CN111796092B

CN111796092B - pH response-based heterochromatic nanoparticles, pathogen detection kit containing nanoparticles and detection method

Info

Publication number: CN111796092B
Application number: CN202010823101.4A
Authority: CN
Inventors: 吴薇; 杨庆利; 颜春蕾
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2020-08-17
Filing date: 2020-08-17
Publication date: 2021-05-11
Anticipated expiration: 2040-08-17
Also published as: AU2021100947A4; CN111796092A

Abstract

The invention discloses heterochromatic nanoparticles based on pH response, a pathogenic bacterium detection kit containing the same and a detection method, and belongs to the technical field of food-borne pathogenic bacterium detection. The heterochromatic nanoparticles are formed by self-assembling pH indicator molecules, BSA and aptamer1 of pathogenic microorganisms; the pathogenic bacterium detection kit containing the nano-particles also comprises a 96-pore plate coated with streptavidin and a nucleic acid aptamer 2 of a target pathogenic microorganism to be detected, wherein the 5' end of the nucleic acid aptamer is modified with biotin; in the pH response-based heterochromatic nanoparticles, aptamer1 specifically recognizes and combines pathogenic bacteria, and OH is added^‑The color change is generated and is larger than that of a blank control group, and the 96-well plate can be used for simultaneously detecting various pathogenic bacteria and has high sensitivity and good specificity.

Description

pH response-based heterochromatic nanoparticles, pathogen detection kit containing nanoparticles and detection method

Technical Field

The invention belongs to the technical field of food-borne pathogenic bacteria detection, and particularly relates to pH response-based heterochromatic nanoparticles, a pathogenic bacteria detection kit containing the same and a detection method.

Background

In recent years, with the acceleration of the progress of economic globalization, food safety has become the focus of today's global public health. Food safety issues arising from food-borne diseases have become a global public health priority. The incidence of food-borne diseases is the front of the total incidence of various diseases, and about 220 million people are lost due to the infection of the food-borne diseases every year around the world. With the rapid development of market economy and the rapid improvement of the living standard of people in China, consumers pay more attention to the food safety problem particularly after China is added to the world trade organization. According to statistical data of the ministry of commerce, the food exported in China has losses of more than 170 billion dollars per year due to food safety problems, and other economic losses caused by food poisoning amount to hundreds of billions of yuan. Pathogenic bacteria are the main cause of food-borne diseases.

Pathogenic bacteria are widely distributed in nature; and with the change of environment, the number and the type of the pathogenic microorganisms are changed correspondingly. In addition, food-borne pathogenic microorganism contamination involves many steps, including breeding, food processing, storage, transportation, sale, cooking, etc. of food materials, and not only these pathogenic microorganisms produce and release toxins to cause food poisoning. Effective monitoring of microbial contamination in the food production and distribution fields has become a real problem facing people. In order to control the pathogenic bacteria pollution in food and integrate pathogenic bacteria limit regulations dispersed in different food standards, GB 29921-2013 'pathogenic bacteria limit in national food Standard for food safety' was issued in 26 12 months in 2013, and has been formally implemented in 1 month 7 in 2014. The national standard stipulates the limits of 5 pathogenic bacteria, namely salmonella, Listeria monocytogenes, Escherichia coli O157: H7, staphylococcus aureus and Vibrio parahaemolyticus in 11 types of food, such as meat products, aquatic products, instant egg products, food products, instant bean products and the like. In view of the characteristics of wide pollution range and great harm of food-borne pathogenic microorganisms, a method which is efficient, sensitive, accurate, economical and suitable for on-site rapid detection is urgently needed to be established so as to reduce or avoid the harm of the pathogenic microorganisms to human bodies.

Pathogenic bacteria related to the current food microbial sanitation detection mainly comprise salmonella, shigella, diarrhea escherichia coli, vibrio parahaemolyticus, yersinia enterocolitica, campylobacter jejuni, staphylococcus aureus, streptococcus hemolyticus, clostridium botulinum, clostridium perfringens, bacillus cereus, listeria monocytogenes and the like. The detection method mainly comprises a conventional detection method, an immunological detection method, a molecular biological detection method and the like. The detection of the pathogenic microorganisms in the food is mainly determined by a traditional culture method, the traditional culture method generally comprises five steps of pre-enrichment, selective plate separation, biochemical screening and serological identification, the biggest problem is that the time consumption is long, the requirement of rapid detection cannot be met, rapid judgment and selection cannot be made, and an ideal identification result cannot be given to some bacteria. The basic principle of immunological detection is antigen-antibody reaction. An antigen-antibody reaction refers to a specific binding reaction that occurs between an antigen and a corresponding antibody. Different microorganisms have specific antigens and can stimulate the body to produce corresponding specific antibodies. In recent years, continuously developed immunological methods such as immunofluorescence technology, enzyme linked immunosorbent assay, radioimmunoassay and the like are gradually applied to detection of pathogenic bacteria, but still have the problems of complex operation, low sensitivity and the like. Compared with the traditional method, the method has high requirements on instruments and equipment, greatly limits the application and popularization of the methods, and greatly reduces the practical value of the methods. Therefore, in order to meet the demand of the modern food industry for rapid detection, an accurate, simple, rapid, sensitive and specific food detection method is urgently needed to be established.

Aptamers are single-stranded DNA or RNA molecules that can recognize and bind to a target molecule. Aptamers are capable of recognizing a variety of highly specific targets, including ions, drugs, mycotoxins, pathogens, or whole cells. Aptamers have good affinity for specific targets with dissociation coefficients (Kd values) ranging between pM to mM. Compared with an antibody, the aptamer has higher stability to temperature, pH, ionic strength and the like, is easier to synthesize and modify, has a longer life cycle and is lower in cost. Based on the advantages of the aptamer, the aptamer has attracted extensive attention in the construction of aptamer-based detection and sensors, and has great potential in pathogen detection and biomolecule screening. The pH indicator has the capability of quickly responding to the change of the pH value of the solution and is used as a sensitive signal molecule in colorimetric analysis. The pH indicator is combined with an aptamer to be assembled into nanoparticles by utilizing the hydrophobicity and the inherent color change effect of the pH indicator, so that a novel synchronous colorimetric detection method of food-borne pathogenic bacteria (escherichia coli, salmonella, staphylococcus aureus, vibrio parahaemolyticus and streptococcus haemolyticus) can be established, and the operation is simple and rapid.

Disclosure of Invention

In view of the problems in the prior art, a first object of the present invention is to provide a self-assembly method for synthesizing heterochromatic nanoparticles based on pH response; a second object of the present invention is to provide the use of heterochromatic nanoparticles based on pH response; the third purpose of the invention is to provide a detection kit for pathogenic bacteria, which contains the heterochromatic nano-particles based on pH response; the fourth purpose of the invention is to provide a method for rapidly detecting food-borne pathogenic bacteria; the method solves the problems of low sensitivity, complex detection method, high requirements on instruments and equipment and the like in the existing technology for detecting the food-borne pathogenic bacteria.

In order to achieve the purpose, the invention adopts the following technical scheme:

the heterochromatic nanoparticles based on pH response are formed by self-assembling pH indicator molecules, BSA and aptamer1 of pathogenic microorganisms; the Aptamer1 is coupled on a pH indicator molecule to serve as an identification unit, BSA blocks non-specific sites, and nanoparticles of the pH-BSA/Aptamer 1 formed through pi-pi accumulation and hydrogen bond interaction are the heterochromatic nanoparticles based on pH response.

On the basis of the scheme, the pH indicator molecule is colorless or light in color under neutral conditions and changes in color under alkaline conditions; preferably, the pH indicator molecule is any one of phenolphthalein, thymolphthalein and curcumin.

On the basis of the scheme, the pathogenic microorganism is one of food-borne pathogenic bacteria escherichia coli, salmonella typhimurium, staphylococcus aureus, vibrio parahaemolyticus and streptococcus haemolyticus;

it is worth to be noted that the heterochromatic nanoparticles based on pH response are suitable for detection of common food-borne pathogenic bacteria in the prior art, are not limited to the pathogenic bacteria mentioned in the invention, and only need to use the aptamers corresponding to the pathogenic bacteria when the heterochromatic nanoparticles are used for detecting other food-borne pathogenic bacteria.

The preparation method of the heterochromatic nano-particles based on pH response comprises the following steps:

(1) dissolving a pH indicator in an organic solvent, dripping aptamer1 into the pH indicator solution, and stirring for 1 h;

(2) dropwise adding the BSA solution into the mixed solution obtained in the step 1), sealing, stirring, centrifuging and washing to obtain the BSA-containing solution.

The heterochromatic nanoparticles based on pH response are applied to preparation of a food-borne pathogenic bacterium detection kit.

A detection kit for food-borne pathogenic bacteria comprises heterochromatic nanoparticles based on pH response, a 96-well plate coated with streptavidin, and a nucleic acid aptamer 2 of a target pathogenic microorganism to be detected, wherein the 5' end of the nucleic acid aptamer 2 is modified with biotin; wherein the heterochromatic nanoparticles based on pH response are formed by self-assembling pH indicator molecules, BSA and aptamer1 of a target pathogenic microorganism to be detected; the Aptamer1 is coupled on a pH indicator molecule to serve as an identification unit, BSA blocks non-specific sites, and nanoparticles of the pH-BSA/Aptamer 1 formed through pi-pi accumulation and hydrogen bond interaction are the heterochromatic nanoparticles based on pH response.

On the basis of the scheme, the pH indicator molecule is colorless or light in color under neutral conditions and changes in color under alkaline conditions, and preferably, the pH indicator molecule is any one of phenolphthalein, thymolphthalein and curcumin.

On the basis of the scheme, the pathogenic microorganism is one of escherichia coli, salmonella typhimurium, staphylococcus aureus, vibrio parahaemolyticus and streptococcus haemolyticus.

It is worth to say that the kit containing the heterochromatic nanoparticles based on pH response is suitable for detecting common food-borne pathogenic bacteria in the prior art, is not limited to the pathogenic bacteria mentioned in the invention, and only needs to adopt the aptamers corresponding to the pathogenic bacteria when detecting other food-borne pathogenic bacteria.

The method for detecting the food-borne pathogenic bacteria by using the kit comprises the following steps:

dripping aptamer 2 of which the 5' -end is modified with biotin on the surface of a 96-well plate coated with streptavidin, incubating for 30min, and washing with SSC-buffer; then adding a sample solution to be tested, and incubating for 2h at 37 ℃; after washing the plate with SSC buffer, heterochromatic nanoparticles based on pH response were added, incubated at 37 ℃ for 90 minutes, and washed with PBS; and finally, adding an alkaline solution into the flat plate, detecting the absorbance of the solution in each hole, and determining the concentration of the pathogenic bacteria to be detected.

The invention has the beneficial effects that:

in the pH response-based heterochromous nanoparticles, aptamer1 specifically recognizes and combines pathogenic bacteria, the pathogenic bacteria are combined with the pH response heterochromous nanoparticles, obvious color change is generated after OH < - > is added, the color change is larger than that of a blank control group, and a 96-pore plate can be used for simultaneously detecting various pathogenic bacteria.

The method has simple and convenient operation, and does not need to carry out complex pretreatment on the sample to be detected; the detection cost is low, the requirement on a detection instrument is low, the result can be observed by naked eyes, and the detection kit can be used for household appliances such as refrigerators, air conditioners and the like.

Particularly, when the invention is used for detecting pathogenic bacteria in food, the pretreatment and target purification of the food are avoided, but the amplification of detection signals is realized, which is beneficial to realizing the qualitative and quantitative analysis of food-borne pathogenic bacteria.

Drawings

FIG. 1 is a schematic diagram of the detection principle of the method of the present invention;

FIG. 2 is a graph of the linear relationship between absorbance signal and log of pathogenic bacteria at various concentrations;

FIG. 3 shows the concentration of 10⁴Comparison of absorbance signal values for different strains of CFU/mL.

Detailed Description

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.

The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1

A detection kit for food-borne pathogenic bacteria comprises heterochromatic nanoparticles based on pH response, a 96-well plate coated with streptavidin, and a nucleic acid aptamer 2 of a target pathogenic microorganism to be detected, wherein the 5' end of the nucleic acid aptamer 2 is modified with biotin; the heterochromatic nanoparticles based on pH response are formed by self-assembling pH indicator molecules, BSA and aptamer1 of a target pathogenic microorganism to be detected; the Aptamer1 is coupled on a pH indicator molecule to serve as an identification unit, BSA blocks non-specific sites, and nanoparticles of the pH-BSA/Aptamer 1 formed through pi-pi accumulation and hydrogen bond interaction are the heterochromatic nanoparticles based on pH response.

The detection principle of the present invention is further explained with reference to fig. 1.

The pH indicator is mixed and stirred with aptamer1 and BSA, aptamer1 is coupled on the surface of a pH indicator molecule to be used as a recognition unit, and the BSA is used for carrying out nonspecific site blocking. The pH-BSA/Aptamer 1 nanoparticles, i.e., pH response-based heterochromatic nanoparticles, are formed by pi-pi stacking and hydrogen bonding interactions. The aptamer 2 was dropped on the surface of a 96-well plate coated with streptavidin, and 5' -modified biotin of the aptamer 2 was immobilized on the 96-well plate by binding to streptavidin. Adding a sample for detection, when pathogenic bacteria exist, firstly specifically identifying and grasping a target by Aptamer 2, washing by PBS to remove unbound pathogenic bacteria, then adding pH-BSA/Aptamer 1 nano particles, specifically identifying and binding target molecules by Aptamer1 of the nano particles by a sandwich method, simultaneously forming a sandwich structure of the Aptamer 2/pathogenic bacteria/pH-BSA/Aptamer 1 nano particles, and removing the unbound nano particles by washing. Adding OH^-The indicator molecules on the nanoparticles change color with alkali, so that obvious color change is generated and is larger than that of a blank control group; when no pathogenic bacteria exist, the Aptamer 2 cannot grasp a target, after the pH-BSA/Aptamer 1 nano particles are added, because no pathogenic bacteria exist, the Aptamer1 cannot be combined with the pathogenic bacteria, a sandwich structure cannot be formed, and then the nano particles are eluted out, OH is added^-No color change will occur. And (3) detecting by using a microplate reader, making a linear relation graph of concentration and absorbance, and calculating the detection sensitivity and the recovery rate.

Example 2

Escherichia coli O157: h7 detection kit

1. Synthesis of heterochromatic nanoparticles based on pH response:

1) 20mg of phenolphthalein indicator was dissolved in 2mL of methanol with stirring at 0 ℃.

2) mu.L of E.coli O157: h7 aptamer1 was sequentially dropped into the indicator solution of step 1).

The Escherichia coli O157: the nucleic acid sequence of H7 aptamer1 is:

5′-CCATGAGTGTTGTGAAATGTTGGGACACTAGGTGGCATAGAGCCG-3′(SEQ ID NO.1)

3) 20mg of BSA was dissolved in water to prepare a solution having a concentration of 1 mg/mL.

4) And 2) mixing the solution in the step 2) with the BSA solution, stirring for 3h, centrifuging (13000rpm for 5min), and washing with ultrapure water for 3 times to obtain a product. The resulting product is finally redispersed in ultrapure water for further use.

2. Streptavidin-coated 96-well plate

1) Streptavidin was coated in buffer (100mM Na)₂HPO₄50mM citric acid, pH 5.0) to a concentration of 5.0. mu.gmL^-1. To each well was added 200. mu.L of the coating solution and incubated at 35 ℃ overnight under block.

2) Each well was washed with 0.01M PBS containing 0.05% Tween-20. After washing, 200. mu.L of 0.1% bovine serum albumin PBS solution was added to each well. The 96-well plate was soaked overnight at room temperature, then washed with 0.01M PBS solution containing 0.05% tween-20 and dried at room temperature.

3) The 96-well plate was coated with a moisture absorbent, and dried and stored at 4 ℃.

3. 5' -end biotin-modified Escherichia coli O157: the nucleic acid sequence of the H7 aptamer 2 is: 5 '-biotin-TGGTCGTGGTGAGGTGCGTGTATGGGTGGTGGATGAGTGTGTGGC-3' (SEQ ID NO.2)

Example 3

Salmonella typhimurium detection kit

1. Synthesis of heterochromatic nanoparticles based on pH response:

1) 8mg thymolphthalein indicator was dissolved in 3.2mL DMSO with stirring at 0 ℃.

2) 10 μ L of Salmonella typhimurium aptamer1 were sequentially dropped into the indicator solution of step 1).

The nucleic acid sequence of the salmonella typhimurium aptamer1 is as follows:

5′-CCCACTCCAAACACGACCAACTCACGCTCTATCAACATCGCTATC-3′(SEQ ID NO.3)

2. Streptavidin-coated 96-well plate

3. The nucleic acid sequence of the salmonella typhimurium aptamer 2 with the 5' -end modified biotin is as follows:

5’-biotin-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3’(SEQ ID NO.4)

example 4

Staphylococcus aureus detection kit

1. Synthesis of heterochromatic nanoparticles based on pH response:

1) 4mg curcumin indicator was dissolved in 4mL DMSO with stirring at 0 ℃.

2) mu.L of Staphylococcus aureus aptamer1 was sequentially dropped into the indicator solution of step 1).

The nucleic acid sequence of the staphylococcus aureus aptamer1 is as follows:

5′-TCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGCTACGT-3′(SEQ ID NO.5)

2. Streptavidin-coated 96-well plate

3. The nucleic acid sequence of the staphylococcus aureus aptamer 2 with the 5' -end modified with biotin is as follows:

5’-biotin-GGGCTGGCCAGATCAGACCCCGGATGATCATCCTTGTGAGAACCA-3’(SEQ ID NO.6)

example 5

Method for detecting pathogenic bacteria by using food-borne pathogenic bacteria detection kit

1) Sample pretreatment: the food sample was ground with a homogenizer (liquid sample does not require this step), and 1g or 1mL of the sample was accurately weighed, diluted with 8mL of phosphate buffered saline PBS, and filtered with filter paper until use.

2) mu.L of 10. mu.M 5' -end-modified biotin aptamer 2 was dropped onto a streptavidin-coated plate surface and incubated in the chamber for 30 minutes.

3) After washing with SSC-buffer, a sample solution to be tested was added to the reaction system, followed by incubation at 37 ℃ for 2h (pH7.4, 200. mu.L).

4) After SSC buffer washing of the plate, the heterochromatic nanoparticles based on pH response prepared according to the present invention described above (100. mu.L, 1mg/mL) were added to the 96-well plate described above, incubated at 37 ℃ for 90 minutes, and then washed with PBS.

5) 100 μ L of NaOH solution was added to each well, shaken for 90 seconds, and the absorbance of the solution was measured.

And (3) detecting by using a microplate reader, making a linear relation graph of concentration and absorbance, and calculating the detection sensitivity and the recovery rate.

Sensitivity and specificity

Sample detection

1) Sample pretreatment: the food sample was ground with a homogenizer (liquid sample does not require this step), and 1g or 1mL of the sample was accurately weighed, diluted with 8mL of phosphate buffered saline PBS, and filtered with filter paper.

2) And (3) product detection: 1mL of the sample is sucked and added into a pathogen detection kit, then the sample is incubated for 2h at 37 ℃, after SSC buffer washing of the plate, the heterochromatic nanoparticles (100 uL, 1mg/mL) prepared according to the pH response in the above examples 2-4 are respectively added into the 96-well plate, and the incubation is carried out for 90 minutes at 37 ℃. After washing with PBS, 100 μ L NaOH solution was added to each well and the assay was read. The result shows that the method can detect 10CFU/mL of pathogenic bacteria, has no cross with other pathogenic bacteria, and has good specificity and sensitivity.

Sensitivity of the reaction

The sensitivity of the kit of example 2 of the invention was tested using various concentrations of E.coli O157: H7 solution. The specific steps are the same as those in the above example 5, except that the "sample solution to be tested" in 3) of example 5 is replaced with: 10¹～10⁷CFU/mL of E.coli O157: H7 solution.

The higher sensitivity indicates that the synthesized nano particles can be used for assembling a pathogen detection kit. As shown in FIG. 2, the linear relationship between the absorbance signal and the logarithm of the E.coli solution concentration is 10¹～10⁷CFU/mL, the linear equation is that y is 0.2051x +0.162, the correlation coefficient is 0.9912, the detection limit is 10CFU/mL, the linearity is good, and the method can be used for detecting unknown samples.

Specificity of

1) The specificity of the kit of example 2 of the present invention was tested using E.coli O157: H7, a nonpathogenic E.coli strain, Vibrio parahaemolyticus, Listeria monocytogenes. The specific steps are the same as those in the above example 5, except that the "sample solution to be tested" in 3) of example 5 is replaced with:

“10⁴CFU/mL of E.coli O157: H7 suspension ";

“10⁴CFU/mL of a nonpathogenic E.coli suspension ";

“10⁴CFU/mL of Vibrio parahaemolyticus suspension ";

“10⁴CFU/mL of listeria monocytogenes suspension ".

The blank control group was replaced with phosphate buffered saline PBS.

As can be seen in FIG. 3, 10⁴Escherichia coli O157: H7 at CFU/mL elicits a strong absorbance signal, whereas 10⁴CFU/mL of nonpathogenic E.coli strains, Vibrio parahaemolyticus, Listeria monocytogenes did not give a clear absorbance signal, similar to that of the blank control (PBS). From these data, it can be seen that the kit of the present invention has high specificity.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. The person skilled in the art can apply it to the detection of other molecules, cells, microorganisms. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Sequence listing

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Claims

1. The heterochromatic nanoparticles based on pH response are characterized by being formed by self-assembling pH indicator molecules, BSA and aptamer1 of pathogenic microorganisms; wherein the aptamer1 is coupled on a pH indicator molecule to serve as an identification unit, BSA blocks a non-specific site, and nanoparticles of the pH-BSA/aptamer 1 formed through pi-pi accumulation and hydrogen bond interaction are heterochromatic nanoparticles based on pH response;

(2) dropwise adding the BSA solution into the mixed solution obtained in the step (1) for sealing, stirring, centrifuging and washing to obtain the BSA-containing solution;

the pH indicator molecule is colorless or light in color under neutral conditions and changes color under alkaline conditions;

the pH indicator molecule is any one of phenolphthalein and thymolphthalein.

2. The pH response based heterochromatic nanoparticle of claim 1, wherein the pathogenic microorganism is one of the food-borne pathogenic bacteria Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Vibrio parahaemolyticus, Streptococcus hemolyticus.

3. Use of the pH response based heterochromatic nanoparticles of claim 1 in the preparation of a food-borne pathogenic bacteria detection kit.

4. A detection kit for food-borne pathogenic bacteria is characterized by comprising heterochromatic nanoparticles based on pH response, a 96-well plate coated with streptavidin, and a nucleic acid aptamer 2 of a target pathogenic microorganism to be detected, wherein the 5' end of the nucleic acid aptamer is modified with biotin; the heterochromatic nanoparticles based on pH response are formed by self-assembling pH indicator molecules, BSA and aptamer1 of a target pathogenic microorganism to be detected; wherein the aptamer1 is coupled on a pH indicator molecule to serve as an identification unit, BSA blocks a non-specific site, and nanoparticles of the pH-BSA/aptamer 1 formed through pi-pi accumulation and hydrogen bond interaction are heterochromatic nanoparticles based on pH response;

the pH indicator molecule is any one of phenolphthalein and thymolphthalein.

5. The food-borne pathogenic bacteria detection kit according to claim 4, wherein the pathogenic microorganisms are one of Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Vibrio parahaemolyticus, and Streptococcus hemolyticus.

6. Method for the detection of food-borne pathogenic bacteria for non-diagnostic purposes using a kit according to claim 4 or 5, characterized in that it comprises the steps of: