Listeria monocytogenes detection method based on combination of CBD magnetic bead enrichment and RPA-quantum dot fluorescence test strip

Abstract

The invention is suitable for the technical field of microbiological detection, and provides a listeria monocytogenes detection method combining CBD magnetic bead enrichment with an RPA-quantum dot fluorescent test strip. The functionalized magnetic beads are constructed by expressing CBD118-500 recombinant protein with high affinity, 86.58% of target bacteria in a sample can be effectively enriched, and after recombinase polymerase amplification (RPA) and quantum dot immunochromatography (QD-LFA) technologies are combined, the detection limit in a labeled meat sample is as low as 9.1 * 100 CFU / mL and is reduced by about two orders of magnitude compared with that before enrichment; detection results of 16 commercially available meat food samples are consistent with PCR results, low-volume enrichment and visual rapid interpretation can be realized in a food complex matrix, an innovative scheme is provided for food-borne Listeria monocytogenes field monitoring, the modular design thought can be expanded to the field of rapid detection of other food-borne pathogens, and the application prospect is wide. The method is of great significance for perfecting a food safety prevention and control network.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular relates to a method for detecting Listeria monocytogenes using CBD magnetic bead enrichment combined with RPA-quantum dot fluorescence test strips. Background Art

[0002] Listeria monocytogenes is a Gram-positive, facultative, anaerobic, foodborne, psychrophilic pathogen that has become a major threat to global food safety and public health due to its unique environmental adaptability and high pathogenicity. The bacterium can cause listeriosis by contaminating foods such as dairy products and ready-to-eat meats, leading to severe clinical symptoms such as sepsis, central nervous system infections, and fetal infections during pregnancy. The bacterium can grow in a wide temperature range of 1 to 45°C, and its psychrophilic nature, which allows it to maintain its ability to proliferate even in a refrigerated environment at 4°C, combined with its significant biofilm-forming ability, significantly increases the difficulty of contamination control in the food production chain. Therefore, establishing an effective detection system for Listeria monocytogenes is of great public health significance.

[0003] At present, traditional culture method is one of the commonly used methods for the detection of Listeria monocytogenes, but the detection cycle of this method is as long as 3 to 5 days and the sensitivity is limited. Although real-time fluorescence PCR in molecular detection technology has improved the detection sensitivity, it has the problem of relying on professional equipment and high detection costs. In recent years, the isothermal amplification characteristics of recombinase polymerase amplification technology (RPA) have successfully gotten rid of the dependence of traditional PCR on thermal cycling instruments. The technical system jointly constructed with quantum dot immunochromatography (QD-LFA) has shown significant advantages. By optimizing the coupling efficiency of amplification products and immune probes, the entire process of nucleic acid amplification and result interpretation can be compressed to within 30 minutes, which greatly breaks through the time bottleneck of traditional molecular detection technology. The quantum dot fluorescence signal amplification effect can also increase the detection sensitivity by 2 orders of magnitude compared with the conventional colloidal gold method.

[0004] However, due to challenges in detecting foodborne pathogens in food, including complex sample matrices, limited sensitivity of existing detection methods, the tendency to miss trace samples, and the time-consuming and equipment-dependent nature of traditional testing, existing detection technologies remain insufficient to meet the demand for rapid and accurate detection of Listeria monocytogenes. To address this, the present invention proposes a method for detecting Listeria monocytogenes using CBD magnetic bead enrichment combined with RPA-quantum dot fluorescence test strips. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting Listeria monocytogenes by combining CBD magnetic bead enrichment with RPA-quantum dot fluorescence test strips, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A CBD118-500 recombinant protein for detecting Listeria monocytogenes, the amino acid sequence of which is shown in SEQ ID No. 1, and the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

[0008] A recombinant expression vector comprises the CBD118-500 recombinant protein encoding gene described above, wherein the gene is inserted into a prokaryotic expression vector pET28a(+) through restriction endonuclease NdeI and XhoI sites, and the His tag on the vector is retained.

[0009] A recombinant strain, characterized in that it is obtained by transforming the above-mentioned recombinant expression vector into Escherichia coli BL21 (DE3) and is used to express CBD118-500 recombinant protein.

[0010] A CBD functionalized magnetic bead is prepared by coupling the above-mentioned CBD118-500 recombinant protein with carboxyl magnetic beads through the carbodiimide method.

[0011] A RPA-quantum dot fluorescent test strip comprises a test strip carrier, a T line coated with streptavidin, a C line coated with goat anti-mouse IgG, quantum dot fluorescent microspheres coupled with digoxigenin antibodies, and an RPA amplification product for detecting Listeria monocytogenes, wherein the RPA amplification product is double-labeled with biotin and digoxigenin.

[0012] An RPA amplification primer pair, characterized in that its sequences are shown as SEQ ID No. 3 and SEQ ID No. 4.

[0013] A method for detecting Listeria monocytogenes, comprising the following steps:

[0014] Using the CBD-functionalized magnetic beads described above to enrich Listeria monocytogenes in the sample;

[0015] Extract the enriched sample DNA;

[0016] Perform RPA amplification using the RPA amplification primer pair described above;

[0017] Digoxin antibody-coupled quantum dot fluorescent microspheres, RPA amplification products, and loading buffer were mixed and added dropwise to the RPA-quantum dot fluorescent test strip described above for chromatography reaction. The fluorescence of the T line and C line was observed to determine whether the sample contained Listeria monocytogenes.

[0018] A Listeria monocytogenes detection system, which combines the above-mentioned CBD functionalized magnetic bead enrichment method and the above-mentioned Listeria monocytogenes detection method, is used for the rapid detection of Listeria monocytogenes in food samples.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This study successfully established a CBD magnetic bead enrichment combined with RPA-quantum dot fluorescence test strips for Listeria monocytogenes detection. This system, constructed by expressing the high-affinity CBD118-500 recombinant protein to construct functionalized magnetic beads, can effectively enrich 86.58% of the target bacteria in the sample. Combining recombinase polymerase amplification (RPA) with quantum dot immunochromatography (QD-LFA) technology, the detection limit in spiked meat samples was as low as 9.1×10 0 The CFU / mL was about two orders of magnitude lower than that before enrichment; the test results of 16 commercially available meat food samples were consistent with the PCR results. Low-capacity enrichment and rapid visual interpretation can be achieved in complex food matrices, providing an innovative solution for on-site monitoring of foodborne Listeria. Its modular design concept can also be extended to the field of rapid detection of other foodborne pathogens, which is of great significance to the improvement of the food safety prevention and control network. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the reaction principle of the RPA-quantum dot fluorescent test strip.

[0022] Figure 2 PCR amplification and sequencing results of CBD118-500; in the figure, a is the PCR amplification result of the CBD118-500 recombinant sequence, where 1: negative control, 2: CBD118-500 fragment, 3: Maker; b is the sequencing result of the CBD118-500 recombinant sequence.

[0023] Figure 3 This is the map of the CBD118-500_PET28a(+) recombinant plasmid.

[0024] Figure 4 Figure 2 is the electrophoresis result of induced expression of CBD118-500 recombinant protein; in the figure, a is the electrophoresis result of induced expression of CBD118-500 recombinant protein, where 1: uninduced whole bacteria, 2: induced supernatant, 3: induced precipitate, 4: Maker; b is the electrophoresis result after purification of CBD118-500 recombinant protein, where 1: purified protein, 2: unpurified protein, 3: Maker.

[0025] Figure 5 The binding activity of CBD118-500 recombinant protein was determined by indirect ELISA.

[0026] Figure 6 The capture efficiency and capture spectrum of CBD functionalized magnetic beads are measured; in the figure, a is the capture efficiency of CBD functionalized magnetic beads for Listeria monocytogenes at different concentrations; b is the capture efficiency of CBD functionalized magnetic beads for Listeria monocytogenes at different serotypes.

[0027] Figure 7 This is the optimization of RPA-quantum dot fluorescent test strip conditions; in the figure, a is the optimization of primer concentration conditions, where 1: primer concentration is 1μmol / L, 2: primer concentration is 1.5μmol / L, 3: primer concentration is 2μmol / L, and 4: negative control; b is the optimization of reaction time conditions, where 1: reaction 5min, 2: reaction 10min, 3: reaction 15min, 4: reaction 20min, and 5: negative control.

[0028] Figure 8 The sensitivity and specificity of the RPA-quantum dot fluorescent test strips are measured. In the figure, a is the sensitivity verification result of the RPA-quantum dot fluorescent test strips, where 1: the bacterial concentration is 3×10 7 CFU / mL, 2: bacterial concentration is 3×10 6 CFU / mL, 3: bacterial concentration is 3×10 5 CFU / mL, 4: bacterial concentration is 3×10 4 CFU / mL, 5: bacterial concentration is 3×10 3 CFU / mL, 6: bacterial concentration is 3×10 2 CFU / mL, 7: bacterial concentration is 3×10 1 CFU / mL; b is the specificity verification result of RPA-quantum dot fluorescence test strip, where 1: Listeria monocytogenes, 2: Klebsiella pneumoniae, 3: Shigella flexneri, 4: Yersinia enterocolitica, 5: DEPC water.

[0029] Figure 9 The sample was spiked after enrichment with RPA-quantum dot fluorescent test strips, where the bacterial concentration was 9.1×10 7 CFU / mL, 2: bacterial concentration is 9.1×10 5 CFU / mL, 3: bacterial concentration is 9.1×10 3 CFU / mL, 4: bacterial concentration is 9.1×10 2 CFU / mL, 5: bacterial concentration is 9.1×10 1 CFU / mL, 6: bacterial concentration is 9.1×10 0 CFU / mL.

[0030] Figure 10The RPA-quantum dot fluorescent test strip is used to detect Listeria monocytogenes in actual samples; in the figure, a is the detection result of the RPA-quantum dot fluorescent test strip, and b is the PCR amplification result. DETAILED DESCRIPTION

[0031] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0032] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0033] Example 1: Design and expression of CBD118-500 recombinant protein;

[0034] The amino acid sequence of the CBD118-500 recombinant protein is shown in SEQ ID No. 1:

[0035] KSLGFEWGGDWSGFVDNPHLQFNYKGYGTDTFGKGASTSNSSKPSADTNTNSLGLVDYMNLNKLDSSFANRKKLATSYGIKNYSGTATQNTTLLAKLKAGKPHTPASKNTYYTENPRKVKTLVQCDLYKSVDFTTKNQTGGTFPPGTVFTISGMGKTKGGTPRLKTKSGYYLTANTK FVKKIGGGGSGGGGSGGGGSHFELCMCVSGEKIPAATHKTNTNINSIRYEGKSLIAHPAYRKWTFNHHHSHVYKVELETSYDHNQYGYKTYIDDKLYYMYKAFADVAKKDRKGRIKVRIKAEKDRVFSVWNNIKIEFGEINKWMHPMKLSVVQLSRGYLELWYPNDRWYYTAKNYFK.

[0036] The nucleotide sequence of the gene encoding the CBD118-500 recombinant protein is shown in SEQ ID No. 2:

[0037]

[0038] 1.1 Main materials;

[0039] Escherichia coli DH5α strains and Listeria monocytogenes serotypes were previously stored in our laboratory; the CBD118-500 recombinant protein sequence and primer sequences were synthesized by Sangon Biotech (Shanghai, China); PCR-related reagents and DNAMaker were purchased from Sangon Biotech (Shanghai, China); carboxyl magnetic beads were purchased from BioMag Scientific (Wuxi, China); and the TwistAmp basic kit was purchased from TwistDx (Hertfordshire, AL, UK).

[0040] 1.2CBD sequence screening and truncation optimization;

[0041] By searching the literature, we have sorted out the CBD proteins of bacteriophage lytic enzymes with lytic activity against Listeria monocytogenes, as shown in Table 1:

[0042] Table 1 Results of literature screening and collation of CBD information

[0043]

[0044] Note: "+++" means extremely high binding, "++" means relatively high binding, and "+" means high binding.

[0045] To achieve enrichment of all serotypes of Listeria monocytogenes, this study identified two candidate proteins: CBD118 and CBD500, after database screening and literature review. The minimum length for an effective targeting protein for CBD118 is 182 amino acids from the C-terminus, while the minimum length for an effective targeting protein for CBD500 is 157 amino acids from the C-terminus. While retaining the shortest possible C-terminal binding structure, the sequences of CBD118 and CBD500 were truncated and linked using a flexible protein linker to express the recombinant CBD118-500 protein, which serves as a capture tool for broad-spectrum enrichment of Listeria monocytogenes.

[0046] 1.3 Construction of CBD protein expression vector;

[0047] After optimizing rare codons in the selected CBD118 and CBD500 gene sequences, the flexible protein linker gene (GGGGS)3 was used to connect the CBD118-500 fusion gene. The ends were then ligated to a cloning vector using restriction endonucleases NedI and XhoI to construct a recombinant cloning plasmid. The plasmid was then transformed into E. coli DH5α cells for culture. After sequencing and alignment, double enzyme digestion was performed, and the target fragment was ligated to the prokaryotic expression vector pET28a(+), retaining the His tag on the pET28a(+) vector to construct a recombinant expression plasmid.

[0048] Results: The size of the selected CBD118 was 546 bp, the size of CBD500 was 471 bp, and the theoretical size of the constructed CBD118-500 was 1074 bp. After the recombinant clone plasmid was transformed into E. coli DH5α cells and cultured, the amplified product was verified by bacterial liquid PCR to be larger than 1000 bp, which was in line with the expected results ( Figure 2 a), and the specific sequences were verified to be consistent with the predictions by sequencing ( Figure 2 b), indicating that the CBD118-500 recombinant fragment has been successfully synthesized. The target fragment was double-digested with NedI and XhoI and ligated to the prokaryotic expression vector pET28a(+), retaining the His tag on the pET28a(+) vector to construct a recombinant expression plasmid, as shown in FIG. Figure 3 The plasmid was transformed into E. coli BL21 (DE3) cells and kept for future use.

[0049] 1.4 Induction expression, purification and concentration of recombinant strains;

[0050] An E. coli Rosetta (DE3) strain containing the recombinant plasmid, which had been correctly identified, was selected for inducible expression experiments. The strain was first activated, and the cells were collected as an uninduced sample. The bacterial solution after IPTG induction was then collected as an induced sample. The cells were disrupted using an ultrasonic disruptor, and after centrifugation, the supernatant and precipitate were collected for subsequent agarose gel electrophoresis to confirm protein expression in the supernatant or inclusion bodies. The E. coli Rosetta (DE3) strain containing the recombinant plasmid was expanded and induced for expression at 0.1 mmol / L IPTG and 16°C for 20 hours. After expression was complete, the strain was ultrasonically disrupted, and the supernatant was centrifuged and purified by nickel gravity affinity chromatography. The eluates at 80 mmol / L, 250 mmol / L, and 300 mmol / L, as well as the eluates at 150 mmol / L, 200 mmol / L, and 220 mmol / L, were collected and dialyzed for impurities.

[0051] Results: As Figure 4As shown in Figure a, the induced sample can be expressed in large quantities in the supernatant under the optimal induction conditions (16°C, 0.1mmol / L IPTG induction reaction for 20h), and the induced sample meets the expected size (45kDa), so the CBD118-500 recombinant protein is expressed in a soluble manner. The CBD118-500 recombinant protein was purified by gradient elution using a nickel ion affinity chromatography column, and a high concentration of CBD118-500 recombinant protein was successfully purified in the expression supernatant, as shown in Figure 4. Figure 4 As shown in b.

[0052] Example 2: Verification of CBD118-500 binding activity and magnetic bead functionalization;

[0053] 2.1 Indirect ELISA to detect binding activity;

[0054] The binding activity of CBD118-500 recombinant protein to target bacteria was verified using an indirect ELISA method. The specific procedure was as follows: different concentrations of Listeria monocytogenes suspension were coated, and CBD118-500 recombinant protein, rabbit anti-His monoclonal antibody, and HRP goat anti-rabbit secondary antibody were added and incubated. Finally, TMB substrate was added, and the absorbance at OD450nm was measured. The P / N ratio was calculated. A P / N ratio greater than 2.1 was considered to indicate binding of CBD118-500 recombinant protein to the target bacteria.

[0055] Results: As Figure 5 As shown in the figure, the OD450 value gradually decreases with the decrease of the coated bacteria concentration. When the coated bacteria concentration is higher, the OD450 absorbance is higher and the P / N is higher, which can reach up to 4.3. The low concentration of coated bacteria (1.35×10 3 CFU / mL), the P / N was still greater than 2.1, indicating that the CBD118-500 recombinant protein had a high binding activity against Listeria monocytogenes (P / N>2.1).

[0056] 2.2 Preparation of CBD-functionalized magnetic beads and determination of their capture efficiency and capture spectrum;

[0057] The CBD118-500 recombinant protein was coupled to carboxyl magnetic beads using the carbodiimide method to prepare functionalized magnetic beads. The specific steps are as follows: take 1 mg of carboxyl magnetic beads and place them in an EP tube, wash the magnetic beads with 500 μL of reaction buffer, and repeat three times. After washing, take 500 μL of EDC solution with a concentration of 4 mg / mL for activation reaction, and add 5.5 mg of sulf-NHS, and shake at room temperature for 15 minutes. After activation, remove the supernatant, wash three times with magnetic bead coupling liquid, add CBD118-500 recombinant protein diluted with coupling liquid, shake at room temperature for 2 hours, and after magnetic separation, use blocking liquid to react for 30 minutes. After the reaction is complete, magnetically separate the supernatant and store the prepared CBD functionalized magnetic beads in preservation liquid.

[0058] 1 mg of CBD-functionalized magnetic beads was mixed with 1 mL of a gradient-diluted Listeria monocytogenes bacterial suspension and incubated at room temperature for 40 minutes on a four-dimensional mixer. After incubation, the successfully captured L. monocytogenes was enriched by magnetic separation and resuspended in 200 μL of PBS. The capture efficiency was determined by culture. Capture efficiency and capture spectrum were determined using different concentrations of L. monocytogenes and different serotypes of L. monocytogenes. Capture efficiency was calculated as follows: Capture efficiency = total CFU in the enriched group / total CFU in the unenriched group × 100% (n = 3).

[0059] Results: 1 mL of 10-fold serial dilutions of Listeria monocytogenes (ATCC19115 standard strain) (1.35×10 4 ~1.35×10 1 CFU / mL) for enrichment analysis, and the bacterial concentration was 1.38×10 4 to 1.38×10 1 CFU / mL, the capture efficiency of CBD functionalized magnetic beads gradually increased with the decrease of bacterial concentration, and the capture rate increased from 56.45% to 86.58% ( Figure 6 The capture analysis of different serotypes of Listeria monocytogenes (1 / 2a, 1 / 2b, 1 / 2c, 4b and 4c) showed that the CBD functionalized magnetic beads had a high capture rate for different serotypes of Listeria monocytogenes, all greater than 75% ( Figure 6 (b)

[0060] Example 3: Optimization and application of RPA-quantum dot fluorescence test strips;

[0061] 3.1RPA-quantum dot fluorescence test strip reaction principle and test strip assembly;

[0062] The reaction principle of the test strip is as follows Figure 1As shown. RPA upstream and downstream primers were designed and modified with biotin and digoxigenin at their 5' ends (details are shown in Table 2). After RPA amplification, digoxigenin antibody-conjugated quantum dot fluorescent microspheres (QDNBs-McAb), the RPA amplification product, and loading buffer were mixed and added dropwise to test strips coated with streptavidin (T line) and goat anti-mouse IgG (C line), respectively. The chromatography reaction was carried out for 5 minutes. If the test sample contains the target gene, the resulting nucleic acid amplification product will form double-stranded DNA labeled with digoxigenin and biotin. One end of this double-stranded DNA will bind to the digoxigenin antibody fluorescent microspheres through digoxigenin, while the other end will bind to the streptavidin protein coated with the T line through biotin, forming a complex with the fluorescent quantum dot that is retained on the T line, causing the T line to display red fluorescence. Excess complexes continue chromatography to the C line, where the fluorescent quantum dot is retained on the C line due to the binding of the digoxigenin antibody to the anti-mouse secondary antibody, causing the C line to display red fluorescence. At this point, both the T and C lines will emit fluorescent signals, and the fluorescence intensity of the T line will increase as the number of genes tested increases. If the test sample does not contain the test gene, RPA will not produce the corresponding amplified product, and the digoxigenin antibody fluorescent microspheres will chromatograph along the T line to the C line. Similarly, the digoxigenin antibody binds to the mouse secondary antibody, causing the fluorescent quantum dots to be retained on the C line, resulting in red fluorescence on the C line and no fluorescence on the T line. In summary, when the test sample is positive, both the C and T lines will fluoresce; when the test sample is negative, only the C line will fluoresce. Visualization is performed using a blue light fluoroscopy (TGreen) or a 365nm UV flashlight.

[0063] Attach the nitrocellulose membrane to the center of a black PVC substrate. Attach an absorbent pad and sample pad to each end (the sample pad is near the T line, and the absorbent pad is near the C line), overlapping the nitrocellulose membrane by 2 mm. Use an XYZ three-dimensional streaking device to evenly apply 1 mg / mL streptavidin and 1 mg / mL goat anti-mouse IgG to the nitrocellulose membrane (NC membrane) along the T and C lines, respectively. Dry at 37°C for 30 minutes, cut into 0.5 cm strips, and seal and store at 4°C until needed.

[0064] 3.2RPA-quantum dot fluorescence test strip primer design, reaction concentration and RPA reaction time optimization;

[0065] Laboratory preliminary Following the primer design requirements in the basic kit guide, multiple primer pairs suitable for RPA reactions were designed using Primer Premier 5.0, using a highly conserved and specific sequence fragment of the Listeria monocytogenes hly gene in GenBank as the target gene. After preliminary experimental screening, primers with strong specificity and high binding affinity were identified. Specific information is shown in Table 2. Biotin and digoxigenin were added to the 5' end for modification.

[0066] Table 2 RPA primer sequences

[0067]

[0068]

[0069] An RPA reaction system was prepared in a total volume of 5 μL, containing 2.95 μL of buffer, 1.12 μL of DEPC water, 0.24 μL of each upstream and downstream primer at a concentration of 10 μmol / L, 0.2 μL of genomic template, a 1 / 10 tube of lyophilized enzyme pellets, and 0.25 μL of 280 mmol / LM MgOAc. Isothermal amplification reactions were performed at 39°C. To improve detection efficiency and sensitivity, the RPA reaction system was optimized. Optimization factors included primer concentrations (1 μmol / L, 1.5 μmol / L, and 2 μmol / L) and RPA reaction times (5 min, 10 min, 15 min, and 20 min). After the reaction using this reaction system, digoxigenin antibody-conjugated quantum dot fluorescent microspheres, the RPA amplification product, and loading buffer were mixed and applied to the sample pad of the test strip. After 5 minutes, the fluorescence of the T and C lines was observed to determine the optimal primer concentration and RPA reaction time.

[0070] Results: The primer concentrations for RPA reaction were optimized, e.g. Figure 7 As shown in Figure a, the fluorescence brightness of the test strip is clearly visible when the primer concentration is 1.5μmol / L and 2μmol / L. In line with the principle of cost saving, the final reaction primer concentration is determined to be 1.5μmol / L. The reaction time is optimized, as shown in Figure 5. Figure 7 As shown in middle b, after 5 minutes of reaction, obvious fluorescent bands can be seen on the test strips. The brightness of the bands at 10 minutes, 15 minutes, and 20 minutes are similar. Therefore, the visualization reaction time is selected as 10 minutes.

[0071] 3.3 Verification of sensitivity and specificity of RPA-quantum dot fluorescence test strips;

[0072] The cultured Listeria monocytogenes was diluted to 3×10 7 ~3×10 1 CFU / mL, DNA was extracted using the boiling method, and RPA-quantum dot fluorescence test strips were used to analyze samples of varying concentrations to evaluate the sensitivity of the method. Using Listeria monocytogenes as the positive control, a bacterial genome extraction kit was used to extract DNA from Klebsiella pneumoniae, Shigella flexneri, and non-pathogenic Yersinia enterocolitica, and the RPA-quantum dot fluorescence test strips were used to evaluate the specificity of the method.

[0073] Results: When evaluating the sensitivity of RPA-quantum dot fluorescence test strips, Figure 8 As shown in a, when the bacterial concentration is 3×10 2When the bacterial concentration was above 3×10 1 CFU / mL, the T line has no obvious fluorescence. It can be seen that the detection limit of the test strip for bacterial solution is 3×10 2 CFU / mL, which corresponds to a genome concentration of 2.4×10 -8 ng / μL.

[0074] In specificity tests, e.g. Figure 8 As shown in (b), the T lines for the three non-target strains and the control group (Klebsiella pneumoniae, Shigella flexneri, Yersinia enterocolitica, and DEPC water) showed no fluorescence after testing. Only the T line for Listeria monocytogenes showed fluorescence. This demonstrates that the RPA-quantum dot fluorescence test strip method has good specificity for detecting Listeria monocytogenes.

[0075] 3.4RPA-quantum dot fluorescence test strip sample spike detection;

[0076] Raw pork samples were selected for spiked experiments to test the detection limit of RPA-quantum dot fluorescence test strips in meat food matrices. The bacterial solution was diluted to 9.1×10 7 ~9.1×10 0 CFU / mL. 1 mL of diluted bacterial solution of varying concentrations was added to 1 g of raw pork sample and homogenized. 8 mL of saline solution was then added to each solution and mixed. CBD-functionalized magnetic beads were then used to enrich Listeria monocytogenes in 1 mL of the mixed solution, and DNA was extracted. Finally, the RPA-quantum dot fluorescence test strip method was used to analyze the detection effect on the spiked sample.

[0077] Results: As Figure 9 As shown, 9.1×10 1 The five experimental groups corresponding to the concentrations of CFU / mL and above all had obvious fluorescence on the T line; 0 CFU / mL concentration, the T line still showed weak fluorescence. This indicates that after enrichment with CBD magnetic beads, the test strip is sensitive to 9.1×10 0 Bacterial concentrations of CFU / mL can still be detected, and the sensitivity is increased by nearly two orders of magnitude compared to the detection of simple bacterial liquid without enrichment.

[0078] 3.5 Application of RPA-quantum dot fluorescence test strips in actual sample detection;

[0079] Sixteen meat samples previously stored in the laboratory were collected. Samples 1-3 were from a local farm processing contaminated meat, samples 4-7 were from a local farmers' market, and samples 8-16 were from a local supermarket. Samples were enriched using CBD-functionalized magnetic beads, and then analyzed for the presence of Listeria monocytogenes using RPA-quantum dot fluorescence test strips.

[0080] Results: As Figure 10 As shown in Figures a and b, samples 1 through 4 tested positive by PCR amplification and also by RPA-quantum dot fluorescence strips. The RPA-quantum dot fluorescence strip method accurately identified Listeria monocytogenes contamination in four of the 16 meat samples, with results consistent with those obtained by PCR. Furthermore, based on source analysis, samples 1 through 3, processed at the slaughterhouse, were positive for Listeria monocytogenes, as was sample 4, a sample from a farmers' market. Samples 9 through 16, processed at a local supermarket, were negative for Listeria monocytogenes.

[0081] discuss:

[0082] In various detection technologies, the detection sensitivity threshold has always been a key factor restricting methodological research. Taking real-time fluorescence quantitative PCR (qPCR) as an example, its theoretical detection limit can reach 10 1 ~10 2 However, in the actual detection of food samples, due to the interference of food matrix, it is often necessary to go through a 12-24h selective bacterial enrichment process to increase the concentration of target bacteria to 10 3 In conventional testing processes, there is a positive correlation between detection sensitivity and time consumption, that is, the higher the detection limit of the method, the longer the pre-treatment time required.

[0083] The present invention successfully constructed and expressed the CBD118-500 recombinant protein, which can achieve efficient enrichment of Listeria monocytogenes in a short time (1 hour) through functionalized magnetic beads. Experimental data showed that when no bacterial enrichment treatment was performed, the detection limit of the RPA quantum dot test strip was 3×10 2 CFU / mL; after enrichment, the detection limit in spiked samples can reach 9.1×10 0 CFU / mL, lowering the detection limit by 10 2 CFU / mL. This enrichment method can effectively enrich the target bacteria from complex matrices, which not only improves the sensitivity of the detection method, but also reduces the time and labor costs of traditional enrichment culture methods. It is worth noting that CBD118-500 has a high concentration of 1.38×10 1CFU / mL), it shows a capture efficiency of up to 86.58%, and this phenomenon may be related to its binding kinetics. At low bacterial concentrations, the contact sites between the magnetic beads and the bacteria are not saturated, and the steric hindrance effect is reduced, which improves the binding efficiency; while at high bacterial concentrations, competitive binding between bacteria or saturation of binding sites on the surface of the magnetic beads may lead to a decrease in capture efficiency. This fully demonstrates that CBD118-500 has significant advantages in trace detection. However, considering that food samples (such as meat and dairy products) contain complex matrix components (such as fat and protein), these components may interfere with the binding of magnetic beads to bacteria. In the future, it is necessary to further optimize the surface modification strategy of the magnetic beads to enhance their anti-interference ability, which is expected to further improve the enrichment rate. In addition, the present invention constructs a broad-spectrum recombinant protein by fusing bacteriophage CBD118 and CBD500, and utilizes a dual-target recognition mechanism and gene sequence optimization design (cutting off non-critical domains and introducing flexible linkers) to achieve efficient capture of Listeria monocytogenes serotypes 1 / 2 and 4, covering major pathogenic serotypes (such as 1 / 2a and 4b), significantly reducing the risk of missed detection in food. Full serotype detection is crucial to food safety: on the one hand, it can adapt to the differences in serotype distribution in different regions (such as European 4b and North American 1 / 2a), meeting the requirements of international trade standardization; on the other hand, it provides precise support for tracing the source of foodborne epidemics and helps to quickly block the transmission chain.

[0084] In addition, the rapid detection system constructed by the present invention based on RPA technology and quantum dot fluorescent test strips can specifically detect Listeria monocytogenes. By optimizing the primer concentration and reaction time, and combining CBD functionalized magnetic beads for enrichment, the system showed high sensitivity (detection limit of 9.1×10 0CFU / mL) and excellent specificity. In the detection of meat samples, its detection results are completely consistent with PCR, which shows that the system has great application potential in clinical and point-of-care testing (POCT). Compared with traditional nucleic acid detection methods (such as PCR or qPCR), the innovation of the present invention lies in combining the rapid amplification advantage of RPA (no need for complex thermal cycling equipment) with the high fluorescence intensity characteristics of quantum dots, which significantly improves the detection efficiency and signal stability. Experimental results show that the RPA reaction system is small, 1 / 10 of the conventional RPA system; the time is only 10 minutes, which is 90% shorter than the conventional PCR reaction time, and has significant advantages in economic cost and time cost. At the same time, the photostability of quantum dots effectively overcomes the defect of easy quenching of traditional organic dyes, making it suitable for detection needs in the field or in resource-limited environments. Moreover, the test strip adopts a visual interpretation method, and only a convenient blue light meter or ultraviolet flashlight is required to observe the results, which further reduces the dependence on equipment and meets the core requirements of POCT. After verification with 16 actual samples, the test results were consistent with the traditional PCR method. According to the analysis of the sources of the 16 meat samples, no Listeria monocytogenes was detected in the meat samples purchased from local supermarkets, which provides certain theoretical guidance for the purchase of edible meat.

[0085] In future practical applications, this detection system can be expanded in multiple dimensions: based on modular design, develop multi-pathogen joint detection technology for Salmonella, Escherichia coli, etc., expand to on-site detection of complex matrices such as dairy products and ready-to-eat foods, and improve the prevention and control network by replacing targeted proteins; combine with portable fluorescence reading devices (such as smartphone adapter modules) to realize intelligent detection and cloud-based data; promote the large-scale production of magnetic bead enrichment kits and test strips and optimize the freeze-drying process, embed them into cold chain logistics nodes to realize real-time pollution monitoring and epidemic warning, and jointly formulate quality control standards for the entire chain with regulatory authorities.

[0086] In summary, the present invention successfully established a CBD magnetic bead enrichment combined with RPA-quantum dot fluorescent test strips for Listeria monocytogenes detection. By expressing the high-affinity recombinant protein CBD118-500, CBD-functionalized magnetic beads were constructed, and the magnetic bead enrichment technology was innovatively combined with the RPA-quantum dot fluorescent test strips constructed by RPA technology and quantum dot fluorescent test strips to develop a CBD enrichment-RPA-quantum dot fluorescent test strip detection system. It can effectively enrich 86.58% of the target bacteria in the sample, and the entire process from bacterial enrichment to result interpretation can be completed within 1.5 hours. The detection limit for spiked meat samples reached 9.1×10 0CFU / mL, demonstrating a two-order-of-magnitude increase in sensitivity compared to unenriched detection. Validated across 16 real-world samples, the results were consistent with those of traditional PCR methods. This detection system provides an innovative solution for on-site detection of pathogenic microorganisms in food. Its modular design can also be expanded to the rapid detection of foodborne pathogens in trace samples from other complex matrices, providing valuable practical value for improving food safety prevention and control networks.

[0087] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A CBD118-500 recombinant protein for detecting Listeria monocytogenes, characterized in that: The amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

2. A recombinant expression vector, characterized in that: The method comprises the CBD118-500 recombinant protein encoding gene according to claim 1, and the gene is inserted into the prokaryotic expression vector pET28a(+) through the restriction endonuclease NdeI and XhoI sites, and the His tag on the vector is retained.

3. A recombinant strain, characterized in that The recombinant expression vector according to claim 2 is transformed into Escherichia coli BL21 (DE3) and used to express the CBD118-500 recombinant protein.

4. A CBD functionalized magnetic bead, characterized in that The CBD118-500 recombinant protein according to claim 1 is coupled with carboxyl magnetic beads by the carbodiimide method.

5. An RPA-quantum dot fluorescence test strip, characterized in that: The invention comprises a test strip carrier, a T line coated with streptavidin, a C line coated with goat anti-mouse IgG, quantum dot fluorescent microspheres coupled with digoxigenin antibodies, and an RPA amplification product for detecting Listeria monocytogenes, wherein the RPA amplification product is double-labeled with biotin and digoxigenin.

6. An RPA amplification primer pair, characterized in that: The sequences thereof are shown in SEQ ID No. 3 and SEQ ID No.

4.

7. A method for detecting Listeria monocytogenes, characterized in that: The following steps are involved: Enriching Listeria monocytogenes in a sample using the CBD-functionalized magnetic beads according to claim 4; Extract the enriched sample DNA; Perform RPA amplification using the RPA amplification primer pair according to claim 6; The digoxin antibody-coupled quantum dot fluorescent microspheres, the RPA amplification product and the loading buffer are mixed and added dropwise to the RPA-quantum dot fluorescent test strip according to claim 5 for chromatography reaction. The fluorescence of the T line and the C line is observed to determine whether the sample contains Listeria monocytogenes.

8. A Listeria monocytogenes detection system, characterized in that: Combined with the CBD functionalized magnetic bead enrichment method described in claim 4 and the Listeria monocytogenes detection method described in claim 7, it is used for the rapid detection of Listeria monocytogenes in food samples.

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