Rapid and accurate urinary tract infection detection method based on bacteriophage

Abstract

The invention relates to the technical field of urinary tract infection detection, and discloses a phage-based rapid and accurate urinary tract infection detection method, which comprises the following steps: S1, constructing a CRISPR / Cas9 system; s2, constructing a homologous recombinant plasmid; s3, constructing three plasmids BL21; s4, constructing, screening, verifying and reporting a phage T4:: Nluc; according to the detection method, a CRISPR / Cas9 system is combined with a homologous recombination technology, so that the efficiency of bacteriophage gene editing is remarkably improved, based on the report bacteriophage T4:: Nluc, rapid, sensitive, specific, accurate, simple and convenient detection on clinical urinary tract infection escherichia coli is realized, and the method can be used for detecting an escherichia coli urinary tract infection sample which is not pretreated, and has a wide application prospect. The kit shows excellent detection timeliness and specificity, is a powerful tool for identifying clinical urinary tract infection escherichia coli, and provides a new technical path for timely and accurate diagnosis and effective treatment of clinical urinary tract infection.

Description

Technical Field

[0001] The present invention relates to the technical field of urinary tract infection detection, and in particular to a bacteriophage-based rapid and accurate detection method for urinary tract infection. Background Art

[0002] Urinary tract infection is one of the most common infectious diseases worldwide, causing considerable economic and public health burden. Escherichia coli accounts for 70-95% of all urinary tract infections.

[0003] At present, the gold standard for diagnosing urinary tract infection is the onset of symptoms accompanied by positive urine culture. The advantage of urine culture as the gold standard is that it can identify and verify live bacteria, but there are many influencing factors, a high rate of missed detection, and a long detection cycle. It takes at least 42 to 78 hours from collecting clinical samples to the results of drug sensitivity tests, which may miss the best treatment time for patients and aggravate antibiotic resistance. Its subsequent biochemical identification requires professional equipment and operators. Other methods for detecting bacteria in urine mainly include microscopic examination after Gram staining, test paper detection, and urine sediment analysis. However, due to various reasons such as sample contamination and improper operation, the above detection methods all have certain false positive and false negative phenomena and lack detection specificity. In addition, lateral flow chromatographic assay (LFCA) and flow cytometry (FCM) are also used for real-time screening of bacteriuria, saving experimental costs, shortening detection time, and achieving sensitive and rapid detection; however, LFCA still requires multicenter prospective studies to identify its accuracy, and FCM only screens bacteriuria and cannot perform pathogen identification and drug sensitivity tests.

[0004] In recent years, with the development of molecular biology technology, MALDI-TOF mass spectrometry, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) and gene chip technology have also been used for the diagnosis of urinary tract infection pathogens. However, the above methods have high costs, complex sample pretreatment, high professional and technical requirements for operators, inability to distinguish between live and dead bacteria, and susceptibility to interference and false positives. As a comprehensive and unbiased detection method, metagenomics next generation sequencing (mNGS) has made up for the shortcomings and limitations of in vitro culture and molecular biology methods to a certain extent. mNGS can widely identify known and unknown microorganisms and even discover new organisms, realize the detection of multi-species infection, and can perform evolutionary tracing, strain identification, virulence assessment and drug resistance prediction. However, there are many problems such as difficulty in unbiased pathogen nucleic acid extraction, large amount of host background nucleic acid interference, time-consuming and labor-intensive library construction process, and high technical cost.

[0005] The reporter phage obtained by genetic engineering has great application potential in bacterial identification. Genetic engineering technologies for phages include traditional homologous recombination technology, λ-Red recombination, bacteriophage recombination of electroporated DNA (BRED), and the newly developed in vitro synthetic restart technology and CRISPR / Cas system-mediated gene editing technology in recent years. Among them, traditional homologous recombination and λ-Red recombination technologies have problems such as low recombination efficiency (about 1%) and difficulty in screening due to lack of recombinant screening markers. BRED technology increases the recombination efficiency to 10%-15%, but this technology depends on host competent cells with high transformation efficiency. In vitro synthetic restart technology effectively improves the production efficiency of engineered phages, but this process depends on the successful transformation of host bacteria, and it is still difficult to engineer phages with too large genomes. The CRISPR / Cas system significantly improves the efficiency and accuracy of gene editing. Among them, the CRISPR / Cas9 system has great application potential and has become a powerful tool for engineering prokaryotic and eukaryotic systems. For this reason, we proposed a rapid and accurate detection method for urinary tract infection based on phages. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a rapid and accurate detection method for urinary tract infection based on bacteriophage, which solves the above-mentioned problems.

[0007] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a method for rapid and accurate detection of urinary tract infection based on bacteriophage, comprising the following steps:

[0008] S1: Construct the CRISPR / Cas9 system. First, construct the correct pCRISPR-cr6 plasmid, transform the pCas9 plasmid and the pCRISPR-cr6 plasmid into Escherichia coli BL21 competent cells in sequence, and perform colony PCR to verify the successful transformation of the plasmids to obtain pCas9-pCRISPR-cr6-BL21, that is, the CRISPR / Cas9 system is successfully constructed;

[0009] S2: construct homologous recombination plasmid, select the upstream 1041bp and downstream 1000bp sequences of the soc gene as the upstream and downstream homology arms, PCR amplify the upstream and downstream homology arms and Nanoluc gene region fragments, and integrate the upstream 1041bp homology arm sequence, Nluc gene region and downstream 1000bp sequence into the pCE-Zero linearized plasmid in sequence to construct a circular homologous recombination plasmid;

[0010] S3: construct three-plasmid BL21, first obtain pCas9-pCRISPR-cr6-BL21 competent cells, then transform the homologous recombination plasmid into pCas9-pCRISPR-cr6-BL21 competent cells to obtain three-plasmid BL21 containing homologous recombination plasmid, pCas9 plasmid and pCRISPR-cr6 plasmid;

[0011] S4: Construction, screening and verification of reporter phage T4::Nluc. First, the T4 phage was infected with three plasmids BL21 by double-layer agar plate method, and PCR templates were prepared for PCR and agarose gel electrophoresis. The PCR reaction procedure was the same as before. At the same time, a control group with T4 phage as template was set up to screen the successfully recombined T4::Nluc. The T4::Nluc that was successfully verified by PCR was further subjected to Sanger sequencing to ensure that T4::Nluc was successfully constructed;

[0012] S5: Evaluate the detection performance of T4::Nluc, including the detection limit of T4::Nluc, the clinical strain detection coverage of T4::Nluc, the detection specificity of T4::Nluc and the effect of urine matrix on the detection performance of T4::Nluc.

[0013] Preferably, the construction of the CRISPR / Cas9 system in S1 specifically includes: in the CRISPR / Cas9 system, the selection of sgRNA is also a major rate-limiting factor for gene editing efficiency, firstly, a crRNA with a high cutting efficiency for the T4 non-essential gene soc, soc-cr6, is selected from the crRNA library, and a BsaI restriction site sequence is added to both ends of the sequence, phosphorylated and annealed into a DNA double strand as the sgRNA to be used;

[0014] The pCRISPR circular plasmid was digested with BsaI enzyme overnight at 37°C, and the success of the digestion was confirmed by agarose gel electrophoresis. The cr6 and the obtained pCRISPR linearized plasmid were connected with T4 DNA ligase at 4°C overnight. The ligation product was transformed into Escherichia coli BL21 competent cells and spread on solid LB agar plates containing a final concentration of 50 μg / mL kanamycin. The plates were cultured at 37°C overnight, and the monoclonal colonies after culture were picked for colony PCR and Sanger sequencing to ensure that the pCRISPR-cr6 plasmid was constructed correctly. The PCR reaction procedure was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 40 s, 72°C final extension for 5 min after 35 cycles, and then cooled to 4°C for storage.

[0015] The verified pCRISPR-cr6-BL21 was cultured overnight, and the pCRISPR-cr6 plasmid was extracted using the FastPure Plasmid Mini Kit and stored at -20°C for later use;

[0016] The pCas9 plasmid and pCRISPR-cr6 plasmid were transformed into Escherichia coli BL21 competent cells in sequence, and colony PCR was performed to verify the successful transformation of the plasmids. The PCR reaction procedure was the same as before, and pCas9-pCRISPR-cr6-BL21 was obtained, that is, the CRISPR / Cas9 system was successfully constructed.

[0017] Preferably, constructing the homologous recombination plasmid in S2 specifically includes: selecting the upstream 1041bp and downstream 1000bp sequences of the soc gene as the upstream and downstream homology arms, and designing primers UHA-F, UHA-R, Nluc-F, Nluc-R, DHA-F, and DHA-R with 20bp sequence overlap at both ends for the upstream homology arm sequence, the Nanoluc (Nluc) gene region, and the downstream homology arm sequence;

[0018] PCR amplified the upstream and downstream homology arms and Nanoluc gene region fragments, and agarose gel electrophoresis confirmed that the band position was correct. FastPure Gel DNA Extraction Mini Kit was used for gel recovery, and the gel-recovered product was used as a template for PCR verification again to ensure that the recovered DNA fragment was correct. The PCR reaction procedure was the same as above. ClonExpress UltraOne Step Cloning Kit was used to construct the reaction system according to the kit instructions, and the reaction was carried out at 50°C for 40 minutes. Gibson assembly was performed, that is, the upstream 1041bp homology arm sequence, the Nluc gene region and the downstream 1000bp sequence were sequentially integrated into the pCE-Zero linearized plasmid to construct a circular homologous recombination plasmid, transformed BL21 competent cells, and coated on ampicillin (50ug / mL) and kanamycin (50ug / mL) double resistance plates. After overnight culture at 37°C, single clone colonies were picked for colony PCR. The PCR reaction procedure was the same as above to ensure that the homologous recombination plasmid was constructed correctly and the homologous recombination plasmid was extracted.

[0019] Preferably, constructing the three-plasmid BL21 in S3 specifically comprises: using 0.1 mol / L calcium chloride solution to prepare pCas9-pCRISPR-cr6-BL21 as competent cells, culturing pCas9-pCRISPR-cr6-BL21 at 37°C, 200 rpm shaking incubator to logarithmic phase, ice bathing for 10 min, centrifuging at 4°C, 4000 rpm for 10 min, discarding the supernatant, adding an equal volume of 4°C precooled 0.1 mol / L calcium chloride solution to resuspend the precipitate, ice bathing for 30 min, centrifuging again at 4°C, 4000 rpm for 10 min, discarding the supernatant, adding an appropriate amount of 4°C precooled 0.1 mol / L calcium chloride solution to resuspend the precipitate to obtain pCas9-pCRISPR-cr6-BL21 competent cells;

[0020] The homologous recombination plasmid was transformed into pCas9-pCRISPR-cr6-BL21 competent cells and spread on a triple-resistance plate containing ampicillin (50 μg / mL), kanamycin (50 μg / mL) and chloramphenicol (30 μg / mL). After overnight culture at 37°C, single clones were picked for colony PCR to verify the successful transformation of the homologous recombination plasmid, and a three-plasmid BL21 containing the homologous recombination plasmid, pCas9 plasmid and pCRISPR-cr6 plasmid was obtained.

[0021] Preferably, the construction, screening and verification of the reporter phage T4::Nluc in S4 specifically comprises: infecting the three-plasmid BL21 with the T4 phage by the double-layer agar plate method, that is, mixing 200 μL of the three-plasmid BL21 in the logarithmic phase with 100 μL of the T4 phage of appropriate titer, leaving it at room temperature for 15 minutes, adding it to 0.625% semi-solid LB agar melted at 55°C, mixing it and immediately pouring it on a solid LB plate, and after solidification, placing it in a 37°C incubator for overnight culture;

[0022] 21 plaques were selected from the single plaques produced after overnight culture, 0.5 μL of phage solution in the center of the plaque was taken and added to 10 μL (10 mM / L) EDTA solution, and heat-shocked at 65°C for 10 min, cooled to room temperature, and used as a template for PCR and agarose gel electrophoresis. The PCR reaction procedure was the same as before, and a control group with T4 phage as a template was set up to screen the successfully recombined T4::Nluc. After agarose gel electrophoresis of the PCR product, T4 will produce a band of 2358 bp, while T4::Nluc will produce a band of 2837 bp. The T4::Nluc that was successfully verified by PCR was further subjected to Sanger sequencing to ensure that T4::Nluc was successfully constructed;

[0023] The successfully verified T4::Nluc was co-cultured with pCas9-pCRISPR-cr6-BL21, and the bacteria and their fragments were filtered through a 0.22 μm bacterial filter to obtain the culture medium of T4::Nluc, which was placed at 4°C for use.

[0024] Preferably, the evaluation of the detection limit of T4::Nluc in S5 specifically comprises: culturing BL21 Escherichia coli overnight, counting colonies, and serially diluting 10 times to a concentration of 1×10 1 CFU / mL~1×10 6 CFU / mL of bacterial solution was mixed with an appropriate amount of T4::Nluc to make the final concentration 3×10 6 PFU / mL, co-cultured at 37°C, 200 rpm on a shaker for 7 hours. During this period, 50 μL of co-culture solution was taken out every 10 minutes, 20 μL of Nluc reaction substrate was added, and incubated at room temperature for 3 minutes. The luminescence value was measured by a multifunctional microplate reader, and the time-luminescence curves of different bacterial concentration groups were plotted with time as the horizontal axis and the luminescence value as the vertical axis, where the threshold value = the average background luminescence value plus three times the standard deviation (SD), and the detection limit was the minimum bacterial concentration required to produce a luminescence signal above the threshold.

[0025] Preferably, the evaluation of the clinical strain detection coverage of T4::Nluc in S5 specifically comprises: using a double-layer agar plate spot method and a luminescence assay method to determine the detection coverage of T4::Nluc in 104 strains of Escherichia coli isolated from clinical urine;

[0026] The double-layer agar plate spot method refers to mixing 200 μL of clinical E. coli culture after overnight culture with 0.625% semi-solid LB agar melted at 55°C, and immediately pouring it on the LB solid plate. After solidification, take 3 μL of T4::Nluc culture solution, spot it on the double-layer agar plate, place it in a 37°C incubator for overnight culture, and observe whether plaques are formed. The formation of plaques indicates that T4::Nluc can recognize and lyse the clinical bacteria, otherwise it cannot recognize and lyse;

[0027] The luminescence assay method refers to co-culturing the clinical bacterial culture overnight with an appropriate amount of T4::Nluc for 3.5 hours, taking 50 μL of the co-culture solution, adding 20 μL of substrate, and incubating at room temperature for 3 minutes. The luminescence value is used to determine whether the strain can be identified and lysed.

[0028] Preferably, the evaluation of the detection specificity of T4::Nluc in S5 specifically includes: BL21 (positive control) and Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus hominis isolated from clinical urine are cultured overnight at 37°C and 200rpm in a shaking table, and then mixed with an appropriate amount of T4::Nluc, and the co-culture is continued for 5 hours. During this period, 50 μL of co-culture solution is taken out every 10 minutes, 20 μL of substrate is added, and after incubation at room temperature for 3 minutes, the luminescence value is determined, and the time-luminescence curve is plotted. At the same time, only the T4::Nluc culture solution group is set as a negative control. If T4::Nluc only produces a detectable luminescent signal after co-culture with BL21 Escherichia coli, and no detectable luminescent signal is produced after co-culture with several other common urinary tract infection pathogens, that is, T4::Nluc does not recognize and lyse several other pathogens, there is no cross reaction, and it has extremely high detection specificity.

[0029] Preferably, the evaluation of the effect of urine matrix on T4::Nluc detection performance in S5 specifically includes: collecting clean midstream urine from healthy people from the clinic, filtering and sterilizing with a 0.22 μm bacterial filter, adding the filtered urine to the Escherichia coli solution, so that the proportion of urine matrix in each group is 0%, 20%, 40%, 60%, 80%, 100%, respectively, and ensuring that the concentration of Escherichia coli in each group is consistent (2.72×10 7 CFU / mL), and an LB liquid culture medium group and a urine group were also set up. Appropriate amount of T4::Nluc was added to the above groups, and co-cultured at 37°C, 200 rpm in a shaking incubator for 5 h. Every 10 min, 50 μL of the co-culture solution was taken out, 20 μL of substrate was added, and the cells were incubated at room temperature for 3 min. The luminescence value was measured and the time-luminescence curve was drawn.

[0030] Compared with the prior art, the present invention provides a rapid and accurate detection method for urinary tract infection based on bacteriophage, which has the following beneficial effects:

[0031] 1. This phage-based rapid and accurate detection method for urinary tract infection uses the CRISPR / Cas9 system combined with homologous recombination technology to significantly improve the efficiency and accuracy of phage gene editing;

[0032] In the CRISPR / Cas9 system, the selection of sgRNA is a major rate-limiting factor in gene editing efficiency. Therefore, in the present invention, a CRISPR / Cas9 system was first constructed by selecting a sgRNA with high cleavage efficiency targeting the T4 phage non-essential gene soc. The CRISPR / Cas9 system was combined with classical homologous recombination by infecting three-plasmid BL21 Escherichia coli with T4 phage. That is, when the T4 phage genome was injected into the three-plasmid BL21 bacteria and the phage nucleic acid was synthesized, the T4 phage genome and the homologous recombination plasmid underwent homologous recombination, thereby integrating the Nluc gene into the phage nucleic acid to produce T4::Nluc. In the above process, the CRISPR / Cas9 system cut the wild-type T4 phage genome to produce a large number of broken DNA double strands, thereby significantly improving the efficiency of homologous recombination; at the same time, the CRISPR / Cas9 system played a counter-selective role by cutting the wild-type T4 phage nucleic acid, increasing the proportion of T4::Nluc in the progeny phage, and assisting in screening out the successfully recombined T4::Nluc, thereby significantly improving the efficiency of phage gene editing.

[0033] 2. This phage-based rapid and accurate detection method for urinary tract infection uses a reporter phage T4::Nluc that is highly specific to host bacteria, highly stable in extreme environments, low-cost and easy to produce. Once infected with host bacteria, the reporter gene is expressed to produce a detectable signal, indicating the presence of live host bacteria. In addition, the proliferation process of the reporter phage can amplify the signal, the detection cycle is short, no bacterial separation is required, live bacteria can be distinguished from dead bacteria, and it is not affected by bacterial antibiotic resistance, making it an ideal recognition element for bacterial identification.

[0034] In the present invention, using the reporter phage T4::Nluc, ≥10 4 CFU / mL of BL21 Escherichia coli meets the diagnostic criteria for urinary tract infection—the number of single bacterial colonies is greater than 10 5CFU / mL, and significantly shortened the detection time, the operation was simpler, and the requirements for laboratory equipment conditions and the technical level of operators were low. Among the 104 Escherichia coli isolated from clinical urine, the double-layer agar plate spot method (gold standard) could detect 27 / 104 (25.96%), while the luminescence assay could detect 40 / 104 (38.46%) clinical Escherichia coli, that is, the luminescence assay in the present invention was more sensitive and could detect low-level infection events that were difficult to observe with the naked eye, and the detection coverage rate in clinical strains was higher; when T4::Nluc was compared with several other common clinical urinary tract infection pathogens (Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, etc.), the detection rate of T4::Nluc was 1.34 times that of T4::Nluc, and ... After co-culture of Escherichia coli, Staphylococcus epidermidis and Staphylococcus hominis), no detectable luminescent signal was generated, that is, there was no cross-reaction. T4::Nluc has extremely high host specificity for Escherichia coli and has great application potential in the detection of Escherichia coli in clinical urinary tract infection. The urine Escherichia coli detection method based on T4::Nluc in the present invention is not affected by the urine matrix components, that is, the urine matrix components do not cause additional increase or decrease in the luminescent signal, and there is no need to perform any complex pre-treatment steps on the urine sample. It can be directly used for the detection of urine samples suspected of clinical infection, and the operation is simple and quick. In addition, T4::Nluc only recognizes and detects live Escherichia coli in urine, so it has more advantages in clinical patient disease status assessment and treatment guidance.

[0035] 3. This phage-based rapid and accurate detection method for urinary tract infection overcomes the problems of existing urinary tract infection detection methods, such as long detection cycle, high missed detection rate, high technical requirements, complex operation, and high cost. It can be used to detect Escherichia coli urinary tract infection samples without any pretreatment, and shows excellent detection timeliness and specificity. It realizes rapid, sensitive, specific, accurate and simple detection of Escherichia coli in clinical urinary tract infection. It is a powerful tool for the identification of Escherichia coli in clinical urinary tract infection, and provides a new technical path for the timely and accurate diagnosis and effective treatment of clinical urinary tract infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of the construction of the CRISPR / Cas9 system of the present invention;

[0037] Figure 2 Schematic diagram of the construction of the homologous recombination plasmid of the present invention;

[0038] Figure 3 Schematic diagram of T4::Nluc constructed by homologous recombination of the present invention;

[0039] Figure 4 A schematic diagram of the detection limit evaluation results of the invention T4::Nluc;

[0040] Figure 5This is a schematic diagram of the detection coverage evaluation results of T4::Nluc in clinical Escherichia coli of the present invention;

[0041] Figure 6 This is a schematic diagram of the detection specificity evaluation results of T4::Nluc of the present invention;

[0042] Figure 7 This is a schematic diagram of the evaluation results of the effect of the urine matrix on the detection performance of T4::Nluc of the present invention;

[0043] Figure 8 Schematic diagram of the principle of constructing T4::Nluc using the CRISPR / Cas9 system combined with homologous recombination technology of the present invention. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] See also Figure 1-8 , a rapid and accurate detection method for urinary tract infection based on bacteriophage, comprising the following steps:

[0046] 1. Construction of CRISPR / Cas9 system;

[0047] Since the selection of sgRNA is also a major rate-limiting factor for gene editing efficiency in the CRISPR / Cas9 system, we first selected soc-cr6, a crRNA with high cleavage efficiency for the T4 non-essential gene soc, from the crRNA library established in the literature by Michelle M Duong et al., added BsaI restriction site sequences at both ends of the sequence, phosphorylated and annealed to a DNA double strand, as the sgRNA used in the present invention.

[0048] The pCRISPR circular plasmid was digested with BsaI overnight at 37°C, and the digestion was confirmed by agarose gel electrophoresis ( Figure 1B), cr6 and the obtained pCRISPR linearized plasmid were connected by T4 DNA ligase at 4°C overnight. The ligation product was transformed into E. coli BL21 competent cells and spread on solid LB agar plates containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37°C overnight. The monoclonal colonies after culture were picked for colony PCR (primers: soc-cr6-F, pCRISPR-R, 646bp), and Sanger sequencing was performed to ensure that the pCRISPR-cr6 plasmid was constructed correctly (see Figure 1 C. Figure 1 D). PCR reaction program: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 40 s, after 35 cycles, 72℃ final extension for 5 min, then cool to 4℃ for storage.

[0049] The verified correct pCRISPR-cr6-BL21 was cultured overnight, and the pCRISPR-cr6 plasmid was extracted using FastPure Plasmid Mini Kit (Nanjing Novogene Biotech Co., Ltd.) and stored at -20°C for future use.

[0050] The pCas9 plasmid and pCRISPR-cr6 plasmid were transformed into E. coli BL21 competent cells in sequence, and colony PCR was performed to verify the successful transformation of the plasmid (primers: pCas9-F, pCas9-R, 1335 bp; soc-cr6-F, pCRISPR-R, 646 bp). The PCR reaction procedure was the same as before. pCas9-pCRISPR-cr6-BL21 was obtained, that is, the CRISPR / Cas9 system was successfully constructed.

[0051] 2. Construction of homologous recombination plasmid;

[0052] The upstream 1041bp and downstream 1000bp sequences of the soc gene were selected as the upstream and downstream homology arms. For the upstream homology arm sequence, Nanoluc (Nluc) gene region and downstream homology arm sequence, primers UHA-F, UHA-R, Nluc-F, Nluc-R, DHA-F, and DHA-R with 20bp sequence overlap at both ends were designed respectively.

[0053] PCR amplified the upstream and downstream homology arms and Nanoluc gene region fragments, and agarose gel electrophoresis confirmed the correct band position (see Figure 2 A), gel recovery was performed using FastPure Gel DNA Extraction Mini Kit (Nanjing Novozyme Biotechnology Co., Ltd.), and PCR verification was performed again using the gel-recovered product as a template to ensure that the recovered DNA fragments were correct (see Figure 2B), the PCR reaction procedure was the same as above, using ClonExpress Ultra One Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd.), the reaction system was constructed according to the kit instructions, the reaction was carried out at 50°C for 40 min, and Gibson assembly was performed, that is, the upstream 1041 bp homology arm sequence, the Nluc gene region and the downstream 1000 bp sequence were sequentially integrated into the pCE-Zero linearized plasmid (containing ampicillin and kanamycin dual resistance genes) to construct a circular homologous recombination plasmid (see Figure 2 C), transform BL21 competent cells, spread on ampicillin (50ug / mL) and kanamycin (50ug / mL) double resistance plates, and culture at 37°C overnight; pick single clones for colony PCR (design 7 different primer pairs: UHA-F, DHA-R, 2761bp; UHA-F, M13R, 2840bp; M13F, UHA-R, 1119bp; Nluc-F, M13R, 1788bp; M13F, DHA-R, 2808bp; M13F, Nluc-R, 1801bp; DHA-F, M13R, 1106bp), the PCR reaction procedure is the same as above, ensure that the homologous recombination plasmid is constructed correctly (see Figure 2 D), extract homologous recombination plasmid.

[0054] 3. Construction of three plasmid BL21;

[0055] pCas9-pCRISPR-cr6-BL21 was prepared as competent cells using 0.1 mol / L calcium chloride solution. pCas9-pCRISPR-cr6-BL21 was cultured at 37°C, 200 rpm shaking incubator until the logarithmic phase; ice bathed for 10 min, centrifuged at 4°C, 4000 rpm for 10 min, and the supernatant was discarded; an equal volume of 4°C precooled 0.1 mol / L calcium chloride solution was added to resuspend the precipitate, and ice bathed for 30 min; centrifuged again at 4°C, 4000 rpm for 10 min, and the supernatant was discarded; an appropriate amount of 4°C precooled 0.1 mol / L calcium chloride solution was added to resuspend the precipitate to obtain pCas9-pCRISPR-cr6-BL21 competent cells.

[0056] The homologous recombination plasmid was transformed into pCas9-pCRISPR-cr6-BL21 competent cells and spread on a triple-resistance plate containing ampicillin (50 μg / mL), kanamycin (50 μg / mL) and chloramphenicol (30 μg / mL). After overnight culture at 37°C, single clones were picked for colony PCR to verify the successful transformation of the homologous recombination plasmid (see Figure 3 A) The three-plasmid BL21 containing homologous recombination plasmid, pCas9 plasmid and pCRISPR-cr6 plasmid was obtained.

[0057] 4. Construction, screening and validation of reporter phage T4::Nluc;

[0058] The T4 phage was infected with the three-plasmid BL21 by the double-layer agar plate method, that is, 200 μL of the three-plasmid BL21 in the logarithmic phase was mixed with 100 μL of T4 phage of appropriate titer, left at room temperature for 15 min, added to 0.625% semi-solid LB agar melted at 55°C, mixed and immediately poured onto a solid LB plate, and after solidification, placed in a 37°C incubator for overnight culture.

[0059] From the single plaques produced after overnight culture, 21 plaques were selected each time, 0.5 μL of phage solution in the center of the plaque was taken and added to 10 μL (10 mM / L) EDTA solution, and after heat shock at 65°C for 10 minutes, it was cooled to room temperature and used as a template for PCR and agarose gel electrophoresis (primers: T4 Recombination Validation-F, T4 Recombination Validation-R). The PCR reaction procedure was the same as before, and a control group with T4 phage as the template was set up at the same time to screen the successfully recombined T4::Nluc. After agarose gel electrophoresis of the PCR product, T4 will produce a 2358 bp band, while T4::Nluc will produce a 2837 bp band (see Figure 3 B). The T4::Nluc that was successfully verified by PCR was further subjected to Sanger sequencing to ensure that T4::Nluc was successfully constructed (see Figure 3 D).

[0060] The successfully verified T4::Nluc was co-cultured with pCas9-pCRISPR-cr6-BL21, and the bacteria and their fragments were filtered through a 0.22 μm bacterial filter to obtain the culture medium of T4::Nluc, which was placed at 4°C for use.

[0061] 5. Evaluate the detection performance of T4::Nluc.

[0062] (1) Detection limit of T4::Nluc;

[0063] After overnight culture of BL21 Escherichia coli, colonies were counted and 10-fold serial dilutions were made to a concentration of 1×10 1 CFU / mL~1×10 6 CFU / mL of bacterial solution was mixed with an appropriate amount of T4::Nluc to make the final concentration 3×10 6PFU / mL, co-cultured at 37°C, 200rpm shaker for 7h. During this period, 50μL of co-culture solution was taken out every 10min, 20μL of Nluc reaction substrate was added, incubated at room temperature for 3min, and the luminescence value was measured by a multifunctional microplate reader. The time-luminescence curves of different bacterial concentration groups were drawn with time as the horizontal axis and luminescence value as the vertical axis (see Figure 4 ); threshold = average background luminescence value plus three times standard deviation (SD), and the detection limit is the minimum bacterial concentration required to produce a luminescence signal above the threshold. The results showed that T4::Nluc could detect ≥10 4 CFU / mL of BL21 meets the diagnostic criteria for E. coli urinary tract infection (single bacterial colony count> 10 5 CFU / mL) and significantly shortened the detection time.

[0064] (2) clinical strain detection coverage of T4::Nluc;

[0065] The detection coverage of T4::Nluc in 104 strains of Escherichia coli isolated from clinical urine was determined by double-layer agar plate spot method (gold standard for confirming the sensitivity of bacteria to a certain phage) and luminescence assay. The double-layer agar plate spot method refers to mixing 200 μL of clinical Escherichia coli culture after overnight culture with 0.625% semi-solid LB agar melted at 55°C, immediately pouring it on the LB solid plate, and after solidification, taking 3 μL of T4::Nluc culture solution, spotting it on the double-layer agar plate, placing it in a 37°C incubator for overnight culture, and observing whether plaques are formed. The formation of plaques indicates that T4::Nluc can recognize and lyse the clinical bacteria, otherwise it cannot recognize and lyse. The luminescence assay method refers to co-culturing the clinical bacterial liquid cultured overnight with an appropriate amount of T4::Nluc for 3.5 hours, taking 50μL of the co-culture liquid, adding 20μL of substrate, incubating at room temperature for 3 minutes, and judging whether the strain can be identified and lysed based on the luminescence value. The results of the double-layer agar plate spot method showed that T4::Nluc phage liquid could form visible plaques on 27 / 104 (25.96%) of the Escherichia coli isolated from clinical urine double-layer agar plates, while the luminescence assay method could detect 40 / 104 (38.46%) of clinical Escherichia coli (see Figure 5 ), that is, the luminescence assay is more sensitive and has a higher detection coverage for clinical strains.

[0066] (3) Detection specificity of T4::Nluc;

[0067] BL21 (positive control) and one strain each of Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus hominis isolated from clinical urine were cultured overnight at 37°C and 200rpm in a shaking incubator, then mixed with an appropriate amount of T4::Nluc and co-cultured for 5 hours. During this period, 50 μL of co-culture solution was taken out every 10 minutes, 20 μL of substrate was added, and after incubation at room temperature for 3 minutes, the luminescence value was measured and the time-luminescence curve was plotted. At the same time, the T4::Nluc culture solution group only was set as the negative control. The results showed that T4::Nluc only produced detectable luminescence signals after co-culture with BL21 Escherichia coli, and no detectable luminescence signals were produced after co-culture with several other common urinary tract infection pathogens (see Figure 6 ), that is, T4::Nluc does not recognize and lyse several other pathogens, there is no cross reaction, and it has extremely high detection specificity.

[0068] (4) Effect of urine matrix on T4::Nluc detection performance.

[0069] Clean midstream urine of healthy people was collected from the clinic and sterilized by filtration using a 0.22 μm bacterial filter. The filtered urine was added to the E. coli solution, so that the proportion of urine matrix in each group was 0%, 20%, 40%, 60%, 80%, and 100%, respectively, while ensuring that the concentration of E. coli in each group was consistent (2.72×10 7 CFU / mL), and set up LB liquid culture medium group and urine group only. Add appropriate amount of T4::Nluc to the above groups, and co-culture at 37℃, 200rpm shaker for 5h. Take out 50μL of co-culture solution every 10min, add 20μL of substrate, incubate at room temperature for 3min, measure the luminescence value, and draw the time-luminescence curve (see Figure 7 ). The results showed that the "urine + T4::Nluc" group did not produce additional luminescence, and its luminescence value was not significantly different from that of the "LB + T4::Nluc group (negative control)" (P>0.05); in addition, the 0%, 20%, 40%, 60%, 80%, and 100% urine matrix groups were all detected positive at 30 minutes, and there was no significant difference in luminescence values ​​between the groups (P>0.05). The above results show that the clinical urine matrix has no effect on the detection performance of T4::Nluc.

[0070] The present invention proposes a bacteriophage-based rapid and accurate detection method for urinary tract infection, which significantly improves the efficiency of phage gene editing by combining CRISPR / Cas9 system with homologous recombination technology. Traditional homologous recombination technology, λ-Red recombination, BRED and in vitro synthetic restart technology have the problems of low recombination efficiency, reliance on successful transformation of host bacteria, and difficulty in screening due to lack of recombinant screening markers when engineering phages. In addition, it is still difficult to engineer phages with too large genomes. The present invention adopts CRISPR / Cas9 system combined with homologous recombination technology to significantly improve the efficiency and accuracy of phage gene editing.

[0071] In the CRISPR / Cas9 system, the selection of sgRNA is a major rate-limiting factor in gene editing efficiency. Therefore, in the present invention, the CRISPR / Cas9 system was first constructed by selecting a sgRNA with high cutting efficiency targeting the non-essential gene soc of T4 phage. The CRISPR / Cas9 system was combined with classical homologous recombination by infecting the three-plasmid BL21 Escherichia coli with T4 phage. That is, when the T4 phage genome is injected into the three-plasmid BL21 bacteria and the phage nucleic acid is synthesized, the genome of the T4 phage undergoes homologous recombination with the homologous recombination plasmid, thereby integrating the Nluc gene into the phage nucleic acid to produce T4::Nluc (see Figure 8 In the above process, the CRISPR / Cas9 system cuts the wild-type T4 phage genome, generating a large number of broken DNA double strands, thereby significantly improving the efficiency of homologous recombination; at the same time, the CRISPR / Cas9 system plays a counter-selective role by cutting the wild-type T4 phage nucleic acid, increasing the proportion of T4::Nluc in the progeny phage, and assisting in screening out the T4::Nluc with successful recombination (see Figure 8 ), thereby significantly improving the efficiency of phage gene editing.

[0072] The present invention proposes a bacteriophage-based rapid and accurate detection method for urinary tract infection based on reporter phage T4::Nluc, which realizes rapid, sensitive, specific, accurate and simple detection of clinical urinary tract infection Escherichia coli.

[0073] At present, the gold standard for diagnosing urinary tract infection is the onset of symptoms accompanied by positive urine culture. The traditional series of detection methods based on bacterial culture are time-consuming and labor-intensive, with a high rate of missed detection; and methods for instant screening of bacteriuria, such as flow immunochromatography and flow cytometry, have problems with the accuracy of bacterial species identification; molecular biology methods including PCR, MALDI-TOF mass spectrometry, fluorescence in situ hybridization, and gene chip technology cannot distinguish between live and dead bacteria and have high technical requirements and high costs.

[0074] The reporter phage T4::Nluc constructed by the present invention has high specificity for host bacteria, high stability in extreme environments, low cost and easy production. Once infected with host bacteria, the reporter gene is expressed to produce a detectable signal, thereby indicating the presence of live host bacteria. In addition, the proliferation process of the reporter phage can amplify the signal, the detection cycle is short, no bacterial separation is required, live bacteria and dead bacteria can be distinguished, and it is not affected by bacterial antibiotic resistance, making it an ideal recognition element for bacterial identification.

[0075] In the present invention, using the reporter phage T4::Nluc, ≥10 4 CFU / mL of BL21 Escherichia coli meets the diagnostic criteria for urinary tract infection—the number of single bacterial colonies is greater than 10 5 CFU / mL, and significantly shortened the detection time, the operation is simpler, and the requirements for laboratory equipment conditions and the technical level of operators are relatively low. Among the 104 Escherichia coli isolated from clinical urine collected, the double-layer agar plate spot method (gold standard) can detect 27 / 104 (25.96%), while the luminescence assay can detect 40 / 104 (38.46%) clinical Escherichia coli, that is, the luminescence assay in the present invention is more sensitive, and can detect low-level infection events that are not easy to observe with the naked eye, and the detection coverage in clinical strains is higher. When T4::Nluc is co-cultured with several other common clinical urinary tract infection pathogens (Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus aureus), no detectable luminescent signal is generated, that is, there is no cross reaction. T4::Nluc has extremely high host specificity for Escherichia coli and has great application potential in the detection of Escherichia coli in clinical urinary tract infection. In addition, the urine Escherichia coli detection method based on T4::Nluc in the present invention is not affected by the urine matrix components, that is, the urine matrix components do not cause additional increase or decrease in the luminescent signal, and no complicated pre-treatment steps are required for the urine sample. It can be directly used for the detection of urine samples suspected of clinical infection, and the operation is simple and fast. Moreover, T4::Nluc only recognizes and detects live Escherichia coli in urine, so it has more advantages in clinical patient disease status assessment and treatment guidance.

[0076] In summary, the present invention proposes a rapid and accurate detection method for urinary tract infection based on bacteriophage, which overcomes the problems of long detection cycle, high missed detection rate, high technical requirements, complex operation, high cost and the like of existing urinary tract infection detection methods, and can be used for the detection of Escherichia coli urinary tract infection samples without any pretreatment, and exhibits excellent detection timeliness and specificity, and realizes rapid, sensitive, specific, accurate and simple detection of Escherichia coli for clinical urinary tract infection, and is a powerful tool for the identification of Escherichia coli for clinical urinary tract infection, and provides a new technical path for the timely and accurate diagnosis and effective treatment of clinical urinary tract infection.

[0077] It is worth noting that the present invention proposes to integrate the Nluc gene into the T4 phage to construct the reporter phage T4::Nluc. In addition to the modification at the non-essential gene soc, the non-essential gene hoc site of the T4 phage can also be selected to genetically engineer the phage.

[0078] In addition to the Nluc gene, the reporter gene can also be selected from fluorescent protein genes, hydrolase (such as β-galactosidase) genes, etc., to express the reporter protein, release detectable reporter molecules, and then realize the detection of the target pathogens.

[0079] The present invention proposes a method for rapid and accurate detection of urinary tract infection based on bacteriophage. In addition to using T4 phage for genetic engineering and transformation into a reporter phage, other Escherichia coli phages such as T3, T7, T6, etc. can also be engineered for the detection of Escherichia coli urinary tract infection. Or the phages of other common urinary tract infection pathogens such as Klebsiella pneumoniae and Pseudomonas aeruginosa are transformed to construct reporter phages for the detection of urinary tract infections caused by other pathogens. Alternatively, a reporter phage cocktail is used to broaden the detection coverage of the same urinary tract infection pathogen or to detect different urinary tract infection pathogens at the same time.

[0080] The following is a detailed description with accompanying drawings:

[0081] Figure 1 Middle: (A) PCR verification results of plasmid transformation of pCas9-BL21 (primers: pCas9-F, pCas9-R, 1335bp), 3 repeats; (B) pCRISPR plasmid digestion results, 1. pCRISPR circular plasmid; 2, 3, 4, 5 are the results after pCRISPR digestion; (C) PCR verification results of pCRISPR-cr6 plasmid (primers: soc-cr6-F, pCRISPR-R, 646bp), 3 repeats; (D) Sanger sequencing alignment results of pCRISPR-cr6 plasmid, the upper part is the sequencing result, and the lower part is the target sequence.

[0082] Figure 2Middle: (A) PCR amplification results of upstream 1041bp homology arm, Nluc gene (702bp) and downstream 1000bp homology arm sequence. 1 and 2 are upstream 1041bp homology arm sequence; 3 and 4 are Nluc gene sequence; 5 and 6 are downstream 1000bp homology arm sequence; (B) Gel recovery verification results of upstream 1041bp homology arm, Nluc gene (702bp) and downstream 1000bp homology arm sequence, 1, 4, 7 are PCR amplification results of upstream 1041bp homology arm, Nluc gene (702bp) and downstream 1000bp homology arm sequence; 2, 5, 8 are gel recovery products; 3, 6, 9 are PCR results using gel recovery products as templates; (C) G Schematic diagram of the principle of ibson assembly; (D) PCR verification results of homologous recombination plasmid, 1. Primers: UHA-F, DHA-R, 2761bp; 2. Primers: UHA-F, M13R, 2840bp; 3. Primers: M13F, UHA-R, 1119bp; 4. Primers: Nluc-F, M13R, 1788bp; 5. Primers: M13F, DHA-R, 2808bp; 6. Primers: M13F, Nluc-R, 1801bp; 7. Primers: DHA-F, M13R, 1106bp.

[0083] Figure 3 Middle: (A) PCR verification of the transformation results of the homologous recombination plasmids in the three-plasmid BL21, 1. Primers: M13F, UHA-R, 1119bp; 2. Primers: Nluc-F, M13R, 1788bp; 3. Primers: M13F, DHA-R, 2808bp; 4. Primers: M13F, Nluc-R, 1801bp; 5. Primers: DHA-F, M13R, 1106bp; (B) PCR results of T4::Nluc. The templates for 1, 2, and 3 are T4::Nluc (primers: T4 Recombination Validation-F, T4 Recombination Validation-R, 2837bp), and the templates for 4 and 5 are T4 (primers: T4 Recombination Validation-F, T4Recombination Validation-R, 2358bp); (C) T4::Nluc purity confirmation results. 1 to 20 are T4::Nluc used as templates for PCR after co-culture, and 21 is T4; (D) Sanger sequencing comparison results of T4::Nluc. The upper part is the sequencing result, and the lower part is the target sequence.

[0084] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A rapid and accurate detection method for urinary tract infection based on bacteriophage, characterized in that: The following steps are involved: S1: Construct the CRISPR / Cas9 system. First, construct the correct pCRISPR-cr6 plasmid, transform the pCas9 plasmid and the pCRISPR-cr6 plasmid into Escherichia coli BL21 competent cells in sequence, and perform colony PCR to verify the successful transformation of the plasmids to obtain pCas9-pCRISPR-cr6-BL21, that is, the CRISPR / Cas9 system is successfully constructed; S2: construct homologous recombination plasmid, select the upstream 1041bp and downstream 1000bp sequences of the soc gene as the upstream and downstream homology arms, PCR amplify the upstream and downstream homology arms and Nanoluc gene region fragments, and integrate the upstream 1041bp homology arm sequence, Nluc gene region and downstream 1000bp sequence into the pCE-Zero linearized plasmid in sequence to construct a circular homologous recombination plasmid; S3: construct three-plasmid BL21, first obtain pCas9-pCRISPR-cr6-BL21 competent cells, then transform the homologous recombination plasmid into pCas9-pCRISPR-cr6-BL21 competent cells to obtain three-plasmid BL21 containing homologous recombination plasmid, pCas9 plasmid and pCRISPR-cr6 plasmid; S4: Construction, screening and verification of reporter phage T4::Nluc. First, the T4 phage was infected with three plasmids BL21 by double-layer agar plate method, and PCR templates were prepared for PCR and agarose gel electrophoresis. The PCR reaction procedure was the same as before. At the same time, a control group with T4 phage as template was set up to screen the successfully recombined T4::Nluc. The T4::Nluc that was successfully verified by PCR was further subjected to Sanger sequencing to ensure that T4::Nluc was successfully constructed; S5: Evaluate the detection performance of T4::Nluc, including the detection limit of T4::Nluc, the clinical strain detection coverage of T4::Nluc, the detection specificity of T4::Nluc and the effect of urine matrix on the detection performance of T4::Nluc.

2. A method for rapid and accurate detection of urinary tract infection based on bacteriophage according to claim 1, characterized in that: The construction of the CRISPR / Cas9 system in S1 specifically includes: in the CRISPR / Cas9 system, the selection of sgRNA is also a major rate-limiting factor for gene editing efficiency. First, a crRNA with a high cleavage efficiency for the T4 non-essential gene soc, soc-cr6, is selected from the crRNA library, and a BsaI restriction site sequence is added to both ends of the sequence, which is phosphorylated and annealed into a double-stranded DNA as the sgRNA to be used; The pCRISPR circular plasmid was digested with BsaI enzyme overnight at 37°C, and the success of the digestion was confirmed by agarose gel electrophoresis. The cr6 and the obtained pCRISPR linearized plasmid were connected with T4 DNA ligase at 4°C overnight. The ligation product was transformed into Escherichia coli BL21 competent cells and spread on solid LB agar plates containing a final concentration of 50 μg / mL kanamycin. The plates were cultured at 37°C overnight, and the monoclonal colonies after culture were picked for colony PCR and Sanger sequencing to ensure that the pCRISPR-cr6 plasmid was constructed correctly. The PCR reaction procedure was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 40 s, 72°C final extension for 5 min after 35 cycles, and then cooled to 4°C for storage. The verified pCRISPR-cr6-BL21 was cultured overnight, and the pCRISPR-cr6 plasmid was extracted using the FastPure Plasmid Mini Kit and stored at -20°C for later use; The pCas9 plasmid and pCRISPR-cr6 plasmid were transformed into Escherichia coli BL21 competent cells in sequence, and colony PCR was performed to verify the successful transformation of the plasmids. The PCR reaction procedure was the same as before, and pCas9-pCRISPR-cr6-BL21 was obtained, that is, the CRISPR / Cas9 system was successfully constructed.

3. A rapid and accurate detection method for urinary tract infection based on bacteriophage according to claim 2, characterized in that: The construction of homologous recombination plasmid in S2 specifically includes: selecting the upstream 1041bp and downstream 1000bp sequences of the soc gene as the upstream and downstream homology arms, and designing primers UHA-F, UHA-R, Nluc-F, Nluc-R, DHA-F, and DHA-R with 20bp sequence overlap at both ends for the upstream homology arm sequence, Nanoluc (Nluc) gene region, and downstream homology arm sequence; PCR amplified the upstream and downstream homology arms and Nanoluc gene region fragments, and agarose gel electrophoresis confirmed that the band position was correct. FastPure Gel DNA Extraction Mini Kit was used for gel recovery, and the gel-recovered product was used as a template for PCR verification again to ensure that the recovered DNA fragment was correct. The PCR reaction procedure was the same as above. ClonExpress Ultra OneStep Cloning Kit was used to construct the reaction system according to the instructions of the kit. The reaction was carried out at 50°C for 40 minutes, and Gibson assembly was performed, that is, the upstream 1041bp homology arm sequence, the Nluc gene region and the downstream 1000bp sequence were sequentially integrated into the pCE-Zero linearized plasmid to construct a circular homologous recombination plasmid, transformed BL21 competent cells, and coated on ampicillin (50ug / mL) and kanamycin (50ug / mL) double resistance plates. After overnight culture at 37°C, single clones were picked for colony PCR. The PCR reaction procedure was the same as above to ensure that the homologous recombination plasmid was constructed correctly and the homologous recombination plasmid was extracted.

4. A method for rapid and accurate detection of urinary tract infection based on bacteriophage according to claim 3, characterized in that: The construction of the three-plasmid BL21 in S3 specifically includes: using 0.1 mol / L calcium chloride solution to prepare pCas9-pCRISPR-cr6-BL21 as competent cells, culturing pCas9-pCRISPR-cr6-BL21 at 37°C, 200 rpm shaking incubator to logarithmic phase, ice bathing for 10 minutes, centrifuging at 4°C, 4000 rpm for 10 minutes, discarding the supernatant, adding an equal volume of 4°C pre-cooled 0.1 mol / L calcium chloride solution to resuspend the precipitate, ice bathing for 30 minutes, centrifuging again at 4°C, 4000 rpm for 10 minutes, discarding the supernatant, adding an appropriate amount of 4°C pre-cooled 0.1 mol / L calcium chloride solution to resuspend the precipitate, and obtaining pCas9-pCRISPR-cr6-BL21 competent cells; The homologous recombination plasmid was transformed into pCas9-pCRISPR-cr6-BL21 competent cells and spread on a triple-resistance plate containing ampicillin (50 μg / mL), kanamycin (50 μg / mL) and chloramphenicol (30 μg / mL). After overnight culture at 37°C, single clones were picked for colony PCR to verify the successful transformation of the homologous recombination plasmid, and a three-plasmid BL21 containing the homologous recombination plasmid, pCas9 plasmid and pCRISPR-cr6 plasmid was obtained.

5. A method for rapid and accurate detection of urinary tract infection based on bacteriophage according to claim 4, characterized in that: The construction, screening and verification of the reporter phage T4::Nluc in S4 specifically includes: using a double-layer agar plate method to infect the T4 phage with the three-plasmid BL21, that is, mixing 200 μL of the three-plasmid BL21 in the logarithmic phase with 100 μL of the T4 phage with an appropriate titer, leaving it at room temperature for 15 minutes, adding it to 0.625% semi-solid LB agar melted at 55°C, mixing it and immediately pouring it onto a solid LB plate, and after solidification, placing it in a 37°C incubator for overnight culture; 21 plaques were selected from the single plaques produced after overnight culture, 0.5 μL of phage solution in the center of the plaque was taken and added to 10 μL (10 mM / L) EDTA solution, and heat-shocked at 65°C for 10 min, cooled to room temperature, and used as a template for PCR and agarose gel electrophoresis. The PCR reaction procedure was the same as before, and a control group with T4 phage as a template was set up to screen the successfully recombined T4::Nluc. After agarose gel electrophoresis of the PCR product, T4 will produce a band of 2358 bp, while T4::Nluc will produce a band of 2837 bp. The T4::Nluc that was successfully verified by PCR was further subjected to Sanger sequencing to ensure that T4::Nluc was successfully constructed; The successfully verified T4::Nluc was co-cultured with pCas9-pCRISPR-cr6-BL21, and the bacteria and their fragments were filtered through a 0.22 μm bacterial filter to obtain the culture medium of T4::Nluc, which was placed at 4°C for use.

6. A method for rapid and accurate detection of urinary tract infection based on bacteriophage according to claim 5, characterized in that: The evaluation of the detection limit of T4::Nluc in S5 specifically includes: BL21 Escherichia coli was cultured overnight and colonies were counted, and 10-fold serial dilutions were made to a concentration of 1×10 1 CFU / mL~1×10 6 CFU / mL of bacterial solution was mixed with an appropriate amount of T4::Nluc to make the final concentration 3×10 6 PFU / mL, co-cultured at 37°C, 200 rpm on a shaker for 7 hours. During this period, 50 μL of co-culture solution was taken out every 10 minutes, 20 μL of Nluc reaction substrate was added, and incubated at room temperature for 3 minutes. The luminescence value was measured by a multifunctional microplate reader, and the time-luminescence curves of different bacterial concentration groups were plotted with time as the horizontal axis and the luminescence value as the vertical axis, where the threshold value = the average background luminescence value plus three times the standard deviation (SD), and the detection limit was the minimum bacterial concentration required to produce a luminescence signal above the threshold.

7. A method for rapid and accurate detection of urinary tract infection based on bacteriophage according to claim 5, characterized in that: The evaluation of the clinical strain detection coverage of T4::Nluc in S5 specifically includes: using a double-layer agar plate spot method and a luminescence assay method to determine the detection coverage of T4::Nluc in 104 strains of Escherichia coli isolated from clinical urine; The double-layer agar plate spot method refers to mixing 200 μL of clinical E. coli culture after overnight culture with 0.625% semi-solid LB agar melted at 55°C, and immediately pouring it on the LB solid plate. After solidification, take 3 μL of T4::Nluc culture solution, spot it on the double-layer agar plate, place it in a 37°C incubator for overnight culture, and observe whether plaques are formed. The formation of plaques indicates that T4::Nluc can recognize and lyse the clinical bacteria, otherwise it cannot recognize and lyse; The luminescence assay method refers to co-culturing the clinical bacterial culture overnight with an appropriate amount of T4::Nluc for 3.5 hours, taking 50 μL of the co-culture solution, adding 20 μL of substrate, and incubating at room temperature for 3 minutes. The luminescence value is used to determine whether the strain can be identified and lysed.

8. A method for rapid and accurate detection of urinary tract infection based on bacteriophage according to claim 5, characterized in that: The evaluation of the detection specificity of T4::Nluc in S5 specifically includes: BL21 (positive control) and 1 strain each of Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus hominis isolated from clinical urine are cultured overnight at 37°C and 200rpm in a shaking incubator, and then mixed with an appropriate amount of T4::Nluc, and the co-culture is continued for 5 hours. During this period, 50 μL of the co-culture solution is taken out every 10 minutes, 20 μL of substrate is added, and after incubation at room temperature for 3 minutes, the luminescence value is measured, and the time-luminescence curve is plotted. At the same time, only the T4::Nluc culture solution group is set as a negative control. If T4::Nluc only produces a detectable luminescent signal after co-culture with BL21 Escherichia coli, and no detectable luminescent signal is produced after co-culture with several other common urinary tract infection pathogens, that is, T4::Nluc does not recognize and lyse several other pathogens, there is no cross reaction, and it has extremely high detection specificity.

9. A method for rapid and accurate detection of urinary tract infection based on bacteriophage according to claim 5, characterized in that: The evaluation of the effect of urine matrix on T4::Nluc detection performance in S5 specifically includes: collecting clean midstream urine from healthy people from the clinic, filtering and sterilizing with a 0.22 μm bacterial filter, adding the filtered urine to the Escherichia coli solution, so that the proportion of urine matrix in each group is 0%, 20%, 40%, 60%, 80%, and 100%, respectively, while ensuring that the concentration of Escherichia coli in each group is consistent (2.72×10 7 CFU / mL), and an LB liquid culture medium group and a urine group were also set up. Appropriate amount of T4::Nluc was added to the above groups, and co-cultured at 37°C, 200 rpm in a shaking incubator for 5 h. Every 10 min, 50 μL of the co-culture solution was taken out, 20 μL of substrate was added, and the cells were incubated at room temperature for 3 min. The luminescence value was measured and the time-luminescence curve was drawn.

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