WO2018218510A1 - Trousse et procédé de marquage de la flore bactérienne, flore bactérienne marquée par fluorescence et application associée - Google Patents

Trousse et procédé de marquage de la flore bactérienne, flore bactérienne marquée par fluorescence et application associée Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
fluorescent probe
fluorescent
bacterial
type
probe
Prior art date
Application number
PCT/CN2017/086618
Other languages
English (en)
Chinese (zh)
Inventor
陈兴
王炜
Original Assignee
北京大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京大学 filed Critical 北京大学
Priority to PCT/CN2017/086618 priority Critical patent/WO2018218510A1/fr
Publication of WO2018218510A1 publication Critical patent/WO2018218510A1/fr

Links

Images

Classifications

    • 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

Definitions

  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne une trousse et un procédé de marquage de la flore bactérienne, de la flore bactérienne marquée par fluorescence et une application associée. Le procédé comprend : l'utilisation d'un premier type de sonde de fluorescence et d'un second type de sonde de fluorescence pour marquer des espèces bactériennes dans une flore bactérienne, le premier type de sonde de fluorescence marquant spécifiquement des bactéries à Gram positif, et le second type de sonde de fluorescence marquant spécifiquement des bactéries à Gram négatif, le premier type de sonde de fluorescence et le second type de sonde de fluorescence portant différents marqueurs de fluorescence, respectivement.
PCT/CN2017/086618 2017-05-31 2017-05-31 Trousse et procédé de marquage de la flore bactérienne, flore bactérienne marquée par fluorescence et application associée WO2018218510A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/086618 WO2018218510A1 (fr) 2017-05-31 2017-05-31 Trousse et procédé de marquage de la flore bactérienne, flore bactérienne marquée par fluorescence et application associée

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/086618 WO2018218510A1 (fr) 2017-05-31 2017-05-31 Trousse et procédé de marquage de la flore bactérienne, flore bactérienne marquée par fluorescence et application associée

Publications (1)

Publication Number Publication Date
WO2018218510A1 true WO2018218510A1 (fr) 2018-12-06

Family

ID=64454276

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/086618 WO2018218510A1 (fr) 2017-05-31 2017-05-31 Trousse et procédé de marquage de la flore bactérienne, flore bactérienne marquée par fluorescence et application associée

Country Status (1)

Country Link
WO (1) WO2018218510A1 (fr)

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

Similar Documents

Publication Publication Date Title
US20170253918A1 (en) Combining protein barcoding with expansion microscopy for in-situ, spatially-resolved proteomics
CN108982430B (zh) 标记细菌菌群样品的试剂盒、方法及应用
CN101974609A (zh) 一种病原菌液基薄层涂片检测试剂盒及其检测方法
CN102165071A (zh) 制备均质塑料-粘附适体-磁珠-荧光团和其他夹心分析物的方法
US20070231833A1 (en) Labeled antimicrobial peptides and method of using the same to detect microorganisms of interest
CN105588944B (zh) 检测agp1、serpina3和cdh1含量的***在筛查活动性结核病患者中的应用
CN109507416A (zh) 一种外泌体肿瘤标志物pdl1的快速检测试剂盒
Saravanan et al. Research on Detection of Mycobacterium Tuberculosis from Microscopic Sputum Smear Images Using Image Segmentation
US6803208B2 (en) Automated epifluorescence microscopy for detection of bacterial contamination in platelets
Ingram et al. Rapid detection of Legionella pneumophila by flow cytometry
CN105758832A (zh) 一种基于微球的杯式时间分辨荧光降钙素原分析试剂盒及其制备方法和应用
KR20220020307A (ko) 표지물질이 접합된 콜리스틴 컨쥬게이트를 포함하는 그람 음성균 검출용 조성물 및 이를 이용한 그람 음성균의 검출 방법
Mouton et al. Evaluation of Fluoretec-M for detection of oral strains of Bacteroides asaccharolyticus and Bacteroides melaninogenicus
US6051395A (en) Method and compound for detecting low levels of microorganisms
WO2018218510A1 (fr) Trousse et procédé de marquage de la flore bactérienne, flore bactérienne marquée par fluorescence et application associée
CN108982848A (zh) 一种基于适配体的耐甲氧西林金黄色葡萄球菌荧光检测方法
CN112461630A (zh) 一种荧光染色液及其应用
EP0859791A1 (fr) Procede de detection d'ookystes de cryptosporidium
Karpovich-Tate Detection of fungi in sinus fluid of patients with allergic fungal rhinosinusitis
EP1354202A1 (fr) Procede de detection de maladies pancreatiques et gastro-intestinales
CN211978947U (zh) 念珠菌甘露聚糖检测试剂盒
US20230109906A1 (en) Assay and kit for live antigen detection and monitoring of neurocysticercosis
US20220276245A1 (en) Test system for recognizing legionellae
CN107356761A (zh) 接骨木凝集素及结合珠蛋白相关蛋白的用途
CN116930134A (zh) 一种检测pap的多色荧光诊断试剂及其制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17911868

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 22/06/2020)

122 Ep: pct application non-entry in european phase

Ref document number: 17911868

Country of ref document: EP

Kind code of ref document: A1