A phage labeled with AIE molecules and its preparation method and application

Abstract

The present invention discloses a phage labeled with an AIE molecule, a preparation method and an application thereof; the phage labeled with an AIE molecule of the present invention comprises MASPB and a phage, and MASPB binds to the genetic material inside the phage. The MASPB of the present invention can enter the interior of the phage to label the genetic material of the phage, thereby forming a phage labeled with an AIE molecule, thereby quickly and accurately detecting target bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a phage labeled with an AIE molecule, and a preparation method and application thereof. Background Art

[0002] Bacterial testing can quickly and accurately identify pathogenic bacteria, providing clinicians with critical information to choose appropriate treatments, reduce unnecessary medication use and the development of antibiotic resistance.

[0003] Existing detection technologies provide effective solutions in different aspects, but there are still some shortcomings: traditional culture methods take a long time, PCR (polymerase chain reaction) and real-time PCR technology require expensive equipment and complex operations, and ELISA (enzyme-linked immunosorbent reaction) lacks sensitivity and specificity.

[0004] Fluorescence imaging technology is an imaging method widely used in the fields of biomedicine, materials science and chemistry. Its basic principle is to use the fluorescence emitted by fluorescent molecules after being irradiated with excitation light for imaging. It can provide high-sensitivity and high-resolution images, and accurately locate and quantitatively analyze target molecules in complex biological samples.

[0005] AIE (aggregation-induced emission) molecules have significant advantages over traditional fluorescent molecules, the core of which is the significant luminescence enhancement in the aggregated state. Traditional fluorescent molecules often reduce luminescence intensity due to light quenching effects under high concentrations or in solid states, while AIE molecules can significantly improve fluorescence quantum yield under these conditions, showing excellent brightness and stability. In addition, AIE molecules have high stability to light and chemical environments, which enables them to exhibit superior performance in applications such as bioimaging, sensors, and optoelectronic devices. This characteristic makes AIE molecules more widely applicable and reliable in practical applications.

[0006] Bacteriophages can accurately identify and target specific bacteria. Compared with traditional methods, phage detection usually has lower background interference and cost. It is also environmentally friendly and does not require the use of harmful chemical reagents. These advantages make phages have broad application prospects in the field of bacterial detection. Currently, the protein shell of phages is mainly fluorescently labeled by chemical coupling. This method is complicated to operate, requires modification of fluorescent molecules, and has low fluorescence intensity. Summary of the invention

[0007] In view of the deficiencies in the prior art, the object of the present invention is to provide a phage labeled with an AIE molecule and a preparation method and application thereof; the AIE molecule of the present invention can enter the interior of the phage to label the genetic material of the phage, forming an AIE molecule-labeled phage, thereby quickly and accurately detecting the target bacteria.

[0008] The purpose of the present invention is achieved through the following technical solutions.

[0009] A bacteriophage labeled with an AIE molecule includes MASPB and a bacteriophage, wherein the MASPB binds to genetic material inside the bacteriophage;

[0010] The structure of the MASPB is as follows:

[0011] .

[0012] The MASPB of the present invention is a positively charged AIE molecule that can enter the interior of the bacteriophage, bind to its genetic material and be stored inside the bacteriophage to maintain activity;

[0013] The MASPB of the present invention has good water solubility and can be fully mixed with the phage solution, and the background after the labeled phage is stained is low;

[0014] After the MASPB-labeled phage of the present invention binds to the target bacteria, the genetic material bound to MASPB enters the interior of the bacteria through the action of the phage, and a large number of MASPBs gather in the bacteria and emit strong fluorescence, which can be observed under a confocal microscope, and the shape of the bacteria is clear.

[0015] Bacteriophages can bind to specific bacteria and inject genetic material; they have specific recognition capabilities.

[0016] Preferably, the phage is selected from the group consisting of Salmonella phage, Escherichia coli phage, Pseudomonas aeruginosa phage, Pasteurella phage, Klebsiella pneumoniae phage, Vibrio cholerae phage, Shigella dysenteriae phage, Acinetobacter baumannii phage, Proteus phage, Enterobacter cloacae phage, Shigella phage, Staphylococcus phage, Streptococcus phage, Clostridium perfringens phage, Mycobacterium phage, and Enterococcus faecalis phage.

[0017] The method for preparing the above-mentioned AIE molecule-labeled phage comprises the following steps:

[0018] MASPB and phage are mixed and incubated in the dark, and MASPB enters the phage and combines with its genetic material, and then is purified to obtain the phage labeled with AIE molecules.

[0019] Preferably, the incubation solvent is SM buffer; the concentration of MASPB is 30-50 μM; the titer of the phage is ≥10 11 PFU / mL.

[0020] Further preferably, the titer of the phage is ≥10 12 PFU / mL.

[0021] Preferably, the incubation is performed at room temperature (20-30°C).

[0022] Preferably, the incubation time is ≥3h.

[0023] Preferably, the purification is by adding a protein to bind remaining unlabeled free MASPB.

[0024] Further preferably, the protein is BSA (bovine serum albumin); the amount of the protein added is a final concentration of 10-20wt%.

[0025] A bacteria detection reagent comprises the above-mentioned AIE molecule-labeled bacteriophage.

[0026] Preferably, the titer of the phage labeled with AIE molecules in the bacterial detection reagent is ≥5×10 10 PFU / mL.

[0027] Further preferably, the titer of the phage labeled with AIE molecules in the bacterial detection reagent is ≥5×10 11 PFU / mL.

[0028] Preferably, the bacteria detection reagent also includes a solvent.

[0029] Further preferably, the solvent is water or SM buffer.

[0030] The preparation method of the above-mentioned bacteria detection reagent comprises the following steps:

[0031] MASPB was dissolved in water to prepare a MASPB stock solution, and the MASPB stock solution was added to the phage solution for incubation in the dark. After the incubation, BSA was added to bind the remaining unlabeled MASPB to prepare a bacterial detection reagent.

[0032] Preferably, the concentration of MASPB in the MASPB mother solution is 10-20 mM; the phage titer of the phage solution is ≥10 11 PFU / mL.

[0033] Further preferably, the phage titer of the phage liquid is ≥10 12 PFU / mL.

[0034] Preferably, the usage ratio of the MASPB mother solution to the phage solution is 3-5 μL:1 mL.

[0035] Preferably, the incubation is performed at room temperature (20-30°C).

[0036] Preferably, the incubation time is ≥3h.

[0037] Application of the above-mentioned AIE molecule-labeled phage or the above-mentioned bacteria detection reagent in detecting bacteria.

[0038] A method for detecting bacteria, comprising the following steps:

[0039] The above-mentioned AIE molecule-labeled phage or the above-mentioned bacterial detection reagent is mixed with the bacterial solution to be tested, incubated, prepared into slices, and the fluorescence is observed under a confocal microscope.

[0040] After incubation, bright fluorescence can be observed in the target bacteria in the test solution that can bind to the phage, while non-target bacteria have no fluorescence.

[0041] Preferably, the incubation time is 20-30 min;

[0042] Preferably, the incubation is carried out in the dark;

[0043] Preferably, the excitation wavelength of the confocal microscope is 470-490 nm, and the emission wavelength is 550-750 nm.

[0044] Further preferably, the excitation wavelength of the confocal microscope is 488 nm, and the emission wavelength is 600-700 nm.

[0045] Preferably, the concentration of the AIE molecule-labeled phage after mixing is ≥ 2.5×10 10 PFU / mL.

[0046] More preferably, the concentration of the AIE molecule-labeled phage after mixing is ≥2.5×10 11 PFU / mL.

[0047] Preferably, the concentration of bacteria after mixing is 10 7 -10 8 CFU / mL.

[0048] Preferably, the incubated liquid is dropped onto a glass slide and covered with a cover glass to prepare a slide.

[0049] The working principle of the present invention is as follows: MASPB has high stability to light and chemical environment, so that it exhibits superior performance in applications such as bio-imaging, sensors and optoelectronic devices. The phage can specifically identify and quickly bind to the host bacteria, and does not recognize other strains. MASPB marks the genetic material of the phage. As the phage specifically binds to the host bacteria such as Salmonella, its genetic material is injected into the bacteria, and MASPB also enters the bacteria. As MASPB accumulates more and more inside the bacteria, it shows enhanced fluorescence and can be observed.

[0050] The beneficial effects of the present invention are as follows:

[0051] Fast detection speed: For bacterial detection in samples, the traditional culture method requires the purification of single bacteria in the sample, and then the detection result is determined by biochemical identification, which takes about 5 days; the PCR method also requires the purification of single bacteria, and then the result is determined by PCR and gel electrophoresis, which takes about 2 days. The AIE-labeled phage of the present invention only needs to be mixed with the sample, and the result can be determined in 20 minutes ( Figure 1 ).

[0052] Simple operation: Traditional culture and PCR methods require professionals to operate in a strict sterile operating environment, and the operation is complicated, requiring plate streaking, picking single colonies for culture, and complex PCR sample addition. The purification method of phage labeled by the coupling method is complicated. This method does not require a strict sterile operating environment, and only requires a simple mixing operation to complete the detection process.

[0053] High accuracy: Traditional culture and PCR methods may cause false positive / false negative results due to complex operations and methodological reasons. This method uses the high specificity of bacteriophages to only identify the target bacteria in the sample, ensuring the high accuracy of the test. In addition, since the fluorescent molecules of the phage labeled by the coupling method are fixed, they can only be aggregated on the surface of the bacteria through the phage, and the degree of aggregation is difficult to further increase, and the fluorescence intensity is low. The MASPB of the present application is combined with the genetic material and is not fixed. After entering the bacteria, it can further aggregate and form bright fluorescence inside the bacteria. The fluorescence intensity is higher, and it has higher recognition and accuracy.

[0054] Low sample requirements: Because bacteriophages have specific recognition functions, accurate identification can be performed whether it is a single bacterial liquid sample, a mixed bacterial liquid sample or a real sample with complex components.

[0055] Long storage time: AIE molecules enter the interior of the bacteriophage, and the bacteriophage has strong resistance to the external environment, so the labeled phage can be stored for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The present invention is a flow chart of detecting bacteria using the traditional culture method and the PCR method.

[0057] Figure 2 This is a staining microscopic examination image of the salmonella detection solution in Example 1 after incubation for 20 minutes for detecting salmonella.

[0058] Figure 3 The following are staining microscopic images of the Salmonella detection solution in Example 2 for detecting Salmonella after incubation for different time periods.

[0059] Figure 4 These are staining microscopic images of different bacteria detected by the Salmonella detection solution in Example 3.

[0060] Figure 5 This is a staining microscopic examination picture of the mixed bacterial solution of Salmonella and Staphylococcus aureus detected by the Salmonella detection solution in Example 4.

[0061] Figure 6 This is a staining microscopic examination picture of the salmonella in simulated feces detected by the salmonella detection solution in Example 5.

[0062] Figure 7 These are staining microscopic images of different bacteria detected by the E. coli detection solution in Example 6.

[0063] Figure 8 This is a staining microscopic examination picture of the mixed bacterial solution of Escherichia coli, Staphylococcus aureus and Salmonella detected by the Escherichia coli detection solution in Example 7.

[0064] Fig. 9 This is the phage titer change curve of the Salmonella detection solution in Example 8 when stored at 4°C.

[0065] Fig.10 This is a staining microscopic picture of Example 9 for determining the time when MASPB labeled phage.

[0066] Fig.11 This is a staining microscopic image of phage labeling by screening different AIE molecules in Example 1. DETAILED DESCRIPTION

[0067] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto.

[0068] The preparation methods of the concentrated Salmonella phage liquid and the concentrated Escherichia coli phage liquid used in the examples are as follows:

[0069] Salmonella phage SHBCC D24898 and Escherichia coli phage SHBCC D24555 were purchased from Shanghai Collection of Microorganisms. The corresponding host bacteria were inoculated into 200 mL TSB liquid culture medium, cultured at 37°C and 180 r / min until the logarithmic growth phase, and then the initial phage solution was added. After culturing at 37°C and 180 r / min for 4-6 hours, the mixture was allowed to stand overnight at room temperature (20-30°C), centrifuged at 7000 r / min for 15 minutes, and the supernatant was taken and filtered using a 0.22 μm filter to obtain the cultured phage solution. After culture, 10% PEG8000 and 2% NaCl were added to the cultured phage solution, and the solution was fully dissolved and stored at 4°C overnight. The next day, the solution was centrifuged at 11000 r / min at 4°C for 40 min, the supernatant was discarded, and the precipitate was fully dissolved by adding 10 mL SM buffer. After adding 10 mL chloroform, the solution was vortexed and mixed. The solution was centrifuged at 7000 r / min for 15 min, and the supernatant (i.e., the upper solution) was obtained to obtain the concentrated phage, which increased the phage titer by 10%.12 -10 15 PFU / mL.

[0070] The preparation method of the single bacterial colony used in the embodiment is as follows:

[0071] After the bacteria stored in glycerol at -80℃ are thawed at room temperature, they are streaked on TSA plates using an inoculation loop and placed upside down in a 37℃ incubator for overnight culture;

[0072] Pick a single colony on the plate and place it in the culture medium, culture it at 37°C and 180 rpm until the logarithmic growth phase to obtain a bacterial solution;

[0073] Use an inoculation loop to dip into the bacterial solution in the logarithmic growth phase after culture, divide it into sections and streak it on a TSA plate, and invert it in a 37°C incubator for overnight culture. The single colony on the plate is the single colony used in subsequent experiments.

[0074] Example 1: Detection of Salmonella by Detection Fluid

[0075] Preparation of Salmonella detection solution: Dissolve MASPB solid in ultrapure water to prepare a 10mM stock solution, take 3μL and add it to 1mL concentrated Salmonella phage solution, incubate at room temperature in the dark for 3h for labeling, add 1mL 20wt% BSA solution for purification, and then prepare the detection solution;

[0076] Sample preparation: Pick a single colony of Salmonella in the culture medium, culture it at 37°C and 180r / min until the logarithmic growth phase, and obtain a bacterial liquid sample (OD600 value is 0.6-0.8);

[0077] Staining: Mix 100 μL bacterial solution with 100 μL detection solution and incubate at room temperature in the dark for 20 min.

[0078] Preparation: Take 2 μL of the incubated solution and drop it on a glass slide, and place a cover glass for preparation;

[0079] Observation results: Observe directly under a confocal microscope (excitation: 488nm; emission: 600-700nm) with a 100× objective lens. Figure 2 The shown image is a staining microscopy image of the Salmonella detection solution after incubation for 20 minutes to detect Salmonella (left: local magnified image; middle: fluorescence field; right: superimposed field).

[0080] Example 2: Determination of detection time

[0081] Preparation of Salmonella detection solution: Dissolve MASPB solid in ultrapure water to prepare a 10mM stock solution, take 3μL and add it to 1mL concentrated Salmonella phage solution, incubate at room temperature in the dark for 3h for labeling, add 1mL 20wt% BSA solution for purification, and then prepare the detection solution;

[0082] Sample preparation: Pick a single colony of Salmonella in the culture medium, culture it at 37℃ and 180r / min until the logarithmic growth phase, and obtain a bacterial liquid sample (OD600 value is 0.6-0.8);

[0083] Staining: Take 100 μL of bacterial solution and 100 μL of detection solution and mix them evenly. Set the incubation time to 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min respectively.

[0084] Preparation: Take 2 μL of the incubated solution and drop it on a glass slide, and place a cover glass for preparation;

[0085] Observation results: Under a confocal microscope (excitation: 488nm; emission: 600-700nm), 100× objective lens observation, direct microscopic examination, the staining effect is as follows Figure 3 The staining microscopy images of the Salmonella detection solution for detecting Salmonella at different incubation times are shown (in the staining microscopy image at each time point, left: fluorescence field; right: overlay field).

[0086] Example 3: Salmonella specific detection verification

[0087] Preparation of Salmonella detection solution: Dissolve MASPB solid in ultrapure water to prepare a 10mM stock solution, take 3μL and add it to 1mL concentrated Salmonella phage solution, incubate at room temperature in the dark for 3h for labeling, add 1mL 20wt% BSA solution for purification, and then prepare the detection solution;

[0088] Sample preparation: Single colonies of Salmonella, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus were picked and placed in culture medium, and cultured at 37°C and 180 r / min until the logarithmic growth phase to obtain bacterial liquid samples (OD600 value was 0.6-0.8);

[0089] Staining: Take 100 μL of bacterial solution and 100 μL of detection solution from each group, mix them and incubate them in the dark at room temperature for 20 min;

[0090] Preparation: Take 2 μL of the incubated solution and drop it on a glass slide, and place a cover glass for preparation;

[0091] Observation results: Under a confocal microscope (excitation: 488nm; emission: 600-700nm), 100× objective lens observation, direct microscopic examination, the staining effect is as follows Figure 4The shown images are of the staining microscopy of the Salmonella test solution for detecting different bacteria (left: fluorescence field; right: overlay field).

[0092] Example 4: Testing of mixed bacterial liquid with test liquid

[0093] Preparation of Salmonella detection solution: Dissolve MASPB solid in ultrapure water to prepare a 10mM stock solution, take 3μL and add it to 1mL concentrated Salmonella phage solution, incubate at room temperature in the dark for 3h for labeling, add 1mL 20wt% BSA solution for purification, and then prepare the detection solution;

[0094] Sample preparation: Single colonies of Salmonella and Staphylococcus aureus were picked up in the culture medium, cultured at 37℃ and 180r / min until the logarithmic growth phase to obtain bacterial solution samples, and the two bacterial solutions were mixed in equal volumes (OD600 value was 0.6-0.8);

[0095] Staining: Mix 100 μL of mixed bacterial solution with 100 μL of detection solution and incubate at room temperature in the dark for 20 min;

[0096] Preparation: Take 2 μL of the incubated solution and drop it on a glass slide, and place a cover glass for preparation;

[0097] Observation results: Under a confocal microscope (excitation: 488nm; emission: 600-700nm), 100× objective lens observation, direct microscopic examination, the staining effect is as follows Figure 5 The shown image is a staining microscopy image of a mixed bacterial solution of Salmonella and Staphylococcus aureus detected by the Salmonella detection solution (left: fluorescence field; middle: overlay field; right: local magnified image).

[0098] Example 5: Detection of Salmonella in Feces

[0099] Preparation of Salmonella detection solution: Dissolve MASPB solid in ultrapure water to prepare a 10mM stock solution, take 3μL and add it to 1mL of concentrated phage solution, incubate at room temperature in the dark for 3h for labeling, add 1mL of 20wt% BSA solution for purification, and then prepare the detection solution;

[0100] Sample preparation: Pick a single colony of Salmonella in the culture medium, culture at 37℃ 180r / min until the logarithmic growth phase, and obtain a bacterial solution (OD600 value is 0.6-0.8). Divide the collected fecal samples into two groups, 5g each, and add 100μL of Salmonella solution to one of the groups. Add 5mL PBS to the two groups of feces respectively, and filter with filter paper to obtain the sample liquid to be tested;

[0101] Staining: Take 100 μL of the liquid sample to be tested and mix it with 100 μL of the test solution in each group, and incubate it in the dark at room temperature for 20 minutes;

[0102] Preparation: Take 2 μL of the incubated solution and drop it on a glass slide, and place a cover glass for preparation;

[0103] Observation results: Under a confocal microscope (excitation: 488nm; emission: 600-700nm), 100× objective lens observation, direct microscopic examination, the staining effect is as follows Figure 6 The staining microscopy images of the Salmonella detection solution detecting Salmonella in simulated feces are shown (A: positive group, Salmonella was added to the feces; B negative group, Salmonella was not added to the feces) (left: fluorescence field; middle: superimposed field; right: local enlarged image).

[0104] Example 6: Detection of Escherichia coli by using test fluid

[0105] Preparation of E. coli detection solution: Dissolve MASPB solid in ultrapure water to prepare a 10mM stock solution, take 3μL and add it to 1mL of concentrated E. coli phage solution, incubate at room temperature in the dark for 3h for labeling, add 1mL of 20wt% BSA solution for purification, and then prepare the detection solution;

[0106] Sample preparation: Pick single colonies of Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Salmonella in the culture medium, culture at 37°C and 180 r / min until the logarithmic growth phase, and obtain bacterial liquid samples (OD600 value is 0.6-0.8);

[0107] Staining: Take 100 μL of bacterial solution and 100 μL of detection solution from each group, mix them and incubate them in the dark at room temperature for 20 min;

[0108] Preparation: Take 2 μL of the incubated solution and drop it on a glass slide, and place a cover glass for preparation;

[0109] Observation results: Under a confocal microscope (excitation: 488nm; emission: 600-700nm), 100× objective lens observation, direct microscopic examination, the staining effect is as follows Figure 7 The shown images are staining microscopy images of E. coli detection solution detecting different bacteria (left: fluorescence field; right: overlay field).

[0110] Example 7: Escherichia coli specific detection verification

[0111] Preparation of E. coli detection solution: Dissolve MASPB solid in ultrapure water to prepare a 10mM stock solution, take 3μL and add it to 1mL of concentrated E. coli phage solution, incubate at room temperature in the dark for 3h for labeling, add 1mL of 20wt% BSA solution for purification, and then prepare the detection solution;

[0112] Sample preparation: Pick single colonies of Escherichia coli, Staphylococcus aureus, and Salmonella in the culture medium, culture at 37℃ and 180r / min until the logarithmic growth phase to obtain bacterial solution samples, and mix the three bacterial solutions in equal volumes (OD600 value is 0.6-0.8);

[0113] Staining: Mix 100 μL of mixed bacterial solution with 100 μL of detection solution and incubate at room temperature in the dark for 20 min;

[0114] Preparation: Take 2 μL of the incubated solution and drop it on a glass slide, and place a cover glass for preparation;

[0115] Observation results: Under a confocal microscope (excitation: 488nm; emission: 600-700nm), 100× objective lens observation, direct microscopic examination, the staining effect is as follows Figure 8 The shown image is a staining microscopy image of a mixed bacterial solution of Escherichia coli, Staphylococcus aureus and Salmonella detected by the Escherichia coli detection solution (left: fluorescence field; middle: superimposed field; right: local magnified image).

[0116] Example 8: Determination of the shelf life of the test solution

[0117] Preparation of Salmonella detection solution: Dissolve MASPB solid in ultrapure water to prepare a 10mM stock solution, take 3μL and add it to 1mL of concentrated phage solution, incubate at room temperature in the dark for 3h for labeling, add 1mL of 20wt% BSA solution for purification, and then prepare the detection solution;

[0118] Materials: Preparation of TSA lower solid culture medium, TSB upper semi-solid culture medium and other commonly used equipment consumables;

[0119] Preparation of bacterial solution: Pick a single colony of Salmonella in the culture medium, culture it at 37℃ and 180r / min until the logarithmic growth phase to obtain a bacterial solution (OD600 value is 0.6-0.8).

[0120] Titer determination: Use the double-layer plate method to determine the phage titer. The test solution is diluted 10 times 10 times, take 10 6 , 10 7 , 10 8 , 10 9 , 10 10 There are five dilution gradient groups in total. Take 100 μL of each group and add it to a 7 mL centrifuge tube. Then add 100 μL of bacterial solution, invert and mix well, add 5 mL of semi-solid culture medium, spread it on a plate with TSA lower layer solid culture medium, and after solidification, invert it in a 37°C incubator overnight. Count the next day, and each group has 3 parallel controls, and the average value is taken.

[0121] The test solution stored at 4℃ was tested for potency at regular intervals. Fig. 9 .

[0122] Example 9: Determination of MSAPB marking time

[0123] Preparation of Salmonella detection solution: Dissolve MASPB solid in ultrapure water to prepare a 10mM stock solution, take 3μL and add it to 1mL of concentrated phage solution, incubate at room temperature in the dark for 1, 3, 5, and 7h for labeling, add 1mL of 20wt% BSA solution for purification, and then prepare the detection solution;

[0124] Sample preparation: Pick a single colony of Salmonella in the culture medium, culture it at 37℃ and 180r / min until the logarithmic growth phase, and obtain a bacterial liquid sample (OD600 value is 0.6-0.8);

[0125] Staining: 100 μL of bacterial solution and 100 μL of detection solution were mixed in each time group and incubated for 20 min;

[0126] Preparation: Take 2 μL of the incubated solution and drop it on a glass slide, and place a cover glass for preparation;

[0127] Observation results: Under a confocal microscope (excitation: 488nm; emission: 600-700nm), 100× objective lens observation, direct microscopic examination, the staining effect is as follows Fig.10 Shown are staining micrographs to determine the time of MASPB-labeled phage (in the staining micrographs for each labeling time point, left: fluorescent field; right: overlay field).

[0128] Comparative Example 1: Screening of marker molecules

[0129] Selection of labeling molecules: Select AIE molecule A0174 (structural formula: )、A0178(Structural formula: )、A0188(Structural formula: ), MASPB, respectively, were dissolved in ultrapure water to prepare a 10 mM stock solution, 3 μL was added to 1 mL of concentrated phage solution, incubated at room temperature in the dark for 3 h for labeling, and 1 mL of 20 wt% BSA solution was added for purification to prepare the detection solution;

[0130] Sample preparation: Single colonies of Salmonella and Staphylococcus aureus were picked up in the culture medium, cultured at 37℃ and 180r / min until the logarithmic growth phase to obtain bacterial solution samples, and the two bacterial solutions were mixed in equal volumes (OD600 value was 0.6-0.8);

[0131] Staining: Mix 100 μL bacterial solution with 100 μL detection solution and incubate at room temperature in the dark for 20 min.

[0132] Preparation: Take 2 μL of the incubated solution and drop it on a glass slide, and place a cover glass for preparation;

[0133] Observation results: Under a confocal microscope (excitation: 488nm; emission: 600-700nm), 100× objective lens observation, direct microscopic examination, the staining effect is as follows Fig.11 The staining microscopy images of comparative example 1 showing screening of different AIE molecules for phage labeling (left: fluorescence field; middle: superimposed field; right: local magnified image).

[0134] Results Analysis

[0135] (1) From Figure 1 It can be seen that the bacteriophage labeled with the AIE molecule of the present invention has fewer steps for detecting bacteria, is simpler to operate, and takes less time to detect, which is conducive to the rapid output of results;

[0136] (2) From Figure 2 , 3 It can be seen that the Salmonella phage labeled with the AIE molecule of the present invention can accurately identify and stain Salmonella in 20 minutes, and the short rod-shaped bright fluorescence is observed under a microscope, and the morphological structure is clear, and the feces are easy to observe;

[0137] (3) From Figure 4 , 5 It can be seen that for different bacterial liquid samples or mixed bacterial liquid samples, the Salmonella phage labeled with AIE molecules can accurately identify and stain Salmonella within 20 minutes. The Salmonella in the sample is short rod-shaped and brightly fluorescent, while Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus have no fluorescence, indicating that the present invention has strong specificity and can accurately fluorescently label Salmonella with high accuracy.

[0138] (4) From Figure 6 It can be seen that in the face of a complex environment such as a simulated real fecal sample, the Salmonella phage labeled with AIE molecules can still accurately identify the Salmonella in the sample, showing short rod-shaped bright fluorescence, while other bacteria and impurities have no fluorescence, indicating that the present invention still has good detection performance in complex samples;

[0139] (5) From Figure 7 , 8 It can be seen that after using this molecule to label E. coli phage, E. coli can be specifically detected, indicating that this method has a wide range of applications in labeling phages for bacterial detection;

[0140] (6) From Fig. 9It can be seen that after the Salmonella phage labeled with AIE molecules was stored in a refrigerator at 4°C for 6 months, the phage titer remained at a high level and could be stored for a long time;

[0141] (7) From Fig.10 It can be seen that after screening the labeling time, MASPB-labeled phages for ≥3h can obtain a good labeling effect;

[0142] (8) From Fig.11 It can be seen that after screening multiple AIE molecules, MASPB labeled phage has the best detection effect, and this molecule was finally selected as the labeling molecule.

Claims

1. A bacteriophage labeled with an AIE molecule, characterized in that: It is composed of MASPB and bacteriophage, and MASPB binds to the genetic material inside the bacteriophage; The MASPB is a positively charged AIE molecule, and the structure of the MASPB is as follows: 。 2. The AIE molecule-labeled phage according to claim 1, characterized in that: The phage is selected from one or more of Salmonella phage, Escherichia coli phage, Pseudomonas aeruginosa phage, Pasteurella phage, Klebsiella pneumoniae phage, Vibrio cholerae phage, Shigella dysenteriae phage, Acinetobacter baumannii phage, Proteus phage, Enterobacter cloacae phage, Shigella phage, Staphylococcus phage, Streptococcus phage, Clostridium perfringens phage, Mycobacterium phage, and Enterococcus faecalis phage.

3. The method for preparing a bacteriophage labeled with an AIE molecule according to any one of claims 1 to 2, characterized in that: The following steps are involved: MASPB and phage are mixed and incubated in the dark, and MASPB enters the phage and combines with its genetic material, and then is purified to obtain the phage labeled with AIE molecules.

4. A bacteria detection reagent, characterized in that: A bacteriophage labeled with the AIE molecule according to any one of claims 1 to 2.

5. The bacteria detection reagent according to claim 4, characterized in that: The titer of the phage labeled with AIE molecules in the bacterial detection reagent is ≥5×10 10 PFU / mL.

6. The method for preparing the bacteria detection reagent according to any one of claims 4 to 5, characterized in that: The following steps are involved: MASPB was dissolved in water to prepare a MASPB stock solution, and the MASPB stock solution was added to the phage solution for incubation in the dark. After the incubation, BSA was added to bind the remaining unlabeled MASPB to prepare a bacterial detection reagent.

7. The method for preparing a bacteria detection reagent according to claim 6, characterized in that: The concentration of MASPB in the MASPB mother solution is 10-20 mM; the phage titer of the phage solution is ≥10 11 PFU / mL; The usage ratio of the MASPB mother solution and the phage solution is 3-5 μL:1 mL.

8. Use of the AIE molecule-labeled phage according to any one of claims 1 to 2 or the bacteria detection reagent according to any one of claims 4 to 5 in detecting bacteria, wherein the application is non-disease diagnosis and treatment.

9. A method for detecting bacteria, the method being a method for non-disease diagnosis and treatment, characterized in that: The following steps are involved: The bacteriophage labeled with the AIE molecule according to any one of claims 1 to 2 or the bacterial detection reagent according to any one of claims 4 to 5 is mixed with the bacterial solution to be tested, incubated, prepared into slices, and the fluorescence is observed under a confocal microscope.

10. The method for detecting bacteria according to claim 9, characterized in that: The incubation time is 20-30 min; The excitation wavelength of the confocal microscope is 470-490 nm, and the emission wavelength is 550-750 nm.

Images