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] The present invention belongs to the field of biological detection technology, and specifically relates to a preparation method and application of a nanozyme for detecting live Staphylococcus aureus by a colorimetric method coupled with a phage cocktail. Background Art

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

[0003] Traditional bacterial detection methods, while reliable, are laborious and time-consuming. In contrast, some rapid detection methods, such as immunological tests (enzyme-linked immunosorbent assay - ELISA) and nucleic acid-based methods (polymerase chain reaction - PCR), can be completed in just a few hours. However, these technologies are expensive, require cumbersome sample preparation procedures, and require high-tech laboratory settings and skilled analysts. Furthermore, these detection methods cannot distinguish between live and dead bacterial cells.

[0004] Bacteriophages are a widespread class of viruses that infect microbial hosts and can specifically recognize their bacterial hosts. They also offer advantages such as low cost, ease of preparation, and strong environmental tolerance. In recent years, several detection technologies using single phages as bacterial recognition agents have been reported. These technologies have been widely used in the detection of Staphylococcus aureus and are capable of distinguishing between live and dead bacteria. However, existing detection methods based on single phages are prone to false positives due to the limited host repertoire of individual phages, which significantly limits the application of phage detection technology in actual food samples. Therefore, the development of a phage cocktail detection method could reduce the risk of false negatives, overcome interference, and enable rapid, simple, specific, and sensitive detection of Staphylococcus aureus. However, currently, most phage cocktail research and development focuses primarily on the field of bacterial therapy. In terms of detection applications, they have only been used for the detection of Klebsiella pneumoniae, and phage cocktails have not yet been reported for the detection of Staphylococcus aureus. In view of this, 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] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a phage cocktail-modified oxidase sensor for detecting Staphylococcus aureus. This method is different from the existing detection methods based on a single phage (virulent or temperate phage). It can not only greatly increase the detection range, but also solve the problems of the prior art such as being laborious and time-consuming, expensive, highly equipment-dependent, complex to operate, and unable to distinguish between live and dead SA bacteria. In addition, it has good specificity, a wide detection range, and reduces the risk of false negatives. It is suitable for the requirements of visual and rapid detection of Staphylococcus aureus in food samples and is expected to be put into practical application.

[0006] The present invention also provides a preparation method and application of the phage cocktail-modified oxidase for detecting Staphylococcus aureus.

[0007] Technical solution: To achieve the above-mentioned objectives, 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, leaving the phage tail exposed. The phages include SapYZU10, SapYZUbeta, and SapYZUM13. The three phages are mixed in equal volumes to prepare a phage cocktail.

[0008] The phage cocktail includes SapYZU10, SapYZUbeta and SapYZUM13. Based on the differences in the host spectra of phages against 91 strains of Staphylococcus aureus, the three phages are mixed in equal volumes to prepare a phage cocktail to achieve full detection of all 91 strains of Staphylococcus aureus.

[0009] The phage cocktail comprises phages SapYZU10, SapYZUbeta and SapYZUM13, which are deposited in the China Center for Type Culture Collection. The deposit number of SapYZUbeta is CCTCC NO: M2022629.

[0010] The method for preparing the nanozyme coupled with the phage cocktail of the present invention comprises the following steps:

[0011] Step S1: preparing phage cocktail;

[0012] Step S2: preparing a PEI@Co-Mn solution;

[0013] Step S3: The phage cocktail obtained in step S1 is mixed with the PEI@Co-Mn solution obtained in step S2 and incubated to obtain a phage cocktail@PEI@Co-Mn solution.

[0014] Wherein, the step S1 is specifically as follows:

[0015] Step S1-1: Add different phage solutions SapYZU10, SapYZUbeta, SapYZUM13 and Staphylococcus aureus suspensions to different sterile centrifuge tubes containing LB semi-solid medium, and immediately pour them onto the bottom LB solid medium to form a double-layer plate, and culture overnight;

[0016] Step S1-2: Pick out individual phage plaques from the double-layer plate in step S1-1 and inoculate them into a Staphylococcus aureus suspension, culture overnight, and then centrifuge to obtain different phage suspensions. Mix equal volumes of the different phage suspensions to prepare a phage cocktail.

[0017] Preferably, the step S1 is specifically as follows:

[0018] Step S1-1: Add 100 μL of phage suspension (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 form a double-layer plate, and culture at 37°C overnight;

[0019] Step S1-2: Pick individual phage plaques from the double-layer plate from step S1-1 and place them in LB tubes containing 100 μL of Staphylococcus aureus YZUstau28 suspension. Incubate overnight at 37°C and 120 rpm, then centrifuge to obtain a phage suspension. Combine the three tubes of phage suspension at a 1:1:1 volume ratio to create a phage cocktail.

[0020] Wherein, the step S2 is specifically as follows:

[0021] Step S2-1: adding KOH to the ethanol solution and stirring to form a uniform solution;

[0022] Step S2-2: CoCl2·6H2O, MnCl2·4H2O and H2O were added under vigorous stirring, and the mixed solution was sonicated and stirred until dissolved;

[0023] Step S2-3: The product in step S2-2 is centrifuged to collect the precipitate, washed and then dried;

[0024] Step S2-4: The powder in step S2-3 is dispersed with deionized water, polyethyleneimine is added and stirred, and a PEI@Co-Mn solution is obtained after washing.

[0025] In step S2-2, the mixture is ultrasonically treated and stirred; and in step S2-4, the molecular weight of the polyethyleneimine is 10,000.

[0026] The step S3 is specifically as follows: PEI@Co-Mn and phage cocktail are mixed at a volume ratio of 1:4-5, and cultured to prepare a phage cocktail@PEI@Co-Mn solution.

[0027] Preferably, the step S2 is specifically as follows:

[0028] Step S2-1: Add KOH to the ethanol solution and stir vigorously to form a uniform solution;

[0029] Step S2-2: CoCl2·6H2O, MnCl2·4H2O and H2O were added under vigorous stirring, and the mixed solution was sonicated and stirred until dissolved;

[0030] Step S2-3: The product in step S2-2 is centrifuged to collect the precipitate, washed, and dried at 110°C;

[0031] Step S2-4: The powder in step S2-3 is dispersed with deionized water, polyethyleneimine is added and stirred, and a PEI@Co-Mn solution is obtained after washing.

[0032] In step S2-2, the mixture is ultrasonically treated and stirred until it is completely dissolved.

[0033] Preferably, in step S2-2, the mixture is ultrasonically treated for 60 minutes; and in step S2-3, the product after centrifugation is washed three times with deionized water.

[0034] Wherein, the molecular weight of the polyethyleneimine in step S2-4 is 10,000-20,000, preferably 10,000.

[0035] Preferably, step S2-1: adding 2.24 g of KOH to 50 mL of ethanol solution and stirring vigorously to form a uniform solution;

[0036] Step S2-2: 4.76 g CoCl2·6H2O, 1.98 g MnCl2·4H2O, and 5 mL H2O were added under vigorous stirring, and the mixed solution was sonicated for 60 min and stirred until completely dissolved;

[0037] Step S2-3: The product in step S2-2 was centrifuged and precipitated, washed three times with deionized water, and then dried at 110°C;

[0038] Step S2-4: The powder obtained in step S2-3 was dissolved in 20 mL of deionized water, 1 g of polyethyleneimine was added and stirred for 0.5 h, and washed with anhydrous ethanol and deionized water 6 times to obtain a PEI@Co-Mn solution.

[0039] Preferably, step S3 is as follows: 1 part of PEI@Co-Mn solution and 3 parts of phage suspension are mixed according to volume ratio, vortexed for 30 seconds, and cultured at 37° C. and 120 rpm for 6 hours to prepare phage cocktail@PEI@Co-Mn solution.

[0040] The concentration of each phage suspension constituting the phage cocktail is not less than 10 8 PFU / mL.

[0041] The nanozyme coupled with the phage cocktail described in the present invention is used in the efficient and specific visual colorimetric detection of live Staphylococcus aureus.

[0042] The application process includes the following steps:

[0043] 1) Add phage cocktail-modified oxidase solution to the buffer;

[0044] 2) Add the sample to be tested, wait for the bacteria and phage in the solution to be fully adsorbed, then add TMB solution, wait for the color reaction to develop, and observe the color with the naked eye or measure the absorbance using a UV spectrophotometer.

[0045] Preferably, the application process includes the following steps:

[0046] 1) Place HAc-NaAc buffer in the centrifuge tubes of the experimental group and the blank control group, and then add the phage cocktail-modified oxidase solution to each tube;

[0047] 2) Add the sample to be tested to the centrifuge tube of the experimental group in 1). After the bacteria and phage in the solution are fully adsorbed, add TMB solution and wait for the color reaction to develop. Observe the color with the naked eye or measure the absorbance using a UV spectrophotometer.

[0048] The concentration of the TMB solution is 0.1-50 mM, and the pH range of the HAc-NaAc buffer is 3.0-9.0.

[0049] Preferably, the concentration of the TMB solution is 2.5 mM, the pH of the HAc-NaAc buffer is 4.0, the adsorption time is 10 min, and the color development reaction time is 15 min.

[0050] This invention provides a colorimetric analysis method for Staphylococcus aureus based on a phage-modified oxidase. Through host spectrum analysis, three strains with a broad host spectrum were selected from 18 S. aureus phage strains. When combined, these strains can recognize all 91 S. aureus strains from food sources such as pork, beef, and lamb. 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 a buffer solution, followed by the addition of 100 μL of TMB solution. The solution color was measured, and the S. aureus concentration was calculated.

[0051] The present invention prepared a phage cocktail-based nanozyme biosensor (phage cocktail@Co-Mn). The phage cocktail was immobilized on the surface of the nanozyme Co-Mn through electrostatic interactions. The resulting phage cocktail@Co-Mn exhibited oxidase-like activity, catalyzing a colorimetric reaction of TMB to produce blue oxidized TMB (TMBox). Upon addition of Staphylococcus aureus, the phage cocktail selectively inhibited the oxidase-like activity of the phage cocktail@Co-Mn through a specific biological interaction with the bacteria.

[0052] The present invention introduces positive charges onto the surface of phage cocktail@Co-Mn via polyethyleneimine (PEI). The phage capsid protein provides negative charges and electrostatically interacts with the positively charged substrate, while the tail fibers remain free for bacterial capture. The phage cocktail-based nanozyme biosensor prepared in this invention offers advantages such as reduced false negative risk, high specificity, a wide detection range, low equipment dependency, simple operation, and low cost.

[0053] Furthermore, the phage cocktail-based nanozyme biosensor (phage cocktail@Co-Mn) prepared by the present invention can distinguish between live and dead S. aureus, with good selectivity and anti-interference performance. This invention, for the first time, proposes immobilizing phage cocktail on the surface of Co-Mn nanomaterials for specific detection of S. aureus. This approach addresses the problems of existing technologies, such as false negative results, time-consuming and labor-intensive methods, high costs, and the inability to distinguish between live and dead S. aureus bacteria.

[0054] The detection effects of the phage cocktail-based nanozyme biosensor (phage cocktail@Co-Mn) prepared by the present invention were compared with those of three other single phage-based nanozyme biosensors (SapYZU10@Co-Mn, SapYZUbeta@Co-Mn, and SapYZUM13@Co-Mn). The results showed that the present invention has a wide detection range, solves the problem of limited detection effect caused by the diversity of bacterial strains and the narrow phage host range in phage detection, improves the accuracy and effectiveness of detection, and provides useful information for the expansion of precise phage detection.

[0055] The detection effects of the phage cocktail-based nanozyme biosensor (phage cocktail@Co-Mn) prepared by the present invention are compared with those of three other single phage nanozyme biosensors (SapYZU10@Co-Mn, SapYZUbeta@Co-Mn, SapYZUM13@Co-Mn), or nanozyme biosensors based on any two phage combinations, or other three phage combinations. The results show that the detection range of the present invention is significantly wide, and the specificity of the phage combination of the present invention is proved. It solves the problem of limited detection effect in phage detection due to the diversity of bacterial strains and the narrow host range of phages, improves the accuracy and effectiveness of detection, and provides useful information for the expansion of precise phage detection.

[0056] This study proposes the use of a phage cocktail for colorimetric detection of Staphylococcus aureus, demonstrating a wider detection range than conventional single-phage nanozymes. Three phage strains were identified through EOP and adsorption rate experiments to demonstrate complementary detection of 91 strains of Staphylococcus aureus, enabling comprehensive detection of all strains.

[0057] In the present invention, three phages with complementary detection ranges form a phage cocktail, which has a wider detection range. 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); and the detection efficiency of the combined phagecocktail@Co-Mn was 100% (91 / 91). The results show that the detection range of phage cocktails is wider than that of single phages.

[0058] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0059] The phage cocktail-modified oxidase (phage cocktail@Co-Mn solution) provided by the present invention has high specificity, good stability, and good phage activity. The detection method of the phage-modified oxidase of the present invention can quickly detect Staphylococcus aureus in the sample by colorimetric analysis, with the minimum detection concentration of 1.05×10 2 CFU / mL. In addition, the detection materials and methods of the present invention can avoid interference from Staphylococcus epidermidis, Staphylococcus xylosus, Staphylococcus albus, Enterobacter sakazakii, Salmonella enteritidis, Vibrio parahaemolyticus, Shigella flexneri, Vibrio influenzae, Enterobacter sakazakii, Bacillus cereus, Yersinia enterocolitica, enteropathogenic Escherichia coli, and mixtures thereof, and have strong specificity for Staphylococcus aureus and a wide recognition range. Therefore, the colorimetric detection of Staphylococcus aureus using phage-modified oxidase proposed in the present invention has the advantages of reducing the risk of false negatives, good specificity, a wide detection range, low equipment dependence, simple operation, and low cost, and is suitable for the requirements of visual rapid detection of Staphylococcus aureus in food samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The plaque morphology of the three phages that make up the phage cocktail.

[0061] Figure 2 In the picture, A is the XPS picture of Co-Mn nanozyme; B is the EDS picture of Co-Mn nanozyme.

[0062] Figure 3 In the figure, A is the TEM image of the three phages that make up the phage cocktail; B is the TEM image of phagecocktail@Co-Mn; C is the laser confocal microscopy image of phage cocktail@Co-Mn, Staphylococcus aureus, and the complex of Phagecocktail@Co-Mn and Staphylococcus aureus.

[0063] Figure 4 The dot-spot images of phage cocktail, Phage cocktail@Co-Mn, and PEI@Co-Mn in Staphylococcus aureus double-layer plates.

[0064] Figure 5 In the picture, A is the full UV-visible spectrum (UV-vis) of the three reaction systems; B is the EPR spectrum of captured free radicals and the catalytic mechanism of the oxidase-like Phage cocktail@Co-Mn.

[0065] Figure 6In the picture, A is the optimization of the buffer pH value in the detection conditions; B is the optimization of the amount of Phage cocktail@Co-Mn in the detection conditions.

[0066] Figure 7 The full UV-Vis spectrum of the prepared Phage cocktail@Co-Mn+TMB colorimetric system with or without Staphylococcus aureus.

[0067] Figure 8 In the figure, A shows the effect of incubation time of Phage cocktail@Co-Mn and Staphylococcus aureus on the color reaction; B shows the stability of Phage cocktail@Co-Mn at room temperature for one month; C shows the change of reaction time on the color development system with or without Staphylococcus aureus; D shows the ratio of absorbance at 652nm and reaction time of the color development system with or without Staphylococcus aureus.

[0068] Figure 9 In the picture, A is the UV-Vis full spectrum of the Phage cocktail@Co-Mn+TMB colorimetric system under different Staphylococcus aureus concentrations; B is the linear fit of the absorbance of the colorimetric system at 652nm and the logarithm of the Staphylococcus aureus concentration; C is the selectivity of the Phage cocktail@Co-Mn+TMB colorimetric system; D is the anti-interference ability of the Phage cocktail@Co-Mn+TMB colorimetric system.

[0069] Figure 10 In the picture, A is the coating result of inactivated and active Staphylococcus aureus; B is the detection result of inactivated and active Staphylococcus aureus by the Phagecocktail@Co-Mn detection system.

[0070] Figure 11 Detection results of 91 strains of Staphylococcus aureus using the Phage cocktail@Co-Mn detection system prepared with three bacteriophages (SapYZU10, SapYZUbeta, and SapYZUM13).

[0071] Figure 12 The detection results of 91 strains of Staphylococcus aureus using the detection system prepared by combining three bacteriophages (SapYZU10, SapYZUbeta, and SapYZUM13) in pairs.

[0072] Figure 13 Detection results of 91 Staphylococcus aureus strains using the detection system prepared for other phage cocktails. DETAILED DESCRIPTION

[0073] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0074] The strains and phages used in the present invention were isolated and screened by Yangzhou University or purchased commercially, and their sources, genome sequences and other information have been made public.

[0075] Staphylococcus aureus YZUstau28, SapYZU10, SapYZUbeta, and SapYZUM13 were all provided by Yangzhou University.

[0076] Among them, SapYZUbeta is deposited in the China Center for Type Culture Collection, and the deposit number of SapYZUbeta is CCTCC NO: M 2022629, which has been disclosed in the applicant's prior application CN116064413A.

[0077] SapYZU10 is a publicly available phage. For details, see 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.

[0078] SapYZUM13 is a publicly available phage. For details, see the literature: Characterization 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.

[0079] The Staphylococcus aureus YZUstau28 used in the present invention is detailed in NCBI JBHESL000000000.

[0080] SapYZUS7 has published phages, 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.

[0081] SapYZUS8 is a publicly available phage. For details, see the literature Nanozyme colourimetry based on temperate bacteriophage for rapid and sensitive detection of Staphylococcus aureus infod matrices, International Journal of Food Microbiology 416(2024)110657.

[0082] SapYZU1 is a publicly available phage. For details, see the literature: Isolation, Identification, Antibacterial Activity and Application of the Broad-spectrum Lytic Staphylococcus aureus Phage SapYZU15, Yangzhou University.

[0083] The Staphylococcus aureus YZUstau28 used in the present invention is detailed in NCBI (JBHESL000000000), YZUstau12 (JBHERW000000000), YZUstau28 (JBHESL000000000), YZUstau32 (JBHESP000000000), YZUstau81 (JBHEUL000000000), and YZUstau89 (JBHEUT000000000), all of which are provided by Yangzhou University.

[0084] Example 1

[0085] Culture of Staphylococcus aureus

[0086] Staphylococcus aureus YZUstau1, YZUstau2, YZUstau4–YZUstau21, YZUstau23-YZUstau74, YZUstau76-YZUstau94 preserved in -20°C refrigerator; Staphylococcus xylosus ATCC 29971 and Staphylococcus albus ATCC 8032 purchased from CICC Culture Collection; Staphylococcus epidermidis CMCC 26069, Enterobacter sakazakii CICC 21569, Salmonella enteritidis CICC 21513, Vibrio parahaemolyticus CICC21617, Shigella flexneri CICC 21534, Vibrio influenzae CICC 21612, Enterobacter sakazakii CICC 21545, Bacillus cereus CICC 21261, Yersinia enterocolitica CICC purchased from CICC Culture Collection 21669. Enteropathogenic Escherichia coli CICC10664 was thawed at 4°C, streaked with a 10 μL inoculation loop, and cultured at 37°C for 36 h. A single colony was then picked and inoculated into 5 mL of LB liquid medium and cultured in a constant temperature shaker at 37°C and 120 rpm for 24 h. To ensure good bacterial activity, the above-obtained bacterial solution was transferred under the same operation and cultured for 24 h. The bacterial solution was centrifuged at 4°C and 8,000 rpm for 10 min, and the supernatant was discarded to obtain a bacterial pellet. The bacteria were resuspended in 1 mL of sterile saline (0.85% NaCl) and centrifuged at 4°C and 8,000 rpm for 10 min to wash away the residual culture medium. This operation was repeated twice. The resulting bacterial suspension was gradiently diluted with sterile saline to obtain a concentration of 10 1 -10 9 CFU / mL of Staphylococcus aureus YZUstau12, YZUstau28, YZUstau32, YZUstau81, and YZUstau89 bacterial suspension.

[0087] Example 2

[0088] Preparation of phage cocktail

[0089] The specific operation is as follows: add 10% of the titer of Staphylococcus aureus YZUstau28 to three sterile centrifuge tubes containing 5 mL of LB semi-solid medium. 8100 μL each of three phage liquids (SapYZU10, SapYZUbeta, SapYZUM13) with a PFU / mL and a suspension of Staphylococcus aureus YZUstau28 cultured to the logarithmic phase were immediately poured onto the bottom LB solid culture medium to make a double-layer plate, which was cultured overnight at 37°C; phage plaques from the double-layer plate were picked and placed in LB test tubes containing 100 μL of the logarithmic phase Staphylococcus aureus YZUstau28 suspension, and cultured overnight at 37°C and 120 r / min. Then, the plates were centrifuged and filtered to obtain three tubes of phage (SapYZU10, SapYZUbeta, SapYZUM13) suspensions.

[0090] The phage plaques formed were counted using the traditional counting method. The results showed that the titer of the suspension of three phages (SapYZU10, SapYZUbeta, SapYZUM13) against the strain YZUstau28 reached about 10 8 PFU / mL or more, and the plaques formed on the plate are bright, clear, and uniform in size (such as Figure 1 The three tubes of phage suspension were mixed at a volume ratio of 1:1:1 to prepare a phage cocktail.

[0091] Example 3

[0092] 1. Preparation and characterization of Co-Mn and phage cocktail@Co-Mn

[0093] The specific steps for preparing Phage cocktail@Co-Mn are as follows: 2.24g of KOH was added to 50mL of ethanol solution and stirred vigorously to form a homogeneous solution. Then, 4.76g of CoCl2·6H2O, 1.98g of MnCl2·4H2O, and 5mL of H2O were added under vigorous stirring. The mixed solution was then ultrasonicated and stirred several times. After reacting for 60 minutes, the solution was centrifuged and repeatedly washed with water to completely remove the Cl in the product. - . Finally, the obtained product was dried at 110°C; the dried powder was dissolved in 20 mL of deionized water, 1 g of polyethyleneimine (molecular weight 10,000) was added and stirred for 0.5 h, and washed 6 times with anhydrous ethanol and deionized water to obtain a PEI@Co-Mn solution. The prepared phage cocktail (Phagecocktail suspension) was mixed with the PEI@Co-Mn solution in a volume ratio of 3:1, vortexed for 30 seconds, and incubated at 37°C and 120 r / min for 6 hours to prepare Phage cocktail@PEI@Co-Mn, and part of the solution was freeze-dried to obtain a solid powder (referred to as "Phage cocktail@Co-Mn").

[0094] 2. Characterization of Co-Mn

[0095] The synthesized Co-Mn was characterized by XPS and EDS. Figure 2 As shown in A, in the XPS spectrum, the peaks of C 1s, O 1s, Mn2p 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 come from Co 3+ The other two peaks at 782.1 and 797.9 eV are labeled as Co 2+ In the spectrum of Mn 2p, the two peaks at 641.6eV and 653.2eV of Mn 2p3 / 2 are closely related to the Mn 2+ Match; the peaks of 644.2eV of Mn 2p3 / 2 and 656.1eV of Mn 2p1 / 2 belong to Mn 3+ The results showed that Co in Co-Mn nanozyme 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. The above results indicate that Co-Mn oxidase was successfully synthesized.

[0096] 3. Characterization of Phage cocktail@Co-Mn

[0097] The microscopic morphology of the three phage strains and Phage cocktail@Co-Mn was observed by transmission electron microscopy (TEM). SapYZU10 and SapYZUbeta both showed typical morphological characteristics of Podoviridae phages, with a polyhedral head and a short tail. SapYZUM13 is a member of the Siphoviridae family ( Figure 3 A); When the phage cocktail was incubated with PEI@Co-Mn, the heads of the three phage strains were all tightly attached to the Co-Mn surface, with their tails facing outwards, and their biological structures were intact ( Figure 3 B). PEI@Co-Mn and Phage cocktail@Co-Mn were labeled with the highly sensitive DNA fluorescent dye SYBR. The specific operation is as follows: the phage concentrate (about 10 11PFU / mL) was mixed with 100-fold diluted PEI@Co-Mn in equal volumes and incubated at 37°C for 6 hours. 200uL was taken in a 1.5mL centrifuge tube and 20uL of 60x SYBR solution (final concentration was 6x) was added in the dark. The cells were stained for 20 minutes and washed 4-8 times with deionized water. After washing, the cells were redissolved in 100uL of deionized water. An appropriate amount of the solution was added dropwise to a glass slide and observed under a confocal microscope. The results are shown in Figure 2. Figure 3 As shown in C, no green fluorescence was observed on 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 spot method was used to verify the activity of the phage in the Phage cocktail@Co-Mn solution. The specific operation was as follows: 10 mL of heated LB solid culture medium was spread on a plate and placed on a sterile operating table to be solidified; 100 μL of Staphylococcus aureus YZUstau28 bacterial suspension cultured to the logarithmic phase was thoroughly mixed with 5 mL of LB semi-solid culture medium, spread on a solidified solid plate, and waited to solidify; 10 μL of PEI@Co-Mn solution, Phagecocktail@Co-Mn solution and phage cocktail suspension were respectively dropped on the above double-layer plate and placed in a 37°C constant temperature incubator for overnight culture. The results are shown in FIG. Figure 4 As shown, phage plaques appeared in the area where the Phage cocktail@Co-Mn solution and phage cocktail were added, and the transparency was similar, while no phage plaques appeared in the area where the PEI@Co-Mn solution was added, indicating that the phage cocktail in Phage cocktail@Co-Mn still had biological activity.

[0098] The above results indicate that the phage cocktail was successfully immobilized on the PEI@Co-Mn surface and Phagecocktail@Co-Mn still retained its biological activity.

[0099] Example 4

[0100] Oxidase-like activity of Phage cocktail@Co-Mn

[0101] TMB (3,3',5,5'-tetramethylbenzidine) was used as a chromogenic substrate to study the oxidase-like activity of Phagecocktail@Co-Mn by colorimetric reaction. The specific operation was as follows: 9 5mL centrifuge tubes were taken and divided into 3 groups (a, b, c groups; n=3), and 2800μL, 2700μL and 2700μL of HAc-NaAc buffer (pH 4, 0.2M) were added to groups a, b and c respectively; then, 100μL of the Phage cocktail@Co-Mn solution prepared in Example 2 was added to groups a and b, and 100μL of TMB solution (10mM, dissolved in ethanol) was added to groups a and c. The reaction was carried out at room temperature for 15min and detected by UV-visible spectrometer. Figure 5 As shown in A, the Phage cocktail@Co-Mn+TMB reaction system has a large absorption peak at 652nm, which is the oxidized TMB (TMBox). In contrast, the other reaction systems did not change color. This shows that Phage cocktail@Co-Mn can accelerate the TMB color development reaction. In order to further study the color development 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 a superoxide anion. Therefore, Phage cocktail@Co-Mn has oxidase-like activity and can oxidize colorless TMB to form blue TMBox.

[0102] Example 5

[0103] Optimization of detection conditions for color development system

[0104] 1. Optimization of buffer pH

[0105] The specific operation is as follows: 5 mL centrifuge tubes were divided into 7 groups (n = 3), and 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) were added to each group. The reaction was carried out for 15 minutes and the results were detected by ultraviolet-visible (UV-vis) spectrometer. Figure 6 As shown in Figure A, the catalytic activity of Phage cocktail@Co-Mn first increases and then decreases, reaching a maximum at pH 4.0. Therefore, the pH value of the buffer solution was set to 4.0 in the following experiments.

[0106] 2. Optimization of Phage cocktail@Co-Mn dosage

[0107] 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 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 3 mL of the system, and the rest was HAc-NaAc buffer (pH 4.0), reacted for 15 minutes, and detected. The results showed ( Figure 6 B) The absorbance of the Phage cocktail@Co-Mn+TMB colorimetric system at 652 nm increased with the amount of Phage cocktail@Co-Mn used. Considering the test cost, 100 μL of Phage cocktail@Co-Mn was used in subsequent experiments.

[0108] Example 6

[0109] Feasibility of Phage cocktail@Co-Mn colorimetric method for detecting Staphylococcus aureus

[0110] The specific operation is as follows: take two groups of 5 mL centrifuge tubes (n=3), add 2700 μL (experimental group) and 2800 μL (blank control group) of HAc-NaAc buffer (pH 4.0, 0.2 M), add 100 μL of Phage cocktail@Co-Mn solution prepared in Example 2 to each group; then add 100 μL of 10% HAc to the experimental group. 9 CFU / mL of Staphylococcus aureus YZUstau28 was incubated at room temperature for 10 minutes; after incubation, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added and reacted at room temperature for 15 minutes, and then color was developed and detected. Figure 7 As shown, after the addition of S. aureus, the absorbance of the Phage cocktail@Co-Mn + TMB colorimetric system at 652 nm dropped dramatically, indicating that S. aureus can inhibit the TMB colorimetric reaction catalyzed by Phagecocktail@Co-Mn. S. aureus is captured by Phage cocktail@Co-Mn, blocking the catalytic site of Phage cocktail@Co-Mn and thus inhibiting its oxidase-like activity.

[0111] Example 7

[0112] Phage cocktail@Co-Mn optimizes the detection conditions of Staphylococcus aureus YZUstau28

[0113] 1Optimization of incubation time of Phage cocktail@Co-Mn and Staphylococcus aureus

[0114] The specific operation is as follows: add 100 μL of 10 M NaAc solution to a centrifuge tube 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. 9 CFU / mL of Staphylococcus aureus (7 groups, n=3) were incubated at room temperature for 2 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min, and 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added and the reaction was carried out at room temperature for 15 min before detection. Figure 8 As shown in Figure A, with the increase of the incubation time of Phagecocktail@Co-Mn and Staphylococcus aureus, the absorbance of the reaction system at 652 nm reached the lowest value at 10 min and then tended to be stable. Therefore, the optimal incubation time was set as 10 min.

[0115] 2 Optimization of reaction time of color development system

[0116] The specific operation is as follows: 100 μL of 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 Phage cocktail@Co-Mn solution prepared in Example 2. After incubation at room temperature for 10 minutes, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added and reacted at room temperature. Detection was performed at reaction times of 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 minutes. The results are shown in the figure. Figure 8 As shown in CD, as the reaction time increases, the absorbance at 652nm of the two Staphylococcus aureus color development systems increases. However, the absorbance ratio at 652nm of the two color development systems decreases within 15min and remains unchanged after 15min. Therefore, the reaction time of the color development system is set to 15min. In summary, the present invention subsequently conducted experiments under the optimal conditions (buffer pH of 4, enzyme dosage of 100μL, incubation time of 10min, and color development reaction time of 20min).

[0117] Example 9

[0118] Stability of Phage cocktail@Co-Mn solution

[0119] The specific operation is as follows: according to the above optimal conditions, the test was performed every 3 days, with 3 parallels; at the same time, the phage titer in the Phage cocktail@Co-Mn solution was measured every 3 days, with 3 parallels. Figure 8 As shown in Figure B, the enzyme activity of the Phage cocktail@Co-Mn solution was stable over the 30 days, indicating that the method has good stability. In addition, the titer of the phage in the Phage cocktail@Co-Mn solution was determined, and the results showed that the titer of the phage SapYZUgamma ranged from 10 8 The PFU / mL fluctuated around 50%, indicating that the phage activity in the Phage cocktail@Co-Mn solution was good.

[0120] Example 10

[0121] Detection of Staphylococcus aureus standard curve

[0122] The specific operation is as follows: Under the optimal conditions, 8 groups of different Staphylococcus aureus YZUstau28 concentrations (the final concentration in the 3mL system is 10 2 -10 8 CFU / mL), 100 μL was taken and added to a centrifuge tube 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. After incubation for 10 minutes, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added and reacted for 15 minutes before detection. The results are shown in Figure 2. Figure 9 As shown in A, with the addition of Staphylococcus aureus, the adsorption peak at 652nm of the color system gradually decreases. Then the absorbance of the color system at 652nm is fitted with the concentration of Staphylococcus aureus. Figure 9 As shown in Figure B, the absorbance of the color system at 652 nm is linearly related to the logarithm of the Staphylococcus aureus concentration, and the equation is: y = -0.057x + 0.58, and the detection range is 10 2 -10 8 CFU / mL, the limit of detection (LOD) was calculated as low as 1.05×10 2 CFU / mL, the detection limit is lower than the national standard requirement.

[0123] Example 11

[0124] Selectivity and interference of Staphylococcus aureus

[0125] The specific operation is as follows: 12 bacterial strains other than Staphylococcus were selected (Staphylococcus xylosus ATCC 29971, Staphylococcus albus ATCC 8032, Staphylococcus epidermidis CMCC 26069, Enterobacter sakazakii CICC 21569, Salmonella enteritidis CICC 21513, Vibrio parahaemolyticus CICC 21617, Shigella flexneri CICC21534, Vibrio influenzae CICC 21612, Enterobacter sakazakii CICC 21545, Bacillus cereus CICC 21261, Yersinia enterocolitica CICC 21669, enteropathogenic Escherichia coli CICC 10664, purchased from CICC Culture Collection), 100 μL of each aliquot or 100 μL of a 1:1 mixture of the aliquots was added to 100 μL of Phage 500. The cocktail@Co-Mn solution was mixed with 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 minutes, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added and reacted at room temperature for 15 minutes before detection. Figure 9 As shown in C, the Phage cocktail@Co-Mn+TMB colorimetric system has good selectivity for Staphylococcus aureus. In addition, the anti-interference results of the Phage cocktail@Co-Mn+TMB colorimetric system are shown in Figure 9 As shown in Figure D, the 12 bacterial strains had no interference with the detection of Staphylococcus aureus. The results showed that the Phage cocktail@Co-Mn+TMB colorimetric system can be used for the detection of Staphylococcus aureus under complex conditions.

[0126] Example 12

[0127] Detection of live and dead Staphylococcus aureus

[0128] The active Staphylococcus aureus strains YZUstau12, YZUstau28, YZUstau32, YZUstau81, and YZUstau89 were inactivated by treatment at 80°C or above for 15 minutes. 100 μL of active or inactivated Staphylococcus aureus was added to 100 μL of Phage cocktail@Co-Mn solution and HAc-NaAc buffer (pH 4.0, 0.2 M) to form a 3 mL system. After incubation at room temperature for 10 minutes, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added and the reaction was carried out at room temperature for 15 minutes before detection. The results are shown in FIG. Figure 10As shown in Figure 2, the Phage cocktail@Co-Mn+TMB colorimetric system cannot detect dead Staphylococcus aureus. The results show that the Phage cocktail@Co-Mn+TMB colorimetric system can distinguish between live and dead Staphylococcus aureus.

[0129] Example 13

[0130] Simulating real sample testing and analysis of influencing factors

[0131] The specific operation is as follows: 4 pre-prepared dishes, including pork, braised pork, spicy chicken, shredded pork with green peppers, and pickled fish, were purchased from the supermarket as simulation samples. They were first sterilized in boiling water, cooled to room temperature, and then 3×10 3 , 3×10 5 and 3×10 7 CFU / mL of Staphylococcus aureus (YZUstau28) suspensions were prepared. Simultaneously, mixed S. aureus suspensions of varying concentrations spiked into simulated samples were subjected to conventional counting methods. These mixed S. aureus suspensions were tested under optimized reaction conditions, with three replicates prepared for each concentration. The concentrations were measured using a UV-visible spectrophotometer. The concentrations were calculated using a simulated sample standard curve and the conventional counting method, and the spiked recoveries and relative standard deviations were calculated. Five common food preservatives (potassium sorbate, 0.0075%; sodium pyrophosphate, 0.5%; sodium hexametaphosphate, 0.5%; sodium tripolyphosphate, 0.5%; and disodium succinate, 2%) were added to 5 ml of LB medium. Five tubes of LB medium were each spiked with varying amounts of NaCl, resulting in NaCl concentrations of 2%, 4%, 6%, 8%, and 10%. Seven tubes of LB medium were then adjusted to pH values ​​ranging from 3 to 9. Then 3×10 2 , 3×10 3 , 3×10 5 and 3×10 7 A suspension of Staphylococcus aureus (YZUstau28) with a concentration of 100 CFU / mL was used. Both colorimetric and traditional methods were used to detect S. aureus in the sample. The results, shown in Tables 1-4 below, show that the recoveries of S. aureus spiked into simulated samples and LB medium ranged from 108.84% to 95.54%, with RSDs ranging from 1.06% to 4.68%. This demonstrates the applicability of this method to the detection of real samples, and that the effects of food additives, NaCl concentration, and pH on the detection were negligible.

[0132] Table 1. Phage cocktail@Co-Mn colorimetric method for the determination of Staphylococcus aureus in food samples

[0133]

[0134] Table 2. Factors affecting the actual measurement of Phage cocktail@Co-Mn colorimetry

[0135]

[0136] Table 3. Factors affecting the actual measurement of Phage cocktail@Co-Mn colorimetry

[0137]

[0138]

[0139] Table 4. Factors affecting the actual measurement of Phage cocktail@Co-Mn colorimetry

[0140]

[0141] Example 14

[0142] Phage cocktail@Co-Mn specifically captures Staphylococcus aureus

[0143] According to the optimal preparation conditions of phage cocktail@Co-Mn mentioned above, single phages SapYZU10n, SapYZUbeta, and SapYZUM13 were used to prepare SapYZU10@Co-Mn, SapYZUbeta@Co-Mn, and SapYZUM13@Co-Mn, respectively.

[0144] Under the 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 operation is as follows: 100 μL of bacterial suspension (YZUstau1, YZUstau2, YZUstau4–YZUstau21, YZUstau23-YZUstau74, YZUstau76-YZUstau94) from 91 strains of Staphylococcus aureus from different sources was taken and added to four color development system solutions containing 100 μL of SapYZU10@Co-Mn, SapYZUbeta@Co-Mn, SapYZUM13@Co-Mn and phage cocktail@Co-Mn, and 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 minutes, 100 μL of TMB solution (2.5 mM, dissolved in ethanol) was added, and the reaction was carried out at room temperature for 15 minutes before detection. The results are shown in Figure 2. Figure 11 As shown in the figure, 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); and the detection efficiency of phage cocktail@Co-Mn was 100% (91 / 91). The results show that the detection range of phage cocktail is wider than that of single phage.

[0145] Example 15

[0146] Referring to the previous optimal preparation conditions, the 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.

[0147] Under the 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 operation is as follows: 100 μL of bacterial liquid from 91 strains of Staphylococcus aureus from different sources (YZUstau1, YZUstau2, YZUstau4–YZUstau21, YZUstau23-YZUstau74, YZUstau76-YZUstau94) was taken and added to four color development system solutions containing 100 μL of SapYZU10+SapYZUbeta@Co-Mn, SapYZUbeta+SapYZUM13@Co-Mn, SapYZU10+SapYZUM13@Co-Mn and phage cocktail@Co-Mn, and then mixed with 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 carried out at room temperature for 15 min for 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); and the detection efficiency of phagecocktail@Co-Mn was 100% (91 / 91). The results show that the detection range of the cocktail composed of three phages is wider than that of other combinations.

[0148] Example 16

[0149] 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 respectively.

[0150] Under the 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 operation is as follows: 100 μL of bacterial suspension of 91 strains of Staphylococcus aureus from different sources (YZUstau1, YZUstau2, YZUstau4–YZUstau21, YZUstau23-YZUstau74, YZUstau76-YZUstau94) was taken and added into four color development system solutions containing 100 μL of SapYZUS7+SapYZU10+SapYZU11@Co-Mn, SapYZU1+SapYZU15+SapYZUM13@Co-Mn, SapYZUS7+SapYZUS8+SapYZUM13@Co-Mn and SapYZU10+SapYZUbeta+SapYZUM13@Co-Mn, and then mixed with 2700 μL 3 mL of HAc-NaAc buffer (pH 4.0, 0.2 M) was added to the system, 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 continued at room temperature for 15 min and the results were detected. 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). The results show that the cocktail composed of the three phages selected in this study (SapYZU10n, SapYZUbeta, SapYZUM13) has a wider detection range than other combinations.

Claims

1. A nanozyme coupled to a phage cocktail, characterized in that: The invention comprises a phage cocktail, polyethyleneimine and Co-Mn. The phage cocktail fixes the phage head on the surface of Co-Mn through polyethyleneimine, and the phage tail is exposed. The phages include SapYZU10, SapYZUbeta and SapYZUM13.

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

3. A method for preparing the nanozyme coupled with a phage cocktail according to claim 1, characterized in that: The steps include: Step S1: preparing phage cocktail; Step S2: preparing a PEI@Co-Mn solution; Step S3: The phage cocktail obtained in step S1 is mixed with the PEI@Co-Mn solution obtained in step S2 and incubated to obtain a phage cocktail@PEI@Co-Mn solution.

4. The preparation method according to claim 3, characterized in that The step S1 is specifically as follows: Step S1-1: Add different phage solutions SapYZU10, SapYZUbeta, SapYZUM13beta and Staphylococcus aureus suspensions to different sterile centrifuge tubes containing LB semi-solid medium, and immediately pour them onto the bottom LB solid medium to form a double-layer plate, and culture overnight; Step S1-2: Pick out individual phage plaques from the double-layer plate in step S1-1 and inoculate them into a Staphylococcus aureus suspension, culture overnight, and then centrifuge to obtain different phage suspensions. Mix equal volumes of the different phage suspensions to prepare a phage cocktail.

5. The preparation method according to claim 2, characterized in that The step S2 is specifically as follows: Step S2-1: adding KOH to the ethanol solution and stirring to form a uniform solution; Step S2-2: CoCl2·6H2O, MnCl2·4H2O and H2O were added under vigorous stirring, and the mixed solution was sonicated and stirred until dissolved; Step S2-3: The product in step S2-2 is centrifuged to collect the precipitate, washed and then dried; Step S2-4: The powder in step S2-3 is dispersed with deionized water, polyethyleneimine is added and stirred, and a PEI@Co-Mn solution is obtained after washing.

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 The step S3 is specifically as follows: PEI@Co-Mn and phage cocktail are mixed at a volume ratio of 1:4-5, and cultured to prepare a 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. Use of the nanozyme coupled to a phage cocktail according to claim 1 in the efficient, specific, visual colorimetric detection of live Staphylococcus aureus.

10. The use according to claim 9, characterized in that The application process includes the following steps: 1) Add phage cocktail-modified oxidase solution to the buffer; 2) Add the sample to be tested, wait for the bacteria and phage in the solution to be fully adsorbed, then add TMB solution, wait for the color reaction to develop, and observe the color with the naked eye or measure the absorbance using a UV spectrophotometer.

Images