VBSsPLWat-Fe3O4 composite material, preparation method thereof and application of vBSsPLWat-Fe3O4 composite material in detection of Shigella sonnei

Abstract

The invention provides a vBSsPLW-coated Fe3O4 composite material, a preparation method thereof and application of the vBSsPLW-coated Fe3O4 composite material in detection of Shigella sonnei, and belongs to the technical field of microbiological detection. The preparation method comprises the following steps: activating prepared magnetic Fe3O4, adding a shigella sonnei bacteriophage vBSsPLW solution, mixing and incubating to obtain a vBSsPLW-coated Fe3O4 composite material, and quantitatively detecting bacteria by utilizing the influence of shigella sonnei on the activity of enzyme-like peroxides of the vBSsPLW-coated Fe3O4 composite material, the lowest detection concentration is 12 CFU / mL, the color development time is only 15 minutes, and other non-specific bacteria cannot be recognized. The kit has the advantages of strong specificity, high sensitivity, short time consumption, visualization and the like, and can meet the detection requirements of shigella sonnei in different food matrixes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganism detection, and particularly relates to a vB_SsP_LW@Fe3O4 composite material, a preparation method thereof and application of the vB_SsP_LW@Fe3O4 composite material in detection of Shigella sonnei. BACKGROUND

[0002] Shigella sonnei is a foodborne pathogenic bacteria that can cause bacterial dysentery, and it has no specific transmission medium. Meat products, dairy products and fruit and vegetable products can be infected by Shigella sonnei. When humans are infected with the bacteria, they enter the intestinal tract, adsorb and invade the intestinal membrane, release endotoxins, and thus cause intestinal flora imbalance and change intestinal cell permeability, causing acute self-limiting diseases characterized by hemorrhagic diarrhea, fever and abdominal pain, and even threatening human life and health. Therefore, it is particularly crucial to provide an effective detection method for Shigella sonnei. The existing detection methods for Shigella sonnei are time-consuming, high-cost and complex to operate, and thus it is necessary to develop a detection method with high specificity, high sensitivity and simple operation.

[0003] Bacteriophages are a general term for viruses that infect bacteria, fungi, algae, actinomycetes or spirochetes and other microorganisms, and exist widely. Due to its high specificity and the characteristic of only infecting living cells, it is considered as an ideal tool for detecting pathogenic bacteria. Compared with other biological recognition elements (antibodies, nucleic acids), bacteriophages have the advantages of easy industrialization, high specificity, low cost and high sensitivity. As a new type of biological recognition tool, bacteriophages can be used to construct biosensors and have great potential in ensuring food quality and safety.

[0004] Fe3O4 is a commonly used nanoenzyme, and due to its peroxidase-like activity, it is applied in food, medicine and other fields. Under acidic conditions (pH 3.0-6.5), Fe3O4 exhibits peroxidase-like activity, and not only has excellent optical, electrical and magnetic properties, but also exhibits good peroxidase-like activity, and is considered as one of the most promising nanoenzymes. With the development of modern science and technology, Fe3O4 is no longer limited to traditional application ranges. Based on the peroxidase activity and excellent biocompatibility of Fe3O4, Fe3O4 has been widely used in many frontier fields. Fe3O4 is increasingly widely used in the field of food safety detection, and can be applied to the detection of food nutrients (vitamins, glucose, etc.), foodborne pathogens (Escherichia coli, Salmonella, Staphylococcus aureus, etc.), biological toxins, agricultural and veterinary drug residues and other components.

[0005] Therefore, it is expected to provide a visual detection method of Sonnei Shigella based on phage and nanozyme to solve the problems of long time consumption, poor specificity and inability to visually determine whether it is contaminated for the existing Sonnei Shigella detection method. SUMMARY

[0006] The purpose of the present application is to provide a visual detection method of Sonnei Shigella based on phage and nanozyme.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The present application provides a preparation method of vB_SsP_LW@Fe3O4 composite material, comprising the following steps: adding magnetic Fe3O4 after activation to Sonnei Shigella phage vB_SsP_LW liquid, mixed incubation, to obtain vB_SsP_LW@Fe3O4 composite material.

[0009] Preferably, the preparation method of the magnetic Fe3O4 is: preparing a mixed solution of ferric chloride hexahydrate and ferrous chloride tetrahydrate, then gradually adding ammonia and ascorbic acid during heating, stirring, washing, drying, to obtain magnetic Fe3O4.

[0010] Preferably, the final concentration of ferric chloride hexahydrate in the mixed solution is 0.200-0.250 mol / mL;

[0011] The final concentration of ferrous chloride tetrahydrate is 0.100-0.150 mol / mL;

[0012] The heating condition is: 65-75 DEG C, 20-40 min;

[0013] The volume ratio of the mixed solution to ammonia and ascorbic acid is: 75-85: 8-12: 2-6;

[0014] The concentration of ascorbic acid is 0.080-0.090 mol / mL;

[0015] The stirring condition is: 85-95 DEG C, 0.5-0.5 h.

[0016] Preferably, the activation method is: dissolving the magnetic Fe3O4 in MES buffer solution, then adding EDC HCl solution and NHS solution, and oscillating;

[0017] Wherein, the mass volume ratio of magnetic Fe3O4 and MES buffer solution is 8-12 mg: 1 mL;

[0018] The concentration of the MES buffer solution is 8-12 mmol / L, and the pH is 5.5-6.0;

[0019] The mass-volume ratio of the magnetic Fe3O4 and EDC HCl solution, NHS solution is 8-12 mg: 0.8-12 mL: 0.8-12 mL;

[0020] The concentration of the EDC HCl solution is 18-22 mg / mL;

[0021] The concentration of the NHS solution is 18-22 mg / mL.

[0022] Preferably, the Shigella sonnei bacteriophage vB_SsP_LW is preserved in Guangdong Microbial Culture Collection Center, address: 5th floor, No. 59 building, Guangzhou Xianlie Middle Road 100, Institute of Microbiology, Guangdong Academy of Sciences, preservation date: June 17, 2025, preservation number: GDMCC No: 66526-B1.

[0023] Preferably, the mass-volume ratio of the magnetic Fe3O4 and Shigella sonnei bacteriophage solution is: 8-10 mg: 15-20 mL;

[0024] The titer of the Shigella sonnei bacteriophage solution in the vB_SsP_LW@Fe3O4 composite material is greater than or equal to 1x10 9 CFU / mL.

[0025] The application provides a vB_SsP_LW@Fe3O4 composite material.

[0026] The application provides application of the vB_SsP_LW@Fe3O4 composite material in detection of Shigella sonnei in food.

[0027] The application provides a method for visually detecting Shigella sonnei in food: adding a sample to be detected into the vB_SsP_LW@Fe3O4 composite material, adsorbing, and then sequentially adding acetic acid-sodium acetate buffer, H2O2 solution and TMB solution, incubating in dark, and determining absorbance value.

[0028] Preferably, the volume ratio of the vB_SsP_LW@Fe3O4 composite material to the sample to be detected is: 9-11: 10-12;

[0029] The adsorption time is: 15-25 min;

[0030] The volume ratio of the vB_SsP_LW@Fe3O4 composite material to acetic acid-sodium acetate buffer, H2O2 solution, TMB solution is: 10-12: 14-160: 18-22: 9-11;

[0031] The H2O2 solution is 80-140 mmol / L, the TMB solution concentration is 5-16 mmol / L, the acetic acid-sodium acetate buffer solution has a pH range of 3.0-5.0, and the concentration is 0.1-0.2 mol / L;

[0032] The light-proof incubation time is 15-20 min, and the wavelength condition for detection is 640-660 nm.

[0033] The present application fixes the phage head on the surface of nano-enzyme through amide reaction to obtain a vB_SsP_LW@Fe3O4 composite material, and then uses the influence of Shigella sonnei on the vB_SsP_LW@Fe3O4 composite material enzyme-like peroxide activity to realize quantitative detection of Shigella sonnei. The detection method provided by the present application has a minimum detection concentration of 12 CFU / mL for Shigella sonnei, a color development time of only 15 min, and cannot recognize other non-specific bacteria, has the advantages of strong specificity, high sensitivity, short time consumption, visualization, etc., and can meet the detection requirements of Shigella sonnei in different food matrices. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is the phage plaque morphology of phage vB_SsP_LW;

[0035] Figure 2 It is the phage plaque picture of pure Fe3O4 liquid (a), vB_SsP_LW@Fe3O4 (b), MES buffer solution (c) and phage vB_SsP_LW (d) on a double-layer plate;

[0036] Figure 3 It is the Fourier infrared spectrum of Fe3O4 and vB_SsP_LW@Fe3O4;

[0037] Figure 4 It is the TEM diagram of Fe3O4 (A) and vB_SsP_LW@Fe3O4 (B);

[0038] Figure 5 It is the TEM diagram of vB_SsP_LW@Fe3O4 adsorbing and lysing Shigella sonnei, the scale is 1 mu m, (A) is the adsorption process, and (B) is the lysis process;

[0039] Figure 6 It is the ultraviolet-visible spectrum diagram of four reaction systems;

[0040] Figure 7 It is the optimization result diagram of acetic acid-sodium acetate buffer solution pH (A), H2O2 concentration (B), TMB concentration (C) and adsorption time (D), respectively;

[0041] Figure 8Fig. 1 is a UV-visible spectrum of vB_SsP_LW@Fe3O4+H2O2+TMB with or without Shigella sonnei;

[0042] Figure 9 Fig. 2 is a UV-visible spectrum of vB_SsP_LW@Fe3O4+H2O2+TMB with different concentrations of Shigella sonnei, wherein a is a UV-visible spectrum of vB_SsP_LW@Fe3O4+H2O2+TMB with different concentrations of Shigella sonnei, and b is a linear relationship between absorbance of vB_SsP_LW@Fe3O4+H2O2+TMB at 652 nm and concentration of Shigella sonnei;

[0043] Figure 10 Fig. 3 is a specific result map of vB_SsP_LW@Fe3O4.

[0044] DEPOSIT INFORMATION

[0045] The Shigella sonnei bacteriophage vB_SsP_LW has a taxonomic name of Shigella sonneiphage, is preserved in the Guangdong Microbial Culture Collection Center, located at No. 59, Building 5, Institute of Microbiology, Guangdong Academy of Sciences, 100, Martyrs' Avenue, Guangzhou, and has a preservation number of GDMCC No: 66526-B1 and a preservation date of June 17, 2025. DETAILED DESCRIPTION

[0046] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0047] Example 1: Activation of Shigella sonnei

[0048] The preserved Shigella sonnei (ATCC 29930) was thawed on ice, and 20 μL of the bacterial solution was inoculated on LB solid medium, and then incubated at 37°C for 12 h. The next day, a single colony was inoculated on LB solid medium, and then incubated at 37°C for 12 h. Then, a single colony was inoculated in 10 mL of LB liquid medium, and then incubated for 8 h to obtain 10 8 CFU / mL of bacterial solution, which was stored at 4°C for later use.

[0049] Example 2: Preparation of bacteriophage solution

[0050] The Shigella sonnei bacteriophage vB_SsP_LW was activated as follows: 2 mL of the bacteriophage solution and 200 μL of the Shigella sonnei bacterial solution (OD 600nm ≈0.6) were added to 20 mL of LB liquid medium, and then incubated at 37°C overnight. Then, the solution was centrifuged at 8000 r / min at 4°C for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the Shigella sonnei bacteriophage vB_SsP_LW solution. The morphology of the bacteriophage plaques was observed by the double-layer plate method.

[0051] Results as shown in Figure 1 Songnia Shiga's bacteriophage vB_SsP_LW formed a clear transparent bacteriophage with a diameter of 9.98 mm on a double-layer plate, and the titer of the bacteriophage liquid was >10 9 PFU / mL measured by the double-layer plate method.

[0052] Example 3: Preparation and characterization of vB_SsP_LW@Fe3O4

[0053] 1. Preparation of vB_SsP_LW@Fe3O4

[0054] 1) Add FeCl3·6H2O and FeCl2·4H2O to 80 mL of ultrapure water, the final concentration of FeCl3·6H2O is 0.205 mol / mL, and the final concentration of FeCl2·4H2O is 0.109 mol / mL, ultrasonic for 2 min to dissolve the solid, and mix the mixture with a magnetic stirrer to mix evenly, when the temperature reaches 70℃, continue to stir at this temperature for 30 min, and obtain a mixed solution; 2· 4H2O, the final concentration of FeCl 2· 2·4H2O is 0.109 mol / mL, ultrasonic for 2 min to dissolve the solid, and mix the mixture with a magnetic stirrer to mix evenly, when the temperature reaches 70℃, continue to stir at this temperature for 30 min, and obtain a mixed solution;

[0055] Add 10 mL of ammonia water (NH4OH) to the mixed solution, keep the temperature unchanged, continue to stir for 30 min, then add 4 mL of 0.085 mol / mL ascorbic acid solution, heat to 90℃, and continue to stir for 1 h, and cool to room temperature (22℃), to obtain a black product, wash the product with ultrapure water until the supernatant is clear, and take the black precipitate to dry at 60℃ for 6 h, to obtain magnetic Fe3O4.

[0056] 2) Take 10 mg of magnetic Fe3O4 in 1 mL of MES buffer (10 mmol / L) with pH of 6.0, ultrasonic for 10 min to disperse the magnetic Fe3O4 in the MES buffer, to obtain a magnetic Fe3O4 suspension, add 1 mL of 20 mg / mL EDC HCl (CAS: 25952-53-8) solution and NHS (CAS: 106627-54-7) solution to the magnetic Fe3O4 suspension, and oscillate at room temperature (22℃) for 30 min, add 18 mL of bacteriophage liquid to the solution, and incubate at 37℃ and 120 r / min overnight, to obtain vB_SsP_LW@Fe3O4 composite material.

[0057] 2. Characterization of vB_SsP_LW@Fe3O4

[0058] 1) The spot method was used to verify whether the bacteriophage vB_SsP_LW and the magnetic Fe3O4 were still active after being connected. Take 200 μL of Shigella sonnei bacterial liquid (OD 600nm≈0.6) with 10 mL LB semi-solid medium, poured into the already prepared nutrient agar plates, after it solidified, respectively, 10 μL pure Fe3O4 liquid (a), vB_SsP_LW@Fe3O4 (b), MES buffer (c) and phage vB_SsP_LW (d) were dropped onto the plates, and incubated at 37°C overnight.

[0059] Results are shown in FIG. 1. Figure 2 As shown in FIG. 1, vB_SsP_LW@Fe3O4 (b) was dropped onto the double-layer agar plates, and phage plaques appeared as the same as pure phage vB_SsP_LW (d), while pure Fe3O4 (a) did not appear phage plaques, indicating that vB_SsP_LW@Fe3O4 could still lyse Shigella sonnei. Figure 2 Figure 2 As shown in FIG. 1, vB_SsP_LW@Fe3O4 (b) was dropped onto the double-layer agar plates, and phage plaques appeared as the same as pure phage vB_SsP_LW (d), while pure Fe3O4 (a) did not appear phage plaques, indicating that vB_SsP_LW@Fe3O4 could still lyse Shigella sonnei. Figure 2

[0060] 2) Respectively, 10 mL of magnetic Fe3O4 liquid and vB_SsP_LW@Fe3O4 liquid were taken into plates, pre-frozen at -80°C for 8 h, and freeze-dried for 3 days. The freeze-dried vB_SsP_LW@Fe3O4 liquid and magnetic Fe3O4 were mixed with potassium bromide (KBr) at 1:100 (w / w), and characterized by Fourier infrared spectroscopy.

[0061] Results are shown in FIG. 2. Figure 3 As shown in FIG. 2, vB_SsP_LW@Fe3O4 appeared C=O stretching vibration (amide I band) and N-H bending (amide II band) at 1640-1680 cm -1 and 1540-1580 cm -1 , specifically, the absorption intensity of vB_SsP_LW@Fe3O4 was higher than that of pure Fe3O4 at 1350 cm -1 and 1550 cm -1 . In addition, compared with pure Fe3O4, the absorption intensity of vB_SsP_LW@Fe3O4 at 1600 cm -1 was reduced, which may be because the coupling of vB_SsP_LW with Fe3O4 consumed part of the carboxyl group. In the range of 3300-3500 cm -10 , the absorption intensity of vB_SsP_LW@Fe3O4 was lower than that of pure Fe3O4, which may be caused by the consumption of amino group. The above analysis indirectly verified that vB_SsP_LW was successfully connected with Fe3O4.

[0062] 3) TEM characterization. Respectively, 10 μL of vB_SsP_LW@Fe3O4 liquid and magnetic Fe3O4 were dropped onto copper mesh, and placed at room temperature for 10 min, the excess sample was absorbed with filter paper, 10 μL of 2% phosphotungstic acid was added for negative staining for 3 min, and the sample was dried for 4 h before being observed by transmission electron microscope. Results are shown in FIG. 3.​​Figure 4 As shown, the magnetic Fe3O4 is a circular nanoparticle with a diameter of about 20 nm Figure 4 A). Figure 4 B It can be seen that the phage vB_SsP_LW head is connected with Fe3O4.

[0063] In addition, 110 μL of vB_SsP_LW@Fe3O4 solution was incubated with 100 μL of bacterial solution for 20 min, and then 10 μL of sample was taken for transmission electron microscopy to observe the process of vB_SsP_LW@Fe3O4 adsorbing Shigella sonnei. Figure 5 As shown, vB_SsP_LW@Fe3O4 first exists outside the bacteria, and is attached to the surface of the bacteria by the adsorption of phage tail protein Figure 5 A), and then the phage releases the holin and Rz protein to lyse the bacteria, causing the contents of the bacteria to overflow Figure 5 B).

[0064] Example 4: Peroxidase-like activity of vB_SsP_LW@Fe3O4

[0065] Four reaction systems were constructed using acetic acid-sodium acetate buffer, H2O2 solution, TMB solution, and vB_SsP_LW@Fe3O4, respectively.

[0066] a: 110 μL of vB_SsP_LW@Fe3O4, 200 μL of H2O2 (100 mmol / L), and 100 μL of TMB solution (10 mmol / L dissolved in anhydrous ethanol) were added to 1500 μL of acetic acid-sodium acetate buffer (pH 4.0, 0.2 mol / L);

[0067] b: Different from system a, this system did not add vB_SsP_LW@Fe3O4;

[0068] c: Different from system a, this system did not add H2O2;

[0069] d: Different from system a, this system did not add TMB solution.

[0070] The above systems were incubated in the dark for 15 min, and the spectra of the four reaction systems at 500-800 nm were determined by ultraviolet-visible spectrophotometry.

[0071] Fe3O4 has peroxidase-like activity and can cause TMB to produce a maximum absorption peak at 652 nm. When Fe3O4 is successfully connected with phage vB_SsP_LW, it should theoretically still have peroxidase-like activity. From Figure 6It can be seen that the highest absorption peak is observed at 652 nm only in the vB_SsP_LW@Fe3O4+H2O2+TMB system, and no obvious absorption peak is observed at 652 nm in the other three systems. This means that the presence of vB_SsP_LW@Fe3O4 is necessary to generate the blue substance TMBox in the H2O2+TMB system, indicating that vB_SsP_LW@Fe3O4 has peroxidase-like activity.

[0072] Example 5: Condition optimization

[0073] 1) pH optimization. 0.2 mol / mL acetic acid-sodium acetate buffer was prepared, and the pH was adjusted with acetic acid and sodium acetate (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, respectively). 110 μL vB_SsP_LW@Fe3O4, 200 μL 100 mmol / L H2O2 solution, and 100 μL 10 mmol / L TMB solution (dissolved with anhydrous ethanol) were sequentially added to 1500 μL acetic acid-sodium acetate buffer with different pH values, and the absorbance at 652 nm was measured after 15 min of reaction in the dark.

[0074] The results are shown in Figure 7 As shown in FIG. A, acidic conditions help vB_SsP_LW@Fe3O4 catalyze TMB color development. With the change of pH, the absorbance of the vB_SsP_LW@Fe3O4+H2O2+TMB reaction system at 652 nm first increases and then decreases, and the absorbance is the highest when the pH is 4.0. In addition, it can be seen from the figure that the peroxidase-like activity of vB_SsP_LW@Fe3O4 in acidic environment is better than that in alkaline environment, and the catalytic activity of vB_SsP_LW@Fe3O4 gradually weakens with the weakening of acidity and the strengthening of alkalinity after pH 4.0.

[0075] 2) H2O2 concentration and TMB concentration optimization. Different concentrations (80, 90, 100, 110, 120, 130, 140 mmol / L) of H2O2 solution and different concentrations (5, 6, 8, 10, 12, 14, 16 mmol / L) of TMB (dissolved with anhydrous ethanol) solution were prepared, and 110 μL vB_SsP_LW@Fe3O4 and different concentrations of H2O2 solution and TMB solution were added to 1500 μL acetic acid-sodium acetate (0.2 mmol / L, pH 4.0), and the absorbance at 652 nm was measured after 15 min of reaction in the dark.

[0076] The results are shown in Figure 7 As shown in FIGS. B and 7C, when the concentrations of H2O2 and TMB are 100 mM and 10 mM, respectively, the absorbance of the system at 652 nm is the highest.

[0077] 3) Incubation time optimization. 100 μL bacteria (10 8 CFU / mL) were incubated with 110 μL vB_SsP_LW@Fe3O4 at room temperature for 0, 5, 10, 15, 20, 25, 30 min, and the unbound bacteria were removed by magnetic attraction. Then, 1500 μL acetic acid-sodium acetate buffer (0.2 mmol / L, pH 4.0), 200 μL H2O2 solution (100 mmol / L), and 100 μL TMB solution (10 mmol / L, dissolved in anhydrous ethanol) were sequentially added, and the reaction was allowed to proceed for 15 min. The absorbance was measured at 652 nm.

[0078] The results are shown in Figure D. With the increase of incubation time, the catalytic activity of vB_SsP_LW@Fe3O4 was gradually inhibited, which was manifested by the gradual decrease of the absorbance of the reaction system at 652 nm. Subsequently, the absorbance tended to be stable after 20 min, and thus the optimal incubation time was 20 min. Figure 7

[0079] Example 6: Feasibility of vB_SsP_LW@Fe3O4 in detecting Shigella sonnei

[0080] 110 μL vB_SsP_LW@Fe3O4 was taken to a 2 mL centrifuge tube, and 100 μL Shigella sonnei bacterial solution (10 8 CFU / mL) was added and incubated at room temperature for 15 min. The unbound bacteria were removed by magnetic attraction, and then 1500 μL acetic acid-sodium acetate buffer (0.2 mmol / L, pH 4.0), 200 μL H2O2 solution (100 mmol / L), and 100 μL TMB solution (10 mmol / L, dissolved in anhydrous ethanol) were sequentially added, and the reaction was allowed to proceed for 15 min. The control group did not add the bacterial solution, and the remaining steps were the same. The absorbance was measured at 500-800 nm.

[0081] The results are shown in Figure D. With the increase of incubation time, the catalytic activity of vB_SsP_LW@Fe3O4 was gradually inhibited, which was manifested by the gradual decrease of the absorbance of the reaction system at 652 nm. Subsequently, the absorbance tended to be stable after 20 min, and thus the optimal incubation time was 20 min. Figure 8 As shown in Figure D, the absorbance of the reaction system without Shigella sonnei was about 0.9 at 652 nm, while the absorbance of the reaction system with Shigella sonnei was also peaked at 652 nm (OD 652nm ≈0.4), but was significantly lower than that of the control group. This indicated that vB_SsP_LW@Fe3O4 could be used for the detection of Shigella sonnei.

[0082] Example 7: Standard curve for vB_SsP_LW@Fe3O4 in detecting Shigella sonnei

[0083] Different concentrations of Shigella sonnei bacterial solution (1.3 x 10 8 ~ 1.3 x 10 2 ​CFU / mL, each number of orders of magnitude is a treatment, a total of 7 groups), 100 μL of different concentrations of bacteria solution was incubated with 110 μL of vB_SsP_LW@Fe3O4 for 15 min, after removing the unbound bacteria, 1500 μL of acetic acid-sodium acetate buffer, 200 μL of H2O2 solution, 100 μL of TMB solution (dissolved with absolute ethanol) were added and reacted for 15 min, and the absorbance at 500-800 nm was measured.

[0084] Under the optimal color development conditions and incubation time, the catalytic activity of vB_SsP_LW@Fe3O4 gradually weakened with the increase of the concentration of added bacteria. As shown in Figure 9 , the absorbance of the reaction system (vB_SsP_LW@Fe3O4+H2O2+TMB) at 652 nm gradually decreased Figure 9 a), the absorbance at this time was linearly fitted with the concentration of S. sonnei (10 8 ~ 10 2 CFU / mL), and a linear relationship was found Figure 9 b). The detection limit was calculated to be 12 CFU / mL by the formula LOD=3S / k (S is the standard deviation of the blank sample, and K is the slope of the standard curve).

[0085] Example 8: Specificity of vB_SsP_LW@Fe3O4

[0086] 1) The S. sonnei ATCC 29930, S. flexneri ATCC 29903, S. boydii ATCC 8700, E. coli GDMCC 1.1917, and Salmonella GDMCC 1.5286 previously stored in the laboratory were taken out from -80℃, 20 μL of bacterial solution was inoculated on LB solid medium, and then single colonies were inoculated on LB liquid medium and incubated at 37℃ overnight. The culture was stored at 4℃ for standby.

[0087] 2) 100 μL of S. sonnei (10 8 CFU / mL), S. flexneri (10 8 CFU / mL), S. boydii (10 8 CFU / mL), E. coli (10 8 CFU / mL), and Salmonella (10 8The 110 μL vB_SsP_LW@Fe3O4 was incubated with 100 μL bacteria (7.57×10

[0088] According to Figure 10 The analysis showed that only S. sonnei had a significant inhibitory effect on vB_SsP_LW@Fe3O4, and the absorbance at 652 nm was about 0.3, while the other four bacteria had little effect on the reaction system, and the absorbance at 652 nm was about 0.65, 0.7, 0.65, and 0.8. This depends on the strong specificity of the bacteriophage vB_SsP_LW, which shows that the bacteriophage is highly specific and can only recognize adsorbed S. sonnei.

[0089] Example 9: Detection of actual samples

[0090] The lettuce, milk and chicken breast were treated as actual samples for detection. The lettuce and chicken breast were washed with pure water, drained, chopped, and then 25 g of sample was weighed and added to 225 mL of physiological saline for homogenization. Finally, the filtrate was obtained by filtering with 0.45 μm and 0.22 μm filter membranes in sequence. The bacteria were added to the filtrate and milk to a final concentration of 7.57×10 5 CFU / mL, 7.57×10 4 CFU / mL, 7.57×10 3 CFU / mL, and detected under the optimal conditions of vB_SsP_LW@Fe3O4 (as in Example 8), while the traditional counting method (GB4789.2-2016) was used as a control to calculate the recovery rate and relative standard deviation.

[0091] Table 1

[0092]

[0093] As shown in Table 1, the recovery rate of the reaction system was in the range of 91.87% to 106%, and the relative standard deviation was in the range of 1.4% to 5.6%, which was consistent with the detection results of the traditional detection method, indicating that the reaction system had good application potential.

[0094] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a vB_SsP_LW@Fe3O4 composite material, characterized in that: The following steps are involved: After the magnetic Fe3O4 was activated, the Shigella sonnei bacteriophage vB_SsP_LW solution was added, mixed and incubated to obtain the vB_SsP_LW@Fe3O4 composite material.

2. The method for preparing the composite material according to claim 1, wherein: The preparation method of the magnetic Fe3O4 comprises the following steps: preparing a mixed solution of ferric chloride hexahydrate and ferrous chloride tetrahydrate, gradually adding ammonia water and ascorbic acid during heating, stirring, washing and drying to obtain the magnetic Fe3O4.

3. The method for preparing the composite material according to claim 2, wherein: The final concentration of ferric chloride hexahydrate in the mixed solution is 0.200-0.250 mol / mL; The final concentration of ferrous chloride tetrahydrate is 0.100-0.150 mol / mL; Heating conditions are: 65-75°C, 20-40 min; The volume ratio of the mixed solution to ammonia water and ascorbic acid is 75-85:8-12:2-6, wherein the concentration of ascorbic acid is 0.080-0.090 mol / mL; The stirring conditions are: 85-95°C, 0.5-0.5h.

4. The method for preparing the composite material according to claim 1, wherein: The activation method is as follows: dissolving the magnetic Fe3O4 in MES buffer, adding EDC HCl solution and NHS solution, and shaking; The mass volume ratio of the magnetic Fe3O4 and MES buffer solution is 8-12 mg:1 mL; The concentration of the MES buffer is 8-12 mmol / L, and the pH is 5.5-6.0; The mass volume ratio of the magnetic Fe3O4, EDC HCl solution and NHS solution is 8-12 mg: 0.8-12 mL: 0.8-12 mL; The concentration of the EDC HCl solution is 18-22 mg / mL; The concentration of the NHS solution is 18-22 mg / mL.

5. The method for preparing the composite material according to claim 4, wherein: The Shigella sonnei phage vB_SsP_LW is deposited in the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is June 17, 2025, and the deposit number is GDMCC No: 66526-B1.

6. The method for preparing the composite material according to claim 5, wherein: The mass volume ratio of the magnetic Fe3O4 and Shigella sonnei phage solution is: 8-10 mg: 15-20 mL; The potency of the Shigella sonnei phage solution in the vB_SsP_LW@Fe3O4 composite material is greater than or equal to 1×10 9 CFU / mL.

7. The vB_SsP_LW@Fe3O4 composite material prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the vB_SsP_LW@Fe3O4 composite material according to claim 7 in detecting Shigella sonnei in food.

9. A method for visually detecting Shigella sonnei in food, characterized in that: The following steps are involved: The sample to be tested is added to the vB_SsP_LW@Fe3O4 composite material for adsorption, and then acetic acid-sodium acetate buffer, H2O2 solution and TMB solution are added in sequence, incubated in the dark, and the absorbance value is measured.

10. The method for visually detecting Shigella sonnei according to claim 9, wherein: The volume ratio of the vB_SsP_LW@Fe3O4 composite material to the sample to be tested is: 9-11:10-12; Adsorption time: 15-20 min; The volume ratio of the vB_SsP_LW@Fe3O4 composite material to acetic acid-sodium acetate buffer, H2O2 solution, and TMB solution is: 10-12:14-160:18-22:9-11; The H2O2 solution is 80-140 mmol / L, the TMB solution concentration is 5-16 mmol / L, the pH range of the acetic acid-sodium acetate buffer is 3.0-5.0, and the concentration is 0.1-0.2 mmol / L; The incubation time in the dark is 15 to 20 minutes; The wavelength condition during detection was 640 to 660 nm.

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