Fluorescent probe for detecting mycobacterium tuberculosis and preparation method and detection method thereof

Abstract

The present application relates to the field of molecular biology technology, and provides a fluorescent probe for detecting Mycobacterium tuberculosis, the fluorescent probe comprising a metabolic group and an aggregation-induced luminescent group, the metabolic group being connected to the aggregation-induced luminescent group by a chemical bond, and the metabolic group being used to synthesize the peptidoglycan of the cell wall of Mycobacterium tuberculosis. The fluorescent probe provided by the present application synthesizes the peptidoglycan of the cell wall of Mycobacterium tuberculosis through the metabolic group contained therein, and aggregates to the cell wall of Mycobacterium tuberculosis in large quantities in a metabolically labeled manner, and the aggregation-induced luminescent group generates a fluorescent signal due to aggregation, thereby realizing the detection of Mycobacterium tuberculosis. In addition, the fluorescent probe can quickly and conveniently detect Mycobacterium tuberculosis, and has strong specificity and high sensitivity.

Description

Technical Field

[0001] The present application belongs to the technical field of molecular biology, and in particular relates to a fluorescent probe for detecting Mycobacterium tuberculosis and a preparation method and a detection method thereof. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium tuberculosis. Mycobacterium tuberculosis can invade various organs of the human body, but mainly invades the lungs, which is called pulmonary tuberculosis. In 2020, 9.87 million people were infected with tuberculosis and 1.28 million people died from tuberculosis worldwide. Therefore, tuberculosis seriously endangers human health and is a major public health problem. The prevention and control of tuberculosis worldwide still faces drug resistance, lack of effective vaccines and early diagnosis methods. Therefore, timely and accurate detection of tuberculosis is crucial for the treatment and prevention of tuberculosis.

[0003] However, current detection methods still have major drawbacks. Tuberculosis culture is the "gold standard" for diagnosing tuberculosis and is inexpensive, but tuberculosis grows slowly and detection takes too long; although acid-fast staining and nucleic acid testing in etiological testing are reliable methods for laboratory diagnosis, the positive rate is very low, and more than half of the patients cannot find etiological evidence; immunological diagnostic methods have the problem of low specificity.

[0004] Therefore, there is an urgent need to develop a new method with strong specificity, high sensitivity, and the ability to quickly and conveniently detect tuberculosis infection. Summary of the invention

[0005] The purpose of the present application is to provide a fluorescent probe for detecting Mycobacterium tuberculosis and a preparation method and a detection method thereof, aiming to solve the problems of low specificity and low sensitivity in the existing detection of Mycobacterium tuberculosis.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present application provides a fluorescent probe for detecting Mycobacterium tuberculosis, wherein the fluorescent probe comprises a metabolic group and an aggregation-induced luminescent group, wherein the metabolic group is connected to the aggregation-induced luminescent group via a chemical bond, and the metabolic group is used to synthesize cell wall peptidoglycan of Mycobacterium tuberculosis.

[0008] In a second aspect, the present application provides a method for preparing a fluorescent probe for detecting Mycobacterium tuberculosis, the preparation method comprising the following steps:

[0009] Providing metabolic groups and aggregation-induced emission groups;

[0010] The metabolic group and the aggregation-induced luminescence group are mixed to undergo a click chemistry reaction to synthesize a fluorescent probe.

[0011] In a third aspect, the present application provides a method for detecting Mycobacterium tuberculosis, wherein the detection method is to use the fluorescent probe provided by the present application or the fluorescent probe prepared by the preparation method provided by the present application for detection, and the detection method comprises the following steps:

[0012] Provide samples to be tested;

[0013] The sample to be tested and the fluorescent probe are mixed and incubated to obtain a mixed solution;

[0014] The mixed solution is subjected to fluorescence spectrum detection or fluorescence imaging detection.

[0015] Compared with the prior art, this application has the following technical effects:

[0016] The first aspect of the present application provides a fluorescent probe for detecting Mycobacterium tuberculosis, which includes a metabolic group and an aggregation-induced luminescent group connected to the metabolic group by a chemical bond, and synthesizes the cell wall peptidoglycan of Mycobacterium tuberculosis through the contained metabolic group, thereby aggregating to the cell wall of Mycobacterium tuberculosis in large quantities in a metabolic labeling manner, and the aggregation-induced luminescent group generates a fluorescent signal due to aggregation, thereby realizing the detection of Mycobacterium tuberculosis. In addition, the fluorescent probe can quickly and conveniently detect Mycobacterium tuberculosis, and has strong specificity and high sensitivity.

[0017] The preparation method of the fluorescent probe provided in the second aspect of the present application is to mix the provided metabolic group with the aggregation-induced luminescent group, and use the click chemistry reaction to achieve an efficient coupling reaction between the metabolic group and the aggregation-induced luminescent group, so as to synthesize a fluorescent probe with the function of detecting Mycobacterium tuberculosis. In addition, the preparation method only uses the splicing of chemical groups, and the synthesis is fast and reliable, with high selectivity and specificity.

[0018] The detection method of Mycobacterium tuberculosis provided in the third aspect of the present application is to detect using the fluorescent probe provided in the present application or the fluorescent probe prepared by the preparation method provided in the present application. The method firstly mixes the sample to be tested and the fluorescent probe for incubation, so that the metabolic groups contained in the fluorescent probe participate in the synthesis of peptidoglycan of the cell wall of Mycobacterium tuberculosis, and aggregate to the cell wall of Mycobacterium tuberculosis in large quantities in the form of metabolic labeling to obtain a mixed solution, and then the mixed solution is subjected to fluorescence spectrum detection or fluorescence imaging detection. The aggregation-induced luminescent group contained in the fluorescent probe generates a fluorescent signal due to aggregation under light radiation, thereby realizing the detection of Mycobacterium tuberculosis. In addition, the detection method provided in the present application has the characteristics of strong specificity and high sensitivity, and has broad application prospects in clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 is a preparation flow chart of the method for preparing the fluorescent probe provided in the embodiments of the present application;

[0021] Figure 2 is a detection flow chart of the method for detecting Mycobacterium tuberculosis provided in the embodiments of the present application;

[0022] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe provided in Example A3 of the present application;

[0023] Figure 4 This is a fluorescence imaging result diagram of free attenuated bovine tuberculosis Mycobacterium tuberculosis specifically labeled with a fluorescent probe provided in Example B1 of the present application;

[0024] Figure 5 This is a graph showing the detection limit results of the fluorescent probe specifically labeling free attenuated bovine tuberculosis Mycobacterium bovis provided in Example B2 of the present application;

[0025] Figure 6 This is a fluorescence imaging result diagram of attenuated bovine tuberculosis Mycobacterium in macrophages labeled with a fluorescent probe provided in Example B3 of the present application. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0027] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0028] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0029] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0030] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0031] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0032] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0033] A first aspect of an embodiment of the present application provides a fluorescent probe for detecting Mycobacterium tuberculosis. The fluorescent probe includes a metabolic group and an aggregation-induced luminescent group. The metabolic group is connected to the aggregation-induced luminescent group through a chemical bond, and the metabolic group is used to synthesize cell wall peptidoglycan of Mycobacterium tuberculosis.

[0034] The fluorescent probe for detecting mycobacterium tuberculosis provided in the embodiment of the present application includes a metabolic group and an aggregation-induced luminescent group connected to the metabolic group by a chemical bond, and the cell wall peptidoglycan of mycobacterium tuberculosis is synthesized by the metabolic group contained, so as to be aggregated to the cell wall of mycobacterium tuberculosis in a large amount in a metabolically labeled manner, and the aggregation-induced luminescent group generates a fluorescent signal due to aggregation, thereby realizing the detection of mycobacterium tuberculosis. In addition, the fluorescent probe can quickly and conveniently detect mycobacterium tuberculosis, and has strong specificity and high sensitivity. In an embodiment, the metabolic group includes at least one modified D-alanine in azide, tetrazine, sulfhydryl, alkyne, trans-cyclooctene, and olefin. Specifically, the metabolic group can be 3-azido-D-alanine, and can also be 3-tetrazine-D-alanine. The D-alanine in the metabolic group contained in the fluorescent probe of the present embodiment is a synthetic raw material for the peptidoglycan of the cell wall of mycobacterium tuberculosis, and can be aggregated to the cell wall of mycobacterium tuberculosis in a large amount by a metabolically labeled manner, thereby realizing specific labeling of mycobacterium tuberculosis.

[0035] In an embodiment, the aggregation-induced luminescent group includes 4-(2-(4-benzhydrylaminephenylethylene)pyridinium bromide modified by at least one of alkynes, trans-cyclooctene, olefins, azides, tetrazines, and thiols. Specifically, the aggregation-induced luminescent group can be 1-butyne-4-(2-(4-benzhydrylaminephenylethylene)pyridinium bromide, or 1-trans-cyclooctene-4-(2-(4-benzhydrylaminephenylethylene)pyridinium bromide. The aggregation-induced luminescent group contained in the fluorescent probe of this embodiment causes the intramolecular spin motion to be greatly restricted after aggregation on the cell wall of Mycobacterium tuberculosis, that is, its non-radiative transition pathway is blocked, and most of the excitons return to the ground state through radiative transition and emit photons, thereby inducing the fluorescent probe to efficiently convert the absorbed energy into a fluorescent signal.

[0036] In an embodiment, the metabolic group and the aggregation-induced emission group are connected by a chemical bond to form a fluorescent probe, and the chemical bond may include any one of trans-cyclooctene-tetrazine, dibenzocyclooctyne-azide, alkynyl-azide, thiol-olefin, and thiol-alkyne.

[0037] In the embodiment, the molecular structure of the fluorescent probe is shown in the following general structural formula I:

[0038]

[0039] The R group in the general structural formula VI includes any one of trans-cyclooctene-tetrazine, dibenzocyclooctyne-azide, alkynyl-azide, mercapto-olefin, and mercapto-alkyne.

[0040] In a specific embodiment, the metabolic group is 3-azido-D-alanine, the aggregation-induced luminescence group is 1-butyne-4-(2-(4-benzhydrylaminephenylethylene)pyridinium bromide, and the molecular structure of the fluorescent probe is:

[0041]

[0042] In this embodiment, the chemical bond for forming the above fluorescent probe molecular structure is alkynyl-azide, and specifically, the azide group in 3-azido-D-alanine is formed by connecting with the butynyl group in 1-butyne-4-(2-(4-benzhydrylaminephenylethylene)pyridine bromide.

[0043] In an embodiment, the excitation light wavelength of the fluorescent probe is 350-500nm; the fluorescence emission spectrum of the fluorescent probe is 500-800nm. Specifically, the excitation light wavelength of the fluorescent probe can be but not limited to 350nm, 380nm, 400nm, 420nm, 440nm, 460nm, 480nm, 500nm. The fluorescence emission spectrum region of the fluorescent probe can be but not limited to 500-700nm, 500-750nm, 500-800nm.

[0044] The second aspect of the present application provides a method for preparing a fluorescent probe for detecting Mycobacterium tuberculosis. Figure 1 As shown, the preparation method comprises the following steps:

[0045] S11: Provide metabolic groups and aggregation-induced luminescence groups;

[0046] S12: The metabolic group is mixed with the aggregation-induced luminescence group to undergo a click chemistry reaction to synthesize a fluorescent probe.

[0047] The preparation method of the fluorescent probe provided in the embodiment of the present application is to mix the provided metabolic group with the aggregation-induced luminescent group, and use the click chemistry reaction to achieve an efficient coupling reaction between the metabolic group and the aggregation-induced luminescent group, thereby synthesizing a fluorescent probe with the function of detecting Mycobacterium tuberculosis. In addition, the preparation method only uses the splicing of chemical groups, and the synthesis is fast and reliable, with high selectivity and specificity.

[0048] In step S11, a metabolic group and an aggregation-induced luminescent group are provided according to a molar ratio of the metabolic group to the aggregation-induced luminescent group of 1: (0.2-5). In a specific embodiment, the molar ratio of the metabolic group to the aggregation-induced luminescent group is but not limited to 1: 0.2, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5. Within the molar ratio range of the metabolic group to the aggregation-induced luminescent group provided in the embodiment of the present application, it is conducive to the click chemistry reaction between the metabolic group and the aggregation-induced luminescent group, thereby achieving efficient coupling of the metabolic group and the aggregation-induced luminescent group.

[0049] In step S12, the metabolic group and the aggregation-inducing luminescent group undergo a click chemistry reaction to form a chemical bond to connect the two; for example, the azide group and the alkyne group form a chemical bond, that is, the metabolic group can be connected by an addition reaction between the azide group contained in the metabolic group and the alkyne group contained in the aggregation-inducing luminescent group, or the tetrazine group and the trans-cyclooctenyl group form a chemical bond, that is, the tetrazine group contained in the metabolic group and the trans-cyclooctenyl group contained in the aggregation-inducing luminescent group undergo an addition reaction to achieve connection.

[0050] When the metabolic group is 3-azido-D-alanine and the aggregation-induced emission group is 1-butyne-4-(2-(4-benzhydrylaminephenylvinyl)pyridinium bromide, 3-azido-D-alanine and 1-butyne-4-(2-(4-benzhydrylaminephenylvinyl)pyridinium bromide are first dissolved in dimethyl sulfoxide to obtain a mixed solution, and then copper bromide is dispersed in trimethylamine to form a dispersion. Finally, the dispersion is added to the mixed solution and stirred, and the azide group in 3-azido-D-alanine and the butynyl group in 1-butyne-4-(2-(4-benzhydrylaminephenylvinyl)pyridinium bromide undergo a Husigen cycloaddition reaction under the catalysis of copper ions to synthesize a cyclic 1,4-disubstituted-1,2,3-triazole.

[0051] When the metabolic group is 3-tetrazine-D-alanine and the aggregation-induced emission group is 1-trans-cyclooctene-4-(2-(4-benzhydrylaminephenylethylene)pyridinium bromide, 3-tetrazine-D-alanine and 1-trans-cyclooctene-4-(2-(4-benzhydrylaminephenylethylene)pyridinium bromide are mixed and treated, and the tetrazine group in 3-tetrazine-D-alanine and the trans-cyclooctene group in 1-trans-cyclooctene-4-(2-(4-benzhydrylaminephenylethylene)pyridinium bromide undergo a Diels-Alder cycloaddition reaction to synthesize a fluorescent probe.

[0052] The third aspect of the embodiment of the present application provides a method for detecting Mycobacterium tuberculosis, wherein the detection method is to use the fluorescent probe provided by the present application or the fluorescent probe prepared by the preparation method provided by the present application for detection, such as Figure 2 As shown, the detection method comprises the following steps:

[0053] S21: Provide samples to be tested;

[0054] S22: mixing the sample to be tested and the fluorescent probe for incubation to obtain a mixed solution;

[0055] S23: performing fluorescence spectrum detection or fluorescence imaging detection on the mixed solution.

[0056] The detection method of Mycobacterium tuberculosis provided in the embodiment of the present application is to detect using the fluorescent probe provided in the present application or the fluorescent probe prepared by the preparation method provided in the present application. The method firstly mixes the sample to be tested and the fluorescent probe for incubation, so that the metabolic groups contained in the fluorescent probe participate in the synthesis of peptidoglycan of the cell wall of Mycobacterium tuberculosis, and aggregate to the cell wall of Mycobacterium tuberculosis in large quantities in the form of metabolic labeling to obtain a mixed solution, and then the mixed solution is subjected to fluorescence spectrum detection or fluorescence imaging detection. The aggregation-induced luminescent group contained in the fluorescent probe generates a fluorescent signal due to aggregation under light radiation, thereby realizing the detection of Mycobacterium tuberculosis. In addition, the detection method provided in the present application has the characteristics of strong specificity and high sensitivity, and has broad application prospects in clinical diagnosis.

[0057] In step S21, the sample to be tested refers to a sample to be tested containing free Mycobacterium tuberculosis or intracellular Mycobacterium tuberculosis.

[0058] In step S22, the method of mixing and incubating is: placing the sample to be tested and the fluorescent probe in a shaker at 37°C for shaking and incubation to obtain a mixed solution. After the step of mixing and incubating, the method further includes: centrifuging, washing, and resuspending the mixed solution in sequence to obtain a bacterial suspension.

[0059] In an embodiment, the incubation time is 0.1-24h. Within the incubation time range provided in the embodiment of the present application, the fluorescent probe can be fully used to specifically label the cell wall of Mycobacterium tuberculosis, thereby facilitating subsequent fluorescence spectrum detection or fluorescence imaging detection. In a specific embodiment, the incubation time can be, but is not limited to, 0.1h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 6h, 12h, 24h.

[0060] In step S23, a fluorescence instrument or an enzyme-labeling instrument may be used to detect the fluorescence spectrum or fluorescence imaging of the bacterial suspension.

[0061] In the embodiment, the detection limit of the fluorescent probe for Mycobacterium tuberculosis is 10 2 -10 3 CFU / mL. Specifically, the detection limit of the fluorescent probe for Mycobacterium tuberculosis can be, but is not limited to, 10 2 CFU / mL, 2.5×10 2 CFU / mL, 5×10 2 CFU / mL, 7.5×10 2 CFU / mL, 10 3 CFU / mL.

[0062] In an embodiment, the concentration of the fluorescent probe is 1-500 μmol / L. Specifically, the concentration of the fluorescent probe can be, but is not limited to, 1 μmol / L, 5 μmol / L, 10 μmol / L, 30 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 300 μmol / L, 400 μmol / L, 500 μmol / L.

[0063] In an embodiment, the mycobacterium tuberculosis includes at least one of human mycobacterium tuberculosis, bovine mycobacterium tuberculosis, mycobacterium africanum, mycobacterium voles, attenuated bovine mycobacterium tuberculosis, mycobacterium caprae, mycobacterium canetti, mycobacterium pinnipedium, mycobacterium mongoose, and mycobacterium antelope. Specifically, the mycobacterium tuberculosis may be human mycobacterium tuberculosis, bovine mycobacterium tuberculosis, or mycobacterium voles.

[0064] The following describes the invention in conjunction with specific embodiments.

[0065] Example A1

[0066] This embodiment provides a method for preparing a fluorescent probe. The preparation method comprises the following steps:

[0067] S11: providing 3-azido-D-alanine and 1-butyne-4-(2-(4-benzhydrylaminephenylethylene)pyridinium bromide in a molar ratio of 1:0.2;

[0068] S12: Dissolve 3-azido-D-alanine and 1-butyne-4-(2-(4-benzhydrylaminephenylethylene)pyridinium bromide in dimethyl sulfoxide to obtain a mixed solution; disperse copper bromide in trimethylamine to form a dispersion; inject the dispersion into the mixed solution, stir at 25°C for 12 hours, extract with dichloromethane, and centrifuge to obtain a solid containing a fluorescent probe; dissolve the solid in dimethyl sulfoxide, filter with a polyethersulfone membrane filter, and evaporate and dry the collected organic layer to obtain a fluorescent probe.

[0069] Example A2

[0070] This embodiment provides a method for preparing a fluorescent probe.

[0071] The preparation method is different from that of Example A1 in that: S11: providing 3-azido-D-alanine and 1-butyne-4-(2-(4-benzhydrylaminephenylethylene)pyridine bromide in a molar ratio of 1:0.34.

[0072] Example A3

[0073] This embodiment provides a method for preparing a fluorescent probe.

[0074] The preparation method is different from that of Example A1 in that: S11: providing 3-azido-D-alanine and 1-butyne-4-(2-(4-benzhydrylaminephenylethylene)pyridinium bromide in a molar ratio of 1:5.

[0075] Example A4

[0076] This embodiment provides a method for preparing a fluorescent probe.

[0077] The preparation method is different from that of Example A1 in that: S11: providing 3-tetrazine-D-alanine and 1-trans-cyclooctene-4-(2-(4-benzhydrylaminephenylethylene)pyridine bromide in a molar ratio of 1:1.

[0078] Example B1

[0079] This embodiment provides a method for detecting Mycobacterium tuberculosis.

[0080] The detection method is to use the fluorescent probe provided in Example A3 for inspection, and the detection method comprises the following steps:

[0081] S21: Take the frozen free attenuated bovine tuberculosis Mycobacterium (BCG) out of the -80℃ refrigerator, wash it twice by centrifugation, place it in 7H9 liquid culture medium, and incubate it in a shaker at 37℃ until OD 600 When the value (absorbance at 600 nm) reaches 0.5, the sample to be tested is obtained;

[0082] S22: 200 μL of the sample to be tested and the fluorescent probe with a concentration of 20 μmol / L were placed in a shaker at 37°C for 2 h to obtain a mixed solution; the mixed solution was then centrifuged at a speed of 7000 rpm for 15 min, washed twice, resuspended in phosphate buffer to obtain a bacterial solution, and the bacterial solution was diluted 10 times to obtain a bacterial suspension;

[0083] S23: The bacterial suspension was placed in an eight-well cell chamber and imaged under a laser confocal microscope.

[0084] Example B2

[0085] This embodiment provides a method for detecting Mycobacterium tuberculosis.

[0086] The difference between this detection method and Example B1 is that in step S22, the bacterial solution is diluted 10 times and 10 times respectively. 2 times, 10 3 times, 10 4 times, 10 5 times, 10 6 times, 10 7 times to obtain a bacterial suspension;

[0087] S23: Add bacterial suspensions of different concentrations into a fluorescent cuvette and place it under a fluorescence meter to detect the fluorescence spectrum of the fluorescent probe.

[0088] Example B3

[0089] This embodiment provides a method for detecting Mycobacterium tuberculosis.

[0090] The detection method is to use the fluorescent probe provided in Example A3 for inspection, and the detection method comprises the following steps:

[0091] S21: Human macrophage Raw264.7 cells were inoculated into two groups of eight-well cell chambers, with 6×105 cells per well and 200 μL of DMEM medium per well. The cells were cultured for 16 hours to allow the cells to adhere to the wall. The original culture medium was removed and replaced with DMEM medium containing BCG (OD 600 =0.01, without double antibody), and continue to culture for 6 hours; discard the supernatant, and wash away the BCG that has not entered the cells with phosphate buffer, and repeat 3 times;

[0092] S22: Remove the supernatant and replace it with DMEM medium containing the fluorescent probe at a concentration of 20 μmol / L. Add DMEM medium without the fluorescent probe to the control group wells and continue culturing for 2 hours. After culturing for 2 hours, discard the supernatant and rinse the fluorescent probe that has not entered the cells with phosphate buffer. Repeat 3 times.

[0093] S32: The eight-well cell chamber was placed under a laser confocal microscope for imaging.

[0094] Related test analysis:

[0095] 1. The fluorescent probe obtained in Example A3 was subjected to nuclear magnetic resonance analysis. The test results are as follows: Figure 3 As shown, from Figure 3 The nuclear magnetic resonance detection results can determine that the fluorescent probe is a cyclic 1,4-disubstituted-1,2,3-triazole, and the molecular structure of the cyclic 1,4-disubstituted-1,2,3-triazole is as follows:

[0096]

[0097] 2. The bacterial suspension provided in Example B1 was observed under a laser confocal microscope and photographed, wherein the fluorescence excitation wavelength was 488 nm and the fluorescence signal collection area was 500-700 nm. The test results are as follows: Figure 4 As shown, green represents the fluorescent signal of the fluorescent probe label. Figure 4 It can be seen that the fluorescent probe is specifically distributed on the bacterial wall of BCG and has a very strong fluorescent signal, indicating that the fluorescent probe can be used to detect free Mycobacterium tuberculosis.

[0098] 3. Using a fluorescence instrument to detect the fluorescence spectrum of the fluorescent probe contained in the bacterial suspension provided in Example B2, wherein the fluorescence excitation wavelength is 468 nm, and the acquisition area of ​​the fluorescence emission spectrum is 500-900 nm. The detection results are as follows: Figure 5 As shown. Figure 5 It can be seen that the linear range of BCG bacterial solution concentration is good in the range of 102-106 CFU / mL, indicating that BCG labeled with fluorescent probe has good sensitivity and can be used for the detection of low concentration of Mycobacterium tuberculosis.

[0099] 4. The bacterial suspension provided in Example B3 was observed under a laser confocal microscope and photographed, wherein the fluorescence excitation wavelength was 488 nm and the fluorescence signal collection area was 500-700 nm. The test results are as follows: Figure 6 As shown, green represents the fluorescent signal of the fluorescent probe label. Figure 6 It can be seen that the experimental group has an extremely strong green fluorescence signal, which is only distributed on the intracellular BCG, while the control group has only a very weak green fluorescence signal, indicating that the fluorescent probe can be used to specifically label intracellular BCG and can therefore be used for the detection of intracellular Mycobacterium tuberculosis.

[0100] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for detecting Mycobacterium tuberculosis, characterized in that: The detection method is to use a fluorescent probe for detection, and the detection method comprises the following steps: Provide samples to be tested; Mixing the sample to be tested and the fluorescent probe for incubation to obtain a mixed solution; Performing fluorescence spectrum detection or fluorescence imaging detection on the mixed solution; Wherein, the molecular structure of the fluorescent probe is shown in the following general structural formula I: ； Wherein, the R group in the general structural formula I includes any one of trans-cyclooctene-tetrazine, dibenzocyclooctyne-azide, and alkynyl-azide; The detection method does not involve the purpose of diagnosis and treatment of diseases.

2. The detection method according to claim 1, characterized in that The excitation wavelength of the fluorescent probe is 350-500nm; The fluorescence emission spectrum of the fluorescent probe is 500-800nm.

3. The detection method according to claim 1, characterized in that The detection limit of the fluorescent probe for Mycobacterium tuberculosis is 10 2 -10 3 CFU / mL.

4. The detection method according to claim 1, characterized in that The incubation time is 0.1-24h; and / or The concentration of the fluorescent probe is 1-500 μmol / L.

5. The detection method according to any one of claims 3 to 4, characterized in that: The Mycobacterium tuberculosis includes at least one of Mycobacterium tuberculosis hominis, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium voles.

6. The detection method according to any one of claims 3 to 4, wherein the Mycobacterium tuberculosis comprises at least one of Mycobacterium caprae, Mycobacterium canetti, Mycobacterium pinnipedium, Mycobacterium mongoose and Mycobacterium antelope.

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