate, biological enzymes, proteinase K, and pepsin.

[0018] Optionally, in step 2), a plurality of detection probes are added to the permeabilized biological sample, and the target capture sequence of each detection probe specifically binds to the target sequence of the target nucleic acid to be detected in the nucleic acid to be detected;

[0019] Preferably, the target capture sequence has 20 to 150 bases.

[0020] Optionally, the detection probe includes a first detection probe and a second detection probe, wherein the first detection probe includes a sequencing adapter sequence and a first target capture sequence; and the second detection probe includes a fixed nucleotide sequence and a second target capture sequence;

[0021] The targeted capture sequence is formed by splicing the first targeted capture sequence and the second targeted capture sequence;

[0022] The step 3) further comprises connecting the first targeted capture sequence and the second targeted capture sequence by a ligase.

[0023] Preferably, the first targeted capture sequence and the second targeted capture sequence both have 10 to 120 bases.

[0024] Optionally, the 3' end of the sequencing adapter sequence in the first detection probe is connected to the 5' end of the first targeted capture sequence; the 3' end of the second targeted capture sequence in the second detection probe is connected to the 5' end of the fixed nucleotide sequence.

[0025] Optionally, the method of releasing the probe in the nucleic acid-probe complex from the biological sample and separating it from the nucleic acid to be detected includes at least one of: cell or cell nucleus lysis release, enzyme digestion release, surfactant treatment release, and heating release.

[0026] Optionally, the matrix includes at least one of gel particles, magnetic particles, polymer particles, and silica particles;

[0027] The particle size of the solid phase carrier is 10 microns to 60 microns.

[0028] Optionally, the coding sequence further includes a cell identification tag and a unique molecule identification tag sequence.

[0029] The present invention also provides a method for detecting nucleic acid in a single cell or a single cell nucleus, comprising the following steps:

[0030] Step 1) preparing a biological sample to be tested into a single cell or single cell nucleus suspension;

[0031] Step 2) permeabilizing a suspension of single cells or single cell nuclei to be detected; the single cells or single cell nuclei are fixed single cells or cell nuclei;

[0032] Step 3) reacting the permeabilized single cell or single cell nucleus suspension to be detected in step 2) with a detection probe, so that the detection probe specifically binds to the nucleic acid to be detected in the single cell or single cell nucleus to form a nucleic acid-probe complex; the detection probe includes a sequencing adapter sequence, a target capture sequence, and an immobilized nucleotide sequence, and the target capture sequence is used to specifically bind to the target sequence of the target nucleic acid to be detected in the nucleic acid to be detected;

[0033] Step 4) dispersing the single cells or cell nuclei in the single cell or cell nucleus suspension into a plurality of regions, such that each region contains at most one cell or cell nucleus; and adding one of the solid phase carriers to each region;

[0034] Step 5) releasing the probe in the nucleic acid-probe conjugate from the single cell or single cell nucleus and separating it from the nucleic acid to be detected; binding the released probe to a solid phase support; the solid phase support includes a substrate having a coding sequence connected to the surface of the substrate, the coding sequence including a PCR primer sequence and a probe capture sequence; the probe capture sequence is used to bind to the fixed nucleotide sequence;

[0035] Step 6) performing a DNA polymerase reaction on the solid phase to obtain a DNA polymerase reaction product, and sequencing the DNA polymerase reaction product to obtain a detection result of the nucleic acid to be detected.

[0036] Optionally, dispersing the single cells or cell nuclei in the single cell or single cell nucleus suspension into multiple regions specifically includes:

[0037] The cells or cell nuclei are captured using a microfluidic chip containing a micropore array, so that each micropore in the micropore array contains at most one cell or cell nucleus; and then one solid phase carrier is captured in each micropore in the micropore array.

[0038] The present invention has the following beneficial effects:

[0039] The present invention provides a method for nucleic acid detection, which comprises: permeabilizing a fixed biological sample so that a detection probe can enter the biological sample and specifically bind to a target sequence of a target nucleic acid to be detected; using a solid phase carrier to capture the detection probe released from the biological sample; after multiple rounds of enzymatic chemical reactions, the captured detection probe is modified by a sequencing adapter, and finally the nucleic acid of the biological sample is measured by sequencing and bioinformatics analysis. In the present invention, as long as the target sequence of mRNA exists, the detection probe will bind to the target sequence to form a complex. When the detection probe is released, the solid phase carrier is used to capture the detection probe, not mRNA. The detection probe belongs to DNA, which is relatively stable relative to RNA and is not easily degraded during the experiment. Therefore, the present invention is suitable for nucleic acid detection of fixed biological samples, and can overcome the effect of cell fixation on the binding of the targeting probe to the target nucleic acid to be detected, resulting in the problem of low binding efficiency of the targeting probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 Schematic diagram of single probe hybridization.

[0042] Figure 2 Schematic diagram of dual-probe hybridization.

[0043] Figure 3 Schematic diagram of probe capture.

[0044] Figure 4 Schematic diagram of the microfluidic chip structure.

[0045] Figure 5 K562 cells after probe reaction observed under a microscope.

[0046] Figure 6 HeLa cells captured by the microwell array of a microfluidic chip observed under a microscope.

[0047] Figure 7 UMAP results of dimensionality reduction clustering of mouse spleen tissue cells. DETAILED DESCRIPTION

[0048] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0049] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intervening value in the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0051] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0052] The present invention provides a method for nucleic acid detection, comprising:

[0053] Step 1) permeabilizing the biological sample to be tested; the biological sample is a biological sample that has been fixed;

[0054] Step 2) reacting the biological sample to be detected after the permeabilization treatment in step 1) with a detection probe, so that the detection probe specifically binds to the nucleic acid to be detected in the biological sample to form a nucleic acid-probe complex; the detection probe includes a sequencing adapter sequence, a target capture sequence and an immobilized nucleotide sequence, and the target capture sequence is used to specifically bind to the target sequence of the target nucleic acid to be detected in the nucleic acid to be detected;

[0055] Step 3) releasing the probe in the nucleic acid-probe conjugate from the biological sample and separating it from the nucleic acid to be detected; binding the released probe to a solid support; the solid support comprising a substrate having a coding sequence attached to its surface, the coding sequence comprising a PCR primer sequence and a probe capture sequence; the probe capture sequence being adapted to bind to the fixed nucleotide sequence;

[0056] Step 4) performing a DNA polymerase reaction on the solid phase to obtain a DNA polymerase reaction product, and sequencing the DNA polymerase reaction product to obtain a detection result of the nucleic acid to be detected.

[0057] The fixed biological sample refers to a biological sample that has been treated with a chemical reagent, which not only reduces or terminates the reaction of exogenous enzymes and endogenous enzymes, prevents cell autolysis, but also maintains a cell structure and chemical composition close to that of a living body. The biological sample can be a tissue organ, a cell or cell cluster, a cell nucleus, etc. The chemical reagent can be a combination of one or more components selected from alcohols (e.g., methanol or ethanol), ketones (e.g., acetone), and aldehydes (e.g., paraformaldehyde, formaldehyde, glutaraldehyde). As an embodiment, the chemical reagent is ethanol, which achieves biological sample fixation based on the dehydration principle, and suitable concentrations for use are 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, etc. For example, 100% ethanol is used to fix the biological sample. As a preferred embodiment, the biological sample is treated with ethanol solutions of different concentrations, for example, 100%, 95%, 85%, and 75% ethanol solutions are used for fixation. In another embodiment, the chemical reagent is paraformaldehyde (PFA), which fixes biological samples by catalyzing the formation of covalent cross-links between protein molecules. Suitable concentrations for use include 40%, 30%, 20%, 10%, 8%, 4%, 1%, 0.5%, 0.1%, and the like. As a commonly used embodiment, the PFA concentration is 4%. Furthermore, after the biological sample, such as tissue, has been fixed with the chemical reagent, such as 4% formalin, the tissue needs to be embedded in paraffin in order to meet the needs of pathological diagnosis and long-term storage of tissue samples, ultimately obtaining formalin-fixed paraffin-embedded (FFPE) tissue.

[0058] Decrosslinking refers to partially or completely reversing the fixation process of the fixed biological sample through physical or chemical methods. The physical method can be a combination of one or more methods such as thermal lysis, photolysis, and sonication. The chemical method refers to a combination of one or more methods such as reducing agents (e.g., DTT, TCEP), surfactants (e.g., Tween-20, Triton X-100, SDS), salt ion solutions (e.g., PBS, TE, citrate), and enzymes to promote partial or complete reversal of crosslinks in the fixed biological sample. In one embodiment, the fixed biological sample is decrosslinked using TE buffer at 90°C. In another embodiment, the fixed biological sample is decrosslinked using TE buffer containing Tween-20 at 85°C. In particular, when the fixed biological sample is paraffin-embedded, the fixed biological sample needs to be dewaxed using a chemical reagent (e.g., xylene, ethanol, isopropanol, etc.) before decrosslinking to remove the paraffin components on the sample surface.

[0059] Permeabilization refers to making the cell membrane and / or nuclear membrane of the biological sample permeable through physical or chemical methods, allowing small molecules (e.g., water molecules, Tris-HCl, short nucleotide fragments, etc.) and macromolecules (e.g., enzymes, proteins, large nucleotide fragments) to enter the cell / nucleus. The physical method can be a combination of one or more methods such as heating, illumination, and sonication. The chemical method refers to a combination of one or more methods such as surfactants (e.g., Tween-20, Triton X-100, SDS, etc.), salt ion solutions (e.g., PBS, TE, citrate, etc.), and biological enzymes (e.g., proteinase K, pepsin, etc.) to promote the permeability of the cell membrane and / or nuclear membrane in the biological sample. As an embodiment, the biological sample is permeabilized using a citrate solution containing a surfactant. The concentration of the surfactant can be 0.005% to 1%. The citrate solution can be 1×SSC, 2×SSC, 3×SSC, 4×SSC, 5×SSC, etc. As a preferred method, the biological sample is permeabilized using 2×SSC containing 0.05% Triton X-100.

[0060] The biological sample fixation, cross-link removal, and permeabilization processes can be performed in separate steps or simultaneously. For example, the biological sample is fixed, cross-linked, and permeabilized sequentially; or the biological sample is fixed, permeabilized, and cross-linked sequentially; any one or more of the aforementioned fixation, cross-link removal, and permeabilization methods can be used. As another embodiment, the biological sample is first fixed and permeabilized simultaneously, and then cross-link removal is performed. For example, the biological sample is fixed and permeabilized in a 4% PFA solution containing 0.01% Tween-20, and then cross-link removal is performed in TE buffer at 90°C. As another embodiment, the biological sample is first fixed, and then cross-link removal and permeabilization are performed simultaneously. For example, the biological sample is fixed using a 4% PFA solution, and then cross-link removal and permeabilization are performed using a TE buffer containing 0.05% Tween-20 at 85°C.

[0061] The detection probe is an oligonucleotide fragment composed of several nucleotides. The detection probe consists of 10 to 150 bases; further, the detection probe consists of 30, 40, 50, 60, 70 or 80 bases. As an embodiment, the detection probe consists of 60 bases. The detection probe contains at least a target capture sequence, which can specifically bind to the target sequence of the target nucleic acid to be detected in the biological sample to complete base complementary pairing. The number of bases in the target capture sequence can be 0, 5, 8, 10, 20, 30, 50, 100, 120, etc. less than that of the detection probe. For example, 25 bases of the detection probe composed of 60 bases are the target capture sequence. Each of the detection probes can only bind to one target sequence, while each of the target sequences can bind to one, two, three, four, five, etc. detection probes. This not only ensures the targeted determination of the target nucleic acid to be tested and improves the effective reads qualification rate of the final sequencing data, but also helps to improve the detection efficiency and sensitivity of the target nucleic acid to be tested. Figure 1 As shown, the detection probe 1000 includes a target capture sequence 1002, a sequencing adapter Read 2 sequence 1003, and an immobilized nucleotide sequence 1004. The target capture sequence 1002 specifically recognizes and binds to the target sequence 1001 of the target nucleic acid 1005 to be detected. The characteristics of the sequencing adapter Read 2 sequence 1003 and the immobilized nucleotide sequence 1004 are described below.

[0062] In one embodiment, the targeting sequence comprises at least a first targeting sequence and a second targeting sequence, and the detection probe comprises at least a first detection probe and a second detection probe, wherein the first detection probe specifically binds to the first targeting sequence, and the second detection probe specifically binds to the second targeting sequence. As a special method, after the first detection probe binds to the first targeting sequence and the second detection probe binds to the second targeting sequence, the first detection probe and the second detection probe can be linked together using one or a combination of methods, such as enzymes and oligonucleotide fragments, to form a complete complementary sequence of the targeting sequence. Thus, the targeting sequence binds to a pair of detection probes.

[0063] As an embodiment, when the target sequence is bound to a pair of detection probes, the first detection probe is complementary to the base near the 3' end of the target sequence, and the second detection probe is complementary to the base near the 5' end of the target sequence. After binding to the target sequence, they can be connected together to form a sequence that is completely complementary to the target sequence. The first detection probe is composed of a first target capture sequence complementary to the target sequence and a sequencing adapter Read 2 sequence; the second detection probe is composed of a second target complementary sequence complementary to the target sequence and a fixed nucleotide sequence. The 3' end of the first target capture sequence has a free hydroxyl group, and the 5' end of the second target capture sequence has a phosphorylation modification, so as to connect the first target capture sequence and the second target capture sequence to form a complete complementary sequence of the targeted gene.

[0064] The number of bases of the first targeted capture sequence and the second targeted capture sequence can be 10 to 120; further, it can be 20 to 60; further, it can be 20 to 30. The number of bases of the first targeted capture sequence and the second targeted capture sequence can be the same or different. As an embodiment, the number of bases of the first targeted capture sequence and the second targeted capture sequence is 25 each. For example, for the genes of the target nucleic acids to be tested, PECAM1, PTPRC, CSNK1A1, FTH1, and CD55, the designed first targeted capture sequence and the second targeted capture sequence are shown in Table 1 (sequences 1 to 12).

[0065] Table 1

[0066]

[0067] The sequencing adapter Read 2 sequence is a base sequence that can be compatible with the sequencer, and the number of bases can be 10 to 50. As an embodiment, in this embodiment of the present invention, the sequencing adapter Read 2 sequence can be compatible with the Illumina second-generation sequencer, and its sequence is: 5'-GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3' (SEQ ID NO: 13).

[0068] The fixed nucleotide sequence is an oligonucleotide fragment containing the same known base sequence information for each of the second detection probes. The fixed nucleotide sequence can have 10 to 100 bases, 20 to 50 bases, 25 to 35 bases, etc. In one embodiment, a segment of the fixed nucleotide sequence contains 30 consecutive A bases.

[0069] The first target capture sequence and the second target capture sequence can be connected together using a cross-linker, a ligase (eg, T4 ligase, Splint R ligase, etc.). As an embodiment, the connection method used is T4 ligase connection.

[0070] As an example, Figure 2 As shown, the targeting sequence of the target nucleic acid 2008 to be detected includes a first targeting sequence 2006 and a second targeting sequence 2007, and the first targeting sequence 2006 and the second targeting sequence 2007 are adjacent. The detection probe includes a first detection probe 2001 and a second detection probe 2000. The first detection probe 2001 is composed of a first targeting capture sequence 2003 complementary to the first targeting sequence 2006 and a sequencing adapter Read 2 sequence 2005; the second detection probe 2000 is composed of a second targeting complementary sequence 2002 complementary to the second targeting sequence 2007 and a fixed nucleotide sequence 2004. After a pair of detection probes are bound to the targeting sequence of the target nucleic acid 2008 to be detected, T4 ligase is used to connect the first detection probe 2001 and the second detection probe 2000 into one, forming a structure as shown in FIG. Figure 3 Targeted detection probe sequence 5005 is shown.

[0071] In order to avoid the problem of being unable to detect due to degradation of the target sequence of mRNA and to improve the detection efficiency of mRNA, probes can be designed for different sequence positions of an mRNA, so that one mRNA corresponds to multiple pairs of detection probes, thereby improving the detection efficiency of the mRNA. As an embodiment, each of the target nucleic acids to be detected can contain one, two, three or more of the target sequences, which can be combined with one or more pairs of detection probes. For example, each of the target nucleic acids to be detected can be combined with two pairs, three pairs, four pairs, five pairs, etc. of detection probes. This method is conducive to improving the detection efficiency and detection sensitivity of the target nucleic acids to be detected. The target nucleic acid to be detected can be DNA, RNA or both. Therefore, when it is necessary to measure the whole transcriptome of the biological sample, the detection probes can be designed based on the whole transcriptome sequence information of the biological sample, and ultimately tens of thousands of pairs of detection probes are obtained.

[0072] The solid phase carrier refers to gel particles, magnetic particles, polymer particles, silica particles, etc. The surface of the solid phase carrier has hundreds of thousands, millions, tens of millions or hundreds of millions of coding sequences. The coding sequence is an oligonucleotide fragment containing at least a PCR primer sequence and a probe capture sequence. Furthermore, the coding sequence is an oligonucleotide fragment containing at least a PCR primer sequence, a cell identification tag, a unique molecular identification tag sequence, and a probe capture sequence. The PCR primer sequence is a known oligonucleotide sequence, and the PCR primer sequence of each coding sequence can be the same or different. As an embodiment, the PCR primer sequence is an oligonucleotide sequence that is partially or completely complementary to or identical to the sequencing primer of the sequencing instrument. In an embodiment of the present invention, the PCR primer sequence is CACGACGCTCTTCCGATCT (SEQ ID NO: 14). The cell identification tag is an oligonucleotide fragment composed of any combination of the four bases A\T\C\G. For example, the cell identification tag is obtained by any arrangement and combination of 1, 2, 3, 4, 5, 10, 20, 30, 60, 70 or 80 bases. As an embodiment, the cell identification tag contains 68 consecutive bases. All cell identification tags on each solid phase carrier are the same, while the cell identification tags between each solid phase carrier are different. The unique molecular identification tag sequence is an oligonucleotide fragment composed of any combination of four bases: A\T\C\G. For example, the unique molecular identification tag sequence is obtained by any arrangement and combination of 1, 2, 3, 4, 5, 8, 10 or 20 bases. As an embodiment, the unique molecular identification tag sequence contains 8 consecutive bases. The probe capture sequence is an oligonucleotide sequence that can undergo base complementary pairing with the detection probe. For example, the probe capture sequence can be complementary to the base of the fixed nucleotide sequence of the second detection probe. As an embodiment, when a section of the fixed nucleotide sequence contains a sequence of 30 consecutive A bases, the probe capture sequence is a sequence containing 30 consecutive T bases. Therefore, when the detection probe is released from the biological sample, it can be captured and enriched by the solid phase carrier.

[0073] The biological sample can release the detection probe in a variety of ways, such as release by cell / nucleus lysis, release by enzymatic digestion, release by heating, or release by a combination of any of these methods. As an embodiment, a chemical reagent such as a surfactant (e.g., Tween-20, Triton X-100, SDS, etc.) is used to lyse cells and then release the detection probe. The reaction concentration of the surfactant can be 0.1%, 0.2%, 0.5%, 1%, 5%, 10%. As another way, a surfactant containing an enzyme (e.g., proteinase K, RNase, etc.) is used to lyse cells and release the detection probe. The reaction concentration of the enzyme can be 0.01U / uL, 0.02U / uL, 0.05U / uL, 0.1U / uL, 0.5U / uL, 1U / uL, 5U / uL, 10U / uL, 20U / uL, 40U / uL, 100U / uL. For example, cells are lysed using 0.5% Triton X-100 containing 1 U / uL RNase.

[0074] As an example, Figure 3 As shown, the solid phase carrier 5000 is a magnetic particle, and a coding sequence 5006 consisting of a PCR primer sequence 5001, a cell identification tag 5002, a unique molecular identification tag sequence 5003, and a probe capture sequence 5004 is attached to its surface. When 0.5% Triton X-100 containing 1U / uL RNase is used to lyse cells, the target detection probe sequence 5005 is released and captured by the probe capture sequence 5004 (combined with the fixed nucleotide sequence 2004).

[0075] After the solid-phase carrier captures the detection probe, a DNA polymerase reaction is performed, generating a large number of DNA fragments containing the detection probe sequence. Each fragment contains a complete set of sequencing adapters, which can be sequenced on a sequencing instrument. Finally, bioinformatics analysis of the generated FastQ file provides nucleic acid detection information for the biological sample.

[0076] The present invention also provides a method for detecting nucleic acid in a single cell or a single cell nucleus, comprising the following steps:

[0077] Step 1) preparing a biological sample to be tested into a single cell or single cell nucleus suspension;

[0078] Step 2) permeabilizing a suspension of single cells or single cell nuclei to be detected; the single cells or single cell nuclei are fixed single cells or cell nuclei;

[0079] Step 3) reacting the permeabilized single cell or single cell nucleus suspension to be detected in step 2) with a detection probe, so that the detection probe specifically binds to the nucleic acid to be detected in the single cell or single cell nucleus to form a nucleic acid-probe complex; the detection probe includes a sequencing adapter sequence, a target capture sequence, and an immobilized nucleotide sequence, and the target capture sequence is used to specifically bind to the target sequence of the target nucleic acid to be detected in the nucleic acid to be detected;

[0080] Step 4) dispersing the single cells or cell nuclei in the single cell or cell nucleus suspension into a plurality of regions, such that each region contains at most one cell or cell nucleus; and adding one of the solid phase carriers to each region;

[0081] Step 5) releasing the probe in the nucleic acid-probe conjugate from the single cell or single cell nucleus and separating it from the nucleic acid to be detected; binding the released probe to a solid phase support; the solid phase support includes a substrate having a coding sequence connected to the surface of the substrate, the coding sequence including a PCR primer sequence and a probe capture sequence; the probe capture sequence is used to bind to the fixed nucleotide sequence;

[0082] Step 6) performing a DNA polymerase reaction on the solid phase to obtain a DNA polymerase reaction product, and sequencing the DNA polymerase reaction product to obtain a detection result of the nucleic acid to be detected.

[0083] The above-mentioned method for detecting nucleic acid of a single cell or a single cell nucleus is consistent with the above-mentioned method for detecting nucleic acid of a biological material, except that the biological material is dispersed into a single cell or a single cell nucleus.

[0084] The biological sample is composed of one or more cells. In particular, the cells are host cells of pathogens or microorganisms such as viruses, bacteria, fungi, and these host organisms are contained in the cells or on the cell surface. When the biological sample is composed of a cell cluster, tissue, or organ composed of multiple cells, it is necessary to prepare the biological sample into a single cell / single cell nucleus suspension by one or more combinations of mechanical shearing, heating, enzymatic digestion, chemical lysis, etc., and then perform treatments such as fixation, cross-linking removal, and permeabilization. In particular, the biological sample can be fixed first, and then perform treatments such as cross-linking removal and permeabilization. For example, when the biological sample is FFPE tissue, after dewaxing using xylene, the tissue block is chopped and homogenized, and the cells are lysed using a surfactant to finally obtain a single cell nucleus suspension.

[0085] Partitioning refers to physically isolating multiple cells / nuclei in a single cell / single cell nucleus suspension into multiple parts, so that each part contains at most one cell. Figure 4As shown, a microfluidic chip 4000 containing a micropore array 4001 is used to separate and capture single cells / single cell nuclei. Specifically, the surface of the microfluidic chip has hundreds, thousands, or tens of thousands of micropores regularly arranged, and the geometric dimensions of each micropore are in the micrometer scale. When the single cell / single cell nucleus suspension is added to the microfluidic chip, based on the Poisson distribution principle and the action of gravity, some / all of the single cells / single cell nuclei fall into the micropores and are captured. The microfluidic chip is cleaned with a cleaning solution such as 1×PBS to remove impurities such as uncaptured single cells / single cell nuclei, cell debris, and free nucleic acids. Subsequently, the solid phase carrier is added to the microfluidic chip in the same manner, so that each micropore captures at most one solid phase carrier, and the uncaptured solid phase carrier will be removed by cleaning. The micropore array is not a single layer, but a double-layer nested structure, that is, the first layer of micropores and the second layer of micropores are geometrically symmetrical (for the specific structure, see Chinese patent CN216149778U, the supplier is Suzhou Deyun Kangrui Biotechnology Co., Ltd.). The first layer of micropores is located at the bottom, with sizes similar to those of cells / nuclei, such as 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, or 50 μm. The second layer of micropores is located at the top, with sizes similar to those of the solid support, such as 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, or 50 μm. This allows for better physical isolation of single cells / nuclei after capture and solid support, preventing them from being carried away during subsequent washing steps. Compared to 10x Genomics' droplet-based microfluidics approach, this not only ensures the capture of sufficient single cells / nuclei to meet research needs, but also allows for multiple rounds of washing after capture to remove clumped cells / nuclei, cell debris, and free nucleic acids, reducing background contamination and improving data quality. It also enables the capture and pairing of single cells / nuclei with solid supports without the need for expensive droplet generation equipment, reducing sample testing costs and making it particularly suitable for resource-constrained environments.

[0086] As previously mentioned, when performing single-cell nucleic acid detection, the coding sequence of the solid phase carrier is an oligonucleotide fragment containing at least a PCR primer sequence, a cell identification tag, a unique molecular identification tag sequence, and a probe capture sequence. The cell identification tag can be used to mark cells / cell nuclei so that different cells / cell nuclei can be identified during subsequent bioinformatics analysis. The unique molecular identification tag sequence can be used to mark the captured detection probe to eliminate the adverse effects of PCR amplification bias and to count the expression levels of the target nucleic acid to be tested during subsequent bioinformatics analysis.

[0087] Example 1: RNA detection of the entire transcriptome of K562 cells

[0088] Cell Fixation, Decrosslinking, and Permeabilization: Resuspend K562 cells in 4% formaldehyde fixative and incubate at room temperature for 1 hour to achieve cell fixation. Centrifuge, remove the fixative, and wash once with 1× PBS. Resuspend the cells in 100 mM TE buffer and incubate in an 85°C thermostat for 30 minutes, then cool to room temperature. Centrifuge, remove the supernatant, and resuspend the cells in 1× PBS containing 0.05% Tween-20 and incubate at room temperature for 15 minutes. Subsequently, centrifuge, remove the supernatant, and wash and resuspend the cells in 1× PBS to obtain a cell suspension.

[0089] The obtained cell suspension was centrifuged and the supernatant was removed. After resuspending the cells with the probe hybridization reaction solution [50nM first detection probe 10uL, 50nM second detection probe 10uL, 3×SSC 80uL], the reaction was carried out at 37°C overnight (18-24h) on a constant temperature oscillating mixer (speed 200rpm). The first detection probe and the second detection probe were designed based on the mRNA of human species, containing a total of approximately 18,000 genes and 52,000 pairs of detection probes. Subsequently, the cells were centrifuged, the supernatant was removed, and the cells were washed 3 times with 3×SSC. Each time the cells were washed, the cells were first resuspended in 3×SSC, incubated at 37°C for 10 minutes, and then centrifuged again to remove the supernatant. After washing, resuspend the cells in the probe ligation reaction solution [10uL of 10×T4 ligase Buffer, 5uL of T4ligase (supplier: Novozymes), 85uL of enzyme-free water] and react at 37°C for 1 hour on a constant temperature shaker (200rpm). After the reaction is completed, remove the cells, centrifuge to remove the supernatant, and finally resuspend the cells in 1×PBS. Use a microscope and cell counter to check the cell quality. Figure 5 As shown, the K562 cells observed under a microscope after the probe reaction had complete morphology and good cell dispersion.

[0090] Take two centrifuge tubes and add one and approximately 1000 magnetic solid phase supports (Supply: Suzhou Deyun Kangrui Biotechnology Co., Ltd.) to each tube, respectively. Then, add 50 μL of freshly prepared cell lysis buffer [2.5 μL Proteinase K (Supply: NEB), 5 μL RNase H (Supply: NEB), 5 μL RNase IF (Supply: NEB), 2.5 μL 10% Triton X-100 (Supply: Sigma), 33 μL enzyme-free water]. Based on the cell concentration determined by a cell counter, immediately add one and approximately 1000 cells (with a volume of no more than 2 μL) to the corresponding centrifuge tubes, so that 1 cell corresponds to 1 solid phase support and 1000 cells correspond to 1000 solid phase supports. At this point, the cells begin to lyse and release the detection probes, which are captured by the probe capture sequences on the solid phase supports. After 5 minutes of reaction, magnetically aspirate until the solution is clear and carefully aspirate the reaction solution. To avoid aspirating the magnetic solid phase supports, a small amount of reaction solution may remain in the centrifuge tube. The solid phase carrier was then washed once with 6×SSC, and the probe extension reaction solution [10×Buffer 5uL, Bst DNA Polymerase (supplier: NEB) 2.5uL, MgSO4 Solution 2.5uL, 10mM dNTP (supplier: Thermo Fisher) 5uL, enzyme-free water 35uL] was immediately added to the tube and reacted at 45°C for 1h.

[0091] After the reaction is complete, remove the centrifuge tube and place it on a magnetic rack. Magnetize until the solution is clear. Carefully aspirate the supernatant and add 50 μL of PCR reaction solution [25 μL of 2× PCR Mix (Supplier: Novozymes), 5 μL of 12 μM PCR Primer, and 20 μL of enzyme-free water]. Perform PCR amplification according to PCR amplification protocol 1 shown in Table 2. After the reaction is complete, remove the tube and purify the DNA once using 1.8× DNA purification magnetic beads (Supplier: Novozymes). Finally, elute the DNA with 20 μL of enzyme-free water to obtain the purified DNA product.

[0092] Table 2

[0093]

[0094]

[0095] The purified DNA product was amplified again according to PCR amplification reaction procedure 2 shown in Table 3 to form a complete sequencing library suitable for second-generation sequencing instruments. The PCR reaction system was: 25 μL of 2× PCR Mix (supplier: Novozymes), 10 μL of purified DNA product, and 15 μL of enzyme-free water. After the reaction was completed, the DNA was removed and purified once using 1× DNA purification magnetic beads. Finally, the DNA was eluted with 30 μL of enzyme-free water to obtain the purified sequencing library product.

[0096] Table 3

[0097]

[0098] The library was sequenced using the Illumina Nova-Seq 6000, a next-generation sequencing instrument, and then analyzed using bioinformatics methods, yielding the data shown in Table 4. Sample IDs K562-1 and K562-1000 represent centrifuge tubes containing one cell and one magnetic solid phase carrier, respectively, and centrifuge tubes containing 1000 cells and 1000 magnetic solid phase carriers. The data demonstrates that the median genes per cell exceed 2000 for both single and multi-cell analyses, demonstrating the excellent gene detection performance of the present invention for fixed biological samples.

[0099] Table 4 K562 cell whole transcriptome RNA detection results

[0100] Sample ID K562-1 K562-1000 Number of Reads 14,924,983 30,171,159 Valid Barcodes 91.67％ 93.08％ Estimated Number of Cells 1 1,356 Fraction Reads in Cells 99.39％ 67.53％ Mean Reads per Cell 14,924,983 22,250 Median UMI Counts per Cell 36,110 5,120 Median Genes per Cell 2,216 2,750 Total Genes Detected 2,216 17,008 Reads Mapped to Probe Set 91.76％ 96.48％ Reads Mapped Confidently to Probe Set 90.12％ 90.49％

[0101] Example 2: Hela single cell whole transcriptome RNA detection

[0102] HeLa cells were resuspended in 4% formaldehyde fixative containing 0.02% Tween-20 and incubated at room temperature for 1 hour to achieve cell fixation and permeabilization. The cells were centrifuged, the fixative was removed, and the cells were washed once with 1× PBS. The cells were resuspended in 100 mM TE buffer and incubated in an 85°C thermostat for 30 minutes, then removed and cooled to room temperature. The cells were centrifuged, the supernatant was removed, and the remaining cell pellet was resuspended in 1× PBS to complete cross-linking.

[0103] To illustrate the capture of Hela cells by the microwell array of a microfluidic chip (supplier: Suzhou Deyun Kangrui Biotechnology Co., Ltd.), a small number of Hela cells were stained with DAPI dye and then added to the microfluidic chip. Due to gravity, the cells will settle in the microwells and be captured. Based on the Poisson distribution principle, the cells are captured by a small number of microwells in the microwell array. Figure 6 As shown, under an inverted fluorescence microscope, HeLa cells can be observed captured by the microwell array of the microfluidic chip. Most microwells contain only a single cell, thus meeting the requirements for single-cell nucleic acid detection. Furthermore, since the cells were washed three times with 1× PBS after capture, the area outside the microwells was free of uncaptured cells and impurities, which helps reduce the impact of background contamination.

[0104] Then, according to the method of Example 1, the remaining cells were reacted with the probe hybridization reaction solution and the probe ligation reaction solution in turn, and finally a cell mother solution was obtained which was resuspended in 1×PBS. The cell quality was inspected using a microscope and a cell counter, and 200uL of a single cell suspension was prepared according to the cell concentration. 200uL of the single cell suspension was added to the microfluidic chip, and after completing the cell capture, it was washed 3 times with 1×PBS. In the same way, 200uL of a magnetic solid phase carrier suspension was added to the microfluidic chip, and after completing the capture, it was washed 3 times with 1×PBS, so that each microwell contained at most one magnetic solid phase carrier, thereby achieving one-to-one pairing of cells and magnetic solid phase carriers. Add cell lysis buffer [Proteinase K (NEB) 10µL, RNase H (NEB) 20µL, RNase If (NEB) 20µL, 10% Triton X-100 (Sigma) 10µL, enzyme-free water 140µL] to the chip to lyse the cells and release the detection probes, which are then captured by the probe capture sequences on the solid phase support. After a 15-minute reaction, the magnetic solid phase support is removed from the microfluidic chip by magnetic attraction into a 0.2mL centrifuge tube and washed once with 2×SSC.

[0105] Similarly, in the manner of Example 1, the recovered magnetic solid phase carrier was reacted with the probe extension reaction solution, and then after two PCR amplification and DNA purification, the single-cell sequencing library construction was completed to obtain a sequencing library purification product. The library was sequenced on both ends using the second-generation sequencing instrument Illumina Nova-Seq 6000, and the data was analyzed using the bioinformatics method to obtain the data results shown in Table 5. As can be seen from the table, a total of 3160 cells were detected in this example, and the gene performance detection indicators such as Median UMI Counts per Cell, Median Genes per Cell, and Reads Mapped to Probe Set were good, proving that single-cell nucleic acid detection can be efficiently completed based on the method described in the present invention.

[0106] Table 5 Hela single cell whole transcriptome RNA detection results

[0107]

[0108]

[0109] Example 3: Transcriptome RNA Detection in Formalin-Fixed Paraffin-Embedded (FFPE) Mouse Spleen Tissue

[0110] Two 50 μm thick tissue sections were cut from formalin-fixed paraffin-embedded (FFPE) mouse spleen tissue using a microtome and placed in a gentleMACS C-tube. The cell nuclei were then extracted according to the instructions of the FFPE Tissue Dissociation Kit (brand: Miltenyi Biotec; catalog number: 130-118-052). Subsequently, the cell nucleus sample was dispersed into single cell nuclei using a microfluidic chip according to the method of Example 2, and the transcriptome gene expression information of the sample was detected using the first detection probe and the second detection probe designed based on mouse mRNA according to the method of Example 2. The results are shown in Table 6. The cells detected in the sample were subjected to dimensionality reduction clustering, and the following was obtained: Figure 7 The UMAP image shown in the figure shows clear cell clusters. Furthermore, this sample successfully detected not only B cells, T cells, and monocytes, which are abundant in spleen tissue, but also relatively fragile neutrophils. This demonstrates the successful application of this invention in detecting FFPE sample types.

[0111] Table 6 Single cell transcriptome RNA detection results of mouse spleen tissue

[0112] Sample ID Spleen Number of Reads 104,238,143 Valid Barcodes 86.06％ Estimated Number of Cells 7320 Fraction Reads in Cells 49.94％ Mean Reads per Cell 14240 Median UMI Counts per Cell 1,335 Median Genes per Cell 901 Total Genes Detected 19,130 Reads Mapped to Probe Set 83.64％ Reads Mapped Confidently to Probe Set 75.63％

[0113] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A nucleic acid detection method, characterized in that: The steps include: Step 1) permeabilizing the biological sample to be tested; the biological sample is a biological sample that has been fixed; Step 2) reacting the biological sample to be detected after the permeabilization treatment in step 1) with a detection probe, so that the detection probe specifically binds to the nucleic acid to be detected in the biological sample to form a nucleic acid-probe complex; the detection probe includes a sequencing adapter sequence, a target capture sequence and an immobilized nucleotide sequence, and the target capture sequence is used to specifically bind to the target sequence of the target nucleic acid to be detected in the nucleic acid to be detected; Step 3) releasing the probe in the nucleic acid-probe conjugate from the biological sample and separating it from the nucleic acid to be detected; binding the released probe to a solid support; the solid support comprising a substrate having a coding sequence attached to its surface, the coding sequence comprising a PCR primer sequence and a probe capture sequence; the probe capture sequence being adapted to bind to the fixed nucleotide sequence; Step 4) performing a DNA polymerase reaction on the solid phase to obtain a DNA polymerase reaction product, and sequencing the DNA polymerase reaction product to obtain a detection result of the nucleic acid to be detected.

2. The nucleic acid detection method according to claim 1, wherein The biological sample to be detected includes at least one of tissue organs, cells, cell clusters, and cell nuclei.

3. The nucleic acid detection method according to claim 1, wherein The step 1) further includes performing a de-crosslinking treatment on the biological sample to be detected; The decrosslinking treatment includes physical decrosslinking treatment or chemical decrosslinking treatment; the physical treatment includes at least one of thermal decomposition, photodecomposition, and sonication decomposition; The chemical cross-linking treatment is to treat the fixed biological sample with a cross-linking reagent; the cross-linking reagent includes at least one of DTT, TCEP, Tween-20, Triton X-100, SDS, PBS, TE, citrate, and biological enzymes; Preferably, before the de-crosslinking treatment, the paraffin on the surface of the fixed biological sample is removed.

4. The nucleic acid detection method according to claim 1, wherein The permeabilization treatment in step 1) includes physical permeabilization treatment or chemical permeabilization treatment; the physical permeabilization treatment includes at least one of heating, light irradiation, and sonication; The chemical permeabilization treatment is to treat the biological sample to be detected with a permeabilization reagent; the permeabilization reagent includes at least one of Tween-20, Triton X-100, SDS, PBS, TE, citrate, biological enzymes, proteinase K, and pepsin.

5. The nucleic acid detection method according to claim 1, wherein In the step 2), a plurality of detection probes are added to the permeabilized biological sample, wherein the target capture sequence of each detection probe specifically binds to the target sequence of the target nucleic acid to be detected in the nucleic acid to be detected; Preferably, the target capture sequence has 20 to 150 bases.

6. The nucleic acid detection method according to claim 1, wherein The detection probe comprises a first detection probe and a second detection probe, wherein the first detection probe comprises a sequencing adapter sequence and a first target capture sequence; the second detection probe comprises a fixed nucleotide sequence and a second target capture sequence; The targeted capture sequence is formed by splicing the first targeted capture sequence and the second targeted capture sequence; The step 3) further comprises connecting the first targeted capture sequence and the second targeted capture sequence by a ligase; Preferably, the first targeted capture sequence and the second targeted capture sequence both have 10 to 120 bases.

7. The nucleic acid detection method according to claim 1, characterized in that The 3' end of the sequencing adapter sequence in the first detection probe is connected to the 5' end of the first targeted capture sequence; the 3' end of the second targeted capture sequence in the second detection probe is connected to the 5' end of the fixed nucleotide sequence.

8. The nucleic acid detection method according to claim 1, wherein The method of releasing the probe in the nucleic acid-probe complex from the biological sample and separating it from the nucleic acid to be detected includes at least one of cell or cell nucleus lysis, enzyme digestion, surfactant treatment, and heating.

9. The nucleic acid detection method according to claim 1, wherein The matrix includes at least one of gel particles, magnetic particles, polymer particles, and silica particles; The particle size of the solid phase carrier is 10 microns to 60 microns.

10. The nucleic acid detection method according to claim 1, characterized in that The coding sequence also includes a cell identification tag and a unique molecular identification tag sequence.

11. A method for detecting nucleic acid in a single cell or a single cell nucleus, characterized in that: The steps include: Step 1) preparing a biological sample to be tested into a single cell or single cell nucleus suspension; Step 2) permeabilizing a suspension of single cells or single cell nuclei to be detected; the single cells or single cell nuclei are fixed single cells or cell nuclei; Step 3) reacting the permeabilized single cell or single cell nucleus suspension to be detected in step 2) with a detection probe, so that the detection probe specifically binds to the nucleic acid to be detected in the single cell or single cell nucleus to form a nucleic acid-probe complex; the detection probe includes a sequencing adapter sequence, a target capture sequence, and an immobilized nucleotide sequence, and the target capture sequence is used to specifically bind to the target sequence of the target nucleic acid to be detected in the nucleic acid to be detected; Step 4) dispersing the single cells or cell nuclei in the single cell or cell nucleus suspension into a plurality of regions, such that each region contains at most one cell or cell nucleus; and adding one of the solid phase carriers to each region; Step 5) releasing the probe in the nucleic acid-probe conjugate from the single cell or single cell nucleus and separating it from the nucleic acid to be detected; binding the released probe to a solid phase support; the solid phase support includes a substrate having a coding sequence connected to the surface of the substrate, the coding sequence including a PCR primer sequence and a probe capture sequence; the probe capture sequence is used to bind to the fixed nucleotide sequence; Step 6) performing a DNA polymerase reaction on the solid phase to obtain a DNA polymerase reaction product, and sequencing the DNA polymerase reaction product to obtain a detection result of the nucleic acid to be detected.

12. The method for detecting nucleic acid of a single cell or a single cell nucleus according to claim 11, characterized in that: The dispersing of the cells to be detected into multiple regions specifically includes: The cells or cell nuclei are captured using a microfluidic chip containing a micropore array, so that each micropore in the micropore array contains at most one cell or cell nucleus; and then one solid phase carrier is captured in each micropore in the micropore array.

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