A streptavidin binding peptide functionalized phage and its preparation method and application in detecting escherichia coli in food

Abstract

The application provides a preparation method of a functionalized phage probe and application of the functionalized phage probe in detection of escherichia coli. The functionalized phage is a recombinant phage (M13KO7@SaBP) in which streptavidin binding peptide (SaBP) is fused and expressed at the N terminal of the main capsid protein P8 protein of M13K07 phage. In the application, the gene encoding SaBP (MDVEAWLGAR) is inserted into the N terminal of the P8 protein gene of M13K07 phage by site-directed mutagenesis to prepare M13K07@SaBP. The stable signal amplification is realized by using the abundant P8 protein (about 2700) of M13 phage and the super strong binding force of biotin-streptavidin, the interference of food sample matrix is reduced by combining the magnetic separation technology, and a detection method of escherichia coli with high sensitivity and small sample matrix interference is constructed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a streptavidin binding peptide functionalized bacteriophage M13K07@SaBP, a preparation method thereof and application thereof in detection of Escherichia coli in food. BACKGROUND

[0002] Escherichia coli, also called Escherichia coli, was discovered by Escherich in 1885. Escherichia coli is a short bacillus with blunt round ends and is gram-negative. Sometimes, individual bacterial bodies appear as similar to a ball rod or a filament due to different environments; Escherichia coli is mostly single or two, but cannot arrange in a long chain shape; most Escherichia coli strains have a capsule or a microcapsule structure, but cannot form spores; most Escherichia coli strains grow with fimbriae, and some of the fimbriae are host-specific fimbriae with adhesion to hosts and other tissues or cells. The biochemical metabolism of Escherichia coli is very active, and can ferment glucose to produce acid and gas, and individual strains do not produce gas. Escherichia coli can also ferment various carbohydrates and can utilize various organic acid salts. In the commonly used biochemical characteristic detection items, the methyl red test of Escherichia coli is positive, indole production and lactose fermentation are positive (individual strains show negative), the V-P test is negative, urease and citrate utilization are negative (extremely individual strains show positive), the nitrate reduction test shows positive, and the oxidase shows negative, and the oxidation-fermentation test shows F type.

[0003] Escherichia coli is a bacterium commonly found in the intestines of humans and warm-blooded animals. Most Escherichia coli strains are harmless. However, some Escherichia coli can cause foodborne diseases. The antigenic components of Escherichia coli are complex, and can be divided into bacterial body antigen (O), flagellar antigen (H) and surface antigen (K). According to the difference of the bacterial body antigen, Escherichia coli can be divided into more than two hundred types, some of which have pathogenicity and can cause diarrhea, collectively referred to as diarrheal Escherichia coli. According to the different biological characteristics, the diarrheal Escherichia coli can be divided into five categories: enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, enteroinvasive Escherichia coli, enteroadherent Escherichia coli and enterohemorrhagic Escherichia coli (EHEC). Enterohemorrhagic Escherichia coli can produce Shiga-like toxin and is also called Shiga toxin-producing Escherichia coli (STEC). Enterohemorrhagic Escherichia coli has broken out in many countries and has become a global public health problem. Enterohemorrhagic Escherichia coli infection mainly causes diarrhea and can cause serious foodborne diseases. It is mainly transmitted to humans through the consumption of contaminated food, such as raw or undercooked ground meat products, raw milk and contaminated raw vegetables and sprouts. Rapid and accurate detection of Escherichia coli in food has become a problem that people often concern

[0004] The existing E. coli detection methods include fermentation method, plate counting method, molecular biology method and immunological method. The fermentation method is a relatively traditional method for detecting E. coli group in food, and this technology has been generally recognized by countries all over the world. At present, the fermentation method for detecting E. coli group in food mainly adopts the national standard GB4789.3-2010 determination of coliform group, and the E. coli needs to be cultured at a suitable temperature. After about 24 hours of culture, the fluorescent substrate is released to estimate the number of E. coli. Since the method is complicated to operate and consumes a lot of material, financial and human resources, it is not suitable for the demand of a large number of detection work.

[0005] The plate counting method is the "gold standard" for E. coli detection. This method is an effective method for counting bacteria in the sample. The operation sequence is to dilute the sample appropriately, so that the microorganisms in the sample are fully dispersed into single cells, take a certain amount of diluent for culture to make the microorganisms grow into visible colonies, and then calculate the number of bacteria through the dilution degree and the sample quantity. This method needs to go through the processes of separation, enrichment, selective culture and biochemical identification, and has a long detection period, cannot feedback the results in time, and cannot quickly respond to sudden foodborne pathogen pollution events.

[0006] The molecular biology method and the immunological method can be used to replace the culture method for early screening of foodborne pathogens. The molecular biology method detects target microorganisms at the gene level, such as polymerase chain reaction (PCR) and other related methods. The DNA or easily degradable RNA of the target bacteria needs to be extracted for long-time PCR amplification. The detection result of this method may be affected by the operation, so professional operators and equipment are needed. The immunological method is a series of detection methods at the protein level based on the specific binding reaction of antigen-antibody. However, it is difficult to obtain paired antibodies, the sensitivity is insufficient, and it cannot distinguish between dead bacteria and live bacteria. SUMMARY

[0007] The purpose of the present application is to overcome the deficiencies in the prior art, and provide a detection method for E. coli.

[0008] The first aspect of the present application is to provide a streptavidin binding peptide functionalized bacteriophage M13K07@SaBP, characterized in that the M13K07@SaBP is a recombinant bacteriophage in which the streptavidin binding peptide SaBP is fused and expressed at the N-terminus of the P8 protein of the M13K07 bacteriophage.

[0009] Preferably, the streptavidin binding peptide SaBP is displayed on the p8 protein of the M13K07 bacteriophage.

[0010] More preferably, the nucleotide sequence of the streptavidin binding peptide functionalized phage M13K07@SaBP is shown as SEQ ID NO: 1.

[0011] The second aspect of the present application is to provide a preparation method of the M13K07@SaBP of the first aspect of the present application: fusing the SaBP to express at the N-terminal of the P8 protein of the M13K07 phage to obtain the M13K07@SaBP.

[0012] Preferably, the preparation method is: taking the M13K07 plasmid as a template, using the primers

[0013] M13K07-SaBP-F:

[0014] 5'-ATGGATGTGGAAGCGTGGCTGGGCGCGCGCGCTGAGGGTGACGATCCCG-3', and

[0015] M13K07-SaBP-R:

[0016] 5'-CCACGCTTCCACATCCATAGCGAAAGACAGCATCGGAA-3'

[0017] carrying out PCR amplification, recovering the amplification product, DpnI digestion of the amplification product, recombination, to obtain the M13K07@SaBP.

[0018] The third aspect of the present application is to provide a probe containing the M13K07@SaBP of the first aspect of the present application.

[0019] The fourth aspect of the present application is to provide a kit containing the probe of the third aspect of the present application.

[0020] Preferably, the kit further contains streptavidin modified magnetic beads.

[0021] Preferably, the kit further contains streptavidin labeled horseradish peroxidase.

[0022] The fifth aspect of the present application is to provide the application of the M13K07@SaBP of the first aspect of the present application, or the probe of the third aspect of the present application, or the kit of the fourth aspect of the present application in detecting Escherichia coli in food.

[0023] Preferably, the M13K07@SaBP is used in combination with magnetic separation technology to reduce the interference of food sample matrix.

[0024] Preferably, the M13K07@SaBP streptavidin modified magnetic beads and streptavidin labeled horseradish peroxidase are used for detection. The phage is amplified in E. coli, the streptavidin modified magnetic beads capture the amplified phage, and the streptavidin labeled horseradish peroxidase is used for signal output, thereby establishing a high-sensitivity and small-sample-matrix-interference detection method for E. coli in food.

[0025] The sixth aspect of the present application provides a detection method for E. coli, which uses the M13K07@SaBP of the first aspect of the present application, or the probe of the third aspect of the present application, or the kit of the fourth aspect of the present application for detection.

[0026] Preferably, the detection method comprises the following specific steps:

[0027] (1) adding the M13K07@SaBP to the sample to be detected, incubating, and amplifying the phage in E. coli; (2) adding streptavidin modified magnetic beads to capture the amplified phage; (3) after magnetic separation, adding streptavidin labeled horseradish peroxidase for reaction to output signal; (4) after magnetic separation, developing color, measuring absorbance, and processing data.

[0028] Preferably, in step (1), incubation is performed at 36-38℃ for 70-80 min.

[0029] Preferably, in step (2), reaction is performed at room temperature for 0.8-1.2 h.

[0030] Preferably, in step (3), reaction is performed at 36-38℃ for 0.8-1.2 h.

[0031] Preferably, in step (4), TMB developing solution is used for color development, and reaction is performed at 36-38℃ for 8-12 min.

[0032] The present application inserts the gene encoding SaBP (MDVEAWLGAR) into the N-terminal of the P8 protein gene of M13K07 phage by site-directed mutagenesis method, prepares phage M13K07@SaBP, uses the abundant P8 protein (about 2700) of M13 phage and the super strong binding force of biotin-streptavidin to realize stable signal amplification, combines with magnetic separation technology to reduce the interference of food sample matrix, and constructs a high-sensitivity and small-sample-matrix-interference detection method for E. coli. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Sequencing results for constructing M13K07@SaBP of the present application.

[0034] Figure 2The construction of phage M13K07@SaBP and the process of detecting Escherichia coli by phage M13K07@SaBP.

[0035] Figure 3 The specific detection results of phage M13K07@SaBP on Escherichia coli.

[0036] Figure 4 The standard curve of M13K07@SaBP phage for detecting Escherichia coli O157:H7 in milk samples.

[0037] Figure 5 The standard curve of M13K07@SaBP phage for detecting Escherichia coli O157:H7 in pork homogenate samples.

[0038] Figure 6 The standard curve of M13K07@SaBP phage for detecting Escherichia coli O157:H7 in lettuce samples. DETAILED DESCRIPTION

[0039] The present application will be further described below with reference to the accompanying drawings and specific examples, so as to better understand the present application. In the examples, the specific techniques or conditions not mentioned are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be commercially available.

[0040] I. Preparation of streptavidin binding peptide functionalized phage M13K07@SaBP

[0041] The M13K07 plasmid was modified to display SaBP (MDVEAWLGAR) on the P8 protein.

[0042] 1. Plasmid extraction: M13K07 strain (Kan resistance) was cultured, and the next day, the plasmid was extracted using the Tiangen plasmid extraction kit, named M13K07, and the concentration was determined by Nanodrop to be 200 ng / μL.

[0043] 2. Primer design: P8 protein is composed of a 23-amino-acid signal peptide and a 50-amino-acid mature protein, so the primer is designed from the mature protein. At the same time, there is no suitable universal primer at the M13K07 modification site, so additional PCR detection and sequencing primers are designed. The specific primer design is as follows:

[0044] Plasmid amplification primer

[0045] Upstream primer M13K07-SaBP-F:

[0046] 5'-ATGGATGTGGAAGCGTGGCTGGGCGCGCGCGCTGAGGGTGACGATCCCG-3'

[0047] Downstream primer M13K07-SaBP-R:

[0048] 5'-CCACGCTTCCACATCCATAGCGAAAGACAGCATCGGAA-3';

[0049] PCR detection and sequencing primers:

[0050] Upstream primer M13K07-PCR-F 969: 5-CGTTGATTTGGGTAATGAATAT-3'

[0051] Downstream primer M13K07-PCR-R 1662: 5'-TCAGCGGAGTGAGAATAGAAA-3'.

[0052] 3. Mutation:

[0053] ① Target plasmid amplification

[0054] Take 1 μL of the original M13K07 plasmid and add 99 μL of sterile water to dilute it 100-fold. Use this template to prepare the PCR reaction system (50 μL) according to the following table:

[0055]

[0056]

[0057] After preparing the above PCR system, perform PCR amplification according to the following table:

[0058]

[0059] After completing the PCR, take 2.5 μL and perform detection by agarose electrophoresis, and recover the amplification product.

[0060] ② DpnI digestion of amplification product: reaction system as follows:

[0061]

[0062] The above mixture is gently blown and mixed, and placed in a 37°C water bath for 1 h.

[0063] ③ Recombination reaction: take 2 μL of DpnI digestion product, gently blow and mix with the following reactants, and incubate in a 37°C water bath for 30 min, then immediately cool on ice.

[0064]

[0065] 4. Detection:

[0066] ① Take 10 μL of the above reaction solution and react with E. coli ER2738 competent cells for heat shock transformation. Larger single colonies and smaller single colonies in the culture dish are selected and added to 20 mL of LB liquid medium containing kan, and incubated overnight;

[0067] ② The next day, about 3 mL of the remaining bacterial solution is added to 100 mL of liquid 2YT medium containing kan, and incubated at 220 rpm and 37°C for 5-8 h until the bacterial concentration is high. Then the incubation conditions are changed to 250 rpm and 30°C for overnight incubation;

[0068] ③ The next day, centrifuge at 8000 rpm and 4°C for 10 min, and pour the supernatant into three new 50 mL centrifuge tubes (33 mL / tube);

[0069] ④ Add 1 / 5 volume (7 mL) of 30% PEG-NaCl solution, and place at 4°C for about 5 h or overnight;

[0070] ⑤ Centrifuge at 8000 rpm and 4°C for 10 min, and pour the supernatant into a conical flask;

[0071] ⑥ Resuspend the precipitate in the centrifuge tube with 1 mL of PBS, add to a 1.5 mL centrifuge tube, blow evenly, add 1 / 5 volume (200 μL) of 30% PEG-NaCl solution, and place at 4°C for about 2 h;

[0072] ⑦ Centrifuge at 10,000 rpm for 5 min, and discard the supernatant;

[0073] ⑧ Resuspend the precipitate with 250 μL of PBS, and place for about 30 min to allow the phage to completely dissolve. Then centrifuge at 10,000 rpm for 5 min, and take the supernatant (the precipitate is bacterial body, etc.), which is the amplification product.

[0074] ⑨ Send the product to the company for sequencing, and the sequencing result is shown in SEQ ID NO: 1. The result shows that the coding sequence of SaBP (MDVEAWLGAR) is inserted into the M13K07 plasmid ( Figure 1 ), and M13K07@SaBP is obtained.

[0075] II. Preparation of streptavidin-modified magnetic beads

[0076] 1. Activation: Take 20 μL magnetic beads, wash with 1 mL PBS (0.01 mol / L, pH 7.4) for three times, resuspend with 780 μL PBS (0.01 mol / L, pH 7.4). Take NHSS 7.2 mg dissolved in 1 mL 1* PBS, take out 100 μL and add to the system; take EDC 6.4 mg dissolved in 1 mL 1* PBS, take out 100 μL and add to the system, keep the magnetic beads in resuspended state in the homogenizer, activate at 37°C for 1 h.

[0077] 2. Coupling: Recover the magnetic beads with the magnetic stand, wash with 1 mL PBS (0.01 mol / L, pH 7.4) for three times, resuspend the magnetic beads in 1 mL PBS (0.01 mol / L, pH 7.4), add streptavidin 80 μg, i.e. take 16 μL (the original concentration of SA is 5 mg / mL), couple at room temperature for 2 h in the homogenizer.

[0078] 3. Blocking: After coupling, add 1 mL BSA with a final concentration of 1% to the product at room temperature for blocking for 45 min. Recover the magnetic beads with the magnetic stand, wash with 1 mL PBS (0.01 mol / L, pH 7.4) for three times, resuspend the streptavidin magnetic beads in 1 mL PBS (0.01 mol / L, pH 7.4) to a final concentration of 1 mg / mL, and store in the refrigerator at 4°C for standby.

[0079] Three, specificity of phage to E. coli

[0080] Bacillus cereus (ATCC 14597), Escherichia coli (ATCC 25922), Pseudomonas fluorescens (ATCC 13525), Pseudomonas aeruginosa (ATCC 9027), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 29213), Staphylococcus aureus (ATCC 25923), Escherichia coli O157:H7 (CMCC 44102), Escherichia coli TG1, Listeria monocytogenes (ATCC 13932), Escherichia coli ER2738, S. enteritidis ) The specificity of the phage to Escherichia coli was detected.

[0081] Preparation of phosphate buffer solution (PBS, 0.01M, pH 7.4): NaCl 8g, Na2HPO4 1.44g, KH2PO4 0.24g, KCl 0.2g were dissolved in 1L ultrapure water. Adjust the pH to 7.4 with 0.1M NaOH.

[0082] 1. Dilute the bacterial solution: dilute other bacteria to 10 5 cfu / mL with LB liquid medium. Dilute the four kinds of Escherichia coli to 10 4 cfu / mL with LB liquid medium, 300μL per tube, three parallel gradients for each.

[0083] 2. Dilute the phage: dilute M13K07@SaBP phage to 10 4 pfu / mL with LB medium, take 10μL and add to each centrifuge tube containing 300μL bacterial solution, incubate at 37℃ for 1.5h.

[0084] 3. Dilute the prepared streptavidin magnetic beads (SA@MB) to 0.125μg / mL with PBS, add 40μL (i.e. 5μg streptavidin magnetic beads) to the reaction solution, mix on a vortex mixer at room temperature for 1h, and then wash with PBST for 3 times.

[0085] 4. Dilute SA-HRP: Add streptavidin-labeled horseradish peroxidase (SA-HRP) dilution buffer (using 1×PBS, pH=7.4, diluted 1:625), 100 μL to each centrifuge tube, and incubate at 37°C for 1 h.

[0086] 5. Color development: After washing 3 times with PBST, add 100 μL of TMB color development solution and react at 37℃ for 10 min.

[0087] 6. Termination: Terminate the reaction with 10% H2SO4, 50 μL per tube.

[0088] 7. Detection: Add 140 μL to each tube onto an ELISA plate and measure the OD. 450 Absorbance value at the location.

[0089] Experimental results are as follows Figure 3 As shown, the results indicate that the bacteriophage of the present invention can specifically detect Escherichia coli 25922, Escherichia coli O157:H7, Escherichia coli TG1, and Enterobacter ER2738, but does not react to Bacillus cereus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes, etc.

[0090] IV. Application of Bacteriophage M13K07@SaBP in Detection

[0091] (a) Detection of E. coli O157:H7 in milk samples

[0092] 1. Dilution: Using a sterile pipette, pipette 2.5 mL of sterile milk into a sterile Erlenmeyer flask containing 22.5 mL of PBS. Shake thoroughly to mix well, preparing a 1:10 sterile milk solution. The pH of the diluted milk should be between 6.5 and 7.5.

[0093] 2. Dilute E. coli O157:H7 with a 1:10 ratio of sterile milk sample to prepare a 10-fold diluted homogenate, resulting in an E. coli O157:H7 concentration of 10. 7 cfu / mL, 10 6 cfu / mL, 10 5 cfu / mL, 10 4 cfu / mL, 10 3 cfu / mL, 10 2 cfu / mL, 10 1 cfu / mL, 10 0 CFU / mL, 0 CFU / mL. Use a new 1 mL sterile pipette tip for each dilution. The entire process, from sample homogenization to inoculation, should not exceed 15 minutes. 300 μL per tube.

[0094] 3. Dilute the phage: Dilute the M13K07@SaBP phage to 10⁻⁶ with PBS. 4 Take 10 μL of pfu / mL and add it to each centrifuge tube containing 300 μL of E. coli O157:H7, and incubate at 37°C for 75 min.

[0095] 4. Dilute the prepared SA@MB to 0.125 μg / mL, add 40 μL to the reaction solution, and mix at room temperature for 1 h. After mixing, wash 3 times with PBST.

[0096] 5. Add SA-HRP dilution buffer (using 1×PBS, pH=7.4, diluted 1:625), 100 μL to each centrifuge tube, and incubate at 37°C for 1 h.

[0097] 6. Display: After washing 3 times with PBST, transfer the magnetic beads to a 96-well plate, add 100 μL of TMB colorimetric solution, and react at 37°C for 10 min.

[0098] 7. Termination: Terminate the reaction with 10% H2SO4, 50 μL per tube.

[0099] 8. Detection: Measurement of OD 450 Calculate the absorbance value. Create a standard curve and calculate the detection limit. The results are as follows: Figure 4 As shown, the detection limit for Escherichia coli O157:H7 in milk samples is 4 cfu / mL.

[0100] (II) Detection of E. coli O157:H7 in pork samples

[0101] 1. Dilution: Weigh 25g of pork and place it in a sterile homogenizing bag. Add 225mL of PBS and mix thoroughly to make a 1:10 mixture. Place the mixture in a clean bench under UV light for 30min. After sterilization, add sterile PBS to make a 1:100 mixture. Centrifuge to remove undissolved parts.

[0102] 2. Dilute E. coli O157:H7 with a 1:100 ratio of sterile pork sample to prepare homogenates of tenfold dilution. The concentration of E. coli O157:H7 is 10. 7 cfu / mL, 10 6 cfu / mL, 10 5 cfu / mL, 10 4 cfu / mL, 10 3 cfu / mL, 10 2 cfu / mL, 10 1 cfu / mL, 10 00 cfu / mL. Each dilution, change 1 mL sterile tip. From the preparation of the sample homogenate to the end of the sample inoculation, the whole process should not exceed 15 min. 300 μL per tube.

[0103] 3. Dilution of phage: M13K07@SaBP phage was diluted with PBS to 10 4 pfu / mL, 10 μL was added to each centrifuge tube containing 300 μL of E. coli O157:H7, and incubated at 37°C for 75 min.

[0104] 4. The prepared SA@MB was diluted to 0.125 μg / mL, 40 μL was added to the reaction solution, and mixed on a mixer for 1 h at room temperature. After mixing, PBST was used for washing 3 times.

[0105] 5. SA-HRP diluent was added (1 x PBS, pH = 7.4, diluted 1:625), 100 μL was added to each centrifuge tube, and incubated at 37°C for 1 h.

[0106] 6. Display: after washing 3 times with PBST, the magnetic beads were transferred to a 96-well plate, 100 μL of TMB color developing solution was added, and reacted at 37°C for 10 min.

[0107] 7. Termination: the reaction was terminated with 10% H2SO4, 50 μL per tube.

[0108] 8. Detection: the OD 450 value at 450 nm was measured. A standard curve was made, the detection limit was calculated, and the results are shown in Table 1. The detection limit of E. coli O157:H7 in milk samples was 4 cfu / mL. Figure 5

[0109] (Three) Detection of E. coli O157:H7 in lettuce samples

[0110] 1. Dilution: 25 g of lettuce was weighed into a sterile homogenization bag, 225 mL of PBS was added, and mixed thoroughly to make a 1:10 mixture, which was sterilized under a UV lamp in a super-clean bench for 30 min.

[0111] 2. E. coli O157:H7 was diluted with 1:10 sterile lettuce sample, and ten-fold dilution sample homogenate was prepared in turn. The concentration of E. coli O157:H7 was 10 7 cfu / mL, 10 6 cfu / mL, 10 5 cfu / mL, 10 4 cfu / mL, 10 3 cfu / mL, 10 2 cfu / mL, 10 1 ​cfu / mL, 10 0 cfu / mL, 0 cfu / mL. Each dilution, change 1 mL sterile tip. From the preparation of the sample homogenate to the end of the sample inoculation, the whole process should not exceed 15 min. 300 μL per tube.

[0112] 3, dilution of phage: M13K07@SaBP phage was diluted with PBS to 10 4 pfu / mL, 10 μL was added to each centrifuge tube containing 300 μL of E. coli O157:H7, and incubated at 37°C for 75 min.

[0113] 4, the prepared SA@MB was diluted to 0.125 μg / mL, 40 μL was added to the reaction solution, and mixed on the mixer for 1 h at room temperature. After mixing, PBST was used for washing 3 times.

[0114] 5, SA-HRP diluent (using 1×PBS, pH = 7.4, diluted 1:625) was added, 100 μL was added to each centrifuge tube, and incubated at 37°C for 1 h.

[0115] 6, display: after washing with PBST for 3 times, the magnetic beads were transferred to a 96-well plate, 100 μL of TMB color developing solution was added, and reacted at 37°C for 10 min.

[0116] 7, termination: the reaction was terminated with 10% H2SO4, 50 μL per tube.

[0117] 8, detection: the OD 450 value was measured at 450 nm. The standard curve was made, the detection limit was calculated, and the results are shown in Table 1. The detection limit of E. coli O157:H7 in milk samples was 3 cfu / mL. Figure 6

[0118] The specific embodiments of the present application are described in detail above, but it is only as an example, and the present application is not limited to the specific embodiments described above. Any equivalent modification and substitution to the utility made by those skilled in the art is also within the scope of the present application. Therefore, any equivalent transformation and modification made without departing from the spirit and scope of the present application should be covered within the scope of the present application.​

Claims

1. A method for detecting Escherichia coli in food, characterized in that, The specific steps are as follows: (1) Add streptavidin-binding peptide-functionalized phage M13K07@SaBP to the sample to be tested and incubate to amplify the phage; (2) Add streptavidin-modified magnetic beads to capture the amplified phage; (3) After magnetic separation, horseradish peroxidase labeled with streptavidin is added to react and output a signal; (4) After magnetic separation, color development is performed, absorbance is measured, and data is processed; The M13K07@SaBP is a recombinant phage fused with the streptavidin-binding peptide SaBP and expressed at the N-terminus of the major capsid protein P8 of the M13K07 phage. The nucleotide sequence of the M13K07@SaBP is shown in SEQ ID NO:

1.

2. The method for detecting Escherichia coli in food according to claim 1, characterized in that, The preparation method of the M13K07@SaBP includes: Using the M13K07 plasmid as a template, primers were used... M13K07-SaBP-F: 5'-atggatgtggaagcgtggctgggcgcgcgcGCTGAGGGGTGACGATCCCG-3', and M13K07-SaBP-R: 5'-ccacgcttccacatccatAGCGAAAGACAGCATCGGAA-3' PCR amplification was performed, the amplification product was recovered, the amplification product DpnI was digested and recombined to obtain the M13K07@SaBP.

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