CN113416733A

CN113416733A - Screening and application of group of sulfonamide antibiotics broad-spectrum specific aptamers

Info

Publication number: CN113416733A
Application number: CN202110701824.1A
Authority: CN
Inventors: 付凤富; 黄琳; 杨玮娟
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2021-06-24
Filing date: 2021-06-24
Publication date: 2021-09-21
Anticipated expiration: 2041-06-24
Also published as: CN113416733B

Abstract

The invention discloses screening and application of a group of sulfonamide antibiotic broad-spectrum specificity aptamers, wherein the aptamers are screened based on SELEX technology, positive screening is carried out by taking sulfamethoxypyridazine, sulfathiazole, sulfamethazine and sulfapyridine as target molecules, negative target molecules are tetracycline, kanamycin sulfate, cefotaxime, enrofloxacin, oxacillin and ampicillin, the 1 st to 3 rd rounds are positive screening, and the 4 th to 11 th rounds are negative screening. The aptamer screened by the method has the advantages of high specificity and high affinity binding with various sulfonamide antibiotics, and can be applied to establishing a colorimetric method for simultaneously and rapidly detecting various sulfonamide antibiotic residues in marine products.

Description

Screening and application of group of sulfonamide antibiotics broad-spectrum specific aptamers

Technical Field

The invention belongs to the field of food safety and antibiotic detection, and particularly relates to screening and application of a group of aptamer with broad-spectrum specificity of sulfonamide antibiotics.

Background

Sulfonamide Antibiotics (SAs) are a class of traditional artificially synthesized antibacterial drugs, and have been widely used in livestock and aquaculture due to their low price, stable chemical properties, broad-spectrum activity, and the like. However, driven by economic benefits, the exceeding of sulfonamide antibiotics is violated or exceeded, so that the residue of the sulfonamide antibiotics in livestock and poultry meat and aquatic products is always exceeded, and great potential safety hazards are directly brought to human health. Therefore, the enhancement of screening and detection of sulfonamide antibiotic residues in animal-derived food including seafood is of great significance for ensuring safe consumption of livestock and poultry meat and aquatic products.

At present, the traditional methods for simultaneously detecting various sulfonamides antibiotics mainly comprise High Performance Liquid Chromatography (HPLC), Ultra High Performance Liquid Chromatography (UHPLC), high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), immunoassay and the like. However, the methods are high in cost and long in time consumption, and cannot meet the requirement of on-site rapid screening of sulfonamide antibiotic residues in livestock and poultry meat and aquatic products.

The aptamer is a single-stranded oligonucleotide screened in vitro by a systematic evolution of ligands by evolution (SELEX) technology of exponential enrichment, not only has high affinity and specificity similar to or even better than that of an antibody, but also has good stability and is easy to synthesize and modify, and the aptamer is also tried to be used for constructing a rapid detection method of sulfonamide antibiotics. However, the existing method for detecting sulfonamide antibiotics based on nucleic acid aptamers can detect single sulfonamide antibiotics, and the detection requirements of actual samples are difficult to meet. Therefore, screening of the aptamer with broad-spectrum specificity of the sulfonamide antibiotics and application of the aptamer to establishment of a method for simultaneously, rapidly and accurately detecting a plurality of sulfonamide antibiotics become the direction of research efforts of researchers.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a method for screening broad-spectrum specific aptamers of sulfonamides antibiotics, and correspondingly provides the aptamers obtained by adopting the method, wherein the aptamers can be simultaneously combined with multiple sulfonamides with high specificity and high affinity, can be applied to a detection method for developing sulfonamides of marine products, and particularly relates to a method for rapidly detecting sulfaquinoxaline, sulfadimethoxine, sulfamethazine, 5-methoxypyrimidine, sulfamethoxypyridazine and sulfamethazine based on the recognition of the aptamers.

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

a group of sulfonamide antibiotics broad-spectrum specificity aptamers, wherein the aptamers are one, or 2 or 3 of nucleotide sequences of Sull04, Sull34 and Sull 43; the nucleotide sequence of the aptamer is as follows:

Sull04：

5’-CTTACGACACGGGGTCTTGGGGTGAGTCCTGTTGTGTCAGTGTGTCGTAAG-3’;

Sull34：

5’-CTTACGACACGGGGTCTTGGGGTGAGTCCTGCTGTGTCAGTGTGTCGTAAG-3’;

Sull43：

5’-CTTACGGCACGGGGTCTTGGGGTGAGTCCTGTTGTGTCAGTGTGTCGTAAG-3’。

the screening method of the group of broad-spectrum specificity aptamer of the sulfonamide antibiotics is based on SELEX technology for screening, nucleic acid is used as the aptamer, sulfamethoxypyridazine, sulfathiazole, sulfamethazine and sulfapyridine are used as target molecules for forward screening, tetracycline (Tet), kanamycin sulfate (Kan), cefotaxime (Cef), enrofloxacin (Enr), oxacillin (Oxa) and ampicillin (Amp) 6 other antibiotics are used as anti-target molecules for reverse screening, rounds 1 to 3 are forward screening, and rounds 4 to 11 are reverse screening.

The group of sulfonamide antibiotic broad-spectrum specificity aptamer is applied to establishing a method for simultaneously detecting multiple sulfonamide antibiotics in marine products. Wherein the sulfanilamide antibiotic is one or more of sulfaquinoxaline, sulfadimethoxine, sulfanilamide-5-methoxypyrimidine, sulfamethoxypyridazine and sulfamethazine.

Compared with the prior art, the invention has the following advantages:

(1) the sulfonamide antibiotic broad-spectrum specificity aptamer is simple and convenient to chemically synthesize and short in screening period;

(2) the 3 broad-spectrum specificity sulfanilamide antibiotic aptamer sequences obtained by screening have higher affinity and specificity to 10 common sulfanilamide antibiotics (sulfamethoxypyridazine, sulfaquinoxaline, sulfanilamide-5-methoxypyrimidine, sulfachloropyridazine, sulfadiazine, sulfadimetrazine, sulfadimethoxine, sulfamonomethoxine and sulfamethazine), overcome the defect that the existing sulfanilamide antibiotic aptamer can only recognize and combine a single sulfanilamide antibiotic, and can be used for developing and constructing various new methods and new technologies for quickly and inexpensively screening the residue of various sulfanilamide antibiotics;

(3) the colorimetric rapid detection method for simultaneously detecting multiple sulfonamide antibiotics (sulfaquinoxaline, sulfadimethoxine, sulfanilamide-5-methoxypyrimidine, sulfamethoxypyridazine and sulfamethazine) in marine products, which is developed by the invention, has the advantages of simple operation, rapidness, good stability, low cost and the like, can be directly used for rapidly screening the 5 sulfonamide antibiotics residues in the marine products, and does not need other additional treatment processes.

Drawings

FIG. 1 is a secondary structure prediction map of the aptamer Sull 04.

FIG. 2 is a secondary structure prediction map of the aptamer Sull 34.

FIG. 3 is a secondary structure prediction map of the aptamer Sull 43.

FIG. 4 is a PAGE analysis of the affinity of 3 broad spectrum specific sulfonamide aptamers to 19 commonly used sulfonamides and other types of antibiotics.

FIG. 5 shows the results of the fluorometric analysis of the affinities of 3 broad spectrum specific sulfonamide aptamers to 19 common sulfonamides and other types of antibiotics.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1: screening, cloning, separating and sequencing of sulfanilamide antibiotic broad-spectrum specific aptamer and prediction of single-stranded DNA secondary structure

(1) Mixing the raw materials in a ratio of 1: 5 molar ratio to prepare a 5 μ L DNA library (5' -CGAGCATA)GGCAGAACTTACGAC (N30) GTCGTAAGAGCGAGTCATTC-3 ') and the cDNA-Biotin sequence (5 ' -TTTTTGTCGTAAGTTCTGCCATTTT/Biotin/-3 '), to which 50 μ L of 5 Xselection buffer (50 mM Tris-HCl, 2.5mM MgCl) was added₂100mM NaCl, pH 7.4), and then preparing a 250 μ L mixed solution with DNase/RNase free-water. After the mixture was metal-bathed at 95 ℃ for 10min, it was cooled at room temperature for more than 30min to ensure hybridization of the DNA library and the cDNA-biotin sequence, thereby obtaining a DNA library-cDNA-biotin conjugate.

(2) To 0.5mL of polyethylene Bio-microcolumn (Bio-Rad, Micro Bio-spin column), 300. mu.L (200. mu.L from the second round) of streptavidin-coated agarose magnetic bead (beads) solution was added, followed by 250. mu.L of 1 Xselection buffer (10 mM Tris-HCl, 0.5mM MgCl)₂20mM NaCl, pH 7.4) were washed 6 times to prepare beads microgravity exchange columns. Circulating the DNA library-cDNA-Biotin conjugate solution in the 250 mu L step (1) through a beads microgravity exchange column for 4 times to fully bind and immobilize the beads, and then selecting a buffer solution (10 mM Tris-HCl, 0.5mM MgCl) with 250 mu L of 1X₂20mM NaCl, pH 7.4) the column was washed 10 times (note: the resin was not allowed to dry, no pressure was applied, and the resin was naturally drained by gravity for washing). Respectively dissolving 750 muL of solution with the concentration of 500 muM in 1X selection buffer (10 mM Tris-HCl, 0.5mM MgCl)₂20mM NaCl, pH 7.4) is added into a beads microgravity exchange column, the target sulfonamide antibiotics (sulfamethoxypyridazine, sulfathiazole, sulfamethazine and sulfapyridine) are added in 3 times, 250 muL is added each time, 3 tubes of eluent is collected by a centrifuge tube according to each 250 muL/tube, and then the eluent is centrifugally filtered and concentrated by the centrifuge tube through ultrafiltration of 3kDa for PCR amplification of the next step.

(3) 900 μ L of PCR Mix solution containing 1 μ M forward primer (F: 5'-CGAGCATAGGCAGAACTTAC-3'), 1 μ M Biotin-modified reverse primer (R: 5 '-/Biotin/GAATGACTCGCTCTTACGAC-3'), and 450 μ L GoTaq Hot Start was prepared with PCR-only water. 27 μ L of PCR Mix solution was taken in 2 PCR tubes, one tube was used as a cathode control, and the other tube was added with 3 μ L of initial DNA library (100 nM) as an anode control. And (3) uniformly mixing the residual PCR Mix solution with the DNA library-target sulfonamide antibiotic conjugate concentrated solution obtained in the step (2), and subpackaging the mixture into 90 mu L/tube by using a PCR tube. And (3) putting the cathode, the anode control and the PCR tube filled with 90 muL of mixed liquor into a PCR instrument, and carrying out amplification on a Thermal Cycler PCR instrument under the amplification conditions: heating at 95 deg.C for 2 min; heating at 95 ℃ for 15s, at 58 ℃ for 30s, at 72 ℃ for 45s, and at 72 ℃ for 5min (11 cycles); infinite circulation at 12 ℃. The PCR products were electrophoretically separated at 100V for 30min by 3wt% agarose gel electrophoresis, and the gel electrophoresis bands were visualized by gel imaging after staining with SYBR Gold to confirm the PCR amplification effect and ensure the absence of byproducts.

(4) Preparing a new beads microgravity exchange column, and circulating the PCR product obtained in the step (3) through the column for 3 times to ensure that the PCR product is fully combined with the beads and is fixed on the beads. After washing the column with 250 μ L of 1 XNo Mg selection buffer (10 mM Tris-ms, 20mM NaCl, pH 7.4) for 6 times, the column bottom cover was closed, 300 μ L of 0.2M NaOH solution was added, incubation was performed at room temperature for 10min to melt the PCR product and generate single-stranded DNA, and single-stranded DNA eluate was collected. After the column was drained, 100 μ L of 0.2M NaOH solution was added to the column to elute the residual DNA on the column. The 2 eluates were combined and neutralized with 0.2M HCl to pH = 8. And then, the combined eluent is centrifugally filtered and concentrated by an ultrafiltration centrifugal tube with the molecular weight of 3kDa, and the concentration of the single-stranded DNA is determined on an enzyme-labeling instrument. The single-stranded DNA obtained in the first round is used as the initial pool of the second round, and each subsequent round is the single-stranded DNA screened in the previous round. For each round (4 to 11 rounds) starting from the fourth round, a counter-selectivity (250 μ L, 300 μ M each dissolved in 1X selection buffer (10 mM Tris-HCl, 0.5mM MgCl) on a column for library DNA immobilization was performed before the target selection step₂20mM NaCl, pH 7.4) to remove non-specific DNA sequences. This experiment was performed for 11 screening rounds.

(5) The 10nM DNA enrichment library collected from round 11 was mixed with PCR Mix, 1 μ M forward primer (F: 5'-CGAGCATAGGCAGAACTTAC-3') and 1 μ M reverse primer (R: 5 '-/Biotin/GAATGACTCGCTCTTACGAC-3') in equal volumes to a final volume of 50 μ L. Then, 9 times of PCR were carried out according to the SELEX procedure under the same PCR conditions as in step (3). The PCR-treated sample was sent to a sequencing service (Biotechnology engineering (Shanghai) Co., Ltd.) for sequencing. After obtaining sequencing data, the sequences obtained by sequencing are compared, the sequence characteristics of each aptamer are analyzed, and secondary structure prediction is carried out by utilizing a DNA sequence analysis design website (http:// www.nupack.org/partition/new). 3 broad-spectrum specificity nucleic acid aptamers of sulfonamide antibiotics, namely Sull04, Sull34 and Sull43 are obtained by screening, and the sequences are as follows:

Sull04：

5’-CTTACGACACGGGGTCTTGGGGTGAGTCCTGTTGTGTCAGTGTGTCGTAAG-3’;

Sull34：

5’-CTTACGACACGGGGTCTTGGGGTGAGTCCTGCTGTGTCAGTGTGTCGTAAG-3’;

Sull43：

5’-CTTACGGCACGGGGTCTTGGGGTGAGTCCTGTTGTGTCAGTGTGTCGTAAG-3’。

FIG. 1 is a secondary structural diagram of the aptamer Sull04, FIG. 2 is a secondary structural diagram of the aptamer Sull34, and FIG. 3 is a secondary structural diagram of the aptamer Sull 43.

Example 2: PAGE analysis of the affinities of 3 broad-spectrum specific aptamers to 19 commonly used sulfonamides and other types of antibiotics

(1) 90pmol of the 11 th round screened DNA enrichment library was mixed in equal volumes with 450pmol of a cDNA-Biotin sequence (5 '-TTTTTGTCGTAAGTTCTGCCATTTT/Biotin/-3') solution in 227 μ L of 1 Xselection buffer (10 mM Tris-HCl, 0.5mM MgCl)₂20mM NaCl, 0.01wt% Tween 20, pH 7.4), incubated at 95 ℃ for 10min, and then cooled at room temperature for more than 30min to ensure formation of a DNA-enriched library-cDNA-biotin conjugate. Meanwhile, 250 muL of beads solution is added into one centrifuge tube, 1.5mL of 1X selection buffer solution is used for washing for 3 times, and then the washing solution is discarded through centrifugation. Adding the DNA-enriched library-cDNA-biotin conjugate into a centrifuge tube, and incubating for 30min at room temperature on a sample mixer to enrich the library with DNAThe cDNA-biotin conjugate was immobilized on beads, then centrifuged, allowed to stand for 5min and the supernatant removed. After the Beads are washed for 5 times by 1.5mL of 1X selection buffer solution, the Beads are subjected to constant volume to 3.0mL by the 1X selection buffer solution, and are uniformly subpackaged into 20 tubes according to 100 mu L/tube, and the rest are stored for later use.

(2) Standing the 20-tube beads solution obtained in the step (1) for 10min, discarding 90 muL of supernatant, and then adding 25 muL of 1 Xselection buffer (10 mM Tris-HCl, 0.5mM MgCl) into one tube₂20mM NaCl, pH 7.4) as a blank control, adding 25 μ L of 500 μ M of 19 common sulfa antibiotics (sulfadimethoxine, sulfadimetridine, sulfamethazine, sulfadiazine, sulfathiazole, sulfanilamide-5-trimethoprim, sulfapyridine, sulfisoxazole, sulfaquinoxaline, sulfamonomethoxine, sulfachloropyridazine, sulfadiazine, sulfamethoxazole, sulfaamidine, sulfapyrazole, sulfadoxine, sulfabenzoyl, sulfacetamide) and 6 other antibiotics (Tet, Kan, Cef, Enr, Oxa and Amp) into the other 19 tubes, incubating the tubes on a mixing instrument in the dark at room temperature for 1h, centrifuging and standing for 10min, and taking 20 μ L of supernatant (supernatant S). 25 μ L of 98wt% formamide (containing 10mM EDTA) was added to the remaining solution in each tube and mixed well, and 10 μ L of supernatant (supernatant B) was centrifuged after heating in a metal bath at 95 ℃ for 10 min.

(3) Analyzing the target-eluted aptamer solution (supernatant S) and the formamide-treated DNA solution (supernatant B) obtained in step (2) by 15wt% denaturing polyacrylamide gel electrophoresis (PAGE), calculating the concentrations of DNA in the supernatants S and B according to the standard concentrations in the gel, and calculating the elution rates for evaluating the affinity of the DNA sequences for the 19 sulfonamides using the following formula:

wherein, is the DNA sequence elution rate, C_SConcentration of target elution Strand in supernatant S, C_BConcentration of target elution chain in supernatant B, V₁Volume of solution (35 μ L), V, before collection of supernatant₂Is the volume (20 mu L), V, of the supernatant S₃The volume of the solution after formamide addition was 40. mu.L.

As shown in FIG. 4, it is demonstrated that 3 sulfa antibiotic broad-spectrum specific aptamers Sull04, Sull34 and Sull43 have strong binding ability to sulfadimethoxine, sulfamethazine, sulfamethoxypyridazine, sulfadiazine, sulfathiazole, sulfam-5-methoxypyrimidine, sulfapyridine, sulfamethoxazole, sulfaquinoxaline, sulfamonomethoxine, sulfachloropyridazine and sulfadoxine.

Example 3: fluorescent method analysis of affinity of 3 sulfanilamide antibiotic broad-spectrum specificity aptamers to 19 common sulfanilamide antibiotics and other types of antibiotics

(1) 30 μ L of 19 common sulfonamide antibiotics of the same type as in example 2 and 6 other antibiotics of the same type were added to a mixture containing 30 μ L of 20 μ M quenching probe BHQ-cDNA sequence (5 '-GTCGTAAGTTCTG/BHQ 1-3') and the aptamers Sull-04, Sull-34, Sull-43 sequence at a final concentration of 0.4 μ M, respectively.

(2) And (2) incubating the mixed solution obtained in the step (1) at 72 ℃ for 5min, further incubating at 41 ℃ for 5min, then incubating at 25 ℃ for 45min, taking out 40 mu L of the mixed solution, testing the fluorescence spectrum (excitation wavelength of 480 nm) of the mixed solution on an enzyme label plate on an enzyme label instrument, and determining the affinity of the nucleic acid aptamers Sull-04, Sull-34 and Sull-43 to various antibiotics according to the fluorescence intensity gain value of the mixed solution of 100 mu M antibiotics at 530 nm.

As shown in FIG. 5, it is demonstrated that 3 sulfa antibiotic broad-spectrum specific aptamers Sull04, Sull34 and Sull43 have strong binding ability to sulfaquinoxaline, sulfamethoxypyridazine, sulfanilamide-5-methoxypyrimidine, sulfachlorpyridazine, sulfapyridine, sulfamethazine, sulfadimethoxine, sulfamethazine, sulfamonomethoxine and sulfamethazine.

Example 4: comparing the recovery rate of the invention and HPLC-MS method

The method comprises the following specific processes:

(1) weighing 0.1g of dried fish sample (sea bass), placing the fish sample (sea bass) in a 10mL polypropylene centrifuge tube, adding 2mL of ethyl acetate, carrying out ultrasonic extraction for 10min after uniformly mixing by vortexing for 30s, separating and collecting supernatant after centrifuging for 10min at 10000r/min, adding 2mL of ethyl acetate into residues, extracting once again according to the method, separating and collecting supernatant, combining the supernatants obtained in the 2 extraction processes, passing through a 0.22 mu L filter membrane, and carrying out nitrogen blowing concentration by a nitrogen blower until the supernatants are nearly dried. Adding 1mL of 1mM formic acid methanol solution into nearly dry residues, performing ultrasonic dissolution for 5min, then adding 2mL of n-hexane for extraction to remove grease, standing, discarding the upper n-hexane layer, centrifuging the lower layer solution at 10000r/min for 5min, separating and collecting supernatant, adding 1mL of 1mM formic acid methanol solution into the residues, performing ultrasonic dissolution extraction again according to the method, separating and collecting supernatant, combining the supernatants obtained in the 2 extraction processes, and passing through a 0.22 mu L filter membrane to obtain a final solution for detecting sulfonamide residues.

(2) Cy7 with a final concentration of 2.5 μ M, an aptamer sequence (Sull-04 or Sull-34 or Sull-43) with a final concentration of 4 μ M, 0.01% Tween, 10mM Tris-HCl, 0.5mM MgCl were added in order in 100 μ L of ultrapure water₂20mM NaCl and 50 muL standard solutions (0, 0.5, 1, 2, 5, 10, 20, 50, 100 and 200 muM) of sulfonamide antibiotics (sulfaquinoxaline, sulfadimethoxine, sulfasoximine, sulfamethoxypyridazine and sulfanilamide-5-methoxypyrimidine) at different concentrations were mixed well. And (3) taking 80 mu L of the mixed solution to a transparent flat-bottom 384-hole plate, and measuring the ultraviolet-visible spectrum by using an enzyme-labeling instrument at room temperature within the range of 450-900 nm. According to the absorbance ratio (A) of the solution at the wavelengths of 670nm and 760nm₆₇₀/A₇₆₀) And establishing a standard curve with the absorbance as an abscissa and the absorbance as an ordinate, wherein the linear relation, the correlation coefficient and the detection limit of the 5 kinds of sulfanilamide antibiotics are shown in tables 1, 2 and 3.

(3) Cy7, 4M aptamer sequence (Sull-04 or Sull-34 or Sull-43), 0.01wt% Tween, 10mM Tris-HCl, 0.5mM MgC were added in order to a final concentration of 2.5 μ M in 100 μ L of ultrapure water_l220mM NaCl and 50 μ L of sample solution, and mixingAnd (4) homogenizing. And (3) taking 80 mu L of the mixed solution to a transparent flat-bottom 384-hole plate, and measuring the ultraviolet-visible spectrum by using an enzyme-labeling instrument at room temperature within the range of 450-900 nm. According to the absorbance ratio (A) of the solution at the wavelengths of 670nm and 760nm₆₇₀/A₇₆₀) And (3) carrying out quantitative calculation by referring to the standard working curve in the step (2), wherein the residue condition (microgram/mL), recovery rate and relative standard deviation result of the sulfonamide antibiotics in the sea bass fish meat sample are shown in tables 1, 2 and 3.

TABLE 1 Linear relationship, correlation coefficient, detection limit, detection concentration, relative standard deviation and recovery rate of 5 sulfonamides by Sull04 method for nucleic acid aptamer

TABLE 2 Linear relationship, correlation coefficient, detection limit, detection concentration, relative standard deviation and recovery rate of 5 sulfonamides by aptamer Sull34 method

TABLE 3 Linear relationship, correlation coefficient, detection limit, detection concentration, relative standard deviation and recovery rate of aptamer Sull43 method 5 sulfonamides

HPLC-MS/MS measurement was performed with reference to International standards for ICS 67.050 (B50) of Ministry of agriculture 1077, Notification-1-2008. The linear relationship, correlation coefficient, detection limit, detection concentration (mug/mL), recovery rate and relative standard deviation result of the 5 kinds of sulfonamide antibiotics are shown in the table 4.

TABLE 4 Linear relationship, correlation coefficient, detection limit, detection concentration, relative standard deviation and recovery rate of 5 sulfonamides by HPLC-MS method

As shown in tables 1, 2 and 3, the results show that the spiking recovery rates of the 3 sulfonamide antibiotic broad-spectrum specific aptamers to sulfaquinoxaline, sulfadimethoxine, sulfasozine, sulfamethoxypyridazine and sulfanilamide-5-methoxypyrimidine are 82% -93%, the Relative Standard Deviation (RSD) of the determination results is 1% -5%, and the difference between the results and the detection results of HPLC-MS in the table 4 is small, so that the method has good precision and accuracy. Therefore, the nucleic acid aptamer has wide application potential and value in the aspect of developing a method for simultaneously detecting multiple sulfonamides antibiotics.

Although the present invention has been described in detail, it will be appreciated by those skilled in the art that the present invention is not limited to the details of the foregoing description, but is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

SEQUENCE LISTING

<110> Fuzhou university

<120> screening of a group of broad-spectrum specific aptamers of sulfonamides antibiotics and application thereof

<130> 8

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 51

<212> DNA

<213> Artificial sequence

<400> 1

cttacgacac ggggtcttgg ggtgagtcct gttgtgtcag tgtgtcgtaa g 51

<210> 2

<211> 51

<212> DNA

<213> Artificial sequence

<400> 2

cttacgacac ggggtcttgg ggtgagtcct gctgtgtcag tgtgtcgtaa g 51

<210> 3

<211> 51

<212> DNA

<213> Artificial sequence

<400> 3

cttacggcac ggggtcttgg ggtgagtcct gttgtgtcag tgtgtcgtaa g 51

<210> 4

<211> 43

<212> DNA

<213> Artificial sequence

<400> 4

cgagcatagg cagaacttac gacgtcgtaa gagcgagtca ttc 43

<210> 5

<211> 25

<212> DNA

<213> Artificial sequence

<400> 5

tttttgtcgt aagttctgcc atttt 25

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

cgagcatagg cagaacttac 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

gaatgactcg ctcttacgac 20

<210> 8

<211> 13

<212> DNA

<213> Artificial sequence

<400> 8

gtcgtaagtt ctg 13

Claims

1. A group of sulphonamide antibiotics broad spectrum specificity aptamer is characterized in that: the aptamer is 1 strip, or 2 strips, or 3 strips in the nucleotide sequences of Sull04, Sull34 and Sull 43;

the nucleotide sequence of the aptamer is as follows:

Sull04：

5’-CTTACGACACGGGGTCTTGGGGTGAGTCCTGTTGTGTCAGTGTGTCGTAAG-3’;

Sull34：

5’-CTTACGACACGGGGTCTTGGGGTGAGTCCTGCTGTGTCAGTGTGTCGTAAG-3’;

Sull43：

5’-CTTACGGCACGGGGTCTTGGGGTGAGTCCTGTTGTGTCAGTGTGTCGTAAG-3’。

2. the method for screening for an aptamer according to claim 1, wherein: the method comprises the steps of taking a nucleic acid sequence as an aptamer, taking 4 sulfa antibiotics of sulfamethoxypyridazine, sulfathiazole, sulfamethazine and sulfapyridine as target molecules for forward screening, and taking 6 other antibiotics of tetracycline, kanamycin sulfate, cefotaxime, enrofloxacin, oxacillin and ampicillin as anti-target molecules for anti-screening, wherein the 1 st to 3 rd rounds of forward screening and the 4 th to 11 th rounds of anti-screening are performed.

3. Use of the aptamer according to claim 1 for establishing a method for simultaneous detection of sulfonamide antibiotics in seafood.

4. Use according to claim 3, characterized in that: the sulfonamide antibiotics are one or more of sulfaquinoxaline, sulfadimethoxine, sulfanilamide-5-methoxypyrimidine, sulfamethoxypyridazine and sulfamethazine.