A long-period fiber grating based on toxic strain imprinted artificial antibody combined with phage modification and its preparation method and application

Abstract

The present invention provides a long-period fiber grating based on toxic strain imprinting artificial antibody combined with bacteriophage modification and its preparation method and application, which belongs to the technical field of toxic live strain detection. The preparation method of the long-period fiber grating based on toxic strain imprinting artificial antibody combined with bacteriophage modification provided by the present invention comprises the following steps: sequentially performing surface silanization treatment on the long-period fiber grating, modifying the polydopamine coating, modifying the toxic strain imprinting artificial antibody, and modifying the bacteriophage, and after sealing, obtaining the long-period fiber grating based on toxic strain imprinting artificial antibody combined with bacteriophage modification. The long-period fiber grating modified by toxic strain imprinting artificial antibody combined with bacteriophage prepared by the method can shorten the detection time to 5 to 10 minutes, improve the sensitivity of detecting specific toxic live strains, and has the advantages of high sensitivity, high accuracy, strong specificity, low detection limit, and green environmental protection.

Description

Technical Field

[0001] The present invention relates to the technical field of virulent live strain detection, and in particular to a long-period fiber grating based on virulent strain imprinting artificial antibody combined with bacteriophage modification, and a preparation method and application thereof. Background Art

[0002] Microorganisms are ubiquitous in nature, and they exist in drinking water, fruits, and other foods. They can cause diseases. For example, staphylococcal disease is an acute or chronic infectious disease caused by Staphylococcus aureus, and clinically has many types such as arthritis, tenosynovitis, foot pad swelling, navel inflammation, staphylococcal sepsis, etc. Therefore, it is necessary to develop a sensitive, rapid, and on-site detection method for the detection of virulent live strains to achieve practical emergency detection.

[0003] Traditionally, the detection of virulent live strains is based on pathogenic bacteria culture. Several microbiological and molecular biology techniques have been used to detect virulent live strains. Other ultrasensitive detection methods based on nucleic acid amplification, such as ligase chain reaction, strand displacement amplification, polymerase chain reaction (PCR), antibody-based immunoassays and immuno-PCR assays, usually require DNA to be isolated from virulent strains in advance, nucleic acid amplification equipment and complex enzyme reactions, which are expensive, complex, and time-consuming, and cannot meet the needs of rapid and simple on-site detection.

[0004] In recent years, the use of optical methods to detect pathogens has received increasing attention. Long-period fiber grating is a passive optical fiber device with periodic modulation of the core refractive index. It has been widely used in the fields of optical fiber communication and optical fiber sensing due to its advantages such as easy manufacturing, low dielectric loss, and compact structure. Fiber grating biosensing utilizes the refractive index sensitivity of long-period fiber grating. When the refractive index of the external environment changes, its resonant wavelength will drift significantly, thereby achieving the detection of biological molecules. When it is combined with biorecognition components (such as antibodies, nucleic acid probes), it is particularly suitable for constructing ultra-sensitive optical fiber biosensors, which are suitable for the detection of virulent live strains.

[0005] At present, most of the fiber optic biosensors for detecting live toxic strains achieve the purpose of qualitative and quantitative detection of pathogens by fixing antibody proteins on the surface to adsorb specific toxic strains to produce signal deviations. However, antibody proteins are expensive and the analysis time of this method is generally 20 to 40 minutes. For actual emergency detection, the detection efficiency is still low. Molecular imprinting technology is one of the best methods for synthesizing artificial antibodies. The synthetic artificial antibodies show binding affinity and selectivity similar to natural antibodies. They can even have better properties than natural antibodies, including practicality and operability, high stability under harsh chemical and physical conditions, and some even have super repeatability, and are inexpensive. Summary of the invention

[0006] The purpose of the present invention is to provide a long-period fiber grating based on toxic strain imprinted artificial antibodies combined with phage modification, and use it for the detection of toxic live strains, so as to provide a detection method for toxic live strains with high accuracy, low detection limit and strong specificity for non-diagnostic purposes.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a long-period fiber grating based on toxic strain imprinted artificial antibody combined with phage modification, comprising the following steps:

[0009] The long-period fiber grating is subjected to surface silanization treatment, polydopamine coating modification, toxic strain imprinting artificial antibody modification, and phage modification in sequence, and after sealing, a long-period fiber grating based on toxic strain imprinting artificial antibody combined with phage modification is obtained.

[0010] Preferably, the long period fiber grating is made of single mode optical fiber and is obtained by recording using femtosecond laser direct writing technology;

[0011] During the recording, the repetition frequency of the femtosecond laser pulse is 0.8-1.2 kHz, and the energy is 0.7 μJ-1.0 μJ;

[0012] The grating period of the long-period fiber grating is 320 μm-400 μm, the total grating length is 15 mm-20 mm, the grating diameter is 100 μm-120 μm, and the loss peak intensity is 25 dB-35 dB.

[0013] Preferably, the surface silanization treatment method is: immersing the long period fiber grating in a hydrochloric acid methanol solution, a sodium hydroxide solution and an APTES acetonitrile solution in sequence;

[0014] The hydrochloric acid-methanol solution is obtained by mixing analytically pure hydrochloric acid and analytically pure methanol in a volume ratio of 0.5-1.5:0.5-1.5, and the long-period fiber grating is immersed in the hydrochloric acid-methanol solution for 1-5 hours;

[0015] The concentration of the sodium hydroxide solution is 0.5-1.2 mol / L, and the long-period fiber grating is immersed in the sodium hydroxide solution for 5-12 hours;

[0016] The volume concentration of the APTES acetonitrile solution is 1.5-2.5%, and the long-period fiber grating is immersed in the APTES acetonitrile solution for 12-20 hours.

[0017] Preferably, the method for modifying the polydopamine coating is: coating a dopamine hydrochloride solution on the long-period fiber grating after surface silanization treatment and reacting for 1 to 2 hours;

[0018] The dopamine hydrochloride solution is obtained by mixing dopamine hydrochloride and Tris-HCl at a mass volume ratio of 8-12 mg:0.8-1.2 mL, wherein the concentration of Tris-HCl is 8-12 mmol / L and the pH value is 7.5-8.5.

[0019] Preferably, the method for modifying the toxic strain imprinted artificial antibody is: wrapping the toxic strain solution on the long-period fiber grating after the modified polydopamine coating, incubating at 15°C to 25°C for 20 to 40 minutes, and then catalyzing with an ammonia tetraethyl orthosilicate solution (the ammonia in the ammonia tetraethyl orthosilicate solution plays a catalytic role) to form the toxic strain imprinted artificial antibody, and finally treating with ammonia to remove the toxic strain;

[0020] The concentration of the toxic strain solution is 10 5 CFU / mL~10 8 CFU / mL;

[0021] The volume ratio of the ammonia solution to the tetraethyl orthosilicate in the ammonia solution is 0.1-0.3:1-3, the volume concentration of the ammonia solution is 20-30%, and the treatment time of the ammonia solution is 20-50 minutes;

[0022] The pH of the ammonia water is 10-12, and the ammonia water treatment time is 5-10 minutes.

[0023] Preferably, the method for modifying the bacteriophage is: the long-period fiber grating modified with the artificial antibody imprinted with the toxic strain is subjected to surface silanization treatment and polydopamine coating modification in sequence, and then coated with a bacteriophage solution, incubated at 15°C to 35°C for 20 to 50 minutes, and repeatedly coated with a bacteriophage solution until the number of bacteriophages modified on the surface of the long-period fiber grating reaches saturation;

[0024] The concentration of the phage solution is 10 4 CFU / mL~10 7 CFU / mL.

[0025] Preferably, the blocking method is: incubating the long-period fiber grating modified with phage with a BSA solution having a mass concentration of 3-7% at 15° C.-25° C. for 5-12 hours.

[0026] The present invention also provides a long-period fiber grating based on the toxic strain imprinted artificial antibody combined with phage modification obtained by the above preparation method.

[0027] The present invention also provides an application of the long-period fiber grating modified by combining the artificial antibody imprinted with the phage to prepare a detection reagent for detecting the toxic strain.

[0028] The present invention also provides a method for detecting toxin-causing strains for non-diagnostic purposes, comprising the following steps:

[0029] (1) Detecting the initial resonance wavelength signal λ of the long-period fiber grating modified by the artificial antibody imprinted with the toxic strain combined with the bacteriophage 0 ;

[0030] (2) The initial resonance wavelength signal λ recorded in step (1) 0 Based on the toxic strain imprinted artificial antibody combined with phage-modified long-period fiber grating, the sample toxic strain is adsorbed and the resonance wavelength signal λ of the sample toxic strain is detected. i ;

[0031] (3) The resonance wavelength signal λ of the sample toxic strain i The initial resonance wavelength signal λ 0 Subtracting them, we get the resonance wavelength shift Δλ;

[0032] (4) Based on the standard curve drawn with the concentration of the virulent strain as the horizontal axis and the resonance wavelength shift as the vertical axis, the concentration of the virulent live strain in the sample is calculated.

[0033] The toxic strain imprinted artificial antibody combined with phage-modified long-period fiber grating provided by the present invention can show stronger adsorption when in contact with the toxic live strain through the specific binding of the phage to the toxic live strain. The time for adsorbing the specific toxic live strain is shortened to 5 to 10 minutes, which is suitable for actual emergency detection. The toxic strain imprinted artificial antibody combined with phage-modified long-period fiber grating provided by the present invention can monitor the amount of phage modified on its surface, improve the sensitivity of detecting specific toxic live strains, and has the advantages of high sensitivity, high accuracy, strong specificity, low detection limit, and green environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The schematic diagram of Example 1 for preparing a long-period fiber grating based on Aeromonas sobria imprinted artificial antibody combined with phage modification;

[0035] Figure 2 for Figure 1 The middle is an enlarged image of the long-period fiber grating modified by artificial antibody combined with bacteriophage based on Aeromonas sobria, in which the resonance wavelength shifts to the left longer as the concentration of Aeromonas sobria solution increases;

[0036] Figure 3This is a microscope photo (500 times) of the long-period fiber grating modified by the artificial antibody bound to bacteriophage based on Aeromonas sobria imprinting in Example 1;

[0037] Figure 4 This is a microscope photo (500 times) of the long-period fiber grating after adsorbing live Aeromonas sobria bacteria in Example 2;

[0038] Figure 5 This is a comparison chart of the resonant wavelength shift of different toxic strains detected by the long-period fiber grating modified with the artificial antibody imprinted with Aeromonas sobria combined with the bacteriophage at the same concentration in Example 3. DETAILED DESCRIPTION

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention. Example 1

[0040] (1) Fabrication of long-period fiber gratings: Using femtosecond laser direct writing technology, high-sensitivity long-period fiber gratings were fabricated in the laboratory using single-mode optical fibers. The optical fiber was written using an optical parametric amplifier system with a central wavelength of 800 nm. Femtosecond laser pulses with a repetition rate of 1 kHz and an energy of 0.8 μJ were focused into the fiber core using a 40x objective lens with an NA (numerical aperture) of 0.6. A long-period fiber grating with a grating period of Λ of 360 μm, a duty cycle of 50%, and a total of 500 grating elements was inscribed along the fiber core, with a total grating length of 18 mm. During the writing of the long-period fiber grating, the transmission spectrum evolution from 1100 nm to 1700 nm was monitored in real time using a supercontinuum source and a spectrum analyzer. The fabricated long-period fiber grating has a diameter of 114 μm and a loss peak intensity of 30 dB.

[0041] (2) Surface silanization treatment of long-period fiber grating: soak the long-period fiber grating prepared in step (1) in a hydrochloric acid methanol solution for 2 hours, wherein the hydrochloric acid methanol solution is prepared by mixing a hydrochloric acid solution and a methanol solution in a volume ratio of 1:1, wherein the hydrochloric acid solution is analytically pure hydrochloric acid with a concentration of 37%, and the methanol solution is analytically pure methanol with a concentration of 99%. After soaking, wash it with deionized water, and after drying, soak it in a 1 mol / L sodium hydroxide solution for 10 hours, and then wash it with deionized water. After drying, soak it in a 2% APTES acetonitrile solution with a volume concentration of 12 hours, and then wash it with deionized water and dry it.

[0042] (3) Modification of the long-period fiber grating surface with polydopamine coating: 10 mg of dopamine hydrochloride was dissolved in 1 mL of 10 mmol / L Tris-HCl (pH = 8.0) to obtain a dopamine hydrochloride solution, which was coated on the long-period fiber grating prepared in step (2). After reacting for 1 hour, the solution was washed with deionized water and dried.

[0043] (4) Long-period fiber grating surface modified with artificial antibody of Aeromonas sobria (toxic strain): 7 CFU / mL Aeromonas sobria solution (the solvent is PBS buffer solution) is coated on the long-period fiber grating prepared in step (3), incubated at 20°C for 30 minutes, washed with PBS buffer solution, and dried; 0.2mL of 25% ammonia water and 2mL of ethyl orthosilicate are stirred and mixed and then coated on the surface of the modified long-period fiber grating, catalyzed by ammonia water in the solution, and reacted for 30 minutes to form a surface silica shell on the surface of the long-period fiber grating coated with the Aeromonas sobria solution, that is, the Aeromonas sobria imprinted artificial antibody is formed, and then washed with methanol (analytical grade), and then washed with deionized water, and dried; the surface of the modified long-period fiber grating is then immersed in 5 mL of NH4OH with a pH of 11. 3 ·H 2 O solution for 10 min to remove Aeromonas sobria, and then rinse the sensing area 5 times with 5 mL PBS buffer and dry it.

[0044] (5) Surface modification of long-period fiber grating with phage: Repeat steps (2) and (3) with the long-period fiber grating obtained in step (4), i.e., perform surface silanization treatment and modify the polydopamine coating on its surface; then, add 10 5 A phage solution (solvent is PBS buffer solution) with a concentration of CFU / mL was coated on the long-period fiber grating area and incubated at 20°C for 30 minutes, then washed and dried with PBS buffer solution. This step was repeated 4 times. After each drying, a supercontinuum spectrum source and a spectrum analyzer were used to record the shift position of the resonance wavelength of the transmission spectrum to determine that the surface modification of the long-period fiber grating with phage reached saturation (i.e., the resonance wavelength basically no longer shifted), thereby monitoring the amount of phage modified on its surface.

[0045] (6) Surface sealing of long-period fiber grating: 5% mass concentration of BSA solution was coated on the long-period fiber grating prepared in step (5) and incubated at 20°C for 6 hours, washed with PBS buffer solution, and dried. Thus, a long-period fiber grating modified by Aeromonas sobria imprinted artificial antibody combined with phage was prepared. The preparation principle diagram is shown in the figure. Figure 1As shown in FIG. 1 , the long-period fiber grating based on the modified artificial antibody of Aeromonas sobria combined with phage finally obtained shifts the resonance wavelength to the left longer as the concentration of Aeromonas sobria solution increases, as shown in FIG. Figure 2 .

[0046] The surface structure of the long-period fiber grating modified by the artificial antibody combined with the bacteriophage based on Aeromonas sobria imprinting was observed under a polarizing microscope with a magnification of 500 times, such as Figure 3 As shown, from Figure 3 It can be seen that there are many "pits" with a particle size of about 1~2 μm on the surface of the modified long-period fiber grating, which is consistent with the particle size of the artificial antibody imprinted by the toxic strain. Example 2

[0047] A standard curve with the concentration of Aeromonas sobria as the horizontal axis and the resonance wavelength offset as the vertical axis was drawn using the long-period fiber grating modified by the Aeromonas sobria imprinted artificial antibody combined with phage prepared in Example 1, and the content of Aeromonas sobria in the sample was detected.

[0048] (1) Preparation of standard mild Aeromonas live bacteria solution: Prepare a mild Aeromonas live bacteria solution with a concentration of 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 CFU / mL, the preparation solvent is PBS buffer solution.

[0049] (2) Recording of initial signal: The sensing area of ​​the long-period fiber grating modified by the Aeromonas sobria imprinted artificial antibody combined with bacteriophage obtained in Example 1 was immersed in a PBS buffer solution, and the transmission spectrum position was recorded, which was recorded as the initial signal position (λ 0 ).

[0050] (3) Target adsorption: The long-period fiber grating sensing area dried in step (2) is immersed in a prepared solution with a concentration of 10 2 CFU / mL standard mild Aeromonas live bacteria solution, and after 10 minutes, rinse the sensing area with 5 mL PBS buffer for 5 times, then immerse the sensing area in PBS buffer solution, and record the transmission spectrum position, which is recorded as the target signal position (λ i ), and λ i With λ 0 Subtracting them, we get the resonant wavelength shift (Δλ).

[0051] (4) Target elution: Immerse the sensing area of ​​the long-period fiber grating in 5 mL of NH4OH with a pH of 11. 3 ·H 2O solution for 5 min, and then rinse the sensing area 5 times with 5 mL PBS buffer.

[0052] (5) Data processing: According to the above steps (2), (3) and (4), the concentration of the standard Aeromonas sobriae solution was determined to be 10 3 , 10 4 , 10 5 , 10 6 , 10 7 The target signal position of CFU / mL was obtained, and the data of the resonance wavelength shift in the above step (3) were obtained, and a standard curve was drawn, as shown in Table 1:

[0053]

[0054] (6) Real sample processing and determination: 1 mL of tap water sample (from Jiangdong Water Plant, Ningbo City, Zhejiang Province) was placed in a 1.5 mL centrifuge tube and centrifuged at 10,000 r / min for 5 minutes. The supernatant was removed and the sample was resuspended in 1 mL of PBS buffer. The above steps (2), (3) and (4) were repeated to obtain the data of the sample resonance wavelength shift, and the data were substituted into the standard curve equation to calculate the concentration of viable Aeromonas sobria in the sample. The resonance wavelength shift values ​​of the three parallel samples were all less than 0.10 nm, which was much lower than the concentration of 10 viable Aeromonas sobria solution. 2 The resonance wavelength shift of CFU / mL was 0.43 nm. A spike recovery experiment was performed, with 3 parallel samples at each concentration, and the relative standard deviation was calculated. The results are shown in Table 2. This method has a good recovery rate.

[0055]

[0056] As can be seen from Table 2, the recovery rates of the detection methods provided by the present invention are all above 95%, and the relative deviations are all below 5%, indicating that the detection methods are accurate and reliable.

[0057] During the experiment, after the live Aeromonas sobria bacteria in the real sample were adsorbed by the long-period fiber grating based on the artificial antibody imprinted with Aeromonas sobria combined with the phage, it was photographed under a polarizing microscope at a magnification of 500 times, as shown in the figure. Figure 4 It was found that many "granular" substances suspected of bacteria appeared on the surface of the long-period fiber grating adsorbed with mild Aeromonas viable bacteria, with a particle size between 1 and 2 μm. Figure 3 The "pit" in the image was almost covered up, indicating that the virulent strain Aeromonas sobriae had been adsorbed on the long-period fiber grating in large quantities within 10 minutes and could be further detected. Example 3

[0058] The anti-interference ability of the long-period fiber grating modified by the artificial antibody based on Aeromonas sobria imprinting combined with bacteriophage prepared in Example 1 was tested for other virulent live strains, including Staphylococcus aureus, Salmonella, and Listeria. The concentration of each virulent live strain was 10 7 CFU / mL, the results are as follows Figure 5 As shown. Figure 5 It can be seen that the long-period fiber grating based on the imprinted artificial antibody of Aeromonas sobria combined with phage modification can only produce obvious resonance wavelength shift for live Aeromonas sobria bacteria, and the resonance wavelength is almost not shifted for the other several virulent live strains, indicating that the long-period fiber grating based on the imprinted artificial antibody of Aeromonas sobria combined with phage modification of the present invention has strong specificity, and other virulent live strains will not interfere with the detection system.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a long-period fiber grating based on toxic strain imprinted artificial antibody combined with phage modification, characterized in that: The steps include: The long-period fiber grating is subjected to surface silanization treatment, polydopamine coating modification, toxic strain imprinting artificial antibody modification, and bacteriophage modification in sequence, and after sealing, a long-period fiber grating based on toxic strain imprinting artificial antibody combined with bacteriophage modification is obtained; The method for modifying the polydopamine coating is as follows: coating a dopamine hydrochloride solution on a long-period fiber grating after surface silanization treatment and reacting for 1 to 2 hours; the dopamine hydrochloride solution is obtained by mixing dopamine hydrochloride with Tris-HCl at a mass volume ratio of 8 to 12 mg: 0.8 to 1.2 mL, wherein the concentration of the Tris-HCl is 8 to 12 mmol / L and the pH value is 7.5 to 8.5; The method for modifying the toxic strain imprinted artificial antibody is as follows: wrapping the toxic strain solution on the long-period fiber grating after the modified polydopamine coating, incubating at 15°C to 25°C for 20 to 40 minutes, then catalyzing with ammonia ethyl orthosilicate solution to form the toxic strain imprinted artificial antibody, and finally treating with ammonia to remove the toxic strain; the concentration of the toxic strain solution is 10 5 CFU / mL~10 8 CFU / mL; the volume ratio of ammonia solution to tetraethyl orthosilicate in the ammonia tetraethyl orthosilicate solution is 0.1-0.3:1-3, the volume concentration of the ammonia solution is 20-30%, and the treatment time of the ammonia tetraethyl orthosilicate solution is 20-50 min; the pH of the ammonia solution is 10-12, and the treatment time of the ammonia solution is 5-10 min; The method for modifying the bacteriophage is as follows: the long-period fiber grating modified with the artificial antibody imprinted with the toxic strain is subjected to surface silanization treatment and modified with a polydopamine coating in sequence, and then coated with a bacteriophage solution, incubated at 15° C. to 35° C. for 20 to 50 minutes, and repeatedly coated with the bacteriophage solution until the number of bacteriophages modified on the surface of the long-period fiber grating reaches saturation; The concentration of the phage solution is 10 4 CFU / mL~10 7 CFU / mL.

2. The preparation method according to claim 1, characterized in that The long-period fiber grating is made of single-mode optical fiber and is obtained by recording using femtosecond laser direct writing technology; During the recording, the repetition frequency of the femtosecond laser pulse is 0.8-1.2 kHz, and the energy is 0.7 μJ-1.0 μJ; The grating period of the long-period fiber grating is 320 μm-400 μm, the total grating length is 15 mm-20 mm, the grating diameter is 100 μm-120 μm, and the loss peak intensity is 25 dB-35 dB.

3. The preparation method according to claim 2, characterized in that: The surface silanization treatment method is: immersing the long period fiber grating in hydrochloric acid methanol solution, sodium hydroxide solution and APTES acetonitrile solution in sequence; The hydrochloric acid-methanol solution is obtained by mixing analytically pure hydrochloric acid and analytically pure methanol in a volume ratio of 0.5-1.5:0.5-1.5, and the long-period fiber grating is immersed in the hydrochloric acid-methanol solution for 1-5 hours; The concentration of the sodium hydroxide solution is 0.5-1.2 mol / L, and the long-period fiber grating is immersed in the sodium hydroxide solution for 5-12 hours; The volume concentration of the APTES acetonitrile solution is 1.5-2.5%, and the long-period fiber grating is immersed in the APTES acetonitrile solution for 12-20 hours.

4. The preparation method according to claim 3, characterized in that: The blocking method is: incubating the long-period fiber grating modified with phage with a BSA solution having a mass concentration of 3-7% at 15° C.-25° C. for 5-12 hours.

5. A long-period fiber grating based on toxic strain imprinted artificial antibody combined with phage modification obtained by the preparation method according to any one of claims 1 to 4.

6. Use of the long-period fiber grating based on toxic strain imprinted artificial antibody combined with bacteriophage modified according to claim 5 in preparing a detection reagent for detecting toxic strains.

7. A method for detecting toxin-causing strains for non-diagnostic purposes, characterized in that: The steps include: (1) Detecting the initial resonance wavelength signal λ0 of the long-period fiber grating modified by the toxic strain imprinted artificial antibody combined with the bacteriophage as described in claim 5; (2) The sample toxic strain is adsorbed by the artificial antibody based on the toxic strain imprinting combined with the phage-modified long-period fiber grating that records the initial resonance wavelength signal λ0 in step (1), and the resonance wavelength signal λ0 of the sample toxic strain is detected. i ; (3) The resonance wavelength signal λ of the sample toxic strain i Subtract the initial resonance wavelength signal λ0 to obtain the resonance wavelength shift Δλ; (4) Based on the standard curve drawn with the concentration of the virulent strain as the horizontal axis and the resonance wavelength shift as the vertical axis, the concentration of the virulent live strain in the sample is calculated.

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