WO2011079839A2

WO2011079839A2 - Soiling

Info

Publication number: WO2011079839A2
Application number: PCT/DE2010/001518
Authority: WO
Inventors: Robert Simmoteit; Hans-Peter Wendel
Original assignee: 3Mach Gmbh
Priority date: 2009-12-29
Filing date: 2010-12-23
Publication date: 2011-07-07
Also published as: DE102009060836B4; WO2011079839A3; DE102009060836A1

Abstract

The invention relates to soiling having a marker, characterized in that the soiling contains a marker, which is a natural or synthetic nucleic acid, DNA, RNA, or Spiegelmer or an analog and is applied to surfaces, wherein the marker is used at least to demonstrate the depletion of soiling having a marker after cleaning in order to enable at least one evaluation of the cleaning success or of spreading or of decontamination or of substance release. The production of a soiling surface is used in particular on test specimen surfaces to examine a cleaning method, spreading, decontamination, or substance release in such a way that, after treatment, the remaining matrix having a marker fraction is made visible by means of a dye or, at least after the marker has been removed, the marker is quantified by means of a PCR method.

Description

A

soiling

Field of the invention

The invention relates to a stain with an arker and a preparation of a stain and a subsequent method for evaluating the cleaning success or a decontamination or carryover or a substance release.

Background of the invention

The determination of the cleaning success especially of reprocessable products in washer-disinfectors is important in many industrial and medical fields and represents a serious problem. In doing so, medical instruments or industrial equipment must be cleaned so that they can be reused and there is no danger emanates for humans. Especially in the field of medical instrument reprocessing, no dirt particles or germs should enter the human body. It is therefore essential to determine the cleaning success quantitatively and qualitatively. However, this determination is difficult due to the different geometries of the products to be cleaned. Furthermore, the products can be heavily, but also hardly visible polluted. In the field of medicine, therefore, primarily protein detection methods are used. These established detection methods are based on protein staining with a subsequent determination of staining. These include z. B. the Biuret, Lowry and the Bradford test. Other methods use the UV light or are based on autofluorescence or require physical detection in a high vacuum (XPS analysis). A quantitative determination of the remaining protein contamination on surfaces is made more difficult because the proteins are exposed to detergents and temperatures of over 90 ° C. during the cleaning process. The latter conditions affect the detection method and make the reproducibility difficult.

Another path was taken with radiolabelled markers. In this method, according to DE 196 31 668 A1, objects are contaminated with a radiolabeled soiling and the cleaning success is documented by the measurement of the remaining radiation intensity along a time axis. Another type of test staining is described in DE 196 02 673 A1. Here, a synthetic test soil containing fibrin or a fibrin precursor is applied to test surfaces and detected by visual inspection after cleaning. Alternatively, DE 2007 017 612 U1 uses a heat-resistant fluorescent compound. This compound is admixed to a test mixture containing at least one polysaccharide, proteins and lipid as well as a selected polymer. Alternatively, soiling can be used in the field of crime prevention or piracy. These are usually based on fluorescent and hidden molecular markers. These labels are nucleic acids or nucleic acid analogs (based on DE 199 14 808), the z. B. are embedded in a curable composition and which melt again below 200 ° C or can be re-liquefied with a solvent.

The known Testanschmutzungen and methods have either the disadvantage that they were radioactively labeled and therefore the method is not suitable for everyday use. The latter methods require expensive devices such. B. a gamma camera or a radiation detector. Protein-based methods, on the other hand, are very vulnerable and the results are very strong. A particular disadvantage is that the detection limits of the known methods are not low enough to be able to make a quantified and reliable statement about the cleanliness. However, this statement is elementary and is currently only indirectly achieved with the support of very expensive and material destructive methods. However, it is important that the reprocessed product is undamaged again and that due to medical or pharmaceutical applications even the smallest impurities can be detected. Furthermore, biological test soils, for example, are susceptible to oxidative processes and influence the analysis and the reproducibility, or test soils are used whose ingredients are unsuitable for analysis. Summary of the invention

The aim of the invention is, in particular to apply a test soiling on various surfaces and in particular to use soils for test specimens, the soiling is sufficiently stable, z. B. in cleaning and thermal decontamination can be used and these processes survive in principle. Furthermore, the goal is to make the detection sensitivity so high that even the smallest contamination of the soiling can be detected.

The soiling should be used in medical instrument reprocessing and hygiene technology, but also in the commercial cleaning, food, pharmaceutical and / or in the laboratory industry and for the production of test bodies and / or for the marking of surfaces. In order to achieve this goal, extensive experimental investigations were carried out and numerous variants of biofuels or contaminants (proteins, dust, germs, fats, paint residues, etc.) were included.

The following points, which are also the subject of the invention, were considered:

1. novel soiling

2. Use of the soiling and inventive production of suitable Anschmut- tion products

3. Novel analytical method at least for checking the effectiveness of a cleaning success of a decontamination, carryover or substance release

These points have also been taken into account in the development and the soiling and the related procedures are coordinated and interrelated.

It was determined experimentally that in particular DNA meets the required conditions. Human, animal, plant or synthetic DNA can be used as a single or double strand. Synthetic single-stranded DNA is preferably used because it has the great advantage of not allowing cross-contamination with naturally occurring DNA. The synthetic DNA is preferably characterized by short sequences less than 1000 nucleotides. For cost reasons, sequences under 100 nucleotides are useful. Both at the 3 'and at the 5' end are defined sequences (primer regions), which are used for the detection and amplification of the DNA in the PCR (see Figure 1). Figure 1 shows such an example of synthetic single-stranded DNA with defined primer regions at the 3 'and 5' ends, where 1 is the sense primer region, 2 is the antisense primer region and 3 is a 20-60 nucleotides / random region designated. Alternatively, however, rare DNA derived from insect, plant, bacterial or rare animal cells or decalcified virus or viral RNA or synthetic viral RNA can be used as a marker.

To enable quantification of the DNA, a procedure had to be developed to allow the applied components to be redissolved. The soiling had to have biocompatible properties in order to be used in medicine. An important requirement was to investigate how well DNA can be redissolved in relation to substances such as proteins (albumin, fibrin, etc.), fats, and other contaminants, when these components are present in a biomatrix or in an artificial or a combination of natural and artificial substances. A possible solidifiable biomatrix from plasma proteins was coagulated by coagulation activators. It has been found that the dissolution behavior of the incorporated DNA in a fibrin and / or protein matrix, at least through the use of an alkaline cleaning agent, behaves similar to that of albumin or fibrin. However, since DNA is everywhere, a way had to be found to determine only the DNA released from the soiling. The solution chosen was a nucleic acid (DNA) or a nucleic acid analog or the like, which may be a single-stranded or double-stranded DNA. Preferably, a DNA which does not occur in nature, or at least a rare DNA or an analog thereof, is used as a marker. In the text of the invention, a DNA that does not occur in nature is defined as synthetic DNA or as a synthetic DNA marker.

In experiments, a synthetic DNA fragment (about 20,000 Da) with defined primer regions, which does not occur in nature, was incorporated as a marker in a biomatrix and thereby detected a high detection sensitivity. This synthetic DNA has no homology to human, animal or plant DNA and therefore no possibility of contamination with foreign DNA.

Detection sensitivity was determined in the femto-gram range. Furthermore, a very linear relationship of the detached DNA with at least 10 base pairs, which does not occur in nature, was found (see Figure 2). FIG. 2 shows a standard calibration curve of a detached synthetic DNA (or synthetic DNA marker). As Figure 2 shows, the linearity between 5 to 0.01 pg / 0.5ml is clearly visible and demonstrates that DNA can still be detected in the femto-gram range. Standard deviations between 0.03 and 0.34 were achieved. The detection sensitivity of the biuret method, on the other hand, is 1-10 pg protein per milliliter and thus many times lower. ger. However, the extraction of the synthetic DNA marker with, for example, an alkaline solution is made more difficult if the biomatrix in which the DNA marker is packaged additionally contains solid constituents.

The invention thus solves the soiling problem by the use of DNA of at least synthetic DNA which is incorporated in a mass of known biomolecules (biomatrix) or other soiling matrix of substances. Biostuffs, other substances or a mixture of the two are preferably used, which can be crosslinked, for example, or bonded together or can be detachably connected to one another. The invention is a soiling which can be used at least for the detection of cleaning processes. In addition, it can be used in all areas of life in which products, foodstuffs, utensils, parts of appliances, furniture parts, buildings or industrial plants must be treated and / or reprocessed, or where evidence must be provided that one or no cleaning or one or more Reprocessing was carried out. It also means applications in which a soiling is used in conjunction with substances to quantify the carryover of these substances. The invention can be used particularly where process validation or developments, in particular in the area of cleaning solutions, are undertaken. This also includes surfaces or building parts that are cleaned. The latter application relates to the detection of cleaning processes, in particular in the field of health care, in which a disinfection and cleaning of floors, furniture surfaces, walls, dishes, laundry and hands is made.

This object has been achieved according to the invention by a soiling with a marker according to claim 1 and their preparation according to claim 9 and a subsequent method at least for checking the effectiveness of a cleaning success, decontamination, carryover or substance release according to claim 13. The following claims relate to the advantageous embodiments of the invention. Inventively, the soiling can also be used alone if quantification of the DNA mark does not make sense, because the remaining residual content of the soiling is visible after the treatment and thus a minimum quantity is already injected. However, in order to make a Qualitative statement, quantification should be used with an RT-PCR method or the like. Furthermore, industrially produced test specimens are available for better quantification.

The inventive solution to the problem is solved with a stain, with a marker, in that the stain contains a marker which is a natural or synthetic nucleic acid, DNA, RNA or spiegelmer or an analogue and can be labeled on surface. The marker is or will be applied, the marker being used at least as a depletion-proof of soiling with a marker after a cleaning in order to allow at least an evaluation of the cleaning success or a carryover or a decontamination or a substance release.

In a further embodiment of the invention, the stain consists of a solidifiable matrix containing solid and / or aqueous solutions in the components as markers at least one artificially produced nucleic acid (synthetic DNA) or synthetic nucleic acid analogs, or RNA or Spiegelmere that in nature are not present or in which as markers a natural DNA, RNA or spiegelmers is included to allow at least an assessment of the cleaning success or a decontamination or carryover by the marker, which is leachable from the solidified matrix by aqueous solutions. As an alternative to cleaning, however, another treatment, for example a treatment of a medical coating, can also be used here in order to check its stability. The soiling matrix or contaminant with the containing marker is in the broadest sense an unpleasant or unhygienic material attachment or an impurity and / or a foreign mixture (which may also be a medical drug or drug mixture) defined by surfaces or depleted depleted or removed shall be. In the medical field, a bio-hardenable matrix is preferably used. The dissolution behavior of the incorporated DNA, RNA or spiegelmers depends on the soiling matrix and is largely influenced by temperature. It has been found that a suitable DNA can already be detached by 30-100 ° C hot water up to 70% of metallic surfaces.

Likewise, stabilized and / or natural DNA, RNA or spiegelmers can be used. Stabilized DNA, RNA or spiegelmers have the advantage that they are less rapidly degraded by nucleases. Stabilizations can e.g. by introducing stable nucleotide analogues, such as phosphorothioates, or by substitution of the 2'-OH by a 2 -NH 2 group. Alternatively, it is also possible to use known DNA, RNA or spiegelmers derived from insects, plants, bacteria or from rare animals. However, these should be chosen so that they do not occur in the field.

The dissolution of the marker, for example, during cleaning, from the biomatrix can not only be used to z. B. to determine a cleaning success. The marker and its dissolution behavior, however, can also be used to detect a release of active substance (example of a decontamination) from a matrix (medical product with a soiling or coating), which is present in particular in vessels, other weaving or organs. Such coatings are used in implants such as stents, etc. The release behavior from the matrix is preferably determined under laboratory conditions.

In this case, a matrix is defined, which is composed of biological substances, a biological mixture or which also consists of artificial substances. A biomatrix preferably consists of a solidifiable structure which may also have slightly non-biological constituents in the mixing. Depending on the field of application and the question, a layered structure can also be an advantage.

In a further development of the invention, a staining with a marker is produced by the fact that at least one solidifiable biomatrix or soiling matrix is present as a solution, paste or spreadable mass and is incorporated in a container or in a pen or the like or is already applied to a surface or is introduced into the room air as an aerosol and only then solidified into a matrix. In this case, hardening of the matrix can preferably be achieved by drying. Alternatively, the fact that film-forming or thickening substances involved in the matrix or involved in the structure of the soiling. The film-forming or thickening substances include, for example, substance groups such as alginates, gelatin, polymethacrylates (Eudragit E, S and RS), hypromelose, 2-hydroxypropulmethyl ester cellulose, sodium carboxymethylcellulose, hydroxypropyl cellulose, methylcellulose, aqueous shellac, polyvinylpyrrolidone (Kollidane), fibrin, Slime or similar Substances that are mixed in or used for film formation to form a lower or upper covering layer or contribute to matrix solidification.

It is envisaged that the level of DNA marker in the soak solution or paste will be at least 0.0001% by volume. Even low concentrations of 5 g are completely sufficient. Preferably, a DNA marker amount of about 0.01 μg / ml in the soak solution or a DNA marker amount of at least 0.01 μg / ml in the eluate should be included for detection. If labeled DNA marker complexes are used, which are later analyzed by visual inspection, the DNA marker portion may also be higher.

The economic goal is to develop specimens with a soiling and a DNA marker, in which a specimen surface has at least one removable soiling. These test specimens are useful for comparing the PCR analyzes (preferably RT-PCR) of many users. The known test specimens of the companies Perek GmbH, Früh-Kontroll GmbH and other companies use test specimens that allow a visual analysis, which is not quantification. Proteins of these specimens are through denaturing the 90 ° C temperature steps in the purification chamber so that the protein determinations are falsified.

It is inventively provided that at least one artificially produced nucleic acid (synthetic DNA) or a nucleic acid analog or the like, which does not occur in nature, is used as a marker. The marker can be incorporated in all possible soiling substances (soluble and non-soluble substances). All possible soiling substances are conceivable which play a role in particular in hygiene and beyond. For example, biofuels are used in hygiene. Alternatively, marker complexes that are used in conjunction with germs or cells and / or biomaterials. Preferably, the inventive soiling is spread or applied by spreading, dipping or spraying. It may be advantageous if the actual soiling is applied to a previously applied film-forming material layer or film-forming or thickening substances are contained in the matrix.

In order to achieve solidification of the biomatrix, it has been found that coagulation, complexation, chemical crosslinking or drying is suitable. This is important because the natural soils that occur in nature or during surgery are at least initiated by coagulation and / or drying. These processes must also be used in the artificially prepared soiling on a surface so that a test specimen can be used to determine, for example, cleaning and decontamination processes.

It is envisaged that the soiling matrix is formed on a surface and that it can be removed or dissolved again completely or at least in part from this matrix. Alternatively, the soiling can also be sprayed in rooms in the form of a soothing arosol, bio-arosol or the like. In this case, at least by spraying a bio-Arosols a solidified biomatrix in which the marker is incorporated, arise. This can then be used to analyze their distribution in rooms or their filtration (air purification) or to check the cleaning of surfaces. Thus, the novel soiling and their production is individually applicable or can be industrially produced and used in controlled processes.

In this case, a soiling solution may consist of two or more components which are added or registered simultaneously or in succession. In this case, a coagulation activator, complexing agent or crosslinker can be applied to an already wetted surface in order to achieve coagulation by enzymes or a complex formation achieved by drying or chemical crosslinking with a biopolymer via an additional crosslinker.

In order to determine the cleaning success, a product or specimen surface already prepared in this way is used to at least deplete this soiling in a subsequent step and to visualize in a subsequent step, at least by adding a dye or an enzyme, the remaining amount of the remaining artificially produced DNA, or to determine these only after detachment from the surface with PCR o. In this process, an alkaline solution with a pH of at least 9 has proven itself. Alternatively, the synthetic DNA can be dissolved out with 30-100 ° C hot aqueous solutions. Mechanical shaking achieves better standardization. Alternatively, film-forming substances (for example Eudragit, Kollidone or the like) may contain or be present as a layer, so that, for. B. by a pH step, a detachment of the residual components of surfaces after cleaning is improved. In order to avoid damage to the DNA marker, the eluate must be reduced to pH 0.7 or to physiological pH values. The eluate may be purified by microconcolation columns to separate mixtures by molecular weight (e.g., YM-30), concentrated, and / or liberated from materials that interfere with the PCR. For example, a 28,000 Da DNA can be purified on a 30,000 Da column.

The production of coded DNA markers and their coding has been described in DE 199 14 808 and coded according to a systematic coding scheme digitally or encrypted. In contrast, in the presented invention, preferably, a non-naturally occurring DNA sequence (synthetic DNA or synthetic DNA marker or analog) or a rare natural DNA is used, which is easy to determine and it is not important here to encrypt them or to code specifically. The invention requires a known sequence in order to multiply these with the PCR method (Polymerase Chain Reaction) in order to clearly prove its origin.

It is inventively provided to incorporate or dissolve the synthetic DNA in a matrix, for example from biomolecules. In this case, preferably fibrinogen or a fibrinhal- tiges mixture is used. Fibrinogen or fibrin-containing blood plasma is converted to fibrin by adding thrombin, thromboplastin, other coagulation activators or by a heparin-binding molecule. Was previously the biosubstance like isch applied with a marker of synthetic DNA on surfaces, so it can be described by z. B. addition of thrombin by the conversion of fibrinogen into fibrin in the here forms coagulation matrix to be incorporated or packaged. The coagulation-activated mixture of biomaterials also serves to protect and stabilize the DNA, since the soiling is exposed to alkaline cleaners and temperatures of over 90 ° C. In addition, the DNA, RNA or the Spiegelmers or their analogues can also be additionally chemically stabilized. This protection is enhanced if there is a high protein content in the biomatrix. The term coagulation in the context of the invention in this case comprises the formation of a fibrin network with or without blood cells, such as platelets, in which mainly other biofuels and / or cell components can be involved.

It is a protein mixture from blood plasma with or without film-forming or thickening substances z. B. coagulant molecules, preferably, if the soiling is to be used in particular in the medical field. However, it is also conceivable that synthetic plasma constituents, such as fibrinogen, and / or chromogenic substrates, such as the customary pNA derivatives (para-nitroaniline) or medical active substances, are contained instead of the blood plasma. For economic or practical reasons, instead of human and animal blood plasma, other biostats and biostatic mixtures such as lipids, mucins, egg yolk, starch, hyaluronic acid and / or hyaluronic derivatives or resins or a combination of these substances may be used. It may further be preferred that the substance mixture, isotopes, dyes or antibodies are admixed in order to allow an additional simple detection of acceptable Restanschmutzungsmen- gene. Finally, it is also conceivable that the marker is chemically linked or combined with these substances (eg isotopes, dyes, antibodies). The addition of dyes has the advantage that the cleaning success is at least visually verifiable directly after cleaning.

One of these possible soiling solutions is made possible by albumin, which aggregates to protein complexes in the presence of heparin. In addition, this aqueous solution can be stabilized with a chemical crosslinker such as EDC, glutaraldehyde or others. The resulting complexes are preferably rebuffered and / or additionally washed, and only then is the synthetic DNA mixed. If the complexes are applied to surfaces by repeated dipping or rinsing, a layered structure results. In these layers and between these layers, the synthetic DNA can be packed. The resulting layered matrix is useful for the preparation of a suitable control body for testing the cleaning success. After drying, it is possible with a simple color test with toluidine blue to detect the coating or soiling. The proof is on the sensitivity - I l of the eye limited. In order to determine the proportion of synthetic DNA, it must first be removed from the surface again before a PCR analysis is possible.

In a further embodiment z. B. the synthetic DNA may be additionally marked with a substance to z. B. to be able to perform a photometric proof or an isotope detection or to stain them visible. Here, a fluorescence or an isotopic label of the DNA is useful. For example, the synthetic DNA has an oligonucleotide sequence of at least 10 base pairs. However, it is also possible to use a synthetic DNA with more than 100,000 base pairs. Preferably, a synthetic DNA with a length of 50-1000 nucleotides (including the primer regions) is used. However, this DNA can also be chemically crosslinked with a biomolecule, such as albumin and / or fibrin or fibrinogen. The cross-linked product, in turn, is more easily determined by a protein or antibody detection test.

For the preparation of the soiling solution or of a soiling aerosol with at least one natural or one synthetic DNA marker, the following substances and mixtures of substances are conceivable as soiling:

1. Albumin solution (human or animal) with or without heparin with or without another dye as a marker.

2. Fibrinogen solutions (human or animal) or solutions with fibrin precursors.

3. Polysaccharide solutions with proteins and / or glycoproteins or lipids.

4. Polysaccharide solutions with proteins and / or glycoproteins or lipids with germs.

5. Blood plasma (human or animal) or plasma constituents with or without thickening substances with or without film-forming substances.

6. Blood plasma with platelets or blood cell components (human or animal)

7. Whole blood (human or animal).

8. Platelet solution with fibrin and / or fibrin precursors (human or animal).

9. Blood or blood plasma with tissue and / or bone components.

10. Soil solution of egg yolk and / or semolina pudding.

1. Contaminant solution containing at least film-forming substances and / or dyes.

12. Contaminant solution or suspensions or emulsions containing solids (dust, particles, germs, abrasion, medical agents, etc.).

13. Contaminant solution containing dyes and / or film-forming substances with or without thickening substances.

14. Contains soiling solution of printable dyes or parts thereof. 15. Soiling solution, emulsions or suspensions of any kind which can be hardened by drying or at least adhere to surfaces.

16. Soiling solution, emulsions or suspensions of any of the above ingredients and compositions.

The possible components of a soiling solution described above can also be applied to other biofuels, such. B. on collagen or cellulose or their derivatives in solution or suspension, expand. The soiling can not only be spread on a solution or sprayed as an aerosol, but conceivable is also a suspension or an emulsion, which are distributed or printed on surfaces by screen printing or pad printing o. The like. Also conceivable is a pasty consistencies, such as with a crayon or lipstick, or a consistency, as found in a lotion or cream. When a stylus is used, the synthetic DNA may be incorporated or packaged in a spreadable matrix, or may be applied to a surface, as in a pen. The use of highlighter pens would be a conceivable application form. As another form, a lipstick would be conceivable in which the consistency of the soiling matrix by Vaseline, gelatin or waxy substances such. As bee and carnauba wax, or oily substances such. As jojoba and almond oil or shea butter, was increased.

In order to be able to produce soiling as souring solutions as a biomatrix, for example, the synthetic DNA is dissolved in a hydrophilic solvent or a buffer. Preferably, a substance or mixture of biocomposites may form the matrix here and / or additionally dyes but also thickening or film-forming substances may be added. Preferably, a human plasma, which may consist of a pool of 5 to 100 plasmas, is used. To prevent the plasma from clotting, citrate and / or heparin (citrate anticoagulant solution) are added. This mixed solution can be bottled and stored. Preferably also at low temperatures (frozen). For use, the mixture is applied to a test surface or product (eg, medical instrument). In order to initiate coagulation, a Ca-containing thrombin or a fibrinogen activation solution is preferably added in a spraying process. The onset of coagulation packs the synthetic DNA into the resulting biomatrix. Alternatively it can be provided that the soiling solution is in a chamber and in a second chamber, a thrombin solution of a spray device. By pressing both solutions can be applied simultaneously and so a soiling on a surface done. The method has the advantage that a even layer is created. This simultaneous application can also be used industrially for the production of control bodies.

Alternatively, blood plasma containing the synthetic DNA marker may also contain platelets and / or other bio-constituents such as bone meal. The latter composition results in a very stable biolayer by cross-linking the bio-constituents. In the absence of water it encrustes and forms a hard-to-remove test soil. In addition, further compositions of a mixture are possible in which z. As gelatin, collogene, tissue components or ingredients of food are used. Thus, solid and soluble components may be included in the biomatrix.

The inventive soiling is after this on appropriate surfaces (product surfaces of any kind but also tissues, films, labels o. Ä.) Was applied or z. B. is present on control surfaces, used in a cleaning process. Alternatively, the soiling can be used to label disposable products to indicate unauthorized cleaning or reprocessing or to confirm a cleaning treatment and its success. This also includes mechanical cleaning methods with a subsequent preferably thermal disinfection. The remainder of synthetic DNA markers and / or natural DNA markers are subjected to subsequent analysis. The following procedures and options are available here:

1. A color-coded matrix, preferably synthetic DNA, is visible on the surface.

2. A protein complex and / or protein-coupled synthetic DNA complex is stained with a color reagent on the surface and thus made visible.

3. An albumin-heparin complex with DNA is stained on the surface by toluidine blue and visualized.

4. An antigen-labeled DNA is detected on the surface with an antibody detection test.

5. A radiolabeled natural or synthetic DNA is detected by a radiation detector on surfaces or in an eluate.

6. The remaining DNA (labeled or unlabeled) is eluted from the surface or the labeled DNA is extracted from the protein complex e.g. B. cleaved enzymatically and then z. B. an RT-PCR method quantified.

7. The remaining DNA (labeled or unlabeled) is eluted from the surface by a pH and / or temperature step and incubated over e.g. B. RT-PCR method quantified. A possible method for evaluating the cleaning success with a soiling and containing a DNA marker is shown in FIG. Representation A, shows a surface 4, on which a film coating 3 is applied. The film coating may alternatively also be applied to a film, an adhesive film, a label or the like. In the case of an adhesive film this z. B. connected via a lower adhesive surface with the surface 4. On this coating is a soiling 1 with a DNA marker 2 contained herein, applied. Representation B has a soiling 1 with a containing DNA marker 2 which is applied to a surface 4. The soiling may additionally contain film-forming and / or thickening substances. Subsequently, in both cases, a manual or mechanical cleaning is carried out with the aim to completely deplete the soiling 1 (shown with arrows a). Representations C and D represent a possible purification result and show that residues of soiling 1 containing DNA marker 2 remain here. These could not be completely depleted by the purification step. In a subsequent step, the detachment of the contamination residues preferably takes place by means of suitable solutions. In the case of FIG. C, the film coating 3, which has, for example, a defined pH solubility, can be easily removed by a suitable pH solution or, in the case of an adhesive film, mechanically removed from the surface. Depending on the choice of soiling matrix, this solution for removing the residual amount (see FIGS. C and D) may be an acidic or alkaline solution in order to remove the remaining amount of soiling from the surface and then adjust the eluate to physiological values. The film coating 3 should not dissolve too easily during the cleaning process. The solutions 5 to replace the Restanschmutzung be collected in a container 6 and can hereafter z. B. an RT-PCR analysis.

The above method thus provides the ability to improve the effectiveness of cleaning solutions and methods such. B. to check the mechanical or mechanical cleaning qualitatively and quantitatively. The results, in turn, can be used to develop better detergents and procedures or to make comparative studies to improve hygiene. The invention further enables the high sensitivity of the detection methods (eg PCR, RT-PCR) to effectively develop and evaluate new soiling bodies, new soiling, improved cleaning solutions or new cleaning machines.

In addition, the invention makes it possible, at least when using natural or synthetic nucleic acid markers (for example DNA, RNA or spiegelmer or their analogues), to also simulate or to make detectable the depletion of viruses and thus to reduce the risk of infection. Alternatively, however, the marker can also be used to simulate or evaluate drug release from a matrix (soiling), or to serve as evidence that decontamination of an article has occurred.

Fields of application and embodiments

It is envisaged to use the inventive soiling in particular in the field of medicine, laboratory, food, pharmaceutical and industrial technology, in order to allow a sensitive proof of a previously performed cleaning and / or disinfection. In this case, the stain filled as a solution, as a paste, as a cream o. The like. Are present, for example, hereby individually produce a Kontrollanschmutzung on surfaces. Preferably, to industrially produce a soiling on control surfaces and to use these in cleaning processes. For practical reasons, a color-visible contaminated surface contamination is preferred. It would also be conceivable a soiling for the clear identification of objects. This could, for example, be heavy or only depleted under certain conditions, or be a soiling for labeling that indicates that depletion has occurred, for example. B. has taken place by a cleaning.

The use is preferred where primarily medical instruments, industrial plants, parts of buildings or in the food industry equipment are contaminated with biomaterials or are. Here it is of interest to prove how well they can be cleaned. The mentioned instruments, devices or surfaces of any kind can be with proteins (albumins, collagens, fibrinogens, enzymes, etc.), bones, germs, cellular components, fats from biological sources or solid or soluble substances, particles or fibers from non-biological sources be contaminated. In the pharmaceutical industry, cells, germs or proteins that are processed in plants and / or propagated or synthesized, can pollute them. Impurities with substances which are deposited in cavities, such as pipes or channels, prove to be particularly difficult. In order to be able to safely remove them again, suitable test contaminants are required whose components can still be detected in the smallest amounts.

The following fields of application and embodiments are suitable for the inventive soiling: 1. Soiling of medical scissors or other medical objects and / or equipment

2. Soiling of items with threads or similar complex surfaces

3. Soiling of test surfaces or specimens

4. Soiling of tubes or instrument cavities

5. Stains from dishes or cutlery

6. Soiling of floors and walls that are subject to cleaning

7. Soiling of room air

8. Soiling of industrial equipment and parts thereof that are being cleaned

9. Soiling of laundry or tissues that are being cleaned

10. Soiling of all objects or products that are being transported or processed to demonstrate carry-over

11. Staining of objects to identify them clearly and / or which is difficult or defined deficient or should be gone after cleaning (hidden label for disposable products that must not be cleaned, decontamination proof).

12. Soiling of hands or other body surfaces (including hair) that are cleaned and / or disinfected

13. Soiling of medical equipment in order to be able to determine and / or rule out the risk of infection before and after treatment in animal experiments

The applications listed above can be extended to other applications, as the cleaning of objects and products is used in many areas of daily life.

The use of the stain always makes the task very sensible if it is applied to known surfaces under constant conditions. It is therefore recommended to apply the soiling in a targeted and controlled manner on control surfaces. Suitable surfaces are smooth or roughened or textured surfaces of metal or plastics. Possible control body surfaces provide screws, scissors, metal mesh, fabric parts, films or labels o. Ä. Alternatively, cavities can be contaminated with the soiling or here a test body be introduced. One of these stains was prepared according to Example 1 with stainless steel screws as a specimen and then cleaned in a washer-disinfector. The screws were used with a DNA amount of about 5 g (recommended 1 - 10 \ g) once in the presence of whole blood as a biomatrix and once without this matrix contaminated and then dried. The DNA still present on the screws after the rinsing procedure in a cleaning machine (standard cleaning method for medical instruments) was quantified by means of real-time RT-PCR.

FIG. 3 shows the result of a PCR analysis of stainless steel screws with and without biomatrix after cleaning (original data). The upper 3 lines (A, B and C) in Figure 3 show DNA levels in whole blood bound as biomatrix, the next 3 lines (D, E and F) show only DNA without blood. The bottom lines (bottom right) are controls. The analysis (see FIG. 3) shows that the biomatrix has an influence on the PRC analysis and that it can be stabilized and DNA amount still contained in the pg range can be dissolved out and analyzed. The result of the cleaned screws thus shows that concentrations of pg DNA quantities are detectable (in principle below the calibration curve, ie <10 femtogram) and that however a clear detection of these minimal traces is also possible (see calibration curve FIG. 4 with 5 pg , 2.5 pg, 1.25 pg, 0.625 pg, 0.3125 pg, 0.156 pg, 0.08 pg, 0.04 pg, 0.02 pg and 0.01 pg DNA concentrations).

FIG. 4 shows analysis results of a calibration curve with DNA concentration amounts of 5 to 0.01 pg (original data, running from left to right). The blood soiling of screws used here can be easily detached and is proof of the efficiency of the cleaning process used. However, for practical reasons, it may be desirable to use a difficult-to-remove biomatrix (eg, a bone meal containing it) to better compare the methods.

In the field of possible applications, however, the soiling can also be printed on paper, plastic or metallic surfaces in a screen or pad printing process, for example, and the coagulation can then be triggered by a spray method. Alternatively, only a spraying or dipping process is available to coat surfaces with soiling. It may be advantageous if the surface tension of the surfaces to be coated has been reduced. In this embodiment, a suitable surface is previously pretreated with a nano-coating or a silicate and only then follows the application of the soiling. The aim here is to achieve a very uniform layer thickness. Alternatively, a film-forming layer of water-soluble substances may first be applied or glued on before the soiling is applied. The latter layer can be used for the complete removal of residual soil. Alternatively, an abatement closing film on the stain or the foreign substances are to determine therefrom a release reaction of a substance contained here.

In another application, the soiling is applied to instrument surfaces with a brush, for example. The coagulation can be started promptly by adding thrombin in a spray process. Alternatively, a first or a cover end or final film-forming layer may be applied beforehand or thereafter by spraying or by brushing. For better solidification, in particular filiform substances may be present in small amounts in soiling. Only after drying of the soiling surface is exposed to a cleaning process.

Other applications result from the use of a soiling of albumin and heparin. Here, cleaned metal flakes are repeatedly dipped in an albumin-heparin solution in which the synthetic DNA marker is contained. Alternatively, silicone tubing or metal tubing or pieces thereof with known diameters and lengths are flushed through with the soil. The thus treated silicone tube is then divided into tube sections. Accordingly, polyurethane materials, from which, for example, catheters are produced, are coated. However, it is advisable to thoroughly clean the surfaces to be coated first.

An interesting field of application is the soiling of room air or of room and furniture surfaces. Here, a soak solution with film-forming substances and / or from mucin or a mucin-protein mixture containing the synthetic DNA marker is sprayed in a room or on furniture surfaces. Sprayed solution mist droplets are air dried allowing them to fall to the ground in areas of low turbulence or low air exchange rates. This creates an Arosol that can be distributed over a large area in the room through the ventilation or the circulating air or on furniture surfaces. Wherever the circulating air is filtered, it is possible to prove how well the filters work without having to burden the room with germs. The fallen to the ground Arosolpartikel can be recorded with a wiping technique and later analyzed by PCR.

Another application is given by the fact that on a label, a print (eg typeface), brushing o. Ä. A visible or hidden soiling area is applied to an object that may not be cleaned or reprocessed. If such a marked object is cleaned, the soiling is completely or partially removed and is therefore no longer or only slightly detectable. Alternatively, however, a soiling of an object can be used in a very targeted manner. which is tailored to demonstrate successful cleaning or treatment. In this case, the residual amount is visualized by means of a color marking and, in the case of relatively small amounts of soiling residues, via a DNA quantity determination, the depletion of the DNA marker is determined precisely. The successful purification is thus characterized by the fact that a dye in the stain or via at least one dye solution and / or a DNA amount determination of the cleaning success at least in the form of a depletion of dye and / or DNA is visible and / or measurable or a Prior cleaning can be excluded because no depletion has occurred.

In another application, the soiling is applied to implant surfaces, such as screws or metal plates, with the aim to examine in the subsequent cleaning process, whether a carryover of uncoated implants takes place with the biomaterials used.

Further advantages of the invention result from the choice of soiling substances, the structure of the soiling matrix and the marker concentrations used, as well as the combination with articles or materials which are to be subjected to at least one purification.

Hereinafter, embodiments of the invention will be described. Examples 1

Preparation of a durable, coagulable soiling solution

0.1 ml of 60 base pair synthetic DNA (about 0.01 μg / ml) is added to 100 ml of fresh normal plasma taken from a plasma pool of at least 5 people, and this solution is mixed. The fresh normal plasma contains 2 units heparin / ml or citrate or another anticoagulant. To prevent bacterial contamination, 0.1-0.0% sodium azide or an antibiotic may be added. Alternatively, the solution may be frozen to avoid the use of sodium azide. Furthermore, also unpolished normal plasma can be used. Preferably, the solution should be stored so that coagulation via thrombin can be initiated. Example 2

Production of a synthetic soiling or soiling surface

1 g of sodium carboxymethylcellulose (alternatively also alginates, methylcellulose, Eudragit E, S or RS) is dissolved in 5-20 ml of ethanol or other aqueous solutions and then stained with about 0.05 g of iron oxide red. The solution is added with 0.5-0.01 ml of a synthetic DNA or natural rare DNA (about 0.01 g / ml). Alternatively, for this approach, a 1-5 ml human plasma solution (undiluted) and / or 1 ml of an aqueous solution with about 10 ⁶ -Keime and / or 1- 10g solid or soluble foreign substances such. B. fibers or particles and / or medicinal agents in (μ - mg amounts) admixed. Subsequently, the solution or suspension or Elmulsion is applied or sprayed on surfaces one or more layers on surfaces.

Example 3

Preparation of a biological soiling solution for test surfaces

2 - 4 g of mucin is dissolved in 100 ml of distilled about 40 ° C warm water and this 0.1 ml of a synthetic DNA with 60 base pairs (about 0.01 mg / ml) stirred. Thereafter, 50-100 ml of fresh normal plasma is added and the solution is applied immediately to test surfaces. Subsequently, the test areas are dried. The whole can also be done under sterile conditions.

Example 4

Preparation of a coagulable soil solution containing insoluble matter To 100 ml of fresh normal plasma, taken from a plasma pool of at least 5 persons, add 0.1 - 0.5 ml of 50 or 100 base pair synthetic DNA (approximately 0.01 g / ml). added and mixed. Alternatively, frozen plasma or fresh and non-pooled plasma may be used. Preferably, human or animal plasma is used. To the solution, 1 - 10 g of bovine or porcine animal bone meal are stirred. The bone meal is previously ground finely through a bone mill. For further use, the solution is frozen or stored sterile and / or cool. Alternatively, a bacterial DNA or an insect DNA can also be used here.

Example 5

Preparation of a coagulable soiling solution with heparin

Solution A: A 1 - 10% albumin solution or a 5-25% human plasma solution is prepared with distilled water and this solution is adjusted to pH 4.0-5.0 with HCl. Solution B is given to this solution. Solution B: A heparin solution with 40-200 IU / ml is prepared with distilled water and adjusted to pH between 2.0-3.0 with HCL. Alternatively, undiluted fresh or frozen human or animal plasma may be used. In order to achieve high soiling concentrations, the resulting aggregates can be concentrated by centrifuging. Thereafter, a synthetic DNA marker is added so that the albumin-heparin solution contains a 0.01 to 0.0001% marker moiety.

Example 6

Production of a test specimen with test stains

A stainless steel sheet in the size of 1 x 6 cm is dipped terminal into a soak solution at room temperature according to Example 1 to 4. The immersion depth is about 3 to 4 cm. When using coagulable soiling agents (fibrinogen-containing solutions), the wetted surfaces are sprayed with a 2000 IU thrombin (human) and Ca solution. For drying, the plates are incubated in a drying oven for at least one hour at 30 ° C.

Example 7

Production of a test specimen with test stains

A stainless steel surface or a plastic surface in the size 1 x 6 cm, immersed terminally in a soiling solution from Example 1 to 5 about 3 - 4 cm and incubated for 5 - 10 minutes at room temperature. Subsequently, the immersed end is briefly immersed in distilled water and then dipped again in the soak solution and finally dried for one hour at RT or 30 ° C. The immersion depth should be 3 - 4 cm as in the first step. After completion of the dipping the stainless steel sheet is dried for at least 1 hour at 30 ° C.

Example 8

Method for checking the effectiveness of a cleaning success or decontamination

A test specimen or a test surface which contains a soiling is subjected to the usual cleaning cycles, for example in a cleaning machine. Thereafter, the test body or the test area is immersed in an alkaline solvent. The alkaline solvent (pH> 9) is a standard alkaline cleaning agent for medical cleaning machines. To aid in the detachment, the solution should be shaken and tempered. The amount of eluate to be used depends on the size of the test specimen or the test area used. After 5 to 10 minutes of elution, preferably at temperatures between 40-80 ° C (preferably also mechanically supported), the eluate is adjusted to a physiological pH with a HCL solution. The eluate can be purified by means of columns according to the molecular weight, concentrated and / or exempt from substances that interfere with the PCR. 1 to 100 μΙ of the eluate are used in an RT-PCR analysis (real time PCR). The PCR is carried out with common laboratory equipment. Two or three determinations are preferred.

Example 9

Double procedure for checking the effectiveness of cleaning success or decontamination

A test specimen or a test surface with a soiling containing, for example, heparin or a color-markable substance is subjected to the usual cleaning cycles in a cleaning machine, for example. In order to visualize the remainder of the soiling after the cleaning has been carried out, the test piece is dipped in a toluidine blue solution (10 mg toluidine blue / liter) or in another suitable dyestuff which can stain a substance in the soiling and after 10 to Rinsed at RT at least once with water for 60 minutes incubation. The blue coloration still indicates a residual soiling in the mg-g range. The sensitivity is dependent on the

Layer structure and to what extent an additional chemical crosslinking of the layers with a chemical crosslinker was made. Subsequently, the test body can be subjected to an RT-PCR method according to Example 8 to determine the remaining soiling via the DNA marker portion.

Claims

claims

1. staining with a marker, characterized in that the stain contains a marker which is a natural or synthetic nucleic acid, DNA, RNA or spiegelmer or an analog and is applied to surfaces or is, wherein the marker at least as a depletion detection of soiling A marker is used after a cleaning to allow at least an assessment of the cleaning success or a carryover or a decontamination or a substance release.

2. Staining with a marker, characterized in that the stain from a solidified Baren matrix contains the solid and / or soluble in aqueous solutions ingredients in which as markers at least one artificially produced nucleic acid (synthetic DNA) or synthetic nucleic acid analogues or RNA or spiegelmer which are not found in nature, or which contain as markers a natural DNA, RNA or spiegelmer, by the marker elutable from the solidified matrix by aqueous solutions, at least an assessment of purification success or decontamination or carryover or a substance release.

3. stain according to any one of the preceding claims, characterized in that the marker is a natural or synthetic single-stranded or double-stranded DNA or a DNA analogue o. The like., And / or from insects, plants, bacteria, viruses or from animals and at least 10 base pairs or the marker has at least 10 base pairs and is additionally associated with a bio-fabric, isotope or dye.

4. stain according to any one of the preceding claims, characterized in that the stain contains solid and / or soluble components and / or germs and / or dyes.

5. stain according to any one of the preceding claims, characterized in that it is at least one solidifiable solution, emulsion or suspension.

6. stain according to one of the preceding claims, characterized in that at least one thickening and / or a film-forming substance and / or a protein and / or protein mixture or an unhygienic substance and / or a medicinal agent is contained.

7. stain according to any one of the preceding claims, characterized in that the soiling is present as a solution, aerosol, paste, suspension, emulsion or spreadable mass and in a container or in a pen o. The like. Contained or applied to a surface.

A soil according to any one of the preceding claims, characterized in that the soiled bone fragments, hydroxyapatite (synthetic or xenogeneic), tricalcium phosphate (eg in combination with hydroxyapatite), calcium sulphate, glass-ceramic, dust, attrition, particles, fibers or polymers as solid constituents and / or fibrin and / or coagulable fibrinogen or a mixture of bio-materials.

9. Preparation of a soiling with a marker according to one of claims 1 to 8, characterized in that the soiling area or the soiling is applied or applied by a coating, dipping, spraying or printing process.

10. Preparation of a soiling according to one of the preceding claims, characterized in that the soiling is applied as a solution, emulision or suspension simultaneously in a one-step process or several times in succession in a multi-layer process on a surface.

11. Preparation of a soiling according to one of the preceding claims, characterized in that the matrix is a biomatrix and this contains at least proteins, germs, cells and / or constituents thereof or the biomatrix contains at least one biostuff or a biostable mixture, which is characterized by coagulation , Complexation, chemical crosslinking or drying solidified.

12. Preparation of a soiling according to one of the preceding claims, characterized in that product or biological surfaces such as containers, disposable products, instruments, silicone tubes, tubes, screws or mesh, tissue, foil, label, specimen surfaces with smooth , structured or roughened surface structures o. Ä. Surfaces, soiling.

13. A method for checking the effectiveness of a cleaning success, decontamination, carryover or substance release, characterized in that an object or specimen with a soiling or soiling according to one of claims 9 to 12 is used to at least deplete them in the first step and in a subsequent second step, at least by adding a dye or an enzyme, the remaining amount of the remaining marker or Marker complex made visible or the marker first detached from the surface and then with PCR o. The like. Is determined.

14. The method according to claim 13, characterized in that in the second step, at least with an acidic, alkaline and / or 30 - 100 ° C warm aqueous solution, the remaining amount of the soiling is removed from the surface.

15. The method according to any one of the preceding claims, characterized in that on a dye in the soiling or on at least one dye solution and / or a DNA amount determination of the cleaning success at least in the form of a depletion of dye and / or visible to DNA and / or measurable or a previous cleaning can be excluded because no depletion has taken place.