Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • 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 is in the field of the detection of microorganisms and the quality check of filterable and / or lightly filterable products and for assessing the hygienic condition of production plants.
• the identification of microorganisms in products could only be carried out by time-consuming cultivation and accompanying amplification, the required results only being available after 1 to 2 weeks.
• the cultivation was carried out, for example, for bacteria, fungi and unicellular algae in the cheapest nutrient media.
• This control checks how many and which microorganisms per unit volume are present in the end product.
• the live microorganisms capable of reproduction which can cause undesired contamination of the intermediate or end product.
• a classic method is, for example, membrane filtration, in which the samples are cultivated and filtered and the microorganisms remain on the membrane. The microorganisms are multiplied and identified on this membrane.
• Other methods are the stand test and partly also the PCR (polymerase chain reaction). However, since the PCR is positive even with "naked" DNA, false positive results are often obtained here.
• DEFT Direct Epifluorescent Filter Technique
• FISH fluorescence in-situ hybridization
• FISH fluorescence in-situ hybridization
• DNA / RNA located in the chromosomes With the aid of a labeled RNA or DNA probe, molecular hybridization with the DNA / RNA located in the chromosomes is carried out (FISH; Amann, RL, W. Ludwig and K.-H. Schleifer, 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbial. Rev. 59, pp. 143-169; see also DE 10160666)
• the specific hybridization of the probe is detected by fluorescence microscopic techniques as described, for example, in DE 19936875.
• the FISH technique is based on the fact that there are certain molecules in bacterial cells that, due to their vital function, have been mutated only slightly 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 can serve as specific markers due to their ubiquitous distribution, their size, and their structural and non-functional constancy.
• the 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 detect 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 deoxyribonulinic 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, then binds them and the cells can be detected in the fluorescence microscope due to their fluorescence.
• the methods for the detection and quantification of microorganisms must become increasingly sensitive and user-friendly in order to meet the requirements for fast detection methods with high efficiency, low detection limits and little effort for as many different microorganisms as possible. It is often sufficient if only an "absence test” is carried out first (yes / no test) and whether there are any microorganisms in the sample before it is determined taxonomically. It is too time-consuming and requires too much Laboratory capacity, if many detection methods have to be used for the most different microorganisms or if the most effective one has to be chosen from the range of different methods on the market.
• the invention is therefore based on the object of providing a system with which both filterable and non-filterable samples and products are investigated and the rapid detection of the quantitative detection of a wide variety of microorganisms is possible both live and dead.
• the system should make it possible to detect microorganisms with a detection limit of ⁇ 10 CFU / g in the selected sample amount.
• the system should not only be applicable specifically for an organism, but should also provide general evidence for microorganisms in samples and products.
• the system should also be able to check the hygienic condition of production plants. Description of the invention
• the invention relates to a quality assurance system for the detection of reproductive microorganisms containing a) a system for the enrichment of microorganisms in a sample in an "overnight culture” corresponding to 8 to 24 hours of cultivation under standard conditions according to international pharmaceutical law (e.g.
• a kit for the detection of living, damaged or dead microorganisms in filterable and / or non-filterable products containing i) at least one reagent containing an inductor and a fluorescent reagent which leads to the formation of a certain enzyme in living cells, which by.
• Reaction with a specific fluorescent reagent releases a fluorescent dye which becomes detectable, ii) at least one nucleic acid probe for the detection of microorganisms via in situ hybridization, the nucleic acid probe being bound to a fluorescent marker at which a detection limit for reproducible microorganisms of ⁇ 10 CFU / g is reached.
• the cultivation in "overnight cultures” corresponds on the one hand to the standard methods which are prescribed in the international pharmaceutical law, food legislation and cosmetics regulations.
• An overnight culture specifically means that the samples between 8 and 24 hours, preferably between 10 and 20 hours and in particular between 12 and
• the standard conditions of cultivation can be found in the legal text. Minor deviations, for example in the concentrations of the nutrient media components, the temperature or other parameters of the standard methods for cultivation should be included in the quality assurance system according to the invention.
• any changes in the legal text for the system according to the invention can be used, but the conditions must be documented in each case a detection limit for reproductive microorganisms can be determined. So it is very important which amount of sample is used.
• An exemplary direct method determines the CO 2 formed during the growth of the microorganisms by adsorption on a membrane and photometric determination in the incubation chamber. This method is marketed for example by the company BioMerieux under the name BacT / ALERT®. The method is very sensitive and indicates potential contamination at the latest after overnight culture, so that the analytical result is available after 24 hours at the latest.
• the material to be examined (possibly diluted) is inserted sterile into a nutrient media ampoule.
• the ampoule is placed in a sample cell within a heatable incubation chamber.
• the ampoule has a gas-permeable membrane that is connected to a detection chamber.
• a detection chamber In the detection chamber there is an indicator that adsorbs the CO 2 generated during the growth of cells over the membrane and whose turnover is measured photometrically in the incubation chamber.
• the measurement signals obtained in this way are documented electronically for each sample cell and converted into "growth curves" (time vs. CO 2 content).
• the method is suitable for all water-miscible and water-immiscible liquids as well as for emulsions, wax-containing pearl luster, oils, pastes and solids.
• a defined amount - usually 1 g - of a sample to be examined is placed under sterile conditions in the liquid chamber and incubated between 15 ° and 40 ° C, preferably at 30 ° C. If the sample contains germs, these will multiply under the evolution of CO 2 . With an infestation of 1 - 100 germs per ml, the gas formation can be registered after about 10 generation cycles and after about 2 hours. In the positive case, the material can be further analyzed immediately using the reagents from bi) or b ii) and the corresponding method As soon as there is a positive indication of contamination, the type of contamination can be verified.
• 1 to 10 g, preferably 5 g, of the sample or product to be examined are suspended in 100 to 1000 ml of standard solution and enriched in the overnight culture.
• This treatment is often necessary because the samples can sometimes have self-inhibiting effects on microorganisms.
• a method is required that can still detect these germs after dilution of the sample.
• this gives rise to the problem of the detection limit, because after dilution the total number of microorganisms is too low for the immediate use of the DEFT or FISH test methods in order to be able to carry out an absence test.
• the first requirement for the quality assurance system to provide an absence test for reproductive microorganisms or, in other words, a yes / no determination for reproductive microorganisms, is given by the fact that the night culture achieves a detection limit of ⁇ 10 CFU / g.
• the sample preparation from 5 to 10 g sample in 100 to 1000 ml standard solution achieves a detection limit of ⁇ 1 CFU / g or ⁇ 1 CFU / 5 g. This means that the minimum detection limit of ⁇ 100 CFU / g required by the international pharmaceutical law is significantly undercut.
• each intermediate can regardless of the consistency during a manufacturing process in each stage
• the detection of microorganisms in the sense of the invention means on the one hand a "yes - no" determination to answer the question of whether there are undesired microorganisms in the samples or products to be examined and on the other hand depending on examined sample or product the exact identification of the detected microorganism. Which evidence is provided depends on the consistency of the sample or product and thus on the reagent to be used and the nucleic acid probe to be used.
• samples and products mean both intermediate products and end products.
• samples can also a portion or part of an intermediate or end product can be understood, for example the liquid or solid portion of a heterogeneous product or intermediate, preferably after defined reaction times in particular to control an entire manufacturing process.
• samples are also understood to mean, for example, residues from cleaning processes in production plants.
• end products mean both the end product for the consumer and the raw product which is for sale and is used for the production of end products for the consumer. Samples can also come from industrial plants for effectiveness testing after disinfection. Industrial plants are regularly disinfected with steam or with chemical agents (hypochlorite or hydrogen peroxide). The effectiveness test has so far been carried out - if at all - using traditional methods. However, experience is often based on effectiveness, because the classic test is too time-consuming.
• filterable sample or product means that these are permeable through filters with a pore diameter of 0.45 ⁇ m. They should therefore not contain any oil droplets or solid particles or the like.
• the reagent from bi) for filterable liquid samples and products or for filterable liquid portions of the samples and products to be examined for the detection of living microorganisms is used from the kit according to the invention.
• Dead microorganisms can also be detected indirectly.
• This method of analysis examines the metabolic path from induction to the formation of an enzyme through the absorption of a specific substance. Induction takes place through a reagent containing an inductor and a fluorescent reagent which can pass through the cell membrane, whereupon the induced enzymes intracellularly give rise to the highly fluorescent compound. Cells without an intact cell membrane or active metabolism cannot form the fluorescent reaction product and show no fluorescence.
• These specific fluorescent reagents include, for example, fluorescine digalactoside for the detection of galactosidase which was induced by galactose as an inducer.
• lactobacilli and coliform bacteria such as Escherichia coli, Aeromonas, Citrobacter, Enterobacter, Klebsiella, pseudomonas and other germs relevant to process water can be detected.
• the fluorescent reagents include 4-methylumbelliferone derivatives which are derivatized specifically for certain enzymes.
• 4-methylumbelliferone heptanoate is used for the detection of lipase or esterase.
• 4-Methylumbelliferone- ⁇ -D-galactoside can also be used for the detection of galactosidase.
• the solution is then filtered through the described microfilters and examined fluorescence optically using an epifluorescence microscope.
• the indicators and fluorescent reagents are added to the residues on the filter.
• the kit uses the nucleic acid probe from b ii) both for filterable liquid samples and products and for non-filterable samples and products as well as for mixtures of filterable and light-filterable samples and products for the detection of living microorganisms.
• 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 exist for a probe of 15 nucleotides over 100% of the sequence. For oligonucleotides with more than 15 nucleotides, one or more mismatching sites are allowed.
• the nucleic acid probes from the kit according to the invention are able to use non-specific nucleic acid probes to detect non-specific microorganisms. So that can often asked question whether there are undesirable microorganisms in samples or products without accurately characterizing the microorganism.
• the hybridization conditions and the duration of the hybridization are adjusted depending on the product and the sample to be examined depending on the nucleic acid probe.
• B. fluorescent groups such. B. 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 Rohe Diagnostics GmbH, Mannheim, Germany), TRITC (available from Molecular Probes from Eugene, USA), 6-FAM or FLUOS-PRTME.
• the quality assurance system according to the invention can be used to detect gram pos. and / or gram-negative bacteria and / or yeasts and / or molds and / or algae are used.
• bacteria also include medically relevant germs such as staphylococci, streptococci, anthrax, tetanus, lactic acid, diphtheria, swine erysipelas or hay bacteria.
• bacteria also count as medically relevant germs such as gonococci, meningococci, legionella, coli, typhoid, Rur and plague bacteria.
• Environmentally relevant and human-associated germs include process-specific germs such as pseudomonas, burkholderia, raouliques, cuttings, corynebacteria and bacillus species.
• targeted inoculated products can be determined by the method according to the invention with the reagent from b i) iesiiüts ⁇ iieN / ⁇ ederfindung ⁇ .
• yeast and mold can also be detected using the method according to the invention.
• Standard germs used for targeted inoculation some of which are also mentioned in international pharmacopoeias, can be used and detected: Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, Staphylococcus aureus, Candida albicans, Aspergillus niger, Sahnonella, Bacillus subtilis.
• Another object of the invention is the use of the quality assurance system according to the invention for the detection of microorganisms and for the quality check of filterable and / or lightly filterable samples or products and for assessing the hygienic condition of production plants.
• the filterable and / or non-filterable samples or products to be examined are selected from the group which is formed from raw products, cosmetic products, pharmaceutical preparations, food preparations, food supplements, textile auxiliaries, washing and cleaning agents as well as paints and varnishes.
• raw products are understood to be products which are used for the production of end products for the consumer.
• These can be surfactants, oil bodies, emulsifiers, pearlescent waxes, consistency agents, thickeners, superfatting agents, stabilizers, polymers, silicone compounds, fats, waxes, lecithins, phospholipids, UV light protection factors, antioxidants, deodorants, antiperspirants, antidandruff agents, film-forming agents, swelling agents , Self-tanners, tyrosine inhibitors (depigmentation agents), hydrotropes, solubilizers, preservatives, perfume oils, non-filterable O / W and W / O emulsions.
• Process water can also be seen as a raw material.
• the cosmetic products can be, for example, ointments, creams, lotions, shampoo, conditioners, shower gels, bath products, decorative cosmetics such as make-up, eye shadows, lipstick, nail polish or the like.
• the pharmaceutical preparations can be in the form of juices, creams, ointments, lotions, suspensions, tinctures, drops or the like.
• the foods are preferably milk or milk products, baked goods or meat products, beverages such as mineral water, beer, lemonade or fruit juice.
• Vitamin solutions, unsaturated fatty acids, in particular conjugated linoleic acids, preservatives or antioxidants are preferably mentioned as food supplements.
• Another object of the invention is a method for the detection of microorganisms in filterable and / or lightly filterable products in which the quality assurance system according to the invention is applied by cultivating the samples a) for enrichment of microorganisms in an "overnight culture” corresponding to 8 to 24 hours under standard conditions in accordance with international pharmaceutical legislation, food legislation and cosmetics regulations and b) use a kit for the detection of living, damaged or dead microorganisms in filterable and / or lightly filterable samples or products by incubating the enriched sample i) with a reagent containing an inductor and a fluorescent reagent , which induces the formation of a special enzyme in the cells and thereby creates a fluorescent compound from a fluorescent reagent, and / or ii) after fixation of the bacteria, incubates them with a nucleic acid probe, which is provided with a fluorescence marker to bring about hybridization and c) the fluorescence of the samples is detected and correlated with the number of cells, the number of
• 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 fixation. However, methanol, mixtures of alcohols, a low-percentage parafonnaldehyde solution or a dilute formaldehyde solution, enzymatic treatments or the like can also be used.
• the fixed bacteria are incubated with fluorescence-labeled nucleic acid probes.
• 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 nucleic acid probes according to the invention can be used with various hybridization solutions. Various organic solvents can be used in concentrations of 0-80%. Compliance with stringent hybridization conditions ensures that the nucleic acid probe actually hybridizes with the target sequence.
• Moderate conditions in the sense of the invention are e.g. B. 0% formamide in a 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 moT / l, preferably 1 ⁇ vo ⁇ rO; 5-mOM ⁇ , particularly preferably 0.9 mol / 1, the salt preferably being sodium chloride. Furthermore, the hybridization solution usually comprises a detergent, such as. B. sodium dodecyl 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%.
• SDS sodium dodecyl sulfate
• Tris-HCl sodium citrate
• PIPES sodium citrate
• HEPES HEPES
• the particularly preferred embodiment of the hybridization solution according to the invention contains 0.02 mol / 1 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 exceed the number of target structures by several orders of magnitude. stride. However, with fluorescence in situ hybridization (FISH), care must be taken to ensure that an excessively high amount of fluorescence-labeled hybridization probe leads to increased background fluorescence.
• the amount of probe should therefore be in a range between 0.5 ng / 1 and 500 ng / 1, preferably between 1.0 ng / 1 and 100 ng / 1 and particularly preferably 1, 0-50 ng / 1.
• the duration of the hybridization is usually between 10 minutes and 12 hours; hybridization is preferably carried out for about 1.5 hours.
• the hybridization temperature is preferably between 44 ° C. and 48 ° C., particularly preferably 46 ° C., the parameter of the hybridization temperature, and also 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 can be optimized to the target sequence in the cell to be detected.
• the non-hybridized and excess nucleic acid probe molecules are removed or washed off using a conventional washing solution.
• this washing solution can contain 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 / 1, preferably 0 , 01-0.05 mol / 1, particularly preferably 0.02 mol / 1, contain.
• the washing solution usually also contains NaCl, the concentration depending on the stringency required being from 0.003 mol / 1 to 0.9 mol / 1, preferably from 0.01 mol / 1 to 0.9 mol / 1.
• the washing solution can contain EDTA in a concentration of up to 0.01 mol / 1, the concentration preferably being 0.005 mol / 1.
• buffer solutions are used for the washing solution which correspond to the hybridization buffer in a lower salt concentration.
• the “washing away” of the unbound nucleic acid probe molecules is usually carried out at a temperature in the range from 30 ° C. to 50 ° C., preferably from 44 ° C. to 50 ° C. and particularly preferably at 46 ° 6 for a duration of 10-40 minutes , Switzerlandsorzugswe e ⁇ für ⁇ rT5 "Min ⁇ jten ⁇ ⁇
• the result when using the kit according to the invention is available after 24 to 48 hours.
• the respective samples or products treated with fluorescent reagent or fluorescent markers are then optically detected with the aid of a microscope, preferably an epifluorescent microscope.
• the analysis time is usually 10-24 hours.
• the samples prepared in this way are incubated overnight at 30 + 5 ° C. If contamination with slow-growing organisms is suspected, the 8-hour pre-enrichment can take up to 24 hours.
• microorganism growth causes clouding in the enrichment broth or CO 2 development in the BacT / ALERT® system can be measured, the sample is further analyzed with the FISH technique and a rough classification into gram positive and gram negative organisms is carried out, possibly analyzed up to the germ species (see Example 2: Non-filterable products). If there is no growth in the enrichment or no CO 2 development in the BacT / ALERT® system, the results are verified using the DEFT method.
• 10 ml - this corresponds to a starting product quantity of 5 g - are removed from the enrichment culture with a sterile syringe and through a 0.45 ⁇ m polycarbonate filter filtered.
• the polycarbonate filter on which the enriched cells lie is placed on a DEFT-PAD containing the fluorescent dye and incubated for 8-15 min at 20-37 ° C in a moist chamber.
• the dye passes from the PAD to the cells, while the background of the polycarbonate filter is not stained.
• the filter is released from the pad and placed on a slide. The color reaction is then stopped with a fixing liquid.
• the preparation is then checked in a fluorescence microscope with 100 ⁇ magnification for the presence of stained cells.
• the filter is scanned completely.
• the background of the filter must be dark.
• the positive and negative controls carried along must provide clear results
• red cells are detected, the specimen was mixed with cells that had already died. Such cells usually come from the environment and are not relevant to the quality of a preparation. Products for parenteral use are excluded. Here the preparation should contain neither red nor green fluorescent cell bodies.
• 10 g of a sample to be examined are weighed in sterile and according to Ph.Eur. 2.6.12. or 2.6.13 in 90 ml THLC1 broth with suitable additives - preferably a standard Stomacher device - homogeneously mixed. 10 ml of this suspension, corresponding to 1 g of the sample to be examined, are pipetted into 100 ml of CASO or Sabouraud-BouiUon.
• the process is to be classified as yes / no. This is usually sufficient for a quality assessment, since the overall goal is "proof of the absence of germs capable of reproduction".

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• 2004
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• 2005
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