CN114921576A - Reagent, kit and detection method for detecting mycobacterium bovis - Google Patents
Reagent, kit and detection method for detecting mycobacterium bovis Download PDFInfo
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Abstract
The invention provides a reagent, a kit and a detection method for detecting mycobacterium bovis, belonging to the technical field of molecular biological detection; the reagent comprises a recognition element based on a CRISPR/Cas12a positive feedback circulation mechanism, a reagent for hyperbranched hybridization chain reaction signal amplification reaction and a reagent for colorimetric analysis; the invention uses CRISPR/Cas12a as a specific recognition element, initiates hyperbranched hybrid chain reaction after autocatalysis, forms a DNA nano structure with n layers of branches, and constructs a multidimensional G-quadruplex-heme DNase matrix on the DNA nano structure, thereby realizing multiple signal amplification and completing the ultra-sensitive detection of Mycobacterium tuberculosis-DNA of cattle. The invention has higher specificity selection on the bovine mycobacterium tuberculosis DNA, the detection limit is 8.9aM (LOD ═ 3 sigma/S), and the ultra-sensitive detection on the bovine mycobacterium tuberculosis DNA is realized.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a reagent, a kit and a detection method for detecting mycobacterium bovis.
Background
Mycobacterium bovis is a member of the mycobacterium tuberculosis complex (MTBC), a group of genetically related mycobacterial species that cause tuberculosis in a variety of mammals. Of all MTBC members, m. bovis has the widest host range and can cause bovine tuberculosis in domestic animals, poultry, wildlife and humans, which is generally characterized by long latency and non-negligible environmental, economic, etc. effects.
At present, the methods for detecting mycobacterium bovis mainly comprise the following steps: microbial culture, immunological methods, molecular detection, and the like. Among them, microbial culture and immunological methods can detect M.bovis infection, but they have low sensitivity and specificity to subclinical or latent infection, and microbial culture also has problems of long incubation time and large detection workload. The molecular detection mainly adopts a PCR technology, and although the PCR technology obviously improves the detection performance and efficiency, the detection sensitivity is low, and false positive and false negative results are easy to exist.
Disclosure of Invention
In view of this, the present invention aims to provide a reagent, a kit and a method for detecting mycobacterium bovis, which have high sensitivity and high specificity.
The invention provides a reagent for detecting mycobacterium bovis, which comprises a recognition element based on a CRISPR/Cas12a positive feedback circulation mechanism, a reagent for hyperbranched hybridization chain reaction signal amplification reaction and a reagent for colorimetric analysis;
the recognition element based on CRISPR/Cas12a positive feedback cycle mechanism comprises: gRNA-T, Cas12a protein, an auxiliary probe and a switchable guide RNA;
the switchable guide RNA is assembled by iDNA-spacer, iDNA-handle and gRNA;
the nucleotide sequence of the gRNA-T is shown in SEQ ID NO. 1;
the auxiliary probe is a double chain formed by base complementary pairing of nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3;
the nucleotide sequence of the iDNA-spacer is shown in SEQ ID NO. 4;
the nucleotide sequence of the iDNA-handle is shown in SEQ ID NO. 5;
the nucleotide sequence of the gRNA is shown in SEQ ID NO. 6;
the probe for the hyperbranched hybridization chain reaction signal amplification reaction comprises H1, SH1, H2, L1, L2 and SH 2; the nucleotide sequences of H1, H2, SH1, SH2, L1 and L2 are respectively shown as SEQ ID NO. 7-SEQ ID NO. 12;
the reagent for colorimetric analysis comprises hemin and H 2 O 2 And ABTS.
Preferably, the mole ratio of the iDNA-spacer, the iDNA-handle and the gRNA is as follows: (1.1-1.3): (1.1-1.3): 1.
the invention also provides a kit for detecting the mycobacterium bovis, which comprises the reagent in the scheme.
Preferably, the kit further comprises a positive control; the positive control substance comprises a target sequence formed by complementary base pairing of nucleotide sequences shown in SEQ ID NO.23 and SEQ ID NO. 24.
Preferably, the concentration of the working solution of the gRNA-T is 50 nM; the working solution concentration of the auxiliary probe is 500 nM; the concentration of the working solution of the switchable guide RNA is 1 mu M; the working solution concentration of the Cas12a protein was 1 μ M.
Preferably, the working solution concentration of H1, SH1, H2, L1, L2 or SH2 is 1 μ M.
The invention also provides a detection method of the non-diagnostic mycobacterium bovis based on the reagent or the kit in the scheme, which comprises the following steps:
2) dissolving the SH1, SH2, H1 and H2 in buffer solution respectively, and performing first incubation respectively to obtain SH1, SH2, H1 and H2 with hairpin structures respectively;
3) mixing the first product, L1 and L2 with SH1, SH2, H1 and H2 of a hairpin structure, and carrying out hyperbranched hybrid chain reaction signal amplification reaction to obtain a second product;
4) mixing the second product with hemin, and carrying out second incubation under a dark condition to obtain a second incubation product; combining said second hatching product with H 2 O 2 Mixing the first hatching product and the ABTS to carry out third hatching to obtain a third hatching product; carrying out solid-liquid separation on the third incubation product, and collecting liquid components;
5) performing ultraviolet spectroscopic detection on the liquid component to obtain a product A 420nm Obtaining the concentration value of the mycobacterium bovis in the sample to be detected according to the standard curve at the maximum absorption peak;
the standard curve takes the concentration of the bovine mycobacterium tuberculosis DNA as an independent variable, and is expressed in A 420nm The corrected maximum absorption peak is a standard curve obtained by a dependent variable;
said at A 420nm The maximum absorption peak after correction is calculated according to the formula shown in formula 1, Δ A 420nm ＝A 420nm -A 0 Formula 1; wherein Δ A 420nm Is at A 420nm A corrected maximum absorption peak at (a); a. the 420nm The maximum absorption peak, A, for the actual measured concentration of a known target of M.bovis DNA 0 The background value of the bovine mycobacterium tuberculosis DNA target concentration is 0;
there is no chronological restriction between step 1) and step 2).
Preferably, the reaction system for the positive feedback recognition cutting reaction is calculated by 20 mu L and comprises the following components: 8 mu L of positive feedback reaction buffer solution, 2 mu L of 50nM gRNA-T, 2 mu L of 1 mu M scgRNA, 2 mu L of 500nM aDNA, 4 mu L of 1 mu M Cas12a protein and 2 mu L of sample to be tested.
Preferably, the reaction procedure of the positive feedback recognition cleavage reaction is as follows: reacting at 37 ℃ for 4h, and treating at 75 ℃ for 15 min.
Preferably, the reaction temperature of the hyperbranched hybridization chain reaction signal amplification reaction is 20-30 ℃, and the reaction time is 2-3 h.
The invention provides a reagent for detecting mycobacterium bovis, which comprises a recognition element based on a CRISPR/Cas12a positive feedback circulation mechanism, a reagent for hyperbranched hybridization chain reaction signal amplification reaction and a reagent for colorimetric analysis; the recognition element based on CRISPR/Cas12a positive feedback cycle mechanism comprises: gRNA-T, Cas12a protein, helper probe (a dna), and switchable guide rna (scgrna); the switchable guide RNA is assembled by iDNA-spacer, iDNA-handle and gRNA, wherein the iDNA-spacer is a composite sequence of DNA and RNA, a tail RNA sequence of the iDNA-spacer is an initiation sequence (initiator), and the scgRNA probe comprises two functional sequences: gRNA sequences and priming sequences; the probe for the hyperbranched hybridization chain reaction signal amplification reaction comprises H1, SH1, H2, L1, L2 and SH 2; the reagent for colorimetric analysis comprises hemin and H 2 O 2 And ABTS.
The schematic diagram of the principle of the reagent for detecting mycobacterium bovis is shown in figure 1, wherein the figure 1 is divided into (a) in figure 1: construction of positive feedback circular cleavage reaction scheme based on CRISPR/Cas12a and (b) in fig. 1: the schematic diagram of the G-quadruplex DNA enzyme colorimetric sensing principle is constructed by multiple nonlinear hyperbranched hybridization chain reactions.
The invention uses CRISPR/Cas12a as a specific recognition element, initiates hyperbranched hybrid chain reaction after autocatalysis, forms a DNA nano structure with n layers of branches, and constructs a multidimensional G-quadruplex-heme DNase matrix on the DNA nano structure, thereby realizing multiple signal amplification and completing the ultra-sensitive detection of Mycobacterium tuberculosis-DNA of cattle.
In the present invention, the iDNA-spacer, iDNA-handle and gRNA can form a structure (scgRNA) carrying two ssDNA bulges by base complementary pairing. When the target sequence exists, the PAM site in the target sequence is identified by the binary complex formed by combining the Cas12a protein and the gRNA-T, the catalytic site of the Cas12a protein is activated through base complementary pairing and combination with the target DNA, then the target DNA chain is subjected to induced cleavage, and then the target double-stranded DNA is cut off by the action domain of the Cas12a proteinCleaving to release cleavage product, exposing catalytic site, activating trans-cleavage activity, cleaving two ssDNA bulge structures in scgRNA, and cleaving due to double-stranded T m The value decreases, resulting in strand unwinding, release of the gRNA and the priming sequence. Due to the strong affinity of the gRNA and the Cas12a protein, the gRNA, the aDNA and the Cas12a protein form a ternary complex, and the trans-cleavage activity of the Cas12a protein is activated again, and the ternary complex also acts on the scgRNA structure.
The released priming sequence can open hairpin H1, and then the hybrid chain reaction occurs by linkage opening of H2, SH1, SH2, L1 and L2. The super hairpins SH1 are provided with 3 XGGG at the 5 'end, 1 XGGG at the 3' end, 1 XGGG at the 5 'end and 3 XGGG at the 3' end, and the super hairpins (SH1 and SH2) are provided with a group of GGGs sealed in the stem region when forming a stem structure, so that the spontaneous formation of an intramolecular G-quadruplex can be effectively avoided, and two bulge loops (c and f for SH1 and G and h for SH2) extend out of the middle of the double-stranded stem of the super hairpins (SH1 and SH 2). Under the condition of no priming sequence, the thermal stability of the super hairpin is important for avoiding high background signal caused by spontaneous opening, the stem is divided into two regions by manually designing two unmatched base pairs, and each region has 8bp, so that the hairpins can stably coexist. The 3 'end of the priming sequence is firstly combined with the 5' end cohesive end of the traditional hairpin H1 and triggers the hybridization chain displacement reaction, the stem sequence of H1 is opened and the 3 'end is released, the 3' end sequence released by the hairpin H1 and the 5 'end cohesive end of the super hairpin SH15 continue to trigger the hybridization chain displacement reaction, and the bulge loop sequences c and f are released, under the action of the single-chain auxiliary L1, the e and d sequences are released, the exposed sequence f and b of the super hairpin SH1 serve as the priming sequences, the hybridization chain reaction can be carried out again with the traditional hairpin H1 to form a one-dimensional linear structure initor H1 SH 1. L1. H1. SH1, the exposed sequence e and d can trigger the hybridization chain displacement reaction with the 5' end cohesive end of the super SH2 to release the bulge loop sequences g and H, and form an assembly initor H1. SH 1. L1. SH 356. SH 3527. 2, and 2, the hybridization chain reaction is triggered, the sequences a, b, H and d are released, the H and d sequences exposed by the super hairpin SH2 serve as initiation sequences, and the hybridization chain reaction can be carried out again with the traditional hairpin H2, and a two-dimensional linear structure [ initiator.H 1.SH 1.L 1.H 1.SH 1] n.SH 2.L 2.H 2.SH 2 is formed in a cycle. Similarly, the sequence a and b exposed by the super hairpin SH2 can hybridize with the cohesive end at the 5' end of SH1, triggering the hybridization chain reaction again, thereby forming a 3-layer branched DNA assembly structure [ [ [ [ [ initiator. H1. SH 1. L1. H1. SH1] n.SH 2. L2. H2. SH2] n.SH 1. L1. H1. SH1] n. As the reaction proceeds, an n-layered branched DNA assembly structure [ [ [ [ initiator. H1. SH 1. L1. H1. SH1] n. SH 2. L2. H2. SH2] n. SH 1. L1. H1. SH1] n. cndot. can be finally formed, as shown in FIG. 1 (b).
Because the 5 'end and the 3' end of the super hairpins SH1 and SH2 both contain GGG sequences, the super hairpins are mutually drawn close and folded through hybrid chain reaction, and after hemin (hemin) is added, G-quadruplex-heme DNase is formed, and the catalytic activity similar to catalase is shown, and the enzyme can catalyze H 2 O 2 Chemical reaction with ABTS to produce ABTS with free radical of green cation ·+ In A at 420nm Has the maximum absorption peak.
The reagent has high specificity selection on the bovine mycobacterium tuberculosis DNA, and the detection limit is 8.9aM (LOD is 3 sigma/S). The detection method of the invention realizes the ultra-sensitive and high-specificity detection of the bovine mycobacterium tuberculosis DNA.
Drawings
FIG. 1 is a schematic diagram of a Mycobacterium tuberculosis detection system for cattle; wherein (a) is a positive feedback cycle cleavage reaction constructed based on CRISPR/Cas12 a; (b) constructing a G-quadruplex-hemin-DNA enzyme colorimetric sensing principle for multiple nonlinear hyperbranched hybridization chain reaction;
FIG. 2 is a feasibility analysis of the Mycobacterium bovis detection method;
FIG. 3 shows that CRISPR/Cas12a has trans-cleavage activity in Mycobacterium bovis detection system;
FIG. 4 shows the specific selection analysis of the target DNA by the Mycobacterium tuberculosis bovis detection system;
FIG. 5 is a performance analysis of a Mycobacterium tuberculosis detection system for cattle; wherein, (a) is a DNA ultraviolet-visible absorption curve of mycobacterium bovis with different concentrations; (b) a saturation curve of a DNA detection signal of the mycobacterium bovis is obtained; (c) calibration curves for DNA response of M.bovis at different concentrations.
Detailed Description
The invention provides a reagent for detecting mycobacterium bovis, which comprises a recognition element based on a CRISPR/Cas12a positive feedback circulation mechanism, a reagent for hyperbranched hybridization chain reaction signal amplification reaction and a reagent for colorimetric analysis;
the recognition element based on CRISPR/Cas12a positive feedback cycle mechanism comprises: gRNA-T, Cas12a protein, an auxiliary probe and a switchable guide RNA;
the switchable guide RNA is assembled by iDNA-spacer, iDNA-handle and gRNA;
the nucleotide sequence of the gRNA-T is shown in SEQ ID NO.1, and specifically comprises the following steps: 5'-uaauuucuacuaaguguagauggucugacgacgcgagaaua-3', respectively;
the auxiliary probe is a double chain formed by complementary base pairing of nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3; the nucleotide sequence shown in SEQ ID NO.2 is specifically as follows: 3 '-ctgctgttttgaaatctagcaatgcggagagagtc-5'; the nucleotide sequence shown in SEQ ID NO.3 is specifically: 5'-gacgacaaaactttagatcgttacgcctctctcag-3';
the nucleotide sequence of the iDNA-spacer is shown in SEQ ID NO.4, and specifically comprises the following steps: 3 '-ctagcaatgctttatttggagagagucgagaug-5';
the nucleotide sequence of the iDNA-handle is shown as SEQ ID NO.5, and specifically comprises the following steps: 3 '-attaaagatgatttatttttcacatcta-5';
the nucleotide sequence of the gRNA is shown as SEQ ID NO.6, and specifically comprises: 5'-uaauuucuacuaaguguagaugaucguuacgccucucucag-3', respectively;
the probe for the hyperbranched hybridization chain reaction signal amplification reaction comprises H1, SH1, H2, L1, L2 and SH 2; the nucleotide sequences of H1, H2, SH1, SH2, L1 and L2 are respectively shown as SEQ ID NO. 7-SEQ ID NO. 12;
the nucleotide sequence shown in SEQ ID NO.7 is specifically: 5'-cctctctcagctctacgaatgctggagtagagctga-3', respectively;
the nucleotide sequence shown in SEQ ID NO.8 is specifically: 5'-tgggcgggaagggagtagagctgagagaggcatcaagctcaggcatgttacctgagcttgatgaatcgatcag ctctactccagcattcagggt-3', respectively;
the nucleotide sequence shown in SEQ ID NO.9 is specifically: 5'-catcaagctcaggcatgttagagcttgatgtatggt-3';
the nucleotide sequence shown in SEQ ID NO.10 is specifically: 5'-tcgattcatcaagctcagg-3', respectively;
the nucleotide sequence shown in SEQ ID NO.11 is specifically: 5'-ggagtagagctgagtcagt-3', respectively;
the nucleotide sequence shown in SEQ ID NO.12 is specifically: 5'-tgggataacatgcctgagcttgatgactgactcagctctactccgaatgctggagtagagctgaaccatacatca agctcagggaagggagggt-3', respectively;
the reagent for colorimetric analysis comprises hemin and H 2 O 2 And ABTS.
In the invention, the molar ratio of the iDNA-spacer, the iDNA-handle and the gRNA is preferably (1.1-1.3): (1.1-1.3): 1, more preferably 1.2: 1.2: 1. in the present invention, the switchable targeting RNA is a triple-stranded structure; the switchable targeting RNA is preferably assembled by the following method: and mixing the iDNA-spacer, the iDNA-handle and the gRNA, and assembling to obtain the switchable guide RNA. In the present invention, the assembled reaction buffer is preferably an annealing buffer; the annealing buffer solution preferably comprises the following components in concentrations with DEPC water as a solvent: 20mM Tris-HCl, 150mM KCl, 1mM EDTA and 50mM MgCl 2 (ii) a The pH value of the annealing buffer solution is preferably 7.5; the concentration of the working solution of the iDNA-spacer is preferably 48 mu M; the working solution concentration of the iDNA-handle is preferably 48 mu M; the working solution concentration of the gRNA is preferably 20 μ M. In the present invention, the procedure of the assembly is preferably: incubating at 95 deg.C for 5min, and cooling to 25 deg.C; the rate of cooling is preferably 0.1 ℃/s; the storage temperature of the switchable guide RNA is preferably 4 ℃.
In the invention, the iDNA-handle and iDNA-spacer sequences are complementarily paired with the gRNA to form a three-strand complex (scgRNA) with a convex structure, wherein the iDNA-spacer sequence contains a priming sequence for priming a downstream hyperbranched hybrid chain reaction, and the priming sequence can be released and the gRNA can complete positive feedback circulation and hyperbranched hybrid chain reaction only after Cas12a is activated to cut the convex structure.
In the present invention, the auxiliary probe is preferably prepared by the following method: and (3) mixing the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3, and carrying out base complementary pairing to obtain the auxiliary probe. In the present invention, the reaction buffer for base complementary pairing is preferably an annealing buffer solution as described in the above embodiment; the procedure for base complementary pairing is preferably: incubating at 95 deg.C for 5min, and cooling to 25 deg.C; the rate of cooling is preferably 0.1 ℃/s; the storage temperature of the auxiliary probe is preferably 4 ℃.
In the present invention, the method for obtaining the target sequence is preferably based on the method for preparing the auxiliary probe.
The invention also provides a kit for detecting the mycobacterium bovis, which comprises the reagent in the scheme.
In the present invention, the kit preferably further comprises a positive control; the positive control product preferably comprises a target sequence (target probe) formed by complementary base pairing of nucleotide sequences shown in SEQ ID NO.23 and SEQ ID NO. 24. In the invention, the nucleotide sequence shown in SEQ ID NO.23 is specifically: 3 '-ggagagccccaaaacccagactgctgcgctcttat-5'; the nucleotide sequence shown in SEQ ID NO.24 is specifically: 5'-cctctcggggttttgggtctgacgacgcgagaata-3' are provided.
In the invention, the concentration of the working solution of the gRNA-T is preferably 50 nM; the working solution concentration of the auxiliary probe is preferably 500 nM; the concentration of the working solution of the switchable guide RNA is preferably 1 mu M; the working solution concentration of the Cas12a protein is preferably 1 μ M. In the present invention, the working solution concentration of H1, SH1, H2, L1, L2 or SH2 is preferably 1 μ M. In the invention, the solvent of the working solution of H1, SH1, H2, L1, L2 or SH2 is preferably 0.01M PBS; the 0.01MPBS uses water as a solvent, and preferably comprises the following components in concentration: 200mM NaCl, 2.5mM MgCl 2 And 20mM KCl; the pH of the 0.01M PBS is preferably 6.2.
In the present invention, the working solution concentration of hemin is preferably 20 μ M; said H 2 O 2 The working solution concentration of (2) is preferably 10 mM; the working solution concentration of ABTS is preferably 10 mM.
In the present invention, the sequences involved in the reagent or the kit are all purchased from Shanghai Bioengineering Co., Ltd. In the invention, the solvent used in the reagent or the kit is DEPC water.
The invention also provides a detection method of the non-diagnostic mycobacterium bovis based on the reagent or the kit in the scheme, which comprises the following steps:
1) mixing a sample to be detected with a recognition element of the CRISPR/Cas12a positive feedback circulation mechanism, and carrying out positive feedback recognition cutting reaction to obtain a first product containing an initiation sequence;
2) dissolving the SH1, SH2, H1 and H2 in a buffer solution respectively, and performing first incubation respectively to obtain SH1, SH2, H1 and H2 with hairpin structures respectively;
3) mixing the first product, L1, L2 and SH1, SH2, H1 and H2 of a hairpin structure, and carrying out hyperbranched hybrid chain reaction signal amplification reaction to obtain a second product;
4) mixing the second product with hemin, and carrying out second incubation under a dark condition to obtain a second incubation product; combining said second hatching product with H 2 O 2 Mixing the first hatching product and the ABTS to carry out third hatching to obtain a third hatching product; carrying out solid-liquid separation on the third incubation product, and collecting liquid components;
5) performing ultraviolet spectroscopic detection on the liquid component to obtain a product A 420nm Obtaining the concentration value of the mycobacterium bovis in the sample to be detected according to the standard curve at the maximum absorption peak;
the standard curve takes the concentration of the bovine mycobacterium tuberculosis DNA as an independent variable, and is expressed in A 420nm The corrected maximum absorption peak is a standard curve obtained by a dependent variable;
said at A 420nm The corrected maximum absorption peak is calculated according to the formula shown in formula 1, Δ A 420nm ＝A 420nm -A 0 Formula 1; wherein Δ A 420nm Is at A 420nm Corrected maximum absorption peak of (a); a. the 420nm The maximum absorption peak of known DNA target concentration of Mycobacterium bovis, A, is actually measured 0 The background value is the background value when the DNA target concentration of the bovine mycobacterium tuberculosis is 0;
there is no chronological restriction between the step 1) and the step 2).
According to the method, a sample to be detected and a recognition element of the CRISPR/Cas12a positive feedback circulation mechanism are mixed, and a positive feedback recognition cutting reaction is carried out to obtain a first product containing an initiation sequence.
In the present invention, the sample to be tested is preferably Mycobacterium bovis genomic DNA or genomic DNA extracted from a test sample.
In the invention, the reaction buffer solution for the positive feedback recognition cutting reaction is preferably a positive feedback reaction buffer solution; the positive feedback reaction buffer solution preferably takes DEPC water as a solvent and comprises the following components in concentration: 20mM Tris-HCl, 100mM KCl, 5mM MgCl 2 1mM DTT, 5% glycerol by volume and 50 mug/mL heparin; the pH value of the positive feedback reaction buffer solution is preferably 7.5; the reaction system for the positive feedback recognition of the cleavage reaction is preferably in 20. mu.L and comprises the following components: 8 μ L of positive feedback reaction buffer solution, 50nM gRNA-T2 μ L, 1 μ M scgRNA 2 μ L, 500nM aDNA2 μ L, 1 μ M Cas12a protein 4 μ L and 2 μ L of sample to be tested. In the present invention, the reaction procedure of the positive feedback recognition cleavage reaction is preferably: reacting at 37 ℃ for 4h, and treating at 75 ℃ for 15 min. Reaction at 37 ℃ for 4h, sufficient to release the priming sequence, and treatment at 75 ℃ for 15min to inactivate the Cas12a protein.
The SH1, SH2, H1 and H2 are respectively dissolved in buffer solution, and first incubation is respectively carried out to respectively obtain SH1, SH2, H1 and H2 with hairpin structures.
In the present invention, the buffer is preferably 0.01M PBS buffer; the 0.01M PBS buffer solution is water as a solvent, and preferably includes the following components in concentration: 200mM NaCl, 2.5mM MgCl 2 And 20mM KCl; the 0.01M PBS buffer solutionThe pH of the liquid is preferably 6.2. In the present invention, the final concentration of each of the probes after dissolution in the buffer is preferably 1. mu.M; the procedure of the first incubation preferably comprises: 95 ℃ for 5 min; the temperature is 25 ℃, the time is 2.5h, and the cooling rate is preferably 0.1 ℃/s; the storage temperature of the hairpin probe is preferably 4 ℃. The invention can slowly anneal to form hairpin structure after incubation at 95 ℃, which can improve the formation ratio of hairpin probe.
The first product, L1, L2 and SH1, SH2, H1, H2 of the hairpin structure are mixed to carry out hyperbranched hybridization chain reaction signal amplification reaction, and a second product is obtained.
In the present invention, the reaction system for recognizing the cleavage reaction by positive feedback is 20. mu.L, and the concentrations of H1, SH1, H2, L1, L2 and SH2 forming the hairpin structure are preferably 1. mu.M, respectively, and the volumes are preferably 5. mu.L, respectively. In the invention, the reaction temperature of the hyperbranched hybrid chain reaction signal amplification reaction is preferably 20-30 ℃, and more preferably 25 ℃; the reaction time of the hyperbranched hybridization chain reaction signal amplification reaction is preferably 2-3 h, and more preferably 2.5 h.
After a second product is obtained, mixing the second product with hemin, and carrying out second incubation under a dark condition to obtain a second incubation product; combining said second hatching product with H 2 O 2 Mixing the first hatching product and the ABTS to carry out third hatching to obtain a third hatching product; and carrying out solid-liquid separation on the third incubation product, and collecting a liquid component.
In the present invention, the reaction system for identifying the cleavage reaction by the positive feedback is 20. mu.L, and the volume of hemin is preferably 5. mu.L. In the present invention, the temperature of the second incubation is preferably 25 ℃; the second incubation time is preferably 25-35 min, and more preferably 30 min; the second hatching product comprises a G-quadruplex-heme dnase.
In the present invention, the third incubation reaction system comprises: second hatching product 10. mu. L, H 2 O 2 45 μ L and ABTS 45 μ L. In the invention, the temperature of the third incubation is preferably 20-30 ℃, and more preferablyPreferably 25 ℃; the time of the third incubation is preferably 5-10 min, and more preferably 8 min.
In the present invention, the solid-liquid separation is preferably centrifugation; the rotating speed of the centrifugation is preferably 8000 r/min; the time of centrifugation is preferably 3-8 min, and more preferably 5 min.
After the liquid component is collected, the ultraviolet light splitting detection is carried out on the liquid component to obtain the component A 420nm Obtaining the concentration value of the mycobacterium bovis in the sample to be detected according to the standard curve at the maximum absorption peak;
the standard curve takes the concentration of bovine mycobacterium tuberculosis DNA as an independent variable and is defined as A 420nm The corrected maximum absorption peak is a standard curve obtained by a dependent variable;
said at A 420nm The maximum absorption peak after correction is calculated according to the formula shown in formula 1, Δ A 420nm ＝A 420nm -A 0 Formula 1; wherein Δ A 420nm Is at A 420nm Corrected maximum absorption peak of (a); a. the 420nm The maximum absorption peak, A, for the actual measured concentration of a known target of M.bovis DNA 0 The background value of the bovine mycobacterium tuberculosis DNA target concentration is 0. In the present invention, the formula shown in formula 1 is more preferably Δ a 420nm ＝0.048C (Mycobacterium tuberculosis of cattle DNA) +0.0071, linear correlation coefficient R 2 The detection limit was 8.9aM (LOD 3 σ/S) at 0.9957.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1 high-sensitivity Mycobacterium bovis colorimetric sensing technology based on CRISPR-Cas12a positive feedback cascade signal amplification and hyperbranched hybridization chain reaction
1. Designing probes
The probes of the present invention were purchased from Shanghai Biotechnology engineering Co., Ltd, and are shown in Table 1.
Note: the substituted bases in SM-2, SM-5, SM-8, SM-11, and SM-14 are underlined.
2. Experimental procedure
(1) Pretreatment of Probe sequences
scgRNA probe: with annealing buffer (pH 7.5, 20mM Tris-HCl, 150mM KCl, 1mM EDTA, 50mM MgCl) 2 ) The probes iDNA-handle, iDNA-spacer and gRNA freeze-dried powder are respectively prepared into solutions of 48 mu M, 48 mu M and 20 mu M. Respectively transferring 48 mu M iDNA-handle, 48 mu MiDNA-spacer and 20 mu M gRNA according to the molar ratio of the iDNA to the gRNA of 1.2: 1, uniformly mixing in a 200 mu L centrifuge tube, incubating for 5min at 95 ℃, then slowly cooling to room temperature, fully assembling a three-chain structure, and storing for later use at 4 ℃.
Target, aDNA probe: with annealing buffer (pH 7.5, 20mM Tris-HCl, 150mM KCl, 1mM EDTA, 50mM MgCl) 2 ) Preparing two single-chain probe freeze-dried powders which can be complementarily paired into mother solutions, and mixing the mother solutions according to the volume ratio of 1: 1, mixing uniformly in a 200 mu L centrifuge tube, incubating for 5min at 95 ℃, slowly cooling to room temperature to fully form DNA double chains, and storing for later use at 4 ℃.
SH1, SH2, H1, H2 probes: with 0.01M PBS (pH 6.2, 200mM NaCl, 2.5mM MgCl) 2 20mM KCl) buffer solution the DNA probes SH1, SH2, H1, H2, L1 and L2 lyophilized powder were all prepared as a 1. mu.M solution. 100 mu L of SH1, 100 mu L of SH2, 100 mu L H1 and 100 mu L of LH2 are respectively transferred into different centrifuge tubes of 200 mu L, incubated for 5min at 95 ℃, then slowly cooled to room temperature, incubated for 2.5h at room temperature to form a hairpin probe, and stored for later use at 4 ℃.
(2) Positive feedback recognition of cleavage reaction formation
mu.L of positive feedback reaction buffer (pH 7.5, 20mM Tris-HCl, 100mM KCl, 5mM MgCl) 2 1mM DTT, 5% glycerol, heparin (50. mu.g/mL)) was mixed with 50nMgRNA-T, 500 nMeDNA, 1. mu.M scgRNA, 1. mu.M Cas12a, and Target probes of different concentrations, in a total reaction volume of 20. mu.L, at 37 ℃ CAfter 4h reaction, the priming sequence was released sufficiently and treated at 75 ℃ for 15min to inactivate the Cas12a enzyme.
(3) Formation of hyperbranched hybrid chain reactions
mu.L of each of six probes, namely 1. mu.M SH1, 1. mu.M SH2, 1. mu. M H1, 1. mu. M H2, 1. mu. M L1 and 1. mu. M L2, are added to the system, mixed uniformly and incubated for 2.5 hours at normal temperature.
(4) Formation and colorimetric analysis of G-quadruplex-heme DNase
Placing the reaction system in dark, adding 5 μ L20 μ M hemin, incubating for 30min in dark to form G-quadruplex-heme DNA enzyme. 10 μ L of the mixture was placed in a fresh sterile centrifuge tube and 45 μ L of 10mM H was added 2 O 2 Mixing with 45 μ L10 mM ATBTS, stirring, incubating at room temperature for 8min, centrifuging, collecting supernatant 50 μ L, detecting with UV-1800 UV spectrophotometer, and detecting at A 420nm Has a maximum absorption peak (definition delta A) 420nm ＝A 420nm －A 0 Wherein A is 420nm As measured on the sample, A 0 Background value when the target concentration of mycobacterium bovis DNA is 0).
3. Feasibility analysis of detection method of mycobacterium tuberculosis of cattle
In order to investigate the feasibility of the method for detecting the target DNA of the mycobacterium tuberculosis of the cattle, 6 groups of experiments are designed for verification, and key influence factors are analyzed. As shown in fig. 2, group 1 is a normal experimental group, group 2 does not add Cas12a protein, group 3 does not add gRNA-T, group 4 does not add SH1, group 5 does not add scgRNA, all of the above variables are replaced by adding an equal volume of annealing buffer solution, and group 6 is a signal background generated by ABTS auto-oxidation. The target concentrations in the feasibility group were all 1 nM.
The conditions not described in the 2 to 5 groups were kept the same as those in the experimental group. Groups 2 and 3 triggered downstream hybridization chain reactions due to a small amount of free priming sequence, forming G-quadruplex-heme DNase, giving a certain background value, groups 4 and 5 failed to form G-quadruplex structures, almost equivalent to the background of the signal generated by ABTS autooxidation in group 6, at A 420nm The signal value at (b) is lower. Group 1The target is normally recognized, and a certain concentration of priming sequence is released through the trans-cleavage of Cas12a, so that the hybridization chain reaction is triggered to form a certain concentration of G-quadruplex-heme DNase, and a higher signal value is generated. The result proves that the signal value is higher only when the target of the mycobacterium bovis exists, and the system can be used for detecting the mycobacterium bovis.
4. Verification of CRISPR/Cas12a trans-cleavage activity
To verify the trans-cleavage activity of CRISPR/Cas12a in the present line, the enzymes were aligned by inactivation at 75 ℃ and not inactivation prior to addition of probes for hyperbranched hybridization chain reaction. As shown in FIG. 3, group 1 was blank, group 2 enzyme-inactivated, and group 3 enzyme-inactivated. The target concentration is 1pM, the blank group target concentration is 0, and a buffer solution with the same volume as the target is added, and the unexplained conditions are consistent with the experimental steps of the invention.
In group 1, a small amount of free priming sequences trigger hybridization chain reaction and have a certain background value, and in group 2, enzyme inactivation is performed at 75 ℃, the CRISPR/Cas12a loses the cutting activity, so that the completeness of a downstream hyperbranched probe is ensured, a G-quadruplex with a certain concentration is formed under the initiation of the priming sequences, and a higher signal value is generated. Group 3 was not inactivated at 75 ℃ so CRISPR/Cas12a also cleaved the downstream hyperbranched hybridization chain reaction probe in trans, failing to form a G-quadruplex, at A 420nm The absorbance was low.
5. Specificity analysis of target by mycobacterium bovis detection system
In order to examine the specificity of the sensing technology for detecting the mycobacterium bovis, single-base substitution is carried out on different positions of a target sequence, wherein the single-base substitution is respectively SM-2, SM-5, SM-8, SM-11 and SM-14, the single-base substitution is compared with mycobacterium bovis DNA, and the concentration of each single-base substitution is 1nM, so that the specificity of a recognition element taking CRISPR/Cas12a as a core is verified. Under the same optimum condition, respectively at A 420nm Measure the respective signal value (background value A) 0 Signal value measured when the concentration of M.tuberculosis DNA was 0), 6 parallel experiments, as shown in FIG. 4, signal value Δ A after completion of the reaction by adding 1nM Target DNA 420nm Is obviously highExperiments show that the invention has higher specificity selection to the bovine mycobacterium tuberculosis DNA.
6. Quantitative analysis
Under the optimal experimental conditions, the quantitative detection of the bovine mycobacterium tuberculosis DNA standard sample is realized. FIG. 5 (a) is a UV-visible absorption curve of M.bovis DNA at concentrations of 0, 1aM, 10aM, 100aM, 1fM, 10fM, 50fM, 100fM, 500fM, and 1pM, respectively. As shown in (b) of FIG. 5, when the bovine Mycobacterium tuberculosis DNA is less than 0.1pM, at Δ A 420nm The peak of the absorbance increases with the increase of the DNA concentration of M.bovis, and after 0.1pM, the peak of the absorbance reaches a maximum, and then the signal value fluctuates slightly, but the total value becomes stable. As shown in FIG. 5 (c), the concentration of M.bovis DNA was in a linear trend between 10aM and 50fM, and the regression equation of the curve was Δ A 420nm ＝0.048C (Mycobacterium tuberculosis of cattle DNA) +0.0071, linear correlation coefficient R2 ═ 0.9957, detection limit 8.9aM (LOD ═ 3 σ/S). The detection method realizes the ultra-sensitive detection of the DNA of the mycobacterium tuberculosis of the cattle, and the DNA concentrations (10aM, 100aM, 1fM, 10fM and 50fM) of the mycobacterium tuberculosis of the cattle are repeated for 6 times in parallel.
7. Detection of Mycobacterium bovis in blood
In order to examine the detection performance of the method in the detection of the actual sample, a standard adding method (inactivated bacteria adding method) is adopted to simulate the actual sample for detection. To contain 10 8 CFU/mL inactivated Mycobacterium bovis bacteria liquid is taken as mother liquid, added into bovine blood, and extracted and prepared into actual samples with the DNA concentrations of the Mycobacterium bovis being 1fmol/L, 10fmol/L and 50fmol/L respectively. The experimental results are shown in the table, the average recovery rate of actual sample detection is 97.0-105.2%, and the Relative Standard Deviation (RSD) is 0.95-6.8%. The sensing technology has better detection performance on the mycobacterium bovis in the bovine blood sample.
TABLE 2 results of spiking recovery experiments in plasma samples
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Sequence listing
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Claims (10)
1. A reagent for detecting mycobacterium bovis comprises a recognition element based on a CRISPR/Cas12a positive feedback circulation mechanism, a reagent for hyperbranched hybridization chain reaction signal amplification reaction and a reagent for colorimetric analysis;
the recognition element based on CRISPR/Cas12a positive feedback cycle mechanism comprises: gRNA-T, Cas12a protein, an auxiliary probe and a switchable guide RNA;
the switchable guide RNA is assembled by iDNA-spacer, iDNA-handle and gRNA;
the nucleotide sequence of the gRNA-T is shown in SEQ ID NO. 1;
the auxiliary probe is a double chain formed by complementary base pairing of nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3;
the nucleotide sequence of the iDNA-spacer is shown in SEQ ID NO. 4;
the nucleotide sequence of the iDNA-handle is shown in SEQ ID NO. 5;
the nucleotide sequence of the gRNA is shown in SEQ ID NO. 6;
the probe for the hyperbranched hybridization chain reaction signal amplification reaction comprises H1, SH1, H2, L1, L2 and SH 2; the nucleotide sequences of H1, H2, SH1, SH2, L1 and L2 are respectively shown as SEQ ID NO. 7-SEQ ID NO. 12;
the reagent bag for colorimetric analysisIncluding hemin, H 2 O 2 And ABTS.
2. The reagent according to claim 1, wherein the molar ratio of iDNA-spacer, iDNA-handle and gRNA is (1.1-1.3): (1.1-1.3): 1.
3. a kit for detecting mycobacterium bovis comprising the reagent of claim 1 or 2.
4. The kit of claim 3, further comprising a positive control; the positive control substance comprises a target sequence formed by complementary base pairing of nucleotide sequences shown in SEQ ID NO.23 and SEQ ID NO. 24.
5. The kit of claim 3, wherein the working solution concentration of the gRNA-T is 50 nM; the working solution concentration of the auxiliary probe is 500 nM; the concentration of the working solution of the switchable guide RNA is 1 mu M; the working solution concentration of the Cas12a protein was 1 μ M.
6. The kit according to claim 3, wherein the working solution concentration of H1, SH1, H2, L1, L2 or SH2 is 1 μ M, respectively.
7. A method for detecting Mycobacterium bovis of non-diagnostic purposes based on the reagent of claim 1 or 2 or the kit of any one of claims 3 to 6, comprising the steps of:
1) mixing a sample to be detected with a recognition element of the CRISPR/Cas12a positive feedback circulation mechanism, and carrying out positive feedback recognition cutting reaction to obtain a first product containing a priming sequence;
2) dissolving the SH1, SH2, H1 and H2 in a buffer solution respectively, and performing first incubation respectively to obtain SH1, SH2, H1 and H2 with hairpin structures respectively;
3) mixing the first product, L1 and L2 with SH1, SH2, H1 and H2 of a hairpin structure, and carrying out hyperbranched hybrid chain reaction signal amplification reaction to obtain a second product;
4) mixing the second product with hemin, and carrying out second incubation under a dark condition to obtain a second incubation product; combining said second hatching product with H 2 O 2 Mixing the first hatching product and the ABTS to carry out third hatching to obtain a third hatching product; carrying out solid-liquid separation on the third incubation product, and collecting a liquid component;
5) performing ultraviolet spectroscopic detection on the liquid component to obtain a product A 420nm Obtaining the concentration value of the mycobacterium bovis in the sample to be detected according to the standard curve at the maximum absorption peak;
the standard curve takes the concentration of the bovine mycobacterium tuberculosis DNA as an independent variable, and is expressed in A 420nm The corrected maximum absorption peak is a standard curve obtained by a dependent variable;
said at A 420nm The maximum absorption peak after correction is calculated according to the formula shown in formula 1, Δ A 420nm ＝A 420nm -A 0 Formula 1; wherein Δ A 420nm Is at A 420nm Corrected maximum absorption peak of (a); a. the 420nm The maximum absorption peak, A, for the actual measured concentration of a known target of M.bovis DNA 0 The background value is the background value when the DNA target concentration of the bovine mycobacterium tuberculosis is 0;
there is no chronological restriction between step 1) and step 2).
8. The detection method according to claim 7, wherein the reaction system for the positive feedback identification cleavage reaction comprises the following components in 20 μ L: 8 mu L of positive feedback reaction buffer solution, 2 mu L of 50nM gRNA-T, 2 mu L of 1 mu M scgRNA, 2 mu L of 500nM aDNA, 4 mu L of 1 mu M Cas12a protein and 2 mu L of sample to be tested.
9. The detection method according to claim 7 or 8, wherein the reaction procedure of the positive feedback recognition cleavage reaction is: reacting at 37 ℃ for 4h, and treating at 75 ℃ for 15 min.
10. The detection method according to claim 7, wherein the reaction temperature of the hyperbranched hybridization chain reaction signal amplification reaction is 20-30 ℃ and the reaction time is 2-3 h.
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