Pseudomonas aeruginosa bacteriophage PAZ1 and use thereof
By using the PAZ1-magnetic bead enrichment system, which is a P. aeruginosa phage coupled with magnetic beads, and combined with the ATP bioluminescence method, the problems of long detection time, high cost and complicated operation in the existing technology have been solved, and rapid and accurate detection of P. aeruginosa has been achieved.
Patent Information
- Application Number
- CN202411086588.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-08
- Publication Date
- 2026-01-02
- Estimated Expiration
- 2044-08-08
AI Technical Summary
Existing technologies for detecting Pseudomonas aeruginosa suffer from problems such as long detection time, high cost, cumbersome operation, and lack of specificity, making it difficult to achieve rapid and accurate detection.
A Pseudomonas aeruginosa phage PAZ1 was coupled with magnetic beads to construct a phage PAZ1-magnetic bead enrichment system, which was then used for specific identification and quantitative detection by ATP bioluminescence method.
It enables rapid and accurate detection of Pseudomonas aeruginosa within 30 minutes, with a detection limit of 607 CFU/mL, and is characterized by its low cost and comprehensive functionality.
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Figure CN118895255B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of foodborne pathogenic bacteria detection in food safety, and particularly relates to a Pseudomonas aeruginosa phage PAZ1 and application thereof. BACKGROUND
[0002] Pseudomonas aeruginosa is one of the most dangerous multi-drug resistant bacteria to human health and safety, and is widely distributed in soil, water, air, food and animals. When the immune system is attacked by Pseudomonas aeruginosa, serious infection may occur, leading to diseases such as gastrointestinal infection, consciousness disorder and shock. Pseudomonas aeruginosa is mainly spread through contamination of various water sources, and is one of the main causes of waterborne diseases. Pseudomonas aeruginosa can be detected in river water, sewage and even disinfected swimming pools. Therefore, specific and rapid detection of waterborne Pseudomonas aeruginosa is of great significance for the prevention and control of food safety hazards caused by Pseudomonas aeruginosa.
[0003] A new foodborne pathogenic bacteria detection method with specificity, rapidity and high sensitivity is the top priority to ensure food quality. At present, the detection of Pseudomonas aeruginosa in food mainly includes traditional culture-based methods and rapid detection methods. Although the traditional culture method has high accuracy, it takes a long time and further biochemical identification is required. The rapid detection based on molecular biology methods such as polymerase chain reaction (PCR) has high specificity and sensitivity, but also has some limitations. For example, the operation requirements are extremely strict, and the cost of instruments and reagents is high. In addition, enzyme-linked immunosorbent assay (ELISA) has good sensitivity and specificity, but the relatively complicated operation steps limit its further development in detection application.
[0004] Phages are the most abundant and diverse species of organisms on earth, and the number of phages in the environment reaches 10 31The first step of phage infecting bacteria is to specifically recognize and bind to the bacterial surface receptor, which makes it have the potential to develop biological recognition elements. Moreover, the preparation method of phage and its maturation also have good stability. Adenosine triphosphate (ATP) is a ubiquitous biological energy source in all living cells. In most bacterial species, the concentration of ATP in cells is roughly constant. The strong fluorescence signal generated by the bioluminescent (BL) system catalyzed by firefly luciferase has been used for high-sensitivity detection of ATP. Therefore, the number of bacteria can be quantified by a simple ATP BL method. Although the BL method lacks sufficient specificity, it can be used in combination with the specific recognition of phage to rapidly and accurately detect viable Pseudomonas aeruginosa.
[0005] The phage PAZ1 is coupled with magnetic beads to construct a phage PAZ1-magnetic bead enrichment system (Phage PAZ1-MBs). The enrichment system has strong specificity and does not recognize other interfering bacteria. In the direct detection of actual samples, the method successfully detects Pseudomonas aeruginosa with different concentrations. It is proved that the method can realize the qualitative and quantitative detection of Pseudomonas aeruginosa in actual samples. SUMMARY
[0006] The purpose of the present application is to overcome the shortcomings of the prior art, and provide a Pseudomonas aeruginosa phage PAZ1 and its application. Based on the characteristics of the Pseudomonas aeruginosa phage PAZ1, a reagent kit is made to quickly and accurately detect Pseudomonas aeruginosa in water samples. The reagent kit is inexpensive and has complete functions, rapid and accurate detection.
[0007] To achieve the above purpose, the technical scheme designed by the present application is as follows:
[0008] The present application provides a Pseudomonas aeruginosa bacteriophage PAZ1, which has a preservation number of CCTCC NO: M 2024545.
[0009] The Pseudomonas aeruginosa bacteriophage PAZ1 described above is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC NO: M 2024545, a preservation date of March 25, 2024, and an address of Wuhan University, Wuhan, China.
[0010] The Pseudomonas aeruginosa bacteriophage PAZ1 is separated and purified from sewage of a farm product market in Wuhan, has strong specificity and does not recognize bacteria of other species; the morphology of the bacteriophage PAZ1 is observed by using a transmission electron microscope, and the bacteriophage PAZ1 belongs to a filamentous bacteriophage; the bacteriophage PAZ1 has the characteristics of rapid adsorption of P. aeruginosa (the maximum adsorption rate can be reached in 15 min); and has high pH stability (6-12) and thermal stability (30-60 DEG C).
[0011] The application further provides a Pseudomonas aeruginosa bacteriophage nano magnetic bead conjugate PhagePAZ1-MBs, which is a complex of the Pseudomonas aeruginosa bacteriophage PAZ1 and carboxyl magnetic beads.
[0012] Further, the diameter of the carboxyl magnetic beads is 200 nm.
[0013] The application further provides a method for preparing PBS buffer containing the Pseudomonas aeruginosa bacteriophage nano magnetic bead conjugate PhagePAZ1-MBs, which comprises the following steps:
[0014] 1) activating the Pseudomonas aeruginosa bacteriophage PAZ1;
[0015] 2) activating the carboxyl magnetic beads to obtain activated carboxyl magnetic beads;
[0016] 3) coupling the Pseudomonas aeruginosa bacteriophage PAZ1 with the activated carboxyl magnetic beads to obtain PBS buffer containing the Pseudomonas aeruginosa bacteriophage nano magnetic bead conjugate PhagePAZ1-MBs, wherein the molar concentration of the PBS is 1 μmol / L, and the mass-volume concentration of bovine serum albumin is 15 g / L,
[0017] The content of the Pseudomonas aeruginosa bacteriophage nano magnetic bead conjugate PhagePAZ1-MBs in each milliliter of the PBS buffer is 50 μL.
[0018] Further, in the step 1), the activation method of the Pseudomonas aeruginosa bacteriophage PAZ1 is as follows:
[0019] The bacteriophage PAZ1 stock solution is mixed with P. aeruginosa CMCC 10104 in a logarithmic growth phase, liquid culture is carried out, 0.22 μm microporous filter membrane filtration is carried out, the bacteriophage PAZ1 liquid is obtained, and the bacteriophage liquid is stored at 4 DEG C; and the use titer of the bacteriophage liquid is: 10 10 PFU / mL.
[0020] The application further provides a kit for enriching and separating Pseudomonas aeruginosa, which comprises the PBS buffer containing the Pseudomonas aeruginosa phage nanometer magnetic bead conjugate PhagePAZ1-MBs.
[0021] Further, the kit further comprises a negative control, a positive control and a PBS buffer solution.
[0022] The negative control is a PBS buffer containing BSA, wherein the mass-volume concentration of the bovine serum albumin is 15 g / L.
[0023] The PBS buffer has a molar concentration of 1 μmol / L.
[0024] The positive control is a Pseudomonas aeruginosa positive control, and the Pseudomonas aeruginosa is Pseudomonas aeruginosa CMCC10104.
[0025] The application further provides a method for enriching and separating Pseudomonas aeruginosa by using the kit, comprising the following steps:
[0026] 1) mixing a sample containing Pseudomonas aeruginosa to be separated with the PBS buffer containing the Pseudomonas aeruginosa phage nanometer magnetic bead conjugate PhagePAZ1-MBs, and performing shaker culture at a temperature of 37 DEG C and a rotating speed of 180 rpm for 20 min to obtain a sample;
[0027] 2) placing the sample in a magnetic separator to fully separate the magnetic beads from the liquid, discarding the supernatant, washing with sterile PBS for three times, and then suspending in 1 mL of sterile PBS.
[0028]
[0029] In the formula, N0 is the concentration of Pseudomonas aeruginosa bacteria liquid in the initial sample, CFU / mL, N a is the concentration of Pseudomonas aeruginosa bacteria liquid not combined with the conjugate, CFU / mL.
[0030] The application further provides a method for rapidly detecting Pseudomonas aeruginosa by using the kit, comprising the following steps:
[0031] 1) mixing a sample to be detected with the PBS buffer containing the Pseudomonas aeruginosa phage nanometer magnetic bead conjugate PhagePAZ1-MBs, and performing shaker culture at a temperature of 37 DEG C and a rotating speed of 180 rpm for 20 min to obtain a sample;
[0032] 2) The sample is placed in a magnetic separator to separate the magnetic beads from the liquid, the supernatant is discarded, and the magnetic beads are resuspended in 100 μL of Tris-HCl buffer, 20 μL of 0.05% CTAB solution is added, and after 4 minutes, the magnetic separation is performed, 50 μL of the supernatant is added to a black enzyme-labeled plate, and 50 μL of luciferin-luciferase solution (0.25 mg / mL luciferin, 0.06 mg / mL luciferase) is added, and the RLUs are immediately measured using a fluorescence spectrophotometer;
[0033] 3) The standard curve is plotted with the logarithmic value of the Pseudomonas aeruginosa concentration as the abscissa and the logarithmic value of the RLU as the ordinate, and after obtaining the accurate RLU value, the total number of viable colonies of Pseudomonas aeruginosa in the sample can be converted.
[0034] Principles of the application:
[0035] Phages (natural viruses that infect bacteria) are a class of organisms that specifically adsorb bacteria, and have the characteristics of high specificity in recognizing host bacteria. Compared with antibodies, phages have the advantages of abundant sources, high natural specificity, and the ability to recognize and adsorb host bacteria. However, the strong lytic activity of phages can lead to rapid destruction of host bacteria, thereby hindering subsequent operations. However, the Pseudomonas aeruginosa phage PAZ1 of the present application is a phage with weak lytic activity, and has the advantage of being a biological recognition element. It is the above advantages of phages that make them have the potential for rapid detection of foodborne pathogenic bacteria.
[0036] Advantages of the application:
[0037] 1. The present application uses phage PAZ1 as a biological recognition element for specifically recognizing Pseudomonas aeruginosa, and couples it with carboxylated nanomagnetic beads to form a bifunctional detection probe with specific recognition of Pseudomonas aeruginosa and fluorescence signal, which can detect Pseudomonas aeruginosa in about 30 minutes, with a detection limit of 607 CFU / mL.
[0038] 2. The present application can be used as an effective means for detecting foodborne pathogenic bacteria, and further expands the application range of phages in the detection of waterborne food. BRIEF DESCRIPTION OF DRAWINGS
[0039] Figure 1 Figure 2 is a morphological feature diagram of phage PAZ1,
[0040] Figure 2A is a plaque diagram of phage PAZ1,
[0041] Figure 2B is a transmission electron micrograph of phage VPP1;
[0042] Figure 2 Figure 3 is a biological property diagram of phage PAZ1,
[0043] Figure A is a schematic diagram of the optimal multiplicity of infection of phage PAZ1,
[0044] Figure B is a graph of the adsorption rate curve of phage PAZ1,
[0045] Figure C is a graph of the one-step growth curve of phage PAZ1,
[0046] Figure D is a schematic diagram of the temperature stability of phage PAZ1,
[0047] Figure E is a schematic diagram of the pH stability of phage PAZ1.
[0048] Figure 3 Figure is a schematic diagram of the optimization of the capture rate conditions of the rapid fluorescent detection kit for Pseudomonas aeruginosa,
[0049] Figure A is a graph of the effect of the titer of phage PAZ1 on the capture efficiency of the conjugate Phage PAZ1-MBs,
[0050] Figure B is a graph of the effect of the amount of conjugate Phage PAZ1-MBs added on the capture efficiency, Figure C is a graph of the effect of the incubation temperature on the capture efficiency of the conjugate Phage PAZ1-MBs, and Figure D is a graph of the effect of the incubation time on the capture efficiency of the conjugate Phage PAZ1-MBs.
[0051] Figure 4 Figure is an analysis graph of the optimization of the ATP bioluminescence method conditions of the rapid fluorescent detection kit for Pseudomonas aeruginosa.
[0052] Figure A is a graph of the effect of the concentration of the extraction agent CTAB on the luminescence intensity,
[0053] Figure B is a graph of the effect of the reaction extraction time on the luminescence intensity,
[0054] Figure C is a graph of the effect of the luciferase concentration on the luminescence intensity,
[0055] Figure D is a graph of the effect of the luciferin concentration on the luminescence intensity.
[0056] Figure 5 Figure is a particle size analysis of the conjugate Phage PAZ1-MBs and a transmission electron micrograph of the capture of Pseudomonas aeruginosa CMCC 10104 by the conjugate Phage PAZ1-MBs.
[0057] Figure A is a schematic diagram of the particle size analysis of the conjugate Phage PAZ1-MBs,
[0058] Figure B is a transmission electron micrograph of the capture of Pseudomonas aeruginosa CMCC 10104 by the conjugate Phage PAZ1-MBs.
[0059] C is a schematic diagram of the capture efficiency of the Pseudomonas aeruginosa rapid fluorescence detection kit for different concentrations of Pseudomonas aeruginosa bacterial liquid.
[0060] D is a bacterial specificity analysis diagram of the Pseudomonas aeruginosa rapid fluorescence detection kit.
[0061] Figure 6 The standard curve diagram of the Pseudomonas aeruginosa rapid fluorescence detection kit for detecting Pseudomonas aeruginosa. DETAILED DESCRIPTION
[0062] The present application will be further described in detail below with specific examples, so that those skilled in the art can understand.
[0063] Example 1 Phage isolation and purification and morphological analysis
[0064] 1. Phage isolation and purification
[0065] The sample sampled from nature was filtered through a 0.22 μm microporous filter, and the filtrate was repeated once according to the above method to obtain the phage stock solution. The purified phage was purified by double-layer plate method for 5-10 times until the size and transparency of the phage plaques appearing on the double-layer plate were consistent, i.e. the purified phage. Figure 1 A).
[0066] 2. Phage morphological analysis
[0067] After the phage was negatively stained with phosphotungstic acid, it was observed under a transmission electron microscope to observe the phage morphology. The specific operation steps are as follows:
[0068] After the copper mesh was immersed in the phage solution for 10 min, the excess liquid was absorbed with filter paper, and then the copper mesh was stained in phosphotungstic acid dye and naturally dried to complete dryness. The prepared copper mesh was observed under a transmission electron microscope to observe the phage morphology, and the size was measured with software Digital Micrograph Demo 3.9.1. Figure 1 B).
[0069] The results show that the phage PAZ1 is a filamentous phage with a length of about 1-2 μm and a width of about 5 nm.
[0070] The above-mentioned Pseudomonas aeruginosa bacteriophage PAZ1 is preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2024545, a preservation date of March 25, 2024, and an address of Wuhan University, Wuhan, China.
[0071] Example 2: Analysis of the biological characteristics of bacteriophage PAZ1
[0072] 1. Analysis of the host spectrum of bacteriophage PAZ1
[0073] The determination of the host spectrum of the bacteriophage was performed using the spot method. 100 μL of a bacterial solution to be determined, which was cultured to the logarithmic phase, was added to a warm semi-solid culture medium, mixed, and then poured onto a pre-prepared LB agar plate. After solidification, 5 μL of bacteriophage with a titer of 10 9 PFU / mL was added to the surface of the upper plate, dried, and then inverted and incubated in a 37°C incubator for 4-6 h. The lysis was observed, and the results are shown in Table 1.
[0074] Table 1. Host spectrum of bacteriophage PAZ1
[0075]
[0076] Note: "+" indicates that a clear plaque was formed on the double-layer agar plate; "-" indicates that no plaque was formed.
[0077] 2. Optimal multiplicity of infection of the bacteriophage
[0078] The multiplicity of infection (MOI) refers to the ratio of the number of bacteriophages to the number of host bacteria at the initial infection. The bacteriophage and the host bacteria were mixed at a certain MOI value (0.001, 0.01, 0.1, 1, 10, 100, 1000), incubated at 37°C for 3.5 h, centrifuged at 11000 r / min for 10 min, and the titer of the bacteriophage in the supernatant of the samples at different MOI values was determined using the double-layer plate method. Three parallel tests were performed.
[0079] The results are shown in Figure 2 A. When the MOI of the bacteriophage was 1, the titer of the bacteriophage reached a maximum, i.e., the optimal multiplicity of infection of the bacteriophage was 1, indicating that when the titer of the bacteriophage and the number of host bacteria were mixed at a ratio of 1, more bacteriophages could be proliferated.
[0080] 3. Adsorption rate
[0081] Fresh bacteriophage solution and host bacterial suspension were mixed in a centrifuge tube at the optimal MOI value, and incubated on a 37°C shaker. Starting at 0 min, the titer of the bacteriophage in the supernatant was determined every 3 min using the double-layer plate method. Three parallel tests were performed. The adsorption rate = 1- (the titer of the unabsorbed bacteriophage at each time point / the titer of the bacteriophage at 0 min) x 100%, and the results of the adsorption rate of the bacteriophage to the host bacteria are shown in Figure 1 B.
[0082] From Figure 2B It can be seen that the optimal adsorption rate of phage PAZ1 is 70.89%, and the time to reach the optimal adsorption rate is 15 min, at which time the phage is adsorbed on the host bacteria in the maximum amount.
[0083] 4. One-step growth curve
[0084] The one-step growth curve of the phage reflects its growth rule. The phage and host bacteria are mixed at the optimal MOI value, incubated at 37°C for about 10 min to allow the phage to be adsorbed on the bacteria, centrifuged at 8000 r / min for 2 min at 4°C, the supernatant is discarded, and the same volume of LB is used for resuspension, which is repeated twice to remove the unadsorbed phage. The above liquid is added to 9 mL of LB liquid medium, and 300 μL is taken every 10 min, centrifuged at 8000 r / min for 2 min, and the titer of the phage in the supernatant is determined by the double-layer plate method. The test is set in triplicate.
[0085] The results are shown in Figure 2 C From the one-step growth curve, the latent period and the burst period of the phage can be seen. The latent period of phage PAZ1 is 20 min, and the burst period is 40 min.
[0086] 5. Stability analysis of phage PAZ1
[0087] The phage stock solution is diluted to 10 6 PFU / mL and divided into 1 mL sterile centrifuge tubes. The centrifuge tubes are placed in constant temperature water baths at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C and 90°C, respectively, to evaluate the temperature stability of the phage.
[0088] As can be seen from Figure 2 D, the phage activity of phage PAZ1 remains at a level comparable to the initial level between 30°C and 60°C, the phage activity decreases after half an hour at 70°C and 80°C, and the phage activity decreases to 0 after 30 min at 90°C. The phage activity gradually decreases until it is completely inactivated as the temperature increases, i.e. the exposure time to high temperature is prolonged.
[0089] Take 100 μL of phage suspension with known titer (10 9 PFU / mL) and add it to 900 μL of PBS buffer with different pH values (2-12), and then place it in a 37°C water bath for 2 h, and then determine the titer of the phage in each centrifuge tube. The test is set in triplicate.
[0090] As can be seen from Figure 2 E, the phage maintains a stable titer, i.e. high activity, at pH 6-12, and the titer is basically reduced to 0 at pH 2, indicating that strong acid directly destroys the activity of the phage.
[0091] Example 3 Preparation of Pseudomonas aeruginosa phage PAZ1 nanomagnetic beads
[0092] 1. Phage activation
[0093] The stored phage PAZ1 stock solution was taken out from -80°C, 100 μL of activated host bacteria suspension (10 8 CFU / mL) and 100 μL of phage stock solution were added into 15 mL of LB medium, which was placed in a constant temperature incubator at 37°C and 180 r / min for about 16 h. Centrifugation was performed at 8000 r / min and 4°C for 20 min. Filtration was performed using a 0.22 μm microporous filter, and the filtrate was the phage stock solution. The phage titer was determined using the double-layer plate method, and 10 10 PFU / mL of phage PAZ1 was obtained, which was stored at 4°C for standby use.
[0094] 2. NHS / EDC activation of carboxylated magnetic beads
[0095] 1) After the carboxyl-modified magnetic beads with a diameter of 200 nm were uniformly dispersed by ultrasonic dispersion, 100 μL of the dispersed magnetic beads were taken into a 1.5 mL centrifuge tube, which was placed on a magnetic separation rack. After solid-liquid separation, the supernatant was removed, and the magnetic beads were retained. Then, 200 μL of 2-(N-morpholino) ethanesulfonic acid (MES) solution was added into the centrifuge tube, which was uniformly mixed and placed on the magnetic separation rack. After solid-liquid separation, the supernatant was removed, and the step was repeated once. Then, N-hydroxysuccinimide (NHS) (100 μL, 20 mg / mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (200 μL, 20 mg / mL) were added to activate the carboxyl groups on the magnetic beads.
[0096] 2) 100 μL of the dispersed magnetic beads were taken into a 1.5 mL centrifuge tube, which was placed on a magnetic separation rack. After solid-liquid separation, the supernatant was removed, and the magnetic beads were retained. Then, 200 μL of 2-(N-morpholino) ethanesulfonic acid (MES) solution was added into the centrifuge tube, which was uniformly mixed and placed on the magnetic separation rack. After solid-liquid separation, the supernatant was removed, and the step was repeated once. Then, N-hydroxysuccinimide (NHS) (100 μL, 20 mg / mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (200 μL, 20 mg / mL) were added to activate the carboxyl groups on the magnetic beads.
[0097] 3) The activated magnetic beads were mixed vertically at 180 r / min at 37°C for 30 min, and then washed with 100 μL of pre-cooled phosphate buffer solution (PBS) for 3 times to remove the excess NHS and EDC, thereby obtaining the activated carboxyl magnetic beads MBs.
[0098] The coupling method of the phage PAZ1 and the activated carboxyl magnetic beads is as follows:
[0099] 1) 200 μL of 1.0 × 10 10 PFU / mL of phage PAZ1 was added to 1 mg of activated carboxyl magnetic beads, which were shaken (180 r / min) at 37°C for 30 min,
[0100] 2) Wash the magnetic beads twice with 200 μL phosphate buffered saline (PBST) to remove excess phage. Then resuspend the PhagePAZl-MBs conjugate in 1 mL of phosphate buffered saline (PBS) containing 3% bovine serum albumin (BSA) and incubate at 37°C with shaking (180 r / min) for 30 min to block the remaining sites. Wash the PhagePAZl-MBs conjugate twice to remove the remaining blocking buffer;
[0101] 3) Finally, resuspend the PhagePAZl-MBs conjugate in PBS (pH 7.4) containing 1.5% bovine serum albumin (BSA) and store at 4°C until use to obtain a PBS buffer containing the Salmonella phage nanomagnetic beads conjugate PhagePAZl-MBs.
[0102] Example 4 Optimization of the use conditions of the Pseudomonas aeruginosa phage nanomagnetic beads conjugate PhagePAZl-MBs
[0103] 1. Optimization of the phage titer of the Pseudomonas aeruginosa phage nanomagnetic beads conjugate PhagePAZl-MBs
[0104] During the preparation of PhagePAZl-MBs, the magnetic beads were conjugated with phage suspensions of different titers (10 1 , 10 3 , 10 5 , 10 7 , 10 9 PFU / mL) respectively. 50 μL of PhagePAZl-MBs were taken and added to a bacterial suspension of 10 5 CFU / mL, incubated at 37°C with shaking (180 r / min) for 30 min, then magnetically separated, the supernatant was discarded, the PhagePAZl-MBs were washed three times with PBS, resuspended in 1 mL of PBS and diluted to a certain number of times for plate counting, and the capture rate was calculated respectively.
[0105] 2. Optimization of the optimal amount of use of the Pseudomonas aeruginosa phage nanomagnetic beads conjugate PhagePAZl-MBs
[0106] Take 25 μL, 50 μL, 75 μL, 100 μL, 125 μL of PhagePAZl-MBs and add them to 100 μL of bacterial culture (10 5 CFU / mL) respectively, incubate at 37°C for 20 min, then place them on a magnetic stand for 5 min to allow the magnetic beads to separate completely from the liquid, dilute to a certain number of times for plate counting, and calculate the capture rate respectively.
[0107] 3. Optimization of the optimal reaction temperature of Pseudomonas aeruginosa phage nanomagnetic bead conjugate PhagePAZ1-MBs
[0108] Take 50 μL of PhagePAZ1-MBs and add to 100 μL of bacterial culture solution (10 5 CFU / mL). Incubate at 4°C, 25°C, 37°C, and 42°C, respectively, for 30 min with shaking, then place on a magnetic stand for 2-3 min to allow the magnetic beads to separate completely from the liquid. Discard the supernatant, wash with PBS three times, resuspend in 1 mL of PBS, dilute by a certain factor, and perform plate counting. Calculate the capture rate.
[0109] 4. Optimization of the optimal incubation time of Pseudomonas aeruginosa phage nanomagnetic bead conjugate PhagePAZ1-MBs
[0110] Take 50 μL of PhagePAZ1-MBs and add to 100 μL of bacterial culture solution (10 5 CFU / mL). Incubate at the optimal temperature for 15 min, 20 min, 25 min, 30 min, and 35 min, respectively, with shaking. After incubation, perform magnetic separation, discard the supernatant, wash with PBS three times, resuspend in 1 mL of PBS, dilute by a certain factor, and perform plate counting. Calculate the capture rate.
[0111] The results, as shown in Figure 3 , indicate that the optimal conditions for PhagePAZ1-MBs to capture Pseudomonas aeruginosa are a phage titer of 10 9 PFU / mL during the preparation of PhagePAZ1-MBs. Add 50 μL of PhagePAZ1-MBs to 1 mL of bacterial solution, and incubate at 25°C for 25 min.
[0112] Example 5. Optimization of the conditions of the rapid detection kit based on ATP bioluminescence method recognized by phage PAZ1
[0113] 1. Optimization of the optimal concentration of CTAB extraction agent for ATP bioluminescence method
[0114] Prepare CTAB solutions with concentrations of 0.01%, 0.02%, 0.05%, 0.1%, and 0.2%. Then, take 20 μL of the CTAB solutions with different concentrations and 100 μL of Pseudomonas aeruginosa (10 7The 50 μL extract was mixed with 50 μL luciferin-luciferase solution (0.25 mg / mL luciferin, 0.08 mg / mL luciferase, and Tris-HCl buffer) and reacted for 3 min. The relative light unit (RLU) of ATP bioluminescence was read by a multifunctional enzyme marker to represent the light intensity.
[0115] 2. Optimization of ATP bioluminescence method for optimal extraction time
[0116] 100 μL of Pseudomonas aeruginosa (10 7 The 50 μL extract was mixed with 50 μL luciferin-luciferase solution (0.25 mg / mL luciferin, 0.08 mg / mL luciferase) and immediately placed in a multifunctional enzyme marker to measure the RLU value.
[0117] 3. Optimization of ATP bioluminescence method for optimal luciferase concentration
[0118] The luciferin concentration was fixed, and other reaction conditions were unchanged. The luciferase concentration was adjusted to 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.10 mg / mL, respectively, and the RLU was measured to obtain the optimal luciferase concentration.
[0119] 4. Optimization of ATP bioluminescence method for optimal D-luciferin concentration
[0120] Other reaction conditions were unchanged, and the D-luciferin concentration was adjusted to 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, 0.30 mg / mL, and 0.35 mg / mL, respectively. The RLU value was measured to obtain the optimal luciferin concentration.
[0121] The results are shown in Table 1. Figure 4 The optimal conditions for the Pseudomonas aeruginosa rapid detection kit ATP bioluminescence method were a CTAB concentration of 0.05%, an extraction time of 4 min, a luciferase concentration of 0.06 mg / mL, and a luciferin concentration of 0.25 mg / mL.
[0122] Example 6 Characterization of the ability of Pseudomonas aeruginosa bacteriophage nanomagnetic bead conjugate PhagePAZ1-MBs to capture bacteria
[0123] 1. Characterization of Pseudomonas aeruginosa bacteriophage nanomagnetic bead conjugate PhagePAZ1-MBs
[0124] The particle size of the uncoupled carboxyl magnetic beads and the coupled PhagePAZl-MBs were measured by nanoparticle size potential analyzer to determine whether the phage PAZl was successfully coupled with the carboxyl magnetic beads. The binding ability of Pseudomonas aeruginosa and PhagePAZl-MBs was observed by transmission electron microscopy. PhagePAZl-MBs were dispersed for 30 min under 96W ultrasonic wave. The copper mesh was inserted into the dispersion for 5-10 min, and the sample was air-dried for testing. TEM samples were prepared by negative staining method.
[0125] The results are shown in Figure 5 A. The particle size of MBs before coupling with phage PAZl was about 175.33 nm, and the particle size of PhagePAZl-MBs after coupling was about 1734.48 nm. The change in particle size proved that the phage PAZl was successfully coupled with the magnetic beads. Figure 5 B is the observation of PhagePAZl-MBs capturing Pseudomonas aeruginosa by transmission electron microscopy. The results show that the phage maintains the recognition ability to the host bacteria after coupling with the magnetic beads, and can capture the target strain in the matrix.
[0126] 2. Characterization of Pseudomonas aeruginosa phage nanomagnetic bead conjugate PhagePAZl-MBs for capturing efficiency of Pseudomonas aeruginosa at different concentrations
[0127] In order to characterize the capture efficiency of phage coupled magnetic beads for Pseudomonas aeruginosa at different concentrations, 50 μL of PhagePAZl-MBs was added to a centrifuge tube containing 1 mL of bacterial solution at different concentrations (10 3 -10 7 CFU / mL), and incubated at 37°C for 25 min with shaking. After incubation, magnetic separation was performed, the supernatant was removed, and the sample was washed with PBS three times, resuspended in 1 mL of PBS, and diluted to a certain multiple for plate counting, and the capture rate was calculated.
[0128] The results are shown in Figure 5 C. PhagePAZl-MBs had good capture ability for Pseudomonas aeruginosa solution at 10 3 -10 5 CFU / mL, with a capture rate of 72.33%-76.69%.
[0129] 3. Specificity analysis of Pseudomonas aeruginosa phage nanomagnetic bead conjugate PhagePAZl-MBs
[0130] Pseudomonas aeruginosa CMCC 10104, Pseudomonas fluorescens ATCC 13525, Shewanella putrefaciens NCTC 10737, Escherichia coli NCTC 12900, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19115, Vibrio parahaemolyticus ATCC 17028. The mixed bacteria solution Mix1 contained Pseudomonas aeruginosa, and the mixed bacteria solution Mix2 did not contain Pseudomonas aeruginosa.
[0131] In the live and dead bacteria detection analysis, Pseudomonas aeruginosa was inactivated at high temperature as the dead bacteria detection group. The concentration of all bacteria solutions was set to 10 6 CFU / mL. 1 mL of the test bacteria was mixed with 50 μL of PhagePAZ1-MBs, and incubated at 37°C with shaking for 25 min. After incubation, magnetic separation was performed, the supernatant was removed, and it was suspended in 100 μL of Tris-HCl buffer. 20 μL of 0.05% CTAB solution was added, and after 4 min, magnetic separation was performed. 50 μL of the supernatant was added to a black enzyme-labeled plate, and then 50 μL of luciferin-luciferase solution (0.25 mg / mL luciferin, 0.06 mg / mL luciferase) was added. The RLUs were immediately measured.
[0132] The results are shown in Table D. PhagePAZ1-MBs had strong specificity for the enrichment of host bacteria, and the specificity was consistent with that of phage PAZ1. Figure 5
[0133] Example 7: Establishment of a standard curve for a rapid detection kit based on the ATP bioluminescence method recognized by phage PAZ1
[0134] 1 mL of Pseudomonas aeruginosa at different concentrations was mixed with 50 μL of PhagePAZ1-MBs. After incubation at 37°C with shaking at 180 r / min for 25 min, magnetic separation was performed, the supernatant was removed, and it was resuspended in 100 μL of Tris-HCl buffer. 20 μL of 0.05% CTAB solution was added, and after 4 min, magnetic separation was performed. 50 μL of the supernatant was added to a black enzyme-labeled plate, and then 50 μL of luciferin-luciferase solution (0.25 mg / mL luciferin, 0.06 mg / mL luciferase) was added. The RLUs were immediately measured. The logarithmic value of the concentration of Pseudomonas aeruginosa was used as the abscissa, and the logarithmic value of the RLUs was used as the ordinate to draw a standard curve. The detection limit was calculated according to the formula LOD = 3.3S b / m.
[0135] In the formula, S b is the standard deviation of the blank group, and m is the slope of the standard curve.
[0136] The results are shown in Table D. PhagePAZ1-MBs had strong specificity for the enrichment of host bacteria, and the specificity was consistent with that of phage PAZ1.Figure 6 RLU values were shown to be in the range of 10 3 -10 7 CFU / mL, increased with the increase of concentration. Linear regression equation was: y = 0.2362x + 2.0506, R 2 = 0.9873. In this experiment, the standard deviation of the blank group was 0.199214, the slope of the standard curve was 0.2362, and the detection limit calculated according to the formula was 607 CFU / mL.
[0137] Example 8 Enrichment and separation of Pseudomonas aeruginosa based on Pseudomonas aeruginosa phage nanometer magnetic bead coupling PhagePAZ1-MBs in different substrates
[0138] The kit for enrichment and separation of Pseudomonas aeruginosa is characterized in that it comprises the PBS buffer containing the Pseudomonas aeruginosa phage nanometer magnetic bead coupling PhagePAZ1-MBs, a negative control, a positive control and a PBS buffer solution; wherein the negative control is a PBS buffer containing BSA, wherein the mass concentration of bovine serum albumin is 15g / L,
[0139] the molar concentration of the PBS buffer is 1μmol / L;
[0140] the positive control is a Pseudomonas aeruginosa positive control, and the Pseudomonas aeruginosa is Pseudomonas aeruginosa CMCC10104.
[0141] 1. Preparation of sample: 100mL of lake water was taken in Nanhu, sterilized at 121℃ for 15min, and used as a negative control. Pseudomonas aeruginosa CMCC 10104 was inoculated to prepare 1mL samples with different degrees of pollution, and vortexed to mix. An equal volume of PBS buffer was added to the control sample.
[0142] 2. The method for enrichment and separation of Pseudomonas aeruginosa using the kit comprises the following steps:
[0143] 1) The sample containing Pseudomonas aeruginosa to be separated was mixed with the PBS buffer containing Pseudomonas aeruginosa phage nanometer magnetic bead coupling PhagePAZ1-MBs, and incubated on a shaker at 37℃ and 180rpm for 20min to obtain a sample;
[0144] 2) The sample was placed in a magnetic separator to fully separate the magnetic beads from the liquid, and the supernatant was retained; wherein the bacterial content in the supernatant was calculated by plate counting method, and the capture efficiency CE of PhageT102-MBs on Salmonella was calculated according to the following formula:
[0145]
[0146] In the formula, N0is the concentration of P. aeruginosa bacterial solution present in the initial sample, CFU / mL, N a is the concentration of P. aeruginosa bacterial solution not combined with the conjugate, CFU / mL.
[0147] Example 9 Application of the ATP bioluminescence rapid detection kit based on phage PAZ1 recognition in spiked samples
[0148] 1. Preparation of samples: the samples were subjected to high-temperature high-pressure sterilization at 121°C for 15 min and were ready for use. P. aeruginosa CMCC10104 was inoculated to prepare 1 mL samples with different degrees of contamination, which were vortexed and mixed. An equal volume of PBS buffer was added to the control group samples.
[0149] 2. 1 mL of actual samples containing different concentrations of bacterial solution (10 3 , 10 5 CFU / mL) were mixed with 50 μL of PhagePAZ1-MBs, respectively, and were incubated at 37°C with shaking at 180 r / min for 25 min, then were subjected to magnetic separation, the supernatant was discarded, and the same volume of Tris-HCl buffer was added to resuspend the bacteria, after vortexing, 100 μL of bacterial suspension was removed, 20 μL of 0.05% CTAB solution was added, 4 min later, magnetic separation was performed, 50 μL of the supernatant was taken and added to a black enzyme-labeled plate, and then 50 μL of luciferin-luciferase solution (0.25 mg / mL luciferin, 0.06 mg / mL luciferase) was added, and the RLUs were immediately measured.
[0150] The results are shown in Table 2, and the ATP bioluminescence rapid detection kit based on P. aeruginosa phage nanomagnetic bead conjugate PhagePAZ1-MBs has good detection efficiency in drinking water, tap water and milk.
[0151] Table 2 Detection results in spiked samples
[0152]
[0153] Example 10 Application of the ATP bioluminescence rapid detection kit based on phage PAZ1 recognition in actual samples
[0154] Collect 5 samples of bottled drinking water, 5 samples of barreled drinking water, 3 samples of sewage, 1 sample of lake water and 1 sample of pond water, totally 20 samples. Take 1 mL of each sample in a 1.5 mL centrifuge tube, add 50 μL of PhagePAZ1-MBs, incubate at 37℃ for 25 min at 180 r / min, then perform magnetic separation, discard the supernatant, resuspend it in 100 μL of Tris-HCl buffer, add 20 μL of 0.05% CTAB solution, after 4 min, perform magnetic separation, take 50 μL of the supernatant and add it to a black enzyme label plate, then add 50 μL of luciferin-luciferase solution (0.25 mg / mL luciferin, 0.06 mg / mL luciferase), and immediately measure the RLU.
[0155] The detection results are shown in Table 3. Pseudomonas aeruginosa was detected only in the sewage sample and the lake water sample, and the RLU values of tap water and drinking water and pond water were all below the detection limit, i.e. below the RLU value, so it was determined that they could not be detected.
[0156] Table 3 Detection results of actual samples
[0157]
[0158] Other parts not described in detail are prior art. Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.
Claims
1. A Pseudomonas aeruginosa bacteriophage ( Pseudomonas aeruginosa The bacteriophage) PAZ1 has the accession number: CCTCCNO:M2024545.
2. A Pseudomonas aeruginosa bacteriophage nanometer magnetic bead conjugate Phage PAZ1-MBs, characterized in that: The conjugate PhagePAZ1-MBs is a complex obtained by coupling the Pseudomonas aeruginosa bacteriophage PAZ1 with carboxyl magnetic beads.
3. The Pseudomonas aeruginosa bacteriophage nanometer magnetic bead conjugate Phage PAZ1-MBs according to claim 2, characterized in that: The diameter of the carboxyl magnetic beads is 200 nm.
4. A method for preparing PBS buffer containing Pseudomonas aeruginosa bacteriophage nanometer magnetic bead conjugate Phage PAZ1-MBs as claimed in claim 2, characterized in that: The method comprises the following steps: 1) activating the Pseudomonas aeruginosa bacteriophage PAZ1; 2) activating the carboxyl magnetic beads to obtain activated carboxyl magnetic beads; 3) coupling the Pseudomonas aeruginosa bacteriophage PAZ1 with the activated carboxyl magnetic beads to obtain the PBS buffer containing the Pseudomonas aeruginosa bacteriophage nano-magnetic bead conjugate PhagePAZ1-MBs, wherein, in the PBS buffer, the molar concentration of PBS is 1 μmol / L, and the mass-volume concentration of bovine serum albumin is 15 g / L, The content of the Pseudomonas aeruginosa bacteriophage nano-magnetic bead conjugate PhagePAZ1-MBs in each milliliter of the PBS buffer is 50 μL.
5. The method of claim 4, wherein: In the step 1), the Pseudomonas aeruginosa bacteriophage PAZ1 is activated by the following method: The phage PAZ1 stock solution was mixed with the logarithmic phase Pseudomonas aeruginosa CMCC 10104, liquid cultured, filtered through a 0.22 μm microporous filter to obtain the phage PAZ1 liquid, which was stored at 4 ℃; and the phage liquid use titer was 10 10 PFU / mL.
6. A kit for enriching isolating Pseudomonas aeruginosa, characterized by: It comprises the PBS buffer containing the Pseudomonas aeruginosa bacteriophage nano-magnetic bead conjugate PhagePAZ1-MBs prepared by the method of claim 4.
7. The kit of claim 6, wherein: The kit further comprises a negative control, a positive control and a PBS buffer solution; The negative control is a PBS buffer containing bovine serum albumin BSA, and the mass-volume concentration of the bovine serum albumin is 15 g / L, and the molar concentration of the PBS buffer is 1 μmol / L; The positive control is a Pseudomonas aeruginosa positive control, and the Pseudomonas aeruginosa is Pseudomonas aeruginosa CMCC 10104.
8. A method for enriching and isolating P. aeruginosa using the kit according to claim 6, characterized by, The method comprises the following steps: 1) mixing a sample containing Pseudomonas aeruginosa to be separated with the PBS buffer containing the Pseudomonas aeruginosa bacteriophage nano-magnetic bead conjugate PhagePAZ1-MBs, and performing shaker culture at a temperature of 37 ℃ and a rotation speed of 180 rpm for a culture time of 20 min to obtain a sample; 2) placing the sample in a magnetic separator to fully separate the magnetic beads from the liquid, discarding the supernatant, washing with sterile PBS for three times, and then suspending in 1 mL of sterile PBS; wherein, The sample was calculated by plate counting method, and the bacterial content of sterile PBS was calculated. The capture efficiency of Phage PAZ1-MBs on Salmonella was calculated according to the following formula CE : In the formula, N 0 is the concentration of P. aeruginosa bacterial solution present in the initial sample, CFU / mL, N a is the concentration of P. aeruginosa bacterial solution not bound to the conjugate, CFU / mL.
9. A method for rapid detection of Pseudomonas aeruginosa using the kit according to claim 8, which is not intended for disease diagnosis, characterized by: The method comprises the following steps: 1) mixing a sample to be detected with the PBS buffer containing the Pseudomonas aeruginosa bacteriophage nano-magnetic bead conjugate PhagePAZ1-MBs, and performing shaker culture at a temperature of 37 ℃ and a rotation speed of 180 rpm for a culture time of 20 min to obtain a sample; 2) placing the sample in a magnetic separator to fully separate the magnetic beads from the liquid, discarding the supernatant, resuspending the sample in 100 μL of Tris-HCl buffer, adding 20 μL of 0.05% CTAB solution, and then performing magnetic separation after 4 min, taking 50 μL of the supernatant and adding it to a black enzyme-labeled plate, adding 50 μL of luciferin-luciferase solution, and immediately measuring the RLU using a fluorescence spectrophotometer; 3) The standard curve is plotted with the logarithmic value of Pseudomonas aeruginosa concentration as the abscissa and the logarithmic value of RLU as the ordinate. After obtaining the accurate RLU value, the total number of viable colonies of Pseudomonas aeruginosa in the sample can be converted.