CN112557651A - Rapid detection method for pathogenic microorganisms with double signal outputs - Google Patents

Rapid detection method for pathogenic microorganisms with double signal outputs Download PDF

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CN112557651A
CN112557651A CN202011238234.1A CN202011238234A CN112557651A CN 112557651 A CN112557651 A CN 112557651A CN 202011238234 A CN202011238234 A CN 202011238234A CN 112557651 A CN112557651 A CN 112557651A
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pathogenic microorganisms
product
solution
phenylenediamine
concentration
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王利华
吴文辉
董军
邵志勇
白向茹
王佳慧
郑思洁
李婷婷
战艺芳
王嫦嫦
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Wuhan Academy of Agricultural Sciences
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    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56938—Staphylococcus
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Abstract

The invention discloses a method for rapidly detecting pathogenic microorganisms with double-signal output, which utilizes the specific combination between pathogenic microorganisms and a nucleic acid aptamer and the destructive effect of double-chain DNA specific nuclease on a structure formed after the combination of the pathogenic microorganisms and the nucleic acid aptamer to release o-phenylenediamine molecules in a mesoporous silicon nano material, and the released o-phenylenediamine molecules are further combined with Ag+The solutions are mixed to generate a yellow oxidation product, the product has a fluorescence signal at the same time, and the concentration of the product is in direct proportion to the concentration of pathogenic microorganisms in the sample to be detected. Based on the method, the indirect qualitative and quantitative determination of pathogenic microorganisms can be realized by the naked eye or a portable fluorescence analyzer through colorimetric and fluorescent dual-signal outputAnd (4) detecting the quantity.

Description

Rapid detection method for pathogenic microorganisms with double signal outputs
Technical Field
The invention relates to the technical field of pathogenic microorganism detection, in particular to a rapid pathogenic microorganism detection method with double signal output, and particularly relates to a rapid pathogenic microorganism detection method based on mesoporous silica nano-materials, nuclease circulating signal amplification and colorimetric and fluorescent double signal output.
Background
Pathogenic microorganisms including staphylococcus aureus, salmonella, escherichia coli, listeria monocytogenes and the like are the leading causes of food-borne diseases, which not only seriously affect the life health of people, but also affect the development of global economy. Therefore, in order to ensure the national civilization, the rapid screening and detection of the types and the contents of microorganisms are very necessary before food or agricultural products enter the market circulation.
At present, the common pathogenic microorganism detection methods mainly comprise three major methods, namely a culture method, a PCR method and an ELISA method. The culture method is used as a gold-labeled method for detecting microorganisms, and although the result is accurate, the method usually needs to be subjected to links such as separation, identification, culture, counting and the like, the operation is complex and time-consuming, and generally needs 3-5 days. The PCR method and the ELISA method, although the detection time is greatly shortened, depend on expensive special instruments and professional technicians, and are difficult to be applied to complex samples. In conclusion, the methods are difficult to meet the urgent requirement of the field rapid detection of pathogenic microorganisms at present. Therefore, a simple, quick, cheap, accurate and high-sensitivity pathogenic microorganism detection method is developed, and the method has great significance for effectively preventing and controlling outbreak of food-borne diseases and reducing the harm of pathogenic microorganisms to human beings.
In recent years, rapid detection of pathogenic microorganisms has been rapidly developed, and various rapid detection methods based on electrochemical, fluorescence, surface enhanced raman spectroscopy and other technologies have been developed. However, the above methods generally detect a target object based on a change in a single signal. The single signal output mode is easily influenced by operators, experimental environments and the like, so that the result is not accurate enough.
Disclosure of Invention
In view of the above, the invention provides a method for rapidly detecting pathogenic microorganisms with dual signal outputs, which does not require expensive instruments and reagents, can perform detection by a colorimetric method and a fluorescence method, and has high accuracy.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a dual-signal output pathogenic microorganism rapid detection method is characterized by comprising the following steps:
s1, mixing the mesoporous silicon with o-phenylenediamine to obtain mesoporous silicon loaded with o-phenylenediamine molecules in mesopores, and marking as a product A;
s2, sealing the product A in the step S1 by adopting a nucleic acid aptamer of a pathogenic microorganism to be detected to obtain a product B;
s3, placing the sample to be detected, the nuclease and the product B obtained in the step S2 in the same reaction system, and centrifuging to obtain a supernatant after reaction;
s4, mixing the supernatant obtained in the step S3 with Ag+Mixing the solutions, and performing qualitative and quantitative analysis on the concentration of pathogenic microorganisms by colorimetric or fluorescent detection;
wherein the mesoporous silicon is mesoporous silicon oxide nanospheres with amino-modified surfaces, the particle size of the mesoporous silicon is 50-200nm, and the pore diameter of the mesoporous silicon is 2-4 nm; the nuclease is a double-stranded DNA specific nuclease.
Further, in the above technical solution, the preparation method of the product a in step S1 is: ultrasonically dispersing mesoporous silica nanospheres in Tris-HCl buffer solution, dropwise adding o-phenylenediamine solution under the stirring condition, and carrying out oscillation reaction at room temperature for 4-6h to ensure that o-phenylenediamine molecules are fully diffused into pores of the mesoporous silica nanospheres to obtain suspension containing a product A; the solvent of the o-phenylenediamine solution is a mixed solution of absolute ethyl alcohol and a Tris-HCl buffer solution; furthermore, the volume ratio of the absolute ethyl alcohol to the Tris-HCl buffer solution is 1:1, and the concentration of the o-phenylenediamine solution is 10-20 mg/mL.
Further, in the above technical solution, the preparation method of the product B in step S2 is: and (3) adding the aptamer solution of the pathogenic microorganism to be detected into the suspension obtained in the step S1, carrying out mild oscillation at 4 ℃ for 6-8h, and then carrying out multiple centrifugation and washing by using a Tris-HCl buffer solution to remove o-phenylenediamine molecules physically adsorbed on the surface of the mesoporous silicon nano material, thereby obtaining a product B. The aptamer has good specificity to pathogenic microorganisms, and the screening of the aptamer is the prior art and is already commercialized.
Further, in the above technical solution, the process in step S3 specifically includes: resuspending the product B into a Tris-HCl buffer solution, adding a mixed solution containing a sample to be detected and double-stranded DNA specific nuclease, reacting at room temperature for 20-30min, and centrifuging to obtain a supernatant; furthermore, the mixed solution is formed by mixing the sample to be detected and the double-stranded DNA specific nuclease stock solution according to a specific proportion, the specific proportion can be adjusted according to actual needs, and the concentration of the double-stranded DNA specific nuclease stock solution is 100-1000U/mL.
Further, in the above technical solution, the Ag in step S4+The solution is AgNO-containing3The citric acid buffer of (1), the AgNO3The concentration of the solution is 0.01-0.1M, the pH of the citric acid buffer solution is 4.5-5.5, and the concentration is 0.05-0.1M; further, the specific parameters of the fluorescence detection in step S4 are: the wavelength of the excitation light is 435nm, the spectral range is 450-700nm, and the average measurement is 3-5 times.
Further, in the above technical solution, the pathogenic microorganism is any one of staphylococcus aureus, salmonella, escherichia coli, listeria monocytogenes, vibrio parahaemolyticus, bacillus cereus, and enterobacter sakazakii.
The concentration of Tris-HCl buffer used in the method is 0.01-0.05M, and the pH is 8.0.
The invention utilizes the space between pathogenic microorganism and nucleic acid aptamerThe specific combination of the two and the destructive effect of the double-chain DNA specific nuclease on the structure formed after the combination of the two, so that the o-phenylenediamine molecules in the mesopores are released, and the released o-phenylenediamine molecules are further combined with Ag+Solution mixing based on Ag+The oxidation of o-phenylenediamine produces a yellow oxidation product which simultaneously has a fluorescent signal. Therefore, the concentration of the pathogenic microorganisms can be simply judged by observing the shade of the color through naked eyes, and the concentration of the pathogenic microorganisms can be accurately determined by constructing a standard curve through a colorimetry or a fluorescence method by adopting an instrument.
The invention has the beneficial effects that: 1) the mesoporous silicon material is adopted as a carrier, and the mesoporous silicon has the characteristics of good biocompatibility, structural repeatability, high load efficiency, adjustable size and easy functionalization, and has the effect of first-level signal amplification; 2) based on the circulating signal amplification strategy of the double-stranded DNA specific nuclease, pathogenic microorganisms in the sample are repeatedly used, o-phenylenediamine molecules are continuously released, and the detection sensitivity is further improved; 3) introduction of Ag+The oxidizing effect on o-phenylenediamine molecules, and a dual-signal output pathogenic microorganism rapid detection method is constructed based on the dual properties of the color and fluorescence of the obtained product, so that the accuracy is greatly improved; in addition, after the analysis of the solution obtained from the reaction was completed, excess Ag contained therein+The method can be further used for inactivating the microbial waste liquid in the reaction system, so that the microbial waste liquid is prevented from entering the environment by mistake, and the biological safety of the experiment is effectively ensured; 4) the construction process of the detection sensor does not need expensive reagents such as antibodies, aptamers modified with signal molecules or functional groups and the like, the detection process does not depend on large-scale instruments and professional operators, and the cost is greatly reduced.
Detailed Description
The technical solutions of the present invention are described in detail below by specific examples, which are only used for explaining the present invention and are not used for limiting the scope of the present invention.
Example 1
The pathogenic microorganism of this example was staphylococcus aureus. The detection method comprises the following steps:
(1) 10mg of the aminated mesoporous silica nanospheres (particle size about 100nm) were ultrasonically dispersed in 1mL of Tris-HCl buffer (10mM, pH8.0), and 1mL of 10mg/mL of o-phenylenediamine in ethanol-Tris-HCl (volume ratio 1:1) was then added dropwise thereto under stirring at room temperature. After mixing well, the mixture was gently shaken at room temperature for 4-6 h.
(2) Adding 5mL of an aptamer solution of Staphylococcus aureus (100. mu.M, sequence 5'-GCTAACCCCCCCAGTCCGTCCTCCCAGCCTCACACCGCCA-3') to the suspension obtained in step (1), gently stirring at 4 ℃ for 6h, centrifuging and washing 5-10 times with a Tris-HCl buffer (10mM, pH8.0), and finally dispersing the material into 2mL of Tris-HCl buffer (10mM, pH 8.0);
(3) and (3) taking 200 mu L of the suspension prepared in the step (2), adding 200 mu L of a mixed solution of a sample to be detected containing staphylococcus aureus and the double-stranded DNA specific nuclease into the suspension, incubating the suspension at room temperature for 30min with gentle shaking, centrifuging the product at 5000rpm for 5min, and resuspending the product into a Tris-HCl buffer solution (10mM, pH8.0) in equal volume. The concentration of Staphylococcus aureus in the sample was from 0 to 108More than 8 concentrations selected in CFU/mL.
(4) Taking 100 mu L of supernatant obtained in the step (3) and 100 mu L of supernatant containing 0.05M AgNO3After the citric acid buffer solution (0.1M, pH 5.5) is mixed and reacted for 30min at room temperature, the concentration of pathogenic microorganisms in the sample is preliminarily determined by observing the color depth of the solution with naked eyes or an ultraviolet lamp; and then taking a small amount of solution after reaction for subsequent colorimetric or fluorescent detection, wherein the parameters detected by a fluorescent method are as follows: excitation light 435nm, spectral range 450 and 700nm, average measurement 3 times.
And drawing a corresponding quantitative standard working curve according to the concentration of the staphylococcus aureus and the change condition of the fluorescence emission peak intensity of the o-phenylenediamine oxidation product at 562nm, wherein the concentration of the detected staphylococcus aureus is as low as 25CFU/mL according to the standard working curve.
Example 2
This example is the concentration test of a staphylococcus aureus mock sample using the standard curve of example 1.
First, normal temperature milk (3000rpm, 5min) purchased from a supermarket is centrifuged to remove impurities. It was then treated by 10-fold dilution with Tris-HCl buffer (10mM, pH 8.0). 1000 and 10000CFU/mL of a Staphylococcus aureus standard solution were added thereto, and the mixture was thoroughly mixed and tested by the same method as in example 1. Firstly, the color of the o-phenylenediamine oxidation product is observed by naked eyes, so that the light color of the o-phenylenediamine oxidation product and the deepened color of the o-phenylenediamine oxidation product can be preliminarily known; then, by measuring the change value of the fluorescence emission peak intensity at 562nm, according to the working curve obtained in example 1, the concentration of staphylococcus aureus in the simulated sample is calculated to be 951.4 and 10323CFU/mL respectively, and the recovery rates of the two samples are calculated to be 95.14% and 103.23% respectively. Therefore, the method can be verified to have good feasibility and accuracy in detection in actual samples.
The above examples are merely illustrative of the present invention, but the embodiments of the present invention are not limited by the above examples. Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art from the above description, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Sequence listing
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Claims (8)

1. A dual-signal output pathogenic microorganism rapid detection method is characterized by comprising the following steps:
s1, mixing the mesoporous silicon with o-phenylenediamine to obtain mesoporous silicon loaded with o-phenylenediamine molecules in mesopores, and marking as a product A;
s2, sealing the product A in the step S1 by adopting a nucleic acid aptamer of a pathogenic microorganism to be detected to obtain a product B;
s3, placing the sample to be detected, the nuclease and the product B obtained in the step S2 in the same reaction system, and centrifuging to obtain a supernatant after reaction;
s4, mixing the supernatant obtained in the step S3 with Ag+Mixing the solutions, and performing qualitative and quantitative analysis on the concentration of pathogenic microorganisms by colorimetric or fluorescent detection;
wherein the mesoporous silicon is mesoporous silicon oxide nanospheres with amino-modified surfaces, the particle size of the mesoporous silicon is 50-200nm, and the pore diameter of the mesoporous silicon is 2-4 nm; the nuclease is a double-stranded DNA specific nuclease.
2. The method for rapidly detecting pathogenic microorganisms according to claim 1, wherein the preparation method of the product A in step S1 comprises the following steps: ultrasonically dispersing mesoporous silica nanospheres in Tris-HCl buffer solution, dropwise adding o-phenylenediamine solution under the stirring condition, and reacting at room temperature for 4-6h to obtain suspension containing a product A; the solvent of the o-phenylenediamine solution is a mixed solution of absolute ethyl alcohol and Tris-HCl buffer solution.
3. The method for rapidly detecting pathogenic microorganisms according to claim 2, wherein the volume ratio of the absolute ethyl alcohol to the Tris-HCl buffer solution is 1:1, and the concentration of the o-phenylenediamine solution is 10-20 mg/mL.
4. The method for rapidly detecting pathogenic microorganisms according to claim 1, wherein the preparation method of the product B in the step S2 comprises the following steps: and (4) adding the aptamer solution of the pathogenic microorganism to be detected into the suspension obtained in the step S1, carrying out mild oscillation at 4 ℃ for 6-8h, and then centrifuging and washing for multiple times by using a Tris-HCl buffer solution to obtain a product B.
5. The method for rapidly detecting pathogenic microorganisms according to claim 1, wherein the process of step S3 specifically comprises: and (3) resuspending the product B into a Tris-HCl buffer solution, adding a mixed solution containing the sample to be detected and the double-stranded DNA specific nuclease, reacting at room temperature for 20-30min, and centrifuging to obtain a supernatant.
6. The method for rapidly detecting pathogenic microorganisms according to claim 1, wherein the Ag in step S4+The solution is AgNO-containing3The citric acid buffer of (1), Ag in the solution+The concentration of (A) is 0.01-0.1M, the pH of the citric acid buffer solution is 4.5-5.5, and the concentration is 0.05-0.1M.
7. The method for rapidly detecting pathogenic microorganisms according to claim 1, characterized in that: the specific parameters of the fluorescence detection in step S4 are: the wavelength of the excitation light is 435nm, the spectral range is 450-700nm, and the average measurement is 3-5 times.
8. The method for rapidly detecting the pathogenic microorganisms according to claim 1, wherein the pathogenic microorganisms are any one of staphylococcus aureus, salmonella, escherichia coli, listeria monocytogenes, vibrio parahaemolyticus, bacillus cereus and enterobacter sakazakii.
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