A preparation method of Salmonella typhimurium aptamer and colorimetric probe thereof

Abstract

The invention discloses a preparation method of a Salmonella typhimurium aptamer and a colorimetric probe thereof, and belongs to the technical field of food safety. The DNA sequence of the Salmonella typhimurium aptamer is shown in SEQ ID No: 1. The steps of using the Salmonella typhimurium aptamer for the preparation of the colorimetric probe are as follows: (1) preparing a gold nanoparticle solution with tetrachloroauric acid solution and distilled water; (2) preparing a Salmonella typhimurium aptamer solution with distilled water; (3) mixing the gold nanoparticle solution and the Salmonella typhimurium aptamer solution in a volume ratio of 5:1.8 to prepare a colorimetric probe solution; the ultraviolet absorption wavelength of the gold nanoparticles modified by the nucleic acid aptamer in the colorimetric probe solution is 523nm. It has been verified that compared with the existing mature nucleic acid aptamers, the colorimetric probe prepared by the invention has a higher linear fitting degree and a lower detection limit; at the same time, the screening method is simple and convenient, and rapid screening can be achieved by only building a simple server.

Description

Technical Field

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

[0002] Salmonella enterica serovar Typhimurium is a common pathogen that can cause murine typhoid fever and pose a threat to human and animal health. In order to effectively detect and treat Salmonella enterica serovar Typhimurium infection, researchers are committed to developing new strategies and technologies, among which the screening method of nucleic acid aptamers has become one of the research hotspots.

[0003] Nucleic acid aptamers are a class of nucleic acid molecules that can bind to specific target molecules with high specificity. They are usually composed of oligonucleotides (oligonucleic acids, such as DNA or RNA), functional nucleotides obtained by screening from random libraries, and are generally about 40 to 60 bp in size. They have some special secondary structures, such as hairpins, stem loops, pseudoknots, G-quadruplexes, etc., which allow aptamers to form a variety of tertiary structures, enabling them to achieve specific binding and recognition with targets. Nucleic acid aptamers have a wide range of applications, including biosensors, drug delivery, molecular diagnosis and treatment.

[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, time-saving, and does not rely on large instruments, but also has good sensitivity, and is expected to become a new trend in the field of rapid detection. Under normal circumstances, when gold nanoparticles prepared by trisodium citrate encounter positively charged cationic surfactants, the electrostatic equilibrium state on the surface of gold nanoparticles is broken, resulting in the aggregation of gold nanoparticles. However, after adding nucleic acid aptamers, the aggregation of gold nanoparticles can be effectively prevented. However, when there is a target (such as bacteria) in the reaction system, nucleic acid aptamers will preferentially bind to the target specifically because of their specific recognition function similar to antibodies, resulting in the re-aggregation of gold nanoparticles. However, cationic surfactants such as hexadecyltrimethylammonium bromide are currently commonly used as salt aggregation factors, which are costly and easily destructive to the bacterial surface, making it impossible for nucleic acid aptamers to bind to the bacterial surface.

[0005] As a type of nucleic acid molecule that can bind to a specific target molecule, the screening methods of aptamers vary depending on the detection target. Several common aptamer screening methods include Cell-SELEX technology and electrophoretic migration method. The above are two common aptamer screening methods. The specific screening method also depends on the detection target and experimental conditions. The traditional screening method not only has a long screening cycle and high experimental cost, but also the binding site between the screened aptamer and the target is unclear. Specifically: Traditional aptamer screening usually requires multiple rounds of screening and amplification processes. These steps involve a lot of experimental operations and time consumption, resulting in a long screening cycle. This limits the rapid development and practical application of aptamers; the experimental materials, reagents and equipment required for traditional screening methods will increase the cost of screening. This makes the screening process less feasible for some research laboratories or institutions with limited resources. The high cost limits the widespread application and commercial development of aptamers; traditional aptamer screening methods often cannot determine the specific binding site between aptamers and target molecules. The lack of knowledge about the binding sites limits the precise design and optimization of aptamers, which makes it difficult to optimize the structure and improve the performance of aptamers.

[0006] Therefore, further research and exploration are needed to find more efficient, economical and accurate screening methods and optimization means. Improving screening efficiency and obtaining high-quality aptamers with high accuracy and high specificity are still important goals of current research, which can promote the progress of accurate detection and treatment research of Salmonella typhimurium. The development of new technologies and methods will help overcome the limitations of traditional methods and provide broader prospects for the application of nucleic acid aptamers. Summary of the invention

[0007] Aiming at the problem that salt aggregation factors in the field of nano-gold particle salt aggregation detection easily destroy active binding sites on the bacterial surface, the present invention provides a Salmonella typhimurium aptamer and a method for preparing a colorimetric probe based on the Salmonella typhimurium aptamer.

[0008] The DNA sequence of a Salmonella typhimurium aptamer is shown in SEQ ID No:1.

[0009] The screening steps for the Salmonella typhimurium aptamer are as follows:

[0010] (1) Construction of the membrane protein structure of Salmonella typhimurium

[0011] Obtain the amino acid sequence and structural information of the membrane protein OmpC of Salmonella typhimurium;

[0012] (2) Performing molecular docking test on the membrane protein of Salmonella typhimurium obtained in step (1)

[0013] 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 OmpC protein of Salmonella typhimurium in step (1) into Surflex-Dock molecular simulation software, and then performing molecular docking between the short-chain oligo DNA fragment library and the surface domain of the membrane protein OmpC protein to obtain the free energy of binding between each short-chain oligo DNA fragment and the membrane protein OmpC protein;

[0014] (3) Optimizing the immune algorithm model based on the molecular docking results obtained in step (2)

[0015] The free energy of the above binding is optimized by combining the improved immune algorithm model, where the algorithm formula of the model is as follows:

[0016]

[0017]

[0018] sim(ab i )=a×aff(ab i )-b×aff(ab j ) (III),

[0019] In the formula, the free energy of binding is taken as antibody, ab i represents the ith antibody, ab j represents the jth antibody, aff(ab i ) represents the affinity of the ith antibody, aff(ab j ) represents the affinity of the jth antibody, aff(ab i ,ab j ) represents the immunoaffinity between the ith antibody and the jth antibody, S(ab i ,ab j ) represents the immunoaffinity evaluation operator between the i-th antibody and the j-th antibody and is used for the similarity between antibodies, N represents the antibody capacity, den(ab i ) represents the antibody concentration between the ith antibody and the jth antibody, a represents the excitation coefficient of the ith antibody, b represents the excitation coefficient of the jth antibody, sim(ab i ) represents the degree of excitation between the ith antibody and the jth antibody;

[0020] According to the results of the optimization treatment, short-chain oligoDNA fragments corresponding to antibodies with high excitation degree were statistically selected to obtain matching nucleic acid aptamers of Salmonella typhimurium.

[0021] The steps for preparing the colorimetric probe based on the Salmonella typhimurium aptamer are as follows:

[0022] (1) Preparation of gold nanoparticle solution

[0023] 1 mL of 1% tetrachloroauric acid solution was added to 100 mL of distilled water and boiled; 2.5 mL of 1% trisodium citrate solution was quickly added and the mixture was boiled for 15 min; the mixture was stirred until it cooled naturally to room temperature to obtain a stable gold nanoparticle solution;

[0024] (2) Preparation of Salmonella typhimurium aptamer solution

[0025] The Salmonella typhimurium aptamer was prepared into a Salmonella typhimurium aptamer solution with a concentration of 10 uM using distilled water;

[0026] (3) Preparation of colorimetric probe solution by covalent coupling

[0027] The gold nanoparticle solution prepared in step (1) and the Salmonella typhimurium aptamer solution prepared in step (2) were mixed in a volume ratio of 5:1.8, and incubated at 37°C for 24 hours; after centrifugation at 4°C and 12000 rpm for 15 minutes, the supernatant was discarded, and 500 uL of 0.01 M phosphate buffer was added to the precipitate, and mixed evenly to obtain a colorimetric probe solution;

[0028] The ultraviolet absorption wavelength of the gold nanoparticles modified with nucleic acid aptamers in the colorimetric probe solution is 523 nm.

[0029] The beneficial technical effects of the present invention are embodied in the following aspects:

[0030] 1. Creativity of the aptamer screening method

[0031] For the first time, targeting the amino acid sequence and structure of the membrane protein OmpC of Salmonella typhimurium, high-throughput screening technology, molecular dynamics simulation and improved immune computer algorithm model were used to screen out nucleic acid aptamers with a binding rate of up to 73.9% with Salmonella typhimurium.

[0032] The Salmonella typhimurium aptamer prepared by the present invention is a new aptamer, and is combined with nano-gold particles to form a colorimetric probe. After verification, the effectiveness and feasibility of the use of the Salmonella typhimurium aptamer prepared by the present invention are known. In the field of aptamers, the number of available effective aptamers is relatively small. The design of the Salmonella typhimurium aptamer prepared by the present invention not only considers the binding rate with Salmonella typhimurium, but also considers the specific binding rate with other common and possible coexisting bacteria.

[0033] It has been verified that, compared with the existing mature nucleic acid aptamers, the colorimetric probe prepared with the aptamer of Salmonella typhimurium of the present invention has a higher degree of linear fitting, with a correlation coefficient of up to 0.98357, and a lower detection limit, which is as low as 4.56CFU / mL. The linear fitting degree of similar colorimetric detection methods in the current prior art generally ranges from 0.82 to 0.96; at the same time, compared with traditional technologies (strand displacement reaction method, polymerase chain reaction amplification method, enzyme-linked immunosorbent assay), their detection time is as long as 12h-3d, and the specificity for some common model strains is cross-linked, while the colorimetric probe prepared with the aptamer of Salmonella typhimurium of the present invention has a lower detection time of only 50min, and has good specificity for common model strains such as Staphylococcus aureus and Escherichia coli.

[0034] 2. Creativity of the method for preparing the fluorescence quenching probe of the present invention

[0035] 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 colorimetric detection of bacteria. 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.

[0036] 3. The inventiveness of the present invention for detection

[0037] After testing, it was found that the linear regression equation established under a standard environment can be directly applied to the detection of pre-prepared prepared foods. There is no need to re-cultivate bacteria to establish a standard curve. Instead, the established linear regression equation can be directly applied, which saves time and is suitable for quickly estimating the preliminary concentration of Salmonella Typhimurium in common samples in emergency environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a secondary structure diagram of the Salmonella typhimurium aptamer of the present invention;

[0039] Figure 2 A three-dimensional structural diagram of the membrane protein matched by the molecular docking of the Salmonella typhimurium aptamer in the present invention;

[0040] Figure 3 It is the application principle diagram of colorimetric detection in the present invention;

[0041] Figure 4 It is the ultraviolet absorption diagram of the nucleic acid aptamer and the nano-gold particles before and after covalent coupling in the colorimetric detection of the present invention;

[0042] Figure 5 It is the ultraviolet absorption diagram of different concentrations of Salmonella typhimurium detected by colorimetry in the present invention;

[0043] Figure 6 The standard curve diagram of different concentrations of Salmonella typhimurium detected by colorimetry in the present invention;

[0044] Figure 7 This is a specific detection diagram of various bacteria detected by colorimetry in the present invention. DETAILED DESCRIPTION

[0045] The present invention is described in detail below in conjunction with specific embodiments.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] It should be noted that all strains used in this application are purchased from the American Type Culture Collection (ATCC), of which Salmonella typhimurium is numbered ATCC 14028, Staphylococcus aureus is numbered ATCC29213, Listeria monocytogenes is numbered ATCC43251, Escherichia coli is numbered ATCC 25922, Cronobacter sakazakii is numbered ATCC 29544, and Pseudomonas aeruginosa is numbered ATCC 15442. In addition, the above strains are purchased through overseas agents of Hefei Xiyue Biotechnology Co., Ltd.

[0051] 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.

[0052] 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.

[0053] Example 1

[0054] The sequence of the Salmonella typhimurium aptamer of the present invention is shown in SEQ ID No:1.

[0055] The Salmonella typhimurium aptamer is a nucleic acid aptamer, and the characterization information is as follows:

[0056] like Figure 1 As shown, its secondary structure is shown in the RNAStructure analysis diagram.

[0057] 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 73.9%, and the result of affinity constant determination is 16.73nM, while the flow cell binding rates with Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Cronobacter sakazakii and Pseudomonas aeruginosa are 8.3%, 11.6%, 21.5%, 17.8%, and 16.2%, respectively. The nucleic acid aptamers we screened are close to the highest level known to the public (such as SEQ ID No: 2 source article DOI: 10.1007 / s00216-022-04166-5), and the nucleic acid aptamer effect we obtained is feasible and meets the use requirements.

[0058] In addition, the screening method of the sequence of the Salmonella typhimurium aptamer is as follows:

[0059] (1) Construction of the membrane protein structure of Salmonella typhimurium

[0060] Obtain the amino acid sequence and structural information of the membrane protein OmpC of Salmonella typhimurium, such as Figure 2 As shown;

[0061] (2) Performing molecular docking test on the membrane protein of Salmonella typhimurium obtained in step (1)

[0062] 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 OmpC protein of Salmonella typhimurium in step (1) into Surflex-Dock molecular simulation software, and then performing molecular docking between the short-chain oligo DNA fragment library and the surface domain of the membrane protein OmpC protein to obtain the free energy of binding between each short-chain oligo DNA fragment and the membrane protein OmpC protein;

[0063] (3) Optimizing the immune algorithm model based on the molecular docking results obtained in step (2)

[0064] The free energy of the above binding is optimized by combining the improved immune algorithm model, where the algorithm formula of the model is as follows:

[0065]

[0066]

[0067] sim(ab i )=a×aff(ab i )-b×aff(ab j ) (III),

[0068] In the formula, the free energy of binding is taken as antibody, ab i represents the ith antibody, ab j represents the jth antibody, aff(ab i ) represents the affinity of the ith antibody, aff(ab j ) represents the affinity of the jth antibody, aff(ab i ,ab j ) represents the immunoaffinity between the ith antibody and the jth antibody, S(ab i ,ab j ) represents the immunoaffinity evaluation operator between the i-th antibody and the j-th antibody and is used for the similarity between antibodies, N represents the antibody capacity, den(ab i ) represents the antibody concentration between the ith antibody and the jth antibody, a represents the excitation coefficient of the ith antibody, b represents the excitation coefficient of the jth antibody, sim(ab i ) represents the degree of excitation between the ith antibody and the jth antibody;

[0069] According to the results of the optimization treatment, short-chain oligoDNA fragments corresponding to antibodies with high excitation degree were statistically selected to obtain matching nucleic acid aptamers of Salmonella typhimurium.

[0070] Example 2

[0071] The steps for using the Salmonella typhimurium aptamer of Example 1 to prepare a colorimetric probe are as follows:

[0072] (1) Preparation of gold nanoparticle solution

[0073] See also Figure 3 A, preparation of gold nanoparticles: add 1 mL of 1% tetrachloroauric acid solution to 100 mL of distilled water and boil; quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 min; continue stirring until naturally cooled to room temperature; prepare a stable gold nanoparticle solution and store it at 4°C for further use.

[0074] (2) Preparation of Salmonella typhimurium aptamer solution

[0075] The Salmonella typhimurium aptamer was prepared into a Salmonella typhimurium aptamer solution with a concentration of 10 uM using distilled water;

[0076] Selection of nucleic acid aptamers: The concentration of the nucleic acid aptamers used was 10 uM; the tested nucleic acid aptamer was shown in SEQ ID NO.1.

[0077] (3) Preparation of colorimetric probes by covalent coupling

[0078] See also Figure 3 In B, the gold nanoparticle solution and the Salmonella typhimurium aptamer solution were mixed at a volume ratio of 5:1.8, and incubated at 37°C for 24h; centrifuged at 4°C and 12000rpm for 15min to remove excess nucleic acid aptamer; 500uL phosphate buffer was added for resuspending to obtain a colorimetric probe solution; and stored at 4°C for further use.

[0079] See also Figure 3 C, detection of the mixture with Salmonella typhimurium and saline solution: PBS buffer was selected as the blank control test solution. At the same time, the bacterial solution of Salmonella typhimurium was centrifuged and washed three times with PBS buffer, and then resuspended and graded diluted with PBS buffer to make concentrations of 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6CFU / mL bacteria-containing test solution. First, take 100uL of the prepared colorimetric probe, and incubate with 100uL of blank control test solution and 100uL of gradient diluted bacteria-containing test solution at 37°C for 40min to obtain an incubation solution. Then, add 40uL of saline solution (magnesium chloride solution, with a molar concentration of 0.1M), incubate for 10min, and measure the relative ultraviolet absorption intensity value at 523nm; use the relative ultraviolet absorption intensity value as the ordinate and the logarithmic value of the bacterial concentration of multiple bacteria-containing test solutions as the abscissa to draw a standard curve, obtain a linear regression equation, and obtain the correlation coefficient and detection limit.

[0080] See also Figure 4 , where "1" represents the UV absorption spectrum curve before covalent coupling, and "2" represents the UV absorption spectrum curve after covalent coupling. When the UV absorption wavelength of gold nanoparticles shifts from 520nm to 523nm, it means that the nucleic acid aptamer in the colorimetric probe is successfully modified to the gold nanoparticles.

[0081] See also Figure 5 , from top to bottom, Figure 5 The "1" curve in the middle represents 10 2 UV absorption spectrum curve of CFU / mL, Figure 5 The "2" curve in the middle represents 10 3 UV absorption spectrum curve of CFU / mL, Figure 5 The "3" curve in the middle represents 10 4 UV absorption spectrum curve of CFU / mL, Figure 5 The "4" curve in the middle represents 10 5 UV absorption spectrum curve of CFU / mL, Figure 5 The "5" curve in the middle represents 10 6 CFU / mL UV absorption spectrum curve. Figure 5 From the perspective of the relative ultraviolet absorption intensity value at 523nm on each ultraviolet absorption spectrum curve is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each ultraviolet absorption spectrum curve is taken as the abscissa, that is, Figure 6 , its linear regression equation is y = -0.09126x + 1.11322, and its correlation coefficient (r 2 ) is 0.98357, and its detection limit is 4.56 CFU / mL. It can be seen that the Salmonella typhimurium aptamer designed by the present invention has a good effect, close to the level of the best existing nucleic acid aptamer (article DOI: 10.1007 / s00216-022-04166-5, where "the detection limit is 13 CFU / mL").

[0082] Next, conduct specific tests:

[0083] The Salmonella typhimurium was replaced with Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa; and the concentrations of 10 6 CFU / mL of the test solution containing bacteria.

[0084] Combination Figure 7 It can be seen that the test solution containing bacteria after adding Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa is Figure 7 As shown in the figure, the relative ultraviolet absorption intensity values ​​are 0.9758, 0.9892, 0.9183, 0.9207 and 0.9568 respectively. Meanwhile, the relative ultraviolet absorption intensity value of the bacteria-containing test solution after adding Salmonella typhimurium under the same conditions is 0.6717. In addition, the relative ultraviolet absorption intensity value of the blank control test solution under the same conditions is 1. This shows that the Salmonella typhimurium nucleic acid aptamer of the present invention has good specificity for Salmonella typhimurium.

[0085] Example 3

[0086] On the basis of Example 2, the detection of real food samples was added, and pre-prepared conditioned foods and the Salmonella typhimurium nucleic acid aptamers designed by the present invention were selected for testing:

[0087] It should be noted that in order to verify whether the above-mentioned standard curve can be effectively applied to the determination under the interference environment (prepared prepared food), the difference between the number of bacteria detected by the probe in the artificial quantitative contamination and the preparatory prepared food was respectively carried out to find out that even in the real sample, the colorimetric probe established by the present invention can be effectively used for detection under the influence of high-protein, high-salt, high-fat and other nutrients.

[0088] It should be noted that the pre-prepared cooked chicken wings involved in this embodiment are purchased from Shandong Dezhou Braised Chicken Co., Ltd. The product standard is GB / T 23586, and its ingredients (per 100g) are as follows: 19.4g protein, 16.0% fat, 700mg salt, 1.5g carbohydrates, and 947KJ energy.

[0089] During the experiment, 1 mL of pre-prepared chicken wings was artificially inoculated with Salmonella typhimurium. The final concentration of Salmonella typhimurium after inoculation was 1.00*10 6CFU / mL, centrifuge at 5000×g for 5 min, discard the supernatant, resuspend the precipitate with 1 mL of PBS buffer as the bacterial test solution. First, take 100uL of the prepared colorimetric probe and incubate it with 100uL of the bacterial test solution at 37°C for 40 min to obtain an incubation solution, then add 40uL of a salt solution (magnesium chloride solution, with a molar concentration of 0.1M), incubate for 10 min, and measure the relative ultraviolet absorption intensity value at 523nm.

[0090] The experimental results show that the concentration of Salmonella Typhimurium measured by the colorimetric probe is calculated to be 0.93*10 6 CFU / mL, almost the same as its actual concentration (1.00*10 6 CFU / mL), it can be seen that even if the corresponding standard curve and linear regression equation in the pre-prepared conditioned food environment are not established, 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 colorimetric probe prepared by the present invention has good versatility and can be quickly applied to different sample detections.

[0091] 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 strain of Salmonella typhimurium ( Salmonella typhimurium ) An aptamer, characterized in that: The DNA sequence of the Salmonella typhimurium aptamer is shown in SEQ ID No:

1.

2. A method for preparing a colorimetric probe based on the Salmonella typhimurium aptamer according to claim 1, characterized in that: The preparation steps are as follows: (1) Preparation of gold nanoparticle solution Add 1 mL of 1% tetrachloroauric acid solution to 100 mL of distilled water and boil; immediately add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 min; continue stirring until naturally cooling to room temperature; a stable gold nanoparticle solution is obtained; (2) Preparation of Salmonella typhimurium aptamer solution The Salmonella typhimurium aptamer described in claim 1 is prepared into a Salmonella typhimurium aptamer solution with a concentration of 10 uM using distilled water; (3) Preparation of colorimetric probe solution by covalent coupling The gold nanoparticle solution prepared in step (1) and the Salmonella typhimurium aptamer solution prepared in step (2) were mixed at a volume ratio of 5:1.8, and incubated at 37°C for 24 hours; centrifuged at 4°C and 12000 rpm for 15 minutes, the supernatant was discarded, and 500 uL of 0.01 M phosphate buffer was added to the precipitate, and mixed evenly to obtain a colorimetric probe solution; The ultraviolet absorption wavelength of the gold nanoparticles modified with nucleic acid aptamers in the colorimetric probe solution is 523 nm.

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