EP1397518A2 - Procedes pour la mise en evidence rapide specifique de bacteries concernant l'eau potable - Google Patents

Procedes pour la mise en evidence rapide specifique de bacteries concernant l'eau potable

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Publication number
EP1397518A2
EP1397518A2 EP02754712A EP02754712A EP1397518A2 EP 1397518 A2 EP1397518 A2 EP 1397518A2 EP 02754712 A EP02754712 A EP 02754712A EP 02754712 A EP02754712 A EP 02754712A EP 1397518 A2 EP1397518 A2 EP 1397518A2
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EP
European Patent Office
Prior art keywords
cac
tcc
tac
cct
ccc
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
EP02754712A
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German (de)
English (en)
Inventor
Claudia Beimfohr
Jiri Snaidr
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vermicon AG
Original Assignee
Vermicon AG
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Publication date
Priority claimed from DE10160666A external-priority patent/DE10160666A1/de
Application filed by Vermicon AG filed Critical Vermicon AG
Publication of EP1397518A2 publication Critical patent/EP1397518A2/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the invention relates to a method for the detection of bacteria in drinking and surface water, in particular a method for the simultaneous specific detection of bacteria of the genus Legionella and the species Legionella pneumophila by in situ hybridization and a method for the specific detection of faecal streptococci by means of in situ hybridization as well as a method for the simultaneous specific detection of coliform bacteria and bacteria of the species Escherichia coli as well as corresponding oligonucleotide probes and kits with which the methods according to the invention can be carried out.
  • Legionella are gram-negative, non-spore-forming rod-shaped bacteria with a length of 0.5 - 20 ⁇ M and a diameter of 0.3 - 0.9 ⁇ m. They are motile due to their polar flagellation with one to three flagella. Legionella are ubiquitous residents of moist soils and all non-marine aquatic habitats. Ideal conditions for their propagation exist at temperatures between 25 ° C and 55 ° C. As a result, they can also be found in appropriate human-made habitats, such as Hot and cold water systems, cooling towers of air conditioning systems and water evaporators. As intracellular parasites of amoebas and ciliates, they can also have unfavorable living conditions such as extreme temperatures and chlorination of water survive.
  • Legionella are pathogens; they cause an acute bacterial pneumonia with an optionally lethal course in humans, which is commonly known as "legionnaires'disease". This name comes from the examination of a conspicuous accumulation of pneumonia cases (189 diseases with 29 deaths) among the approximately 3000 delegates of the annual meeting the "Pennsylvania Division of the American Legion” in July 1976. The investigation led to the isolation of a previously unknown bacterium, L. pneumophila (McDade et al., 1977. Legionnaires' disease: isolation of a bacterium and demonstration of its role in other respiratory disease, N. Engl. J. Med.
  • E. coli as a so-called index organism, is a potential one in the analysis of food, drinking and surface water
  • the coliform bacteria represent an extremely heterogeneous group of bacteria.
  • the genera Escherichia, Enterobacter, Klebsiella and Citrobacter belong to the group of coliforms.
  • the affiliation of bacteria to this group is therefore not defined by taxonomic characteristics, but by the behavior of the bacteria in corresponding detection methods.
  • all gram-negative, aerobic, facultative anaerobic, rods are assigned to the coliforms, which ferment lactose within 48 hours at temperatures between 30 ° C and 37 ° C with the formation of gas and acid.
  • Coliforms that are able to ferment lactose at higher temperatures are also referred to as fecal coliforms, thermotrophic coliforms or presumptive E. coli.
  • E. coli is by no means only used as an index bacterium in microbiological analyzes, but a number of pathogenic strains of this organism are known. These enterovirulent strains are divided into different subgroups (enterotoxin formers, enteropathogens, enterohemorrhagic, enteroinvasive, enteroadherent E. coli). All bacteria this Subgroups trigger diarrheal diseases of various degrees of severity up to life-threatening.
  • E. coli and coliforms are detected by cultivation, which leads to a result within two to four days through several successive cultivation steps on different media.
  • cultivation on Fluorocult LMX broth produces a result after only 30 hours.
  • the membrane filter method for the detection of E. coli (the detection of coliforms is not possible in this way) still takes 22 to 32 hours to get the result.
  • false positive results are not uncommon, since indole-positive Klebsiella oxytoca and Providencia axes are not uncommon in fresh meat.
  • Faecal streptococci are further indicators of faecal contamination of drinking and surface water. Similar to the coliforms, these are also a non-uniform group. Faecal streptococci are phylogenetically assigned to the genera Streptococcus and Enterococcus. These are Gram-positive bacteria, which typically form diplococci or short chains and are common in the intestinal tract of warm-blooded animals.
  • a limit value for faecal streptococci is also laid down in the German regulation for drinking water and water for food businesses, which was valid in 2001. No faecal streptococci should be detectable in 100 ml of drinking water, otherwise the examined water is no longer of drinking water quality.
  • the detection methods recommended in the Drinking Water Ordinance are based on direct cultivation of the water sample or on membrane filtration and subsequent introduction of the filter in 50 ml azide-glucose broth.
  • the cultivation should take place for at least 24 h, if the result is negative for 48 h, at 36 ° C. There is no turbidity or sediment formation in the broth even after 48 hours Ascertainable, the absence of faecal streptococci in the examined sample is considered to be proven.
  • the culture is smeared on enterococcal selective agar according to Slanetz-Barthley and incubated again at 36 ° C. for 24 h. If red-brown or pink colonies are formed, these are examined in more detail.
  • fecal streptococci After transfer to a suitable liquid medium and cultivation for 24 h at 36 ° C, fecal streptococci are considered to have been proven if they multiply in nutrient broth at pH 9.6 and multiply in 6.5% NaCl broth as well as in the case of asculin degradation.
  • the asculin breakdown is checked by adding freshly prepared 7% aqueous solution of iron (II) chloride to the asculin broth. A brown-black color develops when broken down.
  • a Gram stain is often used to distinguish the bacteria from Gram-negative cocci and a catalase test to distinguish between staphylococci. Faecal streptococci are Gram-positive and Catalase-negative. The traditional proof is thus a lengthy (48 - 100 h) and, if suspected, extremely complex procedure.
  • nucleic acid-based detection methods are therefore available.
  • PCR the polymerase chain reaction
  • a characteristic piece of the respective bacterial genome is amplified with 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 for the primers used were present in the examined sample. No further statements are possible; these target sites can originate from a living bacterium as well as from a dead bacterium or from naked DNA. A Differentiation is not possible here. This is particularly problematic when examining samples for ubiquitous germs such as E. coli and coliforms.
  • PCR reaction Since the PCR reaction is positive even in the presence of a dead bacterium or naked DNA, false positive results often occur.
  • quantitative PCR in which an attempt is made to establish a correlation between the amount of bacteria present and the amount of amplified DNA.
  • the advantages of PCR lie in its high specificity, ease of use and in the short amount of time. Significant disadvantages are their high susceptibility to contamination and thus false positive results as well as the aforementioned lack of ability to differentiate between living and dead cells or naked DNA.
  • FISH fluorescence in situ hybridization
  • 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 specific markers (Woese, CR, 1987. Bacterial evolution. Microbiol. Rev. 51, p. 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. Alignment, which is based on knowledge of the Supporting the secondary structure and tertiary structure of these macromolecules, the homologous positions of the ribosomal nucleic acids are brought into harmony with one another.
  • 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. By quickly accessing this data material, newly obtained sequences can be analyzed phylogenetically in a short time.
  • These rRNA databases can be used to construct species- and genus-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.
  • these gene probes which are complementary to a specific region on the ribosomal target sequence, are introduced 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.
  • 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.
  • this is one of the disadvantages of the classic FISH analysis: since only relatively small volumes can naturally be analyzed on a slide, the sensitivity of the method can be unsatisfactory and not sufficient for a reliable analysis.
  • the present invention therefore combines the advantages of classic FISH analysis with those of cultivation.
  • a comparatively short cultivation step ensures that the bacteria to be detected are present in sufficient numbers before the bacteria are detected using specific FISH.
  • “cultivation” means the multiplication of the bacteria contained in the sample in a suitable cultivation medium.
  • the methods suitable for this are well known to the person skilled in the art.
  • fixing the bacteria is understood to mean a treatment with which the bacterial envelope is made permeable to nucleic acid probes. Ethanol is usually used for fixing. If the cell wall cannot be penetrated by the nucleic acid probes with these measures, the person skilled in the art will know Sufficient additional measures are known that lead to the same result. These include, for example, methanol, Mixtures of alcohols, a low paraformaldehyde solution or a dilute formaldehyde solution, enzymatic treatments or the like.
  • the “fixed” bacteria are incubated with fluorescence-labeled nucleic acid probes for the “hybridization”.
  • These nucleic acid probes which consist of an oligonucleotide and a marker attached to it, can then penetrate the cell envelope and bind to the target sequence corresponding to the nucleic acid probe inside the cell.
  • the bond is to be understood as the formation of hydrogen bonds between complementary pieces of nucleic acid.
  • the nucleic acid probe can be complementary to a chromosomal 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 1,000-50,000 times.
  • the rRNA is preferably used as the target site, since the ribosomes in the cell as sites of protein biosynthesis are present thousands of times in each active cell.
  • the nucleic acid probe in the sense of the invention can be a DNA or RNA probe which is generally between 12 and 1000 nucleotides, preferably between 12 and 500, more preferably between 12 and 200, particularly preferably between 12 and 50 and between 15 and 40, and most 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 recorded. Complementarity should be at a probe of 15 Nucleotides must be given over 100% of the sequence. In the case of oligonucleotides with more than 15 nucleotides, one or more mismatching sites are permitted.
  • stringent hybridization conditions ensures that the nucleic acid molecule actually hybridizes with the target sequence.
  • stringent conditions in the sense of the invention mean, for example, 20-80% formamide in the hybridization buffer.
  • “specifically hybridize” means that a molecule binds preferentially to a certain nucleotide sequence under stringent conditions if this sequence is present in a complex mixture of (for example total) DNA or RNA.
  • stringent conditions stands for conditions under which a probe will preferentially hybridize to its target sequence and to a significantly lesser extent or not at all to other sequences. Stringent conditions are in part sequence-dependent and will be different under different circumstances. Longer Sequences hybridize specifically at higher temperatures.
  • stringent conditions are selected so that the temperature is about 5 ° C below the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probe molecules complementary to the target sequence hybridize to the target sequence in an equilibrium state (since the target sequences are usually in excess, 50% of the probes are occupied in equilibrium) .
  • Typical stringent e Conditions where the salt concentration is at least about 0.01 to 1.0 M sodium ion concentration (or other salt) at a pH between 7.0 and 8.3 and the temperature is at least about 30 ° C for short probes (e.g. 10-50 Nucleotides).
  • stringent conditions can be achieved by adding destabilizing agents such as formamide.
  • the inventive method has
  • Nuclein probe molecules the following lengths and sequences (all sequences are given in the 5 '-3' direction).
  • the invention also relates to modifications of the above oligonucleotide sequences which, despite the deviations in the sequence and / or length, show a specific hybridization with target nucleic acid sequences of the respective bacterium and are therefore suitable for use in a method according to the invention.
  • This includes in particular a) nucleic acid molecules which (i) with one of the above oligonucleotide sequences (SEQ ID No. 1 to SEQ ID No.
  • Nucleic acid molecules or one of the probes SEQ ID No. 1 to SEQ ID No. 47 are complementary or hybridize specifically with them under stringent conditions.
  • the degree of sequence identity of a nucleic acid molecule with the probes SEQ ID No. 1 to SEQ JD No. 47 can be determined using conventional algorithms.
  • the program for determining sequence identity is suitable here, which is available at http://www.ncbi.nlm.nih.gov/BLAST (on this page e.g. the link "Standard nucleotide-nucleotide BLAST [blastn]").
  • hybridizing can be synonymous with complementary.
  • those oligonucleotides are also included which are those with the (theoretical) counter strand of an inventive one
  • Oligonucleotide including the modifications of SEQ ID No. 1 to 47, hybridize.
  • the nucleic acid probe molecules according to the invention can be used with various hybridization solutions as part of the detection method.
  • Nucleic acid probe molecule also actually hybridizes to the target sequence.
  • Moderate conditions in the sense of the invention are, for example, 0% formamide in one Hybridization buffer as described below.
  • Stringent conditions in the sense of the invention are, for example, 20-80% formamide in the hybridization buffer.
  • a typical hybridization solution contains 0% - 80% formamide, preferably 20% - 60% formamide, particularly preferably 35% formamide. It also has a salt concentration of 0.1 mol / 1 - 1.5 mol 1, preferably 0.5 mol 1 - 1.0 mol / 1, more preferably 0.7 mol / 1 - 0.9 mol / 1, particularly preferably from 0.9 mol 1, the salt preferably being sodium chloride.
  • the hybridization solution usually comprises a detergent, such as e.g.
  • SDS Sodium dodecyl sulfate
  • Hybridization solution various compounds such as Tris-HCl, sodium citrate, PIPES or HEPES can be used, which are usually used in concentrations of 0.01-0.1 mol / 1, preferably from 0.01 to 0.08 mol / 1, in a pH range of 6.0-9.0, preferably 7.0 to 8.0.
  • the particularly preferred embodiment of the hybridization solution according to the invention contains 0.02 mol / 1 Tris-HCl, pH 8.0.
  • a typical hybridization solution contains 0% -80% formamide, preferably 20% -60% formamide, particularly preferably 35% formamide. It also has a salt concentration of 0.1 mol / 1 - 1.5 mol / 1, preferably 0.5 mol / 1 - 1.0 mol 1, preferably 0.7 mol / 1 - 0.9 mol / 1 , particularly preferably of 0.9 mol / 1, the salt preferably being sodium chloride.
  • the hybridization solution usually comprises a detergent, such as sodium doo decyl sulfate (SDS), in a concentration of 0.001% - 0.2%, preferably in a concentration of 0.005 - 0.05%, more preferably 0.01 - 0.03%, particularly preferably in a concentration of 0.01%.
  • a detergent such as sodium doo decyl sulfate (SDS)
  • SDS sodium doo decyl sulfate
  • Various compounds such as Tris-HCl, sodium citrate, PTPES or HEPES can be used to buffer the hybridization solution, which are usually used in concentrations of 0.01-0.1 mol / 1, preferably from 0.01 to 0.08 mol 1 , in a pH range of 6.0-9.0, preferably 7.0 to 8.0.
  • the particularly preferred embodiment of the hybridization solution according to the invention contains 0.02 mol / l Tris-HCl, pH 8.0.
  • a typical hybridization solution contains 0% -80% formamide, preferably 20% -60% formamide, particularly preferably 50% formamide. It also has a salt concentration of 0.1 mol / l - 1.5 mol / l, preferably 0.7 mol / l - 0.9 mol / l, particularly preferably 0.9 mol l, which is Salt is preferably sodium chloride. Furthermore, the hybridization solution usually comprises a detergent, such as e.g.
  • SDS Sodium dodecyl sulfate
  • Hybridization solution various compounds such as Tris-HCl, sodium citrate, PIPES or HEPES can be used, which are usually used in concentrations of 0.01-0.1 mol / l, preferably from 0.01 to 0.08 mol / l, in a pH range of 6.0-9.0, preferably 7.0 to 8.0.
  • the particularly preferred embodiment of the hybridization solution according to the invention contains 0.02 mol / l Tris-HCl, pH 8.0.
  • the concentration of the probe can vary greatly depending on the marking and the number of target structures to be expected. To enable fast and efficient hybridization, the amount of probes should be the number of
  • the amount of probe should therefore be in a range between 0.5 ng / ⁇ l and 500 ng / ⁇ l, preferably between 1.0 ng / ⁇ l and 100 ng / ⁇ l and particularly preferably 1.0–50 ng / ⁇ l.
  • the preferred concentration in the context of the method according to the invention is 1-10 ng of each nucleic acid molecule used per ⁇ l hybridization solution.
  • the volume of the hybridization solution used should be between 8 ⁇ l and 100 ml, in a particularly preferred embodiment of the method according to the invention it is 40 ⁇ l.
  • the duration of the hybridization is usually between 10 minutes and 12 hours; hybridization is preferably carried out for about 1.5 hours.
  • Hybridization temperature is preferably between 44 ° C and 48 ° C, particularly preferably 46 ° C, the parameter of the hybridization temperature, as well as the concentration of salts and detergents in the hybridization solution depending on the nucleic acid probes, in particular their lengths and the Degree of complementarity to the target sequence in the cell to be detected can be optimized.
  • the person skilled in the art is familiar with the relevant calculations here.
  • This washing solution can, if desired, 0.001-0.1%) of a detergent such as SDS, a concentration of 0.01% being preferred, and Tris-HCl in a concentration of 0.001-0.1 mol / l, preferably 0 01-0.05 mol / l, particularly preferably 0.02 mol / l, the pH of Tris-HCl in the range from 6.0 to 9.0, preferably from 7.0 to 8.0, is particularly preferably 8.0.
  • a detergent may be included, but is not essential.
  • the washing solution usually also contains NaCl, the concentration depending on the stringency required being from 0.003 mol / l to 0.9 mol / l, preferably from 0.01 mol / l to 0.9 mol / l.
  • a NaCl concentration of 0.07 mol / l (method for simultaneous specific detection of bacteria of the genus Legionella of the species L. pneumophila) or 0.07 mol / l (method for specific detection of faecal streptococci) or of 0.018 mol / l (method for the simultaneous specific detection of coliform bacteria and bacteria of the species E. coli).
  • the washing solution can contain EDTA in a concentration of up to 0.01 mol / l, the concentration preferably being 0.005 mol / l. Furthermore, the washing solution can also contain preservatives known to the person skilled in the art in suitable amounts.
  • buffer solutions are used in the washing step, which in principle can look very similar to hybridization buffers (buffered sodium chloride solution), except that the washing step is carried out in a buffer with a lower salt concentration or at a higher temperature.
  • Td 81.5 + 16.6 lg [Na +] + 0.4 x (% GC) - 820 / n - 0.5 X (% FA)
  • the “washing off” of the unbound nucleic acid probe molecules usually takes place at a temperature in the range from 44 ° C. to 52 ° C., preferably from 44 ° C. to 50 ° C. and particularly preferably at 46 ° C. for a period of 10-40 minutes, preferably for 15 minutes.
  • the nucleic acid probe molecules according to the invention are used in the so-called Fast-FISH method for the specific detection of the specified target organisms.
  • the Fast FISH method is known to the person skilled in the art and is described, for example, in German patent application DE 199 36 875.9 and international application WO 99/18234. Reference is hereby expressly made to the disclosure contained in these documents for carrying out the detection methods described there.
  • the specifically hybridized nucleic acid probe molecules can then be detected in the respective cells. The prerequisite for this is that the nucleic acid probe molecule can be detected, for example by the nucleic acid probe molecule being linked to a marker by covalent binding.
  • fluorescent groups such as CY2 (available from Amersham Life Sciences, Inc., Arlington Heights, USA), CY3 (also available from Amersham Life Sciences), CY5 (also available from Amersham Life Sciences), FITC (Molecular Probes Inc., Eugene, USA), FLUOS (available from Röche Diagnostics GmbH, Mannheim, Germany), TRITC
  • chromogens are known for each of these enzymes, which can be converted instead of the natural substrate and can be converted into either colored or fluorescent products. Examples of such chromogens are given in the table below:
  • nucleic acid probe molecules in such a way that a further nucleic acid sequence suitable for hybridization is present at their 5 'or 3' end.
  • This nucleic acid sequence in turn comprises approximately 15 to 1,000, preferably 15-50 nucleotides.
  • This second nucleic acid region can in turn be recognized by a nucleic acid probe molecule which can be detected by one of the means mentioned above.
  • Another possibility is to couple the detectable nucleic acid probe molecules with a hapten, which can then be brought into contact with an antibody recognizing the hapten.
  • Digoxigenin can be cited as an example of such a hapten. The skilled worker is also well known about the examples given.
  • the final evaluation is possible depending on the type of marking of the probe used with a light microscope, epifluorescence microscope, chemiluminometer, fluorometer etc.
  • Another advantage is the simultaneous detection of bacteria of the genus Legionella and the species Legionella pneumophila. So far, only bacteria of the species Legionella pneumophila can be detected with more or less great reliability using methods that have been familiar to date. Epidemiological studies have shown, however, that besides Legionella pneumophila also others Species of the genus Legionella that can cause dangerous Legionnaires' disease, eg Legionella micdadei. The sole detection of Legionella pneumophila must therefore, according to the current state of knowledge, no longer be considered sufficient.
  • Another advantage is the ability to differentiate between bacteria of the genus Legionella and those of the species Legionella pneumophila. This is easily and reliably possible through the use of differently labeled nucleic acid probe molecules.
  • nucleic acid probe molecules used can be used to specifically detect and visualize all species of the genus Legionella, but also highly specifically only the species L. pneumophila. All types of heterogeneous groups of faecal streptococci and coliforms are detected just as reliably, as are all subgroups of the species E. coli. By visualizing the bacteria, simultaneous visual control can take place. False positive results are therefore excluded.
  • the method can be used to easily test large quantities of samples for the presence of the bacteria mentioned.
  • environmental samples can be examined for the presence of Legionella.
  • these samples can be taken, for example, from water or from the ground.
  • the method according to the invention can also be used to examine medical samples. It is suitable, among other things, for the examination of samples from sputum, bronchoalveolar lavage or endotrachial suction. It is also suitable for the examination of tissue samples, eg biopsy material from the lungs, tumor or inflammatory tissue, from secretions such as sweat, saliva, sperm and discharge from the nose, urethra or vagina as well as for urine or stool samples.
  • Another area of application for the present method is the investigation of water, e.g. shower and bath water or drinking water.
  • the food samples are taken from milk or milk products (yoghurt, cheese, curd cheese, butter, buttermilk), drinking water, beverages (lemonades, beer, juices), baked goods or meat products.
  • Another area of application for the method according to the invention is the examination of pharmaceutical and cosmetic products, e.g. Ointments, creams, tinctures, juices, solutions, drops etc. are possible with the method according to the invention.
  • pharmaceutical and cosmetic products e.g. Ointments, creams, tinctures, juices, solutions, drops etc.
  • kits for carrying out the corresponding methods are also made available.
  • the hybridization arrangement included in these kits is e.g. described in German patent application 100 61 655.0.
  • the disclosure contained in this document regarding the in situ hybridization arrangement is hereby expressly incorporated by reference.
  • kits comprise the most important component of the respective hybridization solution with the nucleic acid probe molecules described above for the microorganisms to be detected (referred to as VIT solution).
  • VIT solution the most important component of the respective hybridization solution with the nucleic acid probe molecules described above for the microorganisms to be detected
  • the corresponding hybridization buffer (Solution C) and a concentrate of the corresponding washing solution (Solution D) are also included. It also contains fixation solutions (Solution A and Solution B) and an embedding solution (Finisher). Finishers are commercially available, among other things they prevent the rapid fading of fluorescent probes under the fluorescence microscope. If necessary, solutions for the parallel execution of a positive control and a negative control are included.
  • a suitable aliquot of the fixed cells (preferably 40 ⁇ l) is applied to a slide and dried (46 ° C., 30 min or until completely dry).
  • the dried cells are then completely dehydrated by adding a further fixation solution (Solution B, absolute ethanol, preferably 40 ⁇ l).
  • Solution B absolute ethanol, preferably 40 ⁇ l.
  • the slide is dried again (room temperature, 3 min or until completely dry).
  • the hybridization solution (VIT solution) with the nucleic acid probe molecules described above for the microorganisms to be detected is then applied to the fixed, dehydrated cells.
  • the preferred volume is 40 ul.
  • the slide is then placed in a with hybridization buffer (Solution C, corresponds to the
  • Hybridization solution without probe molecules humidified chamber, preferably the VIT reactor, incubated (46 ° C, 90 min).
  • the slide is then removed from the chamber, the chamber is filled with washing solution (Solution D, 1:10 diluted in distilled water) and the slide is incubated in it (46 ° C, 15 min).
  • washing solution Solution D, 1:10 diluted in distilled water
  • the chamber is then filled with distilled water, the slide is briefly immersed and then air-dried in the lateral position (46 ° C, 30 min or until completely dry).
  • the slide is then embedded in a suitable medium (finisher).

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Abstract

L'invention concerne un procédé pour la mise en évidence de bactéries dans l'eau potable et de surface, notamment un procédé pour la mise en évidence spécifique simultanée de bactéries du genre Legionella et de l'espèce Legionella pneumophila par hybridation in situ. L'invention concerne également un procédé pour la mise en évidence spécifique de streptocoques fécaux par hybridation in situ, ainsi qu'un procédé pour la mise en évidence spécifique simultanée de bactéries coliformes et de bactéries de l'espèce Escherichia coli. L'invention concerne en outre des sondes oligonucléotidiques correspondantes, ainsi que des nécessaires permettant de mettre en oeuvre les procédés selon l'invention.
EP02754712A 2001-06-19 2002-06-19 Procedes pour la mise en evidence rapide specifique de bacteries concernant l'eau potable Ceased EP1397518A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE10129411 2001-06-19
DE10129411 2001-06-19
DE10160666 2001-12-11
DE10160666A DE10160666A1 (de) 2001-06-19 2001-12-11 Verfahren zum spezifischen Schnellnachweis von Trinkwasser relevanten Bakterien
PCT/EP2002/006809 WO2002102824A2 (fr) 2001-06-19 2002-06-19 Procedes pour la mise en evidence rapide specifique de bacteries concernant l'eau potable

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EP1397518A2 true EP1397518A2 (fr) 2004-03-17

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US (1) US20050064444A1 (fr)
EP (1) EP1397518A2 (fr)
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FI116030B (fi) * 2002-11-06 2005-09-15 Kemira Oyj Paperi- ja kartonkikoneiden termofiilisten mikrobien biofilminmuodostuksen esto
DE102004011822A1 (de) * 2004-03-11 2005-09-29 Cognis Deutschland Gmbh & Co. Kg Qualitätssicherungssystem zum Nachweis von Mikroorganismen
US7901932B2 (en) 2005-03-17 2011-03-08 Phigenics, Llc Methods and compositions for rapidly detecting and quantifying viable Legionella
ATE536551T1 (de) 2005-03-17 2011-12-15 Phigenics Llc Methode zum schnellen nachweisen und quantifizieren lebensfähiger legionellen
WO2007047912A2 (fr) * 2005-10-17 2007-04-26 Gen-Probe Incorporated Compositions et procedes destines a detecter des acides nucleiques de legionella pneumophila
US8609829B2 (en) 2005-10-17 2013-12-17 Gen-Probe Incorporated Compositions and methods to detect Legionella pneumophila nucleic acid
DE102007021387A1 (de) 2007-05-04 2008-11-06 Eads Deutschland Gmbh Detektionsvorrichtung zur Detektion von biologischen Mikropartikeln wie Bakterien, Viren, Sporen, Pollen oder biologische Toxine, sowie Detektionsverfahren
ATE525483T1 (de) * 2007-07-30 2011-10-15 Univ Ulm Oligonukleotidsonde und verfahren zum nachweis oder zur unterscheidung von mikroorganismen in einer probe
RU2741464C2 (ru) 2016-08-12 2021-01-26 Колопласт А/С Приспособление для стомического использования

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CA2451053A1 (fr) 2002-12-27
WO2002102824A3 (fr) 2003-09-25
WO2002102824A2 (fr) 2002-12-27
US20050064444A1 (en) 2005-03-24
JP2005515756A (ja) 2005-06-02

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