Phage modified magnetic peroxidase for detecting staphylococcus aureus, and preparation method and application thereof

Abstract

The application discloses a Staphylococcus aureus colorimetric analysis method based on phage modified magnetic peroxidase, and belongs to the technical field of biology. The method first prepares magnetic peroxidase by using one-step precipitation method, then uses electrostatic action to fix phage on the magnetic peroxidase in a directional manner to prepare SapYZU15@Fe3O4, and finally uses TMB colorimetric reaction to quantitatively analyze Staphylococcus aureus in a visualized and rapid manner. Compared with the previously reported Staphylococcus aureus detection method, the application has the characteristics of fast detection speed, good specificity, high sensitivity, low equipment dependence, simple operation, low cost and the like, and is suitable for the visualized and rapid detection requirement of Staphylococcus aureus in food samples.

Description

Technical Field

[0001] This invention relates to a phage-modified magnetic peroxidase for detecting Staphylococcus aureus, its preparation method, and its application, belonging to the field of biotechnology. Background Technology

[0002] Staphylococcus aureus is part of the symbiotic flora of humans and animals, causing everything from minor skin infections to life-threatening illnesses such as pneumonia, toxic shock syndrome, and sepsis. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus aureus (MRDR) are frequently detected in clinical and livestock-related environments and the food chain due to their phenotypic plasticity and adaptability. Therefore, accurate and rapid detection and identification of Staphylococcus aureus is of particular importance not only in public health but also in medical diagnostics, food safety, and environmental monitoring.

[0003] Rapid, effective, and accurate diagnosis of Staphylococcus aureus is crucial for the rapid treatment of infected patients, prevention of infection transmission, and reduction of drug-resistant strains. Traditional culture-based methods for detecting Staphylococcus aureus, while inexpensive, simple, and providing qualitative and quantitative results, are labor-intensive, time-consuming, complex, and often fail to meet practical needs. In recent years, many non-culture techniques for detecting pathogens have rapidly developed and been applied, such as molecular biology methods (e.g., DNA microarrays) and immunological methods (e.g., enzyme-linked immunosorbent assay). Although these methods offer advantages such as high specificity and sensitivity for detecting Staphylococcus aureus, they require expensive equipment, highly trained laboratory analysts, and complex sample preparation processes. Therefore, developing a rapid, simple, specific, and sensitive method for detecting Staphylococcus aureus that overcomes interference is of significant practical importance. Summary of the Invention

[0004] The purpose of this invention is to address the technical problems mentioned above by providing a phage-modified magnetic peroxidase for detecting Staphylococcus aureus, its preparation method, and its application.

[0005] The objective of this invention is achieved through the following technical solution: a phage-modified magnetic peroxidase for detecting Staphylococcus aureus, comprising Staphylococcus aureus phage SapYZUalpha, polyethyleneimine, and Fe3O4. Staphylococcus aureus phage SapYZUalpha uses Staphylococcus aureus ATCC 29213 as the host bacterium. Through electrostatic interaction, the head of Staphylococcus aureus phage SapYZUalpha is fixed to the surface of Fe3O4 using polyethyleneimine, while the tail of Staphylococcus aureus phage SapYZUalpha remains exposed, enabling specific capture of Staphylococcus aureus.

[0006] The preservation number of the Staphylococcus aureus phage SapYZUalpha is CCTCC NO: M 2022023 (the preparation method is based on the method described in Chinese patent document CN 114807058 A).

[0007] A method for preparing a phage-modified magnetic peroxidase, the method being as follows:

[0008] Step S1: Isolation and purification of bacteriophages to obtain bacteriophage suspension;

[0009] Step S2: Prepare PEI@Fe3O4 solution;

[0010] Step S3: Mix the phage suspension obtained in step S1 with the PEI@Fe3O4 solution obtained in step S2, and incubate to obtain SapYZUalpha@PEI@Fe3O4 solution.

[0011] Preferably, step S1 is as follows: Step S1-1: Collect sewage from a vegetable market and its surrounding area in Yangzhou City. Take 50 mL of sewage, centrifuge at 8000 rpm for 10 min in a centrifuge, filter impurities through 0.45 μm and 0.22 μm sterile filter membranes in sequence, and take the supernatant.

[0012] Step S1-2: Take 3 mL of the supernatant from step S1-1 and mix it with an equal volume of 2×LB liquid medium, add 100 μL of Staphylococcus aureus ATCC 29213 bacterial suspension, incubate overnight at 37℃, centrifuge and filter to obtain crude phage lysate;

[0013] Step S1-3: Add 100 μL each of the lysis buffer from step S1-2 and the Staphylococcus aureus ATCC 29213 bacterial suspension from step S1-2 to a sterile centrifuge tube containing 5 mL of LB semi-solid medium. Immediately pour the mixture onto the bottom layer of LB solid medium to make a double-layer plate and incubate overnight at 37°C.

[0014] Step S1-4: Pick a single phage plaque from the double-layer plate in step S1-3 into an LB tube containing 100 μL of Staphylococcus aureus ATCC 29213 suspension, incubate at 37℃ and 125 rpm, remove the tube the next day, centrifuge and filter to obtain phage lysate;

[0015] Step S1-5: Repeat the lysis buffer from step S1-4 with the methods in steps S1-3 and S1-4 until bright, clear, and uniformly sized phage plaques are formed on the double-layer plate to prepare a phage suspension.

[0016] Preferably, step S2 is as follows:

[0017] Step S2-1: Disperse 5.63 g of anhydrous ferric chloride and 5.0 g of ferrous sulfate heptahydrate in 400 mL of deionized water and sonicate for 10 min.

[0018] Step S2-2: The mixture obtained in step S2-1 is subjected to nitrogen gas at 85°C and stirred for 30 min.

[0019] Step S2-3: Add 100 mL of ammonium hydroxide, 0.306 g of trisodium citrate and 10.4 g of polyethyleneimine to step S2-2, and stir for 30 min until completely dissolved;

[0020] Step S2-4: The solution obtained in step S2-3 is magnetically separated using a magnet and the synthesized Fe3O4 is collected. The solution is then washed alternately with ethanol and deionized water 5-8 times to prepare a PEI@Fe3O4 solution.

[0021] Preferably, the molecular weight of the polyethyleneimine in steps S2-3 is 10000.

[0022] Preferably, step S3 is as follows: Take 2 parts of PEI@Fe3O4 solution and 1 part of phage suspension by volume and mix them. Vortex for 30 s and incubate at 37℃ and 125 rpm for 6 h to prepare SapYZUalpha@PEI@Fe3O4 solution.

[0023] Preferably, the concentration of the bacteriophage suspension is 10. 9 PFU / mL.

[0024] A method for detecting Staphylococcus aureus using phage-modified magnetic peroxidase, the method being as follows:

[0025] 1) Place the HAc-NaAc buffer solution into centrifuge tubes of the experimental group and the blank control group, respectively, and then add SapYZUalpha@PEI@Fe3O4 solution to each.

[0026] 2) Add the sample to be tested to the centrifuge tube of the experimental group in 1). After the bacteria and bacteriophages in the solution are fully adsorbed, add H2O2 solution and TMB solution in sequence, wait for the color reaction, and observe the color with the naked eye or measure the absorption light with an ultraviolet spectrophotometer.

[0027] Preferably, the concentration of the TMB solution is 0.1-50 mM, the concentration of the H2O2 solution is 10-1000 mM, and the pH range of the HAc-NaAc buffer solution is 3.0-9.0.

[0028] Preferably, the concentration of the TMB solution is 5 mM, the concentration of the H2O2 solution is 100 mM, the pH of the HAc-NaAc buffer solution is 4.0, the adsorption time is 25 min, and the colorimetric reaction time is 20 min.

[0029] Preferably, the Staphylococcus aureus is the standard strain Staphylococcus aureus ATCC 29213, with a concentration of 10. 9 CFU / mL;

[0030] Preferably, the iron content in the SapYZUalpha@Fe3O4 solution is 1.68 mg / g;

[0031] This invention has the following beneficial effects: the phage activity in the SapYZUalpha@Fe3O4 solution provided by this invention is good; this invention provides a phage-modified magnetic peroxidase for detecting Staphylococcus aureus, its preparation method, and its application. The phage SapYZUalpha is directionally immobilized on the surface of Fe3O4 nanoparticles to prepare the phage-modified magnetic peroxidase SapYZUalpha@Fe3O4. The test solution and the SapYZUalpha@Fe3O4 solution are thoroughly mixed in a buffer solution, then 100 μL of H2O2 and 100 μL of TMB solution are added. The solution color is detected, and the Staphylococcus aureus concentration is calculated. The phage-modified magnetic peroxidase described in this invention can rapidly detect Staphylococcus aureus in samples colorimetrically, with a minimum detection concentration of 3 × 10⁻⁶. 2 CFU / mL. Furthermore, the method described in this invention avoids interference from *Escherichia coli*, *Salmonella*, *Shigella boydii*, *Vibrio parahaemolyticus*, *Listeria monocytogenes*, and mixtures thereof, and exhibits strong specificity for *Staphylococcus aureus*. Therefore, the phage-modified magnetic peroxidase colorimetric detection of *Staphylococcus aureus* proposed in this invention has advantages such as rapid detection speed, high specificity, high sensitivity, 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

[0032] Figure 1 Morphage morphology of Staphylococcus aureus phage SapYZUalpha plaque.

[0033] Figure 2 Image A shows the morphological characteristics of Staphylococcus aureus phage SapYZUalpha; image B shows the XRD patterns of Fe3O4 and SapYZUalpha@Fe3O4; images C and D show the TEM images of PEI@Fe3O4 and SapYZUalpha@Fe3O4, respectively; and the accompanying images in images C and D are the corresponding laser confocal microscope images.

[0034] Figure 3 The empty spots in the images are, respectively, images of Staphylococcus aureus bacteriophages SapYZUalpha, PEI@Fe3O4, SapYZUalpha@Fe3O4, and HAc-NaAc buffer in Staphylococcus aureus double-layer plates.

[0035] Figure 4 Hysteresis regression lines of the prepared PEI@Fe3O4 and SapYZUalpha@Fe3O4.

[0036] Figure 5 In the image, A shows the full UV-Vis spectrum of the four reaction systems; B shows the EPR spectrum of free radical capture and the catalytic mechanism of the peroxidase-like SapYZUalpha@Fe3O4.

[0037] Figure 6 In the image, A represents the optimization of the buffer pH value in the detection conditions; B represents the optimization of the SapYZUalpha@Fe3O4 dosage in the detection conditions.

[0038] Figure 7 UV-Vis full spectrum of the prepared SapYZUalpha@Fe3O4+ H2O2+ TMB colorimetric system with or without Staphylococcus aureus.

[0039] Figure 8 In the images, A, B, and C are TEM images of Staphylococcus aureus, SapYZUalpha@Fe3O4, and the complex of SapYZUalpha@Fe3O4 and Staphylococcus aureus, respectively; D, E, and F are laser confocal microscopy images of the complex of SapYZUalpha@Fe3O4 and Staphylococcus aureus, respectively.

[0040] Figure 9 In the image, A shows the effect of incubation time of SapYZUalpha@Fe3O4 with Staphylococcus aureus on the colorimetric reaction; B shows the stability of SapYZUalpha@Fe3O4 at room temperature for one month; C shows the change in reaction time with or without Staphylococcus aureus in the colorimetric system; and D shows the ratio of absorbance at 652 nm of the colorimetric system with or without Staphylococcus aureus to the reaction time.

[0041] Figure 10In the image, A is the UV-Vis full spectrum of the SapYZUalpha@Fe3O4+ H2O2+ TMB colorimetric system at different Staphylococcus aureus concentrations; B is the linear fit between the absorbance of the colorimetric system at 652 nm and the logarithm of the Staphylococcus aureus concentration; C is the selectivity of the SapYZUalpha@Fe3O4+ H2O2+ TMB colorimetric system; and D is the anti-interference ability of the SapYZUalpha@Fe3O4+ H2O2+ TMB colorimetric system. Detailed Implementation

[0042] The following embodiments will provide a more comprehensive and detailed description of the present invention, but do not limit the scope of the invention.

[0043] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The technical terms and means used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention.

[0044] Example 1

[0045] Culture of Staphylococcus aureus

[0046] Bacterial strains, including Staphylococcus aureus ATCC 29213, Escherichia coli, Salmonella, and Vibrio, were taken from a -80℃ freezer and thawed at 4℃. They were then inoculated into 10 mL of LB liquid medium at a 1% inoculum and cultured at 37℃ and 160 rpm for 18-24 h. To ensure greater bacterial activity, the bacterial suspension was transferred under the same conditions and cultured for 18-24 h. The suspension was then centrifuged at 4℃ and 8,000 rpm for 10 min, and the supernatant was discarded to obtain a bacterial pellet. The pellet was resuspended in 1 mL of sterile physiological saline (0.85% NaCl) and centrifuged at 4℃ and 8,000 rpm for 10 min to wash away any residual culture medium. This process was repeated twice. The resulting bacterial suspension was then serially diluted with sterile physiological saline to obtain a concentration of 10. 1 -10 9 CFU / mL Staphylococcus aureus ATCC 29213 bacterial suspension.

[0047] Example 2

[0048] Isolation and purification of bacteriophages

[0049] The bacteriophage described in this invention uses standard Staphylococcus aureus ATCC 29213 as the host bacterium, isolated from Yangzhou City, Jiangsu Province. Staphylococcus aureus ATCC 29213 was purchased from Beijing Bio-Bio Biotechnology Co., Ltd. The specific operation is as follows: Wastewater samples were collected from a vegetable market and its surrounding area in Yangzhou City. 50 mL of the wastewater sample was centrifuged at 8000 rpm for 10 min to remove impurities. The sample was then filtered sequentially through sterile microporous membranes of 0.45 μm and 0.22 μm to prevent contamination and interference with the experiment. 3 mL of the filtrate was thoroughly mixed with 3 mL of 2×LB liquid medium, and 100 μL of a Staphylococcus aureus ATCC 29213 suspension in the logarithmic growth phase was added. The mixture was incubated overnight at 37 ℃ with shaking at 130 rpm. The next day, the sample was centrifuged at 8000 rpm for 10 min and filtered through a 0.22 µm sterile microporous membrane to obtain crude bacteriophage fluid.

[0050] The purification and preparation of bacteriophages were performed as follows: The crude bacteriophage solution was serially diluted 10-fold (10:10) using sterile SM buffer (1L: NaCl 5.8 g, MgSO4·7H2O 2.0 g, 1M Tris-HCl pH 7.4 50 mL). -1 -10 -7 Take an appropriate dilution of phage suspension and an equal volume (100 μL) of logarithmic growth phase Staphylococcus aureus ATCC 29213 bacterial suspension into a 10 mL sterile centrifuge tube, add 5 mL of LB semi-solid medium, mix well, and quickly pour onto the prepared LB solid medium to make a double-layer plate. After solidification, invert the plate and incubate it in a 37℃ constant temperature incubator.

[0051] Select a single, large, and clearly defined phage plaque from the above double-layer plates and incubate it in LB liquid medium on a shaker. The next day, remove the plaque, centrifuge and filter it. Prepare double-layer plates using the same method, repeating this step 3-5 times until the phage plaques in the double-layer plates are of approximately the same size. This is considered a pure phage suspension (referred to as "phage suspension"). Count the phage plaques using the traditional counting method. The results show that the SapYZUalpha suspensions at various gradients achieve a titer of approximately 10 for strain ATCC 29213. 9 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).

[0052] Example 3

[0053] Preparation and characterization of SapYZUalpha@Fe3O4

[0054] The specific steps for preparing SapYZUalpha@Fe3O4 are as follows: Weigh 5.63 g of anhydrous ferric chloride and 5.0 g of ferrous sulfate heptahydrate, mix them with 400 mL of deionized water in a 1000 mL round-bottom flask, and sonicate for 10 min; mechanically stir the mixture at 85 ℃ under a nitrogen atmosphere for 30 min; add 100 mL of ammonium hydroxide, 0.306 g of trisodium citrate and 10.4 g of PEI (polyethyleneimine, molecular weight 10000) sequentially, and continue stirring for 30 min; use a magnet for magnetic separation, collect the synthesized Fe3O4, and wash it alternately with ethanol and deionized water 5-8 times to remove the weakly magnetic iron in the solution; dilute the precipitate to deionized water to obtain the PEI@Fe3O4 solution; take the prepared phage suspension (10 9 PFU / mL) and PEI@Fe3O4 solution were mixed at a volume ratio of 1:2, vortexed for 30s, and incubated at 37℃ and 125rpm for 6 h to prepare SapYZUalpha@PEI@Fe3O4 (abbreviated as "SapYZUalpha@Fe3O4").

[0055] The synthesized PEI@Fe3O4 was characterized using XRD and Zeta potential. Figure 2 As shown in Figure B, the XRD curve of PEI@Fe3O4 exhibits multiple characteristic peaks, which belong to the standard peaks of Fe3O4 (JCPDS No. 19-0629). The microstructure of bacteriophages SapYZUalpha, PEI@Fe3O4, and SapYZUalpha@Fe3O4 was observed using transmission electron microscopy (TEM). Figure 2 As shown in Figure A, bacteriophage SapYZUalpha possesses a retractable tail sheath and, morphologically, belongs to the family Myotail Phageidae ( Myoviridae PEI@Fe3O4 is spherical with a slightly rough surface and good dispersibility. Figure 2 C); When the bacteriophage SapYZUalpha was incubated with PEI@Fe3O4, the head of SapYZUalpha adhered tightly to the surface of Fe3O4, the tail faced outward, and the complete biological structure was maintained. Figure 2 D). Additionally... Figure 2 Results B showed significant changes in the XRD pattern of SapYZUalpha@Fe3O4, which may be due to the introduced SapYZUalpha and residual culture medium. PEI@Fe3O4 and SapYZUalpha@Fe3O4 were labeled using the highly sensitive DNA fluorescent dye SYBR. The specific procedure was as follows: Phage concentrate (approximately 10...) 11PFU / mL was incubated with PEI@Fe3O4 diluted 100 times at 37℃ for 6 h. 200 μL was transferred to a 1.5 mL centrifuge tube, and 20 μL of 60x SYBR solution (final concentration 6x) was added in the dark. Staining was performed for 20 min, followed by washing 4-8 times with deionized water. After washing, the solution was magnetically separated and redissolved in 100 μL of deionized water. A suitable amount of solution was added to a glass slide and observed under a confocal microscope. The results are as follows: Figure 2 As shown in CD, no green fluorescence was observed in PEI@Fe3O4. In contrast, the green fluorescent spots in the SapYZUalpha@Fe3O4 image were evenly distributed, further indicating the presence of SapYZUalpha on SapYZUalpha@Fe3O4. The spotting method was used to verify the activity of bacteriophages in the SapYZUalpha@Fe3O4 solution. The specific procedure was as follows: 10 mL of heated solid culture medium was spread evenly in a Petri dish and placed in a sterile operating table to solidify; 100 μL of Staphylococcus aureus ATCC 29213 bacterial suspension was thoroughly mixed with 5 mL of semi-solid culture medium and spread evenly in the solidified Petri dish, and allowed to solidify; 10 μL each of PEI@Fe3O4 solution, SapYZUalpha@Fe3O4 solution, bacteriophage suspension, and buffer were dropped into the above double-layer plates and incubated overnight at 37°C. The results are as follows. Figure 3 As shown, phage plaques with similar transparency appeared in the areas where SapYZUalpha@Fe3O4 solution and phage SapYZUalpha were added, while no phage plaques appeared in the PEI@Fe3O4 solution and buffer solution. This indicates that SapYZUalpha in SapYZUalpha@Fe3O4 still has biological activity.

[0056] The above results indicate that the bacteriophage SapYZUalpha was successfully immobilized on the PEI@Fe3O4 surface, and that SapYZUalpha@Fe3O4 retained its biological activity. Furthermore, the magnetic properties of SapYZUalpha@Fe3O4 were also investigated in this invention. Figure 4 As shown, the excellent magnetic properties of SapYZUalpha@Fe3O4 ensure that it can successfully magnetically separate and enrich Staphylococcus aureus from complex samples.

[0057] Example 4

[0058] SapYZUalpha@Fe3O4 peroxidase-like activity

[0059] Using TMB as the chromogenic substrate, the peroxidase-like activity of SapYZUalpha@Fe3O4 was studied by colorimetric reaction. The specific procedure was as follows: Twenty 5 mL centrifuge tubes were divided into four groups (a, b, c, and d; n=3). 2700 μL, 2800 μL, 2800 μL, and 2800 μL of HAc-NaAc buffer (pH 4, 0.2 M) were added to groups a, b, c, and d, respectively. Then, 100 μL of SapYZUalpha@Fe3O4 solution was added to groups a, c, and d; 100 μL of H2O2 solution (100 nM, dissolved in pure water) was added to groups a, b, and c; and 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) solution (5 mM, dissolved in ethanol) was added to groups a, b, and d. The reaction was allowed to proceed for 20 min, and the results were detected using UV-Vis spectrophotometry. Figure 5 As shown in Figure A, the SapYZUalpha@Fe3O4 + H2O2 + 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 SapYZUalpha@Fe3O4 can accelerate the TMB colorimetric reaction in the presence of H2O2. To further investigate the colorimetric mechanism of the SapYZUalpha@Fe3O4 + H2O2 + TMB reaction system, DMPO was used to capture the generated free radicals, which were then detected using EPR. Figure 5 As shown in B, the free radicals generated in the reaction system are hydroxyl radicals. Therefore, SapYZUalpha@Fe3O4 has peroxidase-like activity and can catalyze the decomposition of H2O2 to generate hydroxyl radicals, oxidizing colorless TMB to form blue TMBox.

[0060] Example 5

[0061] Optimization of detection conditions for colorimetric systems

[0062] 1. Optimization of buffer pH

[0063] The specific procedure is as follows: Take 5 mL centrifuge tubes, divide them into 7 groups (n=3), and add 2700 μL of HAc-NaAc buffer (pH 3.0-9.0), 100 μL of SapYZUalpha@Fe3O4 solution, 100 μL of H2O2 solution, and 100 μL of TMB solution to each group, respectively. React for 20 min, and then detect the reaction using a UV-Vis spectrometer. Figure 6 As shown in Figure A, the catalytic activity of SapYZUalpha@Fe3O4 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.

[0064] 2 Optimization of SapYZUalpha@Fe3O4 dosage

[0065] The specific procedure is as follows: Add 10 μL, 50 μL, 100 μL, 150 μL, 200 μL, 250 μL, and 300 μL of SapYZUalpha@Fe3O4 solution (n = 3) to a 3 mL system, react for 20 min, and then detect the results. The results show ( Figure 6 B) The absorbance of the SapYZUalpha@Fe3O4+ H2O2+ TMB colorimetric system at 652 nm increases with the increase of SapYZUalpha@Fe3O4 dosage. Considering the testing cost, 100 μL of SapYZUalpha@Fe3O4 was used in subsequent experiments.

[0066] Example 6

[0067] Feasibility of SapYZUalpha@Fe3O4 Colorimetric Method for Detecting Staphylococcus aureus

[0068] The specific procedure is as follows: Take two sets of 5 mL centrifuge tubes (n = 3), add 2600 μL (experimental group) and 2700 μL (blank control group) of HAc-NaAc buffer to each, respectively. Add 100 μL of SapYZUalpha@Fe3O4 solution to each group. Then add 100 μL of Staphylococcus aureus to the experimental group and incubate for 19 min. After incubation, add 100 μL of H2O2 solution first, followed by 100 μL of TMB solution, react for 20 min, wait for color development, and then detect the results. Figure 7 As shown, after the addition of Staphylococcus aureus, the absorbance of the SapYZUalpha@Fe3O4 + H2O2 + TMB colorimetric system decreased sharply at 652 nm, indicating that Staphylococcus aureus can inhibit the TMB colorimetric reaction catalyzed by SapYZUalpha@Fe3O4. We infer that Staphylococcus aureus is captured by SapYZUalpha@Fe3O4, blocking the catalytic site of SapYZUalpha@Fe3O4, thereby inhibiting its peroxidase-like activity.

[0069] Example 7

[0070] SapYZUalpha@Fe3O4 specifically captures Staphylococcus aureus.

[0071] The specific procedure is as follows: SapYZUalpha@Fe3O4 solution and an equal volume of Staphylococcus aureus ATCC 29213 suspension were thoroughly mixed for 10 min. 20 μL of the mixture was dropped onto a 200-mesh carbon-coated copper grid. After adsorption for 15 min, the copper grid was removed and allowed to air dry for 2-3 min. The grid was then stained with 2% sodium phosphotungstenate solution (pH 7.6), and after 2 min, the moisture was blotted off. The grid was allowed to air dry for 10 min. The samples were then observed using a transmission electron microscope (TEM, Hitachi H600A) at 100 kV. Images of clearly visible Staphylococcus aureus and images of Staphylococcus aureus captured by SapYZUalpha@Fe3O4 were selected for photographic analysis. The results are as follows: Figure 8 As shown in AC, Staphylococcus aureus has a complete biological structure. After adding Staphylococcus aureus to SapYZUalpha@Fe3O4, SapYZUalpha@Fe3O4 tightly surrounds Staphylococcus aureus and lyses Staphylococcus aureus.

[0072] To further confirm this, SapYZUalpha@Fe3O4 and Staphylococcus aureus were labeled with SYBR and DAPI, respectively. The specific procedures were as follows: Staphylococcus aureus was cultured to the logarithmic growth phase. 200 μL of bacterial suspension was placed in a 1.5 mL centrifuge tube, and 600 μL of DAPI solution (10 ug / mL) was added in the dark. Staining was performed for 8 min, followed by washing with deionized water 4-8 times. The precipitate was collected and dissolved in 100 μL of deionized water. Another 200 μL of bacterial suspension was placed in a 1.5 mL centrifuge tube, and 20 μL of 60x SYBR solution (final concentration 6x) was added in the dark. Staining was performed for 10 min, followed by washing with deionized water 4-8 times. The precipitate was collected and dissolved in 100 μL of deionized water. 10 μL of each stained phage and bacteria were taken and allowed to adsorb in the dark for 20 min. A suitable amount of solution was added to a glass slide and observed under a confocal microscope. The results are as follows: Figure 8 As shown in the DF, the green SapYZUalpha@Fe3O4 labeled with SYBR almost completely overlaps with the blue Staphylococcus aureus imprint labeled with DAPI, and the results are consistent with the TEM results, indicating that SapYZUalpha@Fe3O4 has a high capture ability for Staphylococcus aureus.

[0073] Example 8

[0074] Optimization of SapYZUalpha@Fe3O4 detection conditions for Staphylococcus aureus

[0075] 1. Optimization of incubation time between SapYZUalpha@Fe3O4 and Staphylococcus aureus

[0076] The specific procedure is as follows: Add 100 μL of Staphylococcus aureus (7 groups, n = 3) to centrifuge tubes containing 2600 μL of HAc-NaAc buffer and 100 μL of SapYZUalpha@Fe3O4 solution. Add H2O2 solution and TMB solution at 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min of incubation, and detect the results after 20 min of reaction. The results are as follows: Figure 9 As shown in Figure A, with the increase of the incubation time between SapYZUalpha@Fe3O4 and Staphylococcus aureus, the absorbance of the reaction system at 652 nm gradually decreased within 25 min and then tended to stabilize after 25 min. Therefore, the optimal incubation time was set to 25 min.

[0077] 2. Optimization of reaction time for the colorimetric system

[0078] The specific procedure is as follows: 100 μL of Staphylococcus aureus was added to a centrifuge tube containing 2600 μL of HAc-NaAc buffer and 100 μL of SapYZUalpha@Fe3O4 solution. After incubation for 20 min, H2O2 solution and TMB solution were added. Detection was performed at reaction times of 1, 3, 5, ... 25, 27, 29, and 31 min. Results are as follows... Figure 9 As shown in CD, the absorbance at 652 nm increased for both Staphylococcus aureus chromogenic systems with increasing reaction time. However, the absorbance of both systems at 652 nm decreased within 20 min and remained constant after 20 min. Therefore, the reaction time for the chromogenic systems was set to 20 min.

[0079] Example 9

[0080] Stability of SapYZUalpha@Fe3O4 solution

[0081] The specific procedures are as follows: Following the above procedures, perform testing every 3 days, with 3 replicates; simultaneously, every 3 days, take phage samples from the SapYZUalpha@Fe3O4 solution for titer determination, with 3 replicates. Results are as follows... Figure 9 As shown in Figure B, the enzyme activity of the SapYZUalpha@Fe3O4 solution remained stable over 30 days, indicating that the method has good stability. Furthermore, the titer of the bacteriophage in the SapYZUalpha@Fe3O4 solution was determined, showing that the titer range of SapYZUalpha was within 10... 9 The PFU / mL level fluctuated around 100%, indicating that the phage activity in the SapYZUalpha@Fe3O4 solution was relatively good.

[0082] Example 10

[0083] Detection of Staphylococcus aureus standard curve

[0084] The specific procedure is as follows: Under optimal conditions, set up 8 different concentrations of Staphylococcus aureus (with a final concentration of 10 in a 3 mL system). 1 -10 8 (CFU / mL) 100 μL of each solution was added to centrifuge tubes containing 2600 μL of HAc-NaAc buffer and 100 μL of SapYZUalpha@Fe3O4 solution, respectively. After incubation for 20 min, H2O2 solution and TMB solution were added, and the reaction was allowed to proceed for another 20 min before detection. Results are as follows: Figure 10 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 10 As shown in Figure B, the absorbance of the colorimetric system at 652 nm exhibits a linear relationship with the logarithm of the Staphylococcus aureus concentration, with the equation: y = -0.10049x + 1.0284. The calculated limit of detection (LOD) is as low as 1.2 × 10⁻⁶. 2 CFU / mL.

[0085] Example 11

[0086] Selectivity and interference of Staphylococcus aureus

[0087] The specific procedure is as follows: Select 5 bacteria other than Staphylococcus, and add 100 μL of each or a mixture thereof to a 3 mL system containing 100 μL of SapYZUalpha@Fe3O4 solution and HAc-NaAc buffer. Incubate for 20 min, then add H2O2 solution and TMB solution, react for another 20 min, and then detect the results. Figure 10 As shown in Figure C, the SapYZUalpha@Fe3O4+H2O2+ TMB colorimetric system exhibits good selectivity for Staphylococcus aureus. Furthermore, the interference resistance results of the SapYZUalpha@Fe3O4+ H2O2+ TMB colorimetric system are as follows... Figure 10 As shown in Figure D, the five bacteria did not interfere with the detection of Staphylococcus aureus. The results indicate that the SapYZUalpha@Fe3O4+ H2O2+ TMB colorimetric system can be used for the determination of Staphylococcus aureus under complex conditions.

[0088] Example 12

[0089] Simulate real sample testing

[0090] The specific steps are as follows: Purchase ultra-high temperature sterilized milk, fruit juice, and vegetable juice from the supermarket as simulated samples, and add 10g of each to the sample. 1 -10 8 CFU / mL bacterial suspensions were prepared, and Staphylococcus aureus ATCC 29213 bacterial suspensions at different concentrations added to simulated samples were analyzed using the conventional counting method. The ATCC 29213 bacterial suspensions were tested under optimized reaction conditions, with three parallel samples for each ATCC 29213 bacterial suspension concentration. Measurements were performed using a UV-Vis spectrophotometer. The bacterial concentration was calculated based on the simulated sample standard curve, and the recovery rate and relative standard deviation (RSD) were calculated using the conventional counting method. The results are shown in Table 1. The recovery rate of Staphylococcus aureus in the simulated samples ranged from 90.61% to 111.85%, and the RSD ranged from 0.82% to 5.74%. Furthermore, the deviation between the detected colony count and the colony count obtained using the conventional counting method did not exceed 10%, demonstrating that this method can be applied to the detection of real samples.

[0091] Table 1. Determination of Staphylococcus aureus in food samples by SapYZUalpha@Fe3O4 colorimetric method .

Claims

1. A phage-modified magnetic peroxidase for detecting Staphylococcus aureus, characterized in that, The assay comprises Staphylococcus aureus phage SapYZUalpha, polyethyleneimine, and Fe3O4. SapYZUalpha uses Staphylococcus aureus ATCC 29213 as its host bacterium. Through electrostatic interaction, the head of SapYZUalpha is fixed to the Fe3O4 surface using polyethyleneimine, while the tail of SapYZUalpha remains exposed, enabling specific capture of Staphylococcus aureus. SapYZUalpha@PEI@Fe3O4 exhibits peroxidase-like activity, catalyzing the decomposition of H2O2 to generate hydroxyl radicals, which oxidize colorless TMB to form blue TMBox. When Staphylococcus aureus is captured by SapYZUalpha@PEI@Fe3O4, the catalytic site of SapYZUalpha@PEI@Fe3O4 is blocked, thereby inhibiting its peroxidase-like activity. Therefore, Staphylococcus aureus can inhibit the TMB colorimetric reaction catalyzed by SapYZUalpha@PEI@Fe3O4.

2. The method for preparing phage-modified magnetic peroxidase for detecting Staphylococcus aureus as described in claim 1, characterized in that, The method is as follows: Step S1: Isolation and purification of bacteriophages to obtain bacteriophage suspension; Step S2: Prepare PEI@Fe3O4 solution; Step S3: Mix the phage suspension obtained in step S1 with the PEI@Fe3O4 solution obtained in step S2, and incubate to obtain SapYZUalpha@PEI@Fe3O4 solution.

3. The method for preparing phage-modified magnetic peroxidase for detecting Staphylococcus aureus according to claim 2, characterized in that, The specific steps of step S1 are as follows: Step S1-1: Collect sewage from a vegetable market and its surrounding area in Yangzhou City. Take 50 mL of sewage, centrifuge at 8000 rpm for 10 min in a centrifuge, filter impurities through 0.45 μm and 0.22 μm sterile filter membranes in sequence, and take the supernatant. Step S1-2: Take 3 mL of the supernatant from step S1-1 and mix it with an equal volume of 2×LB liquid medium, add 100 μL of Staphylococcus aureus ATCC 29213 bacterial suspension, incubate overnight at 37℃, centrifuge and filter to obtain crude phage lysate; Step S1-3: Add 100 μL each of the lysis buffer from step S1-2 and the Staphylococcus aureus ATCC 29213 bacterial suspension from step S1-2 to a sterile centrifuge tube containing 5 mL of LB semi-solid medium. Immediately pour the mixture onto the bottom layer of LB solid medium to make a double-layer plate and incubate overnight at 37°C. Step S1-4: Pick a single phage plaque from the double-layer plate in step S1-3 into an LB tube containing 100 μL of Staphylococcus aureus ATCC 29213 suspension, incubate at 37℃ and 125 rpm, remove the tube the next day, centrifuge and filter to obtain phage lysate; Step S1-5: Repeat the lysis buffer from step S1-4 with the methods in steps S1-3 and S1-4 until bright, clear, and uniformly sized phage plaques are formed on the double-layer plate to prepare a phage suspension.

4. The method for preparing phage-modified magnetic peroxidase for detecting Staphylococcus aureus according to claim 2, characterized in that, Step S2 is as follows: Step S2-1: Disperse 5.63 g of anhydrous ferric chloride and 5.0 g of ferrous sulfate heptahydrate in 400 mL of deionized water and sonicate for 10 min. Step S2-2: The mixture obtained in step S2-1 is subjected to nitrogen gas at 85°C and stirred for 30 min. Step S2-3: Add 100 mL of ammonium hydroxide, 0.306 g of trisodium citrate and 10.4 g of polyethyleneimine to step S2-2, and stir for 30 min until completely dissolved; Step S2-4: The solution obtained in step S2-3 is magnetically separated using a magnet and the synthesized Fe3O4 is collected. It is then washed alternately with ethanol and deionized water 5-8 times to prepare a PEI@Fe3O4 solution.

5. The method for preparing phage-modified magnetic peroxidase for detecting Staphylococcus aureus according to claim 4, characterized in that, The molecular weight of the polyethyleneimine mentioned in steps S2-3 is 10000.

6. The method for preparing phage-modified magnetic peroxidase for detecting Staphylococcus aureus according to claim 2, characterized in that, The specific steps of step S3 are as follows: Take 2 parts of PEI@Fe3O4 solution and 1 part of phage suspension by volume and mix them. Vortex for 30 s and incubate at 37℃ and 125 rpm for 6 h to prepare SapYZUalpha@PEI@Fe3O4 solution.

7. The method for preparing phage-modified magnetic peroxidase for detecting Staphylococcus aureus according to claim 2, characterized in that, The concentration of the bacteriophage suspension was 10. 9 PFU / mL.

8. The method for detecting Staphylococcus aureus using the phage-modified magnetic peroxidase for detecting Staphylococcus aureus as described in claim 1, characterized in that, The method is as follows: 1) Place the HAc-NaAc buffer solution into the centrifuge tubes of the experimental group and the blank control group respectively, and then add SapYZUalpha@PEI@Fe3O4 solution to each. 2) Add the sample to be tested to the centrifuge tube of the experimental group in 1). After the bacteria and bacteriophages in the solution are fully adsorbed, add H2O2 solution and TMB solution in sequence, wait for the color reaction, and observe the color with the naked eye or measure the absorption light with an ultraviolet spectrophotometer.

9. The method for detecting Staphylococcus aureus using phage-modified magnetic peroxidase according to claim 8, characterized in that, The TMB solution concentration is 0.1-50 mM, the H2O2 solution concentration is 10-1000 mM, and the pH range of the HAc-NaAc buffer is 3.0-9.

0.

10. The method for detecting Staphylococcus aureus using phage-modified magnetic peroxidase according to claim 8, characterized in that, The TMB solution concentration was 5 mM, the H2O2 solution concentration was 100 mM, the pH of the HAc-NaAc buffer was 4.0, the adsorption time was 25 min, and the colorimetric reaction time was 20 min.

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