CN116732211A - Probe set and method for detecting bovine Mycobacterium tuberculosis based on 8-17 DNAzyme and CRISPR-Cas13a trans-cleavage - Google Patents

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

Detection of bovine tuberculosis based on trans-cleavage of 8-17 DNAzyme and CRISPR-Cas13a Mycobacterium probe sets and methods

Technical field

The invention belongs to the technical field of biological analysis and detection, and specifically relates to a probe set and method for detecting bovine Mycobacterium tuberculosis based on 8-17 deoxyribozyme and CRISPR-Cas13a trans-cleavage.

Background technique

Mycobacterium tuberculosis is divided into three main types: Mycobacterium bovis (bovine type), Mycobacterium tuberculosis (human type) and Mycobacterium avium (avian type). Mycobacterium bovis is a subtype of Mycobacterium tuberculosis. Among all Mycobacterium tuberculosis complexes, Mycobacterium bovis has the widest host range and is characterized by long incubation period and disease recurrence. Outbreaks of zoonotic diseases caused by mycobacteria not only cause economic losses to the livestock industry, but also seriously threaten human health. Therefore, establishing a sensitive and rapid method for detecting bovine Mycobacterium tuberculosis is of great significance for disease diagnosis. At present, the main methods for detecting bovine Mycobacterium tuberculosis include: 1. Molecular detection methods, 2. Immunological methods, 3. Microbial culture methods, etc. Although immunological methods and microbial culture methods can detect bovine Mycobacterium tuberculosis, they have shortcomings such as low sensitivity and low specificity. Microbial culture methods also have problems such as long time and heavy detection workload. Therefore, currently The commonly used methods are mainly molecular detection methods, but the most commonly used traditional PCR technology requires expensive temperature control equipment and is prone to problems such as false positives and false negatives.

DNAzyme is a single-stranded DNA fragment with catalytic function obtained using in vitro molecular evolution technology. It has efficient catalytic activity and structure recognition ability. It has been proven to catalyze many enzymes related to RNAse (also known as ribozyme) or protease. The same reactions include RNA/DNA cleavage, ligation, phosphorylation, cleavage of phosphate amide bonds, and porphyrin metallation. Among them, the 8-17 deoxyribozyme consists of a stem-loop structure and an unpaired 4-5nt region. The loop is generally a conserved sequence 5'-AGC-3', and the stem is generally composed of 3 base pairs. This structure can Specific cleavage of 5'-rAG-3'. Yang et al. proposed a new concept of "nanoflare pair", which modified probes on two gold nanoparticles. When TK1 mRNA and survivin mRNA, which are highly expressed in tumor tissues, are present in cells, the two gold nanoparticles can be modified through chain replacement. The probes on the nanoparticles are close to each other to form 8-17 DNAzymes, which can cleave the probes with modified fluorescence quenching groups. This system has developed a multifunctional nanotherapy platform "Nanoflare Pair (NC)". It can be used for in situ composite cancer-related mRNA imaging and subsequent logical control of aggregation of gold nanoparticles to achieve gene therapy and photothermal therapy under infrared light irradiation. The CRISPR gene editing system, which won the Nobel Prize in Chemistry in recent years, is one of the hot spots of research. The detection technology based on the CRISPR-Cas system shows the advantages of high sensitivity, accuracy, no need for thermal cycle equipment, low price, mobility and rapidity. Suitable for developing universal detection technology for grassroots institutions. Among them, CRISPR-Cas13a is mainly used in the field of molecular diagnostic technology due to its sensitivity, specificity and nucleic acid recognition. As the core of some powerful diagnostic technologies, they are completely changing the way of virus detection and opening up a path for biosensing. new roads. For example, Zhang et al. used SHERLOCK to achieve high-sensitivity detection of RNA. They first used RPA to amplify a large number of DNA sequences, and then transcribed them in vitro using T7 to form RNA. Cas13a can recognize the RNA sequence and detect the RNA with modified fluorescent groups. Clusters of RNA probes are cleaved to achieve highly sensitive detection of targets.

A guanine (G)-quadruplex is a highly ordered four-stranded 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. Utilizing the "luminescence" property, G-quadruplex/THT can be used as a molecular beacon to construct a label-free fluorescent biosensing strategy for detecting DNA. Studies have found that the G-quadruplex dimer formed by two series-connected G-quadruplex monomers is a stable DNA structure that can significantly enhance the fluorescence intensity of THT dye. G-quadruplex dimer/THT The fluorescence intensity is approximately nine times that of G-quadruplex monomer/THT and is not affected in high-salt media. Therefore, G-quadruplex dimer/THT, as a new type of fluorescent reporter, has great application potential in more biosensors.

Contents of the invention

In order to solve the above technical problems and overcome the high cost, easy false positives and false negatives in the existing technology for detecting bovine Mycobacterium tuberculosis, the present invention provides a trans-cleavage method based on 8-17 DNAzyme and CRISPR-Cas13a The probe set and method for detecting bovine Mycobacterium tuberculosis uses 8-17 deoxyribozyme to repeatedly cut specific sequences, and uses CRISPR-Cas13a's trans-cutting ability of RNA sequences and the continuous recycling of downstream priming sequences. The signal is amplified, thereby improving the sensitivity of the entire system, and the G-quadruplex dimer formed downstream is combined with the THT dye to be output as a fluorescent signal, making the entire detection process simpler. The system has a constant temperature and can achieve "one Tube type operation, high sensitivity and other advantages.

In order to achieve the above purpose, the present invention first provides a probe set for detecting bovine Mycobacterium tuberculosis based on trans-cleavage of 8-17 DNAzyme and CRISPR-Cas13a, including probe A, probe B, probe HX, Probe gRNA, probe g, probe H1, probe H2, probe a, probe b and LwaCas13a enzyme; the probe A contains 8-17 deoxyribozyme, and the gRNA is combined with the LwaCas13a and g sequences to form CRISPR-Cas13a system; the probe set realizes the detection of the target gene T of Mycobacterium bovis through multiple signal amplification under Pb ²⁺ conditions;

Wherein, the gene sequence of probe A is shown in SEQ ID NO.1, the gene sequence of probe B is shown in SEQ ID NO.2, the gene sequence of probe HX is shown in SEQ ID NO.3, and the gene sequence of probe B is shown in SEQ ID NO.3. The gene sequence of gRNA is shown in SEQ ID NO.4, the gene sequence of probe g is shown in SEQ ID NO.5, the gene sequence of probe H1 is shown in SEQ ID NO.6, and the gene sequence of probe H2 is shown in SEQ ID NO.7 is shown, the gene sequence of probe a is shown in SEQ ID NO.8, the gene sequence of probe b is shown in SEQ ID NO.9, and the gene sequence of target gene T is shown in SEQ ID NO.10 As shown, the functional sequence of 8-17 DNAzyme is shown in SEQ ID NO. 11.

Based on a general inventive concept, the present invention also provides a method for detecting bovine Mycobacterium tuberculosis for non-diagnostic and therapeutic purposes, which includes the following steps:

S1. Probe sequence preprocessing: Configure probe A, probe B, probe HX, probe gRNA, probe g, probe H1, probe H2, probe a, and probe b freeze-dried powder into Mother solution, and then dilute the mother solution with buffer solution respectively; mix the diluted probe A and B mother solutions and incubate them to form probe A-B; mix the diluted probe gRNA and HX mother solutions and incubate them to form probe HX-gRNA; The diluted mother solutions of probes a and b are mixed and incubated to form probes a-b;

S2. Strand displacement releases DNAzyme sequence: mix the sample of Mycobacterium bovis to be tested and probes A-B for reaction;

Cleavage of specific probes by S3, 8-17 deoxyribozyme and LwaCas13a: After the reaction in step S2, add probe HX-gRNA for reaction, then add LwaCas13a and g probes to mix, and add enzyme reaction buffer solution for reaction. ;

S4. Form a G-quadruplex dimer structure: After the S3 reaction, add H1, H2, a-b probe, and THT, add the buffer solution, and then proceed in the dark;

S5. Fluorescence detection: After the S4 reaction is completed, dilute the solution and draw the sample into a cuvette, perform detection on the machine, and calculate the concentration of the sample to be tested based on the target gene T response calibration curve.

Preferably, in the step S1, the concentration ratio of probe A, probe B, probe HX, probe gRNA, probe g, probe H1, probe H2, probe a, and probe b mother liquor after dilution is: 4:4:2:2:1:1:1:2:2; the buffer solution is a tris-hydroxymethylaminomethane hydrochloride (Tris-HCl) buffer solution with pH=7.5 and containing Pb ²⁺ .

Preferably, the molar ratio of probe A to probe B in the probe A-B is 1:1; the molar ratio of probe HX to probe gRNA in the probe HX-gRNA is 6:5; The molar ratio of probe a to probe b in needle a-b is 1:1.1.

Preferably, in step S2, the volume ratio of the bovine Mycobacterium tuberculosis sample to be tested and probes A-B is 2:1, and the reaction time is 1 hour.

Preferably, the molar ratio of HX-gRNA probe, LwaCas13a and g probe in step S3 is 4:1:1; the reaction temperature is 37°C, and the reaction time is 3 hours.

Preferably, the molar ratio of H1, H2, a-b probe and THT in step S4 is 11:11:10:100; the reaction temperature is room temperature, and the reaction time is 1.5 hours.

Preferably, the detection parameters in step S5 are: the spectral bandwidth of the excitation light is 15 nm, the spectral bandwidth of the emission light is 15 nm, and the wavelength of the excitation light is 420 nm.

The detection principle and process of the probe set provided by the present invention for detecting bovine Mycobacterium tuberculosis based on trans-cleavage of 8-1 7 deoxyribozyme and CRISPR-Cas13a are specifically as follows:

The experimental principle is shown in Figure 1. The present invention integrates the cyclic cleavage performance of 8-17 DNAzyme, the trans cyclic cleavage performance of CRISPR-Cas13a, and the sequence recycling mediated by toehold strand replacement to construct a triple recycling signal. Amplification system for high-performance “one-tube” detection of bovine Mycobacterium tuberculosis:

The first signal cycle amplification: through the target T, which is Mycobacterium bovis DNA, and A-B (the A sequence contains the 8-17 DNAzyme functional sequence 5'-TTCTTTTGCTCCGA GCCGGTCGAACTATC-3', the B sequence plays a blocking role in A) Strand displacement releases the A sequence, and further converts the specific probe HX-gRNA (a hybrid dimer of HX and gRNA, which contains two non-complementary single-stranded RNA bulge structures) into the bulge structure (containing 8-17 deoxygenases). Ribozyme recognition site: 5'-GAUAGUUCG-3', 5'-GCAAAAGAA-3', cleavage site: r-AG) cleaves, releasing the DNA sequence H in the HX chain (partial sequence of HX: 5' -CCCCTTCGTTAATGCAGATC-3'), gRNA sequence and A sequence, in which sequence A can be recycled to cleave HX-gRNA, release the H chain, and construct cycle 1;

The second signal cycle amplification: gRNA can combine with LwaCas13a and g sequences to form the LwaCas13a complex with RNA trans-cleaving activity, which can cleave any RNA in the system, thereby cutting the HX-gRNA bulge structure again and releasing Exit the H chain and build loop 2;

The third level of signal cycle amplification: the released H chain can enter the downstream system and repeatedly interact with H1 and H2 to form a "T"-shaped complex to build cycle 3. The free part of the "T" type complex (H1 liberates 5'-AGGGGAAT-3', H2 liberates 5'-TCTTGGAGCGCTACG-3') can be combined with the a-b complex (consisting of a G-quadruplex dimer sequence: 5 The a sequence of '-GGGTTGGGCGGGATGGGGGGTTGGGCGGGATGGG-3' is hybridized with the b sequence that protects the sequence) and strand displacement is performed (the starting point is: 5'-ATTCCCCTCGTAGCGCTCCAAGA-3'), exposing the G-quadruplex in the a sequence. dimer sequence. Thioflavin T (THT), as a water-soluble fluorescent dye, consists of a benzylamine ring and a phenyl sulfide ring. These two rings can rotate freely around the C-C bond, resulting in low fluorescence of THT, but when this rotation is affected by When restricted by certain special structures, the fluorescence of THT will be significantly enhanced, and the G-quadruplex dimer has a strong affinity with the THT dye and can efficiently enhance the fluorescence emission of THT. Therefore, adding THT dye to the entire system can combine with the G-quadruplex dimer sequence in the a sequence to emit fluorescence as a signal output method.

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

(1) This system uses 8-17 deoxyribozyme to repeatedly cut specific sequences, and uses CRISPR-Cas13a's trans-cutting ability of RNA sequences and the continuous recycling of downstream priming sequences to amplify signals, thereby improving the efficiency of the entire system. Sensitivity, and the G-quadruplex dimer formed downstream is combined with the THT dye as a fluorescent signal output, making the entire detection process simpler. The system has the advantages of constant temperature, "one-tube" operation, and high sensitivity. .

(2) The detection system of the present invention has the characteristics of constant temperature and simple operation. In addition, this system constructs a triple cycle signal amplification system: Cycle 1: The A sequence containing 8-17 DNAzyme versus the convex RNA sequence of HX-gRNA Recognition and cyclic cleavage; Cycle 2: The gRNA released after the initial cleavage of HX-gRNA can combine with g and LwaCas13a to form a CRISPR-Cas13a system to achieve cyclic cleavage of HX-gRNA; Cycle 3: After HX-gRNA is cleaved The released H sequence can be recycled to continuously form "T"-shaped complexes. Each cycle has the advantage of high sensitivity and can be used for in vitro high-sensitivity "one-tube" detection of bovine Mycobacterium tuberculosis to achieve accurate detection of bovine Mycobacterium tuberculosis. Quantitative analysis.

(3) The sequence design of the detection system of the present invention is ingenious, making full use of the 8-17 DNAzyme functional sequence to repeatedly cut the specific probe, and of the two sequences released after being cut, one is combined with LwaCas13a to form CRISPR- The Cas13a system cuts the specific probe again, and the other one enters the downstream system for continuous recycling to achieve high-sensitivity detection of triple signal amplification, which makes up for the shortcomings of low detection sensitivity and complicated operation of the conventional system, achieves multiple signal amplification effects, and improves detection performance.

(4) The reaction conditions do not require the use of expensive variable temperature instruments. The reaction conditions are mild and the reaction is carried out at a constant temperature of 37°C. The operation is simple and can achieve "one-tube" operation, which effectively solves the current on-site detection of bovine Mycobacterium tuberculosis at home and abroad. limitations.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the drawings of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1 is a schematic diagram of the probe set detection of bovine Mycobacterium tuberculosis based on the trans-cleavage of 8-1 7 deoxyribozyme and CRISPR-Cas13a according to the present invention;

Figure 2 is the fluorescence spectrum curve of target gene T at different concentrations in Example 1 of the present invention;

Figure 3 is a calibration curve of target gene T response at different concentrations in Example 1 of the present invention;

Figure 4 shows the complete system in Experimental Example 1 of the present invention and the target gene T fluorescence spectrum curve after the key factors are removed from the system;

Figure 5 is an electrophoretic analysis of the target gene T detection method in Experimental Example 1 of the present invention, where a means that AB and T can react smoothly and release the A sequence, and the enzyme can also smoothly amplify the signal upstream; b means metal ions Under the conditions, the 8-17 deoxyribozyme in A can cleave HX-gRNA smoothly, but it cannot cleave it in the absence of metal ions; c is downstream, exposing the G-quadruplex dimer structure in the a sequence. , thereby achieving target detection;

Figure 6 is a saturation curve of the DNA detection signal of the target gene T in the present invention.

Detailed ways

The following examples are used to illustrate the invention but are not intended to limit the scope of the invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the method, steps or conditions of the present invention shall fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are all commercially available.

The percentage sign "%" involved in the present invention, unless otherwise specified, refers to the mass percentage; but the percentage of the solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

The weight parts mentioned in the present invention may be weight units known in the art such as μg, mg, g, kg, etc., or may be multiples thereof, such as 1/10, 1/100, 10 times, 100 times, etc.

The probes involved in the present invention were all purchased from Shanghai Sangon Bioengineering Co., Ltd. The probe sequences used in the following examples and experimental examples are as shown in Table 1:

Among them, the underlined sequence in the A probe sequence is the 8-17 DNAzyme functional sequence;

The underlined sequence in the HX probe sequence is RNA, and the ununderlined sequence is DNA; the fragment near the 3' end is sequence H (CCCCTTCGTTAATGCAGATC), and the AG in italics and bold is the RNA cleavage site;

The bolded and underlined sequences in the H1 and H2 probe sequences form a “T”-shaped complex;

The bold and underlined sequence in a probe sequence is the G-quadruplex dimer sequence, and the italicized one is the foothold of strand replacement.

Example 1

A probe set and method for detecting bovine Mycobacterium tuberculosis based on 8-1 7 deoxyribozyme and CRISPR-Cas13a trans-cleavage to detect the target gene T. The specific steps are as follows:

(1)Probe pretreatment

① Target T: Use dimethylnitrosacetamide water (DEPC water) to prepare the T probe lyophilized powder into a 100 µM stock solution. The stock solution was diluted to different concentrations (1fM, 50fM, 500fM, 1pM, 10pM, 330pM) with Tris-HCl buffer solution (pH 7.5, 150 mM NaCl, 100 uM PbCl ₂ ).

② Probe AB: Use DEPC water to prepare the lyophilized powder of probes A and B into a 100 µM stock solution. Dilute the stock solution to 20 uM with Tris-HCl buffer solution (pH 7.5, 150 mM NaCl, 100 uM PbCl ₂ ). Then mix 15 uL of each A and B probes, incubate at 95°C for 5 minutes and slowly lower to room temperature to form the AB structure.

③ Probe HX-gRNA: Use DEPC water to prepare gRNA, HX probe and lyophilized powder into a 20 µM stock solution. Dilute the stock solution to 10 uM with Tris-Hcl buffer solution (pH 7.5, 150 mM NaCl, 100 uM PbCl ₂ ). Then mix 6 uL HX probe and 5 uL gRNA, incubate at 95°C for 5 minutes and slowly lower to room temperature to form the HX-gRNA structure.

④ Probe g: Use DEPC water to prepare the lyophilized powder of g probe into a 20 µM stock solution. Dilute the stock solution to 5 uM with Tris-HCl buffer solution (pH 7.5, 150 mM NaCl, 100 uM PbCl ₂ ).

⑤ Probes H1 and H2: Use DEPC water to prepare the freeze-dried powder of H1 and H2 probes into a 100 µM stock solution. Dilute the stock solution to 5 uM with Tris-HCl buffer solution (pH 7.5, 150 mM NaCl, 100 uM PbCl ₂ ).

⑥ Probe ab: Use DEPC water to prepare the lyophilized powder of probes a and b into a 100 µM mother solution. Dilute the stock solution to 10 uM with Tris-Hcl buffer solution (pH 7.5, 150 mM NaCl, 100 uM PbCl ₂ ). Then mix 30 uL of a probe and 33 uL of b probe, incubate at 95°C for 5 minutes and slowly lower to room temperature to form an ab structure.

(2) Strand displacement releases DNAzyme sequence

Take 4 µL of different concentrations of target T, 2 µL, and 10uM of A-B, mix them in a 200 µL centrifuge tube, and react for 1 hour.

(3) Cleavage of specific probes by 8-17 DNAzyme and LwaCas13a

After the above reaction, add 2 uL, 10 uM HX-gRNA and react for 1 hour, then add 1uL, 5 uM Cas13a and 1uL, 5 uM g probe to mix, and add enzyme reaction buffer solution to make up the volume to 20 uL. React at 37°C for 3 hours.

(4) Formation of G-quadruplex dimer structure

Add 3.3 uL, 5 uM H1, H2, 3 uL, 5 uM a-b, 3 uL, 50 uM THT to the above solution, make up the total volume with buffer solution to 100 uL, and react at room temperature for 1.5 hours in the dark.

(5) Fluorescence detection

Add 20 uL buffer solution to the above solution for dilution, and then absorb 120 uL sample from the EP tube into a cuvette. Set the fluorescence spectrometer parameters as follows: excitation light spectrum broadband 15 nm, emission light spectrum broadband 15 nm, and excitation light wavelength 420 nm. . Save the data after measuring the parameters.

Figure 2 shows the fluorescence spectrum curves of the target gene T at different concentrations. The results show that as the concentration of the target gene increases, the measured fluorescence intensity increases. The fluorescence intensity is positively correlated with the target gene, and the target range is from 1 fmol/L to 330 pmol/L. Gene T has the strongest fluorescence at 490nm, and is applicable to a wide range of target concentrations for detection;

Figure 3 shows the T response calibration curve of the target gene at different concentrations. When the target concentration, that is, the concentration of Mycobacterium bovis, is in the range of 1 fmol/L-500 fmol/L, the fluorescence intensity increases as the target concentration increases. , showing a good linear relationship, the regression equation is y=53.736×logC+276.871 (y is the fluorescence intensity, C is the target concentration), R ² =0.9813, the detection limit is 0.5 fmol/L (S/N=3) , This result proves that this research method can achieve high sensitivity detection of bovine Mycobacterium tuberculosis.

Experimental example 1

Feasibility analysis of the probe set detection system

1. In order to test the feasibility of the entire system, key factors were removed during the experiment: the A probe with DNAzyme sequence, the HX-gRNA probe that initiates downstream and achieves signal amplification, and the HX-gRNA probe used to combine with H2 to promote G -H1 probe formed by a quadruplex dimer structure.

The results are shown in Figure 4. When the key factors are removed from the entire system, the signal value is greatly different from that of the complete system, and the target cannot be detected. This verifies that the entire system has the ability to detect the bovine Mycobacterium tuberculosis target.

2. In addition, the upstream and downstream systems were characterized through agarose gel electrophoresis, as shown in Figure 5, where 1 in Figure 5(a) is marker, 2 is A, 3 is B, 4 is T, and 5 is AB. , 6 is the AB+T mixture, 7 is the AB+T+HX-gRNA mixture, and 8 is the AB+T+HX-gRNA-g-enzyme mixture. The result diagram shows that AB and T can react smoothly and release the A sequence. Enzymes can also smoothly amplify signals upstream. In Figure 5(b), 1 is the marker, 2 is the AB+T+HX-gRNA mixture under metal ion-free conditions, 3 is the AB+T+HX-gRNA+g+enzyme mixture under metal ion-free conditions, and 4 is Pb ²⁺ AB+T+HX-gRNA mixture at a concentration of 100 µM, 5 is Pb ²⁺ AB+T+HX-gRNA+g+ enzyme mixture at a concentration of 100 µM, as can be seen from Figure 5(b), under metal ion conditions The 8-17 DNAzyme in A below can successfully cleave HX-gRNA, but cannot cleave it in the absence of metal ions (the 8-17 DNAzyme must be able to cleave in the presence of metal ions), so it can It is proved that when the system is complete (containing Pb ²⁺ ), A does achieve its cutting effect. As shown in Figure 5(c), 1 is marker, 2 is HX, 3 is H1, 4 is H2, 5 is ab, 6 is H1+H2+ab, and 7 is HX+H1+H2+ab (the entire downstream system) , 8 is AB+T+HX-gRNA+g+enzyme mixture+H1+H2+ab (the whole system), which proves that the G-quadruplex dimer structure in the a sequence can be exposed through the downstream, thereby achieving the target detection.

Experimental example 2

Examine the DNA detection signal saturation curve of the target gene T

Use UV spectrophotometer to detect the fluorescence intensity of target gene T at different concentrations

Figure 6 is a saturation curve of the DNA detection signal of the target gene T in the present invention. The results show that when the Mycobacterium bovis DNA is lower than 2 pM, its fluorescence intensity increases with the increase of the concentration of Mycobacterium tuberculosis DNA. It tends to saturate after 2 pM and reaches the highest absorption peak, and then the signal value generally tends to be stable.

Experimental example 3

Detection of bovine Mycobacterium tuberculosis in blood

In order to verify the detection performance of the entire system for actual samples of Mycobacterium bovis, this study tested the concentration of actual samples of Mycobacterium bovis within the linear range. Inactivated bovine Mycobacterium tuberculosis liquid containing 10 ⁸ CFU/mL was added to the bovine blood matrix, and DNA was extracted using the nucleic acid extraction method. Finally, the actual samples of 150 fmol/L, 155 fmol/L, and 166 fmol/L were Perform the assay (all conditions remain the same except for target concentration).

The results are shown in Table 2. The recovery rate of actual samples is between 94.4% and 109.0%, and the relative standard deviation is between 1.9% and 4.1%. This phenomenon shows that this study can detect actual samples of bovine Mycobacterium tuberculosis:

Claims (8)

1. A probe set based on trans-cutting of 8-17 deoxyribozyme and CRISPR-Cas13a for detecting bovine Mycobacterium tuberculosis, which is characterized by including probe A, probe B, probe HX, probe gRNA, Probe g, probe H1, probe H2, probe a, probe b and LwaCas13a enzyme; the probe A contains 8-17 deoxyribozyme, and the gRNA combines with the LwaCas13a and g sequences to form a CRISPR-Cas13a system ; The a sequence contains a G-quadruplex dimer sequence; the probe set realizes the detection of the Mycobacterium bovis target gene T through multiple signal amplification under Pb ²⁺ conditions; The gene sequence of probe A is shown in SEQ ID NO.1, the gene sequence of probe B is shown in SEQ ID NO.2, and the gene sequence of probe HX is shown in SEQ ID NO.3. The gene sequence of probe gRNA is shown in SEQ ID NO.4, the gene sequence of probe g is shown in SEQ ID NO.5, the gene sequence of probe H1 is shown in SEQ ID NO.6, the gene sequence of probe H2 is shown As shown in SEQ ID NO.7, the gene sequence of probe a is shown in SEQ ID NO.8, the gene sequence of probe b is shown in SEQ ID NO.9, and the gene sequence of target gene T is shown in SEQ ID NO.10 As shown, the functional sequence of 8-17 DNAzyme is shown in SEQ ID NO.11. 2. A method for detecting bovine Mycobacterium tuberculosis for non-diagnostic and therapeutic purposes, characterized in that, using the probe set according to claim 1, it includes the following steps: S1. Probe sequence preprocessing: Configure probe A, probe B, probe HX, probe gRNA, probe g, probe H1, probe H2, probe a, and probe b freeze-dried powder into Mother solution, and then dilute the mother solution with buffer solution respectively; mix the diluted probe A and B mother solutions and incubate them to form probe A-B; mix the diluted probe gRNA and HX mother solutions and incubate them to form probe HX-gRNA; The diluted mother solutions of probes a and b are mixed and incubated to form probes a-b; S2. Strand displacement releases DNAzyme sequence: mix the sample of Mycobacterium bovis to be tested and probes A-B for reaction; Cleavage of specific probes by S3, 8-17 deoxyribozyme and LwaCas13a: After the reaction in step S2, add probe HX-gRNA for reaction, then add LwaCas13a and g probes to mix, and add enzyme reaction buffer solution for reaction. ; S4. Form a G-quadruplex dimer structure: After the S3 reaction, add H1, H2, a-b probe, and THT, add the buffer solution, and then proceed in the dark; S5. Fluorescence detection: After the S4 reaction is completed, the solution is diluted and the sample is taken into a cuvette and tested on the machine. The concentration of the sample to be tested is calculated based on the target gene T response calibration curve. 3. The detection method according to claim 2, characterized in that in step S1, probe A, probe B, probe HX, probe gRNA, probe g, probe H1, probe H2, probe The concentration ratio of needle a and probe b mother liquor after dilution is 4:4:2:2:1:1:1:1:2:2; the buffer solution is pH=7.5 and contains trihydroxymethylamino of Pb ²⁺ Methane hydrochloride buffer solution. 4. The detection method according to claim 2, characterized in that the molar ratio of probe A and probe B in the probe A-B is 1:1; the molar ratio of probe HX and probe B in the probe HX-gRNA is The molar ratio of gRNA is 6:5; the molar ratio of probe a to probe b among the probes a-b is 1:1.1. 5. The detection method according to claim 2, characterized in that in the step S2, the volume ratio of the sample to be tested for Mycobacterium bovis and probe A-B is 2:1, and the reaction time is 1 hour. 6. The detection method according to claim 2, characterized in that the molar ratio of HX-gRNA probe, LwaCas13a and g probe in step S3 is 4:1:1; the reaction temperature is 37°C, and the reaction time 3 hours. 7. The detection method according to claim 2, characterized in that the molar ratio of H1, H2, a-b probe and THT in step S4 is 11:11:10:100; the reaction temperature is room temperature, and the reaction time is 1.5 Hour. 8. The detection method according to claim 2, characterized in that the detection parameters in step S5 are: the spectral bandwidth of the excitation light is 15 nm, the spectral bandwidth of the emission light is 15 nm, and the wavelength of the excitation light is 420 nm.