Nucleic acid aptamer of G4 structure methicillin-resistant staphylococcus aureus as well as screening method and application of nucleic acid aptamer

Abstract

The invention relates to a nucleic acid aptamer of G4 structure methicillin-resistant staphylococcus aureus and a screening method and application of the nucleic acid aptamer, the nucleic acid aptamer is at least one of MA02, MA05 and MA08, each nucleic acid aptamer is composed of 80 basic groups, and the sequences of the nucleic acid aptamers MA02, MA05 and MA08 are shown as SEQ ID NO.1-3. The nucleic acid aptamer can be combined with MRSA with high affinity and high specificity, has an application prospect in MRSA infection diagnosis, can be used for monitoring existence of MRSA in food and environment (such as water quality, soil and air), can be used for preparing a detection reagent of MRSA, and can be used as a drug carrier for delivery of targeted drugs. The method has wide application prospects in the fields of medicine, food safety, environmental monitoring and the like, and has important scientific, social and economic values.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a group of nucleic acid aptamers against methicillin-resistant Staphylococcus aureus with G4 structures, a screening method thereof and applications thereof. Background Art

[0002] Staphylococcus aureus (SA) is widely distributed globally and is often colonized on the skin, nostrils and mucosal surfaces of healthy individuals. It is one of the most common pathogenic bacteria causing infections and deaths. SA may cause hundreds of thousands to millions of severe invasive infections globally every year, and the number of mild skin infection cases caused by it is even difficult to count. In addition, SA not only has strong pathogenicity but can also infect patients through various routes, and is the main pathogen causing hospital- and community-acquired infections. In short, SA has strong invasive ability and a wide range of infections, posing a threat to the lives of patients and also presenting a huge challenge to public health.

[0003] Bacterial antibiotic resistance (AMR) is one of the major public health threats in the 21st century and is becoming increasingly serious with the abuse of antibiotics. According to a report in The Lancet, SA is one of the main pathogens causing AMR-related deaths, and the number of AMR-related deaths caused by SA infections exceeded 250,000 globally in 2019. Among them, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) has made clinical anti-infection treatment increasingly difficult. Compared with methicillin-sensitive Staphylococcus aureus, infections caused by MRSA have a higher mortality rate. Therefore, rapid and accurate identification of MRSA is of great significance for its prevention, control and treatment.

[0004] Nucleic acid aptamers are single-stranded oligonucleotide fragments (mainly DNA or RNA) with a length of about 25 - 80 bases, often forming complex secondary and even tertiary structures, and can bind to targets with high affinity and specificity. They are also called "chemical antibodies". Nucleic acid aptamers are obtained through iterative rounds of selection by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology. Compared with traditional antibodies, they have the advantages of high cost-effectiveness, easy functional modification, low immunogenicity and small size. Therefore, nucleic acid aptamers have broad application potential in detection and diagnosis.

[0005] The G-quadruplex (G4) is a special higher-order nucleic acid structure formed by the self-assembly of DNA or RNA sequences rich in guanine (G) through Hoogsteen hydrogen bonds. This structure plays an important role in genomic stability, gene expression regulation, and disease occurrence, and has become a research hotspot in the fields of molecular biology, chemical biology, and drug development in recent years. Nucleic acid aptamers based on G-quadruplexes have significant advantages in bacterial detection. They can recognize bacterial targets through their unique structures and achieve efficient detection by combining signal amplification strategies (such as DNAzyme catalysis, hybridization chain reaction, etc.). In addition, G4 aptamers exhibit good stability and anti-interference ability in complex biological environments (such as serum, urine), making them suitable for the rapid analysis of clinical samples. Summary of the Invention

[0006] The object of the present invention is to provide a nucleic acid aptamer against methicillin-resistant Staphylococcus aureus with high affinity, high specificity, and a G4 structure, as well as its screening method and application.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A nucleic acid aptamer against methicillin-resistant Staphylococcus aureus with a G4 structure, wherein the nucleic acid aptamer is at least one of MA02, MA05, and MA08. Each nucleic acid aptamer consists of 80 bases. The sequences of the nucleic acid aptamers MA02, MA05, and MA08 are as follows:

[0009] MA02:

[0010] 5'-AGCAGCACAGAGGTCAGATGGGAGGTGGTGGGAGGTGGGTTGGGGGG GCCTGCCTCGATCTTCACGGTAGCACGCATAGG-3' (SEQ ID NO.1), MA05:

[0011] 5'-AGCAGCACAGAGGTCAGATGGAGGGTAGGCGGGGGCGGGTTGTGGGGA TTCCTCTGGGTCTTCACGGTAGCACGCATAGG-3' (SEQ ID NO.2), MA08:

[0012] 5'-AGCAGCACAGAGGTCAGATGGGAGGTGGTGGGAGGTGGGTTGGGGGG GCCTGGCTCGATCTTCACGGTAGCACGCATAGG-3' (SEQ ID NO.3).

[0013] Preferably, the nucleic acid aptamer is nucleic acid aptamer MA05.

[0014] At 25 °C, 147 mM Na + , 5 mM Mg 2+ Under the conditions of, the secondary structures of the nucleic acid aptamers MA02, MA05, and MA08 are shown as follows:

[0015]

[0016] The three-dimensional structure simulation diagrams of the nucleic acid aptamers MA02, MA05, and MA08 are respectively as Figure 2 shown.

[0017] The 5'-end or 3'-end of at least one of the nucleic acid aptamers MA02, MA05, and MA08 is chemically modified with a fluorescent group, an amino group, biotin, digoxin, or polyethylene glycol.

[0018] The 5'-end of at least one of the nucleic acid aptamers MA02, MA05, and MA08 is modified with a FAM group.

[0019] Use of the nucleic acid aptamer in the preparation of a detection reagent or kit for methicillin-resistant Staphylococcus aureus.

[0020] Use of the nucleic acid aptamer in the preparation of a drug for treating methicillin-resistant Staphylococcus aureus-related diseases.

[0021] Use of the nucleic acid aptamer in the establishment of a detection method for methicillin-resistant Staphylococcus aureus.

[0022] The detection method is the G4-hemin DNAzyme colorimetric method.

[0023] Use of the nucleic acid aptamer in the diagnosis of methicillin-resistant Staphylococcus aureus infection.

[0024] The screening method for the nucleic acid aptamer of methicillin-resistant Staphylococcus aureus with a G4 structure is based on the in vitro Cell-SELEX screening technology of nucleic acid aptamers, using the whole MRSA cells as the target, and screening for nucleic acid aptamers that specifically bind to MRSA from a random ssDNA library.

[0025] Compared with the prior art, the advantages of the present invention are as follows:

[0026] 1. The nucleic acid aptamer is non-toxic, has a small molecular weight, good permeability, and is easy to synthesize and label.

[0027] 2. The synthesis cost of the nucleic acid aptamer is lower than that of antibody preparation, and it has a short cycle and good reproducibility.

[0028] 3. The aptamer can bind to MRSA with high affinity and specificity, and its Kd values are 6.13 nM (MA02), 3.71 nM (MA05), and 12.72 nM (MA08) respectively, showing application prospects in the diagnosis of MRSA infection.

[0029] 4. The aptamer can be used to monitor the presence of MRSA in foods and environments (such as water quality, soil, and air), prepare detection reagents for MRSA, and serve as a drug carrier for targeted drug delivery, showing broad application prospects in multiple fields such as medicine, food safety, and environmental monitoring, and having important scientific, social, and economic values. Brief Description of the Drawings

[0030] Figure 1 It is the secondary structure diagram of the aptamer of the present invention.

[0031] Figure 2 It is the three-dimensional structure simulation diagram of the aptamer of the present invention.

[0032] Figure 3 It is the flow cytometry result diagram of the binding between the screening library of the present invention and MRSA.

[0033] Figure 4 It is the specific analysis result diagram of the aptamer of the present invention.

[0034] Figure 5 It is the affinity detection result diagram of the aptamer of the present invention.

[0035] Figure 6 It is the Cell-SELEX screening strategy diagram of the aptamer of the present invention.

[0036] Figure 7 It is the circular dichroism spectrum characterization of the G4 aptamer structure of the present invention.

[0037] Figure 8 It is the feasibility of the G4-hemin DNAzyme catalyzed TMB color development of the present invention.

[0038] Figure 9 It is the schematic diagram of the G4-hemin DNAzyme colorimetric method for detecting MRSA of the present invention.

[0039] Figure 10 It is the sensitivity and specificity of the G4-hemin DNAzyme colorimetric method for detecting MRSA of the present invention, where a) is the characterization of the colorimetric method sensitivity; (b) is the characterization of the colorimetric method specificity, ***: P < 0.001. Detailed Embodiments

[0040] The following will describe the content of the present invention in detail with reference to the accompanying drawings of the specification and examples:

[0041] A nucleic acid aptamer against methicillin-resistant Staphylococcus aureus with a G4 structure, wherein the nucleic acid aptamer is at least one of MA02, MA05, and MA08, each nucleic acid aptamer consists of 80 bases, and the sequences of the nucleic acid aptamers MA02, MA05, and MA08 are as follows:

[0042] MA02:

[0043] 5’-AGCAGCACAGAGGTCAGATGGGAGGTGGTGGGAGGTGGGTTGGGGGG GCCTGCCTCGATCTTCACGGTAGCACGCATAGG-3’, MA05:

[0044] 5’-AGCAGCACAGAGGTCAGATGGAGGGTAGGCGGGGGCGGGTTGTGGGG ATTCCTCTGGGTCTTCACGGTAGCACGCATAGG-3’, MA08:

[0045] 5’-AGCAGCACAGAGGTCAGATGGGAGGTGGTGGGAGGTGGGTTGGGGGG GCCTGGCTCGATCTTCACGGTAGCACGCATAGG-3’.

[0046] At 25°C, 147 mM Na + , 5 mM Mg 2+ conditions, the simulation results of the secondary structure and three-dimensional structure of the nucleic acid aptamers MA02, MA05, and MA08 are as shown in Figure 1 , 2 shown, where Figure 2 amplify the part with a special structure.

[0047] The screening method of the nucleic acid aptamer is based on the in vitro Cell-SELEX screening technology of nucleic acid aptamers, using the whole MRSA cells as the target, and screening the nucleic acid aptamers specifically binding to MRSA from a random ssDNA library.

[0048] The screening method of the nucleic acid aptamer against methicillin-resistant Staphylococcus aureus with a G4 structure, which includes the following steps (see the Cell-SELEX screening strategy diagram of the nucleic acid aptamer in Figure 6 ):

[0049] ① Incubate common pathogenic bacteria with the ssDNA library for reverse SELEX screening to remove non-specifically binding ssDNA and increase the specificity of the aptamer. Then, separate the bacteria and the ssDNA library in the supernatant by high-speed centrifugation.

[0050] ② The positive SELEX screening was carried out by incubating MRSA with the ssDNA library after reverse screening, and then multiple washes were performed to remove unbound or weakly bound ssDNA.

[0051] ③ The washed MRSA-ssDNA complex was eluted thermally, and the supernatant was taken as a template. The upstream primer P3 modified with fluorescence (FAM) and the downstream primer P4 modified with biotin were added for PCR amplification. The PCR amplification product was incubated with streptavidin magnetic beads to separate the FAM-modified target strand and the biotin-modified complementary strand, which served as the secondary ssDNA library for the next round of SELEX screening.

[0052] The secondary ssDNA library obtained in step ③ was put into the next round for cyclic screening, and reverse screening was only carried out in the 4th - 7th rounds. After the 9th round of SELEX screening, using the library 9ssDNA after the 9th round of SELEX screening as a template, amplification was carried out with primers P1 and P2, and then the product was subjected to high-throughput sequencing.

[0053] Among them, for the ssDNA library, the two ends are 20nt fixed primer sequences, and the middle 40nt is a random sequence, and its sequence is: 5’-AGCAGCACAGAGGTCAGATG-N40-TTCACGGTAGCACGCATAGG-3’;

[0054] The primers P1, P2, P3, and P4 are respectively:

[0055] Primer P1: 5’-AGCAGCACAGAGGTCAGATG-3’

[0056] Primer P2: 5’-CCTATGCGTGCTACCGTGAA-3’

[0057] Primer P3: 5’-FAM-AGCAGCACAGAGGTCAGATG-3’

[0058] Primer P4: 5’-biotin-CCTATGCGTGCTACCGTGAA-3’

[0059] The following specifically describes in detail the in vitro screening of MRSA nucleic acid aptamers of the present invention, the specificity between the nucleic acid aptamers and MRSA, etc. in combination with specific embodiments.

[0060] The formulations of the related reagents involved in the embodiments of the present invention are as follows:

[0061] (1) 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 5 mM MgCl2, pH 7.4.

[0062] (2) LB liquid medium (100 mL): 1 g tryptone, 0.5 g yeast extract, 1 g NaCl.

[0063] The experimental strains involved are as follows:

[0064] Methicillin-resistant Staphylococcus aureus (BNCC 330041) was purchased from Beijing NaChuangLian Biotechnology Co., Ltd. Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853) are the strains preserved in this laboratory.

[0065] The main experimental reagents involved are as follows:

[0066]

[0067] Example 1 In vitro screening of MRSA nucleic acid aptamers

[0068] 1) Synthesize the ssDNA library in vitro. The two ends are 20-nt fixed primer sequences, and the middle is a 40-nt random sequence:

[0069] 5’-AGCAGCACAGAGGTCAGATG-N40-TTCACGGTAGCACGCATAGG-3’, and the library capacity is about 10 24 ;

[0070] Primer P1: 5’-AGCAGCACAGAGGTCAGATG-3’

[0071] Primer P2: 5’-CCTATGCGTGCTACCGTGAA-3’

[0072] Primer P3: 5’-FAM-AGCAGCACAGAGGTCAGATG-3’

[0073] Primer P4: 5’-biotin-CCTATGCGTGCTACCGTGAA-3’

[0074] The above nucleic acid sequences were commissioned to be synthesized by Shanghai Sangon Biotech Co., Ltd. and purified by HPLC.

[0075] 2) Preparation of MRSA bacterial suspension: Pick MRSA colonies and add them to peptone (LB) culture medium. Culture at 37°C and 180 rpm until the logarithmic growth phase. Centrifuge and wash multiple times to remove the culture medium. Adjust the OD 600 to 0.1 (about 10 8 cfu / ml MRSA).

[0076] 3) Dissolve the random ssDNA library in an EP tube with Binding Buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 5 mM MgCl2, pH 7.4). Denature at 95°C for 10 min and slowly cool to room temperature. Mix the ssDNA library with 10 8 cfu of MRSA, incubate on a shaker at room temperature for 1 h, and discard the supernatant after high-speed centrifugation. Wash away the unbound ssDNA with Binding Buffer, add ddH2O, boil in a water bath for 10 min, and centrifuge at 4°C to aspirate the supernatant. Before starting the forward screening in rounds 4 - 7, first incubate the ssDNA library with common pathogenic bacteria for 60 min for reverse screening, and then centrifuge to take the supernatant for forward screening.

[0077] 4) Since the components and concentrations of the SELEX screening products vary in each round, it is necessary to optimize the number of PCR amplification cycles before large-scale PCR amplification of the secondary ssDNA library. Use the supernatant eluted from MRSA by heat as a template and P1 and P2 as primers for qPCR amplification. Among them, the qPCR reaction system (20 μL) is as follows:

[0078]

[0079] The qPCR reaction conditions are pre-denaturation at 95°C for 60 s, denaturation at 95°C for 20 s, annealing at 57°C for 20 s, extension at 72°C for 20 s, for 25 cycles, and then extension at 72°C for 120 s.

[0080] 5) The cycle number that generates 50 - 75% of the maximum fluorescence during qPCR amplification is the optimal cycle number for large-scale amplification. Use the supernatant eluted from MRSA by heat as a template and P3 and P4 as primers for PCR amplification. Among them, the PCR reaction system (1000 μL, aliquoted into 100 μL for PCR amplification) is as follows:

[0081]

[0082] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 90 s, denaturation at 94°C for 20 s, annealing at 57°C for 20 s, extension at 72°C for 20 s, with 10 - 18 cycles, and then extension at 72°C for 120 s.

[0083] 6) Transfer the PCR amplification product to a 1.5 mL EP centrifuge tube, add 100 μL of streptavidin magnetic beads and 350 μL of 4 M NaCl, mix well at room temperature for 30 min, and separate the magnetic phase to discard the supernatant. Add NaOH to denature the DNA, separate the magnetic phase to recover the supernatant, and add HCl and 2×Binding Buffer to the supernatant for neutralization. Mix well to obtain the secondary ssDNA library, and after measuring the concentration, use it for the next round of screening.

[0084] 7) During the screening process, flow cytometry was used to monitor the screening progress. The operation was as follows: Incubate 100 pmol of the ssDNA library (initial, round 1, round 3, round 5, round 7, round 9) with 10^7 cfu of MRSA in 200 μL of Binding Buffer for 60 min. Discard the supernatant by centrifugation, resuspend with Binding Buffer, and detect on a flow cytometer. Statistically analyze the positive rate (for the flow cytometry results of the binding of the screening library to MRSA, see Figure 3 ).

[0085] 8) After the 9th round of screening, perform PCR amplification on the screening product (library 9 ssDNA ) using P1 and P2 as primers. Among them, the PCR reaction system (100 μL) was as follows:

[0086]

[0087] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 90 s, denaturation at 94°C for 20 s, annealing at 57°C for 20 s, extension at 72°C for 20 s, with 15 cycles, and then extension at 72°C for 120 s.

[0088] Send the PCR product to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0089] Perform multiple sequence alignment on the 40 sequences with the highest enrichment degree in the sequencing results using ClustalX2 software, and conduct homology analysis. Simulate the secondary structure of the nucleic acid aptamer on the web page nupack (https: / / www.nupack.org / ), with the conditions of 25°C, 147 mM Na + and 5 mM Mg 2+ .

[0090] The inventors performed specificity and affinity analyses on the 40 sequences with the highest enrichment levels (the analysis methods were similar to those in Examples 2 and 3). After comprehensive analysis, only the sequences MA02, MA05, and MA08 had the best specificity and affinity. Therefore, these 3 sequences with the best affinity were selected for circular dichroism spectroscopy to characterize their structures. To better illustrate the specificity and affinity of the aptamers MA02, MA05, and MA08, the inventors performed the following analyses on the specificity and affinity of the aptamers MA02, MA05, and MA08, as shown in Examples 2 and 3.

[0091] Example 2 Detection of the Specificity of Aptamers by Flow Cytometry

[0092] 1) Entrusted Shanghai Sangon Biotech Co., Ltd. to synthesize FAM-modified aptamers with the following sequences:

[0093] MA02:

[0094] 5’-FAM-AGCAGCACAGAGGTCAGATGGGAGGTGGTGGGAGGTGGGTT GGGGGGGCCTGCCTCGATCTTCACGGTAGCACGCATAGG-3’

[0095] MA05:

[0096] 5’-FAM-AGCAGCACAGAGGTCAGATGGAGGGTAGGCGGGGGCGGGTT GTGGGGATTCCTCTGGGTCTTCACGGTAGCACGCATAGG-3’

[0097] MA08:

[0098] 5’-FAM-AGCAGCACAGAGGTCAGATGGGAGGTGGTGGGAGGTGGGTT GGGGGGGCCTGGCTCGATCTTCACGGTAGCACGCATAGG-3’

[0099] Dilute the aptamers to 25 nM, denature at 95 °C for 10 min, and slowly cool to room temperature.

[0100] 2) Take 10 7 CFU of MRSA, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa respectively, and incubate with the aptamers on a shaker at room temperature for 1 h. After high-speed centrifugation, aspirate and discard the supernatant. Wash away the unbound ssDNA, and resuspend the bacteria in Binding Buffer. Use the corresponding bacteria without adding aptamers as negative controls, and detect them on a flow cytometer to count the positive rate.

[0101] 3) The positive rate of the binding of bacteria to nucleic acid aptamers was plotted using GraphPad Prism 8.0.2 software. As Figure 4 shown, the positive rate of the binding of nucleic acid aptamers to MRSA was higher than that of other pathogenic bacteria, indicating that the nucleic acid aptamers MA02, MA05, and MA08 of the present invention can bind to MRSA with high specificity.

[0102] Example 3 Detection of the affinity of nucleic acid aptamers by flow cytometry

[0103] 1) The nucleic acid aptamers were serially diluted to 8 concentration gradients of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM, denatured at 95 °C for 10 min, and slowly cooled to room temperature.

[0104] 2) Take 10 7 CFU of MRSA and incubate with nucleic acid aptamers at different concentrations on a shaker at room temperature for 1 h. After high-speed centrifugation, the supernatant was aspirated. The unbound ssDNA was washed away, and MRSA was resuspended in Binding Buffer. MRSA without added nucleic acid aptamer was used as a negative control, and the positive rate was counted by flow cytometry.

[0105] 3) The positive rate of the binding of bacteria to nucleic acid aptamers was plotted using GraphPad Prism 8.0.2 software and the Kd value was calculated.

[0106] As Figure 5 shown, the Kd values of the nucleic acid aptamers were 6.13 nM (MA02), 3.71 nM (MA05), and 12.72 nM (MA08), respectively. It can be seen that the nucleic acid aptamer MA05 has the highest affinity.

[0107] Example 4 Detection of MRSA by G4-hemin DNAzyme method

[0108] Experimental method

[0109] (1) The structure of the nucleic acid aptamer was characterized by circular dichroism (CD). First, the nucleic acid aptamer was diluted to 5 μmol / L, thermally denatured at 95 °C for 10 min, and slowly cooled to room temperature. Take 200 μL of the sample and detect it on the machine at 25 °C, and record the spectral data. The instrument parameters were set as follows: wavelength range 200 - 330 nm, bandwidth 0.1 nm, response time 1 s, and each sample was scanned 3 times for repetition. Baseline correction was performed using the buffer before detection, and the CD spectra of all detection results were baseline-subtracted ( Figure 7 Circular dichroism characterization of the G4 nucleic acid aptamer structure).

[0110] (2) To verify that the nucleic acid aptamer and hemin can form a DNAzyme with peroxidase activity and catalyze the color development of TMB, the nucleic acid aptamer was first heated to 95 °C and then slowly cooled to room temperature to form a G-quadruplex. The nucleic acid aptamer and hemin (both with a final concentration of 2 μmol / L) were added to the TMB working solution, and the color reaction was directly observed after incubation at room temperature for 60 min. A control group without the nucleic acid aptamer and only containing the TMB working solution was set up to verify the specificity of the reaction ( Figure 8 Feasibility of color development of TMB catalyzed by G4-hemin DNAzyme).

[0111] (3) This method is based on the binding of a DNA nucleic acid aptamer with a G-quadruplex structure to hemin to form a DNAzyme, and the detection of MRSA is achieved by catalyzing the color reaction of TMB. The design principle is as follows: After a quantitative nucleic acid aptamer specifically binds to MRSA, the MRSA-nucleic acid aptamer complex is separated from the free nucleic acid aptamer. The G4 quadruplex structure of the free aptamer binds to hemin to form a DNzyme with peroxidase activity. Then, the DNAzyme catalyzes the oxidation of TMB by H2O2 to generate oxTMB, which changes from colorless to blue. By detecting the absorbance at 370 nm, MRSA can be quantitatively detected ( Figure 9 Schematic diagram of the colorimetric method for detecting MRSA by G4-hemin DNAzyme).

[0112] To characterize the sensitivity of the G4-hemin DNAzyme colorimetric method, the nucleic acid aptamer was first heated to 95 °C and then slowly cooled to room temperature to form a G-quadruplex. Then, MRSA bacterial suspensions with different concentrations (5×10 7 to 1×10 5 cfu / mL) were prepared, mixed with the nucleic acid aptamer (final concentration of 2 μmol / L), and incubated at room temperature for 30 min. The free nucleic acid aptamer in the supernatant was collected by centrifugation, and hemin and the TMB working solution were added for the color reaction. The absorbance at 370 nm was measured using an enzyme-linked immunosorbent assay reader. With the reciprocal of the MRSA concentration as the abscissa and the absorbance value at 370 nm as the ordinate, a standard curve was plotted to calculate the detection limit. To characterize the specificity of the G4-hemin DNAzyme colorimetric method, four common clinically pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, 10 7 cfu / mL) were mixed with the nucleic acid aptamer, and after the same treatment, the absorbance was measured and compared with the results of MRSA ( Figure 10 Sensitivity and specificity of the G4-hemin DNAzyme colorimetric method for detecting MRSA).

[0113] Experimental results

[0114] (1) The conformational analysis of the candidate nucleic acid aptamers was carried out by circular dichroism spectroscopy (CD). The characteristic spectrum of parallel G-quadruplex with a positive peak at 265 nm and a negative peak at 240 nm was detected, confirming the presence of G-quadruplex in the nucleic acid aptamers. The nucleic acid aptamers MA02, MA05, and MA08 obtained in the present invention are all in the G4 structure. Therefore, the nucleic acid aptamers MA02, MA05, and MA08 of the present invention have significant advantages in bacterial detection, and can recognize bacterial targets through their unique structures and achieve efficient detection by combining signal amplification strategies such as DNAzyme catalysis.

[0115] (2) In the verification experiment of the catalytic activity of G4-hemin DNAzyme, after the nucleic acid aptamer binds to hemin, DNAzyme is formed to catalyze the color development of TMB (the solution changes from colorless to blue), while there is no color reaction in the control group, proving that the G4-hemin complex has specific peroxidase activity.

[0116] (3) Sensitivity: In the range of 5×10 5 ~5×10 7 cfu / mL, the absorbance at 370 nm has a good linear relationship with the reciprocal of the MRSA concentration (R 2 = 0.990). Specificity: The absorbance of MRSA is significantly lower than that of the other four pathogenic bacteria (P<0.001), indicating that the method has high specificity.

[0117] In this study, a colorimetric detection system for MRSA based on G4-hemin DNAzyme was successfully constructed, providing a new strategy for rapid and specific detection.

[0118] The present invention includes but is not limited to the above embodiments. Any equivalent replacement or partial improvement carried out under the spirit and principle of the present invention will be regarded as within the protection scope of the present invention.

Claims

1. A nucleic acid aptamer for methicillin-resistant Staphylococcus aureus with G4 structure, characterized in that: The nucleic acid aptamer is at least one of MA02, MA05, and MA08, and the sequences of the nucleic acid aptamers MA02, MA05, and MA08 are respectively: MA02: 5'-AGCAGCACAGAGGTCAGATGGGAGGTGGTGGGAGGTGGGTTGGGGGG GCCTGCCTCGATCTTCACGGTAGCACGCATAGG-3', MA05: 5'-AGCAGCACAGAGGTCAGATGGAGGGTAGGCGGGGGCGGGTTGTGGGGA TTCCTCTGGGTCTTCACGGTAGCACGCATAGG-3', MA08: 5'-AGCAGCACAGAGGTCAGATGGGAGGTGGTGGGAGGTGGGTTGGGGGG GCCTGGCTCGATCTTCACGGTAGCACGCATAGG-3'.

2. The nucleic acid aptamer according to claim 1, characterized in that: At 25 °C, 147 mM Na + , 5mMMg 2+ Under the conditions, the secondary structures of the nucleic acid aptamers MA02, MA05 and MA08 are respectively as follows:

3. The nucleic acid aptamer according to claim 1, characterized in that: It is the nucleic acid aptamer MA05.

4. The nucleic acid aptamer according to claim 1, characterized in that: The 5' end or 3' end of at least one of the nucleic acid aptamers MA02, MA05 and MA08 is chemically modified with a fluorescent group, an amino group, biotin, digoxigenin or polyethylene glycol.

5. The nucleic acid aptamer according to claim 4, characterized in that: The 5' end of at least one of the nucleic acid aptamers MA02, MA05, and MA08 is modified with a FAM group.

6. Use of the nucleic acid aptamer according to claim 1 in preparing a detection reagent or a kit for methicillin-resistant Staphylococcus aureus.

7. Use of the nucleic acid aptamer according to claim 1 in the preparation of drugs for treating methicillin-resistant Staphylococcus aureus related diseases.

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