Targeted fluorescent bifunctional bacteriophage as well as preparation method and application thereof
By displaying the Vibrio parahaemolyticus nanobody Nb20 and the nanoluciferase Nluc on M13 phage, a targeted fluorescent bifunctional phage M13-Nb20-Nluc is formed, which solves the problems of complex auxiliary materials and assembly effects in the prior art and achieves rapid detection with high sensitivity and good anti-interference.
Patent Information
- Application Number
- CN202510974132.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-15
- Publication Date
- 2025-10-28
AI Technical Summary
In existing M13 bifunctional display methods, the production of auxiliary materials is complex and batch characteristics are unstable, resulting in unsustainable detection methods. Furthermore, the pVIII site displays large molecular proteins that affect phage assembly.
Using phage display technology, the nanobody Nb20 of Vibrio parahaemolyticus was displayed at the N-terminus of the M13 capsid protein pIII, and the nanoluciferase Nluc was displayed at the N-terminus of the pVII protein, forming a targeted fluorescent bifunctional phage M13-Nb20-Nluc.
It achieves rapid detection with strong targeting specificity, good anti-interference and high sensitivity, and is suitable for rapid detection of Vibrio parahaemolyticus in seafood and clinical samples. Moreover, it requires no auxiliary materials and the preparation process is simple and sustainable.
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Figure CN120843446A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of rapid detection technology for pathogenic bacteria, specifically relating to a targeted fluorescent bifunctional bacteriophage, its preparation method, and its application. Background Technology
[0002] Vibrio parahaemolyticus is a halophilic pathogen mainly found in seafood such as shrimp, crab, fish, and clams, as well as related products. This bacterium is a major cause of diarrheal diseases caused by seafood products. Symptoms after ingestion include watery diarrhea, abdominal cramps, nausea, vomiting, and headache, posing a serious threat to public health and safety.
[0003] Phage display is a molecular biology technique based on the genetic modification of phage DNA. This technique inserts a foreign gene sequence into a specific region of the phage capsid protein gene, allowing for co-transcription of the foreign gene and the capsid protein gene without affecting the capsid protein gene's reading frame, thereby achieving the fusion expression of the foreign peptide and the capsid protein. M13 phage is the most commonly used display phage, and its pIII and pVIII proteins are the two most widely used display sites. pIII protein can be used for both short peptide display and the insertion of large antibody fragments, making it an ideal display site for high-affinity antibody screening. pVIII protein is mostly used for short peptide display, generally displaying only 6-8 amino acid residues; excessively long peptide sequences can affect phage assembly.
[0004] Current M13 bifunctional display methods achieve this by displaying antibodies on pIII and short peptides that have undergone in vitro signal modification on pVIII. For example, rapid detection methods for Vibrio parahaemolyticus based on M13 phage display specific antibodies on pIII and short peptides on pVIII to facilitate the use of signal amplification auxiliary materials, such as enzyme-labeled secondary antibodies, quantum dots, or gold / silver nanoparticles. The production processes of these auxiliary materials are complex, batch-to-batch characteristics are unstable, or they are difficult to regenerate during use, leading to the unsustainability of such detection methods.
[0005] Therefore, providing a phage that simultaneously displays a targeted, fluorescent bifunctional macromolecular protein on the surface of M13, along with its preparation method and applications, is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a targeted fluorescent bifunctional bacteriophage, its preparation method, and its applications. This bacteriophage has advantages such as strong targeting specificity, good anti-interference ability, high sensitivity, and continuous rapid preparation, and can be used for the rapid detection of pathogenic microorganism Vibrio parahaemolyticus.
[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a targeted fluorescent bifunctional phage, characterized in that the targeted fluorescent bifunctional phage is obtained by displaying the nanobody Nb20 of Vibrio parahaemolyticus to the N-terminus of the capsid protein pIII of temperate phage M13 and the nanoluciferase Nluc to the N-terminus of the capsid protein pVII of temperate phage M13 through phage display technology, thereby obtaining a targeted fluorescent bifunctional phage with Nb20 displayed at the N-terminus of pIII protein and Nluc displayed at the N-terminus of pVII protein.
[0008] The aforementioned targeted fluorescent bifunctional phage is characterized in that the targeted fluorescent bifunctional phage is Escherichia coliphage M13-Nb20-Nluc, which was deposited at the China Center for Type Culture Collection on April 7, 2025, with accession number CCTCC NO: M 2025711.
[0009] Furthermore, the present invention provides a method for preparing the above-mentioned targeted fluorescent bifunctional phage, characterized by comprising the following steps:
[0010] Step 1: Amplify the linear plasmid pComb3X using the vector_F and vector_R primer pairs and the plasmid pComb3X as templates;
[0011] Step 2: Obtain the Nb20-pIII_C-Nluc_pVII fusion gene fragment through gene synthesis;
[0012] Step 3: Using a seamless cloning kit, the linear plasmid pComb3X obtained in Step 1 was ligated with the Nb20-pIII_C-Nluc_pVII fusion gene fragment obtained in Step 2, transformed into E. coli ER2738, and positive transformants were screened by plating with ampicillin-resistant plates to obtain recombinant E. coli.
[0013] Step 4: Inoculate the recombinant Escherichia coli obtained in Step 3 into LB liquid medium containing ampicillin, shake and culture, then add helper phage M13KO7 and kanamycin to the culture medium, and incubate overnight with shaking after standing in an incubator.
[0014] Step 5: Centrifuge the culture obtained in Step 4 at high speed and transfer the supernatant after centrifugation to a new clean centrifuge tube;
[0015] Step 6: Add PEG / NaCl solution to the supernatant from Step 5 and mix thoroughly.
[0016] Step 7: Centrifuge the mixture from Step 6 at high speed, discard the supernatant after centrifugation, and fully suspend the recombinant phage precipitate using TBS. The suspension is the targeted fluorescent bifunctional phage.
[0017] The method described above is characterized in that, in step one, the vector_F primer sequence is as shown in SEQ ID NO:1, and the vector_R primer sequence is as shown in SEQ ID NO:2;
[0018] SEQ ID NO: 1: 5'-CTAGTTGAACCTGCATggccgcctgggccacggtagcgaaacc-3'; SEQ ID NO: 2: 5'-GCTGGGGGTCAAAGATGAgctagctaattaatttaagcggccgc-3'.
[0019] Furthermore, the present invention provides an application of the above-mentioned targeted fluorescent bifunctional phage in the preparation of a rapid detection reagent for detecting Vibrio parahaemolyticus.
[0020] Compared with the prior art, the present invention has the following advantages:
[0021] 1. This invention obtains a targeted fluorescent bifunctional phage M13-Nb20-Nluc, which displays the nanobody Nb20 on the pIII protein and the nanoluciferase Nluc on the pVII protein, through phage display technology. This phage directly displays catalytically active enzyme molecules on the pVII capsid protein, solving the current problem that "displaying large protein molecules at the pVIII site will affect phage assembly".
[0022] 2. The targeted fluorescent bifunctional bacteriophage of the present invention can directly bind to the target bacteria and catalyze the substrate to generate a fluorescent signal without the need for auxiliary materials. It has the advantages of strong targeting specificity, high sensitivity, good anti-interference ability and continuous rapid preparation. It can be used for the rapid detection of pathogenic microorganism Vibrio parahaemolyticus and is suitable for the rapid detection of Vibrio parahaemolyticus in seafood and clinical samples.
[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0024] Figure 1 This is a schematic diagram illustrating the preparation process of the targeted fluorescent bifunctional phage M13-Nb20-Nluc of this invention.
[0025] Figure 2 The results of the detection of Vibrio parahaemolyticus in shrimp samples using the targeted fluorescent bifunctional bacteriophage M13-Nb20-Nluc of this invention are shown.
[0026] Figure 3 The results show the specificity of the targeted fluorescent bifunctional bacteriophage M13-Nb20-Nluc in detecting different single bacteria.
[0027] Figure 4 The results show the specificity of the targeted fluorescent bifunctional bacteriophage M13-Nb20-Nluc in detecting different mixed bacteria. Detailed Implementation
[0028] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention are described in detail below with reference to examples. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
[0029] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited thereto. Any methodological changes made by those skilled in the art based on these embodiments are included within the scope of protection of the present invention.
[0030] Example 1: A targeted fluorescent bifunctional bacteriophage and its preparation method
[0031] The targeted fluorescent bifunctional phage in this embodiment is obtained by displaying the nanobody Nb20 of Vibrio parahaemolyticus to the N-terminus of the capsid protein pIII of temperate phage M13 and the nanoluciferase Nluc to the N-terminus of the capsid protein pVII of temperate phage M13 through phage display technology, resulting in a targeted fluorescent bifunctional phage M13-Nb20-Nluc with Nb20 displayed at the N-terminus of pIII protein and Nluc displayed at the N-terminus of pVII protein.
[0032] The Nb20 amino acid sequence (SEQ ID NO:3) is as follows:
[0033] MQVQLVESGGGSVQAGGSLRLSCAASGRTHGRYCMAWFRQAPG KEREGVAAIDSAVNMIKLESVKGRFTVSKDNAKNTLYLQMNSLKPEDT AKYYCAADPGYCGSHIRPWITFAYRGQGTQVTVS;
[0034] The Nluc amino acid sequence (SEQ ID NO:4) is as follows:
[0035] MGGGSMVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSMGGGSMVTGYRLFEEIL;
[0036] The specific preparation method is as follows:
[0037] (I) Construction of recombinant phage particle pComb3X-Nb20-pIII_C-Nluc_pVII
[0038] Step 1: Using the vector_F / vector_R primer pair and plasmid pComb3X as a template, amplify the linear plasmid pComb3X. The amplification system is as follows:
[0039] Table 1 Amplification System
[0040] Components Added amount 2×PhantaMaxBuffer 25μL dNTPMix(10mMeach) 1μL PhantaMaxSuper-FidelityDNAPolymerase 1μL vector_F 2μL vector_R 2μL plasmid pComb3X 1μL <![CDATA[ddH2O]]> 18μL
[0041] The vector_F primer sequence is shown in SEQ ID NO:1, and the vector_R primer sequence is shown in SEQ ID NO:2; SEQ ID NO:1: 5'-CTAGTTGAACCTGCATggccgcctgggccacggtagcgaaacc-3'; SEQ ID NO:2: 5'-GCTGGGGGTCAAAGATGAgctagctaattaatttaagcggccgc-3'.
[0042] The uppercase letters represent partial sequences of the Nb20-pIII_C-Nluc_pVII fusion gene fragment, which are used for subsequent homologous recombination to form the circular recombinant phage particle pComb3X-Nb20-pIII_C-Nluc_pVII. The lowercase letters are used to amplify the linear plasmid pComb3X.
[0043] After preparing the amplification system, perform PCR amplification experiments. The amplification program is as follows: a) 95℃, 3 min; b) 95℃, 15 s; c) 55℃, 15 s; d) 72℃, 3 min; e) Repeat steps b)-c) 35 times; f) 72℃, 5 min; f) Incubate at 4℃.
[0044] The PCR amplification products were subjected to agarose gel electrophoresis. The gel block containing the target product was excised and purified using the HiPureGel Pure DNA Mini Kit to obtain the linear plasmid pComb3X fragment.
[0045] Step 2: Obtain the Nb20-pIII_C-Nluc_pVII fusion gene fragment through gene synthesis;
[0046] Step 3: According to the instructions of the ClonExpress II One Step Cloning Kit, the linear plasmid pComb3X obtained in Step 1 and the Nb20-pIII_C-Nluc_pVII fusion gene fragment obtained in Step 2 were recombined and ligated. The ligation product was transformed into E. coli ER2738 competent cells. The resuscitation solution was plated on ampicillin-resistant agar plates to screen for positive transformants. Single colonies that showed positive results by colony PCR were sent to Qingke Biotechnology Co., Ltd. for sequencing. Single clones whose sequencing results were consistent with the expected sequence composition were named recombinant E. coli ER2738 / pComb3X-Nb20-pIII_C-Nluc_pVII.
[0047] (II) Preparation method of targeted fluorescent bifunctional phage M13-Nb20-Nluc
[0048] Step 4: Inoculate the recombinant Escherichia coli ER2738 / pComb3X-Nb20-pIII_C-Nluc_pVII obtained in Step 3 into 100 mL of LB liquid medium containing ampicillin (100 μg / mL) at a ratio of 1% (v / v), and incubate at 37°C with shaking at 250 rpm until the OD value is reached. 600 The concentration was 0.5, and 100 μL of helper phage M13KO7 (titer 10) was added to it. 14 PFU / mL) and kanamycin (to its working concentration of 50 μg / mL) were incubated at 37°C for 1 h, and then incubated overnight at 37°C with shaking at 250 rpm.
[0049] Step 5: Centrifuge the culture obtained in Step 4 at 12000 rpm for 20 min, and transfer the supernatant after centrifugation to a new clean centrifuge tube;
[0050] Step 6: Add 0.2 times the volume of PEG / NaCl solution to the supernatant from Step 5, mix thoroughly, and centrifuge at 12000 rpm for 20 min.
[0051] Step 7: Discard the supernatant after centrifugation in Step 6, and fully suspend the recombinant phage pellet with 1 mL of TBS. Name it as the targeted fluorescent bifunctional phage Escherichia coli phage M13-Nb20-Nluc, and deposit it at the China Center for Type Culture Collection on April 7, 2025, with accession number CCTCC NO: M 2025711.
[0052] Example 2: Application of targeted fluorescent bifunctional bacteriophage in rapid detection of Vibrio parahaemolyticus
[0053] (I) Activation and Counting of Vibrio parahaemolyticus
[0054] In a clean bench, 10 μL of Vibrio parahaemolyticus glycerol suspension was inoculated into a test tube containing 5 mL of 3% sodium chloride alkaline peptone solution. The tube was incubated overnight at 37°C with shaking at 160 rpm. Using an inoculation loop, the overnight activated Vibrio parahaemolyticus suspension was streaked onto TSA solid medium, and the tube was sealed and inverted in a 37°C incubator. Once a single colony appeared, it was picked up with an inoculation loop and transferred to an Erlenmeyer flask containing 3% sodium chloride alkaline peptone solution. The flask was incubated at 37°C with shaking at 160 rpm until the bacterial growth reached OD500. 600 The value is approximately 1. After serially diluting the bacterial culture, 100 μL of each dilution was spread onto TSA solid medium. The plates were sealed with sealing film and incubated upside down in a 37°C incubator. Once single colonies appeared, plates with colony counts between 30 and 300 CFU were selected for counting, and the dilution factor and the corresponding colony count were recorded.
[0055] (II) Rapid quantitative detection of Vibrio parahaemolyticus using targeted fluorescent bifunctional bacteriophages
[0056] Shrimp samples obtained from the local market were confirmed to be free of Vibrio parahaemolyticus using the plate count method, and were then treated as follows: shrimp meat samples were processed according to 10... 3 10 4 10 5 10 6 10 7 10 8 CFU / g of activated Vibrio parahaemolyticus bacterial suspension was inoculated, then homogenized at low speed for 2 min with a stirrer, and centrifuged at 1000 rpm for 5 min to remove particles and lipid layer. The supernatant was transferred to a new centrifuge tube and centrifuged at 8000 rpm for 10 min at 4 °C. The bacterial cells were washed three times with ultrapure water, and finally resuspended in an equal volume of ultrapure water. 50 μL of the targeted fluorescent bifunctional phage M13-Nb20-Nluc (titer 10) prepared in Example 1 was taken. 14Mix 50 μL of resuspended Vibrio parahaemolyticus (PFU / mL) at different dilutions with 50 μL of the resuspended strain, and incubate at room temperature for 1 h. Centrifuge at 12000 rpm for 1 min, discard the supernatant, and resuspend the precipitate in 100 μL of sterile water. Add 100 μL of luciferase nanoparticle substrate solution, incubate at room temperature for 5 min, and detect the chemiluminescence signal value in a microplate.
[0057] The results are as follows Figure 2 As shown, the detection signal value is at 10 3 ~10 8 The four-parameter logistic relationship was well observed within the CFU / mL concentration range, and R0 2 =0.98756, the limit of detection is 10. 3 The CFU / mL result indicates that the targeted fluorescent bifunctional phage M13-Nb20-Nluc has high sensitivity and a wide quantitative range in detecting Vibrio parahaemolyticus.
[0058] (III) Specificity of targeted fluorescent bifunctional phage detection of Vibrio parahaemolyticus
[0059] Take 50 μL of Vibrio parahaemolyticus, Escherichia coli ER2738, Escherichia coli BL21, Escherichia coli TG1, Salmonella typhimurium, Salmonella enteritidis serotype 3, Salmonella enteritidis serotype 4, and Salmonella enteritidis serotype 21 bacterial suspensions, 50 μL of sterile water, and 50 μL of the targeted fluorescent bifunctional phage M13-Nb20-Nluc (titer 10) prepared in Example 1. 14 Mix (PFU / mL) and incubate at room temperature for 1 h. Centrifuge at 12000 rpm for 1 min, discard the supernatant, and resuspend the precipitate in 100 μL of sterile water. Add 100 μL of luciferase nanoparticle substrate solution, incubate at room temperature for 5 min, and detect the chemiluminescence signal value in the microplate.
[0060] The results are as follows Figure 3 As shown, the signal-to-noise ratio (SNR) for detecting Vibrio parahaemolyticus was 3.97, while the SNR for non-target bacteria was basically the same as that for sterile water, indicating that the targeted fluorescent bifunctional phage M13-Nb20-Nluc had high specificity for detecting Vibrio parahaemolyticus.
[0061] (iv) Anti-interference performance of targeted fluorescent bifunctional phage in detecting Vibrio parahaemolyticus
[0062] Mixing with a non-target bacterium: Mix 50 μL of Vibrio parahaemolyticus with 50 μL of Escherichia coli BL21 and 50 μL of Salmonella typhimurium bacterial suspensions, respectively. Centrifuge the suspensions at 12000 rpm for 1 min, discard the supernatant, and suspend the bacterial precipitate in 50 μL of sterile water. Add 50 μL of the targeted fluorescent bifunctional bacteriophage M13-Nb20-Nluc (titer 10) prepared in Example 1 to the suspension. 14 Mix (PFU / mL) and incubate the resulting mixture at room temperature for 1 hour. Centrifuge at 12000 rpm for 1 minute, discard the supernatant, and resuspend the precipitate in 100 μL of sterile water. Add 100 μL of luciferase nanoparticle substrate solution and incubate at room temperature for 5 minutes. Detect the chemiluminescence signal value in a microplate.
[0063] Mix with two non-target bacteria: Mix 50 μL of Vibrio parahaemolyticus, 50 μL of Escherichia coli BL21, and 50 μL of Salmonella typhimurium in the same centrifuge tube until homogeneous. Centrifuge the suspension at 12000 rpm for 1 min, discard the supernatant, and resuspend the bacterial precipitate in 50 μL of sterile water. Add 50 μL of the targeted fluorescent bifunctional bacteriophage M13-Nb20-Nluc (titer 10) prepared in Example 1 to the suspension. 14 Mix (PFU / mL) and incubate the resulting mixture at room temperature for 1 hour. Centrifuge at 12000 rpm for 1 minute, discard the supernatant, and resuspend the precipitate in 100 μL of sterile water. Add 100 μL of luciferase nanoparticle substrate solution and incubate at room temperature for 5 minutes. Detect the chemiluminescence signal value in a microplate.
[0064] The results are as follows Figure 4 As shown, when the Vibrio parahaemolyticus bacterial solution sample contained Escherichia coli BL21 or Salmonella typhimurium, the detection signal values were at the same level as those of the pure Vibrio parahaemolyticus bacterial solution, indicating that the targeted fluorescent bifunctional bacteriophage M13-Nb20-Nluc has strong anti-interference performance in detecting Vibrio parahaemolyticus.
[0065] Example 3: Comparison of the advantages of the targeted fluorescent bifunctional phage of the present invention in rapid detection of Vibrio parahaemolyticus with existing technologies
[0066] To demonstrate the advantages of this invention in rapid detection of Vibrio parahaemolyticus, the method of this invention was compared with existing technologies, and the specific results are shown in the table below:
[0067] Table 2 Comparison of detection results using different detection reagents
[0068]
[0069] The prior art 1 in the table above adopts the detection reagents and methods described in the literature: Wang, P.; Yu, GG; Wei, J.; Liao, XR; Zhang, Y.; Ren, YR; Zhang, C.; Wang, YQ; Zhang, DH; Wang, JL; Wang, YRA single thiolated-phage displayed nanobody-based biosensor for label-free detection offoodborne pathogen. Journal of Hazardous Materials, 2023, 443:130157;
[0070] Existing technology 2 adopts the detection reagents and methods described in the literature: Zhai, Y.; Meng, XJ; Li, L.; Liu, YS; Xu, K.; Zhao, C.; Wang, J.; Song, XL; Li, J.; Jin, MH. Rapid detection of Vibrio parahaemolyticus using magnetic nanobead-based immunoseparation and quantum dot-based immunofluorescence. Rsc Advances, 2021, 11(61): 38638-38647;
[0071] The existing technology 3 adopts the detection reagents and methods described in the literature: Zhou, J.; Zhang, C.; Zhang, X.; Lu, C.; Ming, T.; Li, Y.; Su, X. Immunomagnetic separation-based nanogold enhanced surface plasmon resonance and colloidal gold test strips for rapid detection of Vibrioparahaemolyticus. Archives of Microbiology, 2020, 202(5): 1025-1033;
[0072] The prior art 4 adopts the detection reagents and methods described in the literature: Ding, CC; Li, JW; Liu, X.; Liu, Q. Development of colloidal gold-based immunochromatographic strip test using two monoclonal antibodies for detection of Vibrio parahaemolyticus. Journal of Food Safety, 2018, 38(4).
[0073] As can be seen from Table 2, the targeted fluorescent bifunctional phage of the present invention does not require the use of materials such as gold nanoparticles / quantum dots in the rapid detection of Vibrio parahaemolyticus, nor does it require in vitro modification of the phage. Moreover, its preparation can be achieved through simple culture and collection. It is a green, economical, convenient and sustainable rapid detection technology for pathogenic bacteria.
[0074] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A targeted fluorescent bifunctional phage, characterized in that, The targeted fluorescent bifunctional phage is obtained by displaying the nanobody Nb20 of Vibrio parahaemolyticus to the N-terminus of the capsid protein pIII of temperate phage M13 and the nanoluciferase Nluc to the N-terminus of the capsid protein pVII of temperate phage M13 using phage display technology.
2. The targeted fluorescent bifunctional phage according to claim 1, characterized in that, The targeted fluorescent bifunctional phage is Escherichia coliphage M13-Nb20-Nluc, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025711.
3. A method for preparing the targeted fluorescent bifunctional phage as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Amplify the linear plasmid pComb3X using the vector_F and vector_R primer pairs and the plasmid pComb3X as templates; Step 2: Obtain the Nb20-pIII_C-Nluc_pVII fusion gene fragment through gene synthesis; Step 3: Using a seamless cloning kit, the linear plasmid pComb3X obtained in Step 1 was ligated with the Nb20-pIII_C-Nluc_pVII fusion gene fragment obtained in Step 2, transformed into E. coli ER2738, and positive transformants were screened by plating with ampicillin-resistant plates to obtain recombinant E. coli. Step 4: Inoculate the recombinant Escherichia coli obtained in Step 3 into LB liquid medium containing ampicillin, shake and culture, then add helper phage M13KO7 and kanamycin to the culture medium, and incubate overnight with shaking after standing in an incubator. Step 5: Centrifuge the culture obtained in Step 4 at high speed and transfer the supernatant after centrifugation to a new clean centrifuge tube; Step 6: Add PEG / NaCl solution to the supernatant from Step 5 and mix thoroughly. Step 7: Centrifuge the mixture from Step 6 at high speed, discard the supernatant after centrifugation, and fully suspend the recombinant phage precipitate using TBS. The suspension is the targeted fluorescent bifunctional phage.
4. The method according to claim 3, characterized in that, In step one, the vector_F primer sequence is shown in SEQ ID NO:1, and the vector_R primer sequence is shown in SEQ ID NO:2; SEQ ID NO: 1: 5'-CTAGTTGAACCTGCATggccgcctgggccacggtagcgaaacc-3'; SEQ ID NO: 2: 5'-GCTGGGGGTCAAAGATGAgctagctaattaatttaagcggccgc-3'.
5. The application of a targeted fluorescent bifunctional phage as described in claim 1 or 2 in the preparation of a rapid detection reagent for detecting Vibrio parahaemolyticus.