CN109097365B - Aptamer for vibrio vulnificus recognition, screening method and application - Google Patents
Aptamer for vibrio vulnificus recognition, screening method and application Download PDFInfo
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- CN109097365B CN109097365B CN201810950055.7A CN201810950055A CN109097365B CN 109097365 B CN109097365 B CN 109097365B CN 201810950055 A CN201810950055 A CN 201810950055A CN 109097365 B CN109097365 B CN 109097365B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
Abstract
The present invention relates to a new-type vibrio vulnificusVibrio vulnificus) Specifically recognized ssDNA nucleiAn aptamer, wherein the nucleotide sequence of the aptamer is the nucleotide sequence of a DNA fragment shown in Apt-Vv 01. The invention also provides a screening method of the aptamer for identifying the vibrio vulnificus, and the quality identification and the application of the aptamer according to the affinity of the aptamer for the vibrio vulnificus are carried out. The aptamer of the invention is composed of 149 bases and has a special secondary structure. The aptamer can be applied to the rapid detection of vibrio vulnificus in medical diagnosis, environmental detection, food processing, aquaculture and the like. Effectively protects the water body from being polluted by the pathogenic bacteria, and has wide application value particularly in the aspects of diagnosis, aquaculture and water body antifouling.
Description
Technical Field
The invention relates to an aptamer, in particular to a nucleic acid aptamer used for vibrio vulnificus (Vibrio vulnificus)Vibrio vulnificus) A specific recognition ssDNA aptamer. The invention also relates to a screening method and application of the aptamer, and belongs to the technical field of marine pathogenic microorganism detection.
Background
Vibrio vulnificus (Vibrio vulnificus) Is a gram-negative halophilic bacterium living in estuaries and marine coastal areas, and is one of important marine organism pathogenic bacteria. In 1976, the American centers for disease control isolate Vibrio vulnificus from blood samples for the first time, which can cause human digestive system diseases and death; is also a pathogenic bacterium with serious harm in aquaculture. In aquaculture, the aquaculture environment is deteriorated or aquatic animals can be infected by vibrio vulnificus after being injured, so that diseases are caused and spread of diseases can be caused, the aquatic organisms such as fish, shellfish and the like are greatly damaged, and the economic damage of the aquaculture industry is causedOne of the main causes of loss.
The SELXE technology, namely, the technology of Systematic Evolution of Ligands by exponentiation and Evolution (Systematic Evolution of Ligands by exponentiation) is a novel in vitro screening technology appearing in the nineties of the twentieth century. A random oligonucleotide library (10) can be generated using SELEX technology13~1015) The selected aptamers (aptamers) with high specificity and affinity for accurately identifying specific targets can be widely applied to a plurality of research fields of biological pharmacy, molecular biology, microbiology, medicine and the like. Aptamers possess many superior properties compared to other biomolecules: easy synthesis, easy modification, good stability, high sensitivity, wide target range, strong specificity, wide pH range, low immunogenicity and the like.
The excellent performances of DNA aptamers show that the DNA aptamers can be applied to the rapid detection of Vibrio vulnificus, but research on the rapid detection of Vibrio vulnificus by applying the aptamers to the Vibrio vulnificus as targets is rarely reported at home and abroad.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a ssDNA aptamer for quickly detecting Vibrio vulnificus.
The invention also aims to provide the screening method and the application of the aptamer.
In order to achieve the above purpose, the present invention provides the following technical solutions: the present invention relates to a new-type vibrio vulnificusVibrio vulnificus) The ssDNA aptamer for specific recognition is characterized in that: the nucleotide sequence of the aptamer is the nucleotide sequence of a DNA fragment shown by Apt-Vv 01: Apt-Vv 01:
5’- ctgcagaattctaatacgagtcactataggaagatggcgaaacatcttttctgatgagagttgcgacctcccg
gatgagtgccctggctccgggtttgtctatttttgggggtgttgacagatatccttagtgacggtaagcttggcac-3’。
the invention also provides a vibrio vulnificus (V.vulnificus) as described in the technical schemeVibrio vulnificus) Screening for specifically recognized ssDNA aptamersThe method is characterized by comprising the following specific steps:
(1) synthesizing an initial oligonucleotide library containing 80 random bases;
(2) vibrio vulnificus to be detected: (Vibrio vulnificus) Culturing in LB culture medium;
(3) carrying out gradient dilution on the bacterial liquid and coating a culture medium to detect the concentration of the bacterial liquid;
(4) centrifuging the thalli obtained in the step (2), and reserving a precipitate;
(5) washing the precipitate obtained in the step (4) by using a binding buffer solution and reserving the precipitate;
(6) denaturing the random library obtained in step (1) at 600pM diluted in binding buffer;
(7) uniformly mixing the precipitate obtained in the step (5) and the denatured library obtained in the step (6), and combining for 1 hour;
(8) centrifuging and removing supernatant after the combination is finished, and washing the precipitate for three times by using washing buffer solution;
(9) resuspending and precipitating with deionized water, heating for denaturation to obtain supernatant, and detecting concentration;
(10) asymmetric PCR amplification, and using the product for the next round of screening;
(11) sequencing and analyzing, and synthesizing a nucleic acid aptamer marked by FAM fluorescein; and then measuring the binding rate, specificity and detection line of the aptamer.
As a further scheme of the invention: the initial Vibrio vulnificus cell concentration used in the present invention is 2X 108 CFU per ml.
As a further scheme of the invention: all binding reactions were performed in 1.5 ml sterile centrifuge tubes.
As a further scheme of the invention: the instrument used in the determination of aptamer concentration was an ultraviolettometer (Q5000, usa).
As a further scheme of the invention: the apparatus for the aptamer binding fluorescence intensity detection experiment is a full-wavelength multifunctional microplate detector (M1000 Pro, Switzerland).
As a further scheme of the invention: the apparatus for aptamer binding fluorescence imaging experiments was an olympus inverted microscope (IX 53, japan).
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a vibrio vulnificus aptamer with strong specificity, high affinity and low detection limit, the vibrio vulnificus aptamer consists of 149 basic groups, has a special secondary structure, and has the highest affinity to vibrio vulnificus of 26.8 +/-5.3 nM/L. By detecting the binding rate, fluorescence intensity and fluorescence microscopic imaging of the aptamer to the vibrio vulnificus, the lowest detection bacteria concentration for detecting the vibrio vulnificus is 8CFU/mL, and the detection range is 8CFU/mL to 2 multiplied by 108CFU/mL. The invention details the screening and property identification method of the aptamer. The aptamer can quickly detect the micro vibrio vulnificus somatic cells, can be widely applied to quick detection of vibrio vulnificus pathogenic microorganisms in medical diagnosis, food processing, aquaculture monitoring and environment detection, effectively protects water bodies from being polluted by the pathogenic microorganisms, and has wide application value particularly in the aspects of diagnosis, aquaculture and water body antifouling.
Drawings
FIG. 1 is a diagram showing the prediction of the secondary structure of an aptamer Apt-Vv01 with high specificity and high affinity to Vibrio vulnificus somatic cells, which is screened in the present invention;
FIG. 2 is a graph showing the affinity detection of the aptamer Apt-Vv01 with high specificity and high affinity to Vibrio vulnificus somatic cells screened in the present invention;
FIG. 3 is a graph showing the comparison of fluorescence binding of an aptamer Apt-Vv01 capable of binding with the bacterial cells of Vibrio vulnificus in a specific and high-affinity manner, which is screened in the present invention, during different incubation periods;
FIG. 4 is a comparison chart of the specificity detection of the aptamer Apt-Vv01 with high specificity and high affinity to Vibrio vulnificus somatic cells in 4 control bacteria, which is screened in the invention;
FIG. 5 is a comparison graph of fluorescence binding of the aptamer Apt-Vv01 with high specificity and high affinity to Vibrio vulnificus somatic cells screened in the invention in 4 control bacteria;
FIG. 6 is a line graph showing the detection of the aptamer Apt-Vv01 with high specificity and high affinity to Vibrio vulnificus bacterial cells, which is screened in the present invention.
The specific implementation mode is as follows:
the invention is described in detail below with reference to the figures and the detailed description.
Example 1A pharmaceutical composition for Vibrio vulnificus ((V))Vibrio vulnificus) A specifically recognized ssDNA aptamer, the nucleotide sequence of the aptamer is the nucleotide sequence of a DNA fragment shown as Apt-Vv 01: Apt-Vv 01:
5’- ctgcagaattctaatacgagtcactataggaagatggcgaaacatcttttctgatgagagttgcgacctcccg
gatgagtgccctggctccgggtttgtctatttttgggggtgttgacagatatccttagtgacggtaagcttggcac-3’。
the screening method of the ssDNA aptamer specifically identified comprises the following specific steps:
(1) initial DNA screening libraries containing 80 random bases and forward and backward primers were synthesized (Shanghai Producer).
Initial library: 5 '-GGCGAAACATCTT-N80-TAGTGACGGTAAGCTTGGCAC-3'
Primer 1: 5'-GTGCCAAGCTTACCG-3'
Primer 2: 5'-CTGCAGAATTCTAATACGAGTCACTATAGGAAGATGGCGAAACA-3'
(2) The Vibrio vulnificus was cultured in LB medium until OD600nm was close to 1, and the culture was stopped, and the number of cells per mL was measured and dispensed into 1.5 mL centrifuge tubes (1 mL/tube).
(3) 100 μ L of the 600pM initial library diluted in binding buffer was heated at 95 ℃ for 5 minutes and then iced for 10 minutes. Binding buffer: 100 mM NaCl, 5 mM KCl, 50 mM Tris-HCl, 1 mM MgCl2, pH7.5。
(4) The cells of Vibrio vulnificus were harvested by centrifugation and washed 3 times with washing buffer, and 100. mu.L of binding buffer was used to resuspend the cells. Washing buffer solution: 50 mM NaCl, 2.5 mM KCl, 25 mM Tris-HCl, 0.5 mM MgCl2, pH7.5。
(5) The denatured initial library was mixed with Vibrio vulnificus solution, incubated at 30 ℃ and 1000rpm for 1 hour.
(6) The mixture was centrifuged at 12000g for 5 min, the supernatant discarded, the pellet washed three times with 200. mu.L of wash buffer, and 100. mu.L of deionized water was used to resuspend the cells.
(7) Heating the heavy suspension at 90 ℃ for 10 minutes, carrying out ice bath for 10 minutes, centrifuging 12000g for 5 minutes, collecting supernatant, and recovering DNA by an alcohol precipitation method.
(8) And (3) dissolving the DNA obtained in the step (7) by 20 mu L of deionized water and detecting the concentration.
(9) And (4) carrying out asymmetric PCR amplification by using the DNA obtained in the step (8) as a template, and recovering an amplification product by using a PCR product purification and recovery kit, and diluting the amplification product to 100 mu L for screening the library in the next round.
(10) Taking vibrio anguillarum as a reverse screening bacterium, respectively introducing reverse screening in the 3 rd round, the 6 th round and the 8 th round, carrying out 10 rounds of forward screening in total, and selecting the 10 th round PCR product for clone sequencing.
(11) Characterization of aptamer:
analyzing the sequencing result and synthesizing candidate aptamers with FAM labels. And (4) measuring the binding rate of the selected aptamer, and excluding DNA which is not bound to obtain an aptamer Apt-Vv 01. The secondary structures of the two aptamers were analyzed by DNA secondary structure analysis software, and it was found that the aptamers Apt-Vv01 had loop loops at positions 40, 80, 100, and 140, respectively, as shown in FIG. 1.
And (3) affinity detection:
1) kd values were determined. Using 100 μ L of aptamers with different concentrations (final concentration of 0, 0.5, 1, 2, 5, 10, 20, 30, 40, 50nM) as abscissa under the same thallus concentration, respectively, and combining the fluorescence intensity of the supernatant (F represents the fluorescence value of the supernatant obtained after the resuspension in deionized water, F0Representing the fluorescence value at an aptamer concentration of 0) as the ordinate, using SigmaPlot software and Y = BmaxThe equation (Kd + X) is linearly fitted and gives a Kd value. The result proves that the aptamer Apt-Vv01 has better affinity (K)d=26.8 ± 5.3, see fig. 2).
2) And (4) fluorescence microscopic imaging. 100 mu L of single-chain aptamer Apt-Vv01 with the same concentration is respectively incubated with equivalent vibrio vulnificus for 5 minutes, 15 minutes, 45 minutes, 60 minutes and 90 minutes, centrifuged, resuspended in deionized water, and then coated on a glass slide in trace for fluorescence microscopy imaging. The results showed that the amount of binding of aptamer Apt-Vv01 to Vibrio vulnificus increased with the increase of the binding time, and that aptamer Apt-Vv01 had a strong affinity for Vibrio vulnificus, see FIG. 3.
And (3) specific detection:
affinity detection and fluorescence microscopic imaging results show that the aptamer Apt-Vv01 has strong binding capacity to vibrio vulnificus, and then specificity detection is carried out on the aptamer Apt-Vv01 by using vibrio anguillarum, staphylococcus aureus, escherichia coli and bacillus subtilis.
1) The 4 control strains were cultured under the respective optimum culture conditions, and the number of cells contained per ml was kept approximately the same by OD value and cell count method.
2) The bacterial cells of each strain are obtained by centrifugation, washed, mixed with single-chain aptamer Apt-Vv01 with the same volume and concentration, and incubated and combined for 1 hour at 30 ℃ (the concentration and fluorescence value of the aptamer are respectively measured before combination).
3) And (3) centrifuging 12000g for 5 minutes after the combination is finished, collecting the supernatant to detect the fluorescence value, resuspending the precipitate with deionized water after washing, respectively absorbing trace bacterial suspension to dilute, coating the diluted trace bacterial suspension on a glass slide, and naturally drying the glass slide.
4) Heating, cooling and centrifuging the rest bacterial suspensions, collecting the supernatant, and detecting the respective DNA concentration and fluorescence intensity.
5) Different strains are used as abscissa, and the ratio of aptamer concentration to fluorescence intensity before and after combination (the fluorescence value of the supernatant after combination is F, and the fluorescence value before combination is F)0Fluorescence on binding is Δ F, where Δ F = F0-F) plotting histograms for the ordinate, respectively. The two results mutually demonstrate that the aptamer Apt-Vv01 has strong specificity for Vibrio vulnificus, see FIG. 4.
6) And (4) fluorescence microscopic imaging. The results of observing the binding effect of 5 kinds of bacteria and the aptamer under the bright field and the fluorescence field respectively show that the aptamer Apt-Vv01 has high binding capacity only to the bacterial cells of vibrio vulnificus, and refer to FIG. 5.
Detection limit determination:
1) 2 x 10 to8 CFU per ml vibrio vulnificus suspension is 107，106，105，104，103，102，101Gradient dilution was performed. Dilutions were made below 20 at 20, 15, 10, 8, 5, 1CFU/mL dilutions.
2) 100 μ L of the single-stranded aptamer Apt-Vv01 with a final concentration of 20nM was incubated with the diluted Vibrio vulnificus cells at 30 ℃ for 1 hour (the fluorescence intensity of the nucleic acid aptamer before binding was measured).
3) And after the combination is finished, centrifuging, cleaning and carrying out deionized water heavy suspension once, heating and cooling, collecting the supernatant, and measuring the fluorescence value of the supernatant.
4) Different dilution times are used as abscissa, and fluorescence ratio before and after combination (F before combination)0F) after the deionized water is resuspended is used as a linear fitting graph with the ordinate, and the correlation coefficient R2The value reached 0.9922. The result shows that the detection limit of the aptamer Apt-Vv01 on Vibrio vulnificus can be as low as 8 CFU/ml. The detection range is 8CFU to 2 multiplied by 108 CFU/ml. Refer to fig. 6.
The above is a detailed description of the detection application of the present invention, but the present invention is not limited to the above embodiment, and variations can be made without departing from the gist of the present invention within the knowledge of those skilled in the art.
Sequence listing
<110> Huaihai institute of Industrial and Marine science
<120> aptamer for vibrio vulnificus recognition, screening method and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 149
<212> DNA
<213> Apt-Vv01
<400> 1
ctgcagaatt ctaatacgag tcactatagg aagatggcga aacatctttt ctgatgagag 60
ttgcgacctc ccggatgagt gccctggctc cgggtttgtc tatttttggg ggtgttgaca 120
gatatcctta gtgacggtaa gcttggcac 149
Claims (3)
1. A pharmaceutical composition for treating Vibrio vulnificusVibriovulnificus) A specifically recognized ssDNA aptamer characterized by: the nucleotide sequence of the aptamer is the nucleotide sequence of a DNA fragment shown by Apt-Vv 01: Apt-Vv 01:
5’- ctgcagaattctaatacgagtcactataggaagatggcgaaacatcttttctgatgagagttgcgacctcccg
gatgagtgccctggctccgggtttgtctatttttgggggtgttgacagatatccttagtgacggtaagcttggcac-3’。
2. use of the aptamer according to claim 1 for the detection of Vibrio vulnificus in food processing, aquaculture monitoring, environmental monitoring.
3. Use according to claim 2, characterized in that: the lowest detection bacteria concentration for detecting the vibrio vulnificus is 8CFU/mL, and the detection concentration range is 8CFU/mL to 2 multiplied by 108 CFU/mL。
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| CN110106178B (en) * | 2019-05-17 | 2023-07-18 | 江苏海洋大学 | DNAzyme for identifying and cutting vibrio anguillarum, screening method and application |
| CN112458090B (en) * | 2020-12-04 | 2022-10-11 | 江苏海洋大学 | DNAzyme for vibrio vulnificus specific recognition RNA cutting and application |
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