A rapid detection method of foodborne pathogenic microorganisms

Abstract

The application relates to the technical field of food microorganism detection, and discloses a rapid detection method for foodborne pathogenic microorganisms, which comprises the following steps: extracting target microorganism genomic DNA of a to-be-detected sample; preparing a nucleic acid amplification reaction solution to perform asymmetric PCR to accumulate target single-strand DNA; preparing first and second gold nano probes with different probe surface modifications; mixing the single-strand DNA and the gold nano probes to construct a detection system, so that gold nano particles are cross-linked and aggregated under base complementary pairing to increase the average hydration particle size of the detection system; collecting a scattered light intensity fluctuation signal of particle Brownian motion and calculating the average hydration particle size value, and then inputting the average hydration particle size value into a mathematical model to calculate the equivalent bacterial concentration of the target microorganism. Through the conversion of sequence hybridization recognition into physical increase of particle size, the combination of light scattering signals and equation solving, the fluorescent labeling is removed, the matrix interference is avoided, and rapid and accurate quantitative analysis of microorganisms is realized.

Description

Technical Field

[0001] This invention relates to the field of food microbiology detection technology, specifically a rapid detection method for foodborne pathogenic microorganisms. Background Technology

[0002] Foodborne pathogens are a major cause of food safety problems, and rapid and accurate microbial testing of contaminated food samples is a key step in preventing foodborne disease outbreaks. Conventional pathogen detection methods often rely on traditional biochemical culture, which involves long enrichment and isolation processes, resulting in lengthy testing cycles that fail to meet the demands of modern food processing and distribution for real-time monitoring and rapid response.

[0003] With the development of molecular biology techniques, polymerase chain reaction (PCR) and its derived quantitative fluorescence detection technology have been increasingly applied in the field of microbial detection. While existing fluorescence amplification detection technologies have shortened detection time and improved detection sensitivity, they still have significant limitations in practical applications. These technologies typically rely on expensive, specific fluorescently labeled probes, and the reaction system requires sophisticated and complex optical signal reading equipment, significantly increasing the reagent and instrument costs for grassroots testing institutions. Furthermore, the matrix composition of food samples is complex, and impurities such as pigments, lipids, and large protein molecules can easily quench or interfere with the optical fluorescence signal, leading to deviations in the quantitative results of low-concentration samples.

[0004] Furthermore, some existing technologies have incorporated gold nanoparticles as chromogenic probes for the visualization or absorbance colorimetric detection of microbial nucleic acids. However, traditional colorimetric detection methods primarily rely on optical instruments to read the absorbance values ​​corresponding to the overall color change of the solution. When faced with complex food matrix homogenates that possess their own color or exhibit a certain degree of physical turbidity, the colorimetric signal is easily masked by the matrix background. This macroscopic detection method, which depends on absorbance measurement, suffers from a sensitivity bottleneck in the detection of low-concentration microbial samples, making it difficult to capture subtle changes in the particle state at the microscopic level. Consequently, establishing stable and accurate mathematical models for the quantitative quantification of equivalent bacterial concentrations presents significant challenges. Therefore, existing technologies require a novel approach that can circumvent interference from complex sample matrix backgrounds, eliminate the cost of fluorescent labeling, and achieve efficient and accurate quantitative detection of target pathogenic microorganisms. Summary of the Invention

[0005] The technical problem solved by this invention is that existing methods for detecting foodborne pathogens are cumbersome to operate, have long detection cycles, and rely on complex fluorescently labeled probes or large analytical instruments, making it difficult to achieve efficient and rapid quantitative detection based on low-concentration samples.

[0006] To address the above problems, the present invention provides the following technical solution: This invention provides a rapid detection method for foodborne pathogenic microorganisms, comprising extracting the genomic DNA of the target microorganism from the sample to be tested; Using the target microbial genomic DNA as a template, a nucleic acid amplification reaction solution containing upstream and downstream primers with unequal molar concentrations was prepared, and an asymmetric PCR amplification program was performed to accumulate the target single-stranded DNA in the nucleic acid amplification reaction solution. Prepare a first gold nanoprobe and a second gold nanoprobe based on gold nanoparticles, with the surfaces of a first nucleic acid capture probe and a second nucleic acid capture probe respectively modified. The target single-stranded DNA is mixed with the first and second gold nanoparticle probes to construct a detection system. Under preset hybridization reaction conditions, the target single-stranded DNA undergoes base complementary pairing with the first and second nucleic acid capture probes, respectively, which promotes the cross-linking and aggregation of the gold nanoparticles and increases the average hydration particle size of the detection system. The scattered light intensity fluctuation signal generated by the Brownian motion of the gold nanoparticles in the detection system is collected, the average hydration particle size of the detection system is calculated, and the average hydration particle size is substituted into the pre-established mathematical model for quantitative detection of target microorganisms to calculate the equivalent bacterial concentration of the target microorganism in the sample to be tested.

[0007] The core technical principle of this invention lies in: By establishing an asymmetric nucleic acid amplification reaction, the conventional double-stranded equal-volume amplification mode is changed, and single-stranded DNA targets are accumulated efficiently. Simultaneously, the specific hybridization of single-stranded DNA with double-gold nanoprobes modified with complementary sequences transforms microscopic molecular recognition events into spatial conformational changes in nanoparticles, i.e., the nanoparticles transform from a dispersed state to a cross-linked aggregated state. This cross-linking and aggregation behavior leads to an increase in the average hydration particle size of the system. Since the change in the hydration particle size of nanoparticles has a definite physical correlation with its Brownian motion rate in the liquid environment, dynamic light scattering technology is used to capture the attenuation signal of the scattered light intensity, analyze the particle size evolution process of the system, and then establish a linear correspondence between the hydration particle size and the initial concentration of the target microorganism, achieving quantitative detection.

[0008] Furthermore, the extraction of the target microbial genomic DNA from the sample to be tested includes: The sample to be tested was mechanically pulverized and mixed with buffer solution by shaking to obtain a matrix homogenate; Microbial lysis reagent is added to the matrix homogenate to disrupt the cell structure of the microorganism and release the genomic DNA of the target microorganism, forming a lysis mixture containing free nucleic acid components; A protein precipitation reagent is added to the lysis mixture and centrifugation is applied to precipitate protein molecules and matrix residues, and the liquid supernatant containing free nucleic acid components is extracted. The liquid supernatant is transferred to a nucleic acid binding purification device, whereby the free nucleic acid components adhere to the surface of the solid medium inside. After washing to remove impurities, sterile water is added for elution to obtain the genomic DNA of the target microorganism.

[0009] Furthermore, the execution of the asymmetric PCR amplification procedure, accumulating target single-stranded DNA in the nucleic acid amplification reaction solution, includes: The molar ratio of the upstream primer to the downstream primer in the nucleic acid amplification reaction solution is controlled to be 10:1; The denaturation, annealing, and extension cycles are performed alternately in a thermal cycling amplification device. As the number of cycles increases, once the downstream primer, which is at a low concentration, is consumed, the amplification mode changes from double-stranded amplification to linear amplification guided by the upstream primer, and the target single-stranded DNA accumulates in the nucleic acid amplification reaction solution.

[0010] Furthermore, the preparation of the first gold nanoprobe and the second gold nanoprobe, which are based on gold nanoparticles and respectively surface-modified with a first nucleic acid capture probe and a second nucleic acid capture probe, includes: A colloidal solution of gold nanoparticles was obtained by heating a reducing agent solution to reduce chloroauric acid solution. The first nucleic acid capture probe and the second nucleic acid capture probe are treated with a reducing agent to make the probe ends with thiol modification in a free state; The activated first nucleic acid capture probe and the second nucleic acid capture probe were respectively mixed with the gold nanoparticle colloidal solution and incubated in the dark to fix the first nucleic acid capture probe and the second nucleic acid capture probe onto the surface of the gold nanoparticles through gold-sulfur bonds. During static incubation, sodium chloride solution was added dropwise to the mixed system in batches for aging treatment, thereby reducing the electrostatic repulsion on the surface of the gold nanoparticles and increasing the coverage density of the first and second nucleic acid capture probes. Subsequently, centrifugation and elution were performed to obtain the purified first and second gold nanoparticle probes.

[0011] Furthermore, the step of mixing the target single-stranded DNA with the first and second gold nanoparticle probes to construct a detection system, under preset hybridization conditions, involves the target single-stranded DNA undergoing base complementary pairing with the first and second nucleic acid capture probes, respectively, promoting the cross-linking and aggregation of the gold nanoparticles, and increasing the average hydration particle size of the detection system, including: Hybridization buffer is added to a mixture containing the target single-stranded DNA, the first gold nanoprobe, and the second gold nanoprobe to adjust the salt concentration and pH of the detection system to a set range. The set range is used to promote stable hybridization of the target single-stranded DNA with the first nucleic acid capture probe and the second nucleic acid capture probe, and to inhibit non-specific self-aggregation of the first gold nanoprobe and the second gold nanoprobe. The adjusted detection system was incubated in a constant temperature environment to induce cross-linking of the gold nanoparticles; After the set incubation time is reached, the detection system is transferred to a low-temperature environment to stand, thereby reducing the thermal motion of molecules in the system, maintaining the conformation of the cross-linked and aggregated gold nanoparticles and fixing the hydration particle size state.

[0012] Furthermore, the step of collecting the scattered light intensity fluctuation signal generated by the Brownian motion of the gold nanoparticles within the detection system and calculating the average hydration particle size of the detection system includes: The detection system is illuminated by a monochromatic laser light source, and the scattered light intensity fluctuation signal occurring over time at a set angle is recorded by a photodetector. Autocorrelation mathematical analysis is performed on the scattered light intensity fluctuation signal to generate a time autocorrelation function curve; The translational diffusion coefficient was extracted by fitting the decay rate to the time autocorrelation function curve using the cumulative method. The average hydration particle size is calculated based on the translational diffusion coefficient, the liquid phase kinetic viscosity of the detection system, and the absolute temperature parameter of the environment in which the detection system is located.

[0013] Furthermore, before substituting the average hydration particle size value into the pre-established mathematical model for quantitative detection of target microorganisms to calculate the equivalent bacterial concentration of the target microorganism in the sample to be tested, the process further includes an offline construction process of the mathematical model for quantitative detection of target microorganisms, specifically including: Prepare target microbial standard samples with multiple concentration gradients; For target microbial standard samples at various concentration gradients, genomic DNA extraction, asymmetric PCR amplification, probe hybridization, and light scattering particle size determination were performed to obtain the average hydration particle size corresponding to the equivalent bacterial concentration of each target microorganism. The equivalent bacterial concentration of the target microorganism is converted into a concentration order-of-magnitude parameter as the independent variable, and the corresponding average hydration particle size value is used as the dependent variable for linear regression fitting to obtain the corresponding linear equation as the mathematical model for quantitative detection of the target microorganism. The concentration order-of-magnitude parameter is the decimal logarithmic value of the equivalent bacterial concentration of the target microorganism relative to the preset benchmark concentration.

[0014] In a preferred embodiment of the present invention, when adding a protein precipitation reagent to the lysis mixture and applying centrifugal force to precipitate protein molecules and matrix residues, and extracting the liquid supernatant containing free nucleic acid components: When the matrix of the sample to be tested contains lipid components and floats and separates into layers above the liquid phase under centrifugation, the top lipid structure is avoided when extracting the supernatant of the liquid phase, thereby reducing the inhibitory effect of matrix macromolecules on subsequent specific nucleic acid amplification reactions.

[0015] In a preferred embodiment of the present invention, the step of calculating the average hydration particle size value based on the translational diffusion coefficient, the liquid phase kinetic viscosity of the detection system, and the absolute temperature parameter of the environment in which the detection system is located includes: Based on the Stokes-Einstein equation, the average hydration particle size is obtained by calculating the product of the Boltzmann constant and the absolute temperature parameter of the environment in which the detection system is located, and then dividing the product by three times the product of pi, the liquid phase kinetic viscosity, and the translational diffusion coefficient.

[0016] In a preferred embodiment of the present invention, the step of adding a reducing reagent to treat the first nucleic acid capture probe and the second nucleic acid capture probe, so that the probe ends with thiol modification are in a free state, includes: A solution containing tris(2-carboxyethyl)phosphine and an acidic acetate buffer solution were added to the first nucleic acid capture probe and the second nucleic acid capture probe, respectively. Incubation was performed at room temperature to utilize the reducing properties of the tris(2-carboxyethyl)phosphine component to open the disulfide bonds formed between the first and second nucleic acid capture probe molecules.

[0017] This invention provides a rapid detection method for foodborne pathogenic microorganisms. It has the following beneficial effects: 1. This invention uses target microbial genomic DNA as a template and prepares a nucleic acid amplification reaction solution with unequal molar concentrations of upstream and downstream primers. An asymmetric PCR amplification program is executed during thermal cycling, causing the system to switch to linear amplification after the low-concentration downstream primers are consumed. This results in the accumulation of target single-stranded DNA in the reaction solution. This technique eliminates the need for cumbersome fluorescent labeling of the target nucleic acid, directly utilizing the amplified single-stranded nucleic acid sequence as the ligation sequence for subsequent nanoprobe cross-linking. This simplifies pretreatment and molecular reaction steps and reduces reagent costs for nucleic acid detection.

[0018] 2. This invention utilizes the generated target single-stranded DNA to construct a detection system by mixing it with gold nanoparticles modified with a first nucleic acid capture probe and a second nucleic acid capture probe, respectively. In a preset hybridization buffer environment, base complementary pairing occurs, causing the originally dispersed gold nanoparticles to undergo specific spatial cross-linking and aggregation. This process, which directly transforms the microscopic molecular-level nucleic acid sequence hybridization and recognition process into a physical process of nanoparticle physical conformation change and an increase in overall average hydration particle size, effectively avoids background interference from colored impurities in complex sample matrices on traditional colorimetric detection signals and improves the specificity of the detection signal in complex biochemical environments.

[0019] 3. This invention collects the scattered light intensity fluctuation signal generated by the Brownian motion of cross-linked gold nanoparticles in the detection system and calculates the average hydration particle size of the detection system by combining it with relevant physical equations. Then, the particle size value is directly substituted into a linear mathematical model constructed in advance using standard samples with different concentration gradients. This technical solution, which measures the physical particle size of the liquid phase based on light scattering signals and performs mathematical calculations using linear equations, establishes a direct mapping relationship between the equivalent bacterial concentration of microorganisms and physical measurement indicators. It eliminates the long cycle of conventional microbial culture and counting and realizes efficient and accurate quantitative analysis of target microorganisms in the test sample. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the detection system architecture of the present invention; Figure 2 This is a flowchart of the method of the present invention; Figure 3 This is a flowchart of the sample pretreatment and genomic DNA extraction process of the present invention; Figure 4 This is a flowchart of the asymmetric nucleic acid amplification process of the present invention; Figure 5 This is a flowchart illustrating the preparation process of the gold nanoprobe of the present invention. Figure 6 This is a flowchart of the probe-specific aggregation reaction of the present invention; Figure 7 This is a flowchart illustrating the determination of hydrated particle size and calculation of equivalent bacterial concentration of target microorganisms in the present invention. Figure 8 The figure shows the characterization results of the gold nanoprobe of this invention; In the figure, A is the hydration particle size distribution of unmodified gold nanoparticles, B is the hydration particle size distribution of gold nanoprobes, and C is a comparison of the UV-Vis absorption spectra of the system before and after modification.

[0021] Figure 9 This is a diagram verifying the detection effect of the target single-stranded DNA in this invention; In the diagram, A shows the hydration particle size distribution when 0 pM of target single-stranded DNA is added to the detection system; B shows the hydration particle size distribution when 100 pM of target single-stranded DNA is added to the detection system; C shows the hydration particle size distribution when 10 nM of target single-stranded DNA is added to the detection system; D shows the hydration particle size distribution when 100 nM of target single-stranded DNA is added to the detection system; and E shows a comparison of the UV-Vis absorption spectra of different detection systems. Figure 10 Electrophoresis diagram showing the optimized asymmetric amplification primer concentration of this invention; Figure 11 This is a graph showing the optimized sodium chloride concentration results for the detection system of this invention. In this diagram, A represents the UV-Vis absorption spectra of the blank control (negative) sample at different sodium chloride concentrations; B represents the UV-Vis absorption spectra of the positive sample at different sodium chloride concentrations; C represents the histogram of absorbance difference between the positive and negative detection systems at different sodium chloride concentrations; and D represents the analysis results of the average hydration particle size and particle size difference of the positive and negative detection systems at different sodium chloride concentrations.

[0022] Figure 12 This is a graph showing the pH optimization results of the detection system of the present invention; In this diagram, A represents the UV-Vis absorption spectra of the blank control (negative) sample under different pH conditions; B represents the UV-Vis absorption spectra of the positive sample under different pH conditions; C represents the histogram of absorbance difference between the positive and negative detection systems under different pH conditions; D represents the analysis results of the average hydration particle size of the positive and negative detection systems under different pH conditions; and E represents the curve of the average hydration particle size difference between the positive and negative detection systems under different pH conditions.

[0023] Figure 13 This is a fitting graph for the quantitative detection of target microorganisms in the pure culture medium of the present invention; In this graph, A represents the trend of Aspergillus flavus concentration versus average hydration particle size; B represents the linear regression fitting graph for quantitative detection of Aspergillus flavus. Figure 14 This is a specific experimental analysis diagram of the present invention. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] See attached document Figure 1This invention provides a rapid detection system for foodborne pathogenic microorganisms, comprising a sample pretreatment unit for homogenizing and dissolving the sample matrix and extracting total genomic DNA of the target microorganism from the homogenate. This unit performs preliminary purification of complex samples, providing nucleic acid templates for subsequent molecular operations. A nucleic acid amplification unit receives the nucleic acid template output from the sample pretreatment unit. This unit uses a specific primer combination to perform a thermal cycling amplification program, synthesizing a large number of single-stranded nucleic acid fragments of a specific sequence in the reaction system. A probe hybridization reaction unit accommodates the biochemical reaction between the nucleic acid sequence and nanoparticles. This unit receives the single-stranded nucleic acid fragments generated by the nucleic acid amplification unit and mixes them with gold nanoparticles with complementary sequences modified on their surface. Under set ionic and acid-base conditions, intermolecular base pairing occurs within this unit. A light scattering signal detection unit performs optical measurements on the mixture within the probe hybridization reaction unit. This unit collects the scattering light signal fluctuations of the particles in the system, calculates the particle size data, and outputs the corresponding equivalent bacterial concentration of the target microorganism based on a built-in mathematical model.

[0026] See attached document Figure 2 This invention provides a rapid detection method for foodborne pathogenic microorganisms, which may include: S100, extracting genomic DNA from the sample to be tested. The sample to be tested is homogenized, and buffer solution is added and shaken to fully release the microbial cells in the matrix. Cells are lysed and the target microbial genomic DNA is separated using a nucleic acid extraction reagent. The extracted genomic DNA is transferred to a purification column for impurity elution to obtain a nucleic acid solution that meets the reaction purity requirements.

[0027] S200: Perform asymmetric PCR amplification to obtain target single-stranded DNA. Using the genomic DNA obtained in step S100 as a template, prepare the nucleic acid amplification reaction solution. Add upstream and downstream primers designed specifically for the target gene of the target microorganism to the reaction solution, artificially setting the molar ratio of the upstream and downstream primers to be unequal. Place the reaction solution in a thermal cycling device and perform a cycle program of pre-denaturation, denaturation, annealing, and extension. After the set number of cycles, the target single-stranded DNA with a specific sequence accumulates unidirectionally in the reaction solution. Subsequently, purification reagents are added to remove impurities and excess primers from the amplification product.

[0028] S300, Preparation of Gold Nanoparticles with Surface-Modified Nucleic Acid Probes. A chloroauric acid solution was prepared and heated to boiling. A reducing agent solution was added, and heating and stirring continued until the solution color stabilized and turned a transparent wine-red, yielding a colloidal solution of gold nanoparticles. Nucleic acid capture probes with specific chemical groups modified at both ends were synthesized. A reducing agent was added to treat the nucleic acid probe solution to activate its terminal groups. The activated nucleic acid capture probes were mixed with the colloidal solution of gold nanoparticles and incubated. During incubation, a salt solution was added dropwise to the system in batches, promoting the fixation of the nucleic acid capture probes onto the surface of the gold nanoparticles through chemical bonds, forming gold nanoparticle probes with specific sequence recognition functions.

[0029] S400, initiating the specific aggregation reaction of gold nanoprobes. Take the target single-stranded DNA solution purified in step S200 and mix it thoroughly with the gold nanoprobes prepared in step S300 in a centrifuge tube. Add salt solution and acid-base adjuster to the mixture to bring the ionic strength and pH of the detection system within the set range. Incubate the detection system in a constant temperature environment. During this process, the target single-stranded DNA acts as a bridging sequence, undergoing base-complementary pairing with the nucleic acid capture probes on the surface of the gold nanoparticles. Molecular hybridization shortens the physical distance between the gold nanoparticles and overcomes the electrostatic repulsion on their surface, promoting spatial cross-linking and aggregation of the gold nanoparticles, resulting in a significant increase in the overall hydration particle size within the detection system. The salt solution, acid-base adjuster, and constant temperature incubation conditions together constitute the preset hybridization reaction conditions, which promote stable base-complementary pairing between the target single-stranded DNA and the nucleic acid capture probes on the surface of the gold nanoparticles and inhibit non-specific self-aggregation of the gold nanoprobes in the absence of target single-stranded DNA.

[0030] S500: Determine the hydration particle size of the system and calculate the equivalent concentration of the target microorganism. Transfer the detection system that has completed the aggregation reaction to an optical cuvette. Illuminate the detection system using the light source of a dynamic light scattering detection device, and record the intensity fluctuation signal of scattered light generated by Brownian motion of the particles within the system using a photodetector. The system software calculates the average hydration particle size of the detection system based on the intensity fluctuation data. Retrieve the pre-established quantitative detection mathematical model, which includes a linear fitting relationship between the target microorganism concentration order of magnitude parameter and the average hydration particle size. Substitute the average hydration particle size value measured by the instrument into the above linear fitting relationship to first determine the order of magnitude parameter of the target microorganism concentration corresponding to the sample to be tested, and then convert the target microorganism equivalent concentration data according to the benchmark concentration used in modeling to complete the detection process.

[0031] See attached document Figure 3 The above step S100 is performed by the sample preprocessing unit in the detection system, which is used to convert the sample to be tested into a nucleic acid solution template that meets the purity requirements of the nucleic acid amplification reaction. The specific execution process of step S100 includes: S110, Prepare a matrix homogenate for the test sample. Weigh a fixed amount of solid test sample and mechanically pulverize it. Place the pulverized sample particles in a sterile container and add phosphate buffer for physical mixing. Vortex the mixture to suspend the microbial cells attached to the sample surface in the liquid buffer system, obtaining a matrix homogenate with a fixed liquid-to-solid ratio. In one embodiment of the invention, when dealing with test samples with high lipid content, a matrix homogenate with a volume-to-mass ratio of 10:1 is prepared using phosphate buffer.

[0032] S120, lysing microbial cell structures to release nucleic acid material. A predetermined volume of matrix homogenate is extracted and transferred to a centrifuge container. Microbial lysis buffer is added to the centrifuge container to disrupt the cell wall and cell membrane structure of the microorganisms. After cell rupture, the target genomic DNA contained within the cell is released into a liquid buffer system, forming a lysis mixture containing nucleic acids and organelle fragments.

[0033] S130, Separation of matrix impurities and interfering substances from the lysate mixture. This procedure performs protein and lipid precipitation separation on lysate mixtures containing organic components. A protein precipitation reagent is added to the system, and centrifugation is applied to cause denatured protein molecules to separate from the sample matrix residue and settle to the bottom of the container. The supernatant containing free nucleic acid components is then extracted. When the matrix of the sample contains a large amount of lipids, these lipids typically float and separate into layers on the top of the liquid phase under centrifugation. The top lipid layer should be avoided during supernatant extraction; this step reduces the inhibitory effect of matrix macromolecules on subsequent specific nucleic acid amplification reactions.

[0034] S140, Purify and elute the target total genomic DNA template. Transfer the collected liquid phase supernatant to the appropriate nucleic acid binding purification device. Allow the free nucleic acid molecules in the liquid phase supernatant to attach to the surface of the solid medium inside the device under a high-salt chemical environment. Rinse the solid medium surface multiple times with washing buffer to remove residual salt ions and unbound free macromolecular impurities. Then add sterile water to change the electrostatic environment of the medium surface, causing the nucleic acid molecules to detach from the solid medium surface and dissolve in the elution system. Collect the eluent to obtain the target microbial total genomic DNA.

[0035] It should be noted that the above-described nucleic acid template extraction process combines mechanical homogenization and dispersion, chemical lysis and release, and solid-phase adsorption purification steps. These steps provide a pathway for realizing the state transition of target microbial DNA from the test sample to a liquid nucleic acid template. The micro-volume nucleic acid assay device can detect the target analyte in the eluent, thereby supporting the subsequent asymmetric PCR amplification reaction.

[0036] See attached document Figure 4 Step S200 above is performed by the nucleic acid amplification unit in the detection system, used to convert the extracted genomic DNA template into target single-stranded DNA. The specific execution process of step S200 includes: S210, Prepare the asymmetric nucleic acid amplification reaction system. Add premixed buffer containing DNA polymerase and deoxyribonucleoside triphosphate to a sterile centrifuge tube. Add the total genomic DNA of the target microorganism extracted in step S100 as the amplification template to the reaction tube. Add the upstream and downstream primers targeting the specific gene of the target microorganism to the reaction tube. To achieve asymmetric amplification, the molar concentrations of the upstream and downstream primers are different. Specifically, the molar ratio of the upstream to downstream primers is controlled at 10:1. In one embodiment, the total volume of the reaction system is 50 μL, wherein the final concentration of the upstream primer is configured to be 0.3 μmol / L and the final concentration of the downstream primer is configured to be 0.03 μmol / L. The above primer concentration configuration is used to accumulate single-stranded DNA product during the amplification reaction.

[0037] S220, execute the thermal cycling amplification program. Transfer the configured reaction system to the thermal cycling amplification device and start the set temperature and time control program. The thermal cycling program includes a pre-denaturation stage and subsequent multiple thermal cycling stages. In the pre-denaturation stage, the genomic double-stranded DNA is unstranded and polymerase activity is activated by high-temperature treatment at 95°C for 600s. After entering the thermal cycling stage, alternating operations of 94°C denaturation for 30s, 59°C annealing for 30s, and 72°C extension for 30s are performed, repeated for a total of 40 cycles. In the early stage of the amplification reaction, since both upstream and downstream primers are present in the reaction system, the target fragment generates double-stranded nucleic acid products exponentially. As the number of cycles increases, when the low concentration of downstream primer is consumed, the unconsumed upstream primer remains in the reaction system. At this time, the amplification mode changes from double-stranded amplification to linear amplification dependent on the upstream primer. In the linear amplification stage, the polymerase synthesizes a complementary strand using one nucleic acid strand as a template, accumulating the target single-stranded DNA fragment with a specific sequence in the reaction system. After the cycle is completed, the reaction system is maintained at 72°C for 600 seconds to complete the final fragment extension and repair.

[0038] S230, purifying the amplification product to obtain the target single-stranded DNA. After the thermal cycling program is completed, the reaction tube contains a mixture of target single-stranded DNA, residual double-stranded DNA byproducts, unconsumed upstream primers, and inactivated proteases. The liquid in the reaction tube is mixed with binding buffer, which contains a high concentration of dissociative salts. The mixture is transferred to a centrifugal purification column with a built-in silica matrix membrane. Under centrifugal force, the target single-stranded DNA and other large nucleic acid molecules adhere to the silica matrix membrane. Small molecule fragments such as residual primers and protein impurities permeate the silica matrix membrane and are discharged with the waste liquid. Ethanol-containing washing buffer is added to the centrifugal purification column to wash away residual salt ions. Sterile water is added to alter the electrostatic environment of the matrix membrane, redissolving the attached nucleic acid molecules and collecting them in a collection tube to obtain the purified target single-stranded DNA solution.

[0039] It should be noted that this step involves adjusting the concentration ratio of upstream and downstream primer sets in the amplification system, combined with a two-stage thermal cycling program, to accumulate the target single-stranded DNA in the amplification product. The obtained single-stranded nucleic acid fragment is used for base pairing with the nanoprobe in subsequent steps.

[0040] See attached document Figure 5 The specific execution process of step S300 includes: S310, Preparation of a colloidal solution of gold nanoparticles. A fixed concentration of chloroauric acid solution is measured and placed in a reaction vessel, heated to boiling and stirred. Trisodium citrate solution is added to the boiling chloroauric acid solution as a reducing agent, and the reaction is continued. As the reaction proceeds, the color of the reaction system changes. When the solution color is observed to turn transparent wine-red, the heating operation is stopped. The reaction vessel is allowed to cool naturally to room temperature to obtain the colloidal solution of gold nanoparticles. In one embodiment, the mass concentration of the chloroauric acid solution is prepared as 0.1%, and the mass concentration of the trisodium citrate solution is prepared as 1%.

[0041] S320, Activation of Thiol-Modified Nucleic Acid Capture Probes. First and second nucleic acid capture probes with thiol-modified ends were obtained, each corresponding to a different recognition sequence on the target single-stranded DNA. The nucleic acid capture probes were dissolved in pure water to a predetermined final concentration. Quantitative amounts of the first and second nucleic acid capture probe solutions were taken separately, and tris(2-carboxyethyl)phosphine solution and acidic acetate buffer were added to the solutions. The mixed solutions were incubated at room temperature. The tris(2-carboxyethyl)phosphine reagent in the system has reducing properties, which can break the disulfide bonds formed between nucleic acid capture probe molecules, restoring the thiol groups at the probe ends to a free state. The free thiol groups are used for subsequent coupling reactions with gold nanoparticles.

[0042] S330, Mixing and incubation to initiate surface coupling reactions. Activated first and second nucleic acid capture probes are quantitatively added to the two sets of gold nanoparticle colloidal solutions prepared in step S310. During mixing, the ratio of nucleic acid capture probes to gold nanoparticles is controlled within a preset range. The mixture is placed in a dark environment for static incubation. During incubation, the free thiol groups at the ends of the nucleic acid capture probes contact the surface of the gold nanoparticles, forming gold-sulfur bonds and immobilizing the nucleic acid capture probes on the surface of the gold nanoparticles.

[0043] S340, aging treatment with gradually increasing ionic strength. During the static incubation stage of step S330, sodium chloride solution was added dropwise to the two mixed systems in multiple time batches. As the sodium ion concentration in the system gradually increased, the electrostatic repulsion on the surface of the gold nanoparticles weakened. The change in the charge environment reduced the steric hindrance between nucleic acid molecules, allowing the remaining nucleic acid capture probes in the system to continue to approach and bind to the surface of the gold nanoparticles, thereby increasing the probe coverage density on the surface of individual gold nanoparticles.

[0044] S350, washing and separation to obtain purified gold nanoprobes. After the aging stage, the mixture is transferred to a centrifuge for centrifugation. The gold nanoprobes settle to the bottom of the centrifuge tube under centrifugal force. The supernatant containing unbound probe molecules and free salt ions is aspirated and discarded. The settled gold nanoprobes are washed and resuspended with the prepared elution buffer. The centrifugation and resuspension process is repeated to obtain a first gold nanoprobe solution and a second gold nanoprobe solution with impurities removed, respectively.

[0045] The above gold nanoprobe preparation process involves synthesizing colloidal gold via chemical reduction and coupling two nucleic acid probes with different recognition sequences to the surface of gold nanoparticles using gold-sulfur bonds. An aging treatment with sodium chloride solution is performed to increase the probe loading on the particle surface. The first and second gold nanoprobe solutions obtained through these steps are used to capture different segments of the target single-stranded DNA in subsequent mixed reactions.

[0046] See attached document Figure 6 Step S400 above is executed by the probe hybridization reaction unit in the detection system, used to convert the target single-stranded DNA into a spatial aggregation signal of gold nanoparticles. The specific execution process of step S400 includes: S410, Construct the liquid-phase hybridization reaction system. Extract a predetermined volume of the target single-stranded DNA solution obtained in step S200 into a sterile centrifuge tube. Add the first gold nanoparticle probe solution and the second gold nanoparticle probe solution prepared in step S300 to the centrifuge tube, respectively. Adjust the volume ratio of the target single-stranded DNA to the two sets of gold nanoparticle probes to provide the required number of probe molecules for subsequent binding reactions. Add ultrapure water to make up the volume of the mixed solution to obtain the initial liquid-phase hybridization reaction system.

[0047] S420, adjusting the chemical microenvironment of the detection system. Hybridization buffer is added to the liquid-phase hybridization reaction system. The hybridization buffer contains specific concentrations of sodium chloride and phosphate. The added sodium ions neutralize the negative charge on the surface of the gold nanoparticles, reducing electrostatic repulsion between particles and providing conditions for subsequent particle proximity. The phosphate component maintains the pH of the detection system at a neutral state. By adjusting the amount of hybridization buffer added, the salt concentration and pH of the detection system are kept within a preset range. The preset range is designed to promote stable hybridization between the target single-stranded DNA and the nucleic acid capture probe, and to reduce non-specific self-aggregation of the gold nanoparticle probe. The adjustment of the microenvironment provides the thermodynamic conditions for the formation of hydrogen bonds between nucleic acid molecules.

[0048] S430 performs isothermal hybridization to induce particle cross-linking. The regulated detection system is placed in a constant-temperature water bath and incubated at the preset hybridization temperature and time. During this isothermal incubation, the target single-stranded DNA diffuses in the liquid phase, and two different segments of its sequence undergo base complementarity pairing with the first nucleic acid capture probe on the surface of the first gold nanoprobe and the second nucleic acid capture probe on the surface of the second gold nanoprobe, respectively. A single target single-stranded DNA molecule simultaneously connects to the first and second gold nanoprobes, establishing a cross-linked structure between the particles. The hybridization process spatially pulls the dispersed gold nanoparticles closer together and cross-links them, forming particle aggregates. As the incubation time increases, the overall hydration particle size within the detection system increases, and the solution color changes from red to blue-purple.

[0049] S440 terminates the hybridization reaction and stabilizes the aggregated conformation. After the set incubation time is reached, the detection system is transferred to a low-temperature environment of 4°C and allowed to stand for 5 minutes. The low temperature condition reduces the thermal motion of molecules within the system, minimizing the risk of breakage of existing hydrogen bonds, thus maintaining the conformation of the cross-linked aggregated gold nanoparticles. The cooling operation fixes the hydration particle size state within the system, reducing the risk of signal fluctuations during subsequent detection.

[0050] In the above hybridization reaction examples, a specific sequence of the target single-stranded DNA is utilized to initiate cross-linking between the first and second gold nanoprobes through specific base complementary pairing. This process transforms the sequence recognition of free nucleic acids into a spatial aggregation physical state of gold nanoparticles, thereby increasing the average hydration particle size within the detection system. The average hydration particle size data of the aggregated detection system is used to support the quantitative calculation of the equivalent bacterial concentration of the target microorganism in subsequent steps.

[0051] See attached document Figure 7 Step S500 above is executed by the light scattering signal detection unit in the detection system, used to convert the physical morphological changes of the detection system after the aggregation reaction into equivalent bacterial concentration data of the target microorganism. The specific execution process of step S500 includes: S510: Acquire the scattered light signal from the Brownian motion of the particles. Transfer the detection system, after cooling in step S400, to an optical cuvette. Place the optical cuvette in the sample chamber of the light scattering signal detection unit. Activate the built-in monochromatic laser source to continuously irradiate the detection system. In a constant temperature environment, the cross-linked gold nanoparticle probe aggregates and unreacted free probe molecules within the detection system undergo random Brownian motion in the liquid phase. Brownian motion causes dynamic changes in the spatial position of the particles within the laser irradiation area, resulting in fluctuations in the intensity of light scattered at a specific spatial angle over time. The light scattering signal detection unit uses a photomultiplier tube to receive the scattered light at this specific angle, records, and outputs the time-domain light intensity fluctuation signal.

[0052] S520 analyzes the signal to obtain the average hydration particle size of the detection system. The processor in the light scattering signal detection unit receives the light intensity fluctuation signal, performs autocorrelation mathematical analysis on it, and generates a time autocorrelation function curve. The decay rate is fitted to the time autocorrelation function using the cumulant method to extract the translational diffusion coefficient of the particles in the detection system. Based on the translational diffusion coefficient, combined with the liquid phase kinetic viscosity of the detection system and the absolute temperature parameter of the environment in which the detection system is located, the average hydration particle size of all particles in the detection system is calculated using the Stokes-Einstein equation. The calculation formula is as follows: ; in, Represents the average hydration particle size; Represents the Boltzmann constant; Represents absolute temperature parameter; Represents kinetic viscosity; Represents the translational diffusion coefficient; It represents pi.

[0053] S530, Retrieve Quantitative Detection Mathematical Model. The processor retrieves a pre-established mathematical model for the quantitative detection of the target microorganism from the system's storage module. The offline construction process of this mathematical model includes: configuring target microorganism standard samples with multiple concentration gradients, the concentration of which is determined by microbial counting; for each concentration gradient of target microorganism standard samples, performing the aforementioned genomic DNA extraction, asymmetric PCR amplification, amplification product purification, probe hybridization, and light scattering particle size determination operations to obtain the average hydrated particle size corresponding to the equivalent bacterial concentration of each target microorganism; using the concentration-order-of-magnitude parameter x obtained from the equivalent bacterial concentration of the target microorganism as the independent variable and the corresponding average hydrated particle size as the dependent variable, performing linear regression fitting to obtain the corresponding linear equation as the mathematical model for the quantitative detection of the target microorganism. The formula for this mathematical model is as follows: ; ; in, Represents the average hydration particle size; The concentration order-of-magnitude parameter obtained by converting the equivalent bacterial concentration of the target microorganism; The equivalent bacterial concentration of the target microorganism; The preset baseline concentration used when modeling the standard sample; The slope parameter represents the fitted straight line; The intercept parameter represents the fitted line.

[0054] S540, calculate and output the equivalent bacterial concentration of the target microorganism in the sample to be tested. Substitute the average hydration particle size obtained from step S520 into the mathematical model for quantitative detection of the target microorganism retrieved in step S530. By solving the above linear equation, first obtain the corresponding value of the concentration order-of-magnitude parameter x, and then determine the relationship between x and the equivalent bacterial concentration of the target microorganism. The system calculates the equivalent bacterial concentration of the target microorganism in the test sample by establishing a logarithmic conversion relationship between the two. Based on the sample processing methods used in modeling the standard sample and the preprocessing methods used for the test sample, the system performs corresponding conversions on the calculation results to ensure that the output results are consistent with the concentration expression method of the standard sample. The system then displays the concentration results on a user-friendly interface and outputs a test report including the average hydration particle size parameter.

[0055] Specific application examples: In the above-described particle size determination and concentration calculation embodiments, the light scattering signal detection unit converts the increase in the physical size of the hydrated particles generated by the liquid-phase hybridization reaction into a specific physical value through optical scattering principles and autocorrelation calculations. This value is then further mapped to the equivalent bacterial concentration of the target microorganism using an established linear fitting model. These operations provide a complete data processing path for the non-fluorescent quantitative detection of Aspergillus flavus.

[0056] This embodiment first uses artificially synthesized target single-stranded DNA (Target-ssDNA) as a model target to verify the response effect of the gold nanoprobe detection system. This stage is used to verify that the target single-stranded DNA can simultaneously pair complementaryly with the first and second gold nanoprobes and induce spatial cross-linking and aggregation of the gold nanoprobes. It does not involve peanut matrix spiked detection or bacterial concentration conversion.

[0057] Based on the virulence gene (afIR) of Aspergillus flavus, specific upstream and downstream primers, thiol-modified nucleic acid capture probes, and target single-stranded DNA sequences for response verification were designed. The specific nucleic acid sequences are shown in Table 1.

[0058] Table 1. Primers, probes, and ssDNA sequences used in the experiment Table 1 shows the core nucleic acid sequence set used in the detection system of the present invention, the forward primer Prime (F) and the reverse primer Primer (R) for performing asymmetric PCR amplification; two nucleic acid capture probes Probe1 and Probe2 with thiol (-SH) modified at the ends for coupling with gold nanoparticles; and the target single-stranded DNA sequence Target-ssDNA generated by amplification (or synthesized for verification). This single-stranded DNA can act as a "bridge" to simultaneously pair complementaryly with the above two probes, thereby inducing spatial cross-linking and aggregation of the gold nanoparticle probes.

[0059] Gold nanoparticles were prepared using the trisodium citrate reduction method. Thioylated probes were then coupled to the particle surface via gold-sulfur bonds to prepare gold nanoprobes. (See attached document) Figure 8Intensity represents the signal strength within the particle size distribution. Diameter (nm) represents the diameter in nanometers. Absorbance (au) represents the absorbance, where au is an arbitrary unit. Wavelength (nm) represents the wavelength in nanometers. AuNPs represent gold nanoparticles. Probe1 represents probe 1. Probe2 represents probe 2. The average hydrated particle size of the unmodified gold nanoparticles is approximately 37 nm. After probe coupling, the average hydrated particle size increases to approximately 45 nm. As shown in Figure C, the characteristic absorption peak of the modified gold nanoparticle probe is located near 520 nm, without redshift or peak broadening, indicating that the colloid remains in a stable dispersion state.

[0060] Artificially synthesized target single-stranded DNA was used as the model target for response testing. (See attached document.) Figure 9 Intensity represents the signal strength within the particle size distribution. Diameter (nm) represents the diameter, measured in nanometers. Absorbance (au) represents the absorbance, where au can be any unit. Wavelength (nm) represents the wavelength, measured in nanometers. Control represents the control group. Positive represents the positive group. Without the addition of target single-stranded DNA, the average hydration particle size of the gold nanoprobes was 45 nm. After the addition of target single-stranded DNA, the average hydration particle size of the system increased to 112 nm, 121 nm, and 165 nm, respectively. The characteristic absorption peak at 520 nm in Figure E decreased with increasing concentration. This result verifies that the target single-stranded DNA can induce spatial aggregation of the probes.

[0061] The primer concentration ratio, salt concentration, and pH value of the detection system for asymmetric PCR were optimized. (See attached document.) Figure 10 When the upstream primer to downstream primer concentration ratio was 10:1 (upstream primer final concentration 0.3 μmol / L, downstream primer final concentration 0.03 μmol / L), the electrophoretic bands were the brightest and there were no nonspecific bands. The thermal cycling parameters of this amplification process are shown in Table 2.

[0062] Table 2. Conditions for aPCR amplification reaction Reference Appendix Figure 11In Figures A and B, the horizontal axis represents wavelength (in nm), and the vertical axis represents absorbance. The legend indicates different sodium chloride concentration gradients from 0 to 0.30 mol / L. In Figure C, the horizontal axis represents the molar concentration of sodium chloride, and the vertical axis represents the absorbance difference between the positive and negative systems. In Figure D, the horizontal axis represents sodium chloride concentration, and the vertical axis represents the average hydration particle size (in nm). In the legend, "Positive" represents the positive experimental group, "Negative" represents the negative control group, and ΔD represents the average hydration particle size difference between the positive and negative groups. This set of figures visually demonstrates that when the sodium chloride concentration is 0.20 mol / L, both the absorbance difference and particle size difference of the system reach their maximum, which is the optimal hybridization salt concentration condition. When the sodium chloride concentration is 0.20 mol / L, the absorbance difference and particle size difference between the positive and negative systems reach their maximum values.

[0063] Reference Appendix Figure 12 Wavelength represents wavelength. pH represents acidity or alkalinity. pH=5, pH=6, pH=7, pH=8, and pH=9 represent different pH conditions. ΔA (au) represents the change in absorbance, in arbitrary units. Mean diameter (nm) represents the average diameter, in nanometers. Positive represents the positive group. Negative represents the negative group. ΔMI (nm) represents the change in MI, in nanometers. a, b, c, and d represent statistical significance group markers.

[0064] The results showed that at pH 7, the negative background particle size was stable, while the positive system exhibited a significant increase in particle size and the largest absorbance difference. Therefore, 0.20 mol / L sodium chloride and a pH of 7 were set as the conditions for subsequent hybridization reactions.

[0065] A mathematical model for quantitative detection was constructed using pure Aspergillus flavus culture media with known concentration gradients. (See attached reference.) Figure 13 With the concentration of Aspergillus flavus equivalent bacteria The converted concentration order of magnitude parameter Using the average hydrated particle size as the dependent variable and employing linear regression fitting with the independent variable as the metric, the mathematical model calculation formula corresponding to this embodiment is as follows: ; ; According to the appendix Figure 13 The linear regression fitting results are obtained. =7.423, =44.25; the coefficient of determination R of the fitted equation 2 The value of 0.9870 indicates that the linear fitting relationship has a good fitting effect. In this embodiment, The concentration of Aspergillus flavus is 1.8 CFU / mL. It can be represented as =1.8×10 x CFU / mL. In actual testing, the average hydration particle size will be measured. Substituting into the mathematical model above, we first calculate the concentration order of magnitude parameter. And then according to Calculate the equivalent concentration of Aspergillus flavus .

[0066] To verify the specificity of the detection method, it was compared with non-target microorganisms such as Aspergillus flavus, Salmonella enteritidis, and Staphylococcus aureus. (See attached document.) Figure 14 This figure shows the experimental analysis of the specificity of the detection method. The vertical axis represents the average hydrated particle size (unit: nm); the horizontal axis represents the blank control group, five non-target pathogenic bacteria (Salmonella Enteritidis, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Bacillus cereus), and the target microorganism (Aspergillus flavus); the horizontal dashed line in the figure represents the detection threshold for distinguishing between positive and negative results.

[0067] The Aspergillus flavus target group induced an increase in average hydration particle size to over 70 nm, while the non-target group maintained an average hydration particle size at the baseline level of around 40 nm. Furthermore, the stability of the gold nanoprobes was investigated by storing them at 4°C in the dark; the specific measurement data are shown in Table 3.

[0068] Table 3. Storage Stability During the 28-day storage period, the hydrated particle size remained between 66.32 and 66.63 nm, with an ANOVA P-value greater than 0.05.

[0069] Spiked recovery experiments were conducted by adding different concentrations of Aspergillus flavus spores to peanut substrate, and the measured data are shown in Table 4.

[0070] Table 4. Spike recovery experiments of the AuNPs-DLS method At 1.8×10 1 Up to 1.8×10 6Within the spiking range of CFU / mL, the recovery rate was 98.61% to 102.11%, indicating that the matrix components of the peanut sample did not significantly interfere with the light scattering detection results.

Claims

1. A rapid detection method for foodborne pathogenic microorganisms, characterized in that, This includes extracting the genomic DNA of the target microorganism from the sample to be tested; Using the target microbial genomic DNA as a template, a nucleic acid amplification reaction solution containing upstream and downstream primers with unequal molar concentrations was prepared, and an asymmetric PCR amplification program was performed to accumulate the target single-stranded DNA in the nucleic acid amplification reaction solution. Prepare a first gold nanoprobe and a second gold nanoprobe based on gold nanoparticles, with the surfaces of a first nucleic acid capture probe and a second nucleic acid capture probe respectively modified. The target single-stranded DNA is mixed with the first and second gold nanoparticle probes to construct a detection system. Under preset hybridization reaction conditions, the target single-stranded DNA undergoes base complementary pairing with the first and second nucleic acid capture probes, respectively, which promotes the cross-linking and aggregation of the gold nanoparticles and increases the average hydration particle size of the detection system. The scattered light intensity fluctuation signal generated by the Brownian motion of the gold nanoparticles in the detection system is collected, the average hydration particle size of the detection system is calculated, and the average hydration particle size is substituted into the pre-established mathematical model for quantitative detection of target microorganisms to calculate the equivalent bacterial concentration of the target microorganism in the sample to be tested.

2. The method according to claim 1, characterized in that, The extraction of the target microbial genomic DNA from the sample to be tested includes: The sample to be tested was mechanically pulverized and mixed with buffer solution by shaking to obtain a matrix homogenate; Microbial lysis reagent is added to the matrix homogenate to disrupt the cell structure of the microorganism and release the genomic DNA of the target microorganism, forming a lysis mixture containing free nucleic acid components; A protein precipitation reagent is added to the lysis mixture and centrifugation is applied to precipitate protein molecules and matrix residues, and the liquid supernatant containing free nucleic acid components is extracted. The liquid supernatant is transferred to a nucleic acid binding purification device, whereby the free nucleic acid components adhere to the surface of the solid medium inside. After washing to remove impurities, sterile water is added for elution to obtain the genomic DNA of the target microorganism.

3. The method according to claim 1, characterized in that, The asymmetric PCR amplification procedure, which accumulates target single-stranded DNA in the nucleic acid amplification reaction solution, includes: The molar ratio of the upstream primer to the downstream primer in the nucleic acid amplification reaction solution is controlled to be 10:1; The denaturation, annealing, and extension cycles are performed alternately in a thermal cycling amplification device. As the number of cycles increases, once the downstream primer, which is at a low concentration, is consumed, the amplification mode changes from double-stranded amplification to linear amplification guided by the upstream primer, and the target single-stranded DNA accumulates in the nucleic acid amplification reaction solution.

4. The method according to claim 1, characterized in that, The preparation of the first gold nanoprobe and the second gold nanoprobe, which are based on gold nanoparticles and respectively modified with a first nucleic acid capture probe and a second nucleic acid capture probe, includes: A colloidal solution of gold nanoparticles was obtained by heating a reducing agent solution to reduce chloroauric acid solution. The first nucleic acid capture probe and the second nucleic acid capture probe are treated with a reducing agent to make the probe ends with thiol modification in a free state; The activated first nucleic acid capture probe and the second nucleic acid capture probe were respectively mixed with the gold nanoparticle colloidal solution and incubated in the dark to fix the first nucleic acid capture probe and the second nucleic acid capture probe onto the surface of the gold nanoparticles through gold-sulfur bonds. During static incubation, sodium chloride solution was added dropwise to the mixed system in batches for aging treatment, thereby reducing the electrostatic repulsion on the surface of the gold nanoparticles and increasing the coverage density of the first and second nucleic acid capture probes. Subsequently, centrifugation and elution were performed to obtain the purified first and second gold nanoparticle probes.

5. The method according to claim 1, characterized in that, The method involves mixing the target single-stranded DNA with the first and second gold nanoparticle probes to construct a detection system. Under preset hybridization conditions, the target single-stranded DNA undergoes base complementary pairing with the first and second nucleic acid capture probes, respectively, promoting cross-linking and aggregation of the gold nanoparticles and increasing the average hydration particle size of the detection system. This includes: Hybridization buffer is added to a mixture containing the target single-stranded DNA, the first gold nanoprobe, and the second gold nanoprobe to adjust the salt concentration and pH of the detection system to a set range. The set range is used to promote stable hybridization of the target single-stranded DNA with the first nucleic acid capture probe and the second nucleic acid capture probe, and to inhibit non-specific self-aggregation of the first gold nanoprobe and the second gold nanoprobe. The adjusted detection system was incubated in a constant temperature environment to induce cross-linking of the gold nanoparticles; After the set incubation time is reached, the detection system is transferred to a low-temperature environment to stand, thereby reducing the thermal motion of molecules in the system, maintaining the conformation of the cross-linked and aggregated gold nanoparticles and fixing the hydration particle size state.

6. The method according to claim 1, characterized in that, The process of collecting the scattered light intensity fluctuation signal generated by the Brownian motion of the gold nanoparticles within the detection system and calculating the average hydration particle size of the detection system includes: The detection system is illuminated by a monochromatic laser light source, and the scattered light intensity fluctuation signal occurring over time at a set angle is recorded by a photodetector. Autocorrelation mathematical analysis is performed on the scattered light intensity fluctuation signal to generate a time autocorrelation function curve; The translational diffusion coefficient was extracted by fitting the decay rate to the time autocorrelation function curve using the cumulative method. The average hydration particle size is calculated based on the translational diffusion coefficient, the liquid phase kinetic viscosity of the detection system, and the absolute temperature parameter of the environment in which the detection system is located.

7. The method according to claim 1, characterized in that, Before substituting the average hydration particle size value into the pre-established mathematical model for quantitative detection of target microorganisms to calculate the equivalent bacterial concentration of the target microorganism in the sample to be tested, the process also includes an offline construction process of the mathematical model for quantitative detection of target microorganisms, specifically including: Prepare target microbial standard samples with multiple concentration gradients; For target microbial standard samples at various concentration gradients, genomic DNA extraction, asymmetric PCR amplification, probe hybridization, and light scattering particle size determination were performed to obtain the average hydration particle size corresponding to the equivalent bacterial concentration of each target microorganism. The equivalent bacterial concentration of the target microorganism is converted into a concentration order-of-magnitude parameter as the independent variable, and the corresponding average hydration particle size value is used as the dependent variable for linear regression fitting to obtain the corresponding linear equation as the mathematical model for quantitative detection of the target microorganism. The concentration order-of-magnitude parameter is the decimal logarithmic value of the equivalent bacterial concentration of the target microorganism relative to the preset benchmark concentration.

8. The method according to claim 2, characterized in that, When adding a protein precipitation reagent to the lysis mixture and applying centrifugal force to precipitate protein molecules and matrix residues, and extracting the liquid supernatant containing free nucleic acid components: When the matrix of the sample to be tested contains lipid components and floats and separates into layers above the liquid phase under centrifugation, the top lipid structure is avoided when extracting the supernatant of the liquid phase, thereby reducing the inhibitory effect of matrix macromolecules on subsequent specific nucleic acid amplification reactions.

9. The method according to claim 6, characterized in that, The calculation of the average hydration particle size based on the translational diffusion coefficient, the liquid phase kinetic viscosity of the detection system, and the absolute temperature parameter of the environment in which the detection system is located includes: Based on the Stokes-Einstein equation, the average hydration particle size is obtained by calculating the product of the Boltzmann constant and the absolute temperature parameter of the environment in which the detection system is located, and then dividing the product by three times the product of pi, the liquid phase kinetic viscosity, and the translational diffusion coefficient.

10. The method according to claim 4, characterized in that, The step of adding a reducing agent to treat the first nucleic acid capture probe and the second nucleic acid capture probe, so that the probe ends with thiol modification are in a free state, includes: A solution containing tris(2-carboxyethyl)phosphine and an acidic acetate buffer solution were added to the first nucleic acid capture probe and the second nucleic acid capture probe, respectively. Incubation was performed at room temperature to utilize the reducing properties of the tris(2-carboxyethyl)phosphine component to open the disulfide bonds formed between the first and second nucleic acid capture probe molecules.

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