Fluorescent biological probe for rapidly detecting pseudomonas aeruginosa and preparation method of fluorescent biological probe

Abstract

The invention discloses a fluorescent biological probe for rapidly detecting pseudomonas aeruginosa and a preparation method of the fluorescent biological probe, and belongs to the field of biological detection. A fluorescent quenching agent is used as an indicator, an aptamer of pseudomonas aeruginosa is used as a biological recognition element, and the fluorescent visual biological probe is constructed based on CRISPR / Cas12a and crRNA and is used for rapid detection of food-borne pathogenic bacteria pseudomonas aeruginosa. The method is also suitable for detection of actual water samples and has good stability. The detection method has the remarkable advantages of high efficiency, specificity, convenience, sensitivity and the like, the minimum detection concentration is 10 < 4 > CFU / mL, and the detection method is of great significance in improving the food safety quality, standardizing the food production environment and improving the market supervision efficiency.

Description

Technical Field

[0001] The present invention relates to a fluorescent biological probe for rapid detection of Pseudomonas aeruginosa and a preparation method thereof, belonging to the field of biological detection. Background Art

[0002] Pseudomonas aeruginosa (PA), as a foodborne pathogen, widely exists in daily life and poses a great threat to the ecological environment and public health.

[0003] In terms of food safety, in recent research on the quality analysis of bottled drinking water, it has been found that Pseudomonas aeruginosa, as a common environmental microorganism, shows a specific distribution pattern in the detection of beverage products. Data shows that the relevant parameters of this genus of bacteria account for a significant proportion in the quality observation indicators of bottled drinking water. Its detection frequency reaches 72.92% of the total value of the same category of bacterial flora monitoring, and in the microbial index system of beverages, the detection items of bottled drinking water related to this genus of bacteria account for 35.75%, which verifies the general existence characteristics of this microorganism in the water environment. Moreover, the unqualified rate of Pseudomonas aeruginosa detection shows a trend of being more in summer, followed by autumn, and less in spring and winter. Therefore, Pseudomonas aeruginosa is the main reason for the unqualified microorganisms in bottled drinking water, and a rapid detection method for Pseudomonas aeruginosa needs to be established urgently.

[0004] In recent years, rapid food safety detection technologies have developed vigorously, mainly including nucleic acid amplification technologies, immunoassay technologies, biosensing technologies, etc. ① Nucleic acid amplification technologies, such as Polymerase Chain Reaction (PCR), Loop-Mediated Isothermal Amplification (LAMP), etc., amplify target gene fragments through specific primers, with advantages such as high sensitivity and strong specificity, but are easily affected by interfering substances in the food matrix. ② Immunoassay technologies, such as Enzyme-Linked Immunosorbent Assay (ELISA), colloidal gold immunochromatography, etc., utilize the specific binding reaction of antigen-antibody to achieve rapid detection of the target substance, with simple operation and easy realization of on-site rapid detection, but relatively low sensitivity and specificity. ③ Biosensing technologies, such as Surface Plasmon Resonance (SPR), Quartz Crystal Microbalance (QCM), etc., fix biomolecules on the sensor surface and achieve qualitative and quantitative analysis according to the signal changes caused by the binding of biomolecules to the target substance, with high sensitivity and fast detection speed, but relatively expensive instrument equipment. ④ Rapid detection methods based on emerging technologies such as microfluidic chips and nanomaterials have also gradually attracted attention. For example, a microfluidic chip integrating functions such as PCR and capillary electrophoresis can achieve full-process automation of sample preparation, amplification, separation, and detection, greatly shortening the detection time; while nanomaterials such as carbon nanotubes and gold nanoparticles have characteristics such as large specific surface area and good conductivity, and can be used to construct highly sensitive electrochemical biosensors. Generally speaking, various rapid detection technologies have their own advantages and disadvantages in terms of sensitivity, specificity, speed, cost, etc., but there are certain defects in terms of rapidity.

[0005] The rapid detection and identification of Pseudomonas aeruginosa are crucial for human health, biodefense, and food safety. Therefore, it is necessary to establish a rapid, portable, and reliable method to achieve the rapid detection of Pseudomonas aeruginosa. Summary of the Invention

[0006] Aiming at the deficiencies of the above-mentioned existing technologies, the present invention provides a fluorescent bioprobe for rapid detection of Pseudomonas aeruginosa and its preparation method, aiming to solve the technical problem that there is currently a lack of a rapid, portable, and reliable detection method for Pseudomonas aeruginosa.

[0007] The first technical solution provided by the present invention is a fluorescent biosensor for rapid detection of Pseudomonas aeruginosa. The biosensor includes an aptamer-dsDNA complex, streptavidin-labeled magnetic beads (MBs), and biotinylated ssDNA (bio-ssDNA); the aptamer-dsDNA complex is composed of an oligonucleotide Pseudomonas aeruginosa aptamer, dsDNA-F, and dsDNA-R.

[0008] In some embodiments, the nucleotide sequences of the oligonucleotide Pseudomonas aeruginosa aptamer, dsDNA-F, and dsDNA-R are shown in SEQ ID NO.1-3; the nucleotide sequence of the ssDNA is shown in SEQ ID NO.4.

[0009] The present invention also provides a preparation method of a biosensor for rapid detection of Pseudomonas aeruginosa. The preparation method is that the oligonucleotide Pseudomonas aeruginosa aptamer, dsDNA-F, and dsDNA-R are mixed to form an aptamer-dsDNA complex, the streptavidin-labeled magnetic beads and the biotinylated ssDNA are mixed to form MBs-streptavidin-bio-ssDNA, and the aptamer-dsDNA complex and MBs-streptavidin-bio-ssDNA are mixed to form an MBs-streptavidin-bio-ssDNA-aptamer-dsDNA complex, that is, the biosensor.

[0010] In some embodiments, the preparation method includes the following steps:

[0011] (1) Prepare the aptamer-dsDNA complex by mixing the oligonucleotide Pseudomonas aeruginosa aptamer, dsDNA-F, and dsDNA-R in PBS buffer; then incubate the DNA solution at 95 °C for 5 min and gradually cool it (2 °C / min) to room temperature;

[0012] (2) Add 20 μL of 15 μM bio-ssDNA to the MBs solution and mix, and store it at room temperature for 30 min; wash the MBs twice with PBST buffer to remove the excess bio-ssDNA to obtain MBs-streptavidin-bio-ssDNA;

[0013] (3) Add the aptamer-dsDNA from step (1) (5 μmol / L, 20 μL) to the MBs-streptavidin-bio-ssDNA from step (2) and mix well at room temperature for 20 min; use magnetic separation to wash the prepared MBs-streptavidin-bio-ssDNA-aptamer-dsDNA complex three times with PBST to remove the excess nucleic acid aptamer-dsDNA in the system, and then resuspend the MBs-streptavidin-bio-ssDNA-aptamer-dsDNA complex with 500 μL of PBST. After resuspension, store it in the refrigerator for later use. When aliquoting for use, pipette 20 μL into a centrifuge tube, and ensure that each 20 μL aliquot contains MBs during the above operations, that is, it needs to be mixed well before use.

[0014] In some embodiments, the oligonucleotide Pseudomonas aeruginosa aptamer, dsDNA-F, and dsDNA-R in step (1) are mixed and synthesized into an aptamer-dsDNA complex at a final ratio of 1:1:1.

[0015] In some embodiments, in step (1), the final concentration of the aptamer-dsDNA is 5 μmol / L.

[0016] In some embodiments, in step (2), 20 μL of 10 mg / mL MBs is washed with PBST buffer to remove the residual NaN3 protection solution.

[0017] In some embodiments, 20 μL of streptavidin magnetic beads are selected for the experiment.

[0018] The third technical solution provided by the present invention is a detection system for Pseudomonas aeruginosa, and the detection system includes Cas12a, crRNA, 10×Cas12a Buffer, FQ-ssDNA-BHQ, and the biological probe described in the first technical solution.

[0019] In some embodiments, the nucleotide sequence of crRNA is as shown in SEQ ID NO.5, and the sequence of FQ-ssDNA-BHQ is 5’6-FAM-GATTAGCGTACGCACGTTAC-3’BHQ-1.

[0020] In some embodiments, the final concentrations of Cas12a, crRNA, 10×Cas12a Buffer, FQ-ssDNA-BHQ, and the biological probe described in the first technical solution are 0.1 - 0.2 μM, 0.1 - 0.2 μM, 0.1 - 0.5 μM, 0.1 - 0.2 μM, and 10 - 50 nM, respectively.

[0021] The fourth technical solution provided by the present invention is a detection method for Pseudomonas aeruginosa. The method is to mix Cas12a, crRNA, 10×Cas12a Buffer, FQ-ssDNA-BHQ, and the biological probe described in the first technical solution, add the test sample, form a Cas12a / crRNA detection system, and perform detection.

[0022] In some embodiments, the concentration ratio of Cas12a to crRNA is 1:2 to 2:1.

[0023] In some embodiments, the ratio of Cas12a to crRNA selected is 1:1 as the concentration ratio of Cas12a to crRNA in the final reaction.

[0024] In some embodiments, 0.2 μM is selected as the optimal concentration of Cas12a and crRNA in the CRISPR / Cas12a detection system.

[0025] In some embodiments, 1 μmoL / L is selected as the optimal reaction concentration of FQ-ssDNA-BHQ for the biosensing platform of Pseudomonas aeruginosa based on CRISPR / Cas12a and aptamer.

[0026] In some embodiments, the method adds Cas12a (1 μmol / L), crRNA (1 μmol / L), 10×Cas12a Buffer, FQ-ssDNA-BHQ (5 μmol / L) in a volume ratio of 1:1:1:1, mixes them, adds the biological probe described in the first technical solution, mixes and dilutes 4 times, aspirates 8 μL into a new centrifuge tube, and then adds ultrapure water (10 μL) and the sample (2 μL) to each new centrifuge tube to prepare a 20 μL Cas12a / crRNA detection system for detection.

[0027] In some embodiments, the reaction temperature is 35 - 41 °C and the reaction time is 5 - 30 min.

[0028] In some embodiments, 41 °C is selected as the optimal reaction temperature.

[0029] In some embodiments, 30 min is selected as the optimal time for the reaction of the CRISPR / Cas12a system.

[0030] The fifth technical solution provided by the present invention is a kit, and the kit contains the biological probe described in the first technical solution, or contains the detection system described in the third technical solution.

[0031] The technical effects of the present invention are as follows:

[0032] The present invention uses a fluorescence quencher as an indicator and an aptamer of Pseudomonas aeruginosa as a biorecognition element, and constructs a fluorescence visualization bioprobe based on CRISPR / Cas12a and crRNA for the rapid detection of the foodborne pathogen Pseudomonas aeruginosa. This method is also applicable to the detection in actual water samples and has good stability. This detection method has significant advantages such as high efficiency, specificity, convenience, and sensitivity, and is of great significance for improving food safety quality, standardizing the food production environment, and enhancing the market supervision efficiency. The minimum detection concentration of this system is 10 4 CFU / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a synthetic route diagram of the fluorescence bioprobe for the rapid detection of Pseudomonas aeruginosa in the present invention.

[0034] Figure 2 It is the test result of optimizing the reaction time between magnetic beads and Pseudomonas aeruginosa.

[0035] Figure 3 It is the test result of optimizing the dosage of magnetic beads.

[0036] Figure 4 It is the verification of the cleavage activity of Cas12a / crRNA

[0037] Figure 5 It is the test result of optimizing the ratio of Cas12a to crRNA.

[0038] Figure 6 It is the test result of optimizing the concentrations of Cas12a and crRNA.

[0039] Figure 7 It is the test result of optimizing the concentration of FQ-ssDNA-BHQ.

[0040] Figure 8 It is the test result of optimizing the reaction temperature.

[0041] Figure 9 It is the test result of optimizing the reaction time.

[0042] Figure 10 It is the test result of sensitivity.

[0043] Figure 11 It is the test result of actual samples. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0045] Raw materials used in the embodiments:

[0046] The streptavidin magnetic beads (BeaverBeads TM Streptavidin) were purchased from Suzhou Beaver Biomedical Engineering Co., Ltd. The gene editing protein (Bio--ifesci CRISPR-Cas12a) was purchased from Guangzhou Bolaisi Biotechnology Co., Ltd. The aptamers and primers were both purchased from Sangon Biotech (Shanghai). The PBS buffer was from Shanghai Beyotime Biotechnology Co., Ltd., and the DEPC water (DNase, RNase free) was from Shanghai Beyotime Biotechnology Co., Ltd.

[0047] Sequences involved in the following examples:

[0048] Table 1 Nucleic acid sequences

[0049]

[0050]

[0051] Example 1: Synthesis of a fluorescent bioprobe for rapid detection of Pseudomonas aeruginosa

[0052] I. Overall synthesis route

[0053] As a fluorescent bioprobe for rapid detection of Pseudomonas aeruginosa in the embodiments of the present invention, the principle of the fluorescent bioprobe is as described below:

[0054] It consists of Cas12a protein, crRNA, single-stranded DNA modified with fluorescent groups (Carboxyfluorescein, FAM) and black hole quenching groups (Black Hole BHQ-1) at both ends (abbreviated as FQ-ssDNA-BHQ), and a bioprobe containing aptamers to form a CRISPR / Cas12a Pseudomonas aeruginosa biosensor. When the target exists, it can interact with crRNA, activate the trans-cleavage activity of Cas12a to cause the cleavage of FQ, thereby producing a fluorescence effect.

[0055] The synthesis route of the bioprobe is as Figure 1 , and it is necessary to first synthesize aptamer-dsDNA, and then hybridize it with streptavidin-labeled MBs and biotin-ssDNA to generate the bioprobe.

[0056] II. Preparation of aptamer-dsDNA

[0057] An aptamer-dsDNA complex was prepared by mixing Pseudomonas aeruginosa aptamer (SEQ ID NO.1), dsDNA-F (SEQ ID NO.2) and dsDNA-R (SEQ ID NO.3) in PBS buffer. Then the DNA solution was incubated at 95 °C for 5 min and gradually cooled (2 °C / min) to room temperature. The final concentration of the aptamer-dsDNA was 5 μmol / L.

[0058] III. Construction of Biosensors

[0059] 1. Probe Construction Process

[0060] The biosensor consists of three parts: streptavidin-labeled MBs, biotin-ssDNA, and aptamer-dsDNA. First, 20 μL of 10 mg / mL MBs was washed with PBST buffer to remove the residual NaN3 protection solution. Then, 20 μL of 15 μM bio-ssDNA was added to the MBs solution and mixed, and stored at room temperature for 30 min. The MBs were washed twice with PBST buffer to remove the excess bio-ssDNA. The aptamer-dsDNA (5 μmol / L, 20 μL) was added to the MBs-streptavidin-bio-ssDNA and mixed well at room temperature for 20 min. The prepared MBs-streptavidin-bio-ssDNA-aptamer-dsDNA complex was washed three times with PBST using magnetic separation. After removing the excess nucleic acid aptamer-dsDNA in the system, the MBs-streptavidin-bio-ssDNA-aptamer-dsDNA complex was resuspended with 500 μL of PBST and stored refrigerated for later use, which is the biosensor. When aliquoted for use, 20 μL was pipetted into a centrifuge tube, and it was ensured that each 20 μL aliquot contained MBs, that is, it needed to be mixed well before use.

[0061] 2. Optimization of the Reaction Time between Magnetic Beads and Target

[0062] Optimize the detection of the reaction time between streptavidin magnetic beads and Pseudomonas aeruginosa. Figure 2 As shown, with different mixing and incubation times of the biosensor and the target, the results of fluorescence intensity detection are also different. As the reaction time increases, the fluorescence intensity of the sample group gradually rises and reaches the maximum at 20 min. In the subsequent 25 min group, the fluorescence intensity does not increase significantly. Therefore, the incubation time of 20 min for the biosensor and the target was selected as the final reaction time.

[0063] 3. Optimization of Magnetic Bead Dosage

[0064] Optimize the inspection of the dosage of streptavidin magnetic beads. Figure 3As shown, selecting different amounts of magnetic beads did not significantly improve the fluorescence performance or show a trend. That is, when 20 μL of streptavidin magnetic beads were selected, the magnetic beads had reached an excessive value. Therefore, 20 μL of streptavidin magnetic beads were selected for the experiment to achieve the purpose of saving consumables.

[0065] Example 2: Construction of a Pseudomonas aeruginosa detection system

[0066] I. Verification of the signal transduction process

[0067] Based on the sensitivity test of the fluorescent biological probe for detecting Pseudomonas aeruginosa in Example 1, the following steps are included:

[0068] To verify the cleavage activity of Cas12a / crRNA, the necessity of the components of the Cas12a, crRNA, 10×Cas12a Reaction buffer, and FQ-ssDNA-BHQ system was verified. As shown in Table 2, No. 1 is the full-component system, and the systems of Nos. 2-5 lack different components. After mixing, the reaction was carried out at 39 °C for 20 min. After taking out, it was compared in the dark room and then pipetted and added to a transparent 384-well plate, and the fluorescence value was measured using a microplate reader (λEx: 485 nm, λEm: 528 nm).

[0069] The detection results are as Figure 4 shown. The trans-cleavage activity of Cas12a / crRNA can only be activated when the CRISPR / Cas12a system contains Cas12a, crRNA, 10×Cas12a Reaction buffer, and the biological probe of Example 1 at the same time, so as to cleave the fluorescent probe (FQ-ssDNA-BHQ) and release fluorescence. The experimental results show that the detection system is feasible.

[0070] Table 2 Verification of the cleavage activity of Cas12a / crRNA

[0071]

[0072] II. Optimization of the detection system

[0073] 1. Optimization of the ratio of Cas12a to crRNA

[0074] In the CRISPR / Cas12a system, Cas12a has nuclease activity, while crRNA plays a role in recognizing the target. Therefore, the ratio and concentration of Cas12a and crRNA are very important for the performance of the CRISPR / Cas12a detection system. According to the optimal dosage and optimal reaction time of streptavidin mentioned above, the optimal ratio of the reaction between Cas12a and crRNA was further optimized. To determine the optimal ratio of the reaction between Cas12a and crRNA, multiple groups of experiments were designed and carried out with different ratios of Cas12a to crRNA (Table 3). The total reaction system was 20 μL in total, and three parallels and one negative control were designed for each group. The samples were incubated at 39 °C for 20 min. After taking them out, they were observed and compared in the dark room and then transferred to a transparent 384-well plate, and the fluorescence value was measured using a microplate reader (λEx: 485 nm, λEm: 528 nm).

[0075] Table 3 Ratio of Cas12a to crRNA

[0076]

[0077] As Figure 5 shown, the results detected by the microplate reader indicated that when Cas12a and crRNA were both 2 μL respectively, that is, when the ratio of Cas12a to crRNA was 1:1, there were good negative and positive performances, and the fluorescence intensity ratio between the negative group and the positive group was the largest. Therefore, the ratio of Cas12a to crRNA of 1:1 was selected as the ratio of Cas12a to crRNA for the final reaction.

[0078] 2. Optimization of the concentration of Cas12a and crRNA

[0079] Based on the experimental results of the above-mentioned optimization of the ratio of Cas12a to crRNA, the concentration of Cas12a and crRNA was further optimized. Using the optimal ratio result of the above experiment, that is, when Cas12a:crRNA was 1:1, different dilution multiples were designed, and 1 μM Cas12a and 1 μM crRNA were diluted 1, 2, 3, 4, and 5 times with ultrapure water for experiments (Table 4). The total reaction system was 20 μL in total, and three parallels and one negative control were designed for each group. After taking them out, they were observed and compared in the dark room and then transferred to a transparent 384-well plate, and the fluorescence value was measured using a microplate reader (λEx: 485 nm, λEm: 528 nm).

[0080] Table 4 Concentration of Cas12a and crRNA

[0081]

[0082]

[0083] AsFigure 6 As shown, when diluted 4-fold, that is, when the concentrations of both Cas12a and crRNA are 0.2 μM, the positive fluorescence value is relatively high and the negative fluorescence value is relatively low. Therefore, 0.2 μM is selected as the optimal concentration of Cas12a and crRNA in this CRISPR / Cas12a detection system.

[0084] 3. Optimization of the concentration of the fluorescent probe

[0085] Too low a concentration of FQ-ssDNA-BHQ will affect the detection sensitivity, while too high a concentration of FQ-ssDNA-BHQ will inhibit the cleavage of FQ-ssDNA-BHQ by Cas12a protein. After determining the optimal reaction ratio and concentration of Cas12a and crRNA, the concentration of FQ-ssDNA-BHQ in the system was optimized. According to the above experimental results on the ratio optimization and concentration optimization of Cas12a and crRNA, the concentration of FQ-ssDNA-BHQ was further optimized. Using the optimal ratio result of the above experiment, that is, when Cas12a:crRNA is 1:1 and the concentrations of Cas12a and crRNA are 0.2 μM, different dilution multiples were designed. 5 μM was diluted 1, 2, 3, 4, and 5 times with ultrapure water for experiments (Table 5). The total reaction system was 20 μL, and three parallels and one negative control were designed for each group. After taking them out, observe and compare in the dark room and pipette and add them to a transparent 384-well plate, and use a microplate reader to measure the fluorescence value (λEx: 485 nm, λEm: 528 nm).

[0086] Table 5 Concentration of FQ-ssDNA-BHQ

[0087]

[0088] As Figure 7 shown, the fluorescence detection results of the concentration optimization of FQ-ssDNA-BHQ indicate that when the concentration of FQ-ssDNA-BHQ is too high, the fluorescence detection intensity of the negative control group is also relatively high. When the addition amount of FQ-ssDNA-BHQ is 1 μL, the fluorescence intensity ratio between the negative control and positive control groups is the largest and the difference is the most obvious. Therefore, 1 μL is selected as the optimal reaction addition amount of FQ-ssDNA-BHQ for this biosensing platform of Pseudomonas aeruginosa based on CRISPR / Cas12a and aptamer. The optimal concentration of FQ-ssDNA-BHQ is 0.1 μM.

[0089] 4. Optimization of the reaction temperature

[0090] After the above optimizations of the CRISPR / Cas12a system, the reaction temperature of CRISPR / Cas12a was optimized. Four temperatures, namely 35, 37, 39, and 41 °C, were set. Three negative controls and three positive controls were set for each temperature group. The total reaction system was 20 μL, and the reaction was carried out for 20 min. After the reaction was completed, it was taken out, observed and compared in the dark room, and then aspirated and added to a transparent 384-well plate. The fluorescence value was measured using a microplate reader (λEx: 485 nm, λEm: 528 nm).

[0091] As Figure 8 shown, at 41 °C, the fluorescence intensity of the positive control group was the highest, suggesting that the Cas12a enzyme activity was the highest at this time, and the optimal reaction temperature was 41 °C.

[0092] 5. Optimization of reaction time

[0093] After determining the ratio of Cas12a to crRNA, the concentrations of Cas12a to crRNA, and the concentration of FQ-ssDNA-BHQ in the CRISPR / Cas12a system, as well as the optimal reaction temperature of the CRISPR / Cas12a system, an optimization experiment of the reaction time of the CRISPR / Cas12a system was carried out. The reagents were heated at 41 °C for 5, 10, 15, and 30 min respectively. Three negative controls and positive controls were set for each time group. After heating was completed, it was taken out, observed and compared in the dark room, and then aspirated and added to a transparent 384-well plate. The fluorescence value was measured using a microplate reader (λEx: 485 nm, λEm: 528 nm).

[0094] As Figure 9 shown, as the reaction time increased, the fluorescence value of the positive control group continuously increased. In the time period of 15 to 30 min, the growth rate gradually slowed down. Therefore, 30 min was selected as the optimal reaction time of the CRISPR / Cas12a system.

[0095] Finally, the following optimal detection system was obtained: the molar ratio of Cas12a to crRNA was equimolar, the concentration was 0.2 μmoL / L, the concentration of FQ-ssDNA-BHQ was 1 μmoL / L, and the reaction time and temperature were 30 min and 41 °C respectively.

[0096] Example 3: Sensitivity test

[0097] To evaluate the sensitivity of the method of the present invention, 1 mL of Pseudomonas aeruginosa cultured overnight was centrifuged, washed, and resuspended in 100 μL of bacterial solution, and serially diluted to 10 -9Multiply, respectively take 5 μL and add it into 50 μL of the biological probe in Example 1. Refer to the detection method in Example 2, and heat and react the best CRISPR / Cas12a detection system (Cas12a, crRNA, 10×Cas12a Reaction buffer) added to Example 2. Set 3 negative controls and 3 sample parallels for each gradient group, and the total system is 20 μL. After the reaction is completed, use a microplate reader to detect the fluorescence intensity (λEx: 485 nm, λEm: 528 nm).

[0098] The sensitivity detection results are as Figure 10 shown, indicating that when the concentration of the bacterial solution is 10 9 -10 6 CFU / mL, a significant fluorescence phenomenon is detected, and the fluorescence intensity continuously increases with the increase of the concentration of the bacterial solution. When the concentration of the bacterial solution is 10 3 CFU / mL, there is no obvious difference in the fluorescence intensity from the negative control group, indicating that the minimum detectable concentration of this system is 10 4 CFU / mL.

[0099] Example 4: Detection of actual samples

[0100] In order to detect the feasibility of the method of the present invention in actual applications, a detection test of simulated actual samples was carried out. Respectively take a small amount of lake water from three water sources on campus that are more than 500 meters apart, purchase three different brands of mineral water, and collect a small amount of tap water from laboratory experiments for actual sample inspection.

[0101] Respectively take 5 μL of the collected liquid and add it into 50 μL of the biological probe in Example 1. After incubating at room temperature for 20 min, take 2 μL of the supernatant and add it to the best CRISPR / Cas12a detection system (Cas12a, crRNA, 10×Cas12a Reaction buffer) in Example 2. Set 3 parallels for the negative control group and the sample group. React at 39 °C for 20 min. After taking it out, observe and compare in a dark room and then take and add it to a transparent 384-well plate, and use a microplate reader to measure the fluorescence value (λEx: 485 nm, λEm: 528 nm).

[0102] The detection results are as Figure 11 shown. The fluorescence detection intensity in the lake water is relatively high, which is in line with the actual situation. Pseudomonas aeruginosa grows mostly in humid and dark places and exists in soil, lake water and air. The fluorescence detection intensities of mineral water and tap water are in line with the actual situation. As artificially treated drinking water and domestic water, there should be no detection of Pseudomonas aeruginosa.

[0103] Although the present invention has been disclosed above in its preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A biological probe for rapid detection of Pseudomonas aeruginosa, characterized in that: The biological probe includes an aptamer-dsDNA complex, streptavidin-labeled magnetic beads, and biotin-modified ssDNA; the aptamer-dsDNA complex is composed of an oligonucleotide Pseudomonas aeruginosa aptamer, dsDNA-F, and dsDNA-R; the nucleotide sequences of the oligonucleotide Pseudomonas aeruginosa aptamer, dsDNA-F, and dsDNA-R are shown in SEQ ID NO.1-3; the nucleotide sequence of the ssDNA is shown in SEQ ID NO.

4.

2. A method for preparing a biological probe as described in claim 1, characterized in that, The preparation method is that the aptamer-dsDNA complex formed by mixing the oligonucleotide Pseudomonas aeruginosa aptamer, dsDNA-F, and dsDNA-R, the streptavidin-labeled magnetic beads, and the biotin-modified ssDNA are mixed to form MBs-streptavidin-bio-ssDNA, and the aptamer-dsDNA complex and MBs-streptavidin-bio-ssDNA are mixed to form the MBs-streptavidin-bio-ssDNA-aptamer-dsDNA complex, that is, the probe.

3. The preparation method according to claim 2, characterized in that The preparation method includes the following steps: (1) The oligonucleotide Pseudomonas aeruginosa aptamer, dsDNA-F, and dsDNA-R are mixed and incubated at 95°C for 5 min, and gradually cooled to room temperature to prepare the aptamer-dsDNA complex; (2) The bio-ssDNA is added to the MBs solution and mixed, and stored at room temperature for 30 min; to obtain MBs-streptavidin-bio-ssDNA; (3) The aptamer-dsDNA complex in step (1) is added to the MBs-streptavidin-bio-ssDNA in step (2) at a final concentration of 0.1-0.5 μM and mixed well at room temperature for 20 min to obtain the probe.

4. The preparation method according to claim 3, wherein, In step (1), the concentration ratio of the oligonucleotide Pseudomonas aeruginosa aptamer, dsDNA-F, and dsDNA-R is 1:1:1, and the final concentration of the prepared aptamer-dsDNA is 1-5 μmol / L; In step (2), the final concentration of the added bio-ssDNA is 0.5-1.5 μM, and the addition amount of MBs is at least 20 μL; In step (3), the ratio of the aptamer-dsDNA complex to MBs-streptavidin-bio-ssDNA is 1:

3.

5. A detection system for Pseudomonas aeruginosa, characterized in that: The detection system includes Cas12a, crRNA, 10×Cas12a Buffer, FQ-ssDNA-BHQ, and the biological probe described in claim 1; the nucleotide sequence of the crRNA is shown in SEQ ID NO.5, and the sequence of FQ-ssDNA-BHQ is 5’6-FAM-GATTAGCGTACGCACGTTAC-3’BHQ-1.

6. A detection method for Pseudomonas aeruginosa, characterized in that, The method is to mix Cas12a, crRNA, 10×Cas12a Buffer, FQ-ssDNA-BHQ, and the biological probe described in claim 1, add the test sample, form a Cas12a / crRNA detection system, and perform detection.

7. The detection method according to claim 6, wherein In the detection system, the concentration ratio of the Cas12a to the crRNA is 1:2 to 2:

1.

8. The detection method according to claim 7, characterized in that In the detection system, the final concentrations of the Cas12a, the crRNA, the 10× Cas12a Buffer, the FQ-ssDNA-BHQ, and the biological probe are 0.1 to 0.2 μM, 0.1 to 0.2 μM, 0.1 to 0.5 μM, 0.1 to 0.2 μM, and 10 to 50 nM, respectively.

9. The detection method according to any one of claims 7 to 9, characterized in that The reaction temperature is 35 to 41 °C, and the reaction time is 5 to 30 min.

10. A kit, characterized in that, The kit contains the biological probe described in claim 1, or contains the detection system described in claim 5.

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