CN111139288B - Fluorescent sensor for simultaneously detecting staphylococcal enterotoxins A and B based on aptamer recognition-hybrid chain reaction - Google Patents

Abstract

The invention discloses a fluorescent sensor for simultaneous detection of Staphylococcus aureus enterotoxin A and B based on aptamer recognition-hybridization chain reaction. The detection method comprises: respectively hybridizing enterotoxin A aptamer 2 (SEA‑apt2) and enterotoxin B aptamer 2 (SEB‑apt2) with fluorescently labeled hairpin probes H1 and H2, and on the aptamers A chain reaction of hybridization occurs with the priming sequence to obtain the HCR reaction product; the aptamer 1 of SEA and SEB is modified on the surface of the magnetic beads to recognize the target SEA and SEB in the detection system respectively, and then the HCR reaction product is added to form a sandwich The structured aptamer 1-enterotoxin-aptamer 2/HCR complex realizes the quantitative detection of the target to be tested by measuring the fluorescence value. The present invention fully combines the advantages of high affinity and low cost of aptamers, signal amplification of hybridization chain reaction and high-efficiency separation of magnetic beads, etc., has the advantages of constant temperature amplification, high sensitivity, low cost, simple operation, etc., and has good application prospect.

Description

Simultaneous detection of Staphylococcus aureus intestinal tract based on aptamer recognition-hybridization chain reaction Fluorescent sensors for toxins A and B

technical field

The invention belongs to the field of food safety detection, and in particular relates to a fluorescent sensor detection method based on aptamer recognition-hybridization chain reaction and simultaneous enterotoxin A and B and an application thereof.

Background technique

Staphylococcus aureus (S. au ) is ubiquitous in nature and is one of the main pathogens causing foodborne diseases. It often contaminates milk and dairy products, meat and meat products, poultry and egg products, salads, pastries Waiting for food. Staphylococcus aureus can secrete enterotoxins (Staphylococcalenterotoxins, SEs) when propagated at 20-37°C for 4-8 hours. According to different serotypes, 22 SEs have been discovered and reported, including A, B, and C The five serotypes, D, D and E, are the most common, and they are also the main pathogenic factors causing Staphylococcus aureus food poisoning. According to statistics, the food poisoning incidents caused by Staphylococcus aureus enterotoxin in the world account for about 25%-45% of the total bacterial food poisoning incidents.

At present, the current detection standards of Staphylococcus aureus enterotoxin in my country mainly include: "National Food Safety Standard Food Microbiological Examination of Staphylococcus aureus" (GB 4789.10-2016), "Biotoxin Inspection Regulations at Entry-Exit Ports Part 2 : Staphylococcus aureus enterotoxin B" (SN/T 1763.2-2006), the detection method is based on antigen-antibody recognition technology. Although immunoassay technology is simple and fast, the preparation of antibodies requires immunization of animals, which is costly and easily inactivated. Therefore, it is urgent to develop a rapid detection technology for foodborne pathogens with high sensitivity, low cost and strong practicability.

In recent years, nucleic acid aptamer (Aptamer), as a new type of recognition molecule, has gradually become a research hotspot. or tertiary structure, forming a stable complex by interacting with target molecules through hydrogen bonds, van der Waals forces, etc. Small molecular substances, cells, microorganisms, etc.) are combined. Compared with traditional antibodies, it has a wide range of adaptation; high affinity and high specificity, not limited by immune conditions and immunogenicity; simple preparation, can be artificially synthesized in vitro; denaturation and renaturation are reversible, stable in nature; easy to label and store Etc. At present, nucleic acid aptamers of enterotoxins A, B, and C1 have been screened out (Huang et al., 2014; DeGrasse et al., 2012; Huang et al., 2015).

Hybridization chain reaction (HCR) is an in vitro nucleic acid isothermal signal amplification technology without enzyme participation. Since the HCR reaction does not require enzyme participation, the reaction is easy to control, and the cost is low, the reaction can be carried out at room temperature. The advantages of large-scale instruments and professionals, and diversified output signals make it widely used in the field of analysis and detection.

Contents of the invention

Aiming at the deficiencies of existing detection technologies, a fluorescent sensor based on aptamer recognition-hybridization chain reaction for simultaneous detection of Staphylococcus aureus enterotoxin A and B was proposed to realize the detection of Staphylococcus aureus enterotoxin A and B in food. High sensitivity, low cost, simple and rapid detection.

In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:

A fluorescent sensor for simultaneous detection of Staphylococcus aureus enterotoxin A and B based on aptamer recognition-hybridization chain reaction, comprising the following steps:

(1) Preparation of aptamer-modified magnetic nanoparticles: Aminated Fe ₃ O ₄ magnetic nanoparticles were coupled to streptavidin by the glutaraldehyde method, and grape aureus The aptamer 1 of coccus enterotoxin A and staphylococcus aureus enterotoxin B was modified on the surface of magnetic nanoparticles to obtain aptamer functionalized magnetic nanoparticles MNPs-SEA apt1 and MNPs-SEB apt1;

(2) Synthesis of hairpin probes for hybridization chain reaction system: According to the principle of hybridization chain reaction, the sequences of hairpin probe H1 and hairpin probe H2 for hybridization chain reaction are designed; wherein, H1 includes There are three parts a, b and c, the partial sequence of a is complementary to the sequence c to form a double strand as the stem of the H1 hairpin structure, the sequence b is complementary to the sequence d of the priming strand; H2 includes three parts a', b' and c' , wherein the partial sequence of a' and the sequence of c' are complementary to form a double strand as the stem of the H2 hairpin structure, H1a is complementary to H2a', and H2c' is complementary to H1c;

(3) Preparation of hybridization chain reaction products: Mix aptamer 2 of Staphylococcus aureus enterotoxin A and Staphylococcus aureus enterotoxin B with fluorescently labeled hairpin probe H1 and hairpin probe H2, respectively, at room temperature Hybridization, one end of the aptamer 2 has a short hybridization chain reaction initiating sequence, which acts as the initiator chain to initiate the hybridization chain reaction, and forms the reaction products of SEA apt2-dsDNA and SEB apt2-dsDNA respectively;

(4) Construction of a fluorescent sensor for simultaneous detection of SEA and SEB: MNPs-SEA apt1 and MNPs-SEBapt1 were mixed in hybridization buffer, SEA and SEB standard solutions of different concentrations were added, incubated, magnetically separated, and then SEAapt2- The dsDNA and SEB apt2-dsDNA reaction products were incubated to form aptamer 1-enterotoxin-aptamer 2/dsDNA sandwich structure, magnetically separated, washed, suspended in hybridization buffer, and excited at 495 nm, 588 nm and Under the emission light of 520 nm and 608 nm, detect the fluorescence signals of FAM and ROX, plot the standard concentration abscissa and the relative fluorescence as the ordinate, draw a standard curve, and establish a regression equation to obtain a fluorescence sensor. Measure the target, and calculate the concentration of the analyte according to the fluorescence intensity.

Further, the nucleotide sequence of SEA aptamer 1 in the step (1) is shown in SEQ ID NO.1, and the 5' end of the aptamer is labeled with biotin; the nucleotide sequence of SEB aptamer 1 is shown in SEQ ID NO.1 As shown in ID NO.2, the 5' end of the aptamer is labeled with biotin; in the step (2), the nucleotide sequence of the hairpin probe H1 is shown in SEQ ID NO: 5, and its 5' The end is marked with a fluorescent group; the nucleotide sequence of the hairpin probe H2 is shown in SEQ ID NO: 6, and its 3' is marked with a fluorescent group; the nucleotide sequence of SEA-apt 2 in the step (3) is as follows As shown in SEQ ID NO.3, the nucleotide sequence of SEB-apt 2 is shown in SEQ ID NO.4; wherein, the apt2 ends of SEA and AEB have a small segment of HCR initiation sequence, and the sequence is compatible with the hairpin probe H1 Complementary hybridization of partial sequences.

Further, the particle diameter of Fe ₃ O ₄ nanoparticles in the step (1) is 20-30 nm; the molar ratio of H1:H2:apt 2 in the step (3): 1:1:4-9, The hybridization reaction time is 1-2 h, and the fluorescent marker can be an organic fluorescent dye or a nano-luminescent material; the hybridization buffer in the step (4) is 0.01 mol/L PBS.

Application of fluorescent sensor for simultaneous detection of Staphylococcus aureus enterotoxin A and B based on aptamer recognition-hybridization chain reaction in food safety and environmental monitoring.

The detection principle of the present invention is as follows:

First, mix SEA apt 2 and SEB apt 2 with fluorescently labeled hairpins H1 and H2, respectively, and aptamer 2 has a short HCR initiation sequence at one end, which serves as the initiation chain to initiate the HCR reaction, forming SEA-apt2-dsDNA, respectively and SEBapt2-dsDNA reaction product. Mix MNPs-SEA apt1 and MNPs-SEB apt1 in the hybridization buffer, add the analyte, and then add the HCR reaction product to form a sandwich structure of aptamer 1-SE-aptamer 2/dsDNA, which is realized by the fluorescence value Quantitative detection of analytes.

The present invention has the following beneficial effects:

(1) High sensitivity: By combining the specific surface area and small size effect of nanomaterials, the signal amplification effect of hybridization chain reaction, and the high-efficiency separation effect of magnetic beads, the matrix interference is reduced and the detection sensitivity is maximized.

(2) Simple operation: no expensive instruments and cumbersome detection steps are required, the whole process is carried out under constant temperature conditions, and the operation is simple, which solves the defects of traditional methods such as cumbersome steps, time-consuming and laborious.

(3) Low cost: aptamers and hybrid chains can be artificially synthesized in vitro, which overcomes the shortcomings of long antibody preparation period and high cost, and reduces the production cost of test strips.

Description of drawings

FIG. 1 is a schematic diagram of the detection principle of Example 1.

FIG. 2 is a transmission electron microscope image of the magnetic nanoparticles of Example 1. FIG.

Fig. 3 is a standard curve diagram of detecting SEA and SEB by the fluorescence sensor of Example 1.

Detailed ways

The invention is further elaborated below in conjunction with specific examples, so that those skilled in the art can better understand the present invention and implement it, but the given examples are not intended to limit the scope of the present invention as a limitation of the present invention.

The aptamers (DeGrasse JA, 2013; Huang YK et al, 2015), priming strands, and hairpin DNA used in the following examples were all synthesized by Shanghai Bioengineering Technology Co., Ltd.

Example 1: The construction of a fluorescent sensor for simultaneous detection of Staphylococcus aureus enterotoxin A and B based on aptamer recognition-hybridization chain reaction, including the following specific steps:

(1) Preparation of aptamer-modified magnetic nanoparticles: 6.5 g of 1,6-hexanediamine, 2.0 g of anhydrous sodium acetate (CH ₃ COONa ) and 1.0 g of trichlorotrichloride hexahydrate were added to 30 mL of ethylene glycol FeCl ₃ 6H ₂ O) was stirred at 50°C to obtain a colloidal solution, which was transferred to a 50 mL polytetrafluoroethylene-lined reactor, reacted at 198°C for 6 h, cooled to room temperature, and discarded the upper liquid , deionized water washed out the lower layer of solid matter, collected by magnetic separation, washed twice with deionized water and ethanol, and dried at 50°C for 5-10 h to obtain aminated nano-magnetic beads, as shown in Figure 2;

Quantitatively weigh 10 mg of amino-modified magnetic nanomaterials and disperse them in 0.01 mol/L PBS (pH7.4) to a concentration of 1 mg/mL, ultrasonically disperse for 15 min, add 1.25 mL of 25% glutaraldehyde solution, and mix The solution was shaken and incubated at room temperature for 2 h, magnetically separated, the supernatant discarded, washed three times with 0.01 mol/L PBS, resuspended in PBS, ultrasonically dispersed, and streptavidin was added to make the concentration 0.5 mg/mL, at room temperature Shake and incubate for 12 h, magnetically separate, discard the supernatant, wash with PBS several times, resuspend in 1 mL PBS to obtain streptavidin-modified magnetic nanoparticles (SA-MNPs), and store at 4°C for later use;

Take SA-MNPs at a concentration of 1 mg/mL, add 1 μM biochemical aptamer 1 (SEA apt1 and SEBapt1), react at 37 °C for 2 hours, magnetically separate, discard the supernatant, and wash the polysaccharide with 0.01 mol/L PBS buffer. Resuspend the obtained aptamer functional magnetic nanoparticles (MNPs-SEA apt1 and MNPs-SEB apt1) in 1 mL of 0.1 mol/L PBS buffer and store at 4 °C ;

(2) Synthesis of hairpin probes for hybridization chain reaction (HCR) system: According to the principle of hybridization chain reaction, the sequences of hairpin probe H1 and hairpin probe H2 for HCR were designed; among them, H1 includes There are three parts a, b and c, the partial sequence of a is complementary to the sequence c to form a double strand as the stem of the H1 hairpin structure, the sequence b is complementary to the sequence d of the priming strand; H2 includes three parts a', b' and c' , wherein the partial sequence of a' and the sequence of c' are complementary to form a double strand as the stem of the H2 hairpin structure, H1a is complementary to H2a', and H2c' is complementary to H1c;

(3) Preparation of HCR reaction product: take FAM-H1 and ROX-H2 with a concentration of 1 μmol/L, heat in boiling water for 5 minutes, cool at room temperature to form a hairpin structure, and then mix 0.2 μmol SEA Apt 2 or SEB Apt 2 with FAM-H1 and ROX-H2 hairpin probes were mixed in the same volume ratio, and hybridized at room temperature for 1 hour to obtain the HCR reaction product, which was used for later use;

(4) Construction of a fluorescent sensor for simultaneous detection of SEA and SEB: Take 25 μL of MNPs-SEA apt1 and MNPs-SEB apt1 each, mix them and add them to the wells of the microtiter plate, add 50 μL of SEA+SEB mixed standard solutions of different concentrations ( 0, 0.5, 1, 5, 10, 50, 100, 500 ng/mL), incubate at 37 °C for 30 min, magnetically separate, wash with 10 mM PBS, resuspend in 100 μL PBS solution, add 50 μL of HCR reaction product, 37 °C Incubate for 30 min, magnetically separate, wash with PBS, resuspend in 200 μL PBS solution, detect the fluorescence signals of FAM and ROX under the excitation light of 495 nm and 588 nm and the emission light of 520 nm and 608 nm respectively, and the abscissa of standard concentration, Using relative fluorescence as the ordinate, draw a standard curve and establish a regression equation. Figure 3 shows the standard curve determined by this method. The linear range of SEA is 0.5-100 ng/mL, and the linear range of SEB is 1-100 ng/mL.

Embodiment 2: milk sample detection

Sample pretreatment: Take 10 mL of milk sample, centrifuge at 3500g for 10 min, discard the upper fat layer, and dilute 20 μL of the aspiration solution with distilled water (1:10) to obtain the sample solution to be tested.

Sample detection: Take 50 μL of MNPs-apt1 (1 mg/mL), add it to the well of the microtiter plate, add 200 μL of the sample to be tested, incubate at 37 °C for 30 min, magnetically separate, wash with 10 mM PBS, and resuspend in 100 μL PBS Add 50 μL of HCR reaction product to the solution, incubate at 37°C for 30 min, magnetically separate, wash with PBS, and resuspend in 200 μL PBS solution. Under the excitation light of 495 nm, 588 nm and the emission light of 520 nm, 608 nm, respectively, the fluorescence values of FAM and ROX were detected, substituted into the standard curve, and the contents of SEA and SEB in the sample were calculated.

Embodiment 3: detection of chicken sample

Sample pretreatment: Weigh 10 g of sample and mince, add 15 mL of 10 mM PBS solution, homogenize, shake for 15 min, and centrifuge at 3500 g for 10 min. Draw 5 mL of the upper layer suspension, transfer it to another centrifuge tube, then add 5 mL of heptane, mix well for 5 min, and centrifuge at 3500g for 5 min. Discard the upper organic phase (heptane layer), and take 200 μL of the lower aqueous phase for detection.

Sample detection: Take 50 μL of MNPs-apt1 (1 mg/mL), add it to the well of the microtiter plate, add 200 μL of the sample to be tested, incubate at 37 °C for 30 min, magnetically separate, wash with 10 mM PBS, and resuspend in 100 μL PBS Add 50 μL of HCR reaction product to the solution, incubate at 37°C for 30 min, magnetically separate, wash with PBS, and resuspend in 200 μL PBS solution. Under the excitation light of 495 nm, 588 nm and the emission light of 520 nm, 608 nm, respectively, the fluorescence values of FAM and ROX were detected, substituted into the standard curve, and the contents of SEA and SEB in the sample were calculated.

sequence listing

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Claims (3)

1. a method for preparing a fluorescent sensor based on aptamer recognition-hybridization chain reaction and simultaneously detecting Staphylococcus aureus enterotoxin A, B, is characterized in that: comprise the following steps: (1) Preparation of aptamer-modified magnetic nanoparticles: Aminated Fe ₃ O ₄ magnetic nanoparticles were coupled to streptavidin by the glutaraldehyde method, and grape aureus The aptamer 1 of coccal enterotoxin A and staphylococcus aureus enterotoxin B is modified on the surface of magnetic nanoparticles to obtain aptamer functionalized magnetic nanoparticles MNPs-SEAapt1 and MNPs-SEB apt1; wherein, the SEA adapter The nucleotide sequence of the aptamer 1 is shown in SEQ ID NO.1, and the 5' end of the aptamer is labeled with biotin; the nucleotide sequence of the SEB aptamer 1 is shown in SEQ ID NO.2, and the 5' end of the aptamer labeled with biotin; (2) Synthesis of hairpin probes for hybridization chain reaction system: According to the principle of hybridization chain reaction, the sequences of hairpin probe H1 and hairpin probe H2 for hybridization chain reaction are designed; wherein, H1 includes There are three parts a, b and c, the partial sequence of a is complementary to the sequence c to form a double strand as the stem of the H1 hairpin structure, the sequence b is complementary to the sequence d of the priming strand; H2 includes three parts a', b' and c' , wherein the partial sequence of a' and the c' sequence are complementary to form a double strand as the stem of the H2 hairpin structure, H1a is complementary to H2a', and H2c' is complementary to H1c; wherein, the nucleotide sequence of the hairpin probe H1 As shown in SEQ ID NO: 5, its 5' end is marked with a fluorescent group; the nucleotide sequence of the hairpin probe H2 is shown in SEQ ID NO: 6, and its 3' is marked with a fluorescent group; (3) Preparation of hybridization chain reaction products: Mix aptamer 2 of Staphylococcus aureus enterotoxin A and Staphylococcus aureus enterotoxin B with fluorescently labeled hairpin probe H1 and hairpin probe H2, respectively, at room temperature Hybridization, one end of the aptamer 2 has a short hybridization chain reaction initiating sequence, which serves as the initiator chain to initiate the hybridization chain reaction, respectively forming SEA apt2-dsDNA and SEB apt2-dsDNA reaction products; wherein, the SEA-apt2 The nucleotide sequence is shown in SEQ ID NO.3, and the nucleotide sequence of SEB-apt 2 is shown in SEQ ID NO.4; wherein, the apt2 ends of SEA and AEB have a small segment of HCR initiation sequence, and the sequence is the same as Hairpin probe H1 partial sequence complementary hybridization (4) Construction of a fluorescent sensor for simultaneous detection of SEA and SEB: Mix MNPs-SEA apt1 and MNPs-SEB apt1 in hybridization buffer, add different concentrations of SEA and SEB standard solutions, incubate, magnetically separate, and then add SEA apt2 -dsDNA and SEB apt2-dsDNA reaction product, incubated to form aptamer 1-enterotoxin-aptamer 2/dsDNA sandwich structure, magnetically separated, washed, suspended in hybridization buffer, excited at 495 nm and 588 nm respectively and 520nm, 608nm emission light, detect the fluorescence signal of FAM and ROX, draw the standard curve with the abscissa of the standard substance concentration and the ordinate of the relative fluorescence, and establish the regression equation to obtain the fluorescence sensor. When testing, add For the target to be detected, the concentration of the analyte is calculated according to the fluorescence intensity. 2. The method for preparing a fluorescent sensor based on aptamer recognition-hybridization chain reaction to simultaneously detect Staphylococcus aureus enterotoxin A and B according to claim 1, characterized in that, in the step (1), Fe ₃ O ₄ The particle size of the nanoparticles is 20-30nm; the molar ratio of H1:H2:apt 2 in the step (3) is 1:1:4-9, the hybridization reaction time is 1-2 h, and the fluorescent marker is an organic Fluorescent dyes or nanoluminescent materials; the hybridization buffer in step (4) is 0.01 mol/L PBS. 3. The detection application of the fluorescent sensor based on the aptamer recognition-hybridization chain reaction and the simultaneous detection of Staphylococcus aureus enterotoxin A and B prepared by the method according to any one of claims 1 to 2 in food safety and environmental monitoring .

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