CN111394430A - Detection system based on CRISPR-Cas12a coupling enhanced strand displacement amplification and application thereof - Google Patents
Detection system based on CRISPR-Cas12a coupling enhanced strand displacement amplification and application thereof Download PDFInfo
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Abstract
The invention discloses a detection system based on CRISPR-Cas12a coupling enhanced strand displacement amplification and application thereof, wherein the system comprises a template, L ba Cas12a, crRNA, a report single-stranded DNA molecule, DNA polymerase and endonuclease Nt.BbvCI, wherein the template is complementary with a sequence base to be detected and synthesizes double-stranded DNA complementary with the template, the formed double-stranded DNA comprises two endonuclease Nt.BbvCI recognition sites and obtains an activated strand sequence fragment through shearing and polymerization displacement reaction, the crRNA comprises a fixed sequence and a recognition sequence, the fixed sequence is specifically recognized with L ba Cas12a, and the recognition sequence is complementary with the activated strand sequence fragment.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a detection system based on CRISPR-Cas12a coupling enhanced strand displacement amplification and application thereof.
Background
DNA is one of the most important biomolecules in living cells, and is involved in the regulation of various genetic information. The DNA sensor is a biosensor for detecting specific sequence DNA, is widely applied to the detection of specific sequence DNA with practical significance, and has important significance in the aspects of medical diagnosis, genetic diseases, mutation detection, food control, forensic science, environmental monitoring and the like. In general, a specific sequence DNA associated with a specific disease is usually characterized by ultra trace amount, easy degradation, and complex detection environment. However, current detection techniques for detecting DNA of a specific sequence generally rely on Polymerase Chain Reaction (PCR), which has some disadvantages, such as time consumption, complicated procedures, insufficient detection limit, requirement of specialized instrumentation, etc. Therefore, it is of great significance to develop a detection method capable of simply, ultra-sensitively and accurately detecting a specific sequence DNA. For example, Human Immunodeficiency Virus (HIV) is a related virus of acquired immunodeficiency syndrome (ADIS) and can lead to a collapse of the human immune system and ultimately cell death by destroying and phagocytosing human T4 lymphocytes. To date, there is no effective treatment for hiv. Fortunately, antiretroviral therapies have been found to cure ADIS, and early diagnosis and treatment of HIV infection may be effective in protecting the host immune function and extending survival. Therefore, early and accurate detection of the HIV gene is of great importance for early diagnosis and clinical treatment of infected individuals.
Maintaining genome integrity is a prerequisite for all organisms to complete their gene transcription. Unfortunately, the stability and accuracy of the genome are disturbed by some intrinsic or extrinsic factors, such as some toxic chemicals, ultraviolet radiation and physical radiation, which cause the genome to be mutated and induce various diseases. In organisms, deamination of cytosines may lead to U: the formation of G base pairs ultimately leads to G: c base pair mutation to a: and a T base pair. Thus, repair of such DNA damage is of great importance to maintain the integrity of the organism's genome. In recent years, several specific pathways for DNA repair have been extensively studied, such as mismatch repair (MMR), Nucleotide Excision Repair (NER) and Base Excision Repair (BER). Uracil DNA Glycosylase (UDG) is an important enzyme in base excision repair enzymes. In most organisms from bacteria to humans, base excision repair enzymes are present which characteristically recognize uracil and catalyze the cleavage of the N-glycosidic bond between deoxyribose and uracil, thereby creating a purine/pyrimidine (AP) site to initiate Base Excision Repair (BER) to maintain genome integrity. However, abnormal activity of UDG may prevent the normal progression of uracil-containing DNA base excision repair processes, leading to a variety of pathologies such as chemotherapy resistance, lymphoma, cancer, neurodegenerative diseases, brucm's syndrome, and human immunodeficiency. Given its important biological role, UDG has become a promising therapeutic target and a potential biomarker for diagnosing various diseases. Traditional detection methods such as gel electrophoresis, mass spectrometry, electrochemistry, chemiluminescence, colorimetry, and radioisotope labeling have been used for the active detection of UDG. However, these methods have disadvantages of time consumption, low sensitivity, and complicated operation. Therefore, establishing a detection method capable of accurately, highly sensitively and effectively detecting the activity of UDG has important significance for clinical diagnosis and biomedical research.
The CRISPR/Cas system is an RNA-guided adaptive immune mechanism for specific recognition and cleavage of foreign nucleic acids since its discovery, is expected due to its important role in gene editing furthermore, CRISPR/Cas systems can be used in the field of analysis Doudna finds that CRISPR/Cas12a has indiscriminate cleavage activity (trans-cleavage) after specific recognition of target nucleic acids is activated, which reveals the great potential of CRISPR/Cas in analysis. by combining Cas12assDNase activation with recombinant polymerase isothermal amplification, they create a method called DNA endonuclease targeting CRISPR trans-reporter (detectrr) that can achieve aM-level detection of dna.a. subsequently, Zhang finds that CRISPR/Cas13 can bind to a target single-stranded RNA and activate its indiscriminate trans-cleavage activity (trans-cleavage). their seer L OCK (specific high-enzyme-unlocked) platform based on 13 can detect both carv and carv virus (dezikv) in patient samples, while their potential is still demonstrated by using low-copy-of-DNA (crna) amplification systems, low-cycle amplification of target nucleic acids (crrca) and amplification of all DNA by pcr-mediated amplification of crjs (crna). thus, low-amplification of crjs-mediated amplification of target nucleic acids (crpr) and amplification).
Disclosure of Invention
Aiming at the defects of the existing detection technology, the invention aims to provide a detection system based on CRISPR-Cas12a coupling enhanced strand displacement amplification and application thereof, and solves the problems of low sensitivity, low efficiency, complex operation and the like of the existing specific DNA or UDG activity detection.
In order to solve the technical problems, the invention adopts the following technical scheme that the detection system based on CRISPR-Cas12a coupling enhanced strand displacement amplification comprises a template, L ba Cas12a, a crRNA, a report single-stranded DNA molecule, a DNA polymerase and an endonuclease Nt.BbvCI, wherein the template is complementary with a sequence to be detected in base and synthesizes double-stranded DNA complementary with the template, the formed double-stranded DNA comprises two recognition sites of the endonuclease Nt.BbvCI, the double-stranded DNA is subjected to shearing and polymerization displacement to obtain an activated strand sequence fragment, the crRNA comprises a fixed sequence and a recognition sequence, the fixed sequence is specifically recognized with the L ba Cas12a, and the recognition sequence is complementary with the activated strand sequence fragment, and the crRNA guides L ba Cas12a to be combined with the activated strand sequence fragment to activate the trans-cleavage activity of the L ba Cas12a, so that the report single-stranded DNA molecule is cleaved and fluorescence is generated.
Further, the detection system also comprises additives required for DNA amplification; the additive comprises deoxyribonucleoside triphosphates (dNTPs), a buffer, a diluent, magnesium ions, manganese ions or a cofactor.
The invention also provides application of the detection system based on CRISPR-Cas12a coupled enhanced strand displacement amplification in detection of specific sequence DNA, screening of UDG activity inhibitors or detection of UDG activity biological samples. The method specifically comprises the following steps:
(1) initiating enhanced strand displacement amplification in the presence of a DNA polymerase and an endonuclease nt.bbvci to produce an activated strand sequence fragment;
(2) activating CRISPR/Cas12a system trans-cleavage activity by complementary pairing of the activation strand sequence fragment obtained in the step (1) and a crRNA recognition region;
(3) and (3) cutting the reporter single-stranded DNA molecule added into the system by using the activated CRISPR/Cas12a system in the step (2), and detecting the fluorescence intensity in the system.
The invention also provides a method for detecting HIV-1 by using the detection system based on CRISPR-Cas12a coupled enhanced strand displacement amplification, which is used for non-diagnostic or therapeutic purposes and comprises the following steps:
s1, designing a crRNA sequence with specific recognition region aiming at the L ba Cas12a sequence and the activation strand sequence fragment, wherein the crRNA sequence is shown as SEQ ID NO: 1, and then constructing a crRNA in-vitro transcription vector and carrying out in-vitro transcription and purification, or directly synthesizing;
s2: synthesizing a template-1, wherein the sequence of the template-1 is shown as SEQ ID NO: 2, the template-1 can be complementary with an HIV-1 sequence to be detected to synthesize double-stranded DNA;
s3: mixing the template-1 synthesized in the step S2, HIV-1 with different concentrations, dNTPs, DNA polymerase and endonuclease Nt.BbvCI in a buffer solution according to a proper proportion to obtain a reaction system, placing the reaction system at 37 ℃ for strand displacement amplification reaction, ending the reaction, and inactivating the enzyme at high temperature to obtain an amplification product;
s4, adding a certain concentration of reporter single-stranded DNA molecule, L basAs 12a and the crRNA molecule obtained in the step S1 into the amplification product obtained in the step S3, mixing the mixture in a buffer solution according to a proper proportion to obtain a reaction system, placing the reaction system in a shearing reaction at 37 ℃, terminating the reaction after the reaction is carried out for a period of time, and reading a fluorescence detection signal;
s5: and (3) making a standard curve of HIV-1 concentration-fluorescence intensity according to standard solutions of HIV-1 with different concentrations, calculating a regression equation, and calculating the concentration of HIV-1 in the solution to be detected according to the fluorescence intensity of the solution to be detected.
The invention also provides a method for detecting the activity of the UDG by using the detection system based on CRISPR-Cas12a coupled enhanced strand displacement amplification, which is used for non-diagnostic or therapeutic purposes and comprises the following steps:
1) aiming at L basaCas 12a sequence and activation strand sequence fragment, designing a crRNA sequence with specific recognition region, wherein the crRNA sequence is shown as SEQ ID NO: 1, and then constructing a crRNA in-vitro transcription vector and performing in-vitro transcription and purification, or directly synthesizing;
2) synthesizing a template-2 sequence and a UDG primer, wherein the template-2 sequence is shown as SEQ ID NO: 3, the sequence of the UDG primer is shown as SEQ ID NO: 4 is shown in the specification; the 3' end of the UDG primer is modified with uracil, an object to be detected UDG enzyme can recognize and process the uracil in a UDG primer chain into an AP site, and the AP site can be cut by endonuclease IV to obtain a pretreated UDG primer; the template-2 can be complementary with the base of the pretreated UDG primer to synthesize double-stranded DNA;
3) mixing the template-2 synthesized in the step 2), the UDG primer, the UDG enzyme with different concentrations and the endonuclease IV, and reacting at the constant temperature of 37 ℃ for 40-80 min;
4) adding dNTPs, DNA polymerase and endonuclease Nt.BbvCI into the reaction solution obtained in the step 3) and mixing the mixture in a buffer solution according to a proper proportion to obtain a reaction system, placing the reaction system in a strand displacement amplification reaction at 37 ℃, after the reaction is finished, inactivating the enzyme at a high temperature to obtain an amplification product;
5) adding the amplification product obtained in the step 4) into a report single-stranded DNA molecule, L basas 12a and the crRNA molecule obtained in the step (1), mixing the mixture in a buffer solution according to a proper proportion to obtain a reaction system, placing the reaction system in a shearing reaction at 37 ℃, terminating the reaction after reacting for a period of time, and reading a fluorescence detection signal;
6) according to standard solutions of UDG enzyme with different concentrations, making a standard curve of activity-fluorescence intensity of the UDG enzyme, and calculating a regression equation; and calculating the activity of the UDG enzyme in the solution to be detected according to the fluorescence intensity of the solution to be detected.
Preferably, the strand displacement amplification reaction time is 60-100 min, and the shearing reaction time is 15-35 min.
Preferably, the concentrations of the DNA polymerase and the endonuclease Nt.BbvCI in the reaction system are respectively and independently selected from 0.15-0.35U/. mu. L.
Preferably, the reporter single-stranded DNA molecule is a 7-base single-stranded DNA molecule with HEX and BHQ1 groups at two ends, and the sequence is shown as HEX-NNNNN-BHQ 1.
Preferably, the fluorescence detection is fluorescence excitation at a wavelength of 496nm and fluorescence intensity detection at a wavelength of 556 nm.
The detection principle of the invention is that under the guide of crRNA, L bas 12a protein is combined with an activation strand sequence fragment to activate the trans-cleavage activity of L bas 12a, and simultaneously a single-stranded fluorescent reporter molecule (a commonly used single-stranded reporter molecule is an oligonucleotide sequence, one end of which is provided with a luminescent group and the other end of which is provided with a quenching group, under the normal condition, the complete single-stranded reporter molecule cannot be detected due to the quenching effect, and when the oligonucleotide molecule is hydrolyzed, a free fluorescent signal can be detected), the conversion of sequence information to be detected into the fluorescent signal can be realized by virtue of the CRISPR-Cas12a enzyme trans-cleavage activity, and the multistage amplification of enhanced strand displacement amplification (E-SDA) and enzymatic cascade (Cas enzyme completion) can be realized by coupling the E-SDA and the CRISPR-Cas12a, so that the detection method has good sensitivity.
Compared with the prior art, the invention has the following beneficial effects:
1. the specific sequence nucleic acid detection system aimed by the invention combines a CRIPSR/Cas12a nucleic acid recognition regulation system and enhanced strand displacement amplification (E-SDA), and develops a novel multi-step biological amplification technology. The efficiency of amplification of E-SDA is greatly improved compared to conventional SDA, since the intermediate product is capable of inducing the initiation of another SDA. The activating strand sequence fragment generated in E-SDA can act as an activator to unlock the trans-cleavage activity of CRISPR/Cas12a, thereby indiscriminate cleavage system adding a single-stranded reporter DNA molecule. On the basis, a DNA primer chain capable of detecting the activity of the UDG is designed according to the characteristic that the UDG can recognize and process the AP site generated by uracil in the DNA primer chain, and the rapid detection of the DNA with a specific sequence or the activity of the UDG is realized by detecting the fluorescence intensity. The system provides a new idea and a new choice for detecting the activity of the specific sequence DNA and the UDG, plays a key role in clinical diagnosis and biomedical research, and has good application prospect.
2. The detection system provided by the invention can detect HIV-1 with the concentration as low as 87.3aM or HIV-1 with the concentration as low as 3.1 × 10 under the optimal experimental conditions by the proposed detection method-5The activity of the UDG of U/m L can detect sequence-specific DNA or the activity of the UDG with ultra-sensitivity, and the aM sensitivity to the DNA with a specific sequence can be realized within 2.5 hours, or the ultra-low activity detection of the UDG can be realized within 3.5 hours.
3. The detection system has the advantages of low background, high response speed, high accuracy, good repeatability, simple and convenient operation, strong anti-interference capability, higher sensitivity and specificity, overcomes the dependence of the traditional nucleic acid detection on instruments, and is simple in result reading.
4. The invention firstly utilizes the trans-cutting of the CRISPR/Cas12a system to detect non-nucleic acid substance UDG, can be used for UDG activity detection or UDG inhibitor screening, is used for detecting other biomolecules except nucleic acid, enlarges the detection and application range of the CRISPR/Cas12a system, and provides a theoretical basis of feasibility.
Drawings
FIG. 1 is a schematic diagram of the detection system of the present invention for detecting HIV-1 with specific sequences.
FIG. 2 is a diagram of the optimization of the conditions of the detection system of the present invention; a is the cleavage time of CRISPR/Cas12a, B is the amplification incubation time of E-SDA, C is the concentration of Klenow fragment (3'-5' exo-), and D is the concentration of Nt.BbvCI; wherein the left side of each set of experiments is the experimental group and the right side is the control group.
FIG. 3 is a diagram of an HIV-1 assay with the detection system of the present invention; FIG. A is a graph showing the fluorescence spectra of HIV-1 at various concentrations; FIG. B shows the fluorescence values at 556nm for various concentrations of HIV-1, with the inset being the corresponding log-fit curve for HIV-1 concentrations between 100aM and 5 pM.
FIG. 4 is a diagram showing the specific detection of HIV-1 by the detection system of the present invention; panel A shows the fluorescence spectra at a concentration of 20pM for HIV-1 and various analogs as the target; panel B is a graph of the fluorescence response of the target HIV-1 and various analogs.
FIG. 5 is a schematic diagram of the detection system of the present invention for detecting UDG activity.
FIG. 6 is an analysis chart of the detection system of the present invention for UDG, wherein A is a fluorescence spectrum chart of different concentrations of UDG, and B is a fluorescence value corresponding to 556nm for different concentrations of UDG, and the inset is a graph of the fluorescence value when UDG is 5.0 × 10-5Corresponding to 0.1U/m L, a log fit curve was taken.
FIG. 7 shows the specific detection of UDG by the detection system of the present invention, panel A shows the fluorescence spectrum of UDG and its various analogs at 0.1U/m L, and panel B shows the fluorescence response of UDG and its various analogs.
FIG. 8 shows the detection of UDG inhibitors by the detection system of the present invention, wherein FIG. A shows the fluorescence spectra of 0.1U/m L UDG with different concentrations of UGI added thereto, and FIG. B shows the inhibition of UGI on the activity of 0.1U/m L UDG.
FIG. 9 shows fluorescence intensities of different solutions of the detection system of the present invention, from left to right, He L a cell lysate, He L a cell lysate +0.30U/m L UDI, and lysis buffer are sequentially added.
Detailed Description
The present invention will be described in further detail with reference to examples.
The following examples all nucleic acid sequences were synthesized by Shanghai biological Co., Ltd and purified by high performance liquid chromatography, detailed information of all nucleic acid sequences is shown in Table 1. HiScribere T7 Quick high fidelity RNA synthesis kit was purchased from NEB, MiRcute miRNA isolation and purification kit was purchased from Beijing Tiangen, enzyme, N.BbvCI enzyme, dNTPs, &lTtTtranslation = L "&gTtL &lTt/T &gTtAsbC 12a and uracil glycosylase inhibitor UGI was ordered from NEB, Uracil Glycosyltransferase (UGD), endonuclease IV, Ha L e cell lysate was purchased from Shanghai biological Co., Ltd.
TABLE 1
The fluorescence intensity of the system was measured with an L S-55 spectrophotometer (P.E.USA) during which the excitation wavelength was set at 495nm, the optimum emission wavelength was 556nm, and the excitation and emission gaps were set at 12nm and 8nm, respectively.
Detection system based on CRISPR-Cas12a coupling enhanced strand displacement amplification
The detection system comprises a template, L bas Cas12a, a crRNA, a report single-stranded DNA molecule, a DNA polymerase and an endonuclease Nt.BbvCI, wherein the template is complementary to a sequence base to be detected or a UDG primer base treated by the UDG and the endonuclease IV, double-stranded DNA complementary to the template strand is synthesized in the presence of the DNA polymerase and deoxyribonucleoside triphosphates, the formed double-stranded DNA comprises two endonuclease Nt.BbvCI recognition sites, the double-stranded DNA is sheared at a specific site under the treatment of the endonuclease, an activated strand sequence fragment capable of being complementarily paired with a crRNA recognition region is polymerized and replaced under the action of a polymerase, the crRNA comprises a fixed sequence and a recognition sequence, the fixed sequence is specifically recognized with L bas 12 Cas a, the recognition sequence is complementary to the activated strand sequence fragment, the crRNA guides L bas 12a to be combined with the activated strand sequence fragment, and the trans-cleavage activity of L bas 12 Cas 64 is activated, so that the report single-stranded DNA molecule is cleaved, and fluorescent single-stranded Cas is generated.
Second, detection method of HIV-1 based on CRISPR-Cas12a coupling enhanced strand displacement amplification
As shown in fig. 1, a method for specific sequence DNA (e.g., HIV-1) detection based on CRISPR/Cas12a system in combination with enhanced strand displacement amplification (E-SDA). DNA of a specific sequence (HIV-1) is used as a primer for inducing DNA polymerization to synthesize a double strand in the presence of a polymerase and dNTPs. Two recognition sites of endonuclease Nt.BbvCI are designed in the synthetic template-1, and after the endonuclease Nt.BbvCI is added, the recognition sites are cut. Subsequent strand displacement synthesis extends the 3' end at the nick and displaces the downstream DNA strands (d and c). Subsequently, a cyclic reaction of polymerization and shearing will produce d and c continuously. d (activating strand sequence fragment) is complementary to the recognition region of crRNA, and c can also bind to template-1, thereby initiating another strand displacement amplification. Thus, a large number of trans-cuts are generated that can unlock the CRISPR/Cas12a system. When it is added to the CRISPR/Cas12a system, the trans-cleavage of CRISPR/Cas12a is activated, indiscriminately cleaving the single-stranded reporter DNA molecule added to the system and ultimately generating a fluorescent signal.
Optimization of experiments
Example 1
1) Setting up experimental group and control group, wherein the experimental group and control group respectively comprise 6 samples, each sample in the experimental group is prepared by mixing 2 mu L10 × NEB buffer 2.0(500mM NaCl, 100mM Tris-HCl, 100mM MgCl2，10mM DTT，pH7.9@25℃)，2μL 10×CutSmart(500mM KAc，200mM Tris-Ac，100mM Mg(Ac)21000. mu.g/M L BSA, pH7.9@25 ℃), 1. mu. L1. mu.M template-1 and the same concentration of HIV-1 were added to 12.5. mu. L ddH2And (4) in O. The mixture was heated at 95 ℃ for 5 minutes and then cooled to 25 ℃ at a rate of 0.1 ℃/S and held for 20 minutes; samples from the control group were not added HIV-1, and were otherwise identical to those from the experimental group.
2) To the reaction product of step 1) was added 0.5. mu. L10 mM dNTPs, 0.5. mu. L10U Klenow fragment (3'-5' exo-) and 0.5. mu. L10 UNt. BbvCI. by ddH2O final volume was adjusted to 20. mu. L the reaction mixtures of the experimental group and the control group were incubated at 37 ℃ for 40min, 60min, 80min, 100min, 120min and 140min, respectively, and then heated at 95 ℃ for 5min to inactivate the enzyme, to obtain an amplification product.
3) Adding 2 μ L1 μ M L bCas12a, 2 μ L1 μ M CrRNA, 10 μ L10 × NEB buffer 2.1(500mM NaCl, 100mM Tris-HCl, 100mM MgCl) into the amplification product of step 2)21000. mu.g/M L BSA, pH7.9@25 ℃ C. 2. mu. L10. mu.M single-stranded reporter molecule was added to the mixture and ddH was used2O adjusted the final volume to 100 μ L after incubation at 37 ℃ for 25 minutes, the reaction of the mixture was stopped by heating at 95 ℃ for 5 minutes the fluorescence spectrum of the reaction mixture was recorded by a L S-55 spectrophotometric fluorometer (p.e. usa) exciting the system at 495nm and emitting at 556nm the slits for excitation and emission were set to 12nm and 8nm respectively during the measurement and the experimental results are shown in fig. 2B.
As can be seen from the figure, the net difference Δ F of the fluorescence signals (Δ F ═ F-F0, F and F0 represent the fluorescence intensities of the experimental group and the control group, respectively) increases with the increase of the E-SDA incubation time, but decreases when the E-SDA incubation time reaches 80 min. Therefore, the optimal incubation time for E-SDA is 80 min.
Example 2
The shearing time of the CRISPR/Cas12A is 10min, 20min, 30min, 40min, 50min and 60min respectively, other experimental steps are the same as example 1, and the experimental result is shown in FIG. 2A.
As can be seen, as the shearing time of the CRISPR/Cas12a reaction increased from 5min to 25min, the net difference Δ F in fluorescence signal (Δ F ═ F-F0, F and F0 represent the fluorescence intensity of the experimental group sample and the control sample, respectively) also increased, while the Δ F value did not change much as the shearing time of CRISPR/Cas12a continued to increase. Therefore, 25min was chosen as the optimal cleavage time for CRISPR/Cas12 a.
Example 3
The concentrations of the Klenow fragment (3'-5' exo-) of the DNA polymerase were 0.05U/. mu. L, 0.1U/. mu. L, 0.15U/. mu. L, 0.2U/. mu. L, 0.25U/. mu. L, and 0.3U/. mu. L, respectively, and the other experimental procedures were the same as in example 1, and the results are shown in FIG. 2C.
As can be seen from the figure, the net difference in fluorescence signal Δ F increases with increasing concentration of Klenow fragment (3'-5' exo-), and Δ F reaches a maximum when the concentration of Klenow fragment (3'-5' exo-) reaches 0.25U/. mu. L, and then increases the concentration of Klenow fragment (3'-5' exo-), and the change in Δ F value is small.
Example 4
The concentrations of endonuclease Nt. BbvCI were 0.05U/. mu. L, 0.10U/. mu. L, 0.15U/. mu. L, 0.2U/. mu. L, 0.25U/. mu. L and 0.30U/. mu. L, respectively, and the other experimental procedures were the same as in example 1, and the results are shown in FIG. 2D.
It can be seen from the figure that the net difference in fluorescence signal Δ F increases with increasing concentration of endonuclease nt.bbvci, which reaches a maximum when the concentration of endonuclease nt.bbvci reaches 0.25U/. mu. L, and then increases the concentration of Klenow fragment (3'-5' exo-), which changes little.
EXAMPLE 5 sensitivity test
1) Mu. L10 × NEB buffer 2.0(500mM N)aCl，100mM Tris-HCl，100mM MgCl2，10mMDTT，pH 7.9@25℃)，2μL 10×CutSmart(500mM KAc，200mM Tris-Ac，100mM Mg(Ac)21000. mu.g/M L BSA, pH7.9@25 ℃), 1. mu. L1. mu.M template-1 and various concentrations of HIV-1(100aM, 1fM, 10fM, 500fM, 5pM, 50pM, 5nM and 50nM) were added to 12.5. mu. L ddH2And (4) in O. The mixture was heated at 95 ℃ for 5 minutes and then cooled to 25 ℃ at a rate of 0.1 ℃/S and held for 20 minutes.
2) To the reaction product of step 1) was added 0.5. mu. L10 mM dNTP, 0.5. mu. L10U Klenow fragment (3'-5' exo-) and 0.5. mu. L10 UNt. BbvCI. by ddH2O final volume was adjusted to 20. mu. L the reaction mixture was incubated at 37 ℃ for 80 minutes and then heated at 95 ℃ for 5 minutes to inactivate the enzyme, yielding an amplification product.
3) Adding 2 μ L1 μ M L bCas12a, 2 μ L1 μ M CrRNA, 10 μ L10 × NEB buffer 2.1(500mM NaCl, 100mM Tris-HCl, 100mM MgCl) into the amplification product of step 2)21000. mu.g/M L BSA, pH7.9@25 ℃ C. 2. mu. L10. mu.M single-stranded reporter molecule was added to the mixture and ddH was used2O adjusted the final volume to 100 μ L after incubation at 37 ℃ for 25 minutes, the reaction of the mixture was stopped by heating at 95 ℃ for 5 minutes the fluorescence spectrum of the reaction mixture was recorded by a L S-55 spectrophotometric fluorometer (p.e. usa) exciting the system at 495nm and emitting at 556nm the slits for excitation and emission were set to 12nm and 8nm respectively during the measurement, the results are shown in fig. 3A and 3B.
As can be seen from the fluorescence spectra, the fluorescence intensity of the system increased with increasing HIV-1 concentration. The fluorescence intensity at 556nm increased gradually with increasing HIV-1 content, mainly because the more HIV-1 present, the more d (active chain sequence fragments) were generated. Analysis of the data from the corresponding fitted simulated curves shows that the constructed biosensing platform has a good linear fit (R) between 100aM and 5pM20.99884) and the fitting equation is Y1＝56.04015lgX1-94.65011. Based on the S/N-3 rule (signal-to-noise ratio), the detection limit was calculated to be 87.3 aM. The detection of this method is minimal compared to many other reported sensors (table 2), andthis multi-step amplification strategy can achieve aM sensitivity to sequence-specific DNA within 2.5 hours. In this biosensing platform, E-SDA has higher amplification efficiency compared to conventional SDA, and can produce large amounts of d ″ (active strand sequence fragments) in the presence of the target. Furthermore, d generated can act as an activator to unlock the trans-cleavage activity of the CRISPR/Cas12a system, which itself has significant signal amplification capabilities. Thus, multi-step amplification offers the potential for the proposed biosensing platform to detect targets with ultra-sensitivity.
Table 2 compares the present invention with other previously reported methods (sequence specific DNA)
Example 6 specificity test
In order to verify that the detection method has excellent anti-interference capability when detecting a specific sequence DNA, the specificity of a DNA detection system is verified by adding non-specific nucleic acid sequences different from a trigger sequence, wherein the non-specific nucleic acid sequences comprise nucleic acid sequences shown in SEQ ID NO. 6-8 of DEEV-1, Zikavirus and HIV-2 respectively, and the nucleic acid sequences are not contained and are used as blank control groups. The results are shown in FIGS. 4A and 4B.
As can be seen from the figure, the fluorescence intensity of the experimental group is significantly higher than that of the interfering group, while the fluorescence intensity of the interfering group is comparable to that of the blank group and is negligible. This is because the other interfering DNA is not fully complementary to the template and therefore does not trigger E-SDA, and the fluorescence intensity of the system is almost vanished. The detection system of the DNA biosensing platform has good specificity when detecting sequence-specific DNA (HIV-1).
Example 7 practical application
Under optimized experimental conditions, standard labeling experiments were performed on normal human serum spiked with different concentrations of HIV-1(1fM, 10fM and 500fM) to study that the proposed biosensing platform can be used for detection of HIV-1 in real samples. The procedure was as in example 5, and the results are shown in Table 3.
Table 3 detection of HIV-1 in normal human serum using the biosensor platform (n ═ 3)
As can be seen from the results, the detection system of the present invention achieves satisfactory recovery rates for detection of HIV-1, ranging from 91.25% to 114.75%. All RSDs measured by this method were below 6.44%. The detection system of the invention provides a reliable platform for detecting the sequence specific DNA in the real sample.
Second, UDG activity detection based on CRISPR-Cas12a coupling enhanced strand displacement amplification
The action mechanism is as follows: as shown in FIG. 5, first, a UDG primer having a uracil lesion in the vicinity of the 3' tail was designed, and the primer front portion was allowed to hybridize with template-2 to form a double strand. Under the action of UDG, the N-glycosidic bond between deoxyribose and uracil in the UDG primer can be recognized and removed to generate AP sites. At the same time, the generated AP site can be cleaved by endonuclease IV, resulting in a complete complementation of the UDG primer with template-2. Thus, the UDG primer treated by UDG and endonuclease IV can act as a primer to induce initiation of enhanced strand displacement amplification (E-SDA), generating an activating strand sequence fragment to unlock the trans-cleavage of the CRISPR/Cas12a system. The activated CRISPR/Cas12a system indiscriminately cleaves single-stranded reporter molecules added to the system and ultimately generates a fluorescent signal.
Example 8 sensitivity test
1) Mu. L1. mu.M template-2, 1. mu. L1. mu.M UDG primer, 5. mu. L ddH2O, 1 μ L10 × UDG buffer (200MmTris-HCl, 10mM EDTA, 100mM NaCl, pH8.2@25 ℃), 1 μ L10 × endonuclease IV buffer (500MmTris-acetate, 500mM KCl, 10mM EDTA, 0.5% (v/v) Triton X-100, pH 7.5) was added to a 200 μ L centrifuge tube, the mixture was heated at 95 ℃ for 5 minutes after mixing, and then cooled to 25 ℃ at a rate of 0.1 ℃/SAnd held at temperature for 20 minutes.
2) The reaction system of step 1) was added with UDG (0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.01, 0.1 and 1U/m L) and 0.5. mu. L10U endonuclease IV at different concentrations and incubated at 37 ℃ for 1h to sufficiently repair the UDG primer.
3) The reaction product of step 2) was added with 2. mu. L10 × NEB buffer 2.0(500mM NaCl, 100mM Tris-HCl, 100mM MgCl210mM DTT, pH7.9@25 ℃, 2 μ L10 × CutSmart (500mM KAc, 200mM Tris-Ac, 100mM Mg (Ac)2, 1000 μ g/m L BSA, pH7.9@25 ℃), 0.5 μ L10 mM dNTP, 0.5 μ L10U Klenow fragment (3'-5' exo-) and 0.5 μ L10 UNt. BbvCI were added to the mixture, and enhanced strand displacement amplification (E-SDA) was induced at 37 ℃ for 80min to give an amplified product.
4) Adding 2 mu L1 mu M L bCas12a, 2 mu L1 mu M CrRNA, 10 mu L10 × NEB buffer 2.1(500mM NaCl, 100mM Tris-HCl, 100mM MgCl) into the amplification product obtained in step 3)21000. mu.g/M L BSA, pH7.9@25 ℃ and 2. mu. L10. mu.M single-stranded reporter DNA molecules were added to the mixture and passed through ddH2O adjusted the final volume to 100 μ L the mixture was then incubated at 37 ℃ for 25min and terminated by heating at 95 ℃ for 5min the fluorescence spectra of the reaction mixture were recorded by a L S-55 spectrophotometric fluorometer (p.e. usa) exciting the system at 495nm and emitting at 556nm the slits for excitation and emission were set to 12nm and 8nm respectively during the measurement, the results are shown in fig. 6A and 6B.
As can be seen from the fluorescence spectrum in the figure, the fluorescence intensity of the system at 556nm gradually increased with the increase of the amount of UDG enzyme. This is because only in the presence of UDG, the N-glycosidic bond between deoxyribose and uracil in the UDG primer can be recognized and removed to generate the AP site. After addition of exonuclease IV, the UDG primer is completely cleaved at its AP site. Thus, the treated UDG primer can act as a primer to induce priming of E-SDA, resulting in a large number of d (active strand sequence fragments). The generated d then acts as an activator to unlock the trans-cleavage of the CRISPR/Cas12a system. Thus, the fluorescence intensity of the system is directly proportional to the UDG activity. As can be seen from the corresponding fitting simulation data, fluorescenceLogarithm of light intensity to UDG concentration from 5.0 × 10-5A good linear relationship (R) was obtained up to 0.1U/m L20.99609) with a corresponding regression equation of Y2＝97.36126lgX2+454.10471, detection limit (L OD) was estimated to be 3.1 × 10 according to the rule of S/N-3-5U/m L, which is the lowest value we know (Table 4).
Table 4 compares the present invention with other previously reported methods (UDG Activity)
Example 9 specificity test
To verify that the detection system of the present invention has excellent anti-interference ability in detecting UDG activity, the target UDG was replaced by other equally concentrated interferents for comparative study during the experiment, and the other procedures were the same as the UDG assay (example 8). The interfering enzymes include hoGG 1, hAGG and BSA, and the results are shown in FIGS. 7A and 7B.
As can be seen from the figure, the fluorescence intensity of the experimental group is significantly higher than that of the interfering group, and the fluorescence intensity of the interfering group is comparable to that of the blank group, with negligible fluorescence increment, indicating that the proposed detection method for detecting UDG activity based on E-SDA coupled with CRISPR/Cas12a has high selectivity and specificity.
Application of CRISPR-Cas12 a-based coupled enhanced strand displacement amplification in screening of UDG inhibitors
To confirm that the proposed detection method can be used for screening of UDG inhibitors and for further studies of the interaction between UDG and its inhibitors, UDG inhibition assays were performed. UDG may be present in a stoichiometric ratio of 1: 1M, form a tight and irreversible complex, thereby inhibiting UDG enzymatic activity.
1) Mu. L1. mu.M template-2, 1. mu. L1. mu.M UDG primer, 5. mu. L ddH2O, 1 μ L10 × UDG buffer (200MmTris-HCl, 10mM EDTA, 100mM NaCl, pH8.2@25 ℃), 1 μ L10 × endonuclease IV buffer (500MmTris-acetate, 500mM KCl, 10mM EDTA, 0.5% (v/v) Triton X-100, pH75) into a 200 μ L centrifuge tube after mixing the mixture was heated at 95 ℃ for 5 minutes and then cooled to 25 ℃ at a rate of 0.1 ℃/S and held for 20 minutes.
2) UGI with different concentrations, UDG (0.1U/m L) with the same concentration and endonuclease IV of 0.5 mu L10U are added into the reaction system in the step 1), and the reaction system is incubated for 1h at 37 ℃ to sufficiently repair the UDG primer.
3) The reaction product of step 2) was added with 2. mu. L10 × NEB buffer 2.0(500mM NaCl, 100mM Tris-HCl, 100mM MgCl210mM DTT, pH7.9@25 ℃, 2 μ L10 × CutSmart (500mM KAc, 200mM Tris-Ac, 100mM Mg (Ac)2, 1000 μ g/m L BSA, pH7.9@25 ℃), 0.5 μ L10 mM dNTP, 0.5 μ L10U Klenow fragment (3'-5' exo-) and 0.5 μ L10 UNt. BbvCI were added to the mixture, and enhanced strand displacement amplification (E-SDA) was induced at 37 ℃ for 80min to give an amplified product.
4) Adding 2 mu L1 mu M L bCas12a, 2 mu L1 mu M CrRNA, 10 mu L10 × NEB buffer 2.1(500mM NaCl, 100mM Tris-HCl, 100mM MgCl) into the amplification product obtained in step 3)21000. mu.g/M L BSA, pH7.9@25 ℃ and 2. mu. L10. mu.M single-stranded reporter DNA molecules were added to the mixture and passed through ddH2O adjusted the final volume to 100 μ L the mixture was then incubated at 37 ℃ for 25min and terminated by heating at 95 ℃ for 5min the fluorescence spectra of the reaction mixture were recorded by a L S-55 spectrophotometric fluorometer (p.e. usa) exciting the system at 495nm and emitting at 556nm the slits for excitation and emission were set to 12nm and 8nm respectively during the measurement, the results are shown in fig. 8A and 8B.
As can be seen from the figure, the fluorescence intensity decreases rapidly with increasing UGI concentration. The inhibitory efficiency of the inhibitor can be expressed as the half maximal Inhibitory Concentration (IC)50) Which represents the concentration of inhibitor required to reduce enzyme activity by 50%. FIG. 8B demonstrates that UGI inhibition of UDG is dose-dependent and its IC for UGI50Estimated to be 0.079U/m L, these results demonstrate that the detection system proposed by the present invention can be used for screening of UDG inhibitors and for further studies of the interaction between UDG and its inhibitors.
Fourth, practical application the detection system of the invention is used for detecting UDG activity in He L a lysate
The method comprises the steps of dissolving He L a lysate by using a cell lysate to obtain He L a cell lysate, adding an inhibitor UGI of UDG into the He L a cell lysate to obtain He L a + UGI, using the cell lysate as a negative control, and performing UDG activity detection by using the three solutions as detection results, wherein the experimental steps are the same as those in example 8, and the results are shown in figure 9.
As can be seen from the figure, compared with a negative control, the fluorescence intensity of the He L a cell lysate is obviously higher, which shows that the He L a cell lysate has higher UDG activity, but after UGI is added into the He L a cell lysate, the fluorescence intensity of He L a + UGI is obviously reduced, and the difference between the fluorescence intensity of the He L a + UGI and the fluorescence intensity of the negative control is not large, which shows that the UDG in the He L a cell lysate is inhibited after the UGI is added.
Taken together, this platform, using the CRISPR/Cas12a system in combination with enhanced strand displacement amplification (E-SDA), can detect sequence-specific DNA (e.g., HIV-1) or UDG activity with ultrasensitivity. Under optimal experimental conditions, the proposed detection method shows good linearity (R) from 100aM to 5pM for HIV-120.99884, the limit of detection (L OD) is estimated to be 87.3 aM. in the serum of normal human, and the established detection method has extremely important practical value in detecting specific sequence DNA20.99609) achieves a value of from 5.0 × 10-5UDG activity assay of U/m L to 0.1U/m L, detection limit (L OD) of 3.1 × 10- 5The U/m L, which is the lowest known UDG inhibition assay, shows that the proposed biosensing platform can be used for screening of UDG inhibitors and further study of interaction between UDG and its inhibitors, and its detection of UDG activity in He L a lysates demonstrates that the proposed biosensing platform can be used for detection of UDG activity in real samples.
The above description is only exemplary of the present invention and should not be taken as limiting, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (10)
1. A detection system based on CRISPR-Cas12a coupling enhanced strand displacement amplification is characterized by comprising a template, L bas Cas12a, a crRNA, a report single-stranded DNA molecule, a DNA polymerase and an endonuclease Nt.BbvCI, wherein the template is base-complementary to a sequence to be detected and synthesizes double-stranded DNA complementary to the template, the formed double-stranded DNA comprises two recognition sites of the endonuclease Nt.BbvCI, the double-stranded DNA is subjected to shearing and polymerization displacement to obtain an activated strand sequence fragment, the crRNA comprises a fixed sequence and a recognition sequence, the fixed sequence is specifically recognized by L bas Cas12a, the recognition sequence is complementary to the activated strand sequence fragment, and the crRNA guides L bas 12a to be combined with the activated strand sequence fragment to activate the trans-cleavage activity of L bas 12a, so that the report single-stranded DNA molecule is cleaved and fluorescence is generated.
2. The CRISPR-Cas12 a-coupled enhanced strand displacement amplification-based detection system according to claim 1, further comprising additives required for DNA amplification; the additive comprises deoxyribonucleoside triphosphates (dNTPs), a buffer, a diluent, magnesium ions, manganese ions or a cofactor.
3. Use of a CRISPR-Cas12a coupled enhanced strand displacement amplification based detection system according to claim 1 or 2 for the detection of specific sequence DNA, screening of inhibitors of UDG activity or detection of biological samples of UDG activity.
4. The use according to claim 3, characterized in that it comprises the following steps:
(1) initiating enhanced strand displacement amplification in the presence of a DNA polymerase and an endonuclease nt.bbvci to produce an activated strand sequence fragment;
(2) activating CRISPR/Cas12a system trans-cleavage activity by complementary pairing of the activation strand sequence fragment obtained in the step (1) and a crRNA recognition region;
(3) and (3) cutting the reporter single-stranded DNA molecule added into the system by using the activated CRISPR/Cas12a system in the step (2), and detecting the fluorescence intensity in the system.
5. A method for detecting HIV-1 based on CRISPR-Cas12a coupled enhanced strand displacement amplification detection system according to claim 1, which is used for non-diagnostic or therapeutic purposes, comprising the following steps:
s1, designing a crRNA sequence with specific recognition region aiming at the L ba Cas12a sequence and the activation strand sequence fragment, wherein the crRNA sequence is shown as SEQ ID NO: 1, and then constructing a crRNA in-vitro transcription vector and carrying out in-vitro transcription and purification, or directly synthesizing;
s2: synthesizing a template-1, wherein the sequence of the template-1 is shown as SEQ ID NO: 2, the template-1 can be complementary with an HIV-1 sequence to be detected to synthesize double-stranded DNA;
s3: mixing the template-1 synthesized in the step S2, HIV-1 with different concentrations, dNTPs, DNA polymerase and endonuclease Nt.BbvCI in a buffer solution according to a proper proportion to obtain a reaction system, placing the reaction system at 37 ℃ for strand displacement amplification reaction, ending the reaction, and inactivating the enzyme at high temperature to obtain an amplification product;
s4, adding a certain concentration of reporter single-stranded DNA molecule, L basAs 12a and the crRNA molecule obtained in the step S1 into the amplification product obtained in the step S3, mixing the mixture in a buffer solution according to a proper proportion to obtain a reaction system, placing the reaction system in a shearing reaction at 37 ℃, terminating the reaction after the reaction is carried out for a period of time, and reading a fluorescence detection signal;
s5: and (3) making a standard curve of HIV-1 concentration-fluorescence intensity according to standard solutions of HIV-1 with different concentrations, calculating a regression equation, and calculating the concentration of HIV-1 in the solution to be detected according to the fluorescence intensity of the solution to be detected.
6. A method for detecting UDG activity based on CRISPR-Cas12a coupled enhanced strand displacement amplification detection system according to claim 1, wherein the method is used for non-diagnostic or therapeutic purposes, comprising the following steps:
1) aiming at L basaCas 12a sequence and activation strand sequence fragment, designing a crRNA sequence with specific recognition region, wherein the crRNA sequence is shown as SEQ ID NO: 1, and then constructing a crRNA in-vitro transcription vector and performing in-vitro transcription and purification, or directly synthesizing;
2) synthesizing a template-2 sequence and a UDG primer, wherein the template-2 sequence is shown as SEQ ID NO: 3, the sequence of the UDG primer is shown as SEQ ID NO: 4 is shown in the specification; the 3' end of the UDG primer is modified with uracil, an object to be detected UDG enzyme can recognize and process the uracil in a UDG primer chain into an AP site, and the AP site can be cut by endonuclease IV to obtain a pretreated UDG primer; the template-2 can be complementary with the base of the pretreated UDG primer to synthesize double-stranded DNA;
3) mixing the template-2 synthesized in the step 2), the UDG primer, the UDG enzyme with different concentrations and the endonuclease IV, and reacting at the constant temperature of 37 ℃ for 40-80 min;
4) adding dNTPs, DNA polymerase and endonuclease Nt.BbvCI into the reaction solution obtained in the step 3) and mixing the mixture in a buffer solution according to a proper proportion to obtain a reaction system, placing the reaction system at 37 ℃ for carrying out a strand displacement amplification reaction, and then inactivating the enzyme at a high temperature to obtain an amplification product;
5) adding the amplification product obtained in the step 4) into a report single-stranded DNA molecule, L basas 12a and the crRNA molecule obtained in the step (1), mixing the mixture in a buffer solution according to a proper proportion to obtain a reaction system, placing the reaction system in a shearing reaction at 37 ℃ for carrying out a shearing reaction, terminating the reaction after reacting for a period of time, and reading a fluorescence detection signal;
6) according to standard solutions of UDG enzyme with different concentrations, making a standard curve of activity-fluorescence intensity of the UDG enzyme, and calculating a regression equation; and calculating the activity of the UDG enzyme in the solution to be detected according to the fluorescence intensity of the solution to be detected.
7. The detection method according to claim 5 or 6, wherein the strand displacement amplification reaction time is 60 to 100min, and the shear reaction time is 15 to 35 min.
8. The detection method according to claim 5 or 6, wherein the concentrations of the DNA polymerase and the endonuclease Nt.BbvCI in the reaction system are independently selected from 0.15-0.35U/μ L.
9. The detection method according to claim 5 or 6, wherein the reporter single-stranded DNA molecule comprises a 7-base single-stranded DNA molecule having HEX and BHQ1 groups at its two ends, and the sequence is as follows: HEX-NNNNNNN-BHQ 1.
10. A method of detection as claimed in claim 5 or claim 6 wherein the fluorescence detection is excitation at a wavelength of 496nm and detection of fluorescence intensity at a wavelength of 556 nm.
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| CN114196752A (en) * | 2021-12-08 | 2022-03-18 | 福州市讯刊生物科技有限公司 | miR-21 detection kit based on Cas14 and strand displacement amplification and application thereof |
| CN114196752B (en) * | 2021-12-08 | 2023-08-08 | 福州市讯刊生物科技有限公司 | miR-21 detection kit based on Cas14 and strand displacement amplification and application thereof |
| CN114231530A (en) * | 2021-12-20 | 2022-03-25 | 大连理工大学 | Cas12 a-based on regulation and control of nucleic acid ribozyme and circular guide RNACcrRNA system and application thereof |
| CN114231530B (en) * | 2021-12-20 | 2024-03-15 | 大连理工大学 | Cas12a- C CrRNA system and application thereof |
| CN114324506A (en) * | 2021-12-31 | 2022-04-12 | 军事科学院军事医学研究院军事兽医研究所 | Electrochemical biosensing composition, working solution, sensor, device and application thereof |
| CN114410790A (en) * | 2022-01-27 | 2022-04-29 | 湖南大学 | Biosensing detection system for detecting ctDNA and detection method thereof |
| CN114410790B (en) * | 2022-01-27 | 2024-04-12 | 湖南大学 | Biosensing detection system for detecting ctDNA and detection method thereof |
| CN114540466A (en) * | 2022-03-21 | 2022-05-27 | 重庆创芯生物科技有限公司 | miRNA-21 detection method and kit based on CRISPRCs 12a system |
| CN116411048A (en) * | 2023-02-14 | 2023-07-11 | 重庆大学 | Polynucleotide kinase activity detection method based on CRISPR/Cas12a |
| CN116574848A (en) * | 2023-06-20 | 2023-08-11 | 中国农业科学院兰州兽医研究所 | Primer and detection method for detecting porcine circovirus type 2 based on LAMP-CRISPRCas12a |
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