WO2000010702A1 - Method and device for concentrating selected groups of microorganisms - Google Patents

Method and device for concentrating selected groups of microorganisms

Info

Publication number
WO2000010702A1
WO2000010702A1 PCT/US1999/018618 US9918618W WO2000010702A1 WO 2000010702 A1 WO2000010702 A1 WO 2000010702A1 US 9918618 W US9918618 W US 9918618W WO 2000010702 A1 WO2000010702 A1 WO 2000010702A1
Authority
WO
Grant status
Application
Patent type
Prior art keywords
beads
target
enclosure
device
microorganisms
Prior art date
Application number
PCT/US1999/018618
Other languages
French (fr)
Inventor
Ruth F. Eden
Original Assignee
Eden Ruth F
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04Cell isolation or sorting
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02Separating microorganisms from their culture media
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911Bacteria
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911Bacteria
    • G01N33/56916Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia

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

Background ~ Field of Invention.

This application is based on provisional patent application 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 for aiding 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 foods, beverages, pharmaceuticals, cosmetics, water, and body fluids for microbial contamination, especially with certain pathogenic bacteria. Recent outbreaks of foodborne illness, implicating a variety of foods contaminated with pathogenic bacteria, such as E. coli 0157:H7, Salmonella, Listeria, Campylobacter jejuni, and Cyclospora, have underscored the need for rapid methods for microbiological analysis. Microbiological analysis is critical for assessment of safety and quality, to determine efficiency of manufacturing, and conformance with regulations.

The increased scope, significance, and need for microbiological testing served to reveal the limitations and drawbacks of conventional methods. Classical methods for determining the presence of pathogenic bacteria in samples are taking typically several days to perform. It is desired to provide rapid detection of especially pathogenic bacteria causing illnesses.

Since the desired sensitivity for most assays for pathogenic bacteria is less than one such organism in 25 grams of product, most testing methods rely on an initial enrichment step. The indigenous microflora that is usually present in many foods at high levels often interferes with the selective isolation and identification of pathogenic bacteria. Food processing such as heating, cooling, drying, freezing, addition of preservatives and other causes can sub-lethally injure bacterial cells. These injured cells are extremely sensitive to the ingredients used in selective microbiological media. Therefore, in many assays the process starts with pre-enrichment, in which the sample is incubated in a nutritious, non- selective medium to allow the resuscitation of injured or stressed bacteria. This step is followed by a selective enrichment step where the bacteria of interest are allowed to grow while the indigenous microflora is suppressed. The enrichment procedure is followed either by conventional plating methodology or a variety of more modern and rapid methods such as DNA amplification or immunoassay. It is therefore desired to separate at an early stage the target organisms from the other flora present in the product. One such approach is the utilization of the immuno-magnetic separation technique, involving the utilization of immuno-magnetic particles specific for the target organisms. Magnetic beads with antibodies affixed to their surfaces are mixed with the sample containing the target organism. This organism will bind to the bead surfaces via the antibodies. The organism-bead complex is pulled out of the solution by a magnet, to concentrate the microorganisms.

U.S. patent 4,230,685 describes magnetically responsive microspheres having protein A associated with the outer surface. The microspheres are reacted with antibodies selective to cells, bacteria or viruses to be separated from a mixed population. The microorganism will attach to the antibody and thereby to the microspheres, and the microspheres are then used in a magnetic separation procedure. The preferred microspheres are prepared from a mixture of albumin, Protein A, and magnetic particles. The microspheres are prepared so that the Protein A is present in the exterior surface of the antibody binding. U.S. patent 4,695,393 describes a process for the preparation of such magnetic beads, which can be used in separation of microorganisms.

U.S. patents 5,491 ,068 and 5,695,946 describe a method characterized by antibody capture of the organism of interest by the application of specialized magnetic beads. It entails the incubation of the capture cells to form colonies; removal of material from the colonies with colony lift membrane; and detection of the colony material on 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 is 50-100 fold lower than the desired sensitivity for most food pathogens.

U.S. patent 4,677,055 describes a process for concentrating bacteria utilizing magnetic gel to which anti-specific antigenic determinant antibodies are coupled. It involves the steps of obtaining medium containing the organisms possessing specific antigenic determinants and bringing them in contact with particles of the magnetic gel. This step is followed by the separation of the gel from the medium by magnetic means and inoculation into new medium. In general there are a number of problems associated with magnetic beads.

One such problem results from the small size of such 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 such a large volume. Therefore, many procedures either use a lower sample volume (thereby reducing the sensitivity of the assay) or allow some time (8-18 hours) of pre-enrichment followed by the removal of 1-5 ml of solution for concentration of the target organisms. Another problem associated with the magnetic beads is the fact that they get coated with fat and proteins making it difficult to be collected with a magnet. The process of separating the beads from the medium and washing the unattached bacteria is labor intensive, and creates a contamination hazard of both laboratory surfaces and the beads.

Objects and Advantages.

It is, therefore, an object of the invention to provide a method and device that can be utilized with a large volume of media, to concentrate a target organism. It is another object of the invention to provide a method that is less labor intensive, more rapid and will lend itself to automation.

Still further objects and advantages will become apparent from a consideration of the ensuing description and accompanying drawings.

Brief Description of the Drawings. Fig. 1 shows a side view of the preferred device utilized to concentrate target organisms. Fig. 2 shows a side view of another design of the device utilized to concentrate target organisms.

Preferred Embodiment -- Description.

Figure 1 shows the preferred embodiment of a device for the separation of the target organisms from a suspension containing a mixture of organisms. Beads 1 are made of materials such as nylon, polystyrene or glass. The beads are coated with antibodies to specific microorganisms such as Salmonella, E. coli 0157:H7 and Listeria. A cylindrical enclosure 2 is designed to contain the beads. The enclosure is constructed from a frame 3 supporting a grid 4 covering the frame. The grid's pore size is smaller than the size of the beads to assure that the beads stay within the enclosure 2. However, the pore size is large enough to allow bacteria to freely pass into the enclosure. A rod 5 is attached 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 the device. The beads 11 coated with antibodies are contained in the enclosure 12 made of a grid 13, shaped like a tea bag. A non-wicking string 14 is attached to the upper part of the enclosure 12 allowing movement of the enclosure 12 in the solution, while disallowing the solution containing bacteria to wick up the string. The grid's 13 pore size is smaller than the size of the beads to assure that the beads stay within the enclosure. However, the pore size is large enough to allow bacteria to freely pass into 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. Upon the beginning of the incubation period, or alternatively after several hours of incubation, the enclosure 2 is immersed into the broth containing the sample thereby exposing the beads having immobilized thereon monoclonal or polyclonal antibodies to the selected bacteria of interest. This is accomplished by lowering the device 2 into the solution and agitating it for at least 30 minutes and up to several hours. This step allows cell capture by the beads, and the creation of bead-target microbial cell complexes. The next step involves the separation of the beads with the bound target cells from the suspension containing the food particles and other mixed flora. This is accomplished by pulling the whole device out of the solution, using the rod 5. The device is subsequently washed several times in sterile saline or buffer solution. The washing solution is changed after each wash to remove non-bound organisms. Addition of detergents such as Tween-20 (0.51-0.1 % w/v) or protamine to the incubation broth mixture usually decreases the non-specific adsorption. Tween-20 can be also used in the washing procedure to remove non- specifically bound cells. After the wash step a number of methods can be utilized to detect the presence of the target organism. Several detection procedures can be used in conjunction with the current invention to detect the presence of the microorganism of interest. For example, the device can be inserted into a new growth broth that includes a dye indicator and the changes in the dye characteristics can be utilized to determine presence or absence of the target organism. The microorganisms do not need to be detached from the beads since attachment to the beads has no effect on their growth. Therefore cells can continue to multiply in the appropriate medium. Alternatively the beads can be removed from the enclosure and inoculated onto the surface of appropriate selective or differential agar. Another approach is to utilize an immunoassay. Most immunoassays require lO O5 cells ml"l, therefore the beads should contain enough cells to perform a direct immunoassay. Similarly, this method can be combined with DNA hybridization and amplification techniques such as PCR.

As can be seen from the above disclosure, the method of the invention is particularly characterized by the use of immunological beads contained in an enclosure to select out target microorganisms from the sample. The beads must be capable of effectively capturing the target microorganisms from the test sample, while not capturing significant numbers of other organisms that might be present at much higher numbers. However, the antibody used need not 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 microorganism, to remain contained in the enclosure, while allowing the target organism to enter the enclosure and attach to the beads. The contact time between the beads and the target organism must be long enough to allow strong interaction. Several hours of interaction was found to yield the best results, i.e. the creation of strong interactions to produce high capture efficiency. After the completion of the incubation step the beads are removed from the solution, by the removal of the enclosure in which they are contained. The enclosure and the beads are washed several times, and the beads are transferred into the detection system.

Conclusions, Ramifications, and Scope.

Accordingly, it can be seen that the new method and device can be utilized with a large volume of media, to concentrate a target organism, without the need to utilize 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, more rapid and lends itself to automation. Many different designs, for containing the beads during the various steps of the assay, can be utilized.

Obviously, many modifications and variations of the present invention are possible in light of the above techniques. Although the description above contains many specificities, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of this invention. The invention may be practiced otherwise than as specifically described.

Claims

What is claimed is:
1. A device for separating specific target microorganisms from suspension containing mixed groups of microorganisms, comprising: a plurality of beads coated with at least one antibody material to capture the target microorganisms; and an enclosure made of a grid material enclosing said beads, with a pore size smaller than the size of said beads and larger than the size of the microorganisms.
2. The device of claim 1 wherein said beads are made of resinous material.
3. The device of claim 1 wherein said beads are made of non resinous material.
4. The device of claim 1 further comprising means to agitate said enclosure in the suspension.
5. A method of separating target microorganisms from a suspension containing mixed groups of microorganisms, comprising: immersing a plurality of beads coated with at least one antibody material into the suspension, said beads being held by an enclosure made of a grid with a pore size smaller than the size of said beads and larger than the size of the microorganisms, thereby allowing the capture of the target microorganisms by said beads; and washing said beads to remove organisms not bounded to said beads, after pulling said enclosure from the suspension.
6. The method of claim 5 wherein at least one detergent is applied in said washing.
7. The method of claim 5 further comprising agitation of said enclosure holding said beads in the suspension.
PCT/US1999/018618 1998-08-24 1999-08-23 Method and device for concentrating selected groups of microorganisms WO2000010702A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US9762798 true 1998-08-24 1998-08-24
US60/097,627 1998-08-24

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US09763578 US7071005B1 (en) 1998-08-24 1999-08-23 Method and device for concentrating selected groups of microorganisms
CA 2342108 CA2342108A1 (en) 1998-08-24 1999-08-23 Method and device for concentrating selected groups of microorganisms
EP19990941184 EP1117476A4 (en) 1998-08-24 1999-08-23 Method and device for concentrating selected groups of microorganisms

Publications (1)

Publication Number Publication Date
WO2000010702A1 true true WO2000010702A1 (en) 2000-03-02

Family

ID=22264348

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/018618 WO2000010702A1 (en) 1998-08-24 1999-08-23 Method and device for concentrating selected groups of microorganisms

Country Status (4)

Country Link
EP (1) EP1117476A4 (en)
CN (1) CN1320060A (en)
CA (1) CA2342108A1 (en)
WO (1) WO2000010702A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006053187A3 (en) * 2004-11-12 2006-12-07 Promega Corp Device and method for purification of biological materials

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101215598B (en) 2008-01-11 2010-11-17 中华人民共和国吉林出入境检验检疫局 Method for detecting bacteria by using immunity enrichment thread, and immunity enrichment brush
WO2010114725A1 (en) * 2009-04-03 2010-10-07 3M Innovative Properties Company Microorganism concentration process and device

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US3840345A (en) * 1972-06-23 1974-10-08 Co Ind De Procedes & D Applic Self agglomerating fluidized bed reacting apparatus
US4931401A (en) * 1988-09-01 1990-06-05 La Societe De Recherche Snc Inc. Bioreactor
US5009852A (en) * 1987-12-11 1991-04-23 Charbonnages De France Cooled fluidization grid
US5186824A (en) * 1991-09-04 1993-02-16 Large Scale Biology Corporation System for solid phase reactions
US5776710A (en) * 1992-10-30 1998-07-07 Becton Dickinson And Co. Assay of blood or other biologic samples for target analytes

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US3970518A (en) * 1975-07-01 1976-07-20 General Electric Company Magnetic separation of biological particles
US4230685A (en) * 1979-02-28 1980-10-28 Northwestern University Method of magnetic separation of cells and the like, and microspheres for use therein
DE3617672C2 (en) * 1986-05-26 2000-02-17 Claus Heuck A process for the preparation of a reagent, then a prepared reagent and its use for attachment of polymers and microorganisms from aqueous solutions
US4910148A (en) * 1987-02-10 1990-03-20 Dynal A. S. Magnetic separation of magnetized particles from biological fluids
US5175093A (en) * 1989-11-07 1992-12-29 Lehigh University Bioactive cells immobilized in alginate beads containing voids formed with polyethylene glycol

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3840345A (en) * 1972-06-23 1974-10-08 Co Ind De Procedes & D Applic Self agglomerating fluidized bed reacting apparatus
US5009852A (en) * 1987-12-11 1991-04-23 Charbonnages De France Cooled fluidization grid
US4931401A (en) * 1988-09-01 1990-06-05 La Societe De Recherche Snc Inc. Bioreactor
US5186824A (en) * 1991-09-04 1993-02-16 Large Scale Biology Corporation System for solid phase reactions
US5776710A (en) * 1992-10-30 1998-07-07 Becton Dickinson And Co. Assay of blood or other biologic samples for target analytes

Non-Patent Citations (1)

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Title
See also references of EP1117476A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006053187A3 (en) * 2004-11-12 2006-12-07 Promega Corp Device and method for purification of biological materials

Also Published As

Publication number Publication date Type
CA2342108A1 (en) 2000-03-02 application
EP1117476A4 (en) 2007-08-22 application
CN1320060A (en) 2001-10-31 application
EP1117476A1 (en) 2001-07-25 application

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