CN108753789B - Screening method of aptamer and aptamer specifically binding to pseudomonas aeruginosa - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a screening method of aptamer and the aptamer specifically binding to pseudomonas aeruginosa. The aptamer provided by the invention has good specificity and sensitivity, and the biosensor prepared from the aptamer can realize accurate detection of pseudomonas aeruginosa with the detection range from 10¹CFU/ml to 10⁷CFU/ml, with detection limits as low as 9CFU/ml, showed good detection capability in detecting contamination by Pseudomonas aeruginosa in beverages.

Description

Screening method of aptamer and aptamer specifically binding to pseudomonas aeruginosa

Technical Field

The invention relates to the field of biotechnology, in particular to a screening method of aptamer and the aptamer specifically binding to pseudomonas aeruginosa.

Background

Aptamer (Aptamer) is a single strand or sequence that can be screened from a large-capacity nucleic acid library by using an Exponential Enrichment of Ligands by Systematic Evolution technology (Systematic Evolution of Ligands by amplification) and can be bound with a target molecule with high specificity and high affinity. However, Sanger sequencing used in the SELEX routine does not detect most of the sequences due to its low quantitativity, and the screened aptamers are not truly representative. In contrast, modern High-throughput Sequencing (HTS) technology can provide a large amount of data in gigabytes, thereby ensuring sufficient sequence coverage, and thus being capable of accurately identifying the most abundant sequences in PCR products in any round of SELEX, and opening up a new way for aptamer screening. However, little report has been made on the application of HTS in the study of a microorganism-specific aptamer based on the Whole bacterium SELEX (white-bacteria SELEX, WB-SELEX).

Carbon Dots (CDs) are fluorescent nano materials and have the advantages of high biocompatibility, low toxicity, high water solubility, wide excitation spectrum, moderate quantum yield, photobleaching resistance, easiness in large-scale synthesis, low cost and the like. Carbon dots are a new class of high performance fluorescent agents and can give aptamer sensors higher sensitivity. Graphene Oxide (GO) has excellent fluorescence quenching capability for a plurality of fluorescent groups due to in-chip energy or electron transfer, and can solve the problem that a fluorescence-labeled biosensor has too strong background signal.

Pseudomonas aeruginosa is a gram-negative bacterium, a ubiquitous opportunistic pathogen commonly present in moist environments, and is the main pathogenic bacterium of post-operative wound infection, bedsores, abscesses, suppurative otitis media, particularly infection after burns, which can cause blooding and disseminated, and bacteremia and septicemia. The World Health Organization (WHO) defines it as a safety index for drinking water. The european community and the food code committee emphasized that pseudomonas aeruginosa could not be detected in drinking water. However, the development of aptamer sensor-based pseudomonas aeruginosa field detection technology has not attracted much research attention, resulting in a great challenge in this field. Therefore, the monitoring of the pseudomonas aeruginosa in the water area and the food is of great significance to the food safety and the environmental safety, and the clinical rapid diagnosis of the pseudomonas aeruginosa is of great significance to the diagnosis and treatment of patients.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a screening method for aptamers and aptamers specifically binding to pseudomonas aeruginosa, wherein the screening method provided by the present invention can rapidly and accurately screen out aptamers with high specificity.

The screening method of the aptamer specifically binding to the pseudomonas aeruginosa comprises the following steps:

constructing a random library, and preparing a secondary library by using a primer pair of a nucleotide sequence shown in SEQ ID NO. 3-4; taking escherichia coli, bacillus subtilis and staphylococcus aureus as reverse screening strains, taking pseudomonas aeruginosa as forward screening strains, and alternately performing forward screening and reverse screening for 15 times in total;

sequencing the ssDNA library obtained by screening, selecting the one with higher abundance and lower free energy and Kd value, targeting pseudomonas putida, enterococcus faecalis and clostridium perfringens, excluding the aptamer with lower specificity, and screening to obtain the aptamer specifically binding to pseudomonas aeruginosa;

the random library consists of a5 'end fixed region, a random region and a 3' end fixed region which are connected in sequence; the nucleotide sequence of the 5 'end fixed region is shown as SEQ ID NO.1, and the nucleotide sequence of the 3' end fixed region is shown as SEQ ID NO. 2.

The length of the random region is 40 bp.

Each base in the random region is independently selected from A, T, C, G.

In the invention, the screening is performed in the 1 st round of reverse screening and in the 2 nd to 8 th rounds of forward screening; the 9 th round is reverse screening; screening in 10 th to 12 th rounds; the 13 th to 14 th rounds are reverse screening; round 15 is positive screening.

The screening conditions for each round are shown in table 1:

TABLE 1 screening conditions for each round

In the invention, the ssDNA library obtained by screening is the ssDNA library obtained by screening in 9 th, 12 th and 15 th rounds.

In the present invention, the Escherichia coli used is ATCC25922, Bacillus subtilis is ATCC6051 and Staphylococcus aureus is ATCC 12600.

The sequencing adopts a high-throughput sequencing technology, and comprehensively analyzes the library obtained by screening to obtain a more reliable and representative aptamer.

In particular, sequencing using the Illumina Hiseq 3000 platform yielded data volumes of 1 Gb.

The invention provides an aptamer specifically binding to pseudomonas aeruginosa, and the nucleotide sequence of the aptamer is shown as SEQ ID No. 5.

The aptamer provided by the invention has good affinity, can specifically identify pseudomonas aeruginosa, can be used for various applications such as identification of clinical isolated strains, monitoring of drinking water safety, monitoring of beverage safety and the like, and has high sensitivity, and the detection threshold can reach 9 CFU/mL.

The invention also provides a biosensor, which is prepared from the aptamer, the carbon dot and graphene oxide, wherein the aptamer is provided with a 5-end modified amino group.

According to the biosensor provided by the invention, the carbon quantum dots are firstly used for marking the nucleic acid aptamer with the 5' end for amino modification, and then the graphene oxide is added to quench the fluorescence of the carbon quantum dots. The sensor is incubated with a sample to be detected, the aptamer specifically recognizes the target thallus during incubation, and the carbon quantum dots marked on the aptamer recover fluorescence.

The preparation method of the biosensor comprises the following steps:

EDC and NHS are used for activating carbon point carboxyl, and then mixed and incubated with the aptamer provided by the invention with 5-end modified amino to prepare aptamer-CDs;

and mixing the aptamer-CDs with graphene oxide, and incubating to obtain the biosensor.

The activation is carried out by mixing the charcoal dot solution with PBS buffer solution, then adding EDC and NHS solution, and carrying out ultrasonic treatment for 30min at 25 ℃.

The concentration of the carbon dot solution is 8 mug/mL; the concentration of the PBS buffer solution is 5 mM; the EDC and NHS solution has EDC concentration of 1mg/μ L and NHS concentration of 1mg/μ L.

The volume ratio of EDC and NHS solution to charcoal dot solution was 30 μ L: 100 μ L.

The invention also provides a pseudomonas aeruginosa detection kit, which comprises the biosensor or the biosensor prepared by the preparation method.

The invention also provides a detection method of pseudomonas aeruginosa, a sample to be detected is mixed with the biosensor, the mixture is incubated for 0.5 to 2 hours at the temperature of between 35 and 40 ℃, a fluorescence spectrum is recorded within the range of 380 to 600nm of emission wavelength under the excitation wavelength of 350nm, and whether the pseudomonas aeruginosa exists is judged according to the relative fluorescence intensity;

(F) relative fluorescence intensity_450nm-F0_450nm)/F0_450nm；

Wherein, F_450nmIndicating the fluorescence signal output of the sample to be tested, F0_450nmFluorescence signal output values for the blank.

In the invention, if the relative fluorescence intensity is judged to be greater than or equal to zero, the sample contains pseudomonas aeruginosa.

In the invention, the volume ratio of the sample to be detected to the biosensor is 100: 75.

In the invention, the sample to be detected is mineral water, milk or cola.

The invention provides a method for screening pseudomonas aeruginosa aptamer and screened aptamer, wherein the aptamer has good specificity and sensitivity, and a biosensor prepared from the aptamer can realize the purpose of matchingThe pseudomonas aeruginosa is accurately detected, and the detection range is from 10¹CFU/ml to 10⁷CFU/ml, with detection limits as low as 9CFU/ml, showed good detection capability in detecting contamination by Pseudomonas aeruginosa in beverages.

Drawings

FIG. 1 shows HTS-assisted aptamer screening and identification (FIG. 1-A) and detection of Pseudomonas aeruginosa using the aptamer-CDs/GO system (FIG. 1-B);

FIG. 2 shows A) HRTEM characterization of ceCDs; B) uv-vis spectrum of ceCD (curve a, black), PL excitation (curve b, blue) and PL emission spectrum (curve C, red) C) agarose gel electrophoresis of ssDNA aptamer candidates and ssDNA-ceCD; lanes 1-5: ssDNA aptamer candidates from Pa-A1 to Pa-A5, respectively; lanes 6-10: Pa-A1-ceCD is Pa-A5-ceCD respectively; m: DL2000DNA mark, 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, from bottom to top; D) a red-shift of the PL emission peak of ssDNA-ceCD;

FIG. 3A) the working principle of the ssDNA-CDs/GO system for Pseudomonas aeruginosa; B) the selectivity of three aptamer candidate ssDNA-CD/GO systems for seven bacteria including Pseudomonas aeruginosa; (P <0.05), (P < 0.01): the difference between the two groups is obvious; C) optimizing the concentration of GO in a reaction system; D) optimizing the hatching time;

FIG. 4 is a linear relationship between fluorescence recovery and Pseudomonas aeruginosa concentration.

Detailed Description

The invention provides a screening method of aptamer and the aptamer specifically binding to pseudomonas aeruginosa, and the method can be realized by appropriately improving process parameters by referring to the content in the text by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 aptamer screening

1. Design of random library sequences:

5 '-TGGACCTTGCGATTGCGATTGACAGC (40nt) GCAGACATGAGTCT-CAGGAC-3' middle random region is 40 nucleotides, two ends are respectively fixed regions of 20 nucleotides, and the total length of the sequence is 80 nucleotides.

The primer sequences used for screening were:

an upstream primer: TGGACCTTGCGATTGACAGC

A downstream primer 1: GTCCTGAGACTCATGTCTGC

A downstream primer 2: 5' -Biotin GTCCTGAGACTCATGTCTGC

The above nucleic acid sequences were all accomplished by Shanghai Biometrics Ltd

2. Screening of pseudomonas aeruginosa aptamers by WB-SELEX technology

The process of screening for a Pseudomonas aeruginosa aptamer is shown in FIG. 1-A.

1) Screening used strains and culturing and collecting the strains:

aptamer screening was performed using pseudomonas aeruginosa ATCC 27853. Coli ATCC25922, bacillus subtilis ATCC6051 and staphylococcus aureus ATCC 12600 were used for counter-selection. Pseudomonas putida ATCC12633, enterococcus faecalis ATCC19433, Clostridium perfringens ATCC13124 and the counter-selecting bacteria described above were used to assess the specificity of the aptamers. All aerobic strains were grown in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L NaCl, pH 7.2-7.4) for 16 h at 37 ℃. Anaerobic bacteria were cultured in thioglycollic acid liquid medium (Qingdao Aikb) and anaerobic bottles (BUGBOX, Ruskinn, Bridgend, UK) at 37 ℃ for 48 h. Cells were harvested by centrifugation and washed twice with binding buffer (50mM Tris-HCl, 100mM NaCl, 5mM KCl, 1mM MgCl 2, pH7.5) before use.

2) The first round of screening process is negative screening, and the specific steps are as follows:

1 synthetic library 1.3nmol dissolved in 130 u l2x binding buffer, 95 ℃ heating for 5min, ice bath 10 min.

2 taking Escherichia coli, bacillus subtilis and staphylococcus aureus liquid for centrifugal washing, and then carrying out heavy suspension by using 2x combined buffer solution so that the concentration of each bacterium is 1x 10⁷CFU/ml。

3 mixing the prepared library and bacterial liquid in 300. mu.l 2x binding buffer, and incubating at 37 ℃ for 30min in a shaking table to allow the library and bacteria to bind well.

4, centrifuging the mixed solution at 12000r/min for 10min after the combination is finished, taking the supernatant as a template to carry out PCR amplification and preparing a secondary library. ssDNA bound to the countersieve bacteria was removed by centrifugation.

5, amplification system: 2.5. mu.l each of the forward (10. mu.M) and reverse (10. mu.M) primers, 0.5. mu.l of high fidelity Q5 enzyme, 2. mu.l of template, 10. mu.l of 5xBuffer, 1. mu.l of dNTP (10mM), sterilized deionized water (ddH)₂0)31.5μl。

And (3) amplification procedure: the total cycle number is 30, the pre-denaturation temperature is 98 ℃, the time duration is 30s, the denaturation temperature is 98 ℃, the time duration is 10s, the annealing temperature is 66 ℃, the time duration is 30s, the extension temperature is 72 ℃, the time duration is 20

And s. After the end of the cycle, the final extension temperature was 72 ℃ for 2 min.

3) Secondary library preparation:

1PCR amplification product using polyacrylamide gel electrophoresis, then using Solebao PAGE gel DNA recovery kit recovery correct strip, dissolved in 100u l magnetic bead buffer.

2 the resuspended beads were shaken well and 50ul were taken in a 1.5ml Ep tube. Magnetic separation for 1-2min, removing supernatant, and removing Ep tube from magnetic separator.

3 adding 500ul buffer1, shaking thoroughly, magnetic separating for 1-2min, and removing supernatant. And repeating the steps once.

4 the PCR amplified gel recovery product was diluted to 500ul with Buffer1 and resuspended with magnetic beads. Shaking at room temperature for 90 min. Magnetic separation for 1-2min, and removing supernatant by suction.

5 the beads were washed 2 times with 500ul buffer 1.

6 100ul of freshly prepared NaOH was added and incubated at 37 ℃ or 95 ℃ for 15 min.

7 magnetic separation is carried out for 1-2min, and the supernatant is taken to obtain 100 ulsssDNA.

About 10ul of 1M HCl solution was added to 8 to adjust the pH to about 7.5.

9 the ssDNA content was determined using a Nanodrop 2000.

4) And (4) screening in other rounds:

from the second round, normal screening was performed, which differs from the reverse screening in that after the library had bound to the target strain, unbound ssDNA was removed by centrifugation, the cells were washed several times with 1 Xbinding buffer, and 100. mu. lddH was added₂O resuspending the thallus, heating at 95 ℃ for 10min, centrifuging at 12000r/min for 10min, taking the supernatant as a template to perform PCR amplification and prepare a secondary library. Screening was performed for a total of 15 rounds, and the screening conditions for each round are shown in table 1.

TABLE 1 screening conditions for each round

a volumes of each counterselection bacterium (E.coli ATCC25922, Bacillus subtilis ATCC6051 and Staphylococcus aureus ATCC 12600) were added to the first round of reaction system. The volume was equal to 50. mu.l, and the cell density of each counterselection culture was 1X 10⁷CFU/ml。

b in the other rounds of counterselection, the reaction system was supplemented with volumes of each counterselection bacterium (E.coli ATCC25922, Bacillus subtilis ATCC6051 and Staphylococcus aureus ATCC 12600). The volume was equal to 100. mu.l, and the cell density of each counterselection culture was 1X 10⁷CFU/ml。

3. Identification of candidate aptamers

HTS performed sequencing analysis of ssDNA libraries in rounds 9,12 and 15. Mu.g of ssDNA enriched from the PCR product from the previous round, produced by Novogene Co.Ltd. (Beijing, China) was sequenced using the Illumina Hiseq 3000 platform to generate data volumes of 1 Gb. The number of occurrences of each sequence was counted to reveal the abundance of each sequence within the ssDNA pool. ssDNA with abundance ranking in the first five positions was selected as aptamer candidates. The free energy of aptamer candidates was analyzed using DNAMAN software and the secondary structure of aptamer candidates was predicted by the Mfold program (http:// Mfold. rna. albany. edu/.

TABLE 2 HTS method for the identification of Pseudomonas aeruginosa aptamer candidate sequences, free energy and equilibrium constants.

CD was synthesized using a hydrothermal method. To prepare aptamer-CDs nanoconjugates, 100. mu.L of CD solution (8. mu.g/mL) was sonicated for 30min at 25 ℃ with 30. mu. L N- (3-dimethylaminopropyl) -ethylcarbodiimide hydrochloride (EDC) (1 mg/. mu.l) and N-hydroxysuccinimide (NHS) (1 mg/. mu.l) to activate the carboxyl groups in 5mM PBS buffer. Thereafter, 10. mu.L of the sonicated mixture was added to 90. mu.L of the amino group-modified candidate aptamer at a concentration of 3.3. mu.M in 5mM PBS buffer, and incubated at 25 ℃ for 2 hours. The synthesized CD and aptamer-CD were characterized using HRTEM and agarose gel electrophoresis, UV-vis scanning and fluorescence spectroscopy scanning. The results are shown in FIG. 2. Kd values for candidate aptamers can be determined using the synthesized aptamer-ceCds. As shown in table 1.

To identify the best aptamers, an aptamer-ceCDs/GO system was established based on synthetic aptamer-ceCD. As described above, the present study employed an "on-off" strategy to detect Pseudomonas aeruginosa with the aptamer-ceCDs/GO system. As shown in FIG. 3-A, the fluorescence of aptamer-ceCD is evident in the absence of Graphene Oxide (GO) (FIG. 3-A, curve a, blue). When they are mixed together, aptamer-ceCDs are adsorbed onto the GO surface by pi-pi stacking interactions, and GO quenches the fluorescence of the aptamer-ceCDs by FRET. By this operation, the background fluorescence signal was reduced to a minimum (FIG. 3-A, curve c, black). After the pseudomonas aeruginosa and the aptamer-CDs/GO system are incubated together, the aptamer-CDs are preferentially combined with the pseudomonas aeruginosa, and the pi-pi accumulation between the aptamer-ceCDs and GO is interrupted. In this way, aptamer-CD is released from GO surface and fluorescence is restored (fig. 3-a, curve b, red). Thus, P.aeruginosa acts like a "switch" that turns on fluorescence. By reading the recovery of fluorescence, the presence of P.aeruginosa can be identified. The method is the working principle of the saptamer-CDs/GO system for detecting the pseudomonas aeruginosa.

Through identification, the combination of Pa-A1 on pseudomonas aeruginosa is not affected by other strains, and has good specificity, while Pa-A2-Pa-A5 on other strains have different degrees of combination and poor specificity.

Example 2 detection of Pseudomonas aeruginosa

1. Firstly, aptamer-CD/GO biosensor is synthesized

CD was synthesized using a hydrothermal method, and then 100. mu.L of a CD solution (8. mu.g/mL) was sonicated for 30min at 25 ℃ in 5mM PBS buffer to activate the carboxyl groups with 30. mu. L N- (3-dimethylaminopropyl) -ethylcarbodiimide hydrochloride (EDC) (1 mg/. mu.l) and N-hydroxysuccinimide (NHS) (1 mg/. mu.l). Thereafter, 10. mu.L of the sonicated mixture was added to 90. mu.L of the amino group-modified candidate aptamer dissolved in 5mM PBS buffer at a concentration of 3.3. mu.M, and incubated at 25 ℃ for 2 hours to prepare aptamer-CD. aptamer-CD/GO biosensors were prepared by adding 50. mu.L of GO (1mg/mL) to 25. mu.L of the aforementioned aptamer-CDs solution (100nM) to quench the fluorescence, and then incubating the mixture at 37 ℃ for 1 hour.

2. Detection of Pseudomonas aeruginosa

100 mu.L of Pseudomonas aeruginosa (1X 10)⁷CFU/ml) was added to the reaction and incubated at 37 ℃ for 1 hour. The reaction without addition of P.aeruginosa cells served as a control. The fluorescence spectrum of the aptamer-CDs/GO system was recorded at an excitation wavelength of 350nm and an emission wavelength range of 380-600 nm. According to the relative fluorescence intensity [ (F)_450nm-F0_450nm)/F0_450nm]Calculating the fluorescence signal output value, wherein F_450nmAnd F0_450nmRepresents the fluorescence intensity of aptamer sensors prepared in the presence and absence of pseudomonas aeruginosa at an excitation wavelength of 350nm, respectively. When (F)_450nm-F0_450nm)/F0_450nmThe presence of Pseudomonas aeruginosa is indicated by the value of 0 or more.

In the scheme, an aptamer-CDs/GO system is used for detecting 7 different bacteria. The bacterial strains tested were pseudomonas aeruginosa, pseudomonas putida, enterococcus faecalis, clostridium perfringens, escherichia coli, bacillus subtilis and staphylococcus aureus. The result shows that the biosensor can accurately identify the pseudomonas aeruginosa, has poor signals for other bacteria, does not generate the change of relative fluorescence intensity, and has the accuracy of 100 percent.

3. Sensitivity detection

To assess the sensitivity of the aptamer-CDs/GO system, the assay was performed as described above for a range of different concentrations of Pseudomonas aeruginosa ranging from 10CFU/ml to 1X 10⁷CFU/ml. Under the optimized condition, the fluorescence recovery rate of the Pa-A1-ceCDs/GO system prepared in the presence of different concentrations of pseudomonas aeruginosa is determined. As shown in figure 4, the concentration range of the aptamer sensor for detecting the pseudomonas aeruginosa is 10-10⁸CFU/ml, showing good responsiveness. The fluorescence recovery rate is linearly related to the concentration of pseudomonas aeruginosa, and the standard curve is (F-F0)/F0 ═ 0.0582log [ C (CFU/ml)]0.057 and a correlation coefficient of 0.993(F and F0 represent the fluorescence intensity of the biosensor in the presence and absence, respectively, of Pseudomonas aeruginosa). According to this equation, a limit of detection (LOD) of 9CFU/ml is obtained

4. Analysis of actual samples

Mineral water (Nestle (China) Co., Ltd.), milk (Yili Co., Ltd., China) and Coca Cola (China) Co., Ltd.) were randomly purchased from supermarkets to perform evaluation of the application of the aptamer-CD/GO system to the actual samples. The aptamer-CD/GO system was prepared as described above. The samples were artificially contaminated with different concentrations of Pseudomonas aeruginosa, ranging from 10¹～10³CFU/ml, out of visual identification range. Contaminated samples were also processed using the same procedure for the aptamer-CD/GO system. Sterilized distilled water was used as a negative control.

From the results in table 3, it can be seen that the concentration of bacteria detected using the aptamer sensor system is close to the cell concentration obtained by microscopic counting in mineral water and milk, indicating that neutral and high protein conditions have no significant adverse effects on the operation of the aptamer sensor system. However, the detection effect of coca-cola is greatly limited. This is probably due to the acidic pH environment of Coca Cola, which affects the normal function of the normal folding of the Aptamer

TABLE 3 detection results of Pseudomonas aeruginosa in the actual samples

RSD:Relative standard deviation(Lian et al.,2015).

The result shows that the method provided by the invention can realize accurate detection of the pseudomonas aeruginosa in the beverage.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

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Claims (10)

1. A method for screening for an aptamer that specifically binds to pseudomonas aeruginosa, comprising:

constructing a random library, and preparing a secondary library by using a primer pair of a nucleotide sequence shown in SEQ ID NO. 3-4; taking escherichia coli, bacillus subtilis and staphylococcus aureus as reverse screening strains, taking pseudomonas aeruginosa as forward screening strains, and alternately performing forward screening and reverse screening for 15 times in total;

sequencing the ssDNA library obtained by screening, selecting the one with higher abundance and lower free energy and Kd value, targeting pseudomonas putida, enterococcus faecalis and clostridium perfringens, excluding the aptamer with lower specificity, and screening to obtain the aptamer specifically binding to pseudomonas aeruginosa;

the random library consists of a5 'end fixed region, a random region and a 3' end fixed region which are connected in sequence; the nucleotide sequence of the 5 'end fixed region is shown as SEQ ID NO.1, and the nucleotide sequence of the 3' end fixed region is shown as SEQ ID NO. 2.

2. The screening method according to claim 1, wherein the screening is performed in round 1 for reverse screening and in rounds 2 to 8 for forward screening; the 9 th round is reverse screening; screening in 10 th to 12 th rounds; the 13 th to 14 th rounds are reverse screening; round 15 is positive screening.

3. The screening method of claim 1 or 2, wherein the ssDNA library obtained by screening is the ssDNA library obtained by 9 th, 12 th or 15 th round screening.

4. An aptamer specifically binding to pseudomonas aeruginosa, and the nucleotide sequence of the aptamer is shown as SEQ ID No. 5.

5. A biosensor, which is prepared from the aptamer according to claim 4 with a 5-terminal modified amino group, a carbon dot and graphene oxide.

6. A method for producing the biosensor in accordance with claim 5, comprising:

activating carbon point carboxyl with EDC and NHS, and mixing and incubating with an aptamer of claim 4 with a modified amino at the 5 terminal to obtain aptamer-CDs;

and mixing the aptamer-CDs with graphene oxide, and incubating to obtain the biosensor.

7. A Pseudomonas aeruginosa detection kit, comprising the biosensor according to claim 5 or the biosensor obtained by the preparation method according to claim 6.

8. The detection method of pseudomonas aeruginosa for non-treatment or diagnosis purposes is characterized in that a sample to be detected is mixed with the biosensor of claim 5 or the biosensor prepared by the preparation method of claim 6, the mixture is incubated at 35-40 ℃ for 0.5-2 h, the fluorescence spectrum is recorded within the range of 380-600nm of emission wavelength under 350nm of excitation wavelength, and whether pseudomonas aeruginosa exists is judged according to the relative fluorescence intensity;

the relative fluorescence intensity = (F)_450nm-F0_450nm）/ F0_450nm

Wherein, F_450nmIndicating the fluorescence signal output of the sample to be tested, F0_450nmFluorescence signal output values for the blank.

9. The detection method according to claim 8, wherein if the relative fluorescence intensity is determined to be zero or more, the sample contains Pseudomonas aeruginosa.

10. The detection method according to claim 8, wherein the volume ratio of the sample to be detected to the biosensor is 100: 75.

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