Folic acid-o-phenylenediamine co-doped carbon quantum dot, Escherichia coli O157: H7 detection probe, detection reagent and application

Abstract

The invention belongs to the technical field of food safety detection, and particularly relates to folic acid-o-phenylenediamine co-doped carbon quantum dots, an Escherichia coli O157: H7 detection probe, a detection reagent and application. The folic acid-o-phenylenediamine co-doped carbon quantum dots are prepared by heating folic acid and o-phenylenediamine to react. When the detection probe or the detection reagent based on the folic acid-o-phenylenediamine co-doped carbon quantum dots is used for detecting Escherichia coli O157: H7, the detection limit is low, the specificity is excellent, and the practicability is high.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a folic acid-o-phenylenediamine co-doped carbon quantum dot, a detection probe for Escherichia coli O157:H7, detection reagents, and applications. Background Technology

[0002] Foodborne pathogen contamination is a major threat to global food safety, with *Escherichia coli* O157:H7 being a typical highly pathogenic strain that poses a serious risk to public health. This strain is characterized by extremely low infectious doses (≤10 CFU are sufficient to cause disease) and high pathogenicity, capable of causing serious complications including hemorrhagic diarrhea and hemolytic uremic syndrome, with a high mortality rate. It is widely present in various foods such as meat, dairy products, and vegetables, and contamination can occur during food processing, storage, and transportation. Therefore, establishing a rapid, accurate, and sensitive detection method for *Escherichia coli* O157:H7 is of significant practical importance and application value for the timely prevention and control of foodborne diseases, ensuring food safety, and protecting public health.

[0003] Currently, detection methods for Escherichia coli O157:H7 are mainly divided into three categories, but all have significant limitations: Traditional culture methods, as the "gold standard," are highly reliable, but require steps such as enrichment, isolation, and biochemical identification, with a detection cycle of 5-7 days, which cannot meet the timeliness requirements of modern food safety supervision; Instrumental analysis methods, such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and real-time quantitative PCR, have high sensitivity (detection limits can reach 10² CFU / mL), but rely on expensive equipment and are complex to operate, requiring professional technicians, making it difficult to promote in grassroots testing institutions and on-site rapid testing; Among immunological and aptamer sensing methods, aptamers have attracted attention due to their advantages such as easy synthesis, good stability, and chemical modification. However, in existing technologies, commonly used carbon quantum dots are mostly prepared using a single carbon source, resulting in low fluorescence quantum yield (usually <30%) and weak resistance to matrix interference, which limits the overall performance improvement of detection methods.

[0004] The existing detection methods mentioned above have shortcomings in terms of detection efficiency, ease of operation, sensitivity, and anti-interference ability, making them unable to effectively meet the need for rapid and accurate detection of low concentrations of Escherichia coli O157:H7 in food. Therefore, developing a quantum dot with high fluorescence quantum yield and good fluorescence stability is of great significance for overcoming the technical limitations of detecting Escherichia coli O157:H7 in food and improving the sensitivity, specificity, and practicality of the detection. Summary of the Invention

[0005] Based on this, the present invention provides a folic acid-o-phenylenediamine co-doped carbon quantum dot and a detection probe and reagent based on the folic acid-o-phenylenediamine co-doped carbon quantum dot for the detection of Escherichia coli O157:H7. The detection probe and reagent can achieve efficient and accurate detection of Escherichia coli O157:H7.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: In one aspect, the present invention provides a folic acid-o-phenylenediamine co-doped carbon quantum dot, which is prepared by heating folic acid and o-phenylenediamine.

[0007] Preferably, in the above-mentioned folic acid-o-phenylenediamine co-doped carbon quantum dots, the mass ratio of folic acid to o-phenylenediamine is (1-3):1.

[0008] Preferably, in the folic acid-o-phenylenediamine co-doped carbon quantum dots, the heating reaction temperature is 170℃-190℃.

[0009] Another aspect of the present invention provides a detection probe for Escherichia coli O157:H7, comprising the aforementioned folic acid-o-phenylenediamine co-doped carbon quantum dots and an aminated aptamer; the folic acid-o-phenylenediamine co-doped carbon quantum dots are activated by EDC / NHS and then linked to the aminated Escherichia coli O157:H7 aptamer, the sequence of which is: CCGGACGCTTATGCCTTGCCATCTACAGAGCAGGTGTGACGG.

[0010] In another aspect, the present invention provides a detection reagent for Escherichia coli O157:H7, which includes the above-mentioned detection probe for Escherichia coli O157:H7.

[0011] Preferably, the above-mentioned Escherichia coli O157:H7 detection reagent further includes an Escherichia coli O157:H7 capture probe.

[0012] Preferably, the preparation method of the Escherichia coli O157:H7 capture probe in the above-mentioned Escherichia coli O157:H7 detection reagent includes: (1) The reactants were obtained by heating ferric chloride hexahydrate, anhydrous sodium acetate and polyacrylic acid; (2) The reactants were magnetically separated to obtain a black solid, which is the carboxylated magnetic nanoparticle; (3) Carboxylated magnetic nanoparticles were linked to biotinylated Escherichia coli O157:H7 aptamers via streptavidin to obtain Escherichia coli O157:H7 capture probes.

[0013] More preferably, in the above method for preparing the Escherichia coli O157:H7 capture probe, the heating reaction temperature is 190℃-200℃.

[0014] In another aspect, this invention provides a method for detecting Escherichia coli O157:H7, the method comprising: using the above-mentioned Escherichia coli O157:H7 detection reagent for detection, including: (i) The Escherichia coli O157:H7 bacterial suspension and the Escherichia coli O157:H7 capture probe were mixed and incubated, and then magnetic separation was performed; (ii) After magnetic separation, it is mixed with the Escherichia coli O157:H7 detection probe and incubated before magnetic separation. (iii) After magnetic separation, the fluorescence intensity is detected, and the content of Escherichia coli O157:H7 is calculated based on the standard curve of concentration-fluorescence intensity.

[0015] Preferably, in step (iii) of the above detection method, the fluorescence intensity at 475 nm is detected at an excitation wavelength of 360 nm.

[0016] The beneficial effects of this invention include: (1) The carbon quantum dots prepared by the folic acid-o-phenylenediamine co-doping strategy of the present invention have a fluorescence quantum yield of 58.2%, which is 104% higher than that of carbon quantum dots prepared by a single carbon source (quantum yield 28.5%). Furthermore, the carbon quantum dots have good fluorescence stability in complex food matrices and the decay rate does not exceed 8%.

[0017] (2) Based on the folic acid-o-phenylenediamine co-doped carbon quantum dots of the present invention, the detection limit of Escherichia coli O157:H7 was as low as 10 CFU / mL, which is 100 times higher than the ELISA method (detection limit 10³ CFU / mL), and the detection limit was even lower than that of 10 CFU / mL. 1 -10 6 The linear relationship was good within the CFU / mL concentration range (R²≥0.998).

[0018] (3) Based on the folic acid-o-phenylenediamine co-doped carbon quantum dots of the present invention, the detection of Escherichia coli O157:H7 is carried out in less than 7.5 hours, which is more than 90% shorter than the traditional culture method; the cross-reactivity rate of 10 common interfering bacteria such as Staphylococcus aureus and Salmonella is <1%, and the specificity is significantly improved.

[0019] (4) Based on the folic acid-o-phenylenediamine co-doped carbon quantum dots of the present invention, Escherichia coli O157:H7 can be detected. It is highly practical: no expensive instruments are required, sample pretreatment is simple, and the cost of a single sample detection is less than 50 yuan. It is suitable for grassroots testing institutions and rapid on-site screening. Attached Figure Description

[0020] Figure 1 TEM image of the carboxylated Fe3O4 magnetic nanoparticles prepared in Example 1; Figure 2 The FTIR spectrum of the aptamer-functionalized magnetic nanobead trapping probe prepared in Example 1; Figure 3 The magnetic saturation intensity of the aptamer-functionalized nanomagnetic bead trapping probe prepared in Example 1; Figure 4 TEM image of folic acid-o-phenylenediamine co-doped carbon quantum dots; in the image, A: unmodified aptamer; B: modified aptamer). Figure 5 Fluorescence emission spectrum of aptamer-functionalized carbon quantum dots; Figure 6 FTIR spectra of aptamer-functionalized carbon quantum dots; Figure 7 Standard curve for the detection of Escherichia coli O157:H7; Figure 8 The image shows the results of specificity verification. In the image, 1: Escherichia coli O157:H7 (positive control); 2: Staphylococcus aureus; 3: Salmonella; 4: Listeria monocytogenes; 5: Escherichia coli; 6: Vibrio parahaemolyticus; 7: Bacillus cereus; 8: Cronobacter sakazakii; 9: Yersinia enterocolitica; 10: Shigella; 11: Pseudomonas aeruginosa. Detailed Implementation

[0021] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0022] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0023] In a first aspect, embodiments of the present invention provide a folic acid-o-phenylenediamine co-doped carbon quantum dot, which is prepared by heating folic acid and o-phenylenediamine together.

[0024] It should be noted that the carbon quantum dots prepared by the folic acid-o-phenylenediamine co-doping strategy of this invention have a fluorescence quantum yield of 58.2%, which is 104% higher than that of carbon quantum dots prepared by a single carbon source (quantum yield of 28.5%). Furthermore, they exhibit good fluorescence stability in complex food matrices with a decay rate of no more than 8%.

[0025] In some specific examples, the mass ratio of folic acid to o-phenylenediamine in the above-mentioned folic acid-o-phenylenediamine co-doped carbon quantum dots is (1-3):1.

[0026] It should be noted that in the preparation of folic acid-o-phenylenediamine co-doped carbon quantum dots in this invention, the mass ratio of folic acid to o-phenylenediamine can be (1-3):1, for example 1.5:1, 2:1 or 2.5:1, etc.

[0027] In some specific examples, the temperature of the heating reaction in the above-mentioned folic acid-o-phenylenediamine co-doped carbon quantum dots is 170℃-190℃.

[0028] It should be noted that in the preparation of folic acid-o-phenylenediamine co-doped carbon quantum dots in this invention, the heating reaction temperature is 170℃-190℃, for example, 175℃, 180℃, or 185℃. Furthermore, it should be understood that the reaction of folic acid and o-phenylenediamine is generally carried out in an organic solvent environment, and the organic solvent is known in the art, such as ethanol.

[0029] Secondly, embodiments of the present invention provide a detection probe for Escherichia coli O157:H7, comprising the aforementioned folic acid-o-phenylenediamine co-doped carbon quantum dots and an aminated aptamer; the folic acid-o-phenylenediamine co-doped carbon quantum dots are activated by EDC / NHS and then linked to the aminated Escherichia coli O157:H7 aptamer, the sequence of which is: CCGGACGCTTATGCCTTGCCATCTACAGAGCAGGTGTGACGG.

[0030] It should be noted that the folic acid-o-phenylenediamine co-doped carbon quantum dots in this invention can be linked with an aminated Escherichia coli O157:H7 aptamer to prepare a detection probe for detecting Escherichia coli O157:H7. It should be understood that the method by which the folic acid-o-phenylenediamine co-doped carbon quantum dots are linked with the aminated Escherichia coli O157:H7 aptamer is well known in the art, such as the aforementioned EDC / NHS. Furthermore, the Escherichia coli O157:H7 aptamer exhibits good stability, showing no activity degradation after 6 months of storage at 4°C.

[0031] Thirdly, embodiments of the present invention provide a detection reagent for Escherichia coli O157:H7, which includes the above-mentioned detection probe for Escherichia coli O157:H7.

[0032] It should be noted that the Escherichia coli O157:H7 detection probe of this invention can be prepared into an Escherichia coli O157:H7 detection reagent for detecting Escherichia coli O157:H7. It should be understood that the Escherichia coli O157:H7 detection reagent may also include other auxiliary detection reagents, such as buffer solutions.

[0033] In some specific examples, the above-mentioned Escherichia coli O157:H7 detection reagent also includes an Escherichia coli O157:H7 capture probe.

[0034] It should be noted that the Escherichia coli O157:H7 detection probe in this invention can also be used in conjunction with the Escherichia coli O157:H7 capture probe to form an Escherichia coli O157:H7 detection reagent. In this way, based on the enrichment effect of the Escherichia coli O157:H7 capture probe on Escherichia coli O157:H7, the detection efficiency of Escherichia coli O157:H7 can be improved.

[0035] In some specific examples, the preparation method of the Escherichia coli O157:H7 capture probe in the above-mentioned Escherichia coli O157:H7 detection reagent includes: (1) The reactants were obtained by heating ferric chloride hexahydrate, anhydrous sodium acetate and polyacrylic acid; (2) The reactants were magnetically separated to obtain a black solid, which is the carboxylated magnetic nanoparticle; (3) Carboxylated magnetic nanoparticles were linked to biotinylated Escherichia coli O157:H7 aptamers via streptavidin to obtain Escherichia coli O157:H7 capture probes.

[0036] It should be noted that the Escherichia coli O157:H7 capture probe in this invention uses 1,6-hexanediamine as a multifunctional reagent, which simplifies the magnetic bead preparation process and achieves an enrichment rate of ≥95% for the target bacteria, which is 21.3% higher than that of traditional methods.

[0037] In some specific examples, in the above-mentioned method for preparing the Escherichia coli O157:H7 capture probe, the heating reaction temperature is 190℃-200℃.

[0038] It should be noted that in the above method for preparing the Escherichia coli O157:H7 capture probe, the heating reaction temperature can be 190℃-200℃, such as 193℃, 195℃ or 197℃.

[0039] Fourthly, embodiments of the present invention provide a method for detecting Escherichia coli O157:H7, the method comprising: using the above-mentioned Escherichia coli O157:H7 detection reagent for detection, including: (i) The Escherichia coli O157:H7 bacterial suspension and the Escherichia coli O157:H7 capture probe were mixed and incubated, and then magnetic separation was performed; (ii) After magnetic separation, it is mixed with the Escherichia coli O157:H7 detection probe and incubated before magnetic separation. (iii) After magnetic separation, the fluorescence intensity is detected, and the content of Escherichia coli O157:H7 is calculated based on the standard curve of concentration-fluorescence intensity.

[0040] In some specific examples, in step (iii) of the above detection method, the fluorescence intensity at 475 nm is detected at an excitation wavelength of 360 nm.

[0041] It should be noted that in this invention, the fluorescence intensity at 475nm can be detected at an excitation wavelength of 360nm, thereby enabling the detection of Escherichia coli O157:H7.

[0042] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0043] In the following examples, the Escherichia coli O157:H7 used has the accession number BNCC 192101. In the following examples, The sequence of the aminated Escherichia coli O157:H7 aptamer is as follows: 5'-NH2-CCGGACCGCTTATGCCTTGCCATCTACAGAGCAGGTTGACGG-3'; The biotinylated Escherichia coli O157:H7 aptamer sequence is as follows: 5'-Biotin-CCGGACCGCTTATGCCTTGCCATCTACAGAGCAGGTTGGACGG-3'.

[0044] Preparation Examples Example 1 (I) Preparation of the capture probe (1) Weigh 1.0g FeCl3·6H2O, 2.0g anhydrous sodium acetate and 1.5g polyacrylic acid (PAA) into 30mL ethylene glycol, stir at 50℃ for 30 minutes to form a uniform colloid, transfer to 50mL polytetrafluoroethylene reactor, react at 198℃ for 6 hours; after natural cooling, separate the black product with a 0.8T magnetic field, wash with deionized water and anhydrous ethanol three times alternately, and finally vacuum dry at 50℃ for 10 hours to obtain surface carboxylated Fe3O4 magnetic nanoparticles; (2) Take 10 mg of carboxylated Fe3O4 nanoparticles, wash them with MES buffer at pH 6.0, resuspend them in 4 mL of MES, add freshly prepared EDC (final concentration 10-20 mM) and NHS (final concentration 5-10 mM), and activate them by shaking at room temperature in the dark for 30 minutes; immediately perform magnetic separation, wash twice with ice-cold MES buffer, and react with 125 μg / mL streptavidin solution prepared with 5 mL PBS (pH 7.4) at 4 °C for 12 hours; after the reaction, perform magnetic separation, first block with ethanolamine solution for 1 hour, then block with 1% BSA for 30 minutes, and finally wash thoroughly 4 times with PBS buffer containing 0.05% Tween-20 to obtain streptavidin-coated magnetic particles, resuspend them in storage buffer and store at 4 °C; (3) Take 500 μL of 1 mg / mL streptavidin magnetic beads, discard the supernatant after magnetic separation, and wash once with PBS buffer containing 1 mM MgCl2; then add 500 μL of 10 μM biotinylated aptamer solution (dissolved in PBS+MgCl2 buffer system), and shake at room temperature for 1 hour; after magnetic separation, discard the supernatant, wash 3 times with PBS buffer containing 0.05% Tween-20, and then add 1 mL of 2% BSA solution to block at room temperature for 1 hour; finally, after magnetic separation and washing, resuspend in 500 μL of PBS storage buffer containing 0.05% Tween-20 and store at 4℃ to obtain aptamer-functionalized nanomagnetic bead capture probe.

[0045] (II) Preparation of detection probes (1) Dissolve 0.1g folic acid and 0.05g o-phenylenediamine in 50mL anhydrous ethanol, disperse by ultrasonication (150W power, 30kHz frequency) for 30 minutes to form a clear solution, transfer to 100mL polytetrafluoroethylene reactor and react at 180℃ for 12 hours; after cooling, filter through a 0.22μm filter membrane, dialyze the filtrate in ultrapure water using a 3500Da dialysis bag for 48 hours (changing the water regularly), and finally obtain carbon quantum dot powder by vacuum drying at 60℃ and store at 4℃ in the dark; (2) Dissolve 2 mg of carbon quantum dot powder in 2 mL of PBS solution, add 1 mL of 10 mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) solution and 1 mL of 25 mM NHS (hydroxysuccinimide) solution, and shake at room temperature in the dark for 30 minutes. After the reaction, add 300 μL of 10 μM aminated aptamer solution, shake at 37 °C for 3 hours to obtain the reaction solution. Then dialyze the reaction solution in PBS for 24 hours using a 3500 Da dialysis bag to obtain aptamer-functionalized carbon quantum dots, i.e., detection probes, and store at 4 °C in the dark.

[0046] Characterization test Transmission electron microscopy (TEM) observations of the carboxylated Fe3O4 magnetic nanoparticles prepared in Example 1 are as follows: Figure 1 As shown.

[0047] The FTIR spectrum of the aptamer-functionalized nanobead trapping probe prepared in Example 1 is shown below. Figure 2 As shown, the results indicate that at 583cm -1 A characteristic peak of Fe-O bond appears at 1634 cm⁻¹. -1 A peak of NH shear vibration appears at 1384 cm⁻¹. -1 The presence of a CH asymmetric vibration peak indicates successful amino modification.

[0048] The magnetic saturation intensity (VSM measurement) of the aptamer-functionalized magnetic nanobead trapping probe prepared in Example 1 was 65.8 emu / g (see Example 1). Figure 3 ).

[0049] In addition, the TEM image of the carbon quantum dots prepared in Example 1 is as follows. Figure 4 As shown in the figure, the carbon quantum dots exhibit a relatively uniform dispersion with no obvious agglomeration, indicating that they have good dispersibility in the system. Furthermore, the carbon quantum dots have a relatively uniform particle size, with an average particle size of 1.95 ± 0.2 nm.

[0050] The fluorescence spectrum of the aptamer-functionalized carbon quantum dots (detection probe) prepared in Example 1 is shown below. Figure 5As shown, the results indicate that a strong emission peak appears at 475 nm under 360 nm excitation, with a quantum yield of 58.2%. The FTIR spectrum of the aptamer-functionalized carbon quantum dots (detection probe) prepared in Example 1 is shown below. Figure 6 As shown in the figure, the results indicate that the detection probe is at 3300 cm. -1 -3470cm -1 The peak at 1700 cm⁻¹ is the OH / NH vibration peak. -1 The peak at 1650 cm⁻¹ is the C=O vibration peak. -1 The presence of a characteristic peak for amide bonds indicates successful coupling of the aptamer.

[0051] Detection performance evaluation (a) Establishment of standard curve (1) Inoculate the standard strain of Escherichia coli O157:H7 into LB medium and incubate at 37°C for 24 hours. Prepare 10 μL of the medium with physiological saline. 1 -10 6 CFU / mL concentration gradient bacterial suspensions; (2) Take 500 μL of bacterial suspensions of different concentrations and add 100 μL of capture probe (prepared in Example 1) to each, and incubate at 37°C for 2 hours; (3) After incubation, magnetic separation was performed. After magnetic separation, the sample was washed with water. After washing, 100 μL of detection probe (prepared in Example 1) was added and incubated at 37°C for 2 hours. (4) After incubation, magnetic separation was performed. After magnetic separation, the sample was washed with water and then resuspended in 500 μL PBS solution. The fluorescence intensity at 475 nm was detected using a fluorescence spectrometer, Spectrofluorometer FS5 (UK), at an excitation wavelength of 360 nm. (5) A standard curve was plotted with the logarithm of bacterial concentration on the x-axis and fluorescence intensity on the y-axis, yielding the linear equation Y = 128.5X + 89.2(R²). 2 =0.998).

[0052] Fluorescence curves of O157:H7 standard bacterial suspensions at different concentrations are shown below. Figure 6 As shown, the constructed standard curve is as follows: Figure 7 As shown.

[0053] (ii) Specificity verification Ten common foodborne pathogens, including Staphylococcus aureus (BNCC310011), Salmonella (BNCC 100836), Listeria monocytogenes (BNCC 100777), Escherichia coli (BNCC 102765), and Pseudomonas aeruginosa (BNCC 100010), were selected and prepared according to the method described in (I) above. 6CFU / mL bacterial suspension was tested for fluorescence intensity according to method (I) above; simultaneously, Escherichia coli O157:H7 was used as a positive control (fluorescence intensity set to 100%), and the relative fluorescence intensity of other strains was less than 1%, indicating that the method has good specificity (see Figure 8 ).

[0054] (iii) Limit of detection The detection limit was calculated to be 10 CFU / mL using the 3σ / k method. The specific testing was as follows: First, preliminary data preparation was performed: the fluorescence intensity of a blank sample with the same matrix as the actual detection matrix was measured, with ≥10 parallel experiments. The average fluorescence intensity F0ˉ and standard deviation σ were calculated. Simultaneously, a 10 CFU / mL sample was used as the detection limit. 1 -10 6 Fluorescence intensity was measured using O157:H7 standard bacterial suspension at CFU / mL. Linear regression analysis was performed on the data to obtain the standard curve equation Y = 128.5X + 89.2 (where slope k = 128.5au / (logCFU / mL) and intercept b = 89.2au); subsequently, the standard curve was calculated using formula F... L =F0ˉ+3σ calculates the minimum fluorescence threshold, and substituting the data yields F. L =94.6au; F L Substituting into the standard curve equation, and using the transformed formula X L =(F L -b) / k is used to back-calculate the logarithm of the lowest concentration, and X is calculated. L ≈0.042 (logCFU / mL); then according to formula C L The theoretical concentration calculated using 10XL is approximately 1.1 CFU / mL. Considering the enrichment efficiency of ≥95% during actual sample pretreatment and the impact of subsequent enrichment steps, the theoretical concentration was finally corrected to the actual detection limit of 10 CFU / mL.

[0055] (iv) Repetitiveness At 10 CFU / mL (limit of detection concentration), 10 3 CFU / mL (medium concentration), 10 5 The CFU / mL (high concentration) Escherichia coli O157:H7 standard bacterial solution was used as the sole test object. The stability of the method was verified by intra-batch and inter-batch experiments. The results showed that the intra-batch RSD was <3% and the inter-batch RSD was <5%, indicating that the method has good repeatability.

[0056] (1) Experimental preparation: preparation of standard bacterial solutions with gradient concentrations 10 CFU / mL and 10 CFU / mL solutions were prepared using a 10-fold serial dilution method. 3 CFU / mL, 10 5Standard bacterial solutions of CFU / mL were prepared; three 1mL aliquots of bacterial solutions were taken for each concentration gradient and colony counts were performed using the plate spread method (LB agar medium, 37℃ for 24h) to ensure that the actual concentration deviates from the theoretical value by ≤10% and to guarantee the accuracy of the bacterial solution concentration; at the same time, a blank control of "sterile PBS" was set up. Six aliquots of standard bacterial solutions were prepared for each concentration group (for intra-batch experiments) and nine aliquots were prepared (for inter-batch experiments). (2) Intra-batch repeatability verification Take the 10 CFU / mL and 10 CFU / mL samples prepared in the same batch 3 CFU / mL, 10 5 Six parallel samples were prepared for each concentration of CFU / mL Escherichia coli O157:H7 standard bacterial solution. The standard bacterial solution detection procedure was followed as described in Example 3 (LB broth was added, and the bacteria were incubated at 37°C with shaking for 24 hours, followed by detection using a fluorescence detector (360nm excitation, 475nm emission, calibrated with standard fluorescence solution before the experiment). The fluorescence intensity of each parallel sample was recorded. The results were then compared with the Escherichia coli O157:H7 standard curve (Y=125.3X+87.6, R0). 2 ≥0.995, where X is the logarithm of bacterial concentration and Y is fluorescence intensity), calculate the actual detection concentration for each parallel sample. Data calculation: For each concentration (10 CFU / mL, 10... 3 CFU / mL, 10 5 The detection concentrations of 6 parallel samples at (CFU / mL) were calculated, and the average value (Cˉ) and relative standard deviation (RSD) were calculated for each sample. The formula is RSD = Cˉσ × 100% (where σ is the standard deviation of the 6 detection concentrations). The inter-batch RSDs for all concentrations were <5%, as shown in Table 1 below.

[0057] Table 1. Intra-batch repeatability validation data

[0058] (2) Inter-batch repeatability verification On three different dates (intervals of more than 24 hours, with each experiment using freshly prepared PBS, LB broth, and other reagents), 10 CFU / mL and 10... were re-prepared for each batch. 3 CFU / mL, 10 5 CFU / mL Escherichia coli O157:H7 standard bacterial suspension (preparation method as in step 1, ensuring accurate concentration for each batch); take 3 parallel samples for each concentration, recalibrate the fluorescence detector before each batch experiment, and operate according to the "enrichment-fluorescence detection" procedure consistent with the intra-batch experiment, recording the detection concentration for each batch and each concentration. Data calculation: for each concentration (10 CFU / mL, 10 3 CFU / mL, 10 5The detection concentrations of 9 samples from 3 batches at CFU / mL were calculated, and the overall average (Cˉtotal) and inter-batch RSD (i.e., the relative standard deviation of detection data between different batches) were calculated. The inter-batch RSDs for all concentrations were <5%, as shown in Table 2 below.

[0059] Table 2. Inter-batch repeatability validation data

[0060] (v) Actual sample testing Milk, beef, and lettuce were used as the actual samples for testing. The pretreatment steps are as follows: Milk: Take 25.0 mL of well-mixed milk, add 225.0 mL of sterile PBS (pH 7.2±0.1, sterilized for 20 min), and shake at 37℃ and 150 rpm for 30 min. Beef: Take 25.0 g of skinless lean beef (cut into 1 cm × 1 cm pieces), add 225.0 mL of sterile PBS, and homogenize at 2000 rpm for 2 min. Filter the homogenate through four layers of sterile gauze (sterilized for 30 min). Lettuce: Take 25.0 g of lettuce (cut into 2 cm × 2 cm pieces), rinse three times with sterile saline, and drain for 5 min. Add 225.0 mL of sterile PBS, homogenize at 1800 rpm for 3 min, and centrifuge at 4℃ and 3000 rpm for 5 min. Collect the supernatant. Take the pretreated milk, beef, and lettuce samples, and add 10% PBS solution to each sample. 2 CFU / mL, 10 4 CFU / mL, 10 6 The O157:H7 standard bacterial solution with CFU / mL was tested according to the above method. Three parallel experiments were set up for each concentration gradient. The recovery rate and relative standard deviation (RSD) were calculated. The results are shown in Table 3 below.

[0061] Table 3. Recovery rates and relative standard deviations (RSD) of different samples.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A folic acid-o-phenylenediamine co-doped carbon quantum dot, characterized in that, Folic acid-o-phenylenediamine co-doped carbon quantum dots are prepared by heating folic acid and o-phenylenediamine.

2. The folic acid-o-phenylenediamine co-doped carbon quantum dots according to claim 1, characterized in that, The mass ratio of folic acid to o-phenylenediamine is (1-3):

1.

3. The folic acid-o-phenylenediamine co-doped carbon quantum dots according to claim 1 or 2, characterized in that, The temperature for the heating reaction is 170℃-190℃.

4. A detection probe for Escherichia coli O157:H7, characterized in that, It includes the folic acid-o-phenylenediamine co-doped carbon quantum dots and the aminated aptamer as described in any one of claims 1 to 3; the folic acid-o-phenylenediamine co-doped carbon quantum dots are activated by EDC / NHS and then linked to the aminated Escherichia coli O157:H7 aptamer, the sequence of which is: CCGGACGCTTATGCCTTGCCATCTACAGAGCAGGTGTGACGG.

5. A reagent for detecting Escherichia coli O157:H7, characterized in that, Including the Escherichia coli O157:H7 detection probe as described in claim 4.

6. The Escherichia coli O157:H7 detection reagent according to claim 5, characterized in that, The Escherichia coli O157:H7 detection kit also includes an Escherichia coli O157:H7 capture probe.

7. The Escherichia coli O157:H7 detection reagent according to claim 6, characterized in that, The preparation method of the Escherichia coli O157:H7 capture probe includes: (1) The reactants were obtained by heating ferric chloride hexahydrate, anhydrous sodium acetate and polyacrylic acid; (2) The reactants were magnetically separated to obtain a black solid, which is the carboxylated magnetic nanoparticle; (3) Carboxylated magnetic nanoparticles were linked to biotinylated Escherichia coli O157:H7 aptamers via streptavidin to obtain Escherichia coli O157:H7 capture probes.

8. The Escherichia coli O157:H7 detection reagent according to claim 7, characterized in that, In the preparation method of the Escherichia coli O157:H7 capture probe, the heating reaction temperature is 190℃-200℃.

9. A method for detecting Escherichia coli O157:H7, characterized in that, The detection method includes: using the Escherichia coli O157:H7 detection reagent according to any one of claims 6 to 8, including: (i) The Escherichia coli O157:H7 bacterial suspension and the Escherichia coli O157:H7 capture probe were mixed and incubated, and then magnetic separation was performed; (ii) After magnetic separation, it is mixed with the Escherichia coli O157:H7 detection probe and incubated before magnetic separation. (iii) After magnetic separation, the fluorescence intensity is detected, and the content of Escherichia coli O157:H7 is calculated based on the standard curve of concentration-fluorescence intensity.

10. The detection method according to claim 9, characterized in that, In step (iii), the fluorescence intensity at 475 nm is detected at an excitation wavelength of 360 nm.

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