CN115236050B - Method for detecting salmonella typhimurium by using fluorescent probe based on nitrogen-sulfur co-doped graphene quantum dots - Google Patents
Method for detecting salmonella typhimurium by using fluorescent probe based on nitrogen-sulfur co-doped graphene quantum dots Download PDFInfo
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Abstract
The invention relates to a detection method of salmonella typhimurium by a fluorescent probe based on nitrogen-sulfur co-doped graphene quantum dots, and belongs to the technical field of food safety. Based on sandwich method strategy, the invention uses streptavidin magnetic beads modified by nucleic acid aptamer and nitrogen-sulfur co-doped graphene quantum dots as magnetic separation probes and signal probes; when the to-be-detected object contains salmonella typhimurium, the magnetic separation probe and the signal probe synchronously and specifically identify the salmonella typhimurium to form a sandwich structure, the sandwich structure is adsorbed through the magnetic separation effect, the supernatant is taken, and the fluorescence intensity of the supernatant at 423nm is measured to determine the concentration of the salmonella typhimurium. The method realizes synchronous identification of salmonella typhimurium, has simple and convenient operation, reduces the influence of a sample matrix, has low cost and stable fluorescence characteristic, effectively improves the fluorescence quantum yield of the graphene quantum dots due to doping nitrogen and sulfur elements in the graphene quantum dots, improves the detection sensitivity to 11.9cfu/mL, and has good application and popularization prospects.
Description
Technical Field
The invention belongs to the technical field of food safety, and particularly relates to a detection method of salmonella typhimurium by using a fluorescent probe based on nitrogen-sulfur co-doped graphene quantum dots.
Background
Salmonella typhimurium is one of the major pathogens causing acute gastroenteritis worldwide, and the symptoms of infection generally include fever, nausea, vomiting, diarrhea, and the like. The main route of infection is the ingestion of contaminated dairy products, eggs, meat, poultry and other foods. Among them, dairy products are a kind of foods rich in nutrition and complex in content, but because they contain proteins, carbohydrates and fats, they provide important nutrition for human beings and also provide survival conditions for salmonella typhimurium. Therefore, the establishment of a detection method of salmonella typhimurium in dairy products is of great significance.
In the prior art, the detection of salmonella typhimurium mainly comprises the traditional detection technology, the molecular biological detection technology and the immunological detection technology. The traditional detection technology is regarded as a gold standard for detecting pathogenic bacteria due to high accuracy. However, the traditional culture method is complex in operation, has a detection period of about 5-7 days, requires special staff, and cannot meet the requirement of rapid screening and risk detection of salmonella typhimurium in dairy products. Molecular biological detection techniques including Polymerase Chain Reaction (PCR), recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling Circle Amplification (RCA) and the like cannot meet the requirements of on-site detection due to the need for specific primer design, cumbersome nucleic acid amplification procedures and expensive instrumentation. The immunological detection technology is mainly based on rapid screening of antigen-antibody recognition technology, has the advantages of simplicity, rapidness, high sensitivity, high flux, high specificity and the like, but the antibodies prepared in different batches have large quality difference, the price of the antibodies is high, the activity of the antibodies is easily influenced by external factors, and the popularization and the application of the antibodies are limited.
In recent years, optical detection strategies have become a trend for rapid detection. Traditional organic fluorescent dyes such as carboxyfluorescein (FAM), rhodamine B (Rhodamine B) and the like are widely applied as signal markers in fluorescent detection strategies, but are limited in further popularization due to factors such as poor fluorescence stability, narrow excitation spectrum and the like. Therefore, development of fluorescent nanoparticles with high efficiency and stability is urgently required. The graphene quantum dot as an emerging nano material has the excellent characteristics of easy synthesis, stable fluorescence characteristic, wide spectrum excitation response, good biocompatibility and the like, and has good effects in a plurality of detection methods. Compared with the graphene quantum dots, the surface properties of the graphene quantum dots are obviously changed by doping nitrogen and sulfur, and the absolute fluorescence quantum yield and the light stability of the graphene quantum dots are further improved.
The serious interference of milk matrix to the rapid detection strategy can significantly reduce detection sensitivity and reliability. Although the current sample pre-concentration by a centrifugal means and the microfluidic means of absorbing and decomposing the interfering substances by embedding the corresponding molecules in the channels can correspondingly reduce the interference of the milk matrix on detection, the immunomagnetic separation technology is a main stream method for getting rid of the interference of the milk matrix due to simple operation and high separation efficiency.
In addition, aiming at the limitations of large quality difference, high price, easy influence of external factors and the like of different batches of antibodies in preparation, the aptamer is used as a nucleic acid sequence capable of being specifically combined with a target substance, and has the advantages of easy synthesis and marking, low production cost, high stability, strong specificity and the like, and has successfully replaced the antibody to specifically and efficiently identify the target substance. Therefore, by modifying magnetic beads on the aptamer as a means for getting rid of interference of a milk matrix and combining with the aptamer modified nitrogen-sulfur co-doped graphene quantum dots with excellent luminous performance and high-efficiency recognition characteristics, a novel technology is provided for simply and highly sensitively and rapidly detecting salmonella typhimurium in the milk matrix, and the technology is not reported.
Disclosure of Invention
In order to realize simple and high-sensitivity rapid detection of salmonella typhimurium in a milk matrix, the invention provides a method for detecting salmonella typhimurium by using a fluorescent probe based on nitrogen-sulfur co-doped graphene quantum dots.
A detection method of salmonella typhimurium by using a fluorescent probe based on nitrogen-sulfur co-doped graphene quantum dots;
the signal probe solution is prepared from nitrogen-sulfur co-doped graphene quantum dots modified by an amino aptamer (1); the DNA sequence of the amination aptamer (1) is 5' -NH 2 -(CH 2 ) 6 -TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA G-3′；
The magnetic separation probe solution is prepared from streptavidin magnetic beads modified by biotinylation aptamer (2); the DNA sequence of the biotinylation aptamer (2) is 5'-Biotin-GAG GAA AGT CTA TAG CAG AGG AGA TGT GTG AAC CGA GTA A-3';
standard curve used: the abscissa X is the logarithmic value of the concentration (cfu/mL) of salmonella typhimurium bacteria liquid, and the ordinate Y is I 0 -I, said I 0 To detect the fluorescence intensity at 423nm in the blank solution, I was 10 2 、10 3 、10 4 、10 5 、10 6 、10 7 Fluorescence intensity at 423nm with cfu/mL Salmonella typhimurium solution;
the specific detection operation steps are as follows:
(1) Preparation of liquid to be tested
Taking 1mL of liquid milk at 8000×gCentrifuging for 5min, discarding supernatant, and re-suspending the precipitate with 1mL of sterile PBS buffer solution with a concentration of 0.1M, pH value of 7.4 to obtain a liquid to be detected;
(2) Detection of
(2.1) adding 100 mu L of signal probe solution and 36 mu L of magnetic separation probe solution into 500 mu L of to-be-detected liquid, and incubating for 45min at 37 ℃ to obtain a composite solution;
(2.2) placing the composite solution on a magnetic rack for magnetic separation, and taking supernatant to obtain a solution to be detected;
(2.3) taking the solution to be measured in a quartz cuvette, setting the excitation wavelength of a prism F97Pro fluorescence spectrophotometer to be 349nm, and measuring the fluorescence intensity of the solution to be measured at 423 nm;
(3) Calculating the detection result
Will I 0 Substituting the I value into a standard curve, and calculating to obtain the concentration of salmonella typhimurium in the solution to be detected, thereby completing detection; wherein I is 0 For detecting the fluorescence intensity at 423nm when the blank control solution is detected, I is the fluorescence intensity at 423nm when the solution to be detected is detected; when I 0 When the I value is greater than 104, the detection of salmonella typhimurium in the detected object is indicated; when I 0 And when the I value is less than 104, the salmonella typhimurium is not detected in the tested object.
The further technical scheme is as follows:
the preparation operation steps of the signal probe solution are as follows:
(1) Adding 0.1mL of nitrogen-sulfur co-doped graphene quantum dot stock solution into 9.9mL of ultrapure water to obtain 10mL of nitrogen-sulfur co-doped graphene quantum dot aqueous solution;
(2) Adding 5mg of N-hydroxysuccinimide and 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into 10mL of nitrogen-sulfur co-doped graphene quantum dot aqueous solution, adjusting the pH value to 5.0 by using 10mg/mL sodium hydroxide (NaOH) solution, and incubating for 30min at room temperature in a dark place to obtain an activated nitrogen-sulfur co-doped graphene quantum dot aqueous solution;
(3) And (3) taking the activated nitrogen-sulfur co-doped graphene quantum dot aqueous solution, regulating the pH value to 7.4 by using a 10mg/mL sodium hydroxide (NaOH) solution, adding 20 mu L of 100 mu M of amination aptamer (1) solution, and suspending for 2 hours at room temperature to obtain a signal probe solution.
The preparation operation steps of the nitrogen-sulfur co-doped graphene quantum dot stock solution are as follows:
(1) Uniformly mixing 1.0g of citric acid and 0.3-g L-cysteine to obtain a reaction mixture;
(2) Heating the reaction mixture to 200 ℃ by an oil bath pot to liquefy the reaction mixture, stopping heating when the color of the liquid of the reaction mixture changes from light yellow to orange, and naturally cooling to room temperature to obtain an orange product;
(3) And dissolving the orange product into 100mL of ultrapure water to obtain the nitrogen-sulfur co-doped graphene quantum dot stock solution.
The preparation operation steps of the magnetic separation probe solution are as follows:
(1) Taking 18 mu L of streptavidin magnetic beads with the concentration of 10mg/mL into a siliconizing centrifuge tube, adding 72 mu L of 1 XB & W buffer solution, uniformly mixing, standing on a magnetic rack for 1min, and discarding supernatant when the streptavidin magnetic beads are fully adsorbed on the wall of the centrifuge tube; then adding 72 mu L of 1 XB & W buffer solution to repeat the above operation to obtain treated streptavidin magnetic beads;
(2) The treated streptavidin magnetic beads are resuspended to 36 mu L of 2 XB & W buffer, 36 mu L of 1.5 mu M biotinylated aptamer (2) solution is added, and the mixture is incubated for 30min at room temperature to obtain a coupling solution;
(3) Placing the coupling solution on a magnetic rack, standing for 1min, removing supernatant, adding 50 mu L of Bovine Serum Albumin (BSA) solution with the mass concentration of 1%, and incubating for 30min at room temperature to obtain a closed coupling solution;
(4) Placing the closed coupling solution on a magnetic rack, standing for 1min, discarding supernatant, and re-suspending the precipitate in 36 mu L of sterile PBS buffer solution with the concentration of 0.1 and M, pH value of 7.4 to obtain a magnetic separation probe solution.
The 1 XB & W buffer solution is obtained by diluting 2 XB & W buffer solution with equal volume of ultrapure water; the preparation operation steps of the 2 XB & W buffer solution are as follows:
0.1211g of Tris (hydroxymethyl) aminomethane (Tris), 11.7g of sodium chloride (NaCl) and 0.0372g of ethylenediamine tetraacetic acid (EDTA) are weighed, added with deionized water, fully stirred and dissolved, the pH value is regulated to 7.5 by using hydrochloric acid (HCl) solution with the concentration of 36.5mg/mL, the mixture is uniformly stirred, the volume is fixed to 100mL, the mixture is sterilized at 121 ℃ for 15min and cooled to obtain 2 XB & W buffer solution, and the buffer solution is preserved in a refrigerator at 4 ℃ for standby.
The detection and analysis principle of the method of the invention is as follows:
based on a sandwich method strategy, utilizing the nitrogen-sulfur co-doped graphene quantum dot modified by the amino aptamer (1) as a signal probe, and coupling the biotinylation aptamer (2) onto streptavidin magnetic beads to prepare a magnetic separation probe; when the salmonella typhimurium is contained in the object to be detected, the signal probe and the magnetic separation probe synchronously and specifically identify the salmonella typhimurium to form a sandwich structure, the sandwich structure is adsorbed through the magnetic separation effect, the supernatant is taken, and the fluorescence intensity of the supernatant at 423nm is measured to determine the concentration of the salmonella typhimurium in the solution to be detected. Thus, the content of signal probe in the supernatant from which the sandwich structure is discarded by magnetic separation decreases with the increase of the concentration of salmonella typhimurium in the system. The logarithmic value of the salmonella typhimurium bacterial liquid concentration and the system fluorescence intensity reduction value are in a linear relation, so that a method for detecting salmonella typhimurium by using a fluorescent probe based on nitrogen-sulfur co-doped graphene quantum dots is established.
The beneficial technical effects of the invention are as follows:
(1) The nitrogen-sulfur co-doped graphene quantum dots used in the method of the invention have advantages over traditional fluorescent compounds, such as organic dyes, polymers and the like, because the graphene quantum dots have adjustable optical properties, high water solubility, high stability and organic inertness based on quantum confinement and edge effects. The nitrogen-sulfur co-doped graphene quantum dot is prepared from citric acid and L-cysteine serving as raw materials by adopting a hydrothermal method, and the surface state of the graphene quantum dot can be uniform and the fluorescence quantum yield of the graphene quantum dot can be effectively improved by doping hetero atoms in the graphene quantum dot by using the cysteine in the synthesis process. On the other hand, compared with most high-performance quantum dots which are limited by toxicity of metal elements (such as lead, cadmium and arsenic), the graphene quantum dots have the advantages of low toxicity, good biocompatibility, metabolic degradation in biological application and the like.
(2) According to the invention, immune magnetic beads formed by combining the aptamer and the magnetic beads are utilized to specifically identify and enrich salmonella typhimurium in the sample matrix, so that the load capacity of the identification molecules is effectively increased, the interference of the milk matrix is reduced, and the detection sensitivity and reliability are improved.
(3) The method of the invention uses the aptamer to replace the antibody as the identification element, and reduces the detection cost by about 75%. Meanwhile, the nitrogen-sulfur co-doped graphene quantum dot synthesis raw material is low in price, high in yield and high in cost performance.
(4) According to the invention, two different aptamers are respectively coupled on the nitrogen-sulfur co-doped graphene quantum dot and the streptavidin magnetic beads, so that the defect that the same aptamer is utilized to compete for a binding site in target bacteria in other sandwich structures is avoided.
(5) Based on a reverse detection strategy, the invention utilizes the specificity identification of the aptamer modified streptavidin magnetic beads and the nitrogen-sulfur co-doped graphene quantum dots to the salmonella typhimurium, and converts the target substance into a fluorescent signal to realize quantitative detection. In conventional quantum dot-based immunosensors, the fluorescent signal of a "magnetic bead-target substance-quantum dot" immunoconjugate is directly used as a detection reading, and two significant problems exist with this detection method. First, the fluorescent signal of the quantum dot cannot be effectively measured due to the blocking effect of the magnetic beads. This is because when the volume of the magnetic bead is 6 orders of magnitude greater than the volume of the quantum dot, and the equivalent quantum dot is captured on the surface of the magnetic bead to form a "magnetic bead-target substance-quantum dot" conjugate, the spatially blocked quantum dot cannot be excited, and the fluorescence signal of the corresponding quantum dot is lost and cannot be measured, resulting in only a portion of the quantum dot being excited to generate the fluorescence signal. Secondly, the "magnetic bead-target substance-quantum dot" immunoconjugate requires multiple washing and re-suspension steps, affecting the speed and accuracy of the whole method. In order to solve the problems, the invention outputs the supernatant liquid with the sandwich structure of magnetic separation discarding magnetic beads-target substances-quantum dots as a signal in a reverse detection strategy, thereby avoiding the interference of the magnetic beads and complicated washing steps. Compared with the traditional immunosensor based on quantum dots, the sensitivity of the invention is improved by 2 orders of magnitude, the detection limit of salmonella typhimurium is 11.9cfu/mL, and the whole analysis process can be completed within 50 min.
(6) According to the invention, the nitrogen-sulfur co-doped graphene quantum dots are combined with the magnetic beads for the first time, and the rapid detection of salmonella typhimurium in the dairy product is realized through a sandwich method and a reverse detection strategy. The nitrogen-sulfur co-doped graphene quantum dots in the sandwich structure have high fluorescence emission and photo-bleaching resistance, and can provide high-efficiency and stable fluorescence signals for detection; the immune magnetic beads and salmonella typhimurium perform specific reaction, and target substances are separated from complex matrixes under the action of an external magnetic field, so that the detection sensitivity and reliability can be improved. In addition, the reverse detection strategy avoids the interference of magnetic beads on fluorescent signals and complicated washing steps, and improves the accuracy and speed of the method.
Drawings
FIG. 1 is a schematic diagram of the detection method of the present invention.
FIG. 2 is a fluorescence spectrum of Salmonella typhimurium at different concentrations in example 2 of the present invention.
FIG. 3 is a graph of the logarithmic concentration versus fluorescence intensity for Salmonella typhimurium plotted in example 2 of the present invention.
FIG. 4 is a diagram showing the specificity of the detection method in example 4 of the present invention.
Detailed Description
The invention is further illustrated, but is not limited, by the following examples.
Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The test reagent consumables used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless specified; the quantitative tests in the following examples were all set up three repeated experiments, and the results were averaged; in the following examples, the percentages are by mass unless otherwise indicated.
In the following examples, streptavidin magnetic beads and magnetic separation rack were purchased from Biyun Tian biotechnology Co., ltd; citric acid, L-cysteine, bovine serum albumin, PBS buffer at a concentration of 0.1M, pH value 7.4 and aminated/biotinylated aptamer were purchased from Shanghai Biotechnology Co., ltd; n-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were purchased from Sigma-Aldrich, USA.
Example 1
The nitrogen-sulfur co-doped graphene quantum dot stock solution, the signal probe solution and the magnetic separation probe solution used in the detection method are prepared.
(1) The preparation operation steps of the nitrogen-sulfur co-doped graphene quantum dot stock solution are as follows:
(1.1) uniformly mixing 1.0g of citric acid with 0.3. 0.3g L-cysteine to obtain a reaction mixture;
(1.2) heating the reaction mixture to 200 ℃ by an oil bath pot to liquefy the reaction mixture, stopping heating when the color of the liquid of the reaction mixture changes from light yellow to orange, and naturally cooling to room temperature to obtain an orange product;
(1.3) dissolving the orange product into 100mL of ultrapure water to obtain the nitrogen-sulfur co-doped graphene quantum dot stock solution.
(2) The preparation operation steps of the signaling probe solution are as follows:
(2.1) adding 0.1mL of nitrogen-sulfur co-doped graphene quantum dot stock solution into 9.9mL of ultrapure water to obtain 10mL of nitrogen-sulfur co-doped graphene quantum dot aqueous solution;
(2.2) adding 5mg of N-hydroxysuccinimide and 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into 10mL of nitrogen-sulfur co-doped graphene quantum dot aqueous solution, adjusting the pH value to 5.0 by using 10mg/mL sodium hydroxide (NaOH) solution, and incubating for 30min at room temperature in a dark place to obtain an activated nitrogen-sulfur co-doped graphene quantum dot aqueous solution;
(2.3) taking the activated nitrogen-sulfur co-doped graphene quantum dot aqueous solution, regulating the pH value to 7.4 by using a sodium hydroxide (NaOH) solution with the concentration of 10mg/mL, adding 20 mu L of an amination aptamer (1) solution with the concentration of 100 mu M, and suspending for 2 hours at room temperature to obtain a signal probe solution;
the DNA sequence of the amination aptamer (1) is 5' -NH 2 -(CH 2 ) 6 -TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA G-3′。
(3) The preparation operation steps of the magnetic separation probe solution are as follows:
(3.1) taking 18 mu L of streptavidin magnetic beads with the concentration of 10mg/mL into a siliconizing centrifuge tube, adding 72 mu L of 1 XB & W buffer solution, uniformly mixing, standing on a magnetic rack for 1min, fully adsorbing the streptavidin magnetic beads on the wall of the centrifuge tube, and discarding supernatant; then adding 72 mu L of 1 XB & W buffer solution to repeat the above operation to obtain treated streptavidin magnetic beads;
the 1 XB & W buffer solution is obtained by diluting 2 XB & W buffer solution with equal volume of ultrapure water;
the preparation operation steps of the 2 XB & W buffer solution are as follows:
weighing 0.1211g of Tris (hydroxymethyl) aminomethane (Tris), 11.7g of sodium chloride (NaCl) and 0.0372g of ethylenediamine tetraacetic acid (EDTA), adding deionized water, fully stirring and dissolving, regulating the pH value to 7.5 by using hydrochloric acid (HCl) solution with the concentration of 36.5mg/mL, uniformly stirring, fixing the volume to 100mL, sterilizing at 121 ℃ for 15min, cooling to obtain 2 XB & W buffer solution, and preserving in a refrigerator at 4 ℃ for later use;
(3.2) resuspending the treated streptavidin magnetic beads to 36. Mu.L of 2 XB & W buffer, adding 36. Mu.L of 1.5. Mu.M biotinylated aptamer (2) solution, and incubating at room temperature for 30min to obtain a coupling solution;
the DNA sequence of the biotinylation aptamer (2) is 5'-Biotin-GAG GAA AGT CTA TAG CAG AGG AGA TGT GTG AAC CGA GTA A-3';
(3.3) placing the coupling solution on a magnetic rack, standing for 1min, discarding supernatant, adding 50 mu L of 1% Bovine Serum Albumin (BSA) solution by mass concentration, and incubating for 30min at room temperature to obtain a closed coupling solution;
(3.4) the blocked coupling solution was placed on a magnetic rack, allowed to stand for 1min, the supernatant was discarded, and the pellet was resuspended in 36. Mu.L of 0.1. 0.1M, pH value 7.4 sterile PBS to give a magnetic probe solution.
Example 2
Establishment of detection equation
Referring to fig. 1, the specific detection operation steps are as follows:
(1) Taking 1mL of Salmonella typhimurium stock solution which is cultured in LB broth for 12h to the late logarithmic growth phase, transferring the stock solution into a sterilized centrifuge tube, 8000×gCentrifuging for 5min, discarding supernatant, and re-suspending in 1mL sterile PBS buffer solution with concentration of 0.1M, pH value of 7.4 to obtain bacterial suspension; the bacterial suspension was diluted in a gradient with sterile PBS buffer at a concentration of 0.1 to M, pH at a value of 7.4 to prepare a concentration of 10 2 ，10 3 ，10 4 ，10 5 ，10 6 ，10 7 cfu/mL of Salmonella typhimurium solution; taking sterile PBS buffer solution with the concentration of 0.1M, pH value of 7.4 as a blank control solution, wherein the concentration of salmonella typhimurium in the blank control solution is 0cfu/mL;
(2) Blank solution at 500. Mu.L and concentration of 10 2 ，10 3 ，10 4 ，10 5 ，10 6 ，10 7 cfu/mL of Salmonella typhimurium solution are added with the same 100 mu L of signal probe solution and 36 mu L of magnetic separation probe solution respectively, and incubated at 37 DEG CCulturing for 45min to obtain seven composite solutions;
the signaling probe solution was prepared from example 1;
the magnetic probe solution was prepared from example 1;
(3) Placing the seven composite solutions on a magnetic rack for magnetic separation, and taking supernatant to obtain seven corresponding solutions to be detected;
(4) Respectively placing seven solutions to be tested in a quartz cuvette, setting excitation wavelength of a prism F97Pro fluorescence spectrophotometer to be 349nm, excitation broadband to be 10nm, emission broadband to be 10nm, and measuring the concentration of Salmonella typhimurium to be 0 and 10 respectively in the detection range of 370-600nm 2 ，10 3 ，10 4 ，10 5 ，10 6 ，10 7 The fluorescence intensity of seven solutions to be tested of cfu/mL at 423nm is shown in the attached figure 2; i is as follows 0 -I is an ordinate Y, a standard curve is drawn by taking the logarithmic value of the salmonella typhimurium bacteria concentration (cfu/mL) as an abscissa X, the result is shown in figure 3, and a detection equation is calculated: y= 378.30X-302.45, a correlation coefficient of 0.9976, a detection limit of 11.9cfu/mL;
the I is 0 For the fluorescence intensity at 423nm in the detection of the blank solution according to the invention, I is the detection 10 according to the invention 2 、10 3 、10 4 、10 5 、10 6 、10 7 cfu/mL Salmonella typhimurium solution, fluorescence intensity at 423 nm.
Example 3
Detection and research of salmonella typhimurium in artificially contaminated milk
(1) Preparation of artificial pollution milk to-be-detected liquid
Artificially inoculating Salmonella typhimurium solution of unknown concentration in 1mL of sterile milk at 8000×gCentrifuging for 5min, discarding supernatant, and re-suspending the precipitate with 1mL of sterile PBS buffer solution with a concentration of 0.1M, pH value of 7.4 to obtain milk to-be-detected liquid;
(2) Sample measurement
(2.1) adding 100 mu L of signal probe solution and 36 mu L of magnetic separation probe solution into 500 mu L of milk to-be-detected liquid, and incubating at 37 ℃ for 45min to obtain a composite solution;
the signaling probe solution was prepared from example 1;
the magnetic probe solution was prepared from example 1;
(2.2) placing the composite solution on a magnetic rack for magnetic separation, and taking supernatant to obtain a solution to be detected;
(2.3) taking the solution to be measured in a quartz cuvette, setting the excitation wavelength of a prism F97Pro fluorescence spectrophotometer to be 349nm, and measuring the fluorescence intensity of the solution to be measured at 423 nm;
(3) Calculating the detection result
(3.1) calculating a detection result according to a detection equation, wherein the detection equation is as follows: y= 378.30X-302.45, wherein X represents the logarithmic value of the concentration of salmonella typhimurium bacteria solution and Y is I 0 -I; wherein I is 0 For the fluorescence intensity at 423nm when the blank control solution is detected, I is the fluorescence intensity at 423nm when the solution to be detected is detected;
the detection equation is obtained in example 2;
fluorescence intensity I of the blank solution at 423nm 0 Measured from example 2;
(3.2) calculating to obtain I 0 An I value of 1026.13, which indicates that salmonella typhimurium is detected in the detected artificially contaminated milk, I 0 Substituting the I value into a detection equation to obtain the concentration of the salmonella typhimurium of 3.25X10 3 cfu/mL；
When I 0 When the I value is greater than 104, the detection of salmonella typhimurium in the detected object is indicated.
Example 4
Specific detection method for salmonella typhimurium in milk
(1) Sample processing
Taking the same 1mL sterile milk sample, respectively placing the same 1mL sterile milk samples into seven sterile centrifuge tubes, and respectively and correspondingly adding 1mL sterile milk samples with the concentration of 10 into the seven centrifuge tubes 6 cfu/mL of Salmonella typhimurium, listeria monocytogenes, pseudomonas aeruginosa, escherichia coli, enterobacter sakazakii, staphylococcus aureus, and ParamygdalineVibrio haemolyticus to obtain seven mixed solutions; mixing the seven mixed solutions at 8000× gCentrifuging for 5min, discarding supernatant, and re-suspending and precipitating with 1mL sterile PBS buffer solution with concentration of 0.1 and M, pH value of 7.4 to obtain seven dairy products to be detected containing different pathogenic bacteria;
(2) Detection of
(2.1) respectively adding 500 mu L of corresponding dairy products to be detected containing different pathogenic bacteria into seven sterile centrifuge tubes, respectively adding 100 mu L of signal probe solution and 36 mu L of magnetic separation probe solution, and incubating at 37 ℃ for 45min to obtain seven composite solutions;
the signaling probe solution was prepared from example 1;
the magnetic probe solution was prepared from example 1;
(2.2) placing the seven composite solutions on a magnetic rack for magnetic separation, and taking supernatant to obtain seven solutions to be detected;
(2.3) respectively taking seven solutions to be measured in a quartz cuvette, setting the excitation wavelength of a prism F97Pro fluorescence spectrophotometer to be 349nm, and measuring the fluorescence intensity of the seven solutions to be measured at 423 nm;
(3) Analysis of results
(3.1) calculating a detection result according to a detection equation, wherein the detection equation is as follows: y= 378.30X-302.45, wherein X represents the logarithmic value of the concentration of salmonella typhimurium bacteria solution and Y is I 0 -I; wherein I is 0 For the fluorescence intensity at 423nm when the blank control solution is detected, I is the fluorescence intensity at 423nm when the solution to be detected is detected;
the detection equation is obtained in example 2;
fluorescence intensity I of the blank solution at 423nm 0 Measured from example 2;
(3.2) As shown in FIG. 4, due to the specific recognition of the aptamer to Salmonella typhimurium, when Salmonella typhimurium exists in the system, the fluorescence intensity of the solution to be tested at 423nm is obviously reduced compared with that of the blank control solution, I 0 -I value 1958; when Salmonella typhimurium is not present in the system, listeria monocytogenes, pseudomonas aeruginosaThe fluorescence intensity of the solution to be tested of the cytobacteria, the escherichia coli, the enterobacter sakazakii, the staphylococcus aureus and the vibrio parahaemolyticus at 423nm is not obviously changed compared with that of a blank control solution, and the corresponding I 0 I values of 59, 43, 58, 49, 52 and 43, respectively, thus demonstrating the good specificity of the invention for Salmonella typhimurium;
when I 0 When the I value is greater than 104, the detection of salmonella typhimurium in the detected object is indicated; when I 0 And when the I value is less than 104, the salmonella typhimurium is not detected in the tested object.
Example 5
The invention relates to application in detecting the salmonella typhimurium content in milk
(1) Sample processing
Taking 1mL of commercially available milk at 8000×gCentrifuging for 5min, discarding supernatant, and re-suspending the precipitate with 1mL of sterile PBS buffer solution with a concentration of 0.1M, pH value of 7.4 to obtain a liquid to be detected;
(2) Detection of
(2.1) adding 100 mu L of signal probe solution and 36 mu L of magnetic separation probe solution into 500 mu L of to-be-detected liquid, and incubating for 45min at 37 ℃ to obtain a composite solution;
the signaling probe solution was prepared from example 1;
the magnetic probe solution was prepared from example 1;
(2.2) placing the composite solution on a magnetic rack for magnetic separation, and taking supernatant to obtain a solution to be detected;
(2.3) taking the solution to be measured in a quartz cuvette, setting the excitation wavelength of a prism F97Pro fluorescence spectrophotometer to be 349nm, and measuring the fluorescence intensity of the solution to be measured at 423 nm;
(3) Analysis of results
(3.1) calculating a detection result according to a detection equation, wherein the detection equation is as follows: y= 378.30X-302.45, wherein X represents the logarithmic value of the concentration of salmonella typhimurium bacteria solution and Y is I 0 -I; wherein I is 0 For the fluorescence intensity at 423nm when the blank control solution is detected by the method, I is the fluorescence intensity at 423nm when the solution to be detected is detected by the methodA degree;
the detection equation is obtained in example 2;
fluorescence intensity I of the blank solution at 423nm 0 Measured from example 2;
(3.2) the fluorescence intensity I of the test solution at 423nm was hardly changed from that of the blank solution, I 0 -an I value of 12, indicating that no salmonella typhimurium was detected in the milk;
when I 0 And when the I value is less than 104, the salmonella typhimurium is not detected in the tested object.
The embodiments of the present invention have been described in detail, but the present invention is not limited thereto. Any modification, equivalent replacement, improvement, etc. made within the scope of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A detection method of salmonella typhimurium by using a fluorescent probe based on nitrogen-sulfur co-doped graphene quantum dots is characterized by comprising the following steps of:
the signal probe solution is prepared from nitrogen-sulfur co-doped graphene quantum dots modified by an amino aptamer (1); the DNA sequence of the amination aptamer (1) is 5' -NH 2 -(CH 2 ) 6 -TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA G-3′；
The magnetic separation probe solution is prepared from streptavidin magnetic beads modified by biotinylation aptamer (2); the DNA sequence of the biotinylation aptamer (2) is 5'-Biotin-GAG GAA AGT CTA TAG CAG AGG AGA TGT GTG AAC CGA GTA A-3';
standard curve used: the abscissa X is the logarithmic value of the concentration of salmonella typhimurium bacteria liquid, and the ordinate Y is I 0 -I, said I 0 To detect the fluorescence intensity at 423nm in the blank solution, I was 10 2 、10 3 、10 4 、10 5 、10 6 、10 7 Fluorescence intensity at 423nm with cfu/mL Salmonella typhimurium solution;
the specific detection operation steps are as follows:
(1) Preparation of liquid to be tested
Taking 1mL of liquid milk at 8000×gCentrifuging for 5min, discarding supernatant, and re-suspending the precipitate with 1mL of sterile PBS buffer solution with a concentration of 0.1M, pH value of 7.4 to obtain a liquid to be detected;
(2) Detection of
(2.1) adding 100 mu L of signal probe solution and 36 mu L of magnetic separation probe solution into 500 mu L of to-be-detected liquid, and incubating for 45min at 37 ℃ to obtain a composite solution;
(2.2) placing the composite solution on a magnetic rack for magnetic separation, and taking supernatant to obtain a solution to be detected;
(2.3) taking the solution to be measured in a quartz cuvette, setting the excitation wavelength of a prism F97Pro fluorescence spectrophotometer to be 349nm, and measuring the fluorescence intensity of the solution to be measured at 423 nm;
(3) Calculating the detection result
Will I 0 Substituting the I value into a standard curve, and calculating to obtain the concentration of salmonella typhimurium in the solution to be detected, thereby completing detection; wherein I is 0 For detecting the fluorescence intensity at 423nm when the blank control solution is detected, I is the fluorescence intensity at 423nm when the solution to be detected is detected; when I 0 When the I value is greater than 104, the detection of salmonella typhimurium in the detected object is indicated; when I 0 -when the I value is less than 104, indicating that no salmonella typhimurium is detected in the test object;
the preparation operation steps of the signal probe solution are as follows:
(1) Adding 0.1mL of nitrogen-sulfur co-doped graphene quantum dot stock solution into 9.9mL of ultrapure water to obtain 10mL of nitrogen-sulfur co-doped graphene quantum dot aqueous solution;
(2) Adding 5mg of N-hydroxysuccinimide and 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into 10mL of nitrogen-sulfur co-doped graphene quantum dot aqueous solution, adjusting the pH value to 5.0 by using a sodium hydroxide solution with the concentration of 10mg/mL, and incubating for 30min at room temperature in a dark place to obtain an activated nitrogen-sulfur co-doped graphene quantum dot aqueous solution;
(3) And (3) taking the activated nitrogen-sulfur co-doped graphene quantum dot aqueous solution, regulating the pH value to 7.4 by using a sodium hydroxide solution with the concentration of 10mg/mL, adding 20 mu L of an amination aptamer (1) solution with the concentration of 100 mu M, and suspending for 2 hours at room temperature to obtain a signal probe solution.
2. The method for detecting salmonella typhimurium by using the fluorescent probe based on the nitrogen-sulfur co-doped graphene quantum dots according to claim 1, wherein the preparation operation steps of the nitrogen-sulfur co-doped graphene quantum dot stock solution are as follows:
(1) Uniformly mixing 1.0g of citric acid and 0.3-g L-cysteine to obtain a reaction mixture;
(2) Heating the reaction mixture to 200 ℃ by an oil bath pot to liquefy the reaction mixture, stopping heating when the color of the liquid of the reaction mixture changes from light yellow to orange, and naturally cooling to room temperature to obtain an orange product;
(3) And dissolving the orange product into 100mL of ultrapure water to obtain the nitrogen-sulfur co-doped graphene quantum dot stock solution.
3. The method for detecting salmonella typhimurium by using the fluorescent probe based on the nitrogen-sulfur co-doped graphene quantum dots, which is disclosed in claim 1, is characterized by comprising the following steps: the preparation operation steps of the magnetic separation probe solution are as follows:
(1) Taking 18 mu L of streptavidin magnetic beads with the concentration of 10mg/mL into a siliconizing centrifuge tube, adding 72 mu L of 1 XB & W buffer solution, uniformly mixing, standing on a magnetic rack for 1min, and discarding supernatant when the streptavidin magnetic beads are fully adsorbed on the wall of the centrifuge tube; then adding 72 mu L of 1 XB & W buffer solution to repeat the above operation to obtain treated streptavidin magnetic beads;
(2) The treated streptavidin magnetic beads are resuspended to 36 mu L of 2 XB & W buffer, 36 mu L of 1.5 mu M biotinylated aptamer (2) solution is added, and the mixture is incubated for 30min at room temperature to obtain a coupling solution;
(3) Placing the coupling solution on a magnetic rack, standing for 1min, removing supernatant, adding 50 mu L of bovine serum albumin solution with mass concentration of 1%, and incubating for 30min at room temperature to obtain a closed coupling solution;
(4) Placing the closed coupling solution on a magnetic rack, standing for 1min, discarding supernatant, and re-suspending the precipitate in 36 mu L of sterile PBS buffer solution with the concentration of 0.1 and M, pH value of 7.4 to obtain a magnetic separation probe solution;
the 1 XB & W buffer solution is obtained by diluting 2 XB & W buffer solution with equal volume of ultrapure water;
the preparation operation steps of the 2 XB & W buffer solution are as follows:
0.1211g of tris, 11.7g of sodium chloride and 0.0372g of ethylenediamine tetraacetic acid are weighed, added with deionized water, fully stirred and dissolved, the pH value is regulated to 7.5 by using hydrochloric acid solution with the concentration of 36.5mg/mL, uniformly stirred, the volume is fixed to 100mL, the mixture is sterilized at 121 ℃ for 15min under high pressure, cooled to obtain 2 XB & W buffer solution, and the buffer solution is preserved in a refrigerator at 4 ℃ for standby.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210857738.4A CN115236050B (en) | 2022-07-21 | 2022-07-21 | Method for detecting salmonella typhimurium by using fluorescent probe based on nitrogen-sulfur co-doped graphene quantum dots |
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| CN105861295A (en) * | 2016-05-24 | 2016-08-17 | 长沙医学院 | Biosensor for detecting salmonella typhimurium and preparation and detection methods |
| WO2018182510A1 (en) * | 2017-03-28 | 2018-10-04 | Agency For Science, Technology And Research | Nanomaterial-based bacterial sensors |
| CN111638330A (en) * | 2020-06-10 | 2020-09-08 | 青岛农业大学 | Biosensor for detecting salmonella typhimurium and application thereof |
| CN113912123A (en) * | 2021-09-23 | 2022-01-11 | 山东师范大学 | Salmonella typhimurium multimode test strip based on magnetic molybdenum disulfide catalysis and photothermal effect, and detection method and application thereof |
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| CN109596827A (en) * | 2019-01-17 | 2019-04-09 | 长江师范学院 | Fluorescence detection test strip and its preparation method and application that is a kind of while detecting 4 kinds of pathogenic bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105861295A (en) * | 2016-05-24 | 2016-08-17 | 长沙医学院 | Biosensor for detecting salmonella typhimurium and preparation and detection methods |
| WO2018182510A1 (en) * | 2017-03-28 | 2018-10-04 | Agency For Science, Technology And Research | Nanomaterial-based bacterial sensors |
| CN111638330A (en) * | 2020-06-10 | 2020-09-08 | 青岛农业大学 | Biosensor for detecting salmonella typhimurium and application thereof |
| CN113912123A (en) * | 2021-09-23 | 2022-01-11 | 山东师范大学 | Salmonella typhimurium multimode test strip based on magnetic molybdenum disulfide catalysis and photothermal effect, and detection method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| IMMUNOCAPTURED-LOOP MEDIATED ISOTHERMALAMPLIFICATION ASSAY FOR DETECTION OFVIBRIOPARAHAEMOLYTICUSIN SEAFOOD;YINGWANG YE;Journal of Food Safety;20131111;第34卷(第1期);全文 * |
| 转基因作物中CaMV 35S序列信号双重放大的电化学基因传感方法;郑海松 等;分析测试学报;20211231;第40卷(第7期);全文 * |
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