A cronobacter phage tail fiber protein and its use in bacterial detection

Abstract

The application provides a Cronobacter phage tail fiber protein TFP08 and application thereof in Cronobacter detection; the amino acid and nucleotide sequences of the tail fiber protein are respectively described as SEQ ID NO. 1 and SEQ ID NO. 2; a electrochemical biosensor based on the tail fiber protein can specifically recognize Cronobacter of different species and has good stability, and the detection signal is reduced within 7% when the electrochemical biosensor is stored at room temperature for 7 days; the electrochemical biosensor constructed by using the tail fiber protein can complete the detection of target bacteria within 30 minutes, and the detection limit is 23 CFU / mL within the range of 10 2 -10 7 CFU / mL, and a good linear relationship is shown, and the detection limit is 23 CFU / mL; the electrochemical biosensor can be used for on-site detection and rapid screening of Cronobacter, and can provide an excellent recognition element and a powerful technical support platform for detection of Cronobacter in the fields of food safety, environmental detection and clinical diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and microorganism detection technology, and particularly relates to a Cronobacter phage tail fiber protein and application thereof in bacterial detection BACKGROUND

[0002] Cronobacter is an important foodborne pathogen, which can cause neonatal meningitis, necrotizing enterocolitis and bacteremia, with a mortality rate of 40%-80%. The main infection channel of Cronobacter is infant formula milk powder, which has caused several large-scale milk powder recall events. Cronobacter has been officially recognized as a class A pathogen by FAO-WHO, and is also a pathogenic bacterium that is currently focused on by dairy enterprises. The current national standard of China also clearly stipulates that Cronobacter cannot be detected in formula food for infants aged 0-6 months. Therefore, rapid and accurate detection of Cronobacter is an important means to prevent related food safety incidents.

[0003] The traditional colony counting method is still used as the "gold standard" for detecting Cronobacter, but its detection period is long, usually 3-4 days. Real-time fluorescent quantitative PCR, loop-mediated isothermal nucleic acid amplification, rolling circle amplification and isothermal amplification based on molecular biological techniques effectively improve the sensitivity of Cronobacter detection and shorten the detection time, but in order to reduce sample matrix interference, complex and tedious pretreatment steps are required; enzyme-linked immunosorbent assay, immunomagnetic beads, colloidal gold test strip and immunosensing based on immunological techniques have high sensitivity and strong specificity, especially the immunomagnetic separation technology can selectively capture and separate target bacteria from complex matrix, but immunological techniques rely on antibody recognition, and traditional antibody preparation is complex, with a long production cycle and high cost, which limits the application of immunological techniques. In addition, new antibody substitutes such as aptamers are developing rapidly, but the recognition performance of aptamers is highly dependent on their folding structure and conformation, and such conformation is easily changed in complex matrix, which affects the affinity and specificity of recognition. Therefore, it is urgent to explore new recognition elements to break through the bottleneck of Cronobacter detection technology.

[0004] Phage is a virus that can specifically recognize and infect host bacteria. In recent years, phage as a novel detection method of recognition elements has attracted widespread attention due to its high specificity, accuracy and reduced detection time. Compared with antibodies, phage is easier to produce and resistant to extreme pH, temperature and organic solvents. However, the complex interaction mechanism of intact phage limits the development of new phage recognition elements. Phage tail fiber protein is an important structural protein of phage tail, which is mainly responsible for the specific recognition and adsorption of phage and host bacteria. At present, there is no report on the use of phage tail fiber protein to detect Cronobacter. SUMMARY

[0005] In view of the above problems, the present application successfully obtains a Cronobacter bacteriophage tail fiber protein TFP08 through gene recombination and prokaryotic expression, and constructs an electrochemical biosensor of Cronobacter by taking the Cronobacter bacteriophage tail fiber protein TFP08 as a recognition molecule, so as to be applied to on-site detection and rapid screening of Cronobacter in food, environment and clinical samples.

[0006] Specifically, the present application adopts the following technical solutions:

[0007] Firstly, the present application provides a Cronobacter bacteriophage tail fiber protein, the amino acid sequence of which is shown as SEQ ID No. 1, and the nucleotide sequence of which is shown as SEQ ID No. 2, and the applicant names the tail fiber protein as TFP08.

[0008] Secondly, the present application provides an application of the tail fiber protein with the amino acid sequence shown as SEQ ID No. 1 in detecting Cronobacter for non-medical purposes, such as detecting Cronobacter in the fields of food, environment, clinical diagnosis and the like.

[0009] Specifically, the above application refers to: using the tail fiber protein TFP08 to prepare an electrochemical biosensor, and then using a differential pulse voltammetry method to detect the content of Cronobacter in a sample based on the biosensor, and bringing the peak current signal measured by an electrochemical workstation into a regression equation, so as to obtain the content of Cronobacter in the sample; the regression equation is A=1.57-0.085lgC, wherein A is the peak current signal, and C is the concentration of Cronobacter.

[0010] The preparation method of the electrochemical biosensor is as follows: 1ml of AuNPs is mixed with 1ml of GO (1mg / mL), 40ul of nafion is added, and ultrasonic stirring is performed to obtain AuNPs@GO; 5ul of AuNPs@GO is fixed on the surface of a gold electrode to obtain an AuNPs@GO / gold electrode; then the AuNPs@GO / gold electrode is soaked in an ethanol solution containing 4mM MUA overnight, and then activated by a mixture of EDC (0.4M) and NHS (0.1M); then the electrode is washed with deionized water; 5ul of tail fiber protein TFP08 with a concentration of 5mg / mL is dropped on the top of the electrode for incubation to obtain a TFP 08 / AuNPs@GO / gold electrode; after the TFP 08 / AuNPs@GO / gold electrode is washed with deionized water, the electrode is immersed in a solution containing 100ul of 10mM Ni 2+ , and the electrochemical biosensor is obtained.

[0011] The application utilizes the recognition and adsorption of a bacteriophage tail fiber protein receptor binding protein to specific bacterial host receptors in the recognition process of bacteriophage, the protein is a beta structure-rich trimer, the structures are folded and entangled with each other in the beta helix structure, further improving the stability, not easy to be affected by enzymes, temperature and pH, and having specificity and stability. Compared with the prior art, the application has the beneficial effects that:

[0012] 1、The bacteriophage tail fiber protein TFP08 provided by the application has a wide recognition range, and has good recognition ability for standard strains and clinical isolated strains.

[0013] 2、The bacteriophage tail fiber protein TFP08 provided by the application has high specificity, and has strong recognition ability for Cronobacter, and no recognition ability for non-target bacteria such as Escherichia coli (ATCC 25922, ATCC 36150), Staphylococcus aureus (CMCC26001) and Salmonella (ATCC 13076).

[0014] 3、The electrochemical biosensor prepared based on the bacteriophage tail fiber protein can be effectively used for on-site detection and rapid screening of Cronobacter, has high specificity, and the matrix does not need pretreatment, and the detection time is only about 30 minutes, which can shorten the existing detection time. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The protein detection results are shown in the following table:

[0016] (A) is a gel electrophoresis spectrum of TFP08, wherein 1 is a recombinant plasmid His-ORF08-pET28a, 2 is a double enzyme digestion spectrum of the recombinant plasmid, and M is a DNA molecular weight standard; (B) is a verification result of the transformation process; (C) is an SDS-PAGE spectrum of TFP08, wherein M is a protein molecular weight standard.

[0017] The precipitated protein is shown in the following table: 2 is the supernatant protein, 3 is the purified supernatant protein; (D) is a Western blot analysis spectrum of TFP08, wherein M is a protein molecular weight standard, and 1 is the purified TFP08.

[0018] Figure 2 The detection results of the binding ability of the tail fiber protein TFP08 to host bacteria are shown in the following table.

[0019] Figure 3 The molecular interaction of the tail fiber protein TFP08 to the LPS on the surface of the host bacteria is shown in the following table.

[0020] Figure 4 The construction and determination results of the electrochemical biosensor are shown in the following table.

[0021] Figure 5 Results of specificity and storage stability detection for TFP08 electrochemical sensor.

[0022] Figure 6 Results of linear range and sensitivity detection for TFP08 electrochemical sensor.

[0023] Figure 7 Schematic diagram for preparation of electrochemical biosensor based on phage tail fiber protein TFP08. DETAILED DESCRIPTION

[0024] The raw materials, strains and reagents used in the examples were obtained from conventional commercial channels unless otherwise specified.

[0025] GO and AuNPs were purchased from Nanjing Xianfeng Nanometer Material Technology Co., Ltd.

[0026] Nafion was purchased from Beijing Zhongke Nongan Technology Co., Ltd.

[0027] Cronobacter b2, b29 and b30 were preserved by Jiangsu Academy of Agricultural Sciences, and the strains were conventional strains as disclosed in the literature “Foods 2024, 13, 871.”.

[0028] Staphylococcus aureus (CMCC 26001), Escherichia coli (ATCC 25922, ATCC 36150) and Salmonella (ATCC 13076) were standard strains purchased from China Microbial Culture Collection Center and American Type Culture Collection.

[0029] The Escherichia coli top10 strain was purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0030] LB liquid medium (1 L): add 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride to 950 mL of water. Adjust the pH value to 7.0 with 1M NaOH, and make up to 1 L. Autoclave at 121°C for 20 min, and store at 4°C.

[0031] LB medium with kanamycin resistance: add kanamycin to a final concentration of 50 μg / mL to LB liquid medium.

[0032] Kanamycin-resistant plate: add kanamycin to a final concentration of 50 μg / mL and agar to a final concentration of 1.2% to LB liquid medium.

[0033] Anti-His tag mouse monoclonal antibody and HRP-labeled goat anti-mouse antibody IgG (H+L) were purchased from Biyun Tian Biotechnology Co., Ltd.

[0034] Example 1 Expression of tail fiber protein

[0035] The recombinant plasmid HisORF08-pET28a(+) (Kangshen Biotech) was mixed with 100 μL of E. coli BL21(DE3)pLysS (Kangshen Biotech). The solution was placed in an ice bath for 10 minutes, incubated in a 42°C water bath for 90 seconds, and then transferred to an ice bath for 3 minutes. Next, 1 milliliter of LB liquid medium was added, and the mixture was incubated at 37°C for 45 minutes while shaking at 180 revolutions per minute to recover the cells. 60 μL of the culture was added to a kanamycin-resistant plate, and the plate was inverted and incubated at 37°C overnight.

[0036] A single colony containing the recombinant plasmid was inoculated into 5 milliliters of kanamycin-resistant LB medium and incubated at 37°C overnight. 2 milliliters of the above culture was transferred to 200 milliliters of kanamycin-resistant LB medium and incubated at 37°C with continuous shaking at 180 revolutions per minute. When the OD 600 value reached about 0.6, it was transferred to an ice bath for 10 minutes, and then isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM. After induction and expression for 16 hours in a 16°C shaker, the bacterial cells were collected by centrifugation (10000 rpm, 10 minutes, 4°C) and washed with Tris-HCl (50 mM, pH = 7.2) three times; the bacteria were resuspended in 20 mL of Tris-HCl (50 mM, pH = 7.2), and the cells were subjected to ultrasonic treatment. The cells were centrifuged (12000, 15 minutes, 4°C) to separate the supernatant and the precipitate. The supernatant and the precipitate samples were used for SDS-PAGE to verify the expression, and the detection results are shown in Figure 1 FIG. 1C, where lane 1 is the detection result of the precipitate, and lane 2 is the detection result of the supernatant.

[0037] The synthetic gene was ligated into a recombinant plasmid, which was transformed into the expression strain E. coli top10 strain, and a single colony was picked on a kanamycin-resistant plate for NcoI / BamHI double enzyme digestion verification, and the results are shown in Figure 1 FIG. 1A, where the gene fragment size of the tail fiber protein TFP is about 2600 bp. Subsequently, the recombinant plasmid was transformed into the E. coli expression strain BL21(DE3). The BL21(DE3) after transformation was verified using T7 promoter universal primers, and the detection results are shown in Figure 1 FIG. 1B, where the gene fragment size is also about 2600 bp, indicating that the recombinant plasmid was successfully transformed into the E. coli expression strain BL21(DE3).

[0038] The constructed expression vector was inoculated into kanamycin-resistant LB liquid medium at a seeding amount of 1%, and induced to express at 16°C and 210 rpm for 20 h. The expression was broken by ultrasonic in an ice box (300 W, working for 3 s, intermittent for 4 s, 10 min) for 3 cycles. After ultrasonic, centrifugation was performed at 12,000 rpm and 4°C for 10 min to separate the supernatant and the precipitate. SDS-PAGE electrophoresis (140 V, 60 min) was performed to identify the expression of the protein. The detection results are shown in Figure 1 As shown in Fig. 1C, the target protein mainly exists in the supernatant, indicating that the target protein TFP08 is a soluble protein, which is beneficial to subsequent experiments.

[0039] Then, the protein TFP08 was added to a protein purification column containing 5 mL HisPurTM nickel-NTA resin, and incubated at 4°C for 30 min to allow it to flow through the resin bed. The protein was purified using 25 mM, 50 mM, 100 mM, 250 mM and 500 mM imidazole, respectively. The eluate was collected, and the eluate containing the target protein was determined by SDS-PAGE. The target protein was concentrated by using a 30 KDa ultrafiltration tube at 4000 g for 10 min. The purified protein was obtained, which was a soluble protein. The applicant named it as tail fiber protein TFP08. The amino acid sequence and nucleotide sequence thereof are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. Western blotting was used to analyze the purified TFP08, and the results are shown in Figure 1 As shown in Fig. 1D, there is a target band near 90 kDa. Finally, the BCA kit was used to measure the concentration of the tail fiber protein, which was 5 mg / mL.

[0040] Example 2: Determination of the binding ability of tail fiber protein TFP08 to host bacteria by ELISA method

[0041] Cronobacter b2 and Staphylococcus aureus (CMCC 26001) were cultured in LB liquid medium overnight, centrifuged at 5000 rpm for 3 min, washed with sterile water three times, and finally resuspended with sterile PBS. The b2 strain, CMCC 26001 strain and blank sample (PBST buffer containing 5% mass concentration of skimmed milk powder) were added to a 96-well plate, 100 μL per well, and incubated at 4°C overnight. 0.05% (mass concentration, the same below) PBST (phosphate buffered saline) was used for washing four times, 250 μL of 5% mPBS was added to each well, and the plate was incubated at 37°C for 2 h. Then PBST was used for washing three times, 100 μL of TFP08 (10 μg / mL) was added to each well, and the plate was incubated at 37°C for 1 h. After 1 h, the plate was washed with PBST three times, 100 μL of anti-His tag mouse monoclonal antibody (antibody: PBST was diluted at a volume ratio of 1:3000) was added to each well, and the plate was incubated at 4°C overnight. After overnight incubation, the plate was washed with PBST three times, 100 μL of HRP-labeled goat anti-mouse antibody IgG (H+L) was added to each well, and the plate was incubated at 37°C for 1 h. 10 μL of citrate buffer (CPBS), 10 μL of 3,3',5,5'-tetramethylbenzidine TMB developing solution and 25 μL of H2O2 (0.65%) were added, and the plate was incubated for 15 min. Finally, 50 μL of H2SO4 (2M) was added to terminate the reaction. The OD 450 .

[0042] The detection results are shown in Figure 2 It can be seen that the affinity of the tail fiber protein to the host bacteria b2 is much higher than that of the blank sample mPBS and the non-host bacteria 26001.

[0043] Example 3: Analysis of the binding ability of tail fiber protein TFP08 to host bacteria receptor LPS using a biofilm interferometer

[0044] Previous studies suggest that the receptor of Cronobacter may be on the lipopolysaccharide (LPS) on its surface. Therefore, in this example, the LPS of Cronobacter (b2) was first extracted using an LPS extraction kit (purchased from Beijing Solabio Technology Co., Ltd.), and then the affinity between TFP08 and LPS was analyzed using an Octet Red96 biofilm interferometer. 200 μM of TFP was loaded onto the APS biosensor for 600 s, and the binding of LPS to the immobilized TFP was measured, with an association time of 340 s, followed by a dissociation time of 300 s (the specific steps were performed according to the sensor instruction manual). The results are shown in Figure 3 It can be seen that the tail fiber protein TFP08 has a strong binding force to the LPS on the surface of the host bacteria b2.

[0045] Example 4: Construction of an electrochemical biosensor based on TFP08

[0046] The 1 ml gold nanoparticles (AuNPs) was mixed with 1 ml single-layer graphene oxide (GO, 1 mg / mL), 40 μL nafion was added, and ultrasonic stirring was performed for 30 minutes to obtain AuNPs@GO; 5 μL of AuNPs@GO was fixed on the surface of a gold electrode (dried at room temperature, and the mixture was deposited on the surface of a gold electrode) to obtain an AuNPs@GO / gold electrode; then the AuNPs@GO / gold electrode was soaked in an ethanol solution containing 4 mM 11-mercapto-undecanoic acid (MUA) MUA overnight (the thiol functional group of MUA was combined with AuNPs), and then a mixture of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC, 0.4 M) and N-hydroxysuccinimide sodium salt (NHS, 0.1 M) was used to activate for 30 minutes, followed by three times of washing the electrode with deionized water to remove EDC and NHS; 5 ul of TFP08 solution with a concentration of 5 mg / mL was then dropped on the top of the electrode using a pipette and incubated for 60 minutes, and TFP08 was fixed and modified on the electrode (covalent bonding between the two through an amide bond) to obtain a TFP 08 / AuNPs@GO / gold electrode; after the incubation period, the TFP 08 / AuNPs@GO / gold electrode was washed with deionized water to remove unbound TFP08; finally, the electrode was immersed in a solution containing 100 μL of 10 mM Ni 2+ to connect the His tag on TFP 08 to obtain an electrochemical biosensor, and a preparation flow chart thereof is shown in Figure 7 The sensor preparation method is a conventional method in the art, as disclosed in the document “Talanta, 2024, 270, 125561.”

[0047] The electrochemical biosensor generates a current signal by using Ni 2+ The TFP08 captures bacteria, and GO amplifies the current signal, so that the Cronobacter b2 can be detected by using differential pulse voltammetry (DPV). The detection method is a conventional method in the art, as disclosed in the document “Microchimica Acta, 2024, 191:550.” The specific detection steps of this embodiment are as follows: 5 μL of Cronobacter was dropped on the surface of the TFP08-based electrochemical biosensor electrode prepared in this embodiment, incubated for 30 minutes, and then washed with PBS to remove unbound bacteria, and differential pulse voltammetry (DPV) was used to measure on a CHI 830C electrochemical station. The electrochemical measurement was performed using a three-electrode system--a working electrode, a platinum counter electrode, and a silver / silver chloride reference electrode. The DPV measurement was performed in a PBS solution with a pH value of 7.4. The measurement potential range was set to -600 to -200 mV, the amplitude was 100 mV, and the pulse period was 0.1 second.

[0048] The detection results are shown in Figure 4As shown, no oxidation peak appeared in PBS solution for the bare gold electrode (red line), then TFP08 and Ni 2+ After the combination on the surface of gold electrode, oxidation peak appeared (gold electrode-TFPs-Ni 2+ ), which indicated that TFP08 and Ni2+ successfully combined with the surface of gold electrode. After the combination of AuNPs@GO and TFP08, oxidation peak also appeared (gold electrode-AuNPs@GO-TFPs-Ni 2+ ), and the current signal was significantly enhanced, which indicated that AuNPs@GO could amplify the current signal. After the addition of Cronobacter (b2) on the AuNPs@GO-TFPs-Ni 2+ ), the current signal significantly decreased, which indicated that the target bacteria were successfully captured by TFP08, and the electrochemical biosensor was successfully constructed.

[0049] Example 5 Specificity and storage stability of TFP08 electrochemical sensor

[0050] In order to investigate the specificity of the detection method, this example used the TFP08 electrochemical sensor prepared in Example 4 to detect target bacteria (Cronobacter b2, b29, b30) and non-target bacteria Escherichia coli (ATCC 25922, ATCC 36150), Staphylococcus aureus (CMCC 26001) and Salmonella (ATCC 13076) with a bacterial content of 10 8 CFU / mL.

[0051] The detection results are shown in Figure A of Figure 5 Compared with the blank control, after capturing Cronobacter sakazakii (b29), Cronobacter malonaticus (b30) and Cronobacter dublinensis (b2), the current signal of the TFP08 electrochemical sensor significantly decreased, while the current signals of non-target bacteria such as Escherichia coli, Salmonella and Staphylococcus aureus were basically consistent with the blank control. This indicates that TFP08 has good specificity.

[0052] In addition, the storage stability of the electrochemical biosensor of TFP08 was also evaluated in this example, as shown in Figure B of Figure 5 After the layer-by-layer assembly of AuNPs@GO, TFP08 and Ni 2+ , the sample was stored at room temperature for 7 days. The current signal was determined by DPV. It was found that the TFP08 electrochemical sensor had good stability at room temperature

[0053] Example 6 Linear range and sensitivity of TFP08 electrochemical sensor

[0054] To evaluate the linear range sensitivity of the TFP08 electrochemical sensor prepared in Example 4, Cronobacter (b2) was serially diluted (10... 2 ~10 7 In this embodiment, the TFP08 electrochemical biosensor was used to detect DPV of different concentrations of Cronobacter.

[0055] The results are as follows Figure 6 As shown in Figure A, as the concentration of Cronobacterium increased from 2.3 × 10⁻⁶, the... 1 CFU / mL increased to 3.7 × 10⁻⁶ 7 CFU / mL, Ni 2+ The peak current gradually decreased, and the concentration of Cronobacterium was inversely proportional to the current, indicating that Cronobacterium can inhibit the generation of current. Meanwhile, at 10... 2 -10 7 It showed a good linear relationship in the range of CFU / mL, R 2 =0.9963. The limit of detection (LOD) is 23 CFU / mL. Figure 6 As shown in Figure B, the regression equation is A = 1.57 - 0.085lgC, where A is the peak current signal and C is the concentration of Cronobacter. This indicates that the TFP08 electrochemical sensor has a wide linear range and high sensitivity, meeting the requirements for Cronobacter detection.

[0056] Example 7: Detection of Kronobacter in infant formula and leafy vegetables using the TFP08 electrochemical sensor.

[0057] Infant formula sample preparation: Dissolve the sample in sterile deionized water according to the instructions and set aside;

[0058] Preparation of fresh lettuce samples: First, wash the purchased fresh lettuce with distilled water, then cut it into small pieces, and sterilize it under ultraviolet light for 30 minutes on each side.

[0059] This embodiment refers to the Chinese National Food Safety Standard (GB 4789.40-2024) for the quantitative testing of Cronobacter, after sample dilution: 10 mL and 10 g of skim milk powder or lettuce were mixed with 90 mL of sterile deionized water. Different concentrations of Cronobacter were added to the prepared samples, and then experiments were conducted using the plate count method (GB 4789.2-2022 "National Food Safety Standard for Microbiological Examination of Food - Determination of Total Colony Count") and the TFP08 electrochemical biosensor, respectively. The results of the plate count method were used as a control.

[0060] Table 1. Determination of Kronobacter in different infant formulas and lettuce by the TFP08 electrochemical biosensor.

[0061]

[0062] The detection results are shown in Table 1. The recovery rate of Cronobacter was 99.23%-102.46%, and the RSD was less than 1.17%. This method can be used for the detection of Cronobacter in complex food.

[0063] Example 8 Detection of Cronobacter in farm sewage and bovine serum by TFP08 electrochemical sensor

[0064] The farm sewage and bovine serum were filtered by 0.22 μm filter membrane, then 2 mL sample was mixed with 18.0 mL sterile water, and was ready for use.

[0065] Different concentrations of Cronobacter were added to the prepared samples, and then plate counting method and TFP08 electrochemical biosensor prepared in Example 4 were used for detection, and the results of plate counting method were used as control. The results are shown in Table 2.

[0066] Table 2 Determination of Cronobacter in farm sewage and bovine serum by TFP08 electrochemical biosensor

[0067]

[0068] As can be seen from Table 2, the electrochemical sensor prepared by TFP08 can be used for the detection of Cronobacter in environmental and clinical samples.

Claims

1. Use of a Cronobacter bacteriophage tail fiber protein in detecting Cronobacter for non-diagnostic purposes, wherein the amino acid sequence of the tail fiber protein is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The tail fiber protein is prepared into an electrochemical biosensor to detect Cronobacter in the environment or food.

3. Use according to claim 2, characterized in that, Based on the electrochemical biosensor, the content of Cronobacter in a sample is detected by using differential pulse voltammetry, and the peak current signal obtained is brought into a regression equation, so that the content of Cronobacter in the sample can be obtained; the regression equation is A = 1.57 - 0.085lgC, wherein A is the peak current signal, and its unit is μA; C is the concentration of Cronobacter, and its unit is CFU / mL.

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