Method for specifically detecting microorganisms and in particular gram-positive bacteria

Abstract

The present invention relates to a method for specifically detecting a microorganism or a plurality of microorganisms by in-situ hybridization using a microtiter plate reader, a cell drop cell counter or a flow cytometer.

Description

[0001]METHOD FOR THE SPECIFIC DETECTION OF MICROORGANISMS AND IN PARTICULAR GRAM-POSITIVE BACTERIA. The present invention relates to a method for the specific detection of a microorganism or a group of microorganisms, and in particular of Gram-positive bacteria, by in situ hybridization using flow cytometry. Microbial analysis in the food and pharmaceutical industries is often confronted with a high sample volume (> 100 samples per day), which must be processed quickly and, if possible, in parallel. To realize the processing of such a high sample throughput (high-throughput) with a specific, rapid molecular biological detection system, systems with automatic evaluation and objective result determination are suitable. A modern method of bacterial identification suitable for meeting these requirements isFluorescence in situ hybridization (FISH) has now become established. In its original form, cells on a slide are visualized under the microscope using fluorescently labeled nucleic acid probe molecules (Amann et al., Microbial. Rev. 59 (1995), 143-169). However, the classic FISH technique is particularly plagued by the problem that detection using a microscope requires well-trained laboratory personnel and a considerable amount of time, which naturally severely limits the number of samples analyzed per day. To address this problem, a liquid-phase fluorescence in situ hybridization based on the reaction mechanism of the classic FISH technique was proposed in the following years.in which all steps of the hybridization reaction were transferred virtually unchanged from the slide format to the reaction vessel format, and detection was performed using a flow cytometer (WO 03 / 083131 A1). Within this process, the various hybridization and washing solutions were removed via centrifugation. However, the washing steps required for whole-cell hybridization to increase the signal-to-noise ratio in routine analysis are particularly labor-intensive and require well-trained laboratory personnel to handle. Furthermore, the steps required during centrifugation to remove the aqueous supernatants represent another process parameter that must be considered when evaluating the observed result and the required standardization of the process. DE 102010 012 421 A1 discloses a method for the specific detection of microorganisms.which can be performed quickly and does not require the washing steps required for the classic FISH technique. Specifically, the method involves conducting the hybridization reaction in a microtiter plate and subsequently determining the results using a microtiter plate reader, with the readout fluorescence signal corresponding to the sum of the fluorescently labeled nucleic acid probes specifically bound to the microorganisms. A key prerequisite for this semi-quantitative bacterial determination is the specific fluorescence quenching of unbound fluorescently labeled nucleic acid probe molecules within the reaction space of the microtiter plate in order to enable discrimination between specific and non-specific signals. However, the method has the disadvantage that precise quantification of the cell count is not possible.Because the number of specifically bound fluorescently labeled nucleic acid probe molecules per cell (and thus the contribution of a cell to the measured fluorescence signal intensity) cannot be clearly determined. A single cell can typically bind between 5,000 and 15,000 fluorescently labeled oligonucleotide probes. To overcome the above disadvantages, EP 3673 075 B1 proposed fixing cells of a provided sample with a fixative and separating the fixed cells from the sample. The fixed cells are then homogenized and dried with a chemical homogenizing agent containing i) a monosaccharide or disaccharide, ii) a polyol, and iii) water. The dried cells are then brought into contact with a solution of a fluorescently labeled nucleic acid probe specific for the microorganism to be detected.to provide a first reaction mixture. The first reaction mixture is then incubated to cause the fluorescently labeled nucleic acid probe to bind to the corresponding target nucleic acid sequence in the cells of the microorganism to be detected, before the first reaction mixture is subsequently contacted with a solution of a quencher-labeled nucleic acid probe to provide a second reaction mixture, wherein the quencher-labeled nucleic acid probe comprises a quencher that at least partially quenches the fluorescence of the fluorescently labeled nucleic acid probe and has a nucleic acid sequence that is substantially complementary to the nucleic acid sequence of the fluorescently labeled nucleic acid probe. The second reaction mixture is then incubated,to cause the molecules of the fluorescently labeled nucleic acid probe not bound to the target nucleic acid sequence in the cells of the microorganism to be detected to bind to the quencher-labeled nucleic acid probe, before the second reaction mixture is subsequently introduced into a flow cytometer and the fluorescence emitted by the cells of the microorganism to be detected containing the fluorescently labeled nucleic acid probe is detected. Although this method achieves good results for many fungi and Gram-negative bacteria, it is less suitable or not suitable for Gram-positive bacteria. A further disadvantage of this method is that it is labor- and time-intensive, particularly due to the initial steps of fixing, drying, and homogenizing the cells. Based on this, the present invention was therefore based on the object ofTo provide a method for the quantitative detection of microorganisms, and in particular of Gram-positive bacteria, for example, in food and in the pharmaceutical industry, which is faster and less labor-intensive than the method disclosed in EP 3673 075 B1, and which also yields precise and reliable results, particularly for Gram-positive bacteria. According to the invention, the object is achieved by a method as defined in claim 1. The method according to the invention not only yields precise and reliable results for Gram-positive bacteria, but surprisingly, the method according to the invention allows the first steps of fixing, drying, and homogenizing the cells known from EP 3673 075 B1 to be omitted. This was surprising because the steps of fixing, drying, and homogenizing the cells are essential according to the teaching of EP 3673 075 B1.namely, without these steps in the process, cell clusters are to be obtained which do not allow quantification of the cell number and in particular do not allow quantification of the cell number in a flow cytometer. Despite the omission of these steps, no cell clusters are obtained with the process according to the invention, or, if at all, only very small amounts of cell clusters are obtained which do not interfere with the quantification of the cells. Therefore, the process according to the invention is faster and less labor-intensive than the process known from EP 3 673 075 B1. In addition, the advantages of the process known from EP 3 673 075 B1 are also achieved, namely the rapid and specific detection of microorganisms without the need for a washing step as in the classic FISH technique.as well as the suppression of nonspecific autofluorescence, which occurs in the FISH technique, especially in the classical FISH technique, and which is not without problems during evaluation. Furthermore, the method according to the invention enables a significantly simplified implementation compared to the microtiter plate method described in DE 102010 012 421 A1.by shortening the conventional process steps to the addition of two reaction solutions to the sample mixture, and allowing the reaction to take place in a single reaction vessel. Furthermore, the method according to the invention allows direct quantification of the detected microorganisms. Finally, the present method can be automated, at least in terms of implementation and evaluation. The method according to the invention is therefore particularly suitable for high-throughput analysis. The implementation of the method according to the invention for the specific detection of one or more microorganisms in a sample comprises the following steps: (a) adding an enzyme suitable for permeabilizing the one or more microorganisms to a sample containing the microorganism or the more microorganisms and incubating the mixture thus produced,to obtain a sample with permeabilized cells; (b) contacting the sample obtained in step (a) with permeabilized cells with a solution of a fluorescently labeled nucleic acid probe specific for the microorganism to be detected to provide a first reaction mixture; (c) incubating the first reaction mixture to cause the fluorescently labeled nucleic acid probe to bind to the corresponding target nucleic acid sequence in the cells of the microorganism to be detected; (d) contacting the first reaction mixture following step (c) with a solution of a quencher-labeled nucleic acid probe to provide a second reaction mixture, wherein the quencher-labeled nucleic acid probe comprises a quencher that at least partially quenches the fluorescence of the fluorescently labeled nucleic acid probe and has a nucleic acid sequence,which is substantially complementary to the nucleic acid sequence of the fluorescently labeled nucleic acid probe; (e) incubating the second reaction mixture to cause the molecules of the fluorescently labeled nucleic acid probe not bound to the target nucleic acid sequence in the cells of the microorganism to be detected to bind to the quencher-labeled nucleic acid probe; and (f) quantitatively detecting the fluorescence emitted by the cells of the microorganism to be detected containing the fluorescently labeled nucleic acid probe. The present invention relates to the specific detection of a single microorganism or multiple (i.e., at least two different) microorganisms in a naturally occurring or artificially assembled sample.that when detecting such a microorganism within the scope of the method according to the invention, more than just a single cell is detected. As a rule, the detection of one or more cells takes place, with the detection being based on the detection of the fluorescence emanating from a single cell. The term "microorganism," as used in the context of the present application, encompasses both naturally occurring and artificially produced microorganisms, which may be pathogenic or non-pathogenic in nature and include, among others, bacteria, fungi, microalgae, and protozoa. Preferably, the at least one microorganism to be detected is a bacterium, a fungus, or a unicellular higher organism (protozoan), wherein the bacterium, the fungus, and / or the unicellular higher organism may originate from any taxonomic unit, and the term "taxonomic unit" includes, among others, domains / kingdoms, divisions / phyla, classes,Subclasses, orders, suborders, families, subfamilies, genera, subgenera, species, subspecies, phyla, and subphyla. Good results are achieved in particular when the enzyme added in step (a) is selected from the group consisting of proteinase K, lyticase, achromopeptidase, mutanolysin, and lysozyme. Lysozyme is particularly preferably added as the enzyme in step (a). Specific examples of microorganisms that can be detected by the method according to the invention include, in particular, bacteria, fungi (including yeasts and molds), and protozoa, which are known to damage the quality of water (including wastewater), beverages (e.g., water, beer, fruit juices, and non-alcoholic soft drinks), and foodstuffs (e.g., dairy products such as cheese and yogurt, and meat products such as sausages) and are described, inter alia, in DE 10129 410 A1.DE 101 60 666 A1 and WO 2005 / 031004 A2. The detection of bacteria, yeasts, and molds is particularly preferred in this context. Sample-relevant bacteria include in particular bacteria of the genera Acetobacter, Achromobacter, Acinetobacter, Aerococcus, Aeromonas, Agrobacterium, Alcaligenes, Alicyclobacillus, Aneurinibacillus, Anoxybacillus, Aquabacterium, Arcobacter, Arthrobacter, Arthrobacter, Bacillus, Brevibacillus, Brevibacterium, Brocardia, Brochothrix, Burkholderia, Caldanaerobius, Campylobacter, Carnobacterium, Cellulomonas, Chloroflexi, Chryseobacterium, Chryseobacterium, Citrobacter, Cloacibacterium, Clostridium, Colwellia, Corynebacterium, Cronobacter, Delftia, Desulfotomaculum, Dickeya, Enterobacter, Enterobacteriaceae, Enterococcus, Erwinia, E-scherichia, Facklamia, Flavobacteriaceae, Flavobacterium, Fructobacillus, Geobacillus, Gluconacetobacter, Gluconobac- ter, Janthinobacterium, Jeotgalibacillus, Kocuria,Komagtaeibacter, Kuenenia, Kurthia, Lactobacillus, Lactococcus, Legionella, Lentibacillus, Leuconostoc, Listeria, Lysinibacillus, Macrococcus, Marinilactibacillus, Megasphaera, Microbacteri- um, Micrococcus, Microthrix, Milchsäurebakterien, Moorella, Moraxella, Nitrobacter, Nitrosococcus, Nitrosomonas, Nitrospira, Mycobacterium, Nitrotoga, Nocardia, Nostocoida, Obesumbacterium, Oceanobacillus, Oenococcus, Paenibacillus, Pantoea, Pectinatus, Pectobacterium, Pediococcus, Pedobacter, Photobacterium, Prevotella, Propionibacterium, Pseudoaltero- monas, Pseudomonas, Psychrobacillus, Psychrobacter, Psychroflexus, Rhizobium, Salmonella, Scalindua, Serratia, Shewanella, Shigella, Solibacillus, Sphingobacterium, Sphingomonas, Sporolactobacillus, Sporosarcina, Staphylococcus, Stenotrophomonas, Streptococcus, Streptomyces, Tepidomonas, Thermo- anaerobacterium, Thermophilic bacteria, Thiothrix, Trichococcus, Ureibacillus, Vagococcus, Vibrio, Virgibacillus, Viridibacillus,Weissella, Xanthomonas, Yersinia, and Zymomonas. Sample-relevant fungi that can be detected using the method of the present invention include, in particular, molds and yeasts of the genera Aspergillus, Candida, Debaromyces, Dekkera, Geotrichum, Hanseniaspora, Hyphopichia, Kazachstania, Kloeckera, Kluyveromyces, Lodderomyces, Penicillium, Pichia, Rhodotorula, Saccharomyces, Saccharomycopsis, Schizosaccharomyces, Torulaspora, Wickerhamomyces, Yarrowia, and Zygosaccharomyces. Sample-relevant unicellular higher organisms (protozoa) that can be detected using the method of the present invention include, in particular, Giardia, Cryptosporidium, Amoeba, Trichomonas, Toxoplasma, Balantidium, and Blastocystis. Regarding the detection of microorganisms in water, beverages and food, bacteria of the genera Acinetobacter, Alicyclobacillus, Aquabacterium, Arcobacter, Bacillus, Campylobacter, Enterobacteriaceae,Escherichia, Lactobacillus, Lactococcus, Legionella, Listeria, Microthrix, Mycobacterium, Nitrobacter, Nitrosococcus, Nitrosomonas, Nitrospira, Nitrotoga, Propionibacterium, Salmonella, Shigella, and Streptococcus, as well as fungi of the genera Aspergillus, Candida, Debaromyces, Dekkera, Penicillium, Pichia, Saccharomyces, and Zygosaccharomyces, are of particular relevance, which is why their detection is considered particularly preferred. According to a particularly preferred embodiment of the present invention, the microorganism to be detected in the sample is a bacterium. Particularly preferred examples are bacteria of the genus Acinetobacter, Akkermansia, Alicyclobacillus, Alistipes, Aquabacterium, Arcobacter, Atopobium, Bacillus, Bacteroides, Bifidobacterium, Bilophila, Campylobacter, Clostridium, Corynebacterium, Dialister, Enterobacteriaceae, Enterobacter, Enterococcus, Escherichia, Faecalibacterium, Fusobacterium, Hafnia,Helicobacter, Lactobacillus, Lactococcus, Legionella, Listeria, Megasphaera, Microthrix, Nitrobacter, Nitrosococcus, Nitrosomonas, Nitrospira, Nitrotoga, Parabacteroides, Prevotella, Propionibacterium, Roseburia, Ruminococcus, Salmonella, Shigella, Sphingobacterium, Staphylococcus, Streptococcus, Tannerella, Veillonella, and Vibrio. Most preferably, the microorganism to be detected in the sample is a Gram-positive bacterium. The sample used in the method according to the invention is preferably a liquid sample. It is sufficient if at least some of the individual cells of the microorganism to be detected are present in the liquid phase. In this respect, a partially liquid sample, a suspension, or a dispersion can also be used according to the invention, with a solution being preferred. Particularly preferred is an aqueous sample as the liquid sample,such as, for example, a water sample or a beverage sample, which is submitted for analysis as such (i.e., without the addition of further liquid). The sample to be analyzed in the context of carrying out the method according to the invention can be any primary sample from which a secondary sample is generated, which is then used as a liquid sample in the method according to the invention. A primary sample can be a solid, pasty, liquid, or gaseous sample. Typically, a representative mixture of microorganisms is obtained from the primary sample, which is then transferred into the liquid sample as used in the method according to the invention. According to the invention, in step (a) of the method, an enzyme suitable for permeabilizing the one microorganism or for several microorganisms, preferably proteinase K, lyticase, achromopeptidase, mutanolysin, or lysozyme, and particularly preferably lysozyme, is added to the provided,to be analyzed is added and the mixture thus prepared is incubated to obtain a sample with permeabilized cells. Good results are obtained in particular when 100 to 2,000,000 units, particularly preferably 1,000 to 1,000,000 units, and very particularly preferably 400,000 to 800,000 units, such as 800,000 units, of lysozyme are added to the sample. The term "units" refers to one micromole of substrate conversion per minute under defined conditions. In a further development of the inventive concept, it is proposed to incubate the mixture prepared by adding enzyme and preferably mixing, for example in a vortex mixer, at 23 to 45°C for 1 second to 5 hours. Good results are obtained in particular when the mixture is stored at 30 to 45°C, particularly preferably at 35 to 45°C, very particularly preferably at 37 to 42°C and most preferably at 39 to 41°C, such as 40°C.is incubated. Preferred incubation times are 30 seconds to 2 hours, particularly preferably 1 to 60 minutes, very particularly preferably 10 to 50 minutes, and most preferably 20 to 40 minutes, such as about 30 minutes. Preferably, the incubation is carried out without shaking. According to the invention, in step (b), the sample with permeabilized cells obtained in step (a) is brought into contact with a solution of a fluorescently labeled nucleic acid probe specific for the microorganism to be detected in order to provide a first reaction mixture. This means that the sample with permeabilized cells obtained in step (b), apart from a dilution step or the like, in which no reaction takes place,is subjected to step (b) without an intermediate step. In other words, between steps (a) and (b) of the method according to the invention, no fixation of the cells and no homogenization of the cells, and in particular no homogenization after addition of a homogenizing agent containing mono- or disaccharide, polyol, and water, is carried out. Preferably, the sample obtained in step (a) with permeabilized cells is introduced into a suitable reaction vessel in step (b) and then first mixed with a solution of at least one fluorescently labeled nucleic acid probe specific for the microorganism, i.e., a nucleic acid probe labeled with a fluorescent dye, to provide the first reaction mixture. This first reaction mixture is then incubated under suitable conditions in step (c),to cause the at least one fluorescently labeled nucleic acid probe to bind to the corresponding target nucleic acid sequence in the cells of the microorganism to be detected. Following this step, the first reaction mixture is contacted in step (d) with a solution of at least one quencher-labeled nucleic acid probe, i.e., a nucleic acid probe labeled with a quencher that at least partially quenches the fluorescence of the fluorescent dye of the fluorescently labeled nucleic acid probe, to provide a second reaction mixture, wherein the quencher-labeled nucleic acid probe has a nucleic acid sequence that is substantially complementary, and preferably reversely complementary, to the nucleic acid sequence of the fluorescently labeled nucleic acid probe. The second reaction mixture is then again incubated under suitable conditions.to cause the molecules of the fluorescently labeled nucleic acid probe not bound to the target nucleic acid sequence to bind to the quencher-labeled nucleic acid probe and thus at least partially quench the fluorescence of any free fluorescently labeled nucleic acid probe. This means, on the one hand, that within the scope of the method of the present invention, the solution of the at least one quencher-labeled nucleic acid probe is added directly to the solution of the at least one fluorescently labeled nucleic acid probe, thus avoiding prior separation of any excess fluorescently labeled nucleic acid probe by means of a washing step. Furthermore, only those fluorescently labeled nucleic acid probes that have bound to the target nucleic acid sequence (i.e., in the cells of the microorganism to be specifically detected) carryto the fluorescence signal. The signal of the free fluorescently labeled nucleic acid probes, however, is largely quenched by hybridization with the quencher-labeled nucleic acid probe. This reaction mechanism enables a one-step test system. Regarding the quantitative ratio of the two nucleic acid probes, i.e., the fluorescently labeled nucleic acid probe and the quencher-labeled nucleic acid probe, this depends on the conditions of the specific embodiment of the method according to the invention and can easily be determined by a person skilled in the art using routine tests. However, the required quenching of the fluorescence of the excess fluorescently labeled nucleic acid probe, i.e., the fluorescently labeled nucleic acid probe not bound to the target nucleic acid sequence, by the quencher-labeled nucleic acid probe implies that the quantitative ratio of quencher-labeled nucleic acid probe to fluorescently labeled nucleic acid probe is at least 1:1.and preferably provides an excess of quencher-labeled nucleic acid probe. Regarding the incubation conditions for the first and second reaction mixtures, the incubation time and incubation temperature can be determined by a person skilled in the art of microbial analysis based on the length and GC content of the nucleic acid sequences and checked using simple optimization procedures. However, the incubation temperature for the first reaction mixture obtained after adding the solution of the fluorescently labeled nucleic acid probe to the dried cells is preferably about 60 to 120 minutes, and particularly preferably about 80 to 100 minutes, while the incubation temperature is preferably about 30°C to 50°C, and particularly preferably about 40°C. The incubation temperature for the second reaction mixture obtained after adding the solution of the quencher-labeled nucleic acid probe to the first reaction mixture is preferably about 5 to 30 minutes.and particularly preferably about 10 to 20 minutes, while the incubation temperature is again preferably about 30°C to 50°C, and particularly preferably about 40°C. Fig. 1 schematically illustrates the reaction process underlying the method according to the invention, wherein in the specific example both the fluorescently labeled nucleic acid probe and the quencher-labeled nucleic acid probe are present as single-stranded DNA molecules. In detail, after being added to the suitably fixed and dried cells, the fluorescently labeled nucleic acid probe diffuses to its target sequence and binds to it. The target molecule in the cells of the microorganism to be detected here is an rRNA, wherein the sequence of the rRNA targeted by the fluorescently labeled nucleic acid probe is specific for the microorganism to be detected. Unbound (ie excess) fluorescently labelled nucleic acid probe isIn contrast to the classic FISH technique, in which the latter must be removed during a stringent washing step, the quencher-labeled nucleic acid probe is captured by adding the quencher-labeled nucleic acid probe, forming a non-fluorescent nucleic acid hybrid composed of the fluorescently labeled nucleic acid probe and the quencher-labeled nucleic acid probe. If the fluorescently labeled nucleic acid probe has previously bound to a suitable target nucleic acid sequence, this fluorescently labeled nucleic acid probe no longer forms a hybrid with the quencher-labeled nucleic acid probe. Consequently, after excitation of the fluorescent dye of the fluorescently labeled nucleic acid probe, fluorescence is observed in cases where the fluorescently labeled nucleic acid probe has bound to the target nucleic acid sequence in the microorganism to be detected, while in cases where the fluorescently labeled nucleic acid probe has not bound to the target nucleic acid sequence,as a result of hybridization of the fluorescently labeled nucleic acid probe with the quencher-labeled nucleic acid probe, no fluorescence occurs. Thus, the fluorescence of a particle detected in, for example, a flow cytometer is a direct qualitative and quantifiable signal for the microorganism for which the fluorescently labeled nucleic acid probe is specific. The method according to the invention was described above using nucleic acid probes consisting of deoxyribonucleotides and can therefore also be referred to as DNA probes. However, it is understood that, in principle, other nucleic acid probes can also be used.provided that these nucleic acid probes exhibit the above-described behavior with regard to the formation of hybrids in the microorganism to be detected. Within the scope of the present invention, the fluorescently labeled nucleic acid probe and / or the quencher-labeled nucleic acid probe is preferably a single-stranded nucleic acid probe. However, it is also possible within the scope of the present invention for said nucleic acid probes to be individually and independently double-stranded. In cases in which at least one of the nucleic acid probes is double-stranded, it is preferred,that only a portion of the sequence of the fluorescently labeled nucleic acid probe or a portion of the sequence of the quencher-labeled nucleic acid probe is double-stranded. The extent of the formation of a double strand in the respective probes depends on the requirements of hybridization with the target nucleic acid sequence and in particular the hybridization with the respective complementary probe. Each of the nucleic acid probes to be used within the scope of the present invention, and in particular the fluorescently labeled nucleic acid probe, is preferably designed as a DNA probe, RNA probe, PNS probe, or LNS probe.or as combinations of two or more thereof. The design or selection of the nucleotides forming the respective nucleic acid probes is within the scope of the skill of the person skilled in the art and can be carried out using routine methods and considerations in light of the disclosure provided herein and in particular the presumed mechanism underlying the present invention. As described above, the fluorescently labeled nucleic acid probe is specific for the microorganism to be detected. The generation of corresponding nucleic acid probes is known to a person skilled in the art in the field of microbial analysis and is described in more detail in, among other things, DE 102010 012 421 A1. The specificity is preferably determined by the degree of homology between the nucleic acid probe and its target nucleic acid sequence. Preferably, the degree of homology is at least 70%, more preferably at least 80%, even more preferably at least 95%.and most preferably at least 96%, 97%, 98%, 99%, or 100%. In one embodiment, the nucleic acid probe is thus essentially identical to the target nucleic acid sequence within the specified homology values. Alternatively, the nucleotide sequence of the fluorescently labeled nucleic acid probe can be essentially reverse complementary to the target nucleic acid sequence. Even then, the aforementioned homology values ​​are considered the extent of identity or complementarity of the nucleotide sequence of the nucleic acid probe with or to the target nucleic acid sequence. Alternatively, particularly in the case of a reverse complementary sequence, the extent of homology can also be defined by the conditions under which hybridization of the nucleic acid probe and the target sequence still occurs. The fluorescently labeled nucleic acid probe is preferably reverse complementary to the target sequence,especially when hybridized with the target nucleic acid sequence under moderate or stringent conditions. Corresponding conditions are described, for example, in WO 00 / 68421 A1. What has been said herein regarding the fluorescently labeled nucleic acid probe essentially also applies to the quencher-labeled nucleic acid probe, whereby it is obvious to the person skilled in the art that the quencher-labeled nucleic acid probe is essentially reverse complementary to the fluorescently labeled nucleic acid probe, and the extent of complementarity between the quencher-labeled nucleic acid probe and the fluorescently labeled nucleic acid probe can be defined in the same way as the extent of complementarity between the target nucleic acid sequence in the microorganism to be detected and the fluorescently labeled nucleic acid probe. Target nucleic acid sequences that allow the specific detection of a microorganism are known in the art.For example, reference is made to the publications Clementino et al. (J. Appl. Microbiol. 103 (2007), 141-151), Ni et al. (FEMS Microbiol. Lett. 270 (2007), 58-66), Leaw et al. (J. Clin. Microbiol. 45 (2007), 2220-2229) and Bhardwaj et al. (J. Med. Microbiol. 56 (2007), 185-189). Preferred target nucleic acid sequences in this context are, in particular, 16S rRNA, 23S rRNA, 18S rRNA, tRNA, EF-Tu, mRNA, 16S-23S rRNA spacers, and 23S-5S rRNA spacers, with 16S rRNA and 23S rRNA being particularly preferred. The length of the fluorescently labeled nucleic acid probe and the quencher-labeled nucleic acid probe is, independently of one another, approximately 15 to 31 nucleotides, preferably approximately 17 to 25 nucleotides, more preferably approximately 17 to 23 nucleotides.and most preferably 17 or 18 nucleotides. In a preferred embodiment, the fluorescently labeled nucleic acid probe and the quencher-labeled nucleic acid probe are essentially the same length. Regarding the selection of the length of the two nucleic acid probes, reference is made to the criteria stated in DE 10 2010 012 421 A1. Both nucleic acid probes may comprise additional nucleotides than are necessary to form the aforementioned lengths, although these additional nucleotides preferably do not contribute to or participate in the formation of a double-stranded structure when the fluorescently labeled nucleic acid probe base pairs with the quencher-labeled nucleic acid probe.wherein the base pairing is preferably a Watson-Crick base pairing, and a hybridized complex is formed. In one embodiment, the length of the two nucleic acid probes and the complementary region are of equal length. A fluorescent dye, also referred to herein as a fluorophore, is a molecule that absorbs light at a characteristic wavelength, ideally at its absorption maximum, or is energetically excited by it. This light (photon) is re-emitted after a certain time, e.g., in the form of fluorescence or as vibrational energy (heat). The fluorophore returns to the energetically more favorable unexcited initial state. A quencher (acceptor) is a moleculewhich absorbs energy from an excited fluorophore (donor) and thus quenches its fluorescence emission. Fluorescence quenching generally refers to the attenuation or quenching of a fluorescence signal. For optimal quenching efficiency, a precise match between the fluorescent dye and the corresponding quencher is crucial (Marras et al., Methods in Molecular Biology 335 (2006), 3-16). When selecting a suitable pair of fluorescent dye and quencher, a distinction can be made as to whether the observed quenching is due to static or dynamic quenching. The positioning of the fluorescent dye and the quencher can be determined depending onwhether it is static or dynamic quenching. In so-called static quenching or contact quenching, a non-fluorescent complex forms between the fluorophore and the quencher in the excited state. In static quenching, the donor and acceptor are located very close together (≤ 20 Å). The molecules interact directly through proton-coupled electron transfer via the formation of hydrogen bonds (Förster, Ann. Phys. 2 (1948), 55-75; Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999). However, it is also possible that the dynamic quenching method, so-called FRET quenching (Fluorescence Resonance Energy Transfer),is used. The excited fluorophore transfers its energy to the quencher and then returns to the ground state without radiation. The donor and acceptor are located at a spatial distance of approximately 40 to 100 Å (which corresponds to approximately 3 to 30 nucleotides within a double-stranded DNA). A prerequisite for FRET quenching is that the fluorescence emission spectrum of the donor overlaps with the absorption spectrum of the acceptor. To achieve such quenching, it is preferred according to the inventionthat the fluorescent dye of the fluorescently labeled nucleic acid probe is arranged at the 3' end or near the 3' end of the fluorescently labeled nucleic acid probe, and the quencher of the quencher-labeled nucleic acid probe is arranged at the 5' end or near the 5' end of the quencher-labeled nucleic acid probe. Alternatively, however, the fluorescent dye of the fluorescently labeled nucleic acid probe can also be arranged at the 5' end or near the 5' end of the fluorescently labeled nucleic acid probe, while the quencher of the quencher-labeled nucleic acid probe is arranged at the 3' end or near the 3' end of the quencher-labeled nucleic acid probe. For further details, reference is made in this context to DE 102010 012 421 A1. It is understood thatthat the quenching of the fluorescence emitted by the fluorescent dye of the fluorescently labeled nucleic acid probe by the quencher of the quencher-labeled nucleic acid probe does not necessarily have to be complete. Rather, for the purposes of the present invention, it is sufficient if there is a significant signal difference detectable by the detector system (such as a flow cytometer) between a cell of the microorganism to be detected labeled with the fluorescently labeled nucleic acid probe and the hybridization complex consisting of the fluorescently labeled nucleic acid probe and the quencher-labeled nucleic acid probe. The extent of quenching is usually about 10 to 90%, and preferably at least 50%. The fluorescent dyes used to produce the fluorescently labeled nucleic acid probes are, in particular, thoseas they are also used in the classic FISH technique. The fluorescent dye can, for example, be selected from the group consisting of FAM, TAMRA, CY3, Alexa 350, Pacific Blue, Coumarin, Cy2, Alexa 488, TET, Alexa 532, HEX, Alexa 546, TMR, Cy3.5, Alexa 568, Texas Red, Alexa 594, Alexa 633, Cy5, Cy5.5, Alexa 660, Alexa 680, ATTO 490LS, Rox, and Vic. It goes without saying that there are fundamentally no restrictions regarding the suitability of fluorescent dyes, with the exception that there must be a quencher that at least partially quenches the fluorescence of the fluorescent dye. There are also fundamentally no restrictions regarding the design or selection of the quencher. However, it is understood that the selection of the fluorescent dye and the quencher must be such thatthat a significant quenching of the fluorescence signal of the fluorescent dye occurs either directly or indirectly after its excitation. Sufficient quenching is defined as allowing a distinction between the quenched and the unquenched state. Within the scope of the present invention, it is therefore possible for the quencher itself to be a fluorescent dye. Some exemplary combinations of fluorescent dye and quencher are given in the following table. Fluorescent dye Corresponding quencher FAM Dabcyl, BHQ-1, TAMRA TAMRA BHQ-2 CY3 BHQ-2 Further suitable combinations of fluorescent dye and quencher are described, among others, in Marras et al. (Nucl. Acids Res. 30 (2002), e122).The disclosure of which is hereby explicitly incorporated by reference. To increase the specificity of the method according to the invention, in addition to the fluorescently labeled nucleic acid probe and the quencher-labeled nucleic acid probe, so-called competitor probes can also be added to the reaction. The term "competitor probe," as used in the context of the present application, refers in particular to oligonucleotides that cover potentially occurring unwanted bonds, i.e., in particular, binding sites, of the nucleic acid probes, in particular the fluorescently labeled nucleic acid probe, and thereby exhibit a higher sequence similarity to a non-detectable microorganism than to the microorganism(s) to be detected. The use of competitor probes can preventthat the fluorescently labeled nucleic acid probe binds to the nucleic acid sequence of a non-detectable microorganism, thus leading to false-positive signals. The competitor probe is typically unlabeled and is preferably used before the addition of the fluorescently labeled nucleic acid probe and the quencher-labeled nucleic acid probe. The competitor probe should be substantially complementary to a target nucleic acid sequence of one or more non-detectable microorganisms. The competitor probe, as used in the present invention, can be a DNA or RNA sequence, which generally comprises between 12 and 100 nucleotides, preferably between 15 and 50 nucleotides.and particularly preferably between 17 and 25 nucleotides. By selecting a defined sequence, the hybridization of the fluorescently labeled nucleic acid probe or the quencher-labeled nucleic acid probe to the nucleic acid sequence of a taxonomic unit or artificially assembled group of microorganisms can be blocked. Complementarity to the nucleic acid sequence to be blocked should be over 100% of the sequence for a competitor probe of 15 nucleotides. For competitor probes with more than 15 nucleotides, one or more mismatches are permitted, depending on the length. Such competitor probes are described, for example, in the international patent application WO 2005 / 031004 A2, the disclosure of which is hereby incorporated by reference. Within the scope of the present invention,that in addition to the pair of a fluorescently labeled nucleic acid probe and a quencher-labeled nucleic acid probe (first nucleic acid probe pair), at least one further pair of a fluorescently labeled nucleic acid probe and a quencher-labeled nucleic acid probe matched thereto (second nucleic acid probe pair, third nucleic acid probe pair, etc.) can be used. The design of each further nucleic acid probe pair corresponds to the first nucleic acid probe pair, whereby the fluorescently labeled nucleic acid probe of the second, third, etc. nucleic acid probe pair each addresses a different target nucleic acid sequence in the microorganism to be detected than the fluorescently labeled nucleic acid probe of the first nucleic acid probe pair. This means that in a preferred variant of the method according to the invention, first in step (b) several,specific fluorescent-labeled nucleic acid probes with different nucleic acid sequences for the microorganism to be detected are added to the fixed and dried cells in the form of a single solution or multiple solutions in order to hybridize several fluorescent-labeled nucleic acid probes to target nucleic acid sequences of the microorganism to be detected in parallel, and then, in step (d), a number of different quencher-labeled nucleic acid probes corresponding to the number of fluorescent-labeled nucleic acid probes is added in the form of a single solution or multiple solutions in order to capture the different fluorescent-labeled nucleic acid probes and thus at least partially quench their fluorescence. In a further embodiment of the method according to the invention, the sample to be analyzed contains more than one microorganism, i.e., at least two different microorganisms,which are to be detected in parallel. In this case, it is preferred that the fluorescently labeled nucleic acid probe of the second nucleic acid probe pair is specific for the second microorganism to be detected, the fluorescently labeled nucleic acid probe of the third nucleic acid probe pair is specific for the third microorganism to be detected, etc. However, it is of course also possible here that more than one specific fluorescently labeled nucleic acid probe is used to detect each microorganism to be detected. After the fluorescence of any excess fluorescently labeled nucleic acid probe(s) has been at least partially quenched, the second reaction mixture is subjected to step (f) of a quantitative detection of the fluorescence emitted by the cells of the microorganism to be detected containing the fluorescently labeled nucleic acid probe.The present invention is not particularly limited with regard to the type of quantitative detection of the fluorescence of the second reaction mixture. Good results are achieved in particular when the quantitative detection of the fluorescence of the second reaction mixture is carried out in a flow cytometer, in a fluorescence-detecting microtiter plate reader, or in an automatic cell counter, preferably a cell drop cell counter. Particularly preferably, the detection of the fluorescence emitted by the cells of the microorganism to be detected containing the fluorescently labeled nucleic acid probe is carried out in a flow cytometer. In the microtiter plate reader,The cell droplet counter and the flow cytometer can be commercially available devices. Since the second reaction mixture can be introduced as such (i.e., without further processing and washing steps) into the microtiter plate reader, the cell droplet counter, or the flow cytometer, the workload is reduced and possible sample losses can be prevented. In this respect, the invention relates, in a further aspect, to the use of a microtiter plate reader, a cell droplet counter, or a flow cytometer in the method according to the invention. The fluorescence emitted by the cells can be used directly to quantify the number of cells, in particular whole cells.of the microorganism to be detected. The measured values ​​obtained during this measurement are then visualized on the computer in the form of histograms or dot plots and allow reliable information about the type and quantity of microorganisms contained in the sample. Thus, the method according to the invention allows for direct detection and quantification of one or more microorganisms as whole cells within the framework of whole-cell hybridization. Significant advantages of the method according to the invention are, in particular, its suitability for the rapid and less labor-intensive quantitative detection of Gram-positive bacteria, and, in addition, its very easy handling, as well as the speed, reproducibility, reliability, and objectivity with which the specific detection of microorganisms in a sample is possible. A further advantage is thatthat for the first time, the advantageous method of in situ hybridization in solution can be carried out in such a way that any washing steps can be omitted. This simplifies the handling of the method and thus shortens the time required for the preparation steps. In addition, since the entire process can take place in a single reaction vessel, losses when transferring to new reaction vessels are eliminated, thus enabling more precise quantification. The present invention will now be further explained with the aid of the following examples, comparative examples, and figures. Example: Method for the specific detection of microorganisms using the example of the detection of Lactobacillus rhamnosus. A lyophilized sample to be examined was rehydrated in a suitable manner for at least 1 hour. Subsequently, a 20 µl aliquot was taken,These were placed in a sample container, and 20 µl of a lysozyme solution containing 800,000 units of lysozyme was added. The resulting mixture was mixed in a vortex mixer and then incubated for 30 minutes at 40°C without shaking to obtain a sample containing permeabilized cells. Subsequently, the sample containing permeabilized cells was placed in a reaction tube and mixed with 40 µl of a hybridization solution containing 200 ng of a nucleic acid probe specific for Lactobacillus rhamnosus and labeled with FAM as a fluorescent dye in an aqueous buffer (a solution of 0.9 M NaCl and 0.02 M Tris-HCl (pH 8.0) in a mixture of 65 wt% water and 35 wt% formamide). The fluorescently labeled nucleic acid probe was 20 nucleotides long and exhibited 100% homology to the target nucleic acid sequence. After the resulting reaction mixture was incubated for 1.5 h at 40°C without shaking,40 µl of a quencher solution containing 200 ng of a nucleic acid probe labeled with BHQ1 as a quencher was dissolved in an aqueous buffer (a solution of 0.14 M NaCl and 0.04 M Tris-HCl (pH 8.0) in water) was added to the reaction mixture. The quencher-labeled nucleic acid probe was 20 nucleotides long and showed 100% homology to the nucleic acid sequence of the fluorescently labeled nucleic acid probe. The reaction mixture obtained in this way was incubated for a further 15 minutes at 40°C and then, without further processing, introduced into a flow cytometer (model CyFlow® Cube6 from Sysmex Deutschland GmbH) and analyzed and evaluated at a flow rate of 0.5 µl / sec. Fig. 2 shows the measurement result. The fluorescence signal of the cells is plotted in the dot plot in the Y direction against the scatter, which is a measure of the size of the particle.applied in the X direction. Comparative Example 1 The above-described example was repeated, except that a fixation step, a homogenization step, and then a drying step were performed between the lysozyme treatment and contacting the sample with the hybridization solution. Specifically, 30 µl of 0.1 M CaCl2 / EtOH and 90 µl of EtOH were added to the sample containing permeabilized cells obtained by the lysozyme treatment, before the resulting mixture was incubated at 40°C for 60 minutes. Then, 30 µl of the resulting sample was added to a mixture of 5 µl of the homogenizing agent, which was obtained by mixing a 50 wt% aqueous glucose solution with glycerol in a weight ratio of 8:2. This mixture was mixed in a vortex mixer and dehydrated and dried in a heating block at 80°C for 30 minutes. The resulting sample was then subjected to the following steps: hybridization,Quenching and detection as previously described for Example 1. The results are shown in Fig. 3. Although the initial steps of fixing, drying, and homogenizing of the method known from EP 3673 075 B1 were omitted in Example 1, the same result in terms of avoiding cluster formation was achieved, as can be seen from the comparison of Figure 2 with Figure 3, as in Comparative Example 1, which included the initial steps of fixing, drying, and homogenizing described in EP 3673 075 B1. No large particles, each consisting of clusters of an indeterminate number of connected cells, are visible. Therefore, precise quantification is also possible with the significantly shortened method according to the invention without the steps of fixing, drying, and homogenizing.

Claims

Patent claims 1. A method for detecting one or more microorganisms in a sample, comprising the steps of: (a) adding an enzyme suitable for permeabilizing the one or more microorganisms to a sample containing the microorganism or the more than one microorganism and incubating the mixture thus prepared to obtain a sample with permeabilized cells; (b) contacting the sample with permeabilized cells obtained in step (a) with a solution of a fluorescently labeled nucleic acid probe specific for the microorganism to be detected to provide a first reaction mixture; (c) incubating the first reaction mixture to cause the fluorescently labeled nucleic acid probe to bind to the corresponding target nucleic acid sequence in the cells of the microorganism to be detected;(d) contacting the first reaction mixture following step (c) with a solution of a quencher-labeled nucleic acid probe to provide a second reaction mixture, wherein the quencher-labeled nucleic acid probe comprises a quencher that at least partially quenches the fluorescence of the fluorescence-labeled nucleic acid probe and has a nucleic acid sequence that is substantially complementary to the nucleic acid sequence of the fluorescence-labeled nucleic acid probe; (e) incubating the second reaction mixture to cause the molecules of the fluorescence-labeled nucleic acid probe that are not bound to the target nucleic acid sequence in the cells of the microorganism to be detected to bind to the quencher-labeled nucleic acid probe; and (f) quantitatively detecting the fluorescence emitted by the cells of the microorganism to be detected that contain the fluorescence-labeled nucleic acid probe.

2. The method according to claim 1, wherein the enzyme added in step (a) is selected from the group consisting of proteinase K, lyticase, achromopeptidase, mutanolysin, and lysozyme, and is preferably lysozyme.

3. The method according to claim 1 or 2, wherein the microorganism is a bacterium, a fungus, or a unicellular higher organism (protozoan), and is preferably a Gram-positive bacterium.The method according to claim 3, wherein the bacterium is a bacterium of the genus Acinetobacter, Akkermansia, Alicyclobacillus, Alistipes, Aquabacterium, Arcobacter, Atopobium, Bacillus, Bacteroides, Bifidobacterium, Bilophila, Campylobacter, Clostridium, Corynebacterium, Dialister, Enterobacteriaceae, Enterobacter, Enterococcus, Escherichia, Faecalibacterium, Fusobacterium, Hafnia, Helicobacter, Lactobacillus, Lactococcus, Legionella, Listeria, Megasphaera, Microthrix, Mycobacterium, Nitrobacter, Nitrosococcus, Nitrosomonas, Nitrospira, Nitrotoga, Parabacteroides, Prevotella, Propionibacterium, Roseburia, Ruminococcus, Salmonella, Shigella, Sphingobacterium, Staphylococcus, Streptococcus Tannerella, Veillonella or Vibrio.

5. The method according to any one of the preceding claims, wherein the sample containing the microorganism or the plurality of microorganisms in step (a) contains 100 to 2,000,000 units, preferably 1,000 to 1,000.000 units, and particularly preferably 400,000 to 800,000 units of enzyme are added, and the mixture thus prepared, optionally after mixing, is incubated at 30 to 45°C for 1 to 60 minutes.

6. The method according to claim 5, wherein the incubation takes place at 37 to 42°C for 10 to 50 minutes.

7. The method according to any one of the preceding claims, wherein neither in step (a) nor in step (b) nor between steps (a) and (b) is fixation of the cells and / or homogenization of the cells carried out after addition of a homogenizing agent.

8. The method according to any one of the preceding claims, wherein the target nucleic acid sequence in the cells of the microorganism to be detected is selected from the group consisting of 16S rRNA, 23S rRNA, 18S rRNA, tRNA, EF-Tu, mRNA, 16S-23S rRNA spacer, and 23S-5S rRNA spacer.

9. The method according to any one of the preceding claims, wherein the fluorescently labeled nucleic acid probe is (i) substantially identical or (ii) substantially reverse complementary to the target nucleic acid sequence in the cells of the microorganism to be detected.Method according to one of the preceding claims, wherein the fluorescently labeled nucleic acid probe is selected from a fluorescently labeled DNA probe, RNA probe, PNS probe and LNS probe.

11. The method according to any one of the preceding claims, wherein (i) the fluorescent dye of the fluorescently labeled nucleic acid probe is arranged at the 3' end or near the 3' end of the fluorescently labeled nucleic acid probe and the quencher of the quencher-labeled nucleic acid probe is arranged at the 5' end or near the 5' end of the quencher-labeled nucleic acid probe, or (ii) the fluorescent dye of the fluorescently labeled nucleic acid probe is arranged at the 5' end or near the 5' end of the fluorescently labeled nucleic acid probe and the quencher of the quencher-labeled nucleic acid probe is arranged at the 3' end or near the 3' end of the quencher-labeled nucleic acid probe.

12. The method according to any one of the preceding claims, wherein in step (b) a plurality of fluorescently labeled nucleic acid probes with different nucleic acid sequences, each specific for the microorganism to be detected, are added, and in step (d) a number of different quencher-labeled nucleic acid probes corresponding to the number of fluorescently labeled nucleic acid probes is added.

13. The method according to any one of the preceding claims, wherein the sample contains more than one microorganism and a plurality of different microorganisms are detected simultaneously. 14.Method according to one of the preceding claims, wherein the quantitative detection of the fluorescence emitted by the cells of the microorganism to be detected containing the fluorescently labeled nucleic acid probe is carried out in step (f) in a fluorescence-detecting microtiter plate reader or in an automatic cell counter, preferably in a cell drop cell counter, and particularly preferably in a flow cytometer.

15. Use of a fluorescence-detecting microtiter plate reader, a cell drop cell counter, or a flow cytometer in a method according to one of the preceding claims.

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