Lateral flow chromatography detection method for foodborne pathogenic bacteria based on nanozyme recognition by bacteriophage

Abstract

The present invention discloses for the first time a lateral flow chromatography detection method for nanozymes of foodborne pathogenic bacteria based on bacteriophage recognition. Primers are designed based on the gene encoding the tail fiber protein of pathogenic bacteria bacteriophage, and the tail fiber protein of bacteriophage is expressed. A nanozyme probe with dual functions of capture and catalytic activity is combined to assemble a visualized paper-based chromatography sensor to establish a lateral flow chromatography detection method for nanozymes of foodborne pathogenic bacteria based on bacteriophage recognition. The present invention has good sensitivity and specificity in detecting foodborne pathogenic bacteria, and can successfully detect pathogenic bacteria in food, with a detection limit of up to 10 CFU / mL.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensor detection, and specifically relates to a preparation and detection method of a foodborne pathogenic bacteria nanozyme lateral flow chromatography test strip based on phage recognition. Background Art

[0002] Food safety is related to the public's physical health and life safety. In the past decade, foodborne pathogenic bacteria contamination has always ranked first among food safety incidents. Foodborne pathogens are widely present in the food chain and can be ingested into the human body through contaminated food or drinking water during processing, storage, circulation and other links, leading to food poisoning, intestinal infectious diseases and the spread of livestock and poultry infectious diseases. Common foodborne pathogens include Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus, pathogenic Escherichia coli and Staphylococcus aureus. Detection technology is an important bottleneck restricting the safety supervision of foodborne pathogens. In order to ensure food safety, the demand for rapid detection of foodborne pathogens is more urgent, and higher requirements are also put forward.

[0003] Many analytical methods have been used to detect foodborne pathogens, such as traditional live bacterial culture, immunological and molecular biological detection methods. Traditional live bacterial detection methods are considered the gold standard for detecting pathogens. This method is low-cost and highly sensitive, but it is also negatively affected by being laborious and time-consuming. In addition, live bacterial culture does not yet have a fully integrated and automated system. Immunological and molecular biological detection methods have significantly improved the detection resolution and shortened the analysis time, such as enzyme-linked immunosorbent assay, polymerase chain reaction (PCR), real-time PCR and fluorescent biosensors. However, these methods still have problems such as being time-consuming, requiring specialized equipment and well-trained technicians, which greatly limits their practical application.

[0004] Nanozymes have the advantages of simple and controllable synthesis scheme, excellent catalytic activity, and extremely high environmental stability. Typical nanozymes include nanocarbon materials, metals, metal oxides, and nanocomposites. Magnetic nanozymes have the characteristics of easy functionalization, good biocompatibility, high stability, accuracy, rapidity, low cost, and simplicity, and play an important role in the detection of foodborne pathogens. Magnetic nanoparticles of ferroferric oxide (Fe3O4 NPs) show peroxidase catalytic activity, which can enhance the signal output of lateral flow chromatography sensors and improve detection sensitivity.

[0005] Vancomycin (Van), a commercial antibiotic, exhibits highly specific interactions with Gram-positive bacteria, and specifically recognizes Gram-positive bacteria through five hydrogen bonds formed with D-alanyl-D-alanine (D-Ala-D-Ala) molecules on the bacterial surface. Bovine serum albumin (BSA) is widely used as a blocker to reduce nonspecific absorption, which can reduce the binding of non-target molecules to the specific sites of target molecules.

[0006] As a new type of recognition substance, bacteriophages have been extensively studied. They can infect host bacteria with extremely high specificity. Both the tail fiber protein (TFP) and the tail spike protein of bacteriophages can be used for rapid detection of pathogenic bacteria. During the infection process, bacteriophages use TFP to anchor host bacteria, and use the tail spike protein to dissolve the cell wall, inject their nucleic acids into the host bacteria, and use the bacterial replication mechanism to replicate offspring. However, the tail spike protein can destroy the integrity of the host cell in a short period of time. Therefore, the tail fiber protein without lytic activity is considered to be an ideal recognition element for bacterial detection. Summary of the invention

[0007] The main technical problem to be solved by the present invention is to provide a lateral flow chromatography biosensor for detecting foodborne pathogens based on bacteriophage-nanozyme, which has high detection sensitivity, good specificity and convenient operation.

[0008] In order to solve the above technical problems, the present invention also provides a method for preparing a foodborne pathogenic bacteria nanozyme lateral flow chromatography test strip based on phage recognition, as follows:

[0009] 1. Preparation of nanozyme probe: Weigh 20 mg SFe3O4 NPs and dissolve them in sterile PBS solution (0.01 mol / L, pH 7.4). Under the action of a magnetic field, wash the SFe3O4 NPs thoroughly and resuspend the collected SFe3O4 NPs in a PBS solution. Add 6.5 mg NHS and 5.8 mg EDC to the resuspension and activate it in the dark at room temperature for 1 h. Wash and resuspend the activated SFe3O4 NPs again with sterile PBS, add 24 mg bovine serum albumin, react at room temperature for 2 h, wash and resuspend again to obtain a BSA-SFe3O4 NPs solution; add 200 mg activated vancomycin (Van) to the BSA-SFe3O4 NPs solution, mix well, incubate in the dark at room temperature for 6 h, wash under the action of an external magnetic field, and resuspend in a PBS solution to obtain a Van-BSA-SFe3O4 NPs probe.

[0010] Preferably, the nanozyme probes are washed three times with 10 mL of sterile PBS solution and then resuspended in 8 mL of sterile PBS solution.

[0011] 2. Extraction of bacteriophage tail fiber protein (in order to better illustrate the invention, the present invention uses Listeria monocytogenes phage as the experimental object. Technicians in this technical field can choose different materials according to actual conditions, but the equivalent substitutions or changes made are all within the protection scope of the present invention): Use a kit to extract the DNA of Listeria monocytogenes phage A511, query the genome sequence of phage A511 from NCBI (accession number: NC_009811.2), and design primers based on the tail fiber protein gene sequence. Use phage DNA as a template and primers for PCR amplification to obtain the gene fragment encoding the tail fiber protein. Use Beijing Quanshijin Biological Co., Ltd. -Blunt E1 Expression Kit was used to construct the recombinant plasmid, and the recombinant plasmid was amplified by PCR and verified by sequencing. The recombinant plasmid with the correct sequencing results was transferred into Escherichia coli BL21 (DE3) pLysS competent cells, and IPTG inducer was added to induce the expression of tail fiber protein. The target protein band was observed by SDS-PAGE electrophoresis, and the protein was purified by Ni-NTA purification system.

[0012] Preferably, primers are designed according to the tail fiber protein gene sequence: F: 5'-ATGAGTGATGTAGAATC AGG-3', R: 5'-CTGCTTTAGAAAATAGCTGA-3'.

[0013] Preferably, the PCR amplification system is: 35.2 μL of ddH2O, 1 μL of dNTPs (2.5 mmol / L), 5 μL of 10×PCR buffer, 0.3 μL of Taq polymerase (5 U / μL), 2 μL of upstream and downstream primers, and 2 μL of Mg. 2+ The PCR amplification conditions were: 94°C denaturation, 55°C annealing, 72°C extension, 30 cycles, 72°C extension.

[0014] 3. Test strip assembly: Glue the NC membrane to the bottom plate, and then glue the sample pad and the absorption pad from one side of the bottom plate to the other side in sequence, with the sample pad and the absorption pad overlapping the NC membrane by 2 mm respectively. Spray 35 μL of the tail fiber protein solution on the NC membrane of the test strip to form a test line, and place the sprayed test strip at room temperature to wait for the sprayed protein to dry. Use a strip cutter to cut the test strip into 3.8 mm wide, and place the prepared test strip in a packaging bag containing a desiccant and store it at 4°C.

[0015] Preferably, the concentration of the tail fiber protein solution used is 10 mg / mL.

[0016] 4. The present invention also provides a method for preparing a foodborne pathogenic bacteria nanozyme lateral flow chromatography test strip based on phage recognition, comprising the following steps:

[0017] 1) Preparation of SFe3O4 NPs: Anhydrous NaAc (0.015 mol) was added to 15 mL of ethylene glycol for ultrasonic dissolution, and FeCl3·6H2O (0.004 mol), sodium citrate (0.2 g) and PEG 6000 (0.2 g) were dissolved in 15 mL of ethylene glycol and added to the sodium acetate solution. The solution was ultrasonically dissolved and stirred vigorously under a strong magnetic force, and then transferred to a stainless steel reactor with a polytetrafluoroethylene liner for reaction. After cooling to room temperature, the product was washed several times with deionized water and anhydrous ethanol, and then dried in a 60°C oven for 12 h to obtain carboxyl-containing Fe3O4 NPs (SFe3O4 NPs).

[0018] Preferably, the vigorous stirring time during the reaction is 30 min, and the reaction conditions in the stainless steel reactor are 200° C. and 8 h.

[0019] 2) Activation of vancomycin: 200 mg of vancomycin was dissolved in 2 mL of sterile PBS solution, and then 47.89 mg of NHS and 169.3 mg of EDC were added, and the mixture was reacted at room temperature in the dark for 10 min.

[0020] 3) Induced expression of tail fiber protein: Select BL21(DE3)pLysS containing pEASY-gp99 and inoculate it into 20mL LB broth containing ampicillin (Amp), and culture it at 37℃, 120r / min for 16h. Inoculate 1mL of the above bacterial solution into 99mL LB(Amp+) broth, and culture it at 37℃, 200r / min for 3.5h until the OD of the bacterial solution reaches 600 0.4-0.6. Add IPTG inducer to induce expression. Take 1mL of bacterial solution and centrifuge quickly, resuspend the precipitate with 36μL sterile ultrapure water, add 4μL 4×SDS-PAGE Loading Buffer, mix well, and boil for 3-5min to prepare protein electrophoresis loading solution for SDS-PAGE electrophoresis.

[0021] Preferably, the optimal induction concentration of IPTG is 1 mmol / L, and the optimal induction conditions are 37° C., 200 r / min, and 8 h.

[0022] Preferably, the Amp concentration in LB broth is 100 μg / mL.

[0023] Preferably, the centrifugation conditions for the bacterial liquid are 4°C, 12000r / min, and 1min.

[0024] 5. The present invention also provides a lateral flow chromatography detection method for foodborne pathogenic bacteria nanozymes based on phage recognition, comprising the following steps: adding a nanozyme probe to 1 mL of the sample to be tested, placing it in a shaker for incubation, and then performing magnetic separation, and then resuspending the obtained bacteria-Van-BSA-SFe3O4 NPs complex with 100 μL PBS solution. The sample pad is placed in the above-mentioned bacterial solution-Van-BSA-SFe3O4 NPs resuspension, and the resuspension migrates to the absorption pad. After 10 minutes, the test strip is placed in another test tube containing 500 μL NaAc (pH 4.5), 10 μL H2O2 (30%) and 10 μL DAB (10 mg / mL) for 6 minutes, and the test strip is quickly rinsed with deionized water to terminate the reaction. The color development is observed by the naked eye, and the result is judged: negative reaction: the detection line does not develop color; positive reaction: the detection line develops color.

[0025] Preferably, the dosage of the nanozyme probe is 100 μL.

[0026] Preferably, the incubation conditions in the shaker are 37° C., 200 r / min, 60 min, and the magnetic separation time is 7 min.

[0027] 6. Compared with the prior art, the present invention has achieved the following technical effects:

[0028] The present invention uses the tail fiber protein of food-borne pathogenic bacteria bacteriophage as a specific recognition element, which is not only harmless to the human body and the environment, but also has a stable combination of tail fiber protein and pathogenic bacteria, high sensitivity and good specificity. This is also the first time that bacteriophage tail fiber protein has been applied as a recognition element to a nanozyme test strip to perform high-sensitivity, high-specificity, rapid and simple screening and detection of food-borne pathogenic bacteria.

[0029] The invention has an ingenious concept, is convenient and fast to detect, and has visual detection results. It does not require professional equipment and instruments and does not require the skills of professionals, and can be used for on-site rapid detection of foodborne pathogens in the environment and food. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The specific embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings, wherein:

[0031] Figure 1 Schematic diagram of the overall structure of the test strip in an embodiment of the present invention;

[0032] Figure 2 This is a result diagram of the application of the foodborne pathogenic bacteria nanozyme lateral flow chromatography detection method based on phage recognition prepared in the present invention in actual samples;

[0033] Figure 3 This is a graph showing the stability results of the foodborne pathogenic bacteria nanozyme lateral flow chromatography biosensor based on phage recognition prepared in the present invention;

[0034] Figure 4 This is a graph showing the specific results of the foodborne pathogenic bacteria nanozyme lateral flow chromatography biosensor based on phage recognition prepared in the present invention;

[0035] Figure 5 This is a graph showing the sensitivity results of the foodborne pathogenic bacteria nanozyme lateral flow chromatography biosensor based on phage recognition prepared in the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described below by means of specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0037] Example 1

[0038] Experimental Materials

[0039] In this example, a total of 4 kinds of microorganisms were used in the foodborne pathogenic bacteria nanozyme lateral flow chromatography detection method based on phage recognition. The information of the Listeria monocytogenes and non-Listeria monocytogenes strains used is shown in Table 1.

[0040] Table 1

[0041]

[0042] Example 2

[0043] See also Figure 1 , a lateral flow chromatography test strip for foodborne pathogenic bacteria nanozyme based on phage recognition, the test strip includes a bottom plate, an NC membrane is adhered to the bottom plate, and then a sample pad and an absorption pad are adhered in sequence from one side of the bottom plate to the other side, and the NC membrane is coated with the tail fiber protein of the pathogenic bacteriophage as a detection line. After the nanozyme captures the foodborne pathogens, the capture liquid is dropped on the sample pad of the test strip. Driven by capillary action, the nanozyme loaded with pathogenic bacteria migrates along the sample pad to the NC membrane. When the mixture reaches the detection line, the bacteriophage TFP specifically captures the pathogens, and the nanozyme accumulates on the detection line. The enzymatic reaction of SFe3O4 NPs to DAB / H2O2 will produce a brown insoluble product, thereby achieving visual detection of pathogenic bacteria.

[0044] The sample pad is a sample pad of model SB-06 produced by Shanghai Jie Ning Biotechnology Co., Ltd., and the absorption pad is an absorption pad of model Jn8306 produced by Shanghai Jie Ning Biotechnology Co., Ltd., which can be directly purchased on the market.

[0045] Example 3

[0046] The lateral flow chromatography detection method of foodborne pathogenic bacteria nanozyme based on phage recognition in this embodiment includes the following preparation steps:

[0047] 1) Preparation of nanozyme probe: Weigh 20 mg of SFe3O4 NPs and add them into 10 mL of sterile PBS solution (0.01 mol / L, pH 7.4), mix well and set aside. Under the action of an external magnetic field, wash the SFe3O4 NPs thoroughly and resuspend the collected SFe3O4 NPs in 10 mL of sterile PBS solution. Add 6.5 mg NHS and 5.8 mg EDC to the above resuspension and activate it at room temperature in the dark for 1 h. Wash and resuspend the activated SFe3O4NPs with sterile PBS, add 24 mg bovine serum albumin, react at room temperature for 2 h, wash and resuspend again to obtain BSA-SFe3O4NPs solution; add 200 mg activated vancomycin to the BSA-SFe3O4NPs solution, mix well, incubate at room temperature in the dark for 6 h, wash 3 times under the action of an external magnetic field, and resuspend in 10 mL sterile PBS solution to obtain Van-BSA-SFe3O4NPs probe.

[0048] 2) Extraction of bacteriophage tail fiber protein (in order to better illustrate the invention, the present invention uses Listeria monocytogenes phage as the experimental object. Technicians in this technical field can choose different materials according to actual conditions, but the equivalent substitutions or changes made are all within the protection scope of the present invention): Use a kit to extract Listeria monocytogenes phage DNA, query the phage A511 genome sequence from NCBI (accession number: NC_009811.2), and design primers based on the tail fiber protein gene sequence. Use phage DNA as a template and primers for PCR amplification to obtain the tail fiber protein encoding gene fragment. Use Beijing Quanshijin Biological Co., Ltd. -Blunt E1 Expression Kit was used to construct the recombinant plasmid, and PCR amplification and sequencing were performed for verification. The recombinant plasmid with the correct sequencing results was transferred into Escherichia coli BL21 (DE3) pLysS competent cells, and IPTG inducer was added to induce the expression of tail fiber protein. The protein bands were observed by SDS-PAGE electrophoresis, and the protein was purified using Ni-NTA purification system.

[0049] 3) Test strip assembly: Glue the NC membrane to the bottom plate, and then glue the sample pad and the absorption pad from one side of the bottom plate to the other side in sequence, with the sample pad and the absorption pad overlapping the NC membrane by 2 mm respectively. Spray 35 μL of tail fiber protein solution (10 mg / mL) on the NC membrane of the test strip to form a detection line, and place the sprayed test strip at room temperature to wait for the sprayed protein to dry. Use a strip cutter to cut the test strip into 3.8 mm wide, and place the assembled test strip in a packaging bag containing a desiccant and store it at 4°C.

[0050] In the step 2), primers are designed according to the bacteriophage tail fiber protein gene sequence: F: 5'-ATGAGTGATGTAGAATCAGG-3', R: 5'-CTGCTTTAGAAAATAGCTGA-3'.

[0051] The foodborne pathogenic bacteria nanozyme lateral flow chromatography detection method based on phage recognition in this embodiment also includes the following preparation method:

[0052] 4) Preparation of magnetic nanozymes: Anhydrous NaAc (0.015 mol) was added to 15 mL of ethylene glycol for ultrasonic dissolution, and then FeCl3·6H2O (0.004 mol), sodium citrate (0.2 g) and PEG 6000 (0.2 g) were dissolved in 15 mL of ethylene glycol and added to the sodium acetate solution. After ultrasonic dissolution, the solution was vigorously stirred under a strong magnetic force for 30 min, transferred to a stainless steel reactor with a polytetrafluoroethylene liner, and reacted at 200°C for 8 h. After cooling to room temperature, the product was washed several times with deionized water and anhydrous ethanol, and then placed in a 60°C oven for 12 h to obtain carboxyl-containing Fe3O4 NPs (SFe3O4 NPs).

[0053] 5) Activation of vancomycin: 200 mg of vancomycin was dissolved in 2 mL of sterile PBS solution, and then 47.98 mg of NHS and 169.3 mg of EDC were added, and the mixture was reacted at room temperature in the dark for 10 min.

[0054] 6) Induced expression of tail fiber protein: Select BL21 (DE3) pLysS containing pEASY-gp99 and inoculate it into 20 mL LB broth containing 100 μg / mL ampicillin (Amp), and culture it at 37°C and 120 r / min for 16 h. Inoculate 1 mL of the above bacterial solution into 99 mL LB (Amp+) solution, and culture it at 37°C and 200 r / min for 3.5 h until the OD of the bacterial solution reaches 600 0.4-0.6. Add IPTG inducer to induce expression. Take 1mL of bacterial solution and centrifuge it quickly at 4℃ and 12000r / min for 1min, and resuspend the precipitate with 36μL sterile ultrapure water. Add 4μL 4×SDS-PAGE Loading Buffer, mix well, and boil for 3-5min to prepare protein electrophoresis loading solution.

[0055] In the step 6), the optimal induction concentration of IPTG is 1 mmol / L, and the optimal induction expression conditions are 37° C., 200 r / min, and 8 h.

[0056] Example 4

[0057] In this example, the foodborne pathogenic bacteria nanozyme lateral flow chromatography biosensor based on phage recognition prepared in Example 3 is actually applied, and the application method is as follows:

[0058] Four samples (0.9% saline, milk, lettuce juice, PBS buffer solution) were selected and confirmed to be free of Listeria monocytogenes by conventional plate culture methods. Lettuce was soaked in sodium hypochlorite solution (10%) for 10 minutes, and then the lettuce leaves (10 g) were crushed and mixed with sterile PBS buffer (90 mL), and then homogenized with a homogenizer for 5 minutes. The other three samples were directly stored at 4°C.

[0059] The final concentration was 10 4 CFU / mL of Listeria monocytogenes solution (0.1mL) was added to 0.9mL 0.9% saline, PBS buffer, milk and lettuce juice, and then 100μL Van-BSA-SFe3O4 NPs solution was added. The mixture was incubated at 37°C and 200rpm for 60min, and the bacteria-Van-BSA-SFe3O4 NPs complex was obtained by magnetic separation for 7min. The supernatant obtained by magnetic separation was placed in a 2mL centrifuge tube, and the bacteria-Van-BSA-SFe3O4 NPs complex was resuspended with 100μL sterile PBS solution. The supernatant and bacteria-Van-BSA-SFe3O4 NPs resuspension (capture solution) were diluted to appropriate concentrations, and the capture efficiency of Van-BSA-SFe3O4 NPs was calculated by TSA plate counting method (the colony count was 20-200 as valid data). Capture efficiency (%) = total number of colonies in the capture solution / (total number of colonies in the supernatant + total number of colonies in the capture solution) × 100%. Figure 2 .

[0060] from Figure 2 It can be seen that the capture rates of Van-BSA-SFe3O4 NPs in PBS and saline were 95.7% and 93.2%, respectively, with no significant difference between the two (p>0.05). In milk samples and lettuce juice samples, due to the presence of some matrix effects, the capture rate of Listeria monocytogenes was reduced to 82.1% and 77.7%, with no significant difference between the two samples (p>0.05). The above results show that the prepared magnetic nanozyme probe has a good effect in capturing Listeria monocytogenes in actual samples.

[0061] Example 5

[0062] In this example, the performance of the foodborne pathogen nanozyme lateral flow chromatography biosensor based on phage recognition prepared in Example 3 was evaluated, including the stability, specificity and sensitivity of the chromatography test strip.

[0063] (1) Stability of Chromatographic Test Strips

[0064] After the test strips were placed for 1-6 weeks, the final concentration of 10 4 The resuspended liquid of Listeria monocytogenes-Van-BSA-SFe3O4 NPs with CFU / mL was dropped on the sample pad of the test strips placed at different times. After 10 minutes, the test strips were placed in DAB+H2O2 solution for 6 minutes, and the test strips were quickly rinsed with deionized water to terminate the reaction. The color development of the detection line was observed by naked eyes. The results are shown in Figure 2. Figure 3 .

[0065] Figure 3 From left to right are the reaction results of the test strips placed for 1 to 6 weeks. It can be seen that the nanozyme test strips placed for different times have almost the same reaction to Listeria monocytogenes, indicating that the nanozyme test strip has good stability.

[0066] (2) Specificity of Chromatographic Test Strips

[0067] The resuspended liquid of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella, Staphylococcus aureus and negative control ddH2O and Van-BSA-SFe3O4 NPs probe was dropped on the sample pad of the test strip. After 10 minutes, the test strip was placed in DAB+H2O2 solution for 6 minutes. The test strip was quickly rinsed with deionized water to terminate the reaction, and the color development of the detection line was observed by naked eyes. The results are shown in Figure 4 .

[0068] Figure 4 From left to right, this is the reaction result of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella, Staphylococcus aureus and ddH2O on the test strip. It can be seen that when testing Listeria monocytogenes, obvious bands can be observed on the test strip. No bands were observed in other bacterial solutions and ddH2O, and there were no false positive results, indicating that the chromatographic test strip has a high specificity for Listeria monocytogenes.

[0069] (3) Sensitivity of Chromatographic Test Strips

[0070] The final concentrations (0-10 5 CFU / mL) of Listeria monocytogenes-Van-BSA-SFe3O4 NPs resuspension was dropped on the sample pad of the test strip. After 10 minutes, the test strip was placed in DAB+H2O2 solution for 6 minutes. The test strip was quickly rinsed with deionized water to terminate the reaction, and the color development of the detection line was observed by naked eyes. The results are shown in Figure 5 .

[0071] Figure 5-A from left to right indicates that the concentration of Listeria monocytogenes in the samples is 10 5 , 10 4 , 10 3 , 10 2 、10、0CFU / mL, Figure 5 -B is the calibration curve of Listeria monocytogenes and the signal intensity of the test line. The ordinate represents the signal intensity on the test line, and the abscissa represents the logarithm of the concentration of the Listeria monocytogenes solution, which are 0, 10, 10 from left to right. 2 , 10 3 , 10 4 , 10 5 CFU / mL. As can be seen from Figure 5-A, as the concentration of Listeria monocytogenes solution gradually decreases, the signal intensity of the test line also decreases. When the concentration of Listeria monocytogenes solution in the sample is lower than 10CFU / mL, no brown band appears on the test line, so it can be concluded that the detection limit of the test strip is 10CFU / mL. Figure 5 As shown in -B, the intensity of the brown band on the test line is proportional to the concentration of Listeria monocytogenes, which can be expressed by the regression equation y=763.17x+58.33, R 2 =0.9951. The above results show that the nanozyme test strip can be used for quantitative and qualitative detection of foodborne pathogens.

[0072] In summary, the present invention designs a visual paper-based analyte sensor by preparing a capture-catalytic activity bifunctional nanozyme probe, expressing the tail fiber protein of the bacteriophage of foodborne pathogens, and adding it to the test strip detection line as a specific recognition substance. The detection technology prepared by the present invention does not require the proliferation of microorganisms, directly identifies specific pathogens through bacteriophages, does not require labeling, and improves sensitivity through nanozyme catalysis, does not rely on instruments, and directly observes the detection results of pathogens through visual test strips. At the same time, the present invention establishes an application system of test strips in food pathogen detection, which has a very large space for rapid and highly sensitive detection of foodborne pathogens.

[0073] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A lateral flow chromatography detection method for foodborne pathogenic bacteria nanozymes based on phage recognition, wherein the nanozyme probe Van-BSA-SFe3O4 NPs is used as the first recognition element to capture Gram-positive foodborne pathogens in the sample, and the nanozyme probe is composed of carboxylated ferric oxide nanoparticles SFe3O4 NPs, vancomycin Van and bovine serum albumin BSA, and specifically recognizes Gram-positive bacteria through five hydrogen bonds formed by Van and D-alanyl-D-alanine molecules on the bacterial surface; the paper-based chromatographic test strip includes a bottom plate, on which there are a sample pad, an absorption pad and an NC membrane in sequence, and the NC membrane is characterized in that it is coated with a detection line coated with purified foodborne pathogenic bacteria phage tail fiber protein TFP; TFP is used as the second recognition element to specifically capture the pathogenic bacteria to be detected, and the nanozyme will accumulate on the detection line; using SFe3O4 The peroxidase activity of NPs catalyzes DAB / H2O2 to produce brown insoluble matter, and the color intensity is proportional to the concentration of the pathogenic bacteria to be tested in the sample, thereby realizing the visual detection of the pathogenic bacteria to be tested.

2. A lateral flow chromatography detection method for foodborne pathogenic bacteria nanozyme based on phage recognition, the preparation method of the chromatographic test strip comprises the following steps: 1) Preparation of nanozyme probe: Weigh 20 mg of SFe3O4 NPs and dissolve them in 0.01 mol / L sterile PBS solution. Under the action of an external magnetic field, wash the SFe3O4 NPs thoroughly and resuspend the collected SFe3O4 NPs in PBS solution. 6.5 mg NHS and 5.8 mg EDC were added to the resuspension and activated at room temperature in the dark for 1 h. The activated SFe3O4 NPs were washed and resuspended again with sterile PBS, and 24 mg BSA was added. The mixture was reacted at room temperature for 2 h, washed and resuspended to obtain a BSA-SFe3O4 NPs solution. 200 mg of activated Van was added to the BSA-SFe3O4 NPs solution, mixed evenly, incubated at room temperature in the dark for 6 h, washed and resuspended in a PBS solution under the action of an external magnetic field to obtain a Van-BSA-SFe3O4 NPs nanozyme probe. 2) Extraction of bacteriophage tail fiber protein: DNA of Listeria monocytogenes bacteriophage A511 was extracted using a kit, and the genome sequence of bacteriophage A511 was queried from NCBI, with the registration number NC_009811.

2. Primers were designed based on the tail fiber protein gene sequence; PCR amplification was performed using phage DNA as a template and primers to obtain the gene fragment encoding the tail fiber protein; -Blunt E1 Expression Kit was used to construct the recombinant plasmid, and the recombinant plasmid was amplified by PCR and verified by sequencing; the recombinant plasmid with the correct sequencing result was transferred into Escherichia coli BL21 (DE3) pLysS competent cells, and IPTG inducer was added to induce the expression of tail fiber protein, the protein bands were observed by SDS-PAGE electrophoresis, and the protein was purified by Ni-NTA purification system; 3) Assembling the chromatography test strip: stick the NC membrane to the bottom plate, and then stick the sample pad and the absorption pad from one side of the bottom plate to the other side, with the sample pad and the absorption pad overlapping the NC membrane by 2 mm respectively; Spray 35 μL of tail fiber protein solution on the NC film of the test strip to form a detection line, place the sprayed test strip at room temperature and wait for the sprayed protein to dry; cut the test strip into 3.8 mm wide using a strip cutter, place the prepared test strip in a packaging bag containing a desiccant and store it at 4°C.

3. The method for lateral flow chromatography detection of foodborne pathogenic bacteria nanozymes based on phage recognition according to claim 2, characterized in that: In the step 2), primers are designed according to the bacteriophage tail fiber protein gene sequence: F: 5'-ATGAGTGATGTAGAATCAGG-3', R: 5'-CTGCTTTAGAAAATAGCTGA-3'.

4. The method for lateral flow chromatography detection of foodborne pathogenic bacteria nanozymes based on phage recognition according to claim 2, characterized in that: In the step 3), the concentration of the tail fiber protein solution used is 10 mg / mL.

5. The method for lateral flow chromatography detection of foodborne pathogenic bacteria nanozymes based on phage recognition according to claim 2, characterized in that: The preparation method also includes: 4) Preparation of SFe3O4 NPs: 0.015 mol of anhydrous NaAc was added to 15 mL of ethylene glycol for ultrasonic dissolution, and then 0.004 mol of FeCl3·6H2O, 0.2 g of sodium citrate and 0.2 g of PEG 6000 were dissolved in 15 mL of ethylene glycol and added to the sodium acetate solution. After ultrasonic dissolution, the mixture was vigorously stirred under a strong magnetic force and transferred to a stainless steel reactor with a polytetrafluoroethylene liner for reaction; after cooling to room temperature, the product was washed several times with deionized water and anhydrous ethanol, and then dried in a 60°C oven for 12 h to obtain carboxyl-containing Fe3O4 NPs, namely SFe3O4 NPs; 5) Activation of vancomycin: 200 mg of vancomycin was dissolved in 2 mL of sterile PBS solution, and then 47.89 mg of NHS and 169.3 mg of EDC were added, and the mixture was reacted at room temperature in the dark for 10 minutes.

6. The method for lateral flow chromatography detection of foodborne pathogenic bacteria nanozymes based on phage recognition according to claim 2, characterized in that: The application method of the chromatographic test strip is as follows: add the nanozyme probe to 1 mL of the sample to be tested, incubate it in a shaker, perform magnetic separation, and then resuspend it with 100 μL PBS solution to obtain the bacteria-Van-BSA-SFe3O4 NPs complex; place the sample pad in the above-mentioned bacteria-Van-BSA-SFe3O4 NPs resuspension, and the resuspension migrates to the absorption pad; after 10 minutes, place the test strip in another test tube containing 500 μL NaAc, 10 μL 30% H2O2 and 10 μL 10 mg / mL DAB to react for 6 minutes, and quickly rinse the test strip with deionized water to terminate the reaction; observe the color development by naked eye and judge the result: negative reaction: the detection line does not show color; positive reaction: the detection line shows color.

7. The method for lateral flow chromatography detection of foodborne pathogenic bacteria nanozymes based on phage recognition according to claim 6, characterized in that: The shaking incubation conditions were 37°C, 200 r / min, 60 min, and the magnetic separation time was 7 min.

8. The method for lateral flow chromatography detection of foodborne pathogenic bacteria nanozymes based on phage recognition according to claim 6, characterized in that: The amount of nanozyme probe used was 100 μL.

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