WO2000066267A1

WO2000066267A1 - Preventing cross-contamination in a multi-well plate

Info

Publication number: WO2000066267A1
Application number: PCT/US2000/011505
Authority: WO
Inventors: Darin P. O'brien; Ellen M. Heath
Original assignee: Gentra Systems, Inc.
Priority date: 1999-04-30
Filing date: 2000-04-28
Publication date: 2000-11-09
Also published as: AU4676700A

Abstract

A system that prevents cross-contamination in a multi-well sample processing system. The system includes a multi-holed filter or absorbent matrix member positioned between a top flow-through plate and a bottom collection plate of the system. The top plate has openings and nozzles and the bottom plate has openings which match and correspond to the openings in the matrix member. The sample can go through the top plate openings and nozzles to the bottom plate wells, however, the matrix member prevents cross contamination between the bottom plate wells or across the nozzles. Thus, a user prevents cross-contamination between the unsealed nozzles of the top plate and the unsealed wells of the bottom plate.

Description

PREVENTING CROSS-CONTAMINATION IN A MULTI-WELL PLATE

Field of Invention

The present invention relates generally to sample processing equipment and more specifically to multi-well purification devices.

Background Multi-well plates are used to assay or test a series of samples at one time. Having many wells is a time saving device, and can reduce the errors that occur during processing. Multi-tier multi-well plate systems have a top plate in which the sample is placed to be purified and isolated. The top plate has holes which permit the flow-through of the samples or waste from processing. Multi-tier systems also have a collection reservoir, which is commonly either a vacuum manifold or a bottom plate with wells, to collect either the waste or the final product. Such a design permits faster extraction and purification of samples than a single plate system because the user does not have to transfer the sample from one well to another during the process, the sample and waste product simply go through the flow-through plate into the collection plate or collection reservoir.

Multi-well plates can be used to purify specific components from biological, environmental, or pharmaceutical samples. These components can be proteins, lipids, nucleic acid or carbohydrates as well as metabolytes or environmental elements, or combinations thereof. One such use for multi-well plates is to purify deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from biological materials. DNA is a nucleic acid molecule that is the carrier of coded genetic information. DNA and RNA are used in many applications, including diagnosis of certain infections, forensic sciences, clinical applications, recombinant DNA research, cloning, sequencing and the like. However, the DNA or RNA molecules need to be separated or purified from body tissue or fluid samples.

A problem that arises when using multi-well plates is that cross- contamination may occur among adjacent wells of the plate. In multi-tier systems, the cross-contamination also occurs between the openings and nozzles of the flow-through top plate when the sample is exiting the plate. Cross- contamination occurs when a sample from one well becomes mixed with a sample in another well. This compromises sample integrity and may lead to ruined samples and inaccurate and misleading diagnoses. The cross- contamination can occur during many steps of the purification and testing process, such as when transferring samples between wells, adding a reagent, centrifuging, and the like. The ability to process multiple samples at the same time without cross-contaminating the samples is crucial for clinical diagnostic and forensic laboratories. This is especially true for molecular diagnostics, where extremely sensitive PCR (Polymerase Chain Reaction) and RT-PCR assays and other amplification protocols employed can detect as little as a single molecule of cross-contamination.

U.S. Patent 4,680,269 to Naylor discloses a method to prevent cross- contamination in single plate systems in which the plate does not have a flow- through design. Naylor describes a single plate antimicrobial test kit in which cross-contamination is prevented by use of an impregnated filter paper which covers the plate's well openings and is held down by a lid. The impregnated filter attracts and absorbs the volatile microbes. However, the Naylor invention is only described in terms of a single tier multi-well sealable system, not multi-tier systems that have a top plate with a flow-through design, in which case the nozzles or openings are unsealable during processing. Also, Naylor requires an impregnated filter, which can increase the system's complexity. Therefore, the Naylor solution is not applicable to flow-through plates, because in such systems the nozzles cannot be sealed, and the lower plate's collection reservoir cannot be sealed, and that is a potential source of cross-contamination.

Thus what is needed is an easy to use, inexpensive system to prevent cross-contamination between the unsealed nozzles and wells of a flow-through plate of a multi-tier multi-well plate system while still permitting flow-through of waste or samples to the unsealed wells of a collection reservoir or collection well plate. Summary of Invention

The present invention provides an easy to use system that prevents cross- contamination of samples in a multi-well testing system. The system includes a multi-holed matrix member positioned against a flow-through plate of the system. The plate has openings and nozzles that match and correspond to the openings in the matrix member. The sample or waste product goes through the unsealed nozzles, while the matrix member prevents any cross contamination among wells or between nozzles. Thus, a user can prevent any cross-contamination between the unsealed nozzles and wells of the flow-through plate. In a further embodiment, the system includes a collection reservoir or base plate, to be used for DNA sample collection after it has been isolated in the top plate. The matrix member also prevents cross-contamination among the base plate's unsealed wells during sample processing.

The present system provides the advantage of preventing cross- contamination of components such as nucleic acids in a multi-well plate kit used for purification and sample preparation. The system is simple to use for multi-tier multi-well plate systems, advantageously providing sample and waste flow- through to an unsealed collection reservoir, while still preventing cross- contamination. Moreover, the system prevents cross-contamination during all the steps of the purification process, including centrifuging the multi-well plate, heating the plate, or vacuum aspirating the sample through the multi-well plate.

Brief Description of the Drawings

Fig. 1 is an exploded perspective view of one embodiment of a multi-well sample processing system according to the invention.

Fig. 2 is an assembled view of the system of Fig. 1.

Fig. 3 A is a top and side view of one embodiment of a flow-through plate according to the invention. The side view is along the line 3A-3A.

Fig. 3B is a top and side view of another embodiment of a flow-through plate according to the invention. The side view is along the line 3B-3B.

Fig. 4A is a top and side view of one embodiment of a matrix member according to the invention. The side view is along the line 4A-4A.

Fig. 4B is a view of another embodiment of a matrix member according to the invention applied to a plate.

Fig. 5 A is a top and side view of one embodiment of a base plate according to the invention. The side view is along the line 5A-5A.

Fig. 5B is a top and side view of another embodiment of a base plate according to the invention. The side view is along the line 5B-5B.

Fig. 5C is a top and side view of another embodiment of a base plate according to the invention. The side view is along the line 5C-5C.

Detailed Description In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that the embodiments may be combined or that other embodiments may be utilized and that structural changes may be made without departing from the spirit and scope of the present invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims and their equivalents.

Figures 1 -5 show one embodiment of a multi-well sample processing system 100. System 100 includes a flow-through top plate 105. In this embodiment, plate 105 contains ninety-six holes or openings 106. At a bottom surface of plate 105 there is a plurality of drip directors or nozzles 107. Each nozzle 107 is mounted directly beneath a corresponding hole or opening of flow- through plate 105. Nozzles 107 control the flow-through rate and direction of waste product and samples through plate 105. Figure 3B shows another embodiment of plate 105 without any nozzles. In this example, the flow- through rate of samples can be regulated by the shape and size of holes 106 themselves. The use of the two-tier plate kit and certain nucleic acid isolation techniques are described in co-pending application serial no. 09/154830, entitled Apparatus and Methods for Isolating Nucleic Acids, filed on 8/17/1998, and assigned to the assignee of this application. This co-pending application is incorporated by reference herein. System 100 also includes a filter or guard, such as matrix member 103 for absorbing volatile components, thus preventing cross-contamination among samples when they leave nozzles 107 or are in the wells of a collection reservoir, such as a vacuum manifold or a base plate 101. Figure 4A shows one embodiment of a matrix member 103. In this embodiment, matrix member 103 is made from a filter material such as Whatman Chromatography Paper 3mm Chr (Cat. No. 3030917). Alternative materials include any absorbent or adsorbent filter stock that permits binding or interception of volatile nucleic acid, such as nitrocellulose and nylon transfer membranes, chromatography paper, ion- exchanger membranes, PVDF membranes, cellulose acetate membranes, PTFE and polycarbonate filters, and glass micro fiber and quartz filters. Matrix member 103 is cut into a shape substantially equivalent to the shape and dimensions of plate 105, in this embodiment, for example, the shape would be approximately 8 cm x 12 cm. Matrix member 103 has a plurality of openings 104 which match and correspond to openings 106 in plate 105. Openings 104 are substantially the diameter of the outside diameter of nozzles 107, approximately 1/8 inch in diameter, although those skilled in the art will recognize that other sizes can be used depending on the size of the nozzles or plate openings. Openings 104 are punched to correspond to the 8 x 12 ninety-six well format described above for the top plate. In this embodiment, openings 104 in matrix member 103 surround drip directors 107. When matrix member 103 is positioned against plate 105 the matrix member 103 intercepts and absorbs volatile nucleic acids leaving nozzles 107, or coming up from the wells of plate 101.

In one embodiment, openings 104 in matrix member 103 are cut to a size slightly smaller than the diameter of nozzles 107. This permits matrix member 103 to be positioned against plate 105 by pushing the nozzles through the openings of the matrix member. Because the openings are slightly smaller than the nozzles, the matrix member is seated in position such that the matrix member surrounds the base of nozzles 107. Alternatively, matrix member 103 can have an adhesive, such as 3M Super 77 Spray Adhesive (manufactured by 3M, St. Paul, MN), applied to the side which is to face the top plate. When pressed against the plate, the adhesive holds the matrix member in position.

Figure 4B shows another embodiment of a matrix member 403. In this embodiment, the matrix member includes a series of horizontal strips 401 and a series of vertical strips 402. Strips 401 and 402 are made from the same material as the matrix member described above. However, in this embodiment, the material is cut in strips and then adhesively applied to plate 105. Strips 401 and 402 are applied so that each nozzle 107 is surrounded by absorbent material.

Figure 5 A shows a top and side view of one embodiment of a receptacle plate or base plate 101. Base plate 101 has a plurality of wells 102. Wells 102 provide a cavity or space to capture waste from the samples or a place to capture a purified sample from nozzles 107. Wells 102 run down substantially the whole depth of plate 101 as shown in Figure 5 A. In this embodiment, base plate 101 has ninety-six wells in an 8 x 12 configuration, corresponding and matching the configuration of matrix member openings 104 and top plate openings 106. Each well 102 has a well opening 108 at the top surface of base plate 101. Those skilled in the art will realize that other types of base plates with different size and configuration of wells are possible to use with the system as described above. For example, if the system is used to purify DNA, the user can use two base plates. The first as in Figure 5A, the second as in Figure 5B. Base plate 501 in Figure 5B is used for storing samples of DNA after they have been purified. The important thing is that wells 502 in base plate 501 match and correspond to the openings 104 in matrix member 103 and the openings 106 in top plate 105, thus allowing the matrix member to prevent cross-contamination. Figure 5C shows another embodiment of a base plate. In this embodiment, a base plate well 508 includes an individual tube 509, thus permitting the user to individually handle collected samples. Furthermore, those skilled in the art will realize that no base plate is necessarily needed as a waste collection reservoir because the top flow-through plate and matrix member assembly can also be used above a vacuum manifold or other conduit to collect waste product.

Use of the system described above will next be described in the context of purification and isolation of DNA, however it is to be understood that this example is not a limitation, but merely an example of use of the system. The specific details of many techniques for purification and isolation of DNA are known in the art, thus just an outline of the system will be described. Those skilled in the art will appreciate that the system may be practiced in other situations and using various techniques, such as microbiological assays and the like. The following DNA purification technique is one example of the cross- contamination prevention technique.

To purify DNA without cross-contamination, matrix member 103 is positioned and mounted against the surface of flow-through top plate 105 containing nozzles 107. As described above, the matrix member may be either press fit or adhesively mounted against the plate. The plate/matrix member assembly is next positioned upon a first base plate 101, as shown in Figure 2. Samples from which DNA is to be isolated, such as from whole blood, bone marrow, buffy coat, or body fluids, are placed in each opening 106 of plate 105. A purification solution is added to each opening. A seal is placed over the top openings of the top plate. The completed two-plate assembly is then centrifuged. More purification solution is added and the assembly is centrifuged again. Then an elution solution is added to the top plate openings, and the assembly is centrifuged again. The solutions and centrifuging result in DNA being extracted from the samples, with waste product going through unsealed nozzles 107 of the flow-through top plate into unsealed wells 102 of the first base plate. Matrix member 103 prevents any volatile nucleic acids from transferring across nozzles 107 or from one well to another, thus preventing cross-contamination. First base plate 101 is then disposed and a second base plate 501 is put in its place, the second base plate fitting like the first base plate. Those skilled in the art will realize that the above procedure can also be accomplished using a vacuum manifold to remove waste product. After the samples are purified, elution solution is added to the top flow- through plate, another seal is attached over the top plate openings, the assembly is heated, and then it is centrifuged again. This process results in purified DNA going through unsealed nozzles 107, and the uncontaminated DNA is stored in second base plate 501 and is ready to be amplified. Again, the advantage of the present system is that throughout the process of adding solutions, centrifuging, changing base plates, and heating, the matrix member positioned against the nozzle surface of the flow-through plate effectively prevents any cross- contamination across nozzles or among or between wells. Example of Use and Test Results

The system and process described above was effective in preventing cross-contamination during a test of the system. It was tested by application of the matrix member to a GENERATION^® Capture Plate (Gentra Systems, Inc., Minneapolis, MN) used for the batch purification of genomic DNA from whole blood from 96 samples simultaneously in a 96-well plate. Two plate systems were ran simultaneously, one with the matrix member applied and one without the matrix member. To test for cross-contamination, 200 μl of whole blood were loaded in alternate wells of the top flow-through plates starting with the first well in the first row of the plate. Wells not loaded with blood were loaded with a solution not containing DNA, phosphate buffered saline. All samples purified from both plate systems were analyzed for cross-contamination by Polymerase Chain Reaction (PCR) amplification of the HLA-H locus. Detection of DNA in wells not loaded with blood was used as a measure of cross-contamination. It should be noted that under the conditions employed, this assay can routinely detect as little as one cell equivalent of contaminating genomic DNA. In the plate system processed without the matrix member, amplified DNA was detected in 48 of the 48 wells loaded with blood, as expected. However, faint but detectable DNA bands were observed in 25 of the 48 wells not loaded with blood, demonstrating a measurable level of cross-contamination in the absence of the matrix member. In contrast, in the plate system processed with the matrix member, DNA was detected in 48 of the 48 wells loaded with blood but was not detected in any of the 48 wells loaded with PBS. Thus, application of the matrix member to the plate resulted in a reduction of cross-contamination, to a level undetectable by PCR analysis.

The system described above provides the advantage of preventing cross- contamination in a multi-well kit. The system is simple to use in multi-tier multi-well plates, providing flow-through access to a collection reservoir while still preventing cross-contamination between unsealed nozzles and wells of the flow-through top plate and between wells of the unsealed collection reservoir.

The embodiments and examples described above may be combined or other embodiments may be utilized and structural changes may be made without departing from the spirit and scope of the present invention. The detailed description above is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

What is claimed is:

1. A system for multi-well sample processing, comprising: a plate having a plurality of openings; and a matrix member having a plurality of openings, each matrix member opening corresponding to one of the plate openings, the matrix member positioned against a surface of the plate so that the matrix member openings align with the plate openings.

2. The system according to claim 1 , wherein the plate further comprises: a plurality of nozzles beneath the openings in the plate, the plurality of nozzles corresponding to the plurality of matrix member openings.

3. The system according to claim 1, further comprising a collection reservoir situated beneath the plate.

4. The system according to claim 3, wherein the collection reservoir comprises: a second plate having a plurality of wells corresponding to the openings in the matrix member, the second plate positioned so that the matrix member openings and first plate openings align with the wells.

5. A system for multi-well sample processing, comprising: a first plate having a plurality of openings, the plate having a plurality of nozzles attached to a bottom surface of the plate, each nozzle corresponding to one of the plurality of openings thereon; a matrix member positioned against the first plate, the matrix member having a plurality of openings corresponding to the openings of the plate, the matrix member positionable so that the matrix member openings are over the plate openings and surrounding the nozzles; and a second plate having a plurality of wells corresponding to the openings in the matrix member and the openings in the first plate, the second plate positionable so that the matrix member openings are over the plate wells.

6. The system according to claim 5, wherein the matrix member is made from an absorbing material.

7. The system according to claim 5, wherein the diameter of each of the plurality of nozzles is slightly larger than the diameter of each of the plurality of matrix member openings so that the matrix member is mountable on the bottom surface of the second plate.

8. A system for preventing cross-contamination between nozzles in a plate having a plurality of nozzles, comprising: a matrix member having a plurality of openings, the matrix member having a plurality of openings corresponding to the nozzles of the plate, the matrix member positionable so that the matrix member openings surround the nozzles.

9. A multi-well sample processing system, comprising: a first base plate having a plurality of wells, each well having an opening on a surface of the base plate; a top plate having a plurality of openings corresponding to the well openings of the first base plate, each opening running through the top plate from a top surface to a bottom surface, each opening having a nozzle attached at the bottom surface of the plate; a matrix member positionable against the top plate, the matrix member having a plurality of openings corresponding to the nozzles and the openings of the top plate; and a second base plate having a plurality of wells, each well having an opening on a surface of the base plate corresponding to the top plate nozzles.

10. A method for preventing cross-contamination during purification using a multi-well plate system, comprising: mounting a matrix member having a plurality of openings against a first plate having a plurality of openings corresponding to the openings in the matrix member; and positioning first plate with the matrix member attached upon a collection reservoir.

1 1. The method according to claim 10, wherein the collection reservoir comprises: a second plate, the second plate having a plurality of wells, said wells having well openings at a principal surface of the plate corresponding to the first plate openings.

12. A method for purifying a sample in a multi-well kit with reduced cross- contamination, comprising: placing a matrix member having a plurality of openings between a first plate having a plurality of corresponding openings and a collection reservoir, the wells having openings corresponding to the openings in the matrix member and the openings in the first plate; adding a sample to the openings in the first plate; adding a first solution to the openings in the first plate; centrifuging the plate assembly, placing the matrix member between the first plate and a third plate having a plurality of wells, the wells having openings corresponding to the openings in the matrix member and the openings in the first plate; adding a second solution to the openings in the first plate; heating the plate assembly; and centrifuging the plate assembly.