Staphylococcus aureus aptamer and method for preparing detection probe by using staphylococcus aureus aptamer

Abstract

The invention provides an original staphylococcus aureus nucleic acid aptamer and a detailed screening and preparation process thereof, and aims to improve the detection accuracy and efficiency of common and dangerous strains. The specific DNA sequence (SEQ ID No: 1) of the aptamer is obtained through a series of complex bioinformatics and calculation processes, and comprises the following steps: carrying out a docking experiment on evolved oligonucleotide fragments by using molecular simulation software AutoDock, and calculating the affinity between each short-chain fragment and a target staphylococcus membrane protein FnBPA. Thirdly, the data are optimized through an ant colony algorithm, so that the sequence with the highest combination efficiency is identified; after the aptamer sequence with high affinity is obtained, the biosensor based on the aptamer is developed. The construction process of the sensor comprises the working procedures of mixing of activating reactants and preparation of the fluorescent probe and the quenching probe, and finally a sensitive and specific fluorescent quenching probe is formed. The change of the fluorescence emission wavelength and the ultraviolet absorption wavelength indicates that the aptamer is successfully fixed, so that a rapid and reliable method is provided for detecting the staphylococcus aureus. The method has very important application prospects in the fields of food safety monitoring, medical diagnosis and the like, the time required by means such as traditional microbial culture and the like can be remarkably shortened, and meanwhile, the cost selection lower than that of a molecular biological technology is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and in particular relates to a Staphylococcus aureus aptamer and a preparation method thereof for a detection probe. Background Art

[0002] In recent years, the impact of foodborne pathogenic microorganisms on food safety has become increasingly prominent, posing a great threat to people's health. Staphylococcus aureus (S.aure) is an important foodborne pathogenic microorganism and zoonotic pathogen. Staphylococcal food poisoning is caused by ingesting dairy products, meat, and egg products contaminated with enterotoxins. S.aure is widely present in nature. About 20% to 30% of the general population are carriers of Staphylococcus aureus. It has the characteristics of facultative anaerobic, high temperature resistance, and high salt. It is easy to survive and spread with the activities of humans and animals, threatening the lives of humans and animals. According to the World Health Organization, about 600 million people worldwide become ill and about 420,000 people die each year from eating contaminated food. In my country, about 10% to 25% of food poisoning incidents are caused by Staphylococcus aureus, which seriously threatens food safety and national health. The irrational use and even abuse of antibiotics are particularly prominent, leading to the continuous enhancement of strain resistance. It is reported that methicillin-resistant Staphylococcus aureus infection has been listed as one of the three most difficult infectious diseases in the world, along with hepatitis B virus and acquired immunodeficiency syndrome infection. Therefore, rapid detection of Staphylococcus aureus and its biomarkers in food is an important measure to ensure food safety.

[0003] At present, the traditional detection methods of Staphylococcus aureus are mainly bacterial enrichment culture, morphological identification, biochemical identification, etc. The whole identification process takes 3 to 5 days and the operation is complicated. The detection time of the fluorescent polymerase chain reaction detection method is usually more than 1 hour, and the equipment is expensive and the detection cost is high.

[0004] Compared with traditional detection methods, the detection sensing system established by combining nucleic acid aptamers with gold nanoparticles is not only simple to operate but also has good sensitivity, and is expected to become a new trend in the field of rapid detection. Nucleic acid aptamers refer to oligonucleotide fragments that can bind to specific targets with high affinity and specificity, and can bind to target substances. The emerging fluorescent biological probe detection method is widely used in the determination of target substances due to its advantages such as low cost, simple operation, fast response, short time consumption, high stability, and no reliance on large instruments. Among them, fluorescence resonance energy transfer (FRET, As a non-radiative energy transition, fluorescence resonance energy transfer (FRET) is widely used in fluorescent probe detection technology. Silicon nanoparticles are less studied, and they are often prepared by catalytic reduction of silicon-containing coupling agents. Most methods based on fluorescence resonance energy transfer are achieved by shortening the distance between the donor and the acceptor. For example, energy transfer is performed by using the proximity of ratiometric fluorescent probes coupled to the surface of microbial cells. Ratiometric fluorescent probes have the advantages of high sensitivity, simple operation, and short response time to the target. At present, it is necessary to select a suitable first fluorescent probe that can be used as an energy donor and a suitable second fluorescent probe that can be used as an energy acceptor to form an effective FRET-type fluorescence quenching biosensor. However, due to the high failure rate of the double aptamer sandwich method, new nucleic acid aptamers need to be used in combination. It is easy for the bacterial surface to be completely occupied by one of the fluorescent probes, making the other fluorescent probe unusable. Summary of the invention

[0005] In view of the problems that traditional Staphylococcus aureus detection methods are time-consuming, complicated to operate, expensive in equipment, and high in detection cost, the present invention provides a rapid detection method based on the principle of fluorescence quenching; in view of the high failure rate of the double aptamer sandwich method, new nucleic acid aptamers need to be used in combination, and it is easy for the bacterial surface to be completely occupied by one of the fluorescent probes, making the other fluorescent probe unusable. The present invention screened a nucleic acid aptamer targeting the membrane protein FnBPA protein of Staphylococcus aureus, i.e., a first aptamer, based on the ant colony algorithm and molecular docking and other technologies, and its target is inconsistent with the target of the second aptamer, and a detection method based on the aptamer and its fluorescence quenching is used.

[0006] The DNA sequence of a Staphylococcus aureus aptamer is shown in SEQ ID No:1.

[0007] The screening steps are as follows:

[0008] (1) Construction of the membrane protein structure of Staphylococcus aureus

[0009] Obtain the amino acid sequence and structural information of the membrane protein FnBPA of Staphylococcus aureus;

[0010] (2) Performing molecular docking test on the membrane protein of Staphylococcus aureus obtained in step (1)

[0011] The random design capacity is 10 14A short-chain oligo DNA fragment library, wherein the length of the short-chain oligo DNA fragment ranges from 30 bp to 80 bp; importing the structural information of the membrane protein FnBPA protein of Staphylococcus aureus in step (1) into the AutoDock molecular simulation software, and then performing molecular docking between the short-chain oligo DNA fragment library and the surface domain of the membrane protein FnBPA protein to obtain the free energy of binding between each short-chain oligo DNA fragment and the membrane protein FnBPA protein;

[0012] (3) Optimize the molecular docking results obtained in step (2) using the ant colony algorithm model

[0013] The free energy of the above combination is optimized by combining the improved ant colony algorithm model, where the algorithm formula of the model is as follows:

[0014]

[0015] τ(i,j)=(1-ρ)·τ(i,j)+ρ·Δτ(i,j) k (II)

[0016] Where P ij (t) represents the transition probability value of ant k from node i to node j, τ ij (t) represents the pheromone concentration from node i to node j at time t, η ij (t) represents the constant heuristic function from node i to node j at time t, α represents the pheromone enhancement coefficient, β represents the heuristic information coefficient, l represents the specified position of the binding free energy of the short-chain oligoDNA fragment, τ il (t) represents the pheromone concentration from node i to node l at time t, η il (t) represents the constant heuristic function from node i to node l at time t, allowed i represents the set of nodes that node i is allowed to select next, τ(i,j) represents the sum of pheromones released by ant k from node i to node j, ρ represents the pheromone volatility coefficient (0<ρ<1), Δτ(i,j) k represents the increase in the amount of pheromone released by ant k from node i to node j;

[0017] According to the results of optimization processing, statistics P ij (t) The corresponding short-chain oligo DNA fragment was obtained to obtain the matching Staphylococcus aureus nucleic acid aptamer.

[0018] The preparation method of the detection probe based on the Staphylococcus aureus aptamer is characterized in that the operation steps are as follows:

[0019] 1) Preparation of the first activation mixture

[0020] Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1 to obtain a first activation mixture;

[0021] (2) Preparation of the second activation mixture

[0022] Adding pure water to 3-(2-formylethyl)phosphine hydrochloride to obtain a 3-(2-formylethyl)phosphine hydrochloride solution with a final concentration of 2.867 g / L, i.e., the second activated mixed solution;

[0023] (3) Preparation of fluorescent probes

[0024] 6 mL of deionized water was added to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; 8.0 mg of amikacin was added and stirred continuously for 30 min; the mixture was heated at 200°C for 6 h and cooled to room temperature; pure water was added to dilute the mixture 20 times to obtain a reaction solution; 500 uL of the reaction solution and 200 uL of the first activation mixture were vortexed and mixed, and incubated on a shaker at 180 rpm and room temperature for 30 min; 180 uL of the first aptamer solution with a volume concentration of 10 umol / L was added and vortexed, and incubated on a shaker at 180 rpm and room temperature for 2.5 h to obtain a fluorescent probe, which was then wrapped in tin foil and stored at 4°C in a dark place;

[0025] The DNA sequence of the first aptamer in the first aptamer solution is as shown in SEQ ID No: 1, and the 5' end of the first aptamer is modified with a carboxyl group;

[0026] (4) Preparation of quenching probe

[0027] (4.1) Add 1 mL of 1% tetrachloroauric acid solution to 100 mL of distilled water and boil. Quickly add 3 mL of 1% trisodium citrate solution and continue boiling for 15 min. Cool naturally to room temperature to obtain a gold nanoparticle solution.

[0028] (4.2) 80 μL of the second aptamer solution with a volume concentration of 10 μM was placed in a water bath at 95°C for 5 min, and then cooled at 4°C for 5 min to obtain an annealing solution; 10 μL of a 3-(2-formylethyl)phosphine hydrochloride solution with a mass volume concentration of 2.867 g / L was added to the annealing solution, and the mixture was allowed to react at room temperature for 30 min to obtain a reaction solution; 1 mL of the gold nanoparticle solution was added to the reaction solution, and the mixture was incubated at 37°C for 12 h, and then centrifuged at 10,000 rpm for 20 min, and the supernatant was removed to obtain a precipitate;

[0029] The DNA sequence of the second aptamer in the second aptamer solution is as shown in SEQ ID NO: 2, and the 5' end of the second aptamer is modified with a thiol group;

[0030] (4.3) Add 500 μL of PBS solution with a mass concentration of 0.1 M and a pH value of 7.4 to the precipitate, and store it at 4°C wrapped in tin foil and protected from light to prepare a quenching probe;

[0031] (5) Preparation of biosensor

[0032] The fluorescent probe and the quenching probe are mixed in a volume ratio of 1:2 to prepare a fluorescence quenching biosensor;

[0033] When the fluorescence emission wavelength of silicon nanoparticles shifts from 520nm to 524nm, it indicates that the first aptamer in the fluorescence quenching biosensor is successfully modified onto the silicon nanoparticles. When the ultraviolet absorption wavelength of gold nanoparticles shifts from 521nm to 525nm, it indicates that the second aptamer in the fluorescence quenching biosensor is successfully modified onto the gold nanoparticles.

[0034] The beneficial technical effects of the present invention are embodied in the following aspects:

[0035] 1. Creativity of the aptamer screening method

[0036] The Staphylococcus aureus nucleic acid aptamer developed by this technology is a novel aptamer that combines with nanogold particles to form a colorimetric probe. After rigorous verification, its efficacy and practicality in effectively detecting Staphylococcus aureus have been proven. Although existing nucleic acid aptamers are still limited in quantity and effectiveness, the nucleic acid aptamer designed specifically for Staphylococcus aureus in this invention not only optimizes the binding efficiency with the bacterium, but also specifically considers its specific recognition ability for other bacteria that may coexist. This design is intended to ensure that the probe accurately recognizes Staphylococcus aureus, while avoiding nonspecific binding with other bacteria, thereby improving the detection specificity and accuracy of the probe.

[0037] Compared with the nucleic acid aptamer detection probes in the existing mature technology, the new Staphylococcus aureus aptamer probe we developed showed superior performance. This probe achieved a correlation coefficient of up to 0.98125, ensuring the accuracy of the detection, and had a low detection limit, capable of detecting strain concentrations as low as 21.08 CFU / mL. In contrast, the linear fit of the traditional fluorescence quenching detection method usually fluctuates between 0.82 and 0.96. Moreover, when we compared this technology with traditional methods such as strand displacement reaction, polymerase chain reaction or enzyme-linked immunosorbent assay, the latter required a detection time of up to 12 hours to 3 days, and they had specific cross-reactions for some common model strains. Our new probe not only shortened the detection time to only 50 minutes, but also more accurately distinguished common pathogens such as Staphylococcus aureus and Escherichia coli. The traditional strand displacement reaction method has an unsatisfactory reaction efficiency due to the intramolecular electrostatic repulsion caused by the negative charge of the single-stranded DNA phosphate group. In contrast, the aptamer probe of the present invention greatly improves the detection efficiency and specificity, presenting a significant technical advantage.

[0038] 2. Creativity of the method for preparing the fluorescence quenching probe of the present invention

[0039] The present invention uses magnesium chloride, a divalent cationic metal salt, as a salt-polymerizing factor for the first time, which has a better effect than the use of traditional sodium chloride monovalent salt ions. Selecting magnesium chloride from numerous cationic metal salts also requires creative work. This is because cationic surfactants are usually considered in the prior art, and there are few cationic metal salts used for fluorescence quenching detection of bacteria, and cationic surfactants may destroy the protein or polysaccharide structure on the bacterial surface. Generally, the secondary structure of nucleic acid aptamers is combined with the specific position of the protein or polysaccharide on the bacterial surface to achieve covalent coupling, thereby causing nucleic acid aptamers to be unable to be effectively combined with the bacterial surface. In addition, suitable salt ion concentration also affects whether the test can normally form the polymerization of nano gold, and whether nucleic acid aptamers can quickly dissociate from the surface of nano gold particles, which is also determined by the inherent characteristics of nucleic acid aptamers. Therefore, considering the above-mentioned various complex factors, this is not disclosed in the prior art, and it is also that the corresponding common knowledge in this area cannot obtain technical enlightenment.

[0040] After testing, the linear regression equation established under the standard environment can be directly applied to the detection of quick-frozen dumplings. There is no need to re-cultivate bacteria to establish a standard curve. You only need to directly apply the established linear regression equation, which saves time and is suitable for quickly estimating the initial concentration of Staphylococcus aureus in common samples under emergency conditions. For example, in quick-frozen dumplings, under the environment of artificial inoculation of Staphylococcus aureus in the quick-frozen dumplings, the concentration of Staphylococcus aureus measured by the detection probe is 0.98*10 6CFU / mL, almost the same as its real value of 1.00*10 6 The CFU / mL concentrations are close. It can be seen that even if the corresponding standard curve and linear regression equation are not established in the quick-frozen dumplings environment, the standard curve and linear regression equation established in the pure PBS sample environment can still have good versatility.

[0041] 4. The inventiveness of the present invention for detection

[0042] In summary, the Staphylococcus aureus nucleic acid aptamer prepared by the present invention is a new nucleic acid aptamer, which is combined with nano-gold particles to form a detection probe. It has been verified that the use effectiveness and feasibility of the Staphylococcus aureus nucleic acid aptamer prepared by the present invention are known. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a secondary structure diagram of the Staphylococcus aureus aptamer in the present invention;

[0044] Figure 2 A three-dimensional structural diagram of the membrane protein matched by the molecular docking of the Staphylococcus aureus aptamer in the present invention;

[0045] Figure 3 This is an application principle diagram of fluorescence quenching detection in the present invention;

[0046] Figure 4 It is a relative fluorescence intensity diagram of the nucleic acid aptamer and the nano-gold particles before and after covalent coupling in the fluorescence quenching detection of the present invention;

[0047] Figure 5 It is the ultraviolet absorption diagram of the nucleic acid aptamer and the nano-gold particles before and after covalent coupling in the fluorescence quenching detection of the present invention;

[0048] Figure 6 It is a relative fluorescence intensity diagram of Staphylococcus aureus of different concentrations detected by fluorescence quenching in the present invention;

[0049] Figure 7 It is a standard curve diagram of different concentrations of Staphylococcus aureus detected by fluorescence quenching in the present invention;

[0050] Figure 8 This is a specific detection diagram of various bacteria detected by fluorescence quenching in the present invention. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with specific embodiments.

[0052] Unless otherwise defined, 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.

[0053] The raw materials used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified; the quantitative tests in the following examples are all repeated three times, and the results are averaged; the % in the following examples are all mass percentages unless otherwise specified.

[0054] In the following examples, the synthesized aptamers were purchased from Shanghai Biotechnology Co., Ltd.; unless otherwise specified, other raw materials used were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0055] It should be noted that the phosphate buffer used in this application is a sterile PBS buffer with a pH value of 7.4 and a 0.1 M buffer.

[0056] It should be noted that all strains used in this application are purchased from the American Type Culture Collection (ATCC), of which Staphylococcus aureus is numbered ATCC 43251, Listeria monocytogenes is numbered ATCC43251, Salmonella typhimurium is numbered ATCC 14028, Escherichia coli is numbered ATCC25922, Cronobacter sakazakii is numbered ATCC 29544, and Pseudomonas aeruginosa is numbered ATCC15442. In addition, the above strains are purchased through overseas agents of Hefei Xiyue Biotechnology Co., Ltd.

[0057] It should be noted that the instruments, equipment, raw materials, reagents or method steps used in this application are ensured to be processed under sterile conditions.

[0058] It should be noted that the instruments, equipment, raw materials, reagents or method steps not mentioned in this application are conventional or well-known technical methods for those skilled in the art and will not be described in detail in this application.

[0059] Example 1

[0060] The sequence of the Staphylococcus aureus aptamer of the present invention is shown in SEQ ID No:1.

[0061] The Staphylococcus aureus aptamer is a nucleic acid aptamer, and the characterization information is as follows:

[0062] like Figure 1 As shown, its secondary structure is shown in the RNAStructure analysis diagram.

[0063] The binding test is based on the "Screening and identification of aptamers for Vibrio parahaemolyticus based on whole-bacteria SELEX technology_Lou Xiuqin" - 1.5.1-aptamer binding test, 1.5.3 affinity constant determination, write the method, replace this bacterium, its flow cell binding rate is 78.1%, and the result of affinity constant determination is 14.59nM, while the flow cell binding rates of Salmonella, Listeria monocytogenes, Escherichia coli, Cronobacter sakazakii and Pseudomonas aeruginosa are 7.8%, 17.3%, 9.2%, 10.4%, and 15.1%, respectively. Therefore, the nucleic acid aptamer effect we obtained is feasible and meets the use requirements. It is close to the highest level known to the public (such as SEQ ID No: 2 Source article DOI is:

[0064] https: / / doi.org / 10.1016 / j.talanta.2024.125624., the same test showed that its flow cytometric binding rate was 82.9%, and the result of affinity constant determination was 24.21nM).

[0065] In addition, the screening method of the sequence of Staphylococcus aureus aptamer is as follows:

[0066] The screening steps are as follows:

[0067] (1) Construction of the membrane protein structure of Staphylococcus aureus

[0068] Obtain the amino acid sequence and structural information of the membrane protein FnBPA of Staphylococcus aureus;

[0069] (2) Performing molecular docking test on the membrane protein of Staphylococcus aureus obtained in step (1)

[0070] The random design capacity is 10 14 A short-chain oligo DNA fragment library, wherein the length of the short-chain oligo DNA fragment ranges from 30 bp to 80 bp; importing the structural information of the membrane protein FnBPA protein of Staphylococcus aureus in step (1) into the AutoDock molecular simulation software, and then performing molecular docking between the short-chain oligo DNA fragment library and the surface domain of the membrane protein FnBPA protein to obtain the free energy of binding between each short-chain oligo DNA fragment and the membrane protein FnBPA protein;

[0071] (3) Optimize the molecular docking results obtained in step (2) using the ant colony algorithm model

[0072] The free energy of the above combination is optimized by combining the improved ant colony algorithm model, where the algorithm formula of the model is as follows:

[0073]

[0074] τ(i,j)=(1-ρ)·τ(i,j)+ρ·Δτ(i,j) k (II)

[0075] Where P ij (t) represents the transition probability value of ant k from node i to node j, τ ij (t) represents the pheromone concentration from node i to node j at time t, η ij (t) represents the constant heuristic function from node i to node j at time t, α represents the pheromone enhancement coefficient, β represents the heuristic information coefficient, l represents the specified position of the binding free energy of the short-chain oligoDNA fragment, τ il (t) represents the pheromone concentration from node i to node l at time t, η il (t) represents the constant heuristic function from node i to node l at time t, allowed i represents the set of nodes that node i is allowed to select next, τ(i,j) represents the sum of pheromones released by ant k from node i to node j, ρ represents the pheromone volatility coefficient (0<ρ<1), Δτ(i,j) k represents the increase in the amount of pheromone released by ant k from node i to node j;

[0076] According to the results of optimization processing, statistics P ij (t) The corresponding short-chain oligo DNA fragment was obtained to obtain the matching Staphylococcus aureus nucleic acid aptamer.

[0077] Example 2

[0078] The steps for using the Staphylococcus aureus aptamer of Example 1 to prepare a detection probe are as follows:

[0079] (1) Preparation of the first activation mixture

[0080] 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide were mixed at a volume ratio of 1:1 to obtain a first activation mixture.

[0081] (2) Preparation of the second activation mixture

[0082] Pure water was added to 3-(2-formylethyl)phosphine hydrochloride to obtain a 3-(2-formylethyl)phosphine hydrochloride solution with a final concentration of 2.867 g / L, ie, the second activated mixed solution.

[0083] (3) Preparation of fluorescent probes

[0084] See also Figure 3A in the mixture was added with 6 mL of deionized water to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; 8.0 mg of amikacin was added and stirred continuously for 30 min; the mixture was heated at 200°C for 6 h and cooled to room temperature; pure water was added to dilute the mixture 20 times to obtain a reaction solution; 500 uL of the reaction solution and 200 uL of the first activated mixed solution were vortexed and mixed, and the mixture was incubated on a shaker at 180 rpm and room temperature for 30 min; 180 uL of the first aptamer solution with a volume concentration of 10 umol / L was added and vortexed and mixed, and the mixture was incubated on a shaker at 180 rpm and room temperature for 2.5 h to obtain a fluorescent probe, which was then wrapped in tin foil and stored at 4°C in a dark place.

[0085] The DNA sequence of the first aptamer in the first aptamer solution is shown as SEQ ID No: 1, and the 5' end of the first aptamer is modified with a carboxyl group.

[0086] (4) Preparation of quenching probe

[0087] See also Figure 3 In B, the specific operations are as follows:

[0088] (4.1) Add 1 mL of 1% tetrachloroauric acid solution to 100 mL of distilled water and boil. Quickly add 3 mL of 1% trisodium citrate solution and continue boiling for 15 min. Stop boiling and cool naturally to room temperature to obtain a gold nanoparticle solution.

[0089] (4.2) Place 80 μL of a 10 μM second aptamer solution in a water bath at 95°C for 5 min, and cool at 4°C for 5 min to obtain an annealing solution; add 10 μL of a 2.867 g / L 3-(2-formylethyl)phosphine hydrochloride solution to the annealing solution, and allow the reaction to stand at room temperature for 30 min to obtain a reaction solution; add 1 mL of a gold nanoparticle solution to the reaction solution, incubate at 37°C for 12 h, and centrifuge at 10,000 rpm for 20 min, remove the supernatant, and obtain a precipitate.

[0090] The DNA sequence of the second aptamer in the second aptamer solution is shown in SEQ ID NO: 2, and the 5' end of the second aptamer is modified with a thiol group.

[0091] (4.3) Add 500 μL of PBS solution with a mass concentration of 0.1 M and a pH value of 7.4 to the precipitate, and store it at 4°C wrapped in tin foil and protected from light to prepare a quenching probe;

[0092] (5) Preparation of biosensor

[0093] The fluorescent probe and the quenching probe are mixed in a volume ratio of 1:2 to prepare a fluorescence quenching biosensor;

[0094] See also Figure 4 , Figure 4 Curve A in FIG. 1 is a graph showing the fluorescence emission wavelength of silicon nanoparticles that have not been modified with the first aptamer. Figure 4 Curve B in FIG. 1 is a graph of the fluorescence emission wavelength of silicon nanoparticles modified with the second aptamer. When the fluorescence emission wavelength of the silicon nanoparticles shifts from 520 nm to 524 nm, it indicates that the first aptamer in the fluorescence quenching biosensor is successfully modified onto the silicon nanoparticles.

[0095] See also Figure 5 , Figure 5 Curve A in FIG. 1 is the UV absorption graph of gold nanoparticles without modification by the second aptamer. Figure 5 Curve B in FIG. 1 is the UV absorption graph of gold nanoparticles decorated with the second aptamer. When the UV absorption wavelength of the gold nanoparticles shifts from 521 nm to 525 nm, it indicates that the second aptamer in the fluorescence quenching biosensor is successfully modified onto the gold nanoparticles.

[0096] (6) The specific detection steps are as follows:

[0097] Establishment of linear regression equation: prepare a test solution of the test sample without bacteria, set it as a blank control test solution, and artificially contaminate the blank control test solution with Staphylococcus aureus to set a test solution containing bacteria in a linear gradient dilution.

[0098] The fluorescence quenching biosensor was mixed with the blank control test solution and the bacteria-containing test solution at a volume ratio of 1:1, and incubated for 35 minutes to obtain an incubation solution.

[0099] Under an excitation wavelength of 435 nm, the fluorescence intensity of the incubation solution at an emission wavelength of 525 nm was measured, wherein the fluorescence intensity of the blank control test solution was recorded as F0, and the fluorescence intensity of the bacteria-containing test solution was recorded as F; a standard curve was plotted with the fluorescence intensity ratio F / F0 as the ordinate and the logarithm of the bacterial concentration of the bacteria-containing test solution as the abscissa to obtain a linear regression equation, and the correlation coefficient and detection limit were obtained.

[0100] Detection of Staphylococcus aureus in the test sample: prepare a test solution of the test sample, mix the fluorescence quenching biosensor and the test solution at a volume ratio of 1:1, incubate for 35 minutes, and obtain an incubation solution; measure the fluorescence intensity of the incubation solution at an emission wavelength of 525 nm under an excitation wavelength of 430 nm.

[0101] The obtained fluorescence intensity ratio F1 / F0 is substituted into the linear regression equation obtained in step (2) to obtain the concentration of Staphylococcus aureus in the sample to be tested.

[0102] During the experiment, PBS buffer was selected as the blank control test solution. At the same time, the bacterial solution of Staphylococcus aureus was centrifuged and washed three times with PBS buffer, and then resuspended and gradiently diluted with PBS buffer to make concentrations of 10 0 CFU / mL, 10 1 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL bacteria-containing test solution. First, take 100uL of the prepared fluorescence quenching biosensor, and incubate with 100uL of blank control test solution and 100uL of gradient diluted bacteria-containing test solution at 37°C for 35min to obtain an incubation solution. Then, under an excitation wavelength of 435nm, measure the fluorescence intensity of the incubation solution at an emission wavelength of 525nm, where the fluorescence intensity of the blank control test solution is recorded as F0, and the fluorescence intensity of the bacteria-containing test solution is recorded as F; use the fluorescence intensity ratio F / F0 as the ordinate and the logarithm of the bacterial solution concentration of the bacteria-containing test solution as the abscissa to draw a standard curve, obtain a linear regression equation, and obtain the correlation coefficient and detection limit.

[0103] The fluorescent quenching biosensor prepared by the present invention has a fluorescent probe with an obvious fluorescence emission peak at 525nm, and the ratio of the fluorescence intensity value F at 525nm to the fluorescence intensity value F0 at 525nm is linearly related to the logarithm of the bacterial solution concentration of Staphylococcus aureus.

[0104] like Figure 7 As shown, after measurement, the linear regression equation is y = -0.10088x + 1.01777, and its correlation coefficient (r 2 ) was 0.99976, and its detection limit was 2.608 CFU / mL.

[0105] Next, conduct specific tests:

[0106] The fluorescence intensity ratios F / F0 of the bacteria-containing test solutions after adding Listeria monocytogenes, Salmonella, Escherichia coli, Cronobacter sakazakii and Pseudomonas aeruginosa are 0.92147, 0.99403, 0.98155, 0.978393 and 0.94768, respectively. At the same time, the fluorescence intensity ratio F / F0 of the bacteria-containing test solution after adding Staphylococcus aureus under the same conditions is 0.4122. In addition, the fluorescence intensity ratio F0 / F0 of the blank control test solution under the same conditions is 1. This shows that the fluorescence quenching biosensor prepared by the present invention has good specificity for Staphylococcus aureus.

[0107] Example 3

[0108] On the basis of Example 2, the detection of real food samples was added, and the prepared food quick-frozen dumplings and the Staphylococcus aureus nucleic acid aptamer designed by the present invention were selected for testing:

[0109] It should be noted that in order to verify whether the above-mentioned standard curve can be effectively applied to the determination in an interference environment (quick-frozen dumplings), the difference between the number of bacteria detected by the probe in artificial quantitative contamination and quick-frozen dumplings was carried out to find out that even in real samples, the detection probe established by the present invention can effectively perform detection under the influence of nutrients such as high protein and high fat.

[0110] It should be noted that the quick-frozen dumplings involved in this embodiment were purchased from a domestic supermarket, and their ingredients are as follows: energy 164kcal / 100g, fat 7.7g / 100g, protein 10.2g / 100g, carbohydrates 16.8g / 100g, and sodium 585mg / 100g.

[0111] During the experiment, Staphylococcus aureus was artificially inoculated in 1 mL of LB broth, and the final concentration of Staphylococcus aureus after inoculation was 10 6 CFU / mL, centrifuge at 5000×g for 5 min, discard the supernatant, resuspend the precipitate with 1 mL of PBS buffer as the bacteria-containing test solution, then take 500uL of the test solution and mix it with 500uL of the prepared fluorescence quenching biosensor, incubate for 35 min to obtain an incubation solution, then measure the fluorescence intensity of the incubation solution at an emission wavelength of 525nm under an excitation wavelength of 425nm, wherein the fluorescence intensity of the blank control test solution is recorded as F0, and the fluorescence intensity of the bacteria-containing test solution is recorded as F, finally, according to the obtained fluorescence intensity ratio F / F0, it is substituted into the linear regression equation obtained above to obtain the concentration of Staphylococcus aureus in the test sample.

[0112] The test results show that, according to calculations, in the environment of artificial inoculation of Staphylococcus aureus in quick-frozen dumplings, the concentration of Staphylococcus aureus measured by the detection probe is 0.98*10 6 CFU / mL, almost the same as its actual concentration (1.0*10 6 CFU / mL) is close. It can be seen that even if the corresponding standard curve and linear regression equation are not established in the quick-frozen dumplings environment, the standard curve and linear regression equation established in the pure PBS sample environment can still have good versatility. It can be seen that the detection probe prepared by the present invention has good versatility and can be quickly applied to different sample detections.

[0113] It is easy for those skilled in the art to understand that the above embodiments 1-3 are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Staphylococcus aureus aptamer, characterized in that: The DNA sequence of the Staphylococcus aureus aptamer is shown in SEQ ID No: 1; The structure of the membrane protein of Staphylococcus aureus was constructed and a molecular docking test was performed. At the same time, the results of the molecular docking were optimized by the ant colony algorithm model, and the corresponding short-chain oligoDNA fragments were counted to obtain the matching nucleic acid aptamer of Staphylococcus aureus.

2. A method for preparing a detection probe using the Staphylococcus aureus aptamer according to claim 1, characterized in that: The steps are as follows: (1) Preparation of the first activation mixture Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1 to obtain a first activation mixture; (2) Preparation of the second activation mixture Adding pure water to 3-(2-formylethyl)phosphine hydrochloride to obtain a 3-(2-formylethyl)phosphine hydrochloride solution with a final concentration of 2.867 g / L, i.e., the second activated mixed solution; (3) Preparation of fluorescent probes 6 mL of deionized water was added to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; 8.0 mg of amikacin was added and stirred continuously for 30 min; the mixture was heated at 200°C for 6 h and cooled to room temperature; pure water was added to dilute the mixture 20 times to obtain a reaction solution; 500 uL of the reaction solution and 200 uL of the first activation mixture were vortexed and mixed, and incubated on a shaker at 180 rpm and room temperature for 30 min; 180 uL of the first aptamer solution with a volume concentration of 10 umol / L was added and vortexed, and incubated on a shaker at 180 rpm and room temperature for 2.5 h to obtain a fluorescent probe, which was then wrapped in tin foil and stored at 4°C in a dark place; The DNA sequence of the first aptamer in the first aptamer solution is as shown in SEQ ID No: 1, and the 5' end of the first aptamer is modified with a carboxyl group; (4) Preparation of quenching probe (4.1) Add 1 mL of 1% tetrachloroauric acid solution to 100 mL of distilled water and boil. Quickly add 3 mL of 1% trisodium citrate solution and continue boiling for 15 min. Cool naturally to room temperature to obtain a gold nanoparticle solution. (4.2) Place 80 μL of the second aptamer solution with a volume concentration of 10 μM in a water bath at 95°C for 5 min, and cool at 4°C for 5 min to obtain an annealing solution; add 10 μL of 3-(2-formylethyl)phosphine hydrochloride solution with a mass volume concentration of 2.867 g / L to the annealing solution, and allow to react at room temperature for 30 min to obtain a reaction solution; add 1 mL of the gold nanoparticle solution to the reaction solution, incubate at 37°C for 12 h, and centrifuge at 10,000 rpm for 20 min, remove the supernatant, and obtain a precipitate; The DNA sequence of the second aptamer in the second aptamer solution is as shown in SEQ ID NO: 2, and the 5' end of the second aptamer is modified with a thiol group; (4.3) Add 500 μL of 0.1 M PBS solution with a pH of 7.4 to the precipitate, and store it at 4°C wrapped in tin foil and protected from light to prepare a quenching probe; (5) Preparation of biosensors The fluorescent probe and the quenching probe are mixed in a volume ratio of 1:2 to prepare a fluorescence quenching biosensor; When the fluorescence emission wavelength of silicon nanoparticles shifts from 520nm to 524nm, it indicates that the first aptamer in the fluorescence quenching biosensor is successfully modified onto the silicon nanoparticles. When the ultraviolet absorption wavelength of gold nanoparticles shifts from 521nm to 525nm, it indicates that the second aptamer in the fluorescence quenching biosensor is successfully modified onto the gold nanoparticles.

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