Composite nanoprobe for detecting methicillin-resistant staphylococcus aureus, kit and application of composite nanoprobe

Abstract

The invention discloses a composite nanoprobe for detecting methicillin-resistant staphylococcus aureus, a kit and application of the composite nanoprobe and the kit, and belongs to the technical field of biology. The composite nano probe is prepared from a methicillin-resistant staphylococcus aureus (MRSA) nucleic acid aptamer modified by a fluorescent dye Cy7 and gold-palladium-platinum nano particles. The invention develops a fluorescent sensing detection method for sensitively and specifically recognizing and rapidly detecting MRSA based on the principle that a nucleic acid aptamer specifically recognizes the MRSA and the nano-metal surface energy transfer synergistic inner filter effect between an energy transfer donor and an energy transfer acceptor. The method is high in sensitivity, the detection limit is as low as 8 CFU / mL, and the detection linear range is wide and is 101-106 CFU / mL. The method can realize rapid and high-specificity identification and detection of MRSA within 55min, is suitable for rapid detection of drug-resistant bacteria, and has wide application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a composite nanoprobe for detecting methicillin-resistant Staphylococcus aureus, a kit and application thereof. BACKGROUND

[0002] Bacterial infection seriously threatens human health, among which methicillin-resistant Staphylococcus aureus (MRSA) is a common drug-resistant bacteria in clinic and has become one of the important pathogenic bacteria of nosocomial and community infections. The mortality rate of septicemia, pneumonia and other diseases caused by MRSA infection is as high as 60%. However, MRSA is resistant to commonly used antibiotics β-lactams, and vancomycin is the last line of defense. The binding site of methicillin-sensitive Staphylococcus aureus (MSSA) and β-lactam drugs is penicillin-binding protein (PBPs), and the methicillin resistance gene (mecA) of MRSA can encode alternative penicillin-binding protein (PBP2α), which leads to a significant decrease in the affinity of β-lactams, so that the drug cannot normally function. Rapid identification and specific detection of MRSA in a short time can effectively inhibit the spread of MRSA, reduce MRSA infection and timely treatment.

[0003] Rapid and accurate detection of MRSA is the premise of timely treatment of its infection and control of the disease. However, the current clinical detection of MRSA uses plate culture method which needs 48-72h, then the bacteria need to be separated and biochemical identification is needed to determine the type of bacteria infected, and then drug sensitivity test is needed to determine whether it is a drug-resistant bacteria. The whole process needs to improve the timeliness and reduce the detection, identification and identification time. The current MRSA detection also has Polymerase Chain Reaction (PCR) technology and Enzyme-Linked Immunosorbent Assay (ELISA), PCR technology detection needs 5-24h, and the false positive rate is high; ELISA uses antigen-antibody recognition, which is prone to cross-reaction and cannot accurately distinguish MSSA and MRSA, resulting in high false positive rate. Therefore, it is urgent to develop a new method for sensitive, specific identification and rapid detection of MRSA. SUMMARY

[0004] The purpose of the present application is to provide a composite nanoprobe for detecting methicillin-resistant Staphylococcus aureus, a kit and application thereof, so as to solve the problems existing in the prior art. Based on the principle of specific recognition of MRSA by nucleic acid aptamer and nanometal surface energy transfer (NSET) between energy transfer donors and acceptors and inner filter effect (IFE), the present application develops a fluorescent sensing detection method for sensitive, specific recognition and rapid detection of MRSA. The fluorescent sensing detection method of the present application has the advantages of high sensitivity, strong specificity and low detection cost, has broad application potential and prospect, and provides a new strategy for rapid detection and accurate prevention and control of MRSA in clinical practice.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0006] The present application provides a composite nanoprobe for detecting methicillin-resistant Staphylococcus aureus, which is prepared by mixing and standing methicillin-resistant Staphylococcus aureus nucleic acid aptamer modified by fluorescent dye Cy7 and gold palladium platinum nanoparticles.

[0007] The nucleotide sequence of the methicillin-resistant Staphylococcus aureus nucleic acid aptamer is shown in SEQ ID NO. 1.

[0008] Further, the ratio of the final concentrations of the methicillin-resistant Staphylococcus aureus nucleic acid aptamer modified by fluorescent dye Cy7 and the gold palladium platinum nanoparticles is 5.00-30.0 nM: 3.57-12.5 μg / mL.

[0009] Further, the preparation method of the gold palladium platinum nanoparticles is as follows:

[0010] (1) After mixing the gold salt solution and the cetyltrimethylammonium bromide solution, an ice-cold reducing agent solution is added, and after the solution turns brown, a small gold seed solution is obtained;

[0011] (2) After mixing the gold salt solution, the cetyltrimethylammonium chloride solution and the reducing agent solution, the small gold seed solution is added, and after standing, the product is collected by centrifugation, resuspended, and a wine red gold seed solution is obtained;

[0012] (3) The gold seed solution is mixed with the cetyltrimethylammonium chloride solution, the palladium salt solution, the platinum salt solution and the reducing agent solution, and is left to stand and age at 50-70℃ for 2-4h; after aging, the product is collected by centrifugation, washed, and the gold palladium platinum nanoparticles are obtained.

[0013] Further, in step (1), the concentration ratio of the gold salt solution, the cetyltrimethylammonium bromide solution and the reducing agent solution is 0.5:200:10, and the volume ratio is 5:5:0.6;

[0014] In step (2), the concentration ratio of the gold salt solution, the cetyltrimethylammonium chloride solution, the reducing agent solution and the small gold seed solution is 0.5:200:100:0.24, and the volume ratio is 4:4:3:0.05;

[0015] In step (3), the concentration ratio of the gold seed solution, the cetyltrimethylammonium chloride solution, the palladium salt solution, the platinum salt solution and the reducing agent solution is 1:20:20:20:100, and the volume ratio is 0.5:2:0.05:0.05:1.

[0016] Further, in step (1), the reducing agent solution is a sodium borohydride solution;

[0017] In steps (2) and (3), the reducing agent solution is an ascorbic acid solution.

[0018] Further, the preparation method of the composite nanoprobe is as follows: the fluorescent dye modified methicillin-resistant Staphylococcus aureus nucleic acid aptamer and the gold palladium platinum nanoparticle are mixed, and the mixture is placed in dark for 0-25 min to obtain the composite nanoprobe.

[0019] The application further provides application of the composite nanoprobe in preparation of a kit for detecting methicillin-resistant Staphylococcus aureus.

[0020] The application further provides a kit for detecting methicillin-resistant Staphylococcus aureus, wherein the kit comprises the composite nanoprobe.

[0021] The application further provides a use method of the kit for non-disease diagnosis purposes, comprising the following steps:

[0022] S1, the sample to be detected is mixed with the composite nanoprobe to obtain a detection system, and the fluorescence intensity of the detection system at 775 nm wavelength is measured after the mixture is placed in dark for 10-50 min, and the fluorescence intensity is recorded as F1;

[0023] S2, the fluorescence intensity of the fluorescent dye Cy7 modified methicillin-resistant Staphylococcus aureus nucleic acid aptamer at 775 nm wavelength is detected, and the fluorescence intensity is recorded as F0; the fluorescence intensity of the composite nanoprobe at 775 nm wavelength is detected, and the fluorescence intensity is recorded as F;

[0024] S3, the fluorescence recovery efficiency is calculated according to (F1-F) / (F0-F), and the methicillin-resistant Staphylococcus aureus concentration in the sample to be detected is calculated by substituting into a standard curve prepared from a standard methicillin-resistant Staphylococcus aureus solution.

[0025] Further, in the detection system, the final concentration of the fluorescent dye Cy7 modified methicillin-resistant Staphylococcus aureus aptamer is 5.00-30.0 nM, the final concentration of the gold palladium platinum nanoparticle is 3.57-12.5 μg / mL, and the pH is 6.0-8.5.

[0026] The present application discloses the following technical effects:

[0027] The present application designs a nucleic acid aptamer (SEQ ID NO. 1) specifically recognizing the PBP2α site of methicillin-resistant Staphylococcus aureus (MRSA), uses a gold palladium platinum (AuPdPt) nanoparticle as an energy acceptor, uses a fluorescent dye Cy7 modified MRSA nucleic acid aptamer (Cy7-aptamer, Cy7-apt) as an energy donor, and specifically recognizes the MRSA element. When the AuPdPt nanoparticle is close to the Cy7, the Cy7 fluorescence is efficiently quenched by the surface energy transfer of the nano metal and the internal filter effect; when the MRSA exists, the Cy7-apt specifically recognizes the MRSA and pulls away the energy donor-acceptor distance, causing the Cy7 fluorescence signal to recover, thereby constructing a specific recognition and rapid detection fluorescence sensing detection method. The principle of this sensitization and noise reduction makes the detection method of the present application not only break through the limitation of the existing sensitivity, but also accelerate the detection speed, and the linear range of MRSA detection is 10 1 -10 6 CFU / mL, and the detection limit is as low as 8 CFU / mL; and compared with the plate culture method required by the clinic for more than 48 hours, the present application can realize the integration of rapid specific recognition and detection of MRSA within 55 minutes, without being carried out in multiple steps, and the detection time is greatly shortened. In addition, the detection method of the present application has low cost, wide application potential and prospect, and provides a new strategy for the rapid detection and precise prevention and control of MRSA in the clinic. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0029] Figure 1 It is the scanning electron microscope image (a) and the particle size distribution diagram (b) of the AuPdPt nanoparticle;

[0030] Figure 2 It is the transmission electron microscope image of the AuPdPt nanoparticle;

[0031] Figure 3 Figure 4 is a chart of the visible-near infrared absorption spectrum of AuPdPt nanoparticles and the excitation and emission spectrum of Cy7-apt;

[0032] Figure 4 Figure 6 is the results of the feasibility verification of the fluorescent sensing detection method of the present application;

[0033] Figure 5 Figure 7 is the results of the final concentration optimization of AuPdPt nanoparticles in the fluorescent sensing detection method of the present application;

[0034] Figure 6 Figure 8 is the results of the final concentration condition optimization of aptamer in the fluorescent sensing detection method of the present application;

[0035] Figure 7 Figure 9 is the results of the detection system optimization in the fluorescent sensing detection method of the present application;

[0036] Figure 8 Figure 10 is the results of the fluorescence quenching time optimization in the fluorescent sensing detection method of the present application;

[0037] Figure 9 Figure 11 is the results of the fluorescence recovery time condition optimization in the fluorescent sensing detection method of the present application;

[0038] Figure 10 Figure 12 is the standard curve of the fluorescent sensing detection method of the present application;

[0039] Figure 11 Figure 13 is the specificity detection results of the fluorescent sensing detection method of the present application;

[0040] Figure 12 Figure 14 is the anti-interference ability detection results of the fluorescent sensing detection method of the present application. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the present application and should not be construed to limit the scope of the present application, and are understood to be a more detailed description of certain aspects, features and embodiments of the present application.

[0042] It should be understood that the terms used in the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within any stated range or within any stated intermediate value, as well as any other stated value or intermediate value within the stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently from the range.

[0043] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the disclosure would understand. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the case of conflict between the present specification and any incorporated document, the present specification controls.

[0044] Many modifications and variations of the present disclosure described in the detailed description of the specification can be made without departing from the scope or spirit of the present disclosure, which will be apparent to those skilled in the art. Other implementations of the disclosure will be apparent to those skilled in the art from the specification. The specification and examples of the disclosure are illustrative only.

[0045] As used herein, "comprise", "comprising", "have", "having", "include", "including", "contain", "containing", and the like, are open-ended terms that are intended to mean including, but not limited to.

[0046] The technical principle of the present disclosure is as follows:

[0047] The present disclosure designs a nucleic acid aptamer (SEQ ID NO. 1) that specifically recognizes the PBP2α site of MRSA, uses gold-palladium-platinum (AuPdPt) nanoparticles as an energy acceptor, uses fluorescent dye Cy7 to modify the MRSA nucleic acid aptamer (Cy7-aptamer, Cy7-apt) as an energy donor based on the characteristic of AuPdPt nanoparticles with wide absorption of local surface plasmon resonance, and specifically recognizes the MRSA element. The excitation spectrum and emission spectrum of AuPdPt nanoparticles have different degrees of overlap with Cy7. When the distance between the two is close (usually <10 nm), the Cy7 fluorescence is efficiently quenched by the synergistic effect of nanometal surface energy transfer and inner filter effect; when MRSA is present, Cy7-apt can specifically recognize and pull away the donor-acceptor distance, causing the Cy7 fluorescence signal to recover, thereby constructing a fluorescence sensing detection method for specific recognition and rapid detection. The principle of this sensitization and noise reduction makes the detection method of the present disclosure not only break through the limitation of the existing sensitivity, but also accelerate the detection speed. The linear range of MRSA detection is 10 1 -10 6 CFU / mL, the detection range is wide, and the detection limit is as low as 8 CFU / mL, and the rapid high-sensitivity detection and specific recognition of MRSA can be realized within 55 min. In addition, the detection method of the present disclosure has low cost, and has broad application potential and prospect.

[0048] The reagents or materials involved in the following examples, if no special instructions, can be purchased through the conventional channels; the experimental methods involved, if no special instructions, are the conventional technical methods in the art.

[0049] Example 1 Synthesis of gold palladium platinum (AuPdPt) nanoparticles

[0050] Take 5 mL of 0.5 mM chloroauric acid solution and 5 mL of 200 mM cetyltrimethylammonium bromide solution into a 20 mL glass bottle, then quickly add 0.6 mL of 10 mM sodium borohydride solution (ice water preparation), the solution immediately turns light brown, and the initial small gold seed (0.24 mM) is obtained. Then take 4 mL of 0.5 mM chloroauric acid solution, 4 mL of 200 mM cetyltrimethylammonium chloride solution and 3 mL of 100 mM ascorbic acid solution into a 20 mL glass bottle, add 50 μL of 0.24 mM initial small gold seed, and stand at room temperature for 30 min, then centrifuge at 13000 rpm for 70 min, and disperse the lower precipitate into 2 mL of ultrapure water to prepare a wine red gold seed solution (1 mM).

[0051] Then, take 500 μL of 1 mM gold seed, 2 mL of 20 mM cetyltrimethylammonium chloride solution, 50 μL of 20 mM chloroplatinic acid solution, 50 μL of 20 mM chloroplatinic acid solution and 1 mL of 100 mM ascorbic acid solution into a 20 mL glass bottle, and place it in a 60°C water bath for 3h, during which the solution can be observed to slowly turn black. After the reaction is completed, centrifuge at 13000 rpm for 10 min, collect the lower precipitate and wash once with ultrapure water, and finally disperse the product in 1 mL of ultrapure water to prepare AuPdPt nanoparticles. The obtained AuPdPt nanoparticle solution is black, and the concentration is 250 μg / mL.

[0052] Example 2 Structural characterization of AuPdPt nanoparticle material

[0053] Take 4 mL of 15.0 μg / mL AuPdPt nanoparticle solution, add 2.5% glutaraldehyde, and store in a 4°C refrigerator overnight. Dehydrate at 50%, 70%, 85%, 90% and 100% alcohol concentrations for 10 minutes, then spray gold, and observe under a scanning electron microscope (SEM).

[0054] Take 4 mL of 15.0 μg / mL AuPdPt nanoparticle solution, add 2.5% glutaraldehyde, and store in a 4°C refrigerator overnight. Then slice, and observe under a transmission electron microscope (TEM).

[0055] SEM images are shown in FIG. 2a and FIG. 2b. Figure 1 As shown in FIG. 2a, the AuPdPt nanoparticles are uniformly distributed and have good dispersibility. The particle size distribution of the AuPdPt nanoparticles was obtained by using Image J to count the particle size distribution of the AuPdPt nanoparticles, and the particle size distribution graph of the AuPdPt nanoparticles is shown in FIG. 2c. Figure 1 As shown in FIG. 2b, the average particle size of the AuPdPt nanoparticles is 32.9 ± 8.9 nm. The TEM images are shown in FIG. 2d and FIG. 2e. Figure 2 As shown in FIG. 2d, the white dots in the center of the AuPdPt nanoparticles and the dendritic core-shell structure outside the AuPdPt nanoparticles are shown. The dendritic structure outside the AuPdPt nanoparticles is composed of a large number of small branches, and has a core-shell structure, indicating that the AuPdPt nanoparticles are successfully synthesized.

[0056] Example 3 Visible-near infrared absorption spectrum of AuPdPt nanoparticles and excitation spectrum and emission spectrum of Cy7-apt

[0057] The MRSA aptamer was synthesized by Shengwo Bioengineering (Shanghai) Co., Ltd., and the sequence is as follows:

[0058] 5'-CCATCCACACTCCGCAAGGGTGCCCCGGGGGGCTGTTCAGCGTGGTGGTGGGATGCCGTTTTGGTCCTTAGTCTCCGTCGTCGGCTGCCTCTACAT-3' (SEQ ID NO. 1).

[0059] The freeze-dried powder of the fluorescent dye Cy7 modified MRSA aptamer (Cy7-apt) was added with water to prepare a mother liquor with a concentration of 100 μM, and then diluted with a phosphate buffer (10 mM) with a pH of 7.4 to a corresponding concentration for use.

[0060] AuPdPt nanoparticles with a concentration of 31.25 μg / mL were prepared, and their visible-near infrared absorption spectrum in the range of 500-900 nm was determined. A solution of 25 nM Cy7-apt was prepared to determine its excitation spectrum and emission spectrum. The wavelength was taken as the abscissa, the ultraviolet absorption intensity was taken as the left ordinate, and the fluorescence intensity was taken as the right ordinate to plot the graph. The results are shown in FIG. 3. Figure 3 It can be seen that the excitation and emission spectrum of Cy7-apt and the visible-near infrared absorption spectrum of AuPdPt nanoparticles have a certain degree of overlap, which indicates the possibility of nanometal surface energy transfer and inner filter effect of the composite nanoprobe, and lays a foundation for subsequent establishment of a fluorescence sensing detection method based on MRSA aptamer and AuPdPt nanoparticles for detection of MRSA.

[0061] Example 4 Feasibility verification of constructing a fluorescence sensing detection method based on aptamer functionalized AuPdPt nanoparticles

[0062] 1. Establishment of a fluorescence sensing detection method based on AuPdPt nanoparticles functionalized with aptamer

[0063] S1, Preparation of composite nanoprobe: 100 μL of PBS (10 mM, pH 7.4), 50 μL of Cy7-apt (200 nM), and 40 μL of AuPdPt nanoparticles (125 μg / mL) were placed in a brown Ep tube, vortexed to mix, and placed in the dark for 20 min (to quench fluorescence), to obtain the composite nanoprobe.

[0064] S2, Mix the composite nanoprobe with 40 μL of the sample to be tested, add ultrapure water, and vortex to mix. The total volume was supplemented to 400 μL, vortexed to mix, and placed in the dark for 20 min (to restore fluorescence). The fluorescence spectrum of the system in the range of 750-820 nm was measured.

[0065] 2. Feasibility verification experiment

[0066] LB broth medium and LB agar solid medium were prepared. The LB broth liquid medium was sterilized by 121℃ high pressure steam, and cooled to room temperature for use. 10 μL of MRSA strain was inoculated into 5 mL of high-pressure sterilized LB broth medium, and then cultured in a 37℃ constant temperature shaking incubator for a certain time at a speed of 220 rpm. The OD 600 of the bacteria was measured by a micro UV spectrophotometer. 600 When the OD 8 of the bacteria increased to 0.5, the bacteria were in the logarithmic growth phase, i.e., 10 7 colony forming units per milliliter (CFU / mL). The bacteria were diluted to 1×10

[0067] The experimental setup was grouped as follows: Cy7-apt, Cy7-apt+MRSA, Cy7-apt+AuPdPt, MRSA+AuPdPt, MRSA, AuPdPt, and Cy7-apt+AuPdPt+MRSA. Among them, the Cy7-apt group used PBS instead of AuPdPt nanoparticles and the sample to be tested; the Cy7-apt+MRSA group used PBS instead of AuPdPt nanoparticles; the Cy7-apt+AuPdPt group used PBS instead of the sample to be tested; the MRSA+AuPdPt group used PBS instead of Cy7-apt; the MRSA group used PBS instead of AuPdPt nanoparticles and Cy7-apt, and the AuPdPt group used PBS instead of Cy7-apt and the sample to be tested.

[0068] The results are shown in Figure 4It can be seen that the maximum emission wavelength of Cy7-apt is 775 nm, and the fluorescence of Cy7-apt is effectively quenched after adding AuPdPt nanoparticles. When the system exists MRSA, the fluorescence of the system is significantly recovered. The calculated fluorescence quenching efficiency is 83.58%, and the fluorescence recovery efficiency is 70.27%. It is proved that the fluorescence sensing detection method of the application is feasible for quantitative detection of MRSA.

[0069] Example 5 Optimization of detection conditions of fluorescence sensing detection method based on nucleic acid aptamer functionalized AuPdPt nanoparticles

[0070] Based on the research results of Example 4, the detection conditions of the fluorescence sensing detection method are optimized. The experimental setting groups are: Cy7-apt, Cy7-apt+AuPdPt and Cy7-apt+AuPdPt+MRSA. The optimization process is as follows:

[0071] 1. AuPdPt nanoparticle concentration optimization

[0072] By adjusting the concentration of AuPdPt nanoparticle mother liquor in the preparation of composite nanoprobe, the final concentration of AuPdPt nanoparticles in the detection system (400 μL) is 3.57 μg / mL, 4.17 μg / mL, 5.00 μg / mL, 6.25 μg / mL, 8.33 μg / mL, and 12.5 μg / mL, respectively. The fluorescence intensity at 775 nm is detected.

[0073] The results are shown in Figure 5 It can be seen that with the increase of the concentration of AuPdPt nanoparticles, the fluorescence quenching efficiency of the composite nanoprobe gradually increases; but due to the increase of the concentration of the quencher AuPdPt nanoparticles, the fluorescence recovery efficiency gradually decreases when detecting MRSA. When the final concentration of AuPdPt nanoparticles is 8.44 μg / mL, the quenching efficiency reaches 90.1%, and the recovery efficiency is 84.9%, which has good fluorescence quenching and recovery efficiency, so 8.44 μg / mL is selected as the optimal concentration of AuPdPt nanoparticles.

[0074] 2. Cy7-apt concentration optimization

[0075] By adjusting the concentration of Cy7-apt mother liquor in the preparation of composite nanoprobe, the final concentration of Cy7-apt in the detection system (400 μL) is 5.00 nM, 10.0 nM, 15.0 nM, 20.0 nM, 25.0 nM, and 30.0 nM, respectively. The fluorescence intensity at 775 nm is detected.

[0076] The results are shown in Figure 6It can be seen that when the final concentration of Cy7-apt is 15 nM, the quenching efficiency reaches 91.3%, and the recovery efficiency is 91.2%, which has good fluorescence quenching and recovery efficiency, so 15 nM is selected as the concentration of Cy7-apt.

[0077] 3. Optimization of system pH

[0078] When the final concentration of AuPdPt nanoparticles is 8.44 μg / mL and the final concentration of Cy7-apt is 15 nM, adjust the pH of the detection system to 6.0, 6.5, 7.0, 7.4, 8.0, and 8.5, respectively. Detect the fluorescence intensity at 775 nm.

[0079] The results are shown in Figure 7 It can be seen that the fluorescence recovery efficiency of the system decreases at pH 6.0 and 8.5, and the fluorescence intensity of the dye decreases slightly as the pH decreases. At pH 7.0, the fluorescence quenching efficiency reaches 91.5%, and the fluorescence recovery efficiency is 83.5%. At pH 7.4, the fluorescence quenching efficiency reaches 90.1%, and the fluorescence recovery efficiency is 80.6%. Since pH 7.4 is close to the pH of the physiological environment, pH 7.4 is selected as the optimal pH for subsequent experiments.

[0080] 4. Optimization of fluorescence quenching time

[0081] When the final concentration of AuPdPt nanoparticles is 8.44 μg / mL, the final concentration of Cy7-apt is 15 nM, and the pH of the detection system is 7.4, adjust the fluorescence quenching time to 0, 5, 10, 15, 20, and 25 min, respectively. Detect the fluorescence intensity of the composite nanoprobe at 775 nm.

[0082] The results are shown in Figure 8 It can be seen that within 0-25 min, the fluorescence quenching efficiency of the system gradually increases, and at 15 min, the fluorescence quenching efficiency reaches 86.1%, and remains stable. Therefore, 15 min is selected as the optimal time for the fluorescence quenching reaction of the system in the subsequent experiments.

[0083] 5. Optimization of fluorescence recovery time

[0084] When the final concentration of AuPdPt nanoparticles is 8.44 μg / mL, the final concentration of Cy7-apt is 15 nM, the pH of the detection system is 7.4, and the fluorescence quenching time is 15 min, adjust the fluorescence recovery time to 0, 10, 20, 30, 40, and 50 min, respectively. Detect the fluorescence intensity at 775 nm.

[0085] The results are shown in Figure 9It can be seen that the fluorescence recovery efficiency of the system gradually increased within 0-50 min, and reached 80.5% at 40 min, and remained stable, so 40 min was selected as the optimal incubation time for the fluorescence recovery of the system.

[0086] Example 6: Establishment of standard curve

[0087] Based on the optimization results of Example 5, MRSA was detected. Gradient concentration MRSA samples were set: 1 x 10 7 , 5 x 10 6 , 1 x 10 6 , 5 x 10 5 , 1 x 10 5 , 5 x 10 4 , 1 x 10 4 , 5 x 10 3 , 1 x 10 3 , 5 x 10 2 , 1 x 10 2 , 5 x 10 1 , 1 x 10 1 , 5 CFU / mL. The standard curve corresponding to the fluorescence recovery efficiency of the system and the concentration of MRSA was constructed.

[0088] The results are shown in Table 1. Figure 10 It can be seen that for MRSA with a concentration of 1 x 10 1 - 1 x 10 6 CFU / mL, as the concentration of MRSA increased, the fluorescence recovery efficiency of the system increased; the linear regression equation obtained by linear fitting of the fluorescence recovery efficiency of the system and the concentration of MRSA was: (F1-F) / (F0-F) = 0.0803lgc-0.0669(r = 0.991), where F0 is the initial fluorescence intensity of Cy7-apt; F is the quenched fluorescence intensity after adding AuPdPt nanoparticles; F1 is the recovered fluorescence intensity after adding MRSA; c is the concentration of MRSA. The detection limit of MRSA was calculated by the formula LOD = 3.3δ / S (δ is the standard deviation of 10 blank samples, and S is the slope of the standard curve), and the standard deviation of the blank sample signal was 0.00014, which was defined as the noise value. The calculated minimum detection limit was 8 CFU / mL.

[0089] Example 7: Specificity evaluation

[0090] Based on the optimization results of Example 5, specificity evaluation was performed. 10 7 CFU / mL of clinically common pathogenic bacteria Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, and Salmonella typhimurium were detected as interferents.

[0091] Results are shown in Table 1 Figure 11 It can be seen that only the fluorescence of MRSA recovers, and the fluorescence of the rest of the bacteria does not recover obviously, indicating that the method has good specificity.

[0092] Example 8 Anti-interference ability evaluation

[0093] Based on the optimization results of Example 5, the anti-interference ability evaluation was performed. The experimental setting groups were: Mix, MRSA, Mix+MRSA. Among them, the Mix group was the interference bacterial liquid (including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Salmonella typhimurium); the MRSA group was MRSA alone; the Mix+MRSA group was a mixed system in which MRSA was added to the Mix. The concentration of the interference bacterial liquid was 1×10 7 CFU / mL, and the concentration of MRSA was 1×10 7 CFU / mL.

[0094] Results are shown in Table 1 Figure 12 It can be seen that the presence of other bacteria has no obvious effect on the fluorescence signal response of the method for detecting MRSA. Therefore, the fluorescence sensing detection method constructed by the application has strong anti-interference ability for detecting MRSA.

[0095] Example 9 Detection of clinical complex samples

[0096] Low (1×10 3 CFU / mL), medium (1×10 5 CFU / mL), and high (1×10 7 CFU / mL) three groups of different standard concentrations of MRSA were set for experiments, and actual complex wound secretions (patient wound secretions collected from the University City Hospital Affiliated to Chongqing Medical University) were used as samples. The wound swab was placed in a soft plastic test tube and eluted repeatedly with 2 mL of PBS buffer. Actual complex wound secretion samples were prepared. MRSA was added according to the above concentrations. Each group was repeated three times, the fluorescence recovery efficiency was calculated according to the fluorescence intensity change of the system, and the standard addition recovery rate was calculated, which was (determined value of the sample with standard addition - determined value of the sample) / standard addition amount × 100%.

[0097] The results are shown in Table 1. It can be seen that the standard addition recovery rate of the method in the actual complex sample wound secretion is 90.67%-104.70%, and the relative standard deviation obtained by three parallel experiments is 0.69%-2.56% (n=3), indicating that the method has good accuracy in the application of detecting MRSA in actual complex sample wound secretion.

[0098] Table 1 Standard addition detection results of the method in actual complex sample wound secretion

[0099]

[0100] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the present application made by those skilled in the art, without departing from the design spirit of the present application, should fall within the scope of the present application as defined by the claims.

Claims

1. A composite nanoprobe for detecting methicillin-resistant Staphylococcus aureus, characterized in that, The composite nanoprobe is prepared by mixing methicillin-resistant Staphylococcus aureus nucleic acid aptamer modified by fluorescent dye Cy7 and gold palladium platinum nanoparticles after standing; The nucleotide sequence of the methicillin-resistant Staphylococcus aureus nucleic acid aptamer is shown as SEQ ID NO.

1.

2. The composite nanoprobe of claim 1, wherein, The ratio of the final concentrations of the methicillin-resistant Staphylococcus aureus nucleic acid aptamer modified by fluorescent dye Cy7 and the gold palladium platinum nanoparticles is 5.00-30.0 nM:3.57-12.5 μg / mL.

3. The composite nanoprobe of claim 1, wherein, The preparation method of the gold palladium platinum nanoparticles is as follows: (1) After mixing gold salt solution and cetyltrimethylammonium bromide solution, ice-cold reducing agent solution is added, and after the solution turns brown, small gold seed solution is obtained; (2) After mixing gold salt solution, cetyltrimethylammonium chloride solution and reducing agent solution, the small gold seed solution is added, and after standing, the product is collected by centrifugation, resuspended, and a wine red gold seed solution is obtained; (3) The gold seed solution is mixed with cetyltrimethylammonium chloride solution, palladium salt solution, platinum salt solution and reducing agent solution, and after standing at 50-70°C for 2-4 h, the product is collected by centrifugation, washed, and the gold palladium platinum nanoparticles are obtained.

4. The composite nanoprobe of claim 3, wherein, In step (1), the concentration ratio of the gold salt solution, cetyltrimethylammonium bromide solution and reducing agent solution is 0.5:200:10, and the volume ratio is 5:5:0.6; In step (2), the concentration ratio of the gold salt solution, cetyltrimethylammonium chloride solution, reducing agent solution and small gold seed solution is 0.5:200:100:0.24, and the volume ratio is 4:4:3:0.05; In step (3), the concentration ratio of the gold seed solution, cetyltrimethylammonium chloride solution, palladium salt solution, platinum salt solution and reducing agent solution is 1:20:20:20:100, and the volume ratio is 0.5:2:0.05:0.05:

1.

5. The composite nanoprobe of claim 3, wherein, In step (1), the reducing agent solution is sodium borohydride solution; In steps (2) and (3), the reducing agent solution is ascorbic acid solution.

6. The composite nanoprobe of claim 1, wherein, The preparation method of the composite nanoprobe is as follows: the methicillin-resistant Staphylococcus aureus nucleic acid aptamer modified by fluorescent dye is mixed with the gold palladium platinum nanoparticles, and after standing in the dark for 0-25 min, the composite nanoprobe is obtained.

7. The composite nanoprobe of any one of claims 1-6 for use in the preparation of a kit for detecting methicillin-resistant Staphylococcus aureus.

8. A kit for detecting methicillin-resistant Staphylococcus aureus, characterized by, The kit comprises the composite nanoprobe of any one of claims 1-6.

9. A method of using the kit of claim 8 for a non-disease diagnostic purpose, characterized by, The kit comprises the following steps: S1, mixing the sample to be tested with the composite nanoprobe to obtain a detection system, and after standing in the dark for 10-50 min, measuring the fluorescence intensity of the detection system at a wavelength of 775 nm, denoted as F1; S2, detecting the fluorescence intensity of the methicillin-resistant Staphylococcus aureus nucleic acid aptamer modified by fluorescent dye Cy7 at a wavelength of 775 nm, denoted as F0; and detecting the fluorescence intensity of the composite nanoprobe at a wavelength of 775 nm, denoted as F. S3, calculating the fluorescence recovery efficiency according to (F1-F) / (F0-F), substituting into the standard curve prepared by the standard methicillin-resistant Staphylococcus aureus solution, and calculating the methicillin-resistant Staphylococcus aureus concentration in the sample to be measured.

10. The method of use of claim 9, wherein, In the detection system, the final concentration of the methicillin-resistant Staphylococcus aureus aptamer modified by the fluorescent dye Cy7 is 5.00-30.0 nM, the final concentration of the gold palladium platinum nanoparticle is 3.57-12.5 μg / mL, and the pH is 6.0-8.5.

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