Self-assembled label-free detection method and kit for HBV based on signal amplification

Abstract

The invention discloses a method and kit for detecting hepatitis B virus DNA by a self-assembled label-free CRISPR biosensor based on signal amplification, (1) performing RCA amplification on the HBV nucleic acid sample to be detected; (2) mixing the amplified product of step (1) with Cas12a, crRNA and biotin-labeled ssDNA, incubating the reaction and then heating to inactivate Cas12a; (3) adding S1 to the reaction solution treated by step (2) for incubation and hybridization, then adding S2 for incubation and hybridization, adding streptavidin magnetic ball for oscillation incubation after completion, separating the magnetic ball after incubation, adding fluorescent dye SYBR Green I for incubation, and using a fluorescence spectrophotometer to measure the fluorescence intensity after incubation. Due to the powerful signal output performance brought by multiple signal amplification, the designed method has the advantages of high sensitivity and wide linear range.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and in particular relates to a self-assembled label-free CRISPR biosensor based on signal amplification for detecting hepatitis B virus DNA. Background Art

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor with a high mortality rate. Hepatitis B virus (HBV) is one of its main causes. HBV can promote the occurrence of HCC through various mechanisms. HBV can be transmitted through parenteral and sexual routes, and its incubation period is about 1-6 months. So far, the strategy to prevent HBV infection is vaccination. However, vaccination may not be effective for some people. When a person is infected with HBV, the concentration of HBV DNA in the serum increases and can be detected. Therefore, DNA testing may become an important method for early diagnosis of HBV.

[0003] In recent years, a variety of analytical techniques for detecting hepatitis B virus infection have emerged, including electrochemistry, electrochemiluminescence, molecular imprinting, surface enhanced Raman scattering (SERS), fluorescence, colorimetry, etc. Among these methods, the fluorescence method has outstanding advantages such as simplicity, non-radioactivity, low cost and high sensitivity. With these advantages, the development of fluorescence in biosensing has been promoted. However, the sensitivity is low only through the in situ hybridization mode with a 1:1 signal output between the target and the signal output, which cannot meet the actual needs. Therefore, a signal amplification strategy is combined before the signal output to achieve high-sensitivity detection. With the development of nucleic acid amplification strategies, especially those based on isothermal amplification, they can improve the detection sensitivity at a constant temperature and are widely used. For example: polymerase chain reaction (PCR), strand displacement amplification (SDA), rolling circle amplification (RCA), rolling circle transcription (RCT) and hybridization chain reaction (HCR). Therefore, it is very desirable to apply signal amplification strategies to sensing strategies in clinical diagnosis.

[0004] The CRISPR-Cas system is composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), and is a promising new technology in the biological community. With the continuous development of the CRISPR-Cas system, it is not only used in gene editing, but also has started a revolution in the field of biosensing. The CRISPR-Cas system can achieve accurate sequence-based recognition and efficient nucleic acid catalytic cleavage. According to its signal output mode, there are already fluorescence, electrochemistry, electrochemiluminescence, colorimetry, etc. Among the above output modes, most of them are based on labeled probes, and the synthesis steps of fluorescent labeled probes are cumbersome, costly, and prone to signal leakage. Therefore, the fluorescence specificity of the fluorescent dye SYBR Green I (SGI) to double-stranded DNA (dsDNA) has attracted the interest of scientists. However, the fluorescence enhancement efficiency is related to the amount of dsDNA, and a high content of dsDNA needs to be provided to increase and improve the fluorescence enhancement. Summary of the invention

[0005] The technical problems to be solved by the present invention are: the first object of the present invention is to provide a method for detecting hepatitis B virus DNA by a self-assembled label-free CRISPR biosensor based on signal amplification for non-diagnostic purposes; another object of the present invention is to provide a kit for detecting hepatitis B virus DNA by a self-assembled label-free CRISPR biosensor based on signal amplification.

[0006] The technical solution of the present invention is: a method for detecting hepatitis B virus DNA by a self-assembled label-free CRISPR biosensor based on signal amplification for non-diagnostic purposes, comprising the following steps:

[0007] (1) Extracting the DNA of the HBV nucleic acid sample to be tested, cyclizing it with P-DNA and then performing RCA amplification;

[0008] (2) mixing the amplified product of step (1) with Cas12a, crRNA and biotin-labeled ssDNA, incubating the reaction and then heating to inactivate Cas12a;

[0009] (3) S1 is added to the reaction solution treated in step (2) for incubation and hybridization, and then S2 is added for incubation and hybridization. After completion, streptavidin magnetic balls are added for oscillation and incubation. After the incubation is completed, the magnetic balls are separated, and then the fluorescent dye SYBRGreen I is added for incubation. After the incubation is completed, the fluorescence intensity is measured using a fluorescence spectrophotometer.

[0010] in,

[0011] The nucleotide sequence of ssDNA: 5'-TTTTTTTTTTTTGTTTCTGTTCTCCCTGG-3 (SEQ ID No. 1),

[0012] The nucleotide sequence of S1: 5'-GTGTGCCTATTATGTCTCCTCCTGTGTGCCTATTATGTCTCCTCCTCCAGGGAGAACAGAAAC-3' (SEQ ID No. 2),

[0013] The nucleotide sequence of S2: 5'-AGGAGGAGACATAATAGGCACACGTTTCTGTTCTCCCTGG-3' (SEQ ID No. 3),

[0014] The nucleotide sequence of P-DNA is: 5'-ATAACTGAAAGCCAACTCGGCTACCTTGACACTTCTTTCGGGCTTCCTCGTACCACTACATCCAT-3 (SEQ ID No. 4)

[0015] The nucleotide sequence of crRNA is: 5′-UAAUUUCUACUAAGUGUAGAUCCUUGACACUUCUUUCGGGC-3 (SEQ ID No. 5).

[0016] SYBR Green I can specifically bind to dsDNA to generate a strong fluorescent signal. The present invention designs three DNA sequences, in which the biotin-modified ssDNA (denoted as BIO, the same below) can only hybridize with S1 but not with S2 to form partially hybridized dsDNA, the exposed end of S1 can partially hybridize with S2 to form dsDNA, and at the same time, the exposed end of S2 can hybridize with S1, and the two continuously hybridize to form a macromolecular structure, providing a site for SYBR Green I. The "tree-like" structure is formed with the double-stranded part of BIO-S1 as the trunk and the part of S1 and S2 continuously hybridizing as the crown as the signal output, avoiding the modification of the fluorescent group.

[0017] Specifically, the steps include:

[0018] (1) RCA amplification reaction

[0019] 5 μL of the nucleic acid sample to be tested and 5 μL of 2 μM P-DNA were mixed and incubated at 90°C for 5 min, then cooled to room temperature. Then, 1 μL of 40 U / μL T4 DNA ligase, 1.5 μL of 10×T4 DNA ligase buffer solution and 2.5 μL of enzyme-free water were added and mixed evenly. The mixture was incubated at room temperature for 2 h. After the time was up, 1 μL of 2U / μL phi29 DNA polymerase, 2 μL of 10 mM dNTP and 1.5 μL of 10×phi29 DNA polymerase buffer solution were incubated at 30°C for 1.5 h. After the reaction was completed, the reaction mixture was heated to 65°C for 10 min to inactivate phi29 DNA polymerase and T4 DNA ligase.

[0020] (2) Cleavage reaction of Cas12a system

[0021] After the reaction of step (1) is completed, 1 μL 500nM Cas12a, 1 μL 1.5 μM crRNA, 0.5 μL 1 μM ssDNA, and 2.5 μL 10×Cas12a buffer solution are mixed evenly and incubated at 37°C for 1.5h. After the reaction is completed, the reaction mixture is heated to 65°C for 10min to inactivate Cas12a;

[0022] (3) Label-free self-assembly reaction

[0023] 1 μL of 2 μM S1 was added to the mixture of the shear reaction in step (2) and incubated at 37°C for 1 h. Then, 1 μL of 2 μM S2 was added and incubated at 37°C for 2 h. Then, 5 μL of 2 mg / ml streptavidin magnetic beads were added to the reaction system and incubated at 37°C with shaking for 0.5 h. The reaction product was separated from the mixture by a magnetic stand under the action of a magnetic field, and then mixed with SYBR Green I and incubated for 0.5 h. The fluorescence intensity was measured using a fluorescence spectrophotometer.

[0024] The present invention also discloses a kit for detecting hepatitis B virus DNA using a self-assembled label-free CRISPR biosensor based on signal amplification, the kit comprising the following components:

[0025] (1) RCA amplification reagents: P-DNA, T4 DNA ligase, T4 DNA ligase buffer solution, phi29 DNA polymerase, dNTP, phi29 DNA polymerase buffer solution;

[0026] (2) Cas12a detection reagent: Cas12a, crRNA, and biotin-labeled ssDNA;

[0027] (3) Self-assembly reporter reagents: S1, S2, streptavidin magnetic beads, SYBR Green I;

[0028] The nucleotide sequence of the P-DNA is shown in SEQ ID No.4, the nucleotide sequence of crRNA is shown in SEQ ID No.5, the nucleotide sequence of ssDNA is shown in SEQ ID No.1, the nucleotide sequence of S1 is shown in SEQ ID No.2, and the nucleotide sequence of S2 is shown in SEQ ID No.3.

[0029] In the present invention, the rolling circle amplification (RCA) of target transduction carries out one-to-many exponential amplification to target. Amplified product contains activation sequence to activate Cas12a system to cut single-stranded DNA (ssDNA), and ssDNA is modified with biotin (expressed as BIO) at one end. By the self-assembly reaction between DNA, a large amount of dsDNA is generated to provide site for fluorescent dye SYBR Green I (SG I), enhancing fluorescent signal. Simultaneously introduce streptavidin modified magnetic sphere, by biotin and streptavidin specific binding to magnetic sphere, separated by magnetic field, realize the separation of fracture and unfractured BIO, and then realize label-free signal output.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The signal output of the label-free tree structure can produce aggregated fluorescence, enhance the fluorescence signal, and is low in cost. The cyclic amplification of the RCA reaction amplifies the target one-to-many in a short time, and provides sufficient activation chains for the Cas12a system to ensure the activity of the Cas12a system. In addition, the Cas12a system realizes cyclic amplification of signal output due to its unique "random cutting" reaction. Due to the powerful signal output performance brought by multiple signal amplification, the designed method has the advantages of high sensitivity and wide linear range. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of label-free fluorescence detection based on Cas12a system for HBV DNA detection;

[0033] Figure 2 Feasibility of fluorescence-free self-assembly reaction; (A) shows the fluorescence spectra of different combinations of BIO, S1 and S2; (B) non-denaturing PAGE electrophoresis analysis of different combinations of BIO, S1 and S2;

[0034] Figure 3 Label-free fluorescence detection of HBV DNA fluorescence measurement: (a) presence of P-DNA and HBV; (b) presence of P-DNA and no HBV;

[0035] Figure 4Feasibility analysis of label-free fluorescence detection of HBV DNA by gel electrophoresis; (A) agarose gel electrophoresis; (B) non-denaturing PAGE electrophoresis;

[0036] Figure 5 Effect of the concentration ratio between BIO, S1, and S2 in the dendritic structure on the fluorescence intensity;

[0037] Figure 6 Effects of rolling circle amplification (RCA) reaction conditions of target transduction on signal output. (A) Effect of RCA response time on fluorescence intensity; (B) Effect of P-DNA concentration on fluorescence intensity; (C) Effect of phi29 DNA polymerase concentration on fluorescence intensity;

[0038] Figure 7 Effects of Cas12a system cleavage reaction conditions on signal output. (A) Effect of cas12a concentration on fluorescence intensity; (B) Effect of crRNA concentration on fluorescence intensity; (C) Effect of cleavage reaction time on fluorescence intensity;

[0039] Figure 8 Fluorescence response of mismatched bases compared with HBV DNA (10 nM). Error bars indicate standard deviation (n = 3);

[0040] Fig. 9 (A) Fluorescence response of the HBV DNA detection system at different HBV DNA concentrations from 10 pM to 2 μM; (B) Linear relationship between fluorescence response and logarithm of HBV concentration. Error bars represent standard deviation (n=3). DETAILED DESCRIPTION

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0042] 1. Reagents and instruments

[0043] Oligonucleotides were synthesized and purified by Sangon Biotech (Shanghai, China), and their sequences are listed in Table 1. T4 DNA ligase, phi29 DNA polymerase, and deoxynucleotide triphosphates (dNTPs) were provided by New England Biolabs (Beijing, China). LbCas12a protein (Cas12a) was purchased from Guangzhou Meige Biotechnology Co., Ltd. (Guangzhou, China), streptavidin magnetic beads (average diameter 300 nm) were purchased from Biomag Biotech (Jiangsu, China), and SYBR Green I (SG I) (10000×) was purchased from Beijing Solebao Technology Co., Ltd. The water used in the experiment was ultrapure water (18.2 MΩ cm), and all other reagents were analytical grade.

[0044] Fluorescence spectra were recorded on a Hitachi F-7000 fluorescence spectrophotometer (Hitachi, Japan). GelDoc TM The gel electrophoresis images were scanned by XR+ imaging system (Bio-Rad, USA). The water (18.2 MΩ cm) used in the whole experiment was pretreated by Milli-Q ultrapure water treatment system (Millipore, USA).

[0045] Table 1 Nucleotide sequences used in the experiment

[0046]

[0047] 2. Detection principle of the present invention

[0048] 2.1 System composition

[0049] The detection of the present invention mainly includes three processes: rolling circle amplification (RCA) of target transduction to perform one-to-many exponential amplification of the target; Cas12a, crRNA and single-stranded DNA (ssDNA) cleavage system, one end of ssDNA is modified with biotin (expressed as BIO) and a fluorescence-free self-assembly reporter system. Figure 1 As shown in the figure, taking HBV DNA as a model, the working principle of label-free detection of HBV DNA using the Cas12a system is explained. When the target is present, the two ends of the circular template are brought together by simple base complementary pairing, and the loop is closed under the action of T4 DNA ligase, and then the RCA reaction is triggered by phi29 DNA polymerase for large-scale amplification. The amplified product contains a nucleic acid sequence that can activate the activity of the Cas12a system. It is used as an activation chain. After adding Cas12a and crRNA, the Cas12a system is activated to trans-cut BIO, and the biotin end is cut to separate the break of BIO, that is, the break of the "root".

[0050] For the fluorescence-free self-assembly reporter system, the main chain (S1) is hybridized with BIO to lay the foundation for the growth unit - the trunk, and then the side chain (S2) is added for self-assembly to form a tree structure, generating a large number of double-stranded DNA (dsDNA) structures to provide a large number of bonding sites for SGΙ to produce a strong fluorescent signal. Magnetic balls modified with streptavidin are added, and the specific binding of biotin and streptavidin is used to achieve magnetic separation under the action of a magnetic field. When the target is present, the broken tree structure at the biotin end exists in the solution, and the addition of SGΙ produces a strong fluorescent signal.

[0051] On the contrary, when the target does not exist, the RCA reaction cannot be triggered. At the same time, the P-DNA is digested by the exonuclease of phi29 DNA polymerase, and there is no RCA product. Therefore, the Cas12a system cannot be activated, and the BIO cannot be cut "randomly". Therefore, the self-assembled product is enriched on the magnetic ball under the action of the magnetic field, and no obvious fluorescence signal is restored when SGΙ is added to the solution.

[0052] Through the above principle, the conventional fluorescent labeling output mode of the Cas12a system is broken, and the cutting reaction of the Cas12a system is utilized to realize the output of label-free signals.

[0053] 2.2 First, the fluorescence-free self-assembly reporter system was verified

[0054] ssDNA(BIO):5'-TTTTTTTTTTTTGTTTTCTGTTCTCCCTGG-3'

[0055] S1:5'-GTGTGCCTATTATGTCTCCTCCTGTGTGCCTATTATGTCTCCTCCTCCAGGGAGAACAGAAAC-3'

[0056] S2:5'-AGGAGGAGACATAATAGGCACACGTTTCTGTTCTCCCTGG-3'

[0057] SGΙ can specifically bind to dsDNA to produce a strong fluorescent signal. We designed three DNA sequences, in which the biotin-modified ssDNA, namely BIO, can only hybridize with S1 but not with S2 to form partially hybridized dsDNA. The exposed end of S1 can partially hybridize with S2 to form dsDNA, and at the same time, the exposed end of S2 can hybridize with S1. The two continuously hybridize to form a macromolecular structure, providing a site for SGΙ. The double-stranded part of BIO-S1 is formed as the trunk, and the part where S1 and S2 continuously hybridize is the crown. The "tree-like" structure formed is used as the signal output to avoid modification of the fluorescent group.

[0058] Its fluorescence spectrum is Figure 2 As shown in A, BIO, S1, and S2 successfully hybridized (curve a), and their fluorescence intensity increased the most. Figure 2 B verifies the hybridization relationship between BIO, S1, and S2. BIO can hybridize with S1 to form dsDNA ( Figure 2 In B, lane 4), the increase in molecular weight of S1-BIO caused it to migrate faster than BIO ( Figure 2 Middle B, lane 3), S1( Figure 2 B, lane 1) moves slowly, and BIO does not move with S2 ( Figure 2In lane 5, the double strands are hybridized to form double strands, and two bands, BIO and S2, are shown in lane 5. S1 and S2 can hybridize with each other and continue to extend to form structures with different molecular weights ( Figure 2 B, lane 6), when BIO, S1, and S2 coexist, structures with different molecular weights are also formed ( Figure 2 The results of the fluorescence spectrum curve are consistent with those of the middle B, lane 7. Due to the continuous change of air resistance, the migration speed of the bands is different, and lanes 6 and 7 are distributed in a step-like manner. Therefore, it can be concluded that the structures with different molecular weights are formed, with BIO-S1 as the trunk and the crown structures with different molecular weights extending continuously from S1 and S2.

[0059] Based on the above conclusions, fluorescence experiments and native PAGE electrophoresis analysis were performed to study the feasibility of the proposed strategy for detecting HBV DNA. Figure 3 As shown, in the presence of HBV DNA, an obvious high fluorescence signal can be seen ( Figure 3 , curve a), indicating that HBV DNA can complement P-DNA, trigger RCA reaction under the action of T4 DNA ligase and phi29 DNA polymerase, and produce a large amount of ssDNA. SsDNA is assembled in the presence of Cas12a and crRNA to activate the activity of the Cas12a system, start the trans-cleavage reaction of the Cas12a system, and obtain a large number of cut BIO chains. Therefore, S1 and S2 are added to the reaction solution to generate a large number of tree structures. After adding the magnetic ball, the tree structure is retained in the solution due to the breakage of the "roots". When SGI is specifically inserted, a significantly enhanced fluorescence response is obtained. In the control experiment, the fluorescence intensity is low in the system without HBV ( Figure 3 , curve b), this is because when there is no HBV DNA, there is no occurrence of RCA reaction, and the P-DNA present in the solution is degraded under the action of phi29 DNA polymerase.Due to the generation of no amplified product in the system, only in the presence of both Cas12a and crRNA, there is no assembly to form an active Cas12a system, so the trans-shearing reaction of the Cas12a system is suppressed, and the BIO chain remains intact.S1, S2 are added to the reaction solution, a large amount of tree structures are generated, and after adding magnetic sphere, the complete BIO chain is bound to the magnetic sphere by the specific binding of biotin and streptavidin, and SGI is added in the solution, without obvious fluorescent signal.

[0060] In order to verify the feasibility of the proposed experimental process, Figure 4 As shown in Figure A, agarose gel electrophoresis and non-denaturing PAGE analysis were performed. First, the RCA reaction ( Figure 4A) Agarose gel electrophoresis was performed to verify that when HBV DNA was present, it could hybridize with P-DNA to form partially hybridized circular ssDNA with a gap, resulting in enhanced steric hindrance. Therefore, the DNA in lane 3 ran slower than P-DNA (lane 2) and HBV (lane 1), indicating that HBV and P-DNA were successfully hybridized. HBV DNA and P-DNA then triggered an RCA reaction (lane 4). The molecular weight of the generated long-chain DNA was greater than 5000bp, so its movement speed was slow and located at the port where the sample was added. In the absence of HBV DNA, P-DNA could not form circular DNA, and no RCA reaction occurred (lane 6). No long-chain DNA was present in the reaction solution. At the same time, due to the exonuclease activity of phi29 DNA polymerase, P-DNA can be digested, so the P-DNA in the solution is degraded and the P-DNA band becomes significantly lighter.

[0061] Then, the cleavage reaction of Cas12a was analyzed by non-denaturing PAGE. Figure 4 As shown in B, the supernatant contains tree-like structures with broken "roots" of varying molecular weight (lane 5). This is because in the presence of the target, the RCA reaction is triggered to generate an activation chain, which in turn activates the trans-cleavage of the Cas12a system, causing BIO to break. After magnetic separation, the tree-like structure exists in the solution. When there is no target in the reaction system, such as Figure 4 As shown in lane 6 of middle B, the dendritic structure is drastically reduced and the band becomes lighter. When the target is not present, no RCA reaction occurs, and the activity of the Cas12a system is inhibited. The BIO chain is intact and fixed on the magnetic ball through the specific binding of biotin and streptavidin. It is separated from the solution by magnetic separation. The dendritic structures of varying molecular weight in the supernatant are taken away by the magnetic ball, which is the same as the results of the fluorescence experiment.

[0062] 2.3 Optimization of reaction conditions

[0063] In the designed detection strategy, the occurrence of self-assembly reaction is directly related to the signal output. Therefore, the molar ratio of BIO, S1, and S2 assembled into a dendritic structure is one of the important factors affecting the signal, and the concentration ratio is optimized. Figure 5As shown, when SA-MB remains unchanged (10 μL 2 mg / mL), when the ratio of the three increases to 1:4:4, the fluorescence difference is the largest, and 1:4:4 is the best at this time (BIO is 1 μM, S1 is 4 μM, and S2 is 4 μM). This is because as the ratio of BIO, S1, and S2 continues to increase, the dendritic structure primitives become larger, the double-stranded structure increases, and the SGI sites increase, which is beneficial to improve the signal, but at the same time, the steric hindrance increases, which is not conducive to the enrichment of the dendritic structure by the magnetic ball. Therefore, 1:4:4 is selected as the best BIO, S1, and S2 ratio. The optimal conditions obtained by optimizing the comparison are used as signal reporter molecules. The BIO, S1, S2 and corresponding MB at this time are defined as a whole and named reporter. The polymerization reaction time, the concentration of P-DNA, and the concentration of phi29 DNA polymerase affect the RCA reaction process and have an important influence on the activation of the Cas12a system. Therefore, the polymerization reaction time is optimized first. As Figure 6 As shown in Figure A, the fluorescence intensity increases with time. After 90 min, the fluorescence intensity no longer changes significantly and reaches a plateau. Therefore, 90 min was selected as the optimal polymerization reaction time. Subsequently, the concentration of P-DNA was optimized. Figure 6 As shown in Figure B, when the concentration of P-DNA increased from 0.5 μM to 6 μM, the fluorescence intensity gradually increased and reached a plateau at 2 μM. Finally, the concentration of phi29 DNA polymerase was optimized, and the fluorescence intensity reached the maximum value at 2 U / μL ( Figure 6 Therefore, 2 μM P-DNA and 2 U / μL phi29 DNA polymerase were selected as the optimal conditions.

[0064] On this basis, the cleavage reaction of Cas12a was optimized. Figure 7 As shown in Figure A, as the concentration of Cas12a enzyme increases, the fluorescence intensity increases continuously. When the concentration reaches 0.5 μM, the growth rate decreases sharply and tends to be flat. Therefore, 0.5 μM is selected for the next experiment. Then the concentration of cr RNA is optimized, as shown in Figure 1. Figure 7As shown in B, with the increase of concentration, the fluorescence increases continuously, and it stops increasing after reaching 1.5μM, so 1.5μM is selected as the best. Finally, the reaction time of the CRISPR / Cas12a shearing reaction was optimized, and it was found that the fluorescence intensity was optimal when the time reached 90min, so 90min was selected as the best experiment. The optimal reaction conditions finally determined were RCA reaction time of 90min, P-DNA concentration of 2μM, phi29 DNApolymerase concentration of 2U / μL, Cas12a enzyme concentration of 0.5μM, crRNA concentration of 1.5μM, and shearing reaction reaction time of 90min.

[0065] 2.4 Specificity of the HBV sensing platform

[0066] The specificity of the method was investigated by comparing the target DNA signal with mismatch sequences with one base mismatch (MT1) and two base mismatches (MT2) at a concentration of 100 nM. Figure 8 It can be seen that the fluorescence signal generated by the mismatch sequence is very close to the blank signal and is much lower than the signal generated by the target DNA, indicating that the proposed method has reasonable specificity in detecting sequence-specific DNA.

[0067] 2.5 Analytical capabilities of the HBV sensing platform

[0068] like Fig. 9 As shown in A and B, under the optimal reaction conditions, the relationship between different concentrations of HBV and the change in fluorescence intensity. Fig. 9 In Figure A, within the range of 10pM-2μM, the fluorescence intensity of the system increases with the increase of HBV DNA concentration; Fig. 9 As shown in B, the fluorescence intensity and HBV DNA concentration showed a good linear relationship, the linear range was between 10pM and 100nM, and the linear equation was F = 571.8LogC HBV DNA +1804.8, where F is the fluorescence intensity of HBV DNA at a certain concentration, and C is the concentration of HBV DNA. The regression coefficient R of the linear equation 2 The calculated detection limit of the method is 1.28 pM. The detection limit of the proposed detection method is lower than or equivalent to that of the reported HBV DNA detection method, see Table 2.

[0069] Table 2 Comparison of HBV DNA detection methods

[0070]

[0071] 2.6 Detection of HBV in serum

[0072] In order to explore the feasibility of the proposed sensing strategy in practical applications, the HBV DNA spiked with different concentrations was tested. The serum was diluted 300 times for the detection of target HBV. The steps are as follows: different concentrations of HBV were added to the diluted serum to obtain solutions of different concentrations of 100pM, 1nM and 10nM, and the detection was carried out according to the above steps. The recovery rates of 100pM, 1nM and 10nM were measured to be 99%, 105%, and 98.3%, which were between 90% and 110%, and the corresponding standard deviations were 1%, 0.8%, and 1.4%, all less than 2%. The results show that the proposed method has the possibility of practical application.

[0073] In summary, we proposed a label-free fluorescent signal output strategy for the Cas12a system. In our proposed strategy, the signal output of the label-free tree structure can produce aggregated fluorescence, enhance the fluorescent signal, and has low cost. The cyclic amplification of the RCA reaction amplifies the target one-to-many in a short time, and provides sufficient activation chains for the Cas12a system to ensure the activity of the Cas12a system. In addition, the Cas12a system realizes cyclic amplification of signal output due to its unique "random cutting" reaction. Due to the powerful signal output performance brought about by multiple signal amplification, the designed method has the advantages of high sensitivity and wide linear range.

[0074] Example 1

[0075] 1. Rolling circle amplification (RCA) reaction for target transduction

[0076] 5 μL of nucleic acid sample to be tested and 5 μL of 2 μM P-DNA were mixed and incubated at 90°C for 5 min and then cooled to room temperature. Then, 1 μL of 40U / μL T4 DNA ligase, 1.5 μL of 10×T4 DNA ligase buffer solution and 2.5 μL of enzyme-free water were added and mixed evenly and incubated at room temperature for 2 h. After the time was up, 1 μL of 2U / μL phi29 DNA polymerase, 2 μL of 10mM dNTP and 1.5 μL of 10×phi29 DNA polymerase buffer solution were incubated at 30°C for 1.5 h. After the reaction was completed, the reaction mixture was heated to 65°C for 10 min to inactivate phi29 DNA polymerase and T4 DNA ligase.

[0077] 2. Shearing reaction of Cas12a system

[0078] After the above reaction is completed, 1 μL 500nM Cas12a, 1 μL 1.5 μM crRNA, 0.5 μL 1 μM BIO, and 2.5 μL 10×Cas12a buffer solution are mixed evenly and incubated at 37°C for 1.5 h. After the reaction is completed, the reaction mixture is heated to 65°C for 10 min to inactivate Cas12a.

[0079] 3. Label-free self-assembly reaction

[0080] 1 μL of 2 μM S1 was added to the shear reaction mixture and incubated at 37°C for 1 h, followed by 1 μL of 2 μM S2 and incubated at 37°C for 2 h. Streptavidin magnetic beads (MB, 2 mg / ml) were washed 3 times with Tris-HCL buffer solution and resuspended in Tris-HCL buffer solution. Then 5 μL of treated MBs were added to the reaction system and incubated at 37°C with shaking for 0.5 h. The reaction product was separated from the mixture by a magnetic stand under the action of a magnetic field, and then mixed with SGI and incubated for 0.5 h, and its fluorescence intensity was measured using a FL-7000 fluorescence spectrophotometer. Then, the amplified product was analyzed by 12% polyacrylamide gel electrophoresis in 1×TAE buffer solution.

[0081] The nucleotide sequence of the ssDNA is shown in SEQ ID No.1, the nucleotide sequence of S1 is shown in SEQ ID No.2, the nucleotide sequence of S2 is shown in SEQ ID No.3, the nucleotide sequence of P-DNA is shown in SEQ ID No.4, and the nucleotide sequence of crRNA is shown in SEQ ID No.5.

[0082] Example 2 Preparation of a kit for detecting hepatitis B virus DNA using a self-assembled label-free CRISPR biosensor based on signal amplification

[0083] The kit includes the following components:

[0084] (1) RCA amplification reagents: P-DNA, T4 DNA ligase, T4 DNA ligase buffer solution, phi29 DNA polymerase, dNTP, phi29 DNA polymerase buffer solution;

[0085] (2) Cas12a detection reagent: Cas12a, crRNA, and biotin-labeled ssDNA;

[0086] (3) Self-assembly reporter reagents: S1, S2, streptavidin magnetic beads, SYBR Green I.

[0087] The nucleotide sequence of the ssDNA is shown in SEQ ID No.1, the nucleotide sequence of S1 is shown in SEQ ID No.2, the nucleotide sequence of S2 is shown in SEQ ID No.3, the nucleotide sequence of P-DNA is shown in SEQ ID No.4, and the nucleotide sequence of crRNA is shown in SEQ ID No.5. Sequence Listing <110> Liaocheng University <120> Self-assembled label-free detection method and kit for HBV based on signal amplification <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <400> 1 tttttttttt ttgtttctgt tctccctgg 29 <210> 2 <211> 63 <212> DNA <213> Artificial Sequence <400> 2 gtgtgcctat tatgtctcct cctgtgtgcc tattatgtct cctcctccag ggagaacaga 60 aac 63 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <400> 3 aggaggagac ataataggca cacgtttctg ttctccctgg 40 <210> 4 <211> 65 <212> DNA <213> Artificial Sequence <400> 4 ataactgaaa gccaactcgg ctaccttgac acttctttcg ggcttcctcg taccactaca 60 tccat 65 <210> 5 <211> 41 <212> RNA <213> Artificial Sequence <400> 5 uaauuucuac uaaguguaga uccuugacac uucuuucggg c 41

Claims

1. A method for detecting hepatitis B virus DNA using a self-assembled label-free CRISPR biosensor based on signal amplification for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Extract the DNA of the HBV nucleic acid sample to be tested, cyclize it with P-DNA and then perform RCA amplification; (2) mixing the amplified product of step (1) with Cas12a, crRNA and biotin-labeled ssDNA, incubating the reaction and then heating to inactivate Cas12a; (3) S1 is added to the reaction solution treated in step (2) for incubation and hybridization, and then S2 is added for incubation and hybridization. After completion, streptavidin magnetic balls are added for oscillation and incubation. After the incubation is completed, the magnetic balls are separated, and then the fluorescent dye SYBR Green I is added for incubation. After the incubation is completed, the fluorescence intensity is measured using a fluorescence spectrophotometer; (4) Substituting the fluorescence intensity detected in step (3) into the following formula, calculate the concentration of HBV DNA; F = 571.8 LogC HBV DNA + 1804.8, where F is the fluorescence intensity of HBV DNA at a certain concentration, C HBV DNA is the concentration of HBV DNA; 10 pM ≤C HBV DNA ≤100 nM; Among them, the nucleotide sequence of ssDNA is shown in SEQ ID No.1, the nucleotide sequence of S1 is shown in SEQ ID No.2, the nucleotide sequence of S2 is shown in SEQ ID No.3, the nucleotide sequence of P-DNA is shown in SEQ ID No.4, and the nucleotide sequence of crRNA is shown in SEQ ID No.

5.

2. The method according to claim 1, characterized in that the concentration ratio of biotin-labeled ssDNA, S1, and S2 is 1:4:

4.

3. The method according to claim 1, characterized in that The following steps are involved: (1) RCA amplification reaction 5 μL of the nucleic acid sample to be tested and 5 μL of 2 μM P-DNA were mixed and incubated at 90 °C for 5 min, then cooled to room temperature. Then, 1 μL of 40 U / μL T4 DNA ligase, 1.5 μL of 10 × T4 DNA ligase buffer solution and 2.5 μL of enzyme-free water were added and mixed evenly. The mixture was incubated at room temperature for 2 h. After the time was up, 1 μL of 2 U / μL phi29 DNA polymerase, 2 μL of 10 mM dNTP and 1.5 μL of 10 × phi29 DNA polymerase buffer solution were incubated at 30 °C for 1.5 h. After the reaction was completed, the reaction mixture was heated to 65 °C for 10 min to inactivate phi29 DNA polymerase and T4 DNA ligase. (2) Cleavage reaction of Cas12a system After the reaction in step (1) is completed, 1 μL 500 nM Cas12a, 1 μL 1.5 μM crRNA, 0.5 μL 1 μM ssDNA, and 2.5 μL 10 × Cas12a buffer solution are mixed evenly and incubated at 37 °C for 1.5 h. After the reaction is completed, the reaction mixture is heated to 65 °C for 10 min to inactivate Cas12a. (3) Label-free self-assembly reaction 1 μL 2 μM S1 was added to the mixture of the shear reaction in step (2) and incubated at 37 °C for 1 h. Then, 1 μL 2 μM S2 was added and incubated at 37 °C for 2 h. Then, 5 μL 2 mg / ml streptavidin magnetic beads were added to the reaction system and incubated at 37 °C with shaking for 0.5 h. The reaction product was separated from the mixture by the magnetic field using a magnetic stand, and then mixed with SYBRGreen I and incubated for 0.5 h. The fluorescence intensity was measured using a fluorescence spectrophotometer.

4. A kit for detecting hepatitis B virus DNA based on a self-assembled label-free CRISPR biosensor for signal amplification, characterized in that: The kit includes the following components: (1) RCA amplification reagents: P-DNA, T4 DNA ligase, T4 DNA ligase buffer solution, phi29 DNA polymerase, dNTP, phi29 DNA polymerase buffer solution; (2) Cas12a detection reagent: Cas12a, crRNA, biotin-labeled ssDNA; (3) Self-assembly reporter reagents: S1, S2, streptavidin magnetic beads, SYBR Green I; The nucleotide sequence of the ssDNA is shown in SEQ ID No.1, the nucleotide sequence of S1 is shown in SEQ ID No.2, the nucleotide sequence of S2 is shown in SEQ ID No.3, the nucleotide sequence of P-DNA is shown in SEQ ID No.4, and the nucleotide sequence of crRNA is shown in SEQ ID No.5.

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