CN113281507A - Rapid detection method and kit for staphylococcus aureus - Google Patents
Rapid detection method and kit for staphylococcus aureus Download PDFInfo
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Abstract
The invention discloses a rapid detection kit for staphylococcus aureus, which comprises: fe3O4Magnetic bead specificity enrichment biological probe, vancomycin solution, colloidal gold solution and magnet. It passes through Fe3O4The magnetic bead specificity enrichment bioprobe realizes enrichment and separation of staphylococcus aureus, vancomycin is used for inducing aggregation change of colloidal gold after freeze thawing, quantitative detection is carried out on the staphylococcus aureus according to the aggregation color change of colloid, the variation coefficient is small during quantitative detection, the linear range is wide, the method sensitivity is high, the detection quantitative limit is 2.1CFU/mL, in addition, the invention has the advantages of low cost, simple operation, easy implementation, shortened detection time and good stability.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a rapid detection method and a kit for staphylococcus aureus.
Background
Food-borne pathogenic bacteria constitute a significant threat to human health as biological contaminants in food. Common food-borne pathogenic bacteria are Listeria monocytogenes (Listeria monocytogenes), escherichia coli O157: h7 (Escherichia coil), Staphylococcus aureus (Staphylococcus aureus), Salmonella enteritidis (Salmonella) and the like, which are the most common causes of hospitalization and, to a severe extent, can cause death. Staphylococcus aureus, called Staphylococcus aureus for short, belongs to the genus Staphylococcus and is a very common food-borne pathogenic bacterium. The staphylococcus aureus has strong capability of resisting adverse living environment, is widely existed in nature, such as water, air, dust, excrement of human and animals, is easy to cause food pollution, and causes various diseases, such as pneumonia, pseudomembranous colitis, pericarditis and even septicemia. The enterotoxin produced by staphylococcus aureus under specific conditions is one of the main pathogenic factors leading to food poisoning. Enterotoxin can damage the intestinal tract greatly, can cause acute poisoning symptoms such as nausea, repeated vomiting, abdominal pain, diarrhea, shock and the like, and can cause circulatory failure seriously. The staphylococcus aureus does not need abundant nutrition for growth, has strong capability of resisting adverse environment and salt tolerance, and can cause pollution to various foods. Currently, food poisoning by staphylococcus aureus accounts for a large proportion of bacterial food poisoning. Food poisoning cases caused by staphylococcus aureus in China account for about 20-25% of the total number of food poisoning cases, food poisoning cases caused by staphylococcus aureus in the United states account for 33% of the total number of bacterial food poisoning, and food poisoning cases caused by staphylococcus aureus in Canada account for 45%. At present, the detection methods of food-borne pathogenic bacteria mainly comprise a traditional separation culture method, a molecular biological detection method, an immunological method, a biosensing technology and the like, and although the methods can realize the detection of the food-borne pathogenic bacteria, the methods still have respective defects. The staphylococcus aureus index is the most basic and common detection item in the food microorganism test of GB4789 series. The traditional culture method wastes time and labor for the method for measuring the staphylococcus aureus, and a large amount of manual operation is needed. For example, the national standard GB 4789.10-2016 (national food safety Standard food microbiology inspection for Staphylococcus aureus) requires a series of steps such as pre-enrichment, culture, biochemical identification and the like, takes more than 60 hours, needs BPW, TTB, SC enrichment liquid, XLD, BS and HE, a chromogenic medium, and needs a large amount of reagents for subsequent biochemical identification. The culture method is time-consuming and labor-consuming. The PCR technology is complex in operation, high in cost and requires workers with higher operation technology; enzyme-linked immunoassays are expensive and their activity is easily affected by environmental factors such as temperature and pH. Therefore, it is urgently required to develop a detection method which is simple in operation, easy to implement, rapid and high in sensitivity.
The food sample has complex matrix and few target bacteria to be detected, and a novel pretreatment technology and a novel pretreatment method are required. Fe3O4The magnetic nano-particles have good biocompatibility, conductivity, magnetism, no toxicity and easy synthesis. Using Fe3O4The magnetic nanoparticle coupled antibody, nucleic acid and aptamer can be widely applied to the fields of immunological detection, cell separation, protein purification and the like. In the detection of food-borne pathogens, Fe3O4The magnetic nanoparticles are effectively combined with the specificity of antigen-antibody reaction and the magnetic field responsiveness of magnetic beads, can realize rapid separation and enrichment of target bacteria from a complex matrix to improve the detection efficiency, is an effective sample pretreatment method, and has better development and application prospects. Vancomycin serving as a broad-spectrum glycopeptide antibiotic can be combined with peptidoglycan on the cell wall of gram-positive bacteria, and is combined with the gram-positive bacteria based on the combination, so that the aim of detection is fulfilled. In 2017, the Monausia menhadenensis synthesized vancomycin functionalized magnetic beads and the flow cytometry technology carried out the specific detection of the Staphylococcus aureus within 120min, and the detection limit was 3.3 CFU/mL (Meng X, Yang G, Li F, Wei H, Xu H: Sensitive detection of Staphylococcus aureus with vancomycin-conjugated magnetic beads as antigens carriers)combined with flow cytometry, ACS applied material and interfaces2017, 9(25): 21464-. The colloidal gold has good biocompatibility, large specific surface area, photoelectric characteristics and surface plasmon resonance properties, and is widely applied to the field of biosensing. When the size of the gold nanoparticles is larger than 3.5nm, the gold nanoparticles have larger molar absorptivity, and the molar absorptivity is gradually reduced along with the increase of the particle size, so that the ultraviolet visible absorption peak is blue-shifted, and the color change visible to naked eyes appears, thereby being widely used in colorimetric sensing detection.
In conclusion, in order to realize the field screening of staphylococcus aureus, a detection method and a kit which are rapid, high in sensitivity and strong in specificity are developed, and the method and the kit have important significance in the fields of food microorganism detection and the like.
Disclosure of Invention
The invention aims to provide a rapid detection method and a kit for staphylococcus aureus.
A staphylococcus aureus rapid detection kit, comprising: fe3O4Specifically enriching biological probes, vancomycin solution, colloidal gold solution, magnets and washing liquor by using magnetic beads;
said Fe3O4The magnetic bead specific enrichment bioprobe is a ferroferric oxide magnetic bead specific enrichment bioprobe modified by staphylococcus aureus aptamer;
the gene sequence of the staphylococcus aureus aptamer is 5 'NH 2-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';
said Fe3O4The concentration of the magnetic bead specificity enrichment biological probe is 0.1-0.5 mg/mL; the concentration of the vancomycin solution is 7.8125-500 mu M; the grain size of the colloidal gold is 13-48 nm;
said Fe3O4The magnetic bead specific enrichment bioprobe is prepared by the following method:
1) adding ferric chloride hexahydrate and trisodium citrate into an ethylene glycol solution, and after complete dissolution, enabling the final concentration of the ferric chloride hexahydrate to be 0.2mol/L and the final concentration of the trisodium citrate to be 0.034 mol/L;
2) adding sodium acetate into the solution 1), wherein the final concentration of the sodium acetate is 0.73mol/L, and stirring for 30 min;
3) transferring the solution 2) into a reaction kettle, reacting at 200 ℃ for 10h, cooling, taking out a black solution, sucking out black solid ferroferric oxide by a magnet, and alternately washing for 3-5 times by using distilled water and ethanol in sequence to obtain carboxylated Fe3O4Magnetic beads;
4) by taking carboxylated Fe3O4Magnetic beads are dissolved in PBS buffer solution with pH 6, so that the concentration of the magnetic beads reaches 1-4 mg/mL;
5) adding solid 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide salt (EDC) and N-hydroxysuccinimide (NHS) into the solution 4), enabling the concentrations of the buffer solutions in the solution 4) to reach 2.5-6mg/mL, incubating for 60min at 37 ℃, washing for 3 times by PBS, suspending in PBS buffer solution with pH7.4, and adding 10 muL-60 muL of staphylococcus aureus aptamer with the concentration of 6 muM-10 muM;
6) incubating the mixed solution at 4 deg.C overnight, separating the solution with magnet, discarding supernatant, washing with PBS buffer solution with pH7.4 for 3 times, and suspending in PBS buffer solution with pH7.4 to obtain Fe3O4Enriching biological probes by magnetic bead specificity;
the optimal concentration of EDC and NHS is 5 mg/mL;
the optimal concentration of the staphylococcus aureus aptamer is 10 muM, and the optimal volume is 60 muL;
the colloidal gold solution is prepared by the following method: soaking glassware required by synthesis in a mixed solution of nitric acid and hydrochloric acid at a volume ratio of 1:3 for 30min, washing with ultrapure water for three times, blowing by using nitrogen gas for drying, heating 1mM chloroauric acid to boil, quickly adding 4mmol/L trisodium citrate under a stirring state, and obtaining a colloidal gold solution after 20 min.
A staphylococcus aureus rapid detection kit, comprising: fe3O4Magnetic bead specificity enrichment biological probe, vancomycin solution, colloidal gold solution, magnet, 10 inactivated by 1% formaldehyde9A bacteria liquid positive standard substance and negative quality control deionized water; said Fe3O4The optimal concentration of the magnetic bead specificity enrichment bioprobe is 0.3 mg/mL; the above-mentionedThe optimum concentration of the vancomycin solution is 62.5 mu M; the optimal particle size of the colloidal gold is 13 nm;
a rapid detection method of staphylococcus aureus, which comprises the following steps:
1) after grinding a food sample to be detected, taking 5g of homogenate, adding PBS (phosphate buffer solution) 10m L, fully and uniformly mixing, and transferring 425 mu L of leaching liquor into a centrifuge tube;
2) adding 75 μ L of Fe3O4 magnetic bead specific enrichment bioprobe, mixing uniformly by vortex, enriching for 60min at 37 deg.C, magnetic separating, and sucking out supernatant with pipette. Washing with PBS buffer solution for 3 times, magnetically separating, sucking out washing solution, and discarding;
3) adding 50 μ L of 62.5 μ M vancomycin solution, rotary mixing and incubating for 30min, and magnetic enriching;
4) adding 15 μ L of the supernatant into 300uL of colloidal gold solution, freezing at-80 deg.C for 10min, thawing at 70 deg.C, and measuring absorbance;
the enrichment time of the step 2) is 60min, the freezing temperature of the step 4) is-80 ℃, and the freezing time is 10 min.
The invention provides a rapid detection kit for staphylococcus aureus, which comprises: fe3O4Magnetic bead specificity enrichment biological probe, vancomycin solution, colloidal gold solution and magnet. It passes through Fe3O4The magnetic bead specificity enrichment biological probe realizes enrichment and separation of staphylococcus aureus, vancomycin is used for inducing aggregation change of colloidal gold after freeze thawing, quantitative detection is carried out on the staphylococcus aureus according to the aggregation color change of colloid, the variation coefficient is small during quantitative detection, the linear range is wide, the method sensitivity is high, the lower detection limit is 0.21 CFU/mL, and the quantitative limit is 2.1 CFU/mL.
Drawings
FIG. 1 shows ultraviolet spectra, infrared spectra, particle sizes and zeta potential diagrams of ferroferric oxide magnetic nanoparticles and colloidal gold;
FIG. 2 is a zeta potential diagram of a mixed solution of colloidal gold, vancomycin and colloidal gold vancomycin;
FIG. 3 vancomycin dosage optimization;
FIG. 4 (a) particle size optimization of colloidal gold, (b) absorbance normalization value of the colloidal gold after freeze-thawing, (c) absorption spectra of the colloidal gold with different particle sizes, and (d) influence of freeze-thawing on spectra of mixed solution of the colloidal gold and vancomycin with different particle sizes;
FIG. 5 (a-d) optimization of freezing time of mixed indicator solutions of colloidal vancomycin and (e-g) optimization of dissolution temperature of mixed indicator solutions of colloidal vancomycin;
FIG. 6(a, b) aptamer dosage optimization, (c, d) ferroferric oxide magnetic bead specific enrichment bioprobe dosage optimization and (e, f) enrichment time optimization;
FIG. 7 Standard Curve, Linear regression equation and correlation coefficient for Staphylococcus aureus detection;
FIG. 8 Selectivity of Staphylococcus aureus detection.
Detailed Description
Example 1 preparation of carboxylated ferroferric oxide magnetic beads
Adding ferric chloride hexahydrate and trisodium citrate into ethylene glycol, completely dissolving to make the final concentration of ferric chloride hexahydrate be 0.2mol/L and the final concentration of trisodium citrate be 0.034mol/L, adding sodium acetate to make the final concentration of sodium acetate be 0.73mol/L, and stirring for 30 min. Transferring the mixture to a reaction kettle, reacting for 10h at 200 ℃, cooling, taking out a black solution, sucking out black solid ferroferric oxide by using a magnet, alternately washing for 3-5 times by using distilled water and ethanol in sequence to obtain carboxylated ferroferric oxide nanoparticles, namely carboxylated ferroferric oxide magnetic beads, and putting the carboxylated ferroferric oxide nanoparticles into a refrigerator at 4 ℃ for later use. The hydrated particle size of ferroferric oxide is 562nm as shown in figure 1(b), the infrared spectrum and the ultraviolet spectrum are shown in figure 1(c), and the potential is shown in figure 1 (d).
Example 2 preparation of a biological Probe for enriching specificity of ferroferric oxide magnetic beads
Dissolving carboxylated ferroferric oxide magnetic beads in a phosphate buffer solution with the pH value of 6 to enable the concentration of the carboxylated ferroferric oxide magnetic beads to reach 2mg/mL, adding solid EDC and solid NHS to enable the concentration of the solid EDC and the concentration of the solid NHS to reach 5mg/mL, incubating for 60min at 37 ℃, washing for three times with PBS, suspending in the phosphate buffer solution with the pH value of 7.4, and adding 10 mu M of staphylococcus aureus aptamer: 5' NH2-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' 60. mu.L. The mixed solution was incubated at 37 ℃ for 4 hours. The solution was separated with a magnet and the supernatant discarded. Washing the sample with a phosphate buffer solution with the pH value of 7.4 for three times, and then suspending the sample in the phosphate buffer solution with the pH value of 7.4 to obtain the staphylococcus aureus aptamer-modified ferroferric oxide magnetic bead specific enrichment bioprobe (Fe)3O4And (4) enriching the biological probes by the magnetic bead specificity. The potential of the ferroferric oxide magnetic bead specific enrichment biological probe is shown in figure 1 (d).
The preparation method of the phosphate buffer solution comprises the following steps: collecting NaCl8.0g, KCl0.2g and Na2HPO41.44g and KH2PO40.24g was dissolved in 800mL of distilled water, adjusted to pH 6 and 7.4 with NaOH, and dissolved to 1000 mL.
Example 3 preparation of gold nanoparticles (colloidal gold)
Firstly, soaking glassware required by synthesis in aqua regia (nitric acid: hydrochloric acid =1: 3) for 30min, washing for three times by using ultrapure water, drying by using nitrogen, heating 100mL of 1mM chloroauric acid to boiling, rapidly adding 0.1032g of trisodium citrate under the stirring state to enable the concentration of trisodium citrate to reach 0.4mM, changing the solution from light yellow to wine red after 20min to obtain 13nm gold nanoparticles, cooling to room temperature, and storing at the dark condition of 4 ℃. The ultraviolet spectrum of the gold nanoparticles is shown in figure 1(a), the hydrated particle size is shown in figure 1(b), and the potential is shown in figure 1 (d).
EXAMPLE 4 preparation of vancomycin solution
Accurately weighing 0.005g-0.35g of vancomycin, dissolving in deionized water at room temperature to form 7.8125 mu M-500 mu M vancomycin solution, and placing in a refrigerator at 4 ℃ for later use.
Example 5 vancomycin amount optimization experiment
The embodiment provides a rapid and specific detection method for staphylococcus aureus aggregated by vancomycin-induced colloidal gold after freeze thawing, and the optimization of the vancomycin dosage comprises the following steps: taking 300 mu L of gold nanoparticle solution, sequentially adding 15 mu L of 15.625 mu M-500 mu M vancomycin, uniformly mixing the solution, freezing at-80 ℃ for 10min, then thawing at 70 ℃, and measuring the ultraviolet absorption spectrum of the mixed solution by using an ultraviolet-visible spectrophotometer.
As shown in FIG. 3, it can be seen from FIG. 3 that gold nanoparticles begin to aggregate when the amount of vancomycin added is less than 62.5. mu.M.
Example 6 particle size optimization experiment of gold nanoparticles
Gold nanoparticles with particle sizes of 13nm, 14nm, 18nm, 30nm and 48nm are prepared by a sodium citrate reduction method, and ultraviolet absorption spectra are respectively measured by an ultraviolet-visible spectrophotometer; then accurately transferring 300 mu L of gold nanoparticle solution with each particle size, adding 15 mu L of vancomycin with the particle size of 62.5 mu M, freezing at-80 ℃ for 10min, unfreezing at 70 ℃, and scanning an ultraviolet absorption spectrum.
The experimental result is shown in fig. 4, and the hydrated particle size of gold nanoparticles with different particle sizes is shown in fig. 4 (a); from FIG. 4(b) (c), it can be seen that the absorption peaks of gold nanoparticles with different particle sizes are changed from 520nm to 535 nm; as can be seen from the graph (d), 18nm, 30nm and 48nm gold nanoparticles exhibited an aggregated state after freezing and thawing, with 14nm gold nanoparticles having a low aggregation degree and 13nm gold nanoparticles having almost no change. Therefore, 13nm gold nanoparticles are preferred for this experiment.
Example 7 gold nanoparticles and vancomycin freezing time and freezing-thawing temperature optimization experiment
First, gold nano particle and vancomycin freezing temperature and time optimization experiment
(1) Adding 15 μ L of 62.5 μ M vancomycin into 300uL of gold nanoparticle solution, mixing the above solutions, standing at-20 deg.C for 15min, 30min, 60min, 120min and 180min, and thawing at 70 deg.C. And measuring the ultraviolet absorption spectrum of the mixed solution by using an ultraviolet-visible spectrophotometer.
As shown in FIGS. 5(a) (c), it can be seen from FIGS. 5(a) (c) that the solution could not be completely frozen after freezing at-20 ℃ for 15min and 30 min; when the gold nanoparticles are frozen for 60-180min, the aggregation degree of the gold nanoparticles is gradually increased along with the time;
(2) adding 15 μ L of 62.5 μ M vancomycin into 300uL of gold nanoparticle solution, mixing the above solutions, standing at-80 deg.C for 5min, 10min, 20min, 30min and 60min, and thawing at 70 deg.C. And measuring the ultraviolet absorption spectrum of the mixed solution by using an ultraviolet-visible spectrophotometer.
As shown in FIGS. 5(b) and (d), it can be seen from FIGS. 5(b) and (d) that the aggregation degree of gold nanoparticles gradually increases with time when the sample is frozen at-80 ℃ and is similar when the sample is frozen for more than 10 min. The invention preferably freezes at-80 ℃ for 10 min.
Second, gold nano-particle and vancomycin thawing temperature optimization experiment
Adding 15 μ L of 62.5 μ M vancomycin into 300uL of gold nanoparticle solution, mixing the above solutions uniformly, freezing at-80 deg.C for 10min, thawing at 25 deg.C, 37 deg.C, 50 deg.C, 60 deg.C and 70 deg.C, and measuring the ultraviolet absorption spectrum of the mixed solution with ultraviolet-visible spectrophotometer.
As shown in FIGS. 5(e) (f) (g), it can be seen from FIGS. 5(e) (f) (g) that the thawing temperature does not affect the aggregation of gold nanoparticles, and that the absorption peak is hardly changed, but the time required for thawing gradually decreases as the thawing temperature increases. The invention prefers quick thawing at 70 ℃.
Example 8 Fe3O4Optimization experiment for using amount of staphylococcus aureus enriched by magnetic bead specificity enrichment bioprobe
First, the quantity optimization experiment for the aptamer
（1）Fe3O4Preparation of magnetic bead specificity enrichment biological probe
Dissolving carboxylated ferroferric oxide magnetic beads in phosphate buffer solution with pH 6 to enable the concentration to reach 2mg/mL, adding solid EDC and NHS to enable the concentration to reach 5mg/mL, incubating at 37 ℃ for 60min, washing with PBS for three times, suspending in PBS with pH7.4, adding an aptamer, fixing the volume of a system to be 1 mL by PBS with pH7.4, enabling the total concentration of the aptamer to be 0 muM, 0.2 muM, 0.3 muM, 0.4 muM, 0.5 muM, 0.6 muM and 0.7 muM respectively, incubating at 37 ℃ for 4 hours, separating the solution by using a magnet, discarding the supernatant, washing with PBS buffer solution with pH7.4 for three times, and suspending in PBS buffer solution with pH7.4 to obtain 7 Fe modified by different aptamer concentrations3O4The magnetic beads specifically enrich the biological probes, and the biological probes are placed at 4 ℃ for standby.
（2）Fe3O4Enrichment efficiency of magnetic bead specificity enrichment bioprobe on staphylococcus aureus
Respectively and accurately transferring 375 mu L of PBS solution with the pH value equal to 7.4 into 7 microcentrifuge tubes, and sequentially adding 75 mu L of the 7 Fe with different concentrations prepared in the step 13O4Magnetic bead specificity enrichment biological probe, after the solution is mixed evenly, 50 mu L of 3 multiplied by 10 is added7And (3) enriching the CFU/mL staphylococcus aureus at 37 ℃ for 60min, and taking the supernatant after the magnetic adsorption separation. After diluting the supernatant 1000 times, 10. mu.L of the supernatant was spread evenly on an LB solid dish, and the supernatant was cultured for 24 hours and counted.
The experimental results are shown in fig. 6(a) (b), and it can be seen from fig. 6(a) (b) that the enrichment efficiency gradually increases with the increase of the aptamer volume; at an aptamer concentration of 0.6 μ M, the enrichment efficiency was 93.9%, after which the enrichment efficiency did not increase with increasing volume. Therefore, the preferred concentration of aptamer-modified probes according to the invention is 0.6. mu.M.
II, Fe3O4Optimization experiment for magnetic bead specificity enrichment biological probe dosage
Respectively, a 375uLpH solution equal to 7.4 in PBS was accurately removed, and 0, 0.1, 0.2,0.3,0.4 and 0.5mg/mLFe were sequentially added thereto3O475 mu L of magnetic bead specificity enrichment biological probe, after the solution is mixed evenly, 50 mu L of 3 multiplied by 10 is added7Enriching the CFU/mL staphylococcus aureus at 37 ℃ for 60min, and adsorbing the supernatant by a magnet; after diluting the supernatant 1000 times, 10uL of the supernatant was spread evenly on an LB solid culture dish and cultured for 24 hours, and then counted.
The results are shown in FIGS. 6(c) (d), and it can be seen from FIGS. 6(c) (d) that Fe is accompanied by Fe3O4The use amount of the magnetic bead specificity enrichment bioprobe is increased, and the enrichment efficiency is gradually increased; when Fe3O4When the concentration of the magnetic bead specificity enrichment bioprobe is 0.3mg/mL, the enrichment efficiency is 93.7%, and then the enrichment efficiency tends to be stable. Thus Fe according to the invention3O4The preferred concentration of the magnetic bead-specific enrichment bioprobe is 0.3 mg/mL.
Magnetic enrichment time optimization experiment for staphylococcus aureus
(1)105Magnetic enrichment time optimization experiment for CFU/mL staphylococcus aureus
375uL of PBS solution with pH equal to 7.4 were removed accurately, and 75. mu.L of 0.3mg/mL Fe was added3O4Magnetic bead specificity enrichment biological probe, after the solution is mixed evenly, 10 is added5And (3) 50 mu L of CFU/mL staphylococcus aureus is enriched at 37 ℃ for 0min, 10min, 15min, 20min, 30min and 60min, and is adsorbed by a magnet, and the supernatant is taken. After diluting the supernatant 1000 times, 10. mu.L of the supernatant was spread evenly on an LB solid dish, and the supernatant was cultured for 24 hours and counted.
(2)108Magnetic enrichment time optimization experiment for CFU/mL staphylococcus aureus
375. mu.L of PBS solution with pH equal to 7.4 were each accurately pipetted and 75. mu.L of 0.3mg/mL Fe was added to it3O4Magnetic bead specificity enrichment biological probe, after the solution is mixed evenly, 10 is added8 Collecting 50 μ L of CFU/mL Staphylococcus aureus, enriching at 37 deg.C for 0min, 10min, 15min, 20min, 30min, 60min and 120min, and adsorbing with magnet to obtain supernatant; after diluting the supernatant 1000 times, 10uL of the supernatant was spread evenly on an LB solid culture dish and cultured for 24 hours, and then counted.
The results are shown in FIGS. 6(e) (f) (g) (h), and 10 is shown in FIGS. 6(e) (f) (g) (h)5After the CFU/mL staphylococcus aureus is enriched for 60min, the enrichment efficiency is 93.7%; 108After the CFU/mL staphylococcus aureus is enriched for 60min, the enrichment efficiency is 93.4%; therefore, the preferred enrichment time of the present invention is 60 min.
Example 9 Staphylococcus aureus detection assay
A rapid detection method of staphylococcus aureus comprises the following steps:
(1) dilute staphylococcus aureus solution
Preparing a microcentrifuge tube, coding the microcentrifuge tube into codes of 1, 2, 3, 4, 5 and 6 respectively, and adding 300 microliters of 10 into the tube No.16And repeatedly blowing and beating the CFU/mL staphylococcus aureus solution for 5 times by using a gun head, adding 900 microliters of standard diluent into a No. 2-6 microcentrifuge tube, adding 100 microliters of standard diluent into a No. 2 tube from the No.1 tube, and repeating the following processes. Finally, the dilution is finishedThe volume of liquid in 1 tube was 200. mu.l, 900. mu.l in the 2-5 tubes and 1000. mu.l in the 6 tubes. The concentration of the staphylococcus aureus is 10 from large to small6-10 CFU/mL。
（2）Fe3O4Enrichment of magnetic bead specific enrichment bioprobe on staphylococcus aureus
375. mu. LpH in 7.4PBS were accurately removed and 75. mu.L of 0.3mg/mLFe was added3O4Enrichment of biological probes with magnetic bead specificity, 10-106And (3) mixing 50 mu L of CFU/mL staphylococcus aureus uniformly, incubating at 37 ℃ for 60min, adsorbing and separating by using a magnet, discarding supernatant, washing three times by using PBS buffer solution with pH7.4, and adsorbing and removing the supernatant.
(3) Addition of vancomycin
To the separated Fe3O4And adding 50 mu L of 62.5 mu M vancomycin into the staphylococcus aureus adsorbed by the magnetic bead specific enrichment bioprobe, incubating for 30min at 37 ℃, and enriching by using a magnet again.
(4) Addition of colloidal gold solution
And (4) adding 15 mu L of the supernatant obtained in the step (3) into 300 mu L of gold nanoparticle solution, freezing at-80 ℃ for 10min, and then thawing at 70 ℃.
(5) Color development
And indicating the content of the staphylococcus aureus according to the color and the aggregation state of the thawed colloidal gold solution.
(6) Reading number
And (3) scanning an ultraviolet absorption spectrum at 400-800nm by using a visible spectrophotometer, recording the intensity of absorption light at 564nm, correcting a negative sample according to a standard curve, and determining the concentration of the staphylococcus aureus.
The detection of staphylococcus aureus by the colorimetric method of the present invention is shown in fig. 7, wherein fig. 7 is a linear calibration curve when staphylococcus aureus with different concentrations is detected by using gold nanoparticle and vancomycin complex. As can be seen from FIG. 7, the linear range of the method for detecting Staphylococcus aureus is 10-106CFU/mL, linear relation is good, correlation coefficient is 0.94646, detection lower limit is 0.29 CFU/mL, detection quantification limit is 2.1 CFU/mL.
Example 10 specificity test
mu.L of PBS solution with pH equal to 7.4 and 75. mu.L of 0.3mg/mL Fe were accurately pipetted separately3O4Magnetic bead-specific enrichment bioprobe, to which 50. mu.L 10 was sequentially added6CFU/mL Escherichia coli O157H 7, 106CFU/mL Pseudomonas, 106CFU/mL Listeria monocytogenes and 104Mixing the solutions uniformly, enriching at 37 deg.C for 60min, and adsorbing with magnet to remove supernatant; then adding 50 mu L of 62.5uM vancomycin, incubating for 30min at 37 ℃, enriching by using a magnet again, taking 15 mu L of supernatant, adding into 300uL of gold nanoparticle solution, freezing for 10min at-80 ℃, thawing at 70 ℃, and measuring the absorption value.
The experimental results are shown in fig. 8, and it can be seen from fig. 8 that the method can specifically recognize staphylococcus aureus. Therefore, the magnetic enrichment and vancomycin induced colloidal gold aggregation method has high selectivity and good specificity for detecting staphylococcus aureus.
EXAMPLE 11 preparation and use of Staphylococcus aureus kits
A staphylococcus aureus rapid detection kit, comprising: fe3O4Magnetic bead specificity enrichment biological probe, colloidal gold, vancomycin solution, magnet, 10 inactivated by 1% formaldehyde9Bacteria liquid positive standard substance and negative quality control deionized water.
The application steps of the staphylococcus aureus rapid detection kit are as follows:
after grinding a food sample to be detected, taking 5g of homogenate, adding PBS (phosphate buffer solution) 10m L, fully and uniformly mixing, transferring 375 mu L of leaching liquor into a centrifuge tube, and adding Fe into the centrifuge tube3O4And (3) specifically enriching 75 mu L of biological probe by using magnetic beads, uniformly mixing by using a vortex, enriching for 60min at 37 ℃, performing magnetic separation, and sucking out a supernatant by using a pipette. Washing with PBS buffer solution for 3 times, removing washing solution after magnetic separation, removing, adding 50 μ L vancomycin solution, rotary mixing and incubating for 30min, enriching with magnet, adding supernatant 15 μ L into 300uL gold nanoparticle solution, freezing at-80 deg.C for 10min, thawing at 70 deg.C, and determining its absorption value. By usingThe absorbance was measured with a visible spectrophotometer and was also observed visually. And detecting a negative quality control product and a positive quality control product sample provided by the kit according to the same method, and making a standard curve by using the standard bacterial liquid, wherein if the absorption value of the positive quality control product sample is greater than that of the negative quality control product, the kit is invalid.
Claims (10)
1. A staphylococcus aureus rapid detection kit, comprising: fe3O4Specifically enriching a biological probe, a vancomycin solution, a colloidal gold solution and a magnet by using magnetic beads; said Fe3O4The magnetic bead specific enrichment biological probe is a ferroferric oxide magnetic bead specific enrichment biological probe modified by staphylococcus aureus aptamer.
2. The rapid staphylococcus aureus detection kit according to claim 1, wherein the kit comprises: the staphylococcus aureus aptamer gene sequence is SEQ ID NO. 1.
3. The rapid staphylococcus aureus detection kit according to claim 2, wherein the kit comprises: said Fe3O4The concentration of the magnetic bead specificity enrichment biological probe is 0.1-0.5 mg/mL; the concentration of the vancomycin solution is 7.8125-500 mu M; the grain diameter of the colloidal gold is 13-48 nm.
4. The rapid detection kit for staphylococcus aureus according to claim 3, wherein: said Fe3O4The magnetic bead specific enrichment bioprobe is prepared by the following method:
1) adding ferric chloride hexahydrate and trisodium citrate into ethylene glycol, and after complete dissolution, enabling the final concentration of ferric chloride hexahydrate to be 0.2mol/L and the final concentration of trisodium citrate to be 0.034 mol/L;
2) adding sodium acetate into the solution 1), wherein the final concentration of the sodium acetate is 0.73mol/L, and stirring for 30 min;
3) transferring the solution 2) into a reaction kettle, reacting at 200 ℃ for 10h, cooling, and taking out blackThe color solution is sucked out by a magnet to obtain black solid ferroferric oxide, and the black solid ferroferric oxide is washed for 3 to 5 times by distilled water and ethanol in turn to obtain carboxylated Fe3O4Magnetic beads;
4) by taking carboxylated Fe3O4Magnetic beads are dissolved in PBS buffer solution with pH 6, so that the concentration of the magnetic beads reaches 1-4 mg/mL;
5) adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide salt and N-hydroxysuccinimide into the solution 4), enabling the concentrations of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide salt and the N-hydroxysuccinimide salt in the buffer solution of the solution 4) to reach 2.5-6mg/mL, incubating for 60min at 7 ℃, washing for 3 times by PBS, suspending in PBS buffer solution with pH of 7.4, and adding 10 mu L-60 mu L of staphylococcus aureus aptamer with the concentration of 6 mu M-10 mu M;
6) incubating the mixed solution at 4 deg.C overnight, separating the solution with magnet, discarding supernatant, washing with PBS buffer solution with pH7.4 for 3 times, and suspending in PBS buffer solution with pH7.4 to obtain Fe3O4And (4) enriching the biological probes by the magnetic bead specificity.
5. The rapid detection kit for staphylococcus aureus according to claim 4, wherein: the concentration of EDC and NHS is 5 mg/mL.
6. The rapid detection kit for staphylococcus aureus according to claim 1, 2, 3, 4 or 5, wherein: the concentration of the staphylococcus aureus aptamer is 10 mu M, and the volume of the staphylococcus aureus aptamer is 60 mu L.
7. The rapid detection kit for staphylococcus aureus according to claim 6, wherein: the colloidal gold solution is prepared by the following method: soaking glassware required by synthesis in a mixed solution of nitric acid and hydrochloric acid at a volume ratio of 1:3 for 30min, washing with ultrapure water for three times, blowing by using nitrogen gas for drying, heating 1mM chloroauric acid to boil, quickly adding 4mmol/L trisodium citrate under a stirring state, and obtaining a colloidal gold solution after 20 min.
8. A staphylococcus aureus rapid detection kit, comprising: fe3O4Magnetic beadSpecific enrichment of bioprobe, vancomycin solution, colloidal gold solution, magnet, 10 inactivated with 1% formaldehyde9A bacteria liquid positive standard substance and negative quality control deionized water; said Fe3O4The concentration of the magnetic bead specificity enrichment biological probe is 0.3 mg/mL; the concentration of the vancomycin solution is 62.5 mu M; the grain diameter of the colloidal gold is 13 nm.
9. A method for rapidly detecting staphylococcus aureus in food comprises the following steps:
1) after grinding a food sample to be detected, taking 5g of homogenate, adding PBS (phosphate buffer solution) 10m L, fully and uniformly mixing, and transferring 425 mu L of leaching liquor into a centrifuge tube;
2) adding Fe3O4And (3) specifically enriching 75 mu L of biological probe by using magnetic beads, uniformly mixing by using a vortex, enriching for 60min at 37 ℃, performing magnetic separation, and sucking out a supernatant by using a pipette. Washing with PBS buffer solution for 3 times, magnetically separating, sucking out washing solution, and discarding;
3) adding 50 μ L vancomycin solution, rotary mixing and incubating for 30min, and magnetic enriching;
4) adding 15 μ L of the supernatant into 300uL of colloidal gold solution, freezing at-80 deg.C for 10min, thawing at 70 deg.C, and measuring the absorption value.
10. The method for rapidly detecting staphylococcus aureus according to claim 9, wherein the method comprises the following steps: the enrichment time of the step 2) is 60min, the freezing temperature of the step 4) is-80 ℃, and the freezing time is 10 min.
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| CN117110607A (en) * | 2023-09-16 | 2023-11-24 | 鲁东大学 | Composition for detecting vibrio parahaemolyticus and application thereof |
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