Application of a reagent substrate and a kit for detecting intracranial Staphylococcus infection

Abstract

This application relates to the field of biotechnology, and specifically discloses a kit for detecting intracranial Staphylococcus infection and its application. The kit substrate includes a fluorescent substance containing an NHS ester reaction group and a glycopeptide antibiotic; its preparation method is as follows: the fluorescent substance containing an NHS ester reaction group and the glycopeptide antibiotic are respectively dissolved in DMSO solution; after dissolution is complete, the two are stirred and mixed to obtain a mixture; the stirred mixture is purified to obtain a fluorescent substance-glycopeptide antibiotic fluorescent targeting probe, and the fluorescent substance-glycopeptide antibiotic fluorescent targeting probe is the kit substrate. The kit substrate of this application has strong specificity, and the generated fluorescent substance-glycopeptide antibiotic fluorescent targeting probe can be used to specifically identify intracranial Staphylococcus infection.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, it relates to a kit for detecting intracranial staphylococcus aureus infection and its application. Background Art

[0002] Intracranial infection is one of the main sites of nosocomial infection in neurosurgical patients and one of the most serious complications in clinical craniotomy patients, with an infection rate as high as 17.86% and a mortality rate as high as 21%. Therefore, antibacterial drug treatment for intracranial infection should be started as soon as possible. Cerebrospinal fluid (CSF) bacterial culture is the gold standard for the diagnosis of intracranial infection and can guide clinical treatment through bacterial identification and drug sensitivity test results. However, the positive rate of CSF culture is no more than 10%, and it lacks specificity, some indicators have insufficient sensitivity, and the contamination rate is relatively high, which brings certain difficulties to clinical treatment.

[0003] Due to the insidious onset and rapid progression of intracranial infection, untimely treatment often leads to serious sequelae or even death. Therefore, early diagnosis and timely treatment are of great significance for improving the long-term prognosis of patients. Staphylococcus aureus plays an important role in intracranial infection, and the incidence of MRSA infection is high, which has become a difficult problem in clinical practice. According to the report of the Centers for Disease Control and Prevention in the United States, infections caused by staphylococcus aureus rank second in the world, second only to Escherichia coli. In 2017, the World Health Organization listed staphylococcus aureus as a "high-risk" monitoring object. In recent years, China has gradually strengthened the monitoring of staphylococcus aureus.

[0004] Cerebrospinal fluid bacterial culture is the gold standard for the diagnosis of intracranial infection and can guide clinical treatment through bacterial identification and drug sensitivity test results. However, the positive rate of CSF culture is no more than 10%, and it still lacks specificity, some indicators have insufficient sensitivity, and the contamination rate is relatively high, which brings certain difficulties to clinical treatment. Therefore, early diagnosis of cerebrospinal fluid bacteria is of great significance for improving the long-term prognosis of patients and shows great clinical application prospects.

[0005] Related technologies have disclosed a method for separating staphylococcus aureus, including coupling magnetic nanoparticles with polyethylene glycol, coating magnetic nanoparticles complexed with polyethylene glycol with vancomycin, and capturing target bacteria in a sample solution with magnetic nanoparticles complexed with polyethylene glycol and vancomycin. Through the action of an external magnetic field, the captured target bacteria are separated from the sample solution and resuspended, etc. The target bacteria captured by magnetic separation can be directly analyzed for subsequent steps. Compared with traditional bacterial magnetic separation methods, this method can perform magnetic separation on Gram-positive bacteria in blood, not only improving the separation efficiency of Staphylococcus aureus in the sample but also reducing costs. However, this method mainly separates staphylococcus aureus already present in the blood. There is little introduction on how to detect and verify the presence of staphylococcus aureus in the blood.

[0006] Therefore, there is still a need in the art for a method for detecting Staphylococcus intracranial infections to facilitate the rapid and specific detection of Staphylococcus from cerebrospinal fluid. Summary of the Invention

[0007] To solve the defects existing in the prior art, the present application provides the use of a reagent substrate and a kit for detecting Staphylococcus intracranial infections to facilitate the rapid and specific detection of Staphylococcus from cerebrospinal fluid.

[0008] In a first aspect, the present application provides a reagent substrate for detecting coccus infections, adopting the following technical solution: a kit substrate for detecting coccus infections, including a fluorescent substance containing a succinimidyl ester (NHS ester) reactive group and a glycopeptide antibiotic.

[0009] By adopting the above technical solution, glycopeptide antibiotics can be used to treat bacterial infections, especially for Gram-positive cocci, with targeting properties. Fluorescent substances containing NHS ester reactive groups are widely used in angiography. At the same time, fluorescent substances containing NHS ester reactive groups react rapidly and specifically with primary amines (-NH2), and can label antibodies and amine-modified oligonucleotides containing amines (-NH2) in a short time.

[0010] In the present application, glycopeptide antibiotics are used as ligands to bind to cocci. Fluorescent substances containing NHS ester reactive groups have a fluorescent effect and play a role in tracing bacteria. Glycopeptide antibiotics themselves carry NH2, and this group is coupled and linked to the NHS group in the fluorescent substance containing NHS ester reactive groups to form a single entity. After purification of the obtained product, it directly targets cocci, thereby detecting and identifying cocci.

[0011] Optionally, the fluorescent substance containing NHS ester reactive groups is indocyanine green-succinimidyl ester (ICG NHS ester), and the glycopeptide antibiotic is one of vancomycin, norvancomycin, and teicoplanin.

[0012] Preferably, the glycopeptide antibiotic is vancomycin.

[0013] By adopting the above technical solution, ICG NHS ester is a near-infrared fluorescent substance, which can effectively absorb near-infrared wavelengths around 800 nm and has a low absorption rate for wavelengths in the visible light range. Therefore, the noise caused by autofluorescence is low. In addition, the binding reaction rate between ICG NHS ester and glycopeptide antibiotics is fast, and direct linking can be carried out without introducing other groups.

[0014] Vancomycin can interfere with the cross-linking of peptidoglycan in the cell wall of cocci, thereby causing the dissolution of coccus cells and killing cocci.

[0015] Second aspect, the present application provides a method for preparing a kit substrate for detecting coccus infection, adopting the following technical solution:

[0016] A method for preparing a kit substrate for detecting intracranial Staphylococcus infection, comprising the following steps: dissolving a fluorescent substance containing an NHS ester reaction group and a glycopeptide antibiotic in DMSO solution respectively; after dissolution, stirring and mixing the two to obtain a mixture; purifying the stirred mixture to obtain a fluorescent substance-glycopeptide antibiotic fluorescent targeting probe, and the fluorescent substance-glycopeptide antibiotic fluorescent targeting probe is the kit substrate.

[0017] Wherein, when the fluorescent substance containing an NHS ester reaction group is ICG NHS ester and the glycopeptide antibiotic is vancomycin, the reaction between ICG NHS ester and vancomycin is specifically:

[0018]

[0019] By adopting the above technical solution, dimethyl sulfoxide (DMSO) solution is a polar aprotic solvent, which can dissolve the fluorescent substance containing an NHS ester reaction group and the glycopeptide antibiotic. In addition, DMSO is a cryoprotectant, which can quickly penetrate into cells, lower the freezing point and maintain cell activity.

[0020] After dissolving the fluorescent substance containing an NHS ester reaction group and the glycopeptide antibiotic in DMSO solution respectively, the fluorescent substance containing an NHS ester reaction group and the glycopeptide antibiotic react with the DMSO solution respectively, and finally after purification, a pure fluorescent substance-glycopeptide antibiotic fluorescent targeting probe is obtained.

[0021] Optionally, the stirring temperature of the mixture is 20°C - 30°C.

[0022] By adopting the above technical solution, setting the stirring temperature of the mixture to 20°C - 30°C, when the stirring temperature is lower than 20°C, the reaction rates of the fluorescent substance containing an NHS ester reaction group and the glycopeptide antibiotic with the DMSO solution are relatively slow; when the stirring temperature exceeds 30°C, the fluorescent substance containing an NHS ester reaction group is prone to decomposition and loses its activity.

[0023] Third aspect, the present application provides an application of a kit substrate, adopting the following technical solution:

[0024] A kit substrate can be used to detect coccus infection; the cocci include but are not limited to Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus.

[0025] Optionally, the kit substrate can be used to detect intracranial Staphylococcus infection.

[0026] Fourth aspect, the present application provides a method for detecting Staphylococcus in the intracranial region, adopting the following technical solution:

[0027] A method for detecting Staphylococcus in the intracranial region, comprising the following steps: taking 2 - 5 ml of cerebrospinal fluid, removing the cells in the cerebrospinal fluid; adding a fluorescent substance - glycopeptide antibiotic fluorescent targeting probe to the cerebrospinal fluid and incubating for 1 - 30 min; removing the free fluorescent substance - glycopeptide antibiotic fluorescent targeting probe by centrifugation to obtain a sample; if the bacteria in the sample exhibit red fluorescence, then the cerebrospinal fluid contains Staphylococcus.

[0028] By adopting the above technical solution, the NH2 on the glycopeptide antibiotic reacts with the fluorescent substance containing an NHS - ester reaction group, and the obtained product forms a fluorescent substance - glycopeptide antibiotic fluorescent targeting probe after purification. Five hydrogen bonds on the surface of the fluorescent substance - glycopeptide antibiotic fluorescent targeting probe are connected to the D - alanyl - D - alanine (D - Ala - D - Ala) molecules on the surface of Staphylococcus in the cerebrospinal fluid. When observing the bacteria and finding that the surface of the bacteria exhibits red fluorescence, it indicates that the cerebrospinal fluid contains Staphylococcus.

[0029] Fourth aspect, the present application provides a method for detecting the quantity of Staphylococcus in the intracranial region, adopting the following technical solution: A method for detecting the quantity of Staphylococcus in the intracranial region, comprising the following steps: taking 2 - 5 ml of cerebrospinal fluid, removing the cells in the cerebrospinal fluid; adding the fluorescent substance - glycopeptide antibiotic fluorescent targeting probe as described in any one of claims 3 - 4 to the cerebrospinal fluid and incubating for 1 - 30 min; removing the free fluorescent substance - glycopeptide antibiotic fluorescent targeting probe by centrifugation to obtain a sample; irradiating the sample with a laser to lyse the bacteria, adding the sample solution to a luciferin - luciferase solution, and measuring its bioluminescence signal.

[0030] By adopting the above technical solution, the present application uses the ATPS bioluminescence method to determine the quantity of Staphylococcus, that is, irradiating the sample with a laser to lyse the bacteria, releasing the ATP inside the bacteria, and then combining the sample solution with the luciferin - luciferase solution. Among them, luciferase is an active protein that converts chemical energy into light energy, that is, a biocatalyst. Under the catalysis of luciferase, ATP reacts with luciferin to release fluorescence. The ATP in the bacteria is detected by the fluorescence intensity, and then the total number of bacteria is detected.

[0031] In summary, the present application has the following beneficial effects:

[0032] 1. In this application, a glycopeptide antibiotic and a fluorescent substance containing an NHS ester reactive group are used as the main components and dissolved in a DMSO solution. The fluorescent substance containing an NHS ester reactive group is a dye molecule, and the glycopeptide antibiotic acts as a ligand and has targeting properties for bacteria. The glycopeptide antibiotic is coupled with the fluorescent substance containing an NHS ester reactive group, and after purification, a fluorescent substance - glycopeptide antibiotic fluorescent targeting probe is obtained. The fluorescent substance - glycopeptide antibiotic fluorescent targeting probe directly binds to bacteria, and after incubation, the presence of the fluorescent substance is observed to determine the presence of cocci.

[0033] 2. In this application, the fluorescent substance containing an NHS ester reactive group is ICG NHS ester, and the glycopeptide antibiotic is vancomycin. The NH2 on vancomycin is coupled with the NHS group in the fluorescent substance containing an NHS ester reactive group to form a single entity, obtaining an ICG - vancomycin fluorescent targeting probe. The ICG - vancomycin fluorescent targeting probe binds to the D - alanyl - D - alanine (D - Ala - D - Ala) molecules on the surface of Staphylococcus aureus in cerebrospinal fluid, emitting red fluorescence to achieve specific recognition.

[0034] 3. In this application, Staphylococcus aureus in cerebrospinal fluid is lysed by laser irradiation to release ATP inside the Staphylococcus aureus, and then by adding a luciferin - luciferase solution, ATP reacts with luciferin to release fluorescence. The total number of Staphylococcus aureus in cerebrospinal fluid is determined by detecting the intensity of the red fluorescence. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is an electron micrograph of Staphylococcus aureus provided by this application;

[0036] Figure 2 is a microscopic imaging diagram of Staphylococcus aureus provided by this application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The following further elaborates on this application in detail with reference to the drawings and embodiments.

[0038] Synthesis of the Fluorescent Targeting Probe

[0039] 1. Dissolve indocyanine green - succinimidyl ester (ICG NHS ester) and vancomycin separately in a dimethyl sulfoxide (DMSO) solution;

[0040] 2. Stir - mix the two using a high - speed mixer to obtain a mixture, and place the mixture at 20 - 30 °C for reaction for 18 h; 3. Finally, purify the mixture using a high - performance liquid chromatograph (HPLC) to obtain an ICG - vancomycin fluorescent targeting probe.

[0041] Fluorescent Microscopic Detection of Staphylococcus Aureus in Cerebrospinal Fluid

[0042] 1. Take 2 - 5 ml of cerebrospinal fluid and place it in a high - speed centrifuge. Set the rotation speed to 1000 r / min and the time to 7 min. Remove the cells in the cerebrospinal fluid by high - speed centrifugation.

[0043] 2. Add the ICG - vancomycin fluorescent targeting probe to the centrifuged cerebrospinal fluid and incubate for 1 - 30 min.

[0044] 3. Centrifuge the incubated ICG - vancomycin fluorescent targeting probe. Set the rotation speed to 8000 r / min and the time to 3 - 10 min. Remove the free ICG - vancomycin fluorescent targeting probe by centrifugation to obtain the sample.

[0045] 4. Take a drop of the sample and observe it under a fluorescence microscope or a flow cytometer. If there is red fluorescence on the surface of the bacteria, it is Staphylococcus. Refer to Figure 1 and Figure 2 for comparison. Figure 1 and Figure 2 It can be seen that: Figure 2 Staphylococcus in

[0046] all have attached red fluorescence. That is, the kit prepared by this application can accurately determine the presence or absence of Staphylococcus.

[0047] 1. Take 2 - 5 ml of cerebrospinal fluid and place it in a high - speed centrifuge. Set the rotation speed to 1000 r / min and the time to 7 min. Remove the cells in the cerebrospinal fluid by high - speed centrifugation.

[0048] 2. Add the ICG - vancomycin fluorescent targeting probe to the centrifuged cerebrospinal fluid and incubate for 1 - 30 min.

[0049] 3. Centrifuge the incubated ICG - vancomycin fluorescent targeting probe. Set the rotation speed to 8000 r / min and the time to 3 - 10 min. Remove the free ICG - vancomycin fluorescent targeting probe by centrifugation to obtain the sample.

[0050] 4. Irradiate the sample with an 808 nm infrared laser for 5 min to lyse the bacteria and release ATP.

[0051] 5. Add the sample solution after bacterial lysis to the luciferin - luciferase solution and immediately measure the bioluminescence signal with an enzyme - linked immunosorbent assay (ELISA) reader.

[0052] This specific embodiment is only an interpretation of this application and is not a limitation of this application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of this application, it is protected by the patent law.

Claims

1. A kit substrate for detecting coccus infection, characterized in that, It includes a fluorescent substance containing an NHS ester reactive group and a glycopeptide antibiotic; the fluorescent substance containing an NHS ester reactive group is ICG NHS ester, and the glycopeptide antibiotic is vancomycin; The preparation method of the kit substrate for detecting coccus infection includes the following steps: separately dissolve the fluorescent substance containing an NHS ester reactive group and the glycopeptide antibiotic in DMSO solution; after complete dissolution, stir and mix the two to obtain a mixture; purify the stirred mixture to obtain a fluorescent substance-glycopeptide antibiotic fluorescent targeting probe, and the fluorescent substance-glycopeptide antibiotic fluorescent targeting probe is the kit substrate.

2. A preparation method of the kit substrate for detecting coccus infection according to claim 1, characterized in that: The stirring temperature of the mixture is 20°C - 30°C.

3. An application of the kit substrate according to claim 2, characterized in that: The application of the kit substrate in the preparation of a composition for detecting coccus infection.

4. An application of the kit substrate according to claim 3, characterized in that: The application of the kit substrate in the preparation of a composition for detecting intracranial Staphylococcus infection.

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