Targeting molecule for multi-drug resistant pathogenic bacteria and application thereof

Abstract

The application discloses a kind of targeting molecule of multiple drug-resistant pathogenic bacteria and its application, belong to biological medicine technical field.The targeting molecule is single-stranded DNA nucleic acid aptamer that can specifically recognize target bacteria.The targeting molecule includes double nucleic acid aptamer that respectively combines cell wall anchor surface protein Esp and collagen / fibrillar collagen binding MSCRAMM surface protein Ace, and its end can be connected functional modification group such as fluorescent group, biotin, amino, sulfydryl, nanogold or quantum dot.The targeting molecule can be used to prepare detection reagent, kit, detection probe, biosensor, nucleic acid aptamer chip and magnetic separation material, realize the detection, identification, enrichment, separation and quantification of target bacteria in food, environment, clinical, breeding and drug sensitive monitoring sample.The fluorescence quenching type biosensor constructed based on the targeting molecule has good linear correlation, and the detection limit is 8.17 CFU / mL, and has good detection specificity.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a targeting molecule for multidrug-resistant pathogens and its application. Background Technology

[0002] Enterococcus faecalis is one of the most common opportunistic pathogens in clinical infections, food contamination, and environmental transmission. In immunocompromised individuals, it easily causes various diseases such as urinary tract infections, bacteremia, endocarditis, and wound infections. Some Enterococcus faecalis strains exhibit multidrug resistance, particularly to antibiotics like vancomycin, increasing the difficulty of clinical treatment and raising the risk of nosocomial infection transmission and food safety. Current detection methods for Enterococcus faecalis mainly include plate culture, biochemical identification, immunological detection, and molecular biological detection. While traditional culture and identification methods provide relatively reliable results, they generally suffer from long detection cycles, cumbersome procedures, and stringent experimental requirements, making them unsuitable for rapid screening and on-site testing. Immunological detection and routine nucleic acid detection, although possessing certain sensitivity, still have limitations such as complex sample pretreatment, susceptibility to matrix interference, strong dependence on equipment, and difficulty in balancing detection speed, specificity, and cost control.

[0003] In recent years, nucleic acid aptamers have attracted widespread attention in the fields of pathogen identification and biosensing detection due to their advantages such as ease of synthesis, convenient modification, good stability, high affinity, and the ability to couple with various signaling materials. However, there is still a lack of target molecules for multidrug-resistant Enterococcus faecalis, especially those capable of stably and specifically recognizing Enterococcus faecalis ATCC51299. Furthermore, detection systems based on single recognition sites are prone to problems such as recognition instability, increased false negative risk, or insufficient specificity due to differences in target expression on bacterial surfaces.

[0004] Therefore, developing a targeting molecule that can specifically recognize multidrug-resistant Enterococcus faecalis and is suitable for constructing rapid, sensitive, and highly specific detection systems, especially a nucleic acid aptamer that can achieve dual-site recognition, has important application value. Summary of the Invention

[0005] To address the problems in existing technologies for the detection and identification of multidrug-resistant Enterococci, especially Enterococcus faecalis ATCC51299, such as lack of target molecules, insufficient recognition specificity, limited detection sensitivity, susceptibility to interference from non-target bacteria, and decreased detection stability and increased false negative risk due to differences in target expression on bacterial surface at a single recognition site, this application provides a target molecule for multidrug-resistant pathogens and its application.

[0006] This application adopts the following technical solution: a targeting molecule for multidrug-resistant pathogens, wherein the multidrug-resistant pathogen is Enterococcus faecalis ATCC51299, and the DNA sequence of its targeting molecule is shown in SEQ ID No: 1.

[0007] Preferably, the targeting molecule is a single-stranded DNA aptamer that can specifically recognize and bind to Enterococcus faecalis ATCC51299.

[0008] Preferably, the targeting molecule specifically binds to the cell surface structure, surface protein, or membrane-associated components of Enterococcus faecalis ATCC51299.

[0009] Preferably, the targeting molecule specifically binds to the cell wall anchoring surface protein Esp of Enterococcus faecalis ATCC51299.

[0010] Preferably, the 5' or 3' end of the target molecule is connected to a functional modification group, which includes one or more of the following: fluorescent group, quenching group, biotin, amino group, carboxyl group, thiol group, phosphate group, magnetic microsphere, gold nanoparticle, quantum dot, or other functional groups used for immobilization, labeling, enrichment, or signal amplification.

[0011] The application of the target molecule for multidrug-resistant pathogens, wherein the target molecule is used to prepare products for the detection, identification, enrichment, isolation or quantification of Enterococcus faecalis ATCC51299; the products include detection reagents, detection kits, detection probes, biosensors, nucleic acid aptamer chips, magnetic separation materials, fluorescence detection systems, colorimetric detection systems or electrochemical detection systems; the detection samples include food samples, environmental samples, clinical samples, aquaculture samples or drug susceptibility monitoring samples.

[0012] Preferably, the method for preparing the target molecule for the detection probe is as follows: (1) Preparing the first activation mixture: mixing 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide at a volume ratio of 1:1 to obtain the first activation mixture; (2) Preparing the second activation mixture: adding pure water to 3-(2-formylethyl)phosphine hydrochloride to obtain a 3-(2-formylethyl)phosphine hydrochloride solution with a final concentration of 2.867 g / L, which is the second activation mixture; (3) Preparing the fluorescent probe: adding pure water to 1 mL N-(β-aminoethyl-γ-aminopropyl)methyl Add 6 mL of deionized water to dimethoxysilane, then add 42.0 mg of citric acid and 27.0 mg of L-cysteine, stir continuously for 30 min, heat at 180 °C for 6 h, cool to room temperature, and dilute 20 times with pure water to obtain a reaction solution with co-doped carbon dots. Vortex mix 500 μL of the reaction solution and 250 μL of the first activation mixture, and incubate on a shaker at 180 rpm and room temperature for 30 min. Then add 150 μL of amino-modified first targeting molecule solution with a molar concentration of 10 μmol / L, vortex mix, and incubate on a shaker at 180 rpm and room temperature for 2.5 h. Subsequently, add 30 μL of 0.5 mol / L ethanolamine for blocking for 30 min, and purify by ultrafiltration to obtain the fluorescent probe; the DNA sequence of the first targeting molecule in the first targeting molecule solution is shown in SEQ ID. No: 1 shows that the 5' end of the first target molecule is modified with an amino group; (4) Preparation of quenching probe: 1 mL of 1% chloroauric acid solution was added to 100 mL of distilled water and boiled; 3 mL of 1% trisodium citrate solution was quickly added and boiled for 20 min, and then cooled naturally to room temperature to obtain a gold nanoparticle solution; 100 μL of the second target molecule solution with a molar concentration of 10 μM was placed in a water bath at 95 °C for 5 min and cooled at 4 °C for 5 min to obtain an annealing solution; 10 μL of 2.867 g / L 3-(2-formylethyl)phosphine hydrochloride solution was added to the annealing solution and allowed to stand for 30 min at room temperature to obtain a reaction solution; 1 mL of gold nanoparticle solution was added to the reaction solution and incubated at 37 °C for 12 h, centrifuged at 8000 rpm for 20 min, and the supernatant was removed to obtain a precipitate; The DNA sequence of the second target molecule in the second target molecule solution is as shown in SEQ ID As shown in No:2, the 5' end of the second target molecule is modified with a thiol group; 500 μL of PBS solution with a molar concentration of 0.1M and a pH of 7.4 is added to the precipitate to prepare the quenching probe; (5) Preparation of biosensor: The fluorescent probe and the quenching probe are mixed at a volume ratio of 1:1.5 to prepare a fluorescent quenching biosensor.

[0013] Preferably, the second targeting molecule is a single-stranded DNA aptamer that can specifically recognize and bind to Enterococcus faecalis ATCC51299.

[0014] Preferably, the second targeting molecule specifically binds to the collagen / laminar adhesion protein MSCRAMM surface protein Ace of Enterococcus faecalis ATCC51299.

[0015] Compared with the prior art, this application has at least the following beneficial effects: (1) The target molecule provided in this application is a single-stranded DNA nucleic acid aptamer for multidrug-resistant Enterococcus faecalis ATCC51299, which can specifically recognize and bind to the target bacteria, and has good targeting and recognition capabilities. (2) This application preferably adopts a dual nucleic acid aptamer combination strategy, wherein the first nucleic acid aptamer preferably recognizes the cell wall anchoring surface protein Esp on the surface of the target bacteria, and the second nucleic acid aptamer preferably recognizes the collagen / laminar adhesion protein binding MSCRAMM surface protein Ace on the surface of the target bacteria. The two form dual-site recognition for different surface target structures, which can effectively reduce the detection fluctuation and false negative risk caused by single-site recognition, thereby improving the stability and specificity of the detection system. (3) The first and second nucleic acid aptamers obtained in this application both have high affinity for the target bacteria. The binding rate of the first nucleic acid aptamer to the target bacteria is 74.36%, and the affinity constant Kd is 13.20 nM. The binding rate of the second nucleic acid aptamer to the target bacteria is 72.15%, and the affinity constant Kd is 10.86 nM. This indicates that the target molecule provided in this application has good binding performance. (4) The non-specific binding rate of the target molecule described in this application to non-target strains such as Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa is low, indicating that it has good selectivity and can effectively reduce background interference and improve detection accuracy. (5) The target molecule described in this application can be further connected with functional modification groups such as fluorescent groups, biotin, amino groups, thiol groups, magnetic microspheres, gold nanoparticles, and quantum dots. This facilitates the construction of detection reagents, detection kits, detection probes, biosensors, nucleic acid aptamer chips, and magnetic separation materials, and has good versatility and application expansion value. (6) The fluorescence quenching biosensor constructed based on the dual nucleic acid aptamers of this application can realize rapid quantitative detection of target bacteria. Its linear regression equation is y=-0.09462x+1.01235, and the correlation coefficient R is 1. 2The limit of detection (LOD) was 0.99891, and the limit of detection (LOD) was 8.17 CFU / mL, indicating that the detection system has good linearity and high detection sensitivity. (7) The target molecule and the detection system constructed in this application can be used for the detection, identification, enrichment, separation and quantification of Enterococcus faecalis ATCC51299 in food samples, environmental samples, clinical samples, aquaculture samples and drug sensitivity monitoring samples, and has good practical application prospects. Attached Figure Description

[0016] Figure 1 This is a molecular docking diagram between the first nucleic acid aptamer obtained from the screening in this application and the Enterococcus faecalis cell wall anchoring surface protein Esp.

[0017] Figure 2 This is a molecular docking diagram between the second nucleic acid aptamer obtained from this application and the Enterococcus faecalis collagen / laminoid protein binding to the surface protein Ace of MSCRAMM.

[0018] Figure 3 This is the infrared spectrum of the co-doped carbon dot reaction solution prepared in this application.

[0019] Figure 4 This is a standard curve diagram of the fluorescence quenching biosensor prepared in this application. Detailed Implementation

[0020] The present application will be further described in detail below with reference to specific embodiments. However, these embodiments are only for illustrating the present application and do not constitute any limitation on the scope of protection of the present application. For those skilled in the art, any simple deductions or substitutions made without departing from the concept of the present application shall be deemed to fall within the scope of protection defined by the claims of the present application.

[0021] In the following embodiments, both the first and second aptamers were synthesized by Shanghai Sangon Biotech (Shanghai) Co., Ltd.; unless otherwise specified, all other raw materials used were purchased from Sinopharm Chemical Reagent Co., Ltd. The phosphate buffer used in this application was 0.1M sterile PBS buffer with a pH of 7.4. The first targeting molecule is also referred to as the first nucleic acid aptamer; the second targeting molecule is also referred to as the second nucleic acid aptamer.

[0022] All strains used in this application were commercially available standard test strains. The target strain was *Enterococcus faecalis* ATCC51299; non-target strains used for specificity verification included *Staphylococcus aureus* ATCC29213, *Salmonella typhimurium* ATCC14028, *Escherichia coli* ATCC25922, *Cronobacter akazakii* ATCC29544, *Pseudomonas aeruginosa* ATCC15442, and *Listeria monocytogenes* ATCC43251. All of the above strains were purchased from the American Center for Type Culture Collection (ATCC) or its authorized agents.

[0023] In this application, *Enterococcus faecalis* ATCC51299 is used as a representative strain of multidrug-resistant pathogens. To maintain its drug-resistant phenotype, the target strain was cultured in BHI medium with vancomycin added to a final concentration of 4 μg / mL, and cultured aerobically at 37°C for 24 h. After culture, the culture was washed with sterile PBS buffer and resuspended for subsequent nucleic acid aptamer binding assays, specificity verification experiments, or fluorescence quenching detection experiments. Other non-target strains were activated and cultured according to their standard recommended culture conditions. Specifically, *Staphylococcus aureus* was cultured in tryptone soybean broth at 37°C under aerobic conditions for 24 hours; *Salmonella typhimurium* was cultured in tryptone soybean broth at 37°C under aerobic conditions for 24 hours; *Escherichia coli* was cultured in tryptone soybean broth at 37°C under aerobic conditions for 24 hours; *Cronobacter sakazakii* was cultured in tryptone soybean broth at 37°C under aerobic conditions for 24 hours; *Pseudomonas aeruginosa* was cultured in tryptone soybean broth at 37°C under aerobic conditions for 24 hours; and *Listeria monocytogenes* was cultured in tryptone soybean broth at 37°C under aerobic conditions for 24 hours.

[0024] It should be noted that the instruments, equipment, raw materials, reagents, strain culture, cell washing, bacterial suspension preparation, probe incubation, and detection operations used in this application were all performed under aseptic conditions in accordance with microbiological experimental standards.

[0025] It should be noted that the instruments, equipment, raw materials, reagents, culture conditions, centrifugation conditions, washing conditions, incubation conditions or detection steps not specifically limited in this application can all be achieved by conventional technical means in the field by those skilled in the art, and will not be described in detail in this application.

[0026] It should be noted that 10 6 CFU / mL bacterial suspension was incubated with 100 nmol / LFAM-labeled aptamer in binding buffer at 37°C for 30 min, washed three times with PBS, and then detected by flow cytometry. Kd was obtained by nonlinear regression fitting, and the experiment was repeated three times.

[0027] This application determined the binding characteristics of the first nucleic acid aptamer (first target molecule) and the second nucleic acid aptamer (second target molecule) to various test strains using flow cytometry. To facilitate flow cytometry detection, fluorescently labeled first and second nucleic acid aptamers were used in the flow cytometry validation experiment. The results showed that the first nucleic acid aptamer exhibited a binding rate of 74.36% to the target bacterium *Enterococcus faecalis* ATCC51299, with an affinity constant Kd of 13.20 nM. The non-specific binding rates (under the same conditions) to *Staphylococcus aureus*, *Listeria monocytogenes*, *Salmonella typhimurium*, *Escherichia coli*, *Cronobacter sakazakii*, and *Pseudomonas aeruginosa* were 8.47%, 6.92%, 9.35%, 12.64%, 10.28%, and 7.56%, respectively. The second nucleic acid aptamer showed a binding rate of 72.15% to the target bacterium *Enterococcus faecalis* ATCC51299, with an affinity constant Kd of 10.86 nM. Under the same conditions, the non-specific binding rates (for *Staphylococcus aureus*, *Listeria monocytogenes*, *Salmonella typhimurium*, *Escherichia coli*, *Cronobacter sakazakii*, and *Pseudomonas aeruginosa* were 7.26%, 8.14%, 10.72%, 11.38%, 9.43%, and 8.69%, respectively. These results indicate that both the first and second nucleic acid aptamers obtained in this application can bind efficiently to the target bacterium *Enterococcus faecalis* ATCC51299, and exhibit low non-specific binding rates to common non-target strains, demonstrating that both aptamers possess good target recognition ability and binding specificity. The first nucleic acid aptamer (sequence: 5'-AGCAGCACAGAGGTCAGATGCGTCCATGTTGGTGACCGCATCTGACCTCTGTGCTGCT-3') preferentially recognizes the cell wall anchoring surface protein Esp of Enterococcus faecalis ATCC51299 (e.g., Figure 1As shown), the second nucleic acid aptamer (sequence: 5'-TGCACGTTGACCTAGGATCGTACCGTTAGCGGTACGATCCTAGGTCAACGTGCA-3') preferentially recognizes the collagen / laminus adhesion protein binding MSCRAMM surface protein Ace (as shown) of Enterococcus faecalis ATCC51299. Figure 2 (As shown). Since the first and second nucleic acid aptamers recognize different binding sites on the surface of the target bacteria, their combined use can reduce the risk of false negatives caused by single-site recognition and improve the stability and specificity of the fluorescence quenching biosensor for detecting the target bacteria.

[0028] The following details the DNA sequence screening methods for the first and second nucleic acid aptamers in this application: (1) Target protein structure construction: The amino acid sequences of Esp, the cell wall anchoring surface protein, and Ace, the collagen / laminar adhesion protein binding MSCRAMM surface protein, from Enterococcus faecalis ATCC51299 were obtained, and three-dimensional structural models of Esp and Ace proteins were constructed respectively. The obtained three-dimensional structural models were subjected to energy minimization and structural optimization. After removing unstable conformations, the surface exposed regions, charged amino acid residue distribution, hydrophilic / hydrophobic regions, and potential binding pockets of Esp and Ace proteins were analyzed to determine candidate binding regions that can be used for nucleic acid aptamer molecular docking. (2) Random single-stranded DNA library construction: A single-stranded DNA sequence library was randomly designed. The single-stranded DNA sequence library consisted of short oligonucleotide sequences with a length of 40-80 nt. The short oligonucleotide sequences were randomly composed of four bases: A, T, C, and G, and candidate sequences that could form stable stem-loop, inner loop, or convex loop structures were preferentially retained. Random sequences were initially screened using secondary structure prediction software to remove sequences with excessively long consecutive bases, over-folded secondary structures, or too many free ends, resulting in a set of candidate single-stranded DNA sequences. (3) Screening of candidate sequences for the first nucleic acid aptamer: The three-dimensional structures of the candidate single-stranded DNA sequences were predicted, and the predicted three-dimensional structures of the single-stranded DNA were molecularly docked with the candidate binding regions of the Esp protein. The binding free energy, number of hydrogen bonds, electrostatic interaction, hydrophobic interaction, binding pocket matching degree, conformational stability, and accessibility of the target protein surface exposure site were used as evaluation indicators to screen candidate single-stranded DNA sequences that could form stable complexes with the Esp protein. The candidate single-stranded DNA sequences were further optimized using multi-objective optimization methods, including: reducing the binding free energy between the single-stranded DNA and the Esp protein, increasing the matching degree between the single-stranded DNA and the Esp protein binding region, improving the stability of the secondary structure of the single-stranded DNA, and reducing the possibility of non-specific binding between the single-stranded DNA and the surface proteins of non-target strains. After multiple rounds of iterative screening, candidate sequences for the first nucleic acid aptamer were obtained. The first nucleic acid aptamer is 58 nt in length and is predicted to form a stem-loop secondary structure. The 5' and 3' ends of the aptamer are partially complementary to form a stem region, and the middle bases form an exposed loop region. Molecular docking prediction results show that the first nucleic acid aptamer can embed into the binding pocket on the surface of the Esp protein and interact with multiple surface amino acid residues of the Esp protein through hydrogen bonds, electrostatic interactions and hydrophobic interactions. The predicted binding free energy is -10.23 kcal / mol, indicating that the first nucleic acid aptamer has a relatively stable binding tendency with the Esp protein. (4) Screening of candidate sequences for the second nucleic acid aptamer: Molecular docking is performed between the candidate single-stranded DNA sequence set and the collagen / laminusoidal binding region of the Ace protein.Using the MSCRAMM functional region of the Ace protein as the preferred binding region, the binding free energy, binding site coverage, number of hydrogen bonds, electrostatic complementarity, spatial conformational matching, and exposure of the recognition loop region between candidate single-stranded DNA sequences and the Ace protein were evaluated. Further multi-objective optimization was performed on the candidate sequences, prioritizing those with different binding sites to the first aptamer, lower steric hindrance, stable binding to the Ace protein, and no interference with the first aptamer's recognition of the Esp protein. After multiple rounds of screening and optimization, candidate sequences for the second aptamer were obtained. The second aptamer is 54 nt in length, forming a stem-loop secondary structure. The 5' and 3' ends partially pair to form the stem region, while the middle bases form the exposed recognition loop region. Molecular docking prediction results showed that the second nucleic acid aptamer could bind to the collagen / laminar adhesion protein-MSCRAMM functional region of Ace protein, and form a stable interaction with the amino acid residues on the surface of Ace protein through hydrogen bonds, electrostatic interactions and hydrophobic interactions. The predicted binding free energy was -9.41 kcal / mol, indicating that the second nucleic acid aptamer has a relatively stable binding trend with Ace protein. (5) Synthesis and structural verification of candidate aptamers: The first and second nucleic acid aptamers obtained by screening were sent to a biotechnology company for artificial synthesis. When used for flow cytometry verification, fluorescent labeling groups were attached to the 5' ends of the first and second nucleic acid aptamers, respectively; when used for subsequent probe preparation, the 5' end of the first nucleic acid aptamer was preferably modified with an amino group, and the 5' end of the second nucleic acid aptamer was preferably modified with a thiol group. The synthesized aptamers were annealed and then used for subsequent binding experiments. (6) Verification of aptamer binding performance with target bacteria: The fluorescently labeled first and second nucleic acid aptamers were incubated with a suspension of Enterococcus faecalis ATCC51299, respectively. After washing with PBS buffer to remove unbound aptamers, the fluorescence signal intensity on the surface of the target bacteria was detected by flow cytometry, and the binding rate between the aptamers and the target bacteria was calculated. At the same time, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa were used as non-target strains, and the non-specific binding rate was detected using the same method. The results showed that both the first and second nucleic acid aptamers could specifically bind to the target bacteria Enterococcus faecalis ATCC51299, while the binding rate with non-target strains was low, indicating that the first and second nucleic acid aptamers have good targeting recognition ability and specificity. (7) Screening and confirmation of dual nucleic acid aptamer combinations:

[0029] Furthermore, the first and second nucleic acid aptamers were used in combination to investigate their dual-site recognition capability for Enterococcus faecalis ATCC51299. Since the first aptamer preferentially recognizes the Esp protein and the second aptamer preferentially recognizes the Ace protein, their target sites differ. Using them in combination can reduce detection fluctuations caused by differences in single-target expression, improving the recognition stability and detection specificity of the target bacteria. Therefore, the sequence shown in SEQ ID No: 1 was ultimately selected as the first nucleic acid aptamer, and the sequence shown in SEQ ID No: 2 was selected as the second nucleic acid aptamer to construct a fluorescence-quenched biosensor for detecting Enterococcus faecalis ATCC51299.

[0030] The first and second nucleic acid aptamers obtained by screening were used to prepare a fluorescence quenching biosensor for detecting Enterococcus faecalis ATCC51299. The operation steps are as follows: (1) Preparation of the first activation mixture: 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 5 mM N-hydroxysuccinimide (NHS) were mixed at a volume ratio of 1:1 to obtain the first activation mixture. The first activation mixture was used to activate the carboxyl groups on the surface of the co-doped carbon dots so that they could undergo a coupling reaction with the amino-modified first nucleic acid aptamer. (2) Preparation of the second activation mixture: Pure water was added to 3-(2-formylethyl)phosphine hydrochloride (TCEP·HCl) to obtain a 3-(2-formylethyl)phosphine hydrochloride solution with a final concentration of 2.867 g / L, which is the second activation mixture. The second activation mixture is used to reduce the thiol-modified second nucleic acid aptamer and promote the binding of the second nucleic acid aptamer to gold nanoparticles. (3) Preparation of fluorescent probe: 6 mL of deionized water was added to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane, followed by 42.0 mg of citric acid and 27.0 mg of L-cysteine. The mixture was stirred continuously for 30 min. The mixture was heated at 180 °C for 6 h and cooled to room temperature. The mixture was diluted 20 times with pure water to obtain the co-doped carbon dot reaction solution. Its infrared spectrum is shown in the figure. Figure 3As shown. 500 μL of the co-doped carbon dot reaction solution and 250 μL of the first activation mixture were vortexed and incubated on a shaker at 180 rpm and room temperature for 30 min to activate the carboxyl groups on the surface of the co-doped carbon dots. Then, 150 μL of an amino-modified first nucleic acid aptamer solution with a molar concentration of 10 μmol / L was added, vortexed, and incubated on a shaker at 180 rpm and room temperature for 2.5 h. Subsequently, 30 μL of 0.5 mol / L ethanolamine was added for blocking for 30 min. After purification by ultrafiltration, the co-doped carbon dot fluorescent probe modified with the first nucleic acid aptamer was obtained and stored at 4 °C in the dark. The DNA sequence of the first nucleic acid aptamer in the first nucleic acid aptamer solution is shown in SEQ ID No: 1: 5'-AGCAGCACAGAGGTCAGATGCGTCCATGTTGGTGACCGCATCTGACCTCTGTGCTGCT-3'. The 5' end of the first nucleic acid aptamer is preferably modified with an amino group to undergo an amidation coupling reaction with co-doped carbon dots activated by EDC / NHS. The first nucleic acid aptamer preferably recognizes the cell wall anchoring surface protein Esp of Enterococcus faecalis ATCC51299. (4) Preparation of quenching probe: 1 mL of 1% chloroauric acid solution was added to 100 mL of distilled water and boiled; 3 mL of 1% trisodium citrate solution was quickly added and the mixture was boiled for another 20 min. Boiling was then stopped and the mixture was allowed to cool naturally to room temperature to obtain a gold nanoparticle solution. 100 μL of a 10 μM second nucleic acid aptamer solution was incubated at 95 °C for 5 min, followed by cooling at 4 °C for 5 min to obtain an annealing solution. 10 μL of a 2.867 g / L 3-(2-formylethyl)phosphonic acid hydrochloride solution was added to the annealing solution, and the mixture was allowed to stand at room temperature for 30 min to obtain a reaction solution. 1 mL of gold nanoparticle solution was added to the reaction solution, and the mixture was incubated at 37 °C for 12 h, centrifuged at 8000 rpm for 20 min, and the supernatant was removed to obtain a precipitate. The DNA sequence of the second nucleic acid aptamer in the second nucleic acid aptamer solution is shown in SEQ ID No: 2: 5'-TGCACGTTGACCTAGGATCGTACCGTTAGCGGTACGATCCTAGGTCAACGTGCA-3'. The 5' end of the second nucleic acid aptamer was modified with a thiol group. The second nucleic acid aptamer preferably recognizes the collagen / laminus adhesion protein of Enterococcus faecalis ATCC51299 and the MSCRAMM surface protein Ace. 500 μL of 0.1 M PBS solution (pH 7.4) was added to the resulting precipitate, and the mixture was resuspended and stored at 4°C in the dark to obtain the gold nanoparticle quenching probe modified with the second nucleic acid aptamer.(5) Preparation of a fluorescence-quenched biosensor: The fluorescent probe and the quenching probe are mixed at a volume ratio of 1:1.5 to prepare a fluorescence-quenched biosensor for detecting Enterococcus faecalis ATCC51299. Preferably, when the fluorescence emission wavelength of the co-doped carbon dots shifts from 535 nm to 538 nm, it indicates that the first nucleic acid aptamer has been successfully modified onto the surface of the co-doped carbon dots. Preferably, when the ultraviolet absorption wavelength of the gold nanoparticles shifts from 520 nm to 524 nm, it indicates that the second nucleic acid aptamer has been successfully modified onto the surface of the gold nanoparticles. (6) Specific detection operation steps: Establishment of linear regression equation: Prepare a sterile sample solution to be tested, which is set as a blank control solution; after centrifuging the Enterococcus faecalis ATCC51299 bacterial culture, wash it three times with PBS buffer, then resuspend it with PBS buffer and perform gradient dilution to prepare concentrations of 10. 1 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL and 10 7CFU / mL of bacterial test solution was prepared. 100 μL of the prepared fluorescence-quenched biosensor was mixed with 100 μL of blank control test solution or 100 μL of bacterial test solutions of different concentrations, and incubated at 37℃ for 35 min to obtain the incubation solution. The fluorescence intensity of the incubation solution at the emission wavelength of 538 nm was measured at an excitation wavelength of 410 nm. The fluorescence intensity of the blank control test solution was denoted as F0, and the fluorescence intensity of the bacterial test solution was denoted as F. A standard curve was plotted with the fluorescence intensity ratio F / F0 as the ordinate and the logarithm of the *Enterococcus faecalis* ATCC51299 bacterial concentration in the bacterial test solution as the abscissa to obtain the linear regression equation, correlation coefficient, and detection limit. Detection of Enterococcus faecalis ATCC51299 in the test sample: A test solution was prepared from the test sample. A fluorescence-quenching biosensor was mixed with the test solution at a volume ratio of 1:1 and incubated at 37°C for 35 min to obtain an incubation solution. The fluorescence intensity of the incubation solution at an emission wavelength of 538 nm was measured at an excitation wavelength of 410 nm. Based on the obtained fluorescence intensity ratio F / F0, the concentration of Enterococcus faecalis ATCC51299 in the test sample was calculated by substituting it into the linear regression equation. In the fluorescence-quenching biosensor prepared in this application, co-doped carbon dots modified with a first nucleic acid aptamer serve as the fluorescence donor, and gold nanoparticles modified with a second nucleic acid aptamer serve as the fluorescence acceptor (quencher). When the target bacterium *Enterococcus faecalis* ATCC51299 is present, the first and second aptamers recognize the Esp and Ace proteins on the surface of the target bacterium, respectively, bringing the co-doped carbon dots closer to the gold nanoparticles on the target bacterium surface, thereby enhancing the fluorescence quenching effect. As the concentration of the target bacterium increases, the fluorescence intensity at 538 nm gradually decreases, and the fluorescence intensity ratio F / F0 shows a good linear relationship with the logarithm of the target bacterium concentration.

[0031] like Figure 4 As shown, the fluorescence intensity of different concentrations of Enterococcus faecalis ATCC51299 gradually decreased. The linear regression equation for the detection of Enterococcus faecalis ATCC51299 by the fluorescence-quenched biosensor was determined to be: y = -0.09462x + 1.01235, with a correlation coefficient R0. 2 The limit of detection (LOD) was 0.99891, and the limit of detection (LOD) was 8.17 CFU / mL. These results demonstrate that the fluorescence-quenched biosensor prepared in this application can be used for the rapid quantitative detection of Enterococcus faecalis ATCC51299.

[0032] Next, specificity experiments were performed: Test solutions containing Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa were prepared, and tested under the same conditions as the target bacteria detection (under the same conditions, 10... 6 The fluorescence-quenched biosensor was mixed with the above-mentioned non-target bacterial strains in the test solution (CFU / mL), and incubated at 37°C for 35 min. The fluorescence intensity at 538 nm was then measured. The results showed that the fluorescence intensity ratios (F / F0) of the test solutions containing Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa were 0.93218, 0.94672, 0.92754, 0.90163, 0.91845, and 0.93987, respectively. Under the same conditions, the fluorescence intensity ratio (F / F0) of the test solution containing Enterococcus faecalis ATCC51299 was 0.34864. The fluorescence intensity ratio (F0 / F0) of the blank control test solution was 1. The above results demonstrate that the fluorescence-quenched biosensor prepared in this application exhibits good detection specificity for Enterococcus faecalis ATCC51299. Furthermore, since the first nucleic acid aptamer preferentially recognizes the Esp protein on the surface of Enterococcus faecalis ATCC51299, and the second nucleic acid aptamer preferentially recognizes the Ace protein on the surface of Enterococcus faecalis ATCC51299, the two aptamers form a dual-site recognition targeting different surface target structures. Therefore, background interference caused by non-target strains can be reduced, improving the recognition accuracy and detection stability of the detection system.

[0033] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application. It should not be construed that the specific implementation of this application is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of this application, and all such modifications or substitutions should be considered within the scope of protection defined by the claims. Furthermore, for any issues that may arise with the claims, the embodiments described in the specification shall prevail.

Claims

1. A targeting molecule for multidrug-resistant pathogens, characterized in that: The multidrug-resistant pathogen is Enterococcus faecalis ATCC51299, and the DNA sequence of its target molecule is shown in SEQ ID No:

1.

2. The targeting molecule for multidrug-resistant pathogens according to claim 1, characterized in that: The target molecule is a single-stranded DNA aptamer that can specifically recognize and bind to Enterococcus faecalis ATCC51299.

3. The targeting molecule for multidrug-resistant pathogens according to claim 2, characterized in that: The targeting molecule specifically binds to cell surface structures, surface proteins, or membrane-associated components of Enterococcus faecalis ATCC51299.

4. The targeting molecule for multidrug-resistant pathogens according to claim 3, characterized in that: The targeting molecule specifically binds to the cell wall anchoring surface protein Esp of Enterococcus faecalis ATCC51299.

5. The targeting molecule for multidrug-resistant pathogens according to any one of claims 1-4, characterized in that: The target molecule has a functional modification group attached to its 5' or 3' end. The functional modification group includes one or more of the following: fluorescent group, quencher group, biotin, amino group, carboxyl group, thiol group, phosphate group, magnetic microsphere, gold nanoparticle, quantum dot, or other functional groups used for immobilization, labeling, enrichment, or signal amplification.

6. The application of the targeting molecule for multidrug-resistant pathogens as described in claim 1, characterized in that: The targeted molecule is used to prepare products for the detection, identification, enrichment, isolation, or quantification of Enterococcus faecalis ATCC51299; the products include detection reagents, detection kits, detection probes, biosensors, nucleic acid aptamer chips, magnetic separation materials, fluorescence detection systems, colorimetric detection systems, or electrochemical detection systems; the detection samples include food samples, environmental samples, clinical samples, aquaculture samples, or drug sensitivity monitoring samples.

7. The application of the targeting molecule for multidrug-resistant pathogens according to claim 6, characterized in that: The method for preparing the target molecule for the detection probe is as follows: (1) Preparation of the first activation mixture: 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide are mixed at a volume ratio of 1:1 to obtain the first activation mixture; (2) Preparation of the second activation mixture: Pure water is added to 3-(2-formylethyl)phosphine hydrochloride to obtain a 3-(2-formylethyl)phosphine hydrochloride solution with a final concentration of 2.867 g / L, which is the second activation mixture; (3) Preparation of the fluorescent probe: 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethyl... 6 mL of deionized water was added to an oxysilane, followed by 42.0 mg of citric acid and 27.0 mg of L-cysteine. The mixture was stirred continuously for 30 min, heated at 180 °C for 6 h, cooled to room temperature, and diluted 20 times with pure water to obtain a reaction solution with co-doped carbon dots. 500 μL of the reaction solution and 250 μL of the first activation mixture were vortexed and incubated on a shaker at 180 rpm and room temperature for 30 min. Then, 150 μL of a 10 μmol / L amino-modified first targeting molecule solution was added, vortexed, and incubated on a shaker at 180 rpm and room temperature for 2.5 h. Subsequently, 30 μL of 0.5 mol / L ethanolamine was added for blocking for 30 min. After ultrafiltration purification, the fluorescent probe was obtained. The DNA sequence of the first targeting molecule in the first targeting molecule solution is shown in SEQ ID. No: 1 shows that the 5' end of the first target molecule is modified with an amino group; (4) Preparation of quenching probe: 1 mL of 1% chloroauric acid solution was added to 100 mL of distilled water and boiled; 3 mL of 1% trisodium citrate solution was quickly added and boiled for 20 min, and then cooled naturally to room temperature to obtain a gold nanoparticle solution; 100 μL of the second target molecule solution with a molar concentration of 10 μM was placed in a water bath at 95 °C for 5 min and cooled at 4 °C for 5 min to obtain an annealing solution; 10 μL of 2.867 g / L 3-(2-formylethyl)phosphine hydrochloride solution was added to the annealing solution and allowed to stand for 30 min at room temperature to obtain a reaction solution; 1 mL of gold nanoparticle solution was added to the reaction solution and incubated at 37 °C for 12 h, centrifuged at 8000 rpm for 20 min, and the supernatant was removed to obtain a precipitate; The DNA sequence of the second target molecule in the second target molecule solution is as shown in SEQ ID As shown in No:2, the 5' end of the second target molecule is modified with a thiol group; 500 μL of PBS solution with a molar concentration of 0.1M and a pH of 7.4 is added to the precipitate to prepare the quenching probe; (5) Preparation of biosensor: The fluorescent probe and the quenching probe are mixed at a volume ratio of 1:1.5 to prepare a fluorescent quenching biosensor.

8. The application of the targeting molecule for multidrug-resistant pathogens according to claim 7, characterized in that: The second target molecule is a single-stranded DNA aptamer that can specifically recognize and bind to Enterococcus faecalis ATCC51299.

9. The application of the targeting molecule for multidrug-resistant pathogens according to claim 8, characterized in that: The second targeting molecule specifically binds to the collagen / laminar adhesion protein MSCRAMM surface protein Ace of Enterococcus faecalis ATCC51299.

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