CN113817807A - CRISPR-Cas-based visual detection system triggering non-specific rolling circle amplification and application and method thereof - Google Patents

CRISPR-Cas-based visual detection system triggering non-specific rolling circle amplification and application and method thereof Download PDF

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CN113817807A
CN113817807A CN202111191999.9A CN202111191999A CN113817807A CN 113817807 A CN113817807 A CN 113817807A CN 202111191999 A CN202111191999 A CN 202111191999A CN 113817807 A CN113817807 A CN 113817807A
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冯春凤
张立群
王云霞
熊瑜
刘飞
陈曼
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Second Affiliated Hospital Army Medical University
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Abstract

The invention discloses a CRISPR-Cas-triggered non-specific rolling circle amplification-based visual detection system and application and a method thereof.A CRISPR-Cas, RCA and G-quadruplex/hemin DNAzymes signal amplification technology is adopted to detect a gene to be detected, so that sensitive specificity, low cost, no need of large-scale experimental equipment, simple naked eye interpretation of a detection result can be realized, the detection sensitivity is high, and the minimum detection limit is as low as 20 fmol/L; the Cas/crRNA compound can directly identify the target in serum in the detection process without a nucleic acid extraction step; constant temperature amplification is adopted, precise temperature control equipment is not needed, and the detection method is simple, easy to operate, sensitive, specific, low in cost, simple in equipment, good in universality and high in accuracy.

Description

CRISPR-Cas-based visual detection system triggering non-specific rolling circle amplification and application and method thereof
Technical Field
The invention relates to the field of medical detection, in particular to a CRISPR-Cas12 a-based visual detection system for triggering non-specific rolling circle amplification, and also relates to application and a method of the system.
Background
Epidermal Growth Factor Receptor (EGFR) is a member of the HER/ERBB tyrosine kinase receptor family and plays an important role in various cell signaling pathways such as cell proliferation, differentiation, migration, and apoptosis. Research shows that EGFR is highly expressed in various malignant tumors such as non-small cell lung cancer, ovarian cancer, cervical cancer, colorectal cancer, head and neck cancer, bladder cancer and the like. Of all EGFR kinase domain mutants, approximately 47.0% were in-frame deletion (EGFR19 del) mutations of 15bp for exon 19. Patients carrying the EGFR19del mutation have been shown to exhibit high sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Therefore, EGFR19del gene mutation has become a very important biomarker for predicting the susceptibility of EGFR-TKIs to treatment of non-small cell lung cancer.
Currently, the main methods for detecting EGFR19del include Next Generation Sequencing (NGS), droplet digital polymerase chain reaction (ddPCR), and mutation amplification block system-polymerase chain reaction (ARMS-PCR). However, these methods have the disadvantages of complicated operation, high cost, dependence on thermal cyclers, etc., and have limited their wide application in resource-poor areas. Recently, biosensing technologies such as electrochemical sensing technology and Quartz Crystal Microbalance (QCM) have also been developed for EGFR19del detection. However, these methods often involve multiple washing steps, rely on DNA chemical modification, and require specialized detection equipment to obtain the output signal. Therefore, it is essential and critical to develop a low-cost, simple macroscopic-based EGFR19del colorimetric biosensor.
CRISPR/Cas is an adaptive immune response system for bacteria and archaea, protecting itself from viral and plasmid invasion. In the past few years, this system has been widely used for molecular immunization, genetic engineering, and transcriptional regulation as a powerful gene editing tool. Recent studies found that Cas12, Cas13, and Cas14 effectors exhibit strong trans-cleavage activity after recognizing and cleaving specific targets, which revealed a great potential for CRISPR-Cas in nucleic acid detection. Most of the existing CRISPR-Cas biosensing platforms need to be combined with pre-amplification methods such as PCR, loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA) and the like to realize the hypersensitive detection of targets. However, most of these pre-amplification techniques involve non-specific amplification which is difficult to handle and inevitable. Furthermore, despite the revolutionary breakthrough of these CRISPR-based biosensing platforms in molecular diagnostics, almost all of these technologies require bulky and expensive fluorescent equipment, cumbersome sample handling, and inability to quantify, which limits their application in rapid assays in the field.
RCA has the advantages of simplicity, sensitivity, high efficiency and the like, and is widely applied to detection of DNA, RNA, DNA methylation, protein, cancer cells and the like. The existing RCA detection technology mostly uses target nucleic acid as a template, linear DNA padlocks are connected into a circular DNA template by the base complementary pairing principle, and because a sample contains a nucleic acid fragment close to the target nucleic acid sequence, an impure circular DNA template is easy to generate, thereby causing non-specific RCA reaction.
Disclosure of Invention
In view of the above, the invention aims to provide a CRISPR-Cas12 a-based visual detection system triggering non-specific rolling circle amplification, wherein a CRISPR-Cas system is adopted to induce the cleavage of a circular single-stranded DNA, so that the non-specific amplification reaction of RCA is eliminated, and a label-free and wash-free EGFR19del visual detection platform is constructed; the invention also aims to provide the application of the visual detection system in detecting the target to be detected; the invention also aims to provide a method for detecting a target to be detected by utilizing the visual detection system.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a CRISPR-Cas12 a-based visual detection system triggering non-specific rolling circle amplification comprises single-stranded RNA (ribonucleic acid) containing a stem-loop structure, annular single-stranded nucleic acid, Cas enzyme with trans-cleavage activity, RCA (Rolling circle amplification) reaction reagent and a G-quadruplex/hemin DNAzyme catalytic ABTS (ABTS) color development system;
the single-stranded RNA is homologous with one strand part in the double-stranded DNA target to be detected and can be complementarily combined with the other strand, the single-stranded RNA can be combined with Cas enzyme to form a complex, and the accessory cleavage activity of the complex is activated in the presence of the target to be detected;
the circular single-stranded nucleic acid can be combined with an RCA primer and amplified, and an amplification product can generate a sequence of a G-quadruplex and also has a Cas enzyme auxiliary cutting site sequence;
the G-quadruplex/hemin DNAzyme catalyzed ABTS color development system consists of hemin solution and K+Buffer solution of (1), H2O2Solution and ABTS buffer solution, the amplification product containing K+And hemin solution to form a G-quadruplex/hemin DNAzyme complex, said G-quadruplex/hemin DNAzyme complex with H2O2The solution and ABTS buffer reacted color.
Preferably, the concentration of the single-stranded RNA is 15-35 nmol/L; the concentration of the Cas enzyme is 15-30 nmol/L; the content of phi29 DNA polymerase is 0.1-2U; said group containing K+The pH of the buffer solution is 4-8.
Preferably, the hemin solution in the G-quadruplex/hemin DNAzyme catalysis ABTS color development system is 10 mu mol/Lhemin; said group containing K+The buffer of (1) comprises the following components: 50mmol/L HEPES, 400mmol/L NaCl, 40mmol/L KCl, 0.1% Triton X-100, 2% DMSO, pH 7.0; said H2O2The solution was 40mmol/L H2O2(ii) a The ABTS buffer was prepared by dissolving ABTS in pH 4.5 acetic acid-sodium acetate buffer to a concentration of 7.5 mmol/L.
Preferably, the RCA reaction system comprises RCA primers, phi29 DNA polymerase, dNTPs, and 1 xphi 29 DNA polymerase reaction buffer.
Preferably, the target to be detected is EGFR19del, the single-stranded RNA sequence is shown as SEQ ID NO.1, and the circular single-stranded nucleic acid sequence is shown as SEQ ID NO. 2.
Preferably, the RCA primer is shown as SEQ ID NO. 5.
2. The visual detection system is applied to detection of a target reagent to be detected.
3. The method for detecting the target to be detected by using the visual detection system comprises the following steps:
(1) adding the Cas enzyme and the single-stranded RNA into a buffer solution for incubation to obtain a Cas enzyme \ single-stranded RNA compound solution;
(2) adding a solution to be detected and annular single-stranded nucleic acid into the Cas enzyme \ single-stranded RNA compound solution obtained in the step (1), performing enzyme digestion reaction, and heating to inactivate the Cas enzyme; if the solution to be detected contains the target to be detected, the Cas enzyme is used for enzyme-cutting the target to be detected, and meanwhile, the auxiliary cutting activity is activated, so that the annular single-stranded nucleic acid is cut; if the liquid to be detected does not contain the target to be detected, the attached cleavage activity of the Cas enzyme is not activated, and the ring-shaped single-stranded nucleic acid cannot be cleaved;
(3) adding an RCA reaction reagent into the enzyme-digested product obtained in the step (2), and if the annular single-stranded DNA is not cut, carrying out RCA amplification to obtain an RCA product; if the circular single-stranded DNA is cut, no product is generated after the RCA reaction;
(4) adding hemin solution containing K to RCA product+The buffer of (1) was left for 1h, and the RCA reaction product, if any, was in K+Forming parallel G-quadruplex structure under the induction of (1), combining the formed G-quadruplex structure with hemin to form a stable G-quadruplex/hemin DNAzyme complex with peroxidase activity, and then adding H2O2And ABTS solution, complex catalysis H2O2The mediated oxidation of ABTS, resulting in a color change, which is then observed by a microplate reader, spectrophotometer, or by the naked eye.
Preferably, the Cas enzyme is Cas12, Cas13, or Cas 14;
the circular single-stranded nucleic acid in the present invention may be circular single-stranded DNA, circular single-stranded RNA, or circular single-stranded DNA; if the circular single-stranded nucleic acid is DNA, the Cas enzyme is Cas12 or Cas 14; if the circular single-stranded nucleic acid is RNA, the Cas enzyme is Cas 13.
Preferably, the specific steps are as follows: (1) adding the Cas enzyme and the single-stranded RNA into 1 XNEBuffer, and incubating for 30min at 37 ℃ to obtain a Cas enzyme \ single-stranded RNA compound solution;
(2) adding a solution to be detected and the annular single-stranded DNA into the Cas enzyme \ single-stranded RNA compound solution obtained in the step (1), reacting for 1h at 37 ℃ for enzyme digestion, and then heating to 65 ℃ for reaction for 10min to inactivate the Cas enzyme; if the solution to be detected contains the target to be detected, the Cas enzyme cuts the target to be detected, and simultaneously the auxiliary cutting activity is activated to cut the single-stranded DNA; if the solution to be detected does not contain the target to be detected, the attached cleavage activity of the Cas enzyme is not activated, and the single-stranded DNA cannot be cleaved;
(3) adding an RCA reaction system into the enzyme digestion product obtained in the step (2), reacting for 1h at 30 ℃, then heating to 65 ℃ and reacting for 10min to inactivate phi29 DNA polymerase, and if the annular single-stranded DNA is not cut, carrying out the RCA reaction to obtain an RCA product; if the circular single-stranded DNA is cut, no product is generated after the RCA reaction;
(4) adding hemin solution containing K to RCA product+The buffer of (1) was left for 1h, and if RCA reaction product was present, the RCA reaction product was in K+Forming parallel G-quadruplex structure under the induction of (1), combining the formed G-quadruplex structure with hemin to form a stable G-quadruplex/hemin DNAzyme complex with peroxidase activity, and then adding H2O2And ABTS solution, complex catalysis H2O2The mediated oxidation of ABTS, resulting in a color change, which is then observed by a microplate reader, spectrophotometer, or by the naked eye.
A preferred test target in the present invention is EGFR19 del.
The invention has the beneficial effects that: the invention provides a CRISPR-Cas-triggered non-specific rolling circle amplification-based visual detection system and application and a method thereof, and the CRISPR-Cas, RCA and G-quadruplex/hemin DNAzymes signal amplification technology is adopted to detect a gene to be detected, so that sensitive specificity, low cost, no need of large-scale experimental equipment, simple naked eye interpretation of a detection result can be realized, the detection sensitivity is high, and the minimum detection limit is as low as 20 fmol/L.
The Cas/crRNA compound can directly identify the target in serum in the detection process without a nucleic acid extraction step; the activated Cas enzyme can not only cut a target, but also cut the circular single-stranded nucleic acid in the system into various random short linear fragments which can not initiate RCA reaction, can not generate G-quadruplex sequence and can not form G-quadruplex/hemin DNAzymes, thereby limiting H2O2The oxidation-reduction reaction of the ABTS system can realize the detection of EGFR19del by detecting the absorbance of the solution at the wavelength of 420nm, and the detection can be detected by a common spectrophotometer and a microplate readerAnd (4) measuring, even reading the detection result by naked eyes.
The detection process of the invention adopts a constant temperature RCA technology, and does not need precise temperature control equipment.
The detection system has the advantages of simple method, easy operation, sensitivity, specificity, low cost, simple equipment, good universality and high accuracy.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a diagram showing the results of optimizing conditions of the detection system of the present invention (A is the optimization of crRNA concentration; B is the optimization of LbaCas12a concentration; C is the optimization of phi29 DNA polymerase amount; and D is the optimization of HEPES buffer pH).
FIG. 2 is a graph showing the results of detecting EGFR19del at different concentrations by the detection system of the present invention (A is a spectrum of EGFR19del at different concentrations in the wavelength range of 400 to 500 nm; B is a calibration curve of EGFR19del at a concentration of 50fmol/L to 50nmol/L, and the inset is a corresponding log-fitted linear plot).
FIG. 3 is a graph showing the results of signal changes generated by different substances at the same concentration detected by the detection system (a: blank control without EGFR19del, b: IncRNA HULC, c: IncRNA H19-1, d: Kras G12S, e: Kras G13D, f: wild type EGFR L858R, G: mutant type EGFR L858R, H: wild type EGFR19del, i: double base mismatch EGFR19del, j: single base mismatch EGFR19del, k: EGFR19del, L: mixture of b and k).
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1
(1)EGFR 19del crRNA
EGFR19del crRNA is single-stranded RNA containing a stem-loop structure, and is partially homologous with EGFR19del genome (EGFR19 del-a: 5'-atcgaggatttccttgttggctttcggagatgtcttgatagcgacgggaattttaactttctcaccttctgggat-3' (SQE ID NO. 3);
EGFR19 del-b: 5'-atcccagaaggtgagaaagttaaaattcccgtcgctatcaagacatctccgaaagccaacaaggaaatcctcgat-3' (SQE ID NO. 4). The crRNA can bind to the LbaCas12a, guide the LbaCas12a to cut the EGFR19del genome near the EGFR19del genome pam (tttn), and cut a nonspecific circular DNA template sequence near the LbaCas12a, and the specific sequences are as follows: 5'-uaauuucuacuaaguguagauggagaugucuugauagcgac-3' (SEQ ID NO. 1).
(2) Circular DNA template
The circular DNA template is a circular single-stranded DNA which can be combined with an RCA primer and contains a sequence capable of being replicated to generate a G-quadruplex (C-rich), and also has a LbaCas12a auxiliary cleavage (trans cleavage) site sequence (TTATT), and the specific sequence is as follows: 5'-tattattatttagcacggacatttcccaacccgccctacccacttttattattat-3' (SEQ ID NO. 2).
(3) RCA reaction system
Adding a product obtained after the CRISPR-Cas12a enzyme digestion reaction into a reaction buffer solution system containing a primer (5'-ataaaagtgggtaggg-3' (SEQ ID NO.5), phi29 DNA polymerase, dNTPs and 1 xphi 29 DNA polymerase to initiate RCA reaction, and generating thousands of repeated G-rich single-stranded DNA sequences.
(4) G-quadruplex/hemin DNAzyme catalyzed ABTS color development system:
the RCA reaction product is represented by K+The parallel G-quadruplex structure is combined with hemin (hemin) to form a stable G-quadruplex/hemin DNAzyme complex with peroxidase activity, and the complex can catalyze H2O2Mediated ABTS oxidation, resulting in a color change. This color change can be detected by a microplate reader or a conventional spectrophotometer, and can even be observed by the naked eye.
Example 2
CRISPR-Cas12 a/RCA/G-quadruplex/hemin detection principle:
LbaCas12a and EGFR19del crRNA are combined to assemble a binary complex, the target EGFR19del in a sample to be detected can activate a Cas12a/crRNA complex and release accessory cleavage activity, and non-specific shearing is carried out on a circular DNA template in the system,resulting in failure to initiate the next RCA reaction, the formation of the G-quadruplex/hemin DNAzyme complex, and H restriction2O2Mediated ABTS oxidation, the solution is almost colorless. When the sample to be detected does not contain the target EGFR19del, the Cas12a/crRNA compound is not activated, and the circular DNA template in the system is not sheared by the Cas12a, so that the circular DNA template can be used as an RCA template to start the next RCA reaction to generate a large amount of RCA products rich in G sequences, and the RCA products are subjected to K reaction+And hemin, these RCA products are further folded to form a G-quadruplex/hemin DNAzyem complex with peroxidase activity, promoting H2O2Mediated ABTS oxidation, producing a macroscopic green change. The present invention is therefore a "Turn-off" detection mode.
Example 3
1. Optimization of crRNA concentration:
(1) preparation of Cas12a/crRNA complex: add 0.4. mu.L, 0.6. mu.L, 0.8. mu.L, 1. mu.L, 1.2. mu.L, 1.4. mu.L of 500nmol/L crRNA to 0.5. mu.L of a mixture of 1. mu. mol/L LbaCas12a and 2. mu.L of 10 XNEBuffer 2.1, respectively, by ddH2O the final volume was adjusted to 17. mu.L, mixed and incubated at 37 ℃ for 30min to prepare a Cas12a/crRNA complex solution.
(2) CRISPR-Cas12a enzymatic cleavage: the experimental group and the control group were set. Samples in the experimental group: adding 2 mu L of 1nmol/L target EGFR19del and 1 mu L of 100nmol/L circular DNA template into the step (1), uniformly mixing by vortex, instantly separating the liquid to the bottom of the tube, then placing the tube in a PCR instrument for reaction at 37 ℃ for 1h, and then heating to 65 ℃ for reaction for 10min to inactivate LbaCas12a protease; samples in the control group replaced EGFR19del with the same volume of water, and were otherwise identical to the experimental group.
(3) RCA reaction: to another PCR reaction tube were added 13.6. mu.L of ddH in sequence2O, 2 mu L of 10 XPhi 29 DNA polymerase reaction buffer solution, 1 mu L of 1U/muL of phi29 DNA polymerase, 1 mu L of 10mmol/L dNTPs, 0.4 mu L of the experimental group and the control group CRISPR-Cas12a enzyme digestion product prepared in the step (2) and 2 mu L of 10nmol/L RCA primer, wherein after vortex mixing uniformly, the liquid is instantaneously separated to the bottom of a tube, then the tube is placed in a PCR instrument for reaction at 30 ℃ for 1h, and then the tube is heated to 65 ℃ for reaction for 10min to inactivate the phi29 DNA polymerase, so that the RCA product is prepared.
(4) And (3) detecting absorbance: adding 2. mu.L of 10. mu. mol/L hemin (hemin is ultrasonically dissolved to 1mmol/L by DMSO, HEPES buffer is diluted to 10. mu. mol/L) and 55. mu.L of self-prepared HEPES buffer (50mmol/L HEPES, 400mmol/L NaCl, 40mmol/L KCl, 0.1% Triton X-100, 2% DMSO, pH 7.0) to the RCA product prepared in the step (3), and standing at room temperature for 1 h; then 5. mu.L of 40mmol/L H was added thereto2O2(ddH2O dilution of 35% H2O2To 40mmol/L) and 20. mu.L, 7.5mmol/L ABTS buffer (pH 4.5 acetate-sodium acetate buffer to dissolve ABTS tablets to 7.5 mmol/L); and after rapid and sufficient mixing, immediately placing the mixture in an enzyme-labeling instrument, and detecting the dynamic change of absorbance within 0-30 minutes and the change of absorbance within the wavelength range of 400-500 nm at the wavelength of 420 nm.
The results of this experiment were obtained by taking the decrease in absorbance (Δ a) at a wavelength of 420nm, measured at a wavelength of 420nm, of the experimental group (containing EGFR19 del) relative to the control group (without EGFR19 del), that is, Δ a ═ a (a 19del) as the trend of change in signal0-A)/A0A and A0Represents the absorbance values measured at a wavelength of 420nm for the experimental group containing EGFR19del and the control group without EGFR19del, respectively. As shown in A in FIG. 1, the Δ A gradually increases with the increase of the crRNA concentration, and reaches a maximum value when the final crRNA concentration is 30 nmol/L. Therefore, the optimal concentration of crRNA is 30 nmol/L.
2. LbaCas12a concentration optimization
The LbaCas12a concentrations were 10nmol/L, 15nmol/L, 20nmol/L, 25nmol/L and 30nmol/L, respectively, and the other experimental procedures were the same as in example 1, with the results shown in FIG. 1, B.
As in fig. 1B, Δ a ═ a (a) with increasing concentrations of LbaCas12a [ Δ a ═ B0-A)/A0A and A0Respectively represent the absorbance values measured at a wavelength of 420nm of an experimental group containing EGFR19del and a control group without EGFR19del]The value gradually increased, and Δ a gradually stabilized as the LbaCas12a concentration increased to 25 nmol/L. Therefore, 25nmol/L is the optimal concentration of LbaCas12 a.
3. phi29 DNA polymerase content optimization
The contents of phi29 DNA polymerase were 0.1U, 0.3U, 0.5U, 1U and 2U, respectively, and the results of the other experimental procedures were as in example 1 and are shown in FIG. 1C.
As can be seen from C in FIG. 1, as the content of phi29 DNA polymerase increases, the signal-to-noise ratio S/N (absorbance at 420nm measured in the presence and absence of the circular DNA template for S and N, respectively) gradually increases, and the S/N change stabilizes when the content of phi29 DNA polymerase exceeds 1U. Therefore, the optimal content of phi29 DNA polymerase was 1U.
4. HEPES buffer pH optimization
HEPES buffers were adjusted to pH 4, 5, 6, 7 and 8, and other experimental procedures were performed as in example 1, with the results shown in FIG. 1, item D.
As can be seen from D in FIG. 1, as the pH of the HEPES buffer increases, the signal-to-noise ratio S/N (S and N are absorbance values measured at a wavelength of 420nm in the presence and absence, respectively, of the circular DNA template) gradually increases, and the S/N decreases sharply when the pH of the HEPES buffer exceeds 7. Therefore, the optimal pH of HEPES buffer is 7.
Example 4
And (3) sensitivity test:
(1) the synthetic target EGFR19del was diluted to a concentration of 0mol/L and 0.5X 10-13mol/L、1×10-13mol/L、1×10-12mol/L、1×10-11mol/L、1×10-10mol/L、1×10-9mol/L、1×10-8mol/L、0.5×10-7mol/L。
(2) Preparation of Cas12a/crRNA complex: 13.3. mu.L of ddH was sequentially added to the PCR reaction tube2O, 2. mu.L of 10 XNEBuffer, 1.2. mu.L of 500nmol/L crRNA, 0.5. mu.L of 1. mu.mol/L LbaCas12a, and incubation at 37 ℃ for 30min to prepare a Cas12a/crRNA complex solution.
(3) CRISPR-Cas12a enzymatic cleavage: adding 2 mu L of target EGFR19del prepared in the step (1) with different concentrations and 1 mu L of 100nmol/L circular DNA template into the Cas12a/crRNA complex solution, uniformly mixing by vortex, instantly separating the liquid to the bottom of the tube, then placing the tube in a PCR instrument for reacting at 37 ℃ for 1h, heating to 65 ℃ for reacting for 10min to inactivate LbaCas12a protease, and preparing the CRISPR-Cas12a enzyme digestion product.
(4) RCA reaction: to another PCR reaction tube were added 13.6. mu.L of ddH in sequence2O、2μL 10×phi29And (3) carrying out vortex mixing on the DNA polymerase reaction buffer solution, 1 mu L of 1U/. mu.L phi29 DNA polymerase, 1 mu L of 10mmol/L dNTPs, 0.4 mu L of CRISPR-Cas12a enzyme digestion product prepared in the step (3) and 2 mu L of 10nmol/L RCA primer, then carrying out instantaneous separation on the liquid to the bottom of the tube, subsequently placing the tube in a PCR instrument for reaction at 30 ℃ for 1h, then heating to 65 ℃ for reaction for 10min to inactivate the phi29 DNA polymerase, and preparing the RCA product.
(5) And (3) detecting absorbance: adding 2. mu.L of 10. mu. mol/L hemin (hemin is ultrasonically dissolved to 1mmol/L with DMSO, HEPES buffer is diluted to 10. mu. mol/L) and 55. mu.L of self-prepared HEPES buffer (50mmol/LHEPES, 400mmol/L NaCl, 40mmol/L KCl, 0.1% Triton X-100, 2% DMSO, pH 7.0) to the RCA product prepared in the step (4), and standing at room temperature for 1 h; then 5. mu.L of 40mmol/L H was added thereto2O2(ddH2O dilution of 35% H2O2To 40mmol/L) and 20. mu.L, 7.5mmol/L ABTS buffer (pH 4.5 acetate-sodium acetate buffer to dissolve ABTS tablets to 7.5 mmol/L); and after rapid and sufficient mixing, immediately placing the mixture in an enzyme-labeling instrument, and detecting the dynamic change of absorbance within 0-30 minutes and the change of absorbance within the wavelength range of 400-500 nm at the wavelength of 420 nm.
As a result: the experimental result is measured by the absorbance reduction (delta A) of the experimental group (containing different concentrations of EGFR19 del) relative to the control group (without EGFR19 del) at the wavelength of 420nm, namely, the content of the target in the detection system is measured, namely, the delta A is (A ═ A0-A)/A0A and A0Respectively represent the absorbance values measured at a wavelength of 420nm for the experimental group and the control group. As shown in A and B in FIG. 2, from the measured visible spectrum of 400nm to 500nm (A in FIG. 2), it can be seen that the absorbance of the solution gradually decreases as the concentration of EGFR19del gradually increases. The absorbance at 420nm gradually decreased with increasing concentration of EGFR19del, mainly because more EGFR19del resulted in less circular DNA template for RCA reaction, thereby decreasing the content of G-quadruplex/hemin DNAzyme, failing to catalyze H2O2Mediated ABTS oxidation produces a distinct color change. Analysis of the data from the corresponding fitted calibration curve (B in FIG. 2) shows that the present invention has a good linear fit (R) between 50fmol/L and 50nmol/L20.99992) and the fitting equation is Δ a 0.14721lg cEGFR 19del+0.25098. Based on the 3 sigma/k rule, the detection limit is calculated to be 20fmol/L, which shows that the method has higher sensitivity.
Example 5
And (3) specificity test:
(1) a sample which does not contain a target to be detected and any other interfering nucleic acid sequences is prepared as a control group, and a mixed sample which contains 1nmol/L lncRNA HULC, 1nmol/L lncRNA H19-1, 1nmol/L Kras G12S, 1nmol/L Kras G13D, 1nmol/L wild-type EGFR L858R, 1nmol/L mutant EGFR L858R, 1nmol/L wild-type EGFR19del, 1nmol/L double-base mismatch EGFR19del, 1nmol/L single-base EGFR19del, 1nmol/L target EGFR19del, and contains EGFR19del, lncRNA HULC, lncRNA H19-1, Kras G12S, Kras G13D, wild-type EGFR L858R, mutant EGFR L858R, wild-type EGFR19del, double-base EGFR19del, single-base EGFR19 mismatch 1nmol is prepared as an experiment group.
(2) Preparation of Cas12a/crRNA complex: 13.3. mu.L of ddH was sequentially added to the PCR reaction tube2O, 2. mu.L 10 XNEBuffer 2.1, 1.2. mu.L 500nmol/L crRNA, 0.5. mu.L 1. mu. mol/L LbaCas12a, incubated at 37 ℃ for 30min to prepare a Cas12a/crRNA complex solution.
(3) CRISPR-Cas12a enzymatic cleavage: and (2) adding 2 mu L of the control sample prepared in the step (1), the experimental sample containing the target or interfering nucleic acid and 1 mu L of 100nmol/L circular DNA template into the Cas12a/crRNA complex solution, vortexing and mixing uniformly, then instantly separating the liquid to the bottom of the tube, subsequently placing the tube in a PCR instrument for reacting for 1h at 37 ℃, and then heating to 65 ℃ for reacting for 10min to inactivate LbaCas12a protease, thereby preparing the CRISPR-Cas12a enzyme digestion product.
(4) RCA reaction: to another PCR reaction tube were added 13.6. mu.L of ddH in sequence2O, 2 mu L of 10 XPhi 29 DNA polymerase reaction buffer solution, 1 mu L of 1U/. mu.L phi29 DNA polymerase, 1 mu L of 10mmol/L dNTPs, 0.4 mu L of CRISPR-Cas12a enzyme digestion product prepared in the step (3) and 2 mu L of 10nmol/L RCA primer, wherein after vortex mixing uniformly, the liquid is instantaneously separated to the bottom of a tube, then the tube is placed in a PCR instrument for reaction at 30 ℃ for 1h, and then the tube is heated to 65 ℃ for reaction for 10min to inactivate the phi29 DNA polymerase, so that the RCA product is prepared.
(5) And (3) detecting absorbance: to the RCA product prepared in step (4)Adding 2 μ L, 10 μmol/L hemin (hemin is ultrasonically dissolved to 1mmol/L with DMSO, HEPES buffer is diluted to 10 μmol/L) and 55 μ L of self-prepared HEPES buffer (50mmol/L HEPES, 400mmol/L NaCl, 40mmol/L KCl, 0.1% Triton X-100, 2% DMSO, pH 7.0), standing at room temperature for 1 h; then 5. mu.L of 40mmol/L H was added thereto2O2(ddH2O dilution of 35% H2O2To 40mmol/L) and 20. mu.L, 7.5mmol/L ABTS buffer (pH 4.5 acetate-sodium acetate buffer to dissolve ABTS tablets to 7.5 mmol/L); and after rapid and sufficient mixing, immediately placing the mixture in an enzyme-labeling instrument, and detecting the dynamic change of absorbance within 0-30 minutes and the change of absorbance within the wavelength range of 400-500 nm at the wavelength of 420 nm.
As a result: the result of this experiment is measured as the decrease in absorbance (Δ a) at a wavelength of 420nm of the experimental group (containing EGFR19del or interfering nucleic acid) relative to the control group (without EGFR19del and any interfering nucleic acid), i.e., Δ a ═ a (a0-A)/A0A and A0Respectively represent the absorbance values measured at a wavelength of 420nm for the experimental group and the control group. As shown in FIG. 3, the present invention results in a significant increase in Δ A by the target (k) and only a slight change in Δ A by the interfering nucleic acid (b-j) when the same concentration of target and interfering nucleic acid sequences is detected. In addition, when the detection sample contains both the target and the interfering nucleic acid, the delta A is also obviously increased (l), and the delta A has no obvious difference compared with the experimental group only containing the target, which shows that the method can accurately identify the target from the interfering nucleic acid and has higher specificity.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
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Claims (10)

1. A CRISPR-Cas12 a-based visual detection system for triggering non-specific rolling circle amplification is characterized in that: comprises single-stranded RNA containing a stem-loop structure, annular single-stranded nucleic acid, a Cas enzyme with trans-cleavage activity, an RCA reaction reagent and a G-quadruplex/hemin DNAzyme catalytic ABTS color development system;
the single-stranded RNA is homologous with one strand part in the double-stranded DNA target to be detected and can be complementarily combined with the other strand, the single-stranded RNA can be combined with Cas enzyme to form a complex, and the accessory cleavage activity of the complex is activated in the presence of the target to be detected;
the circular single-stranded nucleic acid can be combined with an RCA primer and amplified, and an amplification product can generate a sequence of a G-quadruplex and also has a Cas enzyme auxiliary cutting site sequence;
the G-quadruplex/hemin DNAzyme catalyzed ABTS color development system consists of hemin solution and K+Buffer solution of (1), H2O2Solution and ABTS buffer solution, the amplification product containing K+And hemin solution to form a G-quadruplex/hemin DNAzyme complex, said G-quadruplex/hemin DNAzyme complex with H2O2The solution and ABTS buffer reacted color.
2. The CRISPR-Cas12 a-based visual detection system for triggering non-specific rolling circle-free amplification according to claim 1, which is characterized in that: the concentration of the single-stranded RNA is 15-35 nmol/L; the concentration of the Cas enzyme is 15-30 nmol/L; the content of phi29 DNA polymerase is 0.1-2U; said group containing K+The pH of the buffer solution is 4-8.
3. The CRISPR-Cas12 a-based visual detection system for triggering non-specific rolling circle-free amplification according to claim 1, which is characterized in that: the hemin solution in the G-quadruplex/hemin DNAzyme catalysis ABTS color development system is 10 mu mol/L hemin; said group containing K+The buffer of (1) comprises the following components: 50mmol/L HEPES, 400mmol/L NaCl, 40mmol/L KCl, 0.1% Triton X-100, 2% DMSO, pH 7.0; said H2O2The solution was 40mmol/L H2O2(ii) a The ABTS buffer solution is prepared by dissolving ABTS in acetic acid-sodium acetate buffer solution with pH of 4.5 to the concentration of 7.5mmol/L。
4. The CRISPR-Cas12 a-based visual detection system for triggering non-specific rolling circle-free amplification according to claim 1, which is characterized in that: the RCA reaction system contains RCA primers, phi29 DNA polymerase, dNTPs and 1 XPhi 29 DNA polymerase reaction buffer solution.
5. The CRISPR-Cas12 a-based visual detection system for triggering non-specific rolling circle-free amplification according to claim 1, which is characterized in that: the target to be detected is EGFR19del, the single-stranded RNA sequence is shown as SEQ ID NO.1, and the annular single-stranded nucleic acid sequence is shown as SEQ ID NO. 2.
6. The CRISPR-Cas12 a-based visual detection system for triggering non-specific rolling circle-free amplification according to claim 4, characterized in that: the RCA primer is shown as SEQ ID NO. 5.
7. Use of the visual inspection system of any one of claims 1 to 6 for the detection of a target agent to be inspected.
8. The method for detecting a target to be detected by using the visual detection system as claimed in any one of claims 1 to 6, which comprises the following steps:
(1) adding the Cas enzyme and the single-stranded RNA into a buffer solution for incubation to obtain a Cas enzyme \ single-stranded RNA compound solution;
(2) adding a solution to be detected and annular single-stranded nucleic acid into the Cas enzyme \ single-stranded RNA compound solution obtained in the step (1), performing enzyme digestion reaction, and heating to inactivate the Cas enzyme; if the solution to be detected contains the target to be detected, the Cas enzyme is used for enzyme-cutting the target to be detected, and meanwhile, the auxiliary cutting activity is activated, so that the annular single-stranded nucleic acid is cut; if the liquid to be detected does not contain the target to be detected, the attached cleavage activity of the Cas enzyme is not activated, and the ring-shaped single-stranded nucleic acid cannot be cleaved;
(3) adding an RCA reaction reagent into the enzyme-digested product obtained in the step (2), and if the annular single-stranded DNA is not cut, carrying out RCA amplification to obtain an RCA product; if the circular single-stranded DNA is not subjected to the RCA cutting reaction, no product is generated;
(4) adding hemin solution containing K to RCA product+The buffer of (1) was left for 1h, and the RCA reaction product, if any, was in K+Forming parallel G-quadruplex structure under the induction of (1), combining the formed G-quadruplex structure with hemin to form a stable G-quadruplex/hemin DNAzyme complex with peroxidase activity, and then adding H2O2And ABTS solution, complex catalysis H2O2The mediated oxidation of ABTS, resulting in a color change, which is then observed by a microplate reader, spectrophotometer, or by the naked eye.
9. The visual inspection system of claim 6, wherein the visual inspection system comprises: the Cas enzyme is Cas12, Cas13, or Cas 14.
10. The visual inspection system of claim 6, wherein the visual inspection system comprises:
(1) adding the Cas enzyme and the single-stranded RNA into 1 XNEBuffer to incubate for 30min at 37 ℃ to obtain a Cas enzyme \ single-stranded RNA compound solution;
(2) adding a solution to be detected and the annular single-stranded DNA into the Cas enzyme \ single-stranded RNA compound solution obtained in the step (1), reacting for 1h at 37 ℃, and then heating to 65 ℃ for reaction for 10min to inactivate the Cas enzyme; if the solution to be detected contains the target to be detected, the Cas enzyme cuts the target to be detected, and simultaneously the auxiliary cutting activity is activated to cut the single-stranded DNA; if the solution to be detected does not contain the target to be detected, the attached cleavage activity of the Cas enzyme is not activated, and the single-stranded DNA cannot be cleaved;
(3) adding an RCA reaction system into the enzyme digestion product obtained in the step (2), reacting for 1h at 30 ℃, then heating to 65 ℃ and reacting for 10min to inactivate phi29 DNA polymerase, and if the annular single-stranded DNA is not cut, carrying out RCA reaction to obtain an RCA product; if the circular single-stranded DNA is cut, no product is generated after the RCA reaction;
(4) adding hemin solution containing K to RCA product+The buffer of (1) was left for 1h, and if RCA reaction product was present, the RCA reaction product was in K+Under the induction of (2) to form a parallel G-quadruplex structure, and a G-quadruplex junction formedThe construct binds hemin to form a stable G-quadruplex/hemin DNAzyme complex with peroxidase activity, followed by the addition of H2O2And ABTS solution, complex catalysis H2O2The mediated oxidation of ABTS, resulting in a color change, which is then observed by a microplate reader, spectrophotometer, or by the naked eye.
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