A detection method for food-borne pathogenic bacteria based on composite nanomaterials
By utilizing the composite nanomaterial PB@DPA-Ce-GMP@Van biosensor system and fluorescence ratio signal output, the problems of high cost and susceptibility to environmental interference in the detection of foodborne pathogens have been solved, achieving rapid detection with high sensitivity and low cost.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HENAN AGRICULTURAL UNIVERSITY
- Filing Date
- 2023-06-16
- Publication Date
- 2026-05-12
AI Technical Summary
Existing methods for detecting foodborne pathogens are costly, complex to operate, and their signals are easily affected by external environmental interference, making it difficult to meet the needs for rapid and accurate on-site testing.
A biosensor system based on the composite nanomaterial PB@DPA-Ce-GMP@Van was used. Detection was performed by combining fluorescence ratio signal output with fluorescence intensity ratio. After incubation of PB@DPA-Ce-GMP@Van with Gram-positive bacteria to form a complex, quenching SC fluorescence and enhancing AR fluorescence, bacterial concentration was analyzed using a linear regression equation.
It achieves highly sensitive detection of foodborne pathogens, with a detection range of 101~107 CFU/mL and a detection limit of 5 CFU/mL, reducing detection costs and improving detection accuracy and speed.
Smart Images
Figure CN116660232B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for detecting foodborne pathogens, specifically a method for detecting foodborne pathogens based on composite nanomaterials, belonging to the field of food safety testing technology. Background Technology
[0002] Foodborne pathogens are pathogenic bacteria that spread through food. They can adhere to food by forming biofilms and survive for extended periods on surfaces in contact with food, leading to food contamination, reduced product quality and shelf life, and potential disease transmission. Food contamination by foodborne pathogens (such as Salmonella, Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus) has developed into a major public health problem, causing significant economic losses worldwide.
[0003] Traditional plate counting methods, with their good specificity and reliability, are widely recognized as the standard method for bacterial detection. However, they typically require time-consuming culturing and laborious separation, as well as stringent laboratory conditions. While detection using large-scale instruments, including gas chromatography, liquid chromatography, and atomic absorption spectrometry, can achieve highly sensitive quantitative detection of target bacteria in a shorter time, these methods are prone to false positives and require relatively expensive equipment and complex operations, making on-site testing impossible. These methods are no longer sufficient to meet the needs of rapid, large-scale food testing and rapid on-site testing of food products both domestically and internationally.
[0004] Effective detection of pathogens in food is a necessary prerequisite for the timely prevention and control of foodborne outbreaks. Currently, fluorescence-based detection technologies have shown great potential in rapid food safety testing due to their advantages such as speed, convenience, ease of operation, visualization, and high sensitivity. The emergence of nanomaterials has further advanced the application of fluorescence methods, with a series of inorganic or organic composite nanomaterials being used as fluorescent biosensors for bacterial detection. For example, the invention patent with publication number CN109856389A discloses the preparation of quantum dot-based magnetic nanoparticles and their application in the detection of multiple foodborne pathogens. This involves combining functionalized magnetic nanoparticles and fluorescently labeled molecules with target pathogens, and antibodies in the functionalized magnetic nanoparticles and antibodies or aptamers in the fluorescently labeled molecules binding to antigens in the target pathogens. Optical detection is performed using a fluorescence spectrometer, determining the type of pathogen by emission wavelength and detecting the quantity of pathogens by emission intensity, thus enabling the detection of multiple foodborne pathogens in food samples. However, the aforementioned patents not only use expensive antibodies as recognition elements, but also only correlate on a single signal channel. This makes them susceptible to factors such as instrument parameters, the microenvironment surrounding the bacteria, changes in the local concentration of the probe, and the excitation light source, which can lead to false positive or false negative results.
[0005] Therefore, there is an urgent need for a detection method for foodborne pathogens based on composite nanomaterials to solve problems such as high detection costs, complex operation, and susceptibility of signals to external environmental interference. Summary of the Invention
[0006] The purpose of this invention is to provide a method for detecting foodborne pathogens based on composite nanomaterials, which has good biocompatibility, high sensitivity, strong anti-interference ability, simple detection operation, low cost, and good linear range and detection limit of the detection results, in order to solve the problems existing in the prior art.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: a method for detecting foodborne pathogens based on composite nanomaterials, comprising the following steps:
[0008] S1. Preparation of test bacterial suspensions for foodborne pathogens:
[0009] Gram-positive bacteria were collected from cryopreservation tubes using an inoculation loop and cultured using the streak inoculation method. After serial dilution, bacterial concentrations ranging from 0 to 10⁻⁶ were obtained. 7 CFU / mL of the test bacterial solution;
[0010] S2. Synthesis of biosensor system material PB@DPA-Ce-GMP@Van:
[0011] Using PB at a concentration of 0.1~0.3 mg / mL as a carrier, after ultrasonic dissolution, it was added to a mixed solution of GMP and Ce(CH3COO)3. After mixing with a mixer, the solution was centrifuged, the supernatant was removed, and the solution was washed with deionized water and reconstituted with deionized water. After mixing evenly, DPA was added. When the color turned into a clear blue, the solution was irradiated under ultraviolet light for 2~4 hours and then freeze-dried under vacuum for later use to obtain PB@DPA-Ce-GMP solution.
[0012] The precipitate was then redissolved in deionized water, and vancomycin (Van) at a concentration of 10-20 μg / mL was added. After ultrasonic treatment, the biosensor system material PB@DPA-Ce-GMP@Van, abbreviated as PCV, was finally obtained.
[0013] S3. Detection of foodborne pathogens:
[0014] The biosensor system material PB@DPA-Ce-GMP@Van synthesized in step S2 above is supplemented with bacteria at a concentration of 0~10 from step S1 above. 7 A CFU / mL culture of Gram-positive bacteria was incubated at room temperature for 2 hours. After centrifugation, the supernatant was collected and incubated to form a PB@DPA-Ce-GMP@Van / Gram-positive bacteria complex.
[0015] In the supernatant, PB@DPA-Ce-GMP@Van quenched the fluorescence of SC and increased the fluorescence intensity of AR. The fluorescence intensity ratio SC / AR was used as the detection signal output, with the fluorescence response of the sensing system as the ordinate and the bacterial concentration of Gram-positive bacteria as the abscissa. The detection data were processed and then linearly fitted to obtain the final linear regression equation.
[0016] In step S1, the specific process of bacterial culture is as follows: streak the bacterial culture medium on nutrient agar medium, incubate at 37°C for 24-48 hours, pick out colonies with good morphology and inoculate them in LB broth with shaking culture, wash with PBS buffer and centrifuge, resuspend in sterile Tris-HCl buffer, take 100 μL of bacterial suspension and plate it for plate counting, and use a UV-Vis spectrophotometer to measure the optical density value (OD) of the bacterial suspension at a wavelength of 600 nm to determine the corresponding bacterial concentration.
[0017] In step S2, the synthesis method of PB is as follows: polyvinylpyrrolidone and potassium ferricyanide are added to hydrochloric acid and stirred at room temperature for 0.5-1 h. Then, the mixture is heated in an oil bath at 80-90°C for 20-25 h. The solution color changes from yellow to blue. After heating, the mixture is cooled to 40-50°C. The reaction solution is centrifuged and the precipitate is washed multiple times with ethanol and water. Finally, the obtained PB is vacuum dried and stored at 4°C for later use.
[0018] In step S2, preferably, the concentration of PB is 0.1 mg / mL, the ultraviolet irradiation time is 2.5 h, and the concentration of vancomycin is 10 μg / mL.
[0019] In step S3, when the Gram-positive bacterium is selected as Staphylococcus aureus, the final linear regression equation of S. aureus bacterial concentration and fluorescence response is: y=0.2151x+0.3117, R²=0.983, where R² represents the correlation coefficient between variables, and the larger the value, the better the fit.
[0020] The beneficial effects of this invention are:
[0021] 1) This invention has high sensitivity for detecting Gram-positive bacteria, and the detection bacterial concentration range can reach 10. 1 ~10 7 With a detection limit of 5 CFU / mL, the linear range is wider and the detection limit is lower than that of many other published bacterial detection methods.
[0022] 2) The PB@DPA-Ce-GMP@Van prepared by the method of the present invention has good dispersibility and uniform particle size distribution. It is a novel composite nanosensor based on lanthanide elements. It has the advantages of simple synthesis method, high sensitivity and low cost. Compared with the antigen targeting technology used in the disclosed enzyme-linked immunosorbent assay, the bacterial targeting material used is relatively inexpensive and has good economic benefits.
[0023] 3) Compared to most developed fluorescence analyses that are usually correlated on a single signal channel and are easily affected by factors such as instrument parameters, the microenvironment around the bacteria, changes in the local concentration of the probe, and the excitation light source, resulting in false positive or false negative results, the method of this invention uses fluorescence ratio as the output signal and reads the signal based on reports from at least two signal channels, providing a reliable way to reduce interference, improve detection accuracy, and enable rapid and sensitive detection of Gram-positive bacteria. Attached Figure Description
[0024] Figure 1 This is a schematic diagram illustrating the synthesis and detection principle of the biosensing system of the present invention;
[0025] Figure 2 The diagram shows the catalytic effect of PCV containing different final concentrations of PB on TMB in the embodiments of the present invention.
[0026] Figure 3 The diagram shows the catalytic effect of PC on TMB under different UV irradiation times in the embodiments of the present invention.
[0027] Figure 4 The diagram shows the antibacterial effect of different concentrations of Van on Staphylococcus aureus in the embodiments of the present invention.
[0028] Figure 5 The TEM, UV-Vis, FT-IR, and XRD characterization spectra of the PCV prepared in the embodiments of the present invention are shown below.
[0029] Figure 6 This is a graph showing the oxidase-like activity evaluation data of the PCV / S prepared in the embodiments of the present invention;
[0030] Figure 7 The spectral data of the PCV prepared in this embodiment of the invention for detecting Staphylococcus aureus are shown in the following experimental data.
[0031] Figure 8 This is a graph showing the optimized experimental conditions for detecting Staphylococcus aureus using the biosensor system prepared in this embodiment of the invention.
[0032] Figure 9 The fluorescence curves and linear equations of the biosensing system prepared in this embodiment of the invention are shown as the fluorescence intensity ratio changes with the concentration of Staphylococcus aureus.
[0033] Figure 10 This is a graph showing the specificity of the biosensor system prepared in this embodiment of the invention for detecting Staphylococcus aureus. Detailed Implementation
[0034] The present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments.
[0035] The instruments used in the following examples include: transmission electron microscope (TEM), scanning electron microscope (SEM), ultraviolet spectrophotometer (UV-Vis), Fourier transform infrared spectrometer (FT-IR), and X-ray diffractometer (XRD). Staphylococcus aureus was selected as the model bacterium in the experiment. The final concentration of PCV reacting with Staphylococcus aureus was 200 μg / mL, and the final concentrations of SC and AR were 5 μM.
[0036] like Figure 1 The diagram shows the synthesis and detection principle of the biosensor system. (a) Synthesis process of PCV; (b) Detection principle diagram; After incubation with bacteria, the fluorescence of SC recovers, while the fluorescence of AR weakens.
[0037] Example: This invention provides a method for detecting foodborne pathogens based on composite nanomaterials, comprising the following steps:
[0038] S1. Preparation of test bacterial suspensions for foodborne pathogens:
[0039] S. aureus strains were isolated and cultured in the laboratory. S. aureus inoculum was collected from cryopreserved tubes using an inoculation loop and cultured using the streak plating method. The inoculum was streaked onto nutrient agar medium and incubated at 37°C for 24–48 h. Morphologically sound colonies were picked and inoculated into LB broth with shaking at 200 rpm for 12 h. After washing with PBS buffer and centrifugation, the colonies were resuspended in sterile 10 mmol / L Tris-HCl buffer (pH 7.5). 100 μL of the bacterial suspension was plated for plate counting. The optical density (OD) of the bacterial suspension at 600 nm was measured using a UV-Vis spectrophotometer to determine the corresponding bacterial concentration, which was approximately 10⁻⁶. 7 When CFU / mL was obtained, the corresponding OD value was approximately 0.2. After serial dilution, bacterial concentrations ranged from 0 to 10. 7 The test bacterial suspension at CFU / mL.
[0040] S2. Synthesis of biosensor system material PB@DPA-Ce-GMP@Van:
[0041] Synthesis of PB: 6 g of polyvinylpyrrolidone and 0.22 g of potassium ferricyanide were added to 80 mL of 0.01 mol / L hydrochloric acid and stirred at room temperature for 1 h. Then, the mixture was heated in an oil bath at 80 °C for 20 h. The solution color changed from yellow to blue. After heating, the mixture was cooled to 40-50 °C. The reaction solution was centrifuged at 11000 rpm for 10 min and the precipitate was washed several times with ethanol and water. Finally, the obtained PB was vacuum dried at 60 °C for 24 h and stored at 4 °C for later use.
[0042] Using PB at a concentration of 0.1~0.3 mg / mL as a carrier, after ultrasonic dissolution, 4 mL was taken into a 50 mL centrifuge tube, and a mixed solution of 10 mM GMP and 10 mM Ce(CH3COO)3 was added. The mixture was shaken and mixed for 10 s, then centrifuged at 8000 rpm for 15 min. The supernatant was removed, and the solution was washed with deionized water and reconstituted with 22 mL of deionized water. After mixing evenly, 10 mM DPA was added. When the color turned into a clear blue, the solution was irradiated under ultraviolet light for 2~4 h, and then freeze-dried under vacuum for later use to obtain PB@DPA-Ce-GMP solution.
[0043] The precipitate was then redissolved in deionized water, and vancomycin (Van) at a concentration of 10-20 μg / mL was added. After ultrasonic treatment for 30 min, the biosensor system material PB@DPA-Ce-GMP@Van was finally obtained.
[0044] S3. Detection of foodborne pathogens:
[0045] The biosensor system material PB@DPA-Ce-GMP@Van synthesized in step S2 above is supplemented with bacteria at a concentration of 0~10 from step S1 above. 7 S. aureus bacterial culture of CFU / mL was incubated at room temperature for 2 h, centrifuged and the supernatant was collected. After incubation, PCV / S complex was formed.
[0046] PCV in the supernatant quenches the fluorescence of SC and increases the fluorescence intensity of AR. The fluorescence intensity ratio SC / AR is used as the detection signal output, with the fluorescence response of the sensing system as the ordinate and the concentration of S. aureus bacterial solution as the abscissa. The detection data are processed and then linearly fitted to obtain the final linear regression equation.
[0047] Experimental Results Verification and Analysis:
[0048] 1. Using 3,3',5,5'-tetramethylbenzidine (TMB) as a catalytic substrate, the catalytic effect of PCV containing different final concentrations of PB on TMB was verified.
[0049] Experimental results are as follows Figure 2As shown, the changes in OxTMB absorbance at 650 nm wavelength of PCV prepared by adding different final concentrations of PB were used to verify the enzyme-like activity of PCV. It can be seen from the figure that the catalytic performance is good and basically stable when the final concentration of PB is 0.1~0.3 mg / mL. Therefore, the final concentration of PB added in the experiment was 0.1 mg / mL.
[0050] 2. Using TMB as a catalytic substrate, the catalytic effect of PC on TMB under different UV irradiation times was verified.
[0051] Experimental results are as follows Figure 3 As shown, the enzyme-like activity of PC prepared by irradiation for different times was verified by the change of OxTMB absorbance at a wavelength of 650 nm. When the irradiation time was 2.5 h, PC showed excellent enzyme-like activity. The enzyme-like activity remained basically unchanged with the increase of time, indicating that irradiation for 2.5 h had achieved good enzyme-like activity. Therefore, 2.5 h was selected as the UV irradiation time in the experiment.
[0052] 3. Verification shows that during the detection process, vancomycin, as an antibiotic, only serves as a recognition unit and will not affect the detection of Staphylococcus aureus.
[0053] Experimental results are as follows Figure 4 As shown in the figure, it can be seen that when the amount of vancomycin added exceeds 20 g / mL, it has an inhibitory effect on Staphylococcus aureus. When the amount of vancomycin added in the preparation material is selected as 10 g / mL, it is far below the minimum inhibitory level, so it will not affect the detection.
[0054] 4. The prepared PCV was characterized by TEM, UV-Vis, FT-IR, and XRD, respectively:
[0055] Its characterization spectrum is as follows Figure 5 As shown, (a) TEM images of PB, Ce, and PCV; (b) UV spectra of PB, Ce, and PCV; (c) FT-IR spectra of Ce, PB, and PCV; and (d) XRD patterns of Ce, PB, and PCV.
[0056] TEM image ( Figure 5 a) The results show that PB has a typical cubic shape, and the network structure formed by the coordination of DPA and GMP molecules with Ce is consistent with the existing disclosed structures. When PB is composited to obtain a PC nanostructure, the vancomycin peak is at 279 nm. The corresponding peak did not appear in the synthesized composite material, possibly due to insufficient doping.
[0057] The corresponding peaks for Ce and PB appear at 360 nm and 680 nm of PCV, respectively. Figure 5 b). Fourier transform infrared spectroscopy analysis of PB, Ce, and PCV ( Figure 5 c). Ce spectrum at 1603 cm⁻¹ -1 The peak at that position is contributed by pyridine nitrogen and participates in the reaction between DPA and Ce. 3 + / 4+ Coordination. 2091 cm⁻¹ in the PB spectrum. -1 The peak at [location] is a characteristic peak of PB caused by the vibration of the cyano bond group. The tensile vibration peak appears at 3144 cm⁻¹. -1 The peak at this location represents the stretching vibration of water molecules. The X-ray diffraction pattern of PB shows typical high-intensity peaks at positions of 17.32°, 24.76°, and 35.16°, which correspond to the (200), (220), and (400) planes in the PB crystal structure, respectively. These peaks conform to all characteristic peaks and indicate good crystallinity.
[0058] The Ce complex did not show strong diffraction peaks. Figure 5 (d) This is because the crystallinity is disrupted after DPA and GMP coordinate with lanthanide ions. The results show that this simple method successfully synthesizes PCV.
[0059] 5. Verification of oxidase-like activity:
[0060] 3,3',5,5'-Tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) were used as catalytic substrates. Different concentrations of Ce and PCV were placed in Tris-HCl buffer (10 mM, pH 6), and TMB and OPD (100 mM) were added respectively to a final volume of 1 mL. After mixing the solutions and letting them stand for 30 s, the UV absorption spectra of TMB and OPD were recorded using a UV-Vis spectrophotometer.
[0061] The results are as follows Figure 6 As shown, (a) are UV spectra of TMB and OPD solutions oxidized by different concentrations of Ce; (b) are UV spectra of TMB and OPD solutions oxidized by different concentrations of PCV.
[0062] Without the addition of DPA-Ce-GMP, both TMB and OPD are colorless and exhibit no obvious absorption peaks. However, under the catalysis of DPA-Ce-GMP, TMB is oxidized to oxTMB, and OPD is oxidized to oxOPD. Similarly, without the addition of PCV, both TMB and OPD are colorless and exhibit no obvious absorption peaks. However, under the catalysis of PCV, TMB is oxidized to blue oxTMB, and OPD is oxidized to yellow oxOPD. Furthermore, with the addition of different concentrations of PCV, the characteristic absorption peaks gradually increase; the characteristic absorption peak of oxTMB at 650 nm gradually exhibits a blue shift, and the blue color of the solution gradually deepens; the characteristic absorption peak of oxOPD at 450 nm gradually increases, and the yellow color of the solution significantly increases. This result fully demonstrates that the combination of PB and vancomycin does not affect the oxidative mimicry ability of Ce, and the synthesized PCV possesses excellent oxidative mimicry ability.
[0063] 6. Feasibility verification of the detection principle:
[0064] 10g of the obtained Staphylococcus aureus suspension 7 CFU / mL diluted to 10 4 CFU / mL, and then samples were added in the following groups: 1) Mix different concentrations of Staphylococcus aureus with PCV aqueous solution with a final concentration of 200 μg / mL, vortex to mix, incubate for 30 min, and centrifuge to collect the supernatant; 2) Only Staphylococcus aureus suspension; 3) Only PCV with a final concentration of 200 μg / mL.
[0065] More than 100 μL of sample was added to Tris-HCl buffer (pH 8, 10 mmol / L) containing AR and SC. The final concentration of AR and SC was 5 μM and the final volume was 1 mL. After incubation for 2 h, the fluorescence intensity changes of SC and AR in the mixed solution were recorded using a fluorescence spectrophotometer.
[0066] The results are as follows Figure 7 As shown, the fluorescence spectra of SC (a, curve 1) and AR (b, curve 1) in the presence of Staphylococcus aureus (curve 2), the fluorescence spectra of SC-PCV before (curve 3) and after (curve 4) culture with Staphylococcus aureus, and the fluorescence spectra of AR-PCV before (curve 3) and after (curve 4) culture with Staphylococcus aureus.
[0067] SC exhibits fluorescence ( Figure 7 a, curve 1), unaffected by Staphylococcus aureus ( Figure 7 a, curve 2), but can be quenched by the prepared PCV ( Figure 7a, Curve 3). After incubation with Staphylococcus aureus, PCV targets Staphylococcus aureus through Van interaction with the bacterial cell membrane. Therefore, when the mixture is centrifuged to remove bacteria, the amount of PCV in the suspension decreases. After incubation with Staphylococcus aureus, PCV targets Staphylococcus aureus through Van interaction with the bacterial cell membrane. Therefore, when the mixture is centrifuged to remove bacteria, the amount of PCV in the suspension decreases, and then the fluorescence of SC recovers ( Figure 7 a, Curve 4).
[0068] Unlike the fluorescence response of SC, AR is a non-fluorescent substrate. Figure 7 b, Curve 1). Staphylococcus aureus had no significant effect on AR fluorescence ( Figure 7 b, curve 2). However, AR can be oxidized by PCV to a fluorescent substrate ( Figure 7 b, Curve 3). After the prepared composite nanomaterials were co-cultured with Staphylococcus aureus bacteria, AR could still be oxidized into fluorescent substrates by residual PCV in the supernatant ( Figure 7 (b, curve 4). Therefore, we constructed a ratiometric fluorescence sensing system with SC and AR as dual-channel readouts for bacterial detection.
[0069] 7. Optimize the pH of the buffer solution, the amount of catalyst, and the reaction time in the reaction system:
[0070] By comparing the SC at 465 nm (FI) in Tris-HCl buffer solutions of different pH values in the presence of PCV solution. 465 ) and AR at 590nm (FI 590 The ratio of fluorescence intensity of SC at 465 nm to that of SC in Tris-HCl buffer solutions of different pH values in the absence of PCV solution (FI) 465 ) and AR at 590nm (FI 590 The difference in fluorescence intensity ratio (ΔFI) 465 / △FI 590 The optimal pH in the reaction system was studied.
[0071] After incubating PCV with Staphylococcus aureus and centrifuging, different volumes of supernatant were used to oxidize SC at 465 nm (FI). 465 ) and AR at 590nm (FI 590 The change in the ratio of fluorescence intensity (FI) 465 / FI 590 The optimal volumetric amount of PCV in the reaction system was investigated.
[0072] The results are as follows Figure 8 As shown, (a) the fluorescence ratio ΔFI of buffer solutions with different pH values. 465 / △FI590 (b) Effects of different incubation times on fluorescence ratio FI 465 / FI 590 Effects of different PCV volumes on fluorescence ratio FI; (c) Effects of different PCV volumes on fluorescence ratio FI 465 / FI 590 The impact;
[0073] As pH increases (from 4 to 8), ΔFI 465 / △FI 590 As the strength increases and the pH value reaches 8, ΔFI 465 / △FI 590 The strength reaches its maximum, and when the pH value continues to increase, ΔFI 465 / △FI 590 The intensity decreases (e.g.) Figure 8 a). Therefore, in order to obtain the lowest detection signal background, we chose Tris-HCl buffer solution with pH 8 as the optimal reaction pH.
[0074] As time goes on, FI 465 / FI 590 The ratio gradually decreased and stabilized after 2 hours (e.g., Figure 8 b). Therefore, 2h was chosen as the optimal catalytic time for the reaction between PCV and SC and AR to ensure the stability of the reaction system.
[0075] As the size of PCV gradually increases, FI 465 / FI 590 Gradually decrease until the volume is 100 μL, FI 465 / FI 590 The basic trend is towards stability (e.g.) Figure 8 b). Therefore, we selected 100 μL as the optimal amount of PCV for subsequent experiments to improve the sensitivity of the reaction system.
[0076] 8. The detection data were processed with the fluorescence response of the sensing system as the ordinate and the concentration of Staphylococcus aureus as the abscissa, and then linear fitting was performed to obtain the linear regression equation.
[0077] 0 to 10 7 After incubating CFU / mL Staphylococcus aureus with PCV solution, centrifuging was performed. The supernatant was then incubated with a mixed solution of SC, AR, and Tris-HCl. The fluorescence spectrum of the reaction system was measured using a fluorescence spectrophotometer.
[0078] The results are as follows Figure 9As shown, the fluorescence curves and linear equations of the fluorescence intensity ratio of the biosensor system as a function of Staphylococcus aureus concentration are presented. (a) Changes in the SC fluorescence spectrum caused by the supernatant after incubation with PCV at different concentrations of Staphylococcus aureus; (b) Changes in the AR fluorescence spectrum caused by the supernatant after incubation with PCV at different concentrations of Staphylococcus aureus; (c) Changes in the AR fluorescence spectrum caused by the supernatant after incubation with PCV at 10 1 Up to 10 7 Standard curve for the detection of Staphylococcus aureus within the CFU / mL range.
[0079] The linear regression equation in the figure is: y=0.2151x+0.3117, R²=0.983, where R² represents the correlation coefficient between variables. The closer the value is to 1, the better. The larger the value, the better the fit.
[0080] 9. Specificity assessment:
[0081] The specificity was verified by using four types of bacteria as model bacteria: Gram-negative bacteria (Escherichia coli and Fluoromandibularia fluorescein) and Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes). The four bacteria (10...) 7 After incubating the CFUm / L samples with PCV, 100 μL of the supernatant was added to the sensing system, and the fluorescence ratios (FI) of the four bacterial pairs were recorded using a fluorescence spectrophotometer. 465 / FI 590 The impact.
[0082] like Figure 10 As shown, the recognition function of PCV for Escherichia coli and Staphylococcus aureus was further verified by scanning electron microscopy. a) Gram-negative bacteria (Escherichia coli and Fluoromandibular syndrome) and Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes) were incubated with PCV, and the supernatant was added to the sensing system to measure the fluorescence ratio FI. 465 / FI 590 The effect of ***p<0.001, **p<0.01; (b) Scanning electron micrographs of Escherichia coli and Staphylococcus aureus after incubation with PCV.
[0083] The fluorescence ratio (FI465 / FI585) of the sensing system was found to increase after incubation with Gram-positive bacteria, while it changed slightly after incubation with Gram-negative bacteria. Figure 10 a). Furthermore, SEM images showed that PCV could be captured on the surface of Gram-positive bacteria, but not on the surface of Escherichia coli. Figure 10 (b) The results are consistent, indicating that the developed sensing system can be used to distinguish between Gram-positive and Gram-negative bacteria.
[0084] 10. Actual sample testing:
[0085] Eggs and milk purchased from a local supermarket were used as real samples. The eggs and milk were pretreated by centrifugation. The suspension was diluted 10-fold with ultrapure water to form a working solution. Different concentrations of Staphylococcus aureus were added to the working solution, and then incubated with PCV. After centrifugation again, SC and AR were added to the supernatant as signal readings for bacterial ratio detection, and the recovery rate was calculated. The results are shown in Table 1 below.
[0086]
[0087] The detection recovery rate was in the range of 83% to 100%, indicating that this method can be applied to the detection of Staphylococcus aureus in real samples.
[0088] This invention provides a method for detecting foodborne pathogens. It can also be applied to the development of test strips. SC and AR are mixed and soaked in test strips of different materials. Different concentrations of bacteria are incubated with PCV and centrifuged. Under UV light, the blue fluorescence of SC increases noticeably with increasing bacterial concentration, while the red fluorescence of AR decreases noticeably with increasing bacterial concentration. Based on the detection principle of this method, corresponding reagent kits can also be developed to meet the needs of rapid and sensitive on-site detection of foodborne pathogens.
[0089] This invention uses fluorescence ratio as the output signal and reports the readout signal based on at least two signal channels, providing a reliable way to reduce interference, improve detection accuracy, and achieve rapid and sensitive bacterial detection with a detection range of up to 10. 1 ~10 7 The detection limit is as low as 5 CFU / mL; compared with many other published bacterial detection methods, it achieves a wider linear range and a lower detection limit.
[0090] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention, as long as they do not depart from the spirit and scope of the technical solution of the present invention, should be covered within the scope of the claims of the present invention.
Claims
1. A method for detecting foodborne pathogens based on composite nanomaterials, characterized in that: Includes the following steps: S1. Preparation of test bacterial suspensions for foodborne pathogens: Gram-positive bacteria were collected from cryopreservation tubes using an inoculation loop and cultured using the streak inoculation method. After serial dilution, bacterial concentrations ranging from 0 to 10⁻⁶ were obtained. 7 CFU / mL of the test bacterial solution; S2. Synthesis of biosensor system material PB@DPA-Ce-GMP@Van: Prussian blue (PB) at a concentration of 0.1–0.3 mg / mL was used as a carrier. After being dissolved by sonication, it was added to a mixed solution of guanosine-5'-monophosphate disodium (GMP) and Ce(CH3COO)3. The mixture was shaken and mixed thoroughly, then centrifuged to remove the supernatant. The solution was washed with deionized water and then reconstituted with deionized water. After mixing thoroughly, 2,6-pyridinedicarboxylic acid (DPA) was added. When the color turned into a clear blue, the solution was irradiated under ultraviolet light for 2–4 hours and then freeze-dried under vacuum for later use to obtain the PB@DPA-Ce-GMP solution. The precipitate was then redissolved in deionized water, and vancomycin (Van) at a concentration of 10~20 μg / mL was added. After ultrasonic treatment, the biosensor system material PB@DPA-Ce-GMP@Van was finally obtained. S3. Detection of foodborne pathogens: The biosensor system material PB@DPA-Ce-GMP@Van synthesized in step S2 above is supplemented with bacteria at a concentration of 0~10 from step S1 above. 7 A CFU / mL culture of Gram-positive bacteria was incubated at room temperature for 2 hours. After centrifugation, the supernatant was collected and incubated to form a PB@DPA-Ce-GMP@Van / Gram-positive bacteria complex. PB@DPA-Ce-GMP@Van in the supernatant quenched the fluorescence of scopolamine (SC) and simultaneously increased the fluorescence intensity of fluorescent red dye (AR). The fluorescence intensity ratio SC / AR was used as the detection signal output, with the fluorescence response of the sensing system as the ordinate and the bacterial concentration of Gram-positive bacteria as the abscissa. The detection data were processed and then linearly fitted to obtain the final linear regression equation.
2. The method for detecting foodborne pathogens based on composite nanomaterials according to claim 1, characterized in that: In step S1, the specific process of bacterial culture is as follows: streak the bacterial culture medium on nutrient agar medium, incubate at 37°C for 24-48 hours, pick out colonies with good morphology and inoculate them in LB broth with shaking culture, wash with PBS buffer and centrifuge, resuspend in sterile Tris-HCl buffer, take 100 μL of bacterial suspension and plate it for plate counting, and use a UV-Vis spectrophotometer to measure the optical density value (OD) of the bacterial suspension at a wavelength of 600 nm to determine the corresponding bacterial concentration.
3. The method for detecting foodborne pathogens based on composite nanomaterials according to claim 1, characterized in that: In step S2, the synthesis method of PB is as follows: polyvinylpyrrolidone and potassium ferricyanide are added to hydrochloric acid and stirred at room temperature for 0.5-1 h. Then, the mixture is heated in an oil bath at 80-90°C for 20-25 h. The solution color changes from yellow to blue. After heating, the mixture is cooled to 40-50°C. The reaction solution is centrifuged and the precipitate is washed multiple times with ethanol and water. Finally, the obtained PB is vacuum dried and stored at 4°C for later use.
4. The method for detecting foodborne pathogens based on composite nanomaterials according to claim 1, characterized in that: In step S2, preferably, the concentration of PB is 0.1 mg / mL, the ultraviolet irradiation time is 2.5 h, and the concentration of vancomycin is 10 μg / mL.
5. The method for detecting foodborne pathogens based on composite nanomaterials according to claim 1, characterized in that: In step S3, when the Gram-positive bacterium is selected as Staphylococcus aureus, the final linear regression equation of S. aureus bacterial concentration and fluorescence response is: y=0.2151x+0.3117, R²=0.983, where R² represents the correlation coefficient between variables, and the larger the value, the better the fit.