DNAzyme-based detection probe sets, kits, and applications

Abstract

This disclosure belongs to the field of biotechnology, specifically relating to DNAzyme-based detection probe sets, kits, and applications. The method, based on an asymmetric MIRA combined with an MNAzyme system, achieves rapid detection of Mycobacterium bovis through nucleic acid amplification and specific signal output under isothermal conditions. Based on the kits and methods provided in this disclosure, the detection process requires no complex instruments, is simple to operate, and has low cost. The detection results can be visualized through fluorescence signals, exhibiting good adaptability for field applications. This method is particularly suitable for on-site diagnosis and point-of-care testing at the grassroots level, and can be used for rapid visual detection of Mycobacterium bovis, showing promising applications in veterinary clinical testing and animal-derived food safety assurance.

Description

Technical Field

[0001] This disclosure belongs to the field of biotechnology, specifically relating to DNAzyme-based detection probe sets, kits, and applications. Background Technology

[0002] Tuberculosis is caused by the tuberculous complex (Mycobacterium tuberculosis) Mycobacterium tuberculosis Mycobacterium tuberculosis complex (MTBC) infection is a chronic, wasting zoonotic infectious disease that seriously threatens global public health security. Within the Mycobacterium tuberculosis complex, Mycobacterium tuberculosis (MTBC) is a major contributor to the disease. Mycobacterium tuberculosis ) and Mycobacterium bovis ( Mycobacterium bovis These are two relatively common zoonotic pathogens. Mycobacterium tuberculosis is the main causative agent of tuberculosis in humans, while Mycobacterium bovis is the main pathogen of tuberculosis in animals, capable of infecting livestock and wild animals, and also spreading to humans.

[0003] In different regions, wild animals can serve as reservoir hosts, spillover hosts, or potential reservoir hosts, participating in the sustained transmission and spread of the Mycobacterium tuberculosis complex. In areas where animal tuberculosis is endemic, Mycobacterium bovis can be cross-transmitted between livestock and wild animals. Animal tuberculosis not only restricts animal trade but also affects wildlife community structure and poses a potential threat to public health, while also imposing a heavy economic burden on the countries and regions involved.

[0004] Currently, traditional methods for diagnosing tuberculosis mainly include bacterial culture, immunological diagnosis, and molecular biological detection. While these methods offer good accuracy and reliability, they still suffer from drawbacks such as long testing cycles, complex procedures, high costs, demanding equipment, and reliance on specialized personnel. These limitations restrict their application in primary laboratories, on-site testing, and resource-constrained environments. Therefore, developing a simple, portable, visual, highly sensitive, and specific rapid on-site detection method for Mycobacterium bovis is of great significance. Summary of the Invention

[0005] This disclosure provides a detection probe set, kit, and application based on the combination of asymmetric multi-enzyme isothermal rapid amplification technology (aMIRA, asymmetric MIRA) and multi-component nuclease (MNAzyme) technology, which can rapidly, specifically, sensitively, and easily detect mycobacteria, and the results can be interpreted visually.

[0006] The first aspect of this disclosure is to provide a probe set.

[0007] The second aspect of this disclosure aims to provide a reagent kit.

[0008] The purpose of this third aspect of the disclosure is to provide a detection method that employs the probe set of the first aspect or the kit of the second aspect.

[0009] The purpose of this fourth aspect of the disclosure is to provide applications of the probe set described in the first aspect of the disclosure or the kit described in the second aspect of the disclosure.

[0010] To achieve the above objectives, the technical solution adopted in this disclosure is as follows: According to a first aspect of this disclosure, a DNAzyme-based detection probe set is provided, comprising: The first probe includes a first target binding arm, a first catalytic core sequence, and a first substrate binding arm; The second probe includes a second target-binding arm, a second catalytic core sequence, and a second substrate-binding arm; and Substrate, The first catalytic core sequence and the second catalytic core sequence are complementary and can pair to form an 8-17 DNAzyme catalytic domain. The first catalytic core sequence includes the nucleotide sequence CCGAGC, and the second catalytic core sequence includes the nucleotide sequence CGGTCGAA. The first target-binding arm and the second target-binding arm comprise nucleotide sequences that are complementary to the 5' and 3' ends of the target to be detected, respectively. The first substrate-binding arm and the second substrate-binding arm each include nucleotide sequences that are complementary to the 3' and 5' ends of the substrate, respectively, and the substrate includes a specific cleavage site of the 8-17 DNAzyme. The first substrate-binding arm includes the nucleotide sequence TCTCTTCT, and the second substrate-binding arm includes the nucleotide sequence ATAGTGC.

[0011] In some embodiments, the 5' end and 3' end of the substrate are respectively attached with a fluorescent group and a fluorescence quenching group.

[0012] In some embodiments, the substrate is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end; and / or The substrate is labeled with a fluorescence quencher group at its 5' end and a fluorescence reporter group at its 3' end.

[0013] In some embodiments, the fluorescent reporter group includes one or more of the following: fluorescein carboxylate (FAM), hexachloro-6-methylfluorescein (HEX), carboxy-X-rhodamine (ROX), cyanine dyes (CY), green fluorescent protein (VIC), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), tetraethylrhodamine (RB200), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), propidium iodide (PI), aminomethylcyclohexanol acetate (AMCA), succinimide ester (Atto), far-infrared fluorescent dye (Alexa Fluor), sulforhodamine 101 (Texas Red), NED, PET, ALEXA, and Quasa.

[0014] In some embodiments, the fluorescence quenching groups include minor groove binders (MGB), carboxytetramethylrhodamine (TAMRA), 4-(4-oxaneaminophenylazo)benzoic acid (DABCYL), BHQ-type compounds, ECLIPSE, and TaqMan. TM One or more of QSY.

[0015] In some embodiments, the substrate comprises a nucleotide sequence as shown in SEQ ID NO: 13, wherein the A at position 18 is an adenine ribonucleotide.

[0016] According to a second aspect of this disclosure, a kit for detecting mycobacteria is provided, comprising the probe set described in the first aspect.

[0017] In some embodiments, the probe assembly includes a first target-binding arm and a second target-binding arm. The first target-binding arm includes a nucleotide sequence as shown in SEQ ID NO: 14, and / or The second target binding arm includes a nucleotide sequence as shown in SEQ ID NO: 15.

[0018] In some embodiments, the first probe comprises a nucleotide sequence as shown in SEQ ID NO: 7, and / or The second probe comprises a nucleotide sequence as shown in SEQ ID NO: 8.

[0019] In some embodiments, the concentration ratio of the first probe to the second probe in the kit is 1:1; In some embodiments, the concentration ratio of the DNAzyme (the concentration of the first probe or the second probe) to the substrate in the kit is 1:2; In some embodiments, the concentration of the DNAzyme (the concentration of the first probe or the second probe) is 0.25-1 μmol / L.

[0020] In some embodiments, the kit also includes metal ions.

[0021] In some embodiments, the metal ions include, but are not limited to, Mg. 2+ Mn 2+ Pb 2+ and Zn 2+ In some implementations, Mg is used. 2+ .

[0022] In some embodiments, the Mg 2+ The concentration is 50-200 mmol / L. In some embodiments, it is 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, 150 mmol / L, 160 mmol / L, 170 mmol / L, 180 mmol / L, 190 mmol / L, 200 mmol / L, or any value between them.

[0023] In some embodiments, the kit also includes a biological buffer solution.

[0024] In some embodiments, the biological buffer solution is used to maintain the pH stability of a neutral to weakly alkaline solution.

[0025] In some embodiments, the biological buffer includes, but is not limited to, Tris-HCl, PBS, borate buffer, etc. In some embodiments, it is Tris-HCl.

[0026] In some embodiments, the concentration of Tris-HCl is 10-50 mmol / L. In some embodiments, it is 10 mmol / L, 12 mmol / L, 14 mmol / L, 16 mmol / L, 18 mmol / L, 20 mmol / L, 22 mmol / L, 24 mmol / L, 26 mmol / L, 28 mmol / L, 30 mmol / L, 32 mmol / L, 34 mmol / L, 36 mmol / L, 38 mmol / L, 40 mmol / L, 42 mmol / L, 44 mmol / L, 46 mmol / L, 48 mmol / L, 50 mmol / L, or any value between them.

[0027] In some embodiments, the kit further includes an asymmetric MIRA reaction primer set, the primer set comprising an upstream primer and a downstream primer; The upstream primer has a nucleotide sequence as shown in SEQ ID NO: 1, and / or the downstream primer has a nucleotide sequence as shown in SEQ ID NO: 2.

[0028] In some embodiments, the kit includes asymmetric MIRA amplification reagents.

[0029] In some embodiments, the asymmetric MIRA amplification reagent includes one or more of recombinases, single-stranded DNA-binding proteins, and DNA polymerases.

[0030] According to a third aspect of this disclosure, a method for detecting mycobacteria is provided, characterized in that the method uses the probe set described in the first aspect or the kit described in the second aspect for detection.

[0031] In some embodiments, the mycobacteria are selected from one or more of the Mycobacterium tuberculosis complex, Mycobacterium leprae, and nontuberculous mycobacteria.

[0032] In some embodiments, the Mycobacterium tuberculosis complex is selected from Mycobacterium tuberculosis (Mycobacterium tuberculosis). M.tuberculosis ), Mycobacterium bovis ( M.bovis Mycobacterium capsulatum ( ) M.caprae ), Mycobacterium vulgatum ( M.microti ), Mycobacterium africanum ( M.africanum ), Mycobacterium carnetti ( M.canettii ) and Mycobacterium pinnipeds ( M.pinnipedii One or more of the following. In some embodiments, it is Mycobacterium bovis (…). M.bovis ).

[0033] In some implementations, the sample to be tested includes a tissue sample or a body fluid sample.

[0034] In some embodiments, the tissue sample or body fluid sample includes one or more of the following: tissue, saliva, blood, serum, plasma, milk, urine, lumbar spine or ventricular CSF, lymph, prostatic fluid, semen, sputum, feces, tears, tumor cells, bronchoalveolar lavage fluid, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion, peritoneal fluid, amniotic fluid, peritoneal fluid, aqueous humor, vitreous humor, and vaginal discharge.

[0035] In some implementations, the upstream primer is in excess relative to the downstream primer, or the downstream primer is in excess relative to the upstream primer.

[0036] In some embodiments, the concentration ratio of the upstream primer to the downstream primer is (1-20):1. In some embodiments, it is (5-10):1. In some embodiments, the concentration ratio of the upstream primer to the downstream primer is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, or any value between them.

[0037] In some embodiments, the concentration of the upstream primer is 0.5-2 μmol / L. In some embodiments, it is 0.8-1.6 μmol / L. In some embodiments, the concentration of the upstream primer is 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L, 1 μmol / L, 1.1 μmol / L, 1.2 μmol / L, 1.3 μmol / L, 1.4 μmol / L, 1.5 μmol / L, 1.6 μmol / L, 1.7 μmol / L, 1.8 μmol / L, 1.9 μmol / L, 2 μmol / L, or any value between them.

[0038] In some embodiments, the method includes the following steps: 1) Extract bacterial DNA from the sample to be tested; 2) Using the bacterial DNA as a template, an asymmetric MIRA reaction was performed using an asymmetric MIRA reaction primer set and an asymmetric MIRA amplification reagent to obtain the amplification product; 3) After inactivating the enzyme in the asymmetric MIRA amplification reagent, add the probe set and substrate to carry out the reaction (MNAzyme reaction). After the reaction, record the fluorescence value using a real-time PCR instrument or a portable constant-temperature fluorescence detector; or use a blue light projector or gel electrophoresis system to detect the endpoint signal and judge the detection result accordingly.

[0039] In some embodiments, the temperature for the asymmetric MIRA reaction in step 2) is 36-45°C. In some embodiments, the incubation temperature can be 36°C, 36.5°C, 37°C, 37.5°C, 38°C, 38.5°C, 39°C, 39.5°C, 40°C, 40.5°C, 41°C, 41.5°C, 42°C, 42.5°C, 43°C, 43.5°C, 44°C, 44.5°C, 45°C, or any value between them. In some embodiments, it is 39-42°C.

[0040] In some embodiments, the asymmetric MIRA reaction time in step 2) is 20-40 min. In some embodiments, the incubation time can be 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, or any value between them.

[0041] In some embodiments, the inactivation treatment in step 3) is performed at a temperature of 55-85°C. In some embodiments, the inactivation can be performed at 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 60.5°C, 61°C, 61.5°C, 62°C, 62.5°C, 63°C, 63.5°C, 64°C, 64.5°C, 65°C, 65.5°C, 66°C, 66.5°C, 67°C, 67.5°C, 68°C, 68.5°C, 69°C, 69.5°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, or any value between them.

[0042] In some embodiments, the inactivation treatment time in step 3) is 4-10 min. In some embodiments, the inactivation time can be 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min, 8 min, 8.5 min, 9 min, 9.5 min, 10 min, or any value between them.

[0043] In some embodiments, the temperature of the MNAzyme reaction in step 3) is 35-42°C. In some embodiments, the incubation temperature can be 35°C, 35.5°C, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, 38.5°C, 39°C, 39.5°C, 40°C, 40.5°C, 41°C, 41.5°C, 42°C, or any value between them.

[0044] In some embodiments, the MNAzyme reaction time in step 3) is 20-80 min. In some embodiments, the incubation time can be 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, or any value between them.

[0045] In some implementations, fluorescence values ​​are detected using a quantitative real-time PCR instrument and / or a portable thermostat.

[0046] In some implementations, a blue light projector and / or a gelation system are used to detect the endpoint signal.

[0047] In some embodiments, the method includes the following steps: 1) Extract bacterial DNA from the sample to be tested; 2) Using the bacterial DNA as a template, an asymmetric MIRA reaction was performed using an asymmetric MIRA reaction primer set and an asymmetric MIRA amplification reagent to obtain the amplification product; 3) After inactivating the enzyme in the asymmetric MIRA amplification reagent, add the probe set and substrate to carry out the MNAzyme reaction. After the reaction, record the fluorescence value using a real-time PCR instrument or a portable constant-temperature fluorescence detector; or use a blue light projector or gel electrophoresis system to detect the endpoint signal and judge the detection result accordingly. When the detection signal forms an amplification curve and the fluorescence value is higher than the threshold, it is determined that the sample to be tested contains Mycobacterium bovis. If the endpoint fluorescence signal of the test sample can be observed and distinguished from that of the negative control, it is determined that the test sample contains Mycobacterium bovis.

[0048] According to the fourth aspect of this disclosure, the use of the probe set described in the first aspect or the kit described in the second aspect in the detection of mycobacteria is provided.

[0049] In some embodiments, the mycobacteria are selected from one or more of the Mycobacterium tuberculosis complex, Mycobacterium leprae, and nontuberculous mycobacteria.

[0050] In some embodiments, the Mycobacterium tuberculosis complex is selected from Mycobacterium tuberculosis (Mycobacterium tuberculosis). M.tuberculosis ), Mycobacterium bovis ( M.bovis Mycobacterium capsulatum ( ) M.caprae ), Mycobacterium vulgatum ( M.microti ), Mycobacterium africanum ( M.africanum ), Mycobacterium carnetti ( M.canettii ) and Mycobacterium pinnipeds ( M.pinnipedii One or more of the following. In some embodiments, it is Mycobacterium bovis (…). M.bovis ). Attached Figure Description

[0051] Figure 1 A schematic diagram of the reaction of the asymmetric MIRA-MNAzyme system of this disclosure is shown. Among them, Figure 1 A is the reaction flow diagram. Figure 1 B is a schematic diagram of the reaction principle.

[0052] Figure 2 The screening results of the primers disclosed herein are shown. Among them, Figure 2 A is a diagram showing the relative positions of the primers at the RD4 region gene of the Mycobacterium tuberculosis complex; Figure 2 B represents the screening results of candidate upstream and downstream primers in Mycobacterium bovis; Figure 2 C represents the screening results of candidate upstream and downstream primers in Mycobacterium tuberculosis; Figure 2 D represents the screening results of candidate upstream and downstream primers in a template-free control; Figure 2 E represents the screening results of aMIRA amplification single-stranded products using Mycobacterium bovis as a template; Figure 2 F represents the screening results of aMIRA amplification single-stranded products using Mycobacterium tuberculosis as a template; Figure 2 G represents the screening results of aMIRA amplification single-stranded products using enzyme-free water as a template.

[0053] Figure 3 The screening results of the MNAzyme system disclosed in this paper are shown. Among them, Figure 3 A represents Mg 2+ Pb 2+ Mn 2+ Zn 2+ The results of DNAzyme cleavage under conditions 8-17; Figure 3 B represents the feasibility analysis results of the MNAzyme system; Figure 3 C represents the screening results for 8-17 DNAzyme splitting sites; Figure 3 D represents the screening results of forward and reverse templates for the RD4 region gene and substrates of different stem lengths; Figure 3 E is a schematic diagram illustrating the selection of DNAzyme splitting sites from 8 to 17; Figure 3 F is a schematic diagram of the final selected configuration 3.

[0054] Figure 4 The screening results for primer concentrations and concentration ratios disclosed in this paper are shown. Among them, Figure 4 A and Figure 4 B represents the aMIRA amplification results as primer concentration increases, using Mycobacterium bovis and enzyme-free water as templates, with upstream and downstream primer concentration ratios of 20:1 and 40:1, respectively. Figure 4 C and Figure 4 D represents the real-time fluorescence detection results as primer concentration increases when the asymmetric MIRA-MNAzyme detection is performed under conditions where the upstream and downstream primer concentration ratios are 20:1 and 40:1, respectively. Figure 4 E represents the real-time fluorescence detection results corresponding to different primer concentration ratios under high-concentration primer conditions when detecting asymmetric MIRA combined with MNAzyme. Figure 4 F represents the optimization of primer concentration and concentration ratio for asymmetric MIRA combined with MNAzyme detection.

[0055] Figure 5 The optimized reaction conditions for the asymmetric MIRA combined with MNAzyme detection of this disclosure are shown. Among them, Figure 5 A represents the optimal reaction temperature for aMIRA. Figure 5 B represents the optimized reaction temperature for MNAzyme. Figure 5 C represents the optimized Tris-HCl concentration in the MNAzyme system buffer. Figure 5 D represents the optimized MgCl2 concentration in the MNAzyme system buffer. Figure 5 E represents the optimal inactivation temperature for aMIRA protein; Figure 5 F represents the optimal concentration ratio of the first or second probe to the modified substrate in the MNAzyme system; Figure 5 G represents the concentration optimization of the first or second probe and the modified substrate in the MNAzyme system.

[0056] Figure 6 The sensitivity analysis results of the asymmetric MIRA combined with MNAzyme detection of this disclosure are shown. Among them, Figure 6 A represents the sensitivity analysis of asymmetric MIRA combined with MNAzyme detection – results from quantitative real-time PCR. Figure 6 B represents the sensitivity analysis of asymmetric MIRA combined with MNAzyme detection – the result of the gel imaging system. Figure 6 C represents the sensitivity analysis of qPCR detection: 1: 10 ng / μL, 2: 1 ng / μL, 3: 100 pg / μL, 4: 10 pg / μL, 5: 1 pg / μL, 6: 100 fg / μL, 7: 10 fg / μL, 8: negative control.

[0057] Figure 7 The specificity analysis results of the asymmetric MIRA combined with MNAzyme detection of this disclosure are shown. Among them, Figure 7A represents the specificity analysis of asymmetric MIRA combined with MNAzyme detection – the result of real-time PCR. Figure 7 B represents the specificity analysis of asymmetric MIRA combined with MNAzyme detection – the result of the gel imaging system.

[0058] Figure 8 The results of this disclosure's asymmetric MIRA combined with MNAzyme detection in simulated milk samples are shown. Among them, Figure 8 A represents the detection results of asymmetric MIRA combined with MNAzyme detection in simulated milk samples - results from a real-time PCR instrument. Figure 8 B represents the detection results of asymmetric MIRA combined with MNAzyme detection in simulated milk samples - gel imaging system detection results; Figure 8 C represents the detection result of qPCR in a simulated milk sample, 1:5×10⁻⁶. 7 CFU / mL, 2: 5×10 6 CFU / mL, 3: 5×10 5 CFU / mL, 4: 5×10 4 CFU / mL, 5: 5 × 10 3 CFU / mL, 6: 5×10 2 CFU / mL, 7: 5×10 1 CFU / mL, 8: negative control. Detailed Implementation

[0059] Tuberculosis is caused by the tuberculous complex (Mycobacterium tuberculosis) Mycobacterium tuberculosis Mycobacterium tuberculosis complex (MTBC) infection is a chronic, wasting zoonotic infectious disease that seriously threatens global public health security. Among the Mycobacterium tuberculosis complex, Mycobacterium tuberculosis and Mycobacterium bovis are two of the most common zoonotic pathogens. Mycobacterium tuberculosis is the main causative agent of tuberculosis in humans, while Mycobacterium bovis is the main pathogen of tuberculosis in animals, infecting livestock, wild animals, and also transmitting to humans.

[0060] Multienzyme isothermal rapid amplification (MIRA) is an isothermal amplification technique for pathogen nucleic acid detection. It typically operates at 37-42°C and completes nucleic acid amplification within 30 minutes, enabling rapid detection of the target nucleic acid. Multicomponent nuclease (MNAzyme) reactions based on deoxyribonucleases (DNAzymes) offer a detection strategy that combines nucleic acid recognition and signal output. The MNAzyme system mainly consists of two RNA-cutting cleaving DNAzyme subunits. Each subunit is modified to include a target-binding arm, a catalytic core, and a substrate-binding arm. When the target nucleic acid sequence is present, it binds to the template-binding arms of the first and second probes, assembling the two subunits into a DNAzyme with RNA-cutting activity—a complete catalytic core with catalytic activity. After binding to the substrate, the DNAzyme, with the participation of metal ions, breaks the phosphodiester bond at the RNA cleavage site in the substrate.

[0061] This disclosure combines asymmetric multienzyme isothermal rapid amplification (aMIRA) with the MNAzyme system, integrating isothermal rapid amplification with nucleic acid-specific recognition and signal amplification. It features lower reaction temperature, shorter detection time, and relatively lower equipment dependence, and can be used for rapid detection of Mycobacterium bovis, especially suitable for field testing at the grassroots level or in resource-constrained scenarios.

[0062] In some embodiments, this disclosure provides a primer pair, method, and application for detecting mycobacteria based on the combination of asymmetric multi-enzyme isothermal rapid amplification (aMIRA) technology and the MNAzyme system. This method enables rapid, specific, sensitive, and convenient detection of *Mycobacterium bovis*, and the results can be visually interpreted. The reaction flow diagram of this disclosure is shown below. Figure 1 As shown in Figure A, the reaction principle is as follows: Figure 1 As shown in B, it is divided into two stages: Stage 1: aMIRA Reaction: The recombinase binds to the primer to form a complex. This complex recognizes homologous sequences in the nucleic acid template and mediates the binding of the primer to the homologous sequence region. Simultaneously, the single-stranded binding protein is stably replaced by the single-stranded region. Subsequently, the recombinase dissociates from the primer, exposing the 3' end of the primer, which is then bound by DNA polymerase. The DNA polymerase extends from the 3' end of the primer, thereby achieving rapid amplification of the target nucleic acid fragment. During aMIRA amplification, excess upstream and downstream primers work together on the template to amplify it. As amplification progresses, the downstream primer is gradually consumed until it is exhausted. At this point, the amplification product accumulates in the form of a single-stranded template, which is the target nucleic acid sequence.

[0063] Phase 2: MNAzyme Reaction: In the MNAzyme system, the target-binding arms of the two split DNAzyme subunits recognize and bind to the single-stranded template (target nucleic acid sequence) accumulated during aMIRA amplification, while the substrate-binding arms recognize and bind to the modified substrate with a stem-loop structure. This induces the self-assembly of the split DNAzyme subunits, forming a complete catalytic core with catalytic activity. Subsequently, the catalytic core cleaves the phosphodiester bond at the 3' end of the adenine ribonucleotide in the modified substrate, separating the fluorophore from the quencher group, thereby generating a detectable fluorescent signal.

[0064] Furthermore, the technology provided in this disclosure only requires a reaction under constant temperature conditions, and the instrument requirements are simple. Only a constant temperature device is needed to complete the nucleic acid amplification process, such as a constant temperature water bath or a portable thermostat. With the help of a blue light projector, the requirements for operators are not high. It is especially suitable for on-site diagnosis and immediate testing at the grassroots level, and has broad application prospects in veterinary clinical practice. It also provides technical support for ensuring the safety of animal-derived food.

[0065] The kit and method for detecting Mycobacterium bovis disclosed herein, by introducing an MNAzyme system, can amplify the detection signal a second time. Furthermore, by simply changing the nucleotide sequence of the split DNAzyme subunit, without redesigning a universal substrate, detection of other targets can be achieved, thereby reducing detection costs and improving the method's versatility. This overcomes the shortcomings of conventional PCR techniques, such as complex operation, long processing time, and high instrument requirements, thus establishing an accurate, intuitive, and convenient rapid detection method for grassroots and field use.

[0066] The present disclosure will be further illustrated below by way of embodiments. It should be understood that the embodiments of the present disclosure are merely illustrative and not intended to limit the present disclosure. Simple modifications to the present disclosure based on its concept are within the scope of protection claimed by the present disclosure. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of the present disclosure. Such structures and techniques have also been described in many publications.

[0067] definition Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0068] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0069] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0070] As used herein, the term "sequence with at least 80% sequence identity" means a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0071] The term "multi-enzyme isothermal amplification technology" or "MIRA" used in this article refers to a rapid isothermal nucleic acid amplification technique that relies on the synergistic action of multiple functional proteins to achieve rapid nucleic acid amplification at room temperature. Specifically, the protein-DNA complex formed by the binding of recombinase and primers searches for homologous sequences in double-stranded DNA. Once the primers locate a homologous sequence, a strand exchange reaction occurs, initiating DNA amplification and exponentially expanding the target region on the template. The replaced DNA strand binds to a single-strand DNA-binding protein (SSB) to prevent further replacement. In this system, the amplification event is initiated by two opposing primers.

[0072] The amplification products of MIRA can be observed by direct electrophoresis, or by using real-time fluorescence monitoring after amplification with a real-time fluorescence PCR instrument and judging the results by the amplification curve.

[0073] The term “MNAzyme” or “multicomponent nuclease” as used in this article refers to two or more oligonucleotide sequences that assemble into an active DNA ribozyme in the presence of “MNAzyme tissue-promoting factors” (e.g., target nucleic acids), thereby enabling the catalysis of modified substrates.

[0074] As used herein, the terms “catalytic nucleic acid molecule,” “catalytic nucleic acid,” “nuclease,” and “catalytic nucleic acid sequence” are used interchangeably and refer to a DNA molecule or a molecule containing DNA (also referred to in the art as a “DNA enzyme,” “deoxyribozyme,” or “DNAzyme”) or an RNA molecule or a molecule containing RNA (also referred to in the art as an “RNAi” or “ribozyme”) or a combination thereof, which is a DNA-RNA hybrid molecule capable of recognizing a substrate and catalyzing modifications of said substrate. The nucleotide residues in the catalytic nucleic acid may include bases A, C, G, T, and U, and their derivatives and analogs.

[0075] The term “Mycobacterium bovis BCG” used in this article is commonly referred to as BCG vaccine. It is a live strain of Mycobacterium bovis that has been attenuated. It is a mutant form of Mycobacterium bovis produced by French microbiologists Calmette and Guerin in 1921 through 13 years of continuous passage of virulent Mycobacterium bovis.

[0076] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0077] The sequence information involved in this disclosure is shown in Table 1 below: Table 1

[0078] Example Example 1: Design and Screening of aMIRA Primers 1. Based on the whole genome sequence of Mycobacterium tuberculosis complex, multiple sequence alignment analysis was performed to screen out RD4 (Region of Difference 4) of Mycobacterium bovis as the detection target. Primers were designed using the sequences flanking RD4 as the primer design regions. Three upstream primers RD4-F1 to RD4-F3 were designed at the N-terminus of the deletion site, and three downstream primers RD4-R1 to RD4-R3 were designed at the C-terminus of the deletion site. Figure 2A). The sequence is shown in Table 1.

[0079] 2. Screening of primer combinations consisting of RD4-F1~RD4-F3 and RD4-R1~RD4-R3.

[0080] Genomic DNA was extracted from Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra (ATCC 25177) using a bacterial genomic DNA extraction kit (catalog number: 60300K-50T, Shanghai Jingnuo Biotechnology) according to the manufacturer's instructions. Using 1 ng / μL of genomic DNA from Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra as templates, MIRA amplification was performed using different primer combinations. Enzyme-free water (template-free group) was used as a negative control. MIRA amplification was performed using a DNA isothermal rapid amplification kit (basic type) (catalog number: WLB8201KIT, Anpu Future (Changzhou)). The amplification reaction system consisted of: 1 / 2 tube of dry powder (containing recombinase, single-stranded binding protein, and DNA polymerase), 14.7 μL of reaction buffer A, 4.25 μL of enzyme-free water, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 2.5 μL of template, and 1.25 μL of reaction buffer B. The reaction program was 39 °C for 30 min. After the reaction, 25 μL of Tris-saturated phenol:chloroform:isoamyl alcohol (25:24:1) DNA extraction buffer was added to the reaction product. After mixing, the mixture was centrifuged at 12000 rpm for 5 min. The supernatant was collected and mixed with 6× DNA Loading buffer (Beijing TransGen Biotech). 8 μL of the mixture was then subjected to agarose gel electrophoresis for detection.

[0081] The results are as follows Figure 2 As shown in B-2D, when Mycobacterium bovis BCG genomic DNA is used as a template, the amplification band of the combination of RD4-F3 (SEQ ID NO: 1) and RD4-R1 (SEQ ID NO: 2) is the brightest. Figure 2 B), indicating that this primer pair has the best amplification effect; using Mycobacterium tuberculosis H37Ra genomic DNA as a template ( Figure 2 C) or use enzyme-free water as a negative control ( Figure 2 In case D), no target band was observed.

[0082] 3. After selecting the optimal primer pair, using genomic DNA of 1 ng / μL Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra as templates, and enzyme-free water (template-free group) as a negative control, the downstream primer was serially diluted 2-fold while keeping the upstream primer concentration constant (0.4 μM) to achieve a concentration ratio of upstream primer to downstream primer of 0.4 μM:0.4 μM (1:1) to 0.4 μM:31.25 nM (320:1) to verify the feasibility of using the optimal primer pair for aMIRA amplification and generating single-stranded DNA bands. The amplification reaction system consisted of: 1 / 2 tube of dry powder (containing recombinase, single-stranded binding protein, and DNA polymerase), 14.7 μL of reaction buffer A, 4.25 μL of enzyme-free water, 1 μL of 10 μM upstream primer RD4-F3 (SEQ ID NO: 1), 1 μL of serially diluted downstream primer RD4-R1 (SEQ ID NO: 2), 2.5 μL of template, and 1.25 μL of reaction buffer B. The reaction program was 39 °C for 30 min. After the reaction, 25 μL of Tris-saturated phenol:chloroform:isoamyl alcohol (25:24:1) DNA extraction buffer was added to the reaction product. After mixing, the mixture was centrifuged at 12000 rpm for 5 min. The supernatant was collected and mixed with 6× DNA loading buffer. 8 μL of the mixture was then analyzed by agarose gel electrophoresis.

[0083] The results are as follows Figure 2 As shown in E-2G, when using Mycobacterium bovis BCG genomic DNA as a template, a single-stranded DNA band generated by asymmetric amplification can be observed when the concentration ratio of the upstream primer to the downstream primer is greater than or equal to 20:1. Figure 2 E); using Mycobacterium tuberculosis H37Ra genomic DNA as a template ( Figure 2 F) or use enzyme-free water as a negative control ( Figure 2 No single-stranded DNA bands were observed in G). These results indicate that the RD4-F3 / RD4-R1 primer combination can be used for aMIRA amplification of Mycobacterium bovis and can obtain single-stranded DNA products under appropriate primer concentration ratios.

[0084] Example 2: Design and Screening of the MNAzyme System 1. During the optimization of the MNAzyme system, the substrate cleavage activity of 8-17 DNAzymes (with nucleotide sequences shown in SEQ ID NO: 9) containing only substrate recognition arms was first systematically evaluated under different metal ion conditions. The cleavage results were detected using 20% ​​polyacrylamide gel electrophoresis. The reaction systems for each lane were prepared according to Table 2, and Tris buffer was added to bring the total volume to 20 μL. The Tris buffer consisted of 20 mM Tris-HCl (pH 7.4) and 100 mM NaCl. Figure 3 As shown in Figure A, the reaction systems for the first three lanes were prepared and stored at 4°C for later use. The reaction systems for lanes 4-11 were placed in a PCR instrument (Hangzhou Baiheng) for reaction at 37°C for 1 hour. After the reaction, each reaction system was mixed with 6×DNA Loading buffer, and 10 μL of the mixture was taken for polyacrylamide gel electrophoresis detection.

[0085] Table 2 Figure 3 The system composition and final concentration of each component in each lane of swim A

[0086] Based on the above results and considering factors such as environmental friendliness, Mg was ultimately selected. 2+ As a reacting ion.

[0087] 2. The catalytic core of the 8-17 DNAzyme is split into two parts, and target-binding arms are connected to each part to construct a first probe and a second probe, thus forming the MNAzyme system. According to existing technology, the activity of the MNAzyme system is also affected by the location of the core split. This is because the paired bases in the 8-17 DNAzyme catalytic core ( Figure 3 The CCG and CGG bases in the catalytic core of F are closely related to its catalytic activity. Therefore, these two sets of paired bases are retained, and site 3 (not located between the paired bases) is selected. Figure 3 E) Feasibility analysis was conducted using it as a cleavage site, resulting in the sequences of the first and second probes shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The first and second probes of this system recognize the single-stranded DNA template (i.e., the target sequence) through their target-binding arms and the substrate through their substrate-binding arms, forming a complete and active catalytic structure that cleaves the substrate and generates a fluorescent signal.

[0088] The feasibility of using 20% ​​polyacrylamide gel electrophoresis to assess the MNAzyme system was analyzed. The reaction mixtures for each lane were prepared according to Table 3, and Tris buffer was added to bring the total volume to 20 μL. Figure 3 As shown in Figure B, the reaction systems for the first six lanes were prepared and stored at 4°C for later use. The reaction systems for lanes 7-15 were placed in a PCR instrument (Hangzhou Baiheng) for reaction at 37°C for 1 hour. After the reaction, each reaction system was mixed with 6×DNALoading buffer, and 10 μL of the mixture was taken for polyacrylamide gel electrophoresis detection.

[0089] Table 3 Figure 3 The system composition and final concentration of each component in each lane of swim grotto B

[0090] like Figure 3 As shown in Figure B, the polyacrylamide gel electrophoresis results indicate that in the control system without a single-stranded DNA template, even in the presence of Mg... 2+ MNAzyme also failed to effectively assemble into an active catalytic structure, and no substrate was observed to be cleaved into half unmodified substrate (lane 8) and half modified substrate (lane 9); this was observed even with the addition of a single-stranded DNA template but without Mg. 2+ In the system containing both single-stranded DNA template and Mg, substrate cleavage was also not observed (lanes 11 and 12); 2+ In this system, MNAzyme can undergo conformational rearrangement to form a complete active catalytic structure, thereby achieving effective cleavage of both unmodified and modified substrates. The fragment length after cleavage of the unmodified substrate is only 18 nt. Due to the short fragment and low product amount, its band brightness is weak, appearing as a thin band (lane 14). The 5' end fragment obtained after cleavage of the modified substrate, carrying a FAM fluorescent group, exhibits a significantly enhanced band signal, making it easier to observe (lane 15). Furthermore, because this cleavage product carries an additional fluorescent group compared to the unmodified substrate cleavage product, its migration rate is slower, resulting in the modified substrate cleavage band being slightly higher than the unmodified substrate cleavage band.

[0091] 3. Optimize the design of DNA cleavage sites. For different cleavage sites ( Figure 3 Five splitting configurations were designed for DNAzyme splitting sites 1-5 (E8-17). Each configuration corresponds to a first probe and a second probe, and the samples were screened using real-time fluorescence. The nucleotide sequences involved are shown in Table 4. The reaction system for each splitting site included: 0.5 μM first probe, 0.5 μM second probe, 0.5 μM single-stranded DNA template (SEQ ID NO: 10), 1 μM modified substrate (SEQ ID NO: 13), and 100 mM Mg. 2+ The volume of the sample was increased to 20 μL with Tris buffer. The reaction program was 37 °C for 30 min, with fluorescence values ​​recorded every 30 s. The reaction was performed on a LineGene 9600 Plus real-time PCR instrument (Hangzhou Borui), and the fluorescence signal was recorded to obtain a real-time fluorescence curve.

[0092] like Figure 3 As shown in C, the split configuration 3 produces the strongest fluorescence signal, and is therefore determined to be the optimal split configuration for the 8-17 DNAzyme.

[0093] Table 4. Nucleotide sequences of the first and second probes corresponding to the five splitting configurations.

[0094] 4. Using the forward and reverse sequences flanking the RD4 deletion site in *Mycobacterium bovis* as templates, substrates with stem lengths of 6 and 8 bases were designed, respectively. The optimal combination was further screened using real-time fluorescence detection. The nucleotide sequences involved are shown in Table 5. Each reaction system included 0.5 μM of the first probe, 0.5 μM of the second probe, 0.5 μM of single-stranded DNA template, 1 μM of substrate, and 100 mM of Mg2+. 2+ The volume of the sample was increased to 20 μL with Tris buffer. The reaction program was 37 °C for 30 min, with fluorescence values ​​recorded every 30 s. The reaction was performed on a LineGene 9600 Plus real-time PCR instrument (Hangzhou Borui), and the fluorescence signal was recorded to obtain a real-time fluorescence curve.

[0095] like Figure 3 As shown in D, based on the intensity of the real-time fluorescence signal, the forward sequences flanking the RD4 deletion site in Mycobacterium bovis were selected as templates, and a substrate with 6 paired bases in the stem region was chosen for subsequent experiments.

[0096] The structure of the finally selected split configuration 3 is as follows: Figure 3 As shown in F, the nucleotide sequences of its first and second probes are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The nucleotide sequence of the substrate with a stem region of 6 paired bases is shown in SEQ ID NO: 13. The nucleotide sequence of the single-stranded DNA template (i.e., the target, which is the forward sequence on both sides of the Mycobacterium bovis RD4 deletion site) is shown in SEQ ID NO: 10.

[0097] Table 5. Screening sequences designed using forward and reverse templates and stem length.

[0098] Example 3: Establishment of an asymmetric MIRA combined with MNAzyme detection system An asymmetric MIRA-MNAzyme detection system was constructed using the DNA Isothermal Rapid Amplification Kit (Basic Type) (Catalog No.: WLB8201KIT, Anpu Future (Changzhou)). The reaction system consisted of an asymmetric MIRA system and an MNAzyme system.

[0099] The aMIRA system includes: 1 / 2 tube of dry powder (containing recombinase, single-stranded binding protein and DNA polymerase), 14.7 μL reaction buffer A, 4.25 μL enzyme-free water, 1 μL of 40 μM upstream primer RD4-F3 (SEQ ID NO: 1), 1 μL of 4 μM downstream primer RD4-R1 (SEQ ID NO: 2), 2.5 μL template, and 1.25 μL reaction buffer B.

[0100] The MNAzyme system consists of: 2 μM first probe (SEQ ID NO: 7), 2 μM second probe (SEQ ID NO: 8), 4 μM modified substrate (SEQ ID NO: 13), 400 mM MgCl2, 4 μL 1M NaCl, 1 μL 1M Tris-HCl (pH=7.4), and enzyme-free water to a final volume of 15 μL.

[0101] In the negative control, the template was mixed with enzyme-free water, while in the positive control, the template was Mycobacterium bovis BCG genomic DNA.

[0102] First, incubate the aMIRA system at 39℃ for 30 minutes, then inactivate it at 65℃ for 5 minutes; after inactivation, add the MNAzyme system and incubate at 37℃ for 30 minutes, then record the fluorescence value using a real-time PCR instrument or a portable constant-temperature fluorescence detector; or after inactivation, add the MNAzyme system and incubate at 37℃ for 60 minutes, then detect the endpoint signal using a blue light projector or a gel electrophoresis system.

[0103] Results description and interpretation: The average fluorescence value of the negative control group plus three times the standard deviation was used as the positive judgment threshold. When the detection signal forms an amplification curve and the fluorescence value is higher than the threshold, the sample is determined to contain Mycobacterium bovis; or, when the endpoint fluorescence signal of the sample is observably distinguishable from that of the negative control, the sample is determined to contain Mycobacterium bovis.

[0104] Example 4: Optimization of reaction conditions for asymmetric MIRA combined with MNAzyme detection 1. Screening of primer concentration and primer concentration ratio To further improve the yield of single-stranded DNA template after aMIRA amplification, the primer concentration was gradually increased, and its feasibility was analyzed by agarose gel electrophoresis. At the same time, the primer concentration range required for subsequent optimization was determined by combining the real-time fluorescence detection results of the MNAzyme system.

[0105] Electrophoresis results as follows Figure 4 As shown in Figures AB, under primer-to-downstream concentration ratios of 20:1 and 40:1, aMIRA amplified single-stranded DNA templates with increasing primer concentrations. Further evaluation of asymmetric MIRA combined with MNAzyme detection was conducted under primer-to-downstream concentration ratios of 20:1 and 40:1, with simultaneous doubling of primer concentrations. Results are as follows... Figure 4As shown in CD, when the final concentrations of excess primers (upstream primer) were 1.6 μM, 1.2 μM, and 0.8 μM, the corresponding fluorescence signals were all higher than those under the initial condition (0.4 μM). Considering that increasing the primer concentration might alter the optimal window for asymmetric amplification, the ratio of upstream to downstream primer concentrations was further re-screened. Figure 4 As shown in Figure E, when the final excess primer concentration was 1.6 μM, the upstream and downstream primer concentration ratios of 1.6 μM:0.32 μM (5:1) and 1.6 μM:0.16 μM (10:1) yielded the highest fluorescence signals. As the primer concentration ratio continued to increase, the fluorescence signal gradually decreased. Based on the initial screening results of primer concentration and concentration ratio, further selections of excess primer final concentrations of 1.6 μM, 1.2 μM, and 0.8 μM were made, and formal optimization was performed using primer ratios of 5:1 and 10:1.

[0106] from Figure 4 The results shown in F indicate that fluorescence signals were detectable when the excess primer (upstream primer) concentration was 0.8-1.6 μM, combined with a primer ratio of 5-10:1. The fluorescence signal steadily increased over time, ultimately determining 1.6 μM:0.16 μM (10:1) as the optimal primer concentration and ratio. The reason for this discrepancy might be that this example was conducted under higher primer concentration conditions, potentially altering the optimal ratio window determined in Example 1. In Example 1, the primer concentration was lower, requiring a ratio greater than or equal to 20:1 to observe single-stranded DNA bands generated by asymmetric amplification. In this example, however, the primer concentration was increased, and an excess of primer was present, allowing for good asymmetric amplification even without an excessively high primer ratio.

[0107] 2. Optimization of reaction conditions This disclosure systematically optimizes the following parameters: aMIRA reaction temperature, MNAzyme reaction temperature, Tris-HCl concentration in the MNAzyme system buffer, MgCl2 concentration in the MNAzyme system buffer, aMIRA protein inactivation temperature, and the concentration ratio and concentration of a single probe to the modified substrate in the MNAzyme system. Specifically: (1) The aMIRA reaction temperature was optimized at 39, 40, 41 and 42 °C, and the results are as follows: Figure 5 As shown in Figure A, it can be seen that the aMIRA reaction can be carried out under conditions of 39-42℃, and 39℃ was finally determined to be the optimal aMIRA reaction temperature.

[0108] (2) The reaction temperature of MNAzyme was optimized at 37, 39, 41 and 43 °C, and the results are as follows: Figure 5 As shown in Figure B, it can be seen that the MNAzyme reaction can be carried out under conditions of 37-43℃, and 37℃ was finally determined to be the optimal temperature for the MNAzyme reaction.

[0109] (3) Since the buffer components have a significant impact on the MNAzyme reaction, the concentrations of Tris-HCl and MgCl2 were further optimized. Three concentration gradients were set for Tris-HCl (10 mM, 25 mM, and 50 mM), and four concentration gradients were set for MgCl2 (50 mM, 100 mM, 150 mM, and 200 mM). Figure 5 The results shown in C indicate that the MNAzyme reaction can proceed at Tris-HCl concentrations ranging from 10 to 50 mM, with 25 mM ultimately determined as the optimal Tris-HCl concentration. From... Figure 5 The results shown in D indicate that the MNAzyme reaction can be carried out when the MgCl2 concentration is between 50 and 200 mM, and 150 mM was finally determined to be the optimal MgCl2 concentration.

[0110] (4) After the aMIRA reaction, heat at 55, 65, 75, and 85 °C for 5 min respectively to determine the optimal inactivation temperature of aMIRA proteins (recombinase, single-stranded DNA-binding protein, and DNA polymerase in the aMIRA reaction). From Figure 5 The results shown in Figure E indicate that fluorescence signals were generated in all experimental groups after treatment at different temperatures, demonstrating that stable detection results could be obtained under the aforementioned temperature conditions. The 65℃ treatment group exhibited a relatively high fluorescence signal, which met the detection requirements after aMIRA protein inactivation. Therefore, 65℃ was determined to be the optimal temperature for aMIRA protein inactivation. This condition reduces heating energy consumption and temperature control requirements while ensuring effective inactivation, making it more suitable for practical applications.

[0111] (5) The concentration ratio of the first or second probe (i.e., a single probe) to the modified substrate in the MNAzyme system was optimized, and four ratios were set: 0.25 μM:1 μM (1:4), 0.5 μM:1 μM (1:2), 0.75 μM:1 μM (3:4), and 1 μM:1 μM (1:1) for the modified substrate. Figure 5 The results shown in F indicate that a single probe concentration to modified substrate concentration ratio of 1:2 is optimal.

[0112] To further improve the fluorescence signal intensity, the concentrations of the probe and substrate were optimized while maintaining a constant 1:2 ratio. The combinations of probe and substrate concentrations were set to 0.5 μM:1 μM, 0.75 μM:1.5 μM, and 1 μM:2 μM. Figure 5 The results shown in G indicate that fluorescence signals can be detected at all three concentrations. The fluorescence signal is strongest when the concentrations are 0.75 μM and 1.5 μM. Therefore, this concentration combination was selected as the optimal concentration of the probe and the modified substrate.

[0113] Example 5: Sensitivity Analysis of Asymmetric MIRA Combined with MNAzyme Detection Using 10-fold serially diluted Mycobacterium bovis BCG genomic DNA as templates (concentration range 10 ng / μL~10 fg / μL), asymmetric MIRA binding MNAzyme detection was performed under optimized optimal conditions (reaction system see Example 3), with a negative control included.

[0114] To conduct methodological comparisons, parallel assays were performed using real-time quantitative PCR (qPCR). The qPCR method was based on a published study (Pinsky BA, et al., Journal of Clinical Microbiology, 2008, 46:2241-2246). The total volume of the qPCR reaction system was 20 μL, including 10 μL of 2×PerfectStart. ® Green qPCRSuperMix (dye-based real-time PCR premix, Beijing TransGen Biotech), 2 μL DNA template, 0.4 μL 10 μM upstream primer, 0.4 μL 10 μM downstream primer, and 7.2 μL enzyme-free water. The reaction program was: 94℃ pre-denaturation for 30 s; 40 cycles of amplification, each cycle being 94℃ for 5 s, 60℃ for 30 s, and 72℃ for 10 s. The reaction was performed on a LineGene 9600 Plus real-time PCR instrument (Hangzhou Bori), and fluorescence signals were recorded to obtain amplification curves. Furthermore, the fluorescence intensity of asymmetric MIRA combined with MNAzyme detection at 30 min was recorded using a real-time PCR instrument and compared with the qPCR results.

[0115] Table 6 Comparison of Asymmetric MIRA-MNAzyme Binding Results with qPCR Detection

[0116] Note: The positive threshold for asymmetric MIRA combined with MNAzyme detection is 540.99.

[0117] The results are as follows Figure 6 A, Figure 6 As shown in C and Table 6, under the conditions of quantitative real-time PCR, the asymmetric MIRA-MNAzyme detection method established in this disclosure has similar detection sensitivity to the qPCR method, and both can detect low concentrations of Mycobacterium bovis genomic DNA. According to the results of this embodiment, the sensitivity of the asymmetric MIRA-MNAzyme detection can reach 100 fg / μL. When using a gel imaging system to detect the endpoint fluorescence signal, the lowest concentration that can be visually interpreted is approximately 1 pg / μL. Figure 6 B).

[0118] Example 6: Specificity analysis of asymmetric MIRA combined with MNAzyme detection To examine the specificity of the method disclosed herein, this embodiment uses Mycobacterium bovis BCG, Mycobacterium tuberculosis H37Ra (ATCC 25177), Mycobacterium avium (CVCC 68202), Mycobacterium marineis strain M (ATCC BAA-535), and Mycobacterium smegmatis mc, respectively. 2 Nucleic acids from *Mycobacterium bovis* 155 (ATCC 700084), *Staphylococcus aureus* (ATCC 29213), and *Escherichia coli* O157:H7 (ATCC23889) were used as reaction templates, and negative controls were set up. Asymmetric MIRA-MNAzyme detection was performed according to the aforementioned method. Inactivated *Mycobacterium bovis* BCG, *Mycobacterium tuberculosis*, *Mycobacterium avium*, *Mycobacterium marineum*, and *Mycobacterium smegmatis* were donated by Professor An Yina of the Key Laboratory of Medical Molecular and Cell Biology, Institute of Biomedical Research, Shanxi University. *Staphylococcus aureus* and *Escherichia coli* were preserved in the Key Laboratory of Precision Nutrition and Food Quality, Department of Nutrition and Health, China Agricultural University. Genomic DNA was extracted from the above seven strains using a bacterial genomic DNA extraction kit (catalog number: 60300K-50T, Shanghai Jingnuo Biotechnology). *Mycobacterium bovis* BCG genomic DNA was diluted to 0.1 ng / μL, and the genomic DNA of the remaining bacteria was diluted to 1 ng / μL for detection.

[0119] The results are as follows Figure 7 As shown in Figures AB, it can be seen that only the experimental group corresponding to Mycobacterium bovis BCG showed a significant increase in fluorescence curve. Figure 7 A), and presents a visual green fluorescent signal (A), Figure 7 (B) No effective fluorescent amplification signals were observed in the remaining bacteria and the negative control group, and the endpoint visualization signal was not obvious. The results indicate that the asymmetric MIRA combined with MNAzyme detection established in this disclosure can achieve specific detection of Mycobacterium bovis without cross-reactivity with other bacteria.

[0120] Example 7: Application Experiment of Asymmetric MIRA Combined with MNAzyme Detection To evaluate the sensitivity and application value of this detection system in real samples, fresh milk was purchased from a local supermarket. A counted *Mycobacterium bovis* (BCG) bacterial suspension was added to the milk and thoroughly mixed. The milk was then serially diluted 10-fold (the bacterial concentrations after dilution are shown in Table 7) to simulate different levels of contamination that may be present in milk samples collected from a ranch. Nucleic acid was extracted from the artificially simulated samples using a bacterial genomic DNA extraction kit (catalog number: 60300K-50T, Shanghai Jingnuo Biotechnology), and detected using asymmetric MIRA combined with MNAzyme detection and qPCR, respectively.

[0121] Table 7 Comparison of Asymmetric MIRA-MNAzyme Binding Results with qPCR Detection

[0122] Note: The positive threshold for asymmetric MIRA combined with MNAzyme detection in milk testing is 617.33.

[0123] The test results show that, under the conditions of quantitative real-time PCR, the asymmetric MIRA-MNAzyme detection method established in this disclosure has similar detection performance to the qPCR method in milk simulated samples, both reaching a detection limit of 5 × 10⁻⁶. 2 CFU / mL Figure 8 A, Figure 8 (C and Table 7). When using a gel imaging system to detect the endpoint fluorescence signal, the lowest concentration that can be visually interpreted is approximately 5 × 10⁻⁶. 3 CFU / mL Figure 8 B).

[0124] While some exemplary embodiments of this application have been described and illustrated, this application is not limited to the disclosed embodiments. Rather, those skilled in the art will recognize that modifications and changes may be made to the described embodiments without departing from the spirit and scope of this application as described in the appended claims.

Claims

1. A DNAzyme-based detection probe set, characterized in that, include: The first probe includes a first target binding arm, a first catalytic core sequence, and a first substrate binding arm; The second probe includes a second target binding arm, a second catalytic core sequence, and a second substrate binding arm. and Substrate, The first catalytic core sequence and the second catalytic core sequence are complementary and can pair to form an 8-17 DNAzyme catalytic domain. The first catalytic core sequence includes the nucleotide sequence CCGAGC, and the second catalytic core sequence includes the nucleotide sequence CGGTCGAA. The first target-binding arm and the second target-binding arm comprise nucleotide sequences that are complementary to the 5' and 3' ends of the target to be detected, respectively. The first substrate-binding arm and the second substrate-binding arm each include nucleotide sequences that are complementary to the 3' and 5' ends of the substrate, respectively, and the substrate includes a specific cleavage site of the 8-17 DNAzyme. The first substrate-binding arm includes the nucleotide sequence TCTCTTCT, and the second substrate-binding arm includes the nucleotide sequence ATAGTGC.

2. The probe assembly according to claim 1, characterized in that, The substrate has a fluorescent group and a fluorescence quencher group attached to its 5' and 3' ends, respectively.

3. The probe assembly according to claim 2, characterized in that, The substrate comprises a nucleotide sequence as shown in SEQ ID NO: 13, wherein the A at position 18 is an adenine ribonucleotide.

4. A kit for detecting mycobacteria, characterized in that, Includes the probe set as described in any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, The first target binding arm includes a nucleotide sequence as shown in SEQ ID NO: 14, and / or The second target-binding arm comprises a nucleotide sequence as shown in SEQ ID NO:

15.

6. The reagent kit according to claim 4, characterized in that, The first probe comprises a nucleotide sequence as shown in SEQ ID NO: 7, and / or The second probe comprises a nucleotide sequence as shown in SEQ ID NO:

8.

7. The reagent kit according to claim 4, characterized in that, The kit also includes an asymmetric MIRA reaction primer set, which includes an upstream primer and a downstream primer; The upstream primer has a nucleotide sequence as shown in SEQ ID NO: 1, and / or The downstream primer has a nucleotide sequence as shown in SEQ ID NO:

2.

8. A method for detecting mycobacteria, characterized in that, The method uses the probe set according to any one of claims 1-3 or the kit according to any one of claims 4-7 for detection.

9. The method according to claim 8, characterized in that, During the detection, the upstream primer is in excess relative to the downstream primer, or the downstream primer is in excess relative to the upstream primer.

10. The method according to claim 9, characterized in that, In the detection, the concentration ratio of the upstream primer to the downstream primer is (1-20):

1.

11. Use of the probe set according to any one of claims 1-3 or the kit according to any one of claims 4-7 in the detection of mycobacteria.

Images