Double-probe detection kit for detecting escherichia coli O157: H7 and preparation method

Abstract

The invention relates to a double-probe detection kit for detecting escherichia coli O157: H7 and a preparation method of the double-probe detection kit, and belongs to the technical field of food safety. The double-probe detection kit consists of a magnetic separation probe reagent and a copper-manganese double-metal nano-enzyme probe reagent which are independently packaged. The magnetic separation probe reagent is a concanavalin agglutinin A modified magnetic bead, and is used for specifically binding escherichia coli O157: H7 in a meat product detection sample and carrying out separation and enrichment treatment; the bimetallic nano-enzyme probe reagent is copper-manganese bimetallic nano-enzyme modified by a nucleic acid aptamer, and is used for specifically combining with the escherichia coli O157: H7 subjected to separation and enrichment treatment and generating an escherichia coli O157: H7 concentration dependent colorimetric signal and a fluorescence signal. According to the invention, mutual verification is carried out between dual-mode signals, so that the accuracy of a detection result is improved; the detection time is within 1.5 h, and the method has good specificity for common type strains. The invention provides a design thought for the development of detection sensors for escherichia coli O157: H7 and other meat-derived pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a dual-probe detection kit for detecting Escherichia coli O157:H7 in meat products and its preparation method. Background Technology

[0002] Escherichia coli O157:H7 is a major vegetative pathogen, belonging to the enterohemorrhagic Escherichia coli (EHEC) serotype. It is a Gram-negative bacillus, non-spore-forming, and possesses capsular flagella and pili structures. Escherichia coli O157:H7 is one of the major pathogenic Escherichia coli strains, characterized by high virulence and low infectious dose; typically, an exposure of only 10-100 CFU is sufficient to cause infection in humans and animals.

[0003] Existing methods for detecting meat-derived pathogens can be broadly categorized into three types: colony counting, immunological detection, and molecular biological detection. Traditional colony counting methods, while simple and low-cost, are often time-consuming. Among immunological detection methods, immunomagnetic bead assays offer high selectivity, sensitivity, and specificity, but are costly and susceptible to affecting biological activity. Enzyme-linked immunosorbent assays (ELISA) offer high specificity and short processing time, but enzyme purity and the reaction process are easily affected by the environment, resulting in poor stability and repeatability, leading to missed detections and false positives. Molecular biological detection methods, including polymerase chain reaction (PCR), gene chip technology, loop-mediated isothermal amplification (LAMP), biosensors, and DNA probe technology, offer advantages over traditional biochemical identification methods, such as high accuracy, low detection limits, and speed. However, they suffer from drawbacks such as false positives, high cost, and complex operational procedures. Developing specific detection methods for *E. coli* O157:H7 is crucial for the rapid detection of this bacterium. In the detection of *E. coli* O157:H7 in meat products, both colorimetric and fluorescence sensing methods have their limitations. While colorimetry allows for rapid initial screening by directly observing color changes with the naked eye, its sensitivity is low, easily affected by the complex background color of meat products, and its quantitative accuracy is insufficient. Fluorescence sensing, although possessing extremely high sensitivity, typically has invisible signals, heavily relying on large, expensive fluorescence detection instruments for result reading, limiting its application in rapid on-site detection scenarios. Furthermore, certain substances in the sample may quench fluorescence, causing false negatives. This poses challenges to the stability and repeatability of the detection. Applications in complex real-world environments have shown that single-signal detection modes suffer from significant deficiencies in detection stability, anti-interference performance, and result accuracy, making it difficult to avoid the risks of false positives or false negatives. Therefore, developing the aforementioned colorimetric-fluorescence dual-modal detection method has become a key and effective solution to overcome the shortcomings of traditional sensors, enabling mutual verification and complementarity among multiple signals.

[0004] Therefore, there is an urgent need to develop a detection technology that is easy to operate, has a rapid response, and combines high sensitivity and accuracy to achieve real-time monitoring of Escherichia coli O157:H7. Although domestic and foreign scholars have made progress in constructing detection systems for foodborne pathogens suitable for on-site analysis in recent years, the current mainstream methods are still mainly based on single signal detection, which generally suffers from limitations such as significant food matrix interference, insufficient signal resolution, and single detection mode, making it difficult to meet the needs of accurate diagnosis in complex scenarios. Summary of the Invention

[0005] To address the problems of high detection difficulty, long detection time, and low sensitivity in existing dual-modal detection technologies for Escherichia coli O157:H7 in food, this invention provides a dual-probe detection kit for detecting Escherichia coli O157:H7, and also provides a method for preparing the dual-probe detection kit for detecting Escherichia coli O157:H7.

[0006] A dual-probe detection kit for detecting Escherichia coli O157:H7 consists of two independent 50 μL magnetic separation probe reagents and 80 μL copper-manganese bimetallic nanozyme probe reagents. The magnetic separation probe reagent is a 0.1 mg / mL solution of concanavalin A-functionalized iron oxide nanospheres, prepared by mixing a 10 mg / mL amino-containing iron oxide nanosphere solution and a 1 mg / mL carboxyl-containing concanavalin A solution at a volume ratio of 1:9. The magnetic separation probe reagent is used to specifically bind to Escherichia coli O157:H7 and to separate and enrich it from the test sample. The copper-manganese bimetallic nanozyme probe reagent is prepared by uniformly mixing an amino-containing copper-manganese bimetallic nanozyme and a 1 mM carboxyl-containing nucleic acid aptamer solution at a volume ratio of 25:8. The DNA sequence of the nucleic acid aptamer is shown in SEQ ID No: 1. The 5' end of the nucleic acid aptamer is modified with a carboxyl group. The nucleic acid aptamer is used to specifically recognize Escherichia coli O157:H7. The copper-manganese bimetallic nanozyme probe reagent is used to specifically bind to and isolate enriched Escherichia coli O157:H7, and generate concentration-dependent fluorescence and colorimetric signals of Escherichia coli O157:H7. The linear detection range of the dual-probe detection kit for Escherichia coli O157:H7 is 10. 1 ~10 6 CFU / mL, where the goodness of fit R 2 The value was 0.986, the limit of detection was 10 CFU / mL, and the recovery rate was 89%–109%.

[0007] The technical solution of the dual-probe detection kit is further as follows: The particle size of the iron oxide nanospheres is 290–310 nm.

[0008] The copper-manganese bimetallic nanozyme has a particle size of 2-3 nm.

[0009] The preparation steps of a dual-probe detection kit for detecting Escherichia coli O157:H7 are as follows: (1) Preparation of activation mixture The 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and the 5 mM N-hydroxysuccinimide were mixed evenly at a volume ratio of 1:1 to obtain an activated mixture. (2) Preparation of magnetic separation probe reagent A magnetic separation probe reagent was assembled by covalent coupling between concanavalin A and amino-containing iron oxide nanospheres. The specific operation is as follows: Add 0.1 mg of magnetic beads with a concentration of 10 mg / mL to 900 μL of activation mixture to activate the carboxyl groups of the magnetic beads, and incubate on a shaker at 37 °C and 180 rpm for 1 h. The magnetic beads are iron oxide nano-magnetic beads; Add 900 μL of 1 mg / mL canavon protein lectin A, and react in a shaker at 37°C and 180 rpm for 4 h with continuous rotation to synthesize targeted magnetic beads through coupling reaction. Unreacted concanavalin A was washed away by magnetic adsorption with 0.1M sterile phosphate solution, and then 1 mL of 0.1M sterile phosphate solution was added to synthesize a 0.1 mg / mL solution of concanavalin A-functionalized iron oxide nanospheres, which is a magnetic separation probe reagent. (3) Preparation of copper-manganese bimetallic nanozyme probe reagent (3.1) Preparation of copper-manganese bimetallic nanozymes Dissolve 0.05g citric acid, 0.07g copper chloride, 0.05g manganese chloride and 100μL ethylenediamine (≥98%) in 5mL ultrapure water and sonicate to react evenly. The mixture was sealed in a reactor and reacted at 180.0℃ for 6.0 h. Centrifuge at 10,000 rpm for 15 minutes to separate the first supernatant, and repeat centrifugation for 15 minutes to separate the second supernatant. In a freeze dryer, the second supernatant is freeze-dried to obtain freeze-dried powder; 10 mL of ultrapure water is added to prepare a nano-enzyme solution. (3.2) Preparation of copper-manganese bimetallic nanozyme probe reagent A 1 mM nucleic acid aptamer solution was placed in a metal bath and cooled at 4 °C for 5 min to denature and anneal the aptamer, thus obtaining a stable aptamer solution. 250 μL of 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide were added to 160 μL of stable aptamer solution to obtain a mixed solution; the solution was shaken and mixed in a shaker; 500 μL of the nanozyme solution was added and the mixture was shaken and mixed for 2.5 h to obtain a copper-manganese bimetallic nanozyme probe reagent. (4) Prepare the dual-probe detection kit The magnetic separation probe reagent and the copper-manganese bimetallic nanozyme probe reagent, which were separately packaged, were paired at a volume ratio of 5:8 and stored at 4°C to prepare a dual-probe detection kit. The amount of magnetic separation probe reagent is 50 μL, and the amount of copper-manganese bimetallic nanozyme probe reagent is 80 μL.

[0010] The technical solution for the further preparation method of the dual-probe detection kit is as follows: In step (3.1), the ultrasonic conditions are: power 300W, time 5min.

[0011] In step (3.1), the freeze-drying conditions are: temperature -50℃, time 24h.

[0012] In step (3.2), the metal bath conditions are: temperature 95℃, time 5min.

[0013] In step (3.2), the shaking conditions are: temperature 37℃, rotation speed 180rpm, and shaking and mixing for 30min; The specific operating steps for the dual-probe detection kit for rapid detection of Escherichia coli O157:H7 are as follows: Add 50 μL of the magnetic separation probe reagent to 200 μL of Escherichia coli O157:H7 solution, then add 80 μL of the targeted copper-manganese bimetallic nanozyme reagent, and finally add 170 μL of acetate-sodium acetate buffer. Incubate at 37 °C for 40 min to obtain the test solution.

[0014] The liquid to be tested was separated by magnetic separation, and the precipitate was resuspended in 500 μL of sterile acetate-sodium acetate buffer to obtain a resuspension solution.

[0015] Take 50 μL of the resuspended solution, add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L 3,3',5,5'-tetramethylbenzidine solution, and react at 60 °C for 10 min to obtain the reaction solution; read the absorbance of the first reaction solution using a UV spectrophotometer. Take another 50 μL of resuspended solution, add 450 μL of sterile acetate-sodium acetate buffer, let stand for 10 min, and read the fluorescence intensity of the second reaction solution using a fluorescence spectrophotometer.

[0016] The detection limit for fluorescence signal in the dual-modal detection technique for Escherichia coli O157:H7 is 10 CFU / mL, where R 2 =0.986.

[0017] The detection limit for the colorimetric signal in the dual-modal detection technique for Escherichia coli O157:H7 is 10 CFU / mL, where R 2 =0.986. The beneficial technical effects of this invention are reflected in the following aspects:

[0018] The innovativeness of the colorimetric-fluorescent dual-probe detection kit This invention is the first to apply concanavalin A as a recognition element to the identification and detection of *Escherichia coli* O157:H7. A magnetic separation probe is assembled by covalently coupling concanavalin A with amino-containing iron oxide nanospheres. This probe is used to screen and separate Gram-negative bacteria, solving the problem of the lack of suitable specific recognition elements for *E. coli* O157:H7 in existing methods. This allows for the first recognition using a dual-probe composition: when *E. coli* O157:H7 is present in the sample solution, the magnetic separation probe binds to the surface of *E. coli* O157:H7, and uses a magnet to separate and enrich *E. coli* O157:H7 from the sample solution, reducing matrix interference. The bimetallic nanozyme probe then performs a secondary recognition of the enriched bacteria through a specific aptamer. The two probes achieve perfect synergy through a "first enrich, then detect" process. The pretreatment of the magnetic separation probe eliminates the inhibition or interference of other substances in the sample on the catalytic activity and fluorescence signal of the nanozyme, allowing the performance of the second probe to be fully utilized. This sequential operation design allows the two probe systems to be both independent and unified in the same detection target, achieving a synergistic enhancement effect.

[0019] This invention achieves a detection limit as low as 10 CFU / mL for *Escherichia coli* O157:H7 in both colorimetric and fluorescence detection modes, meeting the needs of trace detection. The copper-manganese bimetallic nanozyme probe prepared in this invention exhibits a high correlation coefficient of 0.986 in both colorimetric and fluorescence modes, demonstrating a good correlation between concentration and signal response when detecting *E. coli* O157:H7, thus ensuring its practical application. Furthermore, different common foodborne pathogens closely related to *E. coli* O157:H7 and possessing similar antigenic sites were selected to evaluate the specificity of colorimetric and fluorescence detection modes. The dual-probe detection kit of this invention demonstrates significant specificity in both colorimetric and fluorescence modes. By utilizing the combined enzyme-like activity and fluorescence properties of the bimetallic nanozyme probe, a colorimetric-fluorescence dual-mode detection kit was successfully constructed. The linear regression equation established under standard conditions in this invention exhibits good environmental tolerance and can be directly applied to the detection of ham sausages without the need for reculturing bacteria to establish a standard curve, thus saving detection time. It is suitable for rapidly estimating the preliminary concentration of *E. coli* O157:H7 in food samples under emergency conditions. For example, experimental results show that, after calculation, under conditions where *E. coli* O157:H7 is artificially inoculated into ham sausages, the concentration of *E. coli* O157:H7 measured using colorimetric and fluorescence dual-channel methods is 3.8*10... 4 CFU / mL and 3.5*10 4 CFU / mL, almost exactly the same as its actual concentration of 3.5*10 4 When the CFU / mL values ​​are close, the reliability of the results is significantly improved when the results from the two signals corroborate each other; if a deviation occurs, it indicates a possible anomaly requiring verification. This mechanism significantly improves the accuracy and reliability of the test results. It can be seen that even without establishing a corresponding standard curve and linear regression equation under the ham sausage environment, the standard curve and linear regression equation established under the acetate-sodium acetate buffer solution still have good versatility and can be quickly applied to the detection of different samples.

[0020] 2. Inventiveness of the preparation method for the dual-probe detection kit This invention first prepares a magnetic separation probe reagent. By attaching a biorecognition molecule of concanavalin A to magnetite beads, it aims to capture *E. coli* O157:H7. Simultaneously, by attaching an aptamer to a bimetallic nanozyme for specific recognition of *E. coli* O157:H7, a secondary capture is achieved, further improving detection sensitivity. The magnetic separation probe is bound to the surface of *E. coli* O157:H7, and a magnetic rack is used to separate and enrich *E. coli* O157:H7 from the sample solution. This invention utilizes a low-cost combination of citric acid, copper chloride, and manganese chloride to synthesize a targeted bimetallic carbon-based nanozyme with multiple properties, including specific recognition, fluorescence signal, and colorimetric signal, at high temperature. In the preparation process, copper and manganese (Cu and Mn) are creatively co-doped, utilizing their synergistic effect to significantly lower the energy barrier for hydrogen peroxide reduction, improving enzyme-like catalytic efficiency and thus enhancing the colorimetric signal. Furthermore, this synthesis method forms fluorescent carbon dots in situ, achieving a breakthrough by generating two different signal properties with a single probe. Furthermore, both probes were prepared using the EDC / NHS chemical coupling method. This method involves covalent coupling, resulting in a strong, stable, and highly specific connection. This ensures that the biorecognition molecules can be correctly targeted and immobilized on the surface of the nanocarrier, thereby guaranteeing the probe's capture efficiency and stability. Attached Figure Description

[0021] Figure 1 The images show the magnetic probe and transmission electron microscope image of the targeted magnetic beads in this invention.

[0022] Figure 2 The images show the transmission electron microscope (TEM) image, elemental mapping diagram, and zeta potential diagram of the copper-manganese bimetallic nanozyme in this invention.

[0023] Figure 3 This is a graph of the colorimetric fluorescence dual-modal linear detection equation for the dual probes in this invention.

[0024] Figure 4 This study explores the specificity of the bimetallic detection probe in this invention. Detailed Implementation

[0025] The present invention will now be described in detail with reference to specific embodiments.

[0026] Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] Unless otherwise specified, the raw materials used in the following examples are all conventional biochemical reagents; unless otherwise specified, the experimental methods are all conventional methods; unless otherwise specified, the quantitative tests in the following examples are all repeated three times and the results are averaged; unless otherwise specified, the percentages in the following examples are all mass percentages.

[0028] In the following examples, the synthesized aptamers were purchased from Sangon Biotech (Shanghai) Co., Ltd.; unless otherwise specified, all other raw materials used were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0029] It should be noted that the phosphate buffer used in this application is a 0.1M sterile PBS buffer with a pH of 7.4.

[0030] It should be noted that all strain numbers used in this application are consistent with those of the U.S. Culture Collection Center; the number for Staphylococcus aureus is ATCC 29213, for Vibrio parahaemolyticus is ATCC 17802, for Escherichia coli is ATCC 25922, for Cronobacter sakazakii is ATCC 29544, for Pseudomonas aeruginosa is ATCC 15442, for Enterococcus faecalis is ATCC 29212, for Listeria monocytogenes is ATCC 43251, and for Shigella flexneri is ATCC 12022.

[0031] It should be noted that all instruments, equipment, raw materials, reagents, and procedures used in this application are performed under sterile conditions.

[0032] It should be noted that instruments, equipment, raw materials, reagents, or methods not mentioned in this application are conventional or well-known technical methods to those skilled in the art, and will not be described in detail in this application. Example 1

[0033] The preparation steps of a dual-probe detection kit for detecting Escherichia coli O157:H7 are as follows: (1) Preparation of activation mixture The activated mixture was obtained by mixing 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide at a volume ratio of 1:1.

[0034] (2) Preparation of magnetic separation probe reagent A magnetic separation probe reagent was assembled by covalent coupling between concanavalin A and amino-containing iron oxide nanospheres. The specific operation is as follows: Add 0.1 mg of magnetic beads with a concentration of 10 mg / mL to 900 μL of activation mixture to activate the carboxyl groups of the magnetic beads, and incubate on a shaker at 37 °C and 180 rpm for 1 h. The magnetic beads are iron oxide nano-magnetic beads; Add 900 μL of 1 mg / mL canavon protein lectin A, and react in a shaker at 37°C and 180 rpm for 4 h with continuous rotation to synthesize targeted magnetic beads through coupling reaction. Unreacted concanavalin A was washed away by magnetic adsorption with 0.1M sterile phosphate solution, and then 1 mL of 0.1M sterile phosphate solution was added to synthesize a 0.1 mg / mL solution of concanavalin A-functionalized iron oxide nanospheres, which is the magnetic separation probe reagent.

[0035] Magnetic separation probe reagents are used to specifically bind to Escherichia coli O157:H7 and to separate and enrich it from the test sample.

[0036] Following the synthesis of magnetic beads, a binding process was performed to concanavalin A, resulting in a complex of magnetic beads and concanavalin A. The magnetic beads were observed under an electron microscope; the results are recorded below. Figure 1 The magnetic properties of the composite were tested, and the results are as follows: Figure 1 As shown in Figure A, the magnetic separation effect of the magnetic bead@concanavalin A complex is obvious. Transmission electron microscopy observation of the magnetic beads is shown in Figure A. Figure 1 As shown in B, the magnetic beads are about 300 nm in size, and the magnetic bead@concanavalin A complex has a good morphology, indicating that the modification of the magnetic beads and lectin was relatively successful.

[0037] (3) Preparation of copper-manganese bimetallic nanozyme probe reagent (3.1) Preparation of copper-manganese bimetallic nanozymes Dissolve 0.05g citric acid, 0.07g copper chloride, 0.05g manganese chloride and 100μL ethylenediamine (≥98%) in 5mL ultrapure water and sonicate to react evenly; sonication conditions: power 300W, time 5min.

[0038] The mixture was sealed in a reactor and reacted at 180.0℃ for 6.0 h. Centrifuge at 10,000 rpm for 15 minutes to separate the first supernatant, and repeat centrifugation for 15 minutes to separate the second supernatant. In a freeze dryer, the second supernatant was freeze-dried at -50°C for 24 hours to obtain freeze-dried powder; 10 mL of ultrapure water was added to prepare a nanoenzyme solution.

[0039] (3.2) Preparation of copper-manganese bimetallic nanozyme probe reagent A 1 mM nucleic acid aptamer solution was heated in a 95°C metal bath for 5 min and then cooled at 4°C for 5 min to denature and anneal the aptamer, thus obtaining a stable aptamer solution.

[0040] The DNA sequence of the nucleic acid aptamer used is shown in SEQ ID No: 1, and the 5' end of the nucleic acid aptamer is modified with a carboxyl group.

[0041] 250 μL of a mixture containing 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide was added to 160 μL of a stable aptamer solution to obtain a mixed solution. The mixture was shaken for 30 min at 37 °C and 180 rpm in a shaker. Then, 500 μL of the nanozyme solution was added, and the mixture was shaken for 2.5 h to prepare the copper-manganese bimetallic nanozyme probe reagent. The shaker conditions were 37 °C and 180 rpm.

[0042] After synthesis of copper-manganese bimetallic nanozymes, the nanozymes and aptamers were combined to synthesize targeted copper-manganese bimetallic nanozyme complexes. The copper-manganese bimetallic nanozymes and targeted copper-manganese bimetallic nanozymes were observed using transmission electron microscopy, and their potentials were measured using a Malvern Zeta potentiometer, and recorded as follows: Figure 2 The particle size and distribution of copper-manganese bimetallic nanozymes were observed using transmission electron microscopy, as shown in the following results. Figure 2 As shown in Figure A, the copper-manganese bimetallic nanozymes have a particle size of approximately 2-3 nm and are uniformly distributed. The elemental mapping results of the copper-manganese bimetallic nanozymes are shown below. Figure 2 B in Figure 2 As shown in Figure C, copper and manganese elements are widely and uniformly distributed in the bimetallic carbon dot material, proving that both elements have achieved good and uniform bonding with the carbon dots. The potential measurement results of copper-manganese bimetallic nanozymes and copper-manganese bimetallic nanozymes@aptamers are shown below. Figure 2 As shown in D, compared with the copper-manganese bimetallic nanozyme, the aptamer potential of the copper-manganese bimetallic nanozyme after aptamer modification decreased significantly, proving that the aptamer modification was successful.

[0043] The copper-manganese bimetallic nanozyme probe reagent is used to specifically bind to and isolate enriched Escherichia coli O157:H7, and generate concentration-dependent fluorescence and colorimetric signals of Escherichia coli O157:H7.

[0044] (4) Prepare the dual-probe detection kit The magnetic separation probe reagent and the copper-manganese bimetallic nanozyme probe reagent, which are separately packaged, are paired at a volume ratio of 5:8 and stored at 4°C to prepare a dual-probe detection kit.

[0045] In the dual-probe detection kit, the amount of magnetic separation probe reagent is 50 μL, and the amount of copper-manganese bimetallic nanozyme probe reagent is 80 μL.

[0046] The dual-probe detection kit prepared in Example 1 has a linear detection range of 10 for Escherichia coli O157:H7. 1 ~10 6 CFU / mL, where the goodness of fit R 2 The value was 0.986, the limit of detection was 10 CFU / mL, and the recovery rate was 89%–109%. Example 2

[0047] The detection linear equations for the colorimetric and fluorescence modes were established separately. The specific steps are as follows: (1) First, prepare sterile test samples and artificially contaminate them with E. coli. Set up a linear gradient of bacterial solutions with concentrations of 0 CFU / mL, 10 CFU / mL, and 10 CFU / mL. 1 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 Prepare a bacterial solution containing CFU / mL for later use.

[0048] (2) Colorimetric signal detection Add 50 μL of the magnetic separation probe reagent to 200 μL of E. coli O157:H7 solution, then add 80 μL of targeted copper-manganese bimetallic nanozyme reagent, and finally add 170 μL of acetate-sodium acetate buffer. Incubate at 37°C for 40 min to obtain the test solution. The acetate-sodium acetate buffer is a pH 4.5 acetate-sodium acetate buffer purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number R27435-500ml.

[0049] The liquid to be tested was separated by magnetic separation, and the precipitate was resuspended in 500 μL of sterile acetate-sodium acetate buffer to obtain a resuspension solution.

[0050] Take 50 μL of the resuspended solution, add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L 3,3',5,5'-tetramethylbenzidine solution, and react at 60 °C for 10 min to obtain the reaction solution; use a UV spectrophotometer to read the absorbance of the first reaction solution at 653 nm.

[0051] The colorimetric signal detection solution was obtained to investigate the linear relationship between different bacterial concentrations and absorbance. The results are as follows: Figure 3 As shown in Figure A, the linear regression equation is absorbance = 0.173 × lg[E. coli O157:H7 (CFU / mL)] + 0.164. The detection limit for fluorescence signal in the dual-modal detection technology for E. coli O157:H7 is 10 CFU / mL, where R... 2 =0.986.

[0052] (3) Fluorescence signal detection Take another 50 μL of resuspended solution, add 450 μL of sterile acetate-sodium acetate buffer, let stand for 10 min, and use a fluorescence spectrophotometer to read the fluorescence intensity of the second reaction solution at the excitation wavelength of 360 nm.

[0053] The linear relationship between different bacterial concentrations and normalized fluorescence intensity was investigated, and the results are as follows: Figure 3 As shown in B, the linear regression equation is absorbance = 0.141 × lg[E. coli O157:H7 (CFU / mL)] + 0.130. The detection limit for the colorimetric signal in the dual-modal detection technique for E. coli O157:H7 is 10 CFU / mL, where R... 2 =0.986. Example 3

[0054] Testing of real samples The dual probe prepared in Example 1 and the linear regression equation constructed in Example 2 were combined to detect real food samples, with ham sausage samples selected for testing.

[0055] In this embodiment 3, in order to verify whether the linear regression equation constructed according to embodiment 2 can be effectively applied to the determination of interference environments such as ham sausage, artificial quantitative bacterial contamination was performed to explore the difference between the number of bacteria detected by the dual probe in the ham sausage sample, so as to know that even in real food samples, the dual probe established in this application can be effectively detected under the influence of the food matrix.

[0056] The ham sausages involved in this embodiment 3 were purchased from a supermarket and stored at room temperature during the purchase.

[0057] Escherichia coli O157:H7 was inoculated into ham sausages at a concentration of 3.5 x 10⁻⁶. 2 CFU / mL, 3.5*10 4 CFU / mL and 3.5*10 6CFU / mL, inoculated with ham sausage, was allowed to stand at room temperature for 2 hours to obtain the inoculated ham sausage. The inoculated ham sausage was then cut into pieces. In a sterile bag, 25g of the chopped ham sausage was mixed with 25mL of sterile PBS solution and homogenized for 2 minutes to obtain a mixture. The mixture was allowed to stand for 5 minutes, and the supernatant was collected as the test solution. Subsequently, in conjunction with Example 2, a fluorescence-colorimetric dual-modal signal was detected. Based on the obtained fluorescence intensity value and ultraviolet absorption intensity value, respectively, the linear regression equation was substituted to obtain the concentration of Listeria monocytogenes in the real sample.

[0058] The experimental results showed that, after calculation, under the condition of artificial inoculation of E. coli O157:H7 in ham sausage, the concentration of E. coli O157:H7 measured by colorimetric and fluorescence dual-channel methods was 3.8*10. 4 CFU / mL and 3.5*10 4 CFU / mL, almost exactly the same as its actual concentration of 3.5*10 4 The CFU / mL values ​​are close, indicating that even without establishing a corresponding standard curve and linear regression equation under ham sausage sample conditions, the standard curve and linear regression equation established under pure sterile acetate-sodium acetate buffer still have good versatility.

[0059] Therefore, the dual-probe composition prepared by this invention has good versatility and can be quickly applied to the detection of different samples. Example 4

[0060] The other operations in this embodiment 4 are the same as in embodiment 2, except that: Replace Escherichia coli O157:H7 with Bacillus parahaemolyticus, Staphylococcus aureus, Cronobacter, Pseudomonas aeruginosa, Enterococcus faecalis, Listeria monocytogenes, and Shigella flexneri, respectively.

[0061] See Figure 4 A and Figure 4In section B, the horizontal axis number "2" represents the addition of *Bacillus parahaemolyticus*, "3" represents the addition of *Staphylococcus aureus*, "3" represents the addition of *Cronobacter*, "5" represents the addition of *Pseudomonas aeruginosa*, "6" represents the addition of *Enterococcus faecalis*, "7" represents the addition of *Listeria monocytogenes*, and "8" represents the bacterial-containing test solution after the addition of *Shigella flexneri*. The normalized fluorescence intensity and ultraviolet absorption intensity values ​​of *Bacillus parahaemolyticus* are 0.213 and 0.271, respectively, while the normalized fluorescence intensity and ultraviolet absorption intensity values ​​of *Staphylococcus aureus* are 0. The normalized fluorescence intensity and UV absorption intensity values ​​for *Cronobacter* were 0.209 and 0.259, respectively; for *Pseudomonas aeruginosa*, 0.227 and 0.274, respectively; for *Enterococcus faecalis*, 0.226 and 0.253, respectively; for *Listeria monocytogenes*, 0.213 and 0.260, respectively; and for *Shigella flexneri*, 0.225 and 0.265, respectively. Meanwhile, under the same conditions... Figure 4 A and Figure 4 In the diagram, the number "1" in section B indicates the bacterial-containing test solution after adding *E. coli* O157:H7, with normalized fluorescence intensity and ultraviolet absorption intensity values ​​of 0.704 and 0.804, respectively. This demonstrates that the dual-probe composition prepared in this invention exhibits good specificity for *E. coli* O157:H7.

[0062] This invention develops a nucleic acid aptamer for *Escherichia coli* O157:H7 and its screening method, possessing the following technical characteristics: It significantly enhances the detection sensitivity of the target bacterium, achieving accurate identification even in trace samples, thereby effectively avoiding false negative results. Compared with traditional methods, the detection technology based on this aptamer greatly shortens the analysis time and improves detection efficiency, better meeting the needs of rapid response in food safety monitoring. Furthermore, the nucleic acid aptamer used is simple to synthesize and modify, reducing dependence on experimental materials and equipment, helping to control overall detection costs and improving the economic practicality of the technology. The detection process is simple to operate, reducing the need for complex instruments and lowering the operational difficulty, which is beneficial to improving laboratory work efficiency. Simultaneously, this method can simultaneously acquire colorimetric and fluorescence dual-modal signals in a single detection, not only improving detection sensitivity but also reducing misjudgments caused by operational errors through mutual correction between signals. This aptamer technology has good versatility and scalability, applicable not only to the detection of *Escherichia coli* O157:H7 but also to the identification of other pathogenic microorganisms or pollutants, demonstrating broad application potential in multiple fields such as food safety and environmental monitoring.

[0063] Those skilled in the art will readily understand that the above Embodiment 1 is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A dual-probe detection kit for detecting Escherichia coli O157:H7, characterized in that: The double-probe detection kit is composed of 50 μL magnetic separation probe reagent and 80 μL copper-manganese bimetallic nanoscale enzyme probe reagent which are independent of each other; The magnetic separation probe reagent is a 0.1 mg / mL solution of concanavalin A functionalized ferroferric oxide nanomicrospheres, which is prepared by uniformly mixing a 10 mg / mL solution of amino-containing ferroferric oxide nanomicrospheres and a 1 mg / mL solution of carboxyl-containing concanavalin A at a volume ratio of 1:9; The magnetic separation probe reagent is used for specific binding of Escherichia coli O157:H7 and separation and enrichment treatment from a detection sample; The copper-manganese bimetallic nanoscale enzyme probe reagent is prepared by uniformly mixing amino-containing copper-manganese bimetallic nanoscale enzymes and a 1 mM solution of carboxyl-containing aptamer at a volume ratio of 25:8; The DNA sequence of the aptamer is shown as SEQ ID No: 1, and the 5' end of the aptamer is modified with a carboxyl group; the aptamer is used for specific recognition of Escherichia coli O157:H7; The copper-manganese bimetallic nanoscale enzyme probe reagent is used for specific binding of the separated and enriched Escherichia coli O157:H7 and generation of Escherichia coli O157:H7 concentration-dependent fluorescent signals and colorimetric signals; The double-probe detection kit has a linear detection range of 10 1 ~ 10 6 CFU / mL for E. coli O157:H7, wherein the goodness of fit R 2 is 0.986, the lower limit of detection is 10 CFU / mL, and the recovery rate of the standard addition is 89%~109%.

2. The method for preparing a dual-probe detection kit for detecting Escherichia coli O157:H7 according to claim 1, characterized in that, The particle size of the ferroferric oxide nanomicrospheres is 290-310 nm.

3. The double-probe detection kit for detecting Escherichia coli O157:H7 according to claim 1, wherein the double-probe detection kit comprises the oligonucleotide probes of claim 1. The particle size of the copper-manganese bimetallic nanoscale enzyme is 2-3 nm.

4. The method for preparing a dual-probe detection kit for detecting Escherichia coli O157:H7 according to claim 1, characterized in that, The operation steps are as follows: (1) Preparation of an activated mixed solution Mix 10 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide at a volume ratio of 1:1 to obtain an activated mixed solution; (2) Preparation of a magnetic separation probe reagent The magnetic separation probe reagent is assembled by covalent coupling between concanavalin A and amino-containing ferroferric oxide nanomicrospheres, and the specific operation is as follows: Add 0.1 mg of the magnetic beads with a concentration of 10 mg / mL to 900 μL of the activated mixed solution to activate the carboxyl groups of the magnetic beads, and incubate in a shaking incubator at a temperature of 37℃ and a rotation speed of 180 rpm for 1 h; The magnetic beads are ferroferric oxide nanomagnetic beads; Add 900 μL of concanavalin A with a concentration of 1 mg / mL, and react in a shaking incubator at a temperature of 37℃ and a rotation speed of 180 rpm for 4 h with continuous rotation to obtain the target magnetic beads by coupling reaction; Wash away the unreacted concanavalin A by magnetic adsorption with a 0.1 M sterile phosphate solution, and then add 1 mL of a 0.1 M sterile phosphate solution to obtain a 0.1 mg / mL solution of concanavalin A functionalized ferroferric oxide nanomicrospheres, i.e., the magnetic separation probe reagent; (3) Preparation of a copper-manganese bimetallic nanoscale enzyme probe reagent (3.1) Preparation of a copper-manganese bimetallic nanoscale enzyme Dissolve 0.05 g of citric acid, 0.07 g of copper chloride, 0.05 g of manganese chloride and 100 μL of ethylenediamine (≥98%) in 5 mL of ultrapure water, and ultrasonically react uniformly; Seal in a reactor and react at a temperature of 180.0℃ for 6.0 h; In the centrifuge, the rotation speed is 10000 rpm, centrifugation for 15 min, the first supernatant is separated, and the second supernatant is separated after repeating centrifugation for 15 min; In the freeze dryer, the second supernatant is freeze-dried to obtain a freeze-dried powder; 10 mL of ultrapure water is added to prepare a nanoenzyme solution; (3.2) Preparation of copper-manganese bimetallic nanoenzyme probe reagent The nucleic acid aptamer solution with a concentration of 1 mM is subjected to a metal bath, cooled at a temperature of 4℃ for 5 min to denature and anneal the aptamer, and a stable aptamer solution is obtained; In 160 μL of the stable aptamer solution, 250 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride containing 10 mM and N-hydroxysuccinimide containing 5 mM are added to obtain a mixed solution; the mixture is shaken in a shaking bed; 500 μL of the nanoenzyme solution is added, and the mixture is shaken for 2.5 h to prepare a copper-manganese bimetallic nanoenzyme probe reagent; (4) Preparation of double-probe detection kit The magnetic separation probe reagent and the copper-manganese bimetallic nanoenzyme probe reagent are paired by volume ratio of 5:8, respectively packaged, stored at 4℃, and a double-probe detection kit is prepared. The amount of the magnetic separation probe reagent is 50 μL, and the amount of the copper-manganese bimetallic nanoenzyme probe reagent is 80 μL.

5. The method of claim 4, wherein: In step (3.1), the ultrasonic conditions are: power 300 W, time 5 min.

6. The method of claim 4, wherein: In step (3.1), the freeze-drying conditions are: temperature -50℃, time 24 h.

7. The method of claim 4, wherein: In step (3.2), the metal bath conditions are: temperature 95℃, time 5 min.

8. The method of claim 4, wherein: In step (3.2), the shaking bed conditions are: temperature 37℃, rotation speed 180 rpm, shaking mixing time 30 min.

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