A rapid photochromic multi-signal detection kit and method for foodborne pathogens
By combining aptamers for foodborne pathogens, colloidal gold, and fluorescein isothiocyanate, the problem of complex and time-consuming detection methods for foodborne pathogens is solved, enabling rapid and sensitive dual-signal detection and improving detection accuracy and visualization.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-16
- Publication Date
- 2026-03-10
AI Technical Summary
Existing methods for detecting foodborne pathogens are complex, time-consuming, costly, and have low sensitivity, making it difficult to achieve rapid, simple, and accurate on-site screening.
By combining aptamer solution for foodborne pathogens, colloidal gold solution, and fluorescein isothiocyanate solution, specific identification and quantitative detection of foodborne pathogens can be achieved through dual detection of colloidal gold freeze-thaw aggregation changes and fluorescence signals.
It enables rapid, simple, and accurate quantitative detection of foodborne pathogens, with high sensitivity, low cost, and short detection time. Dual-signal detection improves the visualization and accuracy of the detection.
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Figure CN116773442B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biosensing, and particularly relates to a rapid light color multi-signal detection kit and method for foodborne pathogenic bacteria. BACKGROUND
[0002] Bacterial contamination seriously affects human health and is an important issue in the field of public health. Bacteria widely exist in nature, such as water, air, dust, and excreta of humans and animals. They can enter the human body through routes such as droplets, contact, drinking water and food, and have a high infection rate, which poses a great threat to human health. Among them, foodborne pathogenic bacteria as biological contaminants in food pose a major threat to human health. According to the World Health Organization, about 600 million people worldwide suffer from poisoning or illness due to food contamination every year, and 420,000 people die of food poisoning, one-third of whom are children under the age of five. The foodborne pathogenic bacteria that must be detected in the national standard "Limit of Pathogenic Bacteria in Food" of China include Staphylococcus aureus, Salmonella, Vibrio parahaemolyticus, Listeria monocytogenes and Escherichia coli O157:H7. These standards and specifications provide protection for food safety and public health, but preventing food contamination is an eternal challenge for global health care systems and the food industry. It is of great significance to establish a rapid, sensitive and accurate detection method for pathogenic bacteria and take timely countermeasures on this basis.
[0003] Currently, the detection methods for pathogenic bacteria mainly include isolation and culture method, molecular biology detection method, immunological method, etc. Although these methods can realize the detection of foodborne pathogenic bacteria, they still have their own shortcomings. For example, the isolation and culture method is the "gold standard" for foodborne detection and is also the recommended method in the national standard of China, but the experimental period is time-consuming and the operation is complex, which cannot meet the demand for rapid detection of samples; other detection methods, such as molecular biology detection method, have complex technical operation, high cost and require professional staff; enzyme-linked immunoassay needs to use biological enzymes, which are expensive and their activity is easily affected by environmental factors such as temperature and pH. Therefore, it is urgent to develop a simple, easy-to-implement, rapid and sensitive on-site detection method.
[0004] One of the key issues for foodborne pathogen detection is to achieve rapid on-site screening of a large number of samples. Visual colorimetric analysis is the best on-site rapid signal screening method. Colloidal gold material has good biocompatibility, large specific surface area, photoelectric properties, surface plasmon resonance effect. Its molar absorption coefficient gradually decreases with the increase of particle size, the ultraviolet-visible absorption peak is blue-shifted, and the color change is visible to the naked eye. Therefore, colloidal gold is widely used in on-site visual colorimetric sensing detection. However, the visual colorimetric method has single signal and low sensitivity, and cannot realize quantitative detection. Multi-signal analysis method is a new detection method with self-correction and self-verification (Fu, X., Sun, J., Ye, Y., Zhang, Y., Sun, X. (2022). A rapid and ultrasensitive dual detection platform based on Cas12a for simultaneous detection of virulence and resistance genes of drug-resistant Salmonella. Biosensors and Bioelectronics, 195, 113682.). The detection system can generate two or more optical, electrical and color signals through one reaction, and the signals can correct each other and serve as a reference, which can make up for the shortcomings of visual colorimetric analysis for on-site practice. Fluorescein isothiocyanate is an important fluorescent dye molecule, which is low in price, high in light extinction coefficient and good in fluorescence quantum yield, and is widely used for protein fluorescence labeling. Aptamer is an oligonucleotide fragment screened by in vitro exponential enrichment ligand system evolution technology, which can bind to a variety of target substances with high specificity and selectivity, and has great application prospect in medical, biological, environmental and agricultural and forestry detection and analysis fields.
[0005] In summary, in order to realize the on-site screening of foodborne pathogenic bacteria, it is of great significance to develop a rapid, simple, high-accuracy and specific detection method and kit for food microbiological testing and other fields. SUMMARY
[0006] The purpose of the present application is to provide a foodborne pathogenic bacteria light color multi-signal detection method and kit.
[0007] A rapid light color multi-signal detection kit for foodborne pathogenic bacteria, comprising: pathogenic bacteria aptamer solution, colloidal gold solution, fluorescein isothiocyanate solution;
[0008] The concentration of the foodborne pathogen aptamer solution is 10-30 μM; the colloidal gold particle size is 13-40 nm, and the surface functional group is carboxyl; the concentration of the fluorescein isothiocyanate solution is 2.5-20 μM;
[0009] The foodborne pathogen includes Staphylococcus aureus, Listeria and Escherichia coli.
[0010] The Staphylococcus aureus aptamer gene sequence is 5'SH-GCA ATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACT TTG CTA A-3'.
[0011] The colloidal gold solution is prepared by the reaction of chloroauric acid and trisodium citrate.
[0012] The reaction is that 1 mM of chloroauric acid is heated to boiling, and 4 mmol / L of trisodium citrate is quickly added under vigorous stirring, and the reaction is carried out for 20 min.
[0013] The concentration of the aptamer solution is 20 μM; the preferred concentration of fluorescein isothiocyanate FITC is 15 μM; and the colloidal gold has a particle size of 13 nm.
[0014] The kit further comprises negative quality control phosphate buffer and positive quality control inactivated Staphylococcus aureus suspension.
[0015] A rapid colorimetric / fluorescent detection method for foodborne pathogens, comprising: using the rapid colorimetric / fluorescent detection kit for foodborne pathogens according to claim 1;
[0016] 1) After the sample to be tested is added to the buffer and mixed thoroughly, the extraction solution is transferred into a centrifuge tube;
[0017] The volume of the buffer solution is 5-10 mL, and the preferred volume is 5 g of sample plus 10 mL of buffer;
[0018] 2) Add the foodborne pathogen aptamer solution, mix by vortex, and incubate at 37°C for 15-90 min, centrifuge, and use a pipette to aspirate the supernatant;
[0019] 3) Take the supernatant solution and add it to the colloidal gold solution, and freeze at a temperature of -20 to -80 o C for 10-60 min, and incubate at a temperature of 25-70 oC After thawing, the colloidal gold solution was visually observed or scanned at 400-800 nm using a UV-visible spectrometer;
[0020] 4) The dissolved colloidal gold solution was centrifuged to remove aggregated nanoparticles, incubated with a fluorescein solution, and the fluorescence was measured using a fluorescence spectrometer to determine the content of foodborne pathogenic bacteria.
[0021] Step 3) The freezing temperature is -80 o C, the time is 10 min, and the thawing temperature is 70 o C.
[0022] Step 2) The incubation time is 60 min.
[0023] The present application provides a rapid colorimetric / fluorescent detection kit and method for foodborne pathogenic bacteria, which comprises: a foodborne pathogenic bacteria aptamer solution, a colloidal gold solution, a fluorescein isothiocyanate solution, a negative quality control, and a positive quality control. It realizes specific recognition of foodborne pathogenic bacteria through simple centrifugation, uses foodborne pathogenic bacteria aptamer to induce colloidal gold to change after freeze-thaw, and quantitatively detects Staphylococcus aureus according to the color change of colloidal aggregation and the influence of fluorescein isothiocyanate. The coefficient of variation is small during quantitative detection, the method has high sensitivity, wide linear range, low cost, simple operation, is easy to implement, and has short detection time. The colorimetric signal detection limit is 5 CFU / mL, the fluorescent quantitative detection limit is 2 CFU / mL, and the double signal not only realizes on-site visualization, but also improves the detection accuracy. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1 Transmission electron micrograph of gold nanoparticles;
[0025] Figure 2 (a) UV spectrum of colloidal gold; (b) Fluorescence emission spectrum of fluorescein isothiocyanate (FITC);
[0026] Figure 3 (a) Absorption spectrum of colloidal gold of different particle sizes; (b) Effect of freeze-thaw on the spectrum of colloidal gold of different particle sizes mixed with aptamer; (c) Absorbance normalization value of colloidal gold after freeze-thaw;
[0027] Figure 4 (a, b) Optimization of freezing time of colloidal gold vancomycin mixed indicator solution;
[0028] Figure 5 (a-c) Optimization of dissolution temperature of colloidal gold vancomycin mixed indicator solution;
[0029] Figure 6(a) Incubation time optimization of Staphylococcus aureus aptamer with Staphylococcus aureus; (b) Staphylococcus aureus aptamer concentration optimization; (c) Optimization of fluorescein concentration;
[0030] Figure 7 Standard curve for Staphylococcus aureus detection, linear regression equation and correlation coefficient;
[0031] Figure 8 Selectivity of Staphylococcus aureus detection; DETAILED DESCRIPTION
[0032] Example 1 Preparation of gold nanoparticles (colloidal gold)
[0033] The glassware required for synthesis was first immersed in aqua regia (nitric acid: hydrochloric acid = 1:3) for 30 min, washed with ultrapure water three times, and dried with nitrogen. 100 mL of 1 mM chloroauric acid was heated to boiling, and 4 mM trisodium citrate was quickly added under stirring. After 20 min, the solution changed from light yellow to wine red, obtaining gold nanoparticles. After cooling to room temperature, the solution was stored in the dark at 4°C. The UV spectrum of the gold nanoparticles is shown in Figure 2 (a).
[0034] Example 2 Preparation of phosphate buffer (for dilution of bacteria and negative control)
[0035] Buffer: Take NaCl 8.0 g, KCl 0.2 g, Na2HPO4 1.44 g and KH2PO4 0.24 g, dissolve in 800 mL of distilled water, adjust pH to 7.4 with NaOH, and dilute to 1000 mL.
[0036] Example 3 Preparation of fluorescein isothiocyanate (FITC) solution
[0037] Accurately weigh 0.0019 g of FITC and dissolve in deionized water at room temperature to form a 2.5 μM-20 μM FITC solution. The fluorescence emission spectrum of FITC is shown in Figure 2 (b).
[0038] Example 4 Preparation of Staphylococcus aureus aptamer solution
[0039] The aptamer (5'SH-GCA ATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCGCTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACT TTG CTA A-3') dry powder was dissolved in enzyme-free water (water without DNA or RNase) to form a 10 μM-30 μM aptamer solution;
[0040] Example 5: Experiment on optimization of gold nanoparticle size
[0041] Gold nanoparticles with particle sizes of 13 nm, 20 nm, and 40 nm were prepared by sodium citrate reduction. The ultraviolet absorption spectra of each nanoparticle were measured by a UV-Vis spectrophotometer. Then, 100 μL of each nanoparticle solution was accurately transferred, and 25 μL of negative control supernatant was added. The mixture was frozen at -80 °C for 10 min, thawed at 70 °C, and the ultraviolet absorption spectra were scanned.
[0042] Experimental results are as follows Figure 3 As shown, from Figure 3 Figures (a) and (b) show that the absorption peaks of gold nanoparticles of different sizes change in the 520nm-531nm range; Figure (c) shows that 20nm and 40nm gold nanoparticles exhibit an aggregated state after freeze-thaw cycles, while 13nm gold nanoparticles show almost no change. Therefore, 13nm gold nanoparticles are preferred in this experiment.
[0043] Example 6: Optimization Experiment of Freezing Time for Gold Nanoparticles and Aptamers
[0044] Take 100 μL of gold nanoparticle solution, add 25 μL of negative control supernatant, mix the solution thoroughly, and incubate at -80℃ for 5 min, 10 min, 20 min, 30 min, and 60 min, then thaw at 70℃. Measure the UV absorption spectrum of the mixture using a UV-Vis spectrophotometer.
[0045] Experimental results are as follows Figure 4 As shown in (a)(b), from Figure 4 As can be seen from (a) and (b), when frozen at -80°C for 5 min, the gold nanoparticles slightly aggregated, and when frozen for more than 10 min, the state of the gold nanoparticles remained similar. Preferably, in this invention, freezing is performed at -80°C for 10 min.
[0046] Example 7: Optimization Experiment of Melting Temperature of Gold Nanoparticles and Aptamers
[0047] Take 100 μL gold nanoparticle solution, add 25 μL negative control supernatant to it, then mix the above solution uniformly, freeze at -80℃ for 10 min, then thaw at 20℃, 37℃, 50℃, 60℃ and 70℃, and measure the ultraviolet absorption spectrum of the mixed solution by ultraviolet-visible spectrophotometer.
[0048] The experimental results are shown in Figure 5 (a), (b) and (c), and it can be seen from Figure 5 (a), (b) and (c) that the melting temperature does not affect the aggregation of gold nanoparticles, and the absorption peak hardly changes, but as the melting temperature increases, the time required for thawing gradually decreases. The present application preferably uses 70℃ for rapid thawing.
[0049] Example 8 Staphylococcus aureus aptamer incubation time optimization experiment
[0050] After mixing 10 μL of 20 μM Staphylococcus aureus aptamer with 100 μL of 10 5 CFU / mL Staphylococcus aureus uniformly, incubate at 37℃ for 15 min, 30 min, 45 min, 60 min and 90 min, centrifuge at 4000 rpm for 10 min, take 25 μL of supernatant and mix it with 100 μL of gold nanoparticle solution uniformly, then freeze at -80℃ for 10 min, thaw and measure the ultraviolet absorption spectrum of the mixed solution by ultraviolet-visible spectrophotometer.
[0051] The experimental results are shown in Figure 6 (a), and as the incubation time increases, A 650 / A 520 gradually increases; when the incubation time is 60 min, A 650 / A 520 increases gradually, so the preferred incubation time in this experiment is 60 min.
[0052] Example 9 Staphylococcus aureus aptamer concentration optimization experiment
[0053] Mix 10 μL of Staphylococcus aureus aptamer with a concentration of 0 μM, 10 μM, 15 μM, 20 μM, 25 μM and 30 μM respectively with 100 μL of 10 5CFU / mL S. aureus mixed as a positive control group. 10 μL of different concentrations of S. aureus aptamer (0 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM) was mixed with 100 μL of phosphate buffer as a negative control group. After mixing the above solutions uniformly, they were incubated at 37°C for 60 min, centrifuged at 4000 rpm for 10 min, and 25 μL of supernatant was mixed with 100 μL of gold nanoparticle solution uniformly. After freezing at -80°C for 10 min, the mixture was thawed and the ultraviolet absorption spectrum of the mixture was determined by ultraviolet-visible spectrophotometer.
[0054] The experimental results are shown in FIGS. Figure 6 (b) When the concentration of S. aureus aptamer added was 20 μM, the A 650 / A 520 The difference was the largest, and a more obvious colorimetric signal could be obtained. Therefore, the preferred concentration of the aptamer was 20 μM.
[0055] Example 10: Fluorescein isothiocyanate (FITC) concentration optimization experiment
[0056] 10 μL of S. aureus aptamer with a concentration of 20 μM was mixed with 100 μL of phosphate buffer containing 10 5 CFU / mL S. aureus was incubated at 37°C for 1 h. After centrifugation of the mixture at 4000 rpm for 10 min, 25 μL of supernatant was added to 100 μL of gold nanoparticles, and the mixture was frozen and thawed in a refrigerator. After centrifugation of the frozen and thawed gold nanoparticles at 4000 rpm for 10 min, 90 μL of the centrifuged AuNPs solution was incubated with 10 μL of FITC with concentrations of 2.5, 5, 10, 15 and 20 μM at room temperature for 10 min; the negative control group used phosphate buffer instead of S. aureus, and the fluorescence spectrometer was used to determine the fluorescence spectrum of the above mixed solution, and the fluorescence difference ΔF was calculated.
[0057] The experimental results are shown in FIGS. Figure 6 (c) When the concentration of fluorescein isothiocyanate FITC added was 15 μM, the fluorescence difference was the largest. Therefore, the preferred concentration of fluorescein isothiocyanate FITC was 15 μM.
[0058] Example 11: S. aureus detection experiment
[0059] Under the optimal conditions, the steps of the colorimetric / fluorescent dual-mode detection method for S. aureus established by the present application are as follows:
[0060] (1) Dilute the S. aureus solution
[0061] Take 6 1.5 mL microcentrifuge tubes, numbered 1, 2, 3, 4, 5, 6, respectively, add 1000 μL of standard Staphylococcus aureus solution to tube 1, and 900 μL of standard diluent (phosphate buffer) to tubes 2-6, repeatedly blow with a gun head for about 5-10 times (note to control the amplitude not too large to produce bubbles) if there is a vortex instrument, vortex for 1-10 seconds, then change the gun head. Take 100 μL from tube 1 and add to tube 2, and the rest follow the same procedure. After dilution, the liquid in tubes 1-5 is 900 μL, and the liquid in tube 6 is 1000 μL. The concentration of Staphylococcus aureus is from large to small, and the concentration of Staphylococcus aureus is 1 x 10 6 -10 CFU / mL.
[0062] (2) Staphylococcus aureus aptamer binding to Staphylococcus aureus
[0063] Mix 10 μL of Staphylococcus aureus aptamer with a concentration of 20 μM with 10-10 6 CFU / mL of Staphylococcus aureus, incubate at 37°C for 60 min, centrifuge at 4000 rpm for 10 min;
[0064] (3) Addition of colloidal gold solution
[0065] Take 25 μL of supernatant from step (2) and add to 100 μL of gold nanoparticle solution, freeze at -80°C for 10 min, then thaw;
[0066] (4) Color development
[0067] According to the color and aggregation state of the thawed colloidal gold solution, the content of Staphylococcus aureus is indicated.
[0068] (5) Addition of fluorescein isothiocyanate (FITC) solution
[0069] Centrifuge the thawed colloidal gold solution at 4000 rpm for 10 min, take 90 μL of colloidal gold solution and incubate with 10 μL of 15 μM fluorescein isothiocyanate (FITC) solution;
[0070] (6) Reading
[0071] Scan the ultraviolet absorption spectrum at 400-800 nm with a UV-visible spectrophotometer, record the absorption intensity at 520 nm and 650 nm; record the emission spectrum at 500-600 nm under excitation light at 490 nm with a fluorescence spectrophotometer; according to the standard curve, correct the negative sample, and determine the concentration of Staphylococcus aureus.
[0072] The detection results of Staphylococcus aureus using the colorimetric / fluorescence method (absorbance ratio and fluorescence signal) of the present invention are as follows: Figure 7 As shown, where, Figure 7 (a) The scanning spectra of different concentrations of Staphylococcus aureus were detected by freeze-thaw reaction of gold nanoparticles and aptamers. The linear calibration curve is shown in [reference needed]. Figure 7 (b). The linear range of this colorimetric method for the detection of Staphylococcus aureus is 10. 2 -10 6 The linear relationship was good, with a correlation coefficient of 0.994 and a detection limit of 5 CFU / mL.
[0073] Figure 7 (c) shows the fluorescence curves for detecting different concentrations of Staphylococcus aureus based on the interaction between gold nanoparticles and fluorescein isothiocyanate (FITC). The linear correction curve is shown in [reference needed]. Figure 7 (d). The linear range of this fluorescence method for the detection of Staphylococcus aureus is 10. 1 -10 6 The linear relationship was good, with a correlation coefficient of 0.991 and a detection limit of 2 CFU / mL.
[0074] Example 12 Specificity Experiment
[0075] Accurately transfer 100 μL of each of the 10 concentrations. 5 CFU / mL E. coli O157:H7, concentration 10 5 CFU / mL Pseudomonas, concentration of 10 5 CFU / mL Listeria monocytogenes, concentration 10 5 A mixture of CFU / mL Staphylococcus aureus and the above bacteria was prepared. 10 μL of 20 μM Staphylococcus aureus aptamer was added to each tube sequentially. After mixing the solutions thoroughly, the mixture was incubated at 37℃ for 60 min, centrifuged at 4000 rpm for 10 min, and 25 μL of the supernatant was added to 100 μL of gold nanoparticle solution. The mixture was frozen at -80℃ for 10 min, thawed at room temperature, and the absorbance was measured. The thawed colloidal gold solution was centrifuged at 4000 rpm for 10 min, and 90 μL of the colloidal gold solution was incubated with 10 μL of 15 μM fluorescein isothiocyanate solution. The fluorescence spectrum was measured.
[0076] Experimental results are as follows Figure 8 As shown, from Figure 8 As can be seen, this method can specifically identify Staphylococcus aureus. Therefore, the colorimetric / fluorescence dual-mode detection method for Staphylococcus aureus based on aptamer-induced colloidal gold aggregation and the interaction of gold nanoparticles with fluorescein isothiocyanate exhibits high selectivity and specificity.
[0077] Preparation and use of Staphylococcus aureus kit
[0078] A rapid detection kit for Staphylococcus aureus comprises: colloidal gold described in Example 1, phosphate buffer described in Example 2, fluorescein isothiocyanate solution described in Example 3, 10 9 bacterial liquid positive standard, negative quality control phosphate buffer.
[0079] The use steps of the rapid detection kit for Staphylococcus aureus are as follows:
[0080] After the food sample to be tested is ground, 5 g of homogenate is taken, 10 mL of phosphate buffer in the kit is added, and after being fully mixed, 100 μL of the extraction solution is transferred into a centrifuge tube, 10 μL of aptamer in the kit is added to the centrifuge tube, and after being vortexed, it is incubated at 37°C for 60 min, centrifuged at 4000 rpm for 10 min, and the supernatant is aspirated with a pipette. 25 μL of the supernatant is added to 100 μL of gold nanoparticle solution, frozen at -80°C for 10 min, thawed, and the absorbance value is measured. The absorbance value can be measured using a visible spectrophotometer, or it can be observed visually. After thawing, the colloidal gold solution is centrifuged at 4000 rpm for 10 min, 90 μL of the colloidal gold solution is incubated with 10 μL of 15 μM fluorescein isothiocyanate FITC solution, and the fluorescence spectrum is measured. The negative control sample and the positive control sample provided in the kit are tested in the same way as described above, and a standard curve is prepared using the standard bacterial liquid. The A 650 / A 520 The fluorescence value of the positive control sample is less than that of the negative control sample, and the fluorescence value of the negative control sample is less than that of the positive control sample, indicating that the kit is invalid.
Claims
1. A rapid colorimetric / fluorometric test kit for food-borne pathogenic bacteria, comprising: Aqueous solution of foodborne pathogenic bacteria aptamer, colloidal gold solution, and fluorescein isothiocyanate solution; The concentration of the aqueous solution of foodborne pathogenic bacteria aptamer is 10-30 muM; The colloidal gold particle size is 13-40 nm, and the surface functional group is carboxyl; the concentration of the fluorescein isothiocyanate solution is 2.5-20 muM; The foodborne pathogenic bacteria is Staphylococcus aureus; The aptamer gene sequence is: 5'SH-GCAATGGTACGGTACTTCCTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGCTACGTCAAAAGTGCACGCTACTTTGCTAA-3'.
2. The rapid colorimetric / fluorescent detection kit for food-borne pathogenic bacteria according to claim 1, characterized in that: The colloidal gold solution is prepared by the reaction of chloroauric acid and trisodium citrate.
3. The rapid colorimetric / fluorescent test kit for food-borne pathogenic bacteria according to claim 2, characterized in that: The reaction is that 1 mM of chloroauric acid is heated to boiling, and 4 mmol / L of trisodium citrate is quickly added under vigorous stirring, and the reaction is carried out for 20 min.
4. The rapid colorimetric / fluorescent detection kit for food-borne pathogenic bacteria according to claim 3, characterized in that: The concentration of the aqueous solution of aptamer is 20 muM; the concentration of the fluorescein isothiocyanate solution is 15 muM; and the colloidal gold particle size is 13 nm.
5. The rapid colorimetric / fluorescent test kit for food-borne pathogenic bacteria according to claim 4, characterized in that: The kit further comprises negative quality control: phosphate buffer, and positive quality control: inactivated Staphylococcus aureus suspension.
6. The rapid colorimetric / fluorescent test kit for food-borne pathogenic bacteria according to claim 5, characterized in that: The phosphate buffer is prepared by dissolving NaCl 8.0 g, KCl 0.2 g, Na2HPO4 1.44 g, and KH2PO4 0.24 g in 800 mL of distilled water, adjusting the pH to 7.4 with NaOH, and diluting to 1000 mL.
7. A rapid colorimetric / fluorometric detection method for food-borne pathogenic bacteria, comprising: The kit for rapid colorimetric / fluorescence detection of foodborne pathogenic bacteria is used. 1) The sample to be detected is added with buffer, and after being fully mixed, the extraction solution is transferred into a centrifuge tube; The volume of the buffer is 5-10 mL; 2) The aqueous solution of Staphylococcus aureus aptamer is added, and after being vortexed, it is incubated at 37 DEG C for 15-90 min, centrifuged, and the supernatant is aspirated; 3) The supernatant solution is added to the colloidal gold solution, which is frozen at a temperature of -20 to -80 DEG C for 10-60 min, and after being thawed at a temperature of 25-70 DEG C, the colloidal gold solution is visually observed or the scanning spectrum of the colloidal gold solution at 400-800 nm is determined by a UV-visible spectrometer; 4) The thawed colloidal gold solution is centrifuged to remove aggregated nanoparticles, and the fluorescein isothiocyanate solution is added for incubation, and the content of Staphylococcus aureus is determined by measuring the fluorescence using a fluorescence spectrometer.
8. The rapid colorimetric / fluorescent detection method of food-borne pathogenic bacteria according to claim 7, characterized in that: In step 1), the mass of the sample to be detected is 5 g, and the volume of the buffer is 10 mL; in step 3), the freezing temperature is -80 DEG C for 10 min, and the thawing temperature is 70 DEG C.
9. The rapid colorimetric / fluorescent detection method of food-borne pathogenic bacteria according to claim 8, characterized in that: In step 2), the incubation time is 60 min. In step 2), the incubation time is 60 min.