A high-throughput nucleic acid detection chip system

Abstract

This invention discloses a high-throughput nucleic acid detection chip system. It includes a nucleic acid amplification unit and a high-throughput nucleic acid detection chip. The high-throughput nucleic acid detection chip includes an amino-modified glass slide; a graphene oxide quantum dot layer is adsorbed on the amino-modified glass slide; several capture probe bands are disposed on the surface of the graphene oxide quantum dot layer; a DNA detection PDMS layer is disposed above the capture probe bands and reversibly adheres to the amino-modified glass slide; the capture probe bands are arranged parallel to each other and correspond to different target nucleic acids; the DNA detection PDMS layer has multiple independent detection wells, and each detection well is covered with a complete set of capture probe bands; single strands of target DNA are obtained using asymmetric RPA, and DNA detection is achieved through strand hybridization. This invention utilizes the strips to prepare the PDMS layer to complete the patterning of the capture probes, and then uses the detection PDMS layer to complete the sample well separation reaction, avoiding interference between the fabrication microchannels and the detection well structure.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a high-throughput nucleic acid detection chip and detection method based on asymmetric recombinase polymerase amplification, graphene oxide quantum dot barcode microarray, and multi-well PDMS layer. Background Technology

[0002] Nucleic acid testing is a crucial technical means for clinical diagnosis, large-scale population screening, and food microbial contamination safety monitoring. Its speed, sensitivity, and throughput determine the testing efficiency and control effectiveness. Currently, the widely used nucleic acid testing method is based on polymerase chain reaction (PCR) and its derivative amplification technology. This technology has high detection sensitivity and specificity, but its widespread adoption faces two main difficulties: first, it relies on specialized thermal cyclers to achieve nucleic acid denaturation, annealing, and extension, resulting in high equipment costs and failing to meet the needs of on-site testing and limited resources in primary healthcare institutions; second, the amplification time is relatively long, with conventional PCR amplification requiring 1-2 hours, making rapid detection difficult. To overcome the shortcomings of PCR technology, recombinase polymerase amplification (RPA) technology has been developed in recent years. This technology can rapidly amplify nucleic acids under mild isothermal conditions of 37-42℃, without relying on expensive thermal cyclers. It can amplify target DNA to a detectable level in just 10-20 minutes, reducing operational complexity and making it suitable for rapid on-site testing applications. In large-scale screening and multi-pathogen identification scenarios, simply increasing the amplification speed of a single target is still insufficient to meet practical needs. Real-world samples often require the simultaneous detection of multiple pathogens, viruses, or resistance genes, necessitating the processing of large numbers of samples within the same testing batch. Microarray chips can immobilize multiple capture probes within a limited area, while microfluidic chips can achieve the separation, addition, and reaction control of small-volume samples. The combination of these two technologies facilitates the parallel detection of multiple targets and multiple samples.

[0003] However, existing RPA-microarray solutions primarily focus on performing the RPA reaction directly on the microarray surface, or generating labeled amplicones through labeled primers or nucleotides, followed by microarray detection. These solutions are susceptible to competition from different primer systems, variations in labeling efficiency, and the surface reaction environment during multiplex amplification, and may not be suitable for constructing high-density multi-sample detection well arrays. On the other hand, existing graphene oxide quantum dot or graphene oxide barcode microfluidic chips are mostly used for the direct detection of proteins, antibodies, or some RNAs. They do not address the issue of rapid, highly specific hybridization between double-stranded DNA amplification products and surface capture probes, nor have they established a synergistic mechanism for "amplification into single strands—band capture—signal probe readout" applicable to multi-target DNA. Therefore, a nucleic acid detection chip is needed that can simultaneously achieve isothermal rapid amplification, multi-target recognition, multi-sample throughput, and low-background fluorescence readout, enabling rapid, quantitative, and parallel detection of multiple target nucleic acids in multiple samples without relying on complex thermal cycling equipment. Summary of the Invention

[0004] To address the aforementioned limitations of the prior art, the present invention aims to provide a high-throughput nucleic acid detection chip system, comprising a nucleic acid amplification unit and a detection chip. The detection chip uses an amino-modified glass slide as a substrate, on which a graphene oxide quantum dot layer is adsorbed. A PDMS layer is prepared using peelable strips, and multiple parallel capture probe strips are printed on the surface of the graphene oxide quantum dot layer. A DNA detection PDMS layer with multiple independent detection wells is then bonded together, ensuring that each detection well is covered by a complete array of capture probe strips.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a high-throughput nucleic acid detection chip system, comprising a nucleic acid amplification unit and a high-throughput nucleic acid detection chip; the high-throughput nucleic acid detection chip includes an amino-modified glass slide; a graphene oxide quantum dot layer is adsorbed on the amino-modified glass slide; a plurality of capture probe bands are disposed on the surface of the graphene oxide quantum dot layer; a DNA detection PDMS layer is disposed above the capture probe bands and reversibly adheres to the amino-modified glass slide; the capture probe bands are arranged in parallel with each other and correspond to different target nucleic acids; the DNA detection PDMS layer has a plurality of independent detection wells, and each detection well covers a complete set of capture probe bands; the nucleic acid amplification unit includes an asymmetric RPA amplification reagent, a forward primer and a reverse primer corresponding to the target nucleic acid, and a reaction vessel for isothermal amplification; the detection wells receive a target single strand and a fluorescent signal probe, such that a first recognition fragment of the target single strand hybridizes with the corresponding capture probe, and a second recognition fragment of the target single strand hybridizes with the corresponding fluorescent signal probe, thereby forming a capture probe-target single strand-fluorescent signal probe ternary hybridization complex on the corresponding capture probe band.

[0006] Preferably, the high-throughput nucleic acid detection chip is prepared by the following method: (1) The slide was ultrasonically cleaned, dried and then heated in a piranha solution. After cleaning and drying the slide, it was treated with oxygen plasma. Then the slide was immersed in an ethanol solution containing an aminosilane coupling agent to modify the slide with amino to obtain an amino-loaded slide. (2) Immerse the amino-loaded glass slide in the GOQDs dispersion to obtain a glass slide loaded with GOQDs layer; attach the PDMS layer with parallel loop microchannels to the glass slide loaded with GOQDs layer, add various capture probe solutions to the sample port of one of the microchannels respectively, drive the capture probe solutions to flow through the microchannels under negative pressure, so that the capture probes are adsorbed onto GOQDs to form multiple parallel capture probe bands; peel off the PDMS layer and block the non-specific adsorption sites on the surface of GOQDs; after washing, attach it to the DNA detection PDMS layer with multiple detection wells to obtain a high-throughput nucleic acid detection chip.

[0007] Preferably, in step (1), the heating treatment temperature is 100~120℃ and the time is 5~10 min; the oxygen plasma treatment time is 10 min; the concentration of the ethanol solution containing aminosilane coupling agent is 2 vol% and the aminosilane coupling agent is (3-aminopropyl)triethoxysilane; the immersion time is 0.5~2 h.

[0008] Preferably, in step (2), the concentration of the GOQDs dispersion is 1~5 mg / mL, the soaking time is 0.5~2 h; the PDMS layer is removed in 1~3 wt.% BSA / PBS solution and the slide is soaked in the solution for 8~40 min to block the non-specific adsorption sites on the surface of GOQDs.

[0009] Preferably, the DNA detection PDMS layer has 24 to 96 detection wells, each well covered with 5 to 50 capture probe bands.

[0010] The location of the detection well is used to encode the sample number, and the location of the capture probe band is used to encode the target nucleic acid type.

[0011] Preferably, the 5' or 3' end of the fluorescent signal probe is modified with a fluorescent dye, wherein the fluorescent dye is selected from at least one of the Cy3, Cy5, FAM, Texas Red, and Alexa Fluor series dyes.

[0012] Preferably, the asymmetric recombinase polymerase amplification is performed at a temperature of 37-42℃ for 10-30 min; the molar ratio of the forward primer to the reverse primer is determined according to the orientation of the target single strand, so that the amplification system is enriched with the target single strand that can hybridize with the capture probe and the fluorescent signal probe; the molar ratio of the forward primer to the reverse primer is 2:1, 5:1, 10:1, 50:1 or 75:1.

[0013] Preferably, the target nucleic acid includes one or more of the following: conserved genes of pathogens, DNA viral nucleic acids, resistance genes, food microbial contamination marker genes, or environmental microbial marker genes.

[0014] A second aspect of the present invention provides a method for nucleic acid detection using a high-throughput nucleic acid detection chip system, comprising the following steps: S1. Asymmetric recombinase polymerase amplification is performed on the target DNA in the test sample to obtain the target single strand corresponding to the target DNA; S2. After mixing the target single strand with the fluorescent signal probe, add it to the detection well of the DNA detection PDMS layer so that the target single strand hybridizes with the corresponding capture probe and the corresponding fluorescent signal probe at the same time; S3. Remove unbound components and wash the detection chip; S4. Use a microarray chip scanner to read the fluorescence intensity of each captured probe band in each detection well, and obtain the target nucleic acid concentration according to the pre-established target nucleic acid concentration-fluorescence intensity standard curve.

[0015] A third aspect of the present invention provides the application of a high-throughput nucleic acid detection chip system in in vitro pathogenic microorganism nucleic acid detection, wherein the pathogenic microorganism nucleic acid detection includes clinical infection auxiliary diagnosis, food microbial contamination monitoring, environmental pathogen detection, or resistance gene monitoring.

[0016] The beneficial effects of this invention are: (1) This invention utilizes asymmetric RPA to obtain single strands of target DNA and achieves DNA detection through strand hybridization, solving the difficulty of parallel detection of different DNAs by symmetric amplification. Furthermore, isothermal amplification eliminates the need for thermal cycling equipment, significantly shortening the amplification time. A nucleic acid detection chip with multiple parallel capture probe bands and multiple detection wells, each containing multiple capture probe bands, is constructed. A single chip can simultaneously detect 10 types of DNA (i.e., 600 items) from 60 samples, offering high throughput and low sample requirements. By reading the fluorescence signals of the bands, quantitative detection of DNA concentration can be achieved, enabling high detection sensitivity and multi-target parallel detection capabilities in applications such as clinical diagnosis, rapid screening of large-scale populations, and food microbial contamination safety monitoring.

[0017] (2) Compared to directly using double-stranded RPA products or labeled primer products, the asymmetric RPA of the present invention can provide target single strands that can rapidly hybridize with both capture probes and signal probes, reducing the competitive impact of double-stranded refolding on surface hybridization. Compared to conventional microarray surfaces, the graphene oxide quantum dot layer of the present invention simultaneously provides a single-stranded probe adsorption and fixation interface and a background fluorescence modulation interface, which helps to improve the stability of strip formation and the detection signal-to-noise ratio.

[0018] (3) This invention uses asymmetric RPA to amplify target DNA under isothermal conditions, which is suitable for rapid on-site detection. By enriching the target single strand with asymmetric primer ratio, the target single strand can act as a bridging nucleic acid to simultaneously connect the capture probe and the fluorescent signal probe, thereby improving the surface hybridization efficiency. The sample is encoded by the detection well position, and the target nucleic acid type is encoded by the capture probe band position, enabling parallel detection of multiple samples and multiple targets on a single chip. The GOQDs layer has abundant functional groups and adsorption capacity for single-stranded nucleic acids, which can form stable bands and reduce the interference of unbound fluorescent signals on the detection results through background modulation. The capture probe patterning is completed by preparing the PDMS layer using the bands, and the sample well reaction is completed by using the detection PDMS layer, avoiding mutual interference between the microchannel preparation and the detection well structure. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the chip fabrication process; Figure 2 Photograph of a high-throughput nucleic acid detection chip with multiple detection wells; Figure 3 This is a gel electrophoresis image used to verify primer specificity in the examples; Figure 4 This is a schematic diagram of the ternary hybridization reaction of the detection probe-target single-stranded target-fluorescent signal probe within the well; Figure 5 This is a fluorescence scan of the chip; Figure 6 The fluorescence signals and standard curves corresponding to different target DNA concentrations; Figure 7 This is a fluorescence scan of the chip. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0022] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0023] Example 1: Fabrication of a high-throughput nucleic acid detection chip (1) A glass slide with a length of 76 mm, a width of 25 mm, and a thickness of 1 mm was ultrasonically cleaned with water, ethanol, and isopropanol for 5 min in sequence, and then dried with nitrogen. The glass slide was immersed in a piranha solution (piranha solution prepared with a volume ratio of concentrated sulfuric acid and 30% H2O2 aqueous solution of 7:3) and treated at 100℃ for 10 min. After cooling, the glass slide was cleaned with ultrapure water and dried. The glass slide was treated with oxygen plasma for 10 min to generate a large number of hydroxyl groups on the surface. The glass slide was immersed in a 2 vol% (3-aminopropyl)triethoxysilane ethanol solution for 45 min to modify the glass slide with amino groups, and then dried with nitrogen.

[0024] (2) PDMS and RTV615 curing agent were mixed at a mass ratio of 10:1 and stirred evenly. The mixture was then degassed in a vacuum drying oven. The mixture was poured into a mold and heated at 80°C for 2 h to cure it. The mold was then demolded. A PDMS layer containing 10 parallel spiral microchannels (each microchannel is 20 μm wide) was obtained. Through holes were drilled vertically at both ends of the microchannels to serve as the sample inlet and the gas outlet, respectively.

[0025] Amino-modified glass slides were immersed in a 3 mg / mL GOQDs dispersion (water as the dispersant, and graphene quantum dots purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.) for 1 h to allow GOQDs to adsorb onto the surface of the glass slide (GOQDs completely covered the surface of the glass slide). Subsequently, a PDMS layer prepared by strips with 10 parallel loop-shaped microchannels was attached to the glass slide. 3 μL of 10 capture probe solutions with a concentration of 1 μM (dissolved in enzyme-free water, see step (3)) were added to the sample inlet of each of the microchannels. The outlet was connected to a vacuum pump and pumped for 2 h. The capture probe solution was driven by negative pressure (80 torr) to flow through the microchannel, so that the capture probes flowed along the microchannel and adsorbed onto the surface of the GOQDs layer, forming multiple parallel capture probe strips.

[0026] After pumping for 2 h, the PDMS layer was removed in a 3 wt% BSA / PBS solution (0.75 g of BSA was added to 24.25 g of pH 7.4 PBS buffer and dissolved by vortexing) and the slide was soaked for another 20 min to block the non-specific adsorption sites on the surface of the GOQDs layer.

[0027] PDMS and RTV615 curing agent were mixed at a mass ratio of 10:1 and stirred until homogeneous. The mixture was then degassed in a vacuum drying oven. The mixture was poured into a mold and heated at 80°C for 2 hours to cure. The mold was then demolded to obtain a PDMS layer containing 60 detection holes. The detection holes are square, with a side length of 2 mm, a depth of 1 mm, and a single hole volume of 4 mm². 3 The spacing between the capture probe bands was 20 μm; the effective band length covered by each detection well was 2 mm. After washing sequentially with PBS buffer and ultrapure water, the DNA detection PDMS layer with 60 detection wells was attached to a glass slide and aligned with the edge of the slide. Each well contained two sets of capture probe bands (the two sets of capture probe bands in each detection well were repeat detection bands to improve detection reliability), and each set had 10 parallel bands, thus preparing a high-throughput nucleic acid detection chip. The chip fabrication process and chip images are shown below. Figure 1 and Figure 2 As shown.

[0028] (3) Preparation of 10 capture probe solutions: Using the DNA of 10 common pathogenic bacteria (Acinetobacter baumannii, Streptococcus mutans, Listeria monocytogenes, Klebsiella pneumoniae, Streptococcus pneumoniae, Enterococcus faecalis, Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, and Pseudomonas aeruginosa) as targets, we selected their conserved gene sequences (adeS, luxS, prfA, rmpA, ply, gelE, slo, sasG, sodG, and oprL, respectively, in the order of the bacteria mentioned above) as detection targets. We designed capture probes and signal probes for the targets. The 5' end of the signal probe was modified with the fluorescent dye Cy3 (the probe was synthesized and modified by Sangon Biotech (Shanghai) Co., Ltd., and dissolved in enzyme-free water). The sequences are listed in Table 1.

[0029] Table 1. Sequences of capture probes and signal probes for detecting the target in Example 1 Example 2: Preparation of nucleic acid amplification unit (1) RPA primers were designed based on the conserved gene sequences of the above 10 pathogens. The primer sequences are listed in Table 2. RPA was performed using the TwistAmp® Basic kit manufactured by TwistDx Inc. and with reference to the included instruction manual. Ten samples of 10 μL each of the ten target DNAs (1 pM, dissolved in enzyme-free water; NCBI accession numbers are shown in Table 3), 3 μL each of the ten forward primers (10 μM, dissolved in enzyme-free water), 3 μL of the ten reverse primers (dissolved in enzyme-free water) at different concentrations (the forward primer concentration was kept constant at 10 μM; the reverse primers were diluted according to the optimized forward and reverse primer ratios for each gene in Table 4), lyophilized enzyme powder (including recombinase, single-strand binding protein, and strand displacement DNA polymerase), 29 μL of reconstitution buffer, 2.5 μL of 280 mM magnesium acetate solution, and 2.5 μL of ultrapure water were mixed in a test tube and subjected to multiple asymmetric RPA at 40°C for 20 min to obtain single strands of the target DNA. The test tube was then heated to 100°C and held for 5 min to terminate the amplification, yielding the amplicon solution.

[0030] Table 2. RPA primer sequences for 10 genes Table 3. Accession numbers of 10 target DNAs Table 4. Optimized molar ratio of forward and reverse primers for 10 genes in RPA (2) To verify that primers targeting one type of target DNA cannot amplify the other nine types of target DNA, an experiment was conducted to amplify the other nine types of target DNA using primers targeting one type of target DNA. The results are as follows: Figure 3 The gel electrophoresis results are shown in the figure (bands 1-10 in the figure amplified the products of 9 other target DNAs using primers adeS, luxS, prfA, rmpA, ply, gelE, slo, sasG, sodC, and oprL, respectively). The primers for the 10 target DNAs did not amplify the sequences of the other 9 target DNAs, indicating that the primers are specific.

[0031] Example 3: Detection Method (1) Mix 3 μL of the amplicon solution prepared in step (1) of Example 2 and 1 μL of a solution containing 10 fluorescent signal probes prepared in step (3) of Example 1 at a concentration of 0.1 μM. Add the mixture to 60 wells of the DNA detection PDMS layer. The capture probe and signal probe are complementary to a portion of the target DNA amplicon sequence. Hybridize in PBS buffer at 25°C for 20 min. Wash three times with PBS, rinse with ultrapure water, and dry with nitrogen. Attach the signal probes to the chip surface. The hybridization reaction is as follows: Figure 4 As shown.

[0032] (2) Remove the DNA detection PDMS layer and wash the slide surface sequentially with PBS buffer and ultrapure water. Read the fluorescence signal of the bands on the chip using a microarray chip scanner, such as... Figure 5 As shown, there are two sets of fluorescent bands in each of the 60 wells, with 10 parallel fluorescent bands in each set. This indicates that the chip can detect 60 samples, and each sample can detect 10 different DNAs, meaning that a single chip can detect 600 items.

[0033] Example 4: Sensitivity A high-throughput nucleic acid detection chip system was constructed according to Examples 1 and 2, and detection was performed according to the method in Example 3. The difference was that the sample contained only one type of target DNA, and different concentrations of target DNA were amplified. Fluorescence detection was performed on the chip. The concentration of target DNA in the sample was plotted on the x-axis, and the fluorescence signal of the band on the chip was plotted on the y-axis. The detection limit was calculated according to LOD=3σ / k, where σ is the standard deviation of fluorescence intensity of blank sample, and k is the slope of standard curve. Three replicates were set for each concentration.

[0034] like Figure 6 As shown in Table 4, the linear range and detection limit for quantitative detection are obtained, indicating that the chip has high detection sensitivity for the DNA of 10 common pathogens.

[0035] Table 4. Detection range and detection limit for 10 target DNAs Example 5: Chip Detection Specificity Using the 10 target DNAs from Example 2, to verify that the capture probe and signal probe targeting the asymmetric RPA product of one target DNA could not detect the asymmetric RPA products of the other 9 target DNAs, an experiment was conducted to detect the asymmetric RPA products of the other 9 target DNAs using the same capture probe and signal probe targeting the asymmetric RPA product of the one target DNA. The method involved preparing a capture probe band targeting the asymmetric RPA product of the one target DNA on a chip substrate, and adding a signal probe targeting the asymmetric RPA product of the one target DNA and the asymmetric RPA products of the other 9 target DNAs to the wells of the DNA detection PDMS layer. The results are as follows. Figure 7 As shown in the microarray fluorescence image, neither the capture probe nor the signal probe targeting 10 asymmetric RPA products of target DNA could generate fluorescence signals for the detection of the other 9 asymmetric RPA products of target DNA, indicating that the microarray detection is specific.

[0036] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A high-throughput nucleic acid detection chip system, characterized in that, The device includes a nucleic acid amplification unit and a high-throughput nucleic acid detection chip. The high-throughput nucleic acid detection chip includes an amino-modified glass slide. A graphene oxide quantum dot layer is adsorbed onto the amino-modified glass slide. Several capture probe bands are disposed on the surface of the graphene oxide quantum dot layer. A DNA detection PDMS layer, reversibly bonded to the amino-modified glass slide, is disposed above the capture probe bands. The capture probe bands are arranged parallel to each other and correspond to different target nucleic acids. The DNA detection PDMS layer has multiple independent detection wells, and each detection well covers a complete set of capture probe bands. The nucleic acid amplification unit includes asymmetric RPA amplification reagent, forward and reverse primers corresponding to the target nucleic acid, and a reaction vessel for isothermal amplification. The detection wells receive target single-stranded molecules and fluorescent signal probes, causing the first recognition fragment of the target single-stranded molecule to hybridize with the corresponding capture probe, and the second recognition fragment of the target single-stranded molecule to hybridize with the corresponding fluorescent signal probe, thereby forming a capture probe-target single-stranded molecule-fluorescent signal probe ternary hybridization complex on the corresponding capture probe band.

2. The high-throughput nucleic acid detection chip system according to claim 1, characterized in that, The high-throughput nucleic acid detection chip is prepared by the following method: (1) The slide was ultrasonically cleaned, dried and then heated in a piranha solution. After cleaning and drying the slide, it was treated with oxygen plasma. Then the slide was immersed in an ethanol solution containing an aminosilane coupling agent to modify the slide with amino to obtain an amino-loaded slide. (2) Immerse the amino-loaded glass slide in the GOQDs dispersion to obtain a glass slide loaded with GOQDs layer; attach the PDMS layer with parallel loop microchannels to the glass slide loaded with GOQDs layer; add various capture probe solutions to the sample inlet of one of the microchannels respectively; drive the capture probe solutions through the microchannels with negative pressure to adsorb the capture probes onto the GOQDs, forming multiple parallel capture probe bands; peel off the PDMS layer and seal the non-specific adsorption sites on the surface of GOQDs; After washing, it is bonded to a DNA detection PDMS layer with multiple detection wells to obtain a high-throughput nucleic acid detection chip.

3. The high-throughput nucleic acid detection chip system according to claim 2, characterized in that, In step (1), the heating treatment temperature is 100~120℃ and the time is 5~10 min; the oxygen plasma treatment time is 10 min; the concentration of the ethanol solution containing aminosilane coupling agent is 2 vol% and the aminosilane coupling agent is (3-aminopropyl)triethoxysilane; the immersion time is 0.5~2 h.

4. The high-throughput nucleic acid detection chip system according to claim 2, characterized in that, In step (2), the concentration of the GOQDs dispersion is 1~5 mg / mL, and the soaking time is 0.5~2 h; the PDMS layer is removed in 1~3 wt.% BSA / PBS solution and the slide is soaked in the solution for 8~40 min to block the non-specific adsorption sites on the surface of GOQDs.

5. The high-throughput nucleic acid detection chip system according to claim 1, characterized in that, The DNA detection PDMS layer has 24 to 96 detection wells, each well covered with 5 to 50 capture probe bands.

6. The high-throughput nucleic acid detection chip system according to claim 1, characterized in that, The 5' or 3' end of the fluorescent signal probe is modified with a fluorescent dye, which is selected from at least one of the Cy3, Cy5, FAM, Texas Red, and Alexa Fluor series dyes.

7. The high-throughput nucleic acid detection chip system according to claim 1, characterized in that, The molar ratio of the forward primer to the reverse primer is determined according to the orientation of the target single strand, so that the amplification system is enriched with the target single strand that can hybridize with the capture probe and the fluorescent signal probe; the molar ratio of the forward primer to the reverse primer is 2:1, 5:1, 10:1, 50:1 or 75:

1.

8. The high-throughput nucleic acid detection chip system according to claim 1, characterized in that, The target nucleic acid includes one or more of the following: conserved genes of pathogens, DNA viral nucleic acids, resistance genes, food microbial contamination marker genes, or environmental microbial marker genes.

9. A method for nucleic acid detection using the high-throughput nucleic acid detection chip system according to any one of claims 1 to 8, characterized in that, Includes the following steps: S1. Asymmetric recombinase polymerase amplification is performed on the target DNA in the test sample to obtain the target single strand corresponding to the target DNA; S2. After mixing the target single strand with the fluorescent signal probe, add it to the detection well of the DNA detection PDMS layer so that the target single strand hybridizes with the corresponding capture probe and the corresponding fluorescent signal probe at the same time; S3. Remove unbound components and wash the detection chip; S4. Use a microarray chip scanner to read the fluorescence intensity of each captured probe band in each detection well, and obtain the target nucleic acid concentration according to the pre-established target nucleic acid concentration-fluorescence intensity standard curve.

10. The application of the high-throughput nucleic acid detection chip system according to any one of claims 1 to 8 in in vitro nucleic acid detection of pathogenic microorganisms, characterized in that, The pathogenic microorganism nucleic acid detection includes clinical infection auxiliary diagnosis, food microbial contamination monitoring, environmental pathogen detection, or resistance gene monitoring.

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