MXPA01001873A - Method and device for concentrating selected groups of microorganisms. - Google Patents

Abstract

A method and device are described to concentrate target organisms from a mixture of organisms. Beads (1) made of material such as nylon, polystyrene or glass are coated with antibodies of specific microorganisms. The beads (1) are contained in an enclosure (2) surrounded by grid material (4). The pore size of the grid is smaller than the size of the beads, to assure that the beads stay within the grid material and larger than the size of the microorganisms to allow the interaction of the microorganisms with the beads. A rod (5) is attached to the upper part of the enclosure (2) allowing the agitation of the device inside he growth medium containing the target organisms.

Description

METHOD AND DEVICE FOR CONCENTRATING SELECTED GROUPS OF MICROORGANISMS Antecedents-Field of Invention This application is based on the provisional patent application of the US. Serial No. 60 / 097,627 filed August 24, 1998. The present invention relates to products and processes used for the detection of microbes in a sample. More specifically, the present invention provides a method and device to aid in the detection of the presence of specific microbial contamination in food samples, clinical specimens and other products. Background - Prior Art It is necessary to test various substances, such as food, beverages, pharmaceuticals, cosmetics, water and body fluids for microbial contamination, especially with certain pathogenic bacteria. Recent outbreaks of foodborne diseases, involving a variety of foods contaminated with pathogenic bacteria such as E. coli 0157: H7 Salmonella, Listeria, Campylobacter Jejuni, and Ciclospora, have highlighted the need for rapid methods for microbiological analysis. Microbiological analysis is critical for safety and quality assessment, to determine manufacturing efficiency and compliance with regulations. The increased scope, significance and need for microbiological tests serve to reveal the limitations and disadvantages of conventional methods. Classical methods to determine the presence of pathogenic bacteria in samples typically take several days to complete. It is desired to provide rapid detection of especially pathogenic bacteria that cause diseases. Since the sensitivity suitable for most tests for pathogenic bacteria is less than one of these micro-organisms in 25 grams of product, most test methods are based on an initial enrichment step. The indigenous or native micro-flora that is usually present in many foods at high levels often interferes with selective isolation and identification of pathogenic bacteria. In the processing of foods such as heating, cooling, drying, freezing, addition of preservatives and other causes can sub-lethally damage the bacterial cells. These injured cells are extremely sensitive to the ingredients used in selective microbiological media. Therefore, in many trials, the process begins with pre-enrichment, where the sample is incubated in a nutritious, non-selective medium, to allow the resuscitation of injured or under-tensioned bacteria. This stage is followed by a stage of selective enrichment where the bacteria of interest are allowed to grow while the indigenous micro-flora is suppressed. The enrichment procedure is followed either by conventional coating methodology or by a variety of more modern and rapid methods such as DNA amplification or immunoassay. Therefore, it is desired to separate the target organisms from the remaining flora present in the product at an early stage. One such approach is the use of the immuno-magnetic separation technique that involves the use of specific immuno-magnetic particles for the target organisms. Magnetic beads with fixed antibodies on their surfaces are mixed with the sample containing the target organism. This organism will bind to the surfaces of the pearl by means of the antibodies. The organism-pearl complex is extracted from the solution by a magnet to concentrate the micro-organisms. The patent of the U.S.A. No. 4,230,685 discloses magnetic response micro-spheres having protein A associated with the outer surface. The microspheres are reacted with antibodies selective for cells, bacteria or viruses, to separate from a mixed population. The micro-organism will bind to the antibody and in this way to the microspheres, and the microspheres are then used in a magnetic separation process. The preferred microspheres are prepared from a mixture of albumin, protein A and magnetic particles. The microspheres are prepared in such a way that protein A is present on the outer surface of the antibody binding. The US patent . No. 4,695,393 describes a process for the preparation of these magnetic beads, which can be used to separate micro-organisms. The US patents . Nos. 5,491,068 and 5,695,946, describe a method characterized by capture of antibodies from the micro-organism of interest by the application of specialized magnetic beads. Involves the incubation of capture cells to form colonies; removal of colony material with a colony release membrane; and detection of the colony material in the membrane sheet by the use of labeled antibodies, PCR or nucleic acid probes. The main problem with this method is the low sensitivity of one organism per gram. This low sensitivity is inherent in the methodology and 50 to 100 times lower than the sensitivity desired for most food pathogens. The US patent . No. 4,677,055 discloses a process for concentrating bacteria using magnetic gel to which anti-specific antigenic determinant antibodies are coupled. It involves the steps of obtaining media containing organisms that possess antigenic determinants and bringing them into contact with particles of the magnetic gel. This step is followed by separation of the gel from the medium by magnetic means and inoculation in the new medium. In general, there are a number of problems associated with magnetic beads. A problem results from the small size of these beads (3-10 μm) and the large volume of the medium (250-3,000 ml). As a result, it is impossible to remove the magnetic beads from this large volume. Therefore, many procedures either use a smaller sample volume (thus reducing the sensitivity of the assay) or allow some time (8 to 18 hours) of pre-enrichment, followed by removal of 1 to 5 ml of solution for concentration of target organisms. Another problem associated with magnetic beads is the fact that they are coated with fat and protein, making it difficult to collect with a magnet. The process of separating the pearls from the medium and washing the unbound bacteria is labor intensive and creates a danger of contamination of both laboratory surfaces and pearls. OBJECTIVES AND ADVANTAGES Therefore, an object of the invention is to provide a method and device that can be used with a large volume of medium, to concentrate an objective organism. Another object of the invention is to provide a method that is less labor-intensive, faster and conducive to automation. Still further objectives and advantages will be from a consideration of the following description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a side view of the preferred device that is used to concentrate target organisms. Figure 2 shows a side view of another design of the device used to concentrate target organisms. Preferred Modality - Description Figure 1 shows the preferred embodiment of a device for the separation of target organisms from a suspension containing a mixture of organisms. Pearls 1 are made of materials such as nylon, polystyrene or glass. The beads are coated with antibodies to specific micro-organisms such as Salmonella, E. coli 0157: H7 and Listeria. A cylindrical enclosure 2 is designed to house pearls. The enclosure is constructed from a frame 3 that supports a grid 4 that covers the frame. The pore size of the grid is smaller than the size of the beads, to ensure that the beads remain within the enclosure 2. However, the pore size is large enough to allow bacteria to freely pass into the enclosure. A rod 5 is connected to the upper part of the enclosure. The rod 5 allows the enclosure 2 to move in the solution and for subsequent removal of the device from the solution. Figure 2 shows a different design of a device. The beads 11 coated with antibodies are contained in the enclosure 12 prepared from a grid 13, configured as a tea bag. A non-absorbent string by capillary action 14 is connected to the upper part of the enclosure 12 allowing room movement @ 12 in the solution, while not allowing the solution containing bacteria to absorb the string by capillary action. The pore size of the grid 13 is smaller than the size of the beads to ensure that the beads remain within the enclosure. Nevertheless, the pore size is large enough to allow bacteria to freely enter the enclosure. The food sample to be tested for the presence of the target organism is mixed with the appropriate pre-enrichment broth. The pre-enrichment broth is incubated at an appropriate temperature. At the start of the incubation period or alternatively after several hours of incubation, the enclosure 2 is immersed in the broth containing the sample, of this exposing the beads that have immobilized monoclonal or polyclonal antibodies to the selected bacteria of interest ?. This is achieved by lowering device 2 in the solution and shaking it for at least 30 minutes and up to several hours. This stage allows cell capture by the beads and the creation of a microbial-target-pearl cell complex. The next stage involves the separation of the beads with the target cells bound from the suspension containing the food particles and the other mixed flora. This is achieved by pulling the entire device out of the solution, using the rod 5. The device is subsequently washed several times in sterile saline or buffer solution. The wash solution is changed after each wash to remove unbound organisms. The addition of detergents such as TWEEN-20 (0.51-0.1% w / v) or protamine to the incubation broth mixture, usually decreases non-specific absorption. T EEN-20 can also be used in the washing procedure to remove non-specifically bound cells. After the washing step, a number of methods can be used to detect the presence of the target organism. Various detection methods may be reported in conjunction with the current invention to detect the presence of the micro-organism of interest. For example, the device can be inserted into a new culture broth that includes a dye indicator and changes in dye characteristics can be used to determine the presence or absence of the target organism. The micro-organisms do not need to be detached from the pearls since the connection to the pearls has no effect on their growth. Therefore, the cells can continue to multiply in the appropriate medium. Alternatively, the beads can be removed from the enclosure and inoculated onto the appropriate differential or selective agar surface. Another approach is to use an immunoassay. Most immunoassays require 103-105 mi-1 cells, therefore, the beads should contain enough cells to perform a direct immunoassay. Similarly, this method can be combined with DNA amplification and hybridization techniques such as PGR. As can be seen from the above description, the method of the invention is particularly characterized by the use of immunological beads contained in an enclosure for selecting target microorganisms from the sample. Pearls must be able to effectively capture the micro-organisms targeted by the test sample, while not capturing significant numbers of other organisms that may be present in much higher numbers. However, the antibody used does not need to be totally specific to the target organism since an additional selection step is available at the end of the assay. The antibodies must be oriented with their binding sites outward to allow contact between the binding portion of the antibody and the target organism. The size of the beads must be larger than the size of the micro-organism, to remain contained in the enclosure while allowing the target organism to enter the enclosure and connect with the pearls. The contact time between the beads and the target organism must be long enough to allow strong interaction. Several hours of interaction are found to produce the best results, meaning the creation of strong interactions to produce a high capture efficiency. After finishing the incubation stage, the pearls are removed from the solution, by removing the enclosure where they are contained. The enclosure and the beads are washed several times and the beads are transferred to the detection system. Conclusions, Branches and Scope Accordingly, it can be seen that the new method and device can be used with a large volume of medium, to concentrate an objective organism, without the need to use only a portion of the pre-enrichment broth or a small volume of enrichment broth as required for magnetic beads. The invention provides a method and device that is less labor-intensive, faster and conducive to automation. Very different designs to contain the pearls during the various stages of the test, can be used. Obviously, many modifications and variations of the present invention are possible in light of the above techniques. Although the previous description

Claims (13)

1. Contains many specificities, these will not be construed as limiting the scope of the invention but merely provide illustrations of some of the presently preferred embodiments of this invention. The invention may be practiced otherwise than as specifically described. CLAIMS 1. A device for separating specific target micro-organisms from suspension containing mixed groups of micro-organisms, characterized in that it comprises: a plurality of beads coated with at least one antibody material to capture the target micro-organisms; and an enclosure made of a grid material that circumscribes the pearls, with a pore size smaller than the size of the beads and larger than the size of the micro-organisms. The device according to claim 1, characterized in that the beads are made of resinous material. 3. The device according to claim 1, characterized in that the beads are made of non-resinous material. 4. The device according to claim 1, further comprising means for agitating the enclosure in the suspension. 5. The method for separating target micro-organisms from a suspension containing mixed groups of micro-organisms, characterized in that it comprises: immersing a plurality of beads coated with at least one antibody material in the suspension, the beads are held by an enclosure elaborated from a grid with a size of shapes smaller than the size of the pearls and larger than the size of the micro-organisms, in this way allowing the capture of the target micro-organisms by the pearls; and washing the beads to remove organisms not attached to the beads after removing the suspension enclosure. 6. The method according to claim 5, characterized in that at least one detergent is applied in the wash. The method according to claim 5, characterized in that it also comprises agitation of the enclosure containing the beads in the suspension. 8. The method according to claim 7, characterized in that the period of time of agitation is at least 30 minutes. 9. The method according to claim 7, characterized in that the agitation time period is extended by several hours. The method according to claim 5, characterized in that it includes the addition of at least one detergent to the suspension to decrease the absorption of non-specifically bound cells. The method according to claim 5, characterized in that it includes the subsequent step of immersing the enclosure and the beads in a new culture broth. 12. The method according to claim 11, characterized in that it includes the addition of an indicator material to the new culture broth. The method according to claim 5, characterized in that it includes the subsequent step of separating the beads from the enclosure followed by at least one test to reveal the micro-organisms of interest.