A colorimetric detection method for Escherichia coli in milk

Abstract

The present invention relates to a colorimetric detection method for Escherichia coli in milk, and belongs to the technical field of food safety. In the present invention, the double-stranded probe is formed by hybridization of an aptamer and a target DNA. The aptamer in the double-stranded probe specifically binds to the target bacteria, releases the target DNA to hybridize with the hairpin probe, and exponentially amplifies the target DNA under the action of nuclease III. The amplified target DNA displaces the catalytic probe on the multi-walled carbon nanotube composite material. After the carbon nanotube composite material is magnetically adsorbed, the lignin carbon dots doped with potassium chloride in the supernatant catalyze hydrogen peroxide to generate hydroxyl radicals, which then oxidize the colorless substrate 3,3′,5,5′-tetramethylbenzidine to form a blue product, thereby realizing visual detection of Escherichia coli O157:H7. The present invention reduces sample matrix interference, has low cost, and has stable catalytic properties. Through the combination of nuclease III-assisted amplification technology and a new carbon nanozyme, the visual detection limit is further increased to 10 2 cfu / mL, which has very good promotion prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and in particular relates to a colorimetric detection method for Escherichia coli O157:H7 in milk. Background Art

[0002] Food contamination by pathogenic bacteria is a major global health issue. Escherichia coli O157:H7 is a type of serotype of Escherichia coli and is the main cause of foodborne diseases. It can cause hemorrhagic enteritis and has high morbidity and mortality rates. Among them, dairy products are an important contamination route for E. coli O157:H7. Many studies have shown that human digestive tract diseases caused by ingesting dairy products contaminated by E. coli O157:H7 occur from time to time. In addition, the contamination of E. coli O157:H7 not only causes serious harm to human life and health, but also brings huge losses to the dairy industry. Therefore, establishing a highly specific and sensitive detection method is an important technical prerequisite for the effective prevention and control of E. coli O157:H7.

[0003] The detection of E. coli O157:H7 in dairy products usually adopts the traditional microbial culture method, which is highly sensitive and inexpensive, but labor-intensive, requires highly qualified personnel, and takes 3-5 days to obtain test results. Other detection methods include molecular biological detection methods and immunological methods. Although these two technical methods greatly shorten the detection time and obtain higher sensitivity compared with traditional microbial culture methods. However, most molecular detection technologies require the use of large-scale precision instruments, and antibodies, which are the specific recognition elements of immunological methods, are expensive and greatly affected by the external environment. In addition, the reading of the results of these detection methods mostly requires the assistance of large instruments, and visual detection cannot be achieved.

[0004] In recent years, in order to shorten the detection time, reduce the detection cost, simplify the operation steps, and provide visual detection, the detection strategy based on catalase activity has been created and applied. Natural catalase is a biocatalyst that can catalyze hydrogen peroxide (H2O2) to generate hydroxyl radicals, and then oxidize the colorless substrate 3,3′,5,5′-tetramethylbenzidine (TMB) to form a blue product, with excellent catalytic activity and substrate specificity. However, strict storage conditions have prompted researchers to develop new peroxidase mimetics with simple synthesis, more stable structure, easy storage and transportation. In the past few decades, many nanomaterials with catalytic properties similar to peroxidase have been explored, such as metal nanoparticles, metal oxides, metal sulfides, metal organic frameworks, and many composite nanozymes, which have the advantages of good stability and low cost, can overcome the inherent defects of natural enzymes, and provide the possibility of application in different scenarios. But unfortunately, many of the precursors for synthesizing nanozymes reported previously are mostly toxic organics and precious metals, and the synthesis methods are complicated. Carbon-based nanozymes have attracted the interest of many researchers due to their environmental friendliness and ease of synthesis. They mainly include carbon dots, graphene oxides, mesoporous carbon, etc. Among them, carbon dots stand out among these carbon-based nanozymes due to their simple manufacturing technology, low toxicity, high stability and chemical inertness. At present, although the fluorescence properties of carbon dots have been widely studied, the technology based on alkali metal doping to improve the catalytic activity of carbon dots has never been reported in the detection of Escherichia coli O157:H7. In addition, the colorimetric detection strategy based on nanozyme construction is limited by low detection sensitivity and weak anti-interference ability of milk matrix. It is urgently needed to be combined with signal amplification technology and magnetic enrichment technology to achieve ultra-sensitive detection of Escherichia coli O157:H7 in milk. Summary of the invention

[0005] In order to achieve green, rapid and sensitive visual detection of Escherichia coli O157:H7 in milk, the present invention provides a colorimetric detection method for Escherichia coli in milk.

[0006] A colorimetric detection method for Escherichia coli in milk is as follows:

[0007] (1) Preparation of test solution

[0008] Take 1mL of milk at 5000 ×g Centrifuge for 5 min under the same conditions, discard the supernatant, and resuspend the precipitate with 1 mL of 0.1 M, pH 7.4 sterile PBS buffer to obtain the test solution;

[0009] (2) Amplification

[0010] (2.1) Add 200 μL of the test solution to 50 μL of the double-stranded probe solution and incubate at 37°C for 60 min.×g Centrifuge for 5 min under the same conditions, take the supernatant to obtain the composite solution;

[0011] The double-stranded probe is made by hybridizing an aptamer and a target DNA; the DNA sequence of the aptamer is 5′-CCGGAC GCT TAT GCC TTG CCA TCT ACA GAG CAG GTG TGA CGG-3′; the DNA sequence of the target DNA is 5′-GGC AAG GCA TAA GCG TCC GGA AAA AA-3′;

[0012] (2.2) Add 50 μL of 200 nM annealed hairpin probe solution to 60 μL of the complex solution, then add 0.45 μL of 200 U / μL exonuclease III solution and 20 μL of 10× enzyme digestion buffer, and add ultrapure water to a final volume of 200 μL; incubate at 37°C for 75 min, heat at 70°C for 20 min, and cool to room temperature to obtain an amplification solution;

[0013] The annealing hairpin probe is a stem-loop structure with a single-stranded free end at the 3′ end, and the DNA sequence of the annealing hairpin probe is 5′-GGC AAG GCA TAA GCG TCC GGA AAA AAC CGG ACG CTT ATG CCT TGC CAT GCCTTG CC-3′; the DNA sequence of the stem-loop structure in the annealing hairpin probe is 5′-GGC AAG GCA TAA GCG TCCGGA AAA AAC CGG ACG CTT ATG CCT TGC C-3′; the DNA sequence of the single-stranded free end at the 3′ end of the annealing hairpin probe is 5′-AT GCC TTG CC-3′;

[0014] (3) Detection

[0015] (3.1) Add 200 μL of multi-walled carbon nanotube composite probe solution to 200 μL of amplification solution, incubate at 37°C for 50 min, and obtain a conjugate mixture;

[0016] The multi-walled carbon nanotube composite probe is made of a multi-walled carbon nanotube chemically coupled catalytic probe;

[0017] The catalytic probe is made of potassium chloride-doped lignin carbon dots modified with amino cDNA;

[0018] The DNA sequence of the cDNA is 5′-TTT TCC GGA CGC TTA TGC CTT-(CH2)6-NH2-3′;

[0019] (3.2) Place 400 μL of the conjugate mixture on a magnetic rack and let it stand for 1 min. Take the supernatant as the catalytic composite solution.

[0020] (3.3) Add 50 μL of 100 mM hydrogen peroxide (H2O2) solution to the catalytic composite solution, and then add 50 μL of 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution, and react at 25°C in the dark for 15 min to obtain a reaction solution;

[0021] (4) Determination of test results

[0022] Observe the color change of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive;

[0023] Take 200 μL of the positive reaction solution and put it into a cuvette. Use an ultraviolet spectrophotometer to measure the peak absorbance intensity of the reaction solution in the cuvette at 654 nm. Substitute the absorbance intensity value at 654 nm into the detection equation to calculate the concentration of Escherichia coli O157:H7 in the test solution, and the detection is completed.

[0024] The further technical solution is as follows:

[0025] In step (2.1), the preparation steps of the double-stranded probe solution are as follows: 50 μL of 1.8 μM aptamer solution and 50 μL of 1.8 μM target DNA solution are mixed, heated at 95°C for 5 min, and naturally cooled to room temperature to obtain a double-stranded probe solution.

[0026] In step (2.2), the preparation steps of the annealed hairpin probe solution are as follows: 50 μL of a 200 nM hairpin probe solution is heated at 95° C. for 5 min, and naturally cooled to room temperature to obtain an annealed hairpin probe solution.

[0027] In step (3.1), the preparation steps of the multi-walled carbon nanotube composite probe solution are as follows:

[0028] (1) adding 2 mg of carboxylated multi-walled carbon nanotubes to 2 mL of a morpholineethanesulfonic acid monohydrate solution having a concentration of 21.325 g / L and a pH value of 5 to obtain a multi-walled carbon nanotube solution;

[0029] (2) Add 2 mg of N-hydroxysuccinimide and 2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 2 mL of the multi-walled carbon nanotube solution, incubate at 25 °C for 110 min, and obtain an activated multi-walled carbon nanotube solution;

[0030] (3) Add 6 mg of carbonyl iron powder to 2 mL of activated multi-walled carbon nanotube solution and incubate at 25 °C for 60 min to obtain a multi-walled carbon nanotube composite solution;

[0031] (4) Add 2 μL of 7 μM catalytic probe solution to 200 μL of multi-walled carbon nanotube composite solution and incubate at 37 °C for 60 min to obtain a catalytic probe mixed solution;

[0032] (5) Place 202 μL of the catalytic probe mixed solution on a magnetic rack and let it stand for 1 min. Discard the supernatant and add 200 μL of deionized water. Repeat the above operation twice and resuspend the precipitate in 200 μL of deionized water to obtain a multi-walled carbon nanotube composite probe solution.

[0033] The preparation steps of the catalytic probe solution are as follows:

[0034] (1) Add 5 mg of N-hydroxysuccinimide and 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 5 mL of potassium chloride-doped lignin carbon dot solution, and incubate at room temperature in the dark for 30 min to obtain an activated potassium chloride-doped lignin carbon dot solution;

[0035] (2) Take the above activated potassium chloride-doped lignin carbon dot solution, adjust the pH value to 7.4 with a 10 mg / mL sodium hydroxide solution, add 10 μL of a 100 μM amino-modified cDNA solution, and suspend at room temperature for 2 h to obtain a catalytic probe solution.

[0036] The preparation steps of the potassium chloride-doped lignin carbon dot solution are as follows:

[0037] (1) Mix 0.1 g of potassium chloride and 0.1 g of sodium lignin sulfonate, add 20 mL of deionized water and stir well to obtain a pretreatment solution;

[0038] (2) reacting the pre-treated solution at 180°C for 12 hours to obtain a black solution;

[0039] (3) The black solution was heated at 6500 × The mixture was centrifuged at 4000 × g for 10 min, and the supernatant was aspirated; the mixture was filtered through a filter membrane with a pore size of 0.22 μm to obtain a lignin carbon dot solution doped with potassium chloride.

[0040] In step (3.3), the 100 mM hydrogen peroxide (H2O2) solution is prepared by adding 10 μL of 10 M hydrogen peroxide (H2O2) solution to 990 μL of ultrapure water.

[0041] In step (3.3), the 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution is prepared by adding 0.048 g of 3,3′,5,5′-tetramethylbenzidine (TMB) to 10 mL of anhydrous ethanol.

[0042] See attached Figure 1 , the detection and analysis principle of the present invention is described as follows:

[0043] The present invention forms double-stranded DNA by hybridizing the aptamer with the target DNA. When the target bacteria appear, the aptamer in the double-stranded DNA specifically binds to the target bacteria, releases the target DNA, and hybridizes with the free 3' protruding end in the hairpin probe to form a 3' flat-end double-stranded DNA sequence recognized by nuclease III. Nuclease III enzymatically digests the 3' to 5' single nucleotides of the hybridized double-stranded DNA, thereby releasing the enzymatic hydrolysis products of the target DNA and the hairpin probe. Since the remaining sequence of the hairpin probe enzymatic hydrolysis product is exactly the same as the target DNA sequence, the effect of amplifying the target DNA can be achieved. The amplified target DNA hybridizes with the catalytic probe, displacing the catalytic probe on the multi-walled carbon nanotube composite material. After magnetic separation, the catalytic probe in the supernatant catalyzes hydrogen peroxide (H2O2) to generate hydroxyl radicals, which then oxidize the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) to form a blue product, thereby realizing colorimetric detection. Finally, based on the linear relationship between the logarithmic value of the Escherichia coli O157:H7 bacterial concentration and the increase in the system's absorbance intensity, a linear equation between bacterial concentration and absorbance intensity was established to achieve quantitative detection.

[0044] The beneficial technical effects of the present invention are embodied in the following aspects:

[0045] (1) The surface of sodium lignin sulfonate, the raw material for synthesizing lignin carbon dots in the present invention, has oxygen-containing functional groups such as ether bonds and phenolic hydroxyl groups. During the hydrothermal synthesis process, the addition of potassium chloride will change its surface state and convert the oxygen-containing functional groups on the surface of sodium lignin sulfonate salt into carbon-oxygen double bonds and carboxyl groups, thereby increasing the binding sites of hydrogen peroxide. Therefore, compared with natural enzymes, lignin carbon dots doped with potassium chloride have higher catalase-like activity and can improve detection sensitivity. The visual detection limit of the method of the present invention for Escherichia coli O157:H7 is 10 2 cfu / mL, compared with the traditional horseradish peroxidase-based detection of Escherichia coli O157:H7 10 4 The detection limit of cfu / mL was 2 orders of magnitude higher than that of horseradish peroxidase. In addition, compared with the protein nature of horseradish peroxidase, lignin carbon dots doped with potassium chloride have the advantages of being less affected by environmental conditions, high stability, and not easy to inactivate.

[0046] (2) The present invention uses lignin and potassium chloride as precursors for synthesizing nanozymes, avoiding the use of toxic organic matter and precious metals. In addition, lignin is the second largest renewable and sustainable resource in nature after cellulose. Since its surface is rich in various phenolic hydroxyl groups, alcoholic hydroxyl groups, carboxyl groups, carbonyl groups, methoxyl groups and other functional groups, it can create favorable conditions for various reactions and facilitate the functionalization of nucleic acid probes. On the other hand, lignin contains polar hydrophilic side chains and non-polar hydrophobic main chains, so it has a certain surface activity, which is convenient for actual detection in different media. In summary, carbon nanomaterials synthesized from lignin have excellent characteristics such as green, low production cost, easy surface functionalization and good biocompatibility.

[0047] (3) The present invention utilizes exonuclease III-assisted amplification technology to further increase the sensitivity of the sensor. The emerging exonuclease III-assisted amplification technology has the advantages of simplicity and high sensitivity. Compared with sequence-specific nick nucleases, exonuclease III does not require a specific recognition sequence and can directly enzymatically digest any double-stranded DNA with a 3′ blunt end or a concave end, which greatly simplifies the design of nucleic acid probes in the amplification system and is an effective signal amplification method for detecting foodborne pathogens. In the present invention, the hairpin probe in the amplification system is a stem-loop structure with a single-stranded free end at the 3′ end. The characteristics of exonuclease III are utilized to enzymatically digest the 3′ blunt end and concave end of the double-stranded DNA formed after the target DNA and the hairpin probe are hybridized. Since the designed hairpin probe sequence after enzymatic digestion is exactly the same as the target DNA, the target DNA can be amplified in multiples to achieve signal amplification of the detection method. It is worth noting that the amplification system hybridizes the aptamer with the target DNA into a double-stranded DNA, specifically identifies the target bacteria, and avoids the complex primer design and cumbersome specific nucleic acid extraction process when other strategies perform signal amplification through nucleic acid amplification.

[0048] (4) The special hexagonal arrangement of carbon atoms in multi-walled carbon nanotubes gives them π bonds on the surface. Relying on the adsorption effect of π bonds, a large number of nucleic acid probes can be loaded without complex chemical modification, which is more conducive to the biological functionalization of the corresponding target. On this basis, doping multi-walled carbon nanotubes with magnetic substances can not only simplify the separation process of materials, but also enhance the applicability of nanomaterials, and can achieve the separation of detection substances in food matrices to a great extent. In the present invention, multi-walled carbon nanotubes are chemically coupled with carbonyl iron powder, so that the multi-walled carbon nanotube composite material that adsorbs catalytic probes can achieve separation and detection integration, and get rid of the interference of milk matrix. Compared with traditional magnetic beads, multi-walled carbon nanotube composite materials have a larger specific surface area, can load more catalytic probes, and increase catalytic efficiency.

[0049] (5) Traditionally, the synthesis of magnetic multi-walled carbon nanotube composites mostly adopts coprecipitation and solvothermal methods, but this synthesis method often requires reagents with pungent odors such as ammonia water to complete the precipitation oxidation reaction of iron ions, or multiple steps such as high temperature are taken to obtain the desired composite, which adds to the tediousness and danger of the entire process of preparing multi-walled carbon nanocomposites, which is obviously not in line with the development concept of green chemistry. The present invention breaks away from the traditional means of preparing magnetic carbon nanotube composites and obtains a simple, safe and effective composite method. The synthesis method adds N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to an acidic solution of morpholineethanesulfonic acid monohydrate containing carboxylated multi-walled carbon nanotubes, which plays a role in activating the carboxyl groups on the surface of multi-walled carbon nanotubes and improving the stability of nanomaterials. In addition, the use of an acidic morpholineethanesulfonic acid monohydrate solution in the method not only promotes the role of the crosslinking agent, but also provides suitable conditions for the replacement reaction of iron particles. Due to the rapidity and safety of the synthesis method, it will be a promising method for synthesizing magnetic carbon nanocomposites.

[0050] (6) The present invention uses aptamers to replace antibodies as specific recognition elements, reducing the detection cost by about 75%. At the same time, compared with functionalized magnetic beads, multi-walled carbon nanotube composite materials are inexpensive, reducing the cost by about 70%. In addition, sodium lignin sulfonate, a raw material for synthesizing lignin carbon dots, has low cost and high cost-effectiveness, and can be promoted as a new raw material for synthesizing carbon dots. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the detection method of the present invention.

[0052] Figure 2 This is a color diagram of different concentrations of Escherichia coli O157:H7 in Example 2 of the present invention.

[0053] Figure 3 This is the absorption spectrum of Escherichia coli O157:H7 at different concentrations in Example 2 of the present invention.

[0054] Figure 4 This is a standard curve of the logarithm of the concentration of Escherichia coli O157:H7 bacteria-absorbance intensity value in Example 2 of the present invention.

[0055] Figure 5 This is a specific color diagram of the detection method in Example 5 of the present invention.

[0056] Figure 6 This is a graph of the specific absorbance intensity values ​​of the detection method in Example 5 of the present invention. DETAILED DESCRIPTION

[0057] The present invention will be further described below in conjunction with embodiments.

[0058] Unless otherwise defined, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0059] The test reagents and consumables used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified; the quantitative tests in the following examples are all repeated three times, and the results are averaged; the % in the following examples are all mass percentages unless otherwise specified.

[0060] In the following examples, exonuclease III was purchased from Dalian Bao Bioengineering Co., Ltd.;

[0061] 10× enzyme digestion buffer was an aqueous solution containing 78.8 g / L tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 4.7605 g / L magnesium chloride, and 1.54251 g / L dithiothreitol (DTT), purchased from Dalian Bao Bioengineering Co., Ltd.;

[0062] Carbonyl iron powder was purchased from Jilin Zhuochuang Innovation Materials Co., Ltd.;

[0063] Multi-walled carbon nanotubes were purchased from Nanjing Xianfeng Nanomaterial Technology Co., Ltd.;

[0064] Morpholineethanesulfonic acid monohydrate and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Aladdin Reagent Co., Ltd.;

[0065] 10 M hydrogen peroxide (H2O2) solution was purchased from Shanghai Wokai Biotechnology Co., Ltd.;

[0066] N-Hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were purchased from Sigma-Aldrich, USA;

[0067] Sodium lignin sulfonate was purchased from Aladdin Reagent Co., Ltd.;

[0068] Anhydrous ethanol and potassium chloride were purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0069] The filter membrane with a filter diameter of 0.22 μm was purchased from Jinteng Experimental Equipment Co., Ltd.;

[0070] The DNA sequences of cDNA, target DNA, and hairpin probe were designed by the inventors themselves and synthesized by Shanghai Sangon Biotechnology Co., Ltd.; the sequence of aptamer is referenced to “An aptamer-based biosensor for colorimetric detection of Escherichia coliO157:H7” was purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0071] Example 1

[0072] The invention discloses the preparation of double-stranded probe solution, annealed hairpin probe solution, potassium chloride-doped lignin carbon dot solution, catalytic probe solution and multi-walled carbon nanotube composite probe solution.

[0073] (1) The preparation steps of double-stranded probe solution are as follows:

[0074] Mix 50 μL of 1.8 μM aptamer solution with 50 μL of 1.8 μM target DNA solution, heat at 95°C for 5 min, and cool naturally to room temperature to obtain a double-stranded probe solution;

[0075] The DNA sequence of the aptamer in the aptamer solution was 5′-CCG GAC GCT TAT GCC TTG CCA TCT ACAGAG CAG GTG TGA CGG-3′;

[0076] The DNA sequence of the target DNA in the target DNA solution is 5′-GGC AAG GCA TAA GCG TCC GGA AAA AA-3′.

[0077] (2) The steps for preparing the annealed hairpin probe solution are as follows:

[0078] Heat 50 μL of 200 nM hairpin probe solution at 95°C for 5 min and cool naturally to room temperature to obtain an annealed hairpin probe solution;

[0079] The annealing hairpin probe used is a stem-loop structure with a single-stranded free end at the 3′ end, and the DNA sequence of the annealing hairpin probe is 5′-GGC AAG GCA TAA GCG TCC GGA AAA AAC CGG ACG CTT ATG CCT TGC CAT GCCTTG CC-3′. The DNA sequence of the stem-loop structure in the annealing hairpin probe is 5′-GGC AAG GCA TAA GCG TCCGGA AAA AAC CGG ACG CTT ATG CCT TGC C-3′; the DNA sequence of the single-stranded free end at the 3′ end of the annealing hairpin probe is 5′-AT GCC TTG CC-3′.

[0080] (3) The preparation steps of the potassium chloride-doped lignin carbon dot solution are as follows:

[0081] (3.1) Mix 0.1 g of potassium chloride and 0.1 g of sodium lignin sulfonate evenly, add 20 mL of deionized water and stir thoroughly to obtain a pretreatment solution;

[0082] (3.2) reacting the pre-treated solution at 180°C for 12 hours to obtain a black solution;

[0083] (3.3) The black solution was heated to 6500 ×g The mixture was centrifuged for 10 min under the same conditions, and the supernatant was aspirated and filtered through a filter membrane with a pore size of 0.22 μm to obtain a potassium chloride-doped lignin carbon dot solution.

[0084] (4) The preparation steps of the catalytic probe solution are as follows:

[0085] (4.1) Add 5 mg of N-hydroxysuccinimide and 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 5 mL of KCl-doped lignin carbon dot solution, and incubate at room temperature in the dark for 30 min to obtain an activated KCl-doped lignin carbon dot solution;

[0086] (4.2) Take the above activated potassium chloride-doped lignin carbon dot solution, adjust the pH value to 7.4 with a 10 mg / mL sodium hydroxide solution, add 10 μL of a 100 μM amino-modified cDNA solution, and suspend at room temperature for 2 h to obtain a catalytic probe solution;

[0087] The DNA sequence of cDNA in the amination cDNA solution is 5′-TTT TCC GGA CGC TTA TGC CTT-(CH2)6-NH2-3′.

[0088] (5) The preparation steps of the multi-walled carbon nanotube composite probe solution are as follows:

[0089] (5.1) 2 mg of carboxylated multi-walled carbon nanotubes were added to 2 mL of a 21.325 g / L, pH 5, morpholineethanesulfonic acid monohydrate solution to obtain a multi-walled carbon nanotube solution;

[0090] (5.2) Add 2 mg of N-hydroxysuccinimide and 2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 2 mL of the multi-walled carbon nanotube solution, incubate at 25 °C for 110 min, and obtain an activated multi-walled carbon nanotube solution;

[0091] (5.3) Add 6 mg of carbonyl iron powder to 2 mL of activated multi-walled carbon nanotube solution and incubate at 25 °C for 60 min to obtain a multi-walled carbon nanotube composite solution;

[0092] (5.4) Add 2 μL of 7 μM catalytic probe solution to 200 μL of multi-walled carbon nanotube composite solution and incubate at 37°C for 60 min to obtain a catalytic probe mixed solution;

[0093] (5.5) Place 202 μL of the catalytic probe mixed solution on a magnetic rack and let it stand for 1 min. Discard the supernatant and add 200 μL of deionized water. Repeat the above operation twice and resuspend the precipitate in 200 μL of deionized water to obtain a multi-walled carbon nanotube composite probe solution.

[0094] Example 2

[0095] Establishment of detection equation

[0096] (1) Take 1 mL of the E. coli O157:H7 stock solution that has been cultured in LB broth for 12 h until the late logarithmic growth phase, transfer it to a sterilized centrifuge tube, and centrifuge at 5000 ×g The suspension was centrifuged for 5 min under the conditions, the supernatant was discarded, and the suspension was resuspended in 1 mL of sterile PBS buffer with a concentration of 0.1 M and a pH value of 7.4 to obtain a bacterial suspension; the bacterial suspension was gradiently diluted with sterile PBS buffer with a concentration of 0.1 M and a pH value of 7.4 to prepare a 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 cfu / mL of Escherichia coli O157:H7 solution; a sterile PBS buffer solution with a concentration of 0.1M and a pH value of 7.4 was taken as a blank control solution, and the concentration of Escherichia coli O157:H7 in the blank control solution was 0 cfu / mL;

[0097] (2) In 200 μL of blank solution and 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 cfu / mL of E. coli O157:H7 solution, 50 μL of the same double-stranded probe solution was added, and the mixture was incubated at 37°C for 60 min. ×g The mixture was centrifuged for 5 min under the conditions, and the supernatant was taken to obtain seven composite solutions;

[0098] The double-stranded probe solution is prepared in Example 1;

[0099] (3) Add seven identical 50 μL 200 nM annealed hairpin probe solutions to seven 60 μL composite solutions, add 0.45 μL 200 U / μL exonuclease III solution and 20 μL 10× enzyme digestion buffer, and then add ultrapure water to a final volume of 200 μL. Incubate at 37°C for 75 min, heat at 70°C for 20 min, and cool to room temperature to obtain an amplification solution.

[0100] The annealed hairpin probe solution is prepared in Example 1;

[0101] (4) adding seven 200 μL amplification solutions into seven identical 200 μL multi-walled carbon nanotube composite probe solutions respectively, incubating at 37°C for 50 min to obtain seven conjugate mixtures;

[0102] The multi-walled carbon nanotube composite probe solution is prepared as in Example 1;

[0103] (5) Place seven 400 μL conjugate mixtures on a magnetic rack and let stand for 1 min. Take the supernatant solution to prepare seven catalytic composite solutions.

[0104] (6) Add 50 μL of a 100 mM hydrogen peroxide solution to each of the seven catalytic composite solutions, and then add 50 μL of a 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution to each of the seven catalytic composite solutions, and react at 25° C. in the dark for 15 min to obtain seven reaction solutions;

[0105] The 100 mM hydrogen peroxide (H2O2) solution is prepared by adding 10 μL of 10 M hydrogen peroxide (H2O2) solution into 990 μL of ultrapure water; the 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution is prepared by adding 0.048 g of 3,3′,5,5′-tetramethylbenzidine (TMB) into 10 mL of anhydrous ethanol;

[0106] (7) As attached Figure 2 As shown, it was observed that the concentration was 10 2 , 10 3 , 10 4 , 10 5 , 10 6 The reaction solution containing E. coli O157:H7 at a concentration of 10 cfu / mL was blue, indicating a positive result. 1 The reaction solution of E. coli O157:H7 with 10 cfu / mL was colorless and transparent, and the result was negative. Therefore, the visual detection limit was 10 2cfu / mL; Take 200 μL of the five positive reaction solutions in the cuvettes respectively, and measure the peak absorbance intensity of the five positive reaction solutions in the cuvettes at 654 nm by UV spectrophotometer. The results are shown in the attached Figure 3 ; The standard curve was drawn with the peak absorbance intensity of the five positive reaction solutions at 654nm as the ordinate Y and the logarithm of the concentration of Escherichia coli (cfu / mL) as the abscissa X. The results are shown in the attached Figure 4 , the detection equation is obtained: Y=0.1191X+0.1524, and the correlation coefficient is 0.9902.

[0107] Example 3

[0108] Study on the Detection of Escherichia coli O157:H7 in Artificially Contaminated Milk

[0109] (1) Preparation of artificially contaminated milk test solution

[0110] 1 mL of sterile milk was inoculated with an unknown concentration of E. coli O157:H7 solution and the concentration was 5000 ×g Centrifuge for 5 min under the conditions, discard the supernatant, and resuspend the precipitate with 1 mL of sterile PBS buffer with a concentration of 0.1 M and a pH value of 7.4 to obtain the test solution;

[0111] (2) Amplification

[0112] (2.1) Add 200 μL of the test solution to 50 μL of the double-stranded probe solution and incubate at 37°C for 60 min. ×g Centrifuge for 5 min under the same conditions, take the supernatant to obtain the composite solution;

[0113] The double-stranded probe solution is prepared in Example 1;

[0114] (2.2) Add 50 μL of 200 nM annealed hairpin probe solution to 60 μL of the complex solution, then add 0.45 μL of 200 U / μL exonuclease III solution and 20 μL of 10× enzyme digestion buffer, and add ultrapure water to a final volume of 200 μL; incubate at 37°C for 75 min, heat at 70°C for 20 min, and cool to room temperature to obtain an amplification solution;

[0115] The annealed hairpin probe solution is prepared in Example 1;

[0116] (3) Detection

[0117] (3.1) Add 200 μL of multi-walled carbon nanotube composite probe solution to 200 μL of amplification solution, incubate at 37°C for 50 min, and obtain a conjugate mixture;

[0118] The multi-walled carbon nanotube composite probe solution is prepared as in Example 1;

[0119] (3.2) Place 400 μL of the conjugate mixture on a magnetic rack and let it stand for 1 min. Take the supernatant as the catalytic composite solution.

[0120] (3.3) Add 50 μL of 100 mM hydrogen peroxide (H2O2) solution to the catalytic composite solution, and then add 50 μL of 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution, and react at 25°C in the dark for 15 min to obtain a reaction solution;

[0121] The 100 mM hydrogen peroxide (H2O2) solution is prepared by adding 10 μL of 10 M hydrogen peroxide (H2O2) solution into 990 μL of ultrapure water; the 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution is prepared by adding 0.048 g of 3,3′,5,5′-tetramethylbenzidine (TMB) into 10 mL of anhydrous ethanol;

[0122] (4) Determination of test results

[0123] The reaction solution was observed to be blue, and the result was positive. The absorbance intensity of the reaction solution at 654 nm was 0.726, which was put into the detection equation to calculate the concentration of Escherichia coli O157:H7 in the test solution to be 6.55×10 4 cfu / mL.

[0124] Example 4

[0125] Application of the present invention in detecting the content of Escherichia coli O157:H7 in milk

[0126] (1) Sample processing

[0127] Take 1mL of commercially available milk and stir at 5000 ×g Centrifuge for 5 min under the same conditions, discard the supernatant, and resuspend the precipitate with 1 mL of 0.1 M, pH 7.4 sterile PBS buffer to obtain the test solution;

[0128] (2) Amplification

[0129] (2.1) Add 200 μL of the test solution to 50 μL of the double-stranded probe solution and incubate at 37°C for 60 min. ×g Centrifuge for 5 min under the same conditions, take the supernatant to obtain the composite solution;

[0130] (2.2) Add 50 μL of 200 nM annealed hairpin probe solution to 60 μL of the complex solution, then add 0.45 μL of 200 U / μL exonuclease III solution and 20 μL of 10× enzyme digestion buffer, add ultrapure water to a final volume of 200 μL, incubate at 37°C for 75 min, heat at 70°C for 20 min, and cool to room temperature to obtain an amplification solution;

[0131] The annealed hairpin probe solution is prepared in Example 1;

[0132] (3) Detection

[0133] (3.1) Add 200 μL of multi-walled carbon nanotube composite probe solution to 200 μL of amplification solution, incubate at 37°C for 50 min, and obtain a conjugate mixture;

[0134] The multi-walled carbon nanotube composite probe solution is prepared in Example 1;

[0135] (3.2) Place 400 μL of the conjugate mixture on a magnetic rack and let it stand for 1 min. Take the supernatant as the catalytic composite solution.

[0136] (3.3) Add 50 μL of 100 mM hydrogen peroxide (H2O2) solution to the catalytic composite solution, and then add 50 μL of 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution, and react at 25°C in the dark for 15 min to obtain a reaction solution;

[0137] The 100 mM hydrogen peroxide (H2O2) solution is prepared by adding 10 μL of 10 M hydrogen peroxide (H2O2) solution into 990 μL of ultrapure water; the 4.8 g / L 3,3′5,5′-tetramethylbenzidine (TMB) solution is prepared by adding 0.048 g of 3,3′,5,5′-tetramethylbenzidine (TMB) into 10 mL of anhydrous ethanol;

[0138] (4) Determination of test results

[0139] Observe the color of the reaction solution. The reaction solution is colorless and transparent, and the test result is negative.

[0140] Example 5

[0141] Specific Detection Method for Escherichia coli O157:H7 in Milk

[0142] (1) Sample processing

[0143] Take the same 1mL sterile milk sample and place it in seven sterile centrifuge tubes. Add 1mL of 10% 6cfu / mL of Escherichia coli O157:H7, Salmonella typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa, Cronobacter, Staphylococcus aureus, and Vibrio parahaemolyticus to obtain seven mixed solutions; the seven mixed solutions were heated at 5000 ×g The mixture was centrifuged for 5 min under the conditions, the supernatant was discarded, and the precipitate was resuspended with 1 mL of sterile PBS buffer with a concentration of 0.1 M and a pH value of 7.4 to obtain seven dairy test solutions containing different pathogens;

[0144] (2) Amplification

[0145] (2.1) Add 200 μL of the corresponding dairy product test solution containing different pathogens to seven sterile centrifuge tubes, add 50 μL of double-stranded probe solution to each tube, incubate at 37°C for 60 min, and incubate at 5000× g The mixture was centrifuged for 5 min under the above conditions and the supernatant was taken to obtain seven composite solutions;

[0146] The double-stranded probe solution is prepared by Example 1;

[0147] (2.2) Add 50 μL of 200 nM annealed hairpin probe solution, 0.45 μL of 200 U / μL exonuclease III solution and 20 μL of 10× enzyme digestion buffer to each of the seven 60 μL composite solutions, and then add ultrapure water to a final volume of 200 μL. Incubate at 37°C for 75 min, heat at 70°C for 20 min, and cool to room temperature to obtain seven amplification solutions.

[0148] The annealed hairpin probe solution is prepared by Example 1;

[0149] (3) Detection

[0150] (3.1) adding seven 200 μL amplification solutions into seven identical 200 μL multi-walled carbon nanotube composite probe solutions respectively, incubating at 37° C. for 50 min to obtain seven conjugate mixtures;

[0151] The multi-walled carbon nanotube composite probe solution is prepared in Example 1;

[0152] (3.2) Place seven 400 μL conjugate mixtures on a magnetic rack and let stand for 1 min, then take the supernatant to obtain seven catalytic composite solutions;

[0153] (3.3) Add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution to each of the seven catalytic composite solutions, and react at 25°C in the dark for 15 min to obtain seven reaction solutions;

[0154] The 100 mM hydrogen peroxide (H2O2) solution is prepared by adding 10 μL of 10 M hydrogen peroxide (H2O2) solution into 990 μL of ultrapure water; the 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution is prepared by adding 0.048 g of 3,3′,5,5′-tetramethylbenzidine (TMB) into 10 mL of anhydrous ethanol;

[0155] (4) Determination of test results

[0156] The test results are shown in Figure 5, 1 is the reaction solution with Salmonella typhimurium, 2 is the reaction solution with Listeria monocytogenes, 3 is the reaction solution with Pseudomonas aeruginosa, 4 is the reaction solution with Cronobacter, 5 is the reaction solution with Staphylococcus aureus, 6 is the reaction solution with Vibrio parahaemolyticus, 7 is the reaction solution with Escherichia coli O157:H7; it is observed that the reaction solution with Escherichia coli O157:H7 is blue, and the test result is positive; the reaction solution with Salmonella typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa, Cronobacter The reaction solutions of bacteria such as Staphylococcus aureus and Vibrio parahaemolyticus are colorless and transparent, and the test results are negative. As shown in Figure 6, the absorbance intensity values ​​of the seven reaction solutions at 654nm are as follows: Escherichia coli O157:H7 is 0.833; Salmonella typhimurium is 0.01; Listeria monocytogenes is 0.012; Pseudomonas aeruginosa is 0.012; Cronobacter is 0.011; Staphylococcus aureus is 0.014; and Vibrio parahaemolyticus is 0.014. This shows that the present invention has good specificity for Escherichia coli O157:H7.

Claims

1. A colorimetric detection method for Escherichia coli in milk, characterized in that: The detection steps are as follows: (1) Preparation of test solution Take 1mL of milk at 5000 ×g Centrifuge for 5 min under the same conditions, discard the supernatant, and resuspend the precipitate with 1 mL of 0.1 M, pH 7.4 sterile PBS buffer to obtain the test solution; (2) Amplification (2.1) Add 200 μL of the test solution to 50 μL of the double-stranded probe solution and incubate at 37°C for 60 min. ×g Centrifuge for 5 min under the same conditions, take the supernatant to obtain the composite solution; The double-stranded probe is made by hybridizing an aptamer and a target DNA; the DNA sequence of the aptamer is 5′-CCG GACGCT TAT GCC TTG CCA TCT ACA GAG CAG GTG TGA CGG-3′; the DNA sequence of the target DNA is 5′-GGC AAG GCA TAA GCG TCC GGA AAA AA-3′; (2.2) Add 50 μL of 200 nM annealed hairpin probe solution to 60 μL of the complex solution, then add 0.45 μL of 200 U / μL exonuclease III solution and 20 μL of 10× enzyme digestion buffer, and add ultrapure water to a final volume of 200 μL; incubate at 37°C for 75 min, heat at 70°C for 20 min, and cool to room temperature to obtain an amplification solution; The annealing hairpin probe is a stem-loop structure with a single-stranded free end at the 3′ end, and the DNA sequence of the annealing hairpin probe is 5′-GGC AAG GCA TAA GCG TCC GGA AAA AAC CGG ACG CTT ATG CCT TGC CAT GCC TTGCC-3′; the DNA sequence of the stem-loop structure in the annealing hairpin probe is 5′-GGC AAG GCA TAA GCG TCC GGAAAA AAC CGG ACG CTT ATG CCT TGC C-3′; the DNA sequence of the single-stranded free end at the 3′ end of the annealing hairpin probe is 5′-AT GCC TTG CC-3′; (3) Detection (3.1) Add 200 μL of multi-walled carbon nanotube composite probe solution to 200 μL of amplification solution, incubate at 37°C for 50 min, and obtain a conjugate mixture; The multi-walled carbon nanotube composite probe is made of a multi-walled carbon nanotube chemically coupled catalytic probe; The catalytic probe is made of potassium chloride-doped lignin carbon dots modified with amino cDNA; The DNA sequence of the cDNA is 5′-TTT TCC GGA CGC TTA TGC CTT-(CH2)6-NH2-3′; In step (3.1), the preparation steps of the multi-walled carbon nanotube composite probe solution are as follows: (1) adding 2 mg of carboxylated multi-walled carbon nanotubes to 2 mL of a morpholineethanesulfonic acid monohydrate solution having a concentration of 21.325 g / L and a pH value of 5 to obtain a multi-walled carbon nanotube solution; (2) Add 2 mg of N-hydroxysuccinimide and 2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 2 mL of the multi-walled carbon nanotube solution, incubate at 25 °C for 110 min, and obtain an activated multi-walled carbon nanotube solution; (3) Add 6 mg of carbonyl iron powder to 2 mL of activated multi-walled carbon nanotube solution and incubate at 25 °C for 60 min to obtain a multi-walled carbon nanotube composite solution; (4) Add 2 μL of 7 μM catalytic probe solution to 200 μL of multi-walled carbon nanotube composite solution and incubate at 37 °C for 60 min to obtain a catalytic probe mixed solution; (5) Place 202 μL of the catalytic probe mixed solution on a magnetic rack and let it stand for 1 min, discard the supernatant, add 200 μL of deionized water, repeat the above operation twice, and resuspend the precipitate in 200 μL of deionized water to obtain a multi-walled carbon nanotube composite probe solution; The preparation steps of the catalytic probe solution are as follows: (1) Add 5 mg of N-hydroxysuccinimide and 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 5 mL of potassium chloride-doped lignin carbon dot solution, and incubate at room temperature in the dark for 30 min to obtain an activated potassium chloride-doped lignin carbon dot solution; (2) Take the above activated potassium chloride-doped lignin carbon dot solution, adjust the pH value to 7.4 with a 10 mg / mL sodium hydroxide solution, add 10 μL of a 100 μM amino-modified cDNA solution, and suspend at room temperature for 2 h to obtain a catalytic probe solution; The preparation steps of the potassium chloride-doped lignin carbon dot solution are as follows: (1) Mix 0.1 g of potassium chloride and 0.1 g of sodium lignin sulfonate, add 20 mL of deionized water and stir well to obtain a pretreatment solution; (2) reacting the pre-treated solution at 180°C for 12 hours to obtain a black solution; (3) The black solution was heated at 6500 ×g Centrifuge for 10 min under the same conditions and aspirate the supernatant; The solution was filtered through a filter membrane with a pore size of 0.22 μm to obtain a lignin carbon dot solution doped with potassium chloride; (3.2) Place 400 μL of the conjugate mixture on a magnetic rack and let it stand for 1 min. Take the supernatant as the catalytic composite solution. (3.3) Add 50 μL of 100 mM hydrogen peroxide (H2O2) solution to the catalytic composite solution, and then add 50 μL of 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution, and react at 25°C in the dark for 15 min to obtain a reaction solution; (4) Determination of test results Observe the color change of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive; Take 200 μL of the positive reaction solution and put it into a cuvette. Use an ultraviolet spectrophotometer to measure the peak absorbance intensity of the reaction solution in the cuvette at 654 nm. Substitute the absorbance intensity value at 654 nm into the detection equation to calculate the concentration of Escherichia coli O157:H7 in the test solution, and the detection is completed.

2. A colorimetric detection method for Escherichia coli in milk according to claim 1, characterized in that: In step (2.1), the preparation steps of the double-stranded probe solution are as follows: 50 μL of 1.8 μM aptamer solution and 50 μL of 1.8 μM target DNA solution are mixed, heated at 95°C for 5 min, and naturally cooled to room temperature to obtain a double-stranded probe solution.

3. A colorimetric detection method for Escherichia coli in milk according to claim 1, characterized in that: In step (2.2), the preparation steps of the annealed hairpin probe solution are as follows: 50 μL of a 200 nM hairpin probe solution is heated at 95° C. for 5 min, and naturally cooled to room temperature to obtain an annealed hairpin probe solution.

4. A colorimetric detection method for Escherichia coli in milk according to claim 1, characterized in that: In step (3.3), the 100 mM hydrogen peroxide (H2O2) solution is prepared by adding 10 μL of 10 M hydrogen peroxide (H2O2) solution to 990 μL of ultrapure water.

5. A colorimetric detection method for Escherichia coli in milk according to claim 1, characterized in that: In step (3.3), the 4.8 g / L 3,3′,5,5′-tetramethylbenzidine (TMB) solution is prepared by adding 0.048 g of 3,3′,5,5′-tetramethylbenzidine (TMB) to 10 mL of anhydrous ethanol.

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