CN105806819A - Method for simultaneously detecting various food-borne microorganisms based on nanometer fluorescent microscopy hyperspectral imaging technique - Google Patents

Abstract

The invention relates to a method for simultaneously detecting various food-borne microorganisms based on a nanometer fluorescent microscopy hyperspectral imaging technique. The method comprises the following steps of: taking a food-borne microorganism as a research object; constructing a fluorescent microscopy hyperspectral imaging system; constructing a colorful nanometer fluorescent probe with specific recognition food-borne microorganisms by compounding a colorful transformed nanometer material and marking multiple targets for various microorganisms; under a fluorescent microscopy imaging mode, extracting a fluorescence spectrum of an interesting area (ROI), for fluorescence spectrum image data of a to-be-detected object; optimizing a characteristic spectrum image at a specific scale through a data dimension reduction manner and constructing a quantitative determination model for fluorescence intensity change value and food-borne microorganisms in virtue of an image processing manner, thereby realizing the simultaneous detection for various food-borne microorganisms. The method is applicable to the technical field of food safety, environment monitoring, and the like.

Description

A Simultaneous Detection Method of Multiple Foodborne Microorganisms Based on Nano-Fluorescence Microscopic Hyperspectral Imaging Technology

technical field

The invention relates to a simultaneous detection method for multiple food-borne microorganisms based on nano-fluorescent micro-hyperspectral imaging technology, in particular to the combination of hyper-spectral imaging technology, micro-imaging technology and the unique optical properties of nano-crystals, using nano-fluorescence A new idea of micro-hyperspectral imaging detection, establishing a rapid and sensitive method for simultaneous quantitative detection of food-borne microorganisms.

Background technique

Foodborne pathogens are the primary cause of foodborne diseases, causing great harm to human health and a major hidden danger to food safety. Commonly used analysis methods for foodborne pathogens mainly include traditional microbiological testing techniques, molecular biology techniques, instrumental analysis techniques, and immunological techniques. Although these existing analysis methods have their own advantages, they all have certain limitations, such as complicated pre-treatment steps and long time, or large and expensive instruments and equipment, and cannot perform visual analysis on microorganisms. In view of the good working foundation accumulated by the applicant in the field of non-destructive testing of food, especially the in-depth technical research in the field of hyperspectral imaging detection, this project intends to build a fluorescence microscope hyperspectral imaging system for in-depth research on fast, sensitive The method for simultaneous quantitative detection of foodborne microorganisms is applicable to technical fields such as food safety and environmental monitoring.

At present, there is no report on the simultaneous detection of multiple foodborne microorganisms using nanofluorescence microscopy hyperspectral imaging technology. As an emerging quantitative method for food-borne microorganisms, the invention realizes the simultaneous detection and analysis of various food-borne microorganisms.

Contents of the invention

The purpose of the present invention is to provide a simultaneous detection method for multiple food-borne microorganisms based on nano-fluorescence microscopic hyperspectral imaging technology, which has high sensitivity, strong reliability, and fast detection speed, and realizes simultaneous detection of multiple food-borne microorganisms. Detection and analysis, suitable for technical fields such as food safety and environmental monitoring.

In order to achieve the above purpose, the technical solution adopted in the present invention is to construct a set of fluorescent micro-hyperspectral imaging system by taking food-borne microorganisms as the research object, and to construct a micro-organism with specific recognition of food-borne microorganisms by synthesizing multi-color up-conversion nanomaterials. Multi-color nano-fluorescent probes for microorganisms are used for multi-target labeling of various microorganisms; in the fluorescence microscopy imaging mode, the fluorescence spectrum of the region of interest (ROI) is extracted for the fluorescence spectrum image data of the object to be tested, and the data is passed Dimensionality reduction means optimize the characteristic spectral images at a specific scale, and use image processing methods to construct a quantitative detection model of fluorescence intensity changes and food-borne microorganisms to achieve simultaneous detection of various food-borne microorganisms.

The above-mentioned method for simultaneous detection of various food-borne microorganisms based on nano-fluorescence micro-hyperspectral imaging technology, the nano-fluorescence micro-hyperspectral imaging technology is a combination of hyperspectral imaging technology, microscopic imaging technology and nanocrystals Combining unique optical properties, at the same time, the fluorescence microscope hyperspectral imaging system uses a power-adjustable 980nm laser as the light source to irradiate the sample to be tested. The beam expander on the system increases the irradiation area of the laser on the sample. The sample strip passes through the eyepiece of the microscope and the C-Mount interface and finally reaches the slit of the imaging spectrometer, and then passes through the spectroscopic component. The light emitted by the sample strip is scattered in the vertical direction of the sample strip and finally projected to the EMCCD imaging plane, finally Obtain a linear array of fluorescence spectral data.

The above-mentioned method for simultaneous detection of multiple food-borne microorganisms based on nano-fluorescent microscopic hyperspectral imaging technology, the multi-color up-conversion nanomaterials (UCNPs), mainly use oleic acid as a surfactant, by adding gadolinium ( Gd ³⁺ ) rare earth elements regulate the crystal growth on the surface of nanomaterials, and under suitable reaction conditions, 30% Gd ³⁺ doping concentration, 300°C reaction temperature, and 1h reaction time, synthesize six-phase crystals with a size of <100nm and morphology UCNPs with uniformity, good dispersion and high fluorescence efficiency.

The above-mentioned method for simultaneous detection of various food-borne microorganisms based on nano-fluorescent microscopic hyperspectral imaging technology, the multi-color nano-fluorescence probe, the multi-color nano-fluorescence probe is a multi-color up-conversion The nanomaterials bind to immunoglobulins or oligonucleotides to which the corresponding microbes respond specifically.

The above-mentioned method for simultaneous detection of multiple food-borne microorganisms based on nano-fluorescence microscopic hyperspectral imaging technology, and the method for extracting a region of interest (ROI) with a similar angle threshold, any pixel at different wavelengths The spectral data below are combined into a multi-dimensional space vector, and the angle between the pixel vector of the unknown area and the pixel vector of the target area is calculated by analytical method, and the attribution of the pixel in the unknown area is determined according to the size of the angle, so as to Efficient Segmentation of ROIs in Fluorescence Spectral Image Data.

The above-mentioned method for simultaneous detection of multiple food-borne microorganisms based on nano-fluorescent microscopic hyperspectral imaging technology, the data dimensionality reduction means The data dimensionality reduction means involved in the present invention mainly screen out several most The combination of optimal intervals eliminates a large number of variables irrelevant to the detection target in the full spectral region; then, the intelligent search method is used to further optimize the variables from the optimal interval combination, and redundant variables with high collinearity between adjacent wavelengths are eliminated. Therefore, the characteristic spectral image at a specific scale is optimized.

The above-mentioned method for simultaneous detection of multiple food-borne microorganisms based on nano-fluorescent microscopic hyperspectral imaging technology includes the following specific steps:

1) Preparation of food-borne microorganism samples: First, inoculate the microbial strains in Luria-Bertani medium and incubate at 37°C for 24 hours, then centrifuge at 5000g for 5 minutes, discard the supernatant, wash with ultrapure water three times, and re- Dispersed in ultrapure water. Finally, the obtained bacterial liquids were serially diluted 10 times, and 8 gradients of bacterial liquids were obtained for storage. At the same time, the colony plate counting method was used to determine the specific number of bacterial colonies.

2) Preparation of multicolor up-conversion nanomaterials: up-conversion nanoparticles were prepared by pyrolysis method, and the prepared nanoparticles were oleic acid-wrapped nanoparticles. 2ml methanol dissolved rare earth chloride (0.2M, RE=Y/Gd(78%), Yb(20%), Er(2%), Tm(2%) or Ho(2%)), and 3ml oleic acid , 7ml 1-octadecene, were added to a 50ml flask. The above solution was stirred and heated to 160 °C for 30 min, then cooled to room temperature. Then 5 ml of methanol-dissolved NH ₄ F (1.6 mmol) and NaOH (1 mmol) were added and stirred for 30 min. After the methanol was completely evaporated, the above solution was heated to 280-300° C. under the protection of argon for 1.5 h. After the solution was cooled to room temperature, it was centrifuged, and the supernatant liquid was washed with methanol and ethanol for several times. Finally, the precipitate was dried in a vacuum drying oven to obtain a white powder of multicolor up-conversion nanoparticles for storage.

3) Preparation of multicolor nano fluorescent probes: The upconversion nanoparticles are conjugated with food-borne microorganism-specific immunoglobulins or oligonucleotides using the NHS/EDC chemical ligation method. First, the up-converting nanoparticles (1 mg, 5 mg/ml) were activated at room temperature for 3 h with EDC (25 μl, 2 mg/ml) and NHS (12.5 μl, 2 mg/ml). The activated upconversion nanoparticles were then centrifuged at 9350 g for 15 min, and the resulting precipitate was dissolved in 1 ml of ultrapure water. Next, 100 μg of specific immunoglobulin or oligonucleotide was added to the above solution, and incubated overnight on a shaker at 4°C. Centrifuge at 6000 g for 5 min to remove uncoupled upconverting nanoparticles, wash the precipitate with ultrapure water several times, and finally dissolve it in 1 ml of ultrapure water, and store it in a refrigerator at 4°C for future use.

4) Fluorescence spectrum image acquisition: Before image scanning, the fluorescence microscope hyperspectral imaging system needs to be turned on and warmed up for 30 minutes in advance; at the same time, the mixed samples of multi-color nano-fluorescent probes and microorganisms with different dilution factors are placed in the microscope. On the stage, under the irradiation of an external laser light source 980nm laser, microscopic hyperspectral data acquisition is performed. The scanning speed is 0.01mm/s. The sample images pass through the slit field of view of the imaging spectrometer individually and orderly at a moving speed of 0.01mm/s, and the X moving range is 8-20mm. The exposure time of the EMCCD camera was set to 3000ms.

5) Data processing and analysis: first, extract the region of interest (ROI) from the fluorescence image data obtained by the system microscopic hyperspectral imaging system, and then perform data dimension reduction to obtain the characteristic spectral image, and perform calculus fluorescence on the characteristic spectral image Intensity acquisition, the quantitative analysis model of the change value of fluorescence intensity and food-borne microorganisms under different dilution multiples is constructed, and the simultaneous detection of various food-borne microorganisms is realized.

Compared with the prior art, the present invention has the advantages of:

The nano-fluorescence microscopic hyperspectral imaging technology constructed by the present invention combines hyperspectral imaging technology, microscopic imaging technology and the unique optical properties of nanocrystals. At the same time, the fluorescence microscopic hyperspectral imaging system uses a 980nm laser as a light When the sample to be tested is illuminated, the sample strip under the instantaneous field of view passes through the eyepiece of the microscope and the C-Mount interface and finally reaches the slit of the imaging spectrometer, and then passes through the spectrum splitting component, and the light emitted by the sample strip occurs in the vertical direction of the sample strip. Scattering is finally projected to the EMCCD imaging plane, and finally a linear array of fluorescence spectral data is obtained.

The invention relates to a simultaneous detection method of multiple food-borne microorganisms based on nano-fluorescence microscopic hyperspectral imaging technology, which is easy to operate, has high sensitivity and fast detection speed, and is widely used in technical fields such as food safety and environmental monitoring.

The multicolor up-conversion nanomaterials (UCNPs) involved in the present invention mainly use oleic acid as a surfactant, and adjust the crystal growth on the surface of the nanomaterials by adding gadolinium (Gd ³⁺ ) rare earth elements. Under suitable reaction conditions, 30% Gd ^{3 +} Doping concentration, 300°C reaction temperature, and 1h reaction time to synthesize UCNPs with crystal form six phases, size <100nm, uniform shape, good dispersion, and high fluorescence efficiency, including at least up-conversion fluorescent nanoparticles NaY/GdF ₄ : Yb ³⁺ , Er ³⁺ (UCNPs _Er ), NaY/GdF ₄ : Yb ³⁺ , Tm ³⁺ (UCNPs _Tm ), NaY/GdF ₄ : Yb ³⁺ , Ho ³⁺ (UCNPs _Ho ).

The present invention relates to a region of interest (ROI) extraction method for a similar angle threshold, which combines the spectral data of any pixel at different wavelengths into a multi-dimensional space vector, and uses an analytical method to calculate the pixel vector sum and sum of the unknown region. The included angle between the pixel vectors in the target area is used to determine the attribution of the unknown area pixel according to the size of the included angle, so as to effectively segment the ROI in the fluorescence spectral image data.

The data dimensionality reduction method involved in the present invention mainly screens out several optimal interval combinations through target orientation, and eliminates a large number of variables that are irrelevant to the detection target in the full spectrum region; then, applies the intelligent search method to optimize the variables from the optimal interval combination Further preferably, redundant variables with high collinearity between adjacent wavelengths are eliminated, so as to optimize the characteristic spectral image at a specific scale.

Description of drawings

Fig. 1 is the standard curve (a is used for detecting escherichia coli, and b is used for detecting Staphylococcus aureus) that test detects different concentrations of bacterial liquid simultaneously;

Fig. 2 is a device diagram of a fluorescence microscope hyperspectral imaging system;

detailed description

Implementation Example 1

In order to further verify that the method of the present invention detects multiple food-borne microorganisms simultaneously, the examples of the present invention take Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) as examples, and the specific operation steps are as follows:

(1) Preparation of food-borne microorganism samples: First, inoculate the microbial strains in Luria-Bertani medium and incubate at 37°C for 24 hours, then centrifuge at 5000g for 5 minutes, discard the supernatant, and wash with ultrapure water three times, respectively. Redisperse in ultrapure water. Finally, the obtained bacterial liquids were serially diluted 10 times, and 8 gradients of bacterial liquids were obtained for storage. At the same time, the colony plate counting method was used to determine the specific number of bacterial colonies.

(2) Preparation of multicolor up-conversion nanomaterials: up-conversion nanoparticles were prepared by pyrolysis method, and the prepared nanoparticles were oleic acid-wrapped nanoparticles. 2ml methanol dissolved rare earth chloride (0.2M, RE=Y/Gd(78%), Yb(20%), Er(2%), Tm(2%) or Ho(2%)), and 3ml oleic acid , 7ml 1-octadecene, were added to a 50ml flask. The above solution was stirred and heated to 160 °C for 30 min, then cooled to room temperature. Then 5 ml of methanol-dissolved NH ₄ F (1.6 mmol) and NaOH (1 mmol) were added and stirred for 30 min. After the methanol was completely evaporated, the above solution was heated to 300° C. under the protection of argon for 1.5 h. After the solution was cooled to room temperature, it was centrifuged, and the supernatant liquid was washed with methanol and ethanol for several times. Finally, the precipitate was dried in a vacuum drying oven to obtain a white powder of multicolor up-conversion nanoparticles for storage.

(3) Preparation of multi-color nano fluorescent probes: The up-conversion nanoparticles were conjugated with Escherichia coli and Staphylococcus aureus specific immunoglobulins by NHS/EDC chemical ligation method. First, the up-converting nanoparticles (1 mg, 5 mg/ml) were activated at room temperature for 3 h with EDC (25 μl, 2 mg/ml) and NHS (12.5 μl, 2 mg/ml). The activated upconversion nanoparticles were then centrifuged at 9350 g for 15 min, and the resulting precipitate was dissolved in 1 ml of ultrapure water. Next, 100 μg of E.coli antibody was added to the above solution, and incubated overnight on a shaker at 4°C. Centrifuge at 6000 g for 5 min to remove uncoupled upconverting nanoparticles, wash the precipitate with ultrapure water several times, and finally dissolve it in 1 ml of ultrapure water, and store it in a refrigerator at 4°C for future use.

(4) Fluorescence spectrum image acquisition: before image scanning, it is necessary to turn on the fluorescence microscope hyperspectral imaging system (as shown in Figure 1) in advance and warm up for 30 minutes; The mixed sample is placed on the stage of the microscope, under the irradiation of an external laser light source 980nm laser, the microscopic hyperspectral data is acquired. The scanning speed is 0.01mm/s. The sample images pass through the slit field of view of the imaging spectrometer individually and orderly at a moving speed of 0.01mm/s, and the X moving range is 8-20mm. The exposure time of the EMCCD camera was set to 3000ms.

(5) Data processing and analysis: firstly, extract the region of interest (ROI) from the fluorescent image data obtained by the system microscopic hyperspectral imaging system, and then perform data dimensionality reduction to obtain the characteristic spectral image, and perform calculus on the characteristic spectral image The fluorescence intensity is obtained, and the quantitative analysis model of the change value of the fluorescence intensity and the food-borne microorganisms under different dilution factors (as shown in Figure 2) is constructed to realize the simultaneous detection of various food-borne microorganisms.

In the description of this specification, references to the terms "one embodiment," "some embodiments," "exemplary embodiments," "example," "specific examples," or "some examples" are intended to mean that the implementation A specific feature, structure, material, or characteristic described by an embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications, substitutions and modifications can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the invention is defined by the claims and their equivalents.

Claims (7)

1. A method for simultaneous detection of multiple food-borne microorganisms based on nano-fluorescent micro-hyperspectral imaging technology, characterized in that, taking food-borne micro-organisms as the research object, a set of nano-fluorescent micro-hyperspectral imaging system is constructed, through Synthesize multi-color up-conversion nanomaterials, construct a multi-color nano-fluorescence probe with specific recognition of food-borne microorganisms, and perform multi-target labeling on various microorganisms; Spectral image data, extract the fluorescence spectrum of the region of interest, and optimize the characteristic spectral image at a specific scale through data dimensionality reduction methods, and use image processing methods to construct a quantitative detection model for fluorescence intensity changes and food-borne microorganisms to achieve multiple Simultaneous detection of a variety of food-borne microorganisms, the method is applicable to technical fields such as food safety and environmental monitoring. 2. A kind of simultaneous detection method of multiple food-borne microorganisms based on nano-fluorescence micro-hyperspectral imaging technology according to claim 1, characterized in that, the constructed nano-fluorescence micro-hyperspectral imaging system is a combination of hyperspectral Imaging technology, microscopic imaging technology and the unique optical properties of nanocrystals are combined. At the same time, the fluorescence microscopic hyperspectral imaging system uses a power-adjustable 980nm laser as a light source to irradiate the sample to be tested. The beam expander on the system increases the laser For the irradiated area of the sample, the sample strip under the instantaneous field of view passes through the eyepiece of the microscope and the C-Mount interface and finally reaches the slit of the imaging spectrometer, and then passes through the spectrum splitting component. Scattering in the direction is finally projected to the EMCCD imaging plane, and finally a linear array of fluorescence spectrum data is obtained. 3. A kind of method for simultaneously detecting multiple food-borne microorganisms based on nano-fluorescent microscopic hyperspectral imaging technology according to claim 1, characterized in that, the multi-color up-conversion nanomaterial mainly uses oleic acid as Surfactant, by adding gadolinium (Gd ³⁺ ) rare earth elements to adjust the crystal growth on the surface of nanomaterials, synthesize six-phase crystals under appropriate reaction conditions: 30% Gd ³⁺ doping concentration, 300°C reaction temperature, and 1h reaction time Multicolor upconversion nanomaterials with a size <100nm, at least including upconversion fluorescent nanoparticles NaY/GdF ₄ : Yb ³⁺ , Er ³⁺ (UCNPs _Er ), NaY/GdF ₄ : Yb ³⁺ , Tm ³⁺ ( UCNPS _Tm ), NaY/GdF ₄ : Yb ³⁺ , Ho ³⁺ (UCNPS _Ho ). 4. A method for simultaneous detection of multiple food-borne microorganisms based on nano-fluorescent microscopic hyperspectral imaging technology according to claim 1, wherein the multi-color nano-fluorescence probe is made of multi-color Switching nanomaterials bind to immunoglobulins or oligonucleotides that specifically respond to the corresponding microorganisms, including at least UCNPs _Er- Escherichia coli immunoglobulin, UCNPS _Tm -Staphylococcus aureus immunoglobulin, _UCNPS Ho-Salmonella typhimurium immune Globulin, UCNPs _Er- Escherichia coli oligonucleotide, UCNPS _Tm -Staphylococcus aureus oligonucleotide, _UCNPS Ho-Salmonella typhimurium oligonucleotide. 5. a kind of multiple food-borne microorganisms detection method based on nano-fluorescent micro-hyperspectral imaging technology according to claim 1, is characterized in that, adopts a kind of similarity angle threshold method to extract the fluorescence spectrum of interested region, Combine the spectral data of any pixel at different wavelengths into a multi-dimensional space vector, use the analytical method to calculate the angle between the pixel vector in the unknown area and the pixel vector in the target area, and determine the unknown according to the size of the angle Assignment of regional pixels for efficient segmentation of regions of interest in fluorescence spectral image data. 6. A method for simultaneous detection of multiple food-borne microorganisms based on nano-fluorescence microscopic hyperspectral imaging technology according to claim 1, characterized in that, the data dimensionality reduction means mainly screen out several The optimal interval combination eliminates a large number of variables irrelevant to the detection target in the full spectral region; then, the intelligent search method is used to further optimize the variables from the optimal interval combination, and the redundant variables with high collinearity between adjacent wavelengths are eliminated , so as to optimize the characteristic spectral image at a specific scale. 7. A kind of method for simultaneous detection of multiple food-borne microorganisms based on nano-fluorescent microscopic hyperspectral imaging technology according to claim 1, characterized in that the method comprises the following specific steps: Step 1) Preparation of food-borne microorganism samples: first, inoculate the microbial strains in Luria-Bertani medium at 37°C for 24 hours, then centrifuge at a speed of 5000g for 5 minutes, discard the supernatant, and wash three times with ultrapure water, respectively. Redisperse in ultrapure water. Finally, the obtained bacterial liquids were serially diluted 10 times, and 8 gradients of bacterial liquids were obtained for storage. At the same time, the colony plate counting method was used to determine the specific number of bacterial colonies. Step 2) Preparation of multi-color up-conversion nanomaterials: Up-conversion nanoparticles are prepared by pyrolysis method, and the prepared nanoparticles are oleic acid-wrapped nanoparticles. 2ml methanol dissolved rare earth chloride (0.2M, RE=Y/Gd(78%), Yb(20%), Er(2%), Tm(2%) or Ho(2%)), and 3ml oleic acid , 7ml 1-octadecene, were added to a 50ml flask. The above solution was stirred and heated to 160 °C for 30 min, then cooled to room temperature. Then 5 ml of methanol-dissolved NH ₄ F (1.6 mmol) and NaOH (1 mmol) were added and stirred for 30 min. After the methanol was completely evaporated, the above solution was heated to 280-300° C. under the protection of argon for 1.5 h. After the solution was cooled to room temperature, it was centrifuged, and the supernatant liquid was washed with methanol and ethanol for several times. Finally, the precipitate was dried in a vacuum drying oven to obtain a white powder of multicolor up-conversion nanoparticles for storage. Step 3) Preparation of multicolor nano fluorescent probes: The upconversion nanoparticles are conjugated with food-borne microorganism-specific immunoglobulins or oligonucleotides by NHS/EDC chemical ligation method. First, the up-converting nanoparticles (1 mg, 5 mg/ml) were activated at room temperature for 3 h with EDC (25 μl, 2 mg/ml) and NHS (12.5 μl, 2 mg/ml). The activated upconversion nanoparticles were then centrifuged at 9350 g for 15 min, and the resulting precipitate was dissolved in 1 ml of ultrapure water. Next, 100 μg of specific immunoglobulin or oligonucleotide was added to the above solution, and incubated overnight on a shaker at 4°C. Centrifuge at 6000 g for 5 min to remove uncoupled upconverting nanoparticles, wash the precipitate with ultrapure water several times, and finally dissolve it in 1 ml of ultrapure water, and store it in a refrigerator at 4°C for future use. Step 4) Fluorescence spectrum image acquisition: Before image scanning, the fluorescence microscope hyperspectral imaging system needs to be turned on and warmed up for 30 minutes in advance; at the same time, the mixed samples of multicolor nano fluorescent probes and microorganisms with different dilution factors are placed in the microscope Microscopic hyperspectral data acquisition was carried out under the irradiation of an external laser light source with a 980nm laser on the stage. The scanning speed is 0.01mm/s. The sample images pass through the slit field of view of the imaging spectrometer individually and orderly at a moving speed of 0.01mm/s, and the X moving range is 8-20mm. The exposure time of the EMCCD camera was set to 3000ms. Step 5) Data processing and analysis: First, extract the region of interest from the fluorescence image data obtained by the system microscopic hyperspectral imaging system, then perform data dimensionality reduction to obtain the characteristic spectral image, and perform calculus fluorescence intensity acquisition on the characteristic spectral image , construct the quantitative analysis model of the change value of fluorescence intensity and food-borne microorganisms under different dilution factors, and realize the simultaneous detection of various food-borne microorganisms.