CN115931847A - Colorimetric analysis method for staphylococcus aureus based on phage-modified magnetic peroxidase - Google Patents
Colorimetric analysis method for staphylococcus aureus based on phage-modified magnetic peroxidase Download PDFInfo
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Abstract
The invention discloses a colorimetric analysis method for staphylococcus aureus based on phage-modified magnetic peroxidase, belonging to the technical field of biology 3 O 4 Finally, the TMB colorimetric reaction is utilized to rapidly, visually and quantitatively analyze the staphylococcus aureus; compared with the previously reported staphylococcus aureus detection method, the method has the characteristics of high detection speed, good specificity, high sensitivity, low equipment dependence, simplicity in operation, low cost and the like, and is suitable for the visual rapid detection requirement of staphylococcus aureus in food samples.
Description
Technical Field
The invention relates to a colorimetric analysis method for staphylococcus aureus based on phage-modified magnetic peroxidase, belonging to the technical field of biology.
Background
Staphylococcus aureus is part of the human and animal commensal flora and can cause diseases ranging from mild skin infections to life threatening diseases such as pneumonia, toxic shock syndrome and sepsis. MDR and methicillin-resistant staphylococcus aureus (MRSA) are frequently detected in the clinical and livestock-related environment and food chain because of their phenotypic plasticity and adaptability. Therefore, the accurate and rapid detection and identification of staphylococcus aureus is of particular importance not only in public health, but also in medical diagnosis, food safety and environmental monitoring.
The rapid, effective and accurate diagnosis of staphylococcus aureus is of great significance to rapidly treat infected patients, prevent infection transmission and reduce the formation of drug-resistant strains. Conventional culture-based methods for detecting Staphylococcus aureus, while inexpensive, simple and capable of obtaining qualitative and quantitative results, are laborious, time-consuming, complex to operate and difficult to meet practical requirements. In recent years, many non-culture techniques for detecting pathogenic bacteria have been rapidly developed and applied, for example, molecular biological methods (e.g., DNA chips) and immunological methods (e.g., enzyme-linked immunosorbent assay). Although these methods have the advantages of good specificity, high sensitivity, etc., they require expensive equipment and trained laboratory analysts, as well as complex sample preparation procedures. Therefore, the method for preparing the staphylococcus aureus, which can overcome interference and is rapid, simple, convenient, specific and sensitive, has important practical significance.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provide a colorimetric method for staphylococcus aureus based on phage modified magnetic peroxidase.
The purpose of the invention is realized by the following technical scheme: a bacteriophage modified magnetic peroxidase for detecting staphylococcus aureus comprises staphylococcus aureus bacteriophage SapYZUalpha, polyethyleneimine and Fe 3 O 4 The staphylococcus aureus phage SapYZUalpha takes staphylococcus aureus ATCC29213 as host bacteria, and utilizes polyethylene through electrostatic interactionThe head of staphylococcus aureus phage SapYZUalpha is fixed on Fe by using olefine imine 3 O 4 On the surface, the tail part of a staphylococcus aureus bacteriophage SapYZUalpha is exposed outside, and the staphylococcus aureus can be captured specifically.
The preservation number of the staphylococcus aureus bacteriophage SapYZUalpha is CCTCC NO: m2022022022023 (see the method described in chinese patent document CN 114807058A).
A method for preparing phage modified magnetic peroxidase, the method comprises the following steps:
step S1: separating and purifying the phage to obtain phage suspension;
step S2: preparation of PEI @ Fe 3 O 4 A solution;
and step S3: taking the phage suspension obtained in the step S1 and PEI @ Fe obtained in the step S2 3 O 4 Mixing the solutions, incubating to obtain SapYZU15@ PEI @ Fe 3 O 4 And (3) solution.
Preferably, the step S1 specifically includes the following steps: step S1-1: collecting sewage in a certain vegetable market in Yangzhou city and the periphery, taking 50mL of sewage, centrifuging for 10min at 8000rpm in a centrifuge, sequentially filtering impurities by using sterile filter membranes of 0.45 mu m and 0.22 mu m, and taking supernatant;
step S1-2: taking 3mL of the supernatant obtained in the step S1-1, mixing the supernatant with an equal amount of 2 xLB liquid culture medium, adding 100 mu L of staphylococcus aureus ATCC29213 bacterial suspension, carrying out overnight culture at 37 ℃, and carrying out centrifugal filtration to obtain a crude phage lysate;
step S1-3: adding 100 mu L of each of the lysate and the staphylococcus aureus ATCC29213 bacterial suspension obtained in the step S1-2 into a sterile centrifuge tube containing 5mL of LB semisolid culture medium, immediately pouring the lysate and the staphylococcus aureus ATCC29213 bacterial suspension onto the LB solid culture medium at the bottom layer to prepare a double-layer flat plate, and culturing the double-layer flat plate at 37 ℃ overnight;
step S1-4: picking the phage plaques of the double-layer plate in the single step S1-3 into an LB test tube containing 100 mu L of staphylococcus aureus ATCC29213 bacterial suspension, culturing at 37 ℃ and 125rpm, taking out the phage plaques the next day, and performing centrifugal filtration to obtain phage lysate;
step S1-4: and (4) alternately performing the lysate in the step S1-4 according to the method in the step S1-3 and the method in the step S1-4 until bright, clear and uniformly-sized phage plaques are formed on the double-layer plate, and preparing phage suspension.
Preferably, the step S2 is as follows:
step S2-1: dispersing 5.63g of anhydrous ferric chloride and 5.0g of ferrous sulfate heptahydrate in 400mL of deionized water, and dissolving for 10min by ultrasonic waves;
step S2-2: introducing nitrogen into the mixture obtained in the step S2-1 at 85 ℃ and stirring for 30min;
step S2-3: adding 100mL of ammonium hydroxide, 0.306g of trisodium citrate and 10.4g of polyethyleneimine into the step S2-2, and stirring for 30min until the trisodium citrate is completely dissolved;
step S2-4: magnetically separating the solution obtained in step S2-3 with a magnet and collecting the synthesized Fe 3 O 4 Alternately washing with ethanol and deionized water for 5-8 times to obtain PEI @ Fe 3 O 4 And (3) solution.
Preferably, the molecular weight of the polyethyleneimine in step S2-3 is 10000.
Preferably, the step S3 specifically includes the following steps: respectively taking PEI @ Fe according to volume ratio 3 O 4 Mixing the solution 2 parts and phage suspension 1 part, vortexing for 30s, and culturing at 37 deg.C and 125rpm for 6h to obtain SapYZU15@ PEI @ Fe 3 O 4 And (3) solution.
Preferably, the concentration of the bacteriophage suspension is 10 9 PFU/mL。
The method for detecting staphylococcus aureus by using phage modified magnetic peroxidase comprises the following steps:
1) Respectively placing HAc-NaAc buffer solution into the centrifuge tubes of the experimental group and the blank control group, and respectively adding SapYZU15@ PEI @ Fe 3 O 4 A solution;
2) Adding a sample to be detected into the centrifuge tube of the experimental group in the step 1), and sequentially adding H after bacteria and phage in the solution are fully adsorbed 2 O 2 And (3) waiting for a color reaction between the solution and the TMB solution, and observing the color by naked eyes or absorbing light by adopting an ultraviolet spectrophotometer.
Preferably, the concentration of the TMB solution is 0.1-50mM 2 O 2 The concentration of the solution is 10-1000mM, and the pH range of the HAc-NaAc buffer solution is 3.0-9.0.
Preferably, the concentration of the TMB solution is 5mM 2 O 2 The solution concentration is 100mM, the pH of HAc-NaAc buffer solution is 4.0, the adsorption time is 25min, and the color reaction time is 20min.
Wherein, preferably, the staphylococcus aureus is a standard strain staphylococcus aureus ATCC29213 with the concentration of 10 9 CFU/mL；
Among them, sapYZU15@ Fe is preferable 3 O 4 In the solution, the content of iron is 1.68mg/g;
the invention has the following beneficial effects: the invention provides SapYZU15@ Fe 3 O 4 The activity of the phage in the solution is better; the invention provides a colorimetric analysis method for staphylococcus aureus based on bacteriophage modified magnetic peroxidase, which directionally fixes bacteriophage SapYZUalpha on Fe 3 O 4 Preparation of phage-modified magnetic peroxidase SapYZU15@ Fe on surface of nanoparticle 3 O 4 . Mixing the solution to be tested with SapYZU15@ Fe 3 O 4 The solution was mixed well in buffer and 100. Mu.L of H was added 2 O 2 And 100 mu L of TMB solution, detecting the color of the solution, and calculating the concentration of the staphylococcus aureus. The phage modified magnetic peroxidase can be used for quickly detecting staphylococcus aureus of a sample in a colorimetric way, and the lowest detection concentration is 3 multiplied by 10 2 CFU/mL. In addition, the method can avoid the interference of escherichia coli, salmonella, shigella boydii, vibrio parahaemolyticus, listeria monocytogenes and the mixture thereof, and has strong specificity on staphylococcus aureus. Therefore, the colorimetric detection of staphylococcus aureus by using the phage modified magnetic peroxidase has the advantages of high detection speed, high specificity, high sensitivity, low equipment dependence, simplicity in operation, low cost and the like, and is suitable for the visual rapid detection requirement of staphylococcus aureus in food samples.
Drawings
FIG. 1, staphylococcus aureus phage SapYZUalpha plaque morphology.
FIG. 2, A in the picture is a picture of morphological characteristics of Staphylococcus aureus bacteriophage SapYZUalpha; b is Fe 3 O 4 And SapYZU15@ Fe 3 O 4 XRD pictures of (1); c and D are respectively PEI @ Fe 3 O 4 And SapYZU15@ Fe 3 O 4 A TEM image of (B); wherein, the drawings in C and D are corresponding laser confocal microscope pictures.
The plaques in FIG. 3 and the picture are Staphylococcus aureus phages SapYZUalpha and PEI @ Fe respectively 3 O 4 、SapYZU15@Fe 3 O 4 And dot blot pictures of HAc-NaAc buffer in Staphylococcus aureus bi-layer plates.
FIG. 4, PEI @ Fe prepared 3 O 4 And SapYZU15@ Fe 3 O 4 Hysteresis regression line of (2).
FIG. 5, panel A is the ultraviolet-visible spectrum (UV-vis) full spectrum of four reaction systems; b is EPR spectrum for capturing free radical and peroxidase-like SapYZU15@ Fe 3 O 4 The catalytic mechanism of (2).
FIG. 6, panel A is the optimization of buffer pH in the assay conditions; b is SapYZU15@ Fe in detection conditions 3 O 4 And (4) optimizing the dosage.
FIG. 7, sapYZU15@ Fe prepared 3 O 4 +H 2 O 2 + TMB color system UV-Vis full spectrum with or without Staphylococcus aureus.
FIG. 8, panels A, B and C are Staphylococcus aureus, sapYZU15@ Fe, respectively 3 O 4 And SapYZU15@ Fe 3 O 4 And TEM images of staphylococcus aureus complexes; D. e and F are respectively SapYZU15@ Fe 3 O 4 And (3) a laser confocal microscope picture of the staphylococcus aureus compound.
FIG. 9, panel A is SapYZU15@ Fe 3 O 4 The influence of incubation time with staphylococcus aureus on the color reaction; b is SapYZU15@ Fe 3 O 4 Stability at room temperature for one month; c is in reactionTime change of a developing system of staphylococcus aureus or non-staphylococcus aureus; d is the ratio of the absorbance of a chromogenic system of staphylococcus aureus with or without gold at 652nm to the reaction time.
FIG. 10, panel A shows SapYZU15@ Fe at different concentrations of Staphylococcus aureus 3 O 4 +H 2 O 2 + UV-Vis full spectrum of TMB color system; b is linear fitting of the absorbance of the developing system at 652nm and the concentration logarithm of the staphylococcus aureus; c is SapYZU15@ Fe 3 O 4 +H 2 O 2 + selectivity of the TMB color development system; d is SapYZU15@ Fe 3 O 4 +H 2 O 2 + interference immunity of the TMB color system.
Detailed Description
The following examples are intended to more fully and specifically describe the invention without limiting its scope.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The terminology and technical means used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention.
Example 1
Culture of Staphylococcus aureus
Taking out the preserved strains of staphylococcus aureus ATCC29213, escherichia coli, salmonella, vibrio and other bacteria from a refrigerator at the temperature of-80 ℃, unfreezing at the temperature of 4 ℃, inoculating into 10mL of LB liquid culture medium by 1 percent of inoculation amount, and culturing in a constant temperature shaking table at the temperature of 37 ℃ and the rpm of 160 for 18-24 hours; in order to ensure that the bacterial activity is more active, the obtained bacterial liquid is transferred under the same operation and cultured for 18-24h; placing the bacterial liquid in a centrifuge, centrifuging at 4 deg.C and 8,000rpm for 10min, and removing supernatant to obtain thallus precipitate; adding 1mL of sterile physiological saline (0.85% NaCl), resuspending the cells, centrifuging at 4 deg.C and 8,000rpm for 10min to wash off the residual culture medium, and repeating the procedure for 2 times; the obtained bacterial suspension is subjected to gradient dilution by using sterile normal saline to obtain the concentration of 10 1 -10 9 CFU/mL of Staphylococcus aureus ATCC29213 suspension.
Example 2
Isolation and purification of bacteriophages
The bacteriophage of the invention takes standard staphylococcus aureus ATCC29213 as host bacteria, is separated from Yangzhou city of Jiangsu province, and the staphylococcus aureus ATCC29213 is purchased from Beijing Baiohbowei biotechnology limited. The specific operation is as follows: taking 50mL of sewage sample from a certain vegetable market in Yangzhou city and the surrounding sewage, and placing the sewage sample in a high-speed centrifuge for centrifugation at 8000rpm for 10min to remove impurities in the sewage; sequentially filtering with 0.45 μm and 0.22 μm sterile microporous filter membrane to avoid contamination and interference; taking 3mL of filtrate and 3mL of 2 xLB liquid culture medium, fully mixing, simultaneously adding 100 mu L of staphylococcus aureus ATCC29213 bacterial suspension in logarithmic phase, and carrying out shake culture at 37 ℃ overnight at the rotating speed of 130rpm; the next day, the phage was centrifuged at 8000rpm for 10min and filtered through a 0.22 μm sterile microporous membrane to obtain a crude phage solution.
The operation of phage purification preparation was as follows: sterile SM (1L, naCl 5.8g, mgSO 4 ·7H 2 O2.0 g,1M Tris-HCl pH 7.4 50mL) buffer solution was serially diluted 10-fold (10 times by volume) into crude phage solution -1 -10 -7 ) Taking phage suspension with proper dilution and staphylococcus aureus ATCC29213 suspension in logarithmic growth phase in equal volume (100 mu L) in a 10mL sterile centrifuge tube, adding 5mL LB semisolid culture medium, uniformly mixing, quickly pouring onto the prepared LB solid culture medium to prepare a double-layer flat plate, and inversely placing in a constant temperature incubator at 37 ℃ after solidification for culture.
Selecting a single phage plaque with large shape and clear edge in the double-layer plate, performing shake culture in an LB liquid culture medium, taking out the phage plaque after the next day, performing centrifugal filtration, preparing the double-layer plate according to the method, repeating the step for 3-5 times until the size of the phage plaque in the double-layer plate is basically consistent, and considering the phage plaque as a pure phage suspension (referred to as phage suspension for short); the formed phage plaques are counted by a traditional counting method, and the result shows that the titer of the SapYZUalpha suspension of each gradient to the strain ATCC29213 reaches about 10 9 PFU/mL or above, and the plaques formed on the plate are bright, clear, and uniform in size (see FIG. 1)Shown).
Example 3
SapYZU15@Fe 3 O 4 Preparation and characterization of
SapYZU15@Fe 3 O 4 The preparation method comprises the following specific operations: weighing 5.63g of anhydrous ferric chloride and 5.0g of ferrous sulfate heptahydrate, mixing the anhydrous ferric chloride and the ferrous sulfate heptahydrate with 400mL of deionized water in a 1000mL round-bottom flask, and performing ultrasonic treatment for 10min; mechanically stirring the mixture at 85 ℃ for 30min under a nitrogen atmosphere; adding 100mL ammonium hydroxide, 0.306g trisodium citrate and 10.4g PEI (polyethyleneimine, molecular weight 10000) in sequence, and continuing stirring for 30min; magnetic separation with a magnet, collecting the synthesized Fe 3 O 4 Alternately washing the solution with ethanol and deionized water for 5 to 8 times to remove weak magnetic iron in the solution; the sediment is added into deionized water to constant volume, namely PEI @ Fe 3 O 4 A solution; taking the prepared phage suspension (10) 9 PFU/mL) and PEI @ Fe 3 O 4 The solutions were mixed at a volume ratio of 1 3 O 4 (abbreviation "SapYZU15@ Fe) 3 O 4 ”)。
Synthesized PEI @ Fe by using XRD and Zeta potential pair 3 O 4 And (6) performing characterization. As shown in FIG. 2B, PEI @ Fe 3 O 4 Has a plurality of characteristic peaks belonging to Fe 3 O 4 Standard peak of (JCPDSNo.19-0629). Observation of the bacteriophages SapYZUalpha, PEI @ Fe by Transmission microscopy Electron microscopy (TEM) 3 O 4 And SapYZU15@ Fe 3 O 4 The microscopic morphology of (a). As shown in fig. 2A, the bacteriophage SapYZUalpha has a telescopic tail sheath, the morphological feature belonging to the myocaudadae family (Myoviridae); PEI @ Fe 3 O 4 Spherical, slightly rough surface, good dispersibility (fig. 2C); when bacteriophage SapYZUalpha and PEI @ Fe 3 O 4 After incubation, the head of SapYZUalpha was tightly attached to Fe 3 O 4 Surface, tail out, and maintained intact biological structure (fig. 2D). In addition, the results in FIG. 2B show SapYZU15@ Fe 3 O 4 The XRD profile of (a) is greatly changed, which may be caused by the introduced SapYZUalpha and the residual medium. Using GaolingSensitive DNA fluorescent dye SYBR to PEI @ Fe 3 O 4 And SapYZU15@ Fe 3 O 4 The marking is performed. The specific operation is as follows: phage concentrate (about 10) 11 PFU/mL) with 100-fold diluted PEI @ Fe 3 O 4 Incubating at 37 ℃ for 6h, putting 200uL into a 1.5mL centrifuge tube, adding 20uL of SYBR solution (final concentration is 6 x) with concentration of 60x under dark condition, staining for 20min, washing with deionized water for 4-8 times, performing magnetic separation after washing, and dissolving in 100uL of deionized water again; and (3) dropwise adding a proper amount of the solution into a glass slide, and observing under a confocal microscope. The results are shown in FIGS. 2C-D, PEI @ Fe 3 O 4 No green fluorescence is observed, in contrast, at SapYZU15@ Fe 3 O 4 The uniform distribution of the green color spots in the image further illustrates SapYZU15@ Fe 3 O 4 Presence of SapYZUalpha above. Verification of SapYZU15@ Fe by spot method 3 O 4 The activity of the phage in the solution was performed as follows: spreading the heated 10mL solid culture medium in a flat dish, and placing the flat dish in an aseptic operation platform to be solidified; fully and uniformly mixing 100 mu L of staphylococcus aureus ATCC29213 bacterial suspension with 5mL of semi-solid culture medium, and flatly spreading the mixture in a solidified solid plate to be solidified; respectively taking 10 mu L of PEI @ Fe 3 O 4 Solution, sapYZU15@ Fe 3 O 4 The solution, phage suspension and buffer were dropped on the above double-layer plate, and incubated overnight at 37 ℃ under constant temperature. As a result, sapYZU15@ Fe was added dropwise as shown in FIG. 3 3 O 4 The areas of solution and phage SapYZUalpha appeared phage plaque and were similarly transparent, while PEI @ Fe 3 O 4 No phage plaques appeared in the solution and buffer, indicating that SapYZU15@ Fe 3 O 4 The SapYZUalpha in (1) still has biological activity.
These results described above indicate that the phage SapYZUalpha was successfully immobilized on PEI @ Fe 3 O 4 Surface, and SapYZU15@ Fe 3 O 4 Still preserving biological activity. In addition, the invention also relates to SapYZU15@ Fe 3 O 4 The magnetic properties of (2) were investigated. As shown in FIG. 4, sapYZU15@ Fe 3 O 4 Can ensure the excellent magnetic property of the magnetic materialAnd (3) successfully carrying out magnetic separation and enrichment on the staphylococcus aureus in the complex sample.
Example 4
SapYZU15@Fe 3 O 4 Peroxidase-like enzyme activity of (2)
Research on SapYZU15@ Fe by chromogenic reaction with TMB as chromogenic substrate 3 O 4 The peroxidase-like activity of (3). The specific operation is as follows: 20 5mL centrifuge tubes were divided into 4 groups (groups a, b, c, and d; n = 3), and 2700. Mu.L, 2800. Mu.L, and 2800. Mu.L of HAc-NaAc buffer (pH 4, 0.2M) were added to groups a, b, c, and d, respectively; then, 100. Mu.L of SapYZU15@ Fe was added 3 O 4 Solutions in groups a, c and d, 100. Mu.L of H 2 O 2 Solutions (100 nM, dissolved in pure water) in groups a, b and c, 100. Mu.L of TMB (3, 3', 5' -tetramethylbenzidine) solutions (5 mM, dissolved in ethanol) in groups a, b and d, reacted for 20min and detected using UV-Vis spectroscopy. As shown in FIG. 5A, sapYZU15@ Fe 3 O 4 +H 2 O 2 The + TMB reaction system has a large absorption peak at 652nm, which is the oxidized TMB (TMBox). In contrast, the other reaction systems had no color change. This is illustrated in H 2 O 2 In the presence of SapYZU15@ Fe 3 O 4 Can accelerate the color reaction of TMB. To further study SapYZU15@ Fe 3 O 4 +H 2 O 2 And the generated free radicals are captured by DMPO and detected by EPR through the color development mechanism of the TMB reaction system. As shown in fig. 5B, the radical generated in the reaction system is a hydroxyl radical. Thus, sapYZU15@ Fe 3 O 4 Has peroxidase-like activity, and can catalyze H 2 O 2 Decompose to generate hydroxyl radical, oxidize colorless TMB to form blue TMBox.
Example 5
Optimization of detection conditions of color development system
1 optimization of the pH value of the buffer
The specific operation is as follows: 5mL of the centrifuge tubes were divided into 7 groups (n = 3), and 2700. Mu.L of HAc-NaAc buffer (pH 3.0-9.0) and 100. Mu.L of SapYZU15@ Fe were added to each of the tubes 3 O 4 Solution, 100. Mu.L of H 2 O 2 The solution was reacted with 100. Mu.L of TMB solution for 20min and detected by ultraviolet-visible (UV-vis) spectroscopy. As shown in FIG. 6A, sapYZU15@ Fe 3 O 4 The catalytic activity of (a) increases first and then decreases, reaching a maximum at a pH of 4.0. Therefore, the pH of the buffer solution was set to 4.0 in the following experiments.
2SapYZU15@Fe 3 O 4 Optimization of dosage
The specific operation is as follows: 10. Mu.L, 50. Mu.L, 100. Mu.L, 150. Mu.L, 200. Mu.L, 250. Mu.L and 300. Mu.L of SapYZU15@ Fe were added to a 3mL system 3 O 4 Solution (n = 3), react for 20min, detect. The results show (FIG. 6B), sapYZU15@ Fe 3 O 4 +H 2 O 2 + TMB color development Absorbance at 652nm with SapYZU15@ Fe 3 O 4 The dosage is increased by increasing, and 100 mu L of SapYZU15@ Fe is selected in subsequent experiments in consideration of the test cost 3 O 4 。
Example 6
SapYZU15@Fe 3 O 4 Feasibility of colorimetric method for detecting staphylococcus aureus
The specific operation is as follows: 2 groups of 5mL centrifuge tubes (n = 3) were added with 2600. Mu.L (experimental group) and 2700. Mu.L (blank control group) of HAc-NaAc buffer solution, and 100. Mu.L of SapYZU15@ Fe was added to each of the two groups 3 O 4 A solution; then adding 100 mu L of staphylococcus aureus into the experimental group, and incubating for 19min; after incubation, 100. Mu.L of H was added 2 O 2 The solution was added with 100. Mu.L of TMB solution, reacted for 20min, and the color development was waited and detected. As a result, as shown in FIG. 7, sapYZU15@ Fe was obtained after the addition of Staphylococcus aureus 3 O 4 +H 2 O 2 The absorbance of the + TMB color system at 652nm is reduced sharply, which shows that the staphylococcus aureus can inhibit SapYZU15@ Fe 3 O 4 Catalyzed color development of TMB. We concluded that Staphylococcus aureus was SapYZU15@ Fe 3 O 4 Capture, block SapYZU15@ Fe 3 O 4 Thereby inhibiting its peroxidase-like activity.
Example 7
SapYZU15@Fe 3 O 4 Specific capture of Staphylococcus aureus
The specific operation is as follows: fully mixing the SapYZU15@ Fe3O4 solution with the same amount of staphylococcus aureus ATCC29213 bacterial suspension for 10min, dripping 20 mu L of the mixed solution onto a 200-mesh carbon-coated copper net, taking out the copper net after adsorbing for 15min, naturally drying for 2-3min, dyeing with 2% sodium phosphotungstate (pH 7.6) solution, sucking water after 2min, naturally drying for 10min, observing by using a transmission electron microscope (TEM, hitachi H600A) under 100kV voltage, and selecting clear staphylococcus aureus and SapYZU15@ Fe 3 O 4 Images of staphylococcus aureus were captured for photographic analysis. As shown in FIGS. 8A-C, staphylococcus aureus has an intact biological structure as expressed by SapYZU15@ Fe 3 O 4 After addition of Staphylococcus aureus, sapYZU15@ Fe 3 O 4 Tightly surrounding the staphylococcus aureus and lysing it.
To further confirm, sapYZU15@ Fe was labeled with SYBR and DAPI, respectively 3 O 4 And staphylococcus aureus. The specific operation is as follows: culturing staphylococcus aureus to logarithmic phase, placing 200 mu L of bacterial suspension into a 1.5mL centrifuge tube, adding 600 mu L of DAPI solution (10 ug/mL) under dark condition, staining for 8min, washing for 4-8 times with deionized water, collecting precipitate after washing, and dissolving in 100 mu L deionized water; putting another 200 mu L of bacterial suspension into a 1.5mL centrifuge tube, adding 20 mu L of SYBR solution (final concentration is 6 x) with concentration of 60x under dark condition, dyeing for 10min, washing with deionized water for 4-8 times, collecting precipitate after washing, and dissolving in 100 mu L of deionized water; 10 mu L of each stained phage and bacterium are adsorbed for 20min under the dark condition, and a proper amount of solution is dripped into a glass slide and observed under a confocal microscope. As a result, as shown in FIGS. 8D-F, SYBR-labeled green SapYZU15@ Fe 3 O 4 Almost completely coincides with the blue staphylococcus aureus mark marked by DAPI, and the obtained result is consistent with the TEM result, which shows that SapYZU15@ Fe 3 O 4 Has high capture capacity to staphylococcus aureus.
Example 8
SapYZU15@Fe 3 O 4 Optimizing the detection condition of staphylococcus aureus
1SapYZU15@Fe 3 O 4 Optimization of incubation time with Staphylococcus aureus
The specific operation is as follows: in a buffer containing 2600. Mu.L of HAc-NaAc and 100. Mu.L of SapYZU15@ Fe 3 O 4 The centrifuge tubes of the solution were charged with 100 μ L of staphylococcus aureus (7 groups, n = 3), and H was added each time of 2min, 5min, 10min, 15min, 20min, 25min and 30min of incubation 2 O 2 The solution and TMB solution react for 20min and then are detected. The results are shown in FIG. 9A, with SapYZU15@ Fe 3 O 4 And the incubation time with staphylococcus aureus is increased, the absorbance of the reaction system at 652nm gradually decreases within 25min, and becomes stable after 25min, so that the optimal incubation time is set to be 25min.
2 optimization of reaction time of color development System
The specific operation is as follows: mu.L of Staphylococcus aureus was added to a solution containing 2600. Mu.L of HAc-NaAc buffer and 100. Mu.L of SapYZU15@ Fe 3 O 4 Incubating in a centrifuge tube for 20min, and adding H 2 O 2 The solutions and TMB were tested at reaction times of 1, 3, 5, \823025, 27, 29, and 31 min. As shown in FIGS. 9C-D, the absorbance at 652nm of both S.aureus color systems increased with the increase of the reaction time. However, the absorbance ratio at 652nm of both color development systems decreased within 20min and remained unchanged after 20min. Therefore, the reaction time of the color developing system was set to 20min.
Example 9
SapYZU15@Fe 3 O 4 Stability of the solution
The specific operation is as follows: performing detection once every 3 days according to the operation, and performing 3 parallels; at the same time, take SapYZU15@ Fe every 3 days 3 O 4 The phage in solution were titered and 3 replicates were made. As a result, sapYZU15@ Fe was measured in 30 days as shown in FIG. 9B 3 O 4 The enzyme activity of the solution tends to be stable all the time, which shows that the method has good stability; in addition, for SapYZU15@ Fe 3 O 4 The titer of the phage in the solution was determined, and the result showed that the titer of the phage SapYZU15 ranged from 10 9 PFU/mL floated, showing SapYZU15@ Fe 3 O 4 The activity of the phage in solution is better.
Example 10
Detection of standard curve of staphylococcus aureus
The specific operation is as follows: under optimal conditions, 8 different sets of Staphylococcus aureus concentrations were set (final concentration of 10 in a 3mL system) 1 -10 8 CFU/mL), 100. Mu.L of each was added to a solution containing 2600. Mu.L of HAc-NaAc buffer and 100. Mu.L of SapYZU15@ Fe 3 O 4 Incubating in a centrifuge tube for 20min, and adding H 2 O 2 The solution and TMB solution react for 20min and then are detected. As a result, as shown in FIG. 10A, the adsorption peak at 652nm of the coloring system gradually decreased with the addition of Staphylococcus aureus. And then the absorbance of the developing system at 652nm is fitted with the concentration of the staphylococcus aureus. As shown in FIG. 10B, the absorbance of the color development system at 652nm is linearly related to the logarithm of the concentration of Staphylococcus aureus, and the equation is: y = -0.10049x +1.0284, detection Limit (LOD) is calculated as low as 1.2 x 10 2 CFU/mL。
Example 11
Selectivity and interference of Staphylococcus aureus
The specific operation is as follows: selecting 5 bacteria other than Staphylococcus, adding 100 μ L or their mixture into SapYZU15@ Fe containing 100 μ L 3 O 4 In the solution, the solution and HAc-NaAc buffer solution form a 3mL system, and H is added after incubation for 20min 2 O 2 Reacting the solution with TMB solution for 20min, and detecting. As a result, sapYZU15@ Fe was shown in FIG. 10C 3 O 4 +H 2 O 2 The + TMB color development system has good selectivity to staphylococcus aureus. Further, sapYZU15@ Fe 3 O 4 +H 2 O 2 The anti-interference result of the + TMB color development system is shown in FIG. 10D, and 5 bacteria have no interference effect on the detection of staphylococcus aureus. The results show that SapYZU15@ Fe 3 O 4 +H 2 O 2 +TThe MB color development system can be used for detecting staphylococcus aureus under complex conditions.
Example 12
Simulating real sample detection
The specific operation is as follows: purchasing ultra-high temperature sterilized milk, fruit juice and vegetable juice from supermarket as simulation sample, respectively adding 10 1 -10 8 CFU/mL bacterial suspension, and meanwhile, adopting a traditional counting method to operate the staphylococcus aureus ATCC29213 bacterial suspensions with different concentrations added into the simulation sample; and (3) detecting the staphylococcus aureus ATCC29213 suspension according to the optimized reaction conditions, setting three parallel samples for the concentration of each staphylococcus aureus ATCC29213 suspension, measuring by using an ultraviolet-visible spectrophotometer, calculating the concentration of the bacteria liquid according to a simulated sample standard curve, and calculating the standard recovery rate and the relative standard deviation by using a traditional counting method. The results are shown in the following table 1, the recovery rate of the staphylococcus aureus in the simulated sample is between 90.61% and 111.85%, the RSD is between 0.82% and 5.74%, and the deviation of the detected colony number compared with the colony number in the traditional counting method is not more than 10%, which proves that the method can be applied to the detection of the actual sample.
TABLE 1 SapYZU15@ Fe 3 O 4 Colorimetric determination of staphylococcus aureus in food sample
Claims (10)
1. A phage modified magnetic peroxidase for detecting staphylococcus aureus is characterized by comprising staphylococcus aureus phage SapYZUalpha, polyethyleneimine and Fe 3 O 4 The staphylococcus aureus phage SapYZUalpha takes staphylococcus aureus ATCC29213 as host bacteria, and the head of the staphylococcus aureus phage SapYZUalpha is fixed on Fe by using polyethyleneimine through electrostatic interaction 3 O 4 On the surface, the tail of a staphylococcus aureus bacteriophage SapYZUalpha is exposed outside, so that the staphylococcus aureus can be captured specifically.
2. The method for producing a phage-modified magnetic peroxidase-like enzyme according to claim 1, said method comprising:
step S1: separating and purifying the phage to obtain phage suspension;
step S2: preparation of PEI @ Fe 3 O 4 A solution;
and step S3: taking the phage suspension obtained in the step S1 and the PEI @ Fe obtained in the step S2 3 O 4 Mixing the solutions, incubating to obtain SapYZU15@ PEI @ Fe 3 O 4 And (3) solution.
3. The method for producing a phage-modified magnetic peroxidase according to claim 2, wherein the step S1 is specifically as follows: step S1-1: collecting sewage in a certain vegetable market in Yangzhou city and the periphery, taking 50mL of sewage, centrifuging for 10min at 8000rpm in a centrifuge, sequentially filtering impurities by using sterile filter membranes of 0.45 mu m and 0.22 mu m, and taking supernatant;
step S1-2: mixing 3mL of the supernatant obtained in the step S1-1 with an equal amount of 2 xLB liquid culture medium, adding 100 mu L of staphylococcus aureus ATCC29213 bacterial suspension, carrying out overnight culture at 37 ℃, and carrying out centrifugal filtration to obtain a crude phage lysate;
step S1-3: adding 100 mu L of each of the lysate obtained in the step S1-2 and Staphylococcus aureus ATCC29213 bacterial suspension into a sterile centrifuge tube containing 5mL of LB semisolid culture medium, immediately pouring the lysate and Staphylococcus aureus ATCC29213 bacterial suspension onto the LB solid culture medium at the bottom layer to prepare a double-layer flat plate, and performing overnight culture at 37 ℃;
step S1-4: selecting the phage plaques of the double-layer plate in the single step S1-3 into an LB test tube containing 100 mu L of staphylococcus aureus ATCC29213 bacterial suspension, culturing at 37 ℃ and 125rpm, taking out the phage plaques the next day, and performing centrifugal filtration to obtain phage lysate;
step S1-4: and (4) alternately performing the lysate in the step S1-4 according to the method in the step S1-3 and the method in the step S1-4 until bright, clear and uniformly-sized phage plaques are formed on the double-layer plate, and preparing phage suspension.
4. The method for producing a phage-modified magnetic peroxidase according to claim 2, wherein the step S2 is specifically as follows:
step S2-1: dispersing 5.63g of anhydrous ferric chloride and 5.0g of ferrous sulfate heptahydrate in 400mL of deionized water, and dissolving for 10min by ultrasonic waves;
step S2-2: introducing nitrogen into the mixture obtained in the step S2-1 at 85 ℃ and stirring for 30min;
step S2-3: adding 100mL of ammonium hydroxide, 0.306g of trisodium citrate and 10.4g of polyethyleneimine into the step S2-2, and stirring for 30min until the trisodium citrate and the polyethyleneimine are completely dissolved;
step S2-4: performing magnetic separation on the solution obtained in the step S2-3 by using a magnet and collecting the synthesized Fe 3 O 4 Alternately washing with ethanol and deionized water for 5-8 times to obtain PEI @ Fe 3 O 4 And (3) solution.
5. The method for producing a phage-modified magnetic peroxidase-like enzyme according to claim 4, wherein the molecular weight of polyethyleneimine in step S2-3 is 10000.
6. The method for producing a phage-modified magnetic peroxidase according to claim 2, wherein the step S3 is specifically as follows: respectively taking PEI @ Fe according to the volume ratio 3 O 4 Mixing the solution 2 parts and phage suspension 1 part, vortexing for 30s, and culturing at 37 deg.C and 125rpm for 6h to obtain SapYZU15@ PEI @ Fe 3 O 4 And (3) solution.
7. The method for producing a phage-modified magnetic peroxidase-like enzyme according to claim 2, wherein the concentration of the phage suspension is 10 9 PFU/mL。
8. The method for detecting staphylococcus aureus by using the phage-modified magnetic peroxidase according to claim 1, wherein the method comprises:
1) Respectively placing HAc-NaAc buffer solution into centrifuge tubes of an experimental group and a blank control group, and respectively adding SapYZU15@ PEI @ Fe 3 O 4 A solution;
2) Adding a sample to be detected into the centrifuge tube of the experimental group in the step 1), and sequentially adding H after bacteria and phage in the solution are fully adsorbed 2 O 2 And (3) waiting for a color reaction between the solution and the TMB solution, and observing the color by naked eyes or absorbing light by adopting an ultraviolet spectrophotometer.
9. The method for detecting Staphylococcus aureus using phage-modified magnetic peroxidase as in claim 8, wherein the TMB solution has a concentration of 0.1-50mM 2 O 2 The solution concentration is 10-1000mM, and the pH range of HAc-NaAc buffer solution is 3.0-9.0.
10. The method for detecting Staphylococcus aureus by using phage-modified magnetic peroxidase as in claim 8, wherein the concentration of TMB solution is 5mM 2 O 2 The solution concentration is 100mM, the pH of HAc-NaAc buffer solution is 4.0, the adsorption time is 25min, and the color reaction time is 20min.
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