A method for detecting methicillin-resistant Staphylococcus aureus based on CRISPR / Cas12a magnetic relaxation sensor

Abstract

The present invention relates to the technical field of food safety analysis. The present invention provides a method for detecting methicillin-resistant Staphylococcus aureus based on CRISPR / Cas12a magnetic relaxation sensor, comprising the following steps: extracting genomic DNA of a sample to be tested; preparing MNP-ploy-ALP and performing trans-cutting on it using target-activated Cas12a; magnetic separation, adding phosphorylated ascorbate to the supernatant; adding Cu(II), azide-MNP conjugate and alkynyl-MNP conjugate; magnetic separation, unbound azide-MNP conjugate as a signal probe, measuring the transverse relaxation time of the corresponding solution to calculate the target content in the sample to be tested. The present invention can achieve high-sensitivity detection of MRSA without the need for nucleic acid pre-amplification, avoids the cross-contamination risk brought by traditional target nucleic acid amplification, and provides a highly sensitive, accurate and rapid method for the detection of MRSA in food safety.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety analysis, and in particular to a method for detecting methicillin-resistant Staphylococcus aureus based on a CRISPR / Cas12a magnetic relaxation sensor. Background Art

[0002] Infections caused by drug-resistant bacteria have become one of the greatest challenges facing public health. Without efforts to curb antibiotic resistance, this number is projected to reach 10 million by 2050. Methicillin-resistant Staphylococcus aureus (MRSA) is the most common and widespread drug-resistant bacterium, causing significant mortality and morbidity. MRSA strains have been isolated from numerous foods, including milk, raw meat, and eggs, and may spread throughout the food production chain, posing a serious threat to food safety. Rapid and accurate detection of MRSA is a prerequisite for preventing its harm and tracking its epidemiology.

[0003] Currently, MRSA detection methods primarily include molecular and non-molecular testing. Among non-molecular tests, culture-based antimicrobial susceptibility testing offers high accuracy, but this method typically requires a long time (1–2 days). Alternatively, MRSA can be detected using an antigen-antibody latex agglutination test, using anti-PBP2a antibodies to detect the PBP2a protein. This method is simple, time-saving, and easy to identify, but its major drawbacks are its low stability and resolution. Matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most promising non-genotypic techniques for direct identification of pathogens in positive blood cultures. This method identifies MRSA by comparing protein spectra obtained from bacterial or fungal samples with a database of spectra obtained from characteristic microorganisms. However, because the performance of MALDI-TOF MS depends largely on the purity and quantity of the microorganisms, bacterial enrichment and purification procedures from positive blood cultures are required. Among molecular tests, nucleic acid amplification methods are widely used for MRSA detection. Among these, polymerase chain reaction (PCR) remains the gold standard for molecular detection of MRSA. While PCR-based methods offer high sensitivity, they are complex, time-consuming, and require specialized technicians and equipment. Isothermal nucleic acid amplification methods, such as LAMP, do not require a thermal cycler, but their primer design is complex and their sensitivity is insufficient for detecting ultra-low levels of MRSA. Therefore, there is an urgent need to develop rapid, low-cost, and highly sensitive sensing methods to identify MRSA. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting methicillin-resistant Staphylococcus aureus based on CRISPR / Cas12a magnetic relaxation sensor, providing a new detection method for animal-derived food safety.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for detecting methicillin-resistant Staphylococcus aureus using a CRISPR / Cas12a-based magnetic relaxation sensor, comprising the following steps:

[0007] (1) Extracting genomic DNA from the sample to be tested;

[0008] (2) Mix Cas12a protein, crRNA and 1×NE buffer r2.1 and incubate to obtain crRNA-Cas12a complex;

[0009] (3) Preparation of MNP-ploy-ALP using solid-phase RCA;

[0010] (4) mixing MNP-ploy-ALP, genomic DNA of the sample to be tested, and an RNAase inhibitor to obtain material 1;

[0011] (5) mixing the crRNA-Cas12a complex and material 1, incubating and magnetically separating, adding phosphorylated ascorbate to the supernatant, and incubating to obtain material 2;

[0012] (6) mixing material 2, Cu(II) solution, azide-MNP conjugate solution, and alkynyl-MNP conjugate solution, and incubating to obtain material 3;

[0013] (7) Material 3 is magnetically separated, and the unbound azide-MNP conjugate is used as a signal probe. The content of the target substance in the sample to be tested can be calculated by measuring the transverse relaxation time of the corresponding solution.

[0014] Preferably, the final concentration of crRNA after mixing in step (2) is 180-220 nM, the final concentration of Cas12a protein is 230-270 nM, and the incubation condition is 35-39 ° C for 10-20 min.

[0015] Preferably, in step (4), the volume ratio of the MNP-ploy-ALP, the genomic DNA of the sample to be tested, and the RNAase inhibitor is 18-22:4-6:0.2-0.4.

[0016] Preferably, the volume ratio of MNP-ploy-ALP to material 1 in step (5) is 1:2.5-3.5.

[0017] Preferably, the concentration of the phosphorylated ascorbyl ester in step (5) is 90-110 mM; the amount of the phosphorylated ascorbyl ester used is 2-3 times the volume of the MNP-ploy-ALP.

[0018] Preferably, the incubation condition before magnetic separation in step (5) is incubation at 35-39° C. for 25-35 min; and the incubation condition after magnetic separation is incubation at 35-39° C. for 50-70 min.

[0019] Preferably, the concentration of Cu(II) in the Cu(II) solution in step (6) is 0.8-1.2 mM, the concentration of the azide-MNP conjugate in the azide-MNP conjugate solution is 35-45 μg / mL, and the concentration of the alkynyl-MNP conjugate in the alkynyl-MNP conjugate solution is 75-85 μg / mL.

[0020] Preferably, the incubation condition in step (6) is 23-27° C. for 8-12 min.

[0021] Preferably, the incubation in step (6) uses a NaAc-HAc buffer system with a pH of 3.8 to 4.2.

[0022] Preferably, the particle size of the azide-MNP conjugate in step (6) is 25 to 35 nm, and the particle size of the alkynyl-MNP conjugate is 950 to 1050 nm.

[0023] The present invention provides a method for detecting methicillin-resistant Staphylococcus aureus using a magnetic relaxation sensor based on CRISPR / Cas12a, comprising the steps of: extracting genomic DNA of a sample to be tested; preparing MNP-ploy-ALP and performing trans-cutting on it using target-activated Cas12a; magnetic separation, adding phosphorylated ascorbate to the supernatant; adding Cu(II), azide-MNP conjugate and alkynyl-MNP conjugate; magnetic separation, unbound azide-MNP conjugate as a signal probe, measuring the transverse relaxation time of the corresponding solution to calculate the target content in the sample to be tested. The present invention can achieve highly sensitive detection of MRSA without the need for nucleic acid pre-amplification, avoids the risk of cross contamination caused by traditional target nucleic acid amplification, and provides a highly sensitive, accurate and rapid method for the detection of MRSA in food safety. The present invention utilizes the difference in separation speed of superparamagnetic nanoparticles of different particle sizes in a magnetic field, uses small-sized superparamagnetic nanoparticles as signal probes, and enhances the degree of change in the T2 signal caused by the target through cascade solid-phase RCA amplification, CRISPR / Cas12a cleavage, and enzyme-mediated click amplification reaction, thereby achieving highly sensitive target-free amplification detection of MRSA. This method enriches the toolbox of CRISPR detection. By replacing crRNA with the complementary sequence of the corresponding target, the C-MRS biosensor can also be expanded to detect other drug-resistant bacteria, providing a new method for sensitive and accurate detection of drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the technical route and detection principle of the present invention;

[0025] Figure 2 This is the optimization result of MNP30 concentration;

[0026] Figure 3 is the optimization result of Cu(II) concentration;

[0027] Figure 4 Optimization results of the concentration ratio of alkynyl-MNP1000 and azide-MNP30;

[0028] Figure 5 Standard curve and linear range for detecting AA by n-MRS signal readout system;

[0029] Figure 6 is the SEM image of MNP1000 after click reaction;

[0030] Figure 7 Standard curve and linear range for ALP detection by n-MRS signal readout system;

[0031] Figure 8The particle size and potential characterization diagram of MNP-ploy-ALP;

[0032] Figure 9 This is the polyacrylamide gel electrophoresis characterization of MNP-ploy-ALP;

[0033] Figure 10 The stability test results of MNP-ploy-ALP complex;

[0034] Figure 11 is the response relationship between the change in T2 value of the C-MRS sensor and the double-stranded mecA target;

[0035] Figure 12 Schematic diagram of verifying RCA amplification effect on ELISA plate;

[0036] Figure 13 The color development results with and without RCA amplification are shown;

[0037] Figure 14 Optimization results of RCA polymerization time in C-MRS sensor;

[0038] Figure 15 This is the optimization result of MNP-ploy-ALP volume dosage for C-MRS sensor;

[0039] Figure 16 Optimization results of CRISPR / Cas12a cutting time for C-MRS sensor;

[0040] Figure 17 Standard curve and linear range for the C-MRS sensor to detect MRSA plasmid DNA and MRSA bacterial solution;

[0041] Figure 18 The specific results of C-MRS sensor for detecting MRSA;

[0042] Figure 19 Comparison of the detection results of MRSA in spiked samples by C-MRS sensor and qPCR method. DETAILED DESCRIPTION

[0043] The present invention provides a method for detecting methicillin-resistant Staphylococcus aureus based on CRISPR / Cas12a magnetic relaxation sensor, comprising the following steps:

[0044] (1) Extracting genomic DNA from the sample to be tested;

[0045] (2) Mix Cas12a protein, crRNA and 1×NE buffer r2.1 and incubate to obtain crRNA-Cas12a complex;

[0046] (3) Preparation of MNP-ploy-ALP using solid-phase RCA;

[0047] (4) mixing MNP-ploy-ALP, genomic DNA of the sample to be tested, and an RNAase inhibitor to obtain material 1;

[0048] (5) mixing the crRNA-Cas12a complex and material 1, incubating and magnetically separating, adding phosphorylated ascorbate to the supernatant, and incubating to obtain material 2;

[0049] (6) mixing material 2, Cu(II) solution, azide-MNP conjugate solution, and alkynyl-MNP conjugate solution, and incubating to obtain material 3;

[0050] (7) Material 3 is magnetically separated, and the unbound azide-MNP conjugate is used as a signal probe. The content of the target substance in the sample to be tested can be calculated by measuring the transverse relaxation time of the corresponding solution.

[0051] In the present invention, the extraction in step (1) uses a commercial bacterial genomic DNA rapid extraction kit.

[0052] In the present invention, the final concentration of crRNA after mixing in step (2) is preferably 180 to 220 nM, more preferably 200 nM.

[0053] In the present invention, the final concentration of Cas12a protein after mixing in step (2) is preferably 230-270 nM, more preferably 250 nM.

[0054] In the present invention, the incubation condition in step (2) is preferably incubated at 35-39° C. for 10-20 min, and more preferably incubated at 37° C. for 15 min.

[0055] In the present invention, the volume ratio of the MNP-ploy-ALP, the genomic DNA of the sample to be tested, and the RNAase inhibitor in step (4) is preferably 18-22:4-6:0.2-0.4, and more preferably 20:5:0.3.

[0056] In the present invention, the volume ratio of the MNP-ploy-ALP to the material 1 in step (5) is preferably 1:2.5-3.5, more preferably 1:3.

[0057] In the present invention, the concentration of the phosphorylated ascorbyl ester in step (5) is preferably 90-110 mM, more preferably 100 mM.

[0058] In the present invention, the amount of phosphorylated ascorbyl ester used in step (5) is preferably 2 to 3 times the volume of MNP-ploy-ALP, and more preferably 2.5 times.

[0059] In the present invention, the incubation condition before magnetic separation in step (5) is preferably incubated at 35-39° C. for 25-35 min, and more preferably incubated at 37° C. for 30 min.

[0060] In the present invention, the incubation condition after magnetic separation in step (5) is preferably incubated at 35-39° C. for 50-70 min, and more preferably incubated at 37° C. for 60 min.

[0061] In the present invention, the concentration of Cu(II) in the Cu(II) solution in step (6) is preferably 0.8-1.2 mM, more preferably 1.0 mM.

[0062] In the present invention, the concentration of the azide-MNP conjugate in the azide-MNP conjugate solution in step (6) is preferably 35 to 45 μg / mL, more preferably 40 μg / mL.

[0063] In the present invention, the concentration of the alkynyl-MNP conjugate in the alkynyl-MNP conjugate solution in step (6) is preferably 75 to 85 μg / mL, more preferably 80 μg / mL.

[0064] In the present invention, the incubation condition in step (6) is preferably incubated at 23-27° C. for 8-12 min, and more preferably incubated at 25° C. for 10 min.

[0065] In the present invention, the incubation in step (6) preferably uses a NaAc-HAc buffer system with a pH of 3.8 to 4.2, and more preferably a NaAc-HAc buffer system with a pH of 4.0.

[0066] In the present invention, the particle size of the azide-MNP conjugate in step (6) is preferably 25 to 35 nm, more preferably 30 nm.

[0067] In the present invention, the particle size of the alkynyl-MNP conjugate in step (6) is preferably 950 to 1050 nm, more preferably 1000 nm.

[0068] In the present invention, the MNP number-mediated MRS signal readout system is referred to as n-MRS, and the CRISPR / Cas12a-based magnetic relaxation sensor is referred to as C-MRS.

[0069] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0070] It should be noted that when detecting other drug-resistant bacteria, it is only necessary to replace the crRNA with a sequence corresponding to the drug-resistant gene to be tested according to the following example.

[0071] The reagents, materials, solutions and instruments used in this example are as follows:

[0072] Reagents and materials: LbaCas12a (Cpf1, 100 μM), T4 DNA ligase (2,000,000 U / mL), and phi29 DNA polymerase (10,000 U / mL) were obtained from New England Biolabs (USA). Carboxylated magnetic nanoparticles (COOH-MNPs) (1000 nm, 180 nm, and 30 nm, 10 mg / mL) were purchased from Invitrogen (USA). A bacterial genomic DNA rapid extraction kit, RNase A inhibitor, and all nucleic acid sequences were provided by Sangon Biotechnology (Shanghai, China). Alkaline phosphatase (ALP), trisodium 2-phospho-L-ascorbic acid (AAP), ascorbic acid (AA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysulfonic acid succinimide (sulfo-NHS) were purchased from Sigma-Aldrich (USA). Sulfo-SMCC was obtained from Santa Clara (USA). Disodium 4-nitrophenylphosphate hexahydrate (pNPP) was purchased from Aladdin Chemical Co., Ltd. (Shanghai, China). Azidotripolyethylene glycol amino (azido-PEG3-NH2) and alkynetetrapolyethylene glycol amino (alkyne-PEG4-NH2) were purchased from Click Chemistry Tools (USA). All chemicals and reagents used were of analytical grade. Methicillin-resistant Staphylococcus aureus (MRSA) was isolated from clinical specimens collected from a hospital in Wuhan. Ultrapure water (resistivity ≥18.3 MΩ·cm) was used in the experiments. Eggs, milk, and pork were purchased from a supermarket near Huazhong Agricultural University (Wuhan, China).

[0073] Solution preparation:

[0074] Phosphate buffered saline (PBS): Dissolve 8.00 g NaCl, 0.20 g KCl, 0.20 g KH2PO4, and 2.90 g Na2HPO4·12H2O in 1000 mL water and shake well.

[0075] Washing solution (PBST): Add 0.5 mL of Tween-20 to 1000 mL of phosphate buffered saline and shake well to prepare PBST washing solution;

[0076] Sodium acetate buffer (NaAc-Hac, 0.2 M, pH 4.0): 1.3608 g NaAc.3H2O was added to 50 mL water to prepare a 0.2 M NaAc solution; 0.90075 g HAc was added to 50 mL water to prepare a 0.3 M HAc solution; 9 mL of 0.2 M NaAc solution was mixed with 41 mL of 0.3 M HAc solution.

[0077] Buffer A: Dissolve 0.2925 g NaCl and 0.4102 g anhydrous sodium acetate in 50 mL water and shake well.

[0078] MES buffer (10 mM, pH 6.0): Add 0.1066 g MES to 45 mL water and adjust the pH to 6.0.

[0079] TBS (25 mM, pH 7.5) buffer: Dissolve 3.0285 g Tris, 8.00 g NaCl, and 0.20 g KCl in 400 mL water and shake well.

[0080] TBST buffer: Add 0.2 mL of Tween-20 to 400 mL of TBS buffer and shake well to prepare TBST washing solution;

[0081] Instruments: Amicon-3K / 30K ultrafiltration centrifuge tubes from Millipore (Billerica, MA) were used to remove unreacted thiol-DNA during DNA-ALP construction. A qPCR instrument (T100) and gel imager (Gel Doc XR+) were from Bio-Rad (USA). An electrophoresis instrument (JY300HC, Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.) was used to characterize RCA reaction products. A Real-Time PCR system (Jena, Qtower 2.2) was used to detect MRSA in spiked foods. A magnetic separation stand from OceanNanoTech (USA) was used to separate alkynyl-MNP complexes and azide-MNP complexes. A field-emission transmission electron microscope (JEM-2100F, Japan) was used to characterize the magnetic beads. A laser particle size analyzer (Malvern Instruments Ltd., UK) was used to measure the particle size and potential of the magnetic beads. A 0.47T small low-field nuclear magnetic resonance spectrometer from Shanghai Newmai Electronic Technology Co., Ltd. was used to measure transverse relaxation time (T2).

[0082] Example 1 Optimization of n-MRS signal readout system

[0083] Preparation of azide-MNP30 and alkynyl-MNP1000 conjugates: To 200 μL of MNPs30 (5 mg / mL), add 10 μL of 5 mg / mL EDC and 5 μL of 5 mg / mL sulfo-NHS, respectively. The mixture was vortexed at room temperature for 30 minutes for activation. After activation, 500 μL of PBS (pH 7.4, 10 mM) and 20 μL of azide-PEG3-NH2 (10 mg / mL) were added, and the mixture was vortexed at room temperature for 1 hour for conjugation. After conjugation, excess azido-PEG3-NH2 was removed by magnetic separation overnight, and the azide-MNP30 complex was resuspended in PBS and stored at 4°C until further use. Similarly, 200 μL of MNPs1000 (10 mg / mL) was mixed with 20 μL of 5 mg / mL EDC and 10 μL of 5 mg / mL sulfo-NHS and activated by vortexing at room temperature for 20 minutes. After magnetic separation, the MNPs1000 were resuspended in 500 μL of PBS buffer, and 80 μL of alkyne-PEG4-NH2 (10 mg / mL) was added and vortexed at room temperature for 1 hour. After coupling, the magnetic particles were washed three times with PBST, and the prepared alkyne-MNP1000 complex was resuspended in PBS and stored at 4°C until use.

[0084] 50 μL of MNP30 of different concentrations (0.05 μg / mL-1000 μg / mL) were added to different centrifuge tubes, and then 150 μL of PBS was added to them. After mixing, the T2 value was measured to select the concentration of MNP30. The results are shown in Figure 2. Figure 2 As shown;

[0085] Add 50 μL of Cu(II) of different concentrations (0, 0.05, 0.1, 0.5, 1, 2, 5 mM) to different centrifuge tubes, then add 150 μL of PBS to them. After mixing, measure the T2 value to select the concentration of Cu(II).

[0086] The results are as follows Figure 3 As shown;

[0087] 50 μL of alkynyl-MNP1000 conjugate solution and 50 μL of 40 μg / mL azide-MNP30 conjugate solution were mixed at a concentration ratio of 1:1, 2:1, 5:1, and 10:1, respectively. Then, 50 μL of 1 mM Cu(II) and 50 μL of ascorbic acid solutions of different concentrations (0, 10, 20, 30, 40, and 50 mM) were added to the mixed solution. After mixing and reacting at room temperature for 10 minutes, the T2 value was measured to optimize the ratio of alkynyl-MNP1000 conjugate and azide-MNP30 conjugate. The results are shown in FIG. Figure 4 shown.

[0088] The optimization results are as follows: Figure 1-3 As shown, the n-MRS signal readout system has the optimal response effect when the concentration of the azide-MNP30 conjugate is 40 μg / mL, the concentration of Cu(II) is 1 mM, and the concentration ratio of the alkynyl-MNP1000 conjugate to the azide-MNP30 conjugate is 2:1.

[0089] Example 2 Response of n-MRS Signal Readout System to Ascorbic Acid

[0090] 50 μL of 40 μg / mL azide-MNP30 conjugate solution and 50 μL of 80 μg / mL alkynyl-MNP1000 conjugate solution were added to different centrifuge tubes and mixed thoroughly. Then, 50 μL of 1 mM Cu(II) and 50 μL of ascorbic acid solutions of different concentrations (100, 50, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0 mM) were added to the mixed solution. The mixture was reacted at room temperature for 10 minutes. The clear solution obtained after magnetic separation was used to measure the T2 value. The experimental results are shown in Figure 2. Figure 5 As shown in Figure 2, there is a good response relationship between the change in T2 value and the Cu(II) concentration. As the Cu(II) concentration increases, ΔT2 gradually increases, indicating that there is a good response relationship between the change in T2 value and the ascorbic acid concentration. At the same time, SEM characterization of the 1000nm MNP after reaction was performed, and the experimental results are shown in Figure 2. Figure 6 As shown, it is shown that 30nm small magnetic particles can be aggregated on the surface of 1000nm large magnetic particles through click reaction.

[0091] Example 3 Response of the n-MRS Signal Readout System to Alkaline Phosphatase

[0092] Take 50 μL of alkaline phosphatase solution of different concentrations (1000, 500, 200, 100, 10, 1, 0.5, 0.2, 0.1, 0.05, 0 U / L) and add 50 μL of 100 mM AAP solution respectively. The mixed solution is incubated at 37 ° C for 1 hour. After the reaction is completed, 50 μL of 1 mM Cu (II) solution, 50 μL of 40 μg / mL azide-MNP30 conjugate solution and 50 μL of 80 μg / mL alkynyl-MNP1000 conjugate solution are added to the mixed solution in sequence and incubated at room temperature for 10 minutes. After the reaction is completed, magnetic separation is performed and the MNP30 solution that has not been magnetically separated is taken to measure the T2 value. The experimental results are shown in Figure 2. Figure 7 As shown in the figure, the change of T2 value gradually increases with the increase of ALP concentration, indicating that there is a good response relationship between the change of T2 value and ALP concentration.

[0093] Example 4 Response of C-MRS Sensor to MRSA

[0094] Preparation of magnetic nanoparticle-streptavidin complexes: First, add 50 μL of 180 nm (10 mg / mL) MNPs to a 1.5 mL centrifuge tube and magnetically separate the supernatant. Then, add 1.8 μL of 5 mg / mL EDC and 5 μL of 5 mg / mL sulfo-NHS to the MNPs. Activate by vortexing at room temperature for 15 minutes. After magnetic separation, resuspend the MNPs in 500 μL of MES buffer and add 50 μL of streptavidin (1 mg / mL). Vortex at room temperature for 2.5 hours. Next, add 100 μL of PBST (10 mM, pH 7.4, 0.05% Tween-20, containing 1% BSA) for washing and incubate at room temperature for 30 minutes. After magnetic separation and washing, resuspend the MNPs in 1 mL of PBST (10 mM, pH 7.4, containing 0.5% BSA) and store at 4°C.

[0095] Preparation of DNA-ALP: First, 200 μL of thiol-DNA (10 μM), 10 μL of PBS (1 M, pH 5.0), and 5 μL of tris(2-carboxyethyl)phosphine (TCEP, 30 mM) were shaken at 37°C for 60 min. The mixture was then transferred to an Amicon-3K ultrafiltration tube, centrifuged four times (7500 × g, 30 min) in buffer A (0.1 M NaCl, 0.1 M NaAc, pH 7.3), and resuspended in 200 μL of buffer A. Simultaneously, 100 μL of ALP (10 U / mL) was incubated with 0.4 mg of Sulfo-SMCC at room temperature for 60 min. The mixture was then transferred to an Amicon-30K ultrafiltration tube, centrifuged four times (7500 × g, 10 min) in buffer A, and resuspended in 200 μL of buffer A. The activated ALP and thiol-DNA solutions were thoroughly mixed and incubated at 4°C for 2 h. To remove unbound thiol-DNA, the mixture was centrifuged five times with buffer A in an Amicon-30K ultrafiltration tube and finally resuspended in 100 μL buffer A and stored at −20°C.

[0096] Preparation of the MNP-ploy-ALP complex: Hybridize 2 μL of 10 μM biotin-DNA, 2 μL of 10 μM padlock probe, 13 μL of ultrapure water, and 2 μL of 10× Connector Buffer at 90°C for 5 minutes. Then, slowly cool to 25°C using a PCR thermocycler at a gradient rate of 0.1°C / s. Add 1 μL of T4 DNA ligase (2,000,000 U / mL), incubate at room temperature for 2 hours, and inactivate the enzyme in a 65°C water bath. Mix 20 μL of this solution with 100 μL of MNP-SA at room temperature for 60 minutes to initiate the biotin-streptavidin reaction. After washing three times with PBST, the complex is dispersed in 100 μL of ultrapure water. Then, 2 μL (10 mM) of dNTPs, 0.5 μL (10,000 U / mL) of phi29 DNA polymerase, 67.5 μL of ultrapure water, and 20 μL of 10× phi29 buffer were added, and the mixture was reacted at 37°C for 45 min to perform the RCA reaction. The mixture was washed 4 times with TBST (10 mM Tris-HCl, pH 8.0, containing 0.05% Tween-20) buffer and dispersed in 100 μL of TBS (10 mM Tris-HCl, pH 8.0) buffer. 20 μL of DNA-ALP was added and the mixture was reacted at room temperature for 30 min. Finally, the mixture was washed 4 times with TBST to remove unbound DNA-ALP and resuspended in 100 μL of TBS buffer. We characterized the particle size and potential of the prepared MNP-ploy-ALP, and the results are shown in Figure 2. Figure 8 As shown in Figure 2, as the RCA reaction progressed, the particle size of MNP180 increased from 350 nm to 2400 nm, and the Zeta potential of MNP180 decreased from -16.8 mV to -27.5 mV. These results indirectly indicate that the MNP-ploy-ALP was successfully prepared. In addition, the significantly increased molecular weight of ploy-ALP in polyacrylamide gel electrophoresis also verified the formation of MNP-ploy-ALP. The experimental results are shown in Figure 2. Figure 9 We conducted a stability test on the prepared MNP-ploy-ALP, and the experimental results are shown in Figure 10 As shown in the figure, the results showed that the prepared MNP-ploy-ALP could be stably stored at 4°C for more than one week.

[0097] 200 nM Cas12a protein and 250 nM crRNA were mixed in 1× NE buffer r2.1 and pre-incubated at 37°C for 15 minutes. Separately, 30 μL of MNP-ploy-ALP complex, 5 μL of different concentrations of dsmecA target (0 and 100 nM), and 0.3 μL of RNAase inhibitor were added to different centrifuge tubes. The two solutions were mixed and incubated at 37°C for 30 minutes. After magnetic separation, the supernatant was removed and 50 μL of 100 mM AAP solution was added. The mixed solution was incubated at 37°C for 1 hour. After the reaction was completed, 50 μL of 1 mM Cu(II) solution, 50 μL of 40 μg / mL azide-MNP30 conjugate solution, and 50 μL of 80 μg / mL alkynyl-MNP1000 conjugate solution were added to the mixed solution in sequence. The mixture was incubated at room temperature for 10 minutes. After the reaction was completed, magnetic separation was performed and the T2 value of the MNP30 solution that could not be magnetically separated was measured. The experimental results are shown in Figure 2. Figure 11 As shown in Figure 2, there was a significant difference in the T2 change between the 100 nM target and the control group, indicating the feasibility of the constructed C-MRS biosensor for detecting MRSA. We directly hybridized ALP with complementary DNA on magnetic particles to evaluate the amplification effect of solid-phase RCA. Considering the color interference of MNPs, the amplification effect was evaluated on a 96-well microplate. The process is as follows: Figure 12 The experimental results are shown in Figure 13 As shown, the OD405 value of "with RCA" is about twice that of "without RCA", indicating the effectiveness of RCA signal amplification.

[0098] Example 5 Optimization of MRSA Detection Conditions Using C-MRS Sensor

[0099] To separate centrifuge tubes, add 2 μL of 10 μM biotin-DNA, 2 μL of 10 μM padlock probe, 13 μL of ultrapure water, and 2 μL of 10× Connector Buffer. Hybridize at 90°C for 5 minutes. Then, slowly cool to 25°C using a PCR thermocycler at a gradient rate of 0.1°C / s. Add 1 μL of T4 DNA ligase (2,000,000 U / mL), incubate at room temperature for 2 hours, and inactivate in a 65°C water bath. Then, mix 20 μL of this solution with 100 μL of MNP-SA at room temperature for 60 minutes to initiate the biotin-streptavidin reaction. After washing three times with PBST, the complex is dispersed in 100 μL of ultrapure water. Then, 2μL (10mM) of dNTPs, 0.5μL (10,000U / mL) of phi29 DNA polymerase, 67.5μL of ultrapure water, and 20μL of 10×phi29 buffer were added, and the polymerization reaction was carried out at 37°C for 5, 15, 30, 45, and 60min, respectively, to perform the RCA reaction. After washing 4 times with TBST buffer, it was dispersed in 100μL TBS. 20μL of DNA-ALP was added, and the reaction was carried out at room temperature for 30min. After washing 4 times with TBST, the MNP-ploy-ALP complex was resuspended in 100μL TBS buffer. Take 10μL of the MNP-ploy-ALP complex prepared at different polymerization times, add 50μL of 50mM pNPP solution, and react at 37°C in the dark for 1h. Finally, the optical density value (OD405) of the solution at 405nm was measured with a microplate reader to optimize the time of the RCA polymerization reaction. The experimental results are as follows. Figure 14 shown.

[0100] 200nM Cas12a protein and 250nM crRNA were mixed in 1×NE buffer 2.1, and the mixture was pre-incubated at 37°C for 15min; in addition, 10, 20, 30, 40μL MNP-ploy-ALP complex, 5μL different concentrations of dsmecA target (0, 100nM) and 0.3μL RNAase inhibitor were added to different centrifuge tubes. The above two solutions were mixed and incubated at 37°C for 30min. After magnetic separation, the supernatant was removed and 50μL 50mM pNPP solution was added. The mixed solution was incubated at 37°C for 1h, and finally the optical density of the solution at 405nm (OD405) was measured with a microplate reader to optimize the volume of the MNP-ploy-ALP complex. The experimental results are shown in Figure 2. Figure 15 shown.

[0101] 200nM Cas12a protein and 250nM crRNA were mixed in 1×NE buffer r2.1, and the mixture was pre-incubated at 37°C for 15min; in addition, 20μL MNP-ploy-ALP complex, 5μL different concentrations of dsmecA target (0, 100nM) and 0.3μL RNAase inhibitor were added to different centrifuge tubes. The above two solutions were mixed and incubated at 37°C for 10, 20, 30, and 40min respectively. After magnetic separation, the supernatant was removed and 50μL 50mM pNPP solution was added. The mixed solution was incubated at 37°C for 1h. Finally, the optical density of the solution at 405nm (OD405) was measured with a microplate reader to optimize the cutting time of CRISPR / Cas12a. The experimental results are shown in Figure 2. Figure 16 shown.

[0102] The optimization results are as follows: Figure 14-16 As shown, the C-MRS sensor has the optimal response effect when the polymerization time of the RCA reaction is 45 minutes, the volume of the MNP-ploy-ALP complex is 20 μL, and the cutting time of CRISPR / Cas12a is 30 minutes.

[0103] Example 6 Sensitivity of C-MRS Sensor for Detecting MRSA

[0104] (1) 200 nM Cas12a protein and 250 nM crRNA were mixed in 1×NE buffer 2.1, and the mixture was pre-incubated at 37°C for 15 min;

[0105] (2) In addition, 20 μL of MNP-ploy-ALP complex and 5 μL of MRSA plasmid DNA with different concentrations (10, 20, 50, 10 2 ,10 3 ,10 4 ,10 5 ,10 6 ,10 7 ,10 8 copies / μL) or MRSA bacterial solution (10, 20, 50, 10 2 ,10 3 ,10 4 ,10 5 ,10 6 ,10 7 ,10 8 CFU / mL) and 0.3 μL RNAase inhibitor;

[0106] (3) The two solutions were mixed and incubated at 37°C for 30 min. After magnetic separation, the supernatant was removed and 50 μL of 100 mM AAP solution was added. The mixed solution was incubated at 37°C for 1 h.

[0107] (4) After the reaction, 50 μL of 1 mM Cu(II) solution, 50 μL of 40 μg / mL azide-MNP30 conjugate solution, and 50 μL of 80 μg / mL alkynyl-MNP1000 conjugate solution were added to the mixed solution in sequence and incubated at room temperature for 10 minutes.

[0108] (5) After the reaction is completed, magnetic separation is performed and the T2 value of the solution containing MNP30 that cannot be magnetically separated is measured. The experimental results are shown in the figure. Figure 17 As shown in the figure, the change of T2 value gradually increases with the increase of MRSA plasmid DNA and MRSA bacterial liquid concentrations, and this method has good sensitivity and linear range for the detection of MRSA.

[0109] Example 7 Specificity of C-MRS Sensor for Detecting MRSA

[0110] (1) 200 nM Cas12a protein and 250 nM crRNA were mixed in 1×NE buffer r2.1, and the mixture was pre-incubated at 37°C for 15 min;

[0111] (2) Separately, 5 μL of 100 nM dsmecA mutant gene was added to different centrifuge tubes, and different ratios of dsmecA gene (0, 0.001%, 0.015%, 0.1%, 1%, 10%) were mixed into the tubes. Then, 20 μL of MNP-ploy-ALP complex and 0.3 μL of RNAase inhibitor were added to the tubes respectively.

[0112] (3) The two solutions were mixed and incubated at 37°C for 30 min. After magnetic separation, the supernatant was removed and 50 μL of 100 mM AAP solution was added. The mixed solution was incubated at 37°C for 1 h.

[0113] (4) After the reaction, 50 μL of 1 mM Cu(II) solution, 50 μL of 40 μg / mL azide-MNP30 conjugate solution, and 50 μL of 80 μg / mL alkynyl-MNP1000 conjugate solution were added to the mixed solution in sequence and incubated at room temperature for 10 minutes.

[0114] (5) After the reaction is completed, magnetic separation is performed and the T2 value of the solution containing MNP30 that cannot be magnetically separated is measured. The experimental results are shown in the figure. Figure 18The changes in T2 values ​​corresponding to mixing different ratios of ds mecA targets in the ds mecA mutant gene are shown, indicating that this method can separate as low as 0.01% of the mecA target gene from the homologous sequence and has single-base specificity for the detection of MRSA.

[0115] Example 8 Recovery of C-MRS Sensor for Detecting MRSA

[0116] (1) The final MRSA contamination concentration of egg, milk and pork samples was 10 2 , 10 4 and 10 6 CFU / mL or CFU / g. For milk samples, MRSA bacterial solution was added directly, and the contaminated milk samples were equilibrated at 4°C overnight. Under sterile conditions, 1 mL of artificially contaminated milk sample was placed in a sterilized centrifuge tube. Centrifuge at 8000×g for 2 minutes, and then suspend in 100 μL of sterile deionized water to extract DNA. For egg samples, 10 mL of sterile saline was added to the eggshell, and then ground with a mortar. After MRSA contamination, the completely contaminated eggshell was centrifuged at 8000×g for 2 minutes, and then suspended in 100 μL of sterile deionized water to extract DNA. For pork samples, 25 g of pork was added with 225 mL of saline solution for 1 minute, shaken for 20 minutes, inoculated with MRSA overnight, and the precipitate after centrifugation at 8000×g for 2 minutes was resuspended in deionized water to extract DNA.

[0117] (2) Use a bacterial genomic DNA rapid extraction kit to extract the genomic DNA of the contaminated sample.

[0118] (3) 200 nM Cas12a protein and 250 nM crRNA were mixed in 1× NE buffer r2.1, and the mixture was pre-incubated at 37°C for 15 min to obtain the crRNA-Cas12a complex;

[0119] (4) In addition, 20 μL of MNP-ploy-ALP complex, 5 μL of DNA from different contaminated samples, and 0.3 μL of RNAase inhibitor were added to different centrifuge tubes to obtain material 1;

[0120] (5) The crRNA-Cas12a complex and material 1 were mixed and incubated at 37°C for 30 min. After magnetic separation, the supernatant was removed and 50 μL of 100 mM AAP solution was added. The mixed solution was incubated at 37°C for 1 h.

[0121] (6) After the reaction, 50 μL of 1 mM Cu(II) solution, 50 μL of 40 μg / mL azide-MNP30 conjugate solution, and 50 μL of 80 μg / mL alkynyl-MNP1000 conjugate solution were added to the mixed solution in sequence and incubated at room temperature for 10 min.

[0122] (7) After the reaction, magnetic separation was performed, and the T2 value of the solution containing MNP30 that could not be magnetically separated was measured. The experimental results, as shown in Table 1, show the spiked recoveries and coefficients of variation at different MRSA spike levels in eggs, milk, and pork, indicating that this method has good accuracy and precision for the detection of MRSA in complex food matrices.

[0123] Table 1 Recovery and coefficient of variation of MRSA spiked at different levels in eggs, milk and pork

[0124]

[0125] Example 9 Comparison of MRSA detection using C-MRS sensor and qPCR method

[0126] qPCR detection of MRSA

[0127] (1) Take different PCR tubes and add 10 μL TB Green Premix Ex Taq II (Tli RNaseH Plus) (2×conc.), 0.8 μL 10 μM forward primer and 0.8 μL 10 μM reverse primer, 6.4 μL sterile water, and 2 μL DNA extracted from different spiked samples to be tested. Mix well and centrifuge at low speed for 1 min.

[0128] (2) Fluorescence quantification was performed using a Real-Time PCR system (Jena, Qtower 2.2) with the following program: pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds, for 40 cycles. Figure 19 As shown in the figure, the comparison of experimental results shows that the C-MRS biosensor has good consistency with the qPCR method, indicating that the biosensor has good accuracy in actual sample detection.

[0129] As can be seen from the above embodiments, the present invention provides a method for detecting methicillin-resistant Staphylococcus aureus based on CRISPR / Cas12a magnetic relaxation sensor, comprising the following steps: extracting genomic DNA of sample to be tested;Prepare MNP-ploy-ALP and perform trans-cutting on it using target-activated Cas12a;Magnetic separation, adding phosphorylated ascorbate to the supernatant;Add Cu (II), azide-MNP conjugate and alkynyl-MNP conjugate;Magnetic separation, unbound azide-MNP conjugate as signal probe, measure the transverse relaxation time of corresponding solution and calculate the target content in the sample to be tested. The present invention can achieve highly sensitive detection of MRSA without the need for nucleic acid pre-amplification, avoids the risk of cross contamination brought by traditional target nucleic acid amplification, and provides highly sensitive, accurate and rapid methods for the detection of MRSA in food safety.

[0130] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting methicillin-resistant Staphylococcus aureus based on CRISPR / Cas12a magnetic relaxation sensor for non-diagnostic and non-therapeutic purposes, characterized in that: The steps include: (1) Extracting genomic DNA from the sample to be tested; (2) Mix Cas12a protein, crRNA and 1×NE buffer r2.1 and incubate to obtain crRNA-Cas12a complex; (3) Preparation of MNP-ploy-ALP using solid-phase RCA; (4) Mixing MNP-ploy-ALP, genomic DNA of the sample to be tested, and RNAase inhibitor to obtain material 1; (5) Mixing the crRNA-Cas12a complex and material 1, incubating and magnetically separating, adding phosphorylated ascorbate to the supernatant, and incubating to obtain material 2; (6) Mixing material 2, Cu(II) solution, azide-MNP conjugate solution, and alkynyl-MNP conjugate solution, and incubating to obtain material 3; (7) Material 3 is magnetically separated, and the unbound azide-MNP conjugate is used as a signal probe. The content of the target substance in the sample to be tested can be calculated by measuring the transverse relaxation time of the corresponding solution; The preparation method of MNP-ploy-ALP in step (3) is: A, Preparation of magnetic nanoparticle-streptavidin complex; B. Preparation of DNA-ALP: First, 200 μL of 10 μM thiol-DNA, 10 μL of 1 M PBS, and 5 μL of 30 mM tris(2-carboxyethyl)phosphine were shaken at 37°C for 60 min. The mixture was then transferred to an Amicon-3K ultrafiltration tube and centrifuged four times at 7500 × g for 30 min each time using buffer A. The mixture was then resuspended in 200 μL of buffer A. Simultaneously, 100 μL of 10 U / mL ALP was incubated with 0.4 mg of Sulfo-SMCC at room temperature for 60 min. The mixture was then transferred to an Amicon-30K ultrafiltration tube and centrifuged four times at 7500 × g using buffer A for 30 min each time. The activated ALP was obtained by centrifugation at 400 × g for 10 min each time and resuspending in 200 µL of buffer A. The activated ALP and thiol-DNA solution were thoroughly mixed and incubated at 4°C for 2 h. To remove unbound thiol-DNA, the mixture was centrifuged five times with buffer A in an Amicon-30K ultrafiltration tube and finally resuspended in 100 µL of buffer A and stored at -20°C. The buffer A in step B contains 0.1 M NaCl and 0.1 M NaAc, with a pH of 7.3; C. Preparation of MNP-ploy-ALP complex: 2 µL of 10 µM biotin-DNA, 2 µL of 10 µM padlock probe, 13 µL of ultrapure water, and 2 µL of 10× Connector Buffer were hybridized at 90°C for 5 min, then slowly cooled to 25°C at a gradient rate of 0.1°C / s using a PCR thermal cycler system; 1 µL of T4 DNA ligase with an enzyme activity of 2,000,000 U / mL was added, incubated at room temperature for 2 h, and the enzyme was inactivated in a 65°C water bath to obtain a mixed solution; 20 µL of the mixed solution was mixed with 100 µL MNP-SA at room temperature for 60 min to perform a biotin-streptavidin reaction; after washing three times with PBST, the complex was dispersed in 100 µL of ultrapure water; then 2 µL of 10 mM dNTPs and 0.5 µL of phi29 DNA polymerase with an enzyme activity of 10,000 U / mL were added. µL, 67.5 µL ultrapure water, and 20 µL 10× phi29 buffer were reacted at 37°C for 45 min for RCA reaction; washed four times with TBST buffer and dispersed in 100 µL TBS buffer; added 20 µL DNA-ALP and reacted at room temperature for 30 min. Finally, washed four times with TBST to remove unbound DNA-ALP and resuspended in 100 µL TBS buffer to obtain MNP-ploy-ALP; The azide-MNP conjugate solution in step (6) is an azide-MNP30 conjugate solution, and the alkynyl-MNP conjugate solution is an alkynyl-MNP1000 conjugate; The unbound azide-MNP conjugate in step (7) is an unbound azide-MNP30 conjugate.

2. The method according to claim 1, characterized in that The final concentration of crRNA after mixing in step (2) is 180~220 nM, and the final concentration of Cas12a protein is 230~270 nM.

3. The method according to claim 2, characterized in that The volume ratio of the MNP-ploy-ALP, the genomic DNA of the sample to be tested, and the RNAase inhibitor in step (4) is 18-22:4-6:0.2-0.

4.

4. The method according to claim 1, wherein The concentration of the phosphorylated ascorbyl ester in step (5) is 90-110 mM; the amount of the phosphorylated ascorbyl ester used is 2-3 times the volume of the MNP-ploy-ALP.

5. The method according to claim 1, characterized in that The concentration of Cu(II) in the Cu(II) solution in step (6) is 0.8-1.2 mM, the concentration of the azide-MNP conjugate in the azide-MNP conjugate solution is 35-45 μg / mL, and the concentration of the alkynyl-MNP conjugate in the alkynyl-MNP conjugate solution is 75-85 μg / mL.

6. The method according to claim 1, characterized in that During the incubation in step (6), a NaAc-HAc buffer system with a pH of 3.8 to 4.2 is used.

7. The method according to any one of claims 1 to 6, characterized in that The particle size of the azide-MNP conjugate in step (6) is 25-35 nm, and the particle size of the alkynyl-MNP conjugate is 950-1050 nm.

8. The method according to claim 1, characterized in that The preparation method of the magnetic nanoparticle-streptavidin complex in step A is: First, 50 μL of 180 nm 10 mg / mL MNPs were added to a 1.5 mL centrifuge tube, and the supernatant was removed by magnetic separation. 1.8 μL of 5 mg / mL EDC and 5 μL of 5 mg / mL sulfo-NHS were added to the MNPs. The MNPs were activated by vortexing at room temperature for 15 minutes. After magnetic separation, the MNPs were resuspended in 500 μL of MES buffer and 50 microliters of 1 mg / mL streptavidin were added. The MNPs were vortexed at room temperature for 2.5 hours. 100 μL of PBST was then added for washing at room temperature for 30 minutes. After magnetic separation and washing, the MNPs were resuspended in 1 mL of PBST buffer and stored at 4°C. The PBST washing solution in step A has a concentration of 10 mM, a pH of 7.4, and contains 0.05% Tween-20 and 1% BSA; The PBST buffer has a concentration of 10 mM, a pH of 7.4, and contains 0.5% BSA.

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