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
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6841—In situ hybridisation
• • 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
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

• the invention relates to an in situ hybridization arrangement for the specific detection of microorganisms, a method for the specific detection of microorganisms by in situ hybridization and a K t with which the identification and visualization of microorganisms in a sample is possible.
• PCR the polymerase chain reaction
• a specific, characteristic piece of the bacterial genome is amplified using bacteria-specific primers. If the primer finds its destination, a piece of the genetic material multiplies millions of times.
• a qualitative evaluation can take place. In the simplest case, this leads to the statement that the target sites are present in the examined sample. No further statements are permitted since the target sites can come from a living bacterium, from a dead bacterium or from naked DNA. A differentiation is not possible here.
• a further development of this technique is quantitative PCR, in which an attempt is made to establish a correlation between the amount of bacteria present and the amount of DNA obtained and amplified.
• biochemical parameters are also used for bacterial identification: For example, the creation of bacterial profiles based on quinone determinations serves to reproduce an image of the bacterial population that is as distortion-free as possible (Hiraishi, A. 1988. Respiratory quinone profiles as tools for identifying diffemet bacterial populations in activated sludge. J. Gen. Appl. Microbiol. 34: 39-56).
• this method also depends on the cultivation of individual bacteria, since the quinone profiles of the bacteria in pure culture are required to create the reference database.
• the determination of the bacterial quinone profiles does not give a real impression of the actual population relationships in the sample.
• the often large-volume and bulky antibody-fluorescent molecule complex causes problems when entering the target cells.
• the detection is often too specific.
• Antibodies which are expensive to produce, specifically often only detect a specific strain of bacteria, but leave other strains of the same type of bacteria undetected. Often, however, it is not necessary to have a strain-specific detection of the bacteria, but rather to detect a type of bacteria or an entire group of bacteria.
• antibody production is a relatively lengthy and expensive process.
• the fluorescence-in-situ method offers a unique approach to combine the specificity of molecular biological methods such as PCR with the possibility of bacterial visualization, as made possible by the antibody method.
• Hybridization FISH; Amann, RI, W. Ludwig, and K.-H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbial. Rev. 59: 143-169).
• Bacterial species, genera or groups can be visualized and identified directly in the sample in a highly specific manner. This method is the only approach that provides a distortion-free representation of the actual in situ conditions of the biocenosis. Even bacteria that have not yet been cultivated and therefore not described can be identified and also visualized directly in the sample.
• the FISH technique is based on the fact that there are certain molecules in bacterial cells that, due to their vital function, have undergone only little mutation in the course of evolution: the 16S and the 23S ribosomal ribonucleic acid (rRNA). Both are components of the ribosomes, the sites of protein biosynthesis, and due to their ubiquitous distribution, their size, and their structural and functional constancy, they can serve as phylogenetic markers (Woese, CR 1987. Bacterial evolution. Microbiol. Rev. 51: 221-271) , Based on a comparative sequence analysis, phylogenetic relationships can be established based solely on this data. To do this, this sequence data must be aligned.
• Figure 1 shows the secondary structure model of a 16S rRNA.
• phylogenetic calculations can be carried out.
• the use of the latest computer technology makes it possible to carry out large-scale calculations quickly and effectively, and to create large databases that contain the alignment sequences of the 16S rRNA and 23S rRNA. Thanks to the quick access to this data material newly obtained sequences can be analyzed phylogenetically in a short time.
• These rRNA databases can be used to construct specific gene probes. Here, all available rRNA sequences are compared with each other and probes designed for specific sequence sites that specifically record a bacterial species, genus or group.
• FISH fluorescence in situ hybridization
• these gene probes which are complementary to a specific region on the ribosomal target sequence, are smuggled into the cell.
• the gene probes are usually small, 16-20 base long, single-stranded deoxyribonucleic acid pieces and are directed against a target region, which is typical for a type or group of bacteria. If the fluorescence-labeled gene probe finds its target sequence in a bacterial cell, it binds to it and the cells can be detected in the fluorescence microscope due to their fluorescence.
• Figure 2 illustrates the process of in situ hybridization.
• Bacterial population of a sample can be made visible, the FISH technique allows detection of up to 100% of the total bacterial population in many samples.
• the active portion of a population can be determined by the ratio of the probe directed against all bacteria and an unspecific cell staining.
• the bacteria are made visible directly at the place where they act (in situ). Possible interactions between different bacterial populations can thus be recognized and analyzed.
• the detection of bacteria using the FISH technique is much faster than using cultivation. If the identification of bacteria by cultivation often takes several days, only a few hours pass from sampling to bacterial identification even at the species level using the FISH technique.
• the specificity of gene probes can be chosen freely as desired. Individual species can be detected with a probe just as easily as entire genera or groups of bacteria.
• bacterial species or entire bacterial populations can be precisely quantified directly in the sample.
• the detour via cultivation and the associated insufficient quantification need not be taken.
• a bacterium occurring in a sample is examined for its taxonomy, the top-to-bottom approach is used.
• the bacterial sample is analyzed at the beginning of the investigation with gene probes that are as broad as possible, ie, not very specific and only cover entire groups of bacteria. A gradual increase in the specificity of the probes used finally allows the identification of the unknown bacterium.
• the FISH analysis is always carried out on a slide, since during the evaluation the bacteria are visualized, ie made visible, by irradiation with high-energy light.
• the composition of the individual solutions such as hybridization buffer or hybridization solution and wash buffer or wash solution is well known to the person skilled in the art and is e.g. in Snaidr et. al, 1997 (J. Snaidr, R. Amann, I. Huber, W. Ludwig, and K.-H. Schleifer. 1997. Phylogenetic analysis and in situ identification of bacteria in activated sludge. Appl. Env. Microb. 63: 7, 2884-2896).
• Carrying out the FISH for the analysis of microorganisms on a slide usually comprises the steps shown below.
• Ethanol series The slide is immersed in a 50%, 70% and 100% ethanol solution one after the other. 6. Apply a hybridization solution to the recess containing the microorganisms. 7. Apply a probe solution to the same recess.
• the slide is placed horizontally on the pulp in the humid chamber.
• the wet chamber is placed in an incubation oven and incubated for 1-2 hours.
• the moist chamber is opened, the slide is removed and briefly rinsed with distilled water. 12. The damp chamber is discarded.
• a wash buffer solution is introduced into a new plastic tube.
• the rinsed slide is inserted into the plastic tube filled with the wash solution.
• the slide is incubated in the plastic tube in an incubation oven for 10-30 minutes.
• the sample can be fixed differently efficiently. In the subsequent hybridization process, this leads to a differently efficient penetration of the gene probes into the cells and therefore to different brightnesses when the cells are detected under the epifluorescence microscope. However, the cause correlates with the brightness with the ribosome content of the cells. Therefore e.g. in the FISH analysis, the intensity of the fluorescence is a measure of whether the cells were in a good or poor growth state at the time of sampling. This statement is important for an overall assessment of the microbial state of a sample, especially in medical microbiology, but also in food or environmental microbiology. Consequently, differently efficient fixation of the sample to be examined leads to falsified statements about the state of growth and thus about the overall microbial state of a sample.
• the slide must first be rinsed during the washing process and then transferred to another container. Due to this relatively lengthy process, non-specific binding of nucleic acid probe molecules to the cells can take place due to the reduced hybridization temperatures.
• the slide is preferably provided with cutouts (9) into which the sample to be examined and, if appropriate, negative or positive samples can be applied separately from one another.
• the cutouts on the slide are particularly preferably only adjacent in one dimension to further cutouts, e.g. arranged in a row, the recesses can also be offset within the row.
• the cover comprises the fastening means (5) for the slide.
• the cover particularly preferably comprises, as fastening means for the specimen slide, a slot (5) into which one end (7) of the specimen slide can be inserted.
• the slide can of course also engage in fasteners (5) which are part of the container and e.g. in the form of a slit in the bottom of the container, the bottom being in this case opposite the opening of the container.
• the cover is provided with a component (8) which is stable
• the position of the lid with the attached slide enables it to be separated from the arrangement without a lid when the slide is in a horizontal position.
• the lid is particularly preferably designed such that it is possible to stand the lid with the attached slide separately from the arrangement without a lid when the slide is in a horizontal, vertical or lateral position.
• the horizontal position of the slide is understood to mean that the position of the slide is such that it is possible to apply samples or probes to the slide without flowing.
• the lateral position of the slide is understood to mean a position rotated by 90 ° with respect to the horizontal position, the slide being rotated by 90 ° in such a way that drops can run off the slide without possibly running into another recess on the slide , provided that the cutouts are only adjacent to one another in one dimension.
• Invention understood a position rotated by 90 ° with respect to both the horizontal and the lateral position.
• lateral guide rails (11) for the hybridization solution carrier (3) are attached to the container (1) for stabilizing the hybridization solution carrier (3) in the container (1) or such guide rails (11) are part of the Container (1).
• the materials for all components of the arrangement, except for the slide preferably comprise plastics, particularly preferably polyethylene and / or polypropylene. Furthermore, the materials for the above-mentioned components of the arrangement can also comprise metals.
• a method for the specific detection of microorganisms by in situ hybridization comprises the following steps:
• steps a) to c) and optionally d) are carried out with the in situ hybridization arrangement according to the invention.
• fixation and / or drying steps are preferably carried out on the object carrier.
• a mixture of a hybridization solution and a nucleic acid probe molecule solution is applied to the slide in step b).
• the hybridization solution which is required for the moist chamber can be introduced into the arrangement according to the invention in the method according to the invention via cushions located in the carrier for the hybridization solution and soaked with hybridization solution.
• the nucleic acid probe molecule used in step b) is preferably complementary to a chromosomal or episomal DNA, an mRNA or rRNA of a microorganism to be detected.
• the nucleic acid probe molecule is covalently linked to a detectable marker.
• This detectable marker is preferably selected from the group of the following markers:
• Fluorescent marker Fluorescent marker, chemiluminescent marker, radioactive marker, - enzymatically active group,
• the sample is an environmental sample and is taken from water, soil or air; or a food sample, in particular a sample from milk or milk products, drinking water, beverages, baked goods or meat products; or a medical sample, in particular a sample obtained from tissue, secretions or stool; or a sample from waste water, in particular a sample obtained from activated sludge, digested sludge or anaerobic sludge; or a sample obtained from a biofilm, in particular a sample in which the biofilm is obtained from an industrial plant, in which wastewater treatment was formed or is a natural biofilm; or a sample taken from a pharmaceutical or cosmetic product.
• kits for the specific detection of microorganisms by in situ hybridization which contains at least one nucleic acid probe molecule for the specific detection of a microorganism; at least one hybridization solution; if applicable
• the nucleic acid probe molecule in the kit according to the invention is preferably complementary to a cliromosomal or episomal DNA, an mRNA or rRNA of a microorganism to be detected.
• the nucleic acid probe molecule in the kit according to the invention is preferably covalently linked to a detectable marker.
• the detectable marker is particularly preferably selected from the group consisting of fluorescent markers, chemiluminescent markers, radioactive markers, enzymatically active groups, haptens and nucleic acids detectable by hybridization.
• Another object of the present invention is the use of the in situ hybridization arrangement according to the invention for the specific detection of microorganisms by in situ hybridization.
• the present invention furthermore relates to the use of the kit according to the invention in the method according to the invention.
• the arrangement according to the invention comprises a container provided with at least one opening, which is also referred to below as a chamber; a carrier for the hybridization solution, in particular a tub, which can be completely introduced into the container or the chamber or is part of this container; as well as a mounting option for a slide.
• the arrangement further comprises a slide which can be inserted into the chamber for in situ hybridization.
• the arrangement according to the invention preferably further comprises a lid which is suitable for sealing off, in particular for watertight and / or airtight, opening of the container.
• the object holder is preferably attached to this cover, particularly preferably inserted.
• the object holder is firmly and securely in the lid, but can be pulled out of the lid again for final evaluation by hand and without great effort.
• the fixing or fastening of the lid makes it possible to carry out the washing process safely even when the arrangement according to the invention is in a vertical position.
• the cover is provided with a component or the cover comprises a component which enables the cover with a fixed slide to stand stable and separate from the arrangement with the slide in a horizontal or vertical position.
• This stability of the lid which was achieved according to the invention, for example by attaching a support leg, makes it possible to keep the slide in a horizontal position during all reactions taking place.
• the container and / or the lid are constructed in such a way that, when the lid is closed, the arrangement is stable when the slide is in a horizontal position.
• the horizontal storage of the slide, in particular during steps a) and b) of the method, is thus ensured by the construction of the contact surfaces of the components of the in situ hybridization arrangement according to the invention.
• hybridization solution into the reactor using disposable cushions that are located in the tub.
• the disposable pillows are sealed with a freshness seal, which is torn off as soon as the tub is in the chamber, and the hybridization solution can then evaporate in the chamber.
• Both the incubation and the washing process preferably take place in the in situ hybridization arrangement according to the invention.
• microorganisms are preferably not first fixed in a reaction vessel and then immobilized, as is usually the case, but rather the fixing and / or optionally the drying take place directly on the slide.
• fixation on the slide avoids cell loss and is much easier to use and less complicated to carry out.
• fixation on the slide enables the fixation step and the dehydration series to be combined in one work step.
• the hybridization and addition of the nucleic acid probe molecules is preferably not carried out by, as is customary, first a defined amount of a hybridization solution and then a defined amount of one
• drip containers saves the use of expensive pipettes and also makes handling and dosing easier.
• the slide which is preferably attached to the lid, is then inserted into the chamber (see Figure 7).
• Lateral guide lines which are preferably installed in the chamber, ensure problem-free insertion and further fixation or stabilization of the slide in the chamber (see Figure 8).
• the subsequent incubation is preferably carried out in the horizontal position of the slide.
• the subsequent washing of the slide is preferably carried out according to the invention in the chamber and particularly preferably in the vertical position of the slide.
• the lid is preferably constructed so that it can be placed on the side. This is particularly advantageous because the drops can run off the slide without running into another recess in the slide.
• FIG. 9 shows the entire structure of a preferred embodiment of the in situ hybridization arrangement according to the invention, comprising the lid, chamber, tub and supplied slide.
• Figures 10 to 12 show the dimensions of the lid, pan and chamber.
• the envelope can be a lipid envelope that surrounds a virus, around the cell wall of bacteria or around the cell membrane of one cell Eukaryotes. Ethanol is usually used for fixation. If these measures cannot be used to make the cell wall penetrable by nucleic acid probes, the person skilled in the art is sufficiently aware of further measures which lead to the same result. These include, for example, a low-percentage paraformaldehyde solution or a dilute formaldehyde solution, methanol, mixtures of alcohols, enzymatic treatments or the like.
• the nucleic acid probe in the sense of the invention can be a DNA or RNA probe which will generally comprise between 12 and 1000 nucleotides, preferably between 12 and 50, particularly preferably between 17 and 25 nucleotides.
• the nucleic acid probes are selected on the basis of whether a complementary sequence is present in the microorganism to be detected. By selecting a defined sequence, a bacterial species, a bacterial genus or an entire bacterial group can be detected. Complementarity must be given for a probe of 12 to 15 nucleotides in length over 100% of the sequence. With oligonucleotides with more than 15 nucleotides, one or more mismatching sites are allowed. Compliance with stringent hybridization conditions ensures that the nucleic acid probe molecule actually hybridizes with the target sequence. Moderate conditions in the sense of the invention are e.g. 0% formamide in one
• Hybridization solution as described in Example 1.
• Stringent conditions in the sense of the invention are, for example, 20-80% formamide in the hybridization solution.
• the nucleic acid probe should be present in the hybridization solution in an amount of from 15 ng to 1000 ng, this amount being contained in a volume of hybridization solution between 8 ⁇ l and 100 ⁇ l, preferably in 40 ⁇ l.
• the probe concentration is particularly preferably 111 ng / 40 ⁇ l hybridization solution.
• the selection of the respective nucleic acid probes depends on the microorganism to be detected.
• the nucleic acid probe can be complementary to a cliromosomal or episomal DNA, but also to an mRNA or rRNA of the microorganism to be detected. It is advantageous to choose a nucleic acid probe that is complementary to an area that is present in the number of copies of more than 1 in the microorganism to be detected.
• the sequence to be detected is preferably 500-100,000 times per cell, particularly preferably 1000-50,000 times.
• the rRNA is preferably used as the target site, since the ribosomes in the cell, as sites for protein biosynthesis, are present thousands of times in each active cell.
• the nucleic acid probe is incubated with the microorganism fixed in the abovementioned sense, so as to prevent the To allow nucleic acid probe molecules in the microorganism and the hybridization of nucleic acid probe molecules with the nucleic acids of the microorganism. Then the non-hybridized
• the microorganisms are bacteria which occur in the waste water from waste water treatment plants.
• Another area of application for the present method is the investigation of waste water, e.g. Activated sludge, digested sludge or anaerobic sludge.
• waste water e.g. Activated sludge, digested sludge or anaerobic sludge.
• it is suitable to analyze biofilms in industrial plants, as well as to investigate naturally forming biofilms or biofilms that form during waste water purification.
• Examination of pharmaceutical and cosmetic products e.g. Ointments, creams, tinctures, juices etc. are possible with the method according to the invention.
• a kit for carrying out the method for the detection of microorganisms in a sample is provided in a further aspect of the present invention.
• the content of such a kit depends essentially on the nature of the microorganism to be detected. As the most important constituent, it comprises one or more nucleic acid probes specific for the microorganism to be detected in each case and preferably further nucleic acid probes with which a negative or positive control can be carried out. In addition, it preferably comprises a hybridization solution and a washing solution. The choice of hybridization solution depends primarily on the length of the nucleic acid probes used.
• the kit according to the invention comprises at least one nucleic acid probe molecule for the specific detection of a microorganism; at least one hybridization solution; optionally a nucleic acid probe molecule for performing a negative control; optionally a nucleic acid probe molecule for performing a positive control; optionally a washing solution; optionally a fixing solution, optionally an antifading reagent; and the in situ hybridization arrangement according to the invention, the following steps being able to be carried out in the arrangement or in parts of the arrangement:
• Illustration 1
• Figure 3 Top view of the components of a special embodiment of the in situ hybridization arrangement according to the invention: container (1), tub (3), object holder (4), lid (6) with support leg (8) (from left to right)

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• 2000
  • 2000-12-11 DE DE10061655A patent/DE10061655A1/de not_active Ceased
• 2001
  • 2001-12-11 AT AT01991823T patent/ATE298786T1/de active
  • 2001-12-11 EP EP01991823A patent/EP1341897B1/de not_active Expired - Lifetime
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  • 2001-12-11 JP JP2002550112A patent/JP2004523223A/ja active Pending
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