CN111366727B

CN111366727B - Kit for detecting salmonella typhimurium and preparation method thereof

Info

Publication number: CN111366727B
Application number: CN202010196129.XA
Authority: CN
Inventors: 王娟; 赵超; 魏胜男
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2021-05-25
Anticipated expiration: 2040-03-19
Also published as: CN111366727A

Abstract

The invention provides a method for detecting salmonella typhimuriumBelonging to the field of kits. The kit comprises: apt-Fe of Salmonella₃O₄The nano-probe comprises a @ Ag NPs nano-probe, a hydrogen peroxide etching agent, a urease solution and urea/phenol red test paper. The invention also provides a preparation method of the kit for detecting the salmonella typhimurium. The invention utilizes the composite structure of immunomagnetic beads and nano particles to detect the salmonella typhimurium, utilizes an enzyme inhibition method to develop color through test paper, shortens the detection time, has small coefficient of variation during quantitative detection, and has the lowest detection concentration of 21 cfu.mL^‑1The recovery rate reaches 96.18%, and the sensitivity is high and the stability is good.

Description

Kit for detecting salmonella typhimurium and preparation method thereof

Technical Field

The invention belongs to the field of kits, and particularly relates to a kit for detecting salmonella typhimurium and a preparation method thereof.

Background

Salmonella typhimurium (s. typhimurium) is a gram-negative enterobacterium, one of the most common pathogens responsible for outbreaks of food-borne diseases. Salmonella generally contaminates food products, such as eggs, fruits and vegetables, meat, milk, etc., through animal manure. Salmonella infection can cause typical gastroenteritis reactions such as abdominal pain, diarrhea, nausea, vomiting, fever and other symptoms of gastroenteritis. It is reported that food poisoning by salmonella still shows a rising tendency. At present, the bacterium is a necessary pathogenic bacterium for agricultural and sideline products such as eggs, meat, milk and the like.

At present, the traditional detection method of salmonella in China mainly adopts a microbial culture method, the whole process needs 3-5 days, the operation is complicated, the consumed time is long, and the requirements of people on rapidness, simplicity and convenience of food safety detection cannot be met. With the continuous development of the technology, the detection method is promoted to be updated continuously, and more methods are applied to detecting salmonella, such as a PCR (polymerase chain reaction) technology, an enzyme-linked immunosorbent assay (ELISA), a loop-mediated isothermal amplification technology (LAMP) and the like. The PCR technology can amplify detection signals in a short time and has the characteristic of high sensitivity, but the operation process of PCR requires professional personnel and false positive is easy to appear. The ELISA method is based on the specific binding of antigen and antibody for detection, does not need a large-scale instrument, but is long in time consumption and low in sensitivity. LAMP, as a new detection technology, has the advantages of low cost, high sensitivity and high specificity, but is not suitable for field detection of samples because LAMP requires selective enrichment culture. Therefore, it is necessary to develop a rapid, highly sensitive and highly specific detection method to monitor food-borne pathogenic bacteria, so as to ensure food safety.

Disclosure of Invention

The invention aims to provide a kit for detecting salmonella typhimurium and a preparation method thereof, and the kit is based on aptamer-Fe₃O₄The @ Ag nano probe and the urea/phenol red test paper can quickly, sensitively and simply detect the salmonella.

The invention firstly provides a kit for detecting salmonella typhimurium, which comprises:

apt-Fe of Salmonella₃O₄The nano-probe comprises a @ Ag NPs nano-probe, a hydrogen peroxide etching agent, a urease solution and urea/phenol red test paper.

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

the method comprises the following steps: fe₃O₄Preparation of nanoparticles

FeCl is added₃·6H₂O, sodium citrate and CH₃COONa·3H₂Magnetically 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 superparamagnetism₃O₄Nanoparticles;

step two: fe₃O₄Preparation of @ Ag magnetic nano particle

Mixing AgNO₃And polyvinylpyrrolidone k-30 are dissolved in polyethylene glycol, and after full dissolution, the Fe prepared in the step one is added₃O₄Dispersing the nanoparticles therein for reaction, separating brown solid substance from the reaction solution with a permanent magnet, and washing to obtain Fe₃O₄@ Ag magnetic nanoparticles;

step three: apt-Fe₃O₄Preparation of @ Ag NPs nanoprobe

Adding tris (2-carbonylethyl) phosphate solution to SalmonellaThe ligand aqueous solution is mixed and rotated under the room temperature condition to obtain activated aptamer solution, and the Fe obtained in the step two₃O₄Adding the @ Ag nano particles into the activated aptamer solution, and performing mixed spinning overnight at room temperature to obtain apt-Fe₃O₄@ Ag NPs nanoprobe;

step four: preparation of urea/phenol red test paper

Dissolving phenol red and urea in an ethanol solution, soaking filter paper in the solution, and drying the soaked test paper to obtain urea/phenol red test paper;

step five: preparation of etchant

Get H₂O₂Adding into PBS buffer solution, mixing well to obtain H₂O₂Etching agent, keeping away from light for standby;

step six: preparation of urease solutions

And weighing urease, dissolving the urease in a PBS buffer solution to obtain a urease solution, and storing for later use.

Preferably, FeCl is used in the first step₃·6H₂O, sodium citrate and CH₃COONa·3H₂The mass ratio of O is 2.16: 0.5: 3.34.

preferably, the reaction temperature of the first step is 200 ℃ and the reaction time is 10 h.

Preferably, AgNO of the second step₃Polyvinylpyrrolidone k-30, Fe₃O₄The mass ratio of the nano particles is 0.1: 2.5: 0.2.

preferably, the reaction temperature of the second step is 100 ℃, and the reaction time is 12 h.

Preferably, the concentration of the tris (2-carbonylethyl) phosphate solution in the third step is 1mM, and the concentration of the salmonella aptamer aqueous solution is 10. mu.M.

Preferably, the volume ratio of the tris (2-carbonylethyl) phosphate solution to the salmonella aptamer aqueous solution in the third step is 1: 10.

preferably, the volume of the salmonella aptamer aqueous solution in the third step is μ L: fe₃O₄Nanoparticle of @ AgThe mass mg of the seed is 10: 1.

preferably, the apt-Fe₃O₄@ Ag nanoparticle size 130 nm.

Preferably, the mass ratio of phenol red to urea in the step four is 0.1: 3.

the invention has the advantages of

The invention provides a kit for detecting salmonella typhimurium and a preparation method thereof, the kit is prepared by preparing magnetic beads with superparamagnetism, coating silver nano particles on the magnetic beads to form a core-shell structure, coupling the core-shell structure with an aptamer of the salmonella typhimurium to synthesize a nano probe, incubating the salmonella typhimurium and the nano probe to form a magnetic bead-thallus compound, discarding supernatant by using a magnetic frame, adding H, and performing ultrasonic wave amplification to obtain the salmonella typhimurium₂O₂The outer layer of the silver shell of the probe is etched, and the silver shell cannot be etched by H due to the aggregation and protection effects of the thalli on the nano probe₂O₂Oxidizing the mixture into silver ions, and further separating the supernatant by using a magnetic frame, wherein the content of the silver ions in the supernatant is lower than that of the sample without the Salmonella typhimurium. Because the silver ions can inhibit the catalytic activity of urease, the supernatant and the urease are incubated together, and a sample is dripped onto the urea/phenol red test paper, and the urease hydrolyzes urea to increase the pH value, so that the color of the urea phenol red paper is changed from yellow to pink. Thereby realizing the rapid specific detection of the salmonella typhimurium. The invention provides a method for detecting salmonella typhimurium by using an immunomagnetic bead and nanoparticle composite structure, and an enzyme inhibition method is used for developing through test paper, so that the detection time is shortened, the time variation coefficient of quantitative detection is small, and the minimum detection concentration is 21 cfu.mL^-1The recovery rate reaches 96.18%, and the sensitivity is high and the stability is good.

Drawings

FIG. 1 is a graph showing the standard curve of detection of Salmonella in example 5.

FIG. 2 is a graph showing the standard curve of detection of Salmonella in example 6 of the present invention.

FIG. 3 is apt-Fe of the present invention₃O₄A flow chart of detecting the salmonella typhimurium by using the @ Ag NPs and urea phenol red test paper.

Detailed Description

the method comprises the following steps: fe₃O₄Preparation of nanoparticles

FeCl is added₃·6H₂O, sodium citrate and CH₃COONa·3H₂Magnetically 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 10 hours; 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 superparamagnetism Fe₃O₄Nanoparticles; the FeCl₃·6H₂O, sodium citrate and CH₃COONa·3H₂The O mass ratio is preferably 2.16: 0.5: 3.34;

step two: fe₃O₄Preparation of @ Ag magnetic nano particle

Preparation of Fe by liquid phase reduction₃O₄The @ Ag nano-particle specifically comprises: mixing AgNO₃Dissolving polyvinylpyrrolidone k-30 in polyethylene glycol, adding prepared Fe after full dissolution₃O₄Dispersing the nanoparticles therein, preferably stirring at 100 deg.C for 12h, separating brown solid substance from the reaction solution with permanent magnet, washing Fe with acetone and water alternately for multiple times₃O₄@ Ag nano particle to remove glycol and polyvinylpyrrolidone k-30, and finally drying to obtain Fe₃O₄@ Ag magnetic nanoparticles, storing at 4 ℃ in dark for later use; the AgNO₃Polyvinylpyrrolidone k-30, Fe₃O₄The mass ratio of nanoparticles is preferably 0.1: 2.5: 0.2;

step two: salmonella apt-Fe₃O₄Preparation of @ Ag NPs nanoprobe

Adding the tris (2-carbonyl ethyl) phosphate solution into the salmonella aptamer aqueous solution, carrying out mixed spinning at room temperature, preferably for 1h, activating the modified sulfydryl on the aptamer to obtain an activated aptamer solution, taking Fe₃O₄@ Ag nanoparticles, preferably washed with distilled water 2 times, resuspended with distilled water, added with activated aptamer, mixed-spun overnight at room temperature, magnetically separated, supernatant removed, and washed with distilled water three times to obtain apt-Fe₃O₄@ Ag NPs, storing at 4 ℃ in dark for later use; the concentration of the tris (2-carbonylethyl) phosphate solution is preferably 1mM, the concentration of the salmonella aptamer aqueous solution is preferably 10. mu.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: fe₃O₄The mass mg of the @ Ag nanoparticles is preferably 10: 1; the apt-Fe₃O₄@ Ag nanoparticle size is preferably 130 nm;

the sequence of the salmonella-resistant aptamer is as follows: 5' -SH- (CH)₂)₆-TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC CTT GAC ATT ATG ACA G-3', synthesized by Shanghai Biometrics;

step four: preparation of urea/phenol red test paper:

dissolving phenol red and urea in an ethanol solution, soaking neutral cotton filter paper in the solution, preferably soaking at room temperature for 10min, paving the soaked test paper, preferably drying in a 37 ℃ oven, storing the prepared urea/phenol red test paper in a dry and sealed, cool and dark environment, wherein the mass ratio of the phenol red to the urea is preferably 0.1: 3, the concentration of the ethanol solution is preferably 60%;

step five: preparation of etchant

Get H₂O₂Adding into PBS buffer solution, mixing well to obtain H₂O₂An etching agent is stored away from light at 4 ℃ for standby, and the H₂O₂And PBS buffer preferably in a volume ratio of 40: 3960; h₂O₂The concentration of sum is preferably 30%; h₂O₂Etching ofThe concentration of the agent is preferably 100 mM; the PBS buffer solution is 8g of NaCl,0.2g of KCl and 3.63g of Na₂HPO₄·12H₂O,0.24g KH₂PO₄Dissolving in 1L of ultrapure water, and adjusting pH to 7.4;

step six: preparation of urease solutions

Weighing urease, dissolving the urease in a PBS buffer solution to obtain a urease solution, and storing the urease solution at 4 ℃ for later use, wherein the mass mg of the urease is as follows: volume mL of PBS buffer was 50: 1, the concentration of the urease solution is preferably 50U/ml, and the PBS buffer solution is 8g of NaCl,0.2g of KCl and 3.63g of Na₂HPO₄·12H₂O,0.24g KH₂PO₄Dissolved in 1L of ultrapure water and the pH was adjusted to 7.4.

The kit is used for detecting salmonella, and comprises the following specific steps:

taking 900 mu L and 2 mg/mL of samples to be detected^-1apt-Fe₃O₄@ Ag NPs 100. mu.L, and mixed spinning for 45min at room temperature in dark. Magnetic separation, supernatant removal, 100. mu.L of 100mM H₂O₂Reacting the etching agent for 2min, carrying out magnetic separation, incubating 5 mu L of supernatant and 10 mu L of 50U/ml urease for 10min, dropping 2 mu L of reaction liquid to colorimetric test paper, taking a picture by using a smart phone, and carrying out gray scale analysis by using Image J. Image J outputs red (R), green (G) and blue (B) three channel values, the Gray values are calculated according to the Gray value formula (0.30R +0.59G +0.11B), and the Gray values are used as output quantitative signals of salmonella concentration.

The present invention is described in further detail below with reference to specific examples, wherein the starting materials are all commercially available

Example 1 Salmonella apt-Fe₃O₄Preparation of @ Ag NPs

First of all, Fe₃O₄@ Ag, 2.16g FeCl₃·6H₂O, 0.5g sodium citrate and 3.34g CH₃COONa·3H₂O 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 10 hours; 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 obtainFe having superparamagnetism₃O₄Nanoparticles.

Preparation of Fe by liquid phase reduction₃O₄@ Ag nanoparticles 0.1g AgNO₃And 2.5g polyvinylpyrrolidone k-30 in 24mL polyethylene glycol, after sufficient dissolution, 0.2g of the prepared Fe was added₃O₄Dispersing the nanoparticles therein, stirring at 100 deg.C for 12h, separating brown solid substance from the reaction solution with permanent magnet, washing Fe with acetone and water for several times₃O₄@ Ag nanoparticles to remove ethylene glycol and polyvinylpyrrolidone k-30. And finally drying, and storing at 4 ℃ in a dark place for later use.

Adding 1 μ L of 1mM tris (2-carbonylethyl) phosphate solution into 10 μ L of 10 μ M Salmonella aptamer aqueous solution, performing mixed rotation at room temperature for 1h to activate the modified sulfhydryl on the aptamer, and taking 1mg of Fe₃O₄@ Ag nanoparticles were washed 2 times with distilled water, resuspended in 1mL of distilled water, added with the activated aptamer, and vortexed overnight at room temperature. Magnetic separation, supernatant removal and distilled water washing for three times to obtain apt-Fe₃O₄The @ Ag nanoprobe is preserved at 4 ℃ in dark for standby, and 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 GAC ATT ATG ACA G-3', synthesized by Shanghai Biometrics.

Example 2 preparation of urea/phenol red test paper:

0.1g of phenol red and 3g of urea were dissolved in a 60% ethanol solution. Soaking neutral cotton filter paper in the above solution at room temperature for 10 min. The soaked test paper is laid flat and dried in an oven at 37 ℃. And storing the prepared urea/phenol red test paper in a dry, sealed, shady and shady environment.

EXAMPLE 3 preparation of buffer, etchant and urease solution

Weighing 8g NaCl,0.2g KCl and 3.63g Na₂HPO₄·12H₂O,0.24g KH₂PO₄Dissolved in 1L of ultrapure water, and the pH was adjusted to 7.4 to obtain a PBS buffer.

40 μ L of 30% H was taken₂O₂Adding into 3960 μ L PBS buffer, mixing well to obtain 100mM H₂O₂The etching agent is stored in the dark at 4 ℃ for later use.

50mg of urease (1U/mg) was weighed and dissolved in 1mL of PBS buffer to obtain 50U/mL urease solution, which was stored at 4 ℃ for further use.

Example 4 preparation of bacterial liquid Standard

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 12-18 h. 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 solution⁹CFU·mL^-1And storing in a refrigerator at 4 ℃ for later use.

Example 5 based on apt-Fe₃O₄Method for detecting salmonella by using @ Ag NPs and urea/phenol red test paper

The detection process is shown in FIG. 3, and 900 μ L and 2 mg/mL samples are taken^-1apt-Fe₃O₄@ Ag NPs 100. mu.L, and mixed spinning for 45min at room temperature in dark. Magnetic separation, supernatant removal, 100. mu.L of 100mM H₂O₂Reacting the etching agent for 2min, carrying out magnetic separation, incubating 5 mu L of supernatant and 10 mu L of 50U/ml urease for 10min, dropping 2 mu L of reaction liquid to colorimetric test paper, taking a picture by using a smart phone, and carrying out gray scale analysis by using Image J. Image J outputs red (R), green (G) and blue (B) three channel values, the Gray values are calculated according to the Gray value formula (0.30R +0.59G +0.11B), and the Gray values are used as output quantitative signals of salmonella concentration. The number of Salmonella in the sample was determined by reference to the standard graph 1. The concentration range of the bacteria is quantitatively detected to be 10-10⁶CFU/ml.

FIG. 1 is a graph showing the standard curve of the Salmonella detection in example 5, wherein the abscissa is the logarithmic value of the Salmonella concentration and the ordinate is the gray scale value. Detection concentration range: 10-10⁶CFU/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 21 CFU/ml. The method is used for detecting salmonella, Listeria monocytogenes, staphylococcus aureus, vibrio parahaemolyticus, escherichia coli O157: H7 and the like, only the detection result of the salmonella is positive, the rest is negative, the method is high in specificity, false positive and false negative results are not seen, and the results are shown in table 1.

TABLE 1 detection results of the specificity of Salmonella

Note: < + > represents positive salmonella, and < - > represents negative salmonella.

Detection of a simulated sample

Preparing a milk diluent: taking 100 mu L of pure milk, adding 9.9mL of sterile PBS, and carrying out suction filtration on the filtrate by using a 0.22 mu m filter membrane; get 10²、10⁴、10⁶CFU/ml of Salmonella was inoculated into the milk dilution and tested by the test method of the invention.

Taking 900 mu L and 2 mg/mL milk samples to be detected^-1apt-Fe₃O₄@ Ag NPs 100. mu.L, shading at room temperature, and vortex mixing for 45 min. Magnetic separation, supernatant removal, 100. mu.L of 100mM H₂O₂Reacting the etching agent for 2min, carrying out magnetic separation, incubating 5 mu L of supernatant and 10 mu L of 50U/ml urease for 10min, dropping 2 mu L of reaction liquid to colorimetric test paper, taking a picture by using a smart phone, and carrying out gray scale analysis by using Image J. Referring to the standard curve chart 2, the amount of Salmonella in the sample is determined, and the concentration of the Salmonella is quantitatively determined within the range of 10-10⁶CFU/mL. The method has stable detection and the recovery rate of the added standard reaches 96.18 percent.

FIG. 2 is a graph showing the standard curve of detection of Salmonella in example 6 of the present invention. The abscissa is the logarithmic value of the concentration of salmonella in milk and the ordinate is the gray value. Detection concentration range: 10-10⁶CFU/ml。

Claims

1. A preparation method of a kit for detecting Salmonella typhimurium is characterized by comprising the following steps:

the method comprises the following steps: fe₃O₄Preparation of nanoparticles

step two: fe₃O₄Preparation of @ Ag magnetic nano particle

step three: apt-Fe of Salmonella₃O₄Preparation of @ Ag NPs nanoprobe

Adding the tris (2-carbonyl ethyl) phosphate solution into the Salmonella typhimurium aptamer aqueous solution, carrying out mixed rotation at room temperature to obtain an activated aptamer solution, and carrying out Fe extraction in the step two₃O₄Adding the @ Ag nano particles into the activated aptamer solution, and performing mixed spinning overnight at room temperature to obtain apt-Fe of salmonella₃O₄@ Ag NPs nanoprobe;

step four: preparation of urea/phenol red test paper

step five: preparation of etchant

Get H₂O₂Adding into PBS buffer solution, mixing well to obtain H₂O₂Etchant, 2Storing light for later use;

step six: preparation of urease solutions

2. The method for preparing the kit for detecting Salmonella typhimurium according to claim 1, wherein FeCl is used in the step one₃·6H₂O, sodium citrate and CH₃COONa·3H₂The mass ratio of O is 2.16: 0.5: 3.34.

3. the method for preparing a kit for detecting salmonella typhimurium according to claim 1, wherein the reaction temperature in the first step is 200 ℃ and the reaction time is 10 hours.

4. The method for preparing the kit for detecting Salmonella typhimurium according to claim 1, wherein AgNO in the second step₃Polyvinylpyrrolidone k-30, Fe₃O₄The mass ratio of the nano particles is 0.1: 2.5: 0.2.

5. the method for preparing a kit for detecting salmonella typhimurium according to claim 1, wherein the reaction temperature in the second step is 100 ℃ and the reaction time is 12 hours.

6. The method for preparing a kit for detecting Salmonella typhimurium according to claim 1, wherein the concentration of the tris (2-carbonylethyl) phosphate solution in step three is 1mM, and the concentration of the Salmonella typhimurium aptamer aqueous solution is 10. mu.M.

7. The method for preparing a kit for detecting salmonella typhimurium according to claim 1, wherein the volume ratio of the tris (2-carbonylethyl) phosphate solution to the salmonella typhimurium aptamer aqueous solution in the step three is 1: 10.

8. the method for preparing a kit for detecting salmonella typhimurium according to claim 1, wherein the volume of the salmonella typhimurium aptamer aqueous solution in the third step is μ L: fe₃O₄Mass mg of @ Ag nanoparticles is 10: 1.

9. the preparation method of the kit for detecting salmonella typhimurium according to claim 1, wherein the mass ratio of phenol red to urea in the step four is 0.1: 3.