Patent Grant
6910720
The present invention relates to a connector for use in combining the contents of a pair of containers in a closed system. The present invention further relates to a kit comprising the connector and a pair of containers having distinct contents to be combind.
All references referred to herein are hereby incorporated by reference in their entirety. The incorporation of these references, standing alone, should not be construed as an assertion or admission by the inventors that any portion of the contents of all of these references, or any particular reference, is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the inventors reserve the right to rely upon any of such references, where appropriate, for providing material deemed essential to the claimed invention by an examining authority or court. No reference referred to herein is admitted to be prior art to the claimed invention.
Procedures for determining the presence or absence of specific organisms or viruses in a test sample commonly rely upon nucleic acid-based probe testing. To increase the sensitivity of these tests, an amplification step is often included to increase the number of potential nucleic acid target sequences present in the test sample. During amplification, polynucleotide chains containing the target sequence or its complement are synthesized in a template-dependent manner from ribonucleoside or deoxynucleoside triphosphates using nucleotidyltransferases known as polymerases. There are many amplification procedures in common use today, including the polymerase chain reaction (PCR), Q-beta replicase, self-sustained sequence replication (3SR), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), ligase chain reaction (LCR), strand displacement amplification (SDA) and loop-mediated isothermal amplification (LAMP), each of which is well known in the art. See, e.g., Mullis, “Process for Amplifying Nucleic Acid Sequences,” U.S. Pat. No. 4,683,202; Erlich et al., “Kits for Amplifying and Detecting Nucleic Acid Sequences,” U.S. Pat. No. 6,197,563; Walker et al., Nucleic Acids Res., 20:1691-1696 (1992); Fahy et al., “Self-sustained Sequence Replication (3SR): An Isothermal Transcription-Based Amplification System Alternative to PCR,” PCR Methods and Applications, 1:25-33 (1991); Kacian et al., “Nucleic Acid Sequence Amplification Methods,” U.S. Pat. No. 5,399,491; Davey et al., “Nucleic Acid Amplification Process,” U.S. Pat. No. 5,554,517; Birkenmeyer et al, “Amplification of Target Nucleic Acids Using Gap Filling Ligase Chain Reaction,” U.S. Pat. No. 5,427,930; Marshall et al., “Amplification of RNA Sequences Using the Ligase Chain Reaction,” U.S. Pat. No. 5,686,272; Walker, “Strand Displacement Amplification,” U.S. Pat. No. 5,712,124; Notomi et al., “Process for Synthesizing Nucleic Acid,” U.S. Pat. No. 6,410,278; Dattagupta et al., “Isothermal Strand Displacement Amplification,” U.S. Pat. No. 6,214,587; and Helen H. Lee et al., Nucleic Acid Amplification Technologies: Application to Disease Diagnosis (1997).
Because polymerase activity is readily lost at ambient temperature, it is common to manufacture amplification kits which include polymerases that have been freeze-dried in formulations containing other necessary co-factors and substrates for amplification. See, e.g., Shen et al., “Stabilized Enzyme Compositions for Nucleic Acid Amplification,” U.S. Pat. No. 5,834,254. Freeze-drying or lyophilization involves the removal of water from a frozen sample by sublimation under lower pressure. Sublimation is a process by which a solid is evaporated without passing through the liquid stage. Freeze-dried formulations containing polymerases are advantageous because they can be stored at ambient temperature and for prolonged periods of time without substantial losses of enzymatic activity.
Prior to use, dried polymerase formulations must be reconstituted with a reconstitution buffer, such as that disclosed by Shen et al., U.S. Pat. No. 5,834,254. Typically, the lyopholized product is provided in a vacuum-sealed glass bottle, and the buffer is separately provided in a plastic bottle or tube having a re-sealable cap. Reconstitution generally requires manually transferring the buffer from its container to the container holding the dried polymerase formulation, either by pipetting or pouring. The container holding the polymerase formulation is then swirled or otherwise agitated for a period of time sufficient to fully dissolve the dried material, after which time the reconstituted polymerase formulation is transferred back to the container which previously held the buffer. The container holding the reconstituted polymerase formulation is preferably a plastic container having a conically-shaped bottom to minimize waste when pipetting from the container. Plastic containers are preferred because they can be placed in sub-zero freezers for storage and are cheaper to manufacture than glass bottles. The reconstituted polymerase formulation may be used directly in an amplification procedure or sealed and stored for subsequent use.
The manual steps associated with commonly practiced polymerase reconstitution procedures raise two primary concerns. First, each of the manual steps involved in reconstituting dried polymerase formulations presents an opportunity for operator error and variability between reconstitutions, as the accuracy of reconstitutions depends upon precision pipetting or pouring by a practitioner. Second, open containers and manual transfer steps associated with such procedures provide an opportunity for practitioners to inadvertently contaminate reconstituted polymerase solutions with residual test material that may have been picked up from a laboratory workspace. This kind of contamination is especially undesirable since transferring even a minute amount of target-containing material from a workspace to a polymerase-containing solution could lead to the production of billions of target sequences in otherwise negative samples, thereby resulting in false-positives that would have tested negative in the absence of target amplification with the polymerases. Thus, it is an objective of the present invention to provide a manual method for reconstituting dried polymerase formulations in a manner which minimizes opportunities for operator error and contamination.
The present invention satisfies this objective by providing a connector for use in combining the contents of a pair of containers, where the connector comprises an elongated body having top and bottom end portions dimensioned to receive open ends of first and second containers, respectively, in an interference fit, a middle portion positioned between and adjoining the end portions of the body, and an inner surface defining a conduit through the body of the connector. The top end portion includes one or more recesses sized and arranged to facilitate drainage of a fluid from the first container into the second container by means of the conduit when the containers are fitted onto the end portions of the body and the first container is positioned above the second container. To facilitate drainage of a fluid from the first container into the second container, the recesses of the top end portion are preferably generally U-shaped and the end surface of the top end portion is preferably inwardly beveled.
In one embodiment of the present invention, the connector further comprises a collar having wall which extends radially outwardly from and generally perpendicular to an outer surface of the middle portion of the body. The radial wall may have a peripheral end surface adapted for manual manipulation or, alternatively, the collar may further comprise a peripheral end wall, where the radial wall distally terminates at and generally perpendicular to an inner surface of the peripheral end wall. In this latter embodiment, the peripheral end wall has an outer surface which preferably includes a series of spaced-apart ridges for gripping. To strengthen the peripheral end wall for manual manipulation, a plurality of spaced-apart support walls may be included which extend from and generally perpendicular to a top surface and/or a bottom surface of the radial wall and which extend radially outwardly from the outer surface of the middle portion of the body to the inner surface of the peripheral end wall. In the most preferred embodiment, the body has a generally cylindrical shape and the radial wall forms a ring structure which distally terminates at an annular end wall.
In another embodiment of the present invention, at least one of the end portions of the body of the connector proximally terminates at a shelf extending outwardly from the outer surface of the body, where each shelf may be defined by a top or bottom surface of the middle portion. Where the connector includes a collar, the shelf may be the top or bottom surface of the radial wall. Each shelf is sized and arranged so that a top surface at the open end of the first or second container is in touching contact with the shelf when the first and second containers are fitted onto the end portions of the body. In a preferred embodiment, each end portion of the body proximally terminates at a shelf. And in the most preferred embodiment, the shelf at the top end portion is perpendicular to the outer surface of the top end portion and the shelf at the bottom end portion is beveled relative to the outer surface of the bottom end portion.
In still another embodiment of the present invention, a connector is provided for use in combining the contents of a pair of containers, where the connector comprises an elongated body, a collar having a peripheral end wall and a skirt depending from a bottom surface of the peripheral end wall. The body includes an upper end portion dimensioned to receive an open end of a first container in an interference fit and a lower end portion adjoining the upper end portion. The collar includes a wall which extends radially outwardly from and generally perpendicular to an outer surface of the lower end portion and which distally terminates at and generally perpendicular to an inner surface of the peripheral end wall. The skirt is dimensioned to receive an open end of a second container in an interference fit. The upper end portion has an end surface which includes one or more recesses sized and arranged to facilitate drainage of a fluid from the first container into the second container by means of the conduit when the first and second containers are fitted onto the upper end portion and skirt, respectively, and the first container is positioned above the second container. To facilitate drainage of a fluid from the first container into the second container, the end surface of the upper end portion is preferably inwardly beveled and the recesses are preferably generally U-shaped.
In yet another embodiment of the present invention, the peripheral end wall of the skirted connector has an outer surface which includes a series of spaced-apart ridges for gripping. To strengthen the peripheral end wall for manual manipulation, a plurality of spaced-apart support walls are preferably included which extend from and generally perpendicular to a top surface of the radial wall and which extend radially outwardly from the outer surface of the lower end portion of the body to the inner surface of the peripheral end wall. In the most preferred embodiment, the body has a generally cylindrical shape and the radial wall forms a ring structure which distally terminates at an annular end wall.
In a further embodiment of the present invention, the upper end portion of the body proximally terminates at a top shelf, where the top shelf is defined by a top surface of the lower end portion of the body. The top shelf is sized and arranged so that a top surface of the open end of the first container is in touching contact with the top shelf when the first container is fitted onto the top end portion. In this embodiment, the skirt preferably proximally terminates at a bottom shelf, where the bottom shelf is defined by the bottom surface of the peripheral end wall. The top and bottom shelves are preferably generally perpendicular to the outer surfaces of the upper end portion and the skirt, respectively.
In still another embodiment, kits are provided which include any connector according to the present invention in packaged combination with at least one container holding a fluid substance (e.g., solvent or diluent) or solid material (e.g, powder, particles, granules, food product or tissue specimen). Preferably, the kits of this embodiment include a first container holding a lyophilized formulation containing at least one enzyme reagent (i.e., RNA or DNA polymerase) for use in amplifying a nucleic acid sequence and a second container holding a buffer for reconstituting the formulation. An example of a lyophilized formulation for performing a transcription-based amplification is one which comprises reverse transcriptase derived from Moloney murine leukemia virus and/or bacteriophage T7 RNA polymerase in at least one cryoprotectant excipient, such as trehalose or polyvinylpyrrolidone. The container holding the lyophilized formulation may further include, for example, nucleotide triphosphates, metal ions and co-factors necessary for enzymatic activity. Such formulations are disclosed by Shen et al., U.S. Pat. No. 5,834,254. These formulations can be reconstituted with a reconstitution buffer comprising 0.01% (v/v) TRITON® X-100, 41.6 mM MgCl2, 1 mM ZnC2H3O2, 10% (v/v) glycerol, 0.3% (v/v) ethanol, 0.02% (w/v) methyl paraben, and 0.01% (w/v) propyl paraben. Other enzyme-containing formulations and corresponding reconstitution buffers will be readily appreciated by those skilled in the art.
These and other features, aspects, and advantages of the present invention will become apparent to those skilled in the art after considering the following detailed description, appended claims and accompanying drawings.
While the present invention may be embodied in a variety of forms, the following description and accompanying drawings are merely intended to disclose some of these forms as specific examples of the present invention. Accordingly, the present invention is not intended to be limited to the forms or embodiments so described and illustrated. Instead, the full scope of the present invention is set forth in the appended claims.
The figures illustrate preferred connectors 10, 50 of the present invention depicted alone or as part of a system 100, 200 which includes a first container 70 and a second container 80, 90. Connectors according to the present invention can be used to combine the contents of different containers which are preferably kept separate until use for reasons that may be related to, for instance, the stability or toxicity of the combined contents of the different containers. Containers that can be used with or that form a part of the present invention can be of any shape sufficient to hold the material of interest and which allows for attachment of the container to the connector in an interference fit. (As used herein, the term “interference fit” includes, but is not limited to, a frictional fit.) Likewise, the connectors of the present invention can be adapted to accommodate the shapes and sizes of the containers intended for use. The composition of the containers may be, for example, glass or plastic, and is preferably selected to be essentially inert with respect to the materials being combined, whether those materials are in their separated or combined state. The contents of container sets may form, by way of example, a lyophilized material/solvent combination, a concentrated solution/diluent combination or a combination of solutions which can be combined to form a new solution. After assembling a connector and associated containers, mixing may be achieved by repeated hand-inversion, or inversion and swirling, of the closed system.
The top end portion 12 of the connector 10 depicted in
In addition to facilitating attachment of the container 70 to the connector 10, the configuration of the top end portion 12 illustrated in
To guide and accommodate attachment of the container 90 onto the skirt 52 in a frictional fit, a bottom surface 53 of the skirt is configured to include a bevel 54. Additionally, the skirt 52 of this embodiment proximately terminates at an outwardly extending, annular bottom shelf 55 defined by the bottom surface 33 of the annular end wall 18. The bottom shelf 55 functions as a stop to an annular top surface 91 at the mouth of the container 90 when it is fitted onto the skirt 52 of the connector 50. The bottom shelf 55 is preferably substantially perpendicular to an outer surface 56 of the skirt 52. In a preferred embodiment, the width of the bottom shelf 55 is at least as great as the width of the annular top surface 91 of the container 90.
In another embodiment not shown, an inner surface 57 of the skirt 52 may be configured to receive and immobilize a container for mixing. For example, the inner surface 57 of the skirt 52 may be provided with helical threads which are arranged to mate with helical threads 92 positioned on an outer surface 93 of the container 90, where the threads 92 are positioned near the mouth of the container 90, as shown in
For both illustrated connectors 10, 50, attachment of the containers 70, 80, 90 onto the connectors prior to mixing is illustrated in
Connectors of the present invention are preferably formed as a unitary piece from a thermoplastic elastomer (TPE) or a thermoplastic rubber (TPR). TPEs differ from TPRs in that they have higher mechanical strength, but less elongation and fatique resistance. TPEs include elastomeric polyurethane, polyester and nylon. TPRs are generally polyolefin-based or styrene-based. The olefin-based materials have good chemical resistance, while the styrene-based materials are less expensive (but cannot withstand temperatures above about 120° F.). Particularly preferred for the present invention is a TPR sold under the product name KRATON™ G7720-9 (The KRATON Polymer Business; Houston, Tex.). The connectors can be molded using injection molding procedures well-known to those skilled in the art and in accordance with specifications that take into account the dimensions of the containers to be used in conjunction with the connectors, especially surface dimensions that will come into contact with the connectors, as well as the degree of friction desired.
While the present invention has been described and shown in considerable detail with reference to certain preferred embodiments, those skilled in the art will readily appreciate other embodiments of the present invention. Accordingly, the present invention is deemed to include all modifications and variations encompassed within the spirit and scope of the following appended claims.
This application claims the benefit of U.S. Provisional Application No. 60/311,329, filed on Aug. 10, 2001, the contents of which are hereby incorporated by reference herein.
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