WO2018218510A1 - Kit and method for labeling bacterial flora, fluorescence-labeled bacterial flora and application thereof - Google Patents

Kit and method for labeling bacterial flora, fluorescence-labeled bacterial flora and application thereof Download PDF

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WO2018218510A1
WO2018218510A1 PCT/CN2017/086618 CN2017086618W WO2018218510A1 WO 2018218510 A1 WO2018218510 A1 WO 2018218510A1 CN 2017086618 W CN2017086618 W CN 2017086618W WO 2018218510 A1 WO2018218510 A1 WO 2018218510A1
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fluorescent probe
fluorescent
bacterial
type
probe
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PCT/CN2017/086618
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Chinese (zh)
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陈兴
王炜
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北京大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

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  • the present invention relates to the field of bacterial fluorescent labeling, and in particular to a kit, method, fluorescently labeled bacterial flora and application thereof for labeling bacterial bacterial flora samples.
  • bacterial staining, labeling and imaging observations include traditional Gram staining, Fluorescence in situ hybridization (FISH), differential nucleic acid staining (depending on the permeability of different bacterial cell membranes), and wheat germ agglutination.
  • FISH Fluorescence in situ hybridization
  • WGA dye agglutinin
  • the FISH probe kit has the problems of complicated DNA probe design, low rRNA content, cumbersome operation, low signal-to-noise ratio, etc. It can only operate on the bacteria after fixation, and is limited to detecting the presence of primers and the feasibility of primers. Proven bacteria, so their application is still subject to many restrictions.
  • antibiotic fluorescent probe species have been reported in the literature, polymyxin-based probes and vancomycin-based remoraline-based probes are included. These probes are limited to imaging the markers of certain specific bacterial species. Labeling or imaging of bacteria in samples of complex bacterial systems such as gut bacterial flora or complex bacterial samples (referred to as containing two or more bacterial species, of unknown species and possibly containing non-bacterial contaminants) Identification, there is currently no good solution in the prior art.
  • the main object of the present invention is to provide a kit, a method, a fluorescently labeled bacterial flora and the like for labeling bacterial flora, and to solve the problem that it is difficult to accurately mark a complex bacterial system in the prior art.
  • kits for labeling a sample of a bacterial flora comprising a first type of fluorescent probe and a second type of fluorescent probe, the first type of fluorescent probe specific label Gram-positive bacteria in the sample, the second type of fluorescent probe specifically labels Gram-negative bacteria, and the first type of fluorescent probe and the second type of fluorescent probe respectively have different fluorescent labels.
  • the first type of fluorescent probe is selected from one or more of fluorescent probes based on teicoplanin antibiotics.
  • fluorescent probes based on teicoplanin antibiotics include teicoplanin-based fluorescent probes, vancomycin-based fluorescent probes, telavancin-based fluorescent probes, and dalbavancin-based fluorescent probes. Fluorescent probes, oritavancin-based fluorescent probes, epothilone-based fluorescent probes, balchimycin-based fluorescent probes, and ristocetin-based fluorescent probes.
  • the vancomycin-based fluorescent probe is:
  • the fluorescent probe based on Travancin is:
  • the dalbavancin-based fluorescent probe is:
  • Olivin-based fluorescent probes are:
  • the fluorescent probe based on the etodomycin is: Wherein R" is H or Cl;
  • the fluorescent probe based on balchimycin is:
  • the fluorescent probe based on ristocetin is:
  • R 1 to R 18 each independently represent a fluorescent label.
  • the second type of fluorescent probe is any one or more of a tridecapeptide antibiotic-based fluorescent probe and a polymyxin-based antibiotic-based fluorescent probe; preferably, based on a thirteen peptide
  • Fluorescent probes for bacteriocin antibiotics include a threonopeptidin A1-based fluorescent probe, a trichostin B1-based fluorescent probe, and a threomycin C-based fluorescent probe; more preferably, based on The fluorescent probe of the 13-peptide leptin A1 is:
  • Fluorescent probes based on thirteen peptides B1 are:
  • the fluorescent probe based on thirteen peptide C is:
  • R 1 to R 9 each independently represent a fluorescent label
  • the polymyxin-based fluorescent probe is:
  • R' is R" is R 10 to R 14 each independently represent a fluorescent label.
  • the fluorescent label is selected from the group consisting of a coumarin fluorescent label, a naphthalene fluorescent label, a fluoroboron fluorescent label, a xanthene fluorescent label, a cyanine fluorescent label, a squaraine fluorescent label, and an anthraquinone fluorescent label. Any two or more.
  • a fluorescently labeled bacterial flora which is labeled by any of the above kits.
  • a method of labeling a bacterial flora comprising labeling a bacterial species in a bacterial flora with a first type of fluorescent probe and a second type of fluorescent probe, wherein One type of fluorescent probe and the second type of fluorescent probe are the first type of fluorescent probe and the second type of fluorescent probe in any of the above kits.
  • the bacteria in the bacterial flora are simultaneously labeled using the first type of fluorescent probe and the second type of fluorescent probe.
  • kits for diagnosing bacterial pulmonary inflammation, bacterial genitourinary infection, bacterial infectious diarrhea and/or bacterial meningitis are provided.
  • applications include the use of sputum smears to diagnose bacterial lung inflammation; vaginal secretion smears, urethral secretion smears, urine smears or urinary smears for diagnosis of bacterial genitourinary infections; Tablets for the diagnosis of bacterial infectious diarrhea; and/or the use of cerebrospinal fluid smears to diagnose bacterial meningitis.
  • the technical scheme of the present invention by using a fluorescent probe based on narrow-spectrum antibiotics and combining the two types of strains with high specificity, the complementary binding of the two fluorescent labels can be utilized, and mutual confirmation can be avoided, and it can be avoided based on only one type of fluorescent probe.
  • the misjudgment caused by the classification of the two types of bacteria greatly improves the credibility of the mark.
  • the use of two types of probes to label the two types of bacteria it is also more convenient to statistically compare the labeling coverage of the bacterial flora with different fluorescent probe combinations, which is more conducive to the evaluation of fluorescent probe combinations in labeled bacteria The pros and cons of the group.
  • FIG. 1 shows a mouse intestinal flora labeled with an antibiotic probe in a preferred embodiment of the present invention, wherein green indicates a Gram-positive bacteria labeled with teicoplanin probe, Red shows the Gram-negative bacteria labeled with the probe of Triadecaptin A1;
  • Figure 2 shows the results of scatter plots of the bacterial samples of Figure 1 on a flow cytometer using the corresponding wavelengths of the two probes, respectively, showing the clear Gram-positive bacteria (Q3) and Grouping of Gram-negative bacteria (Q1);
  • Figure 3 shows a human sputum bacterial smear labeled with an antibiotic probe in accordance with a preferred embodiment of the present invention, wherein the green color shows the Gram-negative bacteria labeled with the epothilone probe. Red shows Gram-positive bacteria labeled with the probe of Triadecaptin B1.
  • antibiotic-based fluorescent probes have been reported in the prior art, these probes are limited to imaging of specific bacterial species, and have not been widely applied to complex bacterial systems and samples for imaging identification. .
  • the inventors used two types of narrow-spectrum antibiotics (narrow-spectrum antibiotics selective inhibition) for specifically labeling Gram-positive bacteria and Gram-negative bacteria, respectively.
  • kill Gram Probes for negative or positive bacteria are observed by direct labeling fluorescence imaging of these two major types of bacteria in complex bacterial samples.
  • the inventors designed and synthesized fluorescent probes based on teicoplanin antibiotics, including teicoplanin, vancomycin, and telavancin. , dalbavancin, oritavancin, eremomycin, balhimycin, and ristocetin structures.
  • the main mechanism of action of this class of antibiotics is to inhibit the cross-linking and synthesis of peptidoglycan by specifically binding to a D-alanine-D-alanine structure on the bacterial cell wall peptidoglycan. To the role of bacteriostatic sterilization.
  • the bacterial outer membrane mainly composed of lipopolysaccharide (LPS) outside the cell wall is not transparent to teicoplanin antibiotics, so the teicoplanin antibiotics cannot be exposed to the peptide. Glycans, in turn, cannot bind to Gram-negative bacteria.
  • LPS lipopolysaccharide
  • the fluorescent probes designed based on the above antibiotic structure were separately subjected to fluorescent labeling tests, and finally some fluorescent probes of Gram-positive bacteria with the best fluorescence display effect were determined. These narrow-spectrum antibiotic-based probes bind directly to Gram-positive bacteria in complex bacterial samples to achieve specific labeling of Gram-positive bacteria for subsequent imaging analysis.
  • tridecaptin antibiotics including tridepeptidin A1 (tridecaptin A1), tridecapeptide B1 (tridecaptin B1), a probe for tridecaptin C.
  • tridecaptin A1 tridecaptin A1
  • tridecapeptide B1 tridecaptin B1
  • a probe for tridecaptin C can be directly bound to Gram-negative bacteria in complex bacterial samples for specific labeling for subsequent imaging analysis.
  • the antibacterial mechanism of such Gram-negative bacteria antibiotics is specifically binding to lipid II in gram-negative bacteria, and inhibits bacterial cell bactericidal action by blocking cell wall synthesis.
  • the structure of Gram-positive bacteria lipid II is different from the structure of lipid II of Gram-negative bacteria, and tridecapeptide cannot bind to it.
  • a fluorescent probe based on this type of Gram-negative bacteria antibiotic has the advantage of being highly specific for labeling Gram-negative bacteria.
  • the inventors also designed and synthesized a fluorescent probe based on polymyxin antibiotics, which are specific antibiotics for Gram-negative bacteria.
  • the main mechanism of action is By binding to LPS on the outer membrane of the bacteria, the outer membrane swells, destroying cell membrane integrity, leading to osmotic imbalance and cell death.
  • polymyxin-based fluorescent probe can only specifically bind Gram-negative bacteria, but cannot bind Gram-positive bacteria.
  • a kit for labeling a bacterial flora comprising utilizing a first type of fluorescent probe and a second The fluorescent-like probe marks the bacterial species in the bacterial flora, wherein the first fluorescent probe specifically labels the Gram-positive bacteria, and the second fluorescent probe specifically labels the Gram-negative bacteria, and the first The fluorescent-like probe and the second fluorescent probe have different fluorescent labels, respectively.
  • the kit also includes a fluorescent probe that specifically binds two types of bacteria.
  • a fluorescent probe that specifically binds two types of bacteria.
  • the first type of fluorescent probe is used to specifically label Gram-negative bacteria, the bacterial flora is not labeled with the first type of fluorescent probe.
  • the strain should theoretically be Gram-positive bacteria; when the second type of fluorescent probe is used to label Gram-positive bacteria, except for the true Gram-positive bacteria, other Gram-positive bacteria are labeled.
  • Species that are labeled by the first type of fluorescent probe for unknown reasons are also labeled with a second type of fluorescent probe.
  • Such a fluorescent probe of Gram-positive bacteria can verify the labeling result of Gram-negative bacteria, and, The fluorescent probe of Gram-negative bacteria can verify the labeling results of Gram-positive bacteria, and the two types of probes mutually confirm the labeling results, making the labeling results more accurate.
  • the first type of fluorescent probe is selected from the group consisting of fluorescent probes based on teicoplanin-like antibiotics.
  • the design and synthesis method of the fluorescent probe based on the above-mentioned antibiotics is carried out by the existing method, as long as the specific binding property of the antibiotic to the Gram-positive bacteria can be achieved, and the specific marker for the Gram-positive bacteria is realized. can.
  • the fluorescent probe based on teicoplanin antibiotics comprises a teicoplanin-based fluorescent probe, a vancomycin-based fluorescent probe, and a Travancin-based Fluorescent probe (telavancin), fluorescent probe based on dalbavancin, fluorescent probe based on oritavancin, fluorescent probe based on eremomycin, based on Balkh Fluorescent probes of balhimycin, and fluorescent probes based on ristocetin.
  • telavancin Travancin-based Fluorescent probe
  • the fluorescent label in the fluorescent probe is at a design position of the corresponding antibiotic, and the reasonable matching relationship can be appropriately selected according to the type of the selected fluorescent label and the difference in the structure of the antibiotic, as long as the specific recognition and the fluorescence display are satisfied.
  • the teicoplanin-based fluorescent probe is
  • the vancomycin-based fluorescent probe is:
  • Fluorescent probes based on telavancin are:
  • the fluorescent probe of dalbavancin is:
  • Fluorescent probes based on oritavancin are:
  • the fluorescent probe based on the etodomycin is:
  • R" is H or Cl
  • the fluorescent probe based on balchimycin is:
  • the fluorescent probe based on ristocetin is:
  • R 1 to R 18 each independently represent a fluorescent label.
  • the second type of fluorescent probe is one or more of a tripeptide-based antibiotic-based fluorescent probe and a polymyxin-based antibiotic-based fluorescent probe.
  • the thoracic peptide antibiotic-based fluorescent probe comprises a threonopeptin A1-based fluorescent probe, a 13-peptide leptin B1-based fluorescent probe, and a 13-peptide leptin C-based fluorescent probe; More preferably, the fluorescent probe based on the thretin A1 is
  • Fluorescent probes based on thirteen peptides B1 are:
  • the fluorescent probe based on thirteen peptide C is
  • R 1 to R 9 each independently represent a fluorescent label .
  • the polymyxin-based fluorescent probe is:
  • R' is R" is R 10 to R 14 each independently represent a fluorescent label.
  • NHS amine
  • RP-HPLC reverse phase high performance liquid chromatography
  • the above fluorescent probes are designed such that the fluorescent label is designed to be at the position of the amino group labeled by each antibiotic, and the position of the fluorescent label is unique for an antibiotic containing an amino group.
  • antibiotics containing multiple amino groups there may be a result that one probe molecule contains one, two or more fluorophores after the reaction is completed, and a structurally uniform compound cannot be isolated, regardless of the number of fluorescent labels carried. One or more of them do not affect the final markup result.
  • the fluorescent label is selected from any two of coumarins, naphthalenes, fluoroboron fluorescetes, xanthenes, cyanines, squaraines, anthraquinone fluorescent dyes or Two or more.
  • the above fluorescent labels are all commercially available fluorescent labels, and the fluorescent labels are usually fluorescent dyes.
  • the coumarin fluorescent dye has hydroxycoumarin; the naphthalene has dansyl dye; the fluoroboron fluorite has BODIPY; the xanthene has fluorescein, Texas red, Oregon green, rhodamine series , Alexa Fluor series; Cyanine series for cyanine; Seta series, SeTau series for squaric acid; DRAQ series, CyTRAK series for bismuth.
  • the above two types of fluorescent probes are based on two types of antibiotics with the same antibacterial mechanism, so the antibiotic structure can ensure the specificity of each probe to the corresponding species, and the fluorescent labeling of the above fluorescent label specifically binds to it. There is no influence, and therefore, the type of the fluorescent label of the specific fluorescent probe is not particularly limited. There is no significant difference in the performance of labeled bacterial species based on fluorescent probes with different fluorescent labels synthesized by the same antibiotic.
  • a fluorescently labeled bacterial flora is provided, wherein Gram-negative bacteria and Gram-positive bacteria in the bacterial flora are labeled by any of the above kits.
  • the two types of strains can be markedly improved. Marker coverage of bacterial strains, and can improve the accuracy of bacterial taxonomy.
  • the fluorescent probe of the present application is also suitable for live bacterial markers, which can more accurately and accurately distinguish the bacterial group categories.
  • a method of labeling a bacterial flora comprising using a first type of fluorescent probe and a second type of fluorescent probe pair in any of the above kits The bacteria in the bacterial flora are labeled.
  • the order of labeling of the first type of fluorescent probe and the second type of fluorescent probe is not particularly limited. Whether a type of strain is first labeled with a fluorescent probe, and another type of fluorescent probe is used to label another type of strain, or two types of fluorescent probes are simultaneously added to the bacterial flora for simultaneous labeling. Fluorescent-like probes have their own specificity Sexuality has no effect on each other, so it is possible to mark two different types of bacteria separately, improve the coverage and accuracy of the marker in the bacterial flora, and reduce the error rate of the marker.
  • the combination of the first type of fluorescent probe and the second type of fluorescent probe used is different for different bacterial flora samples.
  • the specific number of types of the first type of fluorescent probe in the combination of the first type of fluorescent probe and the second type of fluorescent probe is not particularly limited as long as the color of the fluorescent label carried therein can be combined with the fluorescent probe of the second type.
  • the color of the fluorescent marker carried can be distinguished.
  • the first type of fluorescent probe may have fluorescent probes based on two, three, four, five or more antibiotics.
  • the second type of fluorescent probes may also be based on two or three types. Fluorescent probes of four, five or more antibiotics, as long as the two types of fluorescent probes are used in combination, the fluorescent color can distinguish the species type.
  • a fourth exemplary embodiment of the present application there is also provided the use of the above-described fluorescently labeled bacterial flora in fluorescence microscopy or flow cytometry.
  • the bacterial flora labeled by two different types of fluorescent probes can be identified by fluorescence microscopy imaging. Since the combination of the two types of probes of the present application is also applicable to living bacteria, it can also be different according to actual application requirements.
  • the cells were sorted by flow cytometry for the cells of the two types of bacteria to perform subsequent processing on the selected cells.
  • any one of the above kits providing any of the above kits for diagnosing bacterial lung inflammation, bacterial genitourinary infection, bacterial infection Use in sexual diarrhea and/or bacterial meningitis.
  • diagnosis or auxiliary diagnosis is clinically performed after a preliminary examination of the pathogenic bacteria to determine whether it is a bacterial infection or a Gram-positive infection or a Gram-positive infection. This test is used to guide the accuracy of subsequent medications.
  • the above applications include the use of sputum smears to diagnose bacterial lung inflammation; the use of vaginal secretion smears, urethral secretion smears, urine smears or urinary smears to diagnose bacterial genitourinary system Infection; diagnosis of bacterial infectious diarrhea using fecal smears; and/or diagnosis of bacterial meningitis using cerebrospinal fluid smears.
  • the mouse intestinal bacterial group sample was resuspended in a sterile PBS solution containing 0.5% bovine serum albumin (BSA) (the absorbance of the bacterial liquid density at 600 nm was 0.1-2.0), and the purified labeled gram was added.
  • BSA bovine serum albumin
  • the teicoplanin-rhodamine 110 fluorescent probe of the positive bacteria was added to a final concentration of 0.1-100 ⁇ g/mL, and the sample was incubated at room temperature for 30 min in the dark, and then centrifuged (13,000 rpm, 5 min), and washed three times with PBS.
  • the bacterial sample was resuspended in a sterile PBS solution containing 0.5% BSA (the absorbance of the bacterial liquid density at 600 nm was about 1.0), and the purified gram-negative bacteria of the gram-negative bacteria was added to the thirteen-peptide A1-
  • the Cy5 fluorescent probe was centrifuged to a final concentration of 0.1-100 ⁇ g/mL, and the sample was incubated at room temperature for 30 min in the dark, and then centrifuged (13,000 rpm, 5 min) and washed three times with PBS.
  • the labeled bacterial sample was placed under a fluorescence microscope and the respective wavelengths of the two probes were used (Rhodamine 110, absorption/emission 488/520 nm, shown in Figure 1 as green; Cy5, absorption/emission 645/670 nm, Imaging observation is shown in red in Figure 1.
  • Fig. 1 it can be seen from Fig. 1 that there are still some bacteria in the flora that cannot be labeled by the two types of probes. Therefore, when only one type of probe is used to distinguish the flora, it is difficult to The type of labeled bacteria is distinguished.
  • the bacterial sample treated with the kit of the present application may be a live bacteria or a sample of dead bacteria or paraformaldehyde fixed.
  • the bacterial sample treated in Example 1 was placed on a flow cytometer, and the samples were analyzed by the respective wavelengths of the two probes, and a certain type was used by using Fluorescence activated Cell Sorting.
  • the fluorescence of the probe enables physical separation of Gram-positive or Gram-negative bacteria in complex bacterial samples.
  • Example 3 Gram-positive/negative bacterial staining analysis in clinical sputum smear samples
  • the use of two types of probes in the present invention in clinical bacterial samples is illustrated by taking clinical patient sputum as an example.
  • the sputum sample was mixed in PBS, uniformly applied to a glass slide, naturally dried, and fixed by flame method.
  • a Gram-positive-specific epothilone-Alexa Fluor 647 probe (0.1-100 ⁇ g/mL)
  • a Gram-negative-specific tridecapeptide B1-BODIPY probe 0.1- 100 ⁇ g/mL
  • PBS solution containing 0.5% bovine serum albumin
  • the two types of probes in the kit of the present application have a wide range of application (the combination of the two probes can be applied to most samples); the standard is relatively uniform (no need Consider probe design or synthesis); easy to operate (no need to fix, suitable for live bacteria), after the dyeing mark is completed, other experimental studies can be continued on the sample.
  • the kit of the present application Compared with another method for selectively labeling bacteria by differential nucleic acid staining according to different bacterial cell membrane permeability, the kit of the present application has high specificity and is suitable for fixed samples (often different bacteria pairs after fixation) The distinguishability of nucleic acid dyes is lost) and the advantages of samples containing non-bacterial components (often with the background of nucleic acid dye adsorption), and other experimental studies can be continued on the sample after the dyeing label is completed.
  • the fluorescent probe in the kit of the present application has a comparative bacterial labeling method for labeling the cell wall of Gram-positive bacteria (incorporating N-acetylglucosamine therein) by using wheat germ agglutinin (WGA)
  • WGA wheat germ agglutinin
  • the advantage of higher specificity Since WGA also binds to cell wall components other than Gram-positive bacteria, such as sialic acid (present on the surface of many Gram-negative bacteria) and N-acetylglucosamine in non-bacterial sources, the labeling specificity of this method is reduced.
  • the labeling principle of the kit of the present application is based on a narrow-spectrum antibiotic with high specific binding ability, so the labeling selectivity is better and the anti-interference ability is stronger.
  • the probe of the present application can label most Gram-positive and negative bacteria with high coverage and high specificity.
  • it is possible to complement the conventional Gram staining methods currently used in clinical practice, and to provide more and relatively more accurate information for bacterial diagnosis. It is also possible to make rapid differential labeling and physical separation based on bacterial cell membrane structure characteristics for live bacterial samples.
  • the antibiotics referred to in the present application may also be other compounds which are not marketed or have not been known to function or have similar structures, or compounds which have undergone simple changes in structure, and which have similar labeling effects after being derivatized by fluorescent labeling.
  • other chemical reactions such as using a reactive group such as a carboxyl group in an antibiotic molecule, may be used to carry out the reaction, thereby generating a functionally similar Probe.

Abstract

Provided in the present invention are a kit and method for labeling bacterial flora, fluorescence-labeled bacterial flora and an application thereof. The method comprises: using a first type of fluorescence probe and a second type of fluorescence probe to label bacterial species in bacterial flora, wherein the first type of fluorescence probe specifically labels Gram-positive bacteria, and the second type of fluorescence probe specifically labels Gram-negative bacteria, the first type of fluorescence probe and the second type of fluorescence probe carrying different fluorescence markers, respectively.

Description

标记细菌菌群的试剂盒、方法、带荧光标记的细菌菌群及其应用Kit for labeling bacterial flora, method, fluorescently labeled bacterial flora and application thereof 技术领域Technical field
本发明涉及细菌荧光标记领域，具体而言，涉及一种标记细菌菌群样品的试剂盒、方法、带荧光标记的细菌菌群及其应用。The present invention relates to the field of bacterial fluorescent labeling, and in particular to a kit, method, fluorescently labeled bacterial flora and application thereof for labeling bacterial bacterial flora samples.
背景技术Background technique
针对组成未知的细菌样品进行初步的鉴别观察有很大的科研和临床应用价值。目前常用的细菌染色、标记、成像观察主要有传统革兰氏染色、荧光原位杂交技术(Fluorescence in situ hybridization，FISH)、区分性核酸染色(根据不同细菌细胞膜穿透性不同)、麦胚凝集素(wheat germ agglutinin，WGA)染色标记革兰氏阳性菌细胞壁等方法。Preliminary identification and observation of bacterial samples with unknown composition has great research and clinical application value. At present, the commonly used bacterial staining, labeling and imaging observations include traditional Gram staining, Fluorescence in situ hybridization (FISH), differential nucleic acid staining (depending on the permeability of different bacterial cell membranes), and wheat germ agglutination. A method for staining the cell wall of Gram-positive bacteria by dye agglutinin (WGA).
在医院环境中的临床细菌样品，例如病人的痰液、肺部灌洗液、脓液、体液(血液、尿液、脑脊液)等，目前的常规操作仍是需要进行革兰氏染色。通过革兰氏阳性/阴性菌细胞外膜的结构不同而最终在光学显微镜的明场下呈现不同的颜色(阳性菌为紫色，阴性菌呈红色)，并结合形态学观察以进行初步的细菌学诊断。Clinical bacterial samples in hospital settings, such as sputum, lung lavage, pus, body fluids (blood, urine, cerebrospinal fluid), etc., are still routinely required for Gram staining. Through the different structures of the outer membrane of Gram-positive/negative bacteria, they finally show different colors under the bright field of the optical microscope (positive bacteria are purple, negative bacteria are red), and combined with morphological observation for preliminary bacteriology diagnosis.
在科研环境中，针对复杂细菌样品，包括各类宿主的细菌菌群(例如近年研究火热的哺乳动物肠道细菌)、环境细菌菌群(土壤、水体、原油细菌等)，目前最可行的方式是使用FISH探针，利用针对细菌16s rRNA的含有荧光基团的DNA探针，来对特定的固定后的细菌种类进行标记成像观察(利用荧光显微镜)。In the scientific research environment, the most feasible way for complex bacterial samples, including bacterial colonies of various types of hosts (such as the study of hot mammalian intestinal bacteria in recent years), environmental bacterial flora (soil, water, crude oil bacteria, etc.) Using a FISH probe, a fluorophore-containing DNA probe for bacterial 16s rRNA was used to perform labeling imaging observation on a specific fixed bacterial species (using a fluorescence microscope).
出于各类科学研究的目的，已有一些基于抗生素的细菌标记荧光探针见诸报道。用于细菌成像观察的一类主要是基于万古霉素(特异性抗革兰氏阳性菌)抗生素的荧光探针，文献中的应用主要是用来对某种细菌细胞壁的结构进行成像观察。也有一些报道通过列举比较标记几种常见细菌种类，提到了这类探针可以特异性地标记几个革兰氏阳性菌物种。另一类见诸报道的为基于多粘菌素的探针，主要涉及到的为研究此类抗生素的作用机制与药物动力学方向。其中两种探针Vancomycin-BODIPY和Polymyxin-BODIPY市场曾经有售。For the purpose of various scientific research, some antibiotic-based bacterial labeled fluorescent probes have been reported. One type of bacterial imaging observation is based on the fluorescent probe of vancomycin (specific against Gram-positive bacteria) antibiotics. The application in the literature is mainly used to observe the structure of a certain bacterial cell wall. There are also reports that several common bacterial species are labeled by comparison, and it is mentioned that such probes can specifically label several Gram-positive species. Another type of polymyxin-based probe that has been reported is mainly concerned with the mechanism of action and pharmacokinetics of such antibiotics. Two of the probes, the Vancomycin-BODIPY and Polymyxin-BODIPY markets, were once available for sale.
革兰氏染色这种方法自1884年由Hans Christian Gram发明之后，其基本操作原理一直沿用至今。这种染色方法存在某些细菌种类染色后颜色结果不明确，染色结果受人为操作因素影响较大(染色过程中冲洗的步骤较多，往往需要靠经验判断每一步的操作细节和程度)，仅能对固定之后的细菌进行操作(活的细菌无法应用)，在处理含有非细菌杂质的样品时容易受杂质的染色背景影响等问题。Since Gram's dyeing method was invented by Hans Christian Gram in 1884, its basic operating principle has been used ever since. This kind of dyeing method has some unclear color results after staining of some bacterial species, and the dyeing result is greatly affected by human operation factors (there are many steps in the dyeing process, and it is often necessary to judge the details and degree of operation of each step by experience), only It can handle the bacteria after fixation (the living bacteria cannot be applied), and it is easy to be affected by the staining background of the impurities when processing samples containing non-bacterial impurities.
FISH探针的试剂盒存在DNA探针设计复杂、低rRNA含量、操作繁琐、信噪比低等问题，也是仅能对固定之后的细菌进行操作，并且只限于检测事先知其存在且引物可行性已验证的细菌，故其应用仍受很多限制。 The FISH probe kit has the problems of complicated DNA probe design, low rRNA content, cumbersome operation, low signal-to-noise ratio, etc. It can only operate on the bacteria after fixation, and is limited to detecting the presence of primers and the feasibility of primers. Proven bacteria, so their application is still subject to many restrictions.
尽管文献中已报道的抗生素荧光探针种类包括基于多粘菌素的探针和基于万古霉素/雷莫拉宁的探针。这些探针仅限于对某些特定已知细菌种类的标记成像。而针对如肠道细菌菌群等复杂细菌体系或复杂细菌样品(指含有两种或两种以上细菌物种，且种类未知，且可能含有非细菌类的污染物)样品中的细菌进行标记或成像鉴别，现有技术中目前还没有很好的解决办法。Although antibiotic fluorescent probe species have been reported in the literature, polymyxin-based probes and vancomycin-based remoraline-based probes are included. These probes are limited to imaging the markers of certain specific bacterial species. Labeling or imaging of bacteria in samples of complex bacterial systems such as gut bacterial flora or complex bacterial samples (referred to as containing two or more bacterial species, of unknown species and possibly containing non-bacterial contaminants) Identification, there is currently no good solution in the prior art.
发明内容Summary of the invention
本发明的主要目的在于提供一种标记细菌菌群的试剂盒、方法、带荧光标记的细菌菌群及其应用，以解决现有技术中难以对复杂细菌体系进行准确标记的问题。The main object of the present invention is to provide a kit, a method, a fluorescently labeled bacterial flora and the like for labeling bacterial flora, and to solve the problem that it is difficult to accurately mark a complex bacterial system in the prior art.
为了实现上述目的，根据本发明的一个方面，提供了种标记细菌菌群样品的试剂盒，试剂盒包括第一类荧光探针和第二类荧光探针，第一类荧光探针特异性标记样品中的革兰氏阳性菌，第二类荧光探针特异性标记革兰氏阴性菌，且第一类荧光探针和第二类荧光探针分别带有不同的荧光标记。In order to achieve the above object, according to one aspect of the present invention, there is provided a kit for labeling a sample of a bacterial flora, the kit comprising a first type of fluorescent probe and a second type of fluorescent probe, the first type of fluorescent probe specific label Gram-positive bacteria in the sample, the second type of fluorescent probe specifically labels Gram-negative bacteria, and the first type of fluorescent probe and the second type of fluorescent probe respectively have different fluorescent labels.
进一步地，第一类荧光探针选自基于替考拉宁类抗生素的荧光探针中的一种或多种。Further, the first type of fluorescent probe is selected from one or more of fluorescent probes based on teicoplanin antibiotics.
进一步地，基于替考拉宁类抗生素的荧光探针包括基于替考拉宁的荧光探针、基于万古霉素的荧光探针、基于特拉万星的荧光探针、基于达巴万星的荧光探针、基于奥利万星的荧光探针、基于伊端霉素的荧光探针、基于巴尔赫霉素的荧光探针以及基于瑞斯托霉素的荧光探针。Further, fluorescent probes based on teicoplanin antibiotics include teicoplanin-based fluorescent probes, vancomycin-based fluorescent probes, telavancin-based fluorescent probes, and dalbavancin-based fluorescent probes. Fluorescent probes, oritavancin-based fluorescent probes, epothilone-based fluorescent probes, balchimycin-based fluorescent probes, and ristocetin-based fluorescent probes.
进一步地，基于替考拉宁抗生素的荧光探针为Further, the fluorescent probe based on teicoplanin antibiotic is
Figure PCTCN2017086618-appb-000001
Figure PCTCN2017086618-appb-000001
其中R’为
Figure PCTCN2017086618-appb-000002
Where R' is
Figure PCTCN2017086618-appb-000002
Figure PCTCN2017086618-appb-000003
Figure PCTCN2017086618-appb-000003
基于万古霉素的荧光探针为：
Figure PCTCN2017086618-appb-000004
The vancomycin-based fluorescent probe is:
Figure PCTCN2017086618-appb-000004
基于特拉万星的荧光探针为：
Figure PCTCN2017086618-appb-000005
The fluorescent probe based on Travancin is:
Figure PCTCN2017086618-appb-000005
基于达巴万星的荧光探针为：
Figure PCTCN2017086618-appb-000006
The dalbavancin-based fluorescent probe is:
Figure PCTCN2017086618-appb-000006
基于奥利万星的荧光探针为：
Figure PCTCN2017086618-appb-000007
Olivin-based fluorescent probes are:
Figure PCTCN2017086618-appb-000007
基于伊端霉素的荧光探针为：
Figure PCTCN2017086618-appb-000008
其中R”为H或者Cl；The fluorescent probe based on the etodomycin is:
Figure PCTCN2017086618-appb-000008
Wherein R" is H or Cl;
基于巴尔赫霉素的荧光探针为：
Figure PCTCN2017086618-appb-000009
The fluorescent probe based on balchimycin is:
Figure PCTCN2017086618-appb-000009
基于瑞斯托霉素的荧光探针为:
Figure PCTCN2017086618-appb-000010
The fluorescent probe based on ristocetin is:
Figure PCTCN2017086618-appb-000010
其中，R1至R18各自独立地表示荧光标记。Wherein R 1 to R 18 each independently represent a fluorescent label.
进一步地，第二类荧光探针为基于十三肽菌素类抗生素的荧光探针和基于多粘菌素类抗生素的荧光探针中的任意一种或多种；优选地，基于十三肽菌素类抗生素的荧光探针包括基于十三肽菌素A1的荧光探针、基于十三肽菌素B1的荧光探针和基于十三肽菌素C的荧光探针；更优选地，基于十三肽菌素A1的荧光探针为：Further, the second type of fluorescent probe is any one or more of a tridecapeptide antibiotic-based fluorescent probe and a polymyxin-based antibiotic-based fluorescent probe; preferably, based on a thirteen peptide Fluorescent probes for bacteriocin antibiotics include a threonopeptidin A1-based fluorescent probe, a trichostin B1-based fluorescent probe, and a threomycin C-based fluorescent probe; more preferably, based on The fluorescent probe of the 13-peptide leptin A1 is:
Figure PCTCN2017086618-appb-000011
Figure PCTCN2017086618-appb-000011
其中R’为
Figure PCTCN2017086618-appb-000012
或者
Figure PCTCN2017086618-appb-000013
Where R' is
Figure PCTCN2017086618-appb-000012
or
Figure PCTCN2017086618-appb-000013
基于十三肽菌素B1的荧光探针为：Fluorescent probes based on thirteen peptides B1 are:
Figure PCTCN2017086618-appb-000014
Figure PCTCN2017086618-appb-000014
其中R”为
Figure PCTCN2017086618-appb-000015
或者
Figure PCTCN2017086618-appb-000016
Where R" is
Figure PCTCN2017086618-appb-000015
or
Figure PCTCN2017086618-appb-000016
基于十三肽菌素C的荧光探针为：The fluorescent probe based on thirteen peptide C is:
Figure PCTCN2017086618-appb-000017
Figure PCTCN2017086618-appb-000017
其中R”’为
Figure PCTCN2017086618-appb-000018
其中，R1至R9各自独立地表示荧光标记；Where R"' is
Figure PCTCN2017086618-appb-000018
Wherein R 1 to R 9 each independently represent a fluorescent label;
优选地基于多粘菌素的荧光探针为：Preferably, the polymyxin-based fluorescent probe is:
Figure PCTCN2017086618-appb-000019
Figure PCTCN2017086618-appb-000019
其中，R’为
Figure PCTCN2017086618-appb-000020
R”为
Figure PCTCN2017086618-appb-000021
R10至R14各自独立地表示荧光标记。Where R' is
Figure PCTCN2017086618-appb-000020
R" is
Figure PCTCN2017086618-appb-000021
R 10 to R 14 each independently represent a fluorescent label.
进一步地，荧光标记选自香豆素类荧光标记、萘类荧光标记、氟硼荧类荧光标记、氧杂蒽类荧光标记、菁类荧光标记、方酸类荧光标记以及蒽类荧光标记中的任意两种或两种以上。Further, the fluorescent label is selected from the group consisting of a coumarin fluorescent label, a naphthalene fluorescent label, a fluoroboron fluorescent label, a xanthene fluorescent label, a cyanine fluorescent label, a squaraine fluorescent label, and an anthraquinone fluorescent label. Any two or more.
为了实现上述目的，根据本发明的一个方面，提供了一种带荧光标记的细菌菌群，细菌菌群通过上述任一种试剂盒中进行标记。In order to achieve the above object, according to one aspect of the present invention, there is provided a fluorescently labeled bacterial flora which is labeled by any of the above kits.
根据本发明的另一方面，提供了一种标记细菌菌群的方法，该方法包括利用第一类荧光探针和第二类荧光探针对细菌菌群中的菌种进行标记，其中，第一类荧光探针和第二类荧光探针为上述任一种试剂盒中的第一类荧光探针和第二类荧光探针。According to another aspect of the present invention, there is provided a method of labeling a bacterial flora, the method comprising labeling a bacterial species in a bacterial flora with a first type of fluorescent probe and a second type of fluorescent probe, wherein One type of fluorescent probe and the second type of fluorescent probe are the first type of fluorescent probe and the second type of fluorescent probe in any of the above kits.
进一步地，利用第一类荧光探针和第二类荧光探针同时对细菌菌群中的细菌进行标记。 Further, the bacteria in the bacterial flora are simultaneously labeled using the first type of fluorescent probe and the second type of fluorescent probe.
根据本发明的另一方面，提供了上述任一种带荧光标记的细菌菌群在荧光显微镜观察或流式细胞荧光分选中的应用。According to another aspect of the present invention, there is provided the use of any of the above-described fluorescently labeled bacterial flora in fluorescence microscopy or flow cytometry.
根据本发明的另一方面，提供了上述任一种试剂盒在诊断细菌性肺部炎症、细菌性泌尿生殖***感染、细菌感染性腹泻和/或细菌性脑膜炎中的应用。According to another aspect of the present invention, there is provided the use of any of the above kits for diagnosing bacterial pulmonary inflammation, bacterial genitourinary infection, bacterial infectious diarrhea and/or bacterial meningitis.
进一步地，应用包括利用痰液涂片诊断细菌性肺部炎症；利用***分泌物涂片、尿道分泌物涂片、尿液涂片或尿沉渣涂片诊断细菌性泌尿生殖***感染；利用粪便涂片诊断细菌感染性腹泻；和/或利用脑脊液涂片诊断细菌性脑膜炎。Further, applications include the use of sputum smears to diagnose bacterial lung inflammation; vaginal secretion smears, urethral secretion smears, urine smears or urinary smears for diagnosis of bacterial genitourinary infections; Tablets for the diagnosis of bacterial infectious diarrhea; and/or the use of cerebrospinal fluid smears to diagnose bacterial meningitis.
应用本发明的技术方案，通过采用基于窄谱抗生素的高特异性结合两类菌种的荧光探针，可以利用两种荧光标记的互补结合，相互印证，能够避免仅基于一类荧光探针来对两类细菌分类所导致的误判，从而大大提高标记的可信度。此外，采用两类探针分别对两类菌种进行标记，还有利于更准确地统计不同荧光探针组合对细菌菌群的标记覆盖度，进而更有利于评价荧光探针组合在标记细菌菌群中的优劣。By applying the technical scheme of the present invention, by using a fluorescent probe based on narrow-spectrum antibiotics and combining the two types of strains with high specificity, the complementary binding of the two fluorescent labels can be utilized, and mutual confirmation can be avoided, and it can be avoided based on only one type of fluorescent probe. The misjudgment caused by the classification of the two types of bacteria greatly improves the credibility of the mark. In addition, the use of two types of probes to label the two types of bacteria, it is also more convenient to statistically compare the labeling coverage of the bacterial flora with different fluorescent probe combinations, which is more conducive to the evaluation of fluorescent probe combinations in labeled bacteria The pros and cons of the group.
附图说明DRAWINGS
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：The accompanying drawings, which are incorporated in the claims of the claims In the drawing:
图1示出了根据本发明的一种优选的实施例中的被抗生素探针标记的小鼠肠道菌群，其中，绿色显示的是替考拉宁探针标记的革兰氏阳性菌，红色显示的是十三肽菌素A1(Tridecaptin A1)探针标记的革兰氏阴性菌；1 shows a mouse intestinal flora labeled with an antibiotic probe in a preferred embodiment of the present invention, wherein green indicates a Gram-positive bacteria labeled with teicoplanin probe, Red shows the Gram-negative bacteria labeled with the probe of Triadecaptin A1;
图2示出了图1中的细菌样品在流式细胞仪上分别利用两种探针相应的波长对样品进行分析的散点图结果，可以看出明确的***(Q3)和革兰氏阴性细菌(Q1)的分组；以及Figure 2 shows the results of scatter plots of the bacterial samples of Figure 1 on a flow cytometer using the corresponding wavelengths of the two probes, respectively, showing the clear Gram-positive bacteria (Q3) and Grouping of Gram-negative bacteria (Q1);
图3示出了根据本发明的一种优选的实施例中的被抗生素探针标记的人类痰液细菌涂片，其中，绿色显示的是伊端霉素探针标记的革兰氏阴性菌，红色显示的是十三肽菌素B1(Tridecaptin B1)探针标记的革兰氏阳性菌。Figure 3 shows a human sputum bacterial smear labeled with an antibiotic probe in accordance with a preferred embodiment of the present invention, wherein the green color shows the Gram-negative bacteria labeled with the epothilone probe. Red shows Gram-positive bacteria labeled with the probe of Triadecaptin B1.
具体实施方式detailed description
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。It should be noted that the embodiments in the present application and the features in the embodiments may be combined with each other without conflict. The invention will be described in detail below with reference to the embodiments.
现有技术中虽然有报道基于抗生素的荧光探针，但这些探针仅限于对某些特定细菌种类的标记成像，并没有将其大范围地应用在复杂细菌体系和样品中进行成像鉴别的应用。为了改善现有技术中难以对复杂细菌体系进行准确标记的现状，发明人用两类分别特异性标记***和革兰氏阴性细菌的基于窄谱抗生素(窄谱抗生素指选择性抑制或杀灭革兰氏 阴性菌或阳性菌)的探针，以对复杂细菌样品中的这两大类细菌进行直接的标记荧光成像观察。Although antibiotic-based fluorescent probes have been reported in the prior art, these probes are limited to imaging of specific bacterial species, and have not been widely applied to complex bacterial systems and samples for imaging identification. . In order to improve the current situation in which it is difficult to accurately label complex bacterial systems, the inventors used two types of narrow-spectrum antibiotics (narrow-spectrum antibiotics selective inhibition) for specifically labeling Gram-positive bacteria and Gram-negative bacteria, respectively. Or kill Gram Probes for negative or positive bacteria are observed by direct labeling fluorescence imaging of these two major types of bacteria in complex bacterial samples.
针对革兰氏阳性菌，发明人设计并合成了基于替考拉宁(teicoplanin)类抗生素的荧光探针，包括了基于替考拉宁、万古霉素(vancomycin)、特拉万星(telavancin)、达巴万星(dalbavancin)、奥利万星(oritavancin)、伊端霉素(eremomycin)、巴尔赫霉素(balhimycin)以及瑞斯托霉素(ristocetin)结构的荧光探针。这一类抗生素的主要作用机制是通过特异性地与细菌细胞壁肽聚糖(peptidoglycan)上的一段D-丙氨酸-D-丙氨酸结构结合，抑制肽聚糖交联、合成，进而起到抑菌杀菌的作用。在革兰氏阴性菌中，由于细胞壁外存在的主要由脂多糖(lipopolysaccharide，LPS)构成的细菌外膜对替考拉宁类抗生素不通透，因此替考拉宁类抗生素不能接触到其肽聚糖，进而不能结合革兰氏阴性菌。For Gram-positive bacteria, the inventors designed and synthesized fluorescent probes based on teicoplanin antibiotics, including teicoplanin, vancomycin, and telavancin. , dalbavancin, oritavancin, eremomycin, balhimycin, and ristocetin structures. The main mechanism of action of this class of antibiotics is to inhibit the cross-linking and synthesis of peptidoglycan by specifically binding to a D-alanine-D-alanine structure on the bacterial cell wall peptidoglycan. To the role of bacteriostatic sterilization. In Gram-negative bacteria, the bacterial outer membrane mainly composed of lipopolysaccharide (LPS) outside the cell wall is not transparent to teicoplanin antibiotics, so the teicoplanin antibiotics cannot be exposed to the peptide. Glycans, in turn, cannot bind to Gram-negative bacteria.
通过对基于上述抗生素结构而设计的荧光探针分别进行了荧光标记测试，最终确定了一些荧光显示效果最优的革兰氏阳性菌的荧光探针。这些基于窄谱抗生素的探针皆可与复杂细菌样品中的革兰氏阳性菌直接结合从而实现特异性标记革兰氏阳性菌，以用于后续的成像分析。The fluorescent probes designed based on the above antibiotic structure were separately subjected to fluorescent labeling tests, and finally some fluorescent probes of Gram-positive bacteria with the best fluorescence display effect were determined. These narrow-spectrum antibiotic-based probes bind directly to Gram-positive bacteria in complex bacterial samples to achieve specific labeling of Gram-positive bacteria for subsequent imaging analysis.
同时，针对革兰氏阴性菌，发明人设计并合成了基于十三肽菌素(tridecaptin)类抗生素的荧光探针，包括了基于十三肽菌素A1(tridecaptin A1)，十三肽菌素B1(tridecaptin B1)，十三肽菌素C(tridecaptin C)的探针。这类探针可以与复杂细菌样品中的革兰氏阴性菌直接结合从而实现特异性标记，以用于后续的成像分析。这类革兰氏阴性菌抗生素的抗菌机理为与革兰氏阴性菌中的脂质II(lipid II)特异性结合，通过阻断细胞壁合成起到抑菌杀菌作用。而革兰氏阳性菌脂质II的结构与革兰氏阴性菌的脂质II的结构不同，十三肽菌素不能与其结合。因而，基于该类革兰氏阴性菌抗生素的荧光探针具有标记革兰氏阴性菌特异性高的优点。At the same time, for Gram-negative bacteria, the inventors designed and synthesized fluorescent probes based on tridecaptin antibiotics, including tridepeptidin A1 (tridecaptin A1), tridecapeptide B1 (tridecaptin B1), a probe for tridecaptin C. Such probes can be directly bound to Gram-negative bacteria in complex bacterial samples for specific labeling for subsequent imaging analysis. The antibacterial mechanism of such Gram-negative bacteria antibiotics is specifically binding to lipid II in gram-negative bacteria, and inhibits bacterial cell bactericidal action by blocking cell wall synthesis. The structure of Gram-positive bacteria lipid II is different from the structure of lipid II of Gram-negative bacteria, and tridecapeptide cannot bind to it. Thus, a fluorescent probe based on this type of Gram-negative bacteria antibiotic has the advantage of being highly specific for labeling Gram-negative bacteria.
同样针对革兰氏阴性菌，发明人还设计并合成了基于多粘菌素类抗生素的荧光探针，多粘菌素类抗生素是革兰氏阴性菌专一性的抗生素，其主要作用机制是通过与细菌外膜上的LPS结合，导致外膜膨胀，破坏细胞膜完整性，导致渗透失衡和细胞死亡。而在革兰氏阳性菌中不存在LPS结构，因而基于多粘菌素的荧光探针仅能特异性结合革兰氏阴性菌，而不能结合革兰氏阳性菌。For the Gram-negative bacteria, the inventors also designed and synthesized a fluorescent probe based on polymyxin antibiotics, which are specific antibiotics for Gram-negative bacteria. The main mechanism of action is By binding to LPS on the outer membrane of the bacteria, the outer membrane swells, destroying cell membrane integrity, leading to osmotic imbalance and cell death. However, in the Gram-positive bacteria, there is no LPS structure, and thus the polymyxin-based fluorescent probe can only specifically bind Gram-negative bacteria, but cannot bind Gram-positive bacteria.
基于上述两类荧光探针的研究结果，在本申请一种典型的实施方式中，提供了一种对细菌菌群进行标记的试剂盒，该试剂盒包括利用第一类荧光探针和第二类荧光探针对细菌菌群中的菌种进行标记，其中，第一类荧光探针特异性标记革兰氏阳性菌，第二类荧光探针特异性标记革兰氏阴性菌，且第一类荧光探针和第二类荧光探针分别带有不同的荧光标记。Based on the results of the above two types of fluorescent probes, in an exemplary embodiment of the present application, a kit for labeling a bacterial flora is provided, the kit comprising utilizing a first type of fluorescent probe and a second The fluorescent-like probe marks the bacterial species in the bacterial flora, wherein the first fluorescent probe specifically labels the Gram-positive bacteria, and the second fluorescent probe specifically labels the Gram-negative bacteria, and the first The fluorescent-like probe and the second fluorescent probe have different fluorescent labels, respectively.
该试剂盒同时包括特异性结合两类菌种的荧光探针，当采用第一类荧光探针特异性标记革兰氏阴性菌时，细菌菌群中未被第一类荧光探针标记上的菌种理论上应该是革兰氏阳性菌；而采用第二类荧光探针标记革兰氏阳性菌时，除了真正的革兰氏阳性菌会被标记上外，其他属于革兰氏阳性菌但由于未知原因被第一类荧光探针所标记的菌种，同样会被第二类荧光探针标记上。这样革兰氏阳性菌的荧光探针能够对革兰氏阴性菌的标记结果进行验证，同样， 革兰氏阴性菌的荧光探针能够对革兰氏阳性菌的标记结果进行验证，两类探针相互印证标记结果，使得标记结果更准确。而且，在细菌样品中也存在一些不能被任何一类探针所标记的细菌，其原因可能较复杂，但利用本申请的试剂盒中的探针能够避免现有技术中仅基于一类探针来对两类细菌分类所导致的误判。此外，采用两类探针分别对两类菌种进行标记，还有利于更准确地统计不同荧光探针组合对细菌菌群的标记覆盖度，进而更有利于评价荧光探针组合在标记细菌菌群中的优劣。The kit also includes a fluorescent probe that specifically binds two types of bacteria. When the first type of fluorescent probe is used to specifically label Gram-negative bacteria, the bacterial flora is not labeled with the first type of fluorescent probe. The strain should theoretically be Gram-positive bacteria; when the second type of fluorescent probe is used to label Gram-positive bacteria, except for the true Gram-positive bacteria, other Gram-positive bacteria are labeled. Species that are labeled by the first type of fluorescent probe for unknown reasons are also labeled with a second type of fluorescent probe. Such a fluorescent probe of Gram-positive bacteria can verify the labeling result of Gram-negative bacteria, and, The fluorescent probe of Gram-negative bacteria can verify the labeling results of Gram-positive bacteria, and the two types of probes mutually confirm the labeling results, making the labeling results more accurate. Moreover, there are also some bacteria in the bacterial sample that cannot be labeled by any type of probe, which may be complicated, but the use of the probe in the kit of the present application can avoid the prior art based on only one type of probe. To misjudge the classification of two types of bacteria. In addition, the use of two types of probes to label the two types of bacteria, it is also more convenient to statistically compare the labeling coverage of the bacterial flora with different fluorescent probe combinations, which is more conducive to the evaluation of fluorescent probe combinations in labeled bacteria The pros and cons of the group.
在本申请一种优选的实施例中，第一类荧光探针选自基于替考拉宁(teicoplanin)类抗生素的荧光探针。基于上述类别的抗生素的荧光探针的设计及合成方法采用现有的方法进行，只要能够基于抗生素对革兰氏阳性菌菌种的特异结合性能，实现对革兰氏阳性菌的特异性标记即可。In a preferred embodiment of the present application, the first type of fluorescent probe is selected from the group consisting of fluorescent probes based on teicoplanin-like antibiotics. The design and synthesis method of the fluorescent probe based on the above-mentioned antibiotics is carried out by the existing method, as long as the specific binding property of the antibiotic to the Gram-positive bacteria can be achieved, and the specific marker for the Gram-positive bacteria is realized. can.
在本申请一种优选的实施例中，基于替考拉宁类抗生素的荧光探针包括基于替考拉宁的荧光探针、基于万古霉素(vancomycin)的荧光探针、基于特拉万星(telavancin)的荧光探针、基于达巴万星(dalbavancin)的荧光探针、基于奥利万星(oritavancin)的荧光探针、基于伊端霉素(eremomycin)的荧光探针、基于巴尔赫霉素(balhimycin)的荧光探针，以及基于基于瑞斯托霉素(ristocetin)的荧光探针。In a preferred embodiment of the present application, the fluorescent probe based on teicoplanin antibiotics comprises a teicoplanin-based fluorescent probe, a vancomycin-based fluorescent probe, and a Travancin-based Fluorescent probe (telavancin), fluorescent probe based on dalbavancin, fluorescent probe based on oritavancin, fluorescent probe based on eremomycin, based on Balkh Fluorescent probes of balhimycin, and fluorescent probes based on ristocetin.
具体上述荧光探针中的荧光标记在相应抗生素的设计位置，可以根据所选择的荧光标记的种类的不同以及抗生素结构的差异而进行合理选择适配关系，只要满足特异识别及荧光显示即可。Specifically, the fluorescent label in the fluorescent probe is at a design position of the corresponding antibiotic, and the reasonable matching relationship can be appropriately selected according to the type of the selected fluorescent label and the difference in the structure of the antibiotic, as long as the specific recognition and the fluorescence display are satisfied.
在本申请一种优选的实施例中，基于替考拉宁的荧光探针为In a preferred embodiment of the present application, the teicoplanin-based fluorescent probe is
Figure PCTCN2017086618-appb-000022
Figure PCTCN2017086618-appb-000022
其中R’为
Figure PCTCN2017086618-appb-000023
Figure PCTCN2017086618-appb-000024
Where R' is
Figure PCTCN2017086618-appb-000023
Figure PCTCN2017086618-appb-000024
基于万古霉素的荧光探针为：
Figure PCTCN2017086618-appb-000025
The vancomycin-based fluorescent probe is:
Figure PCTCN2017086618-appb-000025
基于特拉万星(telavancin)的荧光探针为:
Figure PCTCN2017086618-appb-000026
Fluorescent probes based on telavancin are:
Figure PCTCN2017086618-appb-000026
达巴万星(dalbavancin)的荧光探针为:
Figure PCTCN2017086618-appb-000027
The fluorescent probe of dalbavancin is:
Figure PCTCN2017086618-appb-000027
基于奥利万星(oritavancin)的荧光探针为：
Figure PCTCN2017086618-appb-000028
Fluorescent probes based on oritavancin are:
Figure PCTCN2017086618-appb-000028
基于伊端霉素的荧光探针为：
Figure PCTCN2017086618-appb-000029
The fluorescent probe based on the etodomycin is:
Figure PCTCN2017086618-appb-000029
其中R”为H或者Cl；Wherein R" is H or Cl;
基于巴尔赫霉素的荧光探针为：
Figure PCTCN2017086618-appb-000030
The fluorescent probe based on balchimycin is:
Figure PCTCN2017086618-appb-000030
基于瑞斯托霉素的荧光探针为:
Figure PCTCN2017086618-appb-000031
上述各荧光探针中，R1至R18各自独立地表示荧光标记。The fluorescent probe based on ristocetin is:
Figure PCTCN2017086618-appb-000031
In each of the above fluorescent probes, R 1 to R 18 each independently represent a fluorescent label.
在本申请一种优选的实施例中，第二类荧光探针为基于十三肽菌素类抗生素的荧光探针和基于多粘菌素类抗生素的荧光探针中的一种或多种。优选基于十三肽菌素类抗生素的荧光探针包括基于十三肽菌素A1的荧光探针、基于十三肽菌素B1的荧光探针和基于十三肽菌素C的荧光探针；更优选基于十三肽菌素A1的荧光探针为In a preferred embodiment of the present application, the second type of fluorescent probe is one or more of a tripeptide-based antibiotic-based fluorescent probe and a polymyxin-based antibiotic-based fluorescent probe. Preferably, the thoracic peptide antibiotic-based fluorescent probe comprises a threonopeptin A1-based fluorescent probe, a 13-peptide leptin B1-based fluorescent probe, and a 13-peptide leptin C-based fluorescent probe; More preferably, the fluorescent probe based on the thretin A1 is
Figure PCTCN2017086618-appb-000032
Figure PCTCN2017086618-appb-000032
其中R’为
Figure PCTCN2017086618-appb-000033
或者
Figure PCTCN2017086618-appb-000034
Where R' is
Figure PCTCN2017086618-appb-000033
or
Figure PCTCN2017086618-appb-000034
基于十三肽菌素B1的荧光探针为: Fluorescent probes based on thirteen peptides B1 are:
Figure PCTCN2017086618-appb-000035
Figure PCTCN2017086618-appb-000035
其中R”为
Figure PCTCN2017086618-appb-000036
或者
Figure PCTCN2017086618-appb-000037
Where R" is
Figure PCTCN2017086618-appb-000036
or
Figure PCTCN2017086618-appb-000037
基于十三肽菌素C的荧光探针为The fluorescent probe based on thirteen peptide C is
Figure PCTCN2017086618-appb-000038
Figure PCTCN2017086618-appb-000038
其中R”’为
Figure PCTCN2017086618-appb-000039
上述基于基于十三肽菌素A1的荧光探针、基于十三肽菌素B1的荧光探针和基于十三肽菌素C的荧光探针中，R1至R9各自独立地表示荧光标记。Where R"' is
Figure PCTCN2017086618-appb-000039
In the above-described fluorescent probe based on trichostingin A1, fluorescent probe based on triccosin B1, and fluorescent probe based on triccosin C, R 1 to R 9 each independently represent a fluorescent label .
优选地，基于多粘菌素的荧光探针为：Preferably, the polymyxin-based fluorescent probe is:
Figure PCTCN2017086618-appb-000040
Figure PCTCN2017086618-appb-000040
其中，R’为
Figure PCTCN2017086618-appb-000041
R”为
Figure PCTCN2017086618-appb-000042
R10至R14各自独立地表示荧光标记。Where R' is
Figure PCTCN2017086618-appb-000041
R" is
Figure PCTCN2017086618-appb-000042
R 10 to R 14 each independently represent a fluorescent label.
上述荧光探针的典型的合成步骤如下：将某种抗生素(4.0μmol)溶解在300μL磷酸盐缓冲液(phosphate buffered saline，PBS，pH＝7.8)中，然后加入荧光标记的N-羟基琥珀酰亚胺(NHS)活性酯(例如Cy5-NHS，BODIPY-NHS，罗丹明-NHS等)的DMSO溶液(5.0μmol溶于200μL)。反应在室温下避光进行20小时，后用反向高效液相色谱法(reverse phase high performance liquid chromatography，RP-HPLC)将带有荧光基团的抗生素探针产物分离纯化。A typical synthetic procedure for the above fluorescent probe is as follows: an antibiotic (4.0 μmol) is dissolved in 300 μL of phosphate buffered saline (PBS, pH = 7.8), followed by the addition of fluorescently labeled N-hydroxysuccinyl A solution of an amine (NHS) active ester (e.g., Cy5-NHS, BODIPY-NHS, Rhodamine-NHS, etc.) in DMSO (5.0 μmol in 200 μL). The reaction was carried out in the dark at room temperature for 20 hours, and then the antibiotic probe product having a fluorescent group was separated and purified by reverse phase high performance liquid chromatography (RP-HPLC).
上述荧光探针通过将荧光标记设计在各抗生素所标注的氨基基团位置上，对于含有一个氨基基团的抗生素，其荧光标记的位置是唯一的。但对于含有多个氨基的抗生素，则反应完成后可能存在一个探针分子含有一个、两个或多个荧光基团的结果，且无法分离出结构均一的化合物，但无论携带的荧光标记数目为一个或者多个，均不影响最终的标记结果。The above fluorescent probes are designed such that the fluorescent label is designed to be at the position of the amino group labeled by each antibiotic, and the position of the fluorescent label is unique for an antibiotic containing an amino group. However, for antibiotics containing multiple amino groups, there may be a result that one probe molecule contains one, two or more fluorophores after the reaction is completed, and a structurally uniform compound cannot be isolated, regardless of the number of fluorescent labels carried. One or more of them do not affect the final markup result.
在本申请一种优选的实施例中，荧光标记选自香豆素类、萘类、氟硼荧类、氧杂蒽类、菁类、方酸类、蒽类荧光染料中的任意两种或两种以上。In a preferred embodiment of the present application, the fluorescent label is selected from any two of coumarins, naphthalenes, fluoroboron fluorescetes, xanthenes, cyanines, squaraines, anthraquinone fluorescent dyes or Two or more.
上述荧光标记均是现有的市售的荧光标记，荧光标记通常为荧光染料。具体地，香豆素类荧光染料有羟基香豆素；萘类有丹磺酰染料；氟硼荧类有BODIPY；氧杂蒽类有荧光素、德克萨斯红、Oregon green、罗丹明系列、Alexa Fluor系列；菁类有Cyanine系列；方酸类有Seta系列、SeTau系列；蒽类有DRAQ系列、CyTRAK系列。The above fluorescent labels are all commercially available fluorescent labels, and the fluorescent labels are usually fluorescent dyes. Specifically, the coumarin fluorescent dye has hydroxycoumarin; the naphthalene has dansyl dye; the fluoroboron fluorite has BODIPY; the xanthene has fluorescein, Texas red, Oregon green, rhodamine series , Alexa Fluor series; Cyanine series for cyanine; Seta series, SeTau series for squaric acid; DRAQ series, CyTRAK series for bismuth.
上述两类荧光探针分别是基于两类抗菌机制完全相同的抗生素，因此其抗生素结构能确保各探针结合相应菌种类别的特异性，而上述荧光标记的荧光显色性能对其特异性结合并无影响，因而，对具体荧光探针的荧光标记种类并无特殊限定。基于同一抗生素所合成的带有不同荧光标记的荧光探针在标记细菌菌种类别方面的性能并无明显差异。The above two types of fluorescent probes are based on two types of antibiotics with the same antibacterial mechanism, so the antibiotic structure can ensure the specificity of each probe to the corresponding species, and the fluorescent labeling of the above fluorescent label specifically binds to it. There is no influence, and therefore, the type of the fluorescent label of the specific fluorescent probe is not particularly limited. There is no significant difference in the performance of labeled bacterial species based on fluorescent probes with different fluorescent labels synthesized by the same antibiotic.
在本申请另一种典型的实施方式中，提供了一种带荧光标记的细菌菌群，细菌菌群中的革兰氏阴性菌和革兰氏阳性菌通过上述任一种试剂盒进行标记。In another exemplary embodiment of the present application, a fluorescently labeled bacterial flora is provided, wherein Gram-negative bacteria and Gram-positive bacteria in the bacterial flora are labeled by any of the above kits.
利用本申请试剂盒中进行细菌菌群标记，通过特异性标记革兰氏阳性菌的荧光探针和特异性标记革兰氏阴性菌的荧光探针对两类菌种分别进行标记，不仅能提高细菌菌种的标记覆盖度，而且能提高菌群分类的准确性。而且，本申请的荧光探针还适合活菌标记，能够更真实、更准确地区分菌群类别。By using the bacterial flora label in the kit of the present application, by separately labeling the two types of strains by fluorescent labeling of the Gram-positive bacteria and the fluorescent probe of the specific labeling Gram-negative bacteria, the two types of strains can be markedly improved. Marker coverage of bacterial strains, and can improve the accuracy of bacterial taxonomy. Moreover, the fluorescent probe of the present application is also suitable for live bacterial markers, which can more accurately and accurately distinguish the bacterial group categories.
在本申请第三种典型的实施方式中，还提供了一种细菌菌群标记的方法，该方法包括利用上述任一种试剂盒中的第一类荧光探针和第二类荧光探针对细菌菌群中的菌种进行标记。In a third exemplary embodiment of the present application, there is also provided a method of labeling a bacterial flora comprising using a first type of fluorescent probe and a second type of fluorescent probe pair in any of the above kits The bacteria in the bacterial flora are labeled.
本申请的细菌菌群标记的方法中，第一类荧光探针和第二类荧光探针的标记顺序并无特殊限定。无论是先用一类荧光探针标记一类菌种后，再用另一类荧光探针标记另一类菌种，还是将两类荧光探针同时加入细菌菌群中进行同时标记，由于两类荧光探针具有各自的特异 性，相互之间没有影响，因而都能够将两类不同菌种分别标记上，提高细菌菌群中的标记覆盖率和准确性，降低标记的错误率。In the method of labeling a bacterial flora of the present application, the order of labeling of the first type of fluorescent probe and the second type of fluorescent probe is not particularly limited. Whether a type of strain is first labeled with a fluorescent probe, and another type of fluorescent probe is used to label another type of strain, or two types of fluorescent probes are simultaneously added to the bacterial flora for simultaneous labeling. Fluorescent-like probes have their own specificity Sexuality has no effect on each other, so it is possible to mark two different types of bacteria separately, improve the coverage and accuracy of the marker in the bacterial flora, and reduce the error rate of the marker.
具体在不同的标记应用中，针对不同的细菌菌群样品，其所使用的第一类荧光探针和第二类荧光探针的组合不同。第一类荧光探针和第二类荧光探针的组合中第一类荧光探针的具体种类数目并无特殊限定，只要其所携带的荧光标记的颜色能够与第二类的荧光探针中所携带的荧光标记的颜色区分开来即可。比如，第一类荧光探针可以有基于两种、三种、四种、五种甚至更多种抗生素的荧光探针，同样，第二类荧光探针也可以有基于两种、三种、四种、五种甚至更多种抗生素的荧光探针，只要这两类的荧光探针在组合使用时，荧光颜色能够区分菌种类别即可。Specifically, in different labeling applications, the combination of the first type of fluorescent probe and the second type of fluorescent probe used is different for different bacterial flora samples. The specific number of types of the first type of fluorescent probe in the combination of the first type of fluorescent probe and the second type of fluorescent probe is not particularly limited as long as the color of the fluorescent label carried therein can be combined with the fluorescent probe of the second type. The color of the fluorescent marker carried can be distinguished. For example, the first type of fluorescent probe may have fluorescent probes based on two, three, four, five or more antibiotics. Similarly, the second type of fluorescent probes may also be based on two or three types. Fluorescent probes of four, five or more antibiotics, as long as the two types of fluorescent probes are used in combination, the fluorescent color can distinguish the species type.
在本申请第四种典型的实施方式中，还提供了一种上述带有荧光标记的细菌菌群在荧光显微镜观察或流式细胞荧光分选中的应用。两类不同荧光探针所标记的细菌菌群能够通过荧光显微镜成像观察进行菌种鉴定，由于本申请的两类探针的组合同样适用于活菌，因而也可以根据实际应用需求，可通过不同标记经流式细胞仪对两类菌种的细胞进行分选，以便对筛选出的细胞进行后续处理。In a fourth exemplary embodiment of the present application, there is also provided the use of the above-described fluorescently labeled bacterial flora in fluorescence microscopy or flow cytometry. The bacterial flora labeled by two different types of fluorescent probes can be identified by fluorescence microscopy imaging. Since the combination of the two types of probes of the present application is also applicable to living bacteria, it can also be different according to actual application requirements. The cells were sorted by flow cytometry for the cells of the two types of bacteria to perform subsequent processing on the selected cells.
根据本发明的另一方面，提供了上述任一种试剂盒在根据本发明的另一方面，提供了上述任一种试剂盒在诊断细菌性肺部炎症、细菌性泌尿生殖***感染、细菌感染性腹泻和/或细菌性脑膜炎中的应用。According to another aspect of the present invention, there is provided any one of the above kits, according to another aspect of the present invention, providing any of the above kits for diagnosing bacterial lung inflammation, bacterial genitourinary infection, bacterial infection Use in sexual diarrhea and/or bacterial meningitis.
上述诊断或辅助诊断，在临床上，通常是在对病原菌进行初步检查，确定为细菌性感染之后，为进一步确定是革兰氏阴性菌感染还是革兰氏阳性菌感染而进行的检测。该检测用于指导后续用药的准确性。The above-mentioned diagnosis or auxiliary diagnosis is clinically performed after a preliminary examination of the pathogenic bacteria to determine whether it is a bacterial infection or a Gram-positive infection or a Gram-positive infection. This test is used to guide the accuracy of subsequent medications.
具体地，在临床上，上述应用包括利用痰液涂片诊断细菌性肺部炎症；利用***分泌物涂片、尿道分泌物涂片、尿液涂片或尿沉渣涂片诊断细菌性泌尿生殖***感染；利用粪便涂片诊断细菌感染性腹泻；和/或利用脑脊液涂片诊断细菌性脑膜炎。Specifically, clinically, the above applications include the use of sputum smears to diagnose bacterial lung inflammation; the use of vaginal secretion smears, urethral secretion smears, urine smears or urinary smears to diagnose bacterial genitourinary system Infection; diagnosis of bacterial infectious diarrhea using fecal smears; and/or diagnosis of bacterial meningitis using cerebrospinal fluid smears.
下面将结合具体的实施例来进一步说明本申请的有益效果。Advantageous effects of the present application will be further described below in conjunction with specific embodiments.
实施例1：复杂细菌样品荧光标记的成像分析Example 1: Imaging Analysis of Fluorescent Labeling of Complex Bacterial Samples
将小鼠肠道细菌组样品重悬在含有0.5％牛血清白蛋白(BSA)的无菌PBS溶液中(菌液密度在600nm处的吸光值为0.1-2.0)，加入纯化后的标记革兰氏阳性菌的替考拉宁-罗丹明110荧光探针至终浓度0.1-100μg/mL，样品于室温下避光搅动孵育30min，后离心(13,000rpm，5min)，用PBS洗三次。再将细菌样品重悬在含有0.5％BSA的无菌PBS溶液中(菌液密度在600nm处的吸光值约为1.0)，加入纯化后的标记革兰氏阴性菌的十三肽菌素A1-Cy5荧光探针至终浓度0.1-100μg/mL，样品于室温下避光搅动孵育30min，后离心(13,000rpm，5min)，用PBS洗三次。 The mouse intestinal bacterial group sample was resuspended in a sterile PBS solution containing 0.5% bovine serum albumin (BSA) (the absorbance of the bacterial liquid density at 600 nm was 0.1-2.0), and the purified labeled gram was added. The teicoplanin-rhodamine 110 fluorescent probe of the positive bacteria was added to a final concentration of 0.1-100 μg/mL, and the sample was incubated at room temperature for 30 min in the dark, and then centrifuged (13,000 rpm, 5 min), and washed three times with PBS. The bacterial sample was resuspended in a sterile PBS solution containing 0.5% BSA (the absorbance of the bacterial liquid density at 600 nm was about 1.0), and the purified gram-negative bacteria of the gram-negative bacteria was added to the thirteen-peptide A1- The Cy5 fluorescent probe was centrifuged to a final concentration of 0.1-100 μg/mL, and the sample was incubated at room temperature for 30 min in the dark, and then centrifuged (13,000 rpm, 5 min) and washed three times with PBS.
将标记完成后的细菌样品置于荧光显微镜下，分别利用两种探针相应的波长(罗丹明110，吸收/发射488/520nm，图1中显示为绿色；Cy5，吸收/发射645/670nm，图1中显示为红色)进行成像观察。The labeled bacterial sample was placed under a fluorescence microscope and the respective wavelengths of the two probes were used (Rhodamine 110, absorption/emission 488/520 nm, shown in Figure 1 as green; Cy5, absorption/emission 645/670 nm, Imaging observation is shown in red in Figure 1.
观察结果如图1所示。从图1中可以看出，被抗生素探针标记的小鼠肠道菌群中，绿色显示的是替考拉宁探针标记的革兰氏阳性菌，红色显示的是十三肽菌素A1探针结合标记的革兰氏阴性菌。而且，经统计，两种探针的标记信号重合度很低(<10％，显微镜下观察时重合度更低，流式细胞分析时重合度相对高些，原因未知)，两者相加对所有细菌的覆盖率>85％。The observation results are shown in Figure 1. As can be seen from Figure 1, in the intestinal flora of mice labeled with antibiotic probes, green shows Gram-positive bacteria labeled with teicoplanin probe, and red shows tridedomycin A1. The probe binds to the labeled Gram-negative bacteria. Moreover, according to statistics, the coincidence of the labeling signals of the two probes is very low (<10%, the degree of coincidence is lower when observed under the microscope, and the degree of coincidence is relatively high in the flow cytometry analysis, the reason is unknown), and the two are added together. The coverage of all bacteria is >85%.
此外，从图1中也可以看出，菌群中仍有一些不能被两类探针所标记的细菌，因而，当仅采用一类探针对该菌群进行区分时，是难以将其与已标记的细菌的种类进行区分的。In addition, it can be seen from Fig. 1 that there are still some bacteria in the flora that cannot be labeled by the two types of probes. Therefore, when only one type of probe is used to distinguish the flora, it is difficult to The type of labeled bacteria is distinguished.
应用本申请的试剂盒处理的细菌样品可以是活细菌，也可以是死细菌或者多聚甲醛固定后的样品。The bacterial sample treated with the kit of the present application may be a live bacteria or a sample of dead bacteria or paraformaldehyde fixed.
实施例2Example 2
将实施例1处理后的细菌样品，置于流式细胞仪上分别利用两种探针相应的波长对样品进行分析，并使用流式细胞荧光分选技术(Fluorescence activated Cell Sorting)利用某一类探针的荧光实现复杂细菌样品中革兰氏阳性或革兰氏阴性细菌的物理分离。The bacterial sample treated in Example 1 was placed on a flow cytometer, and the samples were analyzed by the respective wavelengths of the two probes, and a certain type was used by using Fluorescence activated Cell Sorting. The fluorescence of the probe enables physical separation of Gram-positive or Gram-negative bacteria in complex bacterial samples.
观察结果如图2所示。从图2中看出明确的***(Q3)和阴性细菌(Q1)的分组及相应的比例。而且，从图2中还可以看出，通过流式细胞仪对两类菌种进行分选时，会有部分两种荧光标记重叠的细菌(Q2)，同样还有少量不能被两类荧光探针所标记的菌种(Q4)。The observation results are shown in Figure 2. The grouping of the Gram-positive bacteria (Q3) and the negative bacteria (Q1) and the corresponding proportions are shown in Fig. 2. Moreover, it can be seen from Fig. 2 that when the two types of bacteria are sorted by flow cytometry, there will be some bacteria (Q2) with overlapping two fluorescent labels, and a small amount can not be detected by two types of fluorescence. The strain labeled by the needle (Q4).
实施例3：临床痰液涂片样品中革兰氏阳性/阴性细菌染色分析Example 3: Gram-positive/negative bacterial staining analysis in clinical sputum smear samples
以临床病人痰液为例说明本发明中的两类探针在临床细菌样品中的应用。将痰液样品混匀于PBS中，均匀涂于载玻片，自然干燥后利用火焰法固定。随后将含有革兰氏阳性菌特异性的伊端霉素-Alexa Fluor 647探针(0.1-100μg/mL)和革兰氏阴性菌特异性的十三肽菌素B1-BODIPY探针(0.1-100μg/mL)的PBS溶液(含有0.5％牛血清白蛋白)加到其上，于室温下孵育30分钟，后用PBS充分清洗后(约2小时)，可直接利用荧光显微镜对两类细菌的染色进行观察。观察结果如图3所示，革兰氏阳性菌可被伊端霉素-Alexa Fluor 647探针结合图中显示为红色，革兰氏阴性菌可被十三肽菌素B1-BODIPY探针结合，图中显示为绿色。结合其形态观察，可以对菌种类别进行区分。The use of two types of probes in the present invention in clinical bacterial samples is illustrated by taking clinical patient sputum as an example. The sputum sample was mixed in PBS, uniformly applied to a glass slide, naturally dried, and fixed by flame method. Subsequently, a Gram-positive-specific epothilone-Alexa Fluor 647 probe (0.1-100 μg/mL) and a Gram-negative-specific tridecapeptide B1-BODIPY probe (0.1- 100 μg/mL) PBS solution (containing 0.5% bovine serum albumin) was added thereto, incubated at room temperature for 30 minutes, and then thoroughly washed with PBS (about 2 hours), which can directly use two kinds of bacteria for fluorescence microscopy. Dyeing was observed. The results are shown in Figure 3. The Gram-positive bacteria can be shown in red by the binding of the iridazim-Alexa Fluor 647 probe, and the Gram-negative bacteria can be bound by the 13-peptidase B1-BODIPY probe. , the picture is shown in green. Combined with its morphological observation, the species types can be distinguished.
从以上的描述中可以看出，在现有技术中，当菌群中存在一些细菌不能被任何一种探针标记(比如，某些细菌表面存在荚膜多糖等情况时)，或者存在细胞结构破损的细菌或其他易吸附物质的干扰的问题时，单一荧光探针对细菌菌群类别进行标记和区分的准确度和可信度比较低。而本申请通过设计合成两大类含有不同荧光标记的基于窄谱抗生素的高特异性探针，可以利用两种荧光标记的互补结合，相互印证，从而大大提高标记的可信度。此外，对临床 细菌样品也提供了传统革兰氏染色方法之外的染色和观察手段，且无需固定，实现了对活细菌的直接标记成像观察，从而提高临床细菌诊断的准确度和覆盖率。As can be seen from the above description, in the prior art, when some bacteria in the flora cannot be labeled by any one of the probes (for example, when some bacteria have capsular polysaccharide on the surface), or there is a cell structure. The sensitivity and confidence of a single fluorescent probe for labeling and distinguishing bacterial flora species is low when there is a problem with the interference of broken bacteria or other easily adsorbable substances. However, by designing and synthesizing two kinds of high-specificity probes based on narrow-spectrum antibiotics containing different fluorescent labels, the present invention can utilize the complementary binding of two fluorescent labels to mutually verify each other, thereby greatly improving the reliability of the label. In addition, for clinical Bacterial samples also provide staining and observation methods other than traditional Gram staining methods, and do not need to be fixed, enabling direct marker imaging observation of live bacteria, thereby improving the accuracy and coverage of clinical bacterial diagnosis.
相比FISH探针所存在的一系列问题，本申请的试剂盒中的两类探针的适用范围广(两种探针的组合使用可适用于绝大部分的样品)；标准比较统一(无需考虑探针设计或合成)；操作简单(无需固定，适用于活细菌)，染色标记完成后还可以对样品继续进行其他实验研究。Compared with the series of problems of FISH probes, the two types of probes in the kit of the present application have a wide range of application (the combination of the two probes can be applied to most samples); the standard is relatively uniform (no need Consider probe design or synthesis); easy to operate (no need to fix, suitable for live bacteria), after the dyeing mark is completed, other experimental studies can be continued on the sample.
相比较另一种根据不同细菌细胞膜穿透性不同而利用差异性核酸染色来选择性标记细菌的方法，本申请的试剂盒具有特异性高，适用于固定后的样品(固定后往往不同细菌对核酸染料的区分性就会丢失)以及含有非细菌性组分(往往有核酸染料吸附背景)的样品的优点，染色标记完成后还可以对样品继续进行其他实验研究。Compared with another method for selectively labeling bacteria by differential nucleic acid staining according to different bacterial cell membrane permeability, the kit of the present application has high specificity and is suitable for fixed samples (often different bacteria pairs after fixation) The distinguishability of nucleic acid dyes is lost) and the advantages of samples containing non-bacterial components (often with the background of nucleic acid dye adsorption), and other experimental studies can be continued on the sample after the dyeing label is completed.
相比较通过使用麦胚凝集素(wheat germ agglutinin，WGA)标记革兰氏阳性菌细胞壁(结合其中的N-乙酰葡萄糖胺)的选择性细菌标记方法，本申请的试剂盒中的荧光探针具有特异性更高的优势。由于WGA也能结合非革兰氏阳性菌细胞壁成分，例如唾液酸(存在于很多革兰氏阴性菌表面)和非细菌来源物质中的N-乙酰葡萄糖胺，导致此方法的标记特异性下降。而本申请的试剂盒的标记原理基于具有高特异性结合能力的窄谱抗生素，故标记的选择性更好，抗干扰能力更强。The fluorescent probe in the kit of the present application has a comparative bacterial labeling method for labeling the cell wall of Gram-positive bacteria (incorporating N-acetylglucosamine therein) by using wheat germ agglutinin (WGA) The advantage of higher specificity. Since WGA also binds to cell wall components other than Gram-positive bacteria, such as sialic acid (present on the surface of many Gram-negative bacteria) and N-acetylglucosamine in non-bacterial sources, the labeling specificity of this method is reduced. However, the labeling principle of the kit of the present application is based on a narrow-spectrum antibiotic with high specific binding ability, so the labeling selectivity is better and the anti-interference ability is stronger.
本申请的探针可以以高覆盖度，高特异性地标记绝大部分革兰氏阳性和阴性菌。应用本发明中的方案，可以很好地对目前临床中所使用的传统革兰氏染色方法形成互补，为细菌诊断提供更多且相对更准确的信息。而且还可以对活的细菌样品做出快速的基于细菌细胞外膜结构特点的区分性标记及物理分离。The probe of the present application can label most Gram-positive and negative bacteria with high coverage and high specificity. By applying the protocol of the present invention, it is possible to complement the conventional Gram staining methods currently used in clinical practice, and to provide more and relatively more accurate information for bacterial diagnosis. It is also possible to make rapid differential labeling and physical separation based on bacterial cell membrane structure characteristics for live bacterial samples.
本申请中涉及到的抗生素还可以是其他未上市的或尚未为我们所知的功能或者结构类似的化合物，或者结构经过简单改变的化合物，利用荧光标记衍生后也可起到类似的标记效果。具体在利用荧光标记进行衍生过程中，除了本申请所提到的合成方式外，也可以利用其他的化学反应，例如利用抗生素分子中的羧基等活性基团等，进行反应，从而生成功能类似的探针。The antibiotics referred to in the present application may also be other compounds which are not marketed or have not been known to function or have similar structures, or compounds which have undergone simple changes in structure, and which have similar labeling effects after being derivatized by fluorescent labeling. Specifically, in the process of derivatization using a fluorescent label, in addition to the synthesis method mentioned in the present application, other chemical reactions, such as using a reactive group such as a carboxyl group in an antibiotic molecule, may be used to carry out the reaction, thereby generating a functionally similar Probe.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。 The above description is only the preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.

Claims (12)

  1. 一种标记细菌菌群样品的试剂盒，其特征在于，所述试剂盒包括第一类荧光探针和第二类荧光探针，第一类荧光探针特异性标记样品中的革兰氏阳性菌，所述第二类荧光探针特异性标记革兰氏阴性菌，且所述第一类荧光探针和第二类荧光探针分别带有不同的荧光标记。A kit for labeling a sample of a bacterial flora, characterized in that the kit comprises a first type of fluorescent probe and a second type of fluorescent probe, and the first type of fluorescent probe specifically labels a Gram-positive sample The second type of fluorescent probe specifically labels Gram-negative bacteria, and the first type of fluorescent probe and the second type of fluorescent probe respectively have different fluorescent labels.
  2. 根据权利要求1所述的试剂盒，其特征在于，所述第一类荧光探针选自基于替考拉宁类抗生素的荧光探针中的一种或多种。The kit according to claim 1, wherein the first type of fluorescent probe is selected from one or more of fluorescent probes based on teicoplanin-based antibiotics.
  3. 根据权利要求2所述的试剂盒，其特征在于，所述基于替考拉宁类抗生素的荧光探针包括基于替考拉宁的荧光探针、基于万古霉素的荧光探针、基于特拉万星的荧光探针、基于达巴万星的荧光探针、基于奥利万星的荧光探针、基于伊端霉素的荧光探针、基于巴尔赫霉素的荧光探针以及基于瑞斯托霉素的荧光探针。The kit according to claim 2, wherein the teicoplanin-based fluorescent probe comprises a teicoplanin-based fluorescent probe, a vancomycin-based fluorescent probe, and a terra Wanxing fluorescent probe, dalbavancin-based fluorescent probe, oritavancin-based fluorescent probe, epothilone-based fluorescent probe, balchimycin-based fluorescent probe, and based on Rees Fluorescent probe for tobramycin.
  4. 根据权利要求3所述的试剂盒，其特征在于，所述基于替考拉宁的荧光探针为The kit according to claim 3, wherein the teicoplanin-based fluorescent probe is
    Figure PCTCN2017086618-appb-100001
    Figure PCTCN2017086618-appb-100001
    其中R’为
    Figure PCTCN2017086618-appb-100002
    Figure PCTCN2017086618-appb-100003
    Where R' is
    Figure PCTCN2017086618-appb-100002
    Figure PCTCN2017086618-appb-100003
    所述基于万古霉素的荧光探针为：
    Figure PCTCN2017086618-appb-100004
    The vancomycin-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100004
    所述基于特拉万星的荧光探针为：
    Figure PCTCN2017086618-appb-100005
    The Travanta-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100005
    所述基于达巴万星的荧光探针为：
    Figure PCTCN2017086618-appb-100006
    The dalbavancin-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100006
    所述基于奥利万星的荧光探针为：
    Figure PCTCN2017086618-appb-100007
    The oritavancin-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100007
    所述基于伊端霉素的荧光探针为：
    Figure PCTCN2017086618-appb-100008
    The epothilone-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100008
    其中R’’为H或者Cl；Wherein R'' is H or Cl;
    所述基于巴尔赫霉素的荧光探针为：
    Figure PCTCN2017086618-appb-100009
    The balcomycin-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100009
    所述基于瑞斯托霉素的荧光探针为:
    Figure PCTCN2017086618-appb-100010
    The ristocetin-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100010
    其中，R1至R18各自独立地表示荧光标记。Wherein R 1 to R 18 each independently represent a fluorescent label.
  5. 根据权利要求1所述的试剂盒，其特征在于，所述第二类荧光探针为基于十三肽菌素类抗生素的荧光探针和基于多粘菌素的荧光探针中的任意一种或多种；优选地，所述基于十三肽菌素类抗生素的荧光探针包括基于十三肽菌素A1的荧光探针、基于十三肽菌素B1的荧光探针和基于十三肽菌素C的荧光探针；更优选地，所述基于十三肽菌素A1的荧光探针为：The kit according to claim 1, wherein the second type of fluorescent probe is a trifluoropeptidic antibiotic-based fluorescent probe and a polymyxin-based fluorescent probe. Or a plurality of; preferably, the threonutridin-based antibiotic-based fluorescent probe comprises a tripeptide peptide A1-based fluorescent probe, a threomycin B1-based fluorescent probe, and a thirteen-peptide Fluorescent probe of bacteriocin C; more preferably, the cephalopeptidin A1-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100011
    其中R’为
    Figure PCTCN2017086618-appb-100012
    或者
    Figure PCTCN2017086618-appb-100013
    Figure PCTCN2017086618-appb-100011
    Where R' is
    Figure PCTCN2017086618-appb-100012
    or
    Figure PCTCN2017086618-appb-100013
    所述基于十三肽菌素B1的荧光探针为：The thoracic peptide B1-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100014
    Figure PCTCN2017086618-appb-100014
    其中R”为
    Figure PCTCN2017086618-appb-100015
    或者
    Figure PCTCN2017086618-appb-100016
    Where R" is
    Figure PCTCN2017086618-appb-100015
    or
    Figure PCTCN2017086618-appb-100016
    所述基于十三肽菌素C的荧光探针为：The threonuclein C-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100017
    Figure PCTCN2017086618-appb-100017
    其中R”’为
    Figure PCTCN2017086618-appb-100018
    其中，R1至R9各自独立地表示荧光标记；    Where R"' is
    Figure PCTCN2017086618-appb-100018
    Wherein R 1 to R 9 each independently represent a fluorescent label;
    优选地所述基于多粘菌素的荧光探针为：Preferably the polymyxin-based fluorescent probe is:
    Figure PCTCN2017086618-appb-100019
    Figure PCTCN2017086618-appb-100019
    其中，R’为
    Figure PCTCN2017086618-appb-100020
    R”为
    Figure PCTCN2017086618-appb-100021
    Where R' is
    Figure PCTCN2017086618-appb-100020
    R" is
    Figure PCTCN2017086618-appb-100021
    R10至R14各自独立地表示荧光标记。R 10 to R 14 each independently represent a fluorescent label.
  6. 根据权利要求1至5中任一项所述的试剂盒，其特征在于，所述荧光标记选自香豆素类荧光标记、萘类荧光标记、氟硼荧类荧光标记、氧杂蒽类荧光标记、菁类荧光标记、方酸类荧光标记以及蒽类荧光标记中的任意两种或两种以上。The kit according to any one of claims 1 to 5, wherein the fluorescent label is selected from the group consisting of a coumarin fluorescent label, a naphthalene fluorescent label, a fluoroboron fluorescent label, and a xanthene-based fluorescent label. Any two or more of a label, a cyanine fluorescent label, a squaric acid fluorescent label, and an anthraquinone fluorescent label.
  7. 一种带荧光标记的细菌菌群，其特征在于，所述细菌菌群通过权利要求1至6中任一项所述的试剂盒中进行标记。A fluorescently labeled bacterial flora characterized in that the bacterial flora is labeled by the kit of any one of claims 1 to 6.
  8. 一种标记细菌菌群的方法，其特征在于，所述方法包括：A method of labeling a bacterial flora, the method comprising:
    利用第一类荧光探针和第二类荧光探针对细菌菌群中的菌种进行标记，其中，所述第一类荧光探针和第二类荧光探针为权利要求1至6中任一项所述的试剂盒中的第一类 荧光探针和第二类荧光探针。Marking the species in the bacterial flora with the first type of fluorescent probe and the second type of fluorescent probe, wherein the first type of fluorescent probe and the second type of fluorescent probe are as claimed in claims 1 to 6. The first category of one of the kits described A fluorescent probe and a second type of fluorescent probe.
  9. 根据权利要求8所述的方法，其特征在于，利用第一类荧光探针和第二类荧光探针同时对细菌菌群中的细菌进行标记。The method according to claim 8, wherein the bacteria in the bacterial flora are simultaneously labeled using the first type of fluorescent probe and the second type of fluorescent probe.
  10. 权利要求7所述的带荧光标记的细菌菌群在荧光显微镜观察或流式细胞荧光分选中的应用。Use of the fluorescently labeled bacterial flora of claim 7 for fluorescence microscopy or flow cytometry.
  11. 权利要求1至6中任一项所述的试剂盒在诊断细菌性肺部炎症、细菌性泌尿生殖***感染、细菌感染性腹泻和/或细菌性脑膜炎中的应用。Use of the kit according to any one of claims 1 to 6 for diagnosing bacterial lung inflammation, bacterial genitourinary infection, bacterial infectious diarrhea and/or bacterial meningitis.
  12. 根据权利要求11所述的应用，其特征在于，所述应用包括The application of claim 11 wherein said application comprises
    利用痰液涂片诊断细菌性肺部炎症；Diagnosis of bacterial lung inflammation with sputum smears;
    利用***分泌物涂片、尿道分泌物涂片、尿液涂片或尿沉渣涂片诊断细菌性泌尿生殖***感染；Diagnosis of bacterial genitourinary infection by vaginal discharge smear, urethral secretion smear, urine smear or urinary sediment smear;
    利用粪便涂片诊断细菌感染性腹泻；和/或Diagnosis of bacterial infectious diarrhea using fecal smears; and/or
    利用脑脊液涂片诊断细菌性脑膜炎。 Diagnosis of bacterial meningitis using cerebrospinal fluid smears.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7754444B2 (en) * 2004-06-24 2010-07-13 The Hong Kong University Of Science And Technology Biofunctional magnetic nanoparticles for pathogen detection
US20170073722A1 (en) * 2015-09-11 2017-03-16 Yogesh Kumar KANHYE Method and apparatus for pathogen testing

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7754444B2 (en) * 2004-06-24 2010-07-13 The Hong Kong University Of Science And Technology Biofunctional magnetic nanoparticles for pathogen detection
US20170073722A1 (en) * 2015-09-11 2017-03-16 Yogesh Kumar KANHYE Method and apparatus for pathogen testing

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