Bacterial membrane probe and detection method for distinguishing gram-positive and gram-negative bacterial infections

Abstract

The invention discloses a bacterial membrane probe and a detection method for distinguishing Gram-positive and Gram-negative bacterial infections, and belongs to the field of biological detection methods, the bacterial membrane probe prepared by the invention detects bacterial types by using a principle that a biological membrane can specifically target homotype cells and derivatives thereof for selective fusion; according to the detection method, the bacterial membrane probe and the extracellular vesicles are selectively fused, so that the types of the Gram pathogenic bacteria are distinguished, a feasible scheme is provided for specific in-situ detection of the target EV-iNOS, rapid, high-sensitivity and high-stability detection of the target EV-iNOS can be realized, the detection efficiency of the EV-iNOS is improved, and the detection cost is reduced. The invention provides a foundation for the specific application of the subsequent M1 type macrophage-derived EV-iNOS in the field of disease diagnosis, and has very high clinical application value.

Description

Technical Field

[0001] The invention belongs to the field of biological detection methods, in particular to a bacterial membrane probe and a detection method for distinguishing Gram-positive and Gram-negative bacterial infections. Background Art

[0002] Using broad-spectrum antibiotics to treat bacterial infections when the pathogens are unknown can easily disrupt the normal balance of microorganisms in the patient's body and cause a large number of mutant strains and drug-resistant strains to appear. Therefore, one of the principles of using antibiotics is to select corresponding narrow-spectrum antibiotics based on the type of pathogens. Therefore, rapid and accurate detection of the type of pathogens is very important for selecting appropriate antibiotics to treat bacterial infections.

[0003] At present, Gram staining is often used to distinguish bacterial types. Although the results of this method are relatively intuitive, it takes a long time and is easily affected by external factors. In recent years, biosensing methods such as electrochemistry and Raman spectroscopy have been tried for bacterial detection, but these methods face problems such as high cost and complex steps, and it is difficult to meet the needs of rapid, accurate and convenient detection of pathogenic bacteria.

[0004] Extracellular vesicles (EVs) are nanoscale membrane vesicles naturally secreted by cells. They can carry a variety of biological molecules, and their composition characteristics are largely affected by the parent cell. If the parent cell is a macrophage, the macrophage will polarize to the M1 type after engulfing bacteria, and will upregulate the expression of inducible nitric oxide synthase (iNOS). EVs secreted by M1 macrophages carry membrane proteins of the bacteria engulfed by the macrophages on their membranes, and contain iNOS inside. When iNOS uses L-arginine and reduced coenzyme II (NADPH) as substrates, it will exert its enzymatic catalytic effect, convert arginine into citrulline, and release a large amount of NO. By converting the detection of iNOS into the detection of NO, the signal amplification output can be achieved. In summary, EV-iNOS derived from M1 macrophages can be used as a biomarker.

[0005] At present, the research on EV-iNOS usually involves steps such as lysing EV, extracting iNOS, and conducting subsequent analysis. These processes are not only cumbersome to operate, but also the samples are susceptible to contamination and degradation during the operation. For this reason, a method for in situ detection of EV-iNOS has been developed. In situ detection can avoid steps such as lysis and extraction, and can effectively prevent the sample from being contaminated or degraded. In order to achieve in situ detection, the EV-iNOS test substance can be delivered into the EV through biological membrane fusion. However, the membrane fusion caused by electrostatic interaction is non-specific, and the interference caused by membrane fusion of non-target EVs cannot be ignored, so that high sensitivity, high stability, and rapid detection of target EV-iNOS cannot be achieved, and the reliability of the detection results is reduced. Therefore, it is necessary to develop new methods to specifically detect the target EV-iNOS in situ. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a bacterial membrane probe and a detection method for distinguishing Gram-positive and Gram-negative bacterial infections in order to address the problem in the prior art that it is difficult to efficiently and reliably distinguish the types of Gram-pathogenic bacteria.

[0007] Technical solution: A bacterial membrane probe, which is a nanoscale vesicle structure of bacterial membrane encapsulating NADPH, L-arginine and NO fluorescent probes, wherein the bacterial membrane comes from Gram-positive bacteria or Gram-negative bacteria.

[0008] The preparation method of the bacterial membrane probe is as follows: Step 1: preparing a bacterial membrane solution using bacteria known to be Gram-positive or Gram-negative; Step 2, preparing NADPH solution, L-arginine solution, and NO fluorescent probe solution; Step 3, adding NADPH solution, L-arginine solution and NO fluorescent probe solution to the bacterial membrane solution, vortexing thoroughly to obtain a mixed solution; Step 4: Add a filter membrane with a pore size of 800 nm to the manual liposome extruder, add the mixed solution and fully squeeze it, then replace it with a filter membrane with a pore size of 400 nm and continue to fully squeeze it; Step 5: Centrifuge the squeezed solution at 25,000 g, and resuspend the precipitate with PBS buffer; centrifuge the resuspended solution at 25,000 g, and resuspend the precipitate with PBS buffer to obtain a bacterial membrane probe.

[0009] Furthermore, the preparation method of the bacterial membrane solution in step 1 is as follows: 2 mL of Gram-positive bacteria or Gram-negative bacteria with a bacterial solution concentration of 1.5×10 8CFU / mL, and then treated with lysozyme, treated with lysis buffer, ultrasonically disrupted, centrifuged at 800 g to collect the supernatant, centrifuged at 25,000 g to obtain the precipitate, and resuspended in 1 mL of PBS to obtain the bacterial membrane solution; The NADPH solution in step 2 is obtained by dissolving NADPH powder in PBS, with a concentration of 2.5 mg / mL; The L-arginine solution in step 2 is obtained by dissolving L-arginine in PBS, with a concentration of 100 μg / mL; The preparation method of the NO fluorescent probe solution in step 2 is as follows: take 1 μL of 5 mM NO fluorescent probe original solution, dilute it in 9 μL of DAF-FM DA diluent, add 40 μL of 0.02 mol / L sodium hydroxide solution to obtain the NO fluorescent probe solution.

[0010] Furthermore, in step three, the volume of the bacterial membrane solution is 1 mL, the volume of the NADPH solution is 50 μL, the volume of the L-arginine solution is 200 μL, and the volume of the NO fluorescent probe solution is 50 μL.

[0011] The present invention discloses a detection method for distinguishing Gram-positive and Gram-negative bacterial infections, which uses a bacterial membrane probe to distinguish Gram-positive and Gram-negative bacterial infections, and comprises the following steps: S1. Extract EVs through macrophage phagocytosis of the bacteria to be tested; S2, the bacterial membrane probe was co-incubated with PBS buffer, EVs extracted by macrophages phagocytosing known Gram-positive bacteria, EVs extracted by macrophages phagocytosing known Gram-negative bacteria, and EVs extracted in S1 in a citric acid-sodium citrate buffer environment, and recorded as the blank control group, positive control group, negative control group, and experimental group, respectively; S3. After co-incubation, place the sample in an ELISA instrument to detect the intensity of the fluorescent signal and determine the type of bacteria to be detected by comparison.

[0012] Furthermore, the method for determining the type of bacteria to be detected by comparison in S3 is as follows: If the bacterial membrane in the bacterial membrane probe in S2 belongs to Gram-positive bacteria, then: the fluorescence signal of the experimental group is higher than that of the blank control group and the negative control group, and is equivalent to that of the positive control group, then the cells to be detected are judged to be Gram-positive bacteria; the fluorescence signal of the experimental group is equivalent to that of the blank control group and the negative control group, and is lower than that of the positive control group, then the cells to be detected are judged to be Gram-negative bacteria; If the bacterial membrane in the bacterial membrane probe in S2 belongs to Gram-negative bacteria, then: the fluorescence signal of the experimental group is higher than that of the blank control group and the positive control group, and is equivalent to that of the negative control group, then the cells to be detected are judged to be Gram-negative bacteria; the fluorescence signal of the experimental group is equivalent to that of the blank control group and the positive control group, and lower than that of the negative control group, then the cells to be detected are judged to be Gram-positive bacteria.

[0013] Beneficial effects: 1) Compared with the prior art, the detection method of the present invention mainly uses the principle that biofilm can specifically target the same type of cells and their derivatives for selective fusion to detect bacterial types. Because the bacterial type of the outer bacterial membrane of the bacterial membrane probe is known, and the EVs secreted by macrophages after engulfing bacteria contain membrane proteins of the corresponding bacteria, the bacterial membrane probe can selectively fuse with EVs carrying membrane proteins of the same type of bacteria. Subsequently, the L-arginine and NADPH carried by the bacterial membrane probe will generate NO under the catalysis of iNOS carried by EV, and finally NO will react with the NO fluorescent probe carried in the bacterial membrane probe to generate a fluorescent signal; if the bacterial membrane carried on the EV membrane If the bacterial type corresponding to the above protein does not match the bacterial type used to prepare the bacterial membrane probe, the bacterial membrane probe and EV will not undergo membrane fusion, and the fluorescent signal cannot be detected. Therefore, the unknown bacterial type can be detected according to the known bacterial type. Therefore, the detection method of the present invention is a feasible solution for specific in situ detection of the target EV-iNOS, which can realize rapid, high-sensitivity and high-stability detection of the target EV-iNOS, improve the detection efficiency of EV-iNOS, and realize the distinction of Gram-type pathogenic bacteria, which provides a basis for the specific application of EV-iNOS derived from M1 macrophages in the field of disease diagnosis, and has a high clinical application value. 2) Compared with the prior art, the bacterial membrane probe prepared by the present invention can be prepared in large quantities at low cost and can be stored at -20°C for about one month. It can be used as needed, and the detection steps are simple and fast, thereby realizing low-cost and rapid detection of pathogenic bacteria types, and the external environment will not affect the detection results, ensuring the accuracy, stability and reliability of the results. 3) Compared with the prior art, the bacterial membrane probe prepared by the present invention can realize high-throughput detection of samples, which can greatly improve the efficiency of clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a schematic diagram of the structure of the bacterial membrane probe in Example 1; Figure 2 This is a schematic diagram of the selective membrane fusion process between the bacterial membrane probe and the EV when the bacterial membrane of the bacterial membrane probe and the bacterial membrane protein on the EV belong to the same type of bacteria in Example 2; Figure 3This is a schematic diagram of the selective membrane fusion process between the bacterial membrane probe and the EV when the bacterial membrane of the bacterial membrane probe and the bacterial membrane protein on the EV do not belong to the same type of bacteria in Example 2; Figure 4 This is a fluorescence signal intensity diagram used to determine the type of bacteria to be detected in Example 2; Figure 5 This is a fluorescence signal intensity diagram used to determine the type of bacteria to be detected in Example 3; Figure 6 The bacterial membrane probe and EV in Example 4 are both derived from Staphylococcus aureus, and the particle size and Zeta potential of the mixed solution after co-incubation; Figure 7 The bacterial membrane probe in Example 4 is derived from Escherichia coli, and the EV is derived from Staphylococcus aureus, and the particle size and Zeta potential of the mixed solution after co-incubation; Figure 8 In Example 4, both the bacterial membrane probe and EV are derived from Escherichia coli, and the particle size and Zeta potential of the mixed solution after co-incubation; Fig. 9 The bacterial membrane probe in Example 4 is derived from Staphylococcus aureus, and the EV is derived from Escherichia coli, and the particle size and Zeta potential of the mixed solution after co-incubation; Fig.10 The fluorescence images of the EVs derived from Staphylococcus aureus and the bacterial membrane probes derived from Staphylococcus aureus and Escherichia coli before and after co-incubation in Example 4; Fig.11 Fluorescence images of the EVs derived from Escherichia coli and the bacterial membrane probes derived from Staphylococcus aureus and Escherichia coli before and after co-incubation in Example 4; Fig.12 The comparison of the detection sensitivity of the bacterial membrane probe derived from Staphylococcus aureus in Example 5 of the present invention and the gold standard ELISA; Fig.13 The comparison of the detection sensitivity of the bacterial membrane probe derived from Escherichia coli in Example 5 of the present invention and the gold standard ELISA; Fig.14 This is a schematic diagram of the research results on the effective time of bacterial membrane probe in Example 6.

[0015] Marks in the accompanying drawings: bacterial membrane-1; bacterial membrane protein-2; NADPH-3; L-arginine-4; NO fluorescent probe-5; bacterial membrane probe-6; EV-7 carrying membrane proteins of the same type of bacteria as the bacterial membrane probe; EV-8 carrying membrane proteins of a different type of bacteria than the bacterial membrane probe. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the embodiments.

[0017] In the examples of the present invention, Staphylococcus aureus and Escherichia coli were purchased from Ningbo Testo Biotechnology Co., Ltd. RAW264.7 (mouse mononuclear macrophage leukemia cells) were purchased from American Type Culture Collection [(ATCC), Manassas, USA]. Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (Saint Louis, USA). Mouse inducible NO synthase (iNOS; NOS2) quantitative detection kit (ELISA) was purchased from Quanzhou Ruixin Biotechnology Co., Ltd. Unless otherwise specified, other chemicals were purchased from Beyotime (Shanghai, China).

[0018] Example 1: Preparation of bacterial membrane probe like Figure 1 As shown, the bacterial membrane probe prepared in this embodiment is a nano-scale vesicle structure of bacterial membrane encapsulating NADPH, L-arginine and NO fluorescent probes, with a size not exceeding 400 nm. The bacterial membrane is from Staphylococcus aureus (a Gram-positive bacterium).

[0019] When preparing bacterial membrane probes, the concentration of Staphylococcus aureus was 1.5×10 8 CFU / mL, use 2mL of bacterial solution, after the bacterial membrane separation step, finally add 1mL of PBS buffer to resuspend and obtain bacterial membrane solution. Take 1mL of bacterial membrane solution; the concentration of NADPH is 2.5mg / mL, take 50μL; the concentration of L-arginine is 100μg / mL, take 200μL; take 50μL of NO fluorescent probe solution, which contains 1μL of NO fluorescent probe original solution with a concentration of 5mM.

[0020] The specific preparation method of bacterial membrane probe is as follows: Step 1: Prepare a bacterial membrane solution using Gram-positive or Gram-negative bacteria; Staphylococcus aureus is selected in this example; the specific steps are as follows: Step 1.1: Centrifuge the Staphylococcus aureus solution and resuspend the bottom precipitate in PBS buffer; measure the OD value to make the concentration of the Staphylococcus aureus solution 1.5×10 8 CFU / mL; Step 1.2, take 2 mL of bacterial solution, add 6 mg of lysozyme to make the final concentration of lysozyme 3 mg / mL, and incubate at 37 ° C with shaking at 200 rpm for 1 hour; Step 1.3, add 800 μL of lysis buffer (RIPA lysis buffer containing 1% protease phosphatase inhibitor cocktail) and let stand on ice for 20 minutes; Step 1.4, ultrasonically disrupt on ice for 2 minutes; Step 1.5: Centrifuge at 800 g for 15 min at 4°C. Collect the supernatant and centrifuge at 25,000 g for 2 h at 4°C. Take the precipitate, resuspend it in 1 mL PBS, and store it at -80°C.

[0021] Step 2, preparing NADPH solution, L-arginine solution, and NO fluorescent probe solution; Step 2.1) Prepare NADPH solution: Take 2.5 mg of NADPH powder, add 1 mL of PBS, vortex thoroughly in a dark environment until completely dissolved, and divide into 20 tubes to obtain 2.5 mg / mL NADPH solution, 50 μL per tube, and store at -80°C in a dark environment.

[0022] Step 2.2), L-arginine solution: Take 2 mg of L-arginine powder, add 20 mL of PBS, vortex thoroughly until completely dissolved to obtain an L-arginine solution with a concentration of 100 μg / mL, and store at room temperature.

[0023] Step 2. 3), NO fluorescent probe solution: Take 1μL of 5mM NO fluorescent probe (DAF-FM DA) original solution, dilute it in 9μL of DAF-FM DA diluent, then add 40μL of 0.02mol / L sodium hydroxide solution, vortex thoroughly, and stand in the dark for 30 minutes to obtain NO fluorescent probe solution.

[0024] Step 3, adding NADPH solution, L-arginine solution and NO fluorescent probe solution to the bacterial membrane solution, vortexing thoroughly to obtain a mixed solution; In step 3, the volume of the bacterial membrane solution is 1 mL, the volume of the NADPH solution is 50 μL, the volume of the L-arginine solution is 200 μL, and the volume of the NO fluorescent probe solution is 50 μL.

[0025] Step 4: Add a filter membrane with a pore size of 800 nm to the manual liposome extruder, add the mixed solution and fully squeeze it, then replace it with a filter membrane with a pore size of 400 nm and continue to fully squeeze it; Step 5: Centrifuge the squeezed solution at 4°C, 25,000 g for 30 min, resuspend the precipitate with 1 mL of PBS buffer, and centrifuge again under the same conditions to fully remove free NADPH, L-arginine or NO fluorescent probes, then resuspend with 1 mL of PBS buffer to obtain the bacterial membrane probe, and store it at -20°C away from light.

[0026] Example 2: Bacterial membrane probe is used to distinguish whether the bacteria A to be detected is Gram-positive bacteria or Gram-negative bacteria The bacterial membrane probe is used to distinguish whether the bacteria A to be detected is Gram-positive or Gram-negative. The specific steps are as follows: S1. Extracting EVs by phagocytosis of the bacteria A to be detected by macrophages, including the following steps: S1.1. Make macrophages engulf the bacteria A to be detected and polarize to M1 type; the specific operation is as follows: When the macrophages in the culture flask reached 80% confluence, the old culture medium was removed and DMEM high-glucose culture medium containing 10% fetal bovine serum (FBS) was added, followed by 200uL of 1.5×10 8 CFU / mL of the bacterial culture of the to-be-tested bacterium A was placed in a 5% CO 2， After being cultured in a sterile environment at 37°C for about 12 hours and observed under a microscope, M1 macrophages accounted for a larger proportion.

[0027] S1.2. Purification of EVs secreted by M1 macrophages; the specific procedures are as follows: When the proportion of M1 macrophages in the culture flask exceeds 80%, remove the old culture medium and add DMEM high-glucose culture medium without FBS. After standing under UV light for 2 hours, collect the supernatant of the culture medium. After centrifugation at 500g and 4°C, take the supernatant and centrifuge it at 10000g and 4°C for 90 minutes, then take the supernatant and centrifuge it at 100000g and 4°C for 2 hours. The resulting precipitate is EV, which is resuspended in PBS and stored at -80°C.

[0028] S2, the bacterial membrane probe was incubated with PBS buffer, EVs extracted by macrophages phagocytosing known Gram-positive bacteria, EVs extracted by macrophages phagocytosing known Gram-negative bacteria, and EVs extracted in S1, respectively, in a citric acid-sodium citrate buffer environment; Preparation of citric acid-sodium citrate buffer: prepare 0.1 mol / L citric acid solution and 0.1 mol / L sodium citrate solution respectively, then mix 4.1 ml of citric acid solution and 5.9 ml of sodium citrate solution evenly to obtain citric acid-sodium citrate buffer.

[0029] S2 comprises the following steps: randomly select four wells in a black 96-well plate: 25 μL of bacterial membrane probe was dropped into one well, and 75 μL of PBS buffer was used as a blank control group; In the second well, 25 μL of bacterial membrane probe, 25 μL of EVs secreted by macrophages engulfing any Gram-positive bacteria, and 50 μL of citric acid-sodium citrate buffer as a positive control group were dropped; Three wells were dripped with 25 μL of bacterial membrane probe, 25 μL of EVs secreted by macrophages engulfing any Gram-negative bacteria, and 50 μL of citric acid-sodium citrate buffer as a negative control group; 25 μL of bacterial membrane probe, 25 μL of EVs secreted by macrophages that engulfed bacteria A to be detected, and 50 μL of citric acid-sodium citrate buffer were dropped into four wells as the experimental group.

[0030] The 96-well plate was then placed in a thermostatic shaker and shaken at 200 rpm at 37°C for 60 minutes.

[0031] In this process, if the bacterial membrane of the bacterial membrane probe and the bacterial membrane protein on the EV belong to the same type of bacteria, the bacterial membrane probe and EV will fuse and emit a fluorescent signal, such as Figure 2 Otherwise, the two will not undergo membrane fusion and no fluorescent signal will be emitted, as shown in Figure 3 shown.

[0032] After co-incubation with S3, place it in an ELISA reader to detect the intensity of the fluorescent signal and determine the type of bacteria.

[0033] Specifically, S3: placing the 96-well plate after constant temperature oscillation in S2 in an ELISA instrument to detect the fluorescence signal intensity of the above four wells.

[0034] If the bacterial membrane in the bacterial membrane probe in S2 belongs to Gram-positive bacteria, then: 1) When the fluorescence signal intensity of well 4 (experimental group) is significantly higher than that of well 1 (blank control group) and well 3 (negative control group), and is equivalent to that of well 2 (positive control group), the bacteria A to be detected is judged to be Gram-positive bacteria; 2) When the fluorescence signal intensity of well 4 (experimental group) is comparable to that of well 1 (blank control group) and well 3 (negative control group), and is significantly lower than that of well 2 (positive control group), the bacteria A to be detected is judged to be Gram-negative bacteria; If the bacterial membrane in the bacterial membrane probe in S2 belongs to Gram-negative bacteria, then: 1) When the fluorescence signal intensity of well 4 (experimental group) is significantly higher than that of well 1 (blank control group) and well 2 (positive control group), and is equivalent to that of well 3 (negative control group), the bacteria A to be detected is judged to be Gram-negative bacteria; 2) When the fluorescence signal intensity of well 4 (experimental group) is comparable to that of well 1 (blank control group) and well 2 (positive control group), and is significantly lower than that of well 3 (negative control group), the bacteria A to be detected is judged to be Gram-positive bacteria.

[0035] The bacterial membrane probe in S2 of this embodiment is prepared in Example 1. The 96-well plate after constant temperature oscillation in S2 is placed in an ELISA instrument to detect the fluorescence signal intensity of the above four wells. After the fluorescence signal intensity is normalized with the blank control group, the results are as follows: Figure 4 As shown by Figure 4 It can be seen that the fluorescence signal intensity of the experimental group is equivalent to that of the positive control group, and is significantly higher than that of the blank control group and the negative control group. Therefore, it can be determined that the bacteria A to be detected is a Gram-positive bacterium.

[0036] Example 3: Bacterial membrane probe is used to distinguish whether the bacteria B to be detected is Gram-positive bacteria or Gram-negative bacteria The bacteria A to be detected in Example 2 was replaced by bacteria B to be detected, and the rest of the operations were the same as in Example 2. The EVs detected in the experimental group were replaced by EVs secreted by macrophages that engulfed bacteria B to be detected. The bacterial membrane probe in S3 in this example was prepared in Example 1.

[0037] The fluorescence signal intensity of the four wells in Example 3 was detected and normalized to the blank control group. The results are as follows: Figure 5 As shown by Figure 5 It can be seen that the fluorescence signal intensity of the experimental group is comparable to that of the blank control and the negative control, and is significantly lower than that of the positive control, thereby judging that the bacteria B to be detected is a Gram-negative bacterium.

[0038] Example 4: Verification of membrane fusion between bacterial membrane probe and EV According to the method in Example 1, a bacterial membrane probe in which the bacterial membrane was derived from Staphylococcus aureus and a bacterial membrane probe in which the bacterial membrane was derived from Escherichia coli (Gram-negative bacteria) were prepared.

[0039] (1) According to the method in Example 2, bacterial membrane probes from Staphylococcus aureus and EVs extracted from macrophages phagocytosing Staphylococcus aureus were co-incubated in a citric acid-sodium citrate buffer environment, and the particle size and Zeta potential of the bacterial membrane probe, EVs, and the mixed solution (co-incubation) were detected. The results are shown in Figure 6As shown in the figure, both the bacterial membrane probe and EV are derived from Staphylococcus aureus, that is, the two have the same membrane. After co-incubation, the particle size and Zeta potential of the mixed solution were detected, and it was found that the particle size and Zeta potential of the bacterial membrane probe and EV before co-incubation had significant changes. The particle size of the mixed solution was approximately the sum of the particle sizes of the bacterial membrane probe and EV, and the Zeta potential of the mixed solution was between the two, indicating that the bacterial membrane probe and EV with the same membrane can undergo effective membrane fusion.

[0040] (2) According to the method in Example 2, the bacterial membrane probe from Escherichia coli and the EV extracted from macrophages phagocytosing Staphylococcus aureus were co-incubated in a citric acid-sodium citrate buffer environment, and the particle size and Zeta potential of the bacterial membrane probe, EV, and the mixed solution (co-incubation) were detected. The results are as follows Figure 7 As shown in the figure, the bacterial membrane probe is derived from Escherichia coli, and the EV is derived from Staphylococcus aureus, that is, the two have non-isotypic membranes. After co-incubation, the particle size and Zeta potential of the mixed solution were detected, and it was found that the particle size and Zeta potential of the mixed solution were not significantly changed compared with the bacterial membrane probe, indicating that the bacterial membrane probe with non-isotypic membrane cannot undergo effective membrane fusion with EV.

[0041] (3) According to the method in Example 2, the bacterial membrane probe from Escherichia coli and the EV extracted from Escherichia coli phagocytosed by macrophages were co-incubated in a citric acid-sodium citrate buffer environment, and the particle size and Zeta potential of the bacterial membrane probe, EV, and the mixed solution (co-incubation) were detected. The results are as follows Figure 8 As shown in the figure, the bacterial membrane probe and EV are both derived from Escherichia coli, that is, the two have the same membrane. After co-incubation, the particle size and Zeta potential of the mixed solution were detected, and it was found that the particle size and Zeta potential of the bacterial membrane probe and EV before co-incubation had changed significantly. The particle size of the mixed solution was approximately the sum of the particle sizes of the bacterial membrane probe and EV, and the Zeta potential of the mixed solution was between the two, indicating that the bacterial membrane probe and EV with the same membrane can undergo effective membrane fusion.

[0042] (4) According to the method in Example 2, bacterial membrane probes from Staphylococcus aureus and EVs extracted from Escherichia coli phagocytosed by macrophages were co-incubated in a citric acid-sodium citrate buffer environment, and the particle size and Zeta potential of the bacterial membrane probe, EVs, and the mixed solution (co-incubation) were detected. The results are shown in Fig. 9 As shown, the bacterial membrane probe is derived from Staphylococcus aureus, and the EV is derived from Escherichia coli, that is, the two have non-isotypic membranes. After co-incubation, the particle size and Zeta potential of the mixed solution were detected, and it was found that the particle size and Zeta potential of the mixed solution were not significantly changed compared with the bacterial membrane probe, indicating that the bacterial membrane probe with non-isotypic membrane cannot undergo effective membrane fusion with EV.

[0043] Figures 6 to 9 In, G +For Staphylococcus aureus, G - For Escherichia coli. Figures 6 to 9 It can be seen that the particle size and Zeta potential of the bacterial membrane probe with the same type of membrane changed significantly after incubation with EV, while the particle size and Zeta potential of the bacterial membrane probe with non-isotype membrane did not change significantly after incubation with EV, which proves that the bacterial membrane probe with the same type of membrane can undergo membrane fusion with EV, while the bacterial membrane probe with non-isotype membrane cannot undergo effective membrane fusion with EV.

[0044] (5) The bacterial membrane probe was stained with a cell membrane red fluorescent probe (DiI), and the EV was stained with a cell membrane green fluorescent probe (DiO), and co-incubated according to the method in Example 2 to investigate the fusion of the bacterial membrane probe and the EV when they were homotypic or heterotypic membranes. The results are shown in Figure 2. Fig.10 and Fig.11 As shown, G + For Staphylococcus aureus, G - For Escherichia coli.

[0045] Fig.10 In the figure, EV originates from Staphylococcus aureus. If the bacterial membrane probe also originates from Staphylococcus aureus, yellow fluorescence will appear when the fluorescence images of the two are merged by the fluorescence photograph method, indicating that membrane fusion of the bacterial membrane probe and EV has occurred. If the bacterial membrane probe originates from Escherichia coli, no yellow fluorescence will be observed when the fluorescence images of EV and the bacterial membrane probe are merged by the fluorescence photograph method, indicating that effective membrane fusion has not occurred between the two.

[0046] Fig.11 In the figure, EV originates from Escherichia coli. If the bacterial membrane probe also originates from Escherichia coli, yellow fluorescence will appear after the fluorescence images of the two are merged by the fluorescence photograph method, indicating that membrane fusion has occurred between the bacterial membrane probe and EV. If the bacterial membrane probe originates from Staphylococcus aureus, no yellow fluorescence will be observed after the fluorescence images of the two are merged by the fluorescence photograph method, indicating that effective membrane fusion has not occurred between the two.

[0047] Depend on Fig.10 and Fig.11 It can be seen that the fluorescence image of the bacterial membrane probe and EV with the same membrane after co-incubation showed yellow fluorescence after being processed by the Merge method of fluorescence photographs, while the fluorescence image of the bacterial membrane probe and EV with non-isotypic membrane showed no yellow fluorescence after being processed by the Merge method of fluorescence photographs, further proving that the bacterial membrane probe and EV with the same membrane can undergo membrane fusion, while the bacterial membrane probe and EV with non-isotypic membrane cannot undergo effective membrane fusion.

[0048] Example 5: Comparison of detection performance between bacterial membrane probe and gold standard ELISA Fig.12 To make the number 1×10 9EVs extracted from macrophages phagocytosing Staphylococcus aureus were diluted to obtain a series of EV solutions with different quantity gradients. All samples were equally divided into two parts, one for incubation with bacterial membrane probe (bacterial membrane from Staphylococcus aureus) to detect fluorescence signals, and the other for detection using mouse inducible NO synthase (iNOS; NOS2) quantitative detection kit (ELISA) according to the manufacturer's instructions.

[0049] Fig.13 To make the number 1×10 9 EVs extracted from Escherichia coli were phagocytosed by macrophages, and a series of EV solutions with gradient quantity were obtained by dilution. All samples were equally divided into two parts, one for incubation with bacterial membrane probe (bacterial membrane from Escherichia coli) to detect fluorescence signal, and the other for detection using mouse inducible NO synthase (iNOS; NOS2) quantitative detection kit (ELISA) according to the manufacturer's instructions.

[0050] Fig.12 and Fig.13 The comparison of the detection performance of the present invention and the gold standard ELISA is shown in the figure. The horizontal axis represents the total number of EVs, and the vertical axis represents the normalized signal response. Fig.12 and Fig.13 It can be seen that the sensitivity of the present invention is improved by an order of magnitude compared to that of ELISA.

[0051] Example 6: Study on the effective time of bacterial membrane probe Homotypic membrane group: The bacterial membrane probe derived from Staphylococcus aureus was stored at -20°C for 0, 5, 10, 15, 20, 25, and 30 days, and then incubated with EVs extracted from macrophages phagocytosing Staphylococcus aureus and the fluorescence signal was detected according to the method in Example 2.

[0052] Non-isotypic membrane group: The bacterial membrane probes from Staphylococcus aureus were stored at -20°C for 0, 5, 10, 15, 20, 25, and 30 days, and incubated with EVs extracted from Escherichia coli phagocytosed by macrophages according to the method in Example 2 to detect the fluorescence signal.

[0053] Control group: The bacterial membrane probe derived from Staphylococcus aureus was stored at -20°C for 0, 5, 10, 15, 20, 25, and 30 days, and then the fluorescence signal was detected.

[0054] The results are as follows Fig.14 As shown, Fig.14 The horizontal axis represents the storage time of the bacterial membrane probe at -20°C, and the vertical axis represents the normalized signal response; Fig.14It can be seen that after the bacterial membrane probe was stored for 30 days and then tested, there was still a significant difference in the fluorescence signal between the isotype membrane group and the non-isotype membrane group, indicating that the bacterial membrane probe of the present invention can be stored at -20°C for about a month.

[0055] The bacterial membrane probe prepared by the present invention detects bacterial types by utilizing the principle that biological membranes can specifically target homotypic cells and their derivatives for selective fusion; by selectively fusing the bacterial membrane probe with extracellular vesicles (EVs), not only the types of Gram-pathogenic bacteria can be distinguished, but also a feasible solution is provided for specific in situ detection of the target EV-iNOS, which can realize rapid, highly sensitive and highly stable detection of the target EV-iNOS, improve the detection efficiency of EV-iNOS, and provide a basis for the subsequent specific application of EV-iNOS derived from M1 macrophages in the field of disease diagnosis, and has high clinical application value.

[0056] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention.

Claims

1. A bacterial membrane probe, characterized in that: The bacterial membrane probe is a nano-scale vesicle structure in which the bacterial membrane encapsulates NADPH, L-arginine and NO fluorescent probes, wherein the bacterial membrane is from Gram-positive bacteria or Gram-negative bacteria.

2. The bacterial membrane probe according to claim 1, characterized in that The preparation method of the bacterial membrane probe is as follows: Step 1: preparing a bacterial membrane solution using bacteria known to be Gram-positive or Gram-negative; Step 2, preparing NADPH solution, L-arginine solution, and NO fluorescent probe solution; Step 3, adding NADPH solution, L-arginine solution and NO fluorescent probe solution to the bacterial membrane solution, vortexing thoroughly to obtain a mixed solution; Step 4: Add a filter membrane with a pore size of 800 nm to the manual liposome extruder, add the mixed solution and fully squeeze it, then replace it with a filter membrane with a pore size of 400 nm and continue to fully squeeze it; Step 5: Centrifuge the squeezed solution at 25,000 g, and resuspend the precipitate with PBS buffer; centrifuge the resuspended solution at 25,000 g, and resuspend the precipitate with PBS buffer to obtain a bacterial membrane probe.

3. The bacterial membrane probe according to claim 2, characterized in that: The preparation method of the bacterial membrane solution in step 1 is as follows: 2 mL of Gram-positive bacteria or Gram-negative bacteria with a bacterial solution concentration of 1.5×10 8 CFU / mL, and then treated with lysozyme, treated with lysis buffer, ultrasonically disrupted, centrifuged at 800 g to collect the supernatant, centrifuged at 25,000 g to obtain the precipitate, and resuspended in 1 mL of PBS to obtain the bacterial membrane solution; The NADPH solution in step 2 is obtained by dissolving NADPH powder in PBS, with a concentration of 2.5 mg / mL; The L-arginine solution in step 2 is obtained by dissolving L-arginine in PBS, with a concentration of 100 μg / mL; The preparation method of the NO fluorescent probe solution in step 2 is as follows: take 1 μL of 5 mM NO fluorescent probe original solution, dilute it in 9 μL of DAF-FM DA diluent, add 40 μL of 0.02 mol / L sodium hydroxide solution to obtain the NO fluorescent probe solution.

4. The bacterial membrane probe according to claim 2, characterized in that In the step 3, the volume of the bacterial membrane solution is 1 mL, the volume of the NADPH solution is 50 μL, the volume of the L-arginine solution is 200 μL, and the volume of the NO fluorescent probe solution is 50 μL.

5. A method for distinguishing Gram-positive and Gram-negative bacterial infections, characterized in that: The detection method utilizes the bacterial membrane probe described in any one of claims 1 to 4 to distinguish between Gram-positive bacteria and Gram-negative bacteria infections.

6. The detection method according to claim 5, characterized in that: The steps include: S1. Extract extracellular vesicles by phagocytosis of the bacteria to be tested by macrophages; S2, the bacterial membrane probe was co-incubated with PBS buffer, extracellular vesicles extracted by macrophage phagocytosis of known Gram-positive bacteria, extracellular vesicles extracted by macrophage phagocytosis of known Gram-negative bacteria, and extracellular vesicles extracted in S1 in the environment of citric acid-sodium citrate buffer, and recorded as blank control group, positive control group, negative control group and experimental group respectively; S3. After co-incubation, place the sample in an ELISA instrument to detect the intensity of the fluorescent signal and determine the type of bacteria to be detected by comparison.

7. The detection method according to claim 6, characterized in that: The method for determining the type of bacteria to be detected by comparison in S3 is as follows: If the bacterial membrane in the bacterial membrane probe in S2 belongs to Gram-positive bacteria, then: If the fluorescence signal of the experimental group is higher than that of the blank control group and the negative control group, and is equivalent to that of the positive control group, the cells to be tested are judged to be Gram-positive bacteria; If the fluorescence signal of the experimental group is comparable to that of the blank control group and the negative control group, and lower than that of the positive control group, the cells to be tested are judged to be Gram-negative bacteria; If the bacterial membrane in the bacterial membrane probe in S2 belongs to Gram-negative bacteria, then: If the fluorescence signal of the experimental group is higher than that of the blank control group and the positive control group, and is equivalent to that of the negative control group, the cells to be tested are judged to be Gram-negative bacteria; If the fluorescence signal of the experimental group is comparable to that of the blank control group and the positive control group, and lower than that of the negative control group, the cells to be detected are judged to be Gram-positive bacteria.

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