Double-probe detection kit for detecting staphylococcus aureus in meat products and preparation method of double-probe detection kit

Abstract

The invention relates to a double-probe detection kit for detecting staphylococcus aureus in meat products and a preparation method of the double-probe detection kit, and belongs to the technical field of food safety. The double-probe detection kit is composed of a magnetic capture probe reagent and a double-signal nano-enzyme probe reagent which are independently packaged and have the same volume; the magnetic capture probe reagent is a vancomycin modified reticular magnetic bead and is used for specifically binding staphylococcus aureus in a meat product detection sample and carrying out separation and enrichment treatment; the double-signal nano-enzyme probe reagent is a blue fluorescent carbon dot modified by a nucleic acid aptamer and is used for specifically combining the staphylococcus aureus subjected to separation and enrichment treatment and generating a staphylococcus aureus concentration dependent colorimetric signal and a fluorescent signal. According to the invention, mutual verification is carried out between dual-mode signals, so that the accuracy of a detection result is improved; according to the method, the detection time is within 1.5 h, the detection time is short, the detection sensitivity is high, and the method has good specificity for common type strains.

Description

Technical Field

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

[0002] Staphylococcus aureus ( Staphylococcus aureus, S. aureus Staphylococcus aureus (S. aureus) is a Gram-positive, clustered, spherical symbiotic bacterium with extremely strong environmental adaptability. It typically parasitizes the skin, nasal cavity, throat, and gastrointestinal tract of humans and animals, and is also widely distributed in air and sewage. This bacterium not only causes skin infections, but in severe cases, it can lead to life-threatening diseases such as meningitis, pneumonia, endocarditis, bacteremia, and sepsis. S. aureus is extremely widespread in nature; its contamination is almost ubiquitous, from various food ingredients to processed foods, with meat products being particularly vulnerable, hence its common name "meat-loving bacteria." S. aureus poses a serious threat to food safety; once contaminated, it can easily cause food poisoning incidents, thus it has long been a key focus of food biocontaminant monitoring. While causing serious harm to human health, S. aureus contamination also incurs huge losses to the meat processing industry. Therefore, establishing a highly specific and sensitive rapid detection method for S. aureus is a crucial step towards timely diagnosis and effective control.

[0003] Methods for detecting Staphylococcus aureus mainly include plate culture, immunoassay based on antigen-antibody specific recognition (such as ELISA), and nucleic acid amplification based on the principle of complementary base pairing (such as PCR). Plate culture is considered the "gold standard" for detection, but it requires multiple steps including bacterial isolation and culture, morphological observation, and biochemical identification, making it cumbersome and time-consuming, thus limiting its use in rapid diagnosis. Immunoassays have high specificity, but the expensive reagent kits hinder large-scale industrial application. Nucleic acid amplification technology, with its ultra-high sensitivity, is an important reference method, but it requires multiple steps of nucleic acid extraction, enrichment, and purification, making it complex and dependent on laboratory conditions. Emerging colorimetric fluorescence detection methods are widely used for target substance determination due to their low cost, ease of operation, fast response, and high stability. Currently, single-colorimetric or single-fluorescence detection techniques have several inherent limitations in pathogen detection. First, single colorimetric methods often struggle to accurately detect low concentrations or weak signal changes, limiting their application in trace analysis. While single fluorescence methods offer higher sensitivity, their signals are susceptible to interference from autofluorescent substances in complex sample matrices, and fluorescent dyes may exhibit photobleaching, affecting signal stability. Furthermore, in complex real-world samples (such as food matrices), the sample's color and turbidity can interfere with colorimetric signals, and fluctuations in environmental factors (such as temperature, pH, and ionic strength) can lead to signal instability. These factors collectively result in insufficient reliability, interference resistance, and accuracy of single-signal detection modes in complex real-world detection environments, making it difficult to avoid false positives or false negatives. Therefore, developing colorimetric-fluorescence dual-modal or multi-modal sensing strategies, through cross-validation and complementarity between signals, has become an important research direction for overcoming these limitations and improving detection reliability.

[0004] Therefore, there is an urgent need to develop a simple, rapid, highly sensitive, and accurate detection method for the real-time detection of Staphylococcus aureus. Although researchers at home and abroad have been committed to exploring detection technologies for foodborne pathogens suitable for on-site analysis in recent years, existing methods are mostly single-modal detection methods and generally suffer from problems such as large matrix interference, poor signal accuracy, and single detection signal. Summary of the Invention

[0005] To achieve simple, rapid, and highly sensitive accurate detection of Staphylococcus aureus in food, this invention provides a dual-probe detection kit for detecting Staphylococcus aureus, and also provides a method for preparing the dual-probe detection kit.

[0006] A dual-probe detection kit for detecting Staphylococcus aureus in meat products consists of individually packaged magnetic capture probe reagents and dual-signal nanozyme probe reagents of equal volume; The concentration of the magnetic capture probe reagent is 2 mg / mL, and the concentration of the dual-signal nanozyme probe reagent is 100 µg / mL. The magnetic capture probe reagent is a vancomycin-modified mesh magnetic bead, used to specifically bind Staphylococcus aureus from meat product test samples and to separate and enrich it. The dual-signal nanozyme probe reagent is a blue fluorescent carbon dot modified with a nucleic acid aptamer, used to specifically bind to the isolated and enriched Staphylococcus aureus and generate a Staphylococcus aureus concentration-dependent colorimetric signal and fluorescence signal. The DNA sequence of the nucleic acid aptamer is shown in SEQ ID No: 1, and the 5' end of the nucleic acid aptamer is modified with a carboxyl group; The dual-probe detection kit exhibits excellent detection performance against Staphylococcus aureus, with a linear detection range of 10. 1 Up to 10 7 CFU / mL (R) 2 =0.993). The detection limit of this kit has been validated to be as low as 10 CFU / mL. In real-world sample testing, its spiked recoveries ranged from 95% to 105%, demonstrating extremely high accuracy.

[0007] The preparation steps of a dual-probe detection kit for detecting Staphylococcus aureus in meat products are as follows: (1) Preparation of activation mixture The activated mixture was prepared by mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride with a molar concentration of 10 mmol / L and N-hydroxysuccinimide with a molar concentration of 5 mmol / L at a volume ratio of 1:1. (2) Preparation of magnetic trapping probe reagents (2.1) 100 µL of 20 mg / mL iron(III) oxide (Fe3O4) magnetic beads were added to 900 µL of activation mixture and incubated at 180 rpm for 1 h at room temperature; then 10 mg of γ-polyglutamic acid was added and incubated at 180 rpm for 4 h at room temperature to carry out the coupling reaction, while continuously rotating the mixture; the mixture was magnetically separated, the supernatant was removed, and the precipitate was dried in a 60 °C oven to obtain a network of magnetic beads; (2.2) Add 2 mg of reticulated magnetic beads to 1 mL of activation mixture and incubate at 180 rpm for 2 h at room temperature to complete activation and obtain a reticulated magnetic bead solution; add 13.5 mg of vancomycin to the reticulated magnetic bead solution and continue to incubate at 180 rpm for 4 h at room temperature; remove the supernatant by magnetic separation and dry the precipitate in a 60 °C oven; resuspend the dried product in ultrapure water to prepare a magnetic capture probe reagent with a mass concentration of 2 mg / mL; (3) Preparation of dual-signal nanozyme probe reagents (3.1) Dissolve 50 mg citric acid, 70 mg copper nitrate and 100 µL ethylenediamine in 5 mL ultrapure water, and react in a reaction vessel at 180 °C for 6 h; centrifuge at 12000 rpm for 10 min, and take the supernatant to prepare a blue fluorescent carbon dot solution with a concentration of 200 µg / mL. (3.2) Add 250 µL of a 5 µM nucleic acid aptamer solution to 250 µL of an activation mixture and incubate at 180 rpm for 30 min at room temperature to obtain a mixed solution; add 500 µL of a blue fluorescent carbon dot solution to the mixed solution and incubate at 180 rpm for 2.5 h at room temperature to obtain a dual-signal nanozyme probe reagent; (4) Prepare the dual-probe detection kit The magnetic capture probe reagent and the dual-signal nanozyme probe reagent, which are separately packaged, are paired at a volume ratio of 1:1 and stored at 4°C to prepare a dual-probe detection kit. The beneficial technical effects of this invention are reflected in the following aspects:

[0008] The inventiveness of this dual-probe detection kit This invention overcomes the limitations of traditional culture, PCR, and ELISA techniques, which are time-consuming and cumbersome, typically requiring 1-2 days. This invention achieves rapid detection within one hour. The core technology lies in the use of interface-enhanced magnetic separation technology driven by mesh-targeted magnetic beads, significantly improving capture efficiency and enrichment effects, thereby greatly enhancing detection sensitivity. Based on this, this invention innovatively constructs a fluorescence-colorimetric dual-signal detection system. This system can simultaneously acquire two signals in a single experiment: the colorimetric signal provides rapid and intuitive on-site screening capabilities, while the fluorescence signal ensures accurate quantification of the target analyte. The two signals can cross-verify, effectively avoiding false positive or false negative results that may occur due to food matrix interference in single-detection modes, significantly enhancing the reliability and accuracy of the results.

[0009] When faced with complex food samples such as sausages, this invention demonstrates strong anti-interference capabilities and high specificity. The key lies in its dual-modal sensing design: when pigments or turbidity in the sample might interfere with the colorimetric signal, the fluorescence signal can serve as a reliable internal correction; conversely, autofluorescence interference that might be encountered in fluorescence detection can also be aided by the colorimetric signal. This mutual reference and compensation mechanism between signals greatly enhances the robustness of the method in complex matrices. Simultaneously, the method exhibits good specificity for common model strains, effectively avoiding cross-reactions with non-target bacteria and ensuring the reliability of the detection results.

[0010] The most valuable innovation of this invention lies in the fact that the standard curve and linear regression equation established in PBS buffer can be directly applied to the detection of complex food samples such as sausages without any correction. Current existing technologies generally have a linear fitting degree ranging from 0.90 to 0.98, while the dual-signal nanozyme probe prepared in this invention exhibits a correlation coefficient as high as 0.993 in both colorimetric and fluorescence modes, demonstrating a high degree of linear fitting. The dual-signal nanozyme probe prepared in this invention can achieve quantitative detection at 10 CFU / mL, exhibiting extremely high sensitivity. This means that in response to food safety emergencies, there is no need to reculture bacteria and draw standard curves for specific samples, greatly saving valuable time. Experimental data fully demonstrates this: in sausage samples, using the standard curve established in PBS, the concentration of Staphylococcus aureus (3.8*10⁻⁶) obtained based on fluorescence and colorimetric signals... 6 CFU / mL and 4.0*10 6 CFU / mL) and the actual concentration (4.1*10) 6 The CFU / mL values ​​are very close. This "one-time calibration, applicable to multiple applications" characteristic highlights the strong versatility and convenience of the dual-signal nanozyme probe and its detection system prepared in this invention, providing a powerful tool for rapid response in food safety.

[0011] 2. The inventiveness of the preparation method of the dual-probe detection kit of the present invention Magnetic separation technology is key to shortening detection time. This invention is the first to apply reticulated magnetic beads as a carrier for the separation and enrichment of Staphylococcus aureus. In step (2), the reticulated magnetic beads, after being modified with γ-polyglutamic acid, have increased specific surface area and abundant modifiable sites, greatly improving the capture efficiency and further reducing the separation time. The capture time of ordinary magnetic beads often takes 1 hour, while the reticulated magnetic beads of this invention reduce the capture time to 30 minutes. Specifically, amino-modified reticulated magnetic beads are further assembled by amidation covalent coupling between γ-polyglutamic acid and iron oxide magnetic beads. This not only greatly improves the flexibility and adaptability of the reticulated magnetic beads in biomolecule capture and application, but also significantly enhances their dispersibility and biocompatibility. A magnetic capture probe was then assembled by covalently coupling vancomycin and reticulated magnetic beads to screen and separate Gram-positive bacteria. This solved the problem of the lack of suitable specific recognition elements for Staphylococcus aureus, thus enabling the first recognition by the dual probe. When Staphylococcus aureus is present in the sample solution, the magnetic capture probe will bind to the surface of Staphylococcus aureus, and the Staphylococcus aureus will be separated and enriched from the sample solution using a magnetic rack, reducing matrix interference from the sample solution and improving the accuracy and sensitivity of the detection.

[0012] In step (3), this invention utilizes a one-step hydrothermal method, employing citric acid and ethylenediamine as carbon and nitrogen sources, and introducing copper metal doping to efficiently synthesize a dual-signal nanozyme probe with both peroxidase-like activity and fluorescence emission characteristics within 6 hours. The nanozyme used in this invention can efficiently catalyze the oxidation of the substrate TMB (3,3',5,5'-tetramethylbenzidine), and its characteristic absorbance at a predetermined concentration reaches 1.8, indicating extremely strong catalytic activity. Even at low concentrations of target bacteria, the dual-signal nanozyme probe can still generate significant and easily interpretable colorimetric signals, effectively ensuring detection sensitivity. Simultaneously, the nanozyme itself can emit stable fluorescence under specific wavelength excitation, thus constructing a fluorescence-colorimetric dual-signal output mode. The two signals can mutually verify each other, effectively avoiding false positive results, and can be flexibly selected according to actual application scenarios. The colorimetric method is suitable for rapid on-site screening, while the fluorescence method is suitable for high-precision quantification. This "one probe, two functions" design significantly demonstrates the reliability and applicability of the dual-signal nanozyme probe. Attached Figure Description

[0013] Figure 1 The images show transmission electron microscopy and magnetic analysis of the mesh magnetic beads prepared in this invention.

[0014] Figure 2 The images show transmission electron microscopy and enzyme activity analysis of the blue fluorescent carbon dots prepared in this invention.

[0015] Figure 3 This is a potential diagram of the magnetic trapping probe and the dual-signal nanozyme probe prepared in this invention.

[0016] Figure 4 This is a standard curve of colorimetric and fluorescence signals of Staphylococcus aureus at different concentrations in this invention.

[0017] Figure 5 This is a diagram illustrating the specific detection of Staphylococcus aureus in this invention. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments.

[0019] 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.

[0020] 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 Examples 2-5 are all repeated three times and the results are averaged; unless otherwise specified, the percentages in the following examples are all mass percentages.

[0021] In the following examples, the nucleic acid aptamers used were purchased from Sangon Biotech (Shanghai) Co., Ltd., and the magnetic beads used were purchased from Tianjin Junyijia Technology Co., Ltd.; unless otherwise specified, all other raw materials used were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0022] It should be noted that the phosphate buffer used in this invention is a 0.01M sterile PBS buffer with a pH of 7.4.

[0023] It should be noted that all the bacterial strains used in this invention can be obtained from the American Type Culture Collection (ATCC); the ATCC numbers are as follows: Staphylococcus aureus 29213, Vibrio parahaemolyticus 17802, Escherichia coli 25922, Cronobacter sakazakii 29544, Pseudomonas aeruginosa 15442, Enterococcus faecalis 29212, Listeria monocytogenes 43251, and Shigella flexneri 12022.

[0024] All instruments, equipment, raw materials, reagents, and methods used in this invention are ensured to be processed under sterile conditions.

[0025] Any instruments, equipment, raw materials, reagents, or method steps not mentioned in this invention are conventional or well-known techniques to those skilled in the art, and will not be described in detail here. Example 1

[0026] The preparation steps of a dual-probe detection kit for detecting Staphylococcus aureus are as follows: (1) Preparation of activation mixture The activated mixture was prepared by mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (10 mmol / L) and N-hydroxysuccinimide (5 mmol / L) at a volume ratio of 1:1.

[0027] (2) Preparation of magnetic trapping probe reagents (2.1) 100 µL of 20 mg / mL iron(III) oxide (Fe3O4) magnetic beads were added to 900 µL of activation mixture and incubated on a shaker at 180 rpm for 1 h at room temperature. Then, 10 mg of γ-polyglutamic acid was added, and the mixture was incubated on a shaker at 180 rpm for another 4 h at room temperature to carry out the coupling reaction, while continuously rotating the mixture. After the coupling reaction was completed, the mixture was magnetically separated, the supernatant was removed, and the precipitate was dried in a 60 °C oven to obtain a network of magnetic beads.

[0028] (2.2) Add 2 mg of magnetic mesh beads to 1 mL of activation mixture and incubate at 180 rpm for 2 h at room temperature to complete activation, obtaining a magnetic mesh bead solution. Add 13.5 mg of vancomycin to the magnetic mesh bead solution and continue incubation at 180 rpm for 4 h at room temperature. Remove the supernatant using magnetic separation, and dry the precipitate in a 60 °C oven. Resuspend the dried product in ultrapure water to prepare a magnetic capture probe reagent with a concentration of 2 mg / mL.

[0029] (3) Preparation of dual-signal nanozyme probe reagents (3.1) Dissolve 50 mg citric acid, 70 mg copper nitrate and 100 µL ethylenediamine in 5 mL ultrapure water and react in a reaction vessel at 180 °C for 6 h. Centrifuge at 12000 rpm for 10 min and take the supernatant to prepare a blue fluorescent carbon dot solution with a concentration of 200 µg / mL.

[0030] (3.2) Add 250 µL of a 5 µM nucleic acid aptamer solution to 250 µL of activation mixture and incubate at 180 rpm for 30 min at room temperature to obtain a mixed solution. Add 500 µL of blue fluorescent carbon dot solution to the mixed solution and continue incubating at 180 rpm for 2.5 h at room temperature to obtain a dual-signal nanozyme probe reagent.

[0031] (4) Prepare the dual-probe detection kit The magnetic capture probe reagent and the dual-signal nanozyme probe reagent, which were separately packaged, were paired at a volume ratio of 1:1 and stored at 4°C to prepare a dual-probe detection kit.

[0032] This dual-probe detection kit exhibits excellent detection performance against Staphylococcus aureus. Its linear detection range is 10-1. 1 Up to 10 7 CFU / mL (R) 2 =0.993). The detection limit of this kit has been validated to be as low as 10 CFU / mL. In real-world sample testing, its spiked recoveries ranged from 95% to 105%, demonstrating extremely high accuracy.

[0033] See Figure 1 , Figure 1 Figure A shows the microstructure of unmodified vancomycin magnetic beads. Figure 1 B and C in the diagram show the magnetic response performance of the magnetic bead. See also Figure 1 In the C-type magnet, the beads completely separated within just 30 seconds after a magnetic field was applied, a stark contrast to the dispersed state. (See...) Figure 1 The number B indicates that the mesh magnetic bead has a rapid magnetic separation capability.

[0034] See Figure 2 , Figure 2 In the image, A represents a transmission electron microscope (TEM) image of a blue fluorescent carbon dot that has not undergone nucleic acid aptamer modification. Figure 2 B in the figure represents the color change under different reaction systems. The solution only turned light blue when blue fluorescent carbon dots, hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine were added simultaneously, while no obvious color change was observed in the other control groups. Figure 2 C in the figure represents the UV absorption spectrum of the blue fluorescent carbon dots. This result is consistent with... Figure 2 The presence of B in the above data confirms that the synthesized carbon dots possess good enzyme-like catalytic activity.

[0035] The potentials of the magnetic capture probe reagent and the dual-signal nanozyme probe reagent prepared in Example 1 are as follows: Figure 3 As shown, Figure 3 Figure A shows that the Zeta potential of the magnetic capture probe reagent decreased from -15mV to -27.6mV, indicating that vancomycin in the magnetic capture probe reagent was successfully modified onto the reticulated magnetic beads; Figure 3 B in the diagram shows that the Zeta potential of the dual-signal nanozyme probe reagent decreased from -3.8 mV to -9.4 mV, indicating that the nucleic acid aptamer in the dual-signal nanozyme probe reagent was successfully modified onto the blue fluorescent carbon dots. Example 2

[0036] The detection method and operation steps of a dual-probe detection kit for detecting Staphylococcus aureus in meat products are as follows: (1) Preparation of the test solution Take 100g of meat products, homogenize them in a homogenizer for 2 minutes, let them stand for 5 minutes, and take the supernatant as the test solution; (2) Preparation of the test solution (2.1) Mix 100µL of magnetic capture probe reagent with 400µL of the above test solution, incubate at room temperature for 30 min at 180 rpm on a shaker, wash three times with a magnetic rack, and resuspend in 400µL of sterile PBS buffer. (2.2) Next, add 100µL of dual-signal nanozyme probe reagent to the above solution, incubate at room temperature on a shaker at 180rpm for 30min, wash three times with a magnetic rack, and resuspend in 500µL of sterile PBS buffer to obtain the test solution; (3) Detection of colorimetric signals (3.1) Take 50µL of the test solution, add 50µL of 100mM hydrogen peroxide (H2O2) solution, then add 50µL of 4.8g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution, and react in the dark at 25℃ for 15min to obtain the reaction solution; (3.2) Observe the color change of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive. (3.3) Take 200 μL of positive reaction solution and put it into a cuvette. Measure the absorbance intensity of the reaction solution in the cuvette at a peak of 654 nm using a UV spectrophotometer. Substitute the absorbance intensity value at 654 nm into the linear regression equation of the colorimetric signal to calculate the concentration of Staphylococcus aureus in the test solution, thus completing the detection of the colorimetric mode. (4) Detection of fluorescence signal Take 200 μL of the test solution and put it into a cuvette. Measure the fluorescence intensity of the test solution in the cuvette at 383 nm under an excitation wavelength of 300 nm using a fluorescence spectrophotometer. Substitute the fluorescence intensity value at 383 nm into the detection equation to calculate the concentration of Staphylococcus aureus in the test solution, thus completing the fluorescence mode detection. Example 3

[0037] Construction of a linear regression equation for the detection of Staphylococcus aureus in meat products using colorimetric and fluorescence dual-modal signals. The establishment of a linear regression equation for the detection of Staphylococcus aureus using the dual-probe detection kit prepared in Example 1 is as follows: (1) Pretreatment First, a sterile template bacterial test solution was prepared, with one set as a blank control test solution. The remaining test solutions were artificially contaminated with Staphylococcus aureus, and linearly serially diluted solutions containing the target bacteria were prepared. In this embodiment, sterile PBS buffer was selected as the blank control test solution. Simultaneously, the Staphylococcus aureus bacterial culture was centrifuged, washed three times with sterile PBS buffer, and then resuspended and serially diluted with sterile PBS buffer to prepare concentrations of 0 CFU / mL, 10 CFU / mL, and 10 CFU / mL, respectively. 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 of the test solution containing the target bacteria; Next, the magnetic capture probe reagent was mixed with the blank control or test solution containing the target bacteria obtained above at a volume ratio of 1:4. The mixture was incubated at room temperature for 30 min, washed three times using a magnetic rack, and resuspended in sterile PBS buffer of the same volume as the blank control or test solution containing the target bacteria. Then, the same volume of dual-signal nanozyme probe reagent as the magnetic capture probe reagent was added, and the mixture was incubated at room temperature for 30 min to obtain the test solution.

[0038] (2) Detection of colorimetric signals Simultaneously, to obtain the precipitate obtained by magnetic separation using a magnetic rack, 400 μL of acetate-sodium acetate buffer solution was added. This acetate-sodium acetate buffer solution, with a pH of 3.0, was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (product number R27435-500 ml). 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide solution with a final concentration of 10 mmol / L were added, and the mixture was reacted at 60°C for 15 min to obtain a colorimetric signal detection solution. The UV absorption intensity value generated at 653 nm was detected using a UV spectrophotometer. The UV absorption intensity value was selected as the ordinate, and the logarithm of the bacterial concentration in the tested solution was used as the abscissa to plot a standard curve. The correlation coefficient and detection limit were obtained based on the linear regression equation. Figure 4 As shown, see Figure 4 In the diagram, A, from left to right, represents: point 0, which indicates the UV absorption peak at a bacterial concentration of 0 CFU / mL (blank control test solution); point 1, which indicates the peak at a bacterial concentration of 10 CFU / mL. 1 The UV absorption peak at CFU / mL and point 2 indicate a bacterial concentration of 10. 2 The UV absorption peak at CFU / mL and point 3 indicate a bacterial concentration of 10. 3 The UV absorption peak at CFU / mL and point 4 indicate a bacterial concentration of 10. 4 The UV absorption peak at CFU / mL and point 5 indicate a bacterial concentration of 10. 5 The UV absorption peak at CFU / mL and point 6 indicate a bacterial concentration of 10. 6 The UV absorption peak at CFU / mL and point 7 indicate a bacterial concentration of 10. 7 The UV absorption peak at CFU / mL; simultaneously, combined with Figure 4 Based on A in the equation, the linear regression equation for the colorimetric signal in the detection of Staphylococcus aureus is y = 0.158x + 0.061, with a detection limit of 10 CFU / mL and an R² value of [missing value]. 2 The value is 0.993.

[0039] (3) Detection of fluorescence signal Next, for the test solution obtained above, the precipitate obtained by magnetic separation using a magnetic rack was used to obtain a fluorescence signal detection solution. The fluorescence intensity value generated at an excitation wavelength of 300 nm was detected using a fluorescence spectrophotometer. The fluorescence intensity value was selected as the ordinate, and the logarithm of the bacterial concentration of the test solution was used as the abscissa to plot a standard curve. The correlation coefficient and detection limit were obtained according to the linear regression equation. Figure 4 As shown, see Figure 4 In the diagram, B, from left to right, represents: point 0, which indicates the fluorescence emission peak at a bacterial concentration of 0 CFU / mL (blank control test solution); point 1, which indicates the peak at a bacterial concentration of 10 CFU / mL. 1 The fluorescence emission peak at CFU / mL and spot number 2 indicate a bacterial concentration of 10. 2 The fluorescence emission peak at CFU / mL and spot number 3 indicate a bacterial concentration of 10. 3 The fluorescence emission peak at CFU / mL and spot number 4 indicate a bacterial concentration of 10. 4 The fluorescence emission peak at CFU / mL and curve 5 indicate a bacterial concentration of 10. 5 The fluorescence emission peak at CFU / mL and curve 6 indicate a bacterial concentration of 10. 6 The fluorescence emission peak at CFU / mL and curve 7 indicate a bacterial concentration of 10. 7 The fluorescence emission peak at CFU / mL; simultaneously, combined with Figure 5 From B in the data, the linear regression equation for the fluorescence signal in the detection of Staphylococcus aureus is y = 0.137x + 0.0836, with a detection limit of 10 CFU / mL and an R² value of [missing value]. 2 The value is 0.993. Example 4

[0040] Testing of real samples The dual-probe detection kit prepared in Example 1 and the linear regression equation constructed in Example 3 were combined to detect real food samples. Sausage samples were selected for testing: In this embodiment 4, in order to verify whether the linear regression equation constructed according to embodiment 3 can be effectively applied to the determination of interference environments such as sausages, artificial quantitative bacterial contamination was performed to explore the difference in the number of bacteria detected by the dual probe detection reagent in sausage samples, so as to know that even in real food samples, the dual probe detection kit established in this application can effectively detect under the influence of high protein.

[0041] The sausage sample involved in Example 4 was purchased from Hema Fresh. The sausage was stored in a refrigerator at 4°C during the purchase. Its nutritional composition per 100g is as follows: energy 1005kJ, protein 15.9g, fat 16.5g, carbohydrates 7.3g, sodium 1010mg.

[0042] Staphylococcus aureus was inoculated into the sausage at a dose of 10. 6The concentration of CFU / g of spiked sausage was determined by homogenizing the sausage for 2 minutes, allowing it to stand for 5 minutes, and then using the supernatant as the test solution. In conjunction with Example 2, colorimetric and fluorescence dual-modal signals were detected. The obtained UV absorption intensity and fluorescence intensity values ​​were substituted into the linear regression equation to obtain the concentration of Staphylococcus aureus in the actual sample.

[0043] The experimental results showed that, after calculation, the concentration of Staphylococcus aureus in sausages artificially inoculated with Staphylococcus aureus was 3.8*10. 6 CFU / mL, 4.0*10 6 CFU / mL corresponds to the detection values ​​of both colorimetric and fluorescence signals, which is almost identical to its actual concentration of 4.1*10. 6 With CFU / mL close to the standard curve, the recovery rates reached 95% and 98%, respectively. It can be seen that even without establishing a corresponding standard curve and linear regression equation under sausage sample conditions, the standard curve and linear regression equation established under pure sterile PBS buffer still demonstrate good versatility. Therefore, the dual-probe detection reagent prepared in this invention has good versatility, enabling rapid application to the detection of different samples. Example 5

[0044] Basically the same as Example 4, except that: Staphylococcus aureus was replaced with Vibrio parahaemolyticus, Escherichia coli O157:H7, Cronobacter sakazakii, Pseudomonas aeruginosa, Enterococcus faecalis, Listeria monocytogenes, and Shigella flexneri, respectively; and concentrations of 10 were prepared for each. 7 CFU / mL of bacterial test solution.

[0045] See Figure 5 A in Figure 5In sample B, the bacterial test solution containing Staphylococcus aureus (1), Vibrio parahaemolyticus (2), Escherichia coli O157:H7 (3), Cronobacter sakazakii (4), Pseudomonas aeruginosa (5), Enterococcus faecalis (6), Listeria monocytogenes (7), and Shigella flexneri (8) was prepared. The UV absorption and fluorescence intensities of sample B were as follows: Sample B: 0.257 and 0.183; Sample B: 0.262 and 0.191; Sample B: 0.253 and 0.192; Sample B: 0.253 and 0.192. The values ​​were 0.261 and 0.187, respectively; the UV absorption intensity and fluorescence intensity values ​​of sample 6 were 0.264 and 0.185, respectively; the UV absorption intensity and fluorescence intensity values ​​of sample 7 were 0.256 and 0.189, respectively; and the UV absorption intensity and fluorescence intensity values ​​of sample 8 were 0.251 and 0.193, respectively. Meanwhile, under the same conditions, when a test solution containing Staphylococcus aureus was added, the UV absorption intensity and fluorescence intensity values ​​were 0.682 and 0.748, respectively. This demonstrates that the dual-probe detection kit prepared in this invention has good specificity for Staphylococcus aureus.

[0046] Those skilled in the art will readily understand that the above embodiments are merely preferred embodiments of the present invention and are 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 Staphylococcus aureus in meat products, characterized in that: The dual-probe detection kit consists of individually packaged magnetic capture probe reagents and dual-signal nanozyme probe reagents of equal volume; The concentration of the magnetic capture probe reagent is 2 mg / mL, and the concentration of the dual-signal nanozyme probe reagent is 100 µg / mL. The magnetic capture probe reagent is a vancomycin-modified mesh magnetic bead, used to specifically bind Staphylococcus aureus from meat product test samples and to separate and enrich it. The dual-signal nanozyme probe reagent is a blue fluorescent carbon dot modified with a nucleic acid aptamer, used to specifically bind to the isolated and enriched Staphylococcus aureus and generate a Staphylococcus aureus concentration-dependent colorimetric signal and fluorescence signal. The DNA sequence of the nucleic acid aptamer is shown in SEQ ID No: 1, and the 5' end of the nucleic acid aptamer is modified with a carboxyl group; The dual-probe detection kit has a linear detection range of 10 for Staphylococcus aureus. 1 ~10 7 CFU / mL, linear fitting correlation coefficient R 2 =0.993, the limit of detection is 10 CFU / mL; the spiked recovery rate in real sample testing is 95% to 105%.

2. The method for preparing a dual-probe detection kit for detecting Staphylococcus aureus in meat products according to claim 1, characterized in that, The operation steps are as follows: (1) Preparation of activation mixture The activated mixture was prepared by mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride with a molar concentration of 10 mmol / L and N-hydroxysuccinimide with a molar concentration of 5 mmol / L at a volume ratio of 1:

1. (2) Preparation of magnetic trapping probe reagents (2.1) 100 µL of 20 mg / mL iron oxide magnetic beads were added to 900 µL of activation mixture and incubated at 180 rpm for 1 h at room temperature; then 10 mg of γ-polyglutamic acid was added and the mixture was incubated at 180 rpm for 4 h at room temperature to carry out the coupling reaction, while the mixture was rotated continuously; the mixture was magnetically separated, the supernatant was removed, and the precipitate was dried in a 60 °C oven to obtain mesh magnetic beads; (2.2) Add 2 mg of reticulated magnetic beads to 1 mL of activation mixture and incubate at 180 rpm for 2 h at room temperature to complete activation and obtain a reticulated magnetic bead solution; add 13.5 mg of vancomycin to the reticulated magnetic bead solution and continue to incubate at 180 rpm for 4 h at room temperature; remove the supernatant by magnetic separation and dry the precipitate in a 60 °C oven; resuspend the dried product in ultrapure water to prepare a magnetic capture probe reagent with a mass concentration of 2 mg / mL; (3) Preparation of dual-signal nanozyme probe reagents (3.1) Dissolve 50 mg citric acid, 70 mg copper nitrate and 100 µL ethylenediamine in 5 mL ultrapure water, and react in a reaction vessel at 180 °C for 6 h; centrifuge at 12000 rpm for 10 min, and take the supernatant to prepare a blue fluorescent carbon dot solution with a concentration of 200 µg / mL. (3.2) Add 250 µL of a 5 µM nucleic acid aptamer solution to 250 µL of an activation mixture and incubate at 180 rpm for 30 min at room temperature to obtain a mixed solution; add 500 µL of a blue fluorescent carbon dot solution to the mixed solution and incubate at 180 rpm for 2.5 h at room temperature to obtain a dual-signal nanozyme probe reagent; (4) Prepare the dual-probe detection kit The magnetic capture probe reagent and the dual-signal nanozyme probe reagent, which are separately packaged, are paired at a volume ratio of 1:1 and stored at 4°C to prepare a dual-probe detection kit.

Images