A pathogenic bacteria multiple raman / visualization dual-mode detection method and kit based on cas12a cis / trans cleavage activity

Abstract

The application discloses a pathogenic bacteria single-tube multiple Raman / visual detection method and kit based on Cas12a and in-situ crRNA synthesis strategy and belongs to the technical field of food safety detection. A single-molecule allosteric hairpin DNA containing a target recognition region, a T7 promoter and a crRNA template is designed, the 16S rRNA of the target bacteria is specifically responded, the recombination of the hairpin structure and the in-situ synthesis of the crRNA are started, the cis and trans cleavage of Cas12a is guided, the rapid visual detection of the total bacterial amount and the SERS identification and quantitative detection of different pathogenic bacteria targets are realized. The problems of insufficient qualitative accuracy and quantitative sensitivity caused by the interference of biological matrix in the existing pathogenic bacteria rapid detection method are solved, the dependence of the CRISPR / Cas12a method on the target PAM site is overcome, and the detection range of the nucleic acid target of the foodborne pathogenic bacteria is greatly widened. The Raman / visual dual-mode detection system is suitable for the scene of on-site rapid detection, the detection limit is as low as the single-cell level, the specificity and reproducibility are excellent, and the system can be used in scenes such as food safety control and live pathogenic bacteria diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a method and kit for the dual-mode detection of pathogenic bacteria based on Cas12a cis / trans cleavage activity using multiple Raman / visualization. Background Technology

[0002] Surface-enhanced Raman spectroscopy (SERS) is a molecular scattering spectroscopy technique based on Raman scattering and the plasmon resonance effect on the surface of noble metal nanomaterials. By introducing an enhancing substrate, the Raman scattering signal intensity of the analyte molecule can be increased by 6 to 15 orders of magnitude, effectively overcoming the limitation of low Raman scattering signal intensity and possessing ultra-high detection sensitivity at the single-cell / single-molecule level. SERS-based detection methods offer advantages such as simple sample pretreatment, fast response speed, molecular fingerprint recognition, narrow half-maximum width, ease of operation, and simultaneous detection of multiple components. Based on these characteristics, SERS technology has attracted widespread attention in various fields, including precise screening of harmful substances in food, monitoring of environmental pollutants, and diagnosis of clinical disease biomarkers, showing broad application prospects in the field of food quality and safety testing. Foodborne pathogens are the main cause of food poisoning incidents, and the types of contaminants are diverse and the infection concentrations are extremely low. Existing detection methods (traditional culture methods, polymerase chain reaction, enzyme-linked immunosorbent assay, etc.) cannot simultaneously meet the comprehensive requirements of sensitivity, timeliness, multiplexing, and field applicability. While SERS technology boasts advantages such as ultra-high sensitivity at the single-cell level and molecular fingerprint recognition, direct SERS methods still struggle to effectively identify and sensitively detect pathogens in complex food matrices due to the extremely low characteristic SERS signal intensity of different pathogens. Labeled SERS methods, by introducing specific tags (aptamers of Raman reporter molecules, antibodies, or nucleic acid probes), significantly improve the sensitivity and specificity of pathogen detection. However, given the complexity of food matrices, the diversity of pathogen species, and extremely low contamination levels (typically below 10 CFU / mL), achieving accurate on-site detection of pathogens at the single-cell level remains a significant challenge. Therefore, establishing a new, highly sensitive multiplex SERS detection method for foodborne pathogens is of great practical importance for improving my country's precise prevention and control capabilities for foodborne pathogens.

[0003] SERS methods targeting pathogenic bacterial nucleic acids have significantly improved detection sensitivity through cascaded signal amplification. Among these, methods based on clustered regularly interspaced short palindromic repeats / CRISPR-associated protein (CRISPR / Cas) have achieved ultra-high sensitivity as low as 10 CFU / mL in real-world sample detection due to their high specificity and signal amplification capabilities. However, many reported methods rely on the trans-cleavage properties of Cas12a / Cas13a, which, while programmable, severely limits single-tube multiplexing capabilities (J. Hazard. Mater. 2025, 488: 137325; Sens. Actuators B Chem. 2024, 399: 134838). To overcome the bottleneck in multiplexing detection, current research relies on spatial segmentation or droplet encoding strategies using microfluidic technology, but still faces technical challenges such as complex high-precision chip fabrication processes, high costs, and insufficient stability of multifunctional chips.

[0004] Cas12a's cis-cleavage activity (i.e., its ability to specifically recognize and cleave target nucleic acids) offers excellent potential for single-tube multiplex analysis. Theoretically, multiple specific CRISPR RNAs (crRNAs) can be designed to differentiate between different target nucleic acids, which can then be coupled with multiplex Raman reporter molecules to achieve single-tube multiplex SERS detection. However, current methods for detecting pathogenic bacteria based on Cas12a's cis-cleavage activity still face two major limitations: First, Cas12a's cis-cleavage activity is highly dependent on the protospacer adjacent motif (PAM) site in the target nucleic acid, limiting the range of target sequences that can be selected. Second, cis-cleavage is a single-target, single-event cleavage, lacking the non-specific signal amplification effect of trans-cleavage, resulting in insufficient sensitivity in detecting low-abundance targets. Due to these limitations, no single-tube multiplex SERS detection method for pathogenic bacteria based on Cas12a's cis-cleavage activity has been reported to date. Summary of the Invention

[0005] To address the technical problems of existing Cas12a-based methods for detecting foodborne pathogens, such as limited single-tube multiplex detection capabilities, reliance on complex microfluidics or chip technology, insufficient signal amplification, and difficulty in balancing sensitivity and specificity, the present invention aims to provide a dual-mode Raman / visualization detection method and kit for pathogens based on Cas12a cis / trans cleavage activity.

[0006] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides a method for the dual-mode detection of pathogenic bacteria based on Cas12a cis / trans cleavage activity for non-disease diagnosis and treatment purposes, comprising the following steps: The target nucleic acid in the sample to be tested is mixed with the reaction system and incubated at an isothermal temperature; the reaction system includes: a transcriptional amplification element, a Cas protein, a Raman probe, and a fluorescent reporter probe. The transcriptional amplification element uses the target nucleic acid as a trigger to generate guide RNA through in situ transcriptional amplification. Once activated, the Cas protein performs both cis and trans cleavage: cis cleavage specifically cleaves the Raman probe and releases the Raman reporter molecule, while trans cleavage non-specifically cleaves the fluorescent reporter probe and generates a fluorescent signal. Rapid visual screening is achieved by detecting fluorescence signals, and qualitative and / or quantitative analysis of pathogens is achieved by detecting Raman signals.

[0007] The transcriptional amplification element includes T7 RNA polymerase and nucleotide substrate; the target nucleic acid is 16S rRNA of pathogenic bacteria; and the specific detection of live bacteria is achieved by targeting RNA.

[0008] The Raman probe uses microspheres as carriers, loads at least one Raman reporter molecule, and is functionalized with nucleic acid strands; the reaction system contains two or more Raman probes loaded with different Raman reporter molecules; the different Raman reporter molecules released by cis-cleavage correspond to different target pathogens, thereby achieving single-tube multiplex detection.

[0009] Preferably, the Raman probe is prepared as follows: using polystyrene (PS) microspheres as a carrier, Raman reporter molecules (such as 4-MBA or 4-NTP) are loaded via physical adsorption or chemical coupling; target-specific nucleic acid chains (NTS-COOH) are covalently linked to the surface of the PS microspheres via an EDC / NHS crosslinking reaction; this nucleic acid chain hybridizes with the complementary strand (TS-Bio) on the magnetic beads to form a double-stranded structure. When Cas12a is activated, its cis-cleavage activity specifically cleaves this double-stranded DNA, releasing the PS microspheres and the loaded Raman reporter molecules. By loading Raman reporter molecules onto microspheres and functionalizing them with nucleic acid chains, an integrated "signal encoding-target recognition" Raman probe is constructed. When the Cas protein performs cis-cleavage, the presence of different targets leads to the specific release of different Raman reporter molecules, thus exhibiting different characteristic peaks in the SERS spectrum. This design allows for the simultaneous detection of two or more pathogenic bacteria in a single reaction system without the need for spatial segmentation or droplet encoding technology using microfluidic chips. Compared with existing CRISPR-SERS methods that rely on trans-cutting, this method completely solves the technical bottleneck of limited multiple detection capabilities, and significantly reduces detection costs and technical barriers.

[0010] The method does not rely on spatial segmentation or droplet encoding of microfluidic chips; in the same reaction system in a single tube, different targets are distinguished by the cis-cleaving activity of Cas12a, and signal amplification is achieved by the trans-cleaving activity.

[0011] The reaction system also includes a reinforcing substrate; the Raman detection is surface-enhanced Raman spectroscopy (SERS); the reinforcing substrate comprises Au@PB nanocomposite material. Specifically, in the Au@PB nanocomposite material, Prussian blue (PB) is present at 2129 cm⁻¹. -1 It exhibits a characteristic Raman peak, located in the Raman silent region of the biological sample (1800-2800 cm⁻¹). -1 Within this region, the food matrix (such as proteins, fats, polysaccharides, etc.) shows no characteristic Raman signals, and is therefore unaffected by sample background interference. (Using 2129 cm⁻¹) -1 The peak intensity is used as an internal standard to calculate the characteristic peak of the target Raman reporter molecule (e.g., 1077 cm⁻¹ for 4-mercaptobenzoic acid). -1 and 1585 cm -1 1335 cm of 4-nitrothiophenol -1 The ratio of the intensity of the laser beam to the internal standard peak can effectively correct systematic errors such as laser power fluctuations, substrate inhomogeneity, and optical path offset, thereby achieving accurate quantitative analysis.

[0012] Secondly, the present invention provides a kit for implementing the method, comprising the following components: (i) Enzyme components: including transcription amplification enzyme and Cas protein; (ii) Nucleic acid components: containing nucleotide substrates and guide RNA or its precursors for recognizing target nucleic acids; (iii) Signal components: including at least one Raman probe and a fluorescent reporter probe; (iv) Enhanced substrate: used for SERS signal detection.

[0013] The transcriptional amplification enzyme is T7 RNA polymerase; the Cas protein is Cas12a; the precursor of the guide RNA is a hairpin DNA probe, which is transcribed into mature crRNA in the reaction system; the Raman probe uses polystyrene microspheres as a carrier, loaded with Raman reporter molecules selected from 4-MBA and / or 4-NTPs; the enhancement substrate is Au@PB nanocomposite material. The enzyme combination of T7 RNA polymerase and Cas12a has been experimentally verified to have good buffer system compatibility, enabling efficient synergistic work in the same reaction tube without the need for stepwise addition. The hairpin DNA / RNA probe, as a precursor of crRNA, is transcribed into mature crRNA by T7 RNA polymerase in the reaction system, achieving "in situ crRNA synthesis" and avoiding the additional costs of pre-synthesizing and purifying crRNA. The Raman probe loaded with 4-MBA / 4-NTPs on polystyrene microspheres features clear signal encoding, controllable release, and good coupling with cis-cleavage. The Au@PB enhancement substrate has dual functions of SERS enhancement and internal standard correction.

[0014] The kit also contains one or more auxiliary components selected from the following: reaction buffer, DTT, MgCl2, tetrahydrofuran, and pathogenic bacterial 16S rRNA standards; the reaction buffer contains NEB Buffer r2.1 and RNAPolReaction Buffer.

[0015] The kit is used for the detection of foodborne pathogens in food samples for non-diagnostic and non-therapeutic purposes; the foodborne pathogens are selected from Escherichia coli O157:H7 and / or Salmonella typhimurium.

[0016] Thirdly, the present invention provides a single-tube multiplexer amplification system based on CRISPR / Cas, comprising: The transcription and amplification module is used to generate guide RNA in situ and perform exponential amplification in response to the presence of target nucleic acids; The Cas enzyme module, whose nuclease activity is activated by a complex formed by the target nucleic acid and the guide RNA generated by transcriptional amplification; The Raman signaling module contains at least one Raman probe that can be released by cis-cleavage of Cas enzyme, and different Raman reporter molecules encode different targets; The fluorescence signal module contains a fluorescent reporter probe that can be trans-cleaved by Cas enzyme; The system simultaneously outputs Raman and fluorescence signals in the same reaction tube, enabling qualitative and quantitative analysis of multiple targets.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a dual-mode detection method for pathogenic bacteria using multiplex Raman / visualization. By integrating transcriptional amplification elements, Cas proteins, Raman probes, and fluorescent reporter probes in the same reaction system, it achieves, for the first time, the synergistic utilization of cis- and trans-cleavage. This method simultaneously converts the recognition and cleavage events of target nucleic acids into Raman and fluorescence signals, outputting two complementary detection results in a single reaction. Compared to existing technologies, this method eliminates the need for step-by-step operations or additional transfer steps, significantly simplifying the detection process. Simultaneously, it utilizes transcriptional amplification elements to pre-amplify signals from low-abundance targets, effectively overcoming the technical deficiency of insufficient sensitivity in direct detection using CRISPR / Cas systems. This provides a novel technical solution for the detection of trace pathogenic bacteria. This method achieves accurate and highly sensitive SERS quantitative detection of multiple bacterial species, solving the core problems faced by classic pathogenic bacteria SERS detection methods, such as overlapping characteristic signals and difficulty in detecting trace pathogenic bacteria in complex food matrices.

[0018] Furthermore, T7 RNA polymerase-mediated transcriptional amplification is characterized by high efficiency, specificity, and isothermal properties, enabling bi-exponential amplification of target RNA under isothermal conditions, significantly improving the detection sensitivity of low-abundance targets. Simultaneously, targeting 16S rRNA effectively distinguishes between live and dead bacteria, as RNA degrades rapidly in dead bacteria. Traditional PCR methods cannot differentiate between live and dead bacteria, easily leading to false positive results. This method achieves a technological breakthrough of "detecting only live bacteria" by targeting RNA, avoiding misjudgments caused by detecting dead bacteria. By cleverly utilizing the cis-cleavage activity of Cas12a and coupling it with an encoding Raman probe, multiplex detection can be achieved in ordinary reaction tubes, completely eliminating the dependence on microfluidic chips. This allows the method to truly be applied to rapid on-site detection scenarios, demonstrating significant practical advantages in resource-constrained conditions such as food safety supervision and environmental monitoring. Au@PB was used as the enhancement substrate, with Prussian blue at 2129 cm⁻¹. -1 Using the intensity of the low biological background characteristic peak as an internal standard, the ratio of the 4-MBA / 4-NTP characteristic peak signal intensity to the internal standard signal intensity is calculated as the identification and quantification standard. This effectively avoids non-specific background interference from the food matrix and solves the problems of traditional matrix interference and insufficient qualitative and quantitative accuracy.

[0019] The kit provided by this invention, which implements the aforementioned dual-mode detection method for pathogenic bacteria using multiplex Raman / visualization, pre-prepares and dispenses the core components required for detection (enzyme components, nucleic acid components, signal components, and enhancement substrate) in kit form. Users do not need to prepare or optimize each component themselves; they only need to add the sample to be tested to complete the detection. The kit format facilitates standardized production, quality control, and commercialization, providing a product carrier for the industrial transformation of the technology. This kit can effectively detect the target bacteria in real food samples such as pasteurized milk, chilled chicken breast, and lettuce, achieving 100% consistency with the RT-qPCR gold standard method, fully verifying the practicality and reliability of the kit in complex food matrices.

[0020] This invention provides a CRISPR / Cas-based single-tube multiplex signal amplification system that organically integrates a transcription amplification module, a Cas enzyme module, a Raman signal module, and a fluorescence signal module, constructing a complete signal transduction chain of "target recognition - signal amplification - dual-mode output." The transcription amplification module solves the "bottleneck" problem of low-abundance targets failing to activate the Cas enzyme. The Raman and fluorescence signal modules utilize the different characteristics of cis- and trans-cutting, respectively, to achieve functional complementarity between "multiple encoding" and "universal amplification." The dual-mode output mechanism allows users to flexibly select the detection mode according to actual needs: fluorescence mode for rapid screening and SERS mode for precise quantification, with neither interfering with the other and providing complementary information. A dual-mode detection system adaptable to all scenarios is constructed, establishing a Raman / visualization dual-mode detection strategy. The visualization mode enables rapid on-site initial screening of total viable bacteria without the need for large instruments, adapting to rapid on-site detection scenarios. The SERS mode achieves precise quantification and species differentiation of multiple pathogens through Raman reporter molecule encoding, overcoming the problems of insufficient sensitivity, poor quantitative accuracy, and insufficient multiplexing capability of traditional SERS methods. This method integrates target recognition, transcriptional amplification, and signal output into a single-pot system, eliminating the need for multi-step pipetting and cap opening, reducing the risk of aerosol contamination, simplifying operation, and meeting the needs of on-site testing. Allosteric hairpin DNA probes (HP) assist T7 RNA polymerase-mediated double-exponential transcriptional amplification to generate a large number of crRNA sequences in situ, overcoming the target PAM site dependence of the classic CRISPR / Cas12a method, and greatly broadening the screening range of nucleic acid targets for foodborne pathogens. Attached Figure Description

[0021] Figure 1This is a schematic diagram illustrating the detection principle of the method of the present invention. (A) Schematic diagram of T7 RNA polymerase-mediated double-exponential transcriptional amplification; the left side shows the closed hairpin structure of the hairpin DNA probe in the absence of a target (including the stem, loop, and T7 promoter sequence); the middle shows the opening of the hairpin structure after hybridization of the target 16S rRNA with the hairpin probe recognition sequence, triggering conformational recombination; the right side shows the exposed T7 promoter initiating T7 RNA polymerase-mediated transcriptional amplification, producing crRNA in situ; (B) Schematic diagram of Cas12a's cis-cleavage of the Raman probe and trans-cleavage of the FL beacon; (C) Schematic diagrams of Raman / visualization detection in the presence of only a single target bacterium, the simultaneous presence of two target bacteria, and the absence of a target bacterium.

[0022] Figure 2 The figures show experimental results for verifying the feasibility of the method of this invention. Among them, (A) polyacrylamide gel electrophoresis verifies T7 RNA polymerase-assisted transcriptional amplification and Cas12a cis / trans cleavage activity; (B) feasibility verification of SERS detection in the presence of only a single target bacterium, in the presence of two target bacteria, and in the absence of target bacteria; (C) feasibility verification of visual detection in the presence of only a single target bacterium, in the presence of two target bacteria, and in the absence of target bacteria.

[0023] Figure 3 The graph shows the effect of four key parameters on detection performance. Among them, (A) the effect of Cas12a concentration on detection performance; (B) the effect of Mg2+ concentration on detection performance; (C) the effect of reaction temperature on detection performance; and (D) the effect of reaction time on detection performance.

[0024] Figure 4 The results of the detection performance analysis of the method of the present invention are as follows: (A) SERS spectra and acquired images of nucleic acids at different concentrations; (B) Standard curve in SERS detection mode; (C) Standard curve in visualization detection mode; (D) Detection specificity; (E) SERS detection reproducibility; (F) Reproducibility of visualization detection.

[0025] Figure 5 The results are actual sample detection results of the method of this invention. Among them, (A) is a schematic diagram of the process of this method and the RT-qPCR detection method; (B) a comparison of sample detection results in SERS mode and visualization mode of this method with RT-qPCR detection results; (C) a confusion matrix of E. coli O157:H7 in SERS detection mode; (D) S. Typhimurium (E) Confusion matrix in SERS detection mode; (E) Confusion matrix in visual detection mode. Detailed Implementation

[0026] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0027] It should be noted that the terms "first," "second," etc., used in the specification and claims are only used to distinguish similar objects and do not necessarily describe a specific order or sequence; it should be understood that these terms can be used interchangeably where appropriate. Furthermore, the terms "comprising," "having," and any variations thereof are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to the expressly listed steps or units, but may include other steps or units not expressly listed or inherent to it.

[0028] like Figure 1 As shown, this invention provides a dual-mode detection method for pathogenic bacteria based on Cas12a cis / trans cleavage activity using multiple Raman / visualization, comprising the following steps: Step 1: Using PS microspheres as a carrier, load the encoded Raman reporter and functionalize it with a non-target chain, then hybridize it with magnetic beads functionalized with a target chain to prepare a Raman probe.

[0029] Step 2: A gold seed solution was prepared using a chemical reduction method. Subsequently, an oxidation treatment was applied to the gold surface using a K3[Fe(CN)6] solution, and K4[Fe(CN)6] and FeCl3·6H2O were added as Prussian blue precursors to prepare an Au@PB reinforced substrate.

[0030] Step 3: Prepare a mixed reaction buffer in advance, and add the following components to the reaction buffer in sequence: T7 RNA polymerase, Cas12a, HP, NTPs, Raman probe, and FL beacon.

[0031] Step 4: Add the extracted total bacterial RNA sample to the above system, mix well, and incubate at 37°C for 35 minutes. After the reaction, use a smartphone to capture sample images and add Au@PB to the reaction system to enhance the SERS signal reading of the samples.

[0032] The average grayscale value in the visualized detection results was acquired and calculated using ImageJ software. The ratio of the acquired SERS spectrum and the intensity of the 4-MBA / 4-NTP characteristic signal to the intensity of the internal standard signal was calculated using BWS software.

[0033] The foodborne pathogens used in the embodiments of this invention are Escherichia coli O157:H7 ATCC35150 and Salmonella Typhimurium ATCC14028, which were purchased from the American Type Culture Collection (ATCC).

[0034] The present invention relates to DNA probe sequences as shown in Table 1.

[0035] Table 1: DNA Sequence Information

[0036] The experimental method for step 2 of this invention is as follows: 200 μL of 1 mM K3[Fe(CN)6] was added to the 50 nm Au NPs seed solution for oxidation treatment, followed by the addition of 1 mL of a mixture of 0.1 mM K4[Fe(CN)6]·3H2O and FeCl3·6H2O, and the mixture was stirred vigorously at room temperature for 3 hours.

[0037] The buffer composition for step 3 of this invention is as follows: 20 μL 1×NEB Buffer r2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg / mL recombinant albumin, pH 7.9), 10 μL 10×RNAPol Reaction Buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8), and 60 μL DEPC water.

[0038] The visualization detection parameters in step 4 of this invention are: a blue-green light source with an excitation wavelength of 418 nm, and an exposure time ranging from 0.2 to 2 s.

[0039] The SERS detection parameters in step 4 of this invention are: excitation wavelength 785 nm, integration time 5 s, integration times 3, and laser power 256 mW.

[0040] The total bacterial RNA sample described in this invention was extracted using a commercially available kit based on the Trizol extraction method.

[0041] The present invention also includes a foodborne pathogen RNA detection kit based on T7 RNA polymerase and CRISPR / Cas12a, the kit comprising T7 RNA polymerase, CRISPR / Cas12a, reaction buffer, NTPs, etc.

[0042] The system described in this invention can be implemented using the aforementioned kit, wherein each component of the kit corresponds to a functional module of the system: the transcription amplification enzyme and Cas protein constitute the Cas enzyme module, HP and NTPs constitute the transcription amplification module, and the Raman probe and fluorescent reporter probe constitute the Raman signal module and the fluorescent signal module, respectively.

[0043] Example 1: Establishment of a Single-Tube Multiplex Detection Method for Pathogenic Bacteria Based on Cas12a and Signal Amplification Raman Tags 1.1 Construction of the reaction system Prepare the mixed reaction buffer in advance: 20 μL 1×NEB Buffer r2.1, 10 μL 10×RNAPolReaction Buffer and 60 μL DEPC water, mix thoroughly and set aside.

[0044] Take 18 μL of the above mixed reaction buffer and add each reaction component sequentially, ensuring the final concentrations meet the following requirements: T7 RNA polymerase 20 U / mL, Cas12a 200 nM, HP 400 nM, NTPs 2.5 mM, FL beacon 1600 nM, DTT 5 mM, RNase inhibitor 1 U / mL, MgCl2 5 mM. Then, add 10 μL of the pre-prepared Raman probe according to the detection requirements and vortex thoroughly to mix.

[0045] 1.2 Dissolution and Annealing of HP Place the lyophilized HP powder in a centrifuge at room temperature and centrifuge at 6000 rpm for 1 min to prevent powder loss. Add an appropriate amount of 1×TE buffer and vortex for 5 min to dissolve completely, preparing a 100 μM stock solution. Store in the refrigerator for later use.

[0046] To obtain the specific hairpin-shaped secondary structure, a slow annealing procedure was adopted: HP stock solution was mixed with annealing buffer (1×TE buffer, 10 mM MgCl2), heated at 95°C for 5 min, then cooled to 25°C at a rate of 1°C / min, and stored in a 4°C refrigerator for later use.

[0047] After the above annealing procedure, the formation of the hairpin structure of *HP* was verified by non-denaturing polyacrylamide gel electrophoresis: the *HP* with the closed hairpin structure migrated significantly faster in the gel than the linearized control, confirming that it formed the expected stem-loop secondary structure. This hairpin structure underwent conformational reorganization in the presence of the target 16S rRNA, and its migration rate decreased after opening, further confirming the target-triggered structural transition.

[0048] 1.3 Transcription amplification and enzyme digestion activity verification Using the 16S rRNA of E. coli O157:H7 as the target, 5 μL of total bacterial RNA sample was added to the above reaction system and incubated at 37℃ for 35 min. A blank control group without the target was set up at the same time.

[0049] Verification was performed using 15% polyacrylamide gel electrophoresis: 1×TBE buffer was pre-cooled to 4°C, electrophoresis was performed at 115 V for 90 min, and imaging was performed after staining with 10000×4S GelRed nucleic acid dye for 15 min. Results showed that the experimental group exhibited both transcript bands and Cas12a cis-cleavage product bands, while the control group showed no corresponding bands, confirming the successful cascade reaction of T7 RNA polymerase-mediated double-exponential transcription and Cas12a cleavage (see...). Figure 2 (A)

[0050] 1.4 Validation of Fluorescence and SERS Detection Fluorescence (visualization) detection: After the reaction, images were captured in a dark chamber using a smartphone under 418 nm light source illumination, and the grayscale values ​​were analyzed using ImageJ software. The results showed that the positive group exhibited significant green fluorescence, with a significantly higher grayscale value than the negative group (P<0.01), confirming the effectiveness of Cas12a trans-cutting and visualization detection (see...). Figure 2 (C)

[0051] SERS detection: After the reaction was completed, the supernatant was collected, and an equal volume of tetrahydrofuran (THF) was added and vortexed to cause the PS microspheres to swell and release a large amount of highly dispersed Raman reporter (4-MBA or 4-NTP). After centrifugation, the supernatant was mixed with the Au@PB enhanced substrate, and the signal was acquired using a portable Raman spectrometer (excitation wavelength 785 nm, integration time 5 s, integration times 3, laser power 256 mW). The results showed that the characteristic peak of the target Raman reporter molecule was observed in the positive group, and its intensity ratio with the Au@PB internal standard peak was significantly increased, confirming the effectiveness of Cas12a cis-cleavage and SERS signal output (see...). Figure 2 (B)

[0052] This embodiment successfully established a single-tube multiplex Raman / visual dual-mode detection method for foodborne pathogens based on Cas12a cis / trans cleavage activity. This method combines T7 RNA polymerase-mediated transcriptional amplification, CRISPR / Cas12a specific recognition and cleavage, and highly sensitive signal output from Raman probes and Au@PB-enhanced substrates, achieving rapid, accurate, dual-mode detection of bacterial RNA samples with good specificity and sensitivity.

[0053] Example 2: Single-tube multiplex nucleic acid detection based on Cas12a and signal amplification Raman tags 2.1 Preparation of core detection components Using the PS Raman tag loaded with 4-mercaptobenzoic acid (4-MBA) / 4-nitrobenzenethiophenol (4-NTP) prepared in the previous section of this invention, and a gold@Prussian blue (Au@PB) enhanced substrate, target detection was performed directly according to the established detection system. To verify the universality and effectiveness of the method in nucleic acid detection, and considering the convenience of experimental operation, this invention synthesized a tag compatible with E. coli O157:H7 and Salmonella typhimurium (…). S. typhimurium The invention includes two DNA fragments, tDNA1 and tDNA2, which are homologous to the 16S rRNA sequence. Since all detection elements in this invention are designed for the 16S rRNA sequences of these two bacteria, and homologous DNA fragments are used as alternative targets, the nucleic acid recognition and signal amplification capabilities of the detection system can be effectively verified without relying on live bacterial culture and RNA extraction. Sequence information is shown in Table 1.

[0054] Nucleic acid lyophilized powder synthesized by Sangon Biotech (Shanghai) Co., Ltd. was dissolved in 1×TE buffer, and its mass concentration (ng / μL) was determined by a micro spectrophotometer. Subsequently, the above nucleic acid stock solution was serially diluted with 1×TE buffer to obtain corresponding concentrations of 8.71×10⁻⁶. -5 ~1.15×10 2 ng / μL target nucleic acid working solution, used for subsequent detection and analysis of sensitivity, specificity and reproducibility.

[0055] 2.2 Precise Quantitative Detection of SERS Add the target nucleic acid working solutions of the above-mentioned gradient concentrations to the constructed multiplex reaction system, and incubate at 37°C for 35 min. Complete SERS signal acquisition and analysis according to the established procedure, using Au@PB 2129 cm⁻¹. -1 The characteristic peak is used as an internal standard; the intensity ratio of the characteristic peak of the target Raman reporter molecule to the internal standard peak is calculated. The results are as follows: Figure 4 Figures A and B show that the target nucleic acid concentration is 8.71 × 10⁻⁶. -5 ~1.15×10 1 Within the range of ng / μL, the characteristic peak intensity ratio showed a good linear relationship with the target nucleic acid concentration, and the coefficient of determination (R0) was high. 2 The limits of detection for the target nucleic acids were 0.98 and 0.97, respectively; and the limits of detection for the target nucleic acids were as low as 1.35 × 10⁻⁶. -5 ng / μL, 1.17×10 - 5 The above results indicate that the method of the present invention achieves highly sensitive and accurate quantitative detection of target nucleic acids. (The detection limit is calculated by dividing the standard deviation of the blank signal by 3 times the slope of the standard curve.)

[0056] 2.3 Visualized Quick Filtering After the reaction, the reaction tubes were placed in a gel imaging system, and fluorescence was excited using a single 418 nm light source in a dark chamber, with images captured using a smartphone. Grayscale values ​​were analyzed using ImageJ software, and the target nucleic acid concentration was found to be 8.71 × 10⁻⁶. -5 ~1.15×10 1 It exhibits a good linear relationship with grayscale value within the range of ng / μL, R 2 The values ​​were 0.93 and 0.94, respectively, and the limits of detection were 6.7 × 10⁻⁶. -5 ng / μL (tDNA1) and 7.8×10 -5 ng / μL (tDNA2) Figure 4 (C) The above results indicate that the method of the present invention is also applicable to rapid visual screening.

[0057] 2.4 Specificity and Reproducibility Verification To further verify the specificity of this method for nucleic acid sequences, five interfering DNA sequences with the same length as the target nucleic acid sequence, similar GC content, and no complex secondary structure were synthesized as interference templates. Dual-mode detection was then performed under the same reaction system and detection conditions. Figure 4 The results showed that only the target nucleic acid exhibited specific fluorescent signals and a significant SERS ratio, confirming the method's excellent specificity.

[0058] In five independent batch experiments, 15 sets of SERS spectra and 50 sets of visual image grayscale values ​​were acquired in parallel for target nucleic acid at a concentration of 11.48 ng / μL, and the relative standard deviation (RSD) was calculated for each batch. Figure 4 Figures E and F show that, under the SERS detection mode, the RSDs of tDNA1 and tDNA2 were 3.07% and 3.65%, respectively; under the visualization detection mode, the RSDs of tDNA1 and tDNA2 were 5.72% and 6.33%, respectively. The RSDs under both modes were less than 7%, confirming that the method of the present invention has good reproducibility for the detection of nucleic acid targets.

[0059] The above results fully demonstrate that the method of the present invention has the advantages of high sensitivity, high specificity, rapid visual screening and accurate SERS quantitative analysis, and is suitable for multiplex detection of nucleic acids of foodborne pathogens.

[0060] Example 3: Detection method based on Cas12a and signal amplification Raman tag for detecting live foodborne pathogens in real samples. 3.1 Sample Pretreatment and RNA Extraction 45 samples were purchased from the local market, including 15 samples each of pasteurized milk, chilled chicken breast, and lettuce. For liquid samples (milk): 1 mL of sample was added to 9 mL of sterile 1×TE buffer, diluted 10-fold, and centrifuged at 4°C and 8000 rpm for 5 min. The precipitate was resuspended in 1 mL of 1×TE buffer. For solid samples (chicken breast and lettuce): 5 g of homogenized sample was weighed, added to 45 mL of sterile 1×TE buffer, vortexed thoroughly for 2 min, and centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was collected. Total RNA was extracted using a column-based bacterial RNA extraction and purification kit (Sangon Biotech, B518655) combined with lysozyme treatment: 1 mL of the treated sample solution was centrifuged at 8000 rpm for 1 min to collect bacterial cells. 100 μL of 3 mg / mL lysozyme solution was added, and the procedure was followed according to the kit instructions. Finally, the RNA was dissolved in 20 μL of 1×TE buffer (pH 8.0) prepared with DEPC water for later use.

[0061] 3.2 Comparison of Actual Sample Testing and Results The above samples were subjected to testing for the target bacterium Escherichia coli (E. coli). E. coli O157:H7 and Salmonella typhimurium ( S. typhimurium After RNA extraction, the Raman / visualization dual-mode detection method established in this invention was used for detection, while real-time quantitative PCR (RT-qPCR) was used as a control and detected according to standard procedures. Figure 5 (A)

[0062] The results are as follows Figure 5 As shown in Figure B, there are 6 positive samples detected by RT-qPCR. Among them, a single... E. coli Three samples were contaminated with O157:H7, and one type of O157:H7 contamination was observed. S. Two samples were found to be contaminated with typhimurium. E. coli O157:H7 and S. One sample with mixed typhimurium contamination was identified. In contrast, the method of this invention exhibited significant fluorescence signals in the visualization detection mode, with image grayscale values ​​exceeding the threshold of the blank group. In SERS mode, it accurately distinguished the species and concentration of the target bacteria. E. coli O157:H7 positive samples showed a 4-MBA characteristic peak (1077 cm⁻¹). -1 and 1585 cm -1 ) and Au@PB internal standard peak (2129 cm) -1 The ratio of ) increases; S. The typhimurium positive sample showed a characteristic peak of 4-NTP (1335 cm⁻¹). -1The ratio of the peak of the two Raman reporter molecules to the internal standard increased; mixed contaminated samples showed characteristic signals of both Raman reporter molecules simultaneously. Furthermore, the remaining 39 negative samples did not show positive signals in either mode. The consistency rate of positive and negative results between the two methods was 100%, confirming the good practicality of the method in actual sample detection. Figure 5 (CE).

[0063] This embodiment successfully verified the application capability of the detection method based on Cas12a and signal amplification Raman tags in detecting live foodborne pathogens in actual food samples (milk, chicken breast, lettuce). The above results fully demonstrate that the method of this invention possesses comprehensive advantages such as high sensitivity, high specificity, dual-mode output, simple operation, and applicability to complex food matrices, and has broad application prospects in the field of rapid on-site detection and precise quantitative analysis of foodborne pathogens in the laboratory.

[0064] Example 4: Composition and usage of a live foodborne pathogen detection kit based on T7 RNA polymerase and CRISPR / Cas12a. 4.1 Kit Components This kit contains the following components, each in an optimized ratio to ensure stable detection performance: Enzyme mixture: T7 RNA polymerase (20 U / mL), Cas12a (200 nM), RNase inhibitor (1 U / mL), lyophilized powder form; Reaction buffer: 1×NEB Buffer r2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg / ml recombinant albumin, pH 7.9), 10 μL 10×RNAPol Reaction Buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8), premixed solution; Nucleic acid reagents: HP (400 nM), NTPs (25 mM), Raman probe (loaded with 4-MBA / 4-NTP), fluorescent probe (1600 nM); Auxiliary reagents: DTT (50 mM), MgCl2 (50 mM), THF, Au@PB to enhance the substrate; Standard products: E. coli O157:H7 16S rRNA standard, S. typhimurium 16S rRNA standard; Instructions for use: including storage conditions, operating procedures, reaction conditions, result interpretation criteria, and precautions.

[0065] 4.2 Instructions for using the reagent kit Sample preparation: Food or clinical samples were processed according to standard methods. Total RNA was extracted using a commercial RNA extraction kit based on the Trizol extraction method and dissolved in DEPC water for testing.

[0066] Reagent preparation: Add the specified volume of reconstitution buffer to the lyophilized enzyme mixture, vortex for 1 min, and let stand at room temperature for 5 min to fully dissolve; dissolve the HP lyophilized powder in DEPC water to prepare a 100 μM stock solution, and dilute it to a working concentration of 400 nM according to the ratio.

[0067] System assembly: In a sterile reaction tube, add 18 μL of mixed reaction buffer, 0.5 μL of enzyme mixture, 0.5 μL of HP working solution, 2 μL of NTPs, 10 μL of Raman probe, 5 μL of fluorescent probe, 1 μL of DTT, and 1 μL of MgCl2 in sequence. Mix gently by pipetting and aspirating. Finally, add 5 μL of RNA sample, for a total volume of 43 μL.

[0068] Constant temperature incubation: Place the reaction tube in a constant temperature environment of 25~37℃ and incubate for 35~55 minutes, avoiding violent shaking during the process.

[0069] Result interpretation: Visual screening (grayscale values ​​≥ threshold indicate suspicious positives); After the reaction was complete, the reaction tube was placed in a dark box and irradiated with a 418 nm blue-green light source. Take images using a smartphone or gel imaging system (exposure time 0.2~2 s, 0.5 s recommended); Use ImageJ software to analyze the average grayscale value; Interpretation criteria: Gray value ≥ negative control gray value + 3 times standard deviation → suspected positive; Gray value < negative control gray value + 3 times standard deviation → negative.

[0070] Suspected positive samples are tested by SERS. The bacterial species are determined based on the characteristic peak shift, and the concentration is calculated by combining the standard curve. Finally, a test report is issued.

[0071] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A dual-mode Raman / visualization method for the detection of pathogenic bacteria based on Cas12a cis / trans cleavage activity for non-disease diagnosis and treatment purposes, characterized in that, Includes the following steps: The target pathogenic bacteria nucleic acid in the sample to be tested is mixed with the reaction system and incubated at an incubator; the reaction system includes: transcriptional amplification element, Cas protein, Raman probe and fluorescent reporter probe; The transcriptional amplification element uses the target nucleic acid as a trigger to generate guide RNA through in situ transcriptional amplification. Once activated, the Cas protein cis-cleaves the Raman probe to release a Raman reporter molecule, and simultaneously trans-cleaves the fluorescent reporter probe to generate a fluorescent signal. The Raman signal generated by the Raman reporter molecule is detected to perform qualitative and / or quantitative analysis of the target pathogenic bacterial nucleic acid; the fluorescence signal is detected for rapid visual screening.

2. The method for detecting pathogenic bacteria using multiple Raman / visual dual-mode detection according to claim 1, characterized in that, The transcriptional amplification element includes T7 RNA polymerase and a nucleotide substrate; the target nucleic acid is 16S rRNA of pathogenic bacteria.

3. The method for detecting pathogenic bacteria using multiple Raman / visual dual-mode detection according to claim 1, characterized in that, The Raman probe uses microspheres as a carrier and loads at least one Raman reporter molecule; the reaction system contains two or more Raman probes loaded with different Raman reporter molecules.

4. The method for detecting pathogenic bacteria using multiple Raman / visual dual-mode detection according to claim 1, characterized in that, The method described is a single-tube, single-reaction system.

5. The method according to claim 1, characterized in that, The reaction system also includes a reinforcing substrate; the reinforcing substrate comprises Au@PB nanocomposite material.

6. A kit for implementing the multiplex Raman / visualization dual-mode detection method for pathogenic bacteria according to any one of claims 1-5, characterized in that, It contains the following components: (i) Enzyme components: including transcription amplification enzyme and Cas protein; (ii) Nucleic acid components: including nucleotide substrates and guide RNA or its precursors for recognizing the nucleic acids of target pathogens; (iii) Signal components: including at least one Raman probe and a fluorescent reporter probe; (iv) Enhanced substrate: used for Raman spectral signal detection.

7. The reagent kit according to claim 6, characterized in that, The transcriptional amplification enzyme is T7 RNA polymerase; the Cas protein is Cas12a; the precursor of the guide RNA is a hairpin DNA probe; the Raman probe uses polystyrene microspheres as a carrier and is loaded with Raman reporter molecules selected from 4-MBA and / or 4-NTP. The reinforcing substrate is an Au@PB nanocomposite material.

8. The reagent kit according to claim 6, characterized in that, The kit also contains one or more auxiliary components selected from the following: reaction buffer, DTT, MgCl2, tetrahydrofuran, and pathogenic bacteria 16S rRNA standards.

9. The reagent kit according to claim 6, characterized in that, The kit is used for the detection of foodborne pathogens in food samples for non-diagnostic and non-therapeutic purposes; the foodborne pathogens are selected from Escherichia coli O157:H7 and / or Salmonella typhimurium.

10. A single-transistor multi-signal amplification system based on CRISPR / Cas, characterized in that, The method for implementing the dual-mode detection of pathogenic bacteria using multiple Raman / visualization as described in claim 1 includes: The transcription and amplification module is used to generate guide RNA in situ and perform exponential amplification in response to the presence of target nucleic acids. The Cas enzyme module, whose nuclease activity is activated by a complex formed by the target nucleic acid and the guide RNA generated by transcriptional amplification; The Raman signaling module contains at least one Raman probe that can be released by cis-cleavage of Cas enzyme, and different Raman reporter molecules encode different targets; The fluorescence signal module contains a fluorescent reporter probe that can be trans-cleaved by Cas enzyme; The system simultaneously outputs Raman and fluorescence signals in the same reaction tube, enabling qualitative and quantitative analysis of multiple targets.

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