CN107858359B - Nucleic acid aptamer capable of specifically recognizing vibrio alginolyticus and application thereof - Google Patents
Nucleic acid aptamer capable of specifically recognizing vibrio alginolyticus and application thereof Download PDFInfo
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Abstract
The invention discloses a ssDNA aptamer capable of specifically recognizing vibrio alginolyticus, a screening method, a detection method and application thereof, wherein the nucleotide sequence of the ssDNA aptamer is 5'-TTTGATATTGCTTTGTCTTCTATTTTATGCTAATCTTTAAATGTACTGGT-3' (SEQ ID NO: 1) or 5 '-GACGCTTACTCAGGTGTGACTCG-TTTGATATTGCTTTGTCTTCTATTTTATGCTAATCTTTAAATGTACTGGT-CGAAGGACGCAGATGAAGTCTC-3' (SEQ ID NO: 2). The ssDNA aptamer of the invention has specificity and high sensitivity to Vibrio alginolyticus, and has no immunogenicity. The ssDNA aptamer has a stable structure, is easy to modify, is convenient to synthesize and store, and can be used for quickly and accurately detecting and diagnosing vibrio alginolyticus.
Description
Technical Field
The invention relates to a ssDNA aptamer, a screening method, a detection method and application thereof, in particular to a ssDNA aptamer capable of specifically identifying aquatic pathogenic bacteria vibrio alginolyticus, and a screening method, a detection method and application thereof.
Background
As a big country for aquaculture, the aquaculture amount accounts for 70 percent of the total aquaculture amount of aquatic products in the world. However, the outbreak and prevalence of bacterial pathogens in recent years have caused huge economic losses to the aquaculture industry in our country. In coastal areas of south China such as Guangxi, vibrio alginolyticus is one of main pathogenic bacteria causing marine cultured fishes to generate bacterial fish diseases, and the fish diseases caused by the vibrio alginolyticus are rapid in onset, high in death rate and wide in prevalence range, and have serious threats to the development of aquaculture industry in south China. At present, the fish bacterial diseases are internationally diagnosed mainly by a traditional observation method, an immunological detection method, a molecular biological technology and the like. However, these methods have the problems of complicated operation, long time consumption, expensive instrument and reagent, low accuracy and the like, and can not rapidly and accurately detect and diagnose vibrio alginolyticus on site.
Exponential enrichment of Ligands phylogenetic technology (Systematic Evolution of Ligands)by Exponential engineering technology, SELEX) is a biological library screening technique using volumes up to 1014-1015The random oligonucleotide library of (a) is subjected to multiple rounds of screening in vitro to finally obtain a single-stranded oligonucleotide, i.e., an aptamer, capable of specifically recognizing the target substance. The aptamer has the advantages of easy screening and obtaining, low cost, easy modification, strong stability, high specificity identification, target substance combination and the like, is developed into a novel detection and treatment tool which is widely concerned at present, and has wide application prospect in the fields of biomedical basic research of major diseases and disease diagnosis.
Disclosure of Invention
The invention aims to provide a ssDNA aptamer for detecting vibrio alginolyticus, which has high specificity, high sensitivity, no immunogenicity, stability and easy modification and is convenient to synthesize and store, so as to at least solve the problem that the existing biological detection technology can not quickly and accurately detect and diagnose the vibrio alginolyticus on site.
The invention aims to provide a ssDNA aptamer capable of specifically recognizing Vibrio alginolyticus, wherein the nucleotide sequence of the ssDNA aptamer is 5'-TTTGATATTGCTTTGTCTTCTATTTTATGCTAATCTTTAAATGTACTGGT-3' (SEQ ID NO: 1).
Further, the nucleotide sequence of the ssDNA aptamer is 5 '-GACGCTTACTCAGGTGTGACTCG-TTTGATATTGCTTTGTCTTCTATTTTATGCTAATCTTTAAATGTACTGGT-CGAAGGACGCAGATGAAGTCTC-3' (SEQ ID NO: 2).
Further, any position on the nucleotide sequence of the ssDNA aptamer can undergo phosphorylation, sulfhydrylation, methylation, amination or isotopic reaction.
Further, a marker is bound to the nucleotide sequence of the ssDNA aptamer.
Still further, the label is selected from one or more of biotin, enzyme, and a luminescent group.
Still further, the luminescent group is selected from one or more of fluorescein isothiocyanate, carboxytetramethylrhodamine, bis (2,2 ' -bipyridine) (4,4 ' -dicarboxy-2, 2 ' -bipyridine) ruthenium ligand.
Another object of the present invention is to provide a method for screening ssDNA aptamers, comprising the steps of:
step 1: synthesizing a single-stranded DNA library and primers shown in the following sequences:
random Library 50:
5’-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC-3’；
FITC labeled 5' primer: 5 '-FITC-GACGCTTACTCAGGTGTGACTCG-3';
biotin labeled 5' primer: 5 '-Biotin-GACGCTTACTCAGGTGTGACTCG-3';
TAMRA labeled 5' primer: 5 '-TAMRA-GACGCTTACTCAGGTGTGACTCG-3';
step 2: dissolving 10nmol of the random library in 300-500 μ l PBS, performing constant-temperature water bath at 80-95 ℃ for 5min, rapidly performing ice bath for 5-20min, and incubating the treated random library and live Vibrio alginolyticus bacteria on ice for 0.5-2 h; after incubation and combination are finished, centrifuging and removing supernatant, taking 10mL PBS to wash live vibrio alginolyticus, carrying out thermostatic water bath for 5-30min at 92 ℃, centrifuging for 1-20min under the condition of 12000g, and collecting supernatant, wherein the supernatant is a ssDNA aptamer library for specifically identifying vibrio alginolyticus;
and step 3: taking 100-: 5min at 94 ℃, 1min at 94 ℃, 30sec at 56 ℃, 1min at 72 ℃, and 5min at 72 ℃ after 15-25 cycles of circulation;
and 4, step 4: incubating 100 mu l of streptavidin labeled magnetic beads and the double-stranded DNA obtained by amplification in the step (3) for 15-30min at normal temperature, absorbing the magnetic beads by using a magnetic separator and removing supernatant, washing the magnetic beads by using 1-4mL of PBS, adding 100-200 mu l of 200mM NaOH solution, reacting for 5-20min at normal temperature, recovering the magnetic beads by using a magnetic separation frame, and reserving supernatant;
and 5: adding the supernatant obtained in the step 4 into sterile water, washing, then carrying out salt separation by a desalting column, naturally dripping under the action of gravity, and adding 300-500 mu l PBS into the collected liquid to obtain a solution containing the DNA single-stranded library;
step 6: replacing the random library in the step 2 with the DNA single-stranded library obtained in the step 5, and repeating the step 2-55-8 times;
and 7: heating the DNA library in the step 6 in a constant-temperature water bath at 80-95 ℃ for 5-20min, then rapidly carrying out ice bath for 5-20min, and taking the solution of the treated DNA single-stranded library and aeromonas hydrophila to incubate on ice for 0.5-2 h; after the incubation and combination are finished, centrifuging and collecting supernatant, and incubating the supernatant solution and the live vibrio alginolyticus bacteria on ice for 0.5-2 h; after finishing incubation and combination, centrifuging to remove supernatant, taking 10mL PBS to wash live vibrio alginolyticus, carrying out thermostatic water bath for 5-30min at 80-95 ℃, centrifuging for 1-20min under the condition of 12000g, and collecting supernatant, wherein the supernatant is a negatively screened ssDNA aptamer library for high-specificity identification of vibrio alginolyticus;
and 8: and (3) taking the supernatant solution obtained in the step (7), and repeating the experimental operation sequence of the step (3), the step (4), the step (5), the step (7), the step (2), the step (3), the step (4) and the step (5) for 8 times in sequence, wherein the finally obtained solution is the ssDNA aptamer.
Further, the primers also include a Biotin-labeled 3' primer: 5' -Biotin-
GAGACTTCATCTGCGTCCTTCG-3’(SEQ ID NO：3)。
Another object of the present invention is to provide a method for ELISA detection of ssDNA aptamers as described above, comprising the steps of:
step 1: biotin labeling the ssDNA aptamer;
step 2: mixing 1-100mg of a sample to be detected with the ssDNA aptamer with the concentration of 100-; after incubation and combination, washing a sample to be detected, adding 100-.
The invention also aims to provide the application of the ssDNA aptamer in detecting the vibrio alginolyticus.
Compared with the prior art, the ssDNA aptamer obtained by screening through SELEX technology and negative screening effectively improve the specificity of the ssDNA aptamer, has higher affinity and specificity compared with the existing protein antibody, has the advantages of no immunogenicity, short preparation period, good reproducibility, small molecular weight and the like, and can be produced in large quantities through in vitro chemical synthesis. In addition, when the ssDNA aptamer is used for carrying out ELISA detection on Vibrio alginolyticus, the operation is simple and quick.
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FIG. 1 is a comparison graph of absorbance values of ELISA detection of ssDNA aptamers in example 1, example 2 and comparative example of the present invention;
FIG. 2 is a photograph of fluorescein isothiocyanate of ssDNA aptamer in example 2 of the present invention and a control example;
FIG. 3 is a photograph of a carboxytetramethylrhodamine label of a ssDNA aptamer in example 2 of the present invention and a control example.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example 1
Example 1 preparation of ssDNA aptamers is as follows:
step 1: synthesis of random Single-stranded DNA libraries and primers shown in the following sequence
Random Library 50:
5’-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC-3’；
5' primer (FITC label): 5 '-FITC-GACGCTTACTCAGGTGTGACTCG-3';
5' primer (Biotin label): 5 '-Biotin-GACGCTTACTCAGGTGTGACTCG-3';
5' primer (TAMRA marker): 5 '-TAMRA-GACGCTTACTCAGGTGTGACTCG-3';
step 2: dissolving 10nmol of the random library in 500 μ l PBS, heating in a constant-temperature water bath at 92 ℃ for 5min, then rapidly inserting into ice, carrying out ice bath for 10min, and incubating the treated random library and Vibrio alginolyticus viable bacteria on the ice for 1 h; after incubation and combination are finished, centrifuging to remove supernatant, washing live vibrio alginolyticus bacteria by using 10mL PBS, carrying out constant-temperature water bath at 92 ℃ for 10min, and centrifuging 12000g to collect supernatant, namely the ssDNA aptamer library for specifically identifying the vibrio alginolyticus;
and step 3: taking 100ul of ssDNA aptamer library for identifying vibrio alginolyticus obtained by screening to perform PCR amplification, wherein the specific amplification strip program is as follows: 5min at 94 ℃, 1min at 94 ℃, 30sec at 56 ℃, 1min at 72 ℃, 5min at 72 ℃ after 20 cycles of circulation; all supernatants obtained after the first round of screening are used for PCR amplification to obtain amplification products;
and 4, step 4: incubating 100 mu l of streptavidin-labeled magnetic beads and the double-stranded DNA obtained by PCR amplification in the step 3 for 20min at normal temperature, binding the double-stranded DNA to the surfaces of the magnetic beads by utilizing the affinity action of biotin on the double-stranded DNA and the streptavidin on the magnetic beads, removing supernatant by utilizing a magnetic separator, washing the magnetic beads by using 2mL of PBS, adding 200 mu l of NaOH solution (200mM) into an EP (ultraviolet) tube, reacting for 10min at normal temperature, and recovering by utilizing a magnetic separation frame to obtain supernatant; washing the desalting column with 10mL of sterile water, adding the supernatant into the desalting column, and naturally dripping under the action of gravity; adding 500. mu.l PBS, and collecting the solution containing the DNA single-stranded library;
and 5: replacing the random library in the step 2 with the DNA single-stranded library obtained in the step 4, and repeating the positive screening process, the PCR amplification process and the single-stranded DNA library preparation process shown in the step 2-4 for 8 times;
step 6: and 5, in the second round and the subsequent rounds of screening, using aeromonas hydrophila as a contrast, and carrying out negative screening on the DNA single-stranded library obtained by screening after the step 5 so as to improve the screening efficiency. The specific negative screening process is as follows: dissolving the screened DNA library, incubating with viable aeromonas hydrophila for 1h on ice after water bath at the constant temperature of 92 ℃ and ice bath, and centrifugally collecting a supernatant solution after incubation is finished; then the supernatant solution and the live vibrio alginolyticus bacteria are combined in an ice bath; after finishing incubation combination, centrifuging to remove supernatant, washing live vibrio alginolyticus bacteria by using 10mL PBS, carrying out constant-temperature water bath at 92 ℃ for 10min, centrifuging 12000g to collect supernatant, wherein the collected supernatant is a ssDNA aptamer library of high-specificity identification vibrio alginolyticus through negative screening;
and 7: after the PCR amplification in the step 3 and the preparation of the single-stranded DNA library in the step 4 are carried out on the supernatant solution collected in the step 6, the processes of the step 6, the step 2, the step 3 and the step 4 are sequentially repeated, a multifunctional microplate reader is used for detecting the enhancement condition of the obtained library on the identification capability of the vibrio alginolyticus, and the identification capability of the library on the vibrio alginolyticus is strongest after 8 rounds of screening; after the amplification product is subjected to clone sequencing analysis, the ssDNA aptamer which can be used for detecting live bacteria of Vibrio alginolyticus in example 1 and comprises ssDNA with the sequence 5'-TTTGATATTGCTTTGTCTTCTATTTTATGCTAATCTTTAAATGTACTGGT-3' (SEQ ID NO.1) is finally obtained.
Example 1 ELISA detection of ssDNA aptamers was as follows:
step 1: biotin labeling of the ssDNA aptamer of example 1;
step 2: mixing 10mg of a sample infected with live vibrio alginolyticus with the ssDNA aptamer with the concentration of 200nM obtained in the step 1, and incubating and combining for 20min on ice; after incubation and combination, washing a sample, adding 200 mu l of streptavidin labeled by horseradish peroxidase, performing incubation and combination again on ice for 40min, washing the sample by using a PBS solution after incubation and combination, adding into a horseradish peroxidase developing kit for TMB developing solution development, reading the light absorption value of the sample at 450nm by using an enzyme-labeling instrument, and recording the result. As shown in FIG. 1, No.1 is the absorbance of the sample in example 1 of the present invention.
Example 2
Example 2 preparation of ssDNA aptamers is as follows:
step 1: synthesis of random Single-stranded DNA libraries and primers shown in the following sequence
Random Library 50:
5’-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC-3’；
5' primer (FITC label): 5 '-FITC-GACGCTTACTCAGGTGTGACTCG-3';
5' primer (Biotin label): 5 '-Biotin-GACGCTTACTCAGGTGTGACTCG-3';
5' primer (TAMRA marker): 5 '-TAMRA-GACGCTTACTCAGGTGTGACTCG-3';
3' primer: 5 '-Biotin-GAGACTTCATCTGCGTCCTTCG-3' (SEQ ID NO: 3);
step 2: dissolving 10nmol of the random library in 500 μ l PBS, heating in a constant-temperature water bath at 92 ℃ for 5min, then rapidly inserting into ice, carrying out ice bath for 10min, and incubating the treated random library and Vibrio alginolyticus viable bacteria on the ice for 1 h; after incubation and combination are finished, centrifuging to remove supernatant, washing live vibrio alginolyticus bacteria by using 10mL PBS, carrying out constant-temperature water bath at 92 ℃ for 10min, and centrifuging 12000g to collect supernatant, namely the ssDNA aptamer library for specifically identifying the vibrio alginolyticus;
and step 3: taking 100ul of ssDNA aptamer library for identifying vibrio alginolyticus obtained by screening to perform PCR amplification, wherein the specific amplification strip program is as follows: 5min at 94 ℃, 1min at 94 ℃, 30sec at 56 ℃, 1min at 72 ℃, 5min at 72 ℃ after 20 cycles of circulation; all supernatants obtained after the first round of screening are used for PCR amplification to obtain amplification products;
and 4, step 4: incubating 100 mu l of streptavidin-labeled magnetic beads and the double-stranded DNA obtained by PCR amplification in the step 3 for 20min at normal temperature, binding the double-stranded DNA to the surfaces of the magnetic beads by utilizing the affinity action of biotin on the double-stranded DNA and the streptavidin on the magnetic beads, removing supernatant by utilizing a magnetic separator, washing the magnetic beads by using 2mL of PBS, adding 200 mu l of NaOH solution (200mM) into an EP (ultraviolet) tube, reacting for 10min at normal temperature, and recovering by utilizing a magnetic separation frame to obtain supernatant; washing the desalting column with 10mL of sterile water, adding the supernatant into the desalting column, and naturally dripping under the action of gravity; adding 500. mu.l PBS, and collecting the solution containing the DNA single-stranded library;
and 5: replacing the random library in the step 2 with the DNA single-stranded library obtained in the step 4, and repeating the positive screening process, the PCR amplification process and the single-stranded DNA library preparation process shown in the step 2-4 for 8 times;
step 6: and 5, in the second round and the subsequent rounds of screening, using aeromonas hydrophila as a contrast, and carrying out negative screening on the DNA single-stranded library obtained by screening after the step 5 so as to improve the screening efficiency. The specific negative screening process is as follows: dissolving the screened DNA library, incubating with viable aeromonas hydrophila for 1h on ice after water bath at the constant temperature of 92 ℃ and ice bath, and centrifugally collecting a supernatant solution after incubation is finished; then the supernatant solution and the live vibrio alginolyticus bacteria are combined in an ice bath; after finishing incubation combination, centrifuging to remove supernatant, washing live vibrio alginolyticus bacteria by using 10mL PBS, carrying out constant-temperature water bath at 92 ℃ for 10min, centrifuging 12000g to collect supernatant, wherein the collected supernatant is a ssDNA aptamer library of high-specificity identification vibrio alginolyticus through negative screening;
and 7: after the PCR amplification in the step 3 and the preparation of the single-stranded DNA library in the step 4 are carried out on the supernatant solution collected in the step 6, the processes of the step 6, the step 2, the step 3 and the step 4 are sequentially repeated, a multifunctional microplate reader is used for detecting the enhancement condition of the obtained library on the identification capability of the vibrio alginolyticus, and the identification capability of the library on the vibrio alginolyticus is strongest after 8 rounds of screening; after the obtained amplification product is subjected to clone sequencing analysis, the ssDNA aptamer which can be used for detecting the live bacteria of the vibrio alginolyticus in the examples 1-3 and comprises ssDNA with the sequence 5 '-GACGCTTACTCAGGTGTGACTCG-TTTGATATTGCTTTGTCTTCTATTTTATGCTAATCTTTAAATGTACTGGT-CGAAGGACGCAGATGAAGTCTC-3' (SEQ ID NO.2) is finally obtained.
Example 2 ELISA detection of ssDNA aptamers was as follows:
step 1: biotin labeling of the ssDNA aptamer of example 2;
step 2: mixing 10mg of a sample infected with live vibrio alginolyticus with the ssDNA aptamer with the concentration of 200nM obtained in the step 1, and incubating and combining for 20min on ice; after incubation and combination, washing a sample, adding 200 mu l of streptavidin labeled by horseradish peroxidase, performing incubation and combination again on ice for 40min, washing the sample by using a PBS solution after incubation and combination, adding into a horseradish peroxidase developing kit for TMB developing solution development, reading the light absorption value of the sample at 450nm by using an enzyme-labeling instrument, and recording the result. As shown in FIG. 1, No.2 is the absorbance of the sample in example 2 of the present invention.
Example 2 fluorescein isothiocyanate labeling assay of ssDNA aptamers was as follows:
step 1: carrying out fluorescein isothiocyanate labeling on the ssDNA aptamer of example 2;
step 2: mixing 10mg of a sample infected with live vibrio alginolyticus with the ssDNA aptamer with the concentration of 200nM obtained in the step 1, and incubating and combining for 20min on ice; after incubation and combination, the sample is washed by PBS solution, the combination effect and the specificity of the ssDNA aptamer marked by fluorescein isothiocyanate on vibrio alginolyticus are detected by a fluorescence microscope, the detection result is shown in figure 2, and No.2 is the detection result of the invention in the embodiment 2.
Example 2 detection of carboxytetramethylrhodamine labels by ssDNA aptamers is as follows:
step 1: carboxytetramethylrhodamine labeling of the ssDNA aptamer of example 2;
step 2: mixing 10mg of a sample infected with live vibrio alginolyticus with the ssDNA aptamer with the concentration of 200nM obtained in the step 1, and incubating and combining for 20min on ice; after incubation and combination, a sample is washed, a PBS solution is used for washing the sample, a fluorescence microscope is used for detecting the combination effect and the specificity of the carboxyl tetramethyl rhodamine marked ssDNA aptamer to vibrio alginolyticus, the detection result is shown in figure 3, and No.2 is the detection result of the invention in the embodiment 2.
Example 3
In the embodiment of the invention, the ssDNA aptamer obtained in the embodiment 2 is used for assembling an ELISA kit to form the ELISA kit for detecting the pathogenic bacteria vibrio alginolyticus of the aquatic product.
Comparative example
Comparative example ssDNA aptamers were prepared as follows:
synthesis of random Single-stranded DNA libraries and primers shown in the following sequence
Random Library 50:
5’-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC-3’；
5' primer (FITC label): 5 '-FITC-GACGCTTACTCAGGTGTGACTCG-3';
5' primer (Biotin label): 5 '-Biotin-GACGCTTACTCAGGTGTGACTCG-3';
5' primer (TAMRA marker): 5 '-TAMRA-GACGCTTACTCAGGTGTGACTCG-3';
3' primer: 5 '-Biotin-GAGACTTCATCTGCGTCCTTCG-3' (SEQ ID NO: 3);
the random single-stranded DNA library described above was used as a control ssDNA aptamer.
Comparative example ELISA detection of ssDNA aptamers was as follows:
step 1: biotin labeling of the control ssDNA aptamer;
step 2: mixing 10mg of a sample infected with live vibrio alginolyticus with the ssDNA aptamer with the concentration of 200nM obtained in the step 1, and incubating and combining for 20min on ice; after incubation and combination, washing a sample, adding 200 mu l of streptavidin labeled by horseradish peroxidase, performing incubation and combination again on ice for 40min, washing the sample by using a PBS solution after incubation and combination, adding into a horseradish peroxidase developing kit for TMB developing solution development, reading the light absorption value of the sample at 450nm by using an enzyme-labeling instrument, and recording the result. As shown in FIG. 1, Con is the absorbance of the control of the present invention.
Control ssDNA aptamer fluorescein isothiocyanate labeling assay was as follows:
step 1: fluorescein isothiocyanate labeling of the control ssDNA aptamer;
step 2: mixing 10mg of a sample infected with live vibrio alginolyticus with the ssDNA aptamer with the concentration of 200nM obtained in the step 1, and incubating and combining for 20min on ice; after incubation and combination, the sample is washed, PBS solution is used for washing the sample, a fluorescence microscope is used for detecting the combination effect and the specificity of the ssDNA aptamer marked by fluorescein isothiocyanate on vibrio alginolyticus, the detection result is shown in figure 2, and Con is the detection result of the comparison example of the invention.
Comparative example detection of carboxyl tetramethyl rhodamine label with ssDNA aptamer was as follows:
step 1: carboxytetramethylrhodamine labeling of control ssDNA aptamers;
step 2: mixing 10mg of a sample infected with live vibrio alginolyticus with the ssDNA aptamer with the concentration of 200nM obtained in the step 1, and incubating and combining for 20min on ice; after incubation and combination, washing the sample with PBS solution, and detecting the combination effect and specificity of the carboxyl tetramethyl rhodamine labeled ssDNA aptamer to vibrio alginolyticus by using a fluorescence microscope, wherein the detection result is shown in FIG. 3, and Con is the detection result of the comparison example of the invention.
As shown in FIG. 1, the ssDNA aptamers of examples 1 and 2 of the present invention can specifically bind to live Vibrio alginolyticus, and the detection results have higher absorbance values, while the control examples have lower absorbance values than those of examples 1 and 2, and do not have the ability to specifically bind to live Vibrio alginolyticus, and cannot specifically identify live Vibrio alginolyticus. In addition, the light absorption value of the sample 1 is obviously higher than that of the sample 2, and the sample has better specific binding performance for the live bacteria of the vibrio alginolyticus.
As shown in FIG. 2, the location of viable bacteria of Vibrio alginolyticus was determined by fluorescence microscopy, and it can be seen that the Vibrio alginolyticus of the comparative example did not exhibit fluorescence during the fluorescence detection, indicating that the ssDNA aptamer of the comparative example failed to specifically bind to Vibrio alginolyticus and failed to effectively detect Vibrio alginolyticus, whereas the ssDNA aptamer of example 2 exhibited fluorescence at the location of Vibrio alginolyticus during the fluorescence detection, indicating that the ssDNA aptamer of example 2 could effectively bind to Vibrio alginolyticus.
As shown in FIG. 3, the location of viable bacteria of Vibrio alginolyticus was determined by a fluorescence microscope, and it can be seen from the figure that the Vibrio alginolyticus of the comparative example did not exhibit fluorescence during the fluorescence detection, indicating that the ssDNA aptamer of the comparative example could not specifically bind to Vibrio alginolyticus and could not effectively detect Vibrio alginolyticus, whereas the location of Vibrio alginolyticus in the fluorescence detection of example 2 exhibited fluorescence, indicating that the ssDNA aptamer of example 2 has a better binding effect with Vibrio alginolyticus. In addition, in the fluorescence detection picture of example 2 in fig. 3, other infectious microbes are clearly visible under the irradiation of visible light, and in the fluorescence detection process, no obvious fluorescence absorption is generated at the position of the infectious microbes, which indicates that the ssDNA aptamer of example 2 can be specifically combined with vibrio alginolyticus, and a false positive result is not easily generated.
Based on the results shown in fig. 1, 2 and 3, it can be proved that the ssDNA aptamer shown in examples 1 and 2 of the present invention can be specifically bound to vibrio alginolyticus after being labeled and modified by fluorescent substances or luminescent materials such as biotin, enzyme, fluorescein isothiocyanate, carboxytetramethylrhodamine, etc., and can be used for detecting vibrio alginolyticus.
The ssDNA aptamers obtained by screening through SELEX technology in the embodiments 1 and 2 of the invention have good affinity and specificity, and the ssDNA aptamers of the embodiments 1 and 2 of the invention have stable structures, still have good affinity and specificity after group marking and modification, and can be applied to ELISA detection kits, and compared with protein antibodies, the ssDNA aptamers of the embodiments 1 and 2 of the invention have the characteristics of short preparation period, good reproducibility and small molecular weight, are convenient for in vitro synthesis, and have good application prospects in the field of detection of aquatic pathogenic bacteria vibrio alginolyticus.
Finally, it should be noted that the above-mentioned embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the modifications and equivalents of the specific embodiments of the present invention can be made by those skilled in the art after reading the present specification, but these modifications and variations do not depart from the scope of the claims of the present application.
Sequence listing
<110> Guangxi academy of sciences
<120> nucleic acid aptamer capable of specifically recognizing vibrio alginolyticus and application thereof
<130> PY1710278
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tttgatattg ctttgtcttc tattttatgc taatctttaa atgtactggt 50
<210> 2
<211> 95
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gacgcttact caggtgtgac tcgtttgata ttgctttgtc ttctatttta tgctaatctt 60
taaatgtact ggtcgaagga cgcagatgaa gtctc 95
Claims (7)
1. A ssDNA aptamer capable of specifically recognizing vibrio alginolyticus, wherein the nucleotide sequence of the ssDNA aptamer is 5'-TTTGATATTGCTTTGTCTTCTATTTTATGCTAATCTTTAAATGTACTGGT-3'.
2. The ssDNA aptamer capable of specifically recognizing the pathogenic bacteria vibrio alginolyticus in the aquatic product is characterized in that the nucleotide sequence of the ssDNA aptamer is 5 '-GACGCTTACTCAGGTGTGACTCG-TTTGATATTGCTTTGTCTTCTATTTTATGCTAATCTTTAAATGTACTGGT-CGAAGGACGCAGATGAAGTCTC-3'.
3. The ssDNA aptamer according to claim 1 or 2, wherein a label is bound to both ends of the nucleotide sequence of the ssDNA aptamer.
4. The ssDNA aptamer according to claim 3, wherein the label is selected from one or more of biotin, an enzyme, and a luminescent group.
5. The ssDNA aptamer according to claim 4, wherein the luminescent group is selected from fluorescein isothiocyanate or carboxytetramethylrhodamine.
6. An ELISA detection method using the ssDNA aptamer according to claim 1 or 2, comprising the steps of:
step 1: biotin labeling the ssDNA aptamer;
step 2: mixing 1-100mg of a sample to be detected with the ssDNA aptamer with the concentration of 100-; after incubation and combination, cleaning a sample to be detected, adding 100-;
the use does not include use in disease diagnosis.
7. Use of the ssDNA aptamer of claim 1 or 2 for detecting Vibrio alginolyticus; the use does not include use in disease diagnosis.
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| CN109207480B (en) * | 2018-09-26 | 2021-08-27 | 广西科学院 | Aptamer specific for trachinotus ovatus nervous necrosis virus and application thereof |
| CN110257387B (en) * | 2019-07-04 | 2022-10-28 | 广西科学院 | Aptamer for identifying grass carp hemorrhagic disease virus as well as construction method and application thereof |
| CN111073892B (en) * | 2019-10-30 | 2023-11-17 | 广西科学院 | Nucleic acid aptamer for identifying garrupa iridovirus infected cells, construction method and application thereof |
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| CN102199667A (en) * | 2011-04-12 | 2011-09-28 | 集美大学 | Oligonucleotide sequence for detecting vibrio alginolyticus and application thereof |
| CN102329862B (en) * | 2011-09-02 | 2013-06-05 | 集美大学 | Three oligonucleotide sequences for identification and detection of vibrio alginolyticus as well as preparation method and application thereof |
| CN102605075B (en) * | 2012-03-22 | 2014-04-16 | 集美大学 | A group of oligonucleotide sequences capable of identifying Vibrio harveyi and Vibrio alginolyticus synchronously and preparation method of the oligonucleotide sequences |
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