A Listeria monocytogenes aptamer and a preparation method thereof for a colorimetric probe

Abstract

The present invention discloses a Listeria monocytogenes aptamer and a colorimetric detection method thereof, belonging to the technical field of food safety. The sequence of the Listeria monocytogenes aptamer is shown as SEQ ID No: 1. The operation steps for using the Listeria monocytogenes aptamer to prepare a colorimetric probe are as follows: (1) Prepare a gold nanoparticle solution with chloroauric acid solution and distilled water; (2) Prepare a Listeria monocytogenes aptamer solution with distilled water; (3) Mix the gold nanoparticle solution and the Listeria monocytogenes aptamer solution according to a volume ratio of 5:1.8 to obtain a colorimetric probe; when the ultraviolet absorption wavelength of the gold nanoparticles shifts from 520 nm to 524 nm, it indicates that the nucleic acid aptamer in the colorimetric probe is successfully modified onto the gold nanoparticles. After verification, compared with the existing mature nucleic acid aptamers, the colorimetric probe prepared by the present invention has a higher degree of linear fitting and a lower detection limit.

Description

Technical Field

[0001] The present invention belongs to the technical field of food detection, and particularly relates to a Listeria monocytogenes aptamer and a colorimetric detection method thereof. Background Art

[0002] A nucleic acid aptamer refers to an oligonucleotide fragment that can bind to a specific target with high affinity and specificity. It is a functional nucleotide screened from a random library through the exponentially enriched ligand system evolution (SELEX) technology. Generally, it is about 40 - 60 bp in size and has some special secondary structures, such as hairpins, stem-loops, pseudoknots, G-quadruplexes, etc., which enable the aptamer to form various tertiary structures and thus bind to the target substance.

[0003] Compared with traditional detection methods, the detection sensing system established by binding nucleic acid aptamers to gold nanoparticles is not only simple to operate, time-consuming short, does not require large instruments, but also has good sensitivity, and is expected to become a new trend in the field of rapid detection. Usually, when gold nanoparticles prepared by sodium citrate encounter a positively charged cationic surfactant, the electrostatic equilibrium state on the surface of the gold nanoparticles is disrupted, resulting in the aggregation of gold nanoparticles. However, after adding nucleic acid aptamers, the aggregation of gold nanoparticles can be effectively prevented. But when there is a target (such as bacteria) in the reaction system, due to its specific recognition function similar to that of antibodies, the nucleic acid aptamer will preferentially bind specifically to the target, resulting in the re-aggregation of gold nanoparticles. But currently, cationic surfactants such as cetyltrimethylammonium bromide are usually used as salt aggregation factors, which are costly and easily damage the surface of bacteria, making it impossible for the nucleic acid aptamer to bind to the surface of bacteria. Summary of the Invention

[0004] Aiming at the problem that the salt aggregation factor in the field of gold nanoparticle salt aggregation detection easily damages the active binding sites on the surface of bacteria, the present invention provides a Listeria monocytogenes aptamer and a colorimetric detection method thereof.

[0005] A Listeria monocytogenes aptamer, the DNA sequence of the Listeria monocytogenes aptamer is shown as SEQ ID No: 1.

[0006] The operation steps for using the Listeria monocytogenes aptamer to prepare a colorimetric probe are as follows:

[0007] (1) Prepare a gold nanoparticle solution

[0008] Add 1 mL of 1% chloroauric acid solution by mass to 100 mL of distilled water and boil; quickly add 2.5 mL of 1% trisodium citrate solution by mass and continue boiling for 15 min; continue stirring until it cools naturally to room temperature; prepare a stable gold nanoparticle solution;

[0009] (2)Prepare the aptamer solution

[0010] The DNA sequence of the aptamer is shown in SEQ ID NO.1; prepare an aptamer solution with a concentration of 10 uM with distilled water;

[0011] (3)Covalently couple to prepare the colorimetric probe

[0012] Mix the gold nanoparticle solution and the aptamer solution at a volume ratio of 5:1.8 and incubate at 37 °C for 24 h; centrifuge at 12000 rpm for 15 min at 4 °C to remove the excess aptamer; add 500 uL of phosphate buffer to resuspend to obtain the colorimetric probe;

[0013] When the ultraviolet absorption wavelength of the gold nanoparticles shifts from 520 nm to 524 nm, it indicates that the aptamer in the colorimetric probe is successfully modified onto the gold nanoparticles.

[0014] The beneficial technical effects of the present invention are embodied in the following aspects:

[0015] 1. It has been verified that compared with the existing mature aptamers, the colorimetric probe prepared with the Listeria monocytogenes aptamer of the present invention has a higher degree of linear fitting, with a correlation coefficient as high as 0.98544, and has a lower detection limit, with the detection limit as low as 22.75 CFU / 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 techniques (strand displacement reaction method, polymerase chain reaction amplification method, enzyme-linked immunosorbent assay method), their detection time is as long as 12 h - 3 d, and there is specific cross-reaction with some common model strains, while the colorimetric probe prepared with the Listeria monocytogenes aptamer of the present invention has a lower detection time of only 50 min and has good specificity for common model strains such as Staphylococcus aureus and Escherichia coli. For example, the traditional strand displacement reaction utilizes the free energy difference of DNA molecular hybridization. Due to the intramolecular electrostatic repulsion of the negatively charged phosphate groups on single-stranded DNA for double-stranded DNA, the efficiency of the strand displacement reaction is relatively low.

[0016] 2. After testing, the linear regression equation established under standard conditions can be directly applied to the detection of LB broth samples. There is no 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 Listeria monocytogenes in common samples in an emergency environment. For example, in an LB broth sample, in an environment where Listeria monocytogenes is artificially inoculated in LB broth, the concentration of Listeria monocytogenes measured by the colorimetric probe is 3.81*10 6 CFU / mL, which is almost close to its true concentration of 3.9*10 6 CFU / mL. It can be seen that even without establishing the corresponding standard curve and linear regression equation in the LB broth sample environment, the standard curve and linear regression equation established in the pure PBS sample environment still have good generality.

[0017] 3. Generally speaking, the Listeria monocytogenes nucleic acid aptamer prepared in the present invention is a new nucleic acid aptamer, which is combined with gold nanoparticles to form a colorimetric probe. After verification, the effectiveness and feasibility of the Listeria monocytogenes nucleic acid aptamer prepared in the present invention are known. In the field of nucleic acid aptamers, the number of available effective nucleic acid aptamers is currently small. The design of the Listeria monocytogenes nucleic acid aptamer prepared in the present invention not only considers the binding rate with Listeria monocytogenes, but also considers the specific binding rate with other common possible co-existing bacteria. At the same time, the present invention uses magnesium chloride, a divalent cation metal salt, as a salt aggregation factor for the first time, which has a better effect than the use of traditional monovalent salt ions of sodium chloride. Selecting magnesium chloride from many cationic metal salts also requires creative labor, because in the prior art, cationic surfactants are usually considered, and there are few uses of cationic metal salts for colorimetric 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 realizes 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 the formation of gold nanoparticles can be normal in the experiment, and the problem of whether the nucleic acid aptamer can quickly dissociate from the surface of gold nanoparticles also belongs to the inherent characteristics of nucleic acid aptamers. Therefore, considering the above various complex factors, this is not disclosed in the prior art and cannot be obtained from the corresponding common general knowledge in the art for technical inspiration. Description of the Drawings

[0018] Figure 1 It is the secondary structure diagram of the Listeria monocytogenes aptamer in the present invention.

[0019] Figure 2 It is the binding rate diagram of the Listeria monocytogenes aptamer in the present invention.

[0020] Figure 3 Specific binding rate graph of the Listeria monocytogenes aptamer in the present invention.

[0021] Figure 4 Schematic diagram of the application principle of colorimetric detection in the present invention.

[0022] Figure 5 Ultraviolet absorption graph before and after covalent coupling of the nucleic acid aptamer and gold nanoparticles for colorimetric detection in the present invention.

[0023] Figure 6 Ultraviolet absorption graph of different concentrations of Listeria monocytogenes for colorimetric detection in the present invention.

[0024] Figure 7 Standard curve graph of different concentrations of Listeria monocytogenes for colorimetric detection in the present invention.

[0025] Figure 8 Specific detection graph of multiple bacteria for colorimetric detection in the present invention. Detailed implementation manners

[0026] The present invention will be further described below in conjunction with specific embodiments.

[0027] Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.

[0028] Unless otherwise specified, the raw materials used in the following embodiments are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods; for the quantitative tests in the following embodiments, unless otherwise specified, three repeated experiments are set, and the results are averaged; the % in the following embodiments, unless otherwise specified, are all mass percentages.

[0029] In the following embodiments, the synthesized aptamers are purchased from Sangon Biotech (Shanghai) Co., Ltd.; unless otherwise specified, other raw materials used are all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0030] It should be reminded that the phosphate buffer solution used in this application is a sterile PBS buffer solution with a concentration of 0.1 M and a pH value of 7.4.

[0031] It should be noted that all the bacterial strains used in this application are purchased from the American Type Culture Collection (ATCC). The strain number of Listeria monocytogenes is ATCC 43251, the strain number of Staphylococcus aureus is ATCC 29213, the strain number of Salmonella is ATCC 14028, the strain number of Escherichia coli is ATCC 25922, the strain number of Cronobacter sakazakii is ATCC 29544, and the strain number of Pseudomonas aeruginosa is ATCC 15442. In addition, the above strains are purchased through overseas agency by Hefei Xiyue Biotechnology Co., Ltd.

[0032] It should be noted that all the instrument and equipment, raw materials, reagents or method steps used in this application are ensured to be processed under aseptic conditions.

[0033] It should be noted that the instrument and 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 elaborated in this application. One can refer to the following paper (Yuwei Ren, Lulu Cao, Xiyan Zhang, Rui Jiao, Dexin Ou, Yang Wang, Danfeng Zhang, Yizhong Shen, Na Ling, Yingwang Ye, A novel fluorescence resonance energy transfer (FRET)-based paper sensor with smartphone for quantitative detection of Vibrio parahaemolyticus, Food Control, Volume 145, 2023, 109412, ISSN 0956-7135, https: / / doi.org / 10.1016 / j.foodcont.2022.109412.).

[0034] Example 1

[0035] The sequence of the aptamer for Listeria monocytogenes of the present invention is shown as SEQ ID No: 1, which is designed according to the peptidoglycan structure of Listeria monocytogenes through a quantum genetic algorithm model.

[0036] The aptamer for Listeria monocytogenes is a nucleic acid aptamer, and its characterization information is as follows:

[0037] As Figure 1 shown, its secondary structure is as shown in the RNAStructure analysis diagram.

[0038] As Figure 2As shown, the binding rate of flow cytometry is 73.1%, approaching the highest known published level (the known binding rate of SEQ ID No:2 is 85.4%). The obtained aptamer effect is feasible and meets the usage requirements.

[0039] As Figure 3 shown, Figure 3 A in Figure 3 is the binding rate to Staphylococcus aureus, Figure 3 B in Figure 3 is the binding rate to Escherichia coli, Figure 3 C in

[0040] Example 2

[0041] The operating steps for using the Listeria monocytogenes aptamer of Example 1 to prepare a colorimetric probe are as follows:

[0042] (1) Preparation of gold nanoparticle solution

[0043] See Figure 4 A in

[0044] For the preparation of gold nanoparticles: Add 1 mL of 1% chloroauric acid solution by mass to 100 mL of distilled water and boil; quickly add 2.5 mL of 1% trisodium citrate solution by mass and continue boiling for 15 min; continue stirring until it cools naturally to room temperature; prepare a stable gold nanoparticle solution and store it at 4 °C for further use.

[0045] Prepare a Listeria monocytogenes aptamer solution with a concentration of 10 uM of Listeria monocytogenes aptamer using distilled water;

[0046] Selection of aptamer: The usage concentration of aptamer is 10 uM; The tested aptamer II is as shown in SEQ ID NO.1, and the DNA sequence of the old aptamer I is:

[0047] tttttttttt atccacgggg cggagatgag ggggaggagg gcgggtaccc ggttgat. Among them, the tested aptamer two was designed by the present invention, and the old aptamer one was an existing publicly disclosed aptamer, which was used for comparison to illustrate the effectiveness of the aptamer screening in this application. It was derived from the following literature: Lisha Zhang, Ru Huang, Weipeng Liu, Hongxing Liu, Xiaoming Zhou, Da Xing, Rapid and visual detection of Listeria monocytogenes based on nanoparticle cluster catalyzed signal amplification, Biosensors and Bioelectronics, Volume 86, 2016, Pages 1 - 7, ISSN 0956 - 5663, https: / / doi.org / 10.1016 / j.bios.2016.05.100.

[0048] (3)Covalent coupling to prepare colorimetric probe

[0049] Mix the gold nanoparticle solution and the Listeria monocytogenes aptamer solution at a volume ratio of 5:1.8, and incubate at 37 °C for 24 h; centrifuge at 12,000 rpm at 4 °C for 15 min to remove the excess aptamer; add 500 μL of phosphate buffer to resuspend and obtain the colorimetric probe; store at 4 °C for further use.

[0050] See Figure 4 in B, for the detection of the mixture of Listeria monocytogenes and salt solution: Select PBS buffer as the blank control test solution. At the same time, centrifuge the Listeria monocytogenes bacterial solution and wash it three times with PBS buffer, then resuspend and serially dilute it with PBS buffer to prepare solutions with concentrations of 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6CFU / mL bacterial solution to be measured. First, take 100 μL of the prepared colorimetric probe and incubate it with 100 μL of the blank control solution to be measured and 100 μL of the serially diluted bacterial solution to be measured at 37 °C for 40 min to obtain the incubation solution. Then, add 40 μL of a salt solution (magnesium chloride solution with a molar concentration of 0.1 M), incubate for 10 min, and measure the relative ultraviolet absorption intensity value at 520 nm. Using the relative ultraviolet absorption intensity value as the ordinate and the logarithm of the bacterial concentration of multiple bacterial solutions to be measured as the abscissa, plot a standard curve, obtain the linear regression equation, and obtain the correlation coefficient and detection limit.

[0051] See Figure 5 A in, where "1" represents the ultraviolet absorption spectral curve before covalent coupling, and "2" represents the ultraviolet absorption spectral curve after covalent coupling. When the ultraviolet absorption wavelength of the gold nanoparticles shifts from 520 nm to 524 nm, it indicates that the aptamer in the colorimetric probe has been successfully modified onto the gold nanoparticles; see Figure 5 B in, where "1" represents the ultraviolet absorption spectral curve before covalent coupling, and "2" represents the ultraviolet absorption spectral curve after covalent coupling. When the ultraviolet absorption wavelength of the gold nanoparticles shifts from 520 nm to 524 nm, it indicates that the second aptamer in the colorimetric probe has been successfully modified onto the gold nanoparticles.

[0052] See Figure 6 A in, from top to bottom ( Figure 6 The "1" curve in A in represents the ultraviolet absorption spectral curve of 0 CFU / mL, Figure 6 The "2" curve in A in represents the ultraviolet absorption spectral curve of 3.9×10 2 CFU / mL, Figure 6 The "3" curve in A in represents the ultraviolet absorption spectral curve of 3.9×10 3 CFU / mL, Figure 6 The "4" curve in A in represents the ultraviolet absorption spectral curve of 3.9×10 4 CFU / mL, Figure 6 The "5" curve in A in represents the ultraviolet absorption spectral curve of 3.9×10 5 CFU / mL, Figure 6 The "6" curve in A in represents the ultraviolet absorption spectral curve of 3.9×10 6 CFU / mL (), and at the same time combined with Figure 7 A in, taking the relative ultraviolet absorption intensity value at 524 nm on each ultraviolet absorption spectral curve as the ordinate and the logarithm of the bacterial concentration corresponding to each of the above ultraviolet absorption spectral curves as the abscissa, the linear regression equation is y = -0.13105x + 1.23119, and its correlation coefficient (r 2) is 0.99114, and its detection limit is 15.23 CFU / mL.

[0053] Meanwhile, referring to Figure 6 B in Figure 6 In B in , the "1" curve represents the ultraviolet absorption spectrum curve of 0 CFU / mL, Figure 6 In B in , the "2" curve represents the ultraviolet absorption spectrum curve of 3.9×10 3 CFU / mL, Figure 6 In B in , the "3" curve represents the ultraviolet absorption spectrum curve of 3.9×10 4 CFU / mL, Figure 6 In B in , the "4" curve represents the ultraviolet absorption spectrum curve of 3.9×10 5 CFU / mL, Figure 6 In B in , the "5" curve represents the ultraviolet absorption spectrum curve of 3.9×10 6 CFU / mL (while combining with Figure 7 B in ), taking the relative ultraviolet absorption intensity value at 524 nm on each ultraviolet absorption spectrum curve as the ordinate, and taking the logarithm value of the corresponding bacterial concentration of each above-mentioned ultraviolet absorption spectrum curve as the abscissa, its linear regression equation is y = -0.13256x + 1.35711, and its correlation coefficient (r 2 ) is 0.98544, and its detection limit is 22.75 CFU / mL. It can be seen from this that the designed Listeria monocytogenes aptamer of the present invention has a good effect, approaching the level of the existing nucleic acid aptamers.

[0054] Next, a specificity test was carried out:

[0055] Listeria monocytogenes was replaced with Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa respectively; and bacterial-containing test solutions with a concentration of 3.9×10 6 CFU / mL were prepared respectively.

[0056] Combining with Figure 8 it can be seen that for the bacterial-containing test solutions after adding Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa, as Figure 8 shown in A in , it is the specificity result diagram of nucleic acid aptamer I, and their relative ultraviolet absorption intensity values are 0.8725, 0.9133, 0.9289, 0.9034, and 0.9152 respectively. Meanwhile, for the bacterial-containing test solution after adding Listeria monocytogenes under the same conditions, its relative ultraviolet absorption intensity value is 0.3650. In addition, for the blank control test solution under the same conditions, its relative ultraviolet absorption intensity value is 0.9395.

[0057] As Figure 8As shown in B of [Figure / Chart / Diagram] [0], the result diagram of aptamer two is presented. Its relative ultraviolet absorption intensity values are 0.8236, 0.8959, 0.9231, 0.9028, and 0.9157 respectively. Meanwhile, under the same conditions, for the bacteria-containing solution to be tested with Listeria monocytogenes added, its relative ultraviolet absorption intensity value is 0.5031. In addition, for the blank control solution to be tested under the same conditions, its relative ultraviolet absorption intensity value is 0.9400.

[0058] Compared with the existing aptamers, it can thus be shown that the Listeria monocytogenes aptamer of the present invention has good specificity for Listeria monocytogenes.

[0059] Example 3

[0060] Based on Example 2, the detection of real food samples was added. An LB broth sample and the Listeria monocytogenes aptamer designed in the present invention were selected for testing:

[0061] 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 (LB broth), the differences between the artificially quantified bacteria inoculation and the number of bacteria detected by the probe in the LB broth sample were respectively carried out, so as to know that even in real samples, the colorimetric probe established in the present invention can effectively detect under the influence of nutrients such as high protein.

[0062] It should be noted that the LB broth involved in this example was purchased from Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park. Its components (g / L) are as follows: tryptone 10.0 g / L, yeast extract powder 5.0 g / L, sodium chloride 10.0 g / L, pH 7.0 ± 0.1.

[0063] During the experiment, Listeria monocytogenes was artificially inoculated into 1 mL of LB broth. The final concentration of Listeria monocytogenes after inoculation was 3.9×10 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 the bacteria-containing solution to be tested. First, 100 μL of the prepared colorimetric probe was taken and incubated with 100 μL of the bacteria-containing solution to be tested at 37 °C for 40 min to obtain the incubation solution. Then, 40 μL of a salt solution (magnesium chloride solution with a molar concentration of 0.1 M) was added, incubated for 10 min, and the relative ultraviolet absorption intensity value was measured at 520 nm.

[0064] The test results showed that after calculation, in the environment of artificially inoculating Listeria monocytogenes in the LB broth sample, the concentration of Listeria monocytogenes measured by the colorimetric probe was 3.81×10 6 CFU / mL, which was almost the same as its true concentration (3.9×10 6CFU / mL), it can be seen that even without establishing the corresponding standard curve and linear regression equation in the LB broth sample environment, the standard curve and linear regression equation established in the pure PBS sample environment still have good generality. Thus, it can be seen that the colorimetric probe prepared by the present invention has good generality and can be quickly applied to different sample detections.

[0065] 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 should be included within the protection scope of the present invention.

Claims

1. A Listeria monocytogenes aptamer, characterized in that: the DNA sequence of the Listeria monocytogenes aptamer is shown in SEQ ID No:

1.

2. A method for preparing a colorimetric probe using the Listeria monocytogenes aptamer according to claim 1, characterized in that the detection operation steps are as follows: (1) Preparation of gold nanoparticle solution Add 1 mL of 1% mass concentration of chloroauric acid solution to 100 mL of distilled water and boil; quickly add 2.5 mL of 1% mass concentration of trisodium citrate solution and continue boiling for 15 min; continue stirring until naturally cooled to room temperature; obtain a stable gold nanoparticle solution; (2) Preparation of Listeria monocytogenes aptamer solution Use distilled water to prepare the Listeria monocytogenes aptamer described in claim 1 into a 10 μM concentration of Listeria monocytogenes aptamer solution; (3) Covalent coupling to prepare a colorimetric probe Mix the gold nanoparticle solution and the Listeria monocytogenes aptamer solution at a volume ratio of 5:1.8, incubate at 37 °C for 24 h; centrifuge at 12,000 rpm at 4 °C for 15 min to remove the excess nucleic acid aptamer; add 500 μL of phosphate buffer to resuspend to obtain a colorimetric probe; When the ultraviolet absorption wavelength of the gold nanoparticles shifts from 520 nm to 524 nm, it indicates that the nucleic acid aptamer in the colorimetric probe is successfully modified onto the gold nanoparticles.

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