A method for detecting salmonella using surface plasmon resonance technology

Abstract

The application relates to a kind of salmonella detection using surface plasmon resonance technology, comprising the following steps: (1) chip preparation, the chip is CM5 chip;(2) bacterial culture, the bacteria is Salmonella enteritidis C50041;(3) bacteria and antibody incubation, the antibody is salmonella PagN (3B3) protein antibody;(4) free antibody separation;(5) surface plasmon resonance technology detection.Relative to prior art, the application has the following beneficial effects: the application can semi-quantitative detection of salmonella in sample;Monoclonal antibody is used as detection target, the application has excellent specificity;Surface plasmon resonance technology is applied to salmonella detection, which can shorten the detection time;Regeneration conditions are optimized, which can ensure the repeatability of detection.

Description

Technical Field

[0001] This invention relates to a method for detecting Salmonella using surface plasmon resonance technology. Background Technology

[0002] Salmonella is a zoonotic foodborne pathogen. It has long been considered one of the most common foodborne pathogens causing public health emergencies such as foodborne microbial safety issues. Despite varying incidence rates, salmonellosis remains a global health threat. Recent studies indicate that Salmonella causes nearly 93.8 million infections and 155,000 deaths annually, severely impacting human health and causing significant social and economic problems. Therefore, rapid and sensitive detection of Salmonella is crucial for preventing outbreaks, implementing early interventions, and providing appropriate treatment.

[0003] Traditional detection methods such as plate culture, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) can detect the species and content of Salmonella, but they still have drawbacks such as being time-consuming and having low sensitivity. For example, plate culture takes approximately 24–52 hours; the detection limit of ELISA is approximately 10. 6 While PCR is rapid and sensitive, it can produce false positives and requires a long processing time. Therefore, how to quickly, accurately, and sensitively detect Salmonella from samples is an urgent problem to be solved.

[0004] With the development of surface plasmon resonance (SPR) technology, it has broad application prospects due to its advantages such as no sample pretreatment required, no labeling, and real-time detection. Currently, although this technology has been widely used for the direct detection of various analytes, including bacterial components, there are no reports on the use of SPR technology to reduce the inhibition of Salmonella detection. Summary of the Invention

[0005] To address the above problems, this application provides a method for detecting Salmonella using surface plasmon resonance technology, which has good sensitivity and specificity.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] A method for detecting Salmonella using surface plasmon resonance (SPR) technology includes the following steps: preparing a Salmonella detection chip; culturing the target bacteria, incubating the target bacteria with an antibody, separating the free antibody, and separating the antibody that has not bound to the bacteria by gradient centrifugation; detecting the response signal of the free antibody using SPR technology, and then calculating the content of the target Salmonella; wherein the antibody is a Salmonella PagN protein monoclonal antibody.

[0008] Furthermore, the Salmonella PagN protein monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0009] The heavy chain variable region contains V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes V L CDR1, V L CDR2 and V L CDR3;

[0010] The V mentioned H The amino acid sequence of CDR1 is shown in SEQ ID NO: 1;

[0011] The V mentioned H The amino acid sequence of CDR2 is shown in SEQ ID NO: 2;

[0012] The V mentioned H The amino acid sequence of CDR3 is shown in SEQ ID NO: 3;

[0013] The V mentioned L The amino acid sequence of CDR1 is shown in SEQ ID NO: 4;

[0014] The V mentioned L The amino acid sequence of CDR2 is shown in SEQ ID NO: 5;

[0015] The V mentioned L The amino acid sequence of CDR3 is shown in SEQ ID NO: 6.

[0016] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0017] Furthermore, the heavy chain amino acid sequence is shown in SEQ ID NO: 9; the light chain amino acid sequence is shown in SEQ ID NO: 10.

[0018] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 17; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 18.

[0019] Furthermore, the nucleotide sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO: 19; the nucleotide sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO: 20.

[0020] Furthermore, the preparation method of the Salmonella detection chip includes: immobilizing Anti mouse IgG onto the surface of the CM5 chip via amino coupling, and then blocking unbound sites with ethanolamine solution; selecting pH 5.0 sodium acetate solution as the coupling buffer.

[0021] Furthermore, the culture of the test bacteria includes: inoculating the test bacteria colonies into LB liquid medium and culturing overnight; then inoculating the bacterial solution into LB liquid medium at a ratio of 1:100, culturing at 37°C and 220 r / min for 3 h; then washing three times with PBS in a 15 mL centrifuge tube and centrifuging at 1500 × g for 10 min.

[0022] Furthermore, the step of incubating the bacteria to be tested with the antibody includes: mixing the Salmonella PagN protein monoclonal antibody with the bacterial culture to be tested, and incubating at room temperature.

[0023] Furthermore, the gradient centrifugation includes: centrifuging the bacterial culture incubated with the antibody at 200, 400, 800, 1200, and 1600 × g for 2 min respectively.

[0024] A method for detecting Salmonella using surface plasmon resonance technology, the method comprising the following steps:

[0025] (1) Chip fabrication, wherein the chip is a CM5 chip;

[0026] (2) Bacterial culture, wherein the bacteria is Salmonella enteritidis C50041;

[0027] (3) Incubation of bacteria with antibody, wherein the antibody is Salmonella PagN protein monoclonal antibody (3B3);

[0028] (4) Free antibody separation: Antibodies that have not bound to bacteria can be separated by gradient centrifugation;

[0029] (5) Surface plasmon resonance technology detection.

[0030] Step (1) is as follows: Take 5 μL of Anti mouse IgG and add it to 162 μL of sodium acetate buffer solution (10 nM sodium acetate) with a pH of 5.0, and mix thoroughly. Take two 1.5 mL EP tubes and add 140 μL of ethanolamine solution, 100 μL of LEDC and 100 μL of NHS mixed solution to each tube. Select File→Open / New Wizard Template to open the wizard dialog box, select Immobilization under Surface Preparation, click New to proceed to the next step, and in the Immobilization Setup wizard window, select CM5 in the Chip type drop-down menu. In this experiment, the ligand will be fixed on the second channel, so check the box before Flow cell 2. Select Amine for Method, enter the name of the ligand: Anti mouse IgG in Ligand, select the Specify contact time and flow rate mode for coupling, Contact time: 480s, Rate: 5 μL / min, click Next to proceed to the next step. In the System Preparations dialog box, check the box before Prime before run. Keep the 25℃ setting unchanged. Click Next to proceed. Place the prepared solutions into the racks according to the sample rack location chart. Return the sample rack to the sample chamber and click Next. The Prepare RunProtocol dialog box will appear. Carefully check the listed items to ensure that the volume of run buffer on the left rack is greater than the minimum requirement displayed in the software. Click Start.

[0031] Step (2) specifically involves: picking a single colony of Salmonella enteritidis C50041 and inoculating it into LB liquid medium for overnight culture. Then, the bacterial solution is inoculated into 6 mL of LB liquid medium at a ratio of 1:100 and cultured at 37°C and 220 rpm for 3 hours. Next, the bacterial solution is washed three times with PBS in a 15 mL centrifuge tube and centrifuged at 1500 × g for 10 minutes. Finally, the bacterial concentration is adjusted.

[0032] Step (3) specifically involves: taking 300 μL of 150 μg / mL Salmonella PagN monoclonal antibody (3B3) and 300 μL of 1×10 8 1×10 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 1×10 1Mix 0 CFU / mL Salmonella enteritidis C50041 bacterial suspension and incubate at room temperature for 1 hour, inverting the container occasionally to mix.

[0033] The specific steps (4) are as follows: centrifuge the bacterial and antibody mixture at 200, 400, 800, 1200, and 1600×g for 2 min respectively, and take 500μL of supernatant for analysis.

[0034] Step (5) is as follows: Open the New Wizard Template, select Kinetics / Affinity in Assay, and click New. In the Injection Sequence dialog box, select 2-1 for Flow path and CM5 for Chip type, and click Next to proceed to the next step. In the setup dialog box, set Startup. Enter Buffer in solution and set Number of Cycles to 3. Click Next to proceed to the next step. In the Injection Parameters dialog box, in Sample, set Contact time to 120s. Set Flow rate to 30μL / min. Set Dissociation time to 300s. In Regeneration, enter Glycine-HCl 1.7 in Solution, set Contact time to 180s, and Flowrate to 20μL / min. Click Next to proceed to the next step. Fill in the sample name and concentration information in the Samples dialog box. Click Next to proceed to the next step. Check the box before Prime before run and keep the 25℃ setting unchanged. Click Next to proceed to the next step. According to the sample rack position table, place the prepared 500μL supernatant in sequence. Return the sample holder to the sample chamber and click Next. The Prepare Run Protocol dialog box will appear. Carefully check the listed items and confirm that the volume of run buffer on the left shelf is greater than the minimum requirement displayed in the software. Click Start.

[0035] Beneficial effects

[0036] 1. This invention utilizes surface plasmon resonance technology, which has advantages such as no sample pretreatment required, no labeling, and real-time detection, thus shortening the detection time;

[0037] 2. Salmonella PagN antibody (3B3) is a monoclonal antibody that can specifically recognize Salmonella, so this invention has excellent specificity;

[0038] 3. This invention is the first to combine surface plasmon resonance technology with the subtraction inhibition method, avoiding the direct detection of Salmonella itself, and instead detecting antibodies that have not bound to Salmonella, effectively reducing the detection limit. This is because the effective detection range of surface plasmon resonance technology is about 300 nm, while the diameter of bacteria is between 0.5 and 7 μm, so only a small part of the bacteria can be detected, resulting in a weak signal, low sensitivity, and a high detection limit. Attached Figure Description

[0039] Figure 1 This is a flowchart of the present invention;

[0040] Figure 2 The results show the electrostatic adsorption response of anti-mouse IgG polyclonal antibodies in coupling buffers at different pH values.

[0041] Figure 3 Results of anti-mouse IgG polyclonal antibody conjugation;

[0042] Figure 4 The result is due to regeneration conditions;

[0043] Figure 5 The results show the optimization of the free antibody separation method;

[0044] Figure 6 Results of concentration optimization for Salmonella PagN monoclonal antibody (3B3); Figure 6 a represents the response value for different concentrations of Salmonella; Figure 6 b represents the inhibition response value of different concentrations of Salmonella; Figure 7 These are results from specific experiments;

[0045] Figure 8 The results of surface plasmon resonance detection are as follows: (A) Response values ​​corresponding to different bacterial concentrations; (B) R / R0 values ​​corresponding to different bacterial concentrations (R: bacterial incubation group, R0: PBS incubation group).

[0046] Figure 9 Results of the experiment on the lysis effect of bacteriophage C3-2 on Salmonella;

[0047] Figure 10 The results of the experiment on the lysis effect of bacteriophage C3-2 on Salmonella. Detailed Implementation

[0048] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. Those skilled in the art can easily understand other advantages and effects of this invention from the content disclosed in this specification. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0050] Material

[0051] Anti-Mouse Antibody (1 mg / mL), sodium acetate buffer (10 mM, pH 5.0), CM5 biosensor chip, ethanolamine (1 mol / L, pH 8.5), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (75 mg / mL), N-hydroxysuccinimide (NHS) (11.5 mg / mL), and glycine-hydrochloric acid (pH 1.7, 10 mM) were purchased from Cytiva, USA; Salmonella PagN monoclonal antibody (3B3) was obtained from the Jiangsu Provincial Key Laboratory of Zoonoses.

[0052] Example 1: Selection of pH for Coupling Buffer

[0053] Prepare four 1.5 mL Eppendorf tubes. Add 2.5 μL of anti-mouse IgG (Cytiva, USA) to each tube, then add 97.5 μL of sodium acetate buffer solution (Cytiva, USA) with pH values ​​of 4.5, 5.0, and 5.5 respectively. Mix thoroughly; remove any air bubbles by centrifugation. Separately, take one 1.5 mL Eppendorf tube and add 200 μL of 50 mM NaOH solution. Cut off the cap of the Eppendorf tube.

[0054] In the Biacore T200 Control Software, select Run→Manual Run. In the dialog box, set the flow rate to 10 μL / min, select Flow path 2, and choose Sample and Reagent Rack1 from the drop-down menu in the upper right corner. Click Start; click Eject rack tray to exit Sample and Reagent Rack1; place the Eppendorf tubes containing sodium acetate buffer (pH 5.5) in position R1D1, those with pH 5.0 in R1D2, and those with pH 4.5 in R1D3, and place the NaOH tubes in position R1E1; return the Sample and Reagent Rack1 to the sample chamber and close the chamber door; click the injection command icon (Sample injection), select the drop-down menu next to Vial / well position, click position R1D1 in the sample position diagram, set the contact time to 120s, and click OK; click the regeneration command icon (Regeneration injection), and select Vial / well... Select position R1E1, regeneration time 30s, and click OK; repeat the above steps, injecting anti-mouse IgG solutions with buffer pH of 5.0 and 4.5 respectively, with injection time of 120s and regeneration time of 30s. After all steps are completed, click the "Stop run" command.

[0055] The optimal pH of the coupling buffer was determined using the results graphs from the Biacore T200 Control Software. Comparative analysis results of coupling buffers at different pH values ​​are presented. Figure 2 The results showed that anti-mouse IgG polyclonal antibody could electrostatically adsorb onto the chip in sodium acetate solutions at pH 4.5, pH 5.0, and pH 5.5, but the adsorption was unstable. However, the adsorption curve was curved in sodium acetate solution at pH 4.5, indicating unstable adsorption. In sodium acetate solution at pH 5.5, the adsorption capacity was comparable to that in sodium acetate solution at pH 4.5, but more stable. In sodium acetate solution at pH 5.0, the electrostatic adsorption curve of anti-mouse IgG polyclonal antibody onto the chip rose faster, indicating the strongest and most stable electrostatic adsorption capacity. Therefore, sodium acetate solution at pH 5.0 was chosen as the coupling buffer.

[0056] Example 2: Preparation of Salmonella Detection Chip

[0057] Add 5 μL of anti-mouse IgG (1 mg / mL) to 162 μL of sodium acetate buffer solution at pH 5.0 and mix thoroughly. Take two 1.5 mL Eppendorf tubes and add 140 μL of ethanolamine solution, 100 μL of EDC and 100 μL of NHS mixed solution to each tube. If there are bubbles, centrifuge to remove them. Cut off the caps of the Eppendorf tubes.

[0058] In the Biacore T200 Control Software, select File→Open / New WizardTemplate to open the wizard dialog box. Select Immobilization under Surface Preparation and click New to proceed to the next step. In the Immobilization Setup wizard window, select CM5 from the Chip type drop-down menu. In this experiment, the ligand is fixed on channel 2 (select Flow cell 2). Select Amine for Method, and enter the ligand name "anti-mouse IgG" in Ligand. Select the Specify contact time and flow rate mode for coupling, with Contact time set to 480s and Rate to 5μL / min. Click Next to proceed to the next step. In the System Preparations dialog box, check the box before Prime before run and keep the 25℃ setting unchanged. Click Next to proceed to the next step. Place the prepared solutions according to the sample rack position table. Return the sample rack to the sample chamber and click Next. The Prepare Run Protocol dialog box will pop up. Carefully check the listed items and confirm that the volume of the run buffer on the left rack is greater than the minimum requirement displayed in the software. Click Start.

[0059] The chips used in this patent are all CM5 chips. The conjugation results of the anti-mouse IgG polyclonal antibody with the chip are as follows: Figure 3 As shown, the process consists of three steps: chip activation, conjugation of anti-mouse IgG polyclonal antibody to the chip, and ethanolamine blocking of unbound sites. First, a mixture of EDC and NHS is used to activate the carboxymethyl dextran on the chip surface (…). Figure 3 a); After activation, rinse the tubing and the chip surface with PBS buffer to remove any remaining EDC and NHS mixture. Figure 3 b); then the anti-mouse IgG polyclonal antibody is adsorbed onto the chip via electrostatic adsorption, and the amino group of the antibody covalently binds to the activated carboxyl amidation group on the chip surface. Figure 3 c); Rinse the tubing and chip surface with PBS buffer to remove unbound antibodies. Figure 3 d); Finally, ethanolamine seals the unbound activated carboxyl sites on the chip surface ( Figure 3 e) Rinse the tubing and chip surface with PBS buffer to remove any remaining ethanolamine. Figure 3 f). The reference channel is channel Fc1, which is not coupled.

[0060] The Salmonella PagN protein monoclonal antibody contains a heavy chain variable region and a light chain variable region;

[0061] The heavy chain variable region contains V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes V L CDR1, V L CDR2 and V L CDR3;

[0062] The V mentioned H The amino acid sequence of CDR1 is GYFMN (SEQ ID NO:1);

[0063] The V mentioned H The amino acid sequence of CDR2 is RINPYNGDTFYNQKFKG (SEQ ID NO:2);

[0064] The V mentioned H The amino acid sequence of CDR3 is SGVAPDY (SEQ ID NO:3);

[0065] The V mentioned L The amino acid sequence of CDR1 is KSSQSLLNSRTRKNYLA (SEQ ID NO:4);

[0066] The V mentioned L The amino acid sequence of CDR2 is WASTRES (SEQ ID NO:5);

[0067] The V mentioned L The amino acid sequence of CDR3 is KQSYNLRT (SEQ ID NO:6).

[0068] The amino acid sequence of the heavy chain variable region is as follows:

[0069] EVQLQQSGPELVKPGASVKISCKASGYSFTGYFMNWVMQSHGKSLEWIGRINPYNGDTFYNQKFKGKATLTVDKSSSTAHMELRSLASEDSAVYYCARSGVAPDYWGQTTLTVSS (SEQ ID NO: 7).

[0070] The amino acid sequence of the light chain variable region is as follows:

[0071] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLRTFGGGTLEIK (SEQ ID NO: 8).

[0072] The heavy chain amino acid sequence is as follows:

[0073] MGWSWIFLFLLSVTAGVFFSEVQLQQSGPELVKPGASVKISCKASGYSFTGYFMNWVMQSHGKSLEWIGRINPYNGDTFYNQKFKGKATLTVDKSSSTAHMELRSLASEDSAVYYCARSGVAPDYWGQTTLTVSS (SEQ ID NO: 9).

[0074] The light chain amino acid sequence is as follows:

[0075] MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLRTFGGGTLEIK (SEQ ID NO: 10).

[0076] The V encoding described H The nucleotide sequence of CDR1 is GGCTACTTTATGAAC (SEQ ID NO:11);

[0077] The V encoding described H The nucleotide sequence of CDR2 is as follows:

[0078] CGTATTAATCCTTACAATGGTGATACTTTCTACAACCAGAAGTTCAAGGGC (SEQ ID NO: 12);

[0079] The V encoding described H The nucleotide sequence of CDR3 is TCGGGAGTAGCCCCTGACTAC (SEQ ID NO:13);

[0080] The V encoding described L The nucleotide sequence of CDR1 is as follows:

[0081] AAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCT (SEQ ID NO: 14);

[0082] The V encoding described L The nucleotide sequence of CDR2 is TGGGCATCCACTAGGGAATCT (SEQ ID NO:15);

[0083] The V encoding described L The nucleotide sequence of CDR3 is AAGCATCTTATAATCTTCGGACG (SEQ ID NO:16).

[0084] The nucleotide sequence encoding the variable region of the heavy chain is as follows:

[0085] GAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC

[0086] AGTGAAGATATCCTGCAAGGTTCTGGTTACTCATTTACTGGCTACTTTATGA

[0087] ACTGGGTGATGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGACGTATT

[0088] AATCCTTACAATGGTGATACTTTCTACAACCAGAAGTTCAAGGGCAGGCCA

[0089] CATTGACTGTAGACAAATCCTCTAGCACAGCCCACATGGAGCTCCGGAGCC

[0090] TGGCATCTGAGGACTCTGCAGTCTATTATTGTGCAAGATCGGGAGTAGCCCCTGACTACTGGGGCCAAGGACCACTCTCACAGTCTCCTCA (SEQ ID NO: 17).

[0091] The nucleotide sequence encoding the variable region of the light chain is as follows:

[0092] GACATTTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAG

[0093] AAGGTCACTATGAGCTCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACC

[0094] CGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAA

[0095] ACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTATCGCTTC

[0096] ACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCA

[0097] AAGCAATCTTATAATCTTCGGACGTTCGGTGGAGGCACCAACTGGAAATCAAA(SEQ ID NO: 18).

[0098] The nucleotide sequence encoding the heavy chain is:

[0099] ATGGGATGGAGCTGGATCTTTCTCTTTCTCCTGTCAGTAACTGCAGGTGTG

[0100] TTCTCTGAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGG

[0101] GGCTTCAGTGAAGATATCCTGCAAGGTTCTGGTTACTCATTTACTGGCTACT

[0102] TTATGAACTGGGTGATGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGA

[0103] CGTATTAATCCTTACAATGGTGATACTTTCTACAACCAGAAGTTCAAGGGC

[0104] AGGCCACATTGACTGTAGACAAATCCTCTAGCACAGCCCACATGGAGCTCC

[0105] GGAGCCTGGCATCTGAGGACTCTGCAGTCTATTATTGTGCAAGATCGGGAGTAGCCCCTGACTACTGGGGCCAAGGACCACTCTCACAGTCTCCTCA (SEQ ID NO: 19).

[0106] The nucleotide sequence encoding the light chain is:

[0107] ATGGATTCACAGGCCCAGGTTCTTATATTGCTGCTGCTATGGGTATCTGGTA

[0108] CCTGTGGGGACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAG

[0109] CAGGAGAGAAGGTCACTATGAGCTCAAATCCAGTCAGAGTCTGCTCAACA

[0110] GTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCA

[0111] GTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCC

[0112] TATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC

[0113] AGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTTCGGACGTTCGGTGGAGGCACCAACTGGAAATCAAA (SEQ ID NO: 20).

[0114] Example 3: Bacterial culture

[0115] Single colonies of *Shigella flexneri* 2a, *Citrobacter freundii*, *Enterobacter cloacae*, *Proteus vulgaris*, *Escherichia coli* ATTC25922, *Vibrio parahaemolyticus*, ΔpagN C50041, *Salmonella typhimurium*, *Salmonella roseum*, *Salmonella London*, *Salmonella infantis*, *Salmonella pullorum*, and *Salmonella enteritidis* C50041 were picked and incubated overnight at 37°C and 220 rpm. The colonies were then inoculated at a 1:100 ratio into 6 mL of LB broth and incubated at 37°C and 220 rpm for 3 h. After incubation, the culture was centrifuged at 1500 × g for 10 min, and the supernatant was discarded. The colonies were resuspended in 10 mL of PBS and washed three times. The OD values ​​were then measured using a UV-Vis spectrophotometer. 600 After counting using the plate count method, the solution was diluted to 1×10⁻⁶ according to the specified ratio. 1 CFU / mL. Then, adjust the bacterial concentration.

[0116] Example 4: Optimization of Regeneration Conditions

[0117] In the Biacore T200 Control Software, open the New Wizard Template. In Assay, select Kinetics / Affinity, click New. In the Injection Sequence dialog box, select 4-1 for Flow path and CM5 for Chip type. Click Next to enter the setup dialog box. Set the Startup, enter PBS in the solution, and set the Number of Cycles to 3. Click Next to enter the Injection Parameters dialog box. In Sample, set the Contact time to 120s, Flow rate to 30μL / min, and Dissociation time to 300s. In Regeneration, enter Glycine-HCl1.7 in Solution, set the Contact time to 180s, and Flow rate to 20μL / min. Click Next. In the Samples dialog box, fill in the monoclonal antibody name and concentration. Click Next, check the "Prime before run" box, and keep the 25℃ setting unchanged. Click Next. According to the minimum sample volume indicated in the sample rack location table, prepare PBS and pH 1.7 in sequence. Place Glycine-HCl and 75 μg / mL Salmonella PagN monoclonal antibody (3B3) in their respective positions and return the sample rack to the sample chamber; click Next, the Prepare Run Protocol dialog box will pop up, carefully check the listed items, confirm that the volume of run buffer on the left rack is greater than the minimum requirement displayed in the software, and click Start.

[0118] To ensure the stable and reusable use of the SPR biosensor, the PagN monoclonal antibody bound to the anti-mouse IgG antibody on the CM5 chip needs to be removed, i.e., chip regeneration. After passing 75 μg / mL of Salmonella PagN monoclonal antibody (3B3) through the chip, a regeneration method using pH 1.7 Gly-HCl solution at a flow rate of 20 μL / min and a regeneration time of 180 s was performed. Ten regeneration experiments were conducted on the chip, and the results were... Figure 4The results showed that after 10 repeated passes of Salmonella PagN monoclonal antibody (3B3) through the chip, the response signal value decreased from 319 RU to 312 RU, a decrease of approximately 2.2%, while the baseline response signal value showed little change (-0.8–1.2 RU). This indicates that using pH 1.7 Gly-HCl as a regeneration solution can completely remove PagN monoclonal antibody (3B3) bound to anti-mouse IgG antibodies on the chip surface without affecting the activity of the anti-mouse IgG antibodies conjugated to the chip surface. These results demonstrate that the SPR biosensor based on subtractive inhibition exhibits good repeatability and stability, and the prepared chip can be repeatedly used for detection, effectively reducing detection costs and inter-group errors.

[0119] Example 5: Optimization of Free Antibody Separation Method

[0120] A single colony of *Salmonella enteritidis* C50041 was picked and incubated overnight at 37°C and 220 rpm in 3 mL LB broth. The colony was then inoculated at a 1:100 ratio into 6 mL LB broth and incubated at 37°C and 220 rpm for 3 h. The culture was then centrifuged at 1500 × g for 10 min, and the supernatant was discarded. The colony was resuspended in 10 mL PBS and washed three times. The OD was then measured using a UV-Vis spectrophotometer. 600 After counting using the plate count method, the solution was diluted to 1×10⁻⁶ according to the specified ratio. 8 CFU / mL; Take 900 μL of 150 μg / mL Salmonella PagN monoclonal antibody (3B3) and mix it with 900 μL of PBS solution and 1×10 8 The bacterial suspension of CFU / mL was thoroughly mixed and incubated at room temperature for 1 hour, inverting occasionally to mix. Then, it was divided into three 600 μL portions and centrifuged using different methods: ① centrifuged at 1600×g for 1 min, ② centrifuged at 200, 400, 800, 1200 and 1600×g for 1 min each, and ③ centrifuged at 200, 400, 800, 1200 and 1600×g for 2 min each. 500 μL of the supernatant (containing unbound free antibodies) was collected as the analyte.

[0121] In the Biacore T200 Control Software, open the New Wizard Template. In Assay, select Kinetics / Affinity, click New. In the Injection Sequence dialog box, select 2-1 for Flow path and CM5 for Chip type. Click Next to enter the setup dialog box. Set the Startup, enter PBS in the solution, and set the Number of Cycles to 3. Click Next to enter the Injection Parameters dialog box. In Sample, set the Contact time to 120s, Flow rate to 30μL / min, and Dissociation time to 300s. In Regeneration, enter Glycine-HCl1.7 in Solution, set the Contact time to 180s, and Flow rate to 20μL / min. Click Next. In the Samples dialog box, fill in the monoclonal antibody name and concentration. Click Next, check the "Prime before run" box, and keep the 25℃ setting unchanged. Click Next. According to the minimum sample volume indicated in the sample rack location table, prepare PBS and pH 1.7 in sequence. Place the Glycine-HCl and supernatant of each group into the corresponding positions, and return the sample rack to the sample chamber; click Next, the Prepare Run Protocol dialog box will pop up, carefully check the listed items, confirm that the volume of the run buffer on the left rack is greater than the minimum requirement displayed in the software, and click Start.

[0122] The response signal value of the PBS group antibody flowing through the CM5 chip is subtracted from the response signal value of the free antibody that did not bind to Salmonella after incubation with the Salmonella suspension flowing through the chip, which is taken as ΔResponse.

[0123] Salmonella PagN monoclonal antibody is a specific antibody against Salmonella and can bind to the bacteria. Salmonella PagN monoclonal antibody (3B3) was incubated with a Salmonella suspension, and the bound PagN monoclonal antibody was removed by centrifugation, thus obtaining unbound free PagN monoclonal antibody. Different centrifugation forces and times resulted in different response values ​​after flowing through the chip (…). Figure 5The corresponding reduction and inhibition response values ​​(ΔResponse) also differ. The reduction and inhibition response value for the group centrifuged at 1600×g for 1 min was 79 RU, the inhibition value for the group centrifuged at 200, 400, 800, 1200, and 1600×g for 1 min each was 99 RU, and the inhibition value for the group centrifuged at 200, 400, 800, 1200, and 1600×g for 2 min each was 112 RU. A higher reduction and inhibition response value indicates a better centrifugation effect, and that the Salmonella PagN monoclonal antibody (3B3) bound to the bacteria can be better removed, indicating that the separation method of centrifuging at 200, 400, 800, 1200, and 1600×g for 2 min each is optimal.

[0124] Example 6: Monoclonal Antibody Concentration Optimization

[0125] Take 300 μL of Salmonella PagN monoclonal antibody (3B3) at concentrations of 150, 100, 50, and 25 μg / mL, respectively, and 300 μL of 1×10⁻⁶ oz. 8 1×10 6 1×10 4 and 1×10 2 The CFU / mL bacterial suspension and PBS solution were thoroughly mixed and incubated at room temperature for 1 hour, with the mixture being inverted occasionally during incubation. Then, the mixture was centrifuged at 200, 400, 800, 1200, and 1600×g for 2 minutes, and 500 μL of the supernatant (containing unbound free antibodies) was collected as the analyte.

[0126] In the Biacore T200 Control Software, open the New Wizard Template. In Assay, select Kinetics / Affinity, click New. In the Injection Sequence dialog box, select 2-1 for Flow path and CM5 for Chip type. Click Next to enter the setup dialog box. Set the Startup, enter PBS in the solution, and set the Number of Cycles to 3. Click Next to enter the Injection Parameters dialog box. In Sample, set the Contact time to 120s, Flow rate to 30μL / min, and Dissociation time to 300s. In Regeneration, enter Glycine-HCl1.7 in Solution, set the Contact time to 180s, and Flow rate to 20μL / min. Click Next. In the Samples dialog box, fill in the monoclonal antibody name and concentration. Click Next, check the "Prime before run" box, and keep the 25℃ setting unchanged. Click Next. According to the minimum sample volume indicated in the sample rack location table, prepare PBS and pH 1.7 in sequence. Place the Glycine-HCl and supernatant of each group into the corresponding positions, and return the sample rack to the sample chamber; click Next, the Prepare Run Protocol dialog box will pop up, carefully check the listed items, confirm that the volume of the run buffer on the left rack is greater than the minimum requirement displayed in the software, and click Start.

[0127] Different concentrations of Salmonella PagN monoclonal antibody (3B3) were incubated with different concentrations of Salmonella, and then passed through a microarray. The results are shown in [Figure 1]. Figure 6 As antibody concentration increases, the response value also increases; the response values ​​for 150 μg / mL and 25 μg / mL Salmonella PagN monoclonal antibody (3B3) are 326 RU and 50 RU, respectively. Figure 6 a) As the concentration of Salmonella increases, the response values ​​also decrease. From Figure 6 b shows that when the concentration of Salmonella during incubation is 1×10⁻⁶, 8 At CFU / mL, the inhibitory response values ​​of Salmonella PagN monoclonal antibody (3B3) at concentrations of 150 μg / mL, 100 μg / mL, 50 μg / mL, and 25 μg / mL were 163 RU, 112 RU, 87 RU, and 33 RU, respectively, representing decreases of 51 RU, 76 RU, and 133 RU compared to the 150 μg / mL group. When the Salmonella concentration during incubation was 1 × 10⁻⁶ CFU / mL... 6At CFU / mL, the inhibitory response values ​​of different concentrations of Salmonella PagN monoclonal antibody (3B3) were 108, 63, 44, and 19 RU, respectively, which were 45, 64, and 89 RU lower than those of the 150 μg / mL group. When the concentration of Salmonella incubated was 1×10⁻⁶, the inhibitory response values ​​were significantly lower. 4 At CFU / mL, the inhibitory response values ​​of different concentrations of Salmonella PagN monoclonal antibody (3B3) were 65 RU, 28 RU, 17 RU, and 10 RU, respectively, which were 37 RU, 48 RU, and 55 RU lower than those of the 150 μg / mL group. When the concentration of Salmonella incubated was 1 × 10⁻⁶ CFU / mL, the inhibitory response values ​​were 65 RU, 28 RU, 17 RU, and 10 RU, respectively. 2 At CFU / mL, the inhibitory response values ​​of different concentrations of Salmonella PagN monoclonal antibody (3B3) were 11, 1.3, 0.9, and 2.5 RU, respectively, which were 9.7, 10.1, and 8.5 RU lower than those of the 150 μg / mL group. These results indicate that the 150 μg / mL Salmonella PagN monoclonal antibody (3B3) exhibits a high response value and a high reduction in inhibitory response value, demonstrating good reduction inhibition effect and high inhibitory sensitivity.

[0128] Example 7: Specificity Experiment

[0129] Take 300 μL of 150 μg / mL Salmonella PagN monoclonal antibody (3B3) and mix it thoroughly with 300 μL of bacterial suspensions of Shigella flexneri 2a, Citrobacter freundii, Enterobacter cloacae, Proteus vulgaris, Escherichia coli ATTC25922, Vibrio parahaemolyticus, ΔpagN C50041, Salmonella typhimurium, Salmonella russinensis, Salmonella London, Salmonella infantis, Salmonella pullorum, Salmonella enteritidis C50041 and PBS solution, and incubate at room temperature for 1 h, inverting the container occasionally to mix. Then, centrifuge at 200, 400, 800, 1200, and 1600×g for 2 min, and collect 500 μL of supernatant (containing unbound free antibody) as analyte.

[0130] In the Biacore T200 Control Software, open the New Wizard Template. In Assay, select Kinetics / Affinity, click New. In the Injection Sequence dialog box, select 2-1 for Flow path and CM5 for Chip type. Click Next to enter the setup dialog box. Set the Startup, enter PBS in the solution, and set the Number of Cycles to 3. Click Next to enter the Injection Parameters dialog box. In Sample, set the Contact time to 120s, Flow rate to 30μL / min, and Dissociation time to 300s. In Regeneration, enter Glycine-HCl1.7 in Solution, set the Contact time to 180s, and Flow rate to 20μL / min. Click Next. In the Samples dialog box, fill in the monoclonal antibody name and concentration. Click Next, check the "Prime before run" box, and keep the 25℃ setting unchanged. Click Next. According to the minimum sample volume indicated in the sample rack location table, prepare PBS and pH 1.7 in sequence. Place the Glycine-HCl and supernatant of each group into the corresponding positions, and return the sample rack to the sample chamber; click Next, the Prepare Run Protocol dialog box will pop up, carefully check the listed items, confirm that the volume of the run buffer on the left rack is greater than the minimum requirement displayed in the software, and click Start.

[0131] PagN protein is an outer membrane protein that is widely distributed and conserved in Salmonella. PagN monoclonal antibody is a specific antibody against the PagN protein of Salmonella spp. Different serotypes of Salmonella and non-Salmonella strains were incubated with PagN monoclonal antibody (3B3) to verify the specificity of this method. The results are shown in Figure (…). Figure 7When Salmonella typhimurium, Salmonella luxius, Salmonella London, Salmonella infantis, Salmonella pullorum, and Salmonella enteritidis C50041 were incubated with PagN monoclonal antibody (3B3), the free antibody response value decreased significantly compared to the PBS group, by approximately 140 RU. However, after strain ΔpagNC50041 was incubated with PagN monoclonal antibody (3B3), there was no significant difference in the free antibody response value. After incubation with PagN monoclonal antibody (3B3), the response values ​​of the free antibody against non-Salmonella spp. Shigella flexneri 2a, Citrobacter freundii, Enterobacter cloacae, Proteus vulgaris, Escherichia coli ATTC25922, and Vibrio parahaemolyticus showed no significant difference compared to the PBS incubation group. This indicates that PagN monoclonal antibody (3B3) specifically recognizes the PagN protein in Salmonella and does not react with other bacterial proteins. PagN monoclonal antibody (3B3) only specifically binds to Salmonella spp. strains and does not bind to non-Salmonella spp. strains, demonstrating that this method can specifically detect different serotypes of Salmonella.

[0132] Example 8: Surface Plasmon Resonance Technology for Detection

[0133] Take 300 μL of 150 μg / mL Salmonella PagN monoclonal antibody (3B3) and 300 μL of 1×10 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 and 1×10 1 The bacterial suspension (CFU / mL) and PBS solution were thoroughly mixed and incubated at room temperature for 1 hour, with the mixture being inverted occasionally during incubation. Then, the mixture was centrifuged at 200, 400, 800, 1200, and 1600×g for 2 minutes, and 500 μL of the supernatant (containing unbound free antibodies) was collected as the analyte.

[0134] In the Biacore T200 Control Software, open the New Wizard Template, select Kinetics / Affinity in Assay, and click New. In the Injection Sequence dialog box, select 2-1 for Flow path and CM5 for Chip type, then click Next to proceed. In the setup dialog box, set the Startup. Enter Buffer in Solution and set Number of Cycles to 3. Click Next to proceed. In the Injection Parameters dialog box, in Sample, set Contact time to 120s, Flow rate to 30μL / min, and Dissociation time to 300s. In Regeneration, enter Glycine-HCl 1.7 in Solution, set Contact time to 180s, and Flow rate to 20μL / min. Click Next to proceed. In the Samples dialog box, fill in the sample name and concentration information. Click Next to proceed. Check the box before Prime before run and keep the 25℃ setting unchanged. Click Next to proceed. According to the sample rack location table, place the prepared 500μL supernatant in sequence. Return the sample holder to the sample chamber and click Next. The Prepare Run Protocol dialog box will appear. Carefully check the listed items and confirm that the volume of run buffer on the left shelf is greater than the minimum requirement displayed in the software. Click Start.

[0135] Salmonella PagN monoclonal antibody (3B3) attenuation inhibition test results ( Figure 8 The results showed that after incubation of 150 μg / mL Salmonella PagN monoclonal antibody (3B3) with different concentrations of Salmonella enteritidis C50041, the free antibody obtained by centrifugation and flowing through the chip showed a lower response value than the PBS incubation group. Furthermore, the response value of the free antibody gradually decreased with increasing bacterial concentration. This indicates that Salmonella PagN monoclonal antibody (3B3) has a significant attenuation and inhibitory effect on Salmonella enteritidis C50041. (1×10⁻⁶) 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 1×10 1The SPR signal values ​​of the Salmonella enterica C50041 group and the PBS group at CFU / mL were 148, 197, 213, 236, 297, 310, 317, and 315 RU, respectively. The SPR signal value of the Salmonella inhibition group was denoted as R, and the SPR signal value of the PBS group was denoted as R0. Data processing was performed using GraphPad 8.0 software, with the fitting method being Analyze→XY analyses→Nonlinear regression (curve fit)→log(inhibitor) vs. response--Variable slope (four parameters). The fitted curve was calculated with the Salmonella enterica C50041 bacterial concentration as X and R / R0 as Y. The curve equation is (…). Figure 8 B) is Y = 0.4107 + 0.6273 / (1 + 10(4.666 - X) * - 0.3392), R² = 0.9704. LOD is defined as the bacterial concentration obtained by subtracting three times the standard deviation from the blank signal; LOD is 300 CFU / mL.

[0136] Example 9: Analysis of Salmonella Lysis by Bacteriophage

[0137] Take 100 μL 1×10 9 Add 900 μL of 1×10 PFU / mL phage C3-2 6 In Salmonella enteritidis C50041 bacterial suspension, the final concentration of bacteriophage C3-2 was 1×10⁻⁶ CFU / mL. 8 PFU / mL was added, and an equal volume of SM buffer was added as a control group. After incubation at 4°C for 1, 2 and 4 h respectively, 100 μL of the mixture was continuously diluted, spread on LB plates for counting, and each group was repeated three times and the average value was taken as the colony count.

[0138] Take 300 μL of 150 μg / mL Salmonella PagN monoclonal antibody (3B3) and 300 μL of 1×10 8 1×10 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 and 1×10 1 The bacterial suspension (CFU / mL) and PBS solution were thoroughly mixed and incubated at room temperature for 1, 2 and 4 hours, respectively, with the mixture being inverted from time to time during incubation. Then, the mixture was centrifuged at 200, 400, 800, 1200 and 1600×g for 2 minutes, and 500 μL of supernatant (containing unbound free antibodies) was collected as the analyte.

[0139] Prepare the PBS, Glycine-HCl 1.7, phage mixture, and control supernatant in sequence according to the minimum sample volume indicated on the sample rack location table. The Biacore T200 Control Software operation is the same as above.

[0140] The data were processed using the same method as in Example 8 above. Using the concentration of Salmonella enteritidis C50041 bacterial culture as X and R / R0 as Y, the results of the bacteriophage C3-2 lysis effect on Salmonella were obtained. Figure 9 , Figure 10 (and Table 1) shows that its curve equation ( Figure 9 The equation is Y = 0.4748 + 0.5762 / (1 + 10). (4.009-X)*-0.3246 R 2 =0.9771. One hour after adding phage C3-2, the response value of the Salmonella and phage mixture was higher than that of the control group, indicating that more PagN monoclonal antibody (3B3) was detected in the experimental group. This suggests that after phage treatment, some Salmonella were lysed, increasing the amount of free antibody. The R / R0 values ​​for the experimental group and the control group at 1 hour were 0.643 and 0.592, respectively. Substituting these values ​​into the fitted curve, the calculated number of Salmonella was 1.6 × 10⁻⁶. 5 and 8.1×10 5 CFU / mL, plate count results were 2×10⁻⁶. 5 and 1.1×10 6 CFU / mL; the R / R0 values ​​of the experimental group and the control group at 2h were 0.732 and 0.618, respectively. Substituting these values ​​into the fitted curve, the Salmonella count was calculated to be 1.7 × 10⁻⁶. 4 and 3.4×10 5 CFU / mL, plate count results were 3.6 × 10⁻⁶. 4 and 9×10 5 CFU / mL; the values ​​for the 4h groups were 0.705 and 0.597, respectively. Substituting these values ​​into the fitted curve, the Salmonella count was calculated to be 3.2 × 10⁻⁶. 4 and 6.6×10 5 CFU / mL, plate count results were 4×10⁻⁶. 4 and 1.1×10 6 CFU / mL. This indicates that the method can rapidly and sensitively detect Salmonella enteritidis C50041 lysed by phage C3-2.

[0141] Table 1. Phage C3-2 lysis of Salmonella enteritidis C50041 (CFU / mL)

[0142]

[0143] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting Salmonella using surface plasmon resonance technology, characterized in that, Includes the following steps: A Salmonella detection chip was prepared; the target bacteria were cultured, incubated with antibodies, and the free antibodies were separated. The antibodies that did not bind to the bacteria were separated by gradient centrifugation; the response signal of the free antibodies was detected by surface plasmon resonance technology, and the content of the target Salmonella was calculated; the antibody was a Salmonella PagN protein monoclonal antibody. The Salmonella PagN protein monoclonal antibody contains a heavy chain variable region and a light chain variable region; The heavy chain variable region contains V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes V L CDR1, V L CDR2 and V L CDR3; The V mentioned H The amino acid sequence of CDR1 is shown in SEQ ID NO: 1; The V mentioned H The amino acid sequence of CDR2 is shown in SEQ ID NO: 2; The V mentioned H The amino acid sequence of CDR3 is shown in SEQ ID NO: 3; The V mentioned L The amino acid sequence of CDR1 is shown in SEQ ID NO: 4; The V mentioned L The amino acid sequence of CDR2 is shown in SEQ ID NO: 5; The V mentioned L The amino acid sequence of CDR3 is shown in SEQ ID NO: 6; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

2. The method for detecting Salmonella using surface plasmon resonance technology according to claim 1, characterized in that, The heavy chain amino acid sequence is shown in SEQ ID NO: 9; the light chain amino acid sequence is shown in SEQ ID NO:

10.

3. The method for detecting Salmonella using surface plasmon resonance technology according to claim 1, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

18.

4. The method for detecting Salmonella using surface plasmon resonance technology according to claim 1, characterized in that, The nucleotide sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO: 19; the nucleotide sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO:

20.

5. The method for detecting Salmonella using surface plasmon resonance technology according to claim 1, characterized in that, The method for preparing the Salmonella detection chip includes: immobilizing Anti mouse IgG onto the surface of the CM5 chip via amino coupling, and then blocking unbound sites with ethanolamine solution; using pH 5.0 sodium acetate solution as the coupling buffer.

6. The method for detecting Salmonella using surface plasmon resonance technology according to claim 1, characterized in that, The culture of the test bacteria includes: inoculating the test bacterial colonies into LB liquid medium and culturing overnight; then, inoculating the bacterial solution into LB liquid medium at a ratio of 1:100, culturing at 37°C and 220 r / min for 3 h; then, washing three times with PBS in a 15 mL centrifuge tube, and centrifuging at 1500× g Centrifuge for 10 min.

7. The method for detecting Salmonella using surface plasmon resonance technology according to claim 1, characterized in that, The step of incubating the bacteria to be tested with the antibody includes: mixing the Salmonella PagN protein monoclonal antibody with the bacterial culture to be tested, and incubating at room temperature.

8. The method for detecting Salmonella using surface plasmon resonance technology according to claim 1, characterized in that, The gradient centrifugation includes centrifuging the bacterial culture incubated with the antibody at 200, 400, 800, 1200, and 1600×g for 2 min respectively.

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