An aptamer biosensor for detecting Salmonella, its preparation method and application

Abstract

An aptamer biosensor for detecting Salmonella, its preparation method and application belong to the technical field of food safety. In order to solve the problems existing in the detection of Salmonella in the prior art, the present invention provides an aptamer biosensor. Its detection principle is that the sandwich structure SMBs-Apt2-AuNPs fluorescence quenching system established by the SMBs-cDNA2 complex, aptamer Apt2 and AuNPs-cDNA3 probe can maintain integrity in the absence of Salmonella, and the AuNPs in the system undergo FRET with AgNCs to quench the fluorescence of AgNCs; in the presence of Salmonella, Salmonella binds to Apt2 as a connecting bridge, and the SMBs-Apt2-AuNPs system is destroyed, and the AuNPs-cDNA3 probe suspended in the supernatant and Salmonella binding Apt2 are removed by magnetic separation, resulting in the fluorescence not being quenched. The aptamer biosensor obtained by the present invention has the characteristics of simplicity, low cost, fast speed and high sensitivity, and can be used for real-time detection of Salmonella contamination in food and food processing, especially Salmonella contamination in dairy product production.

Description

Technical Field

[0001] The present invention relates to an aptamer-based biosensor for detecting Salmonella, its preparation method and application, and belongs to the technical field of food safety. Background Art

[0002] As one of the main causes of food poisoning and acute intestinal diseases, foodborne pathogenic bacteria have had a significant impact on global food safety, especially dairy products.

[0003] Salmonella (S.typhimurium), as one of the common foodborne pathogens, its contamination can cause various diseases in humans. Salmonella will cause symptoms such as diarrhea, fever, and abdominal pain after a latency period of 12 - 72 hours, and in severe cases, it can lead to dehydration, shock, collapse, and even sepsis. According to statistics, about 16 million people are infected with Salmonella worldwide every year, of which about 600,000 die. Salmonella has been listed by the WHO as a foodborne pathogen with severe and moderate hazards. Some studies have shown that when the contamination amount of Salmonella exceeds 10 5 CFU, it can cause infection in healthy humans. For immunocompromised and susceptible populations such as the elderly and children, a contamination amount of only 15 - 20 CFU can cause severe infection. Therefore, it is crucial to prevent and detect Salmonella as early as possible.

[0004] Salmonella belongs to the Enterobacteriaceae family and is a facultative anaerobic Gram-negative bacillus. Its cells are rod-shaped with blunt ends at both ends, and the size is approximately (2 - 5μm) × (0.7 - 1.5μm). Its colonies on the culture medium are colorless, semi-transparent, round, with a smooth surface and a flat edge. Salmonella has a wide growth temperature range (10℃ - 42℃), and it can multiply rapidly when the temperature exceeds 20℃, with 37℃ being its optimal growth temperature. Salmonella has strong environmental adaptability. Although 6.8 - 7.8 is the optimal pH for Salmonella, it can also grow within the pH range of 4.5 - 9.5, and can still survive for a long time under low water activity conditions (water activity Aw < 0.2). In addition, because the cultivation of Salmonella does not require rich nutrients, it can grow even on ordinary culture media.

[0005] Antibodies have problems such as difficulties in preparation and storage, and may also cross-react with different antigens. As a stable and specific recognition element, nucleic acid aptamers can not only specifically recognize target substances, but also be easily conjugated with various molecules and be compatible with various signal transduction modes. Therefore, developing a biosensor based on nucleic acid aptamers is of great significance for realizing rapid and sensitive detection of foodborne pathogenic bacteria.

[0006] At present, the main detection methods for Salmonella include traditional culture methods, molecular biology methods, and immunological methods, such as polymerase chain reaction (PCR), loop-mediated isothermal amplification technology (LAMP), enzyme-linked immunosorbent assay (ELISA), and immunomagnetic separation (IMS), etc. The detection by traditional culture methods is easily interfered by food matrices, has a long cycle, and a cumbersome operation process, which cannot meet the requirements for rapid, simple, and sensitive detection of pathogenic bacteria. Therefore, there is an urgent need for a detection technology with simple operation, rapidity, and accuracy. Molecular biology methods, such as the mainstream PCR technology, although compared with traditional culture methods, PCR technology greatly shortens the detection time and can achieve highly sensitive detection, however, it requires a complex nucleic acid extraction procedure, and since dead cells also have DNA, the number of live cells may be overestimated. Compared with the PCR method, LAMP reduces the need for highly complex equipment, has low cost, and short reaction time. However, due to its too high sensitivity, LAMP may generate aerosols in the environment that cannot be eliminated, resulting in false positive results, and still requires nucleic acid extraction operations. The immunological methods are also limited in their application due to disadvantages such as being easily affected by environmental factors, high detection costs, and low antibody affinity. The problems existing in immunological methods limit the application of technologies such as ELISA and IMS. However, the magnetic separation characteristics of IMS have advantages such as reducing pre-enrichment time and reducing food matrix interference. Therefore, there is an urgent need to combine IMS technology with other detection technologies to develop a simple, economical, rapid, and sensitive pathogenic bacteria detection and analysis system.

[0007] Fluorescent aptamer sensors are the most common optical detection methods, mainly based on the generation or quenching of fluorescence signals after binding to the target, and analyzed by detecting the signal changes in fluorescence intensity. Due to the characteristics of high sensitivity, high detection efficiency, and simple operation, this method has been widely used in the detection of foodborne pathogenic bacteria. Commonly used organic fluorescent dyes have disadvantages such as poor photostability, low absorption, broad emission, and photobleaching characteristics. Quantum dot materials have many advantages compared with organic fluorescent dyes, but most of them contain heavy metals, and their biological toxicity limits their application and development. Therefore, it is very necessary to develop a fluorescent label with more stable optical properties and low toxicity for the detection of foodborne pathogenic bacteria using fluorescent aptamer sensors. Summary of the Invention

[0008] To solve the problems existing in the detection of Salmonella by using traditional microbiological methods, molecular biological methods and immunological methods, the present invention provides an aptamer biosensor for rapid detection of Salmonella, including a sandwich structure SMBs-Apt2-AuNPs established by an SMBs-cDNA2 complex, an aptamer Apt2 and an AuNPs-cDNA3 probe, and silver nanoclusters DNA-AgNCs synthesized with DNA as a template and used as a fluorescence signal; the SMBs-cDNA2 complex is formed by connecting streptavidin magnetic beads with a biotin-modified nucleic acid strand cDNA2, and the nucleotide sequence of cDNA2 is as shown in SEQ ID NO.1; the AuNPs-cDNA3 probe is prepared from an AuNPs solution and a thiol-modified cDNA3, and the nucleotide sequence of cDNA3 is as shown in SEQ ID NO.2; the nucleotide sequence of the aptamer Apt2 is as shown in SEQ ID NO.3; the nucleotide sequence of the DNA template is as shown in SEQ ID NO.4;

[0009] In the absence of Salmonella, the SMBs-Apt2-AuNPs system remains intact. When DNA-AgNCs are added, fluorescence resonance energy transfer occurs between AuNPs and AgNCs in the system to quench the fluorescence of AgNCs; in the presence of Salmonella, the AuNPs-cDNA3 probe suspended in the supernatant and Salmonella bound to Apt2 are removed by magnetic separation, resulting in non-quenching of fluorescence.

[0010] The present invention also provides a preparation method of the above aptamer biosensor, including the following steps:

[0011] (1) Preparation of the SMBs-cDNA2 complex: After being washed with 1×B&W buffer, the SMBs are resuspended in 2×B&W buffer, an equal volume of cDNA2 solution is added for incubation, the supernatant is removed, and after being washed with 1×B&W buffer, it is resuspended in PBS solution to obtain the SMBs-cDNA2 complex;

[0012] (2) Preparation of the AuNPs-cDNA3 probe: The thiol-modified cDNA3 is mixed with the AuNPs solution, incubated, then phosphate buffer A is added and incubated again. Phosphate buffer B containing 2 mol / L NaCl is slowly added in multiple portions for aging until the NaCl concentration in the solution reaches 0.01 mol / L - 2 mol / L. After centrifugation, it is resuspended with deionized water to obtain the AuNP-cDNA3 probe;

[0013] (3) Preparation of SMBs-Apt2-AuNPs system and synthesis of DNA-AgNCs: Mix Apt2, SMBs-cDNA2 complex and AuNPs-cDNA3 probe in PBS solution and incubate. Remove the supernatant by magnetic separation, and then wash three times with PBS solution to obtain the SMBs-Apt2-AuNPs system. Add AgNO3 solution to the PB solution containing the DNA template, incubate in the dark, add NaBH4 and shake vigorously. Stabilize in the dark before use. The concentration of the DNA used is the concentration of the prepared DNA-AgNCs.

[0014] Further defined, the dosage of the SMBs in step (1) is 0.1 - 5 mg, the dosage of 2×B&W buffer is 20 - 500 μL, the dosage of PBS solution is 20 - 500 μL, and the concentration of cDNA2 is 0.5 - 5 μmol / L; the incubation temperature is 25 - 45 °C, and the incubation time is 30 - 120 min; the final concentration of the finally obtained SMBs-cDNA2 complex is 1 - 20 μg / μL.

[0015] Preferably, the dosage of the SMBs is 1 mg, the dosage of 2×B&W buffer is 200 μL, the dosage of PBS solution is 200 μL, and the concentration of cDNA2 is 2 μmol / L; the incubation temperature is 37 °C, and the incubation time is 60 min; the final concentration of the finally obtained SMBs-cDNA2 complex is 5 μg / μL.

[0016] Further defined, step (2) is to mix 1 - 20 μL of thiol-modified cDNA3 with a concentration of 10 - 500 μmol / L with 1 mL of AuNPs solution, incubate at 30 °C - 70 °C for 5 - 24 h, add 50 - 500 μL of phosphate buffer A, incubate at 30 °C - 70 °C for 0.1 - 2 h, and slowly add phosphate buffer B containing 2 mol / L NaCl in multiple times for aging until the NaCl concentration in the solution reaches 0.01 - 2 mol / L. After centrifugation, resuspend with 50 - 500 μL of deionized water to obtain the AuNP-cDNA3 probe.

[0017] Further defined, preparation of phosphate buffer A: Weigh 1.56 g of NaH2PO4·2H2O, 3.58 g of Na2HPO4·12H2O, and 0.1 g of SDS, dissolve them fully in distilled water, adjust the pH to 7.6, and then make up the volume to 100 mL to obtain 0.1 mol / L phosphate buffer A.

[0018] Further limitation: Preparation of phosphate buffer B: Weigh 10 mL of 0.1 M phosphate buffer A, add 11.7 g of NaCl, and make up the volume to 100 mL with distilled water to obtain 0.01 mol / L phosphate buffer B.

[0019] Preferably, the volume of the thiol-modified cDNA3 in step (2) is 8 μL and the concentration is 100 μmol / L; the addition amount of the phosphate buffer A is 100 μL, the incubation temperature before adding the phosphate buffer A is 50 °C, and the incubation time is 12 h; the incubation temperature after adding the phosphate buffer A is 50 °C and the incubation time is 1 h; the end time of the aging is when the NaCl concentration reaches 0.2 mol / L; the usage amount of the deionized water is 200 μL.

[0020] Further limitation: In step (3), 1 - 10 nmol / L Apt2, 1 - 100 μg of the SMBs-cDNA2 complex, and 5 - 50 μL of the AuNPs-cDNA3 probe are mixed in 100 μL of PBS solution and incubated at 25 - 45 °C for 60 - 180 min, then the supernatant is removed by magnetic separation, and then washed three times with PBS solution to prepare the SMBs-Apt2-AuNPs system; 1 - 20 μL of a AgNO3 solution with a concentration of 0.002 - 10 mmol / L is added to 50 - 200 μL of a PB solution containing 0.5 - 5 nmol of DNA template with a concentration of 0.002 - 0.2 mol / L and a pH of 6.4 - 8, and incubated in the dark at 0 - 25 °C for 5 - 50 min, 0.5 - 20 μL of a NaBH4 solution with a concentration of 0.1 - 10 mmol / L is quickly added to the mixture, and shaken vigorously for 1 minute. Before use, the mixture solution is stabilized at 0 - 25 °C in the dark for 5 - 24 h, and the DNA concentration used is the concentration of the prepared DNA-AgNCs.

[0021] The present invention also provides a method for detecting Salmonella using the above aptamer biosensor, comprising the following steps:

[0022] S1. Add the SMBs-Apt2-AuNPs system to the sample and incubate at 25 °C - 45 °C for 60 - 180 min;

[0023] S2. Perform magnetic separation to discard the supernatant and resuspend in 30 μL - 200 μL of distilled water;

[0024] S3. Add 5 μL - 100 μL of DNA-AgNCs with a concentration of 0.1 μmol / L - 5 μmol / L to the solution and incubate for 2 min - 30 min;

[0025] S4. Detect the fluorescence spectrum of DNA-AgNCs using a multi-functional microplate reader. The excitation wavelength is 550 nm to 600 nm, and the emission spectrum from 601 to 750 nm is scanned at intervals of 2 nm.

[0026] Furthermore, it is specified that the volume of the sample in S1 is 20 - 800 μL, the incubation temperature is 37 °C, and the incubation time is 120 min; the volume of the distilled water in S2 is 80 μL; the volume of the DNA-AgNCs in S3 is 20 μL, the concentration is 1.5 μmol / L, and the incubation time is 10 min; the excitation wavelength in S4 is 576 nm.

[0027] The present invention also provides the application of the above aptamer biosensor in the real-time detection of Salmonella contamination in food or food processing.

[0028] The beneficial effects of the present invention:

[0029] The principle of the present invention is that the sandwich structure SMBs-Apt2-AuNPs fluorescence quenching system established by the SMBs-cDNA2 complex, aptamer Apt2, and AuNPs-cDNA3 probe can maintain integrity in the absence of Salmonella. In the system, fluorescence resonance energy transfer (FRET) occurs between AuNPs and AgNCs to quench the fluorescence of AgNCs; in the presence of Salmonella, Salmonella binds to Apt2 acting as a connecting bridge, the SMBs-Apt2-AuNPs system is disrupted, and the AuNPs-cDNA3 probe suspended in the supernatant and Salmonella binding to Apt2 are removed by magnetic separation, resulting in the non-quenching of fluorescence, and thus realizing simple, rapid, and sensitive detection of Salmonella.

[0030] 1) The aptamer biosensor prepared by the method disclosed in the present invention has a fast detection speed, high detection sensitivity, and low detection cost, and can effectively achieve rapid detection of Salmonella.

[0031] 2) The present invention constructs a fluorescent aptamer biosensor based on DNA-AgNCs to solve the problem of low detection performance caused by poor photostability and toxicity of existing fluorescent signal molecules.

[0032] 3) The "sandwich" structure of this method can not only reduce the nucleic acid extraction process, but also effectively reduce the interference of food matrix, greatly improving the detection speed and sensitivity.

[0033] 4) This method does not require expensive instruments and special heating equipment, and is more suitable for on-site detection scenarios with limited resources.

[0034] 5) The method established using the present invention can be used for the real-time detection of Salmonella in foods and food processing, especially for the Salmonella contamination in dairy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the schematic diagram of the invention;

[0036] Figure 2 is the optimization result diagram of the final concentration of NaCl in the preparation process of AuNPs-cDNA3 probe. The final concentrations of NaCl in 1-4 in the figure are 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L respectively;

[0037] Figure 3 is the optimization result diagram of the concentration of Apt2;

[0038] Figure 4 is the optimization result diagram of the addition amount of SMBs-cDNA2 complex;

[0039] Figure 5 is the optimization result diagram of the addition amount of AuNPs-cDNA3 probe;

[0040] Figure 6 is the optimization result diagram of the incubation time of Apt2, SMBs-cDNA2, and AuNPs-cDNA3;

[0041] Figure 7 is the optimization result diagram of the pH of PB solution;

[0042] Figure 8 is for DNA / Ag + optimization result diagram of molar concentration ratio;

[0043] Figure 9 is for NaBH4 / Ag + optimization result diagram of molar concentration ratio;

[0044] Figure 10 is the optimization result diagram of the incubation time of SMBs-Apt2-AuNPs system and Salmonella;

[0045] Figure 11 is the investigation result of the sensitivity of the aptamer biosensor for detecting Salmonella;

[0046] Figure 12 is the linear relationship diagram of the aptamer biosensor for detecting Salmonella;

[0047] Figure 13 is the investigation result diagram of the specificity of the aptamer biosensor for detecting Salmonella;

[0048] Figure 14It is a graph showing the results of sensitivity investigation for detecting artificially contaminated milk using an aptamer biosensor;

[0049] Figure 15 It is a linear relationship graph for detecting Salmonella in artificially contaminated milk using an aptamer biosensor. Specific implementation manners

[0050] The following further describes the present invention in conjunction with specific embodiments and accompanying drawings, but the present invention is not limited by the embodiments.

[0051] The materials, reagents, instruments and methods used in the following examples are all conventional materials, reagents, instruments and methods in the art without special instructions, and can all be obtained through commercial channels. The strains used all originate from the Key Laboratory of Dairy Products of Northeast Agricultural University.

[0052] Example 1: Preparation method of aptamer biosensor

[0053] The schematic diagram of the aptamer biosensor of the present invention is shown in Figure 1 .

[0054] The nucleotide sequence of cDNA2 is shown in SEQ ID NO.1: 5’-CTGTCATAATGTCAAGTCC-3’;

[0055] The nucleotide sequence of cDNA3 is shown in SEQ ID NO.2: 5’-GTCGGGTGACGCCGCCATA-3’;

[0056] The nucleotide sequence of aptamer Apt2 is shown in SEQ ID NO.3: 5’-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3’;

[0057] The nucleotide sequence of the DNA template is shown in SEQ ID NO.4: 5’-AGTGGAAAAACCCCCCCCCCCC-3’.

[0058] (1) Preparation of SMBs-cDNA2 complex:

[0059] Wash 1 mg of SMBs three times with 1×B&W buffer, and then resuspend it in 200 μL of 2×B&W buffer. Subsequently, add an equal volume of 2 μmol / L cDNA2 solution, and then incubate it with shaking at 37 °C for 60 min. After removing the supernatant, wash the SMBs-cDNA2 complex three times with 1×B&W buffer, and finally resuspend it in 200 μL of PBS solution. Take the magnetic bead concentration as the complex concentration, that is, the final concentration is 5 μg / μL. Finally, store the prepared SMBs-cDNA2 complex at 4 °C for later use.

[0060] (2) Preparation of AuNPs-cDNA3 probe:

[0061] Mix 8 μL of thiol-modified cDNA3 with a concentration of 100 μmol / L with 1 mL of AuNPs solution, and then incubate at 50 °C for 12 h. Subsequently, add 100 μL of phosphate buffer A to the mixture, and continue to incubate at 50 °C for 1 h. After that, slowly add phosphate buffer B containing 2 mol / L NaCl in multiple portions for aging until the NaCl concentration in the solution reaches 0.2 mol / L. Centrifuge the solution at 12,000×g for 20 min at 4 °C to remove the excess unbound cDNA3. Finally, resuspend with 200 μL of deionized water to obtain the AuNP-cDNA3 probe, and store it in the dark at 4 °C before use.

[0062] Optimization of NaCl concentration during the preparation of AuNPs-cDNA3 probe:

[0063] Use final NaCl concentrations of 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L respectively, and operate according to step (2). The results are as Figure 2 shown.

[0064] The results show that as the NaCl concentration increases, the color of the prepared AuNPs-cDNA3 probe solution gradually deepens. When the final NaCl concentration is 0.3 mol / L, the color turns dark purple-red, indicating that the salt concentration is too high at this time, resulting in partial aggregation of AuNPs, thus causing color changes. When the final concentration reaches 0.4 mol / L, precipitation appears in the AuNPs solution. Therefore, this study selects 0.2 mol / L as the optimal final NaCl concentration.

[0065] (3) Preparation of SMBs-Apt2-AuNPs system:

[0066] Mix 2.5 nmol / L Apt2, 40 μg of SMBs-cDNA2 complex, and 20 μL of AuNPs-cDNA3 probe in 100 μL of PBS solution, and then incubate at 37 °C for 120 min to prepare the fluorescence quenching system. After magnetic separation to remove the supernatant, wash three times with PBS solution to obtain the SMBs-Apt2-AuNPs system.

[0067] ① Optimization of Apt2 concentration:

[0068] Select the final concentrations of Apt2 to be 1.5 nmol / L, 2.5 nmol / L, 3.5 nmol / L, and 4.5 nmol / L respectively, and conduct the experiment according to step (3). Detect the fluorescence spectrum of DNA-AgNCs by a multifunctional microplate reader (the excitation wavelength is 576 nm, and the emission spectrum from 601 to 750 nm is scanned at 2-nm intervals). The optimization results are as follows Figure 3 shown, where F0 and F represent the fluorescence intensities with peaks at 643 nm before and after the addition of Salmonella typhimurium respectively. The results show that as the final concentration of Apt2 increases, the fluorescence ratio (1 - F0 / F) gradually increases, reaches the maximum value at 2.5 nmol / L, and then decreases (P < 0.05). Therefore, the final concentration of Apt2 at 2.5 nmol / L is finally determined as the optimal concentration.

[0069] ② Optimization of the addition amount of SMBs-cDNA2 complex:

[0070] Select the addition amounts of the SMBs-cDNA2 complex to be 20 μg, 30 μg, 40 μg, and 50 μg respectively, and conduct the experiment according to step (3). Detect the fluorescence spectrum of DNA-AgNCs by a multifunctional microplate reader (the excitation wavelength is 576 nm, and the emission spectrum from 601 to 750 nm is scanned at 2-nm intervals). The optimization results are as follows Figure 4 shown, where F0 and F represent the fluorescence intensities with peaks at 643 nm before and after the addition of Salmonella typhimurium respectively. The results show that the fluorescence ratio (1 - F0 / F) increases as the addition amount of SMBs-cDNA2 increases from 20 μg to 30 μg (P < 0.05), shows no obvious change after 30 μg, and then decreases at 50 μg (P > 0.05). Therefore, the optimal addition amount of the SMBs-cDNA2 complex is finally determined to be 40 μg.

[0071] ③ Optimization of the addition amount of AuNPs-cDNA3 probe:

[0072] Select the addition amounts of the AuNPs-cDNA3 probe to be 5 μL, 10 μL, 15 μL, and 20 μL respectively, and conduct the experiment according to step (3). Detect the fluorescence spectrum of DNA-AgNCs by a multifunctional microplate reader (the excitation wavelength is 576 nm, and the emission spectrum from 601 to 750 nm is scanned at 2-nm intervals). The optimization results are as follows Figure 5 shown, where F0 and F represent the fluorescence intensities with peaks at 643 nm before and after the addition of Salmonella typhimurium respectively. The results show that the fluorescence ratio (1 - F0 / F) increases as the addition amount of the AuNPs-cDNA3 probe increases from 5 μL to 15 μL (P < 0.05), and then remains unchanged at 20 μL (P > 0.05). Therefore, the optimal addition amount of the AuNPs-cDNA3 probe is finally determined to be 15 μL.

[0073] ④ Optimization of the incubation times of SMBs-cDNA2, Apt2, and AuNPs-cDNA3:

[0074] Select the incubation times of the SMBs-Apt2-AuNPs system to be 30 min, 60 min, 90 min, and 120 min respectively, and conduct the experiment according to step (3). Detect the fluorescence spectrum of DNA-AgNCs by a multifunctional microplate reader (the excitation wavelength is 576 nm, and the emission spectrum from 601 to 750 nm is scanned at an interval of 2 nm). The optimization results are as Figure 6 shown, where F0 and F represent the fluorescence intensities with peaks at 643 nm before and after the addition of Salmonella typhimurium respectively. When the fluorescence ratio (1 - F0 / F) does not change significantly after the reaction reaches a certain time, this time is determined to be good, so the optimal incubation time is determined to be 90 min.

[0075] (4) Synthesis of DNA-AgNCs:

[0076] Add 6 μL of 1.5 mmol / L AgNO3 to 89 μL of PB solution with a concentration of 0.02 mol / L and a pH of 7.4 containing 1.5 nmol of DNA, and then incubate at 4 °C in the dark for 20 min. Subsequently, quickly add 5 μL of 1.5 mmol / L NaBH4 to the mixture, and then shake vigorously for 1 min. Before use, stabilize the mixture solution at 4 °C in the dark for 12 hours, and the concentration of the DNA used can be used as the concentration of the prepared DNA-AgNCs.

[0077] ① Optimization of the pH of the PB solution:

[0078] Select the pH of the PB solution to be 6.2, 6.6, 7, and 7.4 respectively, and conduct the experiment according to step (4). Detect the fluorescence spectrum of DNA-AgNCs by a multifunctional microplate reader (the excitation wavelength is 576 nm, and the emission spectrum from 601 to 750 nm is scanned at an interval of 2 nm). The optimization results are as Figure 7 shown. When the pH increases from 6.2 to 7, the fluorescence intensity of the generated DNA-AgNCs gradually increases, while when the pH increases from 7 to 7.4, the fluorescence intensity decreases. Therefore, the optimal pH of the PB solution is determined to be 7.

[0079] ② DNA / Ag + Optimization of the molar concentration ratio:

[0080] Select DNA / Ag +The molar concentration ratios are 1:2, 1:4, 1:6, and 1:8 respectively, and the experiment is carried out according to step (4). The fluorescence spectrum of DNA-AgNCs is detected by a multifunctional microplate reader (the excitation wavelength is 576 nm, and the emission spectrum from 601 to 750 nm is scanned at intervals of 2 nm). The optimization results are as Figure 8 shown. When the DNA / Ag⁺ molar concentration ratio ranges from 1:2 to 1:6, the fluorescence intensity of the generated DNA-AgNCs gradually increases. When the molar concentration ratio ranges from 1:6 to 1:8, the fluorescence intensity decreases. Therefore, the optimal DNA / Ag + molar concentration ratio is determined to be 1:6.

[0081] ③ Selection of the NaBH₄ / Ag + molar concentration ratio

[0082] Select NaBH₄ / Ag + The molar concentration ratios are 1:3, 2:3, 5:6, and 1:1 respectively, and the experiment is carried out according to step (4). The fluorescence spectrum of DNA-AgNCs is detected by a multifunctional microplate reader (the excitation wavelength is 576 nm, and the emission spectrum from 601 to 750 nm is scanned at intervals of 2 nm). The optimization results are as Figure 9 shown. When the DNA / Ag + molar concentration ratio ranges from 1:3 to 5:6, the fluorescence intensity of the generated DNA-AgNCs gradually increases, and when the molar concentration ratio ranges from 5:6 to 1:1, the fluorescence intensity does not change significantly. Therefore, the optimal NaBH₄ / Ag + molar concentration ratio is determined to be 5:6.

[0083] In summary, the optimal reaction conditions: DNA:NaBH₄:Ag + = 1:5:6.

[0084] Example 2: Method for detecting Salmonella using an aptamer biosensor

[0085] (1) Activation and culture of strains

[0086] (1) Activation and culture of the strain

[0087] Using Salmonella typhimurium ATCC 14028 as the standard strain for the experiment, inoculate the bacteria into LB medium at an inoculation amount of 2%, culture at 37 °C and 200 r / min for 12 h, then streak in three zones on an LB plate, and after culturing at 37 °C for 12 - 16 h, single colonies grow. Pick a single colony and inoculate it into LB medium for secondary activation for subsequent experiments.

[0088] (2) Add the target bacteria for detection

[0089] The SMBs-Apt2-AuNPs system obtained in Example 1 was incubated with 200 μL of Salmonella typhimurium ATCC 14028 at 37 °C for 120 min. The supernatant was discarded and resuspended in 80 μL of distilled water. Subsequently, 20 μL of 1.5 μmol / L DNA-AgNCs was added to the solution and incubated for 10 min, and the fluorescence spectrum of DNA-AgNCs was detected by a multifunctional microplate reader (the excitation wavelength was 576 nm, and the emission spectrum from 601 to 750 nm was scanned at 2 nm intervals).

[0090] Optimization of the incubation time of the SMBs-Apt2-AuNPs system with Salmonella:

[0091] The incubation times of the SMBs-Apt2-AuNPs system with Salmonella were selected as 30 min, 60 min, 90 min, and 120 min respectively, and the experiments were carried out according to the above-mentioned target bacteria detection method. The fluorescence spectrum of DNA-AgNCs was detected by a multifunctional microplate reader (the excitation wavelength was 576 nm, and the emission spectrum from 601 to 750 nm was scanned at 2 nm intervals). F0 and F represent the fluorescence intensities with peaks at 643 nm before and after the addition of Salmonella typhimurium respectively. When the fluorescence ratio (1 - F0 / F) did not change significantly after the reaction reached a certain time, this time was determined as the optimal time. Therefore, it can be seen from Figure 10 that the optimal incubation time is 60 min.

[0092] (II) Investigation of the sensitivity of the fluorescence biosensor

[0093] 1 mL of Salmonella typhimurium ATCC 14028 in the late logarithmic phase of culture was taken into a 1.5 mL sterilized centrifuge tube, and the cells were collected by centrifugation at 5000×g for 5 minutes. The cells were washed with an equal volume of PBS and serially diluted, and the number of bacteria was determined to be in the range of 3.7×10 0 to 3.7×10 8 CFU / mL by plate spreading. Under the optimal conditions, the bacterial solutions of each dilution were detected according to the steps in (I)(2). Each experiment was repeated three times. The investigation results are as Figure 11 shown. As the concentration of Salmonella typhimurium increased from 3.7×10 2 to 3.7×10 8 CFU / mL, the fluorescence spectrum gradually increased and the fluorescence intensity increased.

[0094] (III) Linear relationship of the fluorescence biosensor for detecting Salmonella

[0095] The samples were processed and detected according to the steps of sensitivity investigation in (II), and the results are as Figure 12As shown, F0 and F represent the fluorescence intensities with peaks at 643 nm before and after the addition of Salmonella typhimurium, respectively. The fluorescence ratio (1 - F0 / F) shows a linear relationship with the logarithm of the concentration of Salmonella typhimurium in the range of 3.7×10 2 to 3.7×10 5 CFU / mL. The linear regression equation is y = 0.1120x + 0.0307, and the correlation coefficient is R 2 = 0.9858. According to the calculation, the detection limit is 98 CFU / mL (LOD = 3N / S, where N is the standard deviation of the blank sample and S is the slope of the standard curve).

[0096] (IV) Investigation of the specificity of the fluorescence biosensor

[0097] Twelve common foodborne pathogenic bacteria and Salmonella typhimurium (strain information is shown in Table 1) were inoculated into LB liquid medium respectively and cultured overnight until the bacterial liquid concentration reached 10 7 CFU / mL. Specificity test analysis was carried out through the constructed aptamer biosensor. Take 1 mL of freshly cultured Salmonella typhimurium bacterial liquid into a 1.5 mL sterilized centrifuge tube, centrifuge at 5000×g for 5 min to collect the bacterial cells, and resuspend them in an equal volume of PBS solution. The concentration of the test strains is about 10 5 CFU / mL. In addition, sterilized LB broth medium was used as a negative control. Three parallel tests were set for each strain. The investigation results are as Figure 13 shown. The fluorescence ratios (1 - F0 / F) of the twelve common foodborne pathogenic bacteria are much lower than that of Salmonella typhimurium and are similar to those of the negative control group. Therefore, the aptamer biosensor described in the present invention has specificity.

[0098] Table 1 Strain information of twelve common foodborne pathogenic bacteria and Salmonella typhimurium

[0099]

[0100] Example 3: Method for detecting Salmonella in artificially contaminated milk samples using an aptamer biosensor

[0101] (1) Artificially contaminated milk:

[0102] The samples used in this test part were commercial milk purchased in the supermarket, which was tested by "GB 4789.4-2016 National Food Safety Standard Microbiological Examination of Food - Examination of Salmonella" and found to contain no Salmonella. The bacterial liquid of Salmonella typhimurium ATCC14028 was serially diluted, and 1 mL of each dilution gradient was added to 24 mL of milk samples respectively, so that the final concentration range of the bacterial liquid in the samples was 7.6×10 1 to 7.6×10 7CFU / mL.

[0103] (2) Pretreatment of artificially contaminated milk:

[0104] Take 1 mL of the artificially contaminated milk sample, remove the protein and fat in the milk by centrifugation at 8000×g for 5 min (4 °C), wash it three times repeatedly with PBS solution and then resuspend it in an equal volume of PBS solution.

[0105] (3) Investigate the sensitivity of detecting artificially contaminated milk using an aptamer biosensor:

[0106] Gradient dilute the pure cultured Salmonella typhimurium bacterial suspension and add it to the milk sample, and analyze it according to the steps of Example 2(2) under the optimal conditions. Each test is repeated three times. The investigation results are as Figure 14 shown.

[0107] The results show that the fluorescence emission spectrum increases with the increase of the concentration of Salmonella typhimurium in the artificially contaminated milk sample (from 7.6×10 1 to 7.6×10 7 CFU / mL). According to the calculation, the detection limit in the artificially contaminated milk sample is 3.4×10 2 CFU / mL.

[0108] (4) Linear relationship of artificially contaminated milk detection:

[0109] Add Salmonella typhimurium ATCC 14028 with known concentration to the milk sample, process and analyze it according to the method in (3) above, and calculate the corresponding colony count according to the mathematical formula between the bacterial concentration and the fluorescence signal established based on the actual sample detection above. Further compare the results with the plate coating results, and calculate the recovery rate of Salmonella typhimurium in the milk spiked sample by this fluorescence aptamer sensor. The above tests are repeated three times. The results are as Figure 15 shown.

[0110] The results show that there is a good linear relationship (R 3 between the fluorescence ratio (1 - F0 / F) and the logarithm of the Salmonella typhimurium concentration range from 7.6×10 6 to 7.6×10 2 CFU / mL) (R = 0.9754). The linear regression equation is y = 0.1105x – 0.1277, where y is the fluorescence ratio (1 - F0 / F) and x is the logarithmic concentration of Salmonella typhimurium.

[0111] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims. SEQUENCE LISTING <110> Northeast Agricultural University <120> An aptamer biosensor for detecting Salmonella, its preparation method and application <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Synthetic <400> 1 ctgtcataat gtcaagtcc 19 <210> 2 <211> 19 <212> DNA <213> Synthetic <400> 2 gtcgggtgac gccgccata 19 <210> 3 <211> 40 <212> DNA <213> Synthetic <400> 3 tatggcggcg tcacccgacg gggacttgac attatgacag 40 <210> 4 <211> 22 <212> DNA <213> Synthetic <400> 4 agtggaaaaa cccccccccc cc 22

Claims

1. A preparation method of an aptamer biosensor for rapid detection of Salmonella, characterized in that, The aptamer biosensor includes a sandwich structure SMBs-Apt2-AuNPs established by the SMBs-cDNA2 complex, aptamer Apt2, and AuNPs-cDNA3 probe, and silver nanocluster DNA-AgNCs synthesized with DNA as a template and used as a fluorescence signal; the SMBs-cDNA2 complex is formed by linking streptavidin magnetic beads with biotin-modified nucleic acid strand cDNA2, and the nucleotide sequence of cDNA2 is shown in SEQ ID NO.1; the AuNPs-cDNA3 probe is prepared from an AuNPs solution and thiol-modified cDNA3, and the nucleotide sequence of cDNA3 is shown in SEQ ID NO.2; the nucleotide sequence of the aptamer Apt2 is shown in SEQ ID NO.3; the nucleotide sequence of the DNA template is shown in SEQ ID NO.4; In the absence of Salmonella, the SMBs-Apt2-AuNPs system remains intact. When DNA-AgNCs are added, fluorescence resonance energy transfer occurs between AuNPs and AgNCs in the system to quench the fluorescence of AgNCs. In the presence of Salmonella, the AuNPs-cDNA3 probe suspended in the supernatant and Salmonella bound to Apt2 are removed by magnetic separation, resulting in non-quenched fluorescence. The preparation method of the aptamer biosensor includes the following steps: (1) Preparation of the SMBs-cDNA2 complex: After being washed with 1×B&W buffer, SMBs are resuspended in 2×B&W buffer, an equal volume of cDNA2 solution is added for incubation, the supernatant is removed, and after being washed with 1×B&W buffer, it is resuspended in PBS solution to obtain the SMBs-cDNA2 complex; (2) Preparation of the AuNPs-cDNA3 probe: Thiol-modified cDNA3 is mixed with an AuNPs solution, incubated, then phosphate buffer A is added and incubated again. Phosphate buffer B containing 2 mol / L NaCl is slowly added in multiple portions for aging until the NaCl concentration in the solution reaches 0.2 mol / L. After centrifugation, it is resuspended with deionized water to obtain the AuNP-cDNA3 probe; (3) Preparation of SMBs-Apt2-AuNPs system and synthesis of DNA-AgNCs: Mix 2.5 nM Apt2, 40 μg SMBs-cDNA2 complex, and 15 μg AuNPs-cDNA3 probe in 100 μL of PBS solution, incubate at 37 °C for 120 min, remove the supernatant by magnetic separation, and wash three times with PBS solution to obtain the SMBs-Apt2-AuNPs system; add 1 - 20 μL of AgNO3 solution with a concentration of 0.002 - 10 mmol / L to 50 - 200 μL of PB solution with a concentration of 0.002 - 0.2 mol / L and pH of 7 containing 0.5 - 5 nmol of DNA template, incubate at 4 °C in the dark for 20 min, add 0.5 - 20 μL of NaBH4 with a concentration of 0.1 - 10 mmol / L and shake vigorously for 1 min, and stabilize in the dark at 0 - 25 °C for 5 - 24 h before use. The DNA concentration used is the concentration of the prepared DNA-AgNCs; during the synthesis of DNA-AgNCs, the molar concentration ratio of DNA to Ag + is 1:6, and the molar concentration ratio of NaBH4 to Ag + is 5:

6.

2. The preparation method according to claim 1, characterized in that, In step (1), the dosage of SMBs is 0.1 - 5 mg, the dosage of 2×B&W buffer is 20 - 500 μL, the dosage of PBS solution is 20 - 500 μL, and the concentration of the cDNA2 solution is 0.5 - 5 μmol / L; the incubation temperature is 25 - 45 °C, and the incubation time is 30 - 120 min; the final concentration of the SMBs-cDNA2 complex is 1 - 20 μg / µL.

3. The preparation method according to claim 2, characterized in that, The dosage of SMBs is 1 mg, the dosage of 2×B&W buffer is 200 μL, the dosage of PBS solution is 200 μL, and the concentration of the cDNA2 solution is 2 μmol / L; the incubation temperature is 37 °C, and the incubation time is 60 min; the final concentration of the SMBs-cDNA2 complex is 5 μg / µL.

4. The preparation method according to claim 1, characterized in that, The concentration of the mercapto-modified cDNA3 described in step (2) is 10 - 500 μmol / L, the addition amount is 1 - 20 μL, the addition amount of the AuNPs solution is 1 mL, the addition amount of the phosphate buffer A is 50 - 500 μL, and the addition amount of deionized water is 50 - 500 μL; the incubation condition before adding the phosphate buffer A is incubation at 30°C - 70°C for 5 - 24 h, and the incubation condition after adding the phosphate buffer A is incubation at 30°C - 70°C for 0.1 - 2 h.

5. The preparation method according to claim 4, characterized in that, The concentration of the mercapto-modified cDNA3 described in step (2) is 100 μmol / L, the addition amount is 8 μL, the addition amount of the phosphate buffer A is 100 μL, and the addition amount of deionized water is 200 μL; the incubation temperature before adding the phosphate buffer A is 50°C, and the incubation time is 12 h; the incubation temperature after adding the phosphate buffer A is 50°C, and the incubation time is 1 h.

6. A method for detecting Salmonella using the aptamer biosensor described in claim 1, characterized in that, Comprising the following steps: S1. Add the SMBs-Apt2-AuNPs system to the sample, and incubate at 25°C - 45°C for 60 - 180 min; S2. Magnetically separate to discard the supernatant, and resuspend in 30 μL - 200 μL of distilled water; S3. Add 5 μL - 100 μL of DNA-AgNCs with a concentration of 0.1 μmol / L - 5 μmol / L to the solution and incubate for 2 min - 30 min; S4. Detect the fluorescence spectrum of DNA-AgNCs by a multifunctional microplate reader, the excitation wavelength is 550 nm - 600 nm, and scan the emission spectrum of 601 - 750 nm at an interval of 2 nm.

7. The method according to claim 6, characterized in that, The volume of the sample described in S1 is 20 - 800 μL, the incubation temperature is 37°C, and the incubation time is 120 min; the volume of the distilled water described in S2 is 80 μL; the volume of the DNA-AgNCs described in S3 is 20 μL, the concentration is 1.5 μmol / L, and the incubation time is 10 min; the excitation wavelength described in S4 is 576 nm.

8. Use of the aptamer biosensor described in claim 1 in the real-time detection of Salmonella contamination in food or food processing.

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