A Staphylococcus aureus aptamer and a preparation method thereof for a detection probe

Abstract

The present invention provides an original Staphylococcus aureus nucleic acid aptamer and its detailed screening and preparation process, aiming to improve the detection accuracy and efficiency of this common and dangerous strain. The unique DNA sequence (SEQ ID No: 1) of the aptamer was obtained through a series of complex bioinformatics and computational processes, including using the molecular simulation software AutoDock to conduct docking experiments on the evolved oligonucleotide fragments and calculating the affinity between each short-chain fragment and the target staphylococcal membrane protein FnBPA. Next, these data were optimized through the ant colony algorithm to identify the sequence with the highest binding efficiency. After obtaining the aptamer sequence with high affinity, an aptamer-based biosensor was developed. The construction process of the sensor includes the mixing of activated reagents and the preparation process of fluorescent probes and quenching probes, ultimately forming a sensitive and specific fluorescence quenching probe. The changes in fluorescence emission wavelength and ultraviolet absorption wavelength indicate the successful immobilization of the aptamer, thus providing a rapid and reliable method for detecting Staphylococcus aureus. This method has very important application prospects in fields such as food safety monitoring and medical diagnosis, can significantly reduce the time required by traditional microbial culture and other means, and at the same time provide a lower-cost option compared to molecular biology techniques.

Description

Technical Field

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

[0002] In recent years, the impact of foodborne pathogenic microorganism contamination on food safety has become increasingly prominent, posing a great threat to people's health. Staphylococcus aureus (Staphylococcus aure, S. aure) is an important foodborne pathogenic microorganism and a zoonotic pathogen. Staphylococcal food poisoning is caused by ingesting dairy products, meats, and egg products contaminated with enterotoxins. S. aure is widely present in nature. Approximately 20% - 30% of the general population are carriers of Staphylococcus aureus. It has the characteristics of facultative anaerobism, high temperature resistance, and high salt tolerance, and is easy to survive. It spreads with the activities of humans and animals, threatening the lives of humans and animals. According to estimates by the World Health Organization, approximately 600 million people worldwide get sick each year due to eating contaminated food, and approximately 420,000 people die. The unreasonable use, and even abuse, of antibiotics is particularly prominent, leading to an increasing resistance of strains. It is reported that methicillin-resistant Staphylococcus aureus infections have been listed among the three major intractable infectious diseases worldwide, along with hepatitis B virus and acquired immunodeficiency syndrome infections. Therefore, the rapid detection of Staphylococcus aureus and its biomarkers in food is an important measure to ensure food safety.

[0003] Currently, the traditional detection methods for Staphylococcus aureus mainly include bacterial enrichment culture, morphological identification, biochemical identification, etc. The entire identification process takes 3 - 5 days and is complex to operate; the detection time of the fluorescence polymerase chain reaction detection method is usually more than 1 hour, and the equipment is expensive, with a high detection cost.

[0004] Compared with traditional detection methods, the detection sensing system established by binding aptamers to 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. An aptamer refers to an oligonucleotide fragment that can bind to a specific target with high affinity and specificity and can bind to the target substance. The emerging fluorescence biosensor detection method has been widely used in the determination of target substances due to its low cost, simple operation, fast response, short time consumption, high stability, and no dependence on large instruments. Among them, fluorescence resonance energy transfer (FRET, Resonance energy transfer), as a non-radiative energy transition, transfers the energy of the donor excited state to the acceptor excited state through the intermolecular electric dipole interaction and is widely used in fluorescence probe detection technology. There is relatively little research on silicon nanoparticles, which are often prepared by catalytic reduction with a silicon-containing coupling agent. Most of the methods based on fluorescence resonance energy transfer are achieved by shortening the distance between the donor and the acceptor. For example, energy transfer is carried out by the proximity of the ratio-type fluorescence probes coupled on the surface of microbial cells. Ratio-type fluorescence probes have the advantages of high sensitivity, simple operation, short response time to the target, etc. Currently, it is necessary to select a suitable first fluorescence probe that can act as an energy donor and a suitable second fluorescence probe that can act 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, and it is easy to occur that the entire surface of the cell is occupied by one of the fluorescence probes, resulting in the inability to use the other fluorescence probe. Summary of the Invention

[0005] Aiming at the problems of traditional Staphylococcus aureus detection methods, such as long time consumption, complex operation, expensive equipment, and high detection cost, the present invention provides a rapid detection method based on the principle of fluorescence quenching; aiming at the problem that the failure rate of the double aptamer sandwich method is relatively high, new nucleic acid aptamers need to be used in combination, and it is easy to occur that the entire surface of the cell is occupied by one of the fluorescence probes, resulting in the inability to use the other fluorescence probe, the present invention screens a nucleic acid aptamer, namely the first aptamer, targeting the membrane protein FnBPA protein of Staphylococcus aureus based on technologies such as ant colony algorithm and molecular docking. Its target is inconsistent with the target of the second aptamer, based on the aptamer and its fluorescence quenching detection method.

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

[0007] The screening operation steps are as follows:

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

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

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

[0011] Randomly design a capacity of 10 14A library of short-chain oligonucleotide DNA fragments, where the length range of the short-chain oligonucleotide DNA fragments is 30 bp - 80 bp; import the structural information of the membrane protein FnBPA protein of Staphylococcus aureus in step (1) into the AutoDock molecular simulation software, and then perform molecular docking between the short-chain oligonucleotide DNA fragment library and the surface domain of the membrane protein FnBPA protein to obtain the free energy of binding between each short-chain oligonucleotide DNA fragment and the membrane protein FnBPA protein;

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

[0013] Optimize the above-mentioned binding free energy using 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] In the formula, 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 oligonucleotide DNA 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 in the next step, τ(i,j) represents the sum of the pheromones released by ant k passing through node i to node j, ρ represents the pheromone evaporation coefficient (0 < ρ < 1), Δτ(i,j) k represents the increase in the pheromone released by ant k passing through node i to node j;

[0017] According to the results of the optimization process, count the short-chain oligonucleotide DNA fragments corresponding to P ij (t) to obtain the nucleic acid aptamer of the matching Staphylococcus aureus.

[0018] A method for preparing a detection probe based on the Staphylococcus aureus aptamer described above, characterized in that the operation steps are as follows:

[0019] 1) Prepare the first activation mixture

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

[0021] (2) Prepare a second activation mixture

[0022] Add 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, which is the second activation mixture;

[0023] (3) Prepare a fluorescent probe

[0024] Add 6 mL of deionized water to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; then add 8.0 mg of amiphenol and stir continuously for 30 min; heat at 200 °C for 6 h and cool to room temperature; dilute with pure water by 20 times to obtain a reaction solution; vortex-mix 500 μL of the reaction solution and 200 μL of the first activation mixture, incubate on a shaker at 180 rpm at room temperature for 30 min; then add 180 μL of a first aptamer solution with a volume concentration of 10 μmol / L and vortex-mix, incubate on a shaker at 180 rpm at room temperature for 2.5 h to prepare a fluorescent probe, and store it at 4 °C wrapped in tin foil in the dark;

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

[0026] (4) Prepare a quenching probe

[0027] (4.1) Add 1 mL of a 1% mass concentration of chloroauric acid solution to 100 mL of distilled water and boil; quickly add 3 mL of a 1% mass concentration of trisodium citrate solution and continue boiling for 15 min, and naturally cool to room temperature to obtain a gold nanoparticle solution;

[0028] (4.2) Heat 80 μL of a 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 a 3-(2-formylethyl)phosphine hydrochloride solution with a mass volume concentration of 2.867 g / L to the annealing solution, and let it stand and 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, centrifuge at 10000 rpm for 20 min, and remove the supernatant to obtain a precipitate;

[0029] 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;

[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 with tinfoil to avoid light to obtain the quenched probe;

[0031] (5) Prepare the biosensor

[0032] Mix the fluorescent probe and the quenched probe according to a volume ratio of 1:2 to obtain a fluorescence quenching type biosensor;

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

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

[0035] 1. Creativity of the nucleic acid aptamer screening method

[0036] The Staphylococcus aureus nucleic acid aptamer developed by this technology is a novel aptamer. It binds to gold nanoparticles to form a colorimetric probe. After strict verification, its efficacy and practicality in effectively detecting Staphylococcus aureus have been proven. Although the existing nucleic acid aptamers have limitations in terms of quantity and effectiveness, the nucleic acid aptamer specifically designed for Staphylococcus aureus in the present invention not only optimizes the binding efficiency with this bacterium, but also particularly considers its specific recognition ability for other potentially co-existing bacteria. This design aims to ensure the accurate recognition of Staphylococcus aureus by the probe, while avoiding non-specific binding with other bacteria, thereby improving the detection specificity and accuracy of the probe.

[0037] Compared with the nucleic acid aptamer detection probes in existing mature technologies, the novel Staphylococcus aureus aptamer probe we developed exhibits superior performance. This probe achieves a correlation coefficient as high as 0.98125, ensuring the accuracy of detection, and has a low detection limit, capable of detecting strain concentrations as low as 21.08 CFU / mL. In contrast, the linear fitting degree of traditional fluorescence quenching detection methods usually fluctuates between 0.82 and 0.96. Moreover, when we compare this technology with traditional methods such as strand displacement reaction, polymerase chain reaction, or enzyme-linked immunosorbent assay, the latter require detection times ranging from 12 hours to 3 days, and they exhibit specific cross-reactions with some common model strains. Our novel probe not only shortens the detection time to only 50 minutes but also can more accurately distinguish common pathogenic bacteria such as Staphylococcus aureus and Escherichia coli. Due to the intramolecular electrostatic repulsion caused by the negatively charged phosphate groups of single-stranded DNA, the reaction efficiency of traditional strand displacement reaction methods is not ideal. In contrast, the aptamer probe of the present invention greatly improves the detection efficiency and specificity, presenting significant technical advantages.

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

[0039] The present invention first uses magnesium chloride, a divalent cation metal salt, as a salt aggregation factor, which has a better effect than the use of traditional monovalent salt ions such as sodium chloride. Selecting magnesium chloride from numerous cationic metal salts also requires creative labor because in existing technologies, cationic surfactants are usually considered, and there is rarely the use of cationic metal salts for fluorescence quenching detection of bacteria. Cationic surfactants may damage the protein or polysaccharide structure on the surface of bacteria. Generally, the secondary structure of nucleic acid aptamers achieves covalent coupling by binding to specific positions of proteins or polysaccharides on the surface of bacteria, resulting in the inability of nucleic acid aptamers to effectively bind to the surface of bacteria. In addition, the appropriate salt ion concentration also affects whether nanoparticles can be normally formed in the experiment, and the problem of whether nucleic acid aptamers can quickly dissociate from the surface of gold nanoparticles also depends on the inherent characteristics of nucleic acid aptamers. Therefore, considering all these complex factors, this is not disclosed in existing technologies and cannot be obtained as a technical inspiration from the corresponding common general knowledge in this field.

[0040] After testing, the linear regression equation established under standard conditions can be directly applied to the detection of quick-frozen dumplings without the need to re-culture bacteria to establish a standard curve. Just directly apply the already established linear regression equation, which saves time and is suitable for quickly estimating the initial concentration of Staphylococcus aureus in common samples in emergency situations. For example, in quick-frozen dumplings, in an environment where Staphylococcus aureus is artificially inoculated in quick-frozen dumplings, the concentration of Staphylococcus aureus measured by the detection probe is 0.98*10 6CFU / mL, which is almost the same as its true concentration of 1.00*10 6 CFU / mL. It can be seen that even without establishing the corresponding standard curve and linear regression equation in the environment of quick-frozen dumplings, the standard curve and linear regression equation established in the environment of pure PBS samples still have good versatility.

[0041] 4. Creativity of the Invention for Detection

[0042] Generally speaking, the Staphylococcus aureus nucleic acid aptamer prepared by the present invention is a new nucleic acid aptamer, which is combined with gold nanoparticles to form a detection probe. After verification, the 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 It is the secondary structure diagram of the Staphylococcus aureus aptamer in the present invention;

[0044] Figure 2 It is the three-dimensional structure diagram of the membrane protein that is molecularly docked and matched with the Staphylococcus aureus aptamer in the present invention;

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

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

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

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

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

[0050] Figure 8 It is the specific detection diagram of multiple bacteria for fluorescence quenching detection in the present invention. Detailed Embodiments

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

[0052] Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

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

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

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

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

[0057] It should be reminded that all the instrument equipment, raw material reagents or method steps used in this application are ensured to be processed under sterile conditions.

[0058] It should be reminded that the instrument equipment, raw material reagents or method steps not mentioned in this application belong to conventional or well-known technical methods for those skilled in the art and will not be elaborated 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 its characterization information is as follows:

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

[0063] Among them, the binding experiment refers to "Screening and Identification of Vibrio parahaemolyticus Aptamers Based on Whole Bacterium SELEX Technology - Lou Xiuqin" - Replace the method in its 1.5.1 - aptamer binding experiment and 1.5.3 affinity constant determination with this bacterium. The binding rate of flow cytometry is 78.1%, and the result of affinity constant determination is 14.59 nM. At the same time, the binding rates of flow cytometry with 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 we obtained has a feasible effect and meets the usage requirements. It is close to the highest known public level (such as the article DOI of SEQ ID No:2 is:

[0064] https: / / doi.org / 10.1016 / j.talanta.2024.125624., and through the same test, it is known that the binding rate of flow cytometry is 82.9%, and the result of affinity constant determination is 24.21 nM).

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

[0066] The screening operation steps are as follows:

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

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

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

[0070] Randomly design a short-chain oligonucleotide DNA fragment library with a capacity of 10 14 , and the length range of the short-chain oligonucleotide DNA fragments is 30bp - 80bp; Import the structural information of the membrane protein FnBPA of Staphylococcus aureus in step (1) into the AutoDock molecular simulation software, and then conduct molecular docking between the short-chain oligonucleotide DNA fragment library and the surface domain of the membrane protein FnBPA to obtain the free energy of binding between each short-chain oligonucleotide DNA fragment and the membrane protein FnBPA;

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

[0072] Combine the improved ant colony algorithm model to optimize the above-mentioned binding free energy, and 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] In the formula, 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 oligonucleotide DNA 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 the pheromones released by ant k from node i to node j, ρ represents the pheromone evaporation coefficient (0 < ρ < 1), Δτ(i,j) k represents the increase in the pheromone released by ant k from node i to node j;

[0076] According to the results of the optimization process, count the short-chain oligonucleotide DNA fragments corresponding to P ij (t) to obtain the nucleic acid aptamer of the matched Staphylococcus aureus.

[0077] Example 2

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

[0079] (1) Prepare the first activation mixture

[0080] Mix 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride with a concentration of 10 mM and N-hydroxysuccinimide with a concentration of 5 mM in a volume ratio of 1:1 to obtain the first activation mixture.

[0081] (2) Prepare the second activation mixture

[0082] Add 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, which is the second activation mixture.

[0083] (3) Prepare the fluorescent probe

[0084] See Figure 3For A in [the relevant context], add 6 mL of deionized water to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; then add 8.0 mg of amiphenol and stir continuously for 30 min; heat at 200 °C for 6 h, and cool to room temperature; dilute with pure water by 20 times to obtain a reaction solution; vortex-mix 500 μL of the reaction solution and 200 μL of the first activation mixture, incubate on a shaker at 180 rpm and room temperature for 30 min; then add 180 μL of the first aptamer solution with a volume concentration of 10 μmol / L, vortex-mix, and incubate on a shaker at 180 rpm and room temperature for 2.5 h to prepare a fluorescent probe, and store it in the dark at 4 °C wrapped with tin foil.

[0085] 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.

[0086] (4) Preparation of quenching probe

[0087] See Figure 3 in B of [the relevant context], the specific operations are as follows:

[0088] (4.1) Add 1 mL of a 1% mass concentration of chloroauric acid solution to 100 mL of distilled water and boil; quickly add 3 mL of a 1% mass concentration of trisodium citrate solution, continue to boil for 15 min, stop boiling, and cool naturally to room temperature to obtain a gold nanoparticle solution.

[0089] (4.2) Heat 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 a 3-(2-formylethyl)phosphine hydrochloride solution with a mass-volume concentration of 2.867 g / L to the annealing solution, and let it stand and 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, centrifuge at 10000 rpm for 20 min, and remove the supernatant to obtain a precipitate.

[0090] 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 mercapto group.

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

[0092] (5) Preparation of biosensor

[0093] Mix the fluorescent probe and the quenching probe according to a volume ratio of 1:2 to prepare a fluorescence quenching type biosensor;

[0094] See Figure 4 , Figure 4 In Figure 4 , curve A is the fluorescence emission wavelength map of silicon nanoparticles without the modification of the first aptamer. Figure 4 In Figure 4 , curve B is the fluorescence emission wavelength map 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 Figure 5 , Figure 5 In Figure 5 , curve A is the ultraviolet absorption map of gold nanoparticles without the modification of the second aptamer. Figure 5 In Figure 5 , curve B is the ultraviolet absorption map of gold nanoparticles modified with the second aptamer. When the ultraviolet 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 operation steps are as follows:

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

[0098] Mix the fluorescence quenching biosensor with the blank control test solution and the test solutions containing bacteria at a volume ratio of 1:1, incubate for 35 min to obtain the incubation solution.

[0099] Measure the fluorescence intensity of the incubation solution at the emission wavelength of 525 nm under the excitation wavelength of 435 nm, where the fluorescence intensity of the blank control test solution is denoted as F 0 , and the fluorescence intensity of the test solution containing bacteria is denoted as F; Use the fluorescence intensity ratio F / F 0 as the ordinate and the logarithm of the bacterial concentration of the test solution containing bacteria as the abscissa to plot the standard curve, obtain the linear regression equation, and obtain the correlation coefficient and the detection limit.

[0100] Detection of Staphylococcus aureus in the sample to be tested: Prepare the test solution of the sample to be tested, mix the fluorescence quenching biosensor with the test solution at a volume ratio of 1:1, incubate for 35 min to obtain the incubation solution; Measure the fluorescence intensity of the incubation solution at the emission wavelength of 525 nm under the excitation wavelength of 430 nm.

[0101] According to the obtained fluorescence intensity ratio F 1 / F 0 , substitute it 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 serially diluted with PBS buffer to prepare test solutions containing bacteria with 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. First, 100 μL of the prepared fluorescence quenching biosensor was taken and incubated with 100 μL of the blank control test solution and 100 μL of the serially diluted test solution containing bacteria at 37 °C for 35 min to obtain the incubation solution. Then, the fluorescence intensity of the incubation solution was measured at an emission wavelength of 525 nm under an excitation wavelength of 435 nm. The fluorescence intensity of the blank control test solution was denoted as F 0 , and the fluorescence intensity of the test solution containing bacteria was denoted as F; the ratio of fluorescence intensities F / F 0 was used as the ordinate, and the logarithm of the bacterial solution concentration of the test solution containing bacteria was used as the abscissa to plot a standard curve, obtain a linear regression equation, and obtain the correlation coefficient and detection limit.

[0103] For the fluorescence quenching biosensor prepared by the present invention, the fluorescent probe has an obvious fluorescence emission peak at 525 nm. At the same time, the ratio between the fluorescence intensity value F at 525 nm and the fluorescence intensity value F 0 at 525 nm has a linear relationship with the logarithm of the bacterial solution concentration of Staphylococcus aureus.

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

[0105] Then, a specificity test was carried out:

[0106] For the test solutions containing bacteria after adding Listeria monocytogenes, Salmonella, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa, the ratios of the fluorescence intensity ratios F / F 0 were 0.92147, 0.99403, 0.98155, 0.978393, and 0.94768, respectively. At the same time, for the test solution containing bacteria after adding Staphylococcus aureus under the same conditions, the fluorescence intensity ratio F / F 0The ratio between them is 0.4122. In addition, for the blank control test solution under the same conditions, the fluorescence intensity ratio F 0 / F 0 The ratio between them is 1. From this, it can be shown that the fluorescence quenching type 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. Quick-frozen dumplings, a kind of prepared food, and the Staphylococcus aureus nucleic acid aptamer designed by the present invention were selected for testing:

[0109] It should be reminded that in this example, in order to verify whether the above standard curve can be effectively applied to the determination in an interfering environment (quick-frozen dumplings), the differences between the artificially quantitatively inoculated bacteria and the number of bacteria detected by the probe in the quick-frozen dumplings were respectively carried out, so as to know that even in real samples, the detection probe established by the present invention can effectively detect 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 example were purchased from a certain domestic supermarket, and its composition is as follows: energy 164 kcal / 100 g, fat 7.7 g / 100 g, protein 10.2 g / 100 g, carbohydrate 16.8 g / 100 g, sodium 585 mg / 100 g.

[0111] During the experiment, Staphylococcus aureus was artificially inoculated into 1 mL of LB broth, and the final concentration of Staphylococcus aureus after inoculation was 10 6 CFU / mL. It was centrifuged at 5000×g for 5 min, the supernatant was discarded, and the precipitate was resuspended with 1 mL of PBS buffer to obtain a bacteria-containing test solution. Then, 500 μL of the test solution was mixed with 500 μL of the prepared fluorescence quenching type biosensor and incubated for 35 min to obtain an incubation solution. Then, the fluorescence intensity of the incubation solution at an emission wavelength of 525 nm was measured at an excitation wavelength of 425 nm. The fluorescence intensity of the blank control test solution was recorded as F 0 , and the fluorescence intensity of the bacteria-containing test solution was recorded as F. Finally, according to the obtained fluorescence intensity ratio F / F 0 , substituting it into the above-obtained linear regression equation, the concentration of Staphylococcus aureus in the test sample was obtained.

[0112] The test results showed that after calculation, in the environment of artificially inoculating Staphylococcus aureus in quick-frozen dumplings, the concentration of Staphylococcus aureus measured by the detection probe was 0.98*10 6 CFU / mL, which was almost the same as its true concentration (1.0*10 6CFU / mL), it can be seen that even without establishing the corresponding standard curve and linear regression equation in the environment of quick-frozen dumplings, the standard curve and linear regression equation established in the environment of pure PBS samples still have good versatility. Thus, it can be seen that the detection probe prepared by the present invention has good versatility and can be quickly applied to the detection of different samples.

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

2. A method for preparing a detection probe for 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 to protect from light to obtain a quenching probe.

3. The method for preparing the detection probe according to claim 2, characterized in that: Further preparing a biosensor, mixing the fluorescent probe and the quenching probe at 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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