Salmonella live bacteria detection system based on deoxyribozyme activation and method thereof

Abstract

This application belongs to the field of bioanalytical detection technology, specifically disclosing a detection system and method for live Salmonella based on deoxyribonuclease activation. The detection system includes a Sub probe, a deoxyribonuclease probe, a hairpin probe, hyperbranched dendritic nanomolecules, a crRNA / Cas12a binary complex, a fluorescent probe, and magnetic beads. The hyperbranched dendritic nanomolecules include substrate A, substrate B, and a trigger strand B probe. Substrate A consists of an A-F strand probe, an A-Q strand probe, and a helper strand A probe, while substrate B consists of a B-F strand probe, a B-Q strand probe, and a helper strand B probe. This detection system specifically recognizes ribonuclease H2 released only by the metabolism of live Salmonella and utilizes it to activate the Sub-Dz substrate, ensuring from the source that only live bacteria can trigger the subsequent reaction. This effectively solves the problem of interference from dead bacteria, achieving highly sensitive, highly specific, and rapid detection of live Salmonella.

Description

Technical Field

[0001] This application relates to the field of bioanalytical detection technology, specifically to a Salmonella live bacteria detection system and method based on deoxyribonuclease activation. Background Technology

[0002] In the food safety system, foodborne illnesses caused by live pathogens remain a significant threat to public health. Gram-negative bacilli, represented by Salmonella, have become one of the leading pathogens causing human infections due to their strong environmental adaptability in the food processing chain. These microorganisms can maintain metabolic activity for months under adverse conditions such as dryness and low temperatures, and can even persist in animal-derived foods (such as poultry and egg products) through cross-contamination. Epidemiological statistics show that over 70% of Salmonella infection cases can be traced back to the consumption of contaminated animal-derived foods. Since the presence of live bacteria directly determines the risk of infection, developing detection technologies that can specifically identify live microorganisms has become a key breakthrough in improving the effectiveness of food safety early warning systems. However, existing detection systems generally face a common problem: traditional methods cannot effectively distinguish between viable bacterial colonies capable of reproduction and the nucleic acid fragments remaining in dead bacterial cells, making it difficult for monitoring results to accurately reflect the true biohazard level of food.

[0003] Currently, the identification of live pathogenic bacteria mainly relies on two types of technical systems: culture methods based on microbial proliferation characteristics, and molecular detection schemes targeting biomarkers. The plate count method recommended by the International Organization for Standardization (ISO) (such as ISO 6579-1) remains the authoritative method for laboratory confirmation of viable bacteria, but its detection cycle typically takes 3 to 7 days, which cannot meet the timeliness requirements for rapid screening in the food supply chain. In recent years, with the advancement of molecular biology techniques, researchers have turned their attention to biomolecules such as RNA, which degrade rapidly after cell death. By combining reverse transcription amplification (RT-PCR) technology with CRISPR gene editing tools, trace amounts of target bacteria can be identified within hours, with specificity even reaching the level of single-base differential resolution. Although the sensitivity of this type of molecular detection scheme is significantly better than traditional culture methods, the nucleic acid extraction step in its operation requires specialized equipment, and the amplification process is susceptible to interference from inhibitors in the food matrix. These problems severely limit the practical application of this technology in on-site detection scenarios such as slaughterhouses and food processing plants.

[0004] Therefore, developing a rapid detection method with high sensitivity for live Salmonella is of great significance for on-site testing in food processing and other fields. Summary of the Invention

[0005] This application aims to overcome the technical barriers to rapid and highly sensitive detection of live Salmonella by constructing a dual signal amplification system based on deoxyribonuclease activation, HCR-CRISPR / Cas12a, to achieve rapid detection of live Salmonella with high sensitivity and specificity. The core design of this scheme begins with the precise selection of the target: by specifically recognizing ribonuclease H2 released only by the metabolism of live Salmonella, and utilizing it to activate a carefully designed Sub-Dz substrate, it ensures from the source that only live bacteria can trigger the subsequent reaction, effectively solving the problem of interference from dead bacteria.

[0006] To achieve the above objectives, this application first provides a Salmonella live bacteria detection system based on deoxyribozyme activation, comprising a Sub probe, a deoxyribozyme probe, a hairpin probe, a hyperbranched dendritic nanomolecule, a crRNA / Cas12a binary complex, a fluorescent probe, and magnetic beads. The crRNA / Cas12a binary complex is composed of Cas12a protein and crRNA. The hyperbranched dendritic nanomolecule includes substrate A, substrate B, and a trigger strand B probe. Substrate A is composed of an AF strand probe, an AQ strand probe, and a helper strand A probe. Substrate B is composed of a BF strand probe, a BQ strand probe, and a helper strand B probe.

[0007] The sequence of the Sub probe is shown in SEQ ID NO.1:

[0008] In the sequence ACTCATCTGTGACTATGAACTGACTrATGACCTCACTACCAAGTGTCAACTCGTG, rA is an RNA base and a specific cleavage site.

[0009] The sequence of the deoxyribozyme probe is shown in SEQ ID NO.2, the sequence of the trigger strand B probe is shown in SEQ ID NO.4, the sequence of the hairpin strand probe is shown in SEQ ID NO.5, the sequence of the AF strand probe is shown in SEQ ID NO.6, the sequence of the AQ strand probe is shown in SEQ ID NO.7, the sequence of the auxiliary strand A probe is shown in SEQ ID NO.8, the sequence of the BF strand probe is shown in SEQ ID NO.9, the sequence of the BQ strand probe is shown in SEQ ID NO.10, the sequence of the auxiliary strand B probe is shown in SEQ ID NO.11, and the sequence of the crRNA is shown in SEQ ID NO.12.

[0010] Preferably, the sequence of the fluorescent probe is: TCCCCCCT.

[0011] Preferably, the molar ratio of substrate A to substrate B is 1:2, and the molar ratio of the Sub probe to the deoxyribozyme probe is 1:1.

[0012] Based on a general inventive concept, this application also provides a method for detecting live Salmonella bacteria for purposes other than disease diagnosis and treatment, comprising the following steps:

[0013] S1. Probe pretreatment: The lyophilized powders of Sub probe, deoxyribozyme probe, trigger strand B probe, AF strand probe, AQ strand probe, auxiliary strand A probe, BF strand probe, BQ strand probe, auxiliary strand B probe, crRNA and fluorescent probe were respectively prepared into 100 µM solutions with DEPC water; the hairpin strand probe was added to annealing buffer to form a hairpin structure.

[0014] S2. Synthesis of hyperbranched dendritic nanomolecules: AF and AQ chain probe solutions were mixed and reacted with TAE buffer, followed by incubation with auxiliary chain A probe solution to obtain substrate A; BF and BQ chain probe solutions were mixed and reacted with TAE buffer, followed by incubation with auxiliary chain B probe solution to obtain substrate B; substrate A and substrate B solutions were mixed and trigger chain B probe was added to react and obtain hyperbranched dendritic nanomolecule solution.

[0015] S3. Target acquisition and hairpin strand treatment: Mix the Sub probe, deoxyribozyme probe, DEPC water and buffer for hybridization reaction, then add the test solution and incubate. After incubation, heat to inactivate the DNase. Finally, add the hairpin strand probe solution and mix well to obtain the pretreated test solution.

[0016] S4. Signal Detection: Wash the pretreated test solution obtained in step S3 with buffer solution, then add magnetic beads for reaction. After the reaction, remove the supernatant on the magnetic separator and wash the remaining product with HEPES buffer. After washing, add the hyperbranched dendritic nanomolecule solution prepared in step S2 and HEPES buffer for reaction. After the reaction, remove the supernatant on the magnetic separator and wash the remaining product with HEPES buffer. Then add crRNA probe, Cas12a protein and DEPC water for mixing and reaction. Next, add fluorescent probe for reaction in the dark. Finally, inactivate Cas12a protein and measure the fluorescence intensity with a fluorescence spectrophotometer.

[0017] Preferably, the annealing buffer in step S1 contains 50 mM HEPES buffer, 150 mM NaCl, and 15 mM MgCl2.

[0018] Preferably, in step S2, the molar ratio of the AF chain probe, AQ chain probe, and auxiliary chain A probe is 2:3:4; and in step S2, the molar ratio of the BF chain probe, BQ chain probe, and auxiliary chain B probe is 2:3:4.

[0019] Preferably, the magnetic beads in step S4 are modified with streptavidin.

[0020] Preferably, in step S4, the fluorescence spectrophotometer detects the signal value at an emission wavelength EM=520 nm.

[0021] The target identification and amplification principle of the Salmonella live bacteria detection system based on deoxyribozyme activation provided in this application is as follows:

[0022] like Figures 1-3 As shown, this application constructs an intelligent sensing system based on deoxyribozyme activation and CRISPR-Cas12a / hyperbranched hybridization chain reaction (dHCR). Its molecular mechanism comprises a triple cascade reaction module: live Salmonella bacteria can produce RNase H2, which binds to deoxyribozyme (DZ) to activate cleavage activity, thereby specifically cleaving Sub-DZ substrates. The cleavage product first triggers conformational rearrangement of hairpin probes on the magnetic bead surface, exposing pre-designed toehold binding sites and initiating a strand displacement reaction; pre-loaded hyperbranched dendritic nanomolecule structures self-assemble into a three-dimensional network through a dynamic branching migration mechanism, with densely arranged single-stranded DNA (ssDNA) sequences on their surface serving as signal amplification carriers, each branch node carrying more than 100 LbCas12a recognition sites; after recombinant LbCas12a protein forms a binary complex with crRNA, it specifically recognizes and binds to the ssDNA array on the hyperbranched dendritic nanomolecule structure, and multivalent interactions activate the trans-cleavage activity of Cas12a, cleaving FAM-labeled fluorescent probes to generate a fluorescent signal output. The specific principle is as follows:

[0023] (1) Specific recognition of Salmonella

[0024] Only when the ribonuclease (RNase H2) released by live Salmonella bacteria binds to the deoxyribonuclease (DZ) can it cleave the substrate (generated by the binding of chain Sub and chain DZ) to form trigger chain A (as shown in SEQ ID NO.3). The cleavage product of the substrate, trigger chain A, can open the hairpin probe. The hairpin probe is opened and exposes the binding site of the trigger chain B probe, thereby binding to the trigger chain B. The trigger chain B is used to form dendritic nanomolecules, thereby realizing the binding of magnetic beads and hyperbranched dendritic nanomolecules for easy separation and purification.

[0025] (2) Principle of hyperbranched hybridization chain reaction

[0026] This application proposes a self-assembled dendritic nanosystem based on a strand displacement reaction, the core of which consists of two functionally complementary DNA substrates. The AF strand of substrate A is designed with two homologous stem-loop structures (referred to as the m segment), while the BF strand of substrate B contains a similar loop protrusion (the n segment). Under thermodynamic equilibrium conditions, these stem-loop structures remain stably closed, effectively preventing nonspecific unwinding and thus keeping background signals at a low level.

[0027] Activation of the system begins with the binding of the trigger chain B probe to the recognition region of substrate A. This action initiates a chain substitution process, causing the first m-segment of substrate A to unfold and expose its previously hidden binding site. Subsequently, the auxiliary chain A binds to this site via its base anchoring sites, driving the substitution of the AQ chain and the release of byproduct A, while simultaneously triggering a conformational change in the second m-segment. At this point, two parallel active regions are formed on the AF chain of substrate A, which can simultaneously guide structural changes in both substrate B molecules, causing their n-segments to unwind sequentially.

[0028] As the cyclic structure of substrate B opens, the auxiliary chain B intervenes in the reaction, further displacing and releasing byproduct B, while simultaneously generating a new, continuous reaction interface. During this process, the sequence exposed by substrate B can complementaryly pair with the initial recognition domain of substrate A, thus establishing an autocatalytic cycle. Each complete reaction round can generate twice the number of active sites; theoretically, after n cycles, a system with 2... n A tree-like nanostructure with multiple binding sites.

[0029] (3) The Cas12a protein trans-cleaves the FAM chain to generate a fluorescent signal.

[0030] The CRISPR-Cas12a / dendritic nanomolecule coupled sensing system constructed in this application exhibits a four-stage continuous reaction mechanism: the Cas12a-crRNA binary complex specifically recognizes dsDNA targets on the dendritic nanomolecule structure through PAM sequence; the complementary base pairing between crRNA and target DNA triggers local double-strand unwinding and activates the RuvC nuclease domain of Cas12a; after cis-cleavage at a specific position on the target strand, Cas12a undergoes a conformational change to expose the hidden trans-cleavage active site; the activated Cas12a acquires non-specific nuclease activity and can cyclically cleave pre-loaded FAM fluorescently labeled fluorescent probes in solution; the multivalent effect of the dendritic nanomolecules significantly enhances the local reactant concentration, causing a single target molecule to trigger exponential signal amplification, ultimately achieving femtomolar level detection sensitivity.

[0031] The main benefits of this application are as follows:

[0032] 1. The core design of the Salmonella detection system provided in this application begins with the precise selection of the target: by specifically recognizing ribonuclease H2 released only by the metabolism of live Salmonella, and using it to activate a specially designed Sub-Dz substrate, it ensures from the source that only live Salmonella can trigger the subsequent reaction, effectively solving the detection problem of dead bacteria interference.

[0033] 2. At the signal amplification level, the detection system provided in this application employs a cascade strategy: the initial trigger chain generated by deoxyribozyme cleavage first initiates a hyperbranched hybridization chain reaction, self-assembling to form a three-dimensional dendritic DNA nanostructure carrying thousands of identical recognition sites, completing the first exponential amplification of the signal; this structure then acts as an activation platform, efficiently recruiting and activating the CRISPR / Cas12a system, causing it to exhibit trans-cleavage activity, cutting a large number of fluorescent reporter probes in the solution, generating a fluorescent signal, and achieving a second signal amplification. This dual amplification mechanism, combined with the effective removal of impurities by magnetic bead enrichment technology and the simplicity of completing the entire process under constant temperature conditions in a single tube, ultimately forms a tool capable of rapid and accurate detection of live bacteria in the field, providing solid technical support for revolutionizing food safety monitoring models.

[0034] 3. This detection system innovatively constructs a cascaded CRISPR-Cas12a-HCR signal amplification system to address the catalytic activity inhibition problem caused by intramolecular modification of fluorophores and quenchers in traditional DNAzyme detection technologies. By introducing a cascaded amplification mechanism of Cas12a trans-cleavage activity and hybridization chain reaction (HCR), the enzyme activity inhibition effect caused by probe modification in traditional systems is successfully avoided. Based on this, combined with an RNase H2-dependent deoxyribonuclease with specific recognition function, a novel detection strategy that requires no pre-culture, nucleic acid extraction, or amplification is established.

[0035] 4. This detection system designs a CRISPR-Cas12a-HCR signal amplification system based on a three-dimensional dendritic nanostructure. By constructing high-density Cas12a / crRNA complex binding sites on the surface of a dendritic polymer nanocarrier, it integrates CRISPR-Cas12a trans-cleavage activity with hyperbranched hybridization chain reaction (HCR) to construct a dual signal amplification pathway. Its mechanism of action includes the following key steps: When the target ssDNA and the crRNA / Cas12a complex form a ternary complex through base complementarity, the Cas12a nuclease activity is specifically activated. First, it cis-cleaves the ssDNA in the binding region, causing the crRNA / Cas12a-target complex to dissociate and expose the trans-cleavage active site. Subsequently, the activated trans-cleavage enzyme activity non-specifically cleaves the dual-labeled fluorescent probe (FQ probe) in the system, causing spatial separation of the fluorescent group and the quencher group to generate a detectable signal. The design achieves performance optimization through the following mechanisms: ① The hyperbranched amplification effect of HCR significantly increases the signal molecule load, while the Cas12a cascade activation mechanism effectively reduces the background signal caused by spontaneous hybridization of the hairpin probe, thus resolving the inherent contradiction between sensitivity and signal-to-noise ratio in traditional HCR systems; ② The trans-cutting strategy, through a sequence-independent ssDNA cutting method, breaks through the strict dependence of traditional CRISPR systems on PAM sequences, expanding the range of detection targets.

[0036] 5. To address the limitation of traditional DNAzyme detection systems where direct signal output from fluorescent probes is susceptible to matrix interference, this system constructs an HCR signal enhancement system based on a molecular locking mechanism. Its core design incorporates two specific control layers: First, a biotin-labeled hairpin probe (H1) is used as the molecular recognition unit, and its directionality is achieved through streptavidin-coated magnetic beads. Second, RNase H2 is introduced to specifically recognize RNA-DNA hybrid structures in the substrate and achieve site-specific cleavage, releasing the trigger strand only when the target is present. This trigger strand acts on the locking domain of the H1 probe through base complementarity pairing, inducing a hairpin conformational transition and exposing its sticky ends, thereby initiating a hyperbranched HCR reaction to form a three-dimensional dendritic DNA nanostructure. This design enhances detection specificity through the following mechanisms: ① In the inactive state, the stem-loop structure of the lock-type hairpin probe blocks non-specific hybridization through steric hindrance; ② The magnetic bead solidification strategy reduces the free diffusion of the hairpin probe through physical adsorption, effectively suppressing false positive signals caused by random probe collisions; ③ The enzymatic cleavage of RNaseH2 is strictly substrate-dependent, ensuring that the trigger chain is generated only in the presence of the target.

[0037] 6. Compared with traditional detection methods, the detection system provided in this application exhibits three major disruptive advantages: the detection cycle is shortened from 3-4 days in traditional methods to within 7.5 hours, meeting the needs of rapid on-site detection; through closed-chain design and steric hindrance control, it has high specificity; and the detection sensitivity reaches 14 CFU / mL, enabling reliable detection of low-load pathogens. This system employs an isothermal reaction design, eliminating the need for complex temperature control equipment. Combined with a one-tube operation process, it effectively avoids the risks of aerosol contamination and cross-contamination, significantly reducing the technical requirements for operators. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This is a schematic diagram illustrating the detection principle of the Salmonella live bacteria detection system based on deoxyribozyme activation in this application.

[0040] Figure 2 This is a schematic diagram of the hyperbranched hybridization chain reaction principle of this application;

[0041] Figure 3 This is a diagram illustrating the fluorescence cleavage reaction mechanism of this application;

[0042] Figure 4 This is a gel electrophoresis result of the feasibility analysis of the Salmonella live bacteria detection system in Experiment Example 1 of this application;

[0043] Figure 5 This is a graph showing the specificity analysis results of the Salmonella live bacteria detection system for detecting different bacterial targets in Experimental Example 2 of this application;

[0044] Figure 6 This is a fluorescence intensity curve for detecting different concentrations of live Salmonella bacteria in Experiment Example 3 of this application;

[0045] Figure 7 This is a trend graph of fluorescence intensity for detecting different concentrations of live Salmonella bacteria in Experiment Example 3 of this application;

[0046] Figure 8 This is the response calibration curve for detecting different concentrations of live Salmonella bacteria in Experiment Example 3 of this application. Detailed Implementation

[0047] The following embodiments are used to illustrate this application, but are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0048] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0049] Unless otherwise specified, the percentage sign "%" in this application refers to the mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0050] The weight units mentioned in this application may be weight units known in the art such as μg, mg, g, kg, or multiples thereof, such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0051] All probes involved in this application were purchased from Shanghai Sangon Biotech Co., Ltd. The probe sequences used in the following examples and experimental cases are shown in Table 1.

[0052] Table 1. Designed probe sequences

[0053]

[0054] In Table 1, rA in the Sub probe represents an RNA base and is a specific cleavage site;

[0055] In the crRNA probe sequence, the underlined and bolded part "TATTATT" represents DNA bases, while the rest are RNA bases.

[0056] Example 1: Cultivation of Salmonella, the specific steps are as follows:

[0057] (1) Preparation and treatment of culture medium

[0058] Prepare LB liquid medium by including peptone (10g), yeast extract (15g), and NaCl (10g). Adjust the pH to 7.0 using NaOH and bring the volume to 1L. Dispense the solution into ten 250ml Erlenmeyer flasks, each containing 100ml of solution. Add agarose (0.5g) to two of the flasks to prepare LB solid medium. Finally, place all the Erlenmeyer flasks together in an autoclave and sterilize at 125℃ for 25 minutes.

[0059] (2) Bacterial culture

[0060] First, dilute the purchased Salmonella lyophilized powder with sterile water and activate it on solid culture medium (37ºC, 18-24h). After colonies have grown, inoculate it into liquid culture medium and incubate until OD (occurrence limit) is reached. 600 Reached 1.

[0061] Example 2: A Salmonella viable cell detection system based on deoxyribozyme activation was used to detect Salmonella in the test solution. The specific steps are as follows:

[0062] (1) Probe sequence preprocessing

[0063] The lyophilized powders of Sub probe, deoxyribozyme probe, trigger strand B probe, AF strand probe, AQ strand probe, helper strand A probe, BF strand probe, BQ strand probe, helper strand B probe, crRNA, and fluorescent probe listed in Table 1 were each prepared into 100 µM solutions using DEPC water. The hairpin strand probe was prepared into a 1 µM solution using annealing buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 15 mM MgCl2), annealed to form a hairpin structure, and stored at 4ºC for later use.

[0064] (2) Synthesis of hyperbranched dendritic nanomolecules

[0065] Substrate A: Mix 1 μL of AF chain probe (10 μM) and 1.5 μL of AQ chain probe (10 μM), add 2 μL of TAE buffer, react at 85 °C for 5 min, cool to 25 °C and hold for 20 min, then add 2 μL of auxiliary chain A probe solution (10 μM), mix, and incubate at 25 °C for 20 min to obtain substrate A;

[0066] Substrate B: Mix 2 μL of BF chain probe (10 μM) and 3 μL of BQ chain probe (10 μM), add 4 μL of TAE buffer, react at 85 °C for 5 min, cool to 25 °C and hold for 20 min, then add 4 μL of helper chain B probe solution (10 μM), mix, and incubate at 25 °C for 20 min to obtain substrate B;

[0067] Substrate A and substrate B were mixed in a 1:2 molar ratio (6.5 μL of substrate A and 13 μL of substrate B), and 1 μL of trigger chain B (1 μM) was added, for a total volume of 20.5 μL. The mixture was reacted at 25 °C for 60 min to obtain a hyperbranched dendritic nanomolecule solution.

[0068] (3) Target acquisition and hairpin chain processing

[0069] A 36 μL mixed reaction system was constructed, containing 18 μL of 1X buffer (containing 50 mM HEPES, 150 mM NaCl, 15 mM MgCl2, pH=7.5), 4 μL each of the sub probe (10 μM) and deoxyribozyme probe (10 μM), and 6 μL of DEPC water. The mixture was heated to 80 °C and held for 2 min, then cooled to room temperature for 10 min to promote hybridization between the sub probe and Dz. The test solution was then directly added and incubated at 25 °C for 10 min. Finally, the DNase present in the bacterial culture was inactivated at 80 °C for 5 min. The pretreated solution was then mixed thoroughly with 10 μL each of the annealed hairpin strand probe and incubated at 25 °C for 60 min to obtain the pretreated test solution.

[0070] (4) Signal detection

[0071] Take 20 μL of streptavidin-modified magnetic beads, wash them three times with Buffer 1 buffer solution, add 10 μL of the pretreated test solution obtained in step (3) above (the main component is the hairpin chain opened by the target), react at 4 °C for 60 min to allow the hairpin chain to connect to the surface of the magnetic beads, after the reaction is completed, remove the supernatant on the magnetic separator, and wash the remaining product with HEPES buffer by shaking, repeat three times. Then, 8 μL of HEPES buffer and 2 μL of the hyperbranched dendritic nanomolecule solution prepared in step (2) above were added, and the mixture was incubated at 37°C for 60 min to allow the dendritic nanomolecules to bind to the open hairpin strands. After the reaction, the supernatant was aspirated on a magnetic separator, and the mixture was washed three times with HEPES buffer. Then, 19 μL of LEPC water, 4 μL of crRNA (4 μM), and 3 μL of CAS12a (10 μM) were added, mixed well, and reacted at 25°C for 13 min to allow the crRNA and CAS12a to bind to the ssDNA fragments on the dendritic nanomolecules bound to the magnetic beads. Then, 7.5 μL of fluorescent probe (1 μM) was added, and the mixture was reacted at 37°C in the dark for 120 min, followed by a reaction at 75°C for 5 min. The CAS12a protein was inactivated by min. Finally, 20 μL was placed in a new EP tube, 180 μL of water was added and mixed evenly. The mixture was then detected by a fluorescence spectrophotometer RF-6000. The excitation wavelength was set to 520 nm, and the maximum peak value was measured at the emission wavelength EM=520 nm.

[0072] Example 1: Feasibility analysis of a Salmonella live bacteria detection system based on deoxyribonuclease activation.

[0073] The feasibility of the experimental design was verified using PAGE electrophoresis. The experiment consisted of four control groups and three experimental groups. The control groups were lane 1 (trigger chain A only), lane 2 (trigger chain B only), and lanes 4 and 5 (hairpin chains only). The experimental groups were divided into three subgroups to investigate whether the hairpin chain could be opened by trigger chain A (lane 6: hairpin chain and trigger chain A), whether an opened hairpin chain could bind to trigger chain B (lane 7: hairpin chain, trigger chain A, and trigger chain B), and whether trigger chain B could open a closed hairpin chain on its own (lane 3: hairpin chain and trigger chain B). The results are as follows: Figure 4 As shown. Lane 1 is trigger chain A, lane 2 is trigger chain B, lane 3 from top to bottom is a hairpin chain and trigger chain B, lanes 4 and 5 are both hairpin chains, lane 6 is an open hairpin chain, and lane 7 from top to bottom is a combination of an open hairpin chain and trigger chain B, with the open hairpin chain... Figure 4 It is known that the trigger strand A generated by specific cleavage by Salmonella ribonuclease can effectively deconstruct the secondary structure of hairpin DNA. Figure 4 Adding a lane containing both the hairpin strand and trigger strand A linearizes the bond and exposes the binding site of trigger strand B. Notably, the linearized hairpin strand forms a stable complex with trigger strand B. Figure 4 The first band in lane 7), while the untriggered original hairpin chain, due to steric hindrance, cannot specifically bind to the trigger chain B ( Figure 4 Lane 6). Electrophoretic mobility difference analysis further confirmed that the system can only generate an active intermediate that can bind to the dendritic DNA nanostructure when trigger chain A participates in the reaction. The difference in band distribution between the experimental group and the control group under the DNA marker reference provides key molecular interaction evidence for the subsequent construction of a cascade signal amplification system.

[0074] Experimental Example 2: Specificity analysis of the target by the Salmonella live bacteria detection system.

[0075] To examine the specificity of this technology for detecting Salmonella, Staphylococcus aureus, Salmonella, Escherichia coli, and a mixed bacterial solution containing Salmonella, along with sterile water, were selected as controls. The detection method described in Example 2 was used to detect each sample, thus verifying the specificity of the system. Under the same optimal conditions, the signal values ​​were measured at EM=520 nm. Three parallel experiments were conducted, and the results are as follows: Figure 5As shown, the signal values ​​of the Salmonella-added group and the mixed bacterial solution group containing Salmonella after the reaction were significantly higher than those of other groups. The experiment shows that this technique has high specificity for Salmonella.

[0076] Experimental Example 3: Quantitative analysis of a live Salmonella detection system.

[0077] The Salmonella suspension obtained in Example 1 was serially diluted to prepare 10 μL solutions. 8 10 7 10 6 10 5 10 4 10 3 Salmonella suspensions at concentrations of 500, 100, and 50 CFU / mL were used to quantitatively detect live Salmonella standard samples under optimal experimental conditions, following the steps in Example 2. The results are as follows: Figures 6-8 As shown. By Figure 6 The results show that the fluorescence spectral intensity increases with increasing bacterial concentration, corresponding to the following values ​​from top to bottom: 10 8 10 7 10 6 10 5 10 4 10 3 Fluorescence spectra of Salmonella at concentrations of 1, 500, 100, and 50 CFU / mL were obtained. The results showed that when the Salmonella concentration was below 1.0 × 10⁻⁶ CFU / mL... 7 At CFU / mL, the fluorescence intensity at 520 nm was positively correlated with the bacterial concentration (R0). 2 =0.993). By Figures 7-8 It can be seen that the concentration of Salmonella is between 50 and 10. 7 A significant linear correlation was observed between CFU / mL, with the regression equation ΔF520 nm = 518.4 log C + 4367.7 (R² = 0.9939), where ΔF520 nm represents the relative fluorescence intensity at 520 nm; log C (CFU / mL) is the logarithm of the Salmonella concentration. The correlation was significant between 50 and 10 nm. 7 Within the range of CFU / mL, the relative fluorescence intensity value was linearly correlated with the logarithm of Salmonella concentration, and the detection limit was 14 CFU / mL (calculation standard: LOD=3σ / S; σ is the standard deviation of the blank solution, and S is the linear slope).

[0078] Experiment Example 4: Detection of Salmonella in Milk

[0079] To evaluate the detection performance of this Salmonella live bacteria detection system in actual sample testing, a spiking method was used to simulate real samples. Different concentrations of Salmonella were added to 10-fold diluted milk samples, with concentrations of 1×10⁻⁶. 3 CFU / mL, 1×10 4 CFU / mL, 1×10 5 The Salmonella culture used was prepared in the example and was diluted with buffer solution without any pretreatment to simulate the detection environment of the actual sample. The experimental results are shown in Table 2.

[0080] Table 2 Results of spiked recovery experiments in milk samples

[0081]

[0082] As shown in Table 2, the average recovery rate of this Salmonella live bacteria detection system was between 98.6% and 101.2%, demonstrating strong resistance to matrix interference and high detection accuracy.

[0083] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this application.

Claims

1. A method for detecting live Salmonella bacteria not for disease diagnosis or treatment purposes, characterized in that, Includes the following steps: S1. Probe pretreatment: The lyophilized powders of Sub probe, deoxyribozyme probe, trigger strand B probe, AF strand probe, AQ strand probe, auxiliary strand A probe, BF strand probe, BQ strand probe, auxiliary strand B probe, crRNA and fluorescent probe were respectively prepared into 100 µM solutions with DEPC water; the hairpin strand probe was added to annealing buffer to form a hairpin structure. S2. Synthesis of hyperbranched dendritic nanomolecules: AF and AQ chain probe solutions were mixed and reacted with TAE buffer, followed by incubation with auxiliary chain A probe solution to obtain substrate A; BF and BQ chain probe solutions were mixed and reacted with TAE buffer, followed by incubation with auxiliary chain B probe solution to obtain substrate B; substrate A and substrate B solutions were mixed and trigger chain B probe was added to react and obtain hyperbranched dendritic nanomolecule solution. S3. Target acquisition and hairpin chain treatment: Mix the Sub probe, deoxyribozyme probe, DEPC water and buffer for hybridization reaction, then add the test solution and incubate. After incubation, heat to inactivate the deoxyribozyme. Finally, add the hairpin chain probe solution and mix well to obtain the pretreated test solution. S4. Signal Detection: Wash the magnetic beads with buffer solution, then add the pretreated test solution obtained in step S3 to react, so that the open hairpin chain probe in the pretreated test solution connects to the surface of the magnetic beads. After the reaction, remove the supernatant on the magnetic separator and wash the remaining product with HEPES buffer. After washing, add the hyperbranched dendritic nanomolecule solution prepared in step S2 and HEPES buffer to react. After the reaction, remove the supernatant on the magnetic separator and wash the remaining product with HEPES buffer. Then add crRNA probe, Cas12a protein and DEPC water to react. Next, add fluorescent probe to react in the dark. Finally, inactivate Cas12a protein and measure the fluorescence intensity with a fluorescence spectrophotometer. The sequence of the Sub probe is shown in SEQ ID NO.1, the sequence of the deoxyribozyme probe is shown in SEQ ID NO.2, the sequence of the trigger strand B probe is shown in SEQ ID NO.4, the sequence of the hairpin strand probe is shown in SEQ ID NO.5, the sequence of the AF strand probe is shown in SEQ ID NO.6, the sequence of the AQ strand probe is shown in SEQ ID NO.7, the sequence of the auxiliary strand A probe is shown in SEQ ID NO.8, the sequence of the BF strand probe is shown in SEQ ID NO.9, the sequence of the BQ strand probe is shown in SEQ ID NO.10, the sequence of the auxiliary strand B probe is shown in SEQ ID NO.11, and the sequence of the crRNA is shown in SEQ ID NO.

12.

2. The method according to claim 1, characterized in that, The sequence of the fluorescent probe is: TCCCCCCT.

3. The method according to claim 1, characterized in that, In step S2, the molar ratio of substrate A to substrate B is 1:2, and in step S3, the molar ratio of Sub probe to deoxyribozyme probe is 1:

1.

4. The method according to claim 1, characterized in that, The annealing buffer in step S1 contains 50 mM HEPES buffer, 150 mM NaCl, and 15 mM MgCl2.

5. The method according to claim 1, characterized in that, In step S2, the molar ratio of the AF chain probe, AQ chain probe, and auxiliary chain A probe is 2:3:4; in step S2, the molar ratio of the BF chain probe, BQ chain probe, and auxiliary chain B probe is 2:3:

4.

6. The method according to claim 1, characterized in that, In step S4, the magnetic beads are modified with streptavidin.

7. The method according to claim 1, characterized in that, In step S4, the fluorescence spectrophotometer detects the signal value at an emission wavelength EM=520 nm.

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