Acinetobacter baumannii phage-magnetic bead conjugate and method for detecting bacteria

Abstract

The invention discloses an acinetobacter baumannii phage-magnetic bead conjugate and a method for detecting bacteria, and belongs to the technical field of biomedicine. The conjugate is a compound obtained by coupling an acinetobacter baumannii phage vBAbaMB9 and a micron magnetic bead, and when the conjugate is used for bacterial detection, the acinetobacter baumannii can be rapidly and efficiently captured and enriched through the specific recognition of the phage and the adsorption effect of the micron magnetic bead, so that the specific recognition and rapid detection of the acinetobacter baumannii are realized. According to the method, the acinetobacter baumannii can be specifically enriched and separated, a bacteria detection result is directly observed under a confocal microscope, the time consumption is short, the sensitivity is high, the detection efficiency of the acinetobacter baumannii is greatly improved, the treatment of clinical bacterial infection is facilitated, and the method has very important significance on timely diagnosis and treatment of the acinetobacter baumannii.

Description

Technical Field

[0001] The present invention relates to an Acinetobacter baumannii phage-magnetic bead conjugate and a method for detecting bacteria, belonging to the field of biomedical technology. Background Art

[0002] Acinetobacter baumannii is a Gram-negative pathogenic bacterium that widely exists in the environment and often causes hospital-acquired infections. It has a strong environmental adaptability, can effectively resist heat and humidity, ultraviolet rays and conventional chemical disinfectants, and can survive in the hospital environment for a long time. Research shows that Acinetobacter baumannii present in the air of the intensive care unit (ICU) may still cause new infections several months later. Acinetobacter baumannii is highly resistant to a variety of commonly used antibiotics. In a study in Spain, 47.5% of Acinetobacter baumannii isolates were multi-drug resistant strains; the CHINET China Bacterial Resistance Surveillance in 2021 showed that the resistance rates of Acinetobacter baumannii to imipenem and meropenem were 65.6% and 66.5% respectively. Acinetobacter baumannii is one of the most important and deadly multi-drug resistant pathogens in the intensive care unit. In China, the detection rate of Acinetobacter baumannii in the ICU can reach 20%, and the mortality rate of ventilator-associated pneumonia (VAP) caused by it is as high as 40-70%; another Meta-analysis showed that the risk of death from multi-drug resistant bacterial infections in patients infected with Acinetobacter baumannii is 5.90 times that of patients without multi-drug resistance. It can be seen that Acinetobacter baumannii poses a great threat to human health. Timely diagnosis and treatment are important means to block the progression and spread of Acinetobacter baumannii. Therefore, it is urgent and necessary to accelerate the development of new diagnostic technologies and establish a rapid, convenient, specific, sensitive and accurate detection method for Acinetobacter baumannii for human health.

[0003] Immunomagnetic bead separation technology has been widely used in the separation and enrichment of specific pathogens. This technology uses the antigen-antibody reaction to bind magnetic microspheres to target pathogens to form immune complexes. Under the action of a magnetic field, the target pathogens can be quickly attracted and move directionally, thus achieving efficient capture, enrichment and separation, which can significantly improve the sensitivity and accuracy of pathogenic bacteria detection. However, this technology cannot achieve specific detection of pathogenic bacteria. As a kind of bacteriophage, a bacterial virus, it specifically parasitizes inside bacterial cells and precisely recognizes and adsorbs to specific receptors on the surface of host bacteria through specific receptor binding sites at the tail. This adsorption process has extremely high specificity. Combining it with immunomagnetic bead separation technology to capture and enrich Acinetobacter baumannii can achieve specific recognition and rapid detection of Acinetobacter baumannii. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an Acinetobacter baumannii phage-magnetic bead conjugate and a method for detecting bacteria. The Acinetobacter baumannii phage vB_AbaM_B9 is conjugated with micron magnetic beads to obtain the Acinetobacter baumannii phage-magnetic bead conjugate, which can specifically recognize bacteria, realize rapid detection of bacteria, and has high detection efficiency.

[0005] To solve the above technical problems, the technical solution provided by the present invention is: An Acinetobacter baumannii phage-magnetic bead conjugate, which is a complex obtained by conjugating Acinetobacter baumannii phage with micron magnetic beads.

[0006] The Acinetobacter baumannii phage-magnetic bead conjugate is a novel composite material, which can specifically recognize and bind to target bacteria, has a higher bacterial capture efficiency, can rapidly detect Acinetobacter baumannii, and provides a technical basis for the timely diagnosis and treatment of Acinetobacter baumannii.

[0007] Furthermore, the Acinetobacter baumannii phage is Acinetobacter baumannii phage vB_AbaM_B9, and the GenBank accession number of its gene sequence is MH133207. The present invention selects the Acinetobacter baumannii phage strain type vB_AbaM_B9, which is more suitable for application in the field of clinical medical testing, especially has a specific recognition advantage for Acinetobacter baumannii commonly found in hospital environments. Moreover, using phage as the recognition element has many advantages such as high specificity, strong environmental tolerance, strong signal amplification ability, easy access and production, and high safety. The production cost is greatly reduced, and it can effectively distinguish between live bacteria and dead bacteria, solving the problem of dead bacteria interfering with the detection results in traditional methods.

[0008] Furthermore, the particle size of the micron magnetic beads is 3.5 - 4 μm. The present invention selects micron magnetic beads with a relatively large particle size. During magnetic separation, the sedimentation speed of the magnetic beads in the magnetic field is relatively fast, which can quickly separate from the liquid, reducing the separation time and improving the experimental efficiency. In addition, the contact area with cells is relatively small, causing less physical stimulation and damage to cells, and can handle cells more gently, maintaining the activity and function of cells. Moreover, micron magnetic beads can provide more binding sites, have strong binding ability, can effectively and rapidly enrich target substances, and can be quickly attracted and separated under the action of an external magnetic field, improving the repeatability and reliability of the experiment.

[0009] The preparation of an Acinetobacter baumannii phage-magnetic bead conjugate includes the following steps: (1) Activate the carboxyl groups of the micron magnetic beads to obtain activated carboxyl micron magnetic beads; (2) Couple Acinetobacter baumannii phage vB_AbaM_B9 with activated carboxyl micro magnetic beads to obtain an Acinetobacter baumannii phage vB_AbaM_B9-micro magnetic bead conjugate.

[0010] Further, the preparation method of the activated carboxyl micro magnetic beads in step (1) is as follows: Disperse the micro magnetic beads with a particle size of 3.5 - 4 μm by using a vortex device, perform magnetic separation by using a magnetic separation device, then wash with PBS buffer three times, perform magnetic separation, remove the supernatant, retain the magnetic beads, and then add EDC solution and NHS solution, and react at 37 °C for 1 h to obtain activated carboxyl magnetic beads.

[0011] Further, the magnetic separation device is DynaMagTM-2REF 12321D L0T4619002 of Thermo Fisher Scientific Company.

[0012] Further, the concentrations of both the EDC solution and the NHS solution are 20 mg / mL.

[0013] Further, the preparation method of the Acinetobacter baumannii phage vB_AbaM_B9-micro magnetic bead conjugate in step (2) is specifically as follows: (a) Wash three times with PBS buffer to remove the excess NHS solution and EDC solution, and then resuspend with PBS buffer to obtain a suspension; (b) Add Acinetobacter baumannii phage vB_AbaM_B9 to the suspension, gently mix evenly, couple at 37 °C for 4 h; then perform magnetic separation, resuspend with PBS buffer containing 3% BSA (w / v), incubate at 37 °C for 1 h to block the residual sites, perform magnetic separation again to obtain an Acinetobacter baumannii phage vB_AbaM_B9-micro magnetic bead conjugate, wash three times with PBS buffer, and store in PBS buffer containing 0.1% BSA (w / v) at 4 °C for use.

[0014] Further, the molar concentration of the PBS buffer is 0.01 mol / L and the pH is 7.4.

[0015] Note: NHS is N-hydroxysulfosuccinimide, EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, BSA is bovine serum albumin, and PBS buffer is phosphate buffer.

[0016] A method for detecting bacteria by using an Acinetobacter baumannii phage-magnetic bead conjugate includes the following steps: (1) Add the sample to be tested containing Acinetobacter baumannii into the Acinetobacter baumannii phage-magnetic bead conjugate, vortex for 15 s, then place it on a constant temperature mixing device, and incubate at 37 °C and 200 r / min for 30 minutes. Then perform magnetic absorption for 5 minutes, discard the supernatant, and resuspend the remaining precipitate with 40 μL of 4% paraformaldehyde. Wash the precipitate 3 times with 40 μL of PBS buffer to obtain the sample; (2) Take 2 μL of the sample and evenly distribute it on the surface of the gel block. After drying, cover with a cover slip and seal the slide. Perform confocal microscopy imaging and observe the enrichment and separation effect under the microscope.

[0017] Further, in step (1), the volume ratio of the Acinetobacter baumannii phage-magnetic bead conjugate to the sample to be tested containing Acinetobacter baumannii is 1:5.

[0018] The Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate prepared by the present invention can specifically recognize Acinetobacter baumannii through the specific recognition of the phage and the adsorption of micron magnetic beads. It can effectively enrich, separate and detect Acinetobacter baumannii specifically. The detection time is short, the method is simple and convenient, and the sensitivity is high. It greatly improves the efficient diagnostic efficiency for the identification of Acinetobacter baumannii infection in the early stage of disease infection, and can realize the specific recognition and rapid detection of Acinetobacter baumannii, especially with good application in the field of clinical medicine.

[0019] Compared with the prior art, the beneficial effects of the present invention are: (1) The Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate prepared in this invention can specifically enrich and separate Acinetobacter baumannii. By directly observing the bacterial detection results under a confocal microscope, the time consumption is short and the sensitivity is high, which greatly improves the detection efficiency of Pseudomonas aeruginosa and is beneficial to the treatment of clinical bacterial infections. It is of great significance for the timely diagnosis and treatment of Acinetobacter baumannii.

[0020] (2) Acinetobacter baumannii has strong drug resistance and a high fatality rate. The existing detection technologies have limitations such as long time and high cost. Compared with the traditional technologies, the detection method of the present invention has a short detection time, a fast detection speed, and a higher bacterial capture efficiency, and is suitable for clinical medical detection and treatment.

[0021] (3) The present invention optimizes the required phage and selects the Acinetobacter baumannii phage strain vB_AbaM_B9, which is more suitable for application in the field of clinical medical testing, especially having a specific recognition advantage for Acinetobacter baumannii commonly found in hospital environments.

[0022] (4)The present invention optimizes and screens the magnetic beads used, selects micron magnetic beads, which have less separation time, more binding sites, stronger binding ability, can enrich the target substance more effectively, improve the success rate and yield of the experiment; and have relatively less physical stimulation and damage to cells, can process cells more gently, and maintain the activity and function of cells. In addition, the preparation process of micron magnetic beads is relatively simple, the production cost is low, and in large-scale applications, using micron magnetic beads can reduce the experimental cost and improve economic benefits. Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the preparation of Acinetobacter baumannii phage-magnetic bead conjugate and the capture of bacteria; Figure 2 It is a graph showing the capture results of the Acinetobacter baumannii phage-magnetic bead conjugate of Example 1 against different concentrations of Acinetobacter baumannii; Figure 3 It is a graph showing the capture efficiency of the Acinetobacter baumannii phage-magnetic bead conjugate of Example 1 in different concentrations of Acinetobacter baumannii; Figure 4 It is a comparative graph of the results of capturing bacteria by the phage-magnetic bead group, fluorescent phage-magnetic bead group and single magnetic bead control group in Experimental Example 3; Figure 5 It is a comparative graph of the capture efficiency of the phage-magnetic bead group, fluorescent phage-magnetic bead group and single magnetic bead control group against bacteria in Experimental Example 3. Detailed Embodiments

[0024] Example 1 The preparation of an Acinetobacter baumannii phage-magnetic bead conjugate includes the following steps: (1) Vortex and disperse 50 μL of micron magnetic beads with a particle size of 3.5 μm, perform magnetic separation using a magnetic separation device, then wash 3 times with PBS buffer solution, perform magnetic separation, remove the supernatant, retain the magnetic beads, and then add 100 μL of EDC solution and 100 μL of NHS solution, and react at 37 °C for 1 h to obtain activated carboxyl magnetic beads; (2) Wash three times with PBS buffer solution to remove the excess NHS solution and EDC solution, and then resuspend with 100 μL of PBS buffer solution to obtain a suspension; (3) Add 100 μL of Acinetobacter baumannii phage vB_AbaM_B9 to the suspension, gently mix well, and couple at 37 °C for 4 h; then perform magnetic separation, resuspend with 1.5 mL of PBS buffer containing 3% BSA (w / v), incubate at 37 °C for 1 h to block residual sites, perform magnetic separation again to obtain the Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate, wash three times with PBS buffer, and store in 500 μL of PBS buffer containing 0.1% BSA (w / v) at 4 °C for later use.

[0025] Example 2 Preparation of an Acinetobacter baumannii phage-magnetic bead conjugate, comprising the following steps: (1) Disperse 50 μL of micron magnetic beads with a particle size of 4.0 μm using a vortex device, perform magnetic separation using a magnetic separation device, then wash 3 times with PBS buffer, perform magnetic separation, remove the supernatant, retain the magnetic beads, and then add 100 μL of EDC solution and 100 μL of NHS solution, and react at 37 °C for 1 h to obtain activated carboxyl magnetic beads; (2) Wash three times with PBS buffer to remove excess NHS solution and EDC solution, and then resuspend with 100 μL of PBS buffer to obtain a suspension; (3) Add 100 μL of Acinetobacter baumannii phage vB_AbaM_B9 to the suspension, gently mix well, and couple at 37 °C for 4 h; then perform magnetic separation, resuspend with 1.5 mL of PBS buffer containing 3% BSA (w / v), incubate at 37 °C for 1 h to block residual sites, perform magnetic separation again to obtain the Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate, wash three times with PBS buffer, and store in 500 μL of PBS buffer containing 0.1% BSA (w / v) at 4 °C for later use.

[0026] Example 3 A method for detecting bacteria using an Acinetobacter baumannii phage-magnetic bead conjugate, comprising the following steps: (1) Add a sample containing Acinetobacter baumannii to the Acinetobacter baumannii phage-magnetic bead conjugate, vortex and mix well for 15 s, then place it on a constant temperature mixing device, and act at a temperature of 37 °C and a rotation speed of 200 r / min for 30 minutes, then perform magnetic separation for 5 minutes, remove the supernatant, resuspend the remaining precipitate with 40 μL of 4% paraformaldehyde, and wash the precipitate 3 times with 40 μL of PBS buffer to obtain a sample; (2) Take 2 μL of the sample and evenly distribute it on the surface of the gel block. After drying, cover it with a cover slip, seal the slide, and perform confocal microscopy imaging to observe the enrichment and separation effect under the microscope.

[0027] Experimental Example 1 The experimental principle of preparing Acinetobacter baumannii phage-magnetic bead conjugates and their enrichment and capture of bacteria is represented by a schematic diagram.

[0028] As Figure 1 shown, the EDC / NHS method was used to chemically conjugate Acinetobacter baumannii phage vB_AbaM_B9 and micron magnetic beads to prepare Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugates. The specific recognition of Acinetobacter baumannii by Acinetobacter baumannii phage vB_AbaM_B9 was used to capture bacteria, thereby realizing the specific and efficient detection of Acinetobacter baumannii, which is beneficial to the clinical diagnosis and treatment of Acinetobacter baumannii.

[0029] Experimental Example 2 Verification of the capture efficiency of Acinetobacter baumannii phage-magnetic bead conjugates for different concentrations of Acinetobacter baumannii: (1) Pick a single colony of Acinetobacter baumannii into LB medium and culture at 37 °C and 200 rpm for 4 hours until the concentration of Acinetobacter baumannii is 10 5 CFU / mL; (2) Dilute the Acinetobacter baumannii solution in step (1) to concentrations of [10 5 , 10 4 , 10 3 , 10 2 , 10] CFU / mL; (3) Prepare 5 EP tubes, label them as the phage-magnetic bead group, take 40 μL of the Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate from Example 1 and place them in the EP tubes respectively, then add 40 μL of the bacterial dilutions with different concentrations in step (2), label the concentrations, and vortex for 15 s; (4) Place the 5 EP tubes on a constant temperature mixing device, set the incubation temperature to 37 °C, adjust the mixing speed to 200 r / min to evenly disperse the enriched magnetic beads in the solution, and set the incubation time to 30 minutes; (5) Then magnetically attract for 5 minutes, remove the supernatant, resuspend the remaining precipitate with 40 μL of 4% paraformaldehyde, and wash the precipitate 3 times with 40 μL of PBS to obtain the samples; (6) Take 2 μL of the samples with different concentrations obtained in step (5) and evenly distribute them on the surface of the gel block. After drying, cover with a cover slip, seal the slide, image with a confocal microscope, observe the enrichment and separation effect under the microscope, and calculate the capture efficiency of the bacteria.

[0030] The test results are as shown in the appendix Figures 2-3 shown.

[0031] As Figure 2As shown, the Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate was used to enrich and separate different concentrations of Acinetobacter baumannii dilutions, and Acinetobacter baumannii could be quickly captured. Among them, when Acinetobacter baumannii was diluted to 10 CFU / mL, the Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate could still quickly capture Acinetobacter baumannii, indicating that the Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate has good Acinetobacter baumannii capture ability. As Figure 3 shown, the capture efficiency of the Acinetobacter baumannii phage vB_AbaM_B9-magnetic bead conjugate in bacteria at each concentration gradient was calculated. When the concentration of bacteria was as low as 10 CFU / mL, the capture efficiency of the Acinetobacter baumannii phage vB_AbaM_B9-magnetic bead conjugate was still as high as 77.9%, indicating that the Acinetobacter baumannii phage vB_AbaM_B9-magnetic bead conjugate of the present invention has excellent capture ability, still has a high capture efficiency in a low-concentration bacterial environment, has a fast capture speed for bacteria, and a high capture efficiency.

[0032] Experimental Example 3 Comparison of the capture of bacteria by Acinetobacter baumannii phage-magnetic bead conjugate, fluorescent Acinetobacter baumannii phage vB_AbaM_B9-magnetic bead conjugate and single magnetic bead: The Acinetobacter baumannii phage-magnetic bead conjugate of Example 1 (denoted as the phage-magnetic bead group), the fluorescent Acinetobacter baumannii phage vB_AbaM_B9-magnetic bead conjugate (denoted as the fluorescent phage-magnetic bead group) and the single magnetic bead (denoted as the single magnetic bead control group) were respectively added to the Acinetobacter baumannii dilution with a concentration of 10 4 CFU / mL, and the enrichment and separation detection effect was observed under the microscope. The detection method was the same as that in Experimental Example 2. The detection effect is as shown in the appendix Figures 4-5 shown.

[0033] Among them, the preparation method of the fluorescent Acinetobacter baumannii phage vB_AbaM_B9-magnetic bead conjugate is as follows: (1) Culture Acinetobacter baumannii phage vB_AbaM_B9 with a titer of 1x10 pfu / mL to obtain the cultured Acinetobacter baumannii phage vB_AbaM_B9; (2) Prepare a sodium bicarbonate solution with a concentration of 0.3 mol / L containing Cy5.5 NHS ester; the concentration of Cy5.5 NHS ester in the sodium bicarbonate solution is 1 mg / mL; (3) Suspend the cultured Acinetobacter baumannii phage vB_AbaM_B9 in 1 mL of a sodium bicarbonate solution with a concentration of 0.3 mol / L and incubate it in the dark at room temperature for 2 hours to obtain a Cy5.5-labeled phage solution; (4) Add PBS buffer to increase the volume of the Cy5.5-labeled phage solution to 10 mL, then add polyethylene glycol to obtain a mixed solution. Filter and wash to obtain purified Cy5.5-labeled phage; the concentration of PEG in the mixed solution is 10%. (5) Resuspend the purified Cy5.5-labeled phage in 1 mL of PBS buffer and titrate to determine the phage titer to obtain the fluorescent Acinetobacter baumannii phage vB_AbaM_B9. (6) Take 50 μL of magnetic beads (particle size: 3.5 μm), disperse them by vortexing with a vortex device, perform magnetic separation with a magnetic device, wash 3 times with 3 mL of PBS buffer, then perform magnetic separation, remove the supernatant, add 100 μL of EDC solution (20 mg / mL) and 100 μL of NHS solution (20 mg / mL), and react at 37 °C for 1 h to obtain activated carboxyl magnetic beads. (7) Wash three times with PBS buffer (0.01 M, pH 7.4) to remove the excess NHS solution and EDC solution, then resuspend with 100 μL of PBS buffer to obtain a suspension. (8) Add 100 μL of the fluorescent Acinetobacter baumannii phage vB_AbaM_B9 to the suspension, gently mix, and couple at 37 °C for 4 h; after 4 h, perform magnetic separation, resuspend with 1.5 mL of PBS buffer (0.01 M, pH 7.4) containing 3% BSA (w / v), and incubate at 37 °C for 1 h to block the residual sites. Perform magnetic separation again to obtain the fluorescent Acinetobacter baumannii phage vB_AbaM_B9-magnetic bead conjugate. Wash three times with PBS buffer and store in 500 μL of PBS containing 0.1% BSA at 4 °C for later use.

[0034] The preparation method of the single magnetic bead control group is as follows: (1) Take 50 μL of magnetic beads (particle size: 3.5 μm), disperse them by vortexing with a vortex device, perform magnetic separation with a magnetic device, wash 3 times with 3 mL of PBS, then perform magnetic separation, and remove the supernatant; (2) Add 100 μL of EDC solution (20 mg / mL) and 100 μL of NHS solution (20 mg / mL), and react at 37 °C for 1 h to obtain activated carboxyl magnetic beads; (3) Wash three times with PBS buffer (0.01 M, pH 7.4) to remove the excess NHS solution and EDC solution, then resuspend with 100 μL of PBS buffer to obtain a suspension; (4) Perform magnetic separation on the suspension, resuspend it with 1.5 mL of PBS buffer (0.01 M, pH 7.4) containing 3% BSA (w / v), and incubate at 37 °C for 1 h. Perform magnetic separation again to obtain single magnetic bead reagent, then wash it three times with PBS and store it in 500 μL of PBS containing 0.1% BSA at 4 °C for later use.

[0035] As Figures 4-5 shown, conjugates in both the fluorescent phage-magnetic bead group and the phage-magnetic bead group can successfully capture Acinetobacter baumannii. In terms of capture efficiency, the capture efficiencies of the fluorescent phage-magnetic bead group and the phage-magnetic bead group for Acinetobacter baumannii are close, and the difference is not statistically significant; while in the single magnetic bead control group, there are a large number of Acinetobacter baumannii around the magnetic beads, but they cannot be successfully captured. Compared with the phage-magnetic bead group, the capture efficiency of the single magnetic bead control group for Acinetobacter baumannii is significantly reduced, and the difference is statistically significant; this indicates that the capture efficiencies of the fluorescent phage-magnetic bead group and the phage-magnetic bead group for Acinetobacter baumannii are close, and both are significantly better than the unmodified single magnetic bead control group.

[0036] In addition, although the fluorescent phage-magnetic bead group can fluorescently stain the phage and visualize the synthesis effect of the conjugate, as shown in Table 1, the cost of the fluorescent staining agent required in the preparation process of commercially available fluorescent phages is about 2000 - 3500 yuan per 5 mg, and the staining process also takes 2 - 3 h. It can be seen that the fluorescent staining process takes a lot of time and increases the cost. Therefore, for practical applications, the method of the present invention can specifically adsorb Acinetobacter baumannii, has a high capture efficiency for Acinetobacter baumannii, a high detection efficiency, a fast detection speed, and can also omit the staining step, saving economic cost and time cost.

[0037] Table 1. Cost overview of fluorescent staining agents required for commercially available fluorescent phages Experimental Example 4 Select five methods to detect Acinetobacter baumannii and compare their detection times. The results are listed in Table 2.

[0038] Table 2. Detection times of Acinetobacter baumannii As can be seen from Table 2, when different methods are selected to detect Acinetobacter baumannii, there are significant differences in their detection times. Among them, the method of the present invention has the shortest detection time, only 30 - 40 minutes. It can be seen that the method of the present invention has a short detection time and high sensitivity, greatly improving the detection efficiency of Acinetobacter baumannii. Compared with traditional detection methods, the method of the present invention has a short detection time, high efficiency, simple operation, and accurate detection results.

Claims

1. An Acinetobacter baumannii phage-magnetic bead conjugate, characterized in that: The Acinetobacter baumannii phage-magnetic bead conjugate is a complex obtained by conjugating Acinetobacter baumannii phage with micron magnetic beads.

2. The Acinetobacter baumannii phage-magnetic bead conjugate according to claim 1, characterized in that: The Acinetobacter baumannii phage is Acinetobacter baumannii phage vB_AbaM_B9.

3. The Acinetobacter baumannii phage-magnetic bead conjugate according to claim 1, characterized in that: The micron magnetic beads have a particle size of 3.5 - 4 μm.

4. Preparation of the Acinetobacter baumannii phage-magnetic bead conjugate according to claim 1, characterized in that: It includes the following steps: (1) Activate the carboxyl groups of the micron magnetic beads to obtain activated carboxyl micron magnetic beads; (2) Conjugate Acinetobacter baumannii phage vB_AbaM_B9 with the activated carboxyl micron magnetic beads to obtain the Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate.

5. Preparation of the Acinetobacter baumannii phage-magnetic bead conjugate according to claim 4, characterized in that: The preparation method of the activated carboxyl micron magnetic beads in step (1) is as follows: Disperse the micron magnetic beads with a particle size of 3.5 - 4 μm by shaking with a vortex device, perform magnetic separation with a magnetic separation device, then wash 3 times with PBS buffer, perform magnetic separation, remove the supernatant, retain the magnetic beads, add EDC solution and NHS solution, and react at 37 °C for 1 h to obtain activated carboxyl magnetic beads.

6. The preparation of the Acinetobacter baumannii phage-magnetic bead conjugate according to claim 5, wherein: The concentrations of both the EDC solution and the NHS solution are 20 mg / mL.

7. Preparation of the Acinetobacter baumannii phage-magnetic bead conjugate according to claim 4, characterized in that: The preparation method of the Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate in step (2) is specifically as follows: (a) Wash three times with PBS buffer to remove the excess NHS solution and EDC solution, then resuspend with PBS buffer to obtain a suspension; (b) Add Acinetobacter baumannii phage vB_AbaM_B9 to the suspension, gently mix evenly, and conjugate at 37 °C for 4 h; Then perform magnetic separation, resuspend with PBS buffer containing 3% BSA (w / v), incubate at 37 °C for 1 h to block the residual sites, perform magnetic separation again to obtain the Acinetobacter baumannii phage vB_AbaM_B9-micron magnetic bead conjugate, wash three times with PBS buffer, and store in PBS buffer containing 0.1% BSA (w / v) at 4 °C for later use.

8. Preparation of the Acinetobacter baumannii phage-magnetic bead conjugate according to claim 5 or 7, characterized in that: The molar concentration of the PBS buffer is 0.01 mol / L and the pH is 7.

4.

9. A method for detecting bacteria using the Acinetobacter baumannii phage-magnetic bead conjugate according to claim 1, characterized in that: It includes the following steps: (1) Add the sample containing Acinetobacter baumannii to the Acinetobacter baumannii phage-magnetic bead conjugate, vortex and mix evenly for 15 s, then place it on a constant temperature mixing device, act at a temperature of 37 °C and a rotation speed of 200 r / min for 30 minutes, then perform magnetic separation for 5 minutes, remove the supernatant, resuspend the remaining precipitate with 40 μL of 4% paraformaldehyde, and wash the precipitate 3 times with 40 μL of PBS buffer to obtain a sample; (2) Take 2 μL of the sample and evenly distribute it on the surface of the gel block. After drying, cover with a cover slip, seal the slide, perform confocal microscopy imaging, and observe the enrichment and separation effect under the microscope.

Images