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Detection and identification of bacterial strainsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Virus Or BacteriophageDetection and identification of bacterial strains description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070042357, Detection and identification of bacterial strains. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0001] I. Field of the Invention [0002] The present invention relates to a method to detect bacteria, the method comprising the following steps: coupling bacteriophages and/or bacteriophage proteins to a support, incubating the support coupled to the bacteriophages and/or bacteriophage proteins with a sample, optionally removing the sample and the bacteria in the sample not bound to the bacteriophages and/or bacteriophage proteins, optionally adding substances permeabilizing or destroying the bacterial membrane, and detecting the bacteria of the sample bound to the bacteriophages and/or bacteriophage proteins, wherein the bound bacteria are not subjected to any cultivation step. [0003] II. Related Art [0004] The rapid and exact detection of bacteria is the first essential step for the diagnosis and treatment of a bacterial infection in human and animals as well as to initiate preventive measures. Furthermore, the detection is useful to control hygienic and quality of raw materials and processed foodstuff and for the control of hygienic and quality of fresh water and washing water and of water quality of public pools. Additionally, the detection is useful for process monitoring and optimization and for quality control in environmental analytics. Quite in contrast to most of the previously applied procedures, the method described herein also allows a simple detection at the place of need. [0005] The detection of bacteria in biological samples in most cases occurs by means of a combination of cultivating methods by monitoring metabolic activities. For the purpose of phage-typing of bacterial strains of one type of bacteria cultivating methods having a sensitivity for bacteria are coupled to typing bacteriophages. This method involves a dense bacterial lawn on an agar plate of the sample to be analyzed which is overlaid with a suspension of bacteriophages in soft agar, said bacterial lawn having been obtained by isolating a single colony, and subsequent multiplication of said colony. The result is obtained after incubation overnight at the optimum bacterial growth temperature, which usually is 37.degree. C. in most cases, by counting the plaques and by the control of the plaque morphology. A typing variant considers the measurement of adenylate kinase subsequent to phage-mediated cell lysis. In this method, an overnight culture of the bacteria to be analyzed is diluted in buffer, phages are added to it, and lysis is measured by means of specific phages per adenylate kinase activity. [0006] In all methods described thus far, detection does not occur prior to lysis, or detection occurs via lysis. This allows monitoring of sources of infects and detection of sources of infection. This typing has been established for years in regard of numerous bacteria such as Salmonella typhi, Salmonella paratyphi B, Staphylococcus aureus, Pseudomonas aeruginosa as well as a number of further bacteria. These established detection methods yield a result only after several days in most cases. However, on the other hand, it is the rapid and exact determination of the type of bacteria (typing) that is of great importance for a rapid reaction. [0007] Recently, more rapid molecular biological detection methods such as the polymerase chain reaction have been employed, which methods have the drawback, however, that they are more prone to contaminations. Likewise, with these methods the result is regularly available only after one day. [0008] Furthermore, identification of the bacterial genus in some cases even requires the submission of samples to highly specialized reference laboratories, likewise resulting in a time and cost intensive factor. SUMMARY OF THE INVENTION [0009] Accordingly, the invention is based on the object to provide a rapid and economic detection method for bacteria, which method can be carried out by especially microbiologically trained staff in the laboratory on the one hand and on the other hand also in a simplified modification at the place of need and without the corresponding previous knowledge. The object is solved by the subject matter defined in the claims. [0010] One aspect of the present invention is thus a rapid and exact detection system for bacteria providing information on the type of bacteria and the bacterial strain, and optionally allowing a quantification of the bacteria, which detection system is based on the recognition of these bacteria by bacteriophages or bacteriophage proteins. [0011] The conventional detection methods for bacteria based on bacteriophages include time-consuming cultivation steps and the bacteriophage-mediated lysis of the cells. It is true that the method according to the present invention also utilizes the specific recognition of cells by bacteriophages but in contrast to the thus far described methods, subsequent to the step of specific cell recognition and subsequent to the separation of unspecifically bound bacteria a corresponding binding assay is performed, for example measurement of a spectroscopic (e.g., by means of absorption, fluorescence, bio- or chemiluminescence, or circular dichroism) or electric (e.g., by means of measuring the capacity or change of the electric conductivity) signal change. This enables a detection of the bacteria after a few minutes already rather than after hours and days, respectively, as enabled previously. By a targeted coupling, in particular by a covalent fixation of bacteriophages to suitable supporting structures, e.g., to microtiter plates, test stripes, slides, wafer, filter materials, or flow through cell chambers, the procedural step of the binding assay is favored by a reduction of the unspecific background, and enables a broad application for all bacteria. The supporting structures may consist, as an example, of polystyrene, polypropylene, polycarbonate, PMMA, cellulose acetate, nitrocellulose, glass, silicium wafer. Employment of the method according to the present invention furthermore enables the use of lysogenic bacteriophages for the detection of bacteria. [0012] One aspect of the present invention is therefore the provision of a method to detect bacteria, the method comprising the following steps: coupling of bacteriophages and/or bacteriophage proteins to a support, incubating the support coupled to the bacteriophages and/or bacteriophage proteins with a sample, optionally removing the sample and the bacteria of the sample not bound to the bacteriophages and/or bacteriophage proteins, optionally adding substances permeabilizing or destroying the bacterial membrane, and detecting the bacteria in the sample bound to the bacteriophages and/or bacteriophage proteins, wherein the bacteria bound are not subjected to any cultivation step. [0013] Preferred is a method wherein the detection is carried out by means of a colorimetric detection of cellular components and/or products of the phage reproduction, by means of a detection of DNA and/or RNA or by means of an immunoassay. Also preferred is a method wherein the bacteriophages and/or bacteriophage proteins are coupled to the supports by means of adsorption or by means of a chemical bond. An additionally preferred method is a method, wherein the bacteriophages and/or the bacteriophage proteins exhibit modifications. A further preferred method is a method, wherein at least two different bacteriophages and/or bacteriophage proteins recognizing at least two different types and/or genera of bacteria are employed. A further preferred method is a method, wherein the support is, e.g., a microtiter plate, test stripes, slides, wafer, filter material, or a flow-through cell chamber and, e.g., consists of polystyrene, polypropylene, polycarbonate, PMMA, cellulose acetate, nitrocellulose, glass, or silicium wafer. [0014] Bacteriophages specific for the bacteria to be detected desirably are employed for the detection. The phages need not to be specific for only one type of bacteria but may be specific for several types of bacteria or for a bacterial genus. Which phages are employed for the detection depends on which bacteria are to be detected. Furthermore, two or more phages may be used in a single detection method to simultaneously detect several types of bacteria or to type a genus of bacteria exactly. The bacteriophages used may be commercially available bacteriophages from stock collections such as DSM or ATCC, or bacteriophages specifically isolated for this purpose. Both lytic and lysogenic bacteriophages may be employed, the lytic phages being preferred. Their morphologic properties do not limit the phages to be selected, myoviridae (T4-like phages), siphoviridae ((-like phages) or podoviridae (T7-, P22-like phages) being preferred, however. DETAILED DESCRIPTION OF THE INVENTION [0015] Phages bind the corresponding receptors of the bacteria, resulting in a protein-protein or protein-carbohydrate, or protein-lipid interaction. Subsequent to the highly specific recognition of its hosts the phage injects its genetic information (single-stranded or double-stranded DNA or RNA) into the cell and is either present in its lysogenic form or produces, in case of lysis, new phage particles. The bacterial injection of the nucleic acid of the phages causes the binding of the bacteria to the phages, in most cases in an irreversible manner. According to the method of the present invention, after finalization of the recognition step, the detection of the bacteria will follow. This method is basically applicable to all bacteria, for which phages have been described or can be isolated. Preferred bacteria are bacteria that are relevant for food industry, medicine, or environmental analytics, such as lactic acid bacteria, e.g., leuconostoc, pseudomonas, and enterobacteria, e.g., E. coli, salmonella. The step of recognition can be carried out at any temperature ranging from 0.degree. C. to 90.degree. C., preferably at a temperature ranging from 4.degree. C. to 45.degree. C., particularly preferred at a temperature ranging from 15.degree. C. to 37.degree. C., more particularly preferred at a temperature ranging from 20.degree. C. to 37.degree. C., even more particularly preferred at room temperature. [0016] Additionally, it is possible, to isolate and use for the detection distinct phage proteins, e.g., phage receptors, phage adhesines, or portions thereof, e.g., p12 of T4 or p9-tailspike of P22, or variants of these proteins rather than complete phages. Preferred are adhesines irreversibly binding to bacteria, or adhesines the bacterial binding pocket of which has been modified by recombinant or chemical techniques in order to accomplish an irreversible binding. An example for recombinantly modified phage proteins are the "active-site mutants" of the P22-tailspike (cf. Baxa et al., Biophys. J. 71, 2040-2048; 1996). Phage proteins as well as bacteriophages may be used for the method of the present invention. [0017] The bacteriophages and/or bacteriophage proteins used according to the present invention may be adopted to the supporting structures in their host specificity and their binding properties, respectively, by a directed or random mutagenesis. Mutagenesis introduces mutations that can be amino acid additions, deletions, substitutions, or chemical modifications. These mutations have the effect to modify the amino acid sequence in the binding region of the phages or phage proteins aiming at an adaptation of specificity and binding affinity to the assay requirements, e.g. to render the binding of the bacteria to the isolated phage proteins irreversible in order to improve the options to wash. In addition, a recombinant or biochemical modification of the phage proteins may be performed in order to accomplish a switch-off of the enzymatic activity optionally present, thereby improving the binding or rendering it irreversible. [0018] For the purpose of the detection according to the present invention the phages or phage proteins are immobilised on suitable supporting structures, e.g., microtiter plates, test stripes, slides, wafers, filter materials, or flow-through cell chambers. The supporting structures may consist of, e.g., polystyrene, polypropylene, polycarbonate, PMMA, cellulose acetate, nitrocellulose, glass, silicium wafer. The immobilization may be accomplished by adsorption or by covalent binding, wherein the covalent binding is preferred. It is relevant that immobilization is a functional one, that is, the phages and phage proteins, respectively, exhibit structures accessible for bacteria although they are bound to the support material. [0019] In order to suppress an unspecific reaction of the bacteria to be investigated with the support material a blocking with bovine serum albumin or Tween 20 or substances that are likewise employed in ELISAs, such as milk powder, may be performed. Furthermore, to increase the efficiency of the adsorption, the support systems may be pre-coated with suitable proteins (e.g., specific antibodies against phage proteins or unspecific proteins such as BSA) peptides, saccharides, (e.g., mono-, oligo-, or polysaccharides) or detergents (e.g., Tween 20 or octylglucoside). These coatings may occur overnight at a temperature ranging from 4.degree. C. to 20.degree. C. or within a period of 2 h to 4 h at a temperature of 30.degree. C. to 65.degree. C. Subsequently the excess liquid is removed, and the supporting structure dried at about 60-70.degree. C. The basic coating is to guarantee adsorption of functional phages or phage proteins on the one hand and, on the other hand, to prevent an unspecific adsorption of the test bacteria to the supporting structure, thereby increasing the efficiency of the assay. Following the basic coating, the phages or phage proteins are applied by applying an aqueous buffered solution of the phages or phage proteins to the pre-treated supporting structure. After an adsorption at 4-20.degree. C. overnight or at 30-65.degree. C. for a period of 2-4 hrs the coating solution is removed and the supporting structure is dried as described above. In order to increase the coating efficiency, a covalent fixation of the phages or phage proteins with chemical crosslinkers such as glutaric aldehyde may be performed subsequently. [0020] A phage display approach (cf. Gene, 1998, 215, 439-444), wherein peptides are expressed on the phage head protein or on the capsid proteins, which peptides have defined binding properties for particular supporting systems, may be employed with the phages used, e.g., with myoviridae, siphoviridae and podoviridae, in order to improve the functional immobilization. [0021] The immobilization of the phages and phage proteins to the supporting material by means of adsorption may be performed by incubating a phage solution in aqueous buffer, e.g., 100 mM Tris, pH 7.3 or 100 mM sodium phosphate, pH 7.5, over several hours or over night at 5.degree. C. to 45.degree. C., preferably at 15.degree. C. to 37.degree. C., more preferably at 20.degree. C. to 37.degree. C., still more preferably at room temperature. Continue reading about Detection and identification of bacterial strains... 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