A general detection method for foodborne pathogens suitable for long voyage environment

Abstract

The application relates to the technical field of detection, in particular to a general foodborne pathogenic bacteria detection method suitable for a long-distance sailing environment. UiO-66 is used to enrich methylene blue, and is combined with target bacteria aptamer to obtain UiO-66 / MB / Aptamer which simultaneously serves as a signal amplification and recognition probe. A membrane separation method is used to filter out the probes not combined with the bacteria. The electrochemical signal of methylene blue is detected by using a differential pulse voltammetry method, a corresponding linear relationship is constructed, and the concentration of the bacteria is quantitatively detected. The application not only guarantees the sensitivity, detection time, portability and other characteristics of the detection of foodborne pathogenic bacteria, but also provides a general detection method for detecting various target bacteria. Through the reproducibility and stability test under the high-temperature, high-humidity and high-salt environment, it is proved that the application can realize the rapid, sensitive and stable detection of foodborne pathogenic bacteria in a complex long-distance sailing environment.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically, to a universal method for detecting foodborne pathogens suitable for long-distance navigation environments. Background Technology

[0002] Foodborne pathogens causing various diseases remain a major concern. Identifying and diagnosing these diseases is a crucial task for public health and clinical medicine. While numerous studies, particularly on foodborne pathogen detection, have been conducted both domestically and internationally, their application in offshore environments still faces limitations. The unique marine environment, with its high temperature, humidity, and salinity, can significantly impact test results. For example, the commonly used enrichment method for bacterial detection offers high accuracy but requires at least 4-7 days to obtain accurate results. Other methods, such as ELISA relying on antigen-antibody reactions, are easily affected by environmental factors. General detection methods are more susceptible to severe impacts in high-temperature, humid, or high-salinity environments. Furthermore, the diverse and complex environments in which foodborne pathogens exist make the development of rapid food safety detection technologies for complex, long-distance maritime environments a pressing issue that urgently needs to be addressed.

[0003] In the detection of foodborne pathogens, electrochemical detection methods are attracting increasing attention due to their advantages such as high sensitivity, ease of operation, portability, and low cost. These methods primarily include biosensors based on antibodies, enzymes, and aptamers. For the detection of multiple contaminants, two or more detection methods are typically required; therefore, universal detection methods targeting multiple foodborne pathogens warrant further research. Furthermore, the unique marine environment, with its high temperature, high humidity, and high salinity, can all affect sensor performance, and the development of biosensors for foodborne pathogens under these conditions has not yet been reported.

[0004] Aptamers are nucleic acid sequences that can specifically bind to a target and are commonly used as probes to capture that target in electrochemical detection. Their synthesis is now well-established and inexpensive, and they have gradually replaced antibodies in the detection of foodborne pathogens using many electrochemical strategies. In recent years, some materials used to enhance electrochemical signals have been metal ions (e.g., Cd). 2+ Pb 2+ Zn 2+ and Cu 2+ Materials such as methylene blue and ferrocene are often modified onto aptamers to provide electrochemical detection signals. Currently, in most studies, one aptamer modifies only one molecule of the signal material; therefore, improving and innovating upon conventional detection methods is of significant research value.

[0005] Metal-organic frameworks (MOFs) are a type of coordination polymer that has developed rapidly in the last two decades. They possess a three-dimensional porous structure, typically using metal ions as connection points and organic ligands as supports to form a 3D spatial extension. They are a novel class of porous materials with promising applications in catalysis, energy storage, and separation. Their excellent stability and adsorption capacity provide valuable research opportunities for electrochemical detection systems. Among them, the MOF material UiO-66, synthesized using Zr(IV)-containing metal clusters as inorganic nodes and carboxylic acids as organic ligands, has attracted widespread attention due to its good acid resistance, thermal stability, and ease of functionalization. Summary of the Invention

[0006] The purpose of this invention is to provide a universal method for detecting foodborne pathogens suitable for long-distance navigation environments. It is a universal electrochemical detection method for sensitive detection of Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus.

[0007] The method of this invention uses UiO-66 loaded with methylene blue as a signal amplification probe, and the aptamer forms a recognition probe by connecting with UiO-66. In the presence of target bacteria, the aptamer on the probe specifically binds to the target bacteria. Since bacteria are generally larger than 1 μm in size, when filtered using a 0.45 μm filter membrane, the contents of the filtrate after filtration are the recognition probes that have not bound to bacteria. The bacteria are quantitatively detected by measuring the electrochemical signal of methylene blue on the recognition probe in the filtrate on a screen-printed electrode. Currently, aptamer synthesis technology is mature and the preparation cost is low. The method of this invention only requires replacing the aptamer of the target bacteria to achieve universal detection of different foodborne pathogens. The detection limits of Salmonella Typhimurium, Escherichia coli, and Staphylococcus aureus tested by the method of this invention are 3 CFU·mL. -1 4 CFU·mL -1 and 5CFU·mL -1 The detection time was 30 minutes for all tests, while also meeting the requirement for specific detection results. Furthermore, the method of this invention also demonstrated good detection capabilities in various real-world samples, including milk, eggs, vegetables, and shrimp. Finally, the detection system constructed using the method of this invention underwent salt spray testing under high temperature and high humidity conditions, verifying its stability in these environments. In summary, this invention not only ensures high sensitivity, short detection time, and portability for detecting foodborne pathogens, but also provides a universal detection method for detecting multiple target bacteria. Finally, the stability of this detection method in complex environments was verified.

[0008] Based on the above technical solutions, this invention provides a universal method for detecting foodborne pathogens suitable for long-distance navigation environments, comprising the following steps:

[0009] (A) Preparation of recognition probe: Methylene blue was enriched with UiO-66 and bound to the target bacterial aptamer. The resulting UiO-66 / MB / Aptamer served as both a signal amplification and recognition probe.

[0010] (B) The bacterial solution to be tested is mixed with the identification probe solution obtained in (A) and then separated using a filter membrane. Because the target bacteria are too large to pass through the filter membrane, the filtrate contains probes that have not bound to the bacteria.

[0011] (C) Electrochemical detection: The electrochemical signal of methylene blue in the filtrate was detected by differential pulse voltammetry, and the bacterial concentration was quantitatively detected by detecting changes in the signal.

[0012] Furthermore, the foodborne pathogens mentioned are Salmonella typhimurium, Escherichia coli, or Staphylococcus aureus.

[0013] Further, the preparation method of the recognition probe in step (A) is as follows: Take 1 mL of UiO-66 material and mix it with 3 mL of 10 mM methylene blue solution, place it in a shaker at 26℃ and 160 rpm overnight, centrifuge for 5 min, discard the supernatant, resuspend the precipitate at the bottom with 1 mL of ultrapure water, and place it at room temperature for later use; heat the aptamer in a 95℃ water bath for 10 min, and then immediately place it in an ice bath for 10 min; dilute the target bacterial aptamer to 2 μM with phosphate buffered saline (PBS), mix it with the above solution at a 1:1 volume, and place the mixed solution in a shaker at 37℃ and 160 rpm overnight; after rinsing with phosphate buffered saline to remove unbound aptamers, the UiO-66 / MB / Aptamer recognition probe is obtained.

[0014] Because of the high porosity of UiO-66, methylene blue (MB) can be enriched in large quantities, thus effectively amplifying the electrochemical signal. The bacterial aptamer can specifically recognize bacteria; the phosphate group at the end of the aptamer forms a strong Zr-OP coordination bond with the central metal Zr of UiO-66, requiring no complex surface modification and binding stably to UiO-66. Therefore, methylene blue, UiO-66, and the bacterial aptamer are used to construct a signal amplification and recognition probe for electrochemical detection. In the presence of target bacteria, the recognition probe captures the target bacteria through the aptamer. In this invention, only the aptamer of the target bacteria needs to be replaced to achieve specific detection of target foodborne pathogens, meeting the need for universal detection of foodborne pathogens. Currently, the method of using UiO-66 / MB / Aptamer simultaneously as a signal amplification and recognition probe has not been applied to the electrochemical detection of foodborne pathogens, which is a key feature of this invention.

[0015] Furthermore, the filter membrane described in step (B) has a pore size of 0.45 μm.

[0016] The filter membrane used in this invention has a pore size of 0.45 μm, which can filter most common foodborne pathogens. In this embodiment, three common foodborne pathogens were selected: *Salmonella typhimurium* with a volume of approximately (0.6–0.9) × (1–3) μm; *Escherichia coli* with a volume of approximately 0.5 × (1–3) μm; and *Staphylococcus aureus* with a volume of approximately 0.5–1 μm. Apart from these, most bacteria have a volume greater than 0.45 μm; therefore, bacteria meeting the volume requirements can be tested using membrane filtration.

[0017] Further, in step (B), the identification probe solution is taken and mixed with the test bacterial solution at a 1:1 volume ratio. The mixture is shaken in a shaker at 37°C and 160 rpm for 30 min. After removal, 300 μL of the obtained solution is drawn using a 1 mL syringe. A filter with a 0.45 μm pore size is attached to the tip of the syringe, and the syringe plug is pushed to filter slowly, resulting in approximately 200 μL of a clear blue solution. 100 μL of this solution is then uniformly drop-coated onto the surface of the screen-printed electrode for electrochemical detection.

[0018] Furthermore, in step (C), the reduction current signal of the test solution in the range of -0.6V to 0V is tested using the differential pulse voltammetry method. A linear relationship is plotted based on the measured reduction current value and the corresponding bacterial concentration value to obtain a linear equation.

[0019] In this invention, the filtrate used for electrochemical detection is a methylene blue-containing recognition probe that has not bound to bacteria. The lower the concentration of the bacteria being tested, the higher the concentration of the methylene blue-containing probe in the filtrate, and the stronger the resulting electrochemical signal. This reverse detection strategy achieves highly sensitive detection of low concentrations of bacteria, demonstrating the universality and high sensitivity of this method.

[0020] In a second aspect, the present invention provides a universal foodborne pathogen identification probe, wherein a methylene blue-loaded UiO-66 is used as a signal amplification probe, and a bacterial aptamer is used to form an identification probe by connecting with UiO-66.

[0021] Furthermore, the preparation method of the recognition probe is as follows: 1 mL of UiO-66 material is mixed with 3 mL of 10 mM methylene blue solution, and shaken overnight at 26°C and 160 rpm. After centrifugation for 5 min, the supernatant is discarded, and the precipitate at the bottom is resuspended in 1 mL of ultrapure water and kept at room temperature for later use. The aptamer is heated in a 95°C water bath for 10 min, followed immediately by an ice bath for 10 min. The target bacterial aptamer is diluted to 2 μM using phosphate buffered saline (PBS), and mixed with the above solution at a 1:1 volume ratio. The mixed solution is shaken overnight at 37°C and 160 rpm. After rinsing with phosphate buffered saline to remove unbound aptamers, the UiO-66 / MB / Aptamer recognition probe is obtained.

[0022] A third aspect of the present invention provides an application of the identification probe described above in the detection of general foodborne pathogens in long-distance navigation environments.

[0023] The advantages of this invention are:

[0024] 1. The MOF material UiO-66 is characterized by its ease of modification and strong enrichment ability. Conventional bacterial detection methods using methylene blue as an electrochemical indicator often bind only a single molecule of methylene blue to the corresponding recognition probe, resulting in a low enhancement of the electrochemical signal. The detection method of this invention effectively enriches methylene blue based on the high porosity of the UiO-66 structure, thereby effectively amplifying the detection signal and constructing a signal amplification probe in the detection system. Furthermore, by utilizing the phosphate group at the end of the aptamer to form a strong Zr-OP coordination bond with the central metal Zr of UiO-66, no complex surface modification is required. The aptamer and UiO-66 are stably bound, forming UiO-66 / MB / Aptamer, thus realizing the construction of a detection probe with both signal amplification and target recognition functions.

[0025] 2. The aptamer is easy to synthesize and is inexpensive. In the detection method of the present invention, depending on the target bacteria, only the corresponding aptamer needs to be replaced to achieve the identification and detection of the bacteria, which meets the needs of universal detection of a variety of foodborne pathogens.

[0026] 3. The detection method of this invention employs membrane filtration to separate the recognition probe solution that has not bound to bacteria. The recognition probe carries methylene blue, which provides an electrochemical signal. The current signal of the methylene blue is negatively linearly correlated with the concentration of the bacteria being tested. The lower the concentration of the bacteria being tested, the stronger the current signal. This method can effectively improve the detection capability for low-concentration samples and greatly increase the sensitivity of the detection method.

[0027] 4. The detection method of this invention has undergone reproducibility and stability tests under high temperature, high humidity, and high salt environments. The results demonstrate that the method has good reproducibility and good stability after one week of storage, and can achieve effective detection under salt spray conditions. Furthermore, the detection capability for target bacteria in four actual samples was tested, and the results showed good detection capability. The spiked recovery rate and relative standard deviation of the detection both met the detection standards, proving that the detection method of this invention can achieve rapid, sensitive, and stable detection of foodborne pathogens in complex long-distance navigation environments. Attached Figure Description

[0028] Figure 1 The following is a characterization of the materials used in the method of this invention, mainly used to illustrate the enrichment effect of UiO-66 on methylene blue and verify the feasibility of constructing a signal amplification probe. (A) Scanning electron microscope image of UiO-66; (B) Scanning electron microscope image of UiO-66 / MB; (C) Elemental distribution map of UiO-66 / MB.

[0029] Figure 2 In the method of the present invention Figure 1 Based on this, characterization by X-ray diffraction, infrared, and ultraviolet absorption spectroscopy, as well as the zeta potential of the aptamer conjugate with methylene blue and UiO-66, were supplemented. These results collectively illustrate the construction of the signal amplification and recognition probe jointly constructed by the aptamer, methylene blue, and UiO-66. (A) X-ray diffraction patterns of UiO-66 and UiO-66 / MB; (B) Infrared spectra of MB, UiO-66, and UiO-66 / MB; (C) Ultraviolet spectra of MB, UiO-66, and UiO-66 / MB; (D) Zeta potential of UiO-66, UiO-66 / MB, and UiO-66 / MB / Aptamer.

[0030] Figure 3 This is a schematic diagram of the detection method of the present invention, which mainly illustrates the two main methods in this method: preparation of signal probes and membrane separation detection. (A) Synthesis of UiO-66 / MB / Aptamer signal amplification probe and recognition probe; (B) Electrochemical detection based on membrane separation method, in which the methylene blue probe that has not bound to bacteria is filtered out for electrochemical testing.

[0031] Figure 4The linear relationship between different concentrations of Salmonella Typhimurium and electrical signals is shown. (A) Salmonella Typhimurium concentrations of 10⁻¹, 10⁻¹⁰ 2 10 3 10 4 10 5 10 6 CFU·mL -1 (A) Current graph at time; (B) Salmonella typhimurium concentration (10, 10) 2 10 3 10 4 10 5 10 6 CFU·mL -1 The linear relationship between the signal and the current signal.

[0032] Figure 5 The linear relationship between different concentrations of E. coli and the electrical signal is shown. (A) E. coli concentrations of 10, 10 2 10 3 10 4 10 5 10 6 CFU·mL -1 (A) Current graph at different times; (B) E. coli concentration (10, 10) 2 10 3 10 4 10 5 10 6 CFU·mL -1 The linear relationship between the signal and the current signal.

[0033] Figure 6 The linear relationship between different concentrations of Staphylococcus aureus and the electrical signal is shown. (A) Staphylococcus aureus concentrations of 10⁻⁶ and 10⁻⁶. 2 10 3 10 4 10 5 10 6 CFU·mL -1 (A) Current graph at different times; (B) Staphylococcus aureus concentration (10, 10) 2 10 3 10 4 10 5 10 6 CFU·mL -1 The linear relationship between the signal and the current signal.

[0034] Figure 7 This document presents the specificity of this method against Salmonella Typhimurium, Escherichia coli, and Staphylococcus aureus.

[0035] Figure 8The test results demonstrate the reproducibility (A) and stability (B) of this method under salt spray conditions (high temperature, high humidity, and high salt test environment). The test results show that this method can be used to test samples under the above-mentioned complex environment within the test time. Detailed Implementation

[0036] The specific embodiments provided by the present invention will be described in detail below with reference to the examples and accompanying drawings.

[0037] Example 1:

[0038] 1. The features of the universal foodborne pathogen detection method based on MOF and membrane separation of the present invention:

[0039] (1) Due to the high porosity of UiO-66, methylene blue (MB) can be enriched in large quantities, thus effectively amplifying the electrochemical signal. The bacterial aptamer can specifically recognize bacteria, and the phosphate group at the end of the aptamer forms a strong Zr-OP coordination bond with the central metal Zr of UiO-66, requiring no complex surface modification and binding stably to UiO-66. Therefore, methylene blue, UiO-66, and the bacterial aptamer were used to construct a signal amplification and recognition probe for electrochemical detection.

[0040] Figure 1 (A) and (B) are scanning electron microscope images of UiO-66 and UiO-66 / MB, respectively. UiO-66 has a regular octahedral shape with an average diameter of 100 nm. The morphology of UiO-66 after loading with MB remains basically unchanged. Figure 1 (C) shows the elemental analysis of methylene blue after binding with UiO-66. C, O, and Zr are the main elements of UiO-66, while N and S are the main elements of methylene blue. The results demonstrate that methylene blue and UiO-66 are effectively bound together, and the binding amount is relatively high.

[0041] Figure 2 (A) shows the X-ray diffraction patterns of methylene blue and UiO-66, demonstrating that UiO-66 maintains good structure and crystallinity even after enrichment with methylene blue. Figure 2 (B) Infrared spectroscopy and (C) Ultraviolet absorption spectroscopy further proved that methylene blue and UiO-66 successfully combined. Figure 2 (D) The successful construction of the UiO-66 / MB / Aptamer recognition probe was demonstrated by Zeta potential analysis.

[0042] In the presence of target bacteria, the recognition probe captures the target bacteria via an aptamer. This method achieves specific detection of target foodborne pathogens simply by replacing the aptamer for the target bacteria, meeting the need for universal detection of foodborne pathogens. The fact that the method of simultaneously using UiO-66 / MB / Aptamer as both a signal amplification and recognition probe has not been applied to the electrochemical detection of foodborne pathogens is a key feature of this invention.

[0043] (2) This method employs membrane separation to filter the reaction solution. The filter membrane used in this invention has a pore size of 0.45 μm, which can filter most common foodborne pathogens. This invention selected three common foodborne pathogens: *Salmonella typhimurium* with a volume of approximately (0.6–0.9) × (1–3) μm; *Escherichia coli* with a volume of approximately 0.5 × (1–3) μm; and *Staphylococcus aureus* with a volume of approximately 0.5–1 μm. Apart from these, most bacteria have a volume greater than 0.45 μm, therefore, bacteria meeting the volume requirements can be tested using membrane filtration. Furthermore, the filtrate used for electrochemical detection in this method is a methylene blue-containing recognition probe that has not bound to bacteria. The lower the concentration of the bacteria being tested, the higher the concentration of the methylene blue-containing probe in the filtrate, and the stronger the resulting electrochemical signal. This reverse detection strategy achieves highly sensitive detection of low-concentration bacteria. In summary, this method demonstrates its versatility and high sensitivity.

[0044] (3) The detection limits of this method for Salmonella Typhimurium, Escherichia coli, and Staphylococcus aureus are 3 CFU·mL, respectively. -1 4 CFU·mL -1 and 5CFU·mL -1 The detection time is 30 minutes, and it also meets the specificity test requirements. This detection method also demonstrates good detection capability in actual contaminated samples, with both its detection sensitivity and detection time meeting the requirements for rapid detection. Furthermore, within the detection time, the detection system maintains stable detection performance under a salt spray environment (40℃, 99% RH, saturated sodium chloride solution), proving the practicality of this method under complex conditions and its stability in special environments.

[0045] 2. Detection principle:

[0046] The principle of this method is as follows: Figure 3 As shown, this method first enriches methylene blue using UiO-66 and binds it to the target bacterial aptamer, resulting in UiO-66 / MB / Aptamer, which serves as both a signal amplification and recognition probe. Then, a membrane separation method is used to filter out the probe that has not bound to the bacteria. Differential pulse voltammetry is used to detect the electrochemical signal of methylene blue, and a corresponding linear relationship is constructed to quantitatively detect the bacterial concentration. The specific principle is as follows:

[0047] (1) As Figure 3 As shown in (A), a large amount of methylene blue was enriched by synthesized UiO-66, which was then bound to a bacterial aptamer. The aptamer was used to capture the target bacteria, and the enriched methylene blue provided a larger current signal for electrochemical detection, thus improving detection sensitivity.

[0048] (2) Figure 3 As shown in (B), after the target bacteria specifically bind to the aptamer, its volume becomes larger than the pore size of the filter membrane. When the reaction solution is filtered through the membrane, the recognition probes that have not bound to the bacteria are filtered out. The concentration of the target bacteria is then quantified by measuring the methylene blue electrochemical signal using a screen-printed electrode.

[0049] 3. Specific detection operation steps of this method:

[0050] (1) Preparation of the recognition probe: Mix 1 mL of the prepared UiO-66 material with 3 mL of 10 mM methylene blue solution, place in a shaker at 26 °C and 160 rpm overnight, centrifuge for 5 min, discard the supernatant, resuspend the precipitate at the bottom with 1 mL of ultrapure water, and store at room temperature for later use. Heat the aptamer in a 95 °C water bath for 10 min, then immediately place in an ice bath for 10 min. Dilute the aptamer to 2 μM with phosphate buffered saline (PBS), mix with the above solution at a 1:1 volume ratio, and place the mixture in a shaker at 37 °C and 160 rpm overnight. After rinsing with phosphate buffered saline to remove unbound aptamers, the UiO-66 / MB / Aptamer recognition probe is obtained.

[0051] (2) Preparation of bacterial culture: After culturing bacteria in Luria-Bertani (LB) broth at 37°C for 24 h, the cultured bacteria were collected by centrifugation at 7000 rpm and 4°C for 5 min. After washing with sterile water, the bacteria were redispersed in PBS. The bacterial culture was then subjected to 10, 10⁻⁶ ppm precipitates with PBS. 2 10 3 10 4 10 5 10 6 10 7 Serial dilutions were performed using a microplate reader on OD500. 600 The absorbance values ​​of each dilution were measured and recorded. The bacterial count at the optimal concentration was calculated using the plate count method, and a standard curve of bacterial concentration versus absorbance was obtained. Based on the bacterial standard curve, 10... 6 10 4 10 2 CFU·mL -1 The bacterial solution of a certain concentration is stored at 4℃ for later use.

[0052] (3) Preparation of spiked samples: Take sterilized pure milk, dilute it 10 times with phosphate buffer solution, and add it to 10 spiking solution at a 1:1 volume ratio. 6 10 4 10 2 CFU·mL -1 Prepare a concentrated bacterial solution and store at 4°C. Take an egg, thoroughly wipe its surface with 75% alcohol, then under aseptic conditions, thoroughly mix the egg contents, dilute 10 times with phosphate buffer solution, and store at 4°C. Take lettuce, spray with 75% alcohol, take 5g, add 50mL of phosphate buffer solution, grind under aseptic conditions, and store at 4°C. Take frozen shrimp, spray with 75% alcohol, take 5g, add 50mL of phosphate buffer solution, grind under aseptic conditions, and store at 4°C.

[0053] (4) Preparation of detection solution by membrane separation: Take the identification probe solution from (1) above, mix it with a certain concentration of bacterial solution at a volume ratio of 1:1, and shake it in a shaker at 37℃ and 160 rpm for 30 min. After taking it out, use a 1 mL syringe to draw 300 μL of the obtained solution, and put a 0.45 μm pore size filter screen on the front end of the syringe. Push the syringe plug to filter slowly, and about 200 μL of clear blue solution can be obtained. Take 100 μL of this solution and evenly drop it onto the surface of the screen-printed electrode for electrochemical detection.

[0054] (5) Linearity test: The reduction current signal of the test solution in the range of -0.6V to 0V was tested using the differential pulse voltammetry method. A linear relationship was plotted between the measured reduction current value and the corresponding bacterial concentration value to derive the linear equation. Figures 4-6 As shown, the electrical signal corresponds to the concentrations of the three tested bacteria in the range of 10–10⁻⁶. 6 CFU·mL -1 It exhibits a good linear relationship within the range.

[0055] (6) Specificity test: Taking the detection of Salmonella typhimurium as an example, the recognition probe used to detect Salmonella typhimurium is incubated with Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Vibrio parahaemolyticus, and Shigella flexneri respectively before testing. Figure 7 As shown, the current change was significant when Salmonella Typhimurium was detected. The peak current changes were smaller when testing the other four bacteria. Similar tests were performed for specificity against Escherichia coli and Staphylococcus aureus. The results indicate that this method is specific for the detection of Salmonella Typhimurium, Escherichia coli, and Staphylococcus aureus.

[0056] (7) Actual sample testing: Samples containing 10 were prepared according to the spiked recovery method. 6 10 4 10 2CFU·mL -1 Test samples of milk, eggs, vegetables, and frozen shrimp for bacterial concentration. According to Tables 1-3, in the preparation of 10... 6 10 4 10 2 CFU·mL -1 In the spiked samples with varying bacterial concentrations, the recoveries were all between 80% and 130%, which met the requirements.

[0057] (8) Reproducibility and Stability Testing: To verify the stability and reproducibility of the sensors under long-distance voyage conditions, a simulated high-temperature, high-humidity, and high-salt voyage environment (40℃, 99% RH, saturated salt solution) was conducted. To verify reproducibility, five sensors were prepared and used the same detection method to detect pathogenic bacteria. Figure 8 The data shows that the relative standard deviation (RSD) is 2.19%, confirming the reproducibility of the detection method. Simultaneously, the stability of the sensor was studied. The prepared sensor was stored for 7 days, and then steps (4) and (5) were performed to measure its electrochemical signal and analyze the data. As shown in the figure, the RSD is 2.62%, indicating that the sensor has good stability. In summary, this detection method can achieve stable detection of Salmonella Typhimurium, Escherichia coli, and Staphylococcus aureus under long-distance navigation conditions.

[0058] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

[0059] Table 1. Test results of Salmonella Typhimurium in actual samples

[0060]

[0061] a The average of three repeated measurements (rounded).

[0062] Table 2. Test results of Escherichia coli in actual samples

[0063]

[0064] a The average of three repeated measurements (rounded).

[0065] Table 3. Test results of Staphylococcus aureus in actual samples

[0066]

[0067] a The average of three repeated measurements (rounded).

Claims

1. A universal method for detecting foodborne pathogens suitable for long-distance navigation environments, characterized in that, Includes the following steps: (A) Preparation of recognition probe: Methylene blue was enriched with UiO-66 and bound to the target bacterial aptamer. The resulting UiO-66 / MB / Aptamer served as both a signal amplification and recognition probe. (B) The bacterial solution to be tested is mixed with the identification probe solution obtained in (A) and then separated using a filter membrane. Because the target bacteria are too large to pass through the filter membrane, the filtrate contains probes that have not bound to the bacteria. (C) Electrochemical detection: The electrochemical signal of methylene blue in the filtrate was detected by differential pulse voltammetry, and the bacterial concentration was quantitatively detected by detecting changes in the signal.

2. The detection method according to claim 1, characterized in that, The foodborne pathogens mentioned are Salmonella Typhimurium, Escherichia coli, and Staphylococcus aureus.

3. The detection method according to claim 1, characterized in that, The preparation method of the recognition probe in step (A) is as follows: Take 1 mL of UiO-66 material and mix it with 3 mL of 10 mM methylene blue solution, place it in a shaker at 26 ℃ and 160 rpm overnight, centrifuge for 5 min, discard the supernatant, resuspend the precipitate at the bottom with 1 mL of ultrapure water, and place it at room temperature for later use; heat the aptamer in a 95 ℃ water bath for 10 min, and then immediately place it in an ice bath for 10 min; dilute the target bacterial aptamer to 2 μM with phosphate buffer solution, mix it with the above solution at a 1:1 volume, place the mixed solution in a shaker at 37 ℃ and 160 rpm overnight; wash with phosphate buffer solution to remove unbound aptamers, and obtain the UiO-66 / MB / Aptamer recognition probe.

4. The detection method according to claim 1, characterized in that, The filter membrane described in step (B) has a pore size of 0.45 μm.

5. The detection method according to claim 1, characterized in that, In step (B), the identification probe solution is taken and mixed with the test bacterial solution at a volume ratio of 1:

1. The mixture is shaken in a shaker at 37 ℃ and 160 rpm for 30 min. After removal, 300 μL of the obtained solution is drawn using a 1 mL syringe. A filter with a 0.45 μm pore size is attached to the front end of the syringe, and the syringe plug is pushed to filter slowly, resulting in 200 μL of clear blue solution. 100 μL of this solution is then uniformly drop-coated onto the surface of the screen-printed electrode for electrochemical detection.

6. The detection method according to claim 1, characterized in that, In step (C), the reduction current signal of the test solution in the range of -0.6 V to 0 V is tested using the differential pulse voltammetry method. A linear relationship is plotted based on the measured reduction current value and the corresponding bacterial concentration value to obtain the linear equation.

7. A universal foodborne pathogen identification probe, characterized in that, The method involves using methylene blue-loaded UiO-66 as a signal amplification probe, with bacterial aptamers forming a recognition probe by binding to UiO-66. The preparation method of the recognition probe is as follows: 1 mL of UiO-66 material is mixed with 3 mL of 10 mM methylene blue solution and placed in a shaker at 26 ℃ and 160 rpm overnight. After centrifugation for 5 min, the supernatant is discarded, and the precipitate at the bottom is resuspended in 1 mL of ultrapure water and stored at room temperature. The aptamer is heated in a 95 ℃ water bath for 10 min, followed immediately by an ice bath for 10 min. The target bacterial aptamer is diluted to 2 μM using phosphate buffer solution and mixed with the above solution at a 1:1 volume ratio. The mixture is then placed in a shaker at 37 ℃ and 160 rpm overnight. After rinsing with phosphate buffer solution to remove unbound aptamers, the UiO-66 / MB / Aptamer recognition probe is obtained.

8. The application of the identification probe as described in claim 7 in the detection of general foodborne pathogens in long-distance navigation environments.

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