CN111139288B - Fluorescent sensor for simultaneously detecting staphylococcal enterotoxins A and B based on aptamer recognition-hybrid chain reaction - Google Patents
Fluorescent sensor for simultaneously detecting staphylococcal enterotoxins A and B based on aptamer recognition-hybrid chain reaction Download PDFInfo
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Abstract
The invention discloses a fluorescence sensor for simultaneously detecting staphylococcus aureus enterotoxin A and staphylococcus aureus enterotoxin B based on aptamer recognition-hybridization chain reaction. The detection method comprises the following steps: respectively hybridizing an enterotoxin A aptamer 2 (SEA-apt 2) and an enterotoxin B aptamer 2 (SEB-apt 2) with the fluorescence-labeled hairpin probes H1 and H2, and carrying out hybridization chain reaction on a priming sequence on the aptamers to obtain HCR reaction products; modifying the aptamer 1 of SEA and SEB on the surface of a magnetic bead, respectively identifying the target SEA and SEB to be detected in a detection system, then adding an HCR reaction product to form an aptamer 1-enterotoxin-aptamer 2/HCR compound with a sandwich structure, and realizing the quantitative detection of the target to be detected by measuring the fluorescence value. The invention fully combines the advantages of high affinity and low cost of the aptamer, signal amplification of hybrid chain reaction, high-efficiency separation of magnetic beads and the like, has the advantages of constant-temperature amplification, high sensitivity, low cost, simple operation and the like, and has good application prospect.
Description
Technical Field
The invention belongs to the field of food safety detection, and particularly relates to a fluorescence sensor detection method based on aptamer recognition-hybrid chain reaction and enterotoxin A and B and application thereof.
Background
Staphylococcus aureus (1)Staphylococcus aureus, S. au) It is a common pathogen in nature and causes food-borne diseases, and often pollutes milk and dairy products, meat and meat products, poultry and egg products, salad, cakes and other foods. Staphylococcus aureus can Secrete Enterotoxins (SEs) after propagating for 4 to 8 hours at the temperature of 20 to 37 ℃, and 22 SEs are found and reported at present according to the difference of serotypes, wherein five serotypes A, B, C, D and E are the most common and are also main pathogenic factors causing food poisoning of the Staphylococcus aureus. Statistically, the incidence of food poisoning by staphylococcus aureus enterotoxin in the world accounts for approximately 25% -45% of the total incidence of bacterial food poisoning.
At present, the current detection standards of staphylococcus aureus enterotoxin in China mainly comprise: food safety national standard food microbiology test staphylococcus aureus (GB 4789.10-2016), part 2 of the entry and exit port biotoxin test protocol: staphylococcus aureus enterotoxin B (SN/T1763.2-2006), the detection method is based on antigen-antibody recognition technology. Although the immunoassay technology has the characteristics of simplicity and rapidness, the preparation of the antibody needs to immunize animals, the cost is high, and the antibody is easy to inactivate. Therefore, the development of a rapid detection technology for food-borne pathogenic bacteria with high sensitivity, low cost and strong practicability is urgently needed.
In recent years, aptamers (aptamers) have become a focus of research as novel recognition molecules, and essentially a single-stranded oligonucleotide folds into a secondary or tertiary structure such as a hairpin, stem-loop, pseudoknot, or G-quadruplex, and interacts with a target molecule through hydrogen bonds, van der waals forces, or the like to form a stable complex, and the diversity of spatial structures thereof can bind to almost all kinds of target molecules (cytokines, proteins, biotoxins, metal ions, small molecular substances, cells, microorganisms, or the like). Compared with the traditional antibody, the antibody has wide application range; high affinity and high specificity, and is not limited by immune conditions and immunogenicity; the preparation is simple and can be artificially synthesized in vitro; the denaturation and the renaturation are reversible, and the property is stable; easy to mark and store, etc. Currently, aptamers to enterotoxins a, B, C1 have been screened (Huang et al, 2014, deglasse et al, 2012, huang et al, 2015).
The Hybridization Chain Reaction (HCR) is an in vitro nucleic acid isothermal signal amplification technology without enzyme participation, and has the advantages of no need of enzyme participation, easy reaction control, low cost, reaction at normal temperature, no need of large-scale instruments and professionals, diversified output signals and the like, so that the HCR can be widely applied to the field of analysis and detection.
Disclosure of Invention
Aiming at the defects of the prior detection technology, the fluorescent sensor for simultaneously detecting the staphylococcal enterotoxins A and B based on aptamer recognition-hybridization chain reaction is provided, so that the staphylococcal enterotoxins A and B in the food can be detected simply and quickly with high sensitivity and low cost.
In order to solve the technical problems, the invention adopts the following technical scheme:
a fluorescence sensor for simultaneously detecting staphylococcus aureus enterotoxin A and B based on aptamer recognition-hybridization chain reaction comprises the following steps:
(1) Preparing aptamer modified magnetic nanoparticles: amination of Fe by the glutaraldehyde process 3 O 4 Coupling magnetic nanoparticles with streptavidin, and modifying aptamers 1 of staphylococcus aureus enterotoxin A and staphylococcus aureus enterotoxin B on the surfaces of the magnetic nanoparticles through a biotin-avidin system to obtain aptamers functionalized magnetic nanoparticles MNPs-SEA apt1 and MNPs-SEB apt1;
(2) Synthesis of hairpin probes for use in hybrid chain reaction systems: designing sequences of a hairpin probe H1 and a hairpin probe H2 for the hybridization chain reaction according to the principle of the hybridization chain reaction; wherein, H1 comprises three parts of a, b and c, a partial sequence of a and a c sequence are complemented into a double strand as a stem part of an H1 hairpin structure, and a b sequence is complemented with a priming strand d sequence; h2 comprises three parts, namely a ', b ' and c ', wherein a part of the sequence of a ' and the sequence of c ' are complementary into a double strand as a stem of a hairpin structure of H2, H1a is complementary with H2a ', and H2c ' is complementary with H1 c;
(3) Preparation of hybrid chain reaction products: respectively mixing an aptamer 2 of staphylococcus aureus enterotoxin A and staphylococcus aureus enterotoxin B with a fluorescence-labeled hairpin probe H1 and a hairpin probe H2, hybridizing at room temperature, wherein one end of the aptamer 2 is provided with a small segment of hybridization chain reaction initiation sequence which is used as an initiation chain to initiate hybridization chain reaction, and respectively forming SEA apt2-dsDNA and SEB apt2-dsDNA reaction products;
(4) And (3) constructing a fluorescence sensor for simultaneously detecting SEA and SEB: mixing MNPs-SEA apt1 and MNPs-SEB apt1 in a hybridization buffer solution, adding SEA and SEB standard solutions with different concentrations, incubating, performing magnetic separation, then adding a reaction product of SEA apt2-dsDNA and SEB apt2-dsDNA, incubating to form an aptamer 1-enterotoxin-aptamer 2/dsDNA sandwich structure, performing magnetic separation, washing, suspending in the hybridization buffer solution, detecting fluorescence signals of FAM and ROX under excitation light of 495 nm and 588nm and emission light of 520 nm and 608nm respectively, drawing a standard curve by using a standard substance concentration abscissa and using relative fluorescence as an ordinate, establishing a regression equation to obtain the fluorescence sensor, adding a target to be detected during testing, and calculating the concentration of the substance to be detected according to the fluorescence intensity.
Further, in the step (1), the nucleotide sequence of the SEA aptamer 1 is shown as SEQ ID NO.1, and biotin is marked at the 5' end of the aptamer; the SEB aptamer 1 has a nucleotide sequence shown in SEQ ID NO.2, and biotin is marked at the 5' end of the aptamer; in the step (2), the nucleotide sequence of the hairpin probe H1 is shown in SEQ ID NO:5, the 5' end of the derivative is marked with a fluorescent group; the nucleotide sequence of the hairpin probe H2 is shown as SEQ ID NO:6, 3' of which is marked with a fluorescent group; in the step (3), the SEA-apt2 nucleotide sequence is shown as SEQ ID NO.3, and the SEB-apt2 nucleotide sequence is shown as SEQ ID NO. 4; wherein, the apt2 end of SEA and AEB has a small segment of HCR initiation priming sequence, which is complementary hybridized with partial sequence of hairpin probe H1.
Further, fe in the step (1) 3 O 4 The particle size of the nano-particles is 20 to 30 nm; in the step (3), the molar ratio of H1 to H2 to apt2 is 1; the hybridization buffer in the step (4) is 0.01 mol/L PBS.
The fluorescent sensor for simultaneously detecting the staphylococcal enterotoxins A and B based on the aptamer recognition-hybridization chain reaction is applied to the detection of food safety and environmental monitoring.
The detection principle of the invention is as follows:
first, SEA apt2 and SEB apt2 were mixed with fluorescently labeled hairpins H1 and H2, respectively, and aptamer 2 was provided with a small HCR initiation priming sequence at one end, which served as a priming strand to prime the HCR reaction, forming SEA-apt2-dsDNA and SEB apt2-dsDNA reaction products, respectively. MNPs-SEA apt1 and MNPs-SEB apt1 are mixed in a hybridization buffer solution, a substance to be detected is added, then an HCR reaction product is added to form an aptamer 1-SE-aptamer 2/dsDNA sandwich structure, and the quantitative detection of the substance to be detected is realized through a fluorescence value.
The invention has the following beneficial effects:
(1) The sensitivity is high: by combining the specific surface area and small size effect of the nano material, the signal amplification effect of the hybrid chain reaction and the high-efficiency separation effect of the magnetic beads, the matrix interference is reduced, and the detection sensitivity is improved to a greater extent.
(2) The operation is simple: expensive instruments and complicated detection steps are not needed, the whole process is carried out under the constant temperature condition, the operation is simple, and the defects that the steps of the traditional method are complicated, time-consuming and labor-consuming and the like are overcome.
(3) The cost is low: the aptamer and the hybrid chain can be artificially synthesized in vitro, so that the defects of long antibody preparation period and high cost are overcome, and the production cost of the test strip is reduced.
Drawings
Fig. 1 is a schematic diagram of the detection principle of embodiment 1.
FIG. 2 is a transmission electron micrograph of magnetic nanoparticles of example 1.
FIG. 3 is a standard graph of fluorescence sensing assays SEA and SEB of example 1.
Detailed Description
The present invention is further described in the following examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the scope of the present invention.
The aptamers (DeGrasse JA, 2013; huang YK et al, 2015), the trigger strand, and the hairpin DNA used in the following examples were all synthesized by Shanghai bioengineering technology, inc.
Example 1: the construction of the fluorescence sensor for simultaneously detecting the staphylococcal enterotoxins A and B based on the aptamer recognition-hybridization chain reaction comprises the following specific steps:
(1) Preparing aptamer modified magnetic nanoparticles: to 30 mL of ethylene glycol were added 6.5g of 1, 6-hexanediamine, 2.0 g of anhydrous sodium acetate (CH) 3 COONa) and 1.0 g of ferric chloride hexahydrate (FeCl) 3 ·6H 2 O），Stirring at 50 ℃ to obtain a colloidal solution, transferring the solution into a 50 mL reaction kettle with a polytetrafluoroethylene lining, reacting at 198 ℃ for 6 h, cooling to room temperature, discarding the upper layer of liquid, flushing out the lower layer of solid substances by deionized water, carrying out magnetic separation and collection, washing for 2 times by the deionized water and ethanol respectively, and drying at 50 ℃ for 5-10 h to obtain aminated nano magnetic beads, wherein the figure is 2;
quantitatively weighing 10 mg of amino-modified magnetic nano material, dispersing the amino-modified magnetic nano material in 0.01 mol/L PBS (pH7.4) to enable the concentration of the amino-modified magnetic nano material to be 1mg/mL, ultrasonically dispersing for 15 min, adding 1.25 mL of 25% glutaraldehyde solution, oscillating and incubating the mixed solution at room temperature for 2h, magnetically separating, discarding the supernatant, washing the mixture with 0.01 mol/L PBS for three times, suspending the mixture in the PBS, ultrasonically dispersing, adding streptavidin to enable the concentration of the streptavidin to be 0.5 mg/mL, oscillating and incubating at room temperature for 12 h, magnetically separating, discarding the supernatant, washing the PBS for multiple times, and suspending the mixture in 1mL PBS to obtain magnetic nano particles (SA-MNPs) with streptavidin modified on the surfaces, and storing the magnetic nano particles at 4 ℃ for later use;
taking SA-MNPs with the concentration of 1mg/mL, adding 1 mu M of biochemical aptamer 1 (SEA apt1 and SEB apt 1), reacting for 2h at 37 ℃, carrying out magnetic separation, discarding the supernatant, washing for multiple times by using 0.01 mol/L PBS buffer solution to remove redundant aptamer 1, finally suspending the obtained aptamer functional magnetic nanoparticles (MNPs-SEA apt1 and MNPs-SEB apt 1) in 1mL of 0.1 mol/L PBS buffer solution, and storing at 4 ℃;
(2) Synthesis of hairpin probes for Hybrid Chain Reaction (HCR) system: designing sequences of a hairpin probe H1 and a hairpin probe H2 for HCR according to the principle of hybridization chain reaction; wherein, H1 comprises three parts of a, b and c, a partial sequence of a and a c sequence are complemented into a double strand as a stem part of an H1 hairpin structure, and a b sequence is complemented with a priming strand d sequence; h2 comprises three parts of a ', b ' and c ', wherein a part of the sequence of a ' and the sequence of c ' are complementary to form a double strand as a stem part of the hairpin structure of H2, H1a is complementary to H2a ', and H2c ' is complementary to H1 c;
(3) Preparation of HCR reaction product: heating FAM-H1 and ROX-H2 with the concentration of 1 mu mol/L in boiling water for 5 minutes, cooling at room temperature to form a hairpin structure, mixing 0.2 mu mol of SEA Apt2 or SEB Apt2 with FAM-H1 and ROX-H2 hairpin probes in the same volume ratio, and carrying out hybridization reaction at room temperature for 1 hour to obtain an HCR reaction product for later use;
(4) Constructing a fluorescence sensor for simultaneously detecting SEA and SEB: respectively taking 25 mu L of MNPs-SEA apt1 and MNPs-SEB apt1, mixing and adding the mixture into a plate hole of an enzyme-labeled plate, adding 50 mu L of SEA + SEB mixed standard solution (0, 0.5, 1, 5, 10, 50, 100, 500 ng/mL) with different concentrations, incubating at 37 ℃ for 30min, carrying out magnetic separation, washing with 10 mM PBS, suspending in 100 mu L of PBS solution, adding 50 mu L of HCR reaction product, incubating at 37 ℃ for 30min, carrying out magnetic separation, washing with PBS, suspending in 200 mu L of PBS solution, detecting fluorescence signals of FAM and ROX under excitation light of 495 nm and 588nm and emission light of 520 nm and 608nm respectively, drawing a horizontal coordinate of the standard substance concentration and a vertical coordinate of relative fluorescence, drawing a standard curve, establishing a regression equation, wherein the standard curve determined by the method is shown in figure 3, the linear range of the SEA is 0.5-100ng/mL, and the linear range of the SEB is 1-100 ng/mL.
Example 2: milk sample testing
Sample pretreatment: 10 mL of milk sample is taken, 3500g of milk sample is centrifuged for 10 min, an upper fat layer is discarded, 20 mu L of suction liquid is diluted by distilled water (1: 10), and a sample solution to be detected is obtained.
Sample detection: adding 50 mu L of MNPs-apt1 (1 mg/mL) into a hole of an enzyme-labeled plate, adding 200 mu L of a sample to be detected, incubating at 37 ℃ for 30min, carrying out magnetic separation, washing with 10 mM PBS, suspending in 100 mu L of PBS solution, adding 50 mu L of HCR reaction product, incubating at 37 ℃ for 30min, carrying out magnetic separation, washing with PBS, and suspending in 200 mu L of PBS solution. And detecting fluorescence values of FAM and ROX under excitation light at 495 nm and 588nm and emission light at 520 nm and 608nm respectively, substituting the fluorescence values into a standard curve, and calculating the contents of SEA and SEB in the sample.
Example 3: chicken sample testing
Sample pretreatment: weighing 10g of sample, mincing, adding 15 mL of 10 mM PBS, homogenizing, shaking for 15 min, and centrifuging for 10 min at 3500 g. 5 mL of the supernatant was aspirated, transferred to another centrifuge tube, and 5 mL of heptane was added, mixed well for 5 min, and centrifuged at 3500g for 5 min. The upper organic phase (heptane layer) was discarded, and 200. Mu.L of the lower aqueous phase was taken out and examined.
Sample detection: adding 50 mu L of MNPs-apt1 (1 mg/mL) into a hole of an enzyme-labeled plate, adding 200 mu L of a sample to be detected, incubating at 37 ℃ for 30min, carrying out magnetic separation, washing with 10 mM PBS, suspending in 100 mu L of PBS solution, adding 50 mu L of HCR reaction product, incubating at 37 ℃ for 30min, carrying out magnetic separation, washing with PBS, and suspending in 200 mu L of PBS solution. And detecting fluorescence values of FAM and ROX under excitation light at 495 nm and 588nm and emission light at 520 nm and 608nm respectively, substituting the fluorescence values into a standard curve, and calculating the contents of SEA and SEB in the sample.
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Claims (3)
1. A preparation method of a fluorescence sensor for simultaneously detecting staphylococcus aureus enterotoxin A and staphylococcus aureus enterotoxin B based on aptamer recognition-hybridization chain reaction is characterized by comprising the following steps: the method comprises the following steps:
(1) Preparing aptamer modified magnetic nanoparticles: amination of Fe by the glutaraldehyde process 3 O 4 Coupling magnetic nanoparticles with streptavidin, and modifying aptamers 1 of staphylococcus aureus enterotoxin A and staphylococcus aureus enterotoxin B on the surfaces of the magnetic nanoparticles through a biotin-avidin system to obtain aptamer-functionalized magnetic nanoparticles MNPs-SEA apt1 and MNPs-SEB apt1; wherein, the SEA aptamer 1 has a nucleotide sequence shown as SEQ ID NO.1, and biotin is marked at the 5' end of the aptamer; the nucleotide sequence of the SEB aptamer 1 is shown as SEQ ID NO.2, and the 5' end of the aptamerLabeled with biotin;
(2) Synthesis of hairpin probes for use in hybrid chain reaction systems: designing sequences of a hairpin probe H1 and a hairpin probe H2 for the hybridization chain reaction according to the principle of the hybridization chain reaction; the H1 comprises three parts, namely a part sequence a, b and c, wherein the part sequence a is complementary with the sequence c to form a double strand as a stem part of a hairpin structure of the H1, and the sequence b is complementary with the sequence d of the initiating strand; h2 comprises three parts, namely a ', b ' and c ', wherein a part of the sequence of a ' and the sequence of c ' are complementary into a double strand as a stem of a hairpin structure of H2, H1a is complementary with H2a ', and H2c ' is complementary with H1 c; wherein the nucleotide sequence of the hairpin probe H1 is shown in SEQ ID NO:5, the 5' end of the derivative is marked with a fluorescent group; the nucleotide sequence of the hairpin probe H2 is shown as SEQ ID NO:6, 3' of which is marked with a fluorescent group;
(3) Preparation of hybrid chain reaction products: respectively mixing an aptamer 2 of staphylococcus aureus enterotoxin A and staphylococcus aureus enterotoxin B with a fluorescence-labeled hairpin probe H1 and a hairpin probe H2, hybridizing at room temperature, wherein one end of the aptamer 2 is provided with a small segment of hybridization chain reaction initiation sequence which is used as an initiation chain to initiate hybridization chain reaction, and respectively forming SEA apt2-dsDNA and SEB apt2-dsDNA reaction products; wherein, the SEA-apt2 nucleotide sequence is shown as SEQ ID NO.3, and the SEB-apt2 nucleotide sequence is shown as SEQ ID NO. 4; wherein, the apt2 end of SEA and AEB has a small segment of HCR initiation priming sequence, which is complementary hybridized with partial sequence of hairpin probe H1
(4) And (3) constructing a fluorescence sensor for simultaneously detecting SEA and SEB: mixing MNPs-SEA apt1 and MNPs-SEB apt1 in a hybridization buffer solution, adding SEA and SEB standard solutions with different concentrations, incubating, performing magnetic separation, then adding a reaction product of SEA apt2-dsDNA and SEB apt2-dsDNA, incubating to form an aptamer 1-enterotoxin-aptamer 2/dsDNA sandwich structure, performing magnetic separation, washing, suspending in the hybridization buffer solution, detecting fluorescence signals of FAM and ROX under excitation light of 495 nm and 588nm and emission light of 520 nm and 608nm respectively, drawing a standard curve by using a standard substance concentration abscissa and using relative fluorescence as an ordinate, establishing a regression equation to obtain the fluorescence sensor, adding a target to be detected during testing, and calculating the concentration of the substance to be detected according to the fluorescence intensity.
2. The method for preparing the fluorescence sensor for simultaneously detecting the staphylococcal enterotoxins A and B based on the aptamer recognition-hybridization chain reaction according to claim 1, wherein in the step (1), fe 3 O 4 The particle size of the nano-particles is 20 to 30 nm; in the step (3), the molar ratio of H1 to H2 to apt2 is 1; the hybridization buffer in step (4) was 0.01 mol/L PBS.
3. The application of the fluorescent sensor for simultaneously detecting the staphylococcal enterotoxin A and the staphylococcal enterotoxin B based on the aptamer recognition-hybridization chain reaction, which is prepared by the method of any one of claims 1-2, in food safety and environmental monitoring.
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