RCA-Cas12a-DNA tetrahedral sensor for detecting vancomycin in serum and preparation method of RCA-Cas12a-DNA tetrahedral sensor

Abstract

The invention discloses a preparation method of an RCA-Cas12a-DNA (deoxyribonucleic acid) tetrahedron sensor for detecting vancomycin in blood serum. The sensor comprises a target object vancomycin, a DNA chain TSP1, a DNA chain TSP2, a DNA chain TSP3 and a vancomycin aptamer Aptamer which are used for preparing a DNA tetrahedron, a DNA chain T, a DNA chain S, a T4 DNA ligase, exonuclease I and exonuclease III which are used for preparing circular DNA, a DNA chain P and phi29 DNA polymerase which are used for carrying out rolling circle amplification reaction, and LbCas12a nuclease, crRNA and ssDNA-FQ probes which are used for preparing a reporter. The aptamer is specifically combined with a target object, a non-nucleic acid target is converted into nucleic acid detection, and the application range of RCA and Cas12a is expanded; rCA is combined with Cas12a, so that a signal is amplified twice, the detection limit is lower, and the sensitivity is higher; compared with a traditional detection method, expensive instruments and equipment, complex and time-consuming sample pretreatment and skilled operators are not needed, and the method is more convenient and economical.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and in particular relates to a RCA-Cas12a-DNA tetrahedron sensor for detecting vancomycin in serum and a preparation method thereof. Background Art

[0002] RCA technology is an isothermal nucleic acid amplification method with many significant advantages such as rapidity, efficiency, ease of operation, high sensitivity and high specificity, which makes it widely used in biological detection, medical research and industrial applications.

[0003] Cas12a, as a nuclease in the CRISPR system, can specifically recognize and cut specific DNA sequences, and can indiscriminately cleave nearby single-stranded DNA (ssDNA) after binding to the target. This property makes it widely used in molecular biology research and diagnosis.

[0004] Vancomycin is a glycopeptide antibiotic that is mainly used to treat serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria. Its mechanism of action is to inhibit the synthesis of bacterial cell walls, change the permeability of bacterial cell membranes, and selectively inhibit the synthesis of bacterial RNA, thus having potent antibacterial activity. However, the effective therapeutic concentration range of vancomycin is very narrow, so vancomycin therapeutic drug monitoring must be implemented. Summary of the invention

[0005] To solve the above problems, the present invention proposes a RCA-Cas12a-DNA tetrahedron sensor for detecting vancomycin in serum and a preparation method thereof.

[0006] The technical solution adopted by the present invention is as follows: The first aspect of the present invention provides an RCA-Cas12a-DNA tetrahedron sensor for detecting vancomycin in serum, the sensor comprising: a target vancomycin, a DNA chain TSP1, a DNA chain TSP2, a DNA chain TSP3 and a vancomycin aptamer for preparing a DNA tetrahedron, a DNA chain T, a DNA chain S, a T4 DNA ligase, a nuclease I and a nuclease III for preparing a circular DNA, a DNA chain P and a phi29 DNA polymerase for performing a rolling circle amplification reaction, and an LbCas12a nuclease, a crRNA and an ssDNA-FQ probe for preparing a reporter; TSP1: 5'-ATTGGCAGTCTGACTTCCGACTAGGAAGCTACCGAACAATAAGTA CT-3', TSP2: 5'-AGTCAGACTGCCAATTTTGCGGTACCCTCGGCCTACGTACAGCTAAC-3', TSP3: 5'-TAGCTTCCTAGTCGGTGTTAGCTGTACGTAGCCCCGACCCACAATAG-3', Aptamer: 5'-CCGAG GGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGG-3', T: 5'-P-TAGCGTAGAGTACTTATTGTTCGTTTCCAGTATCAGTCTATCTATCTATTGTGGGTCGGGGTACCATCGTGCAATTTCGGTTC-3', S: 5'-AAGTACTCTACGCTAGAACCGAAATTGCAC-3', P: 5'-GTACCATCGTGCAAT-3', crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCAGUAUCAGUCUAUCUAU-3', ssDNA-FQ: 5'-Cy3-TTAATT-BHQ2-3'.

[0007] A second aspect of the present invention provides a method for preparing the above-mentioned RCA-Cas12a-DNA tetrahedron sensor, the steps being as follows: S1: Preparation of TSPA solution: 10 -5 μmol DNA chain TSP1, 10 -5 μmol DNA chain TSP2, 10 -5 μmol DNA strands TSP3 and 10 -5μmol of vancomycin aptamer Aptamer was mixed evenly in 10 μL of 1× TM buffer and reacted to obtain a DNA tetrahedron solution, also known as TSPA solution; S2: Preparation of TSP solution: Dissolve the target vancomycin in ultrapure water to obtain a target solution; add 10 μL of the target solution to the TSPA solution in step S1 to react to obtain a TSP solution; S3: Preparation of TS solution: S3-1: 2×10 -5 μmol DNA chain T and 2×10 -5 μmol of DNA chain S was mixed evenly in 10 μL of 1×TM buffer and reacted to obtain reaction solution 1; S3-2: Add 1 μL of 5U / μL T4 DNA ligase, 3 μL of 10×T4 DNA ligase buffer and 16 μL of ultrapure water to the reaction solution 1 of step S3-1 to carry out a reaction to obtain a reaction solution 2; S3-3: Add 4 μL of 20 U / μL exonuclease I, 0.4 μL of 200 U / μL exonuclease III, 5 μL of 10×ExoⅠBuffer, 5 μL of 10×ExoⅢBuffer and 5.6 μL of ultrapure water to the reaction solution 2 of step S3-2, and react to obtain a circular DNA solution, also called TS solution; S4: Rolling circle amplification reaction: Add 1 μL of 10 U / μL Phi29 DNA polymerase, 1 μL of 10 μM DNA chain P, 1 μL of 0.4 μM circular DNA, 3 μL of 10× Phi29 DNA polymerase buffer, 1.2 μL of 5 mg / mL BSA standard solution, 1 μL of 10 mM dNTPMixture and 1.8 μL of ultrapure water to the TSP solution in step S2 to obtain a rolling circle amplification reaction system, perform a reaction, and obtain a rolling circle amplification reaction product; S5: Preparation of reporter solution: 10 μL of 500 nM LbCas12a nuclease and 10 μL of 500 nM crRNA were mixed evenly for reaction, and then 50 μL of 2 μM ssDNA-FQ probe was added and mixed evenly to obtain a reporter solution; S6: Assembly of the sensor The reporter solution of step S5 and the rolling circle amplification reaction product of step S4 are mixed and reacted to obtain a test solution containing the target object, and the fluorescence intensity in the range of 540 to 650 nm is measured and recorded at an excitation light wavelength of 525 nm.

[0008] In the above step S1, the reaction conditions are: 95°C for 10 min, and 4°C for 15 min.

[0009] In the above step S2, the reaction conditions are: 37° C. for 2 h.

[0010] In the above step S3-1, the reaction conditions are: 95°C for 2 min, 65°C for 2 min, 60°C for 6 min, 20°C for 30 s, and 4°C for 10 min; in the above step S3-2, the reaction conditions are: 16°C for 8 h, 65°C for 10 min; in the above step S3-3, the reaction conditions are: 37°C for 12 h, and 80°C for 15 min.

[0011] In the above step S4, the reaction conditions are: 30° C. 90 min.

[0012] In the above step S5, the reaction conditions are: 25°C for 30 min.

[0013] In the above step S6, the reaction conditions are: 37° C. for 90 min.

[0014] A third aspect of the present invention provides the use of the above-mentioned RCA-Cas12a-DNA tetrahedron sensor in detecting vancomycin, wherein the application is for non-disease diagnosis and treatment purposes.

[0015] Figure 1 It is a schematic diagram of the detection principle of the RCA-Cas12a-DNA tetrahedron sensor of the present invention. When the target does not exist, the DNA tetrahedron formed by DNA chain TSP1, DNA chain TSP2, DNA chain TSP3 and vancomycin aptamer maintains a stable structure, no 3' end exposure, cannot trigger rolling circle amplification, and no signal is generated; and when the target exists, Aptamer binds to the target and falls off from the DNA tetrahedron, and the DNA tetrahedron exposes two 3' ends, which are used as primers to trigger rolling circle amplification (RCA), LbCas12a and crRNA binder recognize specific DNA double-stranded fragments, LbCas12a is activated, and ssDNA-FQ is cut using its trans-cutting activity, and the fluorescent group is away from the quenching group, and the fluorescence is restored.

[0016] The significant advantages of the present invention are: (1) The present invention utilizes aptamers to specifically bind to targets, converting non-nucleic acid targets into nucleic acid detection, thereby expanding the application scope of RCA and Cas12a; (2) The present invention combines RCA with Cas12a, so that the signal is amplified twice, the detection limit is lower, and the sensitivity is higher; (3) Compared with traditional detection methods, the present invention does not require expensive instruments and equipment, complex and time-consuming sample pretreatment, and skilled operators, and is more convenient and economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the detection principle of the RCA-Cas12a-DNA tetrahedron sensor of the present invention.

[0018] Figure 2 It is the fluorescence response diagram and working curve after adding different concentrations of the target in Example 1.

[0019] Figure 3 This is the fluorescence response diagram after adding the target, non-target and their mixture in Example 2. DETAILED DESCRIPTION

[0020] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0021] The nucleotide chain sequences involved in the embodiments of the present invention are as follows: TSP1: 5'-ATTGGCAGTCTGACTTCCGACTAGGAAGCTACCGAACAATAAGTACT-3', TSP2: 5'-AGTCAGACTGCCAATTTTGCGGTACCCTCGGCCTACGTACAGCTAAC-3', TSP3: 5'-TAGCTTCCTAGTCGGTGTTAGCTGTACGTAGCCCCGACCCACAATAG-3', Aptamer: 5'-CCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGG-3', T: 5'-P-TAGCGTAGAGTACTTATTGTTCGTTTCCAGTATCAGTCTATCTATCTATTGTGGGTCGGGGTACCATCGTGCAATTTCGGTTC-3', S: 5'-AAGTACTCTACGCTAGAACCGAAATTGCAC-3', P: 5'-GTACCATCGTGCAAT-3', ssDNA-FQ: 5'-Cy3-TTAATT-BHQ2-3', crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCAGUAUCAGUCUAUCUAU-3'.

[0022] The formula of the 1×TM buffer involved in the embodiment of the present invention is: 10 mM Tris-HCl, 5 mM MgCl2; pH=8.0.

[0023] The LbCas12a nuclease involved in the embodiments of the present invention was purchased from New England Biolabs, catalog number: M0653T.

[0024] Embodiment 1: A method for preparing an RCA-Cas12a-DNA tetrahedron sensor for detecting vancomycin in serum comprises the following steps: S1: Preparation of TSPA solution: 10 -5 μmol DNA chain TSP1, 10 -5 μmol DNA chain TSP2, 10 -5 μmol DNA strands TSP3 and 10 -5 μmol of vancomycin aptamer Aptamer was mixed evenly in 10 μL of 1×TM buffer and reacted to obtain a DNA tetrahedron solution, also known as TSPA solution; wherein, the reaction conditions were: heating at 95°C for 10 minutes, then cooling to 4°C and maintaining for 15 minutes.

[0025] S2: Preparation of TSP solution: The target vancomycin was dissolved in ultrapure water to obtain target solutions of different concentrations; 10 μL of the target solution was added to the TSPA solution of step S1, and the mixture was reacted at 37° C. for 2 h to obtain a TSP solution.

[0026] S3: Preparation of TS solution: S3-1: 2×10 -5 μmol DNA chain T and 2×10 -5 μmol of DNA chain S was mixed evenly in 10 μL of 1×TM buffer and reacted to obtain reaction solution 1; wherein, the reaction conditions were: heating at 95°C for 2 min, then cooling to 65°C and maintaining for 2 min, then cooling to 60°C and maintaining for 6 min, then cooling to 20°C and maintaining for 30 s, and then cooling to 4°C and maintaining for 10 min.

[0027] S3-2: Add 1 μL of 5U / μL T4 DNA ligase, 3 μL of 10×T4 DNA ligase buffer and 16 μL of ultrapure water to the reaction solution 1 of step S3-1 to carry out a reaction to obtain a reaction solution 2; wherein the reaction conditions are: react at 16°C for 8 hours, then heat to 65°C and heat for 10 minutes.

[0028] S3-3: Add 4 μL of 20 U / μL nuclease I, 0.4 μL of 200 U / μL nuclease III, 5 μL of 10×ExoⅠBuffer, 5 μL of 10×ExoⅢBuffer and 5.6 μL of ultrapure water to the reaction solution 2 of step S3-2, and react to obtain a circular DNA solution, also known as TS solution; wherein the reaction conditions are: react at 37°C for 12 hours, then heat to 80°C and heat for 15 minutes.

[0029] S4: Rolling circle amplification reaction: To the TSP solution of step S2, add 1 μL of 10U / μL Phi29 DNA polymerase, 1 μL of 10 μM DNA chain P, 1 μL of 0.4 μM circular DNA, 3 μL of 10×Phi29 DNA polymerase buffer, 1.2 μL of 5 mg / mL BSA standard solution, 1 μL of 10 mM dNTPMixture and 1.8 μL ultrapure water to obtain a rolling circle amplification reaction system, perform reaction, and obtain a rolling circle amplification reaction product.

[0030] S5: Preparation of reporter solution: 10 μL of 500 nM LbCas12a nuclease and 10 μL of 500 nM crRNA were mixed evenly, reacted at 25°C for 30 min, and then 50 μL of 2 μM ssDNA-FQ probe was added and mixed evenly to obtain a reporter solution.

[0031] S6: Assembly of the sensor The reporter solution of step S5 and the rolling circle amplification reaction product of step S4 were mixed, and reacted at 37°C in the dark for 90 min to obtain a test solution containing different concentrations of the target (0, 1fM, 10fM, 1pM, 10pM, 100pM, 1nM, 10nM). The fluorescence intensity in the range of 540-650nm was measured and recorded at an excitation light wavelength of 525nm.

[0032] The results are as follows Figure 2 As shown, Figure 2The relationship between the fluorescence intensity of the RCA-Cas12a-DNA tetrahedron sensor and the concentration of the target vancomycin is shown. As can be seen from the figure, the fluorescence intensity increases with the increase of the concentration of the target vancomycin. At the same time, according to the blank plus 3σ (3 times the standard deviation) method, the detection limit of vancomycin is calculated to be 1fM.

[0033] Embodiment 2: A method for preparing an RCA-Cas12a-DNA tetrahedron sensor for detecting vancomycin in serum comprises the following steps: S1: Same as Example 1.

[0034] S2: Prepare vancomycin solution, kanamycin solution, gentamicin solution, and vancomycin-kanamycin-gentamicin mixed solution with ultrapure water, take 10 μL of each and add them to the TSPA solution in step S1, react at 37° C. for 2 h, and obtain TSP solution.

[0035] S3 to S6: Same as Example 1.

[0036] The results are as follows Figure 3 As shown. From left to right: ultrapure water, vancomycin, kanamycin, gentamicin and vancomycin-kanamycin-gentamicin mixture (the final concentration of each antibiotic in the test solution is 1nM). Compared with other interfering antibiotics, the relative fluorescence signal caused by the target vancomycin has at least an 8-fold increase. Not only that, a strong signal is also shown in the target mixture containing the same concentration of interfering antibiotics, indicating that the RCA-Cas12a-DNA tetrahedron sensor developed by the present invention has good specificity for vancomycin.

[0037] Embodiment 3: The human serum samples were diluted 10 times with ultrapure water, various concentrations of the target vancomycin were added to the diluted serum, and then the recovery experiment was performed using the standard addition method according to the steps described in Example 1. The recovery rate and relative standard deviation (RSD) were calculated through three independent experiments.

[0038] The results are shown in Table 1, with a recovery rate of 91.9% to 124.0% and an RSD of 7.21% to 15.80%. This indicates that the RCA-Cas12a-DNA tetrahedron sensor of the present invention has good accuracy and precision and can be used for actual sample analysis.

[0039] Table 1 The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A RCA-Cas12a-DNA tetrahedron sensor for detecting vancomycin in serum, characterized in that: The sensor comprises: a target substance, vancomycin, DNA chains TSP1, TSP2, TSP3 and Aptamer for preparing DNA tetrahedrons, DNA chains T, S, T4 DNA ligase, exonuclease I and exonuclease III for preparing circular DNA, DNA chain P and phi29 DNA polymerase for performing rolling circle amplification reaction, LbCas12a nuclease, crRNA and ssDNA-FQ probe for preparing reporters; TSP1: 5'-ATTGGCAGTCTGACTTCCGACTAGGAAGCTACCGAACAATAAGTACT-3', TSP2: 5'-AGTCAGACTGCCAATTTTGCGGTACCCTCGGCCTACGTACAGCTAAC-3', TSP3: 5'-TAGCTTCCTAGTCGGTGTTAGCTGTACGTAGCCCCGACCCACAATAG-3', Aptamer: 5'-CCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGG-3', T: 5'-P-TAGCGTAGAGTACTTATTGTTCGTTTCCAGTATCAGTCTATCTATCTATTGTGGGTCGGGGTACCATCGTGCAATTTCGGTTC-3', S: 5'-AAGTACTCTACGCTAGAACCGAAATTGCAC-3', P: 5'-GTACCATCGTGCAAT-3', crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCAGUAUCAGUCUAUCUAU-3', ssDNA-FQ: 5'-Cy3-TTAATT-BHQ2-3'.

2. The preparation method of the RCA-Cas12a-DNA tetrahedron sensor as claimed in claim 1, characterized in that: Here are the steps: S1: Preparation of TSPA solution: 10 -5 μmol DNA chain TSP1, 10 -5 μmol DNA chain TSP2, 10 -5 μmol DNA strands TSP3 and 10 -5 μmol of vancomycin aptamer Aptamer was mixed evenly in 10 μL of 1× TM buffer and reacted to obtain a DNA tetrahedron solution, also known as TSPA solution; S2: Preparation of TSP solution: Dissolve the target vancomycin in ultrapure water to obtain a target solution; add 10 μL of the target solution to the TSPA solution in step S1 to react to obtain a TSP solution; S3: Preparation of TS solution: S3-1: 2×10 -5 μmol DNA chain T and 2×10 -5 μmol of DNA chain S was mixed evenly in 10 μL of 1×TM buffer and reacted to obtain reaction solution 1; S3-2: Add 1 μL of 5U / μL T4 DNA ligase, 3 μL of 10×T4 DNA ligase buffer and 16 μL of ultrapure water to the reaction solution 1 of step S3-1 to carry out a reaction to obtain a reaction solution 2; S3-3: Add 4 μL of 20 U / μL exonuclease I, 0.4 μL of 200 U / μL exonuclease III, 5 μL of 10×ExoⅠ Buffer, 5 μL of 10×ExoⅢ Buffer and 5.6 μL of ultrapure water to the reaction solution 2 of step S3-2, and react to obtain a circular DNA solution, also called TS solution; S4: Rolling circle amplification reaction: Add 1 μL of 10 U / μL Phi29 DNA polymerase, 1 μL of 10 μM DNA chain P, 1 μL of 0.4 μM circular DNA, 3 μL of 10× Phi29 DNA polymerase buffer, 1.2 μL of 5 mg / mL BSA standard solution, 1 μL of 10 mM dNTP Mixture and 1.8 μL of ultrapure water to the TSP solution in step S2 to obtain a rolling circle amplification reaction system, perform a reaction, and obtain a rolling circle amplification reaction product; S5: Preparation of reporter solution: 10 μL of 500 nM LbCas12a nuclease and 10 μL of 500 nM crRNA were mixed evenly for reaction, and then 50 μL of 2 μM ssDNA-FQ probe was added and mixed evenly to obtain a reporter solution; S6: Assembly of the sensor The reporter solution of step S5 and the rolling circle amplification reaction product of step S4 are mixed and reacted to obtain a test solution containing the target object, and the fluorescence intensity in the range of 540-650 nm is measured and recorded at an excitation light wavelength of 525 nm.

3. The preparation method according to claim 2, characterized in that: In step S1, the reaction conditions are: 95°C for 10 min, 4°C for 15 min.

4. The preparation method according to claim 2, characterized in that: In step S2, the reaction conditions are: 37° C. 2 h.

5. The preparation method according to claim 2, characterized in that: In step S3-1, the reaction conditions are: 95°C for 2 min, 65°C for 2 min, 60°C for 6 min, 20°C for 30 s, and 4°C for 10 min; in step S3-2, the reaction conditions are: 16°C for 8 h, 65°C for 10 min; in step S3-3, the reaction conditions are: 37°C for 12 h, and 80°C for 15 min.

6. The preparation method according to claim 2, characterized in that: In step S4, the reaction conditions are: 30° C. for 90 min.

7. The preparation method according to claim 2, characterized in that: In step S5, the reaction conditions are: 25° C. for 30 min.

8. The preparation method according to claim 2, characterized in that: In step S6, the reaction conditions are: 37° C. for 90 min.

9. The use of the RCA-Cas12a-DNA tetrahedron sensor as claimed in claim 1 in detecting vancomycin, characterized in that: The application is for non-disease diagnosis and treatment purposes.

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