Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1998-10-27
2001-02-20
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S283100, C435S030000, C435S004000
Reexamination Certificate
active
06190878
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the field of assay technology, and in particular embodiments, to devices and methods for quantification of analytes, e.g., biological material, in a sample.
Many industries need to detect and quantify the concentration and level of biological material or other analyte in a sample. For example, the determination of bacterial concentration in food and water is an essential part of food and water quality testing. EPA regulations require that no coliform such as
Escherichia coli
can be present in potable water. The “presence/absence” format of a testing medium, such as Colilert® chemical mixture (IDEXX Laboratories, ME) which is used as a testing medium for
Escherichia coli
and all coliform bacteria, is very useful in making this determination. Colilert® chemical mixture is based on the Defined Substrate Technology described in Edberg, “Method and Medium for use in Detecting Target Microbes In Situ in A Specimen Sample of A Possibly Contaminated Material,” U.S. Pat. Nos. 4,925,789 and 5,492,933.
However, there are areas where the quantification, not just the detection, of bacterial concentration is important. Examples of such areas include waste water, incoming water in water purification systems, surface water, and food testing. For example, numerous restaurant chains will only accept raw ground beef or poultry that contains less than a certain concentration of bacterial contamination. Therefore, food processing plants must carry out the necessary microbiological tests to determine the bacterial concentration of these food items before they can be released to customers.
The classical methods of quantification of biological material are the standard plate count method or the multiple tube fermentation (MTF) method. A quantity of sample being tested for microbial contamination is first dispensed in a Petri dish. Then 15 ml of the appropriate media is poured over the sample. The Petri-dish is then swirled to mix the sample in the medium and the Petri-dish is left to solidify at room temperature for approximately 20 minutes. The medium is then incubated at a specific temperature for a specific time, and any resulting colonies are counted.
The multiple tube fermentation method is described in Recles et al., “Most Probable Number Techniques” published in “Compendium of Methods for the Microbiological Examination of Foods”, 3rd ed. 1992, at pages 105-199, and in Greenberg et al., “Standard Methods For the Examination of Water and Wastewater” 8th ed. 1992). In this method, a volume of sample is dispensed into several tubes representing this dilution range. The tubes are then incubated at the appropriate temperature so that the bacteria in each tube are allowed to grow. After incubation at a specific temperature for a specific time, the number of positive tubes is counted. The most probable number can be determined from the formula described in Recles et al., supra.
Water testing is mostly done by membrane filtration, where a certain volume of water is passed through the membrane and the membrane is incubated in a medium for a certain period of time. After appropriate incubation, the colonies are counted.
In many industries there is also a need to qualitatively and/or quantitatively detect the presence of an analyte in a liquid solution. For example, the detection of inorganic ions may be important in a manufacturing process using a test solution.
Heretofore, the methods and devices generally relied upon for measuring an analyte in solution have either required removing all of an aliquot from the test solution or exposing a dip stick to a test solution. Although these methods and devices can detect an analyte in solution, they suffer from a number of disadvantages.
The dip stick-related methods are not quantitative. For example, in the general dip stick embodiments there is no capability for determining or quantifying the presence of an analyte in a unit volume of that solution. Instead, the dip stick is simply brought into contact with the test solution.
Other methods require a user to manually remove an aliquot from a test solution and transfer it to a separate device for detection or quantification of the analyte.
Therefore, despite the ability of these methods and devices to detect an analyte in solution, the methods and devices currently used have proven to have limited accuracy; and/or to be costly, complicated, time consuming; and/or to have a limited range of uses because of the particular assay technology.
Thus, there exists a need for a simple, accurate, and inexpensive method for the determination of an analyte in solution without the disadvantages known in the prior art. In particular, there is a need for devices and methods capable of providing a quantitative assay of an analyte in a particular volume of a solution.
SUMMARY OF INVENTION
The present invention provides devices and methods for detecting and enumerating the presence or absence of biological materials, analytes, and microorganisms in sample solutions.
In one aspect, the invention provides a sterile incubation plate for determining the presence or amount of a biological material in a test sample. The plate is generally described by a flat, horizontal surface containing recessed wells. Each well is adapted to hold an aliquot of liquid, and has a size and shape, and is made of a material, suitable for holding the liquid aliquot within the well by the forces of surface tension. At least one well contains at least one reagent for the detection of the biological material. No positive response is generated in the absence of the target biological material.
In a preferred embodiment, the reagent is deposited into the wells by corona treatment and drying. In another embodiment, the plate may also contain a lid. The well or wells may contain a plurality of reagents, and different wells may contain different reagents or different combinations of reagents, so that numerous assays can be conducted on a single plate. The plate is preferably constructed of plastic, however, it may be constructed of other hydrophobic material(s) which are suitable for conducting the assay. In a preferred embodiment, the plates of the present invention will be rectangular in shape, however, they may also be circular in shape, or of any shape. In a preferred embodiment the wells are about 0.15 inches in diameter. In another preferred embodiment, the wells of the plate hold a total of about 1-milliliter of solution. In another preferred embodiment, each well of the plate holds between 0.1 and 100 &mgr;l of liquid. The well or wells of the plate may be chamfered to aid in the removal of excess fluid. The plate may further comprise a handle portion so that the operator may manipulate the plate and conduct the assay without risk of contacting the test sample.
In another aspect, the invention provides a sterile incubation plate similar to the plate described above, and may be adapted with each of its embodiments, with the further feature that it comprises a lid which contains at least one protrusion(s) which fit(s) into the well(s). In this aspect, the reagent or combination of reagents is contained on the end of the protrusion(s) such that the reagent(s) will dissolve in the test sample upon closing or affixing the lid to the plate. In one embodiment, the protrusions may feature a cavity. The reagent(s) or combination of reagents will be dried onto the end of each protrusion or onto the surface of the cavity. The surface of the cavity can also be corona treated before depositing the reagent. In this aspect as well, a reagent(s) or combination(s) of reagents can be dried on, or the protrusion(s) corona treated, and a combination of reagents may be utilized among multiple protrusions so that multiple assays can be conducted on a single plate. The lower plate portion can be adapted to all of the embodiments with respect to the plate described above.
In another aspect, the invention describes a device for determining the presence or amount of an analyte(s) or microorganism(s) in a test solution. The
Pierson Mark W.
Townsend David
Consalvi Mary S.
Howrey Simon Arnold & White , LLP
IDEXX Laboratories, Inc.
Leary Louise N.
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