Method for detecting salmonella enteritidis

Abstract

The invention provides a method for detecting salmonella enteritidis, which comprises the following steps of: 1, expressing a nano antibody for resisting salmonella enteritidis FliC by using escherichia coli, and verifying the nano antibody; step 2, preparing immunomagnetic beads for resisting FliC protein, and verifying the immunomagnetic beads; step 3, carrying out specific enrichment on the target bacteria; 4, performing rapid splitting decomposition to release nucleic acid; step 5, carrying out isothermal amplification on the target DNA fragment; and step 6, CRISPR / Cas12a mediated signal amplification and detection are carried out. The kit has the effects of ultra-fast detection, high sensitivity, high specificity, portability, easiness in operation, no need of bacterial culture and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection equipment, and particularly relates to a method for detecting Salmonella enteritidis. Background Art

[0002] Salmonella Enteritidis (S.Enteritidis) is one of the major foodborne pathogens globally, widely present in poultry, eggs, and dairy products, and can be transmitted through contaminated food or water, causing severe foodborne diseases. Due to its low infectious dose and high pathogenicity, S.Enteritidis poses a significant threat to public health and food safety, especially in regions with weak food supply chain management and limited detection capabilities. Rapid, sensitive, and portable on-site detection technologies are crucial for controlling the spread of S.Enteritidis and preventing foodborne disease outbreaks.

[0003] Prior Art and Defects Currently, the detection methods for S.Enteritidis mainly include traditional culture methods, immunological methods, and molecular biology methods: Traditional culture methods (such as selective media, biochemical identification): Advantages: High specificity, regarded as the "gold standard" for detection.

[0004] Disadvantages: Long culture period (usually 3 - 7 days), low detection efficiency, unable to meet the requirements of on-site rapid detection.

[0005] Immunological methods (such as ELISA, latex agglutination, immunochromatographic test strips): Advantages: Relatively simple operation, and some methods can achieve relatively fast detection.

[0006] Disadvantages: The sensitivity is affected by antigen - antibody reactions, still requires sample pretreatment and even bacterial culture, and is easily interfered by non - specific reactions.

[0007] Molecular biology methods (such as PCR, qPCR): Advantages: High sensitivity and strong specificity, capable of achieving rapid detection.

[0008] Disadvantages: Dependent on expensive experimental equipment (such as thermal cyclers, fluorescence detectors), complex operation, limited by laboratory environment, not suitable for on - site point - of - care testing.

[0009] In addition, recently emerging isothermal amplification technologies (such as RAA, LAMP) and CRISPR / Cas systems have shown great potential in nucleic acid detection, but still have problems such as non - specific amplification and high false positive rates, affecting the reliability of detection. Summary of the Invention

[0010] Aiming at the limitations of existing detection technologies, the present invention proposes a method for detecting Salmonella enteritidis, which can achieve rapid detection with high sensitivity, strong specificity, without the need for complex instruments, and without bacterial culture. This method can complete the whole detection process within 1 hour through immunomagnetic bead enrichment, RAA nucleic acid amplification, and Cas12a-mediated fluorescence / test strip detection, and the detection limit can reach 1.7×10 2 CFU / mL, which is applicable to multiple application scenarios such as food safety and public health monitoring.

[0011] In view of the on-site rapid detection requirements for Salmonella Enteritidis (S. Enteritidis), the present invention overcomes the deficiencies of on-site detection technologies, including the long time-consuming of traditional culture methods, the limited sensitivity of immunological methods, the dependence on expensive equipment for molecular biology methods, and the non-specific amplification of isothermal amplification technologies.

[0012] The present invention adopts the following technical solutions: A method for detecting Salmonella enteritidis, comprising: Step 1. Expressing nanobody against Salmonella enteritidis FliC using Escherichia coli and verifying it; Step 2. Preparing immunomagnetic beads against FliC protein and verifying them; Step 3. Specific enrichment of target bacteria; Step 4. Rapid lysis to release nucleic acids; Step 5. Isothermal amplification of target DNA fragments; Step 6. CRISPR / Cas12a-mediated signal amplification and detection; Using crRNA to recognize the amplification product, activating Cas12a enzyme, triggering ssDNA cleavage activity, and detecting using a fluorescence probe or a lateral flow test strip; Among them, fluorescence detection generates fluorescence signals; during reagent detection, due to the addition of gold-labeled antibodies, a red band will be formed on the test line.

[0013] Further, Step 1 includes: A1. Inducing the expression of FliC-Nb76 protein Transforming the vector into competent cells; Inoculating the recombinant strain, picking single colonies for inoculation and overnight culture; Inoculating and culturing, adding IPTG for induction of expression.

[0014] A2. Cell lysis and purification of FliC-Nb76 protein Collecting the bacterial cells and washing them with PBS; Breaking the bacterial cells, collecting the supernatant, and extracting the soluble protein; Analyze the expression situation, purify the target protein, collect the eluate, and measure the protein concentration; Identification of A3.FliC-Nb76 protein Collect the purified protein, boil the sample and then perform electrophoresis to detect the protein purification effect and identify the specificity of the fusion protein.

[0015] Furthermore, Step 2 includes: coupling FliC-Nb76 to amino magnetic beads through glutaraldehyde, storing, and incubating successively in anti-6×His antibody and goat anti-mouse secondary antibody.

[0016] Furthermore, Step 2 also includes: verifying whether FliC-His is coupled to the magnetic beads by TMB color development.

[0017] Furthermore, Step 3 includes: adding the sample to the immunomagnetic beads for incubation and magnetic separation to achieve enrichment of S. Enteritidis.

[0018] Furthermore, Step 4 includes: releasing the bacterial genomic DNA by thermal lysis method.

[0019] Furthermore, Step 5 includes: amplifying the conserved gene fragment of S. Enteritidis by recombinase-assisted amplification.

[0020] As described above, the fluorescent probe is an ssDNA probe. The upstream of the ssDNA probe is the fluorescent substance FAM, the downstream is the fluorescent substance BHQ1, and the sequence is 5’-TTTTATTTT-3’.

[0021] For the lateral flow test strip as described above, the upstream of the lateral flow test strip is the fluorescent substance FAM, the downstream is biotin, and the sequence is 5’-TTTTATTTT-3’.

[0022] A crRNA for targeting the conserved gene of S. Enteritidis, its sequence is shown in SEQ ID NO.1, the forward primer of the conserved sequence of Salmonella enteritidis is shown in SEQ ID NO.2, and the reverse primer of the conserved sequence of Salmonella enteritidis is shown in SEQ ID NO.3.

[0023] Application of the Salmonella enteritidis detection method as described above in the monitoring of Salmonella enteritidis in the environment.

[0024] Application of the Salmonella enteritidis detection method as described above in the monitoring of Salmonella carried by animals and humans.

[0025] crRNA sequence: 5’-AAUUUCUACUAAGUGUAGAUCAGAACAUGCUCGCUGCACAAAA-3’, as shown in SEQ ID NO.1.

[0026] The forward primer for the conserved gene sequence of Salmonella enteritidis is: 5’-GAATGGTGAGCAGACAACAGGCTGATTTACTA-3’, as shown in SEQ ID NO.2.

[0027] The reverse primer for the conserved sequence of Salmonella enteritidis is: 5’-GGCGGTTTGATGTGGTTGGTTCGTCACTGATT-3’, as shown in SEQ ID NO.3.

[0028] The primer sequences for the conserved sequence of Salmonella enteritidis: Advantages of the present invention: Improved detection speed: The entire detection process is completed within 1 hour, enabling point-of-care testing (POCT).

[0029] Enhanced detection sensitivity: Ensuring a detection limit as low as 1.7×10 2 CFU / mL, suitable for low-contamination samples.

[0030] Improved detection specificity: Avoiding non-specific amplification and false positive problems of traditional molecular detection methods.

[0031] Achieved portable and instrument-free detection: Realizing visual and instrument-independent reading of detection results, suitable for food safety and on-site monitoring.

[0032] No need for bacterial culture: Reducing the pretreatment steps, making the detection more efficient and easy to operate.

[0033] The present invention (Nanobody-Cas12a) shows significant advantages compared with traditional methods. Its detection time is the shortest (<1 hour), far lower than the 3 - 7 days of traditional culture methods, and also faster than immunological methods (2 - 6 hours) and PCR / qPCR (2 - 4 hours). In terms of sensitivity and specificity, this technology achieves high sensitivity comparable to traditional culture methods and PCR through the bispecific recognition mechanism of nanobody and CRISPR / Cas12a, while the specificity is better than that of immunological methods. In terms of experimental conditions, the present invention does not require microbial culture (necessary for traditional culture methods) and complex equipment (immunology relies on the ELISA platform, and PCR requires special instruments), and only basic experimental conditions are needed to complete, significantly improving the feasibility of on-site detection. Generally speaking, this technology combines rapidity, high precision, low equipment dependence and strong scene adaptability, breaking through the limitations of traditional detection methods in timeliness and operation threshold. Brief Description of the Drawings

[0034] Figure 1 It is the detection flow chart of the present invention; Figure 2 For the expression of Flic-Nb76 and the preparation of MB-FliC-Nb76, A shows the schematic diagram of the coupling of nanobody and amino magnetic beads by glutaraldehyde method, B shows the PAGE diagram of the expression and purification of FliC-Nb76, C shows the WB result diagram of FliC-Nb76, and D shows the verification diagram of the successful coupling of FliC-Nb76 and magnetic beads; Figure 3 For the specific enrichment of Salmonella enteritidis by MB-FliC-Nb76, A shows the successful enrichment of Salmonella enteritidis by MB-FliC-Nb76, B and C show the specific enrichment of Salmonella enteritidis by MB-FliC-Nb76 from the mixed bacterial solution of Salmonella enteritidis and Escherichia coli, D shows that MB-FliC-Nb76 can successfully capture Salmonella enteritidis, E shows that MB-FliC-Nb76 can successfully recover Salmonella enteritidis from the bacterial solution, F shows that MB-FliC-Nb76 can recover bacteria from Salmonella enteritidis solutions with different concentrations, and G and H show that MB-FliC-Nb76 can specifically enrich Salmonella enteritidis under the interference of Escherichia coli; Figure 4 For the specific detection of Salmonella enteritidis by CRISPR / CAS12a, A shows that only when all components are present can the reaction system produce a fluorescence signal, B shows the fluorescence intensity of each reaction in A, C shows that only when all components are present can the test strip produce a visible T band, D shows that only Salmonella enteritidis can make the test strip produce an obvious T band, and other Salmonella or non-Salmonella cannot, E shows that only Salmonella enteritidis can make the RAA reaction produce the target band, and other Salmonella or non-Salmonella cannot, F shows the fluorescence signal intensity of each in G, and G shows that only the RAA product of Salmonella enteritidis can make the fluorescence reaction produce a significant fluorescence signal, and other Salmonella or non-Salmonella cannot; Figure 5 Optimization of experimental conditions, A shows the optimization of the cas12a protein concentration in the system, B shows the optimization of the crRNA concentration in the system, C shows the optimization of the fluorescence probe concentration in the system, D shows the optimization of the cleavage reaction time, and E shows the optimization of the probe concentration in the test strip system; Figure 6To detect standard plasmids, genomic DNA, and bacterial cultures using this method for sensitivity testing, A represents the isothermal amplification electrophoresis patterns using standard plasmids at different concentrations as templates, B represents the fluorescence intensities in D, C represents the results of detecting standard plasmids at different concentrations using test strips, D represents the results of detecting standard plasmids with fluorescence probes at different concentrations, E represents the isothermal amplification electrophoresis patterns using genomic DNA at different concentrations as templates, F represents the fluorescence intensities in H, G represents the results of detecting genomic DNA at different concentrations using test strips, H represents the results of detecting genomic DNA with fluorescence probes at different concentrations, I represents the isothermal amplification electrophoresis patterns using pre-enrichment bacterial cultures at different concentrations as templates, J represents the fluorescence intensities in P, K represents the results of detecting pre-enrichment bacterial cultures at different concentrations using test strips, L represents the results of detecting pre-enrichment bacterial cultures with fluorescence probes at different concentrations, M represents the isothermal amplification electrophoresis patterns using post-enrichment samples at different concentrations as templates, N represents the fluorescence intensities in P, O represents the results of detecting post-enrichment samples at different concentrations using test strips, and P represents the results of detecting post-enrichment samples with fluorescence probes at different concentrations; Figure 7 To test actual samples, A represents the results of detecting Salmonella enteritidis in each sample using the isolation and culture method, B represents the fluorescence results of detecting Salmonella enteritidis in each sample using Nanobody-Cas12a, C represents the results of detecting Salmonella enteritidis in each sample using PCR, D represents the results of detecting Salmonella enteritidis in each sample using qPCR, and E represents the test strip results of detecting Salmonella enteritidis in each sample using Nanobody-Cas12a. Detailed implementation mode

[0035] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be described clearly and completely below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] As Figure 1 shown, a method for detecting Salmonella enteritidis includes: Step 1. Expression of anti-Salmonella enteritidis FliC nanobody The constructed Pet25b-FliC-Nb76 recombinant expression vector was transformed into BL21(DE3) competent cells. The recombinant strain was streaked on LB / AmP solid medium containing 100 μg / mL ampicillin, and a single colony was picked and inoculated into 5 mL of LB / Amp liquid medium, and cultured overnight with shaking at 37°C. Subsequently, the culture was inoculated into 300 mL of LB / Amp liquid medium at a ratio of 1:100 and cultured until the logarithmic growth phase (OD600 = 0.6 - 0.8). IPTG was added to a final concentration of 0.5 mM, and induced expression was carried out at 16°C for 12 hours.

[0037] The induced cells were collected by centrifugation, washed twice with PBS buffer and resuspended in 30 mL of PBS. The cell suspension was sonicated, and the supernatant was collected by centrifugation to extract soluble proteins. SDS-PAGE electrophoresis was used to analyze the protein expression. Subsequently, the target protein was further purified using a Ni-NTA affinity chromatography column, and the eluate was collected to determine the protein concentration.

[0038] Protein identification The purified protein was collected, mixed with 2× loading buffer and boiled, and then SDS-PAGE electrophoresis was carried out to evaluate the protein purification efficiency. At the same time, Western blotting was used for detection, and an anti-His tag antibody was used to specifically recognize the fusion protein to verify the accuracy of its expression and purification.

[0039] Step 2. Preparation of immunomagnetic beads against FliC protein FliC-Nb76 was coupled to amino magnetic beads through glutaraldehyde (GA). Glutaraldehyde is a commonly used bifunctional cross-linking agent, and its two aldehyde groups can form Schiff bases (-N=C-) with amino groups on magnetic beads or biomolecules, thus achieving coupling through a five-carbon bridge.

[0040] The coupling process mainly includes three steps: activation of the magnetic bead surface, protein coupling, and blocking of unbound sites. The prepared magnetic beads were stored at 4°C. By recording the change in the concentration of FliC-Nb76 antibody before and after coupling, the antibody loading amount on the magnetic beads was calculated. Subsequently, the coupled magnetic beads were incubated successively in anti-6×His antibody and goat anti-mouse secondary antibody, and the TMB color reaction was used to verify whether FliC-Nb76 was successfully coupled to the magnetic bead surface.

[0041] Step 3. Specific enrichment of target bacteria The sample (such as food, environmental or clinical sample) was added with immunomagnetic beads and incubated on a shaker at 50 rpm for 20 min, and magnetically separated for 10 s to achieve efficient enrichment of S. Enteritidis and remove non-target bacteria and impurities.

[0042] Step 4. Rapid lysis to release nucleic acids The bacterial genomic DNA was released by thermal lysis, eliminating the need for complex DNA extraction steps.

[0043] Step 5. Isothermal amplification of the target DNA fragment The recombinase-aided amplification (RAA) technique was used to isothermally amplify the conserved gene fragment of S. Enteritidis at 37 - 42 °C, avoiding the dependence on a thermal cycler in traditional PCR.

[0044] Step 6. CRISPR / Cas12a-mediated signal amplification and detection Specific crRNA was designed to recognize the amplification product, activating the Cas12a enzyme and triggering its non-specific single-stranded DNA (ssDNA) cleavage activity. A fluorescence probe (FAM-BHQ1) or a lateral flow test strip (FAM-biotin) was used for signal reading:

[0045] Fluorescence detection: After Cas12a cleaved the probe, FAM was released, generating a 520 nm fluorescence signal under the excitation of the excitation light.

[0046] Reagent detection: After probe cleavage, FAM was separated from biotin and bound to the gold-labeled antibody, forming a red band at the test line (T line), making the result visually observable.

[0047] Example Induced expression of FliC-Nb76 protein: The constructed PET256-SE-Nb recombinant expression vector was transformed into BL21(DE3) competent cells. The recombination was streaked and cultured on an LB (containing 100 μg / mL ampicillin) solid plate, and a single colony was picked and inoculated into 5 mL of LB / Amp liquid medium, cultured overnight at 37 °C on a shaker. It was inoculated into 300 mL of LB / Amp liquid medium at a ratio of 1:100 and cultured until the logarithmic growth phase (OD600 = 0.6 - 0.8). IPTG with a final concentration of 0.5 mM was added, and the culture was induced at 16 °C for 12 h.

[0048] Cell lysis and purification of FliC-Nb76 protein The induced bacterial cells were collected by centrifugation, washed twice with PBS buffer, and resuspended in 30 mL of PBS. The bacterial cell solution was disrupted by sonication, and the supernatant was collected by centrifugation to extract the soluble protein. The supernatant was analyzed by SDS-PAGE electrophoresis for expression, and the target protein was further purified by a Ni-NTA affinity chromatography column. The eluate was collected for protein concentration determination.

[0049] Identification of FliC-Nb76 protein The purified protein collected was boiled with 2×Loading buffer and then subjected to SDS-PAGE electrophoresis to detect the protein purification effect. Meanwhile, Western blot was used to specifically identify the fusion protein with anti-His tag antibody to verify the accuracy of its expression and purification.

[0050] Preparation of immunomagnetic beads against FliC (MB-FliC-Nb76) As Figure 2 shown, FliC-Nb76 was conjugated to amino magnetic beads using the glutaraldehyde method. Primary amines (-NH2) exist at the N-terminus of each polypeptide chain and in the side chains of lysine residues. Since primary amines are positively charged, they are usually on the outer surface of proteins, making them more easily conjugated without changing the protein structure. Glutaraldehyde (GA) is a commonly used homobifunctional crosslinking agent. Its two aldehyde groups can respectively form Schiff bases (-N=C-) with the amino groups of amino magnetic beads or biomolecules, connecting them with a five-carbon bridge, as Figure 2 shown in A. Briefly, the conjugation process mainly includes three main steps: activation of the magnetic bead surface, protein conjugation, and blocking of unbound sites. The prepared magnetic beads were stored at 4°C. Record the change in FliC-Nb76 antibody concentration before and after conjugation, and calculate the amount of antibody conjugated to the magnetic beads. Then, the conjugated magnetic beads were incubated successively with anti-6×His primary antibody and goat anti-mouse secondary antibody, and then developed with TMB to verify that FliC-NB76 was successfully conjugated to the magnetic bead surface.

[0051] Characterization and evaluation of immunomagnetic beads Evaluation of conjugation effect Key parameters include the initial FliC-Nb76 concentration (c1), the volume of FliC-Nb76 used (v1), the FliC-Nb76 concentration after conjugation (c2), the volume of suspended magnetic beads (v2), the magnetic bead concentration in the suspension (c3), and the volume of the MB-FliC-Nb76 suspension (v3). The nanobody concentration in MB-FliC-Nb76 and the nanobody loading on the magnetic beads were calculated respectively as follows. Calculate the nanobody concentration in the MB-FliC-Nb76 suspension and the nanobody loading per unit mass of the magnetic beads (m / m).

[0052] cFliC-Nb76 = v1·(c1 - c2) / v3 Loading capacity = v1·(c1 - c2) / c3·v2 Analysis of enrichment efficiency Salmonella enteritidis ATCC 13076 carrying the mCherry fluorescent plasmid was inoculated into LB medium and cultured overnight at 37 °C and 220 rpm. Subsequently, the culture was serially diluted (10 8 、10 7 、10 6 CFU / mL) and treated with immunomagnetic beads respectively. The fluorescence intensity before and after treatment was observed by a laser confocal microscope, and the enrichment fold was calculated to evaluate the enrichment efficiency of the immunomagnetic beads for the target strain.

[0053] Salmonella enteritidis ATCC 13076 carrying the red fluorescent plasmid was mixed with Escherichia coli (a common strain in the environment) carrying the green fluorescent plasmid in equal proportions as a mixed bacterial sample. The mixture was serially diluted (10 8 、10 7 、10 6 CFU / mL) and enriched with immunomagnetic beads respectively. The signal intensities of the red and green fluorescent channels before and after treatment were measured by a laser confocal microscope, and the enrichment effect of the target bacterium (Salmonella enteritidis) in the presence of the interfering bacterium (Escherichia coli) was analyzed by comparison. The selective enrichment efficiency of the immunomagnetic beads in the mixed bacterial sample was evaluated by calculating the enrichment fold of the red channel and the signal change of the green channel.

[0054] Recovery efficiency determination The recovery rate of the immunomagnetic beads for Salmonella enteritidis in bacterial solutions with different concentrations (10 CFU / mL, 10 CFU / mL, 10³ CFU / mL, 10² CFU / mL) was evaluated by the plate counting method. The specific operation included serially diluting the cultured bacterial solution, and evenly coating the liquids before and after treatment with the immunomagnetic beads on XLT4 plates, culturing at 37 °C for 16 - 18 hours, and then counting the number of colonies formed on the plates. The recovery rate was calculated as the ratio of the number of colonies in the bacterial solution before and after treatment, and the effect of different initial concentrations on the enrichment effect was also evaluated. The sensitivity and enrichment efficiency of the immunomagnetic beads for the target strain in the low-concentration bacterial solution were analyzed to provide data support for the optimization of the subsequent detection system.

[0055] Identification and verification of Salmonella - specific DNA fragments Download the complete genome sequence of Salmonella enteritidis from the NCBI database for comparison, find its conserved sequences, perform BLAST in the database, and design primers for RAA to determine its specificity. Through systematic alignment and analysis of the complete genome sequence of Salmonella enteritidis, highly conserved specific sequence regions were screened and identified. Based on this conserved sequence, RAA (recombinase-mediated amplification) primers targeting the target gene were designed and their specificity was strictly verified. Specifically, a variety of related pathogens were selected as controls, and experiments were conducted to verify whether these primers could specifically bind only to the Salmonella enteritidis genome, thus ensuring the accuracy and reliability of the method.

[0056] Design and feasibility testing of crRNA and ssDNA probes Design crRNA according to the PAM (TTTN) site adjacent to the target RAA amplification product, which is base complementary to the target sequence. Optimize and synthesize crRNA through an online tool, and select the sequence with the highest efficiency for subsequent experiments. Design a fluorescent ssRNA reporter sequence to detect the fluorescence signal intensity of the reaction, and design a lateral flow immunochromatography reporter sequence for test strip color development to ensure that the system has an intuitive detection ability.

[0057] To verify the effectiveness of the biosensor system, the signal changes were observed by removing the key components (target DNA, crRNA, Cas12a protein) in the system one by one, so as to confirm the integrity and functionality of the sensor.

[0058] Design and feasibility testing of crRNA and ssDNA probes To further improve the detection performance, the key parameters of the Cas12a system were systematically optimized, including the Cas12a protein concentration, crRNA and probe concentrations, and the Cas12a cleavage reaction time. The optimization experiments adopted a single-factor regulation strategy, that is, only one parameter was adjusted each time while keeping other conditions unchanged, so as to accurately determine the optimal values of each parameter, maximize the detection sensitivity and specificity, and provide a reliable guarantee for sample detection.

[0059] Sensitivity testing Throughout the detection process, the RAA amplification to the Cas12a cleavage reaction is the key step determining sensitivity. Therefore, the detection sensitivities of this method for three types of samples, namely standard plasmid, genomic DNA, and bacterial suspension, were tested respectively. For the bacterial suspension sample, two treatment strategies were designed: direct detection and detection after enrichment by immunomagnetic beads separation. Through comparative analysis, these two strategies further evaluated the contribution of immunomagnetic beads in improving detection sensitivity and specificity, providing an important basis for optimizing the detection process in actual application scenarios.

[0060] Clinical sample analysis and accuracy assessment To verify the effectiveness of the method, the detection method developed in this study based on nanobody magnetic beads and Cas12a detection technology was systematically compared with existing enrichment culture methods, PCR, and qPCR detection methods. Specifically, the evaluation indicators included four key parameters: detection time, dependence on complex instruments, applicability to on-site detection, and specificity.

[0061] The present invention also demonstrated its comprehensive advantages in shortening the detection time, enhancing portability, and reducing instrument dependence through the detection of simulated contaminated samples, providing a more efficient solution for pathogen detection. To be closer to the actual application scenario, meat (chicken, pork), eggs (chicken eggs), dairy products (milk), and composite foods (sandwiches) were selected and contaminated with Salmonella enteritidis artificially for detection to test the detection performance of each detection method.

[0062] By inducing the PET25b-SE-Nb76-BL21(DE3) recombinant strain with IPTG, a target band with an analytical amount of approximately 20 kDa was observed in SDS-PAGE, indicating the successful expression of the FliC-Nb76 protein in the host cell, as shown in Figure 2 Figure B. In addition, FliC-Nb76 mainly existed in the supernatant of the disrupted bacterial cells in a soluble form. After purification by an NI-NTA affinity chromatography column, an obvious single band was visible in the eluate, indicating a high protein purity. Western blot analysis further verified the specificity of the target protein. The results showed that the His-tag antibody could recognize the FliC-Nb76 band with a molecular weight of approximately 20 kDa, indicating the successful fusion of the His-tag in the recombinant protein, as shown in Figure 2 Figure C.

[0063] As Figure 3 shown, the experimental results indicated that the immunomagnetic beads had a significant enrichment effect on Salmonella enteritidis ATCC13076 carrying the mCherry fluorescent plasmid. Salmonella enteritidis was enriched by the immunomagnetic beads, forming aggregated fluorescent signals, as shown in Figure 3 Figure A. After the gradient-diluted bacterial solutions (10 8 , 10 7 , 10 6 CFU / mL) were treated with the immunomagnetic beads, a significant increase in the fluorescent signal was observed under a laser confocal microscope. Through the quantitative analysis of the fluorescence intensity, the enrichment multiples of the 10 8 , 10 7 , 10 6 CFU / mL bacterial solutions were 27.74-fold, 9.50-fold, and 33.71-fold, respectively, as shown in Figure 3 Figure F. These results indicated that the immunomagnetic beads could effectively enrich the target strain under different bacterial concentration conditions.

[0064] To verify that MB-FliC-Nb76 can specifically enrich Salmonella enteritidis under the interference of other bacteria, Salmonella enteritidis carrying a red fluorescent plasmid was mixed with Escherichia coli carrying a green fluorescent plasmid in equal amounts. As Figure 3 shown in B, after enrichment treatment with MB-FliC-Nb76 and observation by laser confocal microscopy, the signal proportion in the red fluorescence channel increased significantly, while the signal proportion in the green fluorescence channel decreased significantly. As Figure 3 shown in C, it indicates that the immunomagnetic beads can selectively enrich the target bacteria. Similarly, this result was verified at different concentrations. The mixed bacterial solution was serially diluted (10 8 CFU / mL, 10 7 CFU / mL, 10 6 CFU / mL), then treated with MB-FliC-Nb76 respectively, and the fluorescence intensities of the red and green fluorescence channels were observed using the same method. After calculation, at different dilution gradients, the proportion of the fluorescence signal in the red channel increased from approximately 50% before enrichment to over 99% after enrichment, showing a good enrichment effect. As Figure 3 shown in H. At the same time, the proportion of the fluorescence signal in the green channel decreased significantly, further verifying the selective enrichment ability and anti-interference performance of the immunomagnetic beads in the mixed bacterial sample. This result confirmed the high efficiency and reliability of the immunomagnetic beads in enriching the target bacteria in complex samples, providing a good pretreatment method for subsequent detection. The experiment evaluated the recovery rate of the immunomagnetic beads in bacterial solutions with different concentrations by the plate counting method, as Figure 3 shown in E. The results showed that the recovery rates of the immunomagnetic beads in samples with concentrations of 10CFU / mL, 10CFU / mL, and 10³ CFU / mL were approximately 97.83%, 93.44%, and 91.80% respectively, showing a high enrichment efficiency. In the low-concentration sample of 10 2 CFU / ml, the recovery rate was close to 100%. These results indicate that the immunomagnetic beads have good recovery effects on Salmonella enteritidis within different concentration ranges, providing a reliable pretreatment method for subsequent detection.

[0065] As Figure 4As shown, the crRNA is designed to target the RAA amplification product, with its sequence adjacent to the TTTN protospacer adjacent motif (PAM) site. According to the position of the PAM site, a crRNA complementary to the target sequence is designed, optimized through an online tool, and synthesized. The crRNA sequence with the highest efficiency is selected as: 5’-AAUUUCUACUAAGUGUAGAUCAGAACAUGCUCGCUGCACAAAA-3’ (SEQ ID NO.1). The ssDNA reporter sequence for fluorescence detection is designed as 5’-FAM-TTTTATTTT-BHQ1-3’, and the recognition result of the target is indicated by the change in fluorescence intensity. The reporter sequence for lateral flow immunoassay is designed as 5’-FAM-TTTTATTTT-biotin-3’, which is used to develop color on the T band of the test strip and directly read the result. To verify the effectiveness of the biosensor system, a component omission experiment was conducted on various components in the system. The target DNA, crRNA, and Cas12a were removed separately, and the fluorescence signal or the color development result of the test strip was monitored. When any key component was missing in the system, no visible fluorescence signal could be detected. As shown in Figure 4 A and B in, the T band of the test strip did not develop color, and only the signal appeared in the C band, as shown in Figure 4 C in; while when all components were complete, a significant fluorescence signal or color development in the T band of the test strip could be observed, thus verifying the integrity and functionality of the system design.

[0066] Eight Salmonella strains other than Salmonella enteritidis (Salmonella typhimurium, Salmonella pullorum, Salmonella derby, Salmonella heidelberg, Salmonella dublin, Salmonella paratyphi A, Salmonella newport, Salmonella gallinarum) and four non-Salmonella strains (Klebsiella pneumoniae, Proteus vulgaris, Escherichia coli, Staphylococcus aureus) were selected as controls, and the specificity of the designed primers was verified by RAA detection. As shown in Figure 4 E in, and then the RAA products were used for the detection of fluorescence signals and color signals of the test strip, as shown in Figure 4 G and F in. The experiment showed that this detection method only produced specific signals for Salmonella enteritidis, and no amplification or positive signals appeared in the remaining control strains, fully demonstrating the high specificity of the primer design.

[0067] As shown in Figure 5 below, the concentrations of various components in the system and the cleavage reaction time were optimized to improve the method performance. For Cas12a and crRNA, as shown in Figure 5For A and B, we tested them in the recommended final concentration range of 50 - 250 nM. The results showed that there was no significant difference in the reaction effects among different concentrations. Therefore, 50 nM was selected as its working concentration. The optimization experiment for the probe concentration showed that the fluorescence signal intensity increased significantly with the increase of the probe concentration, and at the same time, the background fluorescence of the probe itself was extremely low. Finally, a probe concentration of 2000 nM was determined to facilitate the observation of fluorescence results (for lateral flow immunoassay chromatography, the probe concentration needs to be considered separately), as shown in Figure 5 Figure C. In terms of the cleavage reaction time, the experimental results showed that the fluorescence intensity gradually increased with the reaction time within the first 5 - 6 minutes, and then, due to the full cleavage of the probe, the fluorescence intensity tended to be stable and showed no significant change. Therefore, 6 minutes was selected as the optimal time for the cleavage reaction, as shown in Figure 5 Figure D.

[0068] In the lateral flow immunoassay experiment, the concentration of the probe needs to be discussed separately. The concentrations of other components and the cleavage reaction time are the same as those mentioned above. False positives will occur when the concentration of the probe is too high or too low. The performance of different concentrations of the probe (0 nM, 250 nM, 500 nM, 750 nM, 1000 nM, 1250 nM) in lateral flow immunoassay chromatography was tested respectively. 500 nM was selected as the working concentration of the probe, and no false positive bands appeared at this time, as shown in Figure 5 Figure E.

[0069] As shown in Figure 6 , in order to test the sensitivity of this method, the DNA fragment of 500 bp near the target DNA was inserted into the pUC57 plasmid. The constructed standard plasmid was transformed into the DH5α strain. The plasmid was extracted using a plasmid extraction kit and its concentration was determined to be 126 ng / μL. After calculation, its initial concentration was approximately 3.59×10 13 copies / mL. Subsequently, the plasmid sample was serially diluted 10-fold to 3.59 copies / mL, and each concentration of the standard plasmid was used as a sample. After RAA amplification, it was used for Cas12a detection. The genomic DNA (2.91 × 10 9 –2.91 × 10 0 copies / mL) was serially diluted and the LOD was tested using the same method.

[0070] As shown in Figure 6 , for the bacterial liquid sample, with an initial concentration of 1.7 × 10 9 CFU / ml, the serially diluted bacterial liquid samples were directly detected. The lower limit of detection was approximately 1.7×10 4 CFU / mL, while the lower limit of detection of the sample enriched with immunomagnetic beads was significantly reduced to 1.7×10 2CFU / mL. This indicates that the enrichment effect of immunomagnetic beads significantly improves the detection sensitivity, provides an important advantage for sample pretreatment, and helps to improve the detection performance of Salmonella enteritidis in complex samples.

[0071] As Figure 7 shown, in order to test the compliance of this method with traditional detection methods, Salmonella enteritidis was artificially added to various types of samples (such as eggs, chicken, pork, milk, sandwiches), and this method and traditional enrichment culture, PCR, and qPCR were used for detection respectively, and the detection results were compared. The results showed that the coincidence rates of the detection results of the present invention with enrichment culture, PCR, and qPCR all reached 100%, indicating that the detection performance of this method in various samples is stable and highly reliable.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting enteritidis Salmonella, characterized in that: include: Step 1. Using Escherichia coli to express anti-Salmonella Enteritidis FliC nanoantibodies and verifying them; Step 2. Prepare the immunomagnetic beads against FliC protein and verify them; Step 3. Specific enrichment of target bacteria; Step 4. Rapid lysis to release nucleic acid; Step 5. Isothermal amplification of the target DNA fragment; Step 6. CRISPR / Cas12a-mediated signal amplification and detection; The crRNA is used to recognize the amplification product, activate the Cas12a enzyme, trigger the ssDNA cutting activity, and use a fluorescent probe or lateral flow test strip for detection. Fluorescence detection generates a fluorescent signal. When the test strip is tested, a gold-labeled antibody is added and the test line forms a red strip.

2. The method according to claim 1, characterized in that Step 1 includes: A1. Induced expression of FliC-Nb76 protein Transform the vector into competent cells; Inoculate the recombinant strain, pick a single colony for inoculation and culture overnight; Inoculate, culture, and add IPTG to induce expression; A2. Cell Lysis and FliC-Nb76 Protein Purification Collect the bacteria and wash with PBS; Disrupt the bacteria, collect the supernatant, and extract soluble protein; Analyze the expression, purify the target protein, collect the eluate, and determine the protein concentration; A3. Identification of FliC-Nb76 Protein Collect the purified protein, boil the sample and perform electrophoresis to detect the protein purification effect and identify the specificity of the fusion protein.

3. The method according to claim 1, characterized in that Step 2 includes: FliC-Nb76 is coupled to amino magnetic beads by glutaraldehyde, stored, and incubated in anti-6×His antibody and goat anti-mouse secondary antibody in sequence.

4. The method according to claim 1, characterized in that: Step 2 also includes: verifying whether FliC-His is coupled to the magnetic beads by TMB color development.

5. The method according to claim 1, characterized in that ,Step 3 includes: adding immunomagnetic beads to the sample for incubation and magnetic separation to achieve enrichment of Enteritidis Salmonella.

6. The method according to claim 1, characterized in that ,Step 4 includes: releasing bacterial genomic DNA using thermal lysis method.

7. The method according to claim 1, characterized in that ,Step 5 includes: using recombinase-assisted amplification to amplify the conserved gene fragment of Enteritidis Salmonella.

8. The method according to claim 1, characterized in that , the crRNA, its sequence is shown in SEQ ID NO.1, the forward primer of the conserved gene sequence of Salmonella enteritidis is shown in SEQ ID NO.2, and the reverse primer of the conserved sequence of Salmonella enteritidis is shown in SEQ ID NO.

3.

9. The method according to claim 1, characterized in that: The fluorescent probe is a ssDNA probe, the upstream of the ssDNA probe is the fluorescent substance FAM, the downstream is BHQ1, and the sequence is TTTTATTTT.

10. The method according to claim 1, characterized in that The lateral flow test strip has the fluorescent substance FAM upstream and biotin downstream, and the sequence is TTTTATTTT.

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