CN115856297A - Preparation method of kit for detecting salmonella typhimurium and kit - Google Patents

Abstract

The invention provides a preparation method of a kit for detecting salmonella typhimurium and the kit, and belongs to the technical field of kits. The method comprises the steps of firstly preparing magnetic beads with superparamagnetism, taking the magnetic beads as magnetic cores, coating organic covalent frameworks (COFs) to form core-shell structures, namely MCOF nano magnetic particles, then loading gold nanoparticles on the MCOF nano magnetic particles by using an in-situ reduction method, and finally coupling with aptamers of salmonella typhimurium to synthesize the nanoprobe apt-MCOF-AuNPs with high-emission fluorescence. Formed by incubating salmonella typhimurium and a nano probeAdding quenching agent Fe into the probe-thallus complex ³⁺ Quenching probe fluorescence due to the protection of thallus on the nanoprobe and Fe ³⁺ Competitive binding of (2), the fluorescence of the probe is not affected by Fe ³⁺ Quenching, and under 365nm ultraviolet irradiation, the solution shows bright green fluorescence. Thereby realizing the rapid and specific detection of the salmonella typhimurium.

Description

Preparation method of kit for detecting salmonella typhimurium and kit

Technical Field

The invention relates to the technical field of kits, in particular to a preparation method of a kit for detecting salmonella typhimurium and a kit prepared by the preparation method.

Background

Salmonella typhimurium (s. Typhimurium) is a common food-borne pathogenic bacterium and can cause outbreaks of food-borne diseases. Salmonella typhimurium often contaminates food products, such as milk, eggs, fruits, vegetables, and meat, etc., through animal feces. Salmonella typhimurium infection can cause symptoms typical of food poisoning, such as abdominal pain, diarrhea, nausea, vomiting, fever, and other gastrointestinal symptoms. It has been reported that the food poisoning event by salmonella typhimurium still shows a rising trend. At present, the bacterium is a necessary pathogenic bacterium for agricultural and sideline products such as eggs, meat, milk and the like.

Traditional salmonella typhimurium detection methods include plating, enzyme-linked immunosorbent assay (ELISA) and Polymerase Chain Reaction (PCR). Although these techniques have become the gold standard or recommended test method, there is a need to construct a test method that is more convenient, easy to operate, and results readable in order to meet the needs of field testing for Salmonella typhimurium. Over the past few years, a variety of new biosensors have been widely developed, such as colorimetric sensors, fluorescence sensors, surface Enhanced Raman Scattering (SERS), and electrochemical biosensors. They have the advantages of rapid reaction, convenient use, low cost, good sensitivity, good selectivity and the like. Among them, the fluorescence sensor has the advantages of sensitive detection and insusceptible fluorescence signal, and detects an analyte by a fluorescence color change using a portable optical measuring device. In addition, qualitative and semi-quantitative analysis services can be provided in combination with the image acquisition and image analysis system of the smartphone.

Disclosure of Invention

In view of this, in order to meet the requirement of on-site detection of salmonella typhimurium, on one hand, the invention provides a preparation method of a kit for detecting salmonella typhimurium, which comprises the steps of preparing a magnetic bead with superparamagnetism, using the magnetic bead as a magnetic core, and coating an organic covalent framework (COF) to form a core-shell structureThe structure is MCOF nano magnetic particles, gold nanoparticles are loaded on the MCOF nano magnetic particles by using an in-situ reduction method, and finally the gold nanoparticles are coupled with an aptamer of salmonella typhimurium to synthesize the nanoprobe apt-MCOF-AuNPs with high-emission fluorescence. Incubating salmonella typhimurium and nano probe to form probe-thallus compound, adding quenching agent Fe ³⁺ Quenching probe fluorescence due to the protection of thallus on the nanoprobe and Fe ³⁺ Competitive binding of (3), the fluorescence of the probe is not affected by Fe ³⁺ Quenching, and under 365nm ultraviolet irradiation, the solution shows bright green fluorescence. Thereby realizing the rapid and specific detection of the salmonella typhimurium.

In order to achieve the purpose, the invention provides the following technical scheme:

a preparation method of a kit for detecting salmonella typhimurium comprises the following steps:

the method comprises the following steps: preparation of Fe having superparamagnetism ₃ O ₄ Nanoparticles;

step two: preparation of magnetic organic framework MCOF

Fe prepared in the first step ₃ O ₄ The nano particles are used as magnetic cores, and are coated with organic covalent frameworks (COFs) to form a core-shell structure, so that MCOF magnetic nano particles are obtained;

step three: preparation of MCOF-AuNPs nano composite material

Loading gold nanoparticles on the MCOF magnetic nanoparticles prepared in the second step to obtain an MCOF-AuNPs nano composite material;

step four: preparation of apt-MCOF-AuNPs nano probe

Coupling the MCOF-AuNPs nano composite material obtained in the step three with an aptamer of salmonella typhimurium to synthesize a nano probe apt-MCOF-AuNPs with high-emission fluorescence;

step five: preparation of Fe ³⁺ The quenching agent is sealed and kept away from light for standby.

Preferably, the specific steps are as follows:

the method comprises the following steps: fe ₃ O ₄ Preparation of nanoparticles

FeCl ₃ ·6H ₂ O、CH ₃ COONa·3H ₂ Magnetically stirring O in ethylene glycol at room temperature until the O is completely dissolved, transferring the obtained homogeneous solution into a reaction kettle, placing the reaction kettle in an oven for reaction, separating black brown solid matters from the reaction solution by using a permanent magnet, and cleaning to obtain Fe with superparamagnetism ₃ O ₄ Nanoparticles;

step two: preparation of magnetic organic framework MCOF

Dissolving 1,3, 5-tri (4-aminophenyl) benzene and 2, 5-dimethoxybenzene-1, 4-dimethyl aldehyde in 1, 4-dioxane and n-butanol, adding Fe prepared in the step one ₃ O ₄ Dispersing the nano particles, adding acetic acid to react, separating brown solid substances from a reaction solution by using a permanent magnet, and cleaning to obtain MCOF magnetic nano particles;

step three: preparation of MCOF-AuNPs nano composite material

Adding a chloroauric acid trihydrate solution into the MCOF magnetic nanoparticles obtained in the second step, completely mixing the obtained MCOF magnetic nanoparticles uniformly, adding a trisodium citrate solution to react, separating brown solid substances from the reaction solution by using a permanent magnet, and cleaning the mixture to obtain the MCOF-AuNPs nano composite material;

step four: preparation of apt-MCOF-AuNPs nano probe

Adding the tris (2-carbonylethyl) phosphate solution into the salmonella aptamer aqueous solution, performing mixed spinning at room temperature to obtain an activated aptamer solution, adding the MCOF-AuNPs nanocomposite obtained in the step three into the activated aptamer solution, and performing mixed spinning at room temperature overnight to obtain an apt-MCOF-AuNPs nanoprobe;

step five: preparation of quenchers

Taking FeCl ₃ ·6H ₂ Adding O into double distilled water, and uniformly mixing to obtain Fe ³⁺ And (5) sealing the quenching agent and keeping away from light for later use.

Preferably, in step one, feCl ₃ ·6H ₂ O and CH ₃ COONa·3H ₂ The mass ratio of O is 1.2-3.

Preferably, in step two, 1,3, 5-tris (4-aminophenyl) benzene, 2, 5-bisMethoxybenzene-1, 4-dicarbaldehyde, fe ₃ O ₄ The mass ratio of the nano particles is 102-106:86-88:115-120.

Preferably, in the second step, the volume ratio of the 1, 4-dioxane to the n-butanol is 0.8-1.

Preferably, in the third step, the concentration of the chloroauric acid hydrate solution is 1-1.2mM, and the concentration of the trisodium citrate solution is 100-120mM.

Preferably, in the third step, the volume-to-mass ratio of the chloroauric acid trihydrate solution, the trisodium citrate solution and the MCOF nano particles is 35-40mL:1.5-1.6mL:30-32mg.

Preferably, in step four, the volume of the salmonella aptamer aqueous solution μ L: the mass mg of the MCOF-AuNPs nano composite material is 9-10:0.18-0.2.

Preferably, in step five, the volume of double distilled water mL: feCl ₃ ·6H ₂ The mass mg of O is 95-100:25-27.

On the other hand, the invention also provides a kit prepared by the preparation method.

Compared with the prior art, the invention has the following beneficial effects:

the preparation method of the kit for detecting salmonella typhimurium, provided by the invention, comprises the steps of firstly preparing magnetic beads with superparamagnetism, taking the magnetic beads as magnetic cores, coating organic covalent frameworks (COFs) to form MCOF nano magnetic particles with a core-shell structure, loading the gold nanoparticles on the MCOF nano magnetic particles by using an in-situ reduction method, and finally coupling with aptamers of the salmonella typhimurium to synthesize the nanoprobe apt-MCOF-AuNPs with high-emission fluorescence. Incubating salmonella typhimurium and nano probe to form probe-thallus compound, adding quenching agent Fe ³⁺ Quenching probe fluorescence due to the protection of thallus on the nanoprobe and Fe ³⁺ Competitive binding of (3), the fluorescence of the probe is not affected by Fe ³⁺ Quenching, and under 365nm ultraviolet irradiation, the solution shows bright green fluorescence. Thereby realizing the rapid and specific detection of the salmonella typhimurium.

The invention utilizes the nano composite material apt-MCOF-AuNPs to detect the salmonella typhimurium, utilizes the quenching inhibition effect to realize fluorescence detection, shortens the detection time, has small time variation coefficient of quantitative detection, has the minimum detection concentration of 4CFU/mL, has the simulation sample recovery rate of 97.38-104.56 percent, high sensitivity, good stability and certain applicability. And (3) combining with a smartphone color analysis APP (e.g. ColorColl) to realize image analysis and obtain a semi-quantitative detection result.

Drawings

FIG. 1 is a graph of a Salmonella detection standard;

FIG. 2 is a flow chart of apt-MCOF-AuNPs detection of Salmonella typhimurium provided by the present invention.

Detailed Description

The invention provides a preparation method of a kit for detecting salmonella typhimurium, which comprises the following steps:

the method comprises the following steps: preparation of Fe having superparamagnetism ₃ O ₄ The nano particles are preferably prepared by the following preparation method:

preferably, the mass ratio of 1.35: feCl of 3.6 ₃ ·6H ₂ O and CH ₃ COONa·3H ₂ Magnetically stirring O in ethylene glycol at room temperature until the O is completely dissolved, transferring the obtained homogeneous solution to a reaction kettle, and preferably reacting in an oven at 200 ℃ for 16h; separating dark brown solid substance from the reaction solution with permanent magnet, alternately cleaning with ultrapure water and anhydrous ethanol for 3-5 times to obtain superparamagnetic Fe ₃ O ₄ Nanoparticles;

step two: preparation of magnetic organic framework MCOF

Fe prepared in the first step ₃ O ₄ The nano particle is used as a magnetic core, and is coated with an organic covalent backbone (COF) to form a core-shell structure, so that the MCOF magnetic nano particle is obtained, and the preferable preparation method is as follows:

dissolving 1,3, 5-tri (4-aminophenyl) benzene and 2, 5-dimethoxybenzene-1, 4-diformaldehyde in 1, 4-dioxane and n-butanol, adding Fe prepared in the first step ₃ O ₄ Dispersing the nanoparticles therein, adding acetic acid, stirring preferably at 70 deg.C for 48 hr, and separating green solid substance from the reaction solution with permanent magnetWashing MCOF magnetic nanoparticles with acetone and tetrahydrofuran alternately for multiple times to remove unreacted substances, and finally drying to obtain Fe ₃ O ₄ @ Ag magnetic nanoparticles, storing at 4 ℃ in dark for later use;

wherein, 1,3, 5-tri (4-aminophenyl) benzene, 2, 5-dimethoxybenzene-1, 4-dicarboxaldehyde, fe ₃ O ₄ The mass ratio of nanoparticles is preferably 105.4:87.4:120 of a solvent;

wherein, the volume ratio of the 1, 4-dioxane to the n-butanol is preferably 1;

step three: preparation of MCOF-AuNPs nano composite material

And (3) loading the gold nanoparticles on the MCOF magnetic nanoparticles prepared in the second step to obtain the MCOF-AuNPs nano composite material, wherein the preferable preparation method comprises the following steps:

and (3) adding the chloroauric acid trihydrate solution into the MCOF obtained in the second step, completely mixing, adding the trisodium citrate solution, and preferably stirring and reacting at 20 ℃ for 40min. Separating brown solid matters from the reaction solution by using a permanent magnet, washing the MCOF-AuNPs nano composite material for multiple times by using double distilled water to remove unreacted matters, finally drying to obtain the MCOF-AuNPs nano composite material, and storing the MCOF-AuNPs nano composite material at 4 ℃ in a dark place for later use;

the concentration of the chloroauric acid trihydrate solution is preferably 1mM, and the concentration of the trisodium citrate solution is preferably 100mM; the volume-mass ratio of the chloroauric acid trihydrate solution, the trisodium citrate solution and the MCOF nanoparticles is preferably 40mL:1.6mL:32mg;

step four: preparation of apt-MCOF-AuNPs nano probe

And (3) coupling the MCOF-AuNPs nano composite material obtained in the step (three) with an aptamer of salmonella typhimurium to synthesize a nano probe apt-MCOF-AuNPs with high-emission fluorescence, wherein the preferable preparation method is as follows:

adding a tris (2-carbonyl ethyl) phosphate solution into a salmonella aptamer aqueous solution, carrying out mixed rotation at room temperature, wherein the mixed rotation time is preferably 1h, activating a modified sulfydryl on an aptamer to obtain an activated aptamer solution, taking an MCOF-AuNPs nano composite material, preferably washing the MCOF-AuNPs nano composite material with distilled water for 2 times, carrying out heavy suspension with distilled water, adding the activated aptamer, carrying out mixed rotation overnight at room temperature, carrying out magnetic separation, removing a supernatant, and washing with distilled water for three times to obtain apt-MCOF-AuNPs, and storing the apt-MCOF-AuNPs in a dark place at 4 ℃ for later use;

wherein the concentration of the tris (2-carbonylethyl) phosphate solution is preferably 1mM, the concentration of the salmonella aptamer aqueous solution is preferably 10 μ M, and the volume ratio of the tris (2-carbonylethyl) phosphate solution to the salmonella aptamer aqueous solution is preferably 1:10, volume μ L of salmonella aptamer aqueous solution: the MCOF-AuNPs nanoparticles preferably have a mass mg of 10:0.2;

wherein the size of apt-MCOF-AuNPs nano probe is preferably 300nm;

the sequence of the salmonella-resistant aptamer provided by the invention is as follows: 5' -SH- (CH) ₂ ) ₆ -TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC CTT GACATTTATGACA G-3' synthesized by Shanghai Biotech company;

step five: preparation of Fe ³⁺ The quenching agent is stored in a sealed and dark state for standby, and the preferred preparation method is as follows:

taking FeCl ₃ ·6H ₂ Adding O into double distilled water, and uniformly mixing to obtain Fe ³⁺ And (5) sealing the quenching agent and keeping away from light for later use.

Wherein the volume of double distilled water is mL: feCl ₃ ·6H ₂ O mass mg is 100:27.

the technical solution of the present invention will be clearly explained in detail with reference to the specific embodiments below.

Example 1

The method comprises the following steps: preparation of Fe having superparamagnetism ₃ O ₄ Nanoparticles

Firstly, a hydrothermal method is adopted to prepare Fe ₃ O ₄ Taking 1.35g FeCl ₃ ·6H ₂ O and 3.6g CH ₃ COONa·3H ₂ O was magnetically stirred in 40mL of ethylene glycol at room temperature until completely dissolved. Then transferring the obtained homogeneous solution into a reaction kettle, and reacting in an oven at 200 ℃ for 16h; separating dark brown solid substance from the reaction solution with permanent magnet, alternately cleaning with ultrapure water and anhydrous ethanol for 3 times to obtain superparamagnetism Fe ₃ O ₄ Nanoparticles;

step two: preparation of magnetic organic framework MCOF

Preparation of MCOF Process: 105.4mg of 1,3, 5-tris (4-aminophenyl) benzene and 87.4mg of 2, 5-dimethoxybenzene-1, 4-dicarbaldehyde were dissolved in a mixed solution containing 20mL of 1, 4-dioxahexaalkane and 20mL of n-butanol, and after sufficient dissolution, 120mg of the prepared Fe was added ₃ O ₄ Dispersing the nano particles, adding 0.5mL 12M acetic acid, preferably standing at 70 ℃ for 48h, separating green solid substances from the reaction solution by using a permanent magnet, washing the MCOF magnetic nano particles by using acetone and tetrahydrofuran alternately for multiple times to remove unreacted substances, and finally drying;

step three: preparation of MCOF-AuNPs nano composite material

MCOF-AuNPs are prepared by in situ reduction, 32mg of MCOF is added to 40mL of 1mM HAuCl ₄ The solution was completely dissolved, and 1.6mL of 100mM trisodium citrate solution was added thereto, and the reaction was stirred at 20 ℃ for 40min. Separating brown solid matters from the reaction solution by using a permanent magnet, washing the MCOF-AuNPs nano composite material for multiple times by using double distilled water to remove unreacted matters, finally drying to obtain the MCOF-AuNPs nano composite material, and storing the MCOF-AuNPs nano composite material at 4 ℃ in a dark place for later use;

step four: preparation of apt-MCOF-AuNPs nano probe

Adding 1 mu L of 1mM tris (2-carbonylethyl) phosphate solution into 10 mu L of 10 mu M salmonella aptamer aqueous solution, carrying out mixed spinning for 1h at room temperature to activate the modified sulfydryl on the aptamer, taking 0.2mg MCOF-AuNPs nano composite material, washing the material for 2 times by distilled water, carrying out heavy suspension by 1mL of distilled water, adding the activated aptamer, and carrying out mixed spinning overnight at room temperature. Magnetic separation, supernatant removal and distilled water washing for three times to obtain apt-MCOF-AuNPs nano probe, and keeping the probe away from light at 4 ℃ for later use, wherein the sequence of the used salmonella aptamer is as follows: 5' -SH- (CH) ₂ ) ₆ -TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC CTT GACATTTATGACA G-3' synthesized by Shanghai Biotech.

Step five: quencher (Fe) ³⁺ Standard solution) preparation

27mg FeCl was weighed ₃ ·6H ₂ O, dissolved in 100mL double distilled water to obtain Fe ³⁺ And (5) sealing the quenching agent and keeping away from light for later use.

Examples 2-4 were prepared as in example 1, with the parameter configurations shown in Table 1 below

Table 1 parameter configuration for examples 2-4

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The kits prepared in examples 1 to 4 above were tested by the following methods.

Preparation of bacterial liquid standard substance

Taking a strain stored at minus 80 ℃, activating the strain, streaking and purifying the activated strain on a 3% sodium chloride trypsin vein agar plate, culturing at 37 ℃ for 18-24h, selecting a single colony, inoculating a 3% sodium chloride tryptone liquid culture medium, and performing shake culture at 37 ℃ for 12-18h. And taking 1mL of bacterial liquid by a 10-time dilution plate pouring method for viable bacteria counting. Inactivating the rest bacteria solution with 1% formaldehyde at room temperature for 10min, centrifuging the inactivated bacteria solution at 3000rpm for 3min, collecting thallus, mixing with PBS solution to obtain bacteria suspension, and regulating the bacteria suspension concentration to 10 with PBS solution ⁹ CFU·mL ^-1 And storing in a refrigerator at 4 ℃ for later use.

Method for detecting salmonella typhimurium based on apt-MCOF-AuNPs and quenching agent

The detection process is shown in figure 2, and 800. Mu.L of sample to be detected and 200. Mu.L of apt-MCOF-AuNPs of examples 1-4 with the concentration of 2mg/mL are respectively taken for testing, and are mixed and rotated for 30min at room temperature in a dark place. 200 μ L of 1mM Fe was added ³⁺ React for 3min. Irradiating by 365nm ultraviolet light in a darkroom, taking a picture by a smart phone, performing HSV analysis by using a color analysis APP, and outputting three channel values of Hue (Hue, H), saturation (S) and Value (lightness, V), wherein the Hue Value (Hue Value) is used as an output quantitative signal of the concentration of the salmonella typhimurium. With reference to the standard curve drawn in figure 1,the amount of Salmonella typhimurium in the sample is determined. The concentration range of the bacteria is quantitatively detected to be 10-10 ⁷ CFU/mL。

FIG. 1 is a graph of the above-mentioned Salmonella detection standard, in which the abscissa is the logarithmic value of the concentration of Salmonella typhimurium and the ordinate is the hue value. Detection concentration range: 10-10 ⁷ CFU/mL。

The method has the advantages of stable detection, short detection time, simple operation and good detection effect, and the detection limit can be as low as 4 CFU/ml. The method is used for detecting salmonella typhimurium, listeria monocytogenes, staphylococcus aureus, vibrio parahaemolyticus, escherichia coli O157: H7 and the like, only the detection result of the salmonella in the examples 1-4 is positive, the rest is negative, the method is strong in specificity, false positive and false negative results are not seen, and the results are shown in tables 2-5.

TABLE 2 example 1 specific detection results for Salmonella

TABLE 3 example 2 detection results of the specificity of Salmonella

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Table 4 example 3 results of specific detection of salmonella

TABLE 5 example 4 detection results of Salmonella specificity

Note: the expression < + > represents positive Salmonella typhimurium, and the expression < - > represents negative Salmonella typhimurium.

Detection of a simulated sample

0.5g of chicken was ground and mixed with 50mL of sterile PBS to form a homogenate. 1ml of milk and egg solution was diluted 100-fold with sterile PBS and the filtrate was suction filtered through a 0.22 μm filter. Then, salmonella typhimurium was inoculated into each of the actual samples for culture, diluted, and then plate-counted and detected and compared by the method of the present invention.

800 μ L of food sample to be tested and 200 μ L of apt-MCOF-AuNPs in examples 1-4 were taken and tested, respectively, and the mixture was vortexed at room temperature in a dark place for 30min. Adding Fe ³⁺ React for 3min. Irradiating by 365nm ultraviolet light in a darkroom, taking a picture by a smart phone, carrying out HSV analysis by using APP developed by the owner, and taking a Hue value (Hue value) as an output quantitative signal of the concentration of the salmonella typhimurium. The results of plate counting and the results of the method of the invention are compared with the results of reference table 6, the method of the invention has stable detection, and the recovery rate of standard addition reaches 97.38-104.56%.

TABLE 6 example 1 results of detecting Salmonella typhimurium in food samples

TABLE 7 example 2 results of detecting Salmonella typhimurium in food samples

TABLE 8 example 3 results of detecting Salmonella typhimurium in food samples

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TABLE 9 example 4 results of detecting Salmonella typhimurium in food samples

^* And (3) Log C: salmonella typhimurium concentration (CFU/mL) log.

Comparative example 1

TABLE 10 comparison of this kit with commercially available ELISA kits

	Time of detection	Detection device	Sensitivity of the probe	Coefficient of variation
					Example 1	About 33 minutes	Smart phone	4CFU/mL	Less than 6 percent
Example 2	About 33 minutes	Smart phone	9CFU/mL	Less than 6 percent
					Example 3	About 33 minutes	Smart phone	12CFU/mL	Less than 9 percent
Example 4	About 33 minutes	Smart phone	6CFU/mL	Less than 10 percent
					Commercially available ELISA kit	Greater than 2.5 hours	Precision enzyme mark instrument	10-10 4 CFU/mL	Less than 10-15%

The above description is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto. Any person skilled in the art can substitute or change the technical solution of the present invention and its modified concept equally within the technical scope of the present disclosure; are intended to be covered by the scope of the present invention.

Claims (10)

1. The preparation method of the kit for detecting the salmonella typhimurium is characterized by comprising the following steps of:

the method comprises the following steps: preparation of superparamagnetic Fe ₃ O ₄ Nanoparticles;

step two: preparation of magnetic organic framework MCOF

Fe prepared in the first step ₃ O ₄ The nano particles are used as magnetic cores and coated with organic covalent frameworks (COFs) to form core-shell structures, so that MCOF magnetic nano particles are obtained;

step three: preparation of MCOF-AuNPs nano composite material

Loading gold nanoparticles on the MCOF magnetic nanoparticles prepared in the second step to obtain an MCOF-AuNPs nano composite material;

step four: preparation of apt-MCOF-AuNPs nano probe

Coupling the MCOF-AuNPs nano composite material obtained in the step three with an aptamer of salmonella typhimurium to synthesize a nano probe apt-MCOF-AuNPs with high-emission fluorescence;

step five: preparation of Fe ³⁺ The quenching agent is sealed and kept away from light for standby.

2. The preparation method of the kit for detecting salmonella typhimurium according to claim 1 is characterized by comprising the following steps:

the method comprises the following steps: fe ₃ O ₄ Preparation of nanoparticles

FeCl is added ₃ ·6H ₂ O、CH ₃ COONa·3H ₂ Magnetically stirring O in ethylene glycol at room temperature until the O is completely dissolved, transferring the obtained homogeneous solution into a reaction kettle, placing the reaction kettle in an oven for reaction, separating black brown solid matters from the reaction solution by using a permanent magnet, and cleaning to obtain Fe with superparamagnetism ₃ O ₄ Nanoparticles;

step two: preparation of magnetic organic framework MCOF

Dissolving 1,3, 5-tri (4-aminophenyl) benzene and 2, 5-dimethoxybenzene-1, 4-dimethyl aldehyde in 1, 4-dioxane and n-butanol, adding into the first preparationPrepared Fe ₃ O ₄ Dispersing the nano particles, adding acetic acid to react, separating brown solid substances from a reaction solution by using a permanent magnet, and cleaning to obtain MCOF magnetic nano particles;

step three: preparation of MCOF-AuNPs nano composite material

Adding a chloroauric acid trihydrate solution into the MCOF magnetic nanoparticles obtained in the second step, completely mixing the obtained MCOF magnetic nanoparticles uniformly, adding a trisodium citrate solution to react, separating brown solid substances from the reaction solution by using a permanent magnet, and cleaning the mixture to obtain the MCOF-AuNPs nano composite material;

step four: preparation of apt-MCOF-AuNPs nano probe

Adding the tris (2-carbonylethyl) phosphate solution into the salmonella aptamer aqueous solution, carrying out mixed spinning at room temperature to obtain an activated aptamer solution, adding the MCOF-AuNPs nano composite material obtained in the step three into the activated aptamer solution, and carrying out mixed spinning at room temperature overnight to obtain an apt-MCOF-AuNPs nano probe;

step five: preparation of quenchers

Taking FeCl ₃ ·6H ₂ Adding O into double distilled water, and uniformly mixing to obtain Fe ³⁺ And (5) sealing the quenching agent and keeping away from light for later use.

3. The method for preparing the kit for detecting Salmonella typhimurium according to claim 2, wherein FeCl is added in the step one ₃ ·6H ₂ O and CH ₃ COONa·3H ₂ The mass ratio of O is 1.2-3.

4. The method for preparing a kit for detecting Salmonella typhimurium according to claim 2, wherein in the second step, 1,3, 5-tris (4-aminophenyl) benzene, 2, 5-dimethoxybenzene-1, 4-dicarbaldehyde, fe ₃ O ₄ The mass ratio of the nano particles is 102-106:86-88:115-120.

5. The method for preparing a kit for detecting salmonella typhimurium according to claim 2, wherein in the second step, the volume ratio of 1, 4-dioxane to n-butanol is 0.8-1 to 1.2.

6. The method for preparing a kit for detecting salmonella typhimurium according to claim 2, wherein in the third step, the concentration of the chloroauric acid hydrate solution is 1-1.2mM, and the concentration of the trisodium citrate solution is 100-120mM.

7. The preparation method of the kit for detecting salmonella typhimurium according to claim 2, wherein in the third step, the volume-to-mass ratio of the chloroauric acid hydrate solution, the trisodium citrate solution and the MCOF magnetic nanoparticles is 35-40mL:1.5-1.6mL:30-32mg.

8. The method for preparing a kit for detecting salmonella typhimurium according to claim 2, wherein in the fourth step, the volume of the salmonella aptamer aqueous solution is μ L: the mass mg of the MCOF-AuNPs nano composite material is 9-10:0.18-0.2.

9. The method for preparing a kit for detecting salmonella typhimurium according to claim 2, wherein in the step five, the volume of double distilled water is mL: feCl ₃ ·6H ₂ The mass mg of O is 95-100:25-27.

10. A kit prepared by the method of any one of claims 1 to 9.

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