CN116203235A - Rapid detection kit for staphylococcus aureus and detection method thereof - Google Patents
Rapid detection kit for staphylococcus aureus and detection method thereof Download PDFInfo
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Abstract
The invention discloses a rapid detection kit for staphylococcus aureus based on nano enzyme catalysis multicolor colorimetry, which comprises an immunomagnetic nano probe, a color development liquid A, a color development liquid B, a strong magnet, a sterile PBS buffer solution, 1M hydrochloric acid and 10-10 6 CFU/mL standard bacteria liquid colorimetric card. The invention also discloses a rapid detection method of staphylococcus aureus. The detection kit of the invention mainly carries out specific capture on staphylococcus aureus in a detection sample through an immunomagnetic nano probe, and utilizes the catalytic effect of nano mimic enzyme of the immunomagnetic nano probe and TMB 2+ The directional etching effect on the gold nanorods can realize the efficient and simple quantitative multicolor colorimetric semi-quantitative detection of staphylococcus aureus in the sample. The detection method provided by the invention has the advantages of short time, high accuracy, good sensitivity, simplicity in operation and low cost, is widely applicable to food sample detection, and has the potential of on-site rapid screening.
Description
Technical Field
The invention belongs to the technical field of microorganism detection, and particularly relates to a rapid detection kit and a detection method for staphylococcus aureus based on nano-enzyme catalysis multicolor colorimetric.
Background
With the diversification of people's life and eating patterns, food-borne pathogenic pathogens have become a non-negligible problem. Among them, staphylococcus aureus (Staphylococcus aureus, s. Aureus) is a pathogenic bacterium which is extremely liable to pollute and cause food poisoning by various milk, meat, egg and various products thereof. Staphylococcus aureus is also called staphylococcus aureus, has strong adaptability and wide pollution range, and after being taken into a human body, enterotoxin generated by multiplication of the staphylococcus aureus slightly causes a series of intestinal symptom diseases, and when serious, the staphylococcus aureus can cause middle toxicity, septic shock and the like. Thus, staphylococcus aureus has been currently listed as one of the most important food-borne diseases worldwide. Meanwhile, staphylococcus aureus has hidden dangers of hospital and acquired infection, and in conclusion, a rapid and accurate identification method for staphylococcus aureus is expected to be applied to various fields such as public health, food safety and the like.
Up to now, the following three methods are mainly used for detecting staphylococcus aureus: a method for separating, purifying, culturing and identifying, detecting immunologically and a method for molecular biotechnology. However, the method for separating, purifying, culturing and identifying has high accuracy and takes too long, at least 48 hours and more are needed for one complete and accurate identification, and the possibility of on-site operation is completely avoided; the immunological detection method mainly uses the specific binding reaction of antigen-antibody, but the existing common immunological detection method is extremely easy to generate cross reaction and has complex operation; the molecular biotechnology method is accurate in identification, but the instrument is extremely expensive, a complex pretreatment process is required, and meanwhile, the technical level of operators is extremely high, so that the requirements of on-site quick detection and basic-level personnel detection are difficult to meet.
In view of the defects of the existing detection method, the development of the staphylococcus aureus detection method which is rapid, simple and convenient, has high accuracy and can meet the requirements of on-site screening is particularly important in the aspects of preventing and controlling food-borne diseases.
Disclosure of Invention
The invention aims to provide a rapid detection kit and a detection method for staphylococcus aureus based on nano-enzyme catalysis multicolor colorimetry, which realize rapid, simple and convenient and high-specificity detection for staphylococcus aureus, and the specific technical scheme of the kit is as follows:
a staphylococcus aureus rapid detection kit is characterized by comprising an immunomagnetic nano probe, a color development liquid A, a color development liquid B and 10 1 -10 6 CFU/mL staphylococcus aureus colorimetric card, sterile PBS buffer solution, 1M hydrochloric acid and strong magnet;
the color development liquid A is an aqueous solution of 3, 5' -tetramethyl benzidine (TMB);
the color development liquid B is a gold nanorod solution uniformly dispersed in Cetyl Trimethyl Ammonium Bromide (CTAB);
the immunomagnetic nano probe is carboxylated Fe marked by staphylococcus aureus specific chicken egg yolk antibody 3 O 4 -Ag-MnO 2 Magnetic nanoparticles;
the concentration of the immunomagnetic nano probe is 0.5mg/mL, the concentration of the developing solution A is 1mL, and the concentration of the developing solution B is 0.1mL. The preparation method of the immunomagnetic nano probe is characterized by comprising the following steps:
1) All glassware to be used is cleaned by ultrapure water and dried, poured into aqua regia mixed solution, soaked in a fume hood for 30min, cleaned by ultrapure water for 3-5 times and dried for standby;
2) Weighing 1.08g of ferric trichloride hexahydrate which is ground to be in a uniform state in the beaker in the step 1), adding 20mL of ethylene glycol solution, uniformly mixing, and performing ultrasonic treatment to be in a uniform state;
3) Weighing 1.2g of sodium acetate, 0.2g of trisodium citrate and 0.2g of polyethylene glycol-6000 respectively, adding into the homogenized mixed solution in the step 2), and performing ultrasonic treatment until the homogenized mixed solution is in a homogenized state;
4) Transferring the solution in the step 3) to a high-temperature reaction kettle, reacting for 18 hours at 200 ℃, separating black solid particles in the solution by using a strong magnet, and alternately cleaning the solution by using ultrapure water and absolute ethyl alcohol to obtain carboxylated Fe 3 O 4 Magnetic nanoparticles;
5) 100mM manganese chloride 1mL,0.5% bovine serum albumin 5mL, and stirred at room temperature for 0.5h;
6) Adding 5mL of 70mM sodium hydroxide aqueous solution and 10mL of 100mM silver nitrate into the step 5), and stirring and reacting for 12h at room temperature;
7) Centrifuging the reaction solution obtained in the step 6) at 12000rpm at room temperature, washing with absolute ethanol, re-dispersing the obtained solid particles in pure water, and storing at 4deg.C to obtain Ag-MnO 2 A solution;
8) Weighing Fe in the step 4) 3 O 4 Dispersing magnetic nano particles in the small beaker in the step 1) with pure water, and adding Ag-MnO 2 2mL of solution, so that the final concentration reaches 5mg/mL;
9) Stirring the solution obtained in the step 8) at 80 ℃ for 0.5h, stirring at room temperature for 2h, dispersing the obtained solid particles in ultrapure water, and storing at 4 ℃ to obtain Fe 3 O 4 -Ag-MnO 2 Magnetic nanoparticles;
10 Sucking the magnetic nano particles in the step 9) to disperse in Phosphate Buffer (PBS) so that the proportion reaches 1:1, magnetically adsorbing to remove supernatant;
11 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to the step 10) for activating carboxyl groups on the surface of the magnetic sphere, and the mixture is uniformly mixed and activated for 0.5h;
12 (ii) Fe after activation in step 11) 3 O 4 -Ag-MnO 2 And (3) re-suspending the magnetic nano-particles to 1mL by using PBS buffer solution, adding staphylococcus aureus chicken yolk antibody to a final concentration of 1mg/mL, and reacting for 2 hours at room temperature to obtain the functionalized immunomagnetic nano-probe.
The invention relates to a preparation method of a color development liquid B gold nanorod, which is characterized by comprising the following steps:
1) All glassware to be used is cleaned by ultrapure water and dried, poured into aqua regia mixed solution, soaked in a fume hood for 30min, cleaned by ultrapure water for 3-5 times and dried for standby;
2) 0.2M hexadecyl trimethyl ammonium bromide 5mL,5mM tetrachloroauric acid hydrate 5mL,0.01M sodium borohydride 0.6mL in a small beaker of step 1), vigorously stirred for 2min;
3) Weighing 0.9g of cetyltrimethylammonium bromide and 0.11g of 5' -bromosalicylic acid in a small beaker in the step 1), adding pure water to the total volume of 25mL, adding 4mM of silver nitrate of 1.2mL,1mM of tetrachloroauric acid hydrate of 24mL, slowly stirring for 15min,0.064mM of ascorbic acid of 0.2mL, vigorously stirring for 30s, slowly stirring for 30s, standing at room temperature for 12h;
4) Centrifuging the solution obtained in the step 3) at 8500rpm at room temperature, and precipitating and re-suspending in 10ml of 0.06M hexadecyl trimethyl ammonium bromide and storing at room temperature to obtain the purple red nano rod solution.
The invention also provides a method for detecting staphylococcus aureus by using the detection kit, which is characterized by comprising the following steps:
the invention relates to a detection method of a staphylococcus aureus rapid detection kit, which is characterized by comprising the following steps:
1) Taking fresh staphylococcus aureus, inactivating, diluting to 10 with sterile PBS buffer solution 1 -10 6 CFU/mL staphylococcus aureus standard solution;
2) Adding 0.1mL of immunomagnetic nano probe into standard staphylococcus aureus liquid of each specification, suspending for 0.5h in four dimensions at room temperature, performing magnetic separation by using a strong magnet equipped in a kit, removing the supernatant, adding a chromogenic liquid A for reaction for 5min, adding 0.1mL of 1M hydrochloric acid, taking 0.1mL of supernatant, adding 0.1mL of chromogenic liquid B for etching, reacting for 10min, observing and recording the color of a reaction tube of the standard bacterial liquid, and taking the reaction tube as a standard colorimetric card for comparison of a sample to be detected;
3) And taking the reaction liquid, and measuring the ultraviolet absorption spectrum of the reaction liquid so as to calculate a linear standard curve of the standard bacterial liquid, thereby laying a foundation for the follow-up accurate quantification of the bacterial liquid concentration in the reaction tube.
The detection kit disclosed by the invention realizes rapid enrichment and separation of target bacteria mainly through staphylococcus aureus specificity immunomagnetic nano probes. The immunomagnetic nano particles have good superparamagnetism and ideal biocompatibility because of easy synthesis and easy surface modification, and the immunomagnetic separation technology is to make immunology and magnetismThe nanometer technology is combined, and the enrichment and separation functions are integrated. 3, '5,5' -tetramethyl benzidine (TMB) is catalyzed by utilizing nano mimic enzyme characteristics of immunomagnetic nano probes, and TMB is utilized 2+ For the etching of gold Nanorods (Au nanods, auNRs), combining with the characteristic of longitudinal plasmon resonance of the gold nanorod surface, different etching degrees can make the gold Nanorods exhibit different macroscopic colors and position changes of ultraviolet absorption peak spectra. Thereby realizing rapid and sensitive quantitative multicolor colorimetric semi-quantitative detection for staphylococcus aureus. The quantitative detection has small variation coefficient, wide linear range and detection limit as low as 3.1CFU/mL, and the colorimetric detection limit is 10CFU/mL. When the immune magnetic nano probe is used for capturing staphylococcus aureus, the nano mimic enzyme catalysis site on the surface of the immune magnetic nano probe can be covered, so that the catalysis effect is influenced, and the catalysis effect of TMB is in a proportional relation. The concentration of staphylococcus aureus further influences the etching degree of the gold nanorods, so that the corresponding color change is led to occur. Has high sensitivity and specificity.
The detection method of staphylococcus aureus using the detection kit of the invention has short time, high accuracy, good sensitivity, simple operation and low price. The detection kit mainly realizes rapid enrichment and separation of target bacteria through staphylococcus aureus specificity immunomagnetic nano probes. By utilizing the nano enzyme catalysis of the immunomagnetic nano probe and the longitudinal surface plasma resonance property of the gold nanorod, the rapid and sensitive quantitative multicolor colorimetric semi-quantitative detection of staphylococcus aureus is realized. The quantitative detection has small variation coefficient, wide linear range and detection limit as low as 3.1CFU/mL, and the colorimetric detection limit is 10CFU/mL. In addition, the detection kit has short time for the detection method of staphylococcus aureus, high accuracy, good sensitivity, simple operation and low price.
Drawings
FIG. 1 is a transmission electron microscope image, an element mapping image and an element analysis image of an immunomagnetic nano probe of the invention;
the figure indicates: A-Fe 3 O 4 Magnetic nanoparticle transmission electricityA mirror image; B-Fe 3 O 4 -Ag-MnO 2 A magnetic nano probe transmission electron microscope image; c-magnetic nanoprobe element map (TEM-EDS elemental mapping); an elemental analysis map of the D-magnetic nanoprobe (elemental analysis);
FIG. 2 is a graph showing the verification of the catalytic effect of the magnetic nanoparticles of the present invention;
the figure indicates: a-the magnetic nanoparticles of the invention verify visual effect patterns and ultraviolet absorption spectra for the catalytic effects of 3, '5,5' -Tetramethylbenzidine (TMB) and o-phenylenediamine (OPD); b-comparing the visual effect diagram and the ultraviolet absorption spectrum diagram of the nano enzyme activity verification of different materials (containing the magnetic nano particle material of the invention); c-comparing the catalytic oxidation excess H of different materials 2 O 2 Production of O 2 Picture (1 is H alone) 2 O 2 2 is Fe 3 O 4 3 is Fe 3 O 4 -Ag-MnO 2 4 is HRP);
FIG. 3 is a graph showing the optimized amount of immunomagnetic nanoprobe of the present invention (a: 0.05mg, b:0.1mg, c:0.2mg, d:0.3mg, e:0.4mg, f:0.5mg, g: negative control, h: positive control);
FIG. 4 is a graph showing the effect of detection of Staphylococcus aureus according to the present invention;
the figure indicates: a-visual effect diagram of staphylococcus aureus detection, B-ultraviolet absorption spectrum diagram of staphylococcus aureus detection and C-linear regression diagram of staphylococcus aureus detection;
FIG. 5 is a selective display of a Staphylococcus aureus detection kit of the present invention;
the figure indicates: a selective visual effect diagram of an A-staphylococcus aureus detection kit and a selective histogram of a B-staphylococcus aureus detection kit;
FIG. 6 is a graph of the results of a Staphylococcus aureus food-like assay;
the figure indicates: a-staphylococcus aureus pork food sample detection result visual effect diagram, B-staphylococcus aureus milk food sample detection result visual effect diagram, C-staphylococcus aureus pork food sample detection result linear regression diagram and D-staphylococcus aureus milk food sample detection result linear regression diagram;
FIG. 7 is a chart showing recovery in a Staphylococcus aureus food sample assay.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The preparation of the immunomagnetic nano probe of the embodiment comprises the following steps:
firstly, preparing carboxylated magnetic spheres, weighing 1.08g of ferric trichloride hexahydrate, adding the ferric trichloride into 20mL of ethylene glycol, and carrying out ultrasonic homogenization to obtain a brownish red solution; 1.2g of sodium acetate and 0.2g of trisodium citrate are weighed and added into the brownish red solution formed above, and the solution is homogenized into dark reddish brown transparent solution by ultrasound; finally, adding 0.2g of polyethylene glycol-6000, carrying out ultrasonic homogenization, transferring the obtained homogenized mixed solution into a high-temperature reaction kettle, and reacting for 18h in a 200 ℃ oven. Separating black solid particles by strong magnet, alternately cleaning with ultrapure water and absolute ethanol to obtain carboxylated Fe 3 O 4 Magnetic nanoparticles. 0.195g of manganese chloride was weighed and dissolved in 1mL of pure water, and 0.25g of bovine serum albumin was dissolved in 5mL of pure water, and after mixing, the mixture was vigorously stirred for 0.5h. 0.026g of sodium hydroxide is weighed and dissolved in 5mL of pure water, the mixture is added into the solution for reaction for 5min, 0.017g of silver nitrate is weighed and dissolved in 10mL of pure water, the solution is added dropwise, and the reaction is carried out for 12h. Centrifuging at 12000rpm for 15min, washing with ethanol, and dispersing in pure water to obtain Ag-MnO 2 And (3) nanoparticles. Weighing carboxylated Fe 3 O 4 10mg of magnetic nano particles are dissolved and dispersed by adding water, and Ag-MnO is removed 2 2mL of nanoparticle solution was placed in a beaker and water was added to a final volume of 20mL. Stirring at 80deg.C for 30min, stirring at room temperature for 2 hr, repeatedly washing with pure water, and redispersing in 4mL of pure water. Thus obtaining Fe 3 O 4 -Ag-MnO 2 Magnetic nanoparticles.
The experimental results are shown in fig. 1, and a transmission electron microscope image, element mapping and element analysis are respectively carried out on the magnetic nanoparticles. The magnetic nano particles are characterized by element mapping, element analysis and the like, have Fe, ag and Mn elements, and are characterized by transmission electron microscope TEM and high resolution thereof, so that the size distribution of the magnetic nano particles is uniform, and the magnetic nano particles have good dispersibility.
Experimental results As shown in figure 2, the magnetic nanoparticle has multiple and good nano-enzyme catalytic oxidation activity, and experiments prove that even if H does not exist 2 O 2 In the case of (2), fe 3 O 4 -Ag-MnO 2 The magnetic nanoparticles still have the ideal catalytic effect for TMB.
Preparation of functionalized immunomagnetic nano probe, sucking 0.5mL of Fe 3 O 4 -Ag-MnO 2 Magnetic nanoparticles, 0.5mL of PBS buffer, 1:1, magnetically adsorbing the supernatant, washing with PBS buffer, weighing 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 10mg of N-hydroxysuccinimide (NHS) and dissolving in 1mL of PBS, and re-suspending Fe 3 O 4 -Ag-MnO 2 And (3) suspending the magnetic nano particles for 0.5h to activate carboxyl. After activation, the supernatant was removed by strong magnet adsorption, washed with PBS buffer and finally resuspended in 0.9mL of PBS buffer. Adding staphylococcus aureus specific chicken egg yolk antibody of 0.1mL10mg/mL, and suspending and activating for 2 hours to obtain the functionalized immunomagnetic nano probe.
The PBS buffer solution preparation method comprises the following steps: 8g of sodium chloride, 0.2g of potassium chloride, 3.63g of disodium hydrogen phosphate dodecahydrate and 0.24g of potassium dihydrogen phosphate were dissolved in 1L of ultrapure water, and the pH was adjusted to 7.4.
Example 2
Dosage optimization of immunomagnetic nanoprobe of this embodiment
Sterilizing the obtained immunomagnetic nano probe by ultraviolet irradiation, and culturing staphylococcus aureus at 37 ℃ to logarithmic phase. Dividing the immunomagnetic nano probe into a, b, c, d, e and f, wherein the ratio of the immunomagnetic nano probe to the target bacterial liquid is 0.05mg, b, c, d, e and f is 0.05mg, 0.2mg, 0.3mg, 0.4mg, 0.5mg and 1mL of target bacterial liquid is added, 1mL of target bacterial liquid is complemented by sterile PBS buffer solution, and enrichment is carried out for 0.5h; magnetic separation, taking 50 mu L of supernatant, and counting bacteria in the supernatant by adopting a flat plate coating method. Where g is positive control and h is negative control.
As shown in figure 3, the content of the staphylococcus aureus in the supernatant is sequentially reduced along with the increase of the dosage of the immunomagnetic nano probe, and when the dosage of the immunomagnetic nano probe reaches 0.5mg/mL, the enrichment effect of all the added target staphylococcus aureus can be almost achieved.
Example 3
The preparation of the gold nanorod solution of the color development liquid B in the embodiment comprises the following steps:
all glassware to be used is cleaned by ultrapure water and dried, poured into aqua regia mixed solution, soaked in a fume hood for 30min, cleaned by ultrapure water for 3-5 times, and dried for standby. 0.36g of cetyltrimethylammonium bromide was weighed and dissolved in 5mL of warm water, 0.1mL of tetrachloroauric acid hydrate was mixed with 5mL of pure water, and slowly added dropwise to cetyltrimethylammonium bromide, followed by stirring at constant speed for 1min.0.0004g of sodium borohydride is dissolved in fresh ice water, 0.6mL of sodium borohydride is taken and rapidly added, and after violent stirring is carried out for 2min, the mixture is stood at room temperature for 30min, thus obtaining seed solution. 0.9g of cetyltrimethylammonium bromide and 0.11g of 5' -bromosalicylic acid are weighed into a beaker, pure water is added to the beaker until the total volume is 25mL, 0.002g of silver nitrate is dissolved in 1.2mL of water, 0.985mL of tetrachloroauric acid hydrate is added to 24mL of water for uniform mixing, the solution is added, the mixture is slowly stirred for 15min,0.0023g of ascorbic acid is dissolved in 0.2mL of water, the solution is rapidly added, the solution becomes colorless after intense stirring for 30s, 0.08mL of seed solution is injected, after slow stirring for 30s, the mixture is left stand for 12h at room temperature. The resulting solution was centrifuged at 8500rpm at room temperature for 15min,0.22g of cetyltrimethylammonium bromide was dissolved in 10mL of warm water, and the precipitate was resuspended and stored at room temperature. Thus obtaining the purple-red nanometer rod solution.
Example 4
The preparation of the staphylococcus aureus detection kit of the embodiment comprises the following steps:
taking 0.9mL of sample liquid to be detected, adding 0.1mL of immunomagnetic nano probe, performing four-dimensional suspension reaction at room temperature for 0.5h, magnetically separating supernatant, washing with PBS buffer solution for three times, adding 1mL of color development liquid A, reacting for 5min, adding 0.1mL of 1M hydrochloric acid, taking 0.1mL of color development liquid B, adding 0.1mL of color development liquid B, performing color development for 10min, and measuring an absorption spectrum by an ultraviolet spectrophotometer.
The configuration method of the 1M hydrochloric acid comprises the following steps: the concentrated hydrochloric acid of 87.72mL is measured and put into a beaker containing 900mL of pure water, and the 1M hydrochloric acid can be obtained after the mixture is evenly mixed and quantified by a 1000mL volumetric flask.
The experimental results are shown in FIG. 4, in which the color changes sequentially as the concentration of Staphylococcus aureus increases. When the concentration of the staphylococcus aureus reaches 10CFU/mL, obvious color difference exists between the detection tube and the blank tube. The limit of detection for visual detection of staphylococcus aureus was therefore 10CFU/mL. Scanning the spectrum of the detection solution under an ultraviolet spectrophotometer to obtain spectrograms at different concentrations, and obtaining 10-10 with peak displacement as ordinate and staphylococcus aureus concentration as abscissa 6 In the CFU/mL range, the staphylococcus aureus detection linear curve is Y= -11.952lgC+102.11, where R 2 In this range, data have good consistency and detection result is accurate as shown in=0.993. Capturing the color of the reaction tube with a camera, and printing to obtain 10-10 6 And a standard colorimetric card within the CFU/mL range so as to carry out semi-quantitative colorimetric detection on the sample to be detected with unknown concentration. If the color of the tube to be measured is similar to that of the standard colorimetric card, the concentration of the staphylococcus aureus of the standard tube is defined as the concentration between the two standard tubes.
Example 5
Selectivity of staphylococcus aureus detection kit
Selecting common 4 food-borne pathogenic bacteria: listeria monocytogenes (Listeria monocytogenes), E.coli O157: h7 (Escherichia coliO: H7), vibrio parahaemolyticus (Vibrio parahaemolyticus) and Salmonella (Salmonella), which are detected by the detection method of the present invention. Taking 0.9mL of sample liquid to be detected, adding 0.1mL of immunomagnetic nano probe, suspending at room temperature for reaction for 0.5h, magnetically separating supernatant, washing with PBS buffer solution for three times, adding 1mL of developing solution A, reacting for 5min, adsorbing supernatant with strong magnet, adding 0.1mL of 1M hydrochloric acid, adding 0.1mL of developing solution B for 0.1mL, developing for 10min, and measuring an absorption spectrum in an ultraviolet spectrophotometer.
As shown in FIG. 5A, the color of the detection solution is obviously changed only when the detection system contains the target bacteria staphylococcus aureus, and the color of the detection solution of other bacteria is consistent with that of the blank detection solution. FIG. 5B shows a bar graph obtained according to the ultraviolet spectrum according to the peak displacement of each detection liquid, and the detection liquid with the staphylococcus aureus exists and the detection liquid with the non-target bacteria has no difference, and has obvious color difference with other detection liquids, so that the detection kit has better selectivity on the staphylococcus aureus.
Example 6
The invention detects staphylococcus aureus in pork
1) Preparing pork extract: weighing 5g of pork, adding 10mL of sterilized PBS buffer solution, grinding uniformly, filtering with filter paper, and carrying out suction filtration on the filtrate with a 0.22 mu m filter membrane;
2) The invention is used for detecting staphylococcus aureus:
3) The staphylococcus aureus with different concentrations is prepared and inoculated in the leaching liquid, and the method of the kit is adopted to detect the staphylococcus aureus. Taking 0.9mL of sample liquid to be detected, adding 0.1mL of immunomagnetic nano probe, performing suspension reaction at room temperature for 0.5h, magnetically separating supernatant, washing with PBS buffer solution for three times, adding 1mL of developing solution A, performing reaction for 5min, adsorbing supernatant with strong magnet, adding 0.1mL of 1M hydrochloric acid, adding 0.1mL of developing solution B for 0.1mL, performing development for 5min, and measuring an absorption spectrum in an ultraviolet spectrophotometer.
FIG. 6A shows that the standard bacterial liquid is added into the pork sample at 10-10 6 Visual effect diagram in CFU/mL range, fig. 6C pork sample added with standard bacterial liquid 10-10 6 The experimental results are shown in FIG. 7 after the samples (50 CFU/mL,500CFU/mL,5000 CFU/mL) with different concentrations are added respectively to the linear regression standard curve in the CFU/mL range. And determining the number of staphylococcus aureus in the sample according to a linear regression standard curve. The average addition recovery was calculated to be 106%. This shows that the detection method has good applicability in complex solid food matrix detection.
Example 7
The invention detects staphylococcus aureus in milk:
1) Taking skimmed cow milk 1:100 was diluted to 10mL and the sample solution was suction filtered through a 0.22 μm filter.
2) The invention is used for detecting staphylococcus aureus:
3) The staphylococcus aureus with different concentrations is prepared and inoculated in the leaching liquid, and the method of the kit is adopted to detect the staphylococcus aureus. Taking 0.9mL of sample liquid to be detected, adding 0.1mL of immunomagnetic nano probe, suspending at room temperature for reaction for 0.5h, magnetically separating supernatant, washing with PBS buffer solution for three times, adding 1mL of developing solution A, reacting for 5min, adsorbing supernatant with strong magnet, adding 0.1mL of 1M hydrochloric acid, adding 0.1mL of developing solution B for 0.1mL, developing for 5min, and measuring an absorption spectrum in an ultraviolet spectrophotometer.
FIG. 6B shows that the standard bacterial liquid is added into the milk sample at 10-10 6 Visual effect diagram in CFU/mL range, FIG. 6D shows that the standard bacterial liquid is added in 10-10 for milk sample 6 The experimental results are shown in Table 7 after adding samples of different concentrations (50 CFU/mL,500CFU/mL,5000 CFU/mL) to the linear regression standard curve within the CFU/mL range. And determining the number of staphylococcus aureus in the sample according to a linear regression standard curve. The average addition recovery was calculated to be 106%. This shows that the detection method has good applicability in complex liquid food matrix detection.
Therefore, the rapid detection kit for staphylococcus aureus based on immunomagnetic separation and nanomaterial etching provided by the invention mainly realizes rapid enrichment and separation of target bacteria through the specific immunomagnetic nano probe for staphylococcus aureus. By utilizing the nano enzyme catalysis of the immunomagnetic nano probe and the surface plasma resonance property of the gold nanorod, the rapid and sensitive quantitative multicolor colorimetric semi-quantitative detection of staphylococcus aureus can be realized. The quantitative detection has small variation coefficient, wide linear range and detection limit as low as 3.1CFU/mL, and the colorimetric detection limit is 10CFU/mL. In addition, the detection method for staphylococcus aureus by adopting the detection kit provided by the invention has the advantages of short time, high accuracy, good sensitivity, simplicity in operation and low cost.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A staphylococcus aureus rapid detection kit is characterized by comprising an immunomagnetic nano probe, a color development liquid A, a color development liquid B and 10 1 -10 6 CFU/mL staphylococcus aureus colorimetric card, sterile PBS buffer solution, 1M hydrochloric acid and strong magnet;
the color development liquid A is an aqueous solution of 3, '5,5' -tetramethyl benzidine;
the color development liquid B is gold nanorod solution uniformly dispersed in cetyl trimethyl ammonium bromide;
the immunomagnetic nano probe is carboxylated Fe marked by staphylococcus aureus specific chicken egg yolk antibody 3 O 4 -Ag-MnO 2 Magnetic nanoparticles;
the concentration of the immunomagnetic nano probe is 0.5mg/mL, the concentration of the developing solution A is 1mL, and the concentration of the developing solution B is 0.1mL.
2. The rapid detection kit for staphylococcus aureus according to claim 1, wherein the preparation process of the immunomagnetic nano probe comprises the following steps:
1) All glassware to be used is cleaned by ultrapure water and dried, poured into aqua regia mixed solution, soaked in a fume hood for 30min, cleaned by ultrapure water for 3-5 times and dried for standby;
2) Weighing 1.08g of ferric trichloride hexahydrate which is ground to be in a uniform state in the beaker in the step 1), adding 20mL of ethylene glycol solution, uniformly mixing, and performing ultrasonic treatment to be in a uniform state;
3) Weighing 1.2g of sodium acetate, 0.2g of trisodium citrate and 0.2g of polyethylene glycol-6000 respectively, adding into the homogenized mixed solution in the step 2), and performing ultrasonic treatment until the homogenized mixed solution is in a homogenized state;
4) Transferring the solution in the step 3) to a high-temperature reaction kettle, reacting for 18 hours at 200 ℃, separating black solid particles in the solution by using a strong magnet, and alternately cleaning the solution by using ultrapure water and absolute ethyl alcohol to obtain carboxylated Fe 3 O 4 Magnetic nanoparticles;
5) 100mM manganese chloride 1mL,0.5% bovine serum albumin 5mL, and stirred at room temperature for 0.5h;
6) Adding 5mL of 70mM sodium hydroxide aqueous solution and 10mL of 100mM silver nitrate into the step 5), and stirring and reacting for 12h at room temperature;
7) Centrifuging the reaction solution obtained in the step 6) at 12000rpm at room temperature, washing with absolute ethanol, re-dispersing the obtained solid particles in pure water, and storing at 4deg.C to obtain Ag-MnO 2 A solution;
8) Weighing Fe in the step 4) 3 O 4 Dispersing magnetic nano particles in the small beaker in the step 1) with pure water, and adding Ag-MnO 2 2mL of solution, so that the final concentration reaches 5mg/mL;
9) Stirring the solution obtained in the step 8) at 80 ℃ for 0.5h, stirring at room temperature for 2h, dispersing the obtained solid particles in ultrapure water, and storing at 4 ℃ to obtain Fe 3 O 4 -Ag-MnO 2 Magnetic nanoparticles;
10 Sucking the magnetic nano particles in the step 9) to disperse in phosphate buffer solution so that the proportion reaches 1:1, magnetically adsorbing to remove supernatant;
11 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to the step 10) for activating carboxyl groups on the surface of the magnetic sphere, and the mixture is uniformly mixed and activated for 0.5h;
12 (ii) Fe after activation in step 11) 3 O 4 -Ag-MnO 2 And (3) re-suspending the magnetic nano-particles to 1mL by using PBS buffer solution, adding staphylococcus aureus chicken yolk antibody to a final concentration of 1mg/mL, and reacting for 2 hours at room temperature to obtain the functionalized immunomagnetic nano-probe.
3. The rapid detection kit for staphylococcus aureus according to claim 1, wherein the preparation process of the color development liquid B comprises the following steps:
1) All glassware to be used is cleaned by ultrapure water and dried, poured into aqua regia mixed solution, soaked in a fume hood for 30min, cleaned by ultrapure water for 3-5 times and dried for standby;
2) 0.2M hexadecyl trimethyl ammonium bromide 5mL,5mM tetrachloroauric acid hydrate 5mL,0.01M sodium borohydride 0.6mL in a small beaker of step 1), vigorously stirred for 2min;
3) Weighing 0.9g of cetyltrimethylammonium bromide and 0.11g of 5' -bromosalicylic acid in a small beaker in the step 1), adding pure water to the total volume of 25mL, adding 4mM of silver nitrate of 1.2mL,1mM of tetrachloroauric acid hydrate of 24mL, slowly stirring for 15min,0.064mM of ascorbic acid of 0.2mL, vigorously stirring for 30s, slowly stirring for 30s, standing at room temperature for 12h;
4) Centrifuging the solution obtained in the step 3) at 8500rpm at room temperature, and precipitating and re-suspending in 10ml of 0.06M hexadecyl trimethyl ammonium bromide and storing at room temperature to obtain purple red nano rod solution, which is the color development liquid B of the rapid detection kit.
4. The method for detecting a rapid test kit for staphylococcus aureus according to claim 1, which comprises the following steps:
1) Taking fresh staphylococcus aureus, inactivating, diluting to 10 with sterile PBS buffer solution 1 -10 6 CFU/mL staphylococcus aureus standard solution;
2) Adding 0.1mL of immunomagnetic nano probe into standard staphylococcus aureus liquid of each specification, suspending for 0.5h in four dimensions at room temperature, performing magnetic separation by using a strong magnet equipped in a kit, removing the supernatant, adding a chromogenic liquid A for reaction for 5min, adding 0.1mL of 1M hydrochloric acid, taking 0.1mL of supernatant, adding 0.1mL of chromogenic liquid B for etching, reacting for 10min, observing and recording the color of a reaction tube of the standard bacterial liquid, and taking the reaction tube as a standard colorimetric card for comparison of a sample to be detected;
3) And taking the reaction liquid, and measuring the ultraviolet absorption spectrum of the reaction liquid so as to calculate a linear standard curve of the standard bacterial liquid, thereby laying a foundation for the follow-up accurate quantification of the bacterial liquid concentration in the reaction tube.
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| CN117110607B (en) * | 2023-09-16 | 2024-01-26 | 鲁东大学 | Composition for detecting vibrio parahaemolyticus and application thereof |
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