Visual staphylococcus aureus detection kit based on immunomagnetic beads

Abstract

The application discloses a kind of for visualizing based on immunomagnetic bead detection staphylococcus aureus kit, preparation carboxylated Fe3O4 Nano magnetic beads, the magnetic bead is coupled with anti staphylococcus aureus chicken yolk antibody IgY, and immunomagnetic bead is obtained;Take the liquid to be measured, it is mixed with immunomagnetic bead, is suspended after room temperature on rotating mixer, and is magnetically separated, then appropriate amount of H2O2 Solution is added, and is magnetically separated, and the remaining H2O2 Solution is added 50 μL HRP solution and 50 μL TMB solution color development, and color change of solution can be qualitatively observed with naked eye.The application provides the use of immunomagnetic bead and TMB-HRP-H2O2 color development system combination staphylococcus aureus detection, fast, visual detection staphylococcus aureus, shorten detection time, and the minimum detection concentration is 10 3 CFU·mL ‑1 , with the recovery rate of 102.26%, high sensitivity, good stability, strong specificity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a visualization-based reagent kit for detecting Staphylococcus aureus using immunomagnetic beads. Background Technology

[0002] In recent years, serious food safety incidents have occurred frequently worldwide, severely impacting the economic conditions and social stability of many countries. Globally, billions of people suffer from foodborne illnesses each year, with over 66% caused by foodborne pathogens. In both developed and developing countries, food poisoning caused by Staphylococcus aureus (SFP) accounts for a significant proportion of bacterial food poisoning.

[0003] Staphylococcus aureus is a common foodborne pathogen. While not inherently highly toxic, it can multiply rapidly in food and produce Staphylococcus aureus enterotoxin. This enterotoxin is highly destructive to the intestines, often causing Staphylococcus aureus enteritis. This disease has a rapid onset and severe symptoms, primarily including vomiting, fever, abdominal pain, diarrhea, and shock. In severe cases, it can lead to circulatory failure.

[0004] Currently, laboratory detection methods for Staphylococcus aureus mainly include traditional isolation and culture biochemical identification methods, immunological detection methods, and molecular biological detection methods. However, these methods have drawbacks such as complex operation, long processing time, low sensitivity, poor specificity, and high requirements for experimental conditions and operators. Therefore, establishing a simple, rapid, sensitive, and accurate detection method for Staphylococcus aureus is essential for preventing and controlling food safety problems caused by Staphylococcus aureus. Summary of the Invention

[0005] The purpose of this invention is to provide a visual reagent kit for detecting Staphylococcus aureus based on immunomagnetic beads.

[0006] A visualization-based reagent kit for detecting Staphylococcus aureus using immunomagnetic beads includes: immunomagnetic beads and a chromogenic solution; the immunomagnetic beads are carboxylated Fe3O4 nanoparticles coupled with anti-Staphylococcus aureus chicken egg yolk antibody IgY; the chromogenic solution includes: TMB solution, HRP solution, and H2O2 solution.

[0007] The concentration of the immunomagnetic beads is 0.5~0.8 mg·mL. -1 The concentration of the TMB solution is 1.0~2.5 mg·mL. -1 The HRP solution concentration is 1.0~10 μg·mL. -1 ;

[0008] The concentration of the immunomagnetic beads is 0.6 mg / ml. -1 The TMB solution concentration was 2.0 mg·mL. -1 The HRP solution concentration was 2.5 μg·mL. -1 ;

[0009] The volume of H2O2 solution used is 50 μL, the volume of TMB solution used is 50 μL, and the volume of HRP solution used is 50 μL;

[0010] The carboxylated Fe3O4 nanoparticles were prepared using the following method:

[0011] ① Add 1.08 g FeCl3∙6H2O and 20 mL ethylene glycol to a beaker and sonicate to dissolve for 10 min;

[0012] ② Add 1.2 g sodium acetate and 0.2 g trisodium citrate to ① and sonicate to dissolve for 10 min;

[0013] ③ Add 0.2 g of PEG-6000 to ② and sonicate until completely dissolved;

[0014] ④ Transfer the above solution ③ to the reaction vessel and react at 198℃ for 18 h. After the reaction vessel is cooled to room temperature, transfer the magnetic bead solution to a small beaker and wash the magnetic beads three times with water and anhydrous ethanol alternately to obtain carboxylated Fe3O4 nanoparticles.

[0015] The immunomagnetic beads were prepared using the following method:

[0016] ① Take 5 mg of carboxylated Fe3O4 nanoparticles, add 1 mL of PBS, sonicate for 1-2 min, magnetically separate, wash the obtained magnetic beads twice with PBS, and then resuspend in 1 mL of PBS.

[0017] ② Add 10 mg EDC·HCl and 5 mg NHS to ①, suspend for 30 min, then magnetically separate, wash 3 times with PBS, and resuspend in 1 mL PBS;

[0018] ③ Add 500 μg of anti-Staphylococcus aureus chicken egg yolk antibody IgY to ②, suspend at room temperature for 2 h, and then magnetically separate. Wash the obtained magnetic beads 3 times with PBS.

[0019] ④ Add 1 mL of blocking solution and block for 1 h to obtain functionalized immunomagnetic beads.

[0020] Another object of the present invention is to provide a method for visually detecting Staphylococcus aureus in food.

[0021] A method for visually detecting Staphylococcus aureus in food, which includes:

[0022] ① Take 100 μL of the sample solution to be tested, add the above immunomagnetic beads, the reaction system is 1 mL, suspend and enrich at room temperature for 30~75 min, magnetically separate, remove the supernatant, and wash 3 times with PBS;

[0023] ② Add H2O2 solution to ①, decompose the reaction for 1~4 min, perform magnetic separation, and take the supernatant;

[0024] ③ Add a colorimetric solution with a pH of 4.0~6.0 to ②. The colorimetric solution includes the above HRP solution and TMB solution. React in the dark for 5 minutes and observe the color change with the naked eye or measure the absorption spectrum with an ultraviolet spectrophotometer.

[0025] The enrichment time was 60 min, the H2O2 decomposition reaction time was 2 min, and the pH of the colorimetric solution was 4.5.

[0026] This invention provides a visual detection kit for Staphylococcus aureus based on immunomagnetic beads. The kit involves preparing carboxylated Fe3O4 nanomagnetic beads, conjugating these beads with an anti-Staphylococcus aureus chicken egg yolk antibody IgY, and obtaining immunomagnetic beads. The test solution is mixed with the immunomagnetic beads, suspended at room temperature using a rotary mixer, and then magnetically separated. An appropriate amount of H2O2 solution is added, followed by magnetic separation. The remaining H2O2 solution is then added to 50 μL of HRP solution and 50 μL of TMB solution for color development. The color of the solution is observed, thus achieving rapid and specific detection of Staphylococcus aureus. This invention proposes using an immunomagnetic bead combined with a TMB-HRP-H2O2 colorimetric system for the rapid and visual detection of Staphylococcus aureus, shortening the detection time, with a minimum detection concentration of 10. 3 CFU·mL -1 The spiked recovery rate was 102.26%, with high sensitivity and good stability. It showed no cross-reactivity with Escherichia coli O157: H7, Salmonella, Shigella boydii, Vibrio parahaemolyticus, Listeria monocytogenes, Staphylococcus aureus and their mixtures, and had high specificity. Attached Figure Description

[0027] Figure 1 Results of purity analysis of IgY antibody;

[0028] Figure 2 TEM morphological feature images of MBs and IgY-MBs;

[0029] Figure 3 FTIR absorption spectra of MBs and IgY-MBs;

[0030] Figure 4 Hysteresis loops of MBs and IgY-MBs at room temperature;

[0031] Figure 5Results of determination of immunomagnetic bead concentration;

[0032] Figure 6 Results of determination of immunomagnetic bead enrichment time;

[0033] Figure 7 Transmission electron microscopy images: (A) Staphylococcus aureus (B) Target bacteria enriched by immunomagnetic beads;

[0034] Figure 8 Results of the optimal H2O2 dosage;

[0035] Figure 9 Results of the optimal reaction time between magnetic bead-bacterial complex and H2O2;

[0036] Figure 10 Results of determining the optimal pH value of the reaction system;

[0037] Figure 11 Results of determination of optimal TMB concentration;

[0038] Figure 12 Results of determining the optimal HRP concentration;

[0039] Figure 13 Determination of the detection limit of a detection method: (A) Visual judgment (B) Standard curve (C) Ultraviolet absorption spectrum;

[0040] Figure 14 Specific results of the detection method;

[0041] Figure 15 Simulated sample test results: (A) Visual judgment (B) Standard curve (C) Ultraviolet absorption spectrum. Detailed Implementation

[0042] Example 1: Preparation of Staphylococcus aureus chicken egg yolk antibody

[0043] 1. Strains culture

[0044] Prepare LA and LB media and autoclave. Take Staphylococcus aureus strains stored at -20℃ and inoculate them onto LA (solid) medium using the streak plate method. Incubate at 37℃ for 18 hours. Pick single colonies from the LA medium and inoculate them into Erlenmeyer flasks containing 200 mL of LB liquid medium. Incubate on an air shaker at 37℃ and 180 rpm for 18 hours. Serially dilute 1 mL of the bacterial suspension and count the bacteria using the plate count method, recording the bacterial concentration. Add a 1% formaldehyde solution to the remaining bacterial suspension and inactivate at 4℃ for 24 hours. Simultaneously verify complete inactivation using the plate count method. Centrifuge the inactivated bacterial suspension at 4000 rpm for 15 minutes, discard the supernatant, and resuspend in 0.9% physiological saline. Repeat the centrifugation and resuspension steps twice. Concentrate the prepared bacterial suspension to 4 × 10⁻⁶. 9 CFU·mL -1 Store in a refrigerator at 4℃ for later use.

[0045] 2. Vaccine preparation

[0046] Mix Freund's complete adjuvant with the prepared bacterial suspension at a 1:1 ratio to achieve a final bacterial concentration of 2 × 10⁻⁶. 9 CFU·mL -1 Place the above mixture on a magnetic stirrer and stir, observing the emulsification state of the vaccine. Stir until the vaccine is completely emulsified, and let it stand for 2 days without separating into water and emulsion; this is the vaccine for primary immunization. Take Freund's incomplete adjuvant and mix it with the above bacterial solution at a 1:1 ratio, stirring in the same way until the mixture is completely emulsified; this is the vaccine for subsequent booster immunizations.

[0047] 3. Preparation and extraction of chicken egg yolk antibodies

[0048] The prepared vaccine was sent to SPF-grade high-producing laying hens at the Shandong Provincial Poultry Research Institute to collect eggs. Chicken egg yolk antibody IgY was extracted using the polyethylene glycol precipitation method, with the specific steps as follows:

[0049] (1) Gently wipe away the stains on the surface of the egg with 75% alcohol, gently break the eggshell, use an egg yolk separator to separate the yolk and place it on filter paper and gently roll it, use a syringe to draw out the yolk liquid and record the volume.

[0050] (2) Add PBS buffer and 3.5% PEG-6000 (w / v) twice the volume of egg yolk liquid, shake and mix on a water bath shaker for 15 min, and centrifuge at 10000 rpm and 4℃ for 20 min.

[0051] (3) Filter the supernatant, record the volume, add 8.5% PEG-6000 (w / v), shake and mix on a water bath shaker for 15 min, centrifuge at 10000 rpm and 4℃ for 20 min.

[0052] (4) Discard the supernatant, resuspend the precipitate in 10 mL of PBS buffer, add 12% PEG-6000 (w / v), shake and mix on a water bath shaker for 15 min, centrifuge at 10000 rpm and 4℃ for 20 min.

[0053] (5) Discard the supernatant, add 1 mL of PBS buffer to the precipitate and resuspend. Dialyze with 0.1% saline overnight, then change to PBS buffer and dialyze for 4 hours. This is the prepared chicken egg yolk antibody IgY. Aliquot and store in a -20℃ freezer for later use.

[0054] 4. Antibody titer determination

[0055] The titer of chicken egg yolk antibody IgY was determined using the iELISA method. The specific steps are as follows:

[0056] (1) Antigen coating: The concentration of 1×10 9 CFU·mL -1 The inactivated Staphylococcus aureus culture was diluted 100-fold with coating buffer. 100 μL of the culture was added to each well of a 96-well plate (ELISA plate) and incubated overnight at 4°C.

[0057] (2) Blocking with skim milk powder: Discard the bacterial culture in the plate, add 300 μL of PBST buffer to each well to wash the plate and pat the liquid in the plate dry with a plate patting paper. Repeat the above washing steps twice, add 300 μL of 5% skim milk powder to each well, and block at 37 ℃ for 2 h.

[0058] (3) Adding the antibody to be tested: Dilute the chicken egg yolk antibody IgY to be tested 1:1000 with 5% skim milk powder (blocking buffer), discard the blocking buffer in the plate, wash the plate three times with PBST buffer, and add the antibody to be tested using the serial dilution method. The specific steps are as follows: add 200 μL of the diluted antibody to be tested to the first column, add 100 μL of blocking buffer to each well from the second column onwards, for a total of 9 columns, and the last column is a blank control. Incubate at 37 ℃ for 1 h. The corresponding dilutions from left to right are 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000.

[0059] (4) Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-chicken secondary antibody 1:8000 with blocking buffer, discard the liquid in the plate, wash the plate three times with PBST buffer, add 100 μL of diluted secondary antibody to each well, and incubate at 37 ℃ for 1 h.

[0060] (5) Color development: Discard the liquid in the plate, wash the plate three times with PBST buffer, add 100 μL of color development solution to each well and react at room temperature in the dark for 5 min.

[0061] (6) Termination: Add 50 μL of stop solution (concentrated sulfuric acid) to each well to terminate the reaction, and measure the absorbance at 450 nm using a multi-functional microplate reader.

[0062] The results are shown in Table 1. The absorbance value of the blank control (Control) is 0.086. The antibody concentrations below 1:64000 are all greater than 2.1×0.086=0.1806. Therefore, the titer of the extracted antibody is 1:64000.

[0063]

[0064] 5. Antibody purity identification

[0065] The purity of chicken egg yolk antibody IgY was identified using SDS-PAGE. The specific steps are as follows:

[0066] (1) Install the gel mold with the short plate facing outwards and fill it with double-distilled water to check for leakage.

[0067] (2) Prepare 5 mL of 12% separating gel (the separating gel formula is 1.75 mL of double distilled water, 2.0 mL of Acr-Bis solution, 1.25 mL of separating gel buffer, 0.05 mL of 10% APS solution, and 2.0 µL of TEMED solution). After mixing, quickly pour the mixture between two glass plates, fill the plate with double distilled water, press the separating gel surface flat, remove air bubbles, and let it stand until the separating gel polymerizes. A clear interface will appear between the separating gel and the water layer. Remove the water layer covering the separating gel and use the edge of filter paper to absorb the residual liquid.

[0068] (3) Prepare 2 mL of 5% stacking gel (the stacking gel formula is 1.14 mL of double distilled water, 0.34 mL of Acr-Bis solution, 0.5 mL of stacking gel buffer, 0.02 mL of APS solution, and 2 µL of TEMED solution). After mixing, pour the mixture between the two glass plates until it reaches the top of the glass plates. Quickly insert the comb, being careful to avoid generating air bubbles. Let it stand at room temperature for about 20 minutes and wait for the stacking gel to collect.

[0069] (4) After the stacking gel polymerizes, fix it on the electrophoresis apparatus, add 1× electrophoresis buffer to each of the upper and lower electrophoresis tanks, and carefully pull out the comb to avoid damaging the lanes.

[0070] (5) Mix the protein sample to be tested with 2×SDS loading buffer at a 1:1 ratio, and boil in a boiling water bath for 3-5 min to completely denature the protein. Add 10 µL of the protein sample to be tested to each lane, and add 5 µL of marker as a control to the middle lane.

[0071] (6) Connect the electrophoresis apparatus to the power supply. Apply a voltage of 70 V to the stacking gel for about 20 min. The indicator will enter the separating gel. Continue electrophoresis with a voltage of 90 V for about 90 min until the indicator reaches the bottom of the separating gel. Then turn off the power supply.

[0072] (7) After electrophoresis, remove the electrophoresis apparatus, separate the glass plate, take out the gel intact and put it into a petri dish, add an appropriate amount of Coomassie brilliant blue staining solution, and stain on a horizontal shaker for 30 min.

[0073] (8) Discard the staining solution, add an appropriate amount of destaining solution, and shake on a horizontal shaker overnight for destaining. The destaining solution should be changed several times during the process until clear bands are seen. Use a gel imaging system to take pictures and analyze the results.

[0074] The results are as follows Figure 1 As shown, the antibody has few impurities, clear bands, and high purity, with the heavy chain at approximately 72 kDa and the light chain at approximately 26 kDa.

[0075] 6. Determination of antibody protein content

[0076] The specific steps for detecting antibody protein content using the BCA protein quantification kit are as follows:

[0077] (1) Preparation of BCA working solution: Prepare an appropriate amount of BCA working solution according to the number of samples by mixing 50 volumes of reagent A and 1 volume of reagent B.

[0078] (2) Protein concentration determination: Protein standards were prepared in concentrations of 0, 1, 2, 4, 6, 8, and 10 μL, respectively.

[0079] Add the protein standard solution to seven wells of a 96-well plate, and bring the volume to 10 μL with sterile double-distilled water. Dilute the protein samples to be tested at 1:20, 1:50, and 1:100, respectively, and add 10 μL of each solution to the sample wells. Perform three replicates for each assay. Add 200 μL of BCA working solution (i.e., a sample to working solution volume ratio of 1:20) to each of the sample wells and protein standard wells. Incubate at 37°C for 30 min, then cool to room temperature.

[0080] (3) Result determination: The absorbance value at a wavelength of 562 nm was measured using a multi-functional microplate reader, and a standard curve was plotted. The protein content of the antibody to be tested was calculated to be 21.78 mg·mL from the standard curve. -1 .

[0081] 7. Evaluation of antibody specificity

[0082] The specificity of chicken egg yolk antibodies was evaluated using iELISA, with coating antigens at concentrations of 1×10⁻⁶. 7Nine types of bacteria—Staphylococcus aureus, Escherichia coli O157:H7, Vibrio parahaemolyticus, Shigella boydii, Salmonella, Listeria monocytogenes, Klebsiella pneumoniae, Cronobacter sakazakii, and Proteus mirabilis—were inactivated in a solution diluted 1:1000 with chicken egg yolk antibody IgY. The preparation method for the inactivated solution was the same as for bacterial culture. A positive result was defined as a positive value (P) to negative value (N) ratio greater than 2.1.

[0083] The specificity of the antibody was verified by iELISA. The results are shown in Table 2. Except for the target bacterium, all other pathogenic bacteria showed negative results and no cross-reaction, indicating that the prepared antibody has good specificity.

[0084]

[0085] Example 2 Preparation of Immunomagnetic Beads

[0086] 1. Synthesis of Functionalized Carboxyl Magnetic Beads

[0087] Fe3O4 functionalized nanoparticles with carboxyl groups on their surface were synthesized by a solvothermal method using trisodium citrate as a stabilizer.

[0088] The specific steps are as follows:

[0089] (1) Take 1.08 g FeCl3∙6H2O and 20 mL ethylene glycol and add them to a 50 mL beaker. Sonicate for 10 min until completely dissolved. Then weigh 1.2 g sodium acetate and 0.2 g trisodium citrate and add them to the beaker. Sonicate for 10 min until completely dissolved. Finally, add 0.2 g PEG-6000 and sonicate until completely dissolved.

[0090] (2) Transfer the above solution to a stainless steel high-pressure reactor lined with polytetrafluoroethylene and react at 198°C for 18 h. After the reactor is cooled to room temperature, transfer the solution (magnetic beads) to a small beaker, wash the magnetic beads three times alternately with water and anhydrous ethanol, and wash with water for the last time. Dry in a vacuum drying oven at 60°C overnight and store in a refrigerator at 4°C for later use.

[0091] 2. Preparation of immunomagnetic beads

[0092] Immunomagnetic beads were prepared by coupling the above-mentioned carboxyl-modified Fe3O4 functionalized nanoparticles with chicken egg yolk antibody IgY using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and NN-hydroxysuccinimide (NHS) as coupling agents.

[0093] The specific steps are as follows:

[0094] (1) Weigh 5 mg of Fe3O4 functionalized nanoparticles, add 1 mL of PBS buffer solution to resuspend, sonicate for 1-2 min, magnetically separate, wash twice with PBS and then resuspend in 1 mL of PBS.

[0095] (2) Add 10 mg EDC·HCl and 5 mg NHS, place on a rotary mixer and suspend at room temperature for 30 min, then magnetically separate, wash three times with PBS, and resuspend in 1 mL PBS.

[0096] (3) Add 500 μg of chicken egg yolk antibody IgY, suspend at room temperature for 2 h, then separate magnetically and wash three times with PBS.

[0097] (4) Add 1 mL of 5% skim milk powder (blocking solution), suspend at room temperature for 1 h, wash three times with PBS, and resuspend with 1 mL of PBS to obtain a concentration of 5 mg / mL. -1 The immunomagnetic beads should be stored at 4°C for later use.

[0098] The morphological characteristics of functionalized carboxyl magnetic beads and immunomagnetic beads were observed using transmission electron microscopy, such as... Figure 2 As shown in A and B, the functionalized carboxyl magnetic beads prepared in this invention are spherical with a slightly rough surface, good dispersibility, and an average particle size of 240 nm. The immunomagnetic beads have an average particle size of 281.7 nm, with blurred edges and a larger particle size than the functionalized carboxyl magnetic beads, indicating that the antibody IgY was successfully coupled to the surface of the magnetic beads. Figure 2 C).

[0099] Fourier transform infrared spectroscopy characterization results are as follows Figure 3 As shown, immunomagnetic beads at 586 cm⁻¹ -1 Stretching vibration peaks of the Fe-O bonds in Fe3O4 nanoparticles appeared at 1454 cm⁻¹. -1 3333cm -1 2819 cm -1 and 1612 cm -1 At this location, CO, NH bonds, -CH2 bonds, and characteristic C=O absorption peaks in peptide bonds were observed, proving that IgY was successfully coupled to the surface of carboxyl magnetic beads.

[0100] Characterization results of the magnetic measurement system are as follows Figure 4 As shown, the hysteresis loops indicate that the saturation magnetic susceptibility of the functionalized carboxyl magnetic beads is 59.286 emu / g, and that of the immunomagnetic beads is 52.039 emu / g, both exhibiting superparamagnetism. The coupling of IgY only slightly reduces the saturation magnetic susceptibility of the beads without destroying their magnetic force. The immunomagnetic beads still possess strong magnetization and rapid response to external magnetic fields, laying the foundation for rapid enrichment of target bacteria.

[0101] Example 3: Preparation of Buffer Solution

[0102] 1×PBS buffer: Dissolve 20 mL of 20×PBS buffer in 380 mL of double-distilled water, mix well, and store at room temperature for later use.

[0103] Example 4 Preparation of bacterial suspension standard

[0104] Staphylococcus aureus strains stored at -20℃ were inoculated onto LA (solid) medium using the streak plate method and incubated at 37℃ for 18 hours. Single colonies from the LA medium were picked and inoculated into Erlenmeyer flasks containing 200 mL of LB liquid medium. The flasks were then placed on an air shaker and incubated at 37℃ and 180 rpm for 18 hours. 1 mL of the bacterial suspension was serially diluted and counted using the plate count method, and the bacterial concentration was recorded. A 1% formaldehyde solution was added to the remaining bacterial suspension, and the suspension was inactivated at 4℃ for 24 hours. The complete inactivation of bacteria was verified using the plate count method. The inactivated bacterial suspension was centrifuged at 4000 rpm for 15 minutes, the supernatant was discarded, and the suspension was resuspended in 0.9% physiological saline. This centrifugation and resuscitation process was repeated twice. The prepared bacterial suspension was concentrated to a concentration of 4 × 10⁻⁶. 9 CFU·mL -1 Store in a refrigerator at 4℃ for later use.

[0105] Example 5: Establishment of a Visual Detection Method for Staphylococcus aureus

[0106] First, fresh Staphylococcus aureus bacterial suspension (live bacteria) was prepared. The bacterial suspension was counted using a turbidimeter, and diluted with PBS to obtain bacterial suspensions of different concentrations. Then, immunomagnetic separation technology was used to specifically enrich and separate the Staphylococcus aureus suspensions of different concentrations. Excess unbound material was washed away with PBS buffer solution. An appropriate amount of H2O2 solution was added to the immunomagnetic bead-bacterial complex. The small molecule H2O2 rapidly permeated the cell membrane of Staphylococcus aureus and was decomposed by catalase. After magnetic separation, the remaining H2O2 solution was reacted with HRP and TMB for colorimetric reaction. A UV-Vis spectrophotometer was used to scan the spectrum in the range of 350nm-750nm, thereby achieving rapid visual and quantitative detection of Staphylococcus aureus in solution.

[0107] 1. Optimization of immunomagnetic bead dosage

[0108] Take 100 μL 1×10 5 CFU·mL -1 Staphylococcus aureus bacterial suspensions were added to final concentrations of 0, 0.5, 0.6, 0.7, 0.8, and 0.9 mg·mL, respectively. -1Immunomagnetic beads were used, with each reaction system consisting of 1 mL. The reaction system was suspended at room temperature for 1 hour using a rotary mixer, followed by magnetic separation. The supernatant was then diluted 10-fold, 100-fold, and 1000-fold. 50 μL of the supernatant from each dilution gradient was then added dropwise to disposable bacterial culture dishes containing LA medium (three parallel samples were prepared for each concentration gradient). The spread plate method was used for culture and counting. The enrichment efficiency of different concentrations of immunomagnetic beads for Staphylococcus aureus was calculated to determine the optimal amount of immunomagnetic beads. The enrichment efficiency was calculated using the following formula:

[0109] Enrichment efficiency (%) = (N0 - N) i ) / N0×100%

[0110] Wherein, N0 represents the colony count in the positive control group (immunomagnetic bead concentration is 0), N i This represents the colony count for the remaining concentrations of immunomagnetic beads, expressed in CFU. For example... Figure 5 As shown, (A→F) 0.5, 0.6, 0.7, 0.8, 0.9 mg·mL -1 The enrichment efficiencies of five different concentrations of immunomagnetic beads for the target bacteria were 76.3%, 87.6%, 75.3%, 71.7%, and 66.8%, respectively. Therefore, 0.6 mg / mL -1 This is the optimal dosage for immunomagnetic beads.

[0111] 2. Optimization of enrichment time

[0112] Take 100 μL 1×10 5 CFU·mL -1 Staphylococcus aureus bacterial suspension was added to the optimal amount of immunomagnetic beads (1 mL reaction system), and the suspension was incubated at room temperature for 15 min, 30 min, 45 min, 60 min, and 75 min, respectively. The supernatant was magnetically separated, and the dilution and counting procedures were the same as above. The enrichment efficiency of Staphylococcus aureus at different enrichment times was calculated to determine the optimal enrichment time. 0.6 mg / mL was selected. -1 Immunomagnetic beads optimize enrichment time, such as Figure 6 As shown in (A→F), when the enrichment times are 15 min, 30 min, 45 min, 60 min, and 75 min, the enrichment efficiencies are 60.8%, 67.6%, 70.7%, 91.4%, and 76.6%, respectively. Therefore, the optimal enrichment time is 60 min. Additionally... Figure 7 Image A shows a transmission electron microscope (TEM) image of Staphylococcus aureus. Figure 7 Image B shows a transmission electron microscope image of the target bacteria enriched by immunomagnetic beads, revealing that the target bacteria are adhered to by a large number of immunomagnetic beads.

[0113] 3. Optimization of H2O2 dosage

[0114] Based on the optimized dosage and enrichment time of the immunomagnetic beads, the final concentration was 1×10⁻⁶. 5 CFU·mL -1 Enrichment was performed using the target bacterial culture and PBS solution (blank control), with a reaction volume of 1 mL for each. After washing three times with PBS, 20, 30, 40, 50, and 60 μL of 0.2 mL H₂O₂ solution were added respectively (three parallel samples for each dosage), and the mixture was decomposed for 1 min. The supernatant was then magnetically separated. 100 μL of colorimetric reagent (50 μL of 2 mg·mL⁻¹ TMB solution and 50 μL of 1:400 diluted HRP solution) was added to the supernatant, and the mixture was reacted in the dark for 5 min. The spectrum was scanned in the range of 350 nm–750 nm using a UV-Vis spectrophotometer to determine the peak position. The absorbance difference Δ(A₀-A) between the sample and the blank control under different H₂O₂ dosages was calculated to determine the optimal H₂O₂ dosage. Figure 8 As shown (A0: absorbance when bacterial concentration is 0; A: absorbance when bacterial concentration is 10),... 5 CFU·mL -1 The absorbance difference between the sample and the blank control initially increased and then decreased with increasing H2O2 dosage, reaching its maximum when the H2O2 dosage was 50 μL. Therefore, 50 μL was selected as the optimal H2O2 dosage for obtaining the best visual effect.

[0115] 4. Optimization of H2O2 decomposition reaction time

[0116] Based on the optimized dosage and enrichment time of the immunomagnetic beads, the final concentration was 1×10⁻⁶. 5 CFU·mL -1 Enrichment was performed using the target bacterial culture and PBS solution (blank control), with a reaction volume of 1 mL for each. After washing three times with PBS, the optimized amount of H2O2 was added, and decomposition was carried out for 1, 2, 3, 4, and 5 mins respectively (three parallel samples were set for each decomposition time). The supernatant was magnetically separated, and the chromogenic solution was added. The reaction was carried out in the dark for 5 min, and the absorbance value was measured with a UV-Vis spectrophotometer. The absorbance difference between the sample and the blank control under different H2O2 reaction time conditions was calculated to determine the optimal H2O2 reaction time. Figure 9 As shown (A0: absorbance when bacterial concentration is 0; A: absorbance when bacterial concentration is 10),... 5 CFU·mL -1 The absorbance difference between the sample and the blank control reached its maximum at a reaction time of 2 minutes, after which it decreased. To obtain the best visual effect, 2 minutes was selected as the optimal H2O2 decomposition reaction time.

[0117] 5. Optimization of pH value of the colorimetric solution

[0118] After enriching bacteria and decomposing H2O2 according to the optimized dosage of immunomagnetic beads, enrichment time, H2O2 dosage, and H2O2 decomposition reaction time, the supernatant was magnetically separated and added to colorimetric solutions with pH values ​​of 4.0, 4.5, 5.0, 5.5, and 6.0 (three parallel samples were set up for each pH value). The reaction was carried out in the dark for 5 minutes, and the absorbance was measured using a UV-Vis spectrophotometer. The absorbance difference between the sample and the blank control under different pH conditions was calculated to determine the optimal pH value of the colorimetric solution. Figure 10 As shown (A0: absorbance when bacterial concentration is 0; A: absorbance when bacterial concentration is 10),... 5 CFU·mL -1 The absorbance difference between the sample and the blank control reaches its maximum when the pH of the colorimetric solution is 4.5, and then shows a decreasing trend. Therefore, the optimal pH of the colorimetric solution is 4.5.

[0119] 6. Optimization of TMB solution concentration

[0120] After enriching the target bacteria and decomposing H2O2 according to the optimized dosage of immunomagnetic beads, enrichment time, H2O2 dosage, and H2O2 decomposition reaction time, the supernatant was magnetically separated and then TMB was added at concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 mg·mL. -1 The colorimetric solution (three parallel samples were set up for each TMB solution concentration) was reacted in the dark for 5 min, and the absorbance was measured by a UV-Vis spectrophotometer. The absorbance difference between the samples and the blank control at different TMB concentrations was calculated to determine the optimal TMB solution concentration. Figure 11 As shown (A0: absorbance when bacterial concentration is 0; A: absorbance when bacterial concentration is 10),... 5 CFU·mL -1 The absorbance difference between the sample and the blank control showed a trend of first increasing and then decreasing when the TMB concentration was 2.0 mg·mL⁻¹. -1 The difference is greatest at this time, therefore the optimal concentration of TMB solution is 2.0 mg·mL. -1 .

[0121] 7. Optimization of HRP solution concentration

[0122] After enriching bacteria and decomposing H2O2 according to the optimized dosage of immunomagnetic beads, enrichment time, H2O2 dosage, and H2O2 decomposition reaction time, the supernatant was magnetically separated and HRP solution (original concentration 1 mg·mL⁻¹) was added. -1The colorimetric solutions were diluted 1:100, 1:200, 1:400, 1:800, and 1:1000 (three parallel samples were set up for each HRP solution concentration). The reaction was carried out in the dark for 5 minutes, and the absorbance was measured using a UV-Vis spectrophotometer. The absorbance difference between the samples and the blank control at different HRP concentrations was calculated to determine the optimal HRP solution concentration. Figure 12 As shown (A0: absorbance when bacterial concentration is 0; A: absorbance when bacterial concentration is 10),... 5 CFU·mL -1 The absorbance difference between the sample and the blank control initially increased and then decreased with increasing dilution, reaching its maximum at a dilution of 1:400, at which point the HRP solution concentration was 2.5 μg·mL⁻¹. -1 That is the optimal concentration.

[0123] Example 6: Evaluation of the Visual Detection Method for Staphylococcus aureus

[0124] 1. Determination of the detection limit

[0125] Following the optimized experimental conditions and procedures described above, experiments were conducted on 0 and 10... 3 10 4 10 5 10 6 10 7 CFU·mL -1 Staphylococcus aureus was detected, with three parallel samples for each bacterial concentration. The final product was spectrally scanned using a UV-Vis spectrophotometer to establish a standard curve and determine the detection limit of the method. The results are as follows: Figure 13 As shown in Figure A, the color of the reaction product changes from dark blue to light blue as the bacterial concentration increases. When the bacterial concentration is 10... 3 CFU·mL -1 At that time, the color of the reaction product was significantly lighter than that of the blank control group (bacterial concentration of 0), and the detection result could be determined with the naked eye, meaning the detection limit of this method is 10. 3 CFU·mL -1 Meanwhile, linear fitting showed that the bacterial culture at 10 3 ~10 7 CFU·mL -1 There is a good linear relationship within the concentration range. Figure 13 B), the standard curve is y = -0.256x + 1.864 (R 2 =0.992). Ultraviolet absorption spectrum ( Figure 13 C) shows that the absorption peak of the reaction product is at 654 nm, and gradually decreases with the increase of bacterial concentration.

[0126] 2. Evaluation of accuracy

[0127] Take a concentration of 10 5 CFU·mL- 1 The absorbance of the bacterial culture was measured under optimized experimental conditions and procedures (three parallel samples were set up). The bacterial concentration was calculated based on the standard curve, and the spiked recovery rate and relative standard deviation were calculated to evaluate the accuracy of the method. The calculation formula is as follows:

[0128] Spike recovery rate (%) = (Measured bacterial concentration of spiked sample / Theoretical bacterial concentration of spiked sample) × 100%

[0129] RSD=(S / `χ)

[0130] Where RSD is the relative standard deviation, S is the standard deviation, and χ is the mean.

[0131] According to the established detection methods, 10 5 CFU·mL −1 The absorbance of the bacterial culture was 0.583 at 654 nm, and x = 5.01 was calculated from the standard curve. The recovery rate was 102.26%, RSD was 5.45%, and the relative standard deviation was less than 10%, indicating good accuracy of the method.

[0132] 3. Evaluation of specificity

[0133] Staphylococcus aureus, Escherichia coli O157: H7, Vibrio parahaemolyticus, Shigella boydii, Salmonella, and Listeria monocytogenes strains were selected and cultured at a concentration of 1×10⁻⁶. 6 Fresh bacterial cultures were analyzed using the established detection method. Each bacterial culture, a blank control, and a mixed sample of all cultures were tested (three replicates for each sample) to evaluate the specificity of the method. Results are as follows: Figure 14 As shown in the results (0→7: blank, Escherichia coli O157: H7, Salmonella, Shigella boydii, Vibrio parahaemolyticus, Listeria monocytogenes, Staphylococcus aureus, mixture), compared to the blank control and other bacterial detection results, the absorbance value of Staphylococcus aureus and the reaction product containing the mixed bacterial solution of Staphylococcus aureus showed a significant decrease at 654 nm. This indicates that the established detection method has good specificity and can specifically detect the target bacteria.

[0134] 4. Repeatability evaluation

[0135] Select high, medium, and low (10) 7 10 5 10 3 CFU·mL -1Three concentrations of Staphylococcus aureus bacterial suspensions were tested three times a day at different times according to the established detection method, and the intra-day repeatability of the detection method was calculated. Testing was conducted for three consecutive days according to the established detection method, and the inter-day repeatability of the detection method was calculated. Three parallel samples were set up for each concentration of bacterial suspension. As shown in Table 3, the RSD range of intra-day repeatability for the three concentrations of bacterial suspensions was between 3.59% and 4.94%, and the RSD range of inter-day repeatability was between 5.42% and 8.96%, all of which were below 10%, demonstrating that the established detection method has good repeatability.

[0136]

[0137] Example 7 Detection of Simulated Samples

[0138] Pure milk was purchased from a supermarket to dilute the bacterial culture as a simulated sample, and concentrations of 10 were prepared. 3 10 4 10 5 10 6 10 7 CFU·mL -1 The bacterial suspension was tested under optimized reaction conditions. Three parallel samples were prepared for each bacterial concentration. The final product was spectrally scanned using a UV-Vis spectrophotometer to establish a standard curve simulating the sample and determine the detection limit. Additionally, 10... 5 10 6 10 7 CFU·mL -1 Three target bacterial solutions at different concentrations were used. The bacterial concentrations were calculated based on the simulated sample standard curve, leading to the calculation of spiked recoveries and relative standard deviations, thus evaluating the effectiveness of the method. Visualization results are shown below. Figure 15 As shown in Figure A, with the increase of the target bacteria concentration, the color of the reaction product changes from dark blue to light blue until it becomes almost colorless and transparent. When the bacterial concentration is 10... 3 CFU·mL -1 At that time, the color of the reaction product was significantly lighter than that of the blank control group (bacterial concentration of 0), and the test result could be determined with the naked eye. Figure 15 B shows the target bacteria at 10 3 ~10 7 CFU·mL -1 There is a good linear relationship within the concentration range, and the standard curve is y = -0.369x + 2.690 (R²). 2 =0.987), therefore the detection limit for the simulated sample is 10. 3 CFU·mL -1 Ultraviolet scanning spectrum ( Figure 15C) The absorption peak is located at 654 nm, and the absorbance gradually decreases with increasing bacterial concentration. The spiked recoveries of the target bacteria at three different concentrations were calculated using a standard curve established with a milk simulation sample. The results are shown in Table 4. The spiked recoveries of the three target bacteria concentrations ranged from 96.57% to 104.96%, and the relative standard deviations ranged from 7.28% to 7.78%, all less than 10%. This demonstrates that the method is minimally affected by the complex components of food samples and can be applied to the detection of real samples.

[0139] .

Claims

1. A visualization-based immunomagnetic bead-based reagent kit for the detection of Staphylococcus aureus, comprising: Immunomagnetic beads, solution colorimetric reagent; The immunomagnetic beads are carboxylated Fe3O4 nanoparticles coupled with anti-Staphylococcus aureus chicken egg yolk antibody IgY; The solution colorimetric agents include: TMB solution, HRP solution, and H2O2 solution; The concentration of the immunomagnetic beads was 0.6 mg·mL⁻¹, the concentration of the TMB solution was 2.0 mg·mL⁻¹, and the concentration of the HRP solution was 2.5 μg·mL⁻¹. The volume of H2O2 solution used is 50 μL, the volume of TMB solution used is 50 μL, and the volume of HRP solution used is 50 μL; The carboxylated Fe3O4 nanoparticles were prepared by the following method: 1) 1.08 g FeCl3∙6H2O and 20 mL ethylene glycol were added to a beaker and dissolved by sonication for 10 min; 2) 1.2 g sodium acetate and 0.2 g trisodium citrate were added to 1) and dissolved by sonication for 10 min; 3) 0.2 g PEG-6000 was added to 2) and sonicated until completely dissolved; 4) The above solution 3) was transferred to a reaction vessel and reacted at 198°C for 18 h. After the reaction vessel was cooled to room temperature, the magnetic bead solution was transferred to a small beaker and the magnetic beads were washed three times alternately with water and anhydrous ethanol to obtain carboxylated Fe3O4 nanoparticles. The immunomagnetic beads were prepared by the following method: 1) Take 5 mg of carboxylated Fe3O4 nanoparticles, add 1 mL of PBS, sonicate to resuspend for 1-2 min, magnetically separate, wash the obtained magnetic beads twice with PBS, and then resuspend in 1 mL of PBS; 2) Add 10 mg of EDC·HCl and 5 mg of NHS to 1), suspend for 30 min, magnetically separate, wash 3 times with PBS, and then resuspend in 1 mL of PBS; 3) Add 500 μg of anti-Staphylococcus aureus chicken egg yolk antibody IgY to 2), suspend at room temperature for 2 h, magnetically separate, and wash the obtained magnetic beads 3 times with PBS; 4) Add 1 mL of blocking solution and block for 1 h to obtain functionalized immunomagnetic beads.

2. A method for visually detecting Staphylococcus aureus in food, comprising: 1) Take 100 μL of the sample solution to be tested and add the immunomagnetic beads described in claim 1. The reaction system is 1 mL. The mixture is suspended and enriched at room temperature for 30-75 min. After magnetic separation, the supernatant is removed and the sample is washed with PBS. 2) Add H2O2 solution, decompose for 1-4 minutes, magnetically separate, and take the supernatant; 3) Add a colorimetric solution with a pH of 4.0 to 6.0 to the supernatant in 2). The colorimetric solution includes the HRP solution and TMB solution described in claim 1. React in the dark for 5 minutes and observe the color with the naked eye or measure the absorption light using an ultraviolet spectrophotometer.

3. The method for visually detecting Staphylococcus aureus in food according to claim 2, characterized in that: The enrichment time was 60 min, the H2O2 decomposition reaction time was 2 min, and the pH of the colorimetric solution was 4.5.

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