AviTag engineered bacteriophage, magnetic bacteriophage probe thereof, construction method and application

Abstract

The invention discloses sgRNA, an editing plasmid, a recombinant plasmid, an AviTag engineered bacteriophage, a magnetic bacteriophage probe, and a construction method and application thereof. The invention further discloses an enrichment separation and quantitative detection method for the pseudomonas aeruginosa. The magnetic bacteriophage probe can rapidly and specifically enrich pseudomonas aeruginosa in complex matrixes such as serum, infectious microbes and interference components are removed through magnetic separation, and sensitive quantitative detection of target bacteria is achieved in combination with an ATP bioluminescence system. According to the invention, by optimizing the AviTag-linker configuration, the display and biotinylation efficiency of the AviTag tag on the surface of the bacteriophage is remarkably improved, on the premise of keeping the titer of the bacteriophage and the stability of a host spectrum, the capture capability of the magnetic bacteriophage probe is enhanced, and low detection limit, short detection time and good repeatability are realized.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection of pathogenic microorganisms and bionanotechnology, specifically involving an AviTag engineered bacteriophage, its magnetic bacteriophage probe, construction method and application. Background Technology

[0002] *Pseudomonas aeruginosa* is an opportunistic pathogen widely found in both natural and hospital environments. It can cause a variety of clinical infections, including respiratory tract infections, urinary tract infections, wound infections, and bacteremia, and is a significant nosocomial pathogen in departments such as intensive care units and burn and plastic surgery departments. In recent years, *Pseudomonas aeruginosa* has shown increasing resistance to multiple antibiotics, especially carbapenems, and has become one of the multidrug-resistant bacteria of particular concern to the World Health Organization, posing a serious challenge to clinical treatment and infection control.

[0003] Existing laboratory detection methods for Pseudomonas aeruginosa mainly include traditional bacterial culture and identification, drug susceptibility testing, and molecular biology methods such as polymerase chain reaction (PCR) and real-time quantitative PCR. Although traditional culture methods have good specificity and reliability, the culture cycle usually requires 24–48 hours, which is difficult to meet the clinical needs of severe infections or rapid decision-making. Molecular detection methods have high requirements for instruments and personnel, and they mostly target bacterial nucleic acids, which have limited reflection of the number of viable bacteria, thus having certain limitations in scenarios such as efficacy monitoring and clearance assessment.

[0004] Bacteriophages are a class of viruses that specifically infect bacteria. They recognize specific receptors on the bacterial surface through tail filaments or tail threads, exhibiting high specificity for host bacteria. Bacteriophage-based detection technologies have received widespread attention in recent years. By combining bacteriophages with magnetic nanoparticles, fluorescent nanoparticles, or nucleic acid amplification systems, specific recognition, enrichment, and signal amplification of target bacteria can be achieved. However, existing bacteriophage probes still have the following problems: First, the connection between bacteriophages and nanomaterials often involves random covalent coupling or physical adsorption, resulting in uncontrollable orientation and potential partial obscuring of the recognition domain, thus reducing capture efficiency. Second, chemical modification of bacteriophages often fails to achieve site specificity, potentially affecting the structural stability and infectivity of the bacteriophage. Third, in complex matrices such as blood and serum, non-specific adsorption and matrix interference are significant, requiring further improvement in the enrichment efficiency and detection sensitivity of bacteriophage probes.

[0005] Therefore, it is necessary to provide an engineered strategy for controllable display of functional tags on the surface of bacteriophages, and on this basis, to construct magnetic bacteriophage probes for rapid and specific enrichment of Pseudomonas aeruginosa in complex samples, and to achieve quantitative detection by combining with a sensitive signal transduction system, so as to meet the needs of rapid detection in clinical and field settings. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide an sgRNA and an editing plasmid containing the sgRNA.

[0007] A second objective of this invention is to provide a recombinant plasmid and a method for constructing the same.

[0008] A third objective of this invention is to provide an AviTag engineered phage and a method for constructing it.

[0009] The fourth objective of this invention is to provide a magnetic phage probe and a method for constructing the same.

[0010] The fifth objective of this invention is to provide the application of the sgRNA, the edited plasmid, the recombinant plasmid, the AviTag engineered phage, and the magnetic phage probe in the enrichment, isolation, and / or quantitative detection of Pseudomonas aeruginosa.

[0011] The sixth objective of this invention is to provide a rapid enrichment, isolation, and sensitive detection method for Pseudomonas aeruginosa in complex samples, overcoming the problems of long detection cycles and difficulty in accurately reflecting the viable bacterial load in existing methods.

[0012] Technical solution: In order to achieve the above objectives, the present invention provides an sgRNA, the sequence of which is shown in SEQ ID NO: 1.

[0013] The present invention provides an editing plasmid, which is obtained by cloning the sgRNA into the pTCPLS plasmid.

[0014] The present invention provides a recombinant plasmid, which is obtained by cloning a recombinant fragment into the edit plasmid. The recombinant fragment sequentially includes the nucleotide sequence of the front homologous arm as shown in SEQ ID NO: 2, the nucleotide sequence encoding the linker peptide as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, the nucleotide sequence encoding the AviTag tag as shown in SEQ ID NO: 6, and the nucleotide sequence of the back homologous arm as shown in SEQ ID NO: 7.

[0015] The method for constructing recombinant plasmids according to the present invention includes the following steps:

[0016] 1) Linearization of pTCPLS plasmid with restriction endonuclease SapⅠ;

[0017] (2) The sgRNA fragment is ligated to the linearized pTCPLS fragment obtained in step (1) using DNA ligase to obtain the editing plasmid pTCPLS-sgRNA;

[0018] (3) Linearize the pTCPLS-sgRNA plasmid obtained in step (2) using restriction endonucleases XbaI and XhoI;

[0019] (4) The nucleotide sequence of the front homologous arm shown in SEQ ID NO:2, the nucleotide sequence encoding the linker peptide shown in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, the nucleotide sequence encoding the AviTag tag shown in SEQ ID NO:6, and the nucleotide sequence of the back homologous arm shown in SEQ ID NO:7 are linked together to obtain the front homologous arm-linker-AviTag-back homologous arm coding sequence;

[0020] (5) Use DNA ligase to ligate the coding sequence of the front homologous arm-linker-AviTag-back homologous arm obtained in step (4) to the linearized pTCPLS-sgRNA obtained in step (3) to obtain a recombinant plasmid.

[0021] Among them, four recombinant plasmids were obtained by displaying AviTag for three types of linkers and without linker: pTCPLS-AviTag; pTCPLS-G4S-AviTag; pTCPLS-EA3K-AviTag; and pTCPLS-G4SEA3K-AviTag.

[0022] This invention provides an AviTag engineered phage, which is obtained by introducing the recombinant plasmid into host bacteria and then infecting the host bacteria with the phage.

[0023] This invention provides a method for constructing AviTag engineered phage, wherein the method involves introducing the recombinant plasmid into host bacteria and then infecting the host bacteria with the phage.

[0024] This invention provides a magnetic phage probe, which is obtained by loading engineered phage onto magnetic nanoparticles. Preferably, the magnetic nanoparticles have a particle size of 200 nm to 1000 nm, and more preferably, the particle size is 1000 nm. Preferably, the number of phages loaded per microgram of magnetic particles reaches 10. 8 ~10 9 PFU.

[0025] This invention provides a method for constructing a magnetic phage probe. The method involves biotinylated phage by site-specific biotinylation of the engineered phage with BirA biotin ligase to obtain a biotinylated phage; then, the biotinylated phage is coupled to the surface of magnetic nanoparticles via biotin-streptavidin interaction to form a magnetic phage probe.

[0026] This invention provides the application of the sgRNA, the edited plasmid, the recombinant plasmid, the AviTag engineered phage, and the magnetic phage probe in the enrichment, isolation, and / or quantitative detection of Pseudomonas aeruginosa.

[0027] This invention provides a method for the enrichment, isolation, and quantitative detection of Pseudomonas aeruginosa, comprising the following steps:

[0028] (1) Take the sample to be tested and mix it with the magnetic phage probe to incubate it, so that the probe binds to Pseudomonas aeruginosa in the sample to form a bacterial-magnetic phage probe complex.

[0029] (2) Apply an external magnetic field to the incubation reaction system to separate the bacteria-magnetic phage probe complex, and wash it multiple times with washing buffer to remove unbound bacteria and matrix interference components, so as to obtain enriched Pseudomonas aeruginosa-magnetic phage probe complex.

[0030] (3) Add ATP detection premix to the enriched Pseudomonas aeruginosa-magnetic phage probe complex obtained in step (2) to cause bacterial lysis and release intracellular ATP. The ATP reacts with luciferin and oxygen under the catalysis of luciferase to generate a bioluminescent signal, and the intensity of the bioluminescent signal is detected.

[0031] (4) Substitute the intensity of the luminescent signal obtained in step (3) into the pre-established standard curve to calculate the number of Pseudomonas aeruginosa in the sample to be tested.

[0032] Preferably, in step (1), the incubation temperature of the sample to be tested and the magnetic phage probe is 20-30℃, preferably 25℃; the incubation time is 5-15 min, preferably 10 min.

[0033] Preferably, in step (2), the washing buffer is PBST buffer, specifically PBS + 0.05% Tween-20, and the number of washing cycles is 2 to 5, preferably 3.

[0034] Preferably, in step (3), the ATP detection premix includes bacterial lysate, luciferin and luciferase, and the incubation time is 20 to 60 min, preferably 30 min.

[0035] This invention discloses a method for the enrichment, isolation, and detection of *Pseudomonas aeruginosa*. The method utilizes the lysis phage PT62, which specifically recognizes *P. aeruginosa* PAO1, as the recognition unit. An AviTag tag is displayed by fusing a linker peptide to the C-terminus of the phage's capsid protein using CRISPR, resulting in an engineered phage EPT62 displaying the AviTag tag. After site-specific biotinylation catalyzed by BirA biotin ligase, the phage is coupled to magnetic particles modified with streptavidin to construct a magnetic phage probe MNP@EPT62. The sample to be tested is mixed with the magnetic phage probe and incubated for magnetic separation and enrichment. Subsequently, an ATP detection premix containing bacterial lysis buffer, luciferin, and luciferase is added, causing the enriched *P. aeruginosa* to lyse, releasing intracellular ATP and triggering a bioluminescent reaction. The quantitative analysis of the number of *P. aeruginosa* in the sample is achieved by detecting the intensity of the luminescence signal and combining it with a standard curve. This invention constructs an engineered phage displaying AviTag and combines it with streptavidin magnetic particles and an ATP bioluminescent system to achieve specific enrichment and quantitative detection of Pseudomonas aeruginosa, thereby shortening the detection time and improving detection sensitivity and specificity.

[0036] Furthermore, the phage PT62 is a lytic phage capable of specifically recognizing Pseudomonas aeruginosa, with the preferred recognized strain being Pseudomonas aeruginosa PAO1.

[0037] Furthermore, the nucleotide sequence of the AviTag is shown in SEQ ID NO: 6, and its amino acid sequence is shown in SEQ ID NO: 8.

[0038] Furthermore, the nucleotide sequence of the linker is shown in SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11.

[0039] Furthermore, the insertion site of the AviTag is before the C-terminal stop codon of the PT62 phage capsid protein. The AviTag tag is extended moderately from the capsid surface by a linker peptide, which facilitates recognition by BirA biotin ligase and site-specific biotinylation.

[0040] The preparation method of the above-mentioned magnetic phage probe MNP@EPT62 of the present invention specifically includes the following steps:

[0041] (1) Construction of engineered phage EPT62: Using the nucleotide sequence encoding the capsid protein in the phage PT62 genome as a template, a homologous arm primer for inserting the linker peptide-AviTag fusion fragment before its C-terminal stop codon was designed. The linker peptide-AviTag fusion fragment was inserted into the C-terminus of the capsid protein by CRISPR-Cas9 gene editing technology to obtain engineered phage EPT62 that stably displays the AviTag tag; further, the nucleotide sequence of the capsid protein is shown in SEQ ID NO: 12, and the amino acid sequence is shown in SEQ ID NO: 13.

[0042] (2) Biotinylation of engineered phage EPT62: EPT62 was added to a reaction system containing BirA biotin ligase, ATP and biotin, and incubated at 30°C for 60 min to induce site-specific biotinylation of AviTag lysine residues, thus obtaining biotinylated phage.

[0043] (3) Preparation of magnetic phage probe: Biotinylated phage is mixed with magnetic particles modified with streptavidin and incubated at 25°C for 30 min. The phage is oriented and fixed on the surface of the magnetic particles by the ultra-high affinity between biotin and streptavidin. Unbound phage is removed by magnetic separation and washing to obtain a high-density loaded magnetic phage probe.

[0044] Furthermore, the magnetic particles have a particle size of 1000 nm; the ratio of engineered phage EPT62 to magnetic particles is 10000:1, in order to obtain a magnetic phage probe that has both high loading capacity and good dispersibility.

[0045] The method for enrichment, isolation, and detection of Pseudomonas aeruginosa according to the present invention includes the following steps:

[0046] (1) The sample to be tested is mixed with the magnetic phage probe and incubated to allow the probe to specifically bind to Pseudomonas aeruginosa in the sample to form a bacterial-magnetic phage probe complex.

[0047] (2) Apply an external magnetic field to perform magnetic separation of the bacterial-magnetic phage probe complex, and wash with PBST buffer to remove unbound bacteria and interfering components to obtain enriched Pseudomonas aeruginosa;

[0048] (3) Add ATP detection premix to the enriched Pseudomonas aeruginosa, the premix comprising bacterial lysate, luciferin and luciferase, to cause bacterial lysis and release of intracellular ATP, which reacts with luciferin and oxygen under the catalysis of luciferase to produce bioluminescence.

[0049] (4) Collect the magnetic beads again by magnetic separation and detect the intensity of the luminescence signal in the supernatant;

[0050] (5) The number of Pseudomonas aeruginosa in the sample to be tested is calculated based on the intensity of the luminescent signal and the pre-established standard curve.

[0051] Furthermore, in step (1), the incubation temperature of the sample and the magnetic phage probe is 25°C and the incubation time is 10 min; in step (2), the PBST buffer is washed 3 times.

[0052] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention uses engineered bacteriophage EPT62 to specifically modify the phage capsid protein, avoiding the adverse effects of traditional random chemical modifications on phage structure and infectivity, and ensuring the stability of phage titer and host spectrum; by using a biotin-streptavidin system to densely and directionally immobilize biotinylated phages on the surface of magnetic particles, constructing a magnetic phage probe, significantly improving the capture efficiency and binding stability of *Pseudomonas aeruginosa* in complex samples; magnetic separation effectively removes most matrix interference, and combined with the ATP bioluminescence detection system, converts the number of viable bacteria into a sensitive and quantifiable luminescent signal, enabling rapid quantitative detection of *Pseudomonas aeruginosa* in samples, characterized by a low detection limit, wide linear range, and good repeatability; Furthermore, the AviTag-biotin-streptavidin module used in this invention has good versatility; simply changing the phage targeting different pathogens allows for rapid detection of other bacteria, demonstrating good platformization and application prospects. Attached Figure Description

[0053] Figure 1 This is a schematic diagram illustrating the structure of the Capsid-AviTag fusion protein, predicted using AlphaFold 3 and inserted before the C-terminal stop codon of the phage PT62 capsid protein, as described in an embodiment of the present invention. The figure shows the three-dimensional conformations of the Capsid-AviTag fusion protein under conditions without a linker and with different linker peptides: G4S, EA3K, and G4SEA3K.

[0054] Figure 2 This diagram illustrates the construction and identification of CRISPR-Cas9 knockout / edit plasmids according to an embodiment of the present invention. M: 2000bp molecular weight standard; 1: pTCPLS empty vector; 2: pTCPLS-AviTag; 3: pTCPLS-G4S-AviTag; 4: pTCPLS-EA3K-AviTag; 5: pTCPLS-G4SEA3K-AviTag.

[0055] Figure 3This is a verification diagram of the phage gene modification described in the embodiments of the present invention. M: 2000 bp molecular weight standard; 1: PT62; 2: PT62-AviTag; 3: PT62-G4S-AviTag; 4: PT62-EA3K-AviTag; 5: PT62-G4SEA3K-AviTag.

[0056] Figure 4 This is a transmission electron microscope (TEM) image of the phage layer purified by CsCl density gradient centrifugation according to an embodiment of the present invention. The left image shows the location of the phage aggregation band in the CsCl gradient centrifugation tube; the right image shows the morphological structure of the phage observed under a TEM after the corresponding bands were collected.

[0057] Figure 5 The image shows the titer detection results of PT62, PT62-AviTag, PT62-G4S-AviTag, PT62-EA3K-AviTag, and PT62-G4SEA3K-AviTag described in the embodiments of the present invention.

[0058] Figure 6 This is a verification diagram of the biotinylation effect of bacteriophages according to an embodiment of the present invention. (A) Bacteriophage SDS-PAGE electrophoresis image, showing the bands of each bacteriophage capsid protein; (B) Corresponding Western blot image; (C) Comparative analysis of the gray values ​​of biotinylated bands of different engineered bacteriophages, using the gray value of the biotinylated band of PT62-G4SEA3K-AviTag as the normalization benchmark (denoted as 1), and comparing the ratios of the gray values ​​of the other bacteriophage bands divided by the gray value of PT62-G4SEA3K-AviTag; (D) Comparison of the gray values ​​of the biotinylated Western blot bands of PT62-G4SEA3K-AviTag and pure protein Capsid-AviTag, using the gray value of the biotinylated band of Capsid-AviTag as the normalization benchmark (denoted as 1).

[0059] Figure 7 The images show transmission electron microscopy (TEM) images of the magnetic phage probe MNP@EPT62 described in this embodiment of the invention. (A) shows the binding morphology of magnetic beads with a particle size of approximately 200 nm with phage PT62-G4SEA3K-AviTag; (B) shows the binding morphology of magnetic beads with a particle size of approximately 1000 nm with phage PT62-G4SEA3K-AviTag.

[0060] Figure 8 This is a hydration particle size analysis diagram of PT62-G4SEA3K-AviTag combined with magnetic beads according to an embodiment of the present invention.

[0061] Figure 9This is a quantitative analysis diagram of the binding of PT62-G4SEA3K-AviTag to magnetic beads as described in this embodiment of the invention. The number of phages in the supernatant before and after coupling was quantitatively determined by qPCR, and the phage load and coupling efficiency per unit mass of magnetic beads were calculated.

[0062] Figure 10 This is a transmission electron microscope image of MNP@EPT62 during the enrichment of Pseudomonas aeruginosa PAO1 as described in an embodiment of the present invention.

[0063] Figure 11 This is a verification diagram of the magnetic separation specificity of MNP@EPT62 against Pseudomonas aeruginosa PAO1 described in the embodiments of the present invention.

[0064] Figure 12 This is a graph showing the changes in luminescence signal values ​​of the ATP detection mixture and Pseudomonas aeruginosa PAO1 under different incubation times according to an embodiment of the present invention.

[0065] Figure 13 This is a standard curve for ATP bioluminescence detection as described in this embodiment of the invention. Using different initial concentrations of Pseudomonas aeruginosa PAO1 as the detection target, a linear relationship between bacterial concentration (CFU) and relative luminescence intensity (RLU) was plotted.

[0066] Figure 14 The image shows the detection results of simulated clinical bacterial samples and composite bacterial samples as described in the embodiments of the present invention. Detailed Implementation

[0067] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0068] Those skilled in the art should understand that these embodiments are for illustrative purposes only and are not intended to limit the invention; any equivalent substitutions or modifications made without departing from the spirit and substance of the invention fall within the scope of protection of the invention. Unless otherwise stated, the reagents and consumables used in the embodiments of the present invention are all conventional products in the art and are sourced from commercial channels; the buffer solutions used, unless otherwise specified, are all conventionally prepared PBS or PBST (PBS + 0.05% Tween-20) with a pH of 7.2 to 7.4; the temperature unit is degrees Celsius (°C), the volume unit is mL or μL, and the concentration unit is mol / L, PFU / mL, or CFU / mL.

[0069] Example 1: Construction of engineered bacteriophage EPT62

[0070] This embodiment provides a method for constructing engineered phages that display an AviTag tag at the C-terminus of the PT62 capsid protein, illustrating the process of obtaining engineered phages EPT62 (including PT62-AviTag, PT62-G4S-AviTag, PT62-EA3K-AviTag, and PT62-G4SEA3K-AviTag) in this invention.

[0071] 1. Determination of insertion site and sgRNA design

[0072] A lytic phage, PT62 (NCBI, GenBank accession number: PX674086), capable of specifically recognizing P. aeruginosa PAO1 (Biofeng (http: / / www.biofeng.com / ), strain ATCC 15692), was selected. The nucleotide sequence encoding the capsid protein in its genome is shown in SEQ ID NO: 12. An insertion site was determined before the C-terminal stop codon of the capsid protein coding sequence as the embedding site for the linker-AviTag fusion fragment.

[0073] Using the Benchling online tool (https: / / benchling.com / ), an sgRNA target sequence (shown in SEQ ID NO: 1) was designed near the above insertion site, and sgRNA sequence primers F1 and R1 were designed and synthesized accordingly (primer sequences are shown in Table 1). SEQ ID NO: 1: 5'-cggggtttcgtggcatcacg-3'.

[0074] Table 1 Primers of the present invention

[0075] The double-stranded sgRNA sequences were obtained by annealing the F1 and R1 primers. The annealing system consisted of 10 μL (100 μM) of F1 primer, 10 μL (100 μM) of R1 primer, and 40 μL of DEPC water. The annealing program was as follows: 95℃ for 5 min, followed by incubation at 80℃, 65℃, 50℃, 35℃, 25℃, and 16℃ for 10 min each. After annealing, the samples were stored at -20℃ for later use.

[0076] Meanwhile, to optimize the display configuration of AviTag, AlphaFold 3 (https: / / alphafold.com / ) was used to predict the three-dimensional conformation of the Capsid-AviTag fusion protein after inserting AviTag into the C-terminus of the PT62 capsid protein with different linker peptides (G4S, EA3K, and G4SEA3K) or without linker peptides, providing structural basis for subsequent screening of linker peptide sequences (see [link to AlphaFold 3]). Figure 1Predicted structures indicate that the linker peptides enable the AviTag module to extend moderately from the surface of the capsid protein, increasing its exposure, but there may be folding and hiding phenomena. The G4SEA3K linker peptide provides the optimal spatial extension effect.

[0077] 2. Construction of CRISPR-Cas9 editing vector

[0078] The pTCPLS plasmid (pTCPLS plasmid is derived from the article He L, Liu L, Zhou X, Hu Z, Shen J. Visual Counting of Influenza A Viruses with Magnetic T4 Phage SPR Probe. ACS Sens. 2025 Apr 25;10(4):2928-2937. doi: 10.1021 / acssensors.4c03670. Epub 2025 Mar 27. PMID:40150976.) was linearized using the restriction endonuclease SapⅠ (New England Biolabs, catalog number: R0569S). The digestion system consisted of 1 μg pTCPLS plasmid, 1 μL SapⅠ enzyme, 5 μL 10×rCutSmart buffer, and DEPC water to a final volume of 50 μL. The reaction was carried out at 37℃ for 60 min. The digestion product was linearized by agarose gel electrophoresis and then purified for later use.

[0079] The annealed double-stranded sgRNA fragment was ligated to the linearized pTCPLS plasmid using T4 DNA ligase (Takara, catalog number: 2011A), and the ligation product was transformed into E. coli DH5α competent cells. After antibiotic selection and sequencing, the edited plasmid pTCPLS-sgRNA carrying the sgRNA was obtained.

[0080] Subsequently, the pTCPLS-sgRNA plasmid was linearized by double digestion with restriction endonucleases XbaI (New England Biolabs, catalog number: R0145V) and XhoI (New England Biolabs, catalog number: R0146V). The digestion system consisted of 1 μg pTCPLS-sgRNA plasmid, 1 μL BamHI enzyme, 1 μL HindIII enzyme, 5 μL 10×rCutSmart buffer, and DEPC water to a final volume of 50 μL. The reaction was carried out at 37°C for 60 min. The digestion products were confirmed to be linearized by agarose gel electrophoresis and then purified for later use.

[0081] 3. Homologous arm and donor DNA design

[0082] Homologous arms of approximately 500–1000 bp were designed upstream and downstream of the phage PT62 genome insertion site. The linker to be screened (SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5) and the coding sequence of the AviTag tag (SEQ ID NO: 6) were placed between the two homologous arms to construct the donor homologous arm DNA fragment. The nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO: 2, and the sequence of the downstream homologous arm is shown in SEQ ID NO: 7. Using primers F2 and R2 containing the upstream and downstream homologous arms, and primers F3-1–F3-4 and R3-1–R3-4 containing the linker-AviTag coding sequence (primer sequences are shown in Table 1), PCR amplification was performed using the PT62 genome as a template to obtain donor homologous arm DNA with the complete insert fragment (upstream homologous arm-linker-AviTag-downstream homologous arm). The PCR product was separated by agarose gel electrophoresis and purified for later use. Primers F2 and R3-1, and primers F3-1 and R2 are used to amplify insert fragments without a linker; primers F2 and R3-2 are used to amplify insert fragments containing the G4S linker (GGGGS); primers F2 and R3-3, and primers F3-3 and R2 are used to amplify insert fragments containing the EA3K linker (EAAAK); primers F2 and R3-4, and primers F3-4 and R2 are used to amplify insert fragments containing the G4SEA3K complex linker (GGGGSEAAAK).

[0083] For example, when amplifying a gene insert encoding the G4SEA3K complex linker peptide, PCR amplification using primers F2 and R3-4 yields amplified fragment 1, and PCR amplification using primers F3-4 and R2 yields amplified fragment 2. Then, using amplified fragment 1 and amplified fragment 2 (in a 1:1 volume ratio) as templates, primers F2 and R2 are used to amplify the gene insert. The PCR amplification system is shown in Table 2. The amplification steps and systems for the remaining inserts are the same. Finally, four complete inserts are obtained (pre-homologous arm - linker - AviTag - post-homologous arm).

[0084] Table 2. Preparation of a 50 μL high-fidelity enzyme PCR system

[0085] PCR reaction program: 95℃ for 5 min; 95℃ for 15 s, 56℃ for 15 s, 72℃ for 30-60 sec / kb, for a total of 35 cycles; 72℃ for 5 min.

[0086] The four amplified complete insert fragments (pre-homologous arm-linker-AviTag-post-homologous arm) obtained above were ligated to the linearized pTCPLS-sgRNA plasmid using T4 DNA ligase (Takara, catalog number 2011A). The ligation products were transformed into DH5α competent cells, and the cells were screened for resistance and sequenced to obtain the editing plasmid pTCPLS-PT62 carrying the PT62 homologous arm and the linker-AviTag insert fragment (a total of four plasmids were obtained for the three linkers and for the non-linker AviTag display: pTCPLS-AviTag; pTCPLS-G4S-AviTag; pTCPLS-EA3K-AviTag; pTCPLS-G4SEA3K-AviTag). PCR amplification with primers F4 and R4 was performed. Figure 2 This plasmid can specifically target the region corresponding to wild-type phage PT62, and achieve site-specific insertion modification of the C-terminus of the PT62 capsid protein through CRISPR-Cas9-mediated double-strand cleavage and homologous recombination.

[0087] 4. Editing plasmids and introducing them into the host bacteria

[0088] The four constructed editing plasmids pTCPLS-PT62 were transformed into the phage PT62 host bacteriophage Pseudomonas aeruginosa PAO1 (strain ATCC 15692). Gentamicin resistance markers were used for screening to obtain four stable host strains carrying the CRISPR-Cas9 editing system and donor homologous arms. These host strains were used for subsequent PT62 phage infection and the acquisition of engineered phage EPT62.

[0089] 5. Phage infection and acquisition of recombinants

[0090] Take 10 wild-type PT62 phages 6 PFU was used to infect PAO1 host bacteria carrying the four editing plasmids pTCPLS-PT62, and the cultures were incubated at 37°C and 220 rpm for 6–8 h. Bacterial debris was removed by centrifugation at 8000 g for 10 min, and the supernatant containing the phages was collected. Phage plaques were prepared using the double-layer plate method, and individual plaques were picked. PCR amplification was performed using the phages as templates and the conserved sequences flanking the insertion region as primers F5 and R5 (primer sequences are shown in Table 1, and the amplification system and procedure are shown in step 3). The PCR products were analyzed by agarose gel electrophoresis (see [link to analysis]). Figure 3The amplified band lengths of different engineered phages were consistent with expectations. Sequencing of the amplified products confirmed that the linker peptide-AviTag fusion fragment was correctly inserted before the C-terminal stop codon of the PT62 capsid protein, and that the reading frame was correct. Positive clones were identified as engineered phage EPT62 (four engineered phages were obtained by displaying AviTag with three linkers and without a linker: PT62-AviTag; PT62-G4S-AviTag; PT62-EA3K-AviTag; and PT62-G4SEA3K-AviTag).

[0091] 6. Phage amplification and purification

[0092] Four engineered bacteriophages, EPT62, validated by sequencing, were selected and amplified using fresh logarithmic growth phase PAO1 as the host bacteria. The bacteriophages were added to the host bacterial suspension (OD) at an MOI of 0.0001. 600 =0.6), and cultured at 37℃ and 220 rpm for 6–8 h. Bacterial debris was removed by centrifugation at 8000 g for 10 min, and the supernatant was collected. Concentration and purification were performed using ultracentrifugation (28000 rpm, 2 h) and CsCl density gradient centrifugation (180000 g, 1 h) to obtain a high-titer engineered phage stock solution (see...). Figure 4 The morphological characteristics of bacteriophages were observed using transmission electron microscopy (see...). Figure 4 The titer of EPT62 was determined using the double-layer plate method, and its host profile was compared with that of wild-type PT62. The results showed that the infectivity of EPT62 against Pseudomonas aeruginosa PAO1 was not significantly reduced, and the titer could reach 10. 13 PFU / mL (see Figure 5 ).

[0093] Example 2: Preparation of magnetic phage probes

[0094] This embodiment provides a specific method for preparing the magnetic phage probe of the present invention.

[0095] 1. Biotinylation of engineered bacteriophages

[0096] Four engineered EPT62 stock solutions obtained in Example 1 were used for biotin modification using an AviTag-tagged protein biotin labeling kit (BirA method) (Beyotime, catalog number: P0630S). 1×10⁴ units of each of the four engineered EPT62 stock solutions were added to the reaction system. 11Add PFU, 1 μL of BirA, 10 μL of Biotin Ligase Buffer A, and 10 μL of Biotin Ligase Buffer B to a final volume of 100 μL. Incubate at 30°C for 30 min to induce site-specific biotinylation of the lysine residues on AviTag, resulting in biotinylated phages: PT62-AviTag@Bio, PT62-G4S-AviTag@Bio, PT62-EA3K-AviTag@Bio, and PT62-G4SEA3K-AviTag@Bio.

[0097] After the reaction, excess free biotin and enzymes were removed from the system using a dialysis bag to obtain four purified biotinylated phages, EPT62@Bio. Wild-type PT62 phage was simultaneously biotinylated as a control using the same method.

[0098] Biotin labeling was detected by Western blot using streptavidin-HRP as a probe, and a specific band (approximately 40 kDa) of EPT62@Bio was observed. Figure 6 Under the premise of basically consistent phage capsid protein concentration, the biotinylation effect was evaluated by comparing the gray intensity of the biotinylation-specific bands in each group, thereby determining the display efficiency of AviTag. The results showed that the engineered phage PT62-G4SEA3K-AviTag, which used the G4SEA3K linker peptide to display AviTag, had the strongest biotinylation signal. Its band gray intensity was basically equivalent to that of the biotinylated band of pure protein Capsid-AviTag. Therefore, subsequent experiments were all conducted using PT62-G4SEA3K-AviTag phage.

[0099] 2. Pretreatment of streptavidin magnetic particles

[0100] Magnetic nanoparticles SA-MNP with surface-modified streptavidin and particle sizes of 200 nm (Nanjing Dongna Biotechnology Co., Ltd., catalog number: Mag9101) and 1000 nm (Nanjing Dongna Biotechnology Co., Ltd., catalog number: MB1058) were selected. The magnetic nanoparticles were resuspended in 1×PBS buffer (pH 7.4), gently agitated to homogenize, and then separated on a magnetic rack. The supernatant was discarded. The washing process was repeated 2–3 times to remove preservatives and storage solutions. Finally, the nanoparticles were resuspended in PBS at a concentration of 10 mg / mL for later use.

[0101] 3. Coupling of biotinylated bacteriophages with magnetic particles

[0102] 1×10 11PFU-biotinylated phage PT62-G4SEA3K-AviTag-Bio was added to 10 μL of streptavidin magnetic particle suspension (concentration 10 mg / mL). After thorough mixing, the mixture was slowly incubated at 25°C with rotation for 60 min to allow for stable coupling between biotin and streptavidin through their high affinity. After incubation, the mixture was separated on a magnetic rack, and the supernatant containing unbound phage was discarded. The phage was gently resuspended in PBST buffer (PBS + 0.05% Tween-20, pH 7.4) and washed three times (500 μL each time) to remove non-specifically bound phage. Finally, the phage was resuspended in an appropriate amount of PBS to obtain the magnetic phage probe MNP@EPT62.

[0103] 4. Characterization of magnetic phage probes

[0104] (1) Morphological observation: A small amount of MNP@EPT62 was dropped onto a copper mesh carrier. After negative staining, it was observed under a transmission electron microscope. A large number of phage particles were seen on the surface of the magnetic particles, indicating that the phages were successfully loaded onto the surface of the magnetic particles. Figure 7 ). Figure 7 In this context, A represents the binding morphology of magnetic beads with a particle size of approximately 200 nm to bacteriophage PT62-G4SEA3K-AviTag; Figure 7 In the figure, B represents the binding morphology of magnetic beads with a particle size of approximately 1000 nm with bacteriophage PT62-G4SEA3K-AviTag. Since 1000 nm magnetic beads can load more bacteriophages, magnetic beads with a particle size of 1000 nm were used in subsequent experiments.

[0105] (2) Particle size determination: The hydrated particle size of MNP@EPT62 was determined using a Malvern particle size analyzer. Compared with bare magnetic particles, MNP was slightly larger, and the particle size distribution was relatively concentrated, indicating good probe dispersion. Figure 8 ).

[0106] (3) Loading capacity and coupling efficiency: To quantitatively evaluate the coupling effect between biotinylated engineered phages and streptavidin magnetic particles, qPCR was used to analyze the number of phages before and after the coupling reaction. The primers used were F6 and R6 (primer sequences are shown in Table 1), and the amplification system is shown in Table 3. Using unmodified phage PT62 as a control, the initial amount of phage in the system before coupling (Total), the number of unbound phages in the supernatant after the coupling reaction (Supernatant), and the number of phages loaded on the surface of the magnetic particles (MNP-bound) were detected, and the coupling efficiency was calculated accordingly. The results showed that ( Figure 9After three PBST washes, the number of residual phages in the third wash was reduced by several orders of magnitude compared to the initial amount, indicating that the non-specifically bound phages had been sufficiently diluted and their residual amount would have virtually no impact on the experimental results. Under typical conditions, MNP@EPT62 can achieve a loading of 10 phages per microgram of magnetic particles. 8 ~10 9 PFU-grade phages with a coupling efficiency greater than 90%.

[0107] Table 3 shows the preparation system for real-time quantitative PCR with a total volume of 20 μL.

[0108] qPCR reaction program: 95℃ for 5 min; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles.

[0109] Example 3: Enrichment and isolation of Pseudomonas aeruginosa PAO1

[0110] This embodiment is used to verify the enrichment ability and separation effect of the magnetic phage probe MNP@EPT62 of the present invention on Pseudomonas aeruginosa PAO1.

[0111] 1. Steps for enriching magnetic phage probes

[0112] Take 500 μL of the test sample containing Pseudomonas aeruginosa PAO1, and add 10 μL of pre-prepared MNP@EPT62 probe suspension (containing EPT62 10 μL). 10 After gently mixing with PFU, incubate at 25°C for 10 min. During incubation, the reaction tube can be placed on a rotary mixer and gently shaken to promote full contact and binding between the magnetic phage probe and Pseudomonas aeruginosa in the sample, forming a bacteria-magnetic phage probe complex.

[0113] After incubation, place the reaction system on a magnetic rack and let it stand for 1–2 minutes until the magnetic beads are completely adsorbed onto the tube wall. Carefully discard the supernatant. Add 500 μL of PBST buffer and gently pipette to resuspend the magnetic beads. Place the mixture on the magnetic rack again for separation and discard the supernatant. Repeat the above washing steps three times to thoroughly remove unbound bacteria and components in the sample matrix that may interfere with subsequent detection, ultimately obtaining the enriched *Pseudomonas aeruginosa*–magnetic phage probe complex. Figure 10 ).

[0114] 2. Evaluation of the binding characteristics and separation efficiency of magnetic phage probes to PAO1

[0115] To evaluate the enrichment performance of MNP@EPT62 on Pseudomonas aeruginosa PAO1, standard bacterial suspensions of PAO1 at different initial concentrations (10... 2 103 10 4 10 5 10 6 10 7 10 8 Add an equal volume of MNP@EPT62 probe suspension to the bacterial suspension as described above, incubate at 25°C for 10 min, and then perform magnetic separation and washing. Take the original bacterial suspension before incubation and the supernatant after magnetic separation for qPCR quantification. The primers used are F7 and R7 (primer sequences are shown in Table 1). The amplification system and conditions are as described in Example 2. Measure the original bacterial concentration (C0) and the concentration of the supernatant after magnetic separation (C5). sup The capture rate is calculated using the following formula: Capture rate (%) = (C0 - C) sup ) / C0× 100%.

[0116] Simultaneously, the enriched bacterial-magnetic phage probe complex was quantified by qPCR to assess the actual recovery and reproducibility. The results showed that MNP@EPT62 had a capture rate of over 90% for PAO1 (Table 4).

[0117] Table 4 Capture Rate

[0118] In addition, 10 3 CFU's PAO1 was mixed with equal amounts of non-target bacteria such as Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, Salmonella, and Vibrio parahaemolyticus, and enriched under the same conditions. The changes in the number of different bacterial species before and after enrichment were compared. The results showed that MNP@EPT62 had virtually no specific adsorption for non-target bacteria, indicating that the magnetic phage probe of this invention has good specificity for Pseudomonas aeruginosa. Figure 11 ).

[0119] Example 4: Establishment of a quantitative method for ATP bioluminescence detection

[0120] This embodiment provides a method for quantitative detection of enriched Pseudomonas aeruginosa based on ATP bioluminescence reaction, which is used to establish the integrated detection system of "magnetic bacteriophage enrichment + ATP bioluminescence" of this invention.

[0121] 1. Establishment of ATP detection method

[0122] Referring to the instructions for the commercially available ATP bioluminescence detection kit (Beyotime, catalog number: C0052S), the reaction system was optimized for the application scenario of this invention. The signal value of pure bacterial samples reached a plateau 20 minutes after the addition of the ATP detection premix, and began to decay after 60 minutes at room temperature. Figure 12Therefore, we set the subsequent pyrolysis time to 30 min and the reaction temperature to room temperature / 25℃.

[0123] 2. Drawing the standard curve

[0124] Take a standard bacterial suspension containing a known concentration of Pseudomonas aeruginosa PAO1 (10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 CFU was prepared into a series of dilutions with 1×PBS (pH 7.4) at different concentrations, and 100 μL was added to each well of the reaction plate; PBS was used as a blank control. 100 μL of ATP detection premix (Beyotime, catalog number: C0052L) was added to each well. After gently mixing, the mixture was incubated at room temperature for 30 min to allow the bacteria to lyse and release intracellular ATP. Under the catalysis of luciferase, ATP reacted with luciferin and oxygen to generate excited-state oxidized luciferin, producing a visible light signal.

[0125] The RLU value of each well was measured using a luminescence detector, and a standard curve was plotted with the initial bacterial count (CFU) on the x-axis and the corresponding RLU on the y-axis. Experimental results showed that at 10... 3 ~10 8 Within the CFU range, the luminescence intensity showed a good linear relationship with the concentration of *Pseudomonas aeruginosa*. Figure 13 When the bacterial count reaches 10... 3 For CFU / well, a positive value of >2.1 is considered a positive result, and the bacterial count is considered positive when it reaches 10⁻⁶. 2 The detection value of CFU / bacteria per well can reach 1.5 times that of the blank control, and it is still identified as a positive result (Table 5). Therefore, the detection limit of this invention is 1 CFU / μL.

[0126] Table 5 ATP Detection Sensitivity

[0127] 3. Detection of pooled samples and simulated clinical samples

[0128] To evaluate the applicability and specificity of the method of this invention in complex samples, 10 3 CFU-containing Pseudomonas aeruginosa PAO1 was mixed with equal volumes of Klebsiella pneumoniae KP72, Escherichia coli BL21, Acinetobacter baumannii Aba-3001, Salmonella C5004, and Vibrio parahaemolyticus VP17802 preserved in a laboratory bacterial bank to prepare a mixed bacterial suspension (Mix sample); simultaneously, 10 3CFU of Pseudomonas aeruginosa PAO1 was added to PBS (PBS sample), serum sample, urine sample, and sputum sample to prepare simulated clinical samples.

[0129] The above-mentioned mixed samples and simulated clinical samples were incubated with the MNP@EPT62 probe and subjected to magnetic separation and washing under the conditions of Example 3 to enrich the *Pseudomonas aeruginosa*-magnetic phage probe complex; subsequently, ATP detection premixed solution was added for incubation, and the luminescence signal intensity was measured. The detection results showed that the method of this invention can achieve efficient separation and detection of *Pseudomonas aeruginosa* PAO1 in serum, urine, sputum, and mixed samples. Figure 14 Compared with traditional plate counting methods, the method of this invention significantly shortens the overall detection time, and can complete the entire process from enrichment to quantitative detection within 50 minutes. It is suitable for rapid quantitative detection and screening of Pseudomonas aeruginosa in complex samples.

[0130] SEQ ID NO: 1

[0131] cggggtttcgtggcatcacg

[0132] SEQ ID NO: 2

[0133] ccgacaagatcgtccgcatgcaccgccgcgtagtcgagaccttcatcgaccgcgacctgggcgatgcggtctactccgagggcctgaccccgatgtcgccgcgtgtgttcagcctgctgctggagcacgacaagctgatgaacgtcgagtaccaggcaaccggcgcgaccaacgactacgtgaagtcccgcgtggccatcctcaacggcgtcaaggtgctggagactccgcgcttcgccaccaaggcaatcgcagcccacccgctgggccgtcacttcaacgtgagcgccgaggagtccgagcgccagatcgccctgttcctcccgagcaagaccctgatcaccgcccaagtggcgccggtccaggccaagctgtgggaagacaacgagaaattctcgtgggtcctggataccttccagatgtacaacatcggtgcccgtcgtccggacaccgctggtgccatcgaactgaagggtatcggcgccttcgacatcaccgcg

[0134] SEQ ID NO:3

[0135] G4S:GGTGGTGGTGGTTCT

[0136] SEQ ID NO:4

[0137] EA3K:GAAGCAGCCGCTAAA

[0138] SEQ ID NO:5

[0139] G4SEA3K:GGTGGTGGCGGCTCTGAAGCTGCCGCTAAA

[0140] SEQ ID NO:6

[0141] GGCCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAA

[0142] SEQ ID NO:7

[0143] Tgacgaaaccccgcacttcggtgtggggtttcttcaaagcctaacgacccgcgcagattccctgcgtgggtttttgcgctttaggagaaaccctatgctactactcgacgcagtgaatgtcatcctgcgcaagatcggcgagctgccaatcccgagcatggatgagacgtatccaaccatggccatcgccctcccggagctggaagatcaacgcatccagttgctgacccaaggctggtggttcaacacctggtggaagcacaagctgacacctgatcccacgggccgcatcaacctgcccaagggcaccttggcattctacccggattccccggacctccagtgggacggcctgggagtgcgagatgccaacaccggcgacgaccgcatcggtaagtcggtcgagggccgattggtgctgtctcgggagtgggaccatatcccggagatcgcacagcgcgtcattgcgcaccaggctgcgctcgcggtatacactcacgagattggaccggac

[0144] SEQ ID NO:8

[0145] GLNDIFEAQKIEWHE

[0146] SEQ ID NO:9

[0147] G4S:GGGGS

[0148] SEQ ID NO:10

[0149] EA3K:EAAAK

[0150] SEQ ID NO:11

[0151] G4SEA3K:GGGGSEAAAK

[0152] SEQ ID NO:12

[0153]

[0154] SEQ ID NO:13

[0155] MSFLNDLTRPNYAGKNADVDIHLEEHLGIVDKHFAYTSKFAPLMNIRDLRGSNVVRLDRLGNVEAKGRRAGEELERSRVVNDKWNLTVDTLLYLRHQFDHQDEWTQSFDMRKEVAELDGQELARKFDQACLIQVIKAAAMDAPVDLEDAFSPGVLEKLDLTGLTAKQAADKIVRMHRRVVETFIDRDLGDAVYSEGLTPMSPRVFSLLLEHDKLMNVEYQATGATNDYVKSRVAILNGVKVLETPRFATKAIAAHPLGRHFNVSAEESERQIALFLPSKTLITAQVAPVQAKLWEDNEKFSWVLDTFQMYNIGARRPDTAGAIELKGIGAFDITA。

Claims

1. An sgRNA, characterized in that, The sequence of the sgRNA is shown in SEQ ID NO:

1.

2. An editing plasmid, characterized in that, The editing plasmid is obtained by cloning the sgRNA described in claim 1 into the pTCPLS plasmid.

3. A recombinant plasmid, characterized in that, The recombinant plasmid is obtained by introducing the recombinant fragment into the edit plasmid of claim 2. The recombinant fragment sequentially includes the nucleotide sequence of the front homologous arm as shown in SEQ ID NO: 2, the nucleotide sequence encoding the linker peptide as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, the nucleotide sequence encoding the AviTag tag as shown in SEQ ID NO: 6, and the nucleotide sequence of the back homologous arm as shown in SEQ ID NO:

7.

4. The method for constructing the recombinant plasmid according to claim 3, characterized in that, Includes the following steps: (1) Linearization of pTCPLS plasmid by restriction endonuclease SapⅠ; (2) The sgRNA fragment described in claim 1 is ligated to the linearized pTCPLS fragment obtained in step (1) using DNA ligase to obtain the editing plasmid pTCPLS-sgRNA; (3) Linearize the pTCPLS-sgRNA plasmid obtained in step (2) using restriction endonucleases XbaI and XhoI; (4) The nucleotide sequence of the front homologous arm shown in SEQ ID NO:2, the nucleotide sequence encoding the linker peptide shown in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, the nucleotide sequence encoding the AviTag tag shown in SEQ ID NO:6, and the nucleotide sequence of the back homologous arm shown in SEQ ID NO:7 are linked together to obtain the front homologous arm-linker-AviTag-back homologous arm coding sequence; (5) Use DNA ligase to ligate the coding sequence of the front homologous arm-linker-AviTag-back homologous arm obtained in step (4) to the linearized pTCPLS-sgRNA fragment obtained in step (3) to obtain a recombinant plasmid.

5. An AviTag engineered phage, characterized in that, The engineered phage is obtained by introducing the recombinant plasmid described in claim 3 into host bacteria and then infecting the host bacteria with the phage.

6. A method for constructing AviTag engineered phage, characterized in that, The method for constructing the engineered bacteriophage involves introducing the recombinant plasmid described in claim 3 into host bacteria, and then infecting the host bacteria with the bacteriophage.

7. A magnetic phage probe, characterized in that, The magnetic phage probe is obtained by loading the engineered phage of claim 5 onto magnetic nanoparticles. Preferably, the magnetic nanoparticles have a particle size of 200 nm to 1000 nm, and more preferably, the particle size of the magnetic nanoparticles is 1000 nm. Preferably, the number of phages loaded per microgram of magnetic particles reaches 10. 8 ~10 9 PFU.

8. A method for constructing a magnetic phage probe, characterized in that, The method for constructing the magnetic phage probe is to obtain a biotinylated phage by specifically biotinylating the engineered phage described in claim 5 with BirA biotin ligase; and then coupling the biotinylated phage to the surface of magnetic nanoparticles through biotin-streptavidin interaction to form a magnetic phage probe.

9. The application of the sgRNA of claim 1, the editing plasmid of claim 2, the recombinant plasmid of claim 3, the AviTag engineered phage of claim 5, and the magnetic phage probe of claim 7 in the enrichment, isolation, and / or quantitative detection of Pseudomonas aeruginosa.

10. A method for the enrichment, isolation, and quantitative detection of Pseudomonas aeruginosa, characterized in that, Includes the following steps: (1) Take the sample to be tested and mix it with the magnetic phage probe of claim 7 for incubation, so that the probe binds to Pseudomonas aeruginosa in the sample to form a bacterial-magnetic phage probe complex. (2) Apply an external magnetic field to the incubation reaction system to separate the bacteria-magnetic phage probe complex, and wash it multiple times with washing buffer to remove unbound bacteria and matrix interference components, so as to obtain enriched Pseudomonas aeruginosa-magnetic phage probe complex. (3) Add ATP detection premix to the enriched Pseudomonas aeruginosa-magnetic phage probe complex obtained in step (2) to cause bacterial lysis and release intracellular ATP. The ATP reacts with luciferin and oxygen under the catalysis of luciferase to generate a light-emitting signal, and the intensity of the light-emitting signal is detected. (4) Substitute the intensity of the luminescent signal obtained in step (3) into the pre-established standard curve to calculate the number of Pseudomonas aeruginosa in the sample to be tested. Preferably, in step (1), the incubation temperature of the sample to be tested and the magnetic phage probe is 20-30℃, preferably 25℃; the incubation time is 5-15 min, preferably 10 min. Preferably, in step (2), the washing buffer is PBST buffer, specifically PBS + 0.05% Tween-20, and the number of washing cycles is 2 to 5, preferably 3. Preferably, in step (3), the ATP detection premix includes bacterial lysate, luciferin and luciferase, and the incubation time is 20 to 60 min, preferably 30 min.

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