Preparation method and application of nano-enzyme for broad-spectrum detection of viable bacteria of staphylococcus aureus through colorimetric method coupled with cocktail of bacteriophage

Abstract

The invention discloses a preparation method and application of a nano-enzyme coupled with phage cocktail and used for detecting staphylococcus aureus viable bacteria by a colorimetric method. The phage cocktail modified oxygenase comprises phage cocktail, polyethyleneimine and Co-Mn oxidase, according to the phage cocktail, the head of a phage is fixed on the surface of Co-Mn oxidase through polyethyleneimine, and the tail of the phage is exposed outside; the phage cocktail is prepared from SapYZU10, SapYZUbeta and SapYZUM13, and the phage cocktail is prepared from the SapYZU10, the SapYZUbeta and the According to the prepared phage cocktail modified oxidoid enzyme, the TMB colorimetric reaction is utilized, and staphylococcus aureus is rapidly, visually and quantitatively analyzed; compared with the previously reported staphylococcus aureus detection method, the method has the advantages of high detection speed, good specificity, high sensitivity, low equipment dependence, simple operation, low cost and wide host range, and is suitable for visual rapid detection of staphylococcus aureus in food samples.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a method for preparing a nanozyme for detecting live Staphylococcus aureus using a colorimetric method coupled with a bacteriophage cocktail and its application. Background Technology

[0002] Staphylococcus aureus is one of the most common foodborne pathogens. It can produce a variety of virulence factors, causing not only food poisoning but also serious infections in any animal. Furthermore, Staphylococcus aureus can rapidly acquire resistance and virulence genes through horizontal gene transfer of moving genetic elements. Therefore, Staphylococcus aureus food poisoning has become a significant public health problem in many countries. Thus, rapid, specific, accurate, and timely detection of Staphylococcus aureus in food is crucial for ensuring food safety.

[0003] Traditional bacterial detection methods, while reliable, are labor-intensive and time-consuming. In contrast, some rapid detection methods, such as immunological assays (ELISA) and nucleic acid-based methods (PCR), can be completed in just a few hours. However, these techniques are expensive, require complex sample preparation procedures, and necessitate high-tech laboratory setups and skilled analysts. Furthermore, these methods cannot distinguish between live and dead bacterial cells.

[0004] Bacteriophages are a widely distributed class of viruses that infect host microorganisms. They can specifically recognize bacterial hosts and have advantages such as low cost, ease of preparation, and strong environmental tolerance. In recent years, some detection technologies using single bacteriophages as bacterial recognition agents have been reported. These technologies have been widely used in the detection of Staphylococcus aureus and have the ability to distinguish between dead and live bacteria. However, existing detection methods based on single bacteriophages are prone to false positives due to the limited host spectrum of a single bacteriophage, which greatly limits the application of bacteriophage detection technology in actual food samples. Therefore, the development of a bacteriophage cocktail detection method can reduce the risk of false negatives, overcome interference, and rapidly, simply, specifically, and sensitively detect Staphylococcus aureus. However, at present, most bacteriophage cocktail research and development focuses on the field of bacterial therapy. In terms of detection applications, it has only been used for the detection of Klebsiella pneumoniae, and bacteriophage cocktails have not yet been reported for the detection of Staphylococcus aureus. Therefore, focusing on the research and development of phage cocktail detection technology is of great significance for achieving broad-spectrum detection of Staphylococcus aureus. Summary of the Invention

[0005] Objective of the Invention: To address the problems existing in the prior art, this invention provides a phage cocktail-modified oxidase sensor for detecting Staphylococcus aureus. This method differs from existing detection methods based on a single phage (virulent or temperate phage), significantly increasing the detection range while solving the problems of labor-intensive, time-consuming, expensive, highly dependent on equipment, complex operation, and inability to distinguish between live and dead phages in existing technologies.

[0006] This method addresses the problem of SA bacteria, and its high specificity, broad detection range, and reduced risk of false negatives make it suitable for the visual and rapid detection requirements of Staphylococcus aureus in food samples, with the potential for practical application.

[0007] The present invention also provides a method for preparing and applying the phage cocktail modified oxidase for detecting Staphylococcus aureus.

[0008] Technical solution: To achieve the above objective, the present invention provides a nanozyme coupled with a phage cocktail, comprising a phage cocktail, polyethyleneimine, and Co-Mn. The phage cocktail fixes the phage head to the Co-Mn surface through polyethyleneimine, while the phage tail is exposed. The phages include SapYZU10, SapYZUbeta, and SapYZUM13. The three phages are mixed in equal volumes to prepare the phage cocktail.

[0009] The phage cocktail includes SapYZU10, SapYZUbeta, and SapYZUM13. Based on the differences in the host spectrum of the phages for 91 strains of Staphylococcus aureus, the three phages are mixed in equal volumes to prepare the phage cocktail, so as to achieve the detection of all 91 strains of Staphylococcus aureus.

[0010] The phage cocktail consists of phages SapYZU10, SapYZUbeta, and SapYZUM13, deposited at the China Center for Type Culture Collection (CCTCC). The accession number for SapYZUbeta is CCTCC NO: M2022629.

[0011] The method for preparing the nanozyme coupled with a bacteriophage cocktail according to the present invention includes the following steps:

[0012] Step S1: Prepare a phage cocktail;

[0013] Step S2: Prepare PEI@Co-Mn solution;

[0014] Step S3: Mix the phage cocktail obtained in step S1 with the PEI@Co-Mn solution obtained in step S2 and incubate to obtain the phage cocktail@PEI@Co-Mn solution.

[0015] Specifically, step S1 is as follows:

[0016] Step S1-1: Add different phage suspensions (SapYZU10, SapYZUbeta, SapYZUM13 and Staphylococcus aureus) to different sterile centrifuge tubes containing LB semi-solid medium, respectively, and immediately pour them onto the bottom layer of LB solid medium to make double-layer plates and incubate overnight.

[0017] Step S1-2: Pick a single phage plaque from the double-layer plate in step S1-1 and inoculate it into a Staphylococcus aureus suspension. Incubate overnight, then centrifuge and filter to obtain different phage suspensions. Mix the different phage suspensions in equal volumes to prepare a phage cocktail.

[0018] Preferably, step S1 is as follows:

[0019] Step S1-1: Add 100 μL each of phage fluid (SapYZU10, SapYZUbeta, SapYZUM13) and Staphylococcus aureus YZUstau28 suspension to three sterile centrifuge tubes containing 5 mL of LB semi-solid medium. Immediately pour the suspension onto the bottom layer of LB solid medium to make a double-layer plate and incubate overnight at 37°C.

[0020] Step S1-2: Pick individual phage plaques from the double-layer plate in Step S1-1 and place them into LB tubes containing 100 μL of Staphylococcus aureus YZUstau28 bacterial suspension. Incubate overnight at 37°C and 120 rpm, then centrifuge and filter to obtain phage suspensions. Mix the three phage suspensions in a 1:1:1 volume ratio to prepare a phage cocktail.

[0021] Specifically, step S2 is as follows:

[0022] Step S2-1: Add KOH to the ethanol solution and stir to form a homogeneous solution;

[0023] Step S2-2: Add CoCl2·6H2O, MnCl2·4H2O and H2O under vigorous stirring, sonicate the mixed solution and stir until dissolved;

[0024] Step S2-3: The product from step S2-2 is centrifuged to collect the precipitate, washed, and then dried;

[0025] Step S2-4: Disperse the powder from step S2-3 with deionized water, add polyethyleneimine and stir, then wash to obtain a PEI@Co-Mn solution.

[0026] In step S2-2, the mixture is ultrasonically treated and stirred; in step S2-4, the molecular weight of the polyethyleneimine is 10000.

[0027] Specifically, step S3 involves mixing PEI@Co-Mn and phage cocktail at a volume ratio of 1:4-5 and culturing them to prepare a phage cocktail@PEI@Co-Mn solution.

[0028] Preferably, step S2 is as follows:

[0029] Step S2-1: Add KOH to the ethanol solution and stir vigorously to form a homogeneous solution;

[0030] Step S2-2: Add CoCl2·6H2O, MnCl2·4H2O and H2O under vigorous stirring, sonicate the mixed solution and stir until dissolved;

[0031] Step S2-3: The product from step S2-2 is centrifuged to collect the precipitate, washed, and then dried at 110℃;

[0032] Step S2-4: Disperse the powder from step S2-3 with deionized water, add polyethyleneimine and stir, then wash to obtain a PEI@Co-Mn solution.

[0033] In step S2-2, the mixture is ultrasonically treated and stirred until completely dissolved.

[0034] Preferably, in step S2-2, the mixture is ultrasonically treated for 60 min; in step S2-3, the product after centrifugation is washed three times with deionized water.

[0035] In step S2-4, the molecular weight of the polyethyleneimine is 10,000-20,000, preferably 10,000.

[0036] Preferably, step S2-1 involves adding 2.24g of KOH to 50mL of ethanol solution and stirring vigorously to form a homogeneous solution.

[0037] Step S2-2: Add 4.76g CoCl2·6H2O, 1.98g MnCl2·4H2O and 5mL H2O under vigorous stirring. Sonicate the mixed solution for 60min and stir until completely dissolved.

[0038] Preferably, 4.76g CoCl2·6H2O, 3.96g MnCl2·4H2O and 5mL H2O are added under vigorous stirring, the mixed solution is sonicated for 60min and stirred until completely dissolved; Step S2-3: The product in step S2-2 is centrifuged to collect the precipitate, washed 3 times with deionized water and dried at 110℃.

[0039] Step S2-4: Dissolve the powder obtained in step S2-3 in 20 mL of deionized water, add 1 g of polyethyleneimine and stir for 0.5 h, wash 6 times with anhydrous ethanol and deionized water to obtain PEI@Co-Mn solution.

[0040] Preferably, step S3 is as follows: Take 1 part of PEI@Co-Mn solution and 3 parts of phage suspension by volume and mix them. Vortex for 30 seconds and incubate at 37℃ and 120r / min for 6 hours to prepare phage cocktail@PEI@Co-Mn solution.

[0041] The concentration of each phage suspension constituting the phage cocktail is not less than 10. 8 PFU / mL.

[0042] The present invention relates to the application of nanozymes coupled with bacteriophage cocktails in the highly efficient, specific, and visual colorimetric detection of live Staphylococcus aureus.

[0043] The application process includes the following steps:

[0044] 1) Add phage cocktail modified oxidase solution to the buffer solution;

[0045] 2) Add the sample to be tested, wait for the bacteria and bacteriophages in the solution to be fully adsorbed, add TMB solution, wait for the color reaction, observe the color with the naked eye or measure the absorption light with an ultraviolet spectrophotometer.

[0046] Preferably, the application process includes the following steps:

[0047] 1) Place the HAc-NaAc buffer into the centrifuge tubes of the experimental group and the blank control group respectively, and then add the phage cocktail modified oxidase solution to each.

[0048] 2) Add the sample to be tested to the centrifuge tube of the experimental group in 1), wait for the bacteria and bacteriophages in the solution to be fully adsorbed, add TMB solution, wait for the color reaction, observe the color with the naked eye or measure the absorption light with an ultraviolet spectrophotometer.

[0049] The TMB solution concentration is 0.1-50 mM, and the pH range of the HAc-NaAc buffer is 3.0-9.0.

[0050] Preferably, the TMB solution concentration is 2.5 mM, the HAc-NaAc buffer pH is 4.0, the adsorption time is 10 min, and the colorimetric reaction time is 15 min.

[0051] Preferably, the TMB solution concentration is 2.5 mM, the HAc-NaAc buffer pH is 4.0, the adsorption time is 10 min, and the colorimetric reaction time is 7 min.

[0052] This invention provides a colorimetric analysis method for Staphylococcus aureus based on phage-modified oxidase. Through host spectrum determination, three phages with broad host spectra were screened from 18 Staphylococcus aureus strains, and their combination could recognize all 91 Staphylococcus aureus strains from food sources such as pork, beef, and mutton. Subsequently, this phage cocktail was directionally immobilized on the surface of Co-Mn nanoparticles to prepare the phage-modified oxidase phage cocktail@Co-Mn. The test solution and the phagecocktail@Co-Mn solution were thoroughly mixed in buffer solution, and then 100 μL of TMB solution was added. The solution color was detected, and the Staphylococcus aureus concentration was calculated.

[0053] This invention prepares a phage cocktail-based nanozyme biosensor (phage cocktail@Co-Mn). This phage cocktail is immobilized on the surface of the Co-Mn nanozyme through electrostatic interactions. The resulting phage cocktail@Co-Mn exhibits oxidase-like activity, catalyzing a colorimetric reaction of TMB to generate blue oxidized TMB (TMBox). Upon addition of Staphylococcus aureus, the phage cocktail selectively inhibits the oxidase-like activity of phage cocktail@Co-Mn through specific biological interactions with Staphylococcus aureus.

[0054] This invention introduces a positive charge onto the surface of phage cocktail@Co-Mn using polyethyleneimine (PEI), where the phage capsid protein provides a negative charge and interacts electrostatically with the positively charged substrate, while its tail fibers remain free for bacterial capture. The phage cocktail-based nanozyme biosensor prepared by this invention offers advantages such as reduced false negative risk, high specificity, broad detection range, low device dependence, simple operation, and low cost.

[0055] Furthermore, the phage cocktail-based nanoenzyme biosensor (phage cocktail@Co-Mn) prepared in this invention can distinguish between live and dead Staphylococcus aureus (SA) bacteria; it exhibits good selectivity and anti-interference properties. This invention is the first to propose immobilizing a phage cocktail on the surface of Co-Mn nanomaterials for the specific detection of SA bacteria; and it solves the problems of false negative results, labor-intensive and time-consuming processes, high costs, and the inability to distinguish between live and dead SA bacteria in existing technologies.

[0056] The detection performance of the phage cocktail-based nanozyme biosensor (phage cocktail@Co-Mn) prepared in this invention was compared with that of three other single phage nanozyme biosensors (SapYZU10@Co-Mn, SapYZUbeta@Co-Mn, and SapYZUM13@Co-Mn). This comparison demonstrates that the present invention has a wide detection range, solves the problem of limited detection performance caused by the diversity of bacterial strains and the narrow range of phage hosts in phage detection, improves the accuracy and effectiveness of detection, and provides useful information for the expansion of precise phage detection.

[0057] The detection performance of the phage cocktail-based nanozyme biosensor (phage cocktail@Co-Mn) prepared in this invention was compared with that of three other single-phage nanozyme biosensors (SapYZU10@Co-Mn, SapYZUbeta@Co-Mn, SapYZUM13@Co-Mn), or nanozyme biosensors with any two phage combinations, or other combinations of three phages. This demonstrates that the detection range of this invention is significantly wider, proving the uniqueness of the phage combination. It solves the problem of limited detection performance caused by the diversity of bacterial strains and the narrow host range of phages in phage detection, improving the accuracy and effectiveness of detection, and providing useful information for the expansion of precise phage detection.

[0058] This invention is the first to propose a phage cocktail colorimetric method for detecting Staphylococcus aureus, which offers a wider detection range than conventional single-phage nanozymes. Through EOP and adsorption rate experiments, three complementary phage strains for detecting 91 Staphylococcus aureus strains were screened, enabling comprehensive detection of these strains.

[0059] In this invention, three phages with complementary detection ranges form a phage cocktail, resulting in a wider detection range. For example... Figure 11 As shown, the detection efficiency of SapYZU10@Co-Mn was 70.33% (64 / 91); the detection efficiency of SapYZUbeta@Co-Mn was 76.92% (70 / 91); the detection efficiency of SapYZUM13@Co-Mn was 74.73% (68 / 91); while the detection efficiency of the combination phagecocktail@Co-Mn was 100% (91 / 91). These results indicate that phage cocktails have a wider detection range than single phages.

[0060] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0061] The phage cocktail-modified oxidase (phage cocktail@Co-Mn solution) provided by this invention exhibits high specificity and good stability, and the phages within it demonstrate excellent activity. The detection method for this phage-modified oxidase described in this invention enables rapid colorimetric detection of Staphylococcus aureus in samples, with a minimum detection concentration of 1.05 × 10⁻⁶. 2 CFU / mL. Furthermore, the detection materials and methods of this invention can avoid interference from Staphylococcus epidermidis, Staphylococcus xylosus, Staphylococcus albus, Cronobacter sakazakii, Salmonella enteritidis, Vibrio parahaemolyticus, Shigella flexneri, Vibrio influenzae, Cronobacter sakazakii, Bacillus cereus, Yersinia enterocolitica, enteropathogenic Escherichia coli, and mixtures thereof, exhibiting strong specificity for Staphylococcus aureus and a broad recognition range. Therefore, the phage-modified oxidase colorimetric detection of Staphylococcus aureus proposed in this invention has advantages such as reduced false negative risk, high specificity, broad detection range, low equipment dependence, simple operation, and low cost, making it suitable for the visual and rapid detection requirements of Staphylococcus aureus in food samples. Attached Figure Description

[0062] Figure 1 Plaque morphology of the three bacteriophages that make up the bacteriophage cocktail.

[0063] Figure 2 Image A is an XPS image of Co-Mn nanozyme; image B is an EDS image of Co-Mn nanozyme.

[0064] Figure 3 Image A shows TEM images of the three bacteriophages that make up the phage cocktail; image B shows a TEM image of phagecocktail@Co-Mn; and image C shows laser confocal microscopy images of phage cocktail@Co-Mn, Staphylococcus aureus, and the complex of phagecocktail@Co-Mn and Staphylococcus aureus.

[0065] Figure 4 Images of phage cocktail, Phage cocktail@Co-Mn, and PEI@Co-Mn specks on Staphylococcus aureus bilayer plates.

[0066] Figure 5 Image A shows the full UV-Vis spectra of the three reaction systems; image B shows the EPR spectrum of free radical capture and the catalytic mechanism of the oxidase-like Phage cocktail@Co-Mn.

[0067] Figure 6In the image, A represents the optimization of the buffer pH value in the detection conditions; B represents the optimization of the amount of Phage cocktail@Co-Mn in the detection conditions.

[0068] Figure 7 The UV-Vis full spectrum of the prepared Phage cocktail@Co-Mn+TMB colorimetric system with or without Staphylococcus aureus.

[0069] Figure 8 In Figure A, the effect of incubation time of Phage cocktail@Co-Mn with Staphylococcus aureus on the colorimetric reaction is shown; in Figure B, the stability of Phage cocktail@Co-Mn at room temperature for one month is shown; in Figure C, the effect of reaction time on the colorimetric system with or without Staphylococcus aureus is shown; and in Figure D, the ratio of absorbance at 652 nm of the colorimetric system with or without Staphylococcus aureus to the reaction time is shown.

[0070] Figure 9 In Figure A, the UV-Vis full spectrum of the Phage cocktail@Co-Mn+TMB colorimetric system under different Staphylococcus aureus concentrations is shown; in Figure B, the linear fit between the absorbance of the colorimetric system at 652 nm and the logarithm of the Staphylococcus aureus concentration is shown; in Figure C, the selectivity of the Phage cocktail@Co-Mn+TMB colorimetric system is shown; and in Figure D, the anti-interference ability of the Phage cocktail@Co-Mn+TMB colorimetric system is shown.

[0071] Figure 10 In Figure A, the results of the coating of inactivated and highly active Staphylococcus aureus are shown; in Figure B, the results of the Phage cocktail@Co-Mn detection system for the detection of inactivated and highly active Staphylococcus aureus are shown.

[0072] Figure 11 The detection results of 91 Staphylococcus aureus strains using the Phage cocktail@Co-Mn detection system prepared for three bacteriophages (SapYZU10, SapYZUbeta, and SapYZUM13).

[0073] Figure 12 The detection results of 91 Staphylococcus aureus strains using a detection system prepared by combining three bacteriophages (SapYZU10, SapYZUbeta, and SapYZUM13) in pairs.

[0074] Figure 13 The detection results of 91 Staphylococcus aureus strains using the detection system prepared for other phage cocktails.

[0075] Figure 14The images show the characterization of Co-Mn nanozymes and the results of phage immobilization efficiency measurements. In the images, A is a scanning electron microscope image, B is a transmission electron microscope image, C is a Brunauer-Emmett-Teller image, D and E show the length and width of Co-Mn nanozymes, F shows the phage immobilization efficiency measurement results, and G and H show the increase in surface area and porous structure of the Co-Mn nanozyme material.

[0076] Figure 15 In Figure A, the effect of incubation time of Phage cocktail@Co-Mn with Staphylococcus aureus on the colorimetric reaction (using 3.96 g MnCl2·4H2O); in Figure B, the stability of Phage cocktail@Co-Mn at room temperature for one month (using 3.96 g MnCl2·4H2O); in Figure C, the change in reaction time over time for the colorimetric system with and without Staphylococcus aureus (using 3.96 g MnCl2·4H2O); and in Figure D, the ratio of absorbance at 652 nm of the colorimetric system with and without Staphylococcus aureus to the reaction time.

[0077] Figure 16 In Figure A, the UV-Vis full spectrum of the Phage cocktail@Co-Mn+TMB colorimetric system at different Staphylococcus aureus concentrations (using 3.96g MnCl2·4H2O); in Figure B, the linear fit between the absorbance of the colorimetric system at 652nm and the logarithm of the Staphylococcus aureus concentration (using 3.96g MnCl2·4H2O); in Figure C, the selectivity of the Phage cocktail@Co-Mn+TMB colorimetric system (using 3.96g MnCl2·4H2O); and in Figure D, the anti-interference ability of the Phage cocktail@Co-Mn+TMB colorimetric system (using 3.96g MnCl2·4H2O). Detailed Implementation

[0078] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0079] The strains and bacteriophages used in this invention were isolated and screened by Yangzhou University or purchased commercially, and their sources, genome sequences, and other information have been disclosed.

[0080] Staphylococcus aureus YZUstau28, SapYZU10, SapYZUbeta, and SapYZUM13 were all provided by Yangzhou University.

[0081] SapYZUbeta is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2022629. It has been published in the applicant's earlier application CN116064413A.

[0082] SapYZU10 is a publicly disclosed bacteriophage. For details, please refer to the literature Isolation and characterization of abroad-spectrum phage SapYZU11 and its potential application for biological control of Staphylococcus aureus; Quality Assurance and Safety of Crops & Foods, 2023; 15(2):32–48.

[0083] SapYZUM13 is a publicly disclosed bacteriophage. For details, please refer to the literature: Characterisation of a colourimetric biosensor SapYZUM13@Mn3O4-NH2 reveals the mechanisms underlying its rapid and sensitive detection of viable Staphylococcus aureus in food, Food Chemistry 457 (2024) 140189.

[0084] The Staphylococcus aureus YZUstau28 used in this invention can be found in NCBI JBHESL000000000.

[0085] SapYZUS7 has published phage, please see the literature for details: High-sensitivity and high-specificity colorimetric detection of viable Staphylococcus aureus in ready-to-eat foods using a temperate-bacteriophage-based system with peroxidase-like activity, Sensors & Actuators: B. Chemical 399 (2024) 134810.

[0086] SapYZUS8 has been publicly disclosed as a bacteriophage. For details, please refer to the literature Nanozyme colourimetry based on temperatebacteriophage for rapid and sensitive detection of Staphylococcus aureus infood matrices, International Journal of Food Microbiology 416(2024)110657.

[0087] SapYZU1 has been publicly disclosed as a bacteriophage. For details, please refer to the literature: Isolation, identification, antibacterial activity and application of SapYZU15, a broad-spectrum lytic Staphylococcus aureus bacteriophage, Yangzhou University.

[0088] The Staphylococcus aureus strains used in this invention, YZUstau28 (JBHESL000000000), YZUstau12 (JBHERW000000000), YZUstau28 (JBHESL000000000), YZUstau32 (JBHESP000000000), YZUstau81 (JBHEUL000000000), and YZUstau89 (JBHEUT000000000), were all provided by Yangzhou University and can be found on NCBI.

[0089] Example 1

[0090] Culture of Staphylococcus aureus

[0091] The following strains were collected and stored (at -20°C): *Staphylococcus aureus* YZUstau1, YZUstau2, YZUstau4–YZUstau21, YZUstau23–YZUstau74, YZUstau76–YZUstau94; *Staphylococcus xylose* ATCC 29971 and *Staphylococcus albus* ATCC 8032 purchased from the CICC Culture Collection Center; and *Staphylococcus epidermidis* CMCC 26069, *Cronobacter sakazakii* CICC 21569, *Salmonella enteritidis* CICC 21513, *Vibrio parahaemolyticus* CICC 21617, *Shigella flexneri* CICC 21534, *Vibrio influenzae* CICC 21612, *Cronobacter sakazakii* CICC 21545, *Bacillus cereus* CICC 21261, and *Yersinia enterocolitica* CICC. 21669. Enteropathogenic Escherichia coli CICC10664, after thawing at 4°C, was streaked with a 10 μL loop and cultured at 37°C for 36 h. Single colonies were then picked and inoculated into 5 mL of LB broth and cultured at 37°C and 120 rpm for 24 h in a shaker. To ensure good bacterial viability, the obtained bacterial suspension was transferred under the same procedure and cultured for 24 h. The suspension was centrifuged at 4°C and 8,000 rpm for 10 min, and the supernatant was discarded to obtain a bacterial pellet. The bacterial pellet was resuspended in 1 mL of sterile physiological saline (0.85% NaCl) and centrifuged at 4°C and 8,000 rpm for 10 min to wash away residual culture medium. This process was repeated twice. The resulting bacterial suspension was serially diluted with sterile physiological saline to obtain a concentration of 10. 1 -10 9 CFU / mL of Staphylococcus aureus suspensions of YZUstau12, YZUstau28, YZUstau32, YZUstau81, and YZUstau89.

[0092] Example 2

[0093] Preparation of phage cocktail

[0094] The specific procedure is as follows: Add 10 titers of anti-Staphylococcus aureus YZUstau 28 to three sterile centrifuge tubes containing 5 mL of LB semi-solid culture medium. 8100 μL each of three PFU / mL phage suspensions (SapYZU10, SapYZUbeta, SapYZUM13) and a suspension of Staphylococcus aureus YZUstau28 cultured to the logarithmic phase were immediately poured onto the bottom layer of LB solid medium to prepare double-layer plates and incubated overnight at 37°C. Phage plaques from the double-layer plates were picked into LB tubes containing 100 μL of Staphylococcus aureus YZUstau28 suspension in the logarithmic phase and incubated overnight at 37°C and 120 rpm. Then, the tubes were centrifuged and filtered to obtain three phage suspensions (SapYZU10, SapYZUbeta, SapYZUM13).

[0095] The phage plaques formed were counted using the traditional counting method. The results showed that the titers of the suspensions of the three phage strains (SapYZU10, SapYZUbeta, and SapYZUM13) against strain YZUstau28 reached approximately 10. 8 PFU / mL or higher, and the phage plaques formed on the plate are bright, clear, and uniform in size (e.g., Figure 1 (As shown). Three tubes of phage suspension were mixed in a volume ratio of 1:1:1 to prepare a phage cocktail.

[0096] Example 3

[0097] 1. Preparation and characterization of Co-Mn and phage cocktail@Co-Mn

[0098] The specific procedures for preparing Phage cocktail@Co-Mn are as follows: 2.24 g of KOH was added to 50 mL of ethanol solution and stirred vigorously to form a homogeneous solution. Then, under vigorous stirring, 4.76 g of CoCl₂·6H₂O, 1.98 g of MnCl₂·4H₂O, and 5 mL of H₂O were added. The mixture was then sonicated and stirred several times alternately. After reacting for 60 min, the solution was centrifuged and repeatedly washed with water to completely remove Cl₂ from the product. - Finally, the obtained product was dried at 110℃; the dried powder was dissolved in 20mL of deionized water, 1g of polyethyleneimine (molecular weight 10000) was added and stirred for 0.5h, and washed 6 times with anhydrous ethanol and deionized water to obtain PEI@Co-Mn solution. The prepared phage cocktail suspension was mixed with the PEI@Co-Mn solution at a volume ratio of 3:1, vortexed for 30s, and incubated at 37℃ and 120r / min for 6h to prepare Phage cocktail@PEI@Co-Mn. A portion of the solution was then freeze-dried to obtain solid powder (referred to as "Phage cocktail@Co-Mn").

[0099] 2. Characterization of Co-Mn

[0100] The synthesized Co-Mn was characterized using XPS and EDS. Figure 2 As shown in Figure A, in the XPS spectrum, the peaks of C 1s, O 1s, Mn 2p, and Co 2p are located at 285.08, 531.08, 641.08, and 781.08 eV, respectively. The two peaks of Co 2p3 / 2 at 780.8 eV and 796.5 eV originate from Co. 3+ The two spin orbitals, and two other peaks at 782.1 and 797.9 eV, were labeled as Co. 2+ In the spectrum of Mn 2p, the two peaks at 641.6 eV and 653.2 eV of Mn 2p3 / 2 are related to those of Mn. 2+ Matching; the peaks at 644.2 eV for Mn 2p3 / 2 and 656.1 eV for Mn 2p1 / 2 belong to Mn. 3+ The results showed that Co in Co-Mn nanozymes 2+ / Co 3+ and Mn 2+ / Mn 3+ The redox electron pairs coexist. In the EDX spectrum ( Figure 2 B) Mn and Co are evenly distributed. These results indicate that Co-Mn oxidase was successfully synthesized.

[0101] 3. Characterization of Phage cocktail@Co-Mn

[0102] The micromorphology of three bacteriophages and Phage cocktail@Co-Mn was observed using transmission electron microscopy (TEM). SapYZU10 and SapYZUbeta both exhibited typical morphological characteristics of Podoviridae bacteriophages, with polyhedral heads and short tails. SapYZUM13 is a member of the Siphoviridae bacteriophage family (…). Figure 3 A); When the phage cocktail was incubated with PEI@Co-Mn, the heads of all three phage strains adhered tightly to the Co-Mn surface, with their tails facing outwards, and maintained their complete biological structure. Figure 3 B). PEI@Co-Mn and Phage cocktail@Co-Mn were labeled using the highly sensitive DNA fluorescent dye SYBR. The specific procedure was as follows: Phage concentrate (approximately 10...) 11A mixture of PFU / mL and PEI@Co-Mn diluted 100 times was incubated at 37℃ for 6 h. 200 μL of the mixture was transferred to a 1.5 mL centrifuge tube, and 20 μL of 60x SYBR solution (final concentration 6x) was added in the dark. The mixture was stained for 20 min, washed 4-8 times with deionized water, and then redissolved in 100 μL of deionized water. A suitable amount of the solution was added to a glass slide and observed under a confocal microscope. The results are as follows: Figure 3 As shown in Figure C, no green fluorescence was observed in PEI@Co-Mn. In contrast, the green fluorescent spots in the Phage cocktail@Co-Mn image were evenly distributed, indicating the presence of SapYZUgamma on Phagecocktail@Co-Mn. The dot matrix assay was used to verify the phage activity in the Phage cocktail@Co-Mn solution. The specific procedure was as follows: 10 mL of heated LB solid medium was spread evenly in a Petri dish and placed in a sterile operating table to solidify. 100 μL of Staphylococcus aureus YZUstau28 suspension cultured to the logarithmic growth phase was thoroughly mixed with 5 mL of LB semi-solid medium and spread evenly in the solidified Petri dish. 10 μL of PEI@Co-Mn solution, Phagecocktail@Co-Mn solution, and phage cocktail suspension were respectively dropped into the above double-layer plates and incubated overnight at 37°C. The results are shown below. Figure 4 As shown, phage cocktails and phage cocktails showed phage plaques with similar transparency in the areas where Phage cocktail@Co-Mn solution and phage cocktail were added, while no phage plaques appeared in the PEI@Co-Mn solution area. This indicates that the phage cocktail in Phage cocktail@Co-Mn still has biological activity.

[0103] These results indicate that the phage cocktail was successfully immobilized on the PEI@Co-Mn surface, and that Phagecocktail@Co-Mn retained its biological activity.

[0104] Example 4

[0105] Phage cocktail@Co-Mn oxidase activity

[0106] TMB (3,3',5,5'-tetramethylbenzidine) was used as a chromogenic substrate to study the oxidase-like activity of Phagecocktail@Co-Mn via a colorimetric reaction. The specific procedure was as follows: Nine 5 mL centrifuge tubes were divided into three groups (a, b, and c; n=3). 2800 μL, 2700 μL, and 2700 μL of HAc-NaAc buffer (pH 4, 0.2 M) were added to groups a, b, and c, respectively. Then, 100 μL of the Phagecocktail@Co-Mn solution prepared in Example 2 was added to groups a and b, and 100 μL of TMB solution (10 mM, dissolved in ethanol) was added to groups a and c. The reaction was carried out at room temperature for 15 min, and the results were detected using a UV-Vis spectrophotometer. Figure 5 As shown in Figure A, the Phage cocktail@Co-Mn+TMB reaction system exhibits a large absorption peak at 652 nm, which represents the oxidized TMB (TMBox). In contrast, other reaction systems show no color change. This indicates that Phage cocktail@Co-Mn can accelerate the colorimetric reaction of TMB. To further investigate the colorimetric mechanism of the Phage cocktail@Co-Mn+TMB reaction system, DMPO was used to capture the generated free radicals, and EPR was used for detection. Figure 5 As shown in Figure B, the free radical generated in the reaction system is the superoxide anion. Therefore, Phage cocktail@Co-Mn exhibits oxidase-like activity and can oxidize colorless TMB to form blue TMBox.

[0107] Example 5

[0108] Optimization of detection conditions for colorimetric systems

[0109] 1. Optimization of buffer pH

[0110] The specific procedure is as follows: Take 5 mL centrifuge tubes, divide them into 7 groups (n=3), and add 2800 μL of HAc-NaAc buffer (0.2 M, pH 3.0-9.0), 100 μL of Phage cocktail@Co-Mn solution prepared in Example 2, and 100 μL of TMB solution (2.5 mM, dissolved in ethanol) to each group. React for 15 min, and then detect the reaction using a UV-Vis spectrometer. Figure 6 As shown in Figure A, the catalytic activity of Phage cocktail@Co-Mn first increases and then decreases, reaching its maximum at pH 4.0. Therefore, the pH of the buffer solution was set to 4.0 in subsequent experiments.

[0111] 2. Optimization of Phage cocktail@Co-Mn dosage

[0112] The specific operation is as follows: Using HAc-NaAc buffer (pH 5.0, 0.2M), 10 μL, 50 μL, 100 μL, 150 μL, 200 μL, 250 μL, and 300 μL of the Phage cocktail@Co-Mn solution prepared in Example 2 (n=3) and 100 μL of TMB solution (2.5 mM, dissolved in anhydrous ethanol) were added to a 3 mL system, respectively. The remainder was HAc-NaAc buffer (pH 4.0). The reaction was allowed to proceed for 15 min, and then the reaction was detected. The results showed ( Figure 6 B) The absorbance of the Phage cocktail@Co-Mn+TMB colorimetric system at 652nm increases with the increase of Phage cocktail@Co-Mn dosage. Considering the testing cost, 100μL of Phage cocktail@Co-Mn was used in subsequent experiments.

[0113] Example 6

[0114] Feasibility of Phage Cocktail@Co-Mn Colorimetric Method for Detecting Staphylococcus aureus

[0115] The specific procedures are as follows: Take two sets of 5 mL centrifuge tubes (n=3), and add 2700 μL (experimental group) and 2800 μL (blank control group) of HAc-NaAc buffer (pH 4.0, 0.2 M) to each group. Add 100 μL of the Phage cocktail@Co-Mn solution prepared in Example 2 to each group; then add 100 μL of a 10% concentration solution to the experimental group. 9 Staphylococcus aureus YZUstau28 at CFU / mL was incubated at room temperature for 10 min. After incubation, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the mixture was allowed to react at room temperature for 15 min. Color development was then observed, and the results were as follows: Figure 7 As shown, the absorbance of the Phage cocktail@Co-Mn+TMB colorimetric system at 652 nm decreased sharply after the addition of Staphylococcus aureus, indicating that Staphylococcus aureus can inhibit the TMB colorimetric reaction catalyzed by Phagecocktail@Co-Mn. Staphylococcus aureus is captured by Phage cocktail@Co-Mn, blocking the catalytic site of Phage cocktail@Co-Mn and thus inhibiting its oxidase-like activity.

[0116] Example 7

[0117] Phage cocktail@Co-Mn optimizes the detection conditions for Staphylococcus aureus YZUstau28.

[0118] Optimization of incubation time of 1Phage cocktail@Co-Mn with Staphylococcus aureus

[0119] The specific procedure is as follows: Add 100 μL of a 10% concentration solution to a centrifuge tube containing 2700 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) and 100 μL of the Phage cocktail@Co-Mn solution prepared in Example 2. 9 Staphylococcus aureus at CFU / mL (7 groups, n=3) was incubated at room temperature for 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min. 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added at each incubation time, and the mixture was reacted for 15 min at room temperature before detection. Results are as follows: Figure 8 As shown in Figure A, as the incubation time of Phagecocktail@Co-Mn with Staphylococcus aureus increases, the absorbance of the reaction system at 652 nm reaches its lowest value at 10 min and then tends to stabilize. Therefore, the optimal incubation time is set to 10 min.

[0120] 2. Optimization of reaction time for the colorimetric system

[0121] The specific steps are as follows: Prepare 100 μL of a 10... 9 CFU / mL of Staphylococcus aureus was added to a centrifuge tube containing 2700 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) and 100 μL of the Phage cocktail@Co-Mn solution prepared in Example 2. After incubation at room temperature for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was continued at room temperature. Detection was performed at reaction times of 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 min. Results are as follows: Figure 8 As shown in CD, the absorbance of both Staphylococcus aureus chromogenic systems increased at 652 nm with increasing reaction time. However, the absorbance of the two chromogenic systems at 652 nm decreased within 15 min and remained constant after 15 min. Therefore, the reaction time of the chromogenic system was set to 15 min. In summary, subsequent experiments in this invention were conducted under optimal conditions (buffer pH 4, enzyme volume 100 μL, incubation time 10 min, and chromogenic reaction time 20 min).

[0122] Example 9

[0123] Stability of Phage cocktail@Co-Mn solution

[0124] The specific procedures are as follows: Following the optimal operating conditions described above, perform testing every 3 days, with 3 replicates; simultaneously, every 3 days, collect phage titers from the Phage cocktail@Co-Mn solution for titer determination, with 3 replicates. Results are as follows... Figure 8 As shown in Figure B, the enzyme activity of the Phage cocktail@Co-Mn solution remained stable over 30 days, indicating that the method has good stability. Furthermore, the titer of the bacteriophage in the Phage cocktail@Co-Mn solution was determined, showing that the titer of bacteriophage SapYZUgamma ranged from 10 to... 8 The PFU / mL level fluctuated around 100%, indicating that the phage activity in the Phage cocktail@Co-Mn solution was good.

[0125] Example 10

[0126] Detection of Staphylococcus aureus standard curve

[0127] The specific operation is as follows: Under optimal conditions, set up 8 different concentrations of Staphylococcus aureus YZUstau28 (with a final concentration of 10 in a 3 mL system). 2 -10 8 CFU / mL), 100 μL of each solution was added to centrifuge tubes containing 2700 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) and 100 μL of Phage cocktail@Co-Mn solution prepared in Example 2, respectively. After incubation for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was allowed to proceed for 15 min before detection. The results are as follows. Figure 9 As shown in Figure A, with the addition of Staphylococcus aureus, the adsorption peak at 652 nm of the colorimetric system gradually decreased. The absorbance of the colorimetric system at 652 nm was then fitted to the concentration of Staphylococcus aureus. Figure 9 As shown in Figure B, the absorbance of the colorimetric system at 652 nm is linearly related to the logarithm of the Staphylococcus aureus concentration, with the equation: y = -0.057x + 0.58, and the detection range is 10. 2 -10 8 CFU / mL, with a calculated limit of detection (LOD) as low as 1.05 × 10⁻⁶. 2 CFU / mL, detection limit is lower than the national standard requirement.

[0128] Example 11

[0129] Selectivity and interference of Staphylococcus aureus

[0130] The specific procedure is as follows: Select 12 strains of bacteria other than Staphylococcus (strains purchased from the CICC Culture Collection Center: Staphylococcus xylose ATCC 29971, Staphylococcus albus ATCC 8032, Staphylococcus epidermidis CMCC 26069, Cronobacter sakazakii CICC 21569, Salmonella enteritidis CICC 21513, Vibrio parahaemolyticus CICC 21617, Shigella flexneri CICC 21534, Vibrio influenzae CICC 21612, Cronobacter sakazakii CICC 21545, Bacillus cereus CICC 21261, Yersinia enterocolitica CICC 21669, and enteropathogenic Escherichia coli CICC 10664), and add 100 μL of each strain or 100 μL of a 1:1 mixture thereof to a container containing 100 μL of Phage. A 3 mL system was prepared by mixing 2700 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) with Co-Mn solution at room temperature. After incubation for 10 min at room temperature, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was allowed to proceed for another 15 min at room temperature. The results were then analyzed. Figure 9 As shown in Figure C, the Phage cocktail@Co-Mn+TMB chromogenic system exhibits good selectivity for Staphylococcus aureus. Furthermore, the interference resistance results of the Phage cocktail@Co-Mn+TMB chromogenic system are as follows: Figure 9 As shown in Figure D, the 12 bacterial strains did not interfere with the detection of Staphylococcus aureus. The results indicate that the Phage cocktail@Co-Mn+TMB colorimetric system can be used for the determination of Staphylococcus aureus under complex conditions.

[0131] Example 12

[0132] Detection of live and dead Staphylococcus aureus

[0133] Viable *Staphylococcus aureus* strains YZUstau12, YZUstau28, YZUstau32, YZUstau81, and YZUstau89 were inactivated by treatment at 80°C or above for 15 min. 100 μL of either viable or inactivated *Staphylococcus aureus* were added to 100 μL of Phage cocktail@Co-Mn solution, and then combined with HAc-NaAc buffer (pH 4.0, 0.2 M) to form a 3 mL system. After incubation at room temperature for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was allowed to proceed for another 15 min at room temperature before detection. Results are as follows: Figure 10As shown, the Phage cocktail@Co-Mn+TMB chromogenic system cannot detect dead Staphylococcus aureus. The results indicate that the Phage cocktail@Co-Mn+TMB chromogenic system can distinguish between live and dead Staphylococcus aureus.

[0134] Example 13

[0135] Simulated real sample testing and influencing factor analysis

[0136] The specific steps are as follows: Purchase four pre-cooked dishes from the supermarket: pork, braised pork, spicy chicken, shredded pork with green peppers, and pickled fish as simulated samples. First, sterilize them in boiling water, cool them to room temperature, and then add 3×10 to each dish. 3 3×10 5 and 3×10 7 CFU / mL Staphylococcus aureus (YZUstau28) bacterial suspension was prepared. Simultaneously, mixed Staphylococcus aureus suspensions of different concentrations added to the simulated sample were analyzed using conventional counting methods. The mixed Staphylococcus aureus suspensions were tested under optimized reaction conditions, with three parallel samples for each concentration. Measurements were performed using a UV-Vis spectrophotometer. Bacterial concentrations were calculated based on the simulated sample standard curve and conventional counting methods. Spike recoveries and relative standard deviations were calculated. Five commonly used food preservatives (potassium sorbate, 0.0075%; sodium pyrophosphate, 0.5%; sodium hexametaphosphate, 0.5%; sodium tripolyphosphate, 0.5%; disodium succinate, 2%) were added to 5 ml of LB medium. Five LB liquid culture media were prepared and different amounts of NaCl were added to achieve NaCl concentrations of 2%, 4%, 6%, 8%, and 10%. Seven LB liquid culture media were prepared and their pH adjusted to 3-9. Then inoculate with 3×10 2 3×10 3 3×10 5 and 3×10 7 A CFU / mL Staphylococcus aureus suspension (YZUstau28) was used. Staphylococcus aureus in the samples was detected using both colorimetric and conventional methods. The results are shown in Tables 1-4 below. The recoveries of Staphylococcus aureus in the simulated samples and LB medium ranged from 108.84% to 95.54%, with RSDs ranging from 1.06% to 4.68%, demonstrating that this method can be applied to the detection of real samples, and that the effects of food additives, NaCl concentration, and pH value on the detection are negligible.

[0137] Table 1. Determination of Staphylococcus aureus in food samples by Phage cocktail@Co-Mn colorimetric method

[0138]

[0139]

[0140] Table 3. Factors affecting the measured effects of the Phage cocktail@Co-Mn colorimetric method

[0141]

[0142]

[0143] Table 4. Factors affecting the measured effects of the Phage cocktail@Co-Mn colorimetric method

[0144]

[0145] Example 14

[0146] Phage cocktail@Co-Mn specifically captures Staphylococcus aureus.

[0147] Using the optimal preparation conditions for phage cocktail@Co-Mn described above, SapYZU10@Co-Mn, SapYZUbeta@Co-Mn, and SapYZUM13@Co-Mn were prepared using single phages SapYZU10n, SapYZUbeta, and SapYZUM13, respectively.

[0148] Under optimal conditions, 91 Staphylococcus aureus strains from different sources were detected using the SapYZU10@Co-Mn, SapYZUbeta@Co-Mn, SapYZUM13@Co-Mn, and phagecocktail@Co-Mn colorimetric systems. The specific procedures are as follows: 100 μL of bacterial suspension (YZUstau1, YZUstau2, YZUstau4–YZUstau21, YZUstau23–YZUstau74, YZUstau76–YZUstau94) from 91 Staphylococcus aureus strains from different sources was taken and added to four colorimetric systems containing 100 μL of SapYZU10@Co-Mn, SapYZUbeta@Co-Mn, SapYZUM13@Co-Mn, and phage cocktail@Co-Mn. Each system was further prepared by adding 2700 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) to form a 3 mL system. After incubation at room temperature for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was allowed to proceed for 15 min at room temperature before detection. The results are as follows: Figure 11As shown, the detection efficiency of SapYZU10@Co-Mn was 70.33% (64 / 91); the detection efficiency of SapYZUbeta@Co-Mn was 76.92% (70 / 91); the detection efficiency of SapYZUM13@Co-Mn was 74.73% (68 / 91); while the detection efficiency of phage cocktail@Co-Mn was 100% (91 / 91). These results indicate that phage cocktails have a broader detection range than single phages.

[0149] Example 15

[0150] Referring to the previous optimal preparation conditions, the three bacteriophages (SapYZU10, SapYZUbeta, and SapYZUM13) were combined in pairs to prepare SapYZU10+SapYZUbeta@Co-Mn, SapYZUbeta+SapYZUM13@Co-Mn, and SapYZU10+SapYZUM13@Co-Mn, respectively.

[0151] Under optimal conditions, 91 Staphylococcus aureus strains from different sources were detected using the SapYZU10+SapYZUbeta@Co-Mn, SapYZUbeta+SapYZUM13@Co-Mn, SapYZU10+SapYZUM13@Co-Mn, and phage cocktail@Co-Mn colorimetric systems. The specific procedures are as follows: 100 μL of bacterial suspension (YZUstau1, YZUstau2, YZUstau4–YZUstau21, YZUstau23–YZUstau74, YZUstau76–YZUstau94) from 91 Staphylococcus aureus strains from different sources was taken and added to four colorimetric systems containing 100 μL of SapYZU10+SapYZUbeta@Co-Mn, SapYZUbeta+SapYZUM13@Co-Mn, SapYZU10+SapYZUM13@Co-Mn, and phage cocktail@Co-Mn. A 3 mL system was formed with 2700 μL of HAc-NaAc buffer (pH 4.0, 0.2 M), incubated at room temperature for 10 min, and then 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added. The reaction was allowed to proceed for 15 min at room temperature before detection. The results are as follows Figure 12As shown, the detection efficiency of SapYZU10+SapYZUbeta@Co-Mn was 68.13% (62 / 91); the detection efficiency of SapYZUbeta+SapYZUM13@Co-Mn was 73.63% (67 / 91); the detection efficiency of SapYZU10+SapYZUM13@Co-Mn was 68.13% (62 / 91); while the detection efficiency of phagecocktail@Co-Mn was 100% (91 / 91). The results indicate that the detection range of the three phage cocktails is wider than that of the other combinations.

[0152] Example 16

[0153] Referring to the previous optimal preparation conditions, different phages were selected to prepare phage cocktail detection systems: SapYZUS7+SapYZU10+SapYZU11@Co-Mn, SapYZU1+SapYZU15+SapYZUM13@Co-Mn, SapYZUS7+SapYZUS8+SapYZUM13@Co-Mn, and SapYZU10+SapYZUbeta+SapYZUM13@Co-Mn.

[0154] Under optimal conditions, 91 Staphylococcus aureus strains from different sources were detected using the SapYZUS7+SapYZU10+SapYZU11@Co-Mn, SapYZU1+SapYZU15+SapYZUM13@Co-Mn, SapYZUS7+SapYZUS8+SapYZUM13@Co-Mn, and SapYZU10+SapYZUbeta+SapYZUM13@Co-Mn colorimetric systems. The specific procedures are as follows: 100 μL of bacterial suspension (YZUstau1, YZUstau2, YZUstau4–YZUstau21, YZUstau23–YZUstau74, YZUstau76–YZUstau94) from 91 Staphylococcus aureus strains from different sources was taken and added to four colorimetric systems containing 100 μL of each of the following solutions: SapYZUS7+SapYZU10+SapYZU11@Co-Mn, SapYZU1+SapYZU15+SapYZUM13@Co-Mn, SapYZUS7+SapYZUS8+SapYZUM13@Co-Mn, and SapYZU10+SapYZUbeta+SapYZUM13@Co-Mn. This was then combined with 2700 μL of... A 3 mL system of HAc-NaAc buffer (pH 4.0, 0.2 M) was prepared and incubated at room temperature for 10 min. Then, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was allowed to proceed for another 15 min at room temperature before detection. Results were as follows: Figure 13 As shown, the detection efficiency of SapYZUS7+SapYZU10+SapYZU11@Co-Mn was 79.12% (72 / 91); the detection efficiency of SapYZU1+SapYZU15+SapYZUM13@Co-Mn was 84.62% (77 / 91); the detection efficiency of SapYZUS7+SapYZUS8+SapYZUM13@Co-Mn was 75.82% (69 / 91); and the detection efficiency of SapYZU10+SapYZUbeta+SapYZUM13 was 100% (91 / 91). These results indicate that the cocktail of three bacteriophages (SapYZU10n, SapYZUbeta, and SapYZUM13) selected in this study has a wider detection range than other combinations.

[0155] Example 17

[0156] Referring to the previous optimal preparation conditions, the synthesis method of Co-Mn nanozyme material was optimized by adjusting the formula to 4.76 g CoCl2·6H2O and 3.96 g MnCl2·4H2O. After adjustment, the detection time of the prepared Phage cocktail@Co-Mn was shortened to 7 min, and the detection limit was reduced to 92 CFU / mL. The results were obtained using scanning electron microscopy (SEM). Figure 14 A) Transmission electron microscopy ( Figure 14 B) and Brunauer-Emmett-Teller ( Figure 14 C) Characterization of Co-Mn nanozymes. For example... Figure 14 As shown in Figure A, Co-Mn exhibits a lamellar structure. (Transmission electron microscopy results) Figure 14 B, D, and E) indicate that the length of Co-Mn is 10.50 ± 0.22 nm and the width is 6.54 ± 0.10 nm. For example... Figure 14 As shown in Figure C, the Co-Mn nanozyme has a porous structure with a surface area of ​​16.859 m². 2 / g, pore volume is 0.072cm³ 3 / g. Furthermore, the phage immobilization efficiency assay results ( Figure 14 F) indicates that Co-Mn nanozymes exhibit an immobilization efficiency of 96.42-99.62% for phages SapYZU10, SapYZU11, SapYZUM13, and phage cocktails. These results demonstrate that the increased surface area and porous structure of the Co-Mn nanozyme material significantly contribute to its effectiveness. Figure 14 The addition of G and H components enhances the phage immobilization efficiency. Furthermore, the increased phage content in the detection system enhances its ability to recognize Staphylococcus aureus, resulting in a shorter detection time and a lower detection limit.

[0157] Example 18

[0158] Referring to the previous optimal preparation conditions, the detection conditions of Phage cocktail@Co-Mn against Staphylococcus aureus YZUstau28 were optimized by adjusting the formula to 4.76 g CoCl2·6H2O and 3.96 g MnCl2·4H2O.

[0159] Optimization of incubation time of 1Phage cocktail@Co-Mn with Staphylococcus aureus

[0160] The specific procedure is as follows: Add 100 μL of a 10% concentration solution to a centrifuge tube containing 2700 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) and 100 μL of the Phage cocktail@Co-Mn solution prepared in Example 2. 9 Staphylococcus aureus at CFU / mL (7 groups, n=3) was incubated at room temperature for 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, at which 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was carried out for 7 min at room temperature before detection. Results are as follows: Figure 15 As shown in Figure A, as the incubation time of Phagecocktail@Co-Mn with Staphylococcus aureus increases, the absorbance of the reaction system at 652 nm reaches its lowest value at 10 min and then tends to stabilize. Therefore, the optimal incubation time is set to 10 min.

[0161] 2. Optimization of reaction time for the colorimetric system

[0162] The specific steps are as follows: Prepare 100 μL of a 10... 9 CFU / mL of Staphylococcus aureus was added to a centrifuge tube containing 2700 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) and 100 μL of the Phage cocktail@Co-Mn solution prepared in Example 2. After incubation at room temperature for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was continued at room temperature. Detection was performed at reaction times of 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 min. Results are as follows: Figure 15 As shown in CD, the absorbance of both Staphylococcus aureus chromogenic systems increased at 652 nm with increasing reaction time. However, the absorbance of both systems at 652 nm decreased sharply within 7 minutes, then stabilized after 7 minutes. Therefore, the reaction time for the chromogenic system was set to 7 minutes. In summary, subsequent experiments in this invention were conducted under optimal conditions (buffer pH 4, enzyme volume 100 μL, incubation time 10 minutes, and chromogenic reaction time 7 minutes).

[0163] 3. Stability of Phage cocktail@Co-Mn solution

[0164] The specific procedures are as follows: Following the optimal operating conditions described above, perform testing every 3 days, with 3 replicates; simultaneously, every 3 days, collect phage titers from the Phage cocktail@Co-Mn solution for titer determination, with 3 replicates. Results are as follows... Figure 15 As shown in Figure B, the enzyme activity of the Phage cocktail@Co-Mn solution remained stable over 30 days, indicating that the method has good stability. Furthermore, the titer of the bacteriophage in the Phage cocktail@Co-Mn solution was determined, showing that the titer of bacteriophage SapYZUgamma ranged from 10 to... 8 The PFU / mL level fluctuated around 100%, indicating that the phage activity in the Phage cocktail@Co-Mn solution was good.

[0165] 4. Detection of the standard curve for Staphylococcus aureus

[0166] The specific operation is as follows: Under optimal conditions, set up 8 different concentrations of Staphylococcus aureus YZUstau28 (with a final concentration of 10 in a 3 mL system). 2 -10 8 CFU / mL), 100 μL of each solution was added to centrifuge tubes containing 2700 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) and 100 μL of Phage cocktail@Co-Mn solution prepared in Example 2, respectively. After incubation for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was allowed to proceed for 7 min before detection. The results are as follows. Figure 16 As shown in Figure A, with the addition of Staphylococcus aureus, the adsorption peak at 652 nm of the colorimetric system gradually decreased. The absorbance of the colorimetric system at 652 nm was then fitted to the concentration of Staphylococcus aureus. Figure 16 As shown in Figure B, the absorbance of the colorimetric system at 652 nm is linearly related to the logarithm of the Staphylococcus aureus concentration, with the equation: y = -0.081X + 0.60. The detection range is 10. 2 -10 8 With a CFU / mL limit of detection (LOD) as low as 92 CFU / mL, the LOD is lower than the national standard requirement.

[0167] 5. Selectivity and interference of Staphylococcus aureus

[0168] The specific procedure is as follows: Select 12 strains of bacteria other than Staphylococcus (strains purchased from the CICC Culture Collection Center: Staphylococcus xylose ATCC 29971, Staphylococcus albus ATCC 8032, Staphylococcus epidermidis CMCC 26069, Cronobacter sakazakii CICC 21569, Salmonella enteritidis CICC 21513, Vibrio parahaemolyticus CICC 21617, Shigella flexneri CICC 21534, Vibrio influenzae CICC 21612, Cronobacter sakazakii CICC 21545, Bacillus cereus CICC 21261, Yersinia enterocolitica CICC 21669, and enteropathogenic Escherichia coli CICC 10664), and add 100 μL of each strain or 100 μL of a 1:1 mixture thereof to a container containing 100 μL of Phage. A 3 mL system was prepared by mixing 2700 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) with Co-Mn solution at room temperature. After incubation at room temperature for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was continued at room temperature for 7 min. The results were then analyzed. Figure 16 As shown in Figure C, the Phage cocktail@Co-Mn+TMB chromogenic system exhibits good selectivity for Staphylococcus aureus. Furthermore, the interference resistance results of the Phage cocktail@Co-Mn+TMB chromogenic system are as follows: Figure 16 As shown in Figure D, the 12 bacterial strains did not interfere with the detection of Staphylococcus aureus. The results indicate that the Phage cocktail@Co-Mn+TMB colorimetric system can be used for the determination of Staphylococcus aureus under complex conditions.

[0169] 6. Following the method in Example 12, Phage cocktail@Co-Mn was prepared using 4.76 g CoCl2·6H2O and 3.96 g MnCl2·4H2O. After incubation at room temperature for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was allowed to proceed for 7 min at room temperature. The Phage cocktail@Co-Mn+TMB colorimetric system could not detect dead Staphylococcus aureus. The results indicate that the Phage cocktail@Co-Mn+TMB colorimetric system can distinguish between live and dead Staphylococcus aureus.

[0170] 7. Following the method in Example 14, Phage cocktail@Co-Mn was prepared using 4.76 g CoCl2·6H2O and 3.96 g MnCl2·4H2O. This specifically captured Staphylococcus aureus. After incubation at room temperature for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was allowed to proceed for 7 min at room temperature before detection. The results showed that the detection range of the phage cocktail was broader than that of a single phage.

[0171] 8. Following the method in Example 15, Phage cocktail@Co-Mn was prepared using 4.76 g CoCl2·6H2O and 3.96 g MnCl2·4H2O. Three phages (SapYZU10, SapYZUbeta, and SapYZUM13) were combined in pairs to prepare SapYZU10+SapYZUbeta@Co-Mn, SapYZUbeta+SapYZUM13@Co-Mn, and SapYZU10+SapYZUM13@Co-Mn, respectively. After incubation at room temperature for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was allowed to proceed for 7 min at room temperature before detection. The results showed that the detection range of the cocktail composed of the three phages was wider than that of the other combinations.

[0172] 9. Following the method in Example 16, Phage cocktail@Co-Mn was prepared using 4.76 g CoCl2·6H2O and 3.96 g MnCl2·4H2O. After incubation at room temperature for 10 min, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was allowed to proceed for 7 min at room temperature before detection. The results showed that the cocktail composed of the three phages selected in this study (SapYZU10n, SapYZUbeta, and SapYZUM13) had a wider detection range than other combinations.

Claims

1. A nanozyme coupled to a bacteriophage cocktail, characterized in that, The formulation includes a phage cocktail, polyethyleneimine, and Co-Mn. The phage cocktail fixes the phage head to the surface of Co-Mn with polyethyleneimine, while the phage tail is exposed. The phages include SapYZU10, SapYZUbeta, and SapYZUM13.

2. The nanozyme coupled to a phage cocktail according to claim 1, characterized in that, The bacteriophage is a combination of three bacteriophages: SapYZU10, SapYZUbeta, and SapYZUM13. Preferably, the three bacteriophages are mixed in equal volumes to prepare a bacteriophage cocktail.

3. A method for preparing the nanozyme coupled with a bacteriophage cocktail as described in claim 1, characterized in that, Includes the following steps: Step S1: Prepare a phage cocktail; Step S2: Prepare PEI@Co-Mn solution; Step S3: Mix the phage cocktail obtained in step S1 with the PEI@Co-Mn solution obtained in step S2 and incubate to obtain the phage cocktail@PEI@Co-Mn solution.

4. The preparation method according to claim 3, characterized in that, The specific steps of S1 are as follows: Step S1-1: Add different phage suspensions (SapYZU10, SapYZUbeta, SapYZUM13beta and Staphylococcus aureus) to different sterile centrifuge tubes containing LB semi-solid medium, respectively, and immediately pour them onto the bottom layer of LB solid medium to make double-layer plates and incubate overnight. Step S1-2: Pick a single phage plaque from the double-layer plate in step S1-1 and inoculate it into a Staphylococcus aureus suspension. Incubate overnight, then centrifuge and filter to obtain different phage suspensions. Mix the different phage suspensions in equal volumes to prepare a phage cocktail.

5. The preparation method according to claim 2, characterized in that, Step S2 is as follows: Step S2-1: Add KOH to the ethanol solution and stir to form a homogeneous solution; Step S2-2: Add CoCl2·6H2O, MnCl2·4H2O and H2O under vigorous stirring, sonicate the mixed solution and stir until dissolved; Step S2-3: The product from step S2-2 is centrifuged to collect the precipitate, washed, and then dried; Step S2-4: Disperse the powder from step S2-3 with deionized water, add polyethyleneimine and stir, then wash to obtain a PEI@Co-Mn solution.

6. The preparation method according to claim 5, characterized in that, In step S2-2, the mixture is ultrasonically treated and stirred; in step S2-4, the molecular weight of the polyethyleneimine is 10,000-20,000.

7. The preparation method according to claim 3, characterized in that, Step S3 is as follows: PEI@Co-Mn and phage cocktail are mixed at a volume ratio of 1:4-5 and cultured to prepare phage cocktail@PEI@Co-Mn solution.

8. The preparation method according to claim 7, characterized in that, The concentration of each phage suspension constituting the phage cocktail is not less than 10. 8 PFU / mL.

9. The application of the nanozyme coupled with a bacteriophage cocktail as described in claim 1 in the highly efficient, specific, visual colorimetric detection of live Staphylococcus aureus.

10. The application according to claim 9, characterized in that, The application process includes the following steps: 1) Add phage cocktail modified oxidase solution to the buffer solution; 2) Add the sample to be tested, wait for the bacteria and bacteriophages in the solution to be fully adsorbed, add TMB solution, wait for the color reaction, observe the color with the naked eye or measure the absorption light with an ultraviolet spectrophotometer.

Images