CN116732211B - Probe set and method for detecting mycobacterium bovis based on 8-17 deoxyribozyme and CRISPR-Cas13a trans-cleavage - Google Patents
Probe set and method for detecting mycobacterium bovis based on 8-17 deoxyribozyme and CRISPR-Cas13a trans-cleavage Download PDFInfo
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Abstract
The invention provides a probe set and a method for detecting mycobacterium bovis based on 8-17 deoxyribozyme and CRISPR-Cas13a trans-cutting, which integrate the cyclic cutting performance of 8-17 deoxyribozyme, CRISPR-Cas13a trans-cyclic cutting performance and the cyclic utilization of a sequence mediated by foothold strand displacement, and construct a triple-cycle signal amplification system for high-performance 'one-tube' detection of mycobacterium bovis. The 8-17 deoxyribozyme sequence is innovatively designed in the A sequence, the A sequence is released when a target T exists, the HX-gRNA bulge structure is repeatedly cut, the CRISPR-Cas13a system realizes the cutting of the HX-gRNA bulge structure, the H sequence enters the downstream for recycling and is subjected to hybridization chain reaction with H1 and H2, and the G-quadruplex dimer structure is released and is combined with THT dye to serve as a signal output mode. The design constructs a multiple signal amplification system, has the characteristics of constant temperature, high sensitivity, simple operation, realization of 'one-tube' operation and the like, and provides a novel method for nucleic acid detection.
Description
Technical Field
The invention belongs to the technical field of biological analysis and detection, and particularly relates to a probe set and a method for detecting mycobacterium bovis based on trans-cutting of 8-17 deoxyribozyme and CRISPR-Cas13 a.
Background
Mycobacterium tuberculosis is mainly divided into three types: namely Mycobacterium bovis (bovine type), mycobacterium tuberculosis (human type) and Mycobacterium avium (avian type). Mycobacterium bovis is a subtype of Mycobacterium tuberculosis, and in all Mycobacterium tuberculosis complexes, the Mycobacterium bovis has the characteristics of the widest host range, long latency period, disease recurrence and the like, and the outbreak of zoonotic diseases caused by the Mycobacterium bovis not only can cause economic loss of animal husbandry, but also seriously threatens the health of human beings. Therefore, the establishment of a method for sensitively and rapidly detecting the mycobacterium bovis has important significance for disease diagnosis. At present, the method for detecting the mycobacterium bovis mainly comprises the following steps: 1. molecular detection method, 2, immunological method, 3, microorganism culture method, etc. Although immunological methods and microorganism culture methods can realize detection of bovine mycobacterium tuberculosis, the methods have the defects of low sensitivity, low specificity and the like, and the microorganism culture method also has the problems of long time and large detection workload, so the method commonly used at present is mainly a molecular detection method, but the most traditional PCR technology used at present needs expensive temperature control equipment and is easy to cause problems of false positive, false negative and the like.
Deoxyribozymes are single-stranded DNA fragments with catalytic functions obtained by in vitro molecular evolution technology, have high catalytic activity and structure recognition capability, and have been demonstrated to catalyze a number of reactions identical to that of RNases (also called ribozymes) or proteases, including RNA/DNA cleavage, ligation, phosphorylation, cleavage of phosphoamidic bonds, and porphyrin metallization. Wherein the 8-17 deoxyribozyme consists of a stem-loop structure and an unpaired 4-5nt region, the loop part is generally a conserved sequence of 5'-AGC-3', the stem is generally composed of 3 base pairs, and the structure can specifically cut 5 '-rAG-3'. The Yang et al propose a new concept of ' nanoflare pair ', modify probes on two gold nanoparticles, when TK1 mRNA and survivin mRNA highly expressed in tumor tissues exist in cells, the probes on the two gold nanoparticles are mutually close to form 8-17 deoxyribozymes through strand displacement, so that the probes with modified fluorescence quenching groups are cut, and the system develops a multifunctional nanotreatment platform ' nanoflare pair ' (NC) ', which can be used for in-situ composite cancer related mRNA imaging and subsequent logic control aggregation gold nanoparticles, thereby realizing gene treatment and photothermal treatment under infrared light irradiation. In recent years, a CRISPR gene editing system for obtaining the Nobel chemical prize is one of the hot spots of research, and a detection technology based on the CRISPR-Cas system has the advantages of high sensitivity, high accuracy, no need of a thermal cycling instrument, low price, portability and rapidness, and is suitable for developing a universal detection technology facing a basic mechanism. The CRISPR-Cas13a is mainly used in the technical field of molecular diagnosis due to the characteristics of sensitivity, specificity, nucleic acid identification and the like, and serves as the core of a part of powerful diagnosis technology, the CRISPR-Cas13a is thoroughly changing the mode of virus detection, and a new path is opened up for biosensing. For example, zhang et al uses shirlock to realize high-sensitivity detection of RNA, first uses RPA to amplify a large amount of DNA sequences, and then uses T7 in vitro transcription to form RNA, cas13a can recognize the RNA sequences, and cleaves RNA probes with modified fluorophores, thereby realizing high-sensitivity detection of targets.
Guanine (G) -quadruplex is a highly ordered quadruplex structure derived from a G-rich single stranded nucleic acid sequence, and thioflavin T (THT) is a water-soluble fluorescent dye that specifically binds G-quadruplex with significant fluorescence enhancement. By utilizing the characteristic of luminescence, the G-quadruplex/THT can be used as a molecular beacon to construct a label-free fluorescence biosensing strategy for detecting DNA. The study shows that the G quadruplex dimer formed by two G quadruplex monomers connected in series is a stable DNA structure, can obviously enhance the fluorescence intensity of THT dye, has the fluorescence intensity of G quadruplex dimer/THT about nine times that of G quadruplex monomer/THT, and is not affected in high-salt medium. Therefore, the G-quadruplex dimer/THT as a novel fluorescent reporter has great application potential in more biosensors.
Disclosure of Invention
In order to solve the technical problems, the invention solves the problems of high cost, easy false positive and false negative and the like in the existing technology for detecting the mycobacterium bovis, and provides a probe set and a method for detecting the mycobacterium bovis based on 8-17 deoxyribozyme and CRISPR-Cas13a trans-cutting.
In order to achieve the above purpose, the invention firstly provides a probe set for detecting bovine mycobacterium tuberculosis based on trans-cleavage of 8-17 deoxyribozymes and CRISPR-Cas13a, which comprises a probe A, a probe B, a probe HX, a probe gRNA, a probe g, a probe H1, a probe H2, a probe a, a probe B and LwaCas13a enzyme; the probe A contains 8-17 deoxyribozymes, and the gRNA is combined with LwaCas13a and g sequences to form a CRISPR-Cas13a system; the probe set is in Pb 2+ Under the condition, the detection of the bovine mycobacterium tuberculosis target gene T is realized through multiple signal amplification;
the gene sequence of the probe A is shown as SEQ ID NO.1, the gene sequence of the probe B is shown as SEQ ID NO.2, the gene sequence of the probe HX is shown as SEQ ID NO.3, the gene sequence of the probe gRNA is shown as SEQ ID NO.4, the gene sequence of the probe g is shown as SEQ ID NO.5, the gene sequence of the probe H1 is shown as SEQ ID NO.6, the gene sequence of the probe H2 is shown as SEQ ID NO.7, the gene sequence of the probe a is shown as SEQ ID NO.8, the gene sequence of the probe B is shown as SEQ ID NO.9, the gene sequence of the target gene T is shown as SEQ ID NO.10, and the functional sequence of the 8-17 deoxyribonuclease is shown as SEQ ID NO. 11.
Based on a general inventive concept, the invention also provides a detection method of the mycobacterium tuberculosis of the cattle of the non-diagnosis and treatment purpose, which comprises the following steps:
s1, pretreatment of a probe sequence: preparing freeze-dried powder of the probe A, the probe B, the probe HX, the probe gRNA, the probe g, the probe H1, the probe H2, the probe a and the probe B into mother solutions, and respectively diluting the mother solutions by using buffer solutions; mixing diluted probe A, B mother liquor and incubating to form probes A-B; mixing diluted probe gRNA and HX mother solution, and incubating to form probe HX-gRNA; mixing diluted mother solutions of the probes a and b, and incubating to form probes a-b;
s2, strand displacement releasing deoxyribozyme sequence: uniformly mixing a sample to be tested of mycobacterium bovis with the probes A-B for reaction;
cleavage of specific probes by S3, 8-17 deoxyribozymes and LwaCas13 a: after the S2 step reaction is finished, adding a probe HX-gRNA for reaction, then adding LwaCas13a and g probes for mixing, and adding an enzyme reaction buffer solution for reaction;
s4, forming a G-quadruplex dimer structure: after the S3 reaction is finished, adding H1, H2, a-b probes and THT, adding a buffer solution, and carrying out a reaction in a dark place;
s5, fluorescence detection: and (3) after the S4 reaction is finished, diluting the solution, sucking the sample into a cuvette, performing on-machine detection, and calculating the concentration of the sample to be detected according to a target gene T response calibration curve.
Preferably, in the step S1, the concentration ratio of the diluted mother solutions of the probe a, the probe B, the probe HX, the probe gRNA, the probe g, the probe H1, the probe H2, the probe a, and the probe B is 4:4:2:2:1:1:1:2:2; the buffer solution has a ph=7.5 and contains Pb 2+ Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) buffer solution.
Preferably, the mol ratio of the probe A to the probe B in the probes A-B is 1:1; the molar ratio of the probe HX to the probe gRNA in the probe HX-gRNA is 6:5; the molar ratio of the probe a to the probe b in the probes a-b is 1:1.1.
Preferably, in the step S2, the volume ratio of the sample to be tested of the mycobacterium bovis to the probe A-B is 2:1, and the reaction time is 1h.
Preferably, the molar ratio of HX-gRNA probe, lwaCas13a and g probe in the step S3 is 4:1:1; the reaction temperature was 37℃and the reaction time was 3 hours.
Preferably, in the step S4, the molar ratio of H1, H2, a-b probe and THT is 11:11:10:100; the reaction temperature was room temperature and the reaction time was 1.5 hours.
Preferably, the detection parameters in the step S5 are: excitation light spectral bandwidth 15 nm, emission light spectral bandwidth 15 nm, excitation light wavelength 420 nm.
The detection principle and the detection process of the probe set for detecting the mycobacterium bovis based on the 8-17 deoxyribozyme and CRISPR-Cas13a trans-cutting provided by the invention are as follows:
the experimental principle is shown in figure 1, and the invention integrates the 8-17 deoxyribozyme circular cutting performance, CRISPR-Cas13a trans-circular cutting performance and the sequence circular utilization mediated by foothold strand displacement, so as to construct a triple-cycle signal amplification system for high-performance 'one-tube' detection of mycobacterium bovis:
first heavy signal cyclic amplification: the target T, namely the bovine tuberculosis mycobacterium DNA and A-B (A sequence comprises 8-17 deoxyribozyme functional sequence 5'-TTCTTTTGCTCCGA GCCGGTCGAACTATC-3', B sequence plays a role in blocking A) are subjected to strand displacement to release A sequence, a specific probe HX-gRNA (which is a hybrid dimer of HX and gRNA and comprises two non-complementary single-stranded RNA bulge structures) bulge structures (comprising 8-17 deoxyribozyme recognition sites: 5'-GAUAGUUCG-3',5'-GCAAAAGAA-3', and cleavage sites: r-AG) are further subjected to cleavage, and DNA sequence H (part sequence of HX: 5'-CCCCTTCGTTAATGCAGATC-3'), gRNA sequence and A sequence in HX chain are released, wherein the sequence A can be circularly used for cleaving HX-gRNA to release H chain, and cycle 1 is constructed;
second repeated signal cyclic amplification: the gRNA can be combined with LwaCas13a and g sequences to form an LwaCas13a compound with RNA trans-cutting activity, so that the cutting effect of any RNA in a system is realized, the HX-gRNA bulge structure is cut again, an H chain is released, and cycle 2 is constructed;
third triple signal cycle amplification: the released H chain can enter a downstream system to repeatedly react with H1 and H2 to form a T-shaped compound, so as to construct cycle 3. The "T" complex free portion (H1 free 5'-AGGGGAAT-3', H2 free 5'-TCTTGGAGCGCTACG-3') can be subjected to strand displacement (found by hybridization of the a sequence containing the G-quadruplex dimer sequence: 5'-GGGTTGGGCGGGATGGGGGGTTGGGCGGGATGGG-3' with the b sequence protecting the sequence) with the a-b complex (found in the cut-off: 5'-ATTCCCCTCGTAGCGCTCCAAGA-3') to expose the G-quadruplex dimer sequence in the a sequence. Thioflavin T (THT) as a water-soluble fluorescent dye consists of a benzylamine ring and a phenyl sulfide ring, which can rotate freely around the C-C bond, resulting in very low fluorescence of THT, but when this rotation is limited by some special structure, the fluorescence of THT is significantly enhanced, while G-quadruplex dimer has a strong affinity with THT dye, which can efficiently enhance the fluorescence emission of THT. Therefore, THT dye is added into the whole system, and can be combined with the G-quadruplex dimer sequence in the a sequence to emit fluorescence as a signal output mode.
Compared with the prior art, the invention has the following beneficial effects:
(1) The system repeatedly cuts a specific sequence by utilizing 8-17 deoxyribozymes, and amplifies signals in a mode that CRISPR-Cas13a continuously circulates a reverse cutting capacity of an RNA sequence and a downstream initiation sequence, so that the sensitivity of the whole system is improved, and G-quadruplex dimer formed in the downstream is combined with THT dye to be output as a fluorescent signal, so that the whole detection process is simpler, and the system has the advantages of constant temperature, realization of one-tube operation, high sensitivity and the like.
(2) The detection system has the characteristics of constant temperature and simple operation, and in addition, the system constructs a triple-cycle signal amplification system: cycle 1: the A sequence containing 8-17 deoxyribozyme recognizes the convex RNA sequence of HX-gRNA and circularly cuts the convex RNA sequence; cycle 2: the gRNA released after the initial cutting of HX-gRNA can be combined with g and LwaCas13a to form a CRISPR-Cas13a system, so that the circular cutting of HX-gRNA is realized; cycle 3: the H sequence released after the HX-gRNA is cut can be recycled to continuously form a T-shaped compound, each cycle has the advantages of high sensitivity and the like, can be used for in-vitro high-sensitivity one-tube detection of the mycobacterium tuberculosis, and realizes accurate quantitative analysis of the mycobacterium tuberculosis.
(3) The detection system has ingenious sequence design, makes full use of 8-17 deoxyribozyme functional sequences to repeatedly cut the specific probe and two sequences released after cutting, one of the sequences is combined with LwaCas13a to form a CRISPR-Cas13a system to cut the specific probe again, and the other sequence enters a downstream system to be continuously recycled to realize the high-sensitivity detection of triple signal amplification, so that the defects of low detection sensitivity and complex operation of the conventional system are overcome, the multiple signal amplification effect is realized, and the detection performance is improved.
(4) The reaction condition is mild without expensive temperature-changing instrument, the reaction is carried out at the constant temperature of 37 ℃, the operation is simple, the one-tube operation can be realized, and the limitation of the current domestic and foreign on-site detection of the mycobacterium bovis is effectively solved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the detection of a probe set for detecting Mycobacterium bovis based on trans-cleavage of 8-17 deoxyribose nucleic acid and CRISPR-Cas13 a;
FIG. 2 is a graph showing the T fluorescence spectrum of target genes at different concentrations in example 1 of the present invention;
FIG. 3 is a calibration curve showing T-response of target genes at different concentrations in example 1 of the present invention;
FIG. 4 is a graph showing the T fluorescence spectrum of the target gene after the key factors are removed from the complete system and the system in experimental example 1 of the present invention;
FIG. 5 shows the electrophoresis analysis of the target gene T detection method in experimental example 1 of the present invention, wherein a is AB and T which can react smoothly and release A sequence, and enzyme can amplify upstream smoothly; b is 8-17 deoxyribozyme in A under the condition of metal ion can smoothly cut HX-gRNA, and can not cut under the condition of no metal ion; c is downstream, so that the G-quadruplex dimer structure in the a sequence is exposed, thereby realizing the detection of the target;
FIG. 6 shows saturation curves of DNA detection signals of target gene T in the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated; the reagents used in the examples were all commercially available unless otherwise specified.
The percentage "%" referred to in the present invention refers to mass percent unless otherwise specified; however, the percentage of the solution, unless otherwise specified, refers to the grams of solute contained in the 100 mL solution.
The parts by weight of the present invention may be those known in the art such as mu g, mg, g, kg, or may be multiples thereof such as 1/10, 1/100, 10 times, 100 times, etc.
The probes according to the present invention were purchased from Shanghai Biotechnology Co., ltd, and the probe sequences used in the following examples and experimental examples are shown in Table 1:
wherein, the underlined sequence in the A probe sequence is 8-17 deoxyribozyme functional sequence;
the sequence with the underlined HX probe sequence is RNA, and the sequence without the underlined DNA; fragments near the 3' end are sequence H (CCCCTTCGTTAATGCAGATC), with the italicized AG being the RNA cleavage site;
the bolded and underlined sequences in the H1, H2 probe sequences form a "T" complex;
the bolded and underlined sequence in the a probe sequence is the G-quadruplex dimer sequence, italicized is the stand for strand displacement.
Example 1
The method for detecting the target gene T based on the probe group for detecting the mycobacterium bovis by 8-17 deoxyribozyme and CRISPR-Cas13a trans-cleavage comprises the following specific steps:
(1) Probe pretreatment
(1) Target T: the T probe was prepared with dimethylnitrosoacetamide water (DEPC water) as a 100 μm stock solution from the lyophilized powder. Buffer solution (pH 7.5, 150 mM NaCl, 100 uM PbCl) with Tris-HCl 2 ) The mother liquor was diluted to different concentrations (1 fM, 50fM, 500fM, 1pM, 10pM, 330 pM).
(2) Probes A-B A A, B probe was used to prepare a 100 μm stock solution from lyophilized powder with DEPC water. Buffer solution (pH 7.5, 150 mM NaCl, 100 uM PbCl) with Tris-HCl 2 ) The mother liquor was diluted to 20 uM. Then, A, B probes 15 and uL are mixed, incubated for 5min at 95 ℃ and then slowly cooled to room temperature to form an A-B structure.
(3) Probe HX-gRNA the gRNA, HX probe were prepared with DEPC water as a 20 μm stock solution from lyophilized powder. With Tris-HCl buffer (pH 7.5, 150 mM NaCl, 100 uM PbCl) 2 ) The mother liquor was diluted to 10 uM. After that, 6 uL of HX probe and 5 uL of gRNA were mixed and incubated at 95℃for 5min, and then cooled slowly to room temperature to form HX-gRNA structure.
(4) Probe g: the g probe was used to prepare a 20 μm stock solution of lyophilized powder with DEPC water. Buffer solution (pH 7.5, 150 mM NaCl, 100 uM PbCl) with Tris-HCl 2 ) The mother liquor was diluted to 5 uM.
(5) Probes H1 and H2 the H1 and H2 probes were prepared with DEPC water to prepare a 100 mu M stock solution of lyophilized powder. Buffer solution (pH 7.5, 150 mM NaCl, 100 uM PbCl) with Tris-HCl 2 ) The mother liquor was diluted to 5 uM.
(6) Probes a-b the freeze-dried powder of the probes a and b is prepared into mother liquor of 100 mu M by DEPC water. With Tris-HCl buffer (pH 7.5, 150 mM NaCl, 100 uM PbCl) 2 ) The mother liquor was diluted to 10 uM. Then mixing the a probe 30 uL and the b probe 33 uL, incubating at 95 ℃ for 5min, and slowly cooling to room temperature to form an a-b structure.
(2) Strand displacement release deoxyribose nucleic acid sequence
Taking targets T, 2 mu L and A-B of 10 mu M with different concentrations at a centrifuge tube of 200 mu L, and uniformly mixing, and reacting for 1 hour.
(3) Cleavage of specific probes by 8-17 deoxyribozymes and LwaCas13a
After the reaction was completed, HX-gRNA of 2 uL and 10uM was added to react for 1 hour, then 1uL of 5 uM of Cas13a and 1uL of 5 uM of g probe were added to mix, and an enzyme reaction buffer solution was added to make up the volume to 20 uL, and the reaction was carried out at 37℃for 3 hours.
(4) Forming a G-quadruplex dimer structure
To the above solution, 3.3 uL, 5 uM of H1, H2,3 uL, 5 uM a-b,3 uL, 50 uM THT were added, and the total volume was made up to 100 uL with a buffer solution, and reacted at room temperature for 1.5 hours under dark conditions.
(5) Fluorescence detection
Adding 20 uL buffer solution into the solution for dilution, then sucking a 120 uL sample in an EP tube into a cuvette, and setting the parameters of a fluorescence spectrometer as follows: excitation light spectrum broadband 15 nm, emission light spectrum broadband 15 nm, excitation light wavelength 420 nm. And (5) measuring parameters and then storing data.
FIG. 2 is a graph of T fluorescence spectra of target genes with different concentrations, and the result shows that the fluorescence intensity is increased along with the increase of the concentration of the target genes, the fluorescence intensity is positively correlated with the target genes, and the fluorescence of the target genes T with the concentration of 1 fmol/L to 330 pmol/L is strongest at 490nm, so that the concentration range of the target genes T suitable for detection is wide;
FIG. 3 shows T-response calibration curves of target genes with different concentrations, wherein when the concentration of the target, namely, the concentration of Mycobacterium bovis is in the range of 1 fmol/L-500 fmol/L, the fluorescence intensity is increased along with the increase of the target concentration, a better linear relationship is formed, the regression equation is y= 53.736 ×logC+276.871 (y is fluorescence intensity, C is target concentration), R 2 The detection limit is 0.5 fmol/L (S/n=3) = 0.9813, and the result proves that the research method can realize high-sensitivity detection of mycobacterium tuberculosis bovis.
Experimental example 1
Feasibility analysis of the Probe set detection System
1. In order to test the feasibility of the whole system, key factors are removed in the experimental process: an A probe with a deoxyribozyme sequence, an HX-gRNA probe which starts downstream and realizes signal amplification, and an H1 probe which is used for combining with H2 and promoting the formation of a G-quadruplex dimer structure.
As shown in FIG. 4, when the key factors are removed from the whole system, the signal value is greatly different from that of the whole system, the detection of the target cannot be realized, and the capability of the whole system for detecting the mycobacterium tuberculosis bovis target is verified.
2. In addition, the upstream and downstream systems were also characterized by agarose gel electrophoresis, as shown in FIG. 5, wherein in FIG. 5 (a), 1 is marker,2 is A, 3 is B, 4 is T, 5 is AB, 6 is AB+T mixture, 7 is AB+T+HX-gRNA mixture, 8 is AB+T+HX-gRNA-g-enzyme mixture, and the result shows that AB and T can smoothly react and release A sequence, and enzyme can smoothly play a role in amplifying signal on upstream. In FIG. 5 (b), 1 is a marker,2 is a mixture of AB+T+HX-gRNA in the absence of metal ions, 3 is a mixture of AB+T+HX-gRNA+g+ enzyme in the absence of metal ions, and 4 is Pb 2+ AB+T+HX-gRNA mixture at 100 mu M concentration, 5 Pb 2+ As can be seen from FIG. 5 (b), the 8-17 deoxyribose enzyme in A can cleave the HX-gRNA smoothly under the condition of metal ion, but can not cleave under the condition of no metal ion (8-17 deoxyribose enzyme has to exert cleavage effect in the presence of metal ion), thus proving that in the systemIn the complete case (Pb-containing) 2+ ) A does achieve its cutting action. The detection of the target was demonstrated by exposing the G-quadruplex dimer structure in the a sequence downstream, shown in FIG. 5 (c), where 1 is marker,2 is HX, 3 is H1, 4 is H2, 5 is a-b,6 is H1+H2+a-b,7 is HX+H1+H2+a-b (the entire downstream system), and 8 is AB+T+HX-gRNA+g+enzyme mixture+H1+H2+a-b (the entire system).
Experimental example 2
DNA detection signal saturation curve for investigating target gene T
Detecting fluorescence intensities of target genes T with different concentrations by using ultraviolet spectrophotometer
FIG. 6 shows the saturation curve of DNA detection signal of target gene T in the present invention, and the result shows that when the DNA of Mycobacterium bovis is lower than 2 pM, the fluorescence intensity increases with the increase of the DNA concentration of Mycobacterium bovis, the saturation is carried out after 2 pM, the highest absorption peak is reached, and then the signal value is stabilized as a whole.
Experimental example 3
Detection of mycobacterium bovis in blood
In order to verify the detection performance of the whole system on the actual sample of the mycobacterium bovis, the study is carried out for detecting the concentration of the actual sample of the mycobacterium bovis with the concentration in a linear range. Will contain 10 8 The inactivated mycobacterium bovis solution of CFU/mL is added into bovine blood matrix, DNA is extracted by adopting a nucleic acid extraction method, and finally, the actual samples of 150 fmol/L, 155 fmol/L and 166 fmol/L are detected (other conditions are consistent except for different target concentrations).
The results are shown in Table 2, the recovery rate of the actual sample is 94.4% -109.0%, and the relative standard deviation is 1.9% -4.1%, which shows that the actual sample of the mycobacterium bovis can be detected in the study:
Claims (8)
1. 8-based-17 deoxyribozymes trans-cut with CRISPR-Cas13a to detect mycobacterium bovis, characterized by comprising probe a, probe B, probe HX, probe gRNA, probe g, probe H1, probe H2, probe a, probe B and LwaCas13a enzymes; the probe A contains 8-17 deoxyribozymes, and the gRNA is combined with LwaCas13a and g sequences to form a CRISPR-Cas13a system; the probe a contains a G-quadruplex dimer sequence; the probe set is in Pb 2+ Under the condition, the detection of the bovine mycobacterium tuberculosis target gene T is realized through multiple signal amplification;
the gene sequence of the probe A is shown as SEQ ID NO.1, the gene sequence of the probe B is shown as SEQ ID NO.2, the gene sequence of the probe HX is shown as SEQ ID NO.3, the gene sequence of the probe gRNA is shown as SEQ ID NO.4, the gene sequence of the probe g is shown as SEQ ID NO.5, the gene sequence of the probe H1 is shown as SEQ ID NO.6, the gene sequence of the probe H2 is shown as SEQ ID NO.7, the gene sequence of the probe a is shown as SEQ ID NO.8, the gene sequence of the probe B is shown as SEQ ID NO.9, the gene sequence of the target gene T is shown as SEQ ID NO.10, and the functional sequence of the 8-17 deoxyribonuclease is shown as SEQ ID NO. 11.
2. A method for detecting Mycobacterium tuberculosis in cattle of non-diagnostic purpose, which is characterized by applying the probe set as described in claim 1 and comprising the following steps:
s1, pretreatment of a probe sequence: preparing freeze-dried powder of the probe A, the probe B, the probe HX, the probe gRNA, the probe g, the probe H1, the probe H2, the probe a and the probe B into mother solutions, and respectively diluting the mother solutions by using buffer solutions; mixing diluted probe A, B mother liquor and incubating to form probes A-B; mixing diluted probe gRNA and HX mother solution, and incubating to form probe HX-gRNA; mixing diluted mother solutions of the probes a and b, and incubating to form probes a-b;
s2, strand displacement releasing deoxyribozyme sequence: uniformly mixing a sample to be tested of mycobacterium bovis with the probes A-B for reaction;
cleavage of specific probes by S3, 8-17 deoxyribozymes and LwaCas13 a: after the S2 step reaction is finished, adding a probe HX-gRNA for reaction, then adding LwaCas13a and g probes for mixing, and adding an enzyme reaction buffer solution for reaction;
s4, forming a G-quadruplex dimer structure: after the S3 reaction is finished, adding H1, H2, a-b probes and THT, adding a buffer solution, and carrying out a reaction in a dark place;
s5, fluorescence detection: and (3) after the S4 reaction is finished, diluting the solution, sucking the sample, loading the sample into a cuvette for machine detection, and calculating the concentration of the sample to be detected according to a target gene T response calibration curve.
3. The method according to claim 2, wherein the concentration ratio of probe a, probe B, probe HX, probe gRNA, probe g, probe H1, probe H2, probe a, probe B mother liquor after dilution in step S1 is 4:4:2:2:1:1:1:2:2:2; the buffer solution has a ph=7.5 and contains Pb 2+ Tris hydrochloride buffer solution.
4. The method according to claim 2, wherein the molar ratio of probe a to probe B in probes a-B is 1:1; the molar ratio of the probe HX to the probe gRNA in the probe HX-gRNA is 6:5; the molar ratio of the probe a to the probe b in the probes a-b is 1:1.1.
5. The method according to claim 2, wherein the volume ratio of the sample to be tested of mycobacterium bovis to the probe a-B in the step S2 is 2:1, and the reaction time is 1h.
6. The method according to claim 2, wherein the molar ratio of HX-gRNA probe, lwaCas13a and g probe in step S3 is 4:1:1; the reaction temperature was 37℃and the reaction time was 3 hours.
7. The method according to claim 2, wherein the molar ratio of H1, H2, a-b probe and THT in step S4 is 11:11:10:100; the reaction temperature was room temperature and the reaction time was 1.5 hours.
8. The method according to claim 2, wherein the detection parameters in step S5 are: excitation light spectral bandwidth 15 nm, emission light spectral bandwidth 15 nm, excitation light wavelength 420 nm.
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