Device and method for phylogenetic-based differential cell separation and DNA extraction

Abstract

A device for differential cell separation while maintaining cell viability for downstream applications that employs a filter manifold having a central cavity and a filter support positioned therein and a filter suspended in the central cavity by the filter support. A syringe is interconnected to a first side of the filter manifold to dispense a fluid onto the filter. A recovery tube is interconnected to a second side of the filter manifold so that it is in communication with the cavity of the filter manifold and it is positioned to receive the fluid dispensed from the syringe after the fluid has passed through the filter. The device may be used to implement a facile protocol for differential cell separation that maintains cell viability for downstream applications.

Description

TITLEDEVICE AND METHOD FOR PHYLOGENETIC-BASED DIFFERENTIAL CELL SEPARATION AND DNA EXTRACTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to US Provisional Application No. 63 / 538,104, filed on September 13, 2023.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No.1R41GM140530-01A1 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND OF THE INVENTION1. FIELD OF THE INVENTION

[0003] The present disclosure relates to DNA extraction from samples and, more specifically, to size exclusion and enzymatic separation of prokaryotic cells, including bacterial cells, from eukaryotic and metazoan cells, including human and plant cells, prior to extraction that maintains cell viability for downstream applications.2. DESCRIPTION OF THE RELATED ART

[0004] DNA analyses routinely focus on single organismal targets. However, an unknown biological sample will often contain more biological organisms or organismal components rather than just that of the single target organism. While the DNA from nontargeted organisms may contain valuable data regarding the identity of the sample, these nontarget organisms can complicate downstream analyses.

[0005] For example, biological samples will contain a mixture of plant, bacterial and metazoan DNAs. In forensic analyses, unknown or questioned samples may contain microbial communities that are not the focus of the investigation. For example, within the context of human forensic genetics, a human DNA sample will also contain microbial DNA, and given NGS shotgun sequencing, the shorter and more numerous genomic microbial DNA will likely be preferentially sequenced in whole genome amplification / shotgun sequencing applications. This over representation of the non-target DNAs will pose a challenge to the interpretation of results, whereby the target DNAs are not sequenced at appropriate depths.

[0006] On the other hand, in medical analyses, samples targeting bacteria populations may include human tissues. Research involving the human microbiome has proven of theutmost importance for various scientific fields, but the inability or lack of success in separating the human cells from the bacterial cells has become challenging to researchers

[0007] Another example is present in the agricultural and forestry sectors that can be volatile due to influence by unpredictable environmental, pest, trade index, supply / demand factors. The ability to analyze all biological components in a sample to understand the interactions and / or presence among pests / microorganisms and plant / animal cells would benefit these sectors. A method to better understand and control the unpredictability related to pests and disease would benefit both public and private forestry and agricultural support services.

[0008] In these cases, the samples contain a wealth of potential information beyond the target, for example, the microbial, plant or animal profiles associated with a person, animal, object or place. Capturing this information is a challenge, however, due to many factors, such as time, cost, and data quality. The reliability and accuracy of environmental DNA NGS data presents a more pressing challenge to scientific analysts than cost or time as it is imperative that the data obtained in all aspects of science be accurate and valid. Accordingly, there is a need in the art for a device and an approach that can separate the metazoan, plant and microbial contents of a sample for increased interpretability of any of the contents of the sample.BRIEF SUMMARY OF THE INVENTION

[0009] In a first aspect, the present invention provides a device for differential cell separation while maintaining cell viability for downstream applications. The device for differential cell separation comprises a filter manifold having an inner surface that defines a central cavity and a filter support positioned in the central cavity. A filter is suspended in the central cavity by the filter support. A syringe is interconnected to a first side of the filter manifold to dispense a fluid onto the filter. A recovery tube is interconnected to a second side of the filter manifold and in communication with the cavity of the filter manifold to receive the fluid dispensed from the syringe after the fluid has passed through the filter. The device may include an adaptor interconnecting the syringe to the first side of the filter manifold. The adaptor may include a first set of threads that can engage a corresponding second set of threads of the first side of the filter manifold. The syringe may be connected to the adaptor by a luer lock. The device may include an end cap interconnecting the recovery tube to the second side of the filter manifold. The end cap may include a first set of threads that engage a corresponding second set of threads of the filter manifold. The end cap may also include a tube fitting that frictionally engages an opening of the recovery tube. The filtersupport may extend circumferentially around an inside of the filter manifold that forms the inner cavity. The filter may be secured in a filter cup having an upper rim that engages the filter support. The upper rim of the filter cup may include a series of openings that align with an receive a corresponding series of posts extending from the filter support. The device may further comprise a second filter manifold having a second central cavity and a second filter support positioned in the second central cavity, wherein the second filter manifold is coupled between the filter manifold and the recovery tube so that the second central cavity is in communication with the first filter cavity and the second filter cavity is in communication with the recovery tube.

[0010] In another aspect, the present invention provides a method for differential cell separation that may be implemented for differential cell separation while maintaining cell viability for downstream applications, including by using the device of the present invention for facile implementation. In a first step, the method of separating prokaryotic and eukaryotic cells from a sample containing an amount of prokaryotic cells and an amount of eukaryotic cells comprises providing a first filter manifold with a first central cavity and a first filter suspended in the first central cavity and a first recovery tube connected to the first filter manifold and in communication with the first central cavity downstream of the first filter. In another step, the method comprises suspending a first filter in the first filter manifold. In another step, the method comprises applying an unknown sample to the first filter. In another step, the method comprises pumping air onto the first filter using a syringe coupled to the first manifold. In another step, the method comprises washing the first filter with a first solution. In another step, the method comprises collecting the first solution in the first recovery tube as a first fraction. In another step, the method comprises providing a second filter manifold with a second central cavity and a second filter suspended in the second central cavity and a second recovery tube connected to the second filter manifold and in communication with the second central cavity downstream of the second filter. In another step, the method comprises suspending a second filter in the second filter manifold. In another step, the method comprises applying the first fraction to the second filter. In another step, the method comprises pumping air onto the second fdter using the syringe coupled to the second manifold. In another step, the method comprises washing the second filter with a second solution. In another step, the method comprises collecting the second solution in the second recovery tube as a second fraction containing the prokaryotic (bacterial) cells.

[0011] Any of the eukaryotic cells remaining in the second fraction may be removed by adding a non-bacterial cell lysis reagent to the second fraction, incubating the secondfraction after adding the reagent and centrifuging the second fraction after incubating to provide a first supernatant that is removed and a first pellet that contains the amount of prokaryotic cells. The method may further comprise the steps of cutting the first filter and the second filter into pieces, placing the pieces into a test tube, adding an amount of buffer and an amount of a prokaryotic lysis reagent, mixing the pieces with the amount of buffer and the amount of the prokaryotic lysis reagent, and centrifuging the pieces with the amount of buffer and the amount of the lysis reagent to form a second supernatant that is removed and a second pellet containing the amount of eukaryotic (human) cells. Both pellets may then be treated with Dnase and inactivated prior to DNA extraction.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0012] The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:

[0013] FIG. 1 is an exploded view of an exemplary device for use in cell and DNA separation according to the present invention.

[0014] FIG. 2 is an assembled view of an exemplary device for use in cell and DNA separation according to the present invention.

[0015] FIG. 3 is a side view of an exemplary filter assembly for use in cell and DNA separation according to the present invention.

[0016] FIG. 4 is a top view a filter assembly for use in cell and DNA separation according to the present invention.DETAILED DESCRIPTION OF THE INVENTION

[0017] Referring to the figures, there is seen in FIG. 1, an exemplary device 10 for use in connection with phylogenetic-based differential cell separation and DNA extraction according to the present invention. Device 10 comprises a syringe 12 that may be selectively attached to an adaptor 14, such as by using corresponding Luer lock components. Adaptor 14 serves as a cover for a filter manifold 16 and may be coupled thereto using conventional approaches such as corresponding threads. For example, adaptor 14 may include internal threads on an underside portion that can selectively engage and interlock with outward threads on the top of filter manifold 16. As further seen in FIG. 1, filter manifold 16 is modular and can be attached in series to one or more additional filter manifolds 16 by providing internal threads on the bottom of filter manifold 16 that are dimensioned to engage the outward threads on the top of another filter manifold 16. Device 10 further comprises an end cap 18 that can engage the lower internal threads of the bottom of a single filter manifold16 (or the last filter manifold 16 if additional filter manifold 16 are used in series). End cap 18 includes a series of tube fittings 20. Device 10 additionally comprises a recovery tube 22 that can be selectively attached to one of the series of tube fittings 20. Recovery tube 22 preferably includes a cover 24 for enclosing recovery tube 22 when not recovery tube 22 is not coupled to one of the series of tube fittings 20.

[0018] As seen in FIG. 2, all components of device 10 may be interlocked into an assembled configuration to provide an internal pathway extending from syringe 12 through adaptor 14, each filter manifold 16, end cap 18, and terminating in recovery tube 22. As should be recognized, syringe 12 may therefore be actuator to release a fluid contained therein through adaptor 14 for filtration by each filter manifold 16 with the filtered fluid collected in recovery tube 22.

[0019] Referring to FIGS. 3 and 4, filter manifold 16 includes a filter support 30 extending circumferentially therein that is dimensioned to suspend a filter 32 (such as a filter cup) in filter manifold 16. Filter 32 is removable to provide access to any filtrate caught by filter cup when a fluid is passes through device 10. As seen in FIGS. 3 and 4, an upper rim 34 of filter 32 may include openings 36 spaced therearound that are received on posts 38 positioned around the upper surface 40 of filter support 30 to secure filter 32 in place during use of device 10. Filter manifold 16 can accommodate various size filters 32, including filter 32 having different pore sizes

[0020] Device 10 may be used to implement a facile process for ultimately separating metazoan, plant and microbial cells, while maintaining cell viability for downstream applications including DNA extraction, culturing. Microscopic analyses, sequencing, assessment, and the like. In one step, two filters designated Fl and F2 (such as filters formed as filter cups 32 and positioned in filter manifolds 16) are coated on both sides with a detergent solution dilution. The top sides of filters Fl and F2 are then washed as a part of a prewetting step where 1 mL of PBS is forced through the filter manifold(s) using a syringe. Side B of each filter is placed in a clean tube. A syringe is connected and withdrawn to the 3 ml mark to draw the PBS through the filter. The syringe is then reversed, and the withdrawn fluid pumped back through the filter. This step may be repeated to ensure complete prewetting. This can be applied to device 10 or individual filter manifolds 16, where the number of filter cups 32 or filter manifolds 16 is dependent on the size exclusion and purity of the sample desired.

[0021] To capture the bacteria (microbial) fraction, the pre-wet Fl filter is attached to a clean test tube labeled bacteria Fraction (BF1), which can be filter manifold 16 of device10. The “mix,” defined as the unknown sample to be separated, is then applied to the pre-wet filter (side A) using a pipette (or a syringe or dropper). A syringe is attached to the top of filter manifold 16, air pumped onto the top side of the filter from the syringe, and then the syringe is removed. The filter or filters are then washed with 250 pL PBS (using pipette). The pumping of air steps may then be repeated to force through the PBS. Next, the pre-wet F2 filter is attached to a clean test tube labeled Bacteria Fraction (BF2), which can be filter manifold 16 of device 10. The contents of BF1 are then transferred to the pre-wet filter F2 (top side) by pipetting. The steps are then performed with F2 filter and repeated to force the sample through the filter. The contents of BF2 are saved.

[0022] To remove any residual eukaryotic cells from the Bacteria fraction (BF2), 500 pL to 1 ml of lysis buffer used during DNA isolation, such as Buffer AL available from Qiagen, is added to the tube. The buffer may be scaled down to the appropriate volume (example: if 500 pL sample (BF2), 250 pL AL buffer may be added). BF2 is then incubated for 30 minutes at room temperature with end over end rotation. BF2 is then centrifuged at 10,000 xg for 10 minutes. The supernatant is then removed and saved, with the remaining pellet comprising the residual bacteria. 1 pL DNase, 10 pL DNase buffer and 39 pL PCR grade H2O are added to the pellet on ice. The contents are then mixed by gentle vortexing and incubated at 37 C for 10 minutes without shaking. 1 pL of an EDTA solution is then added, mixed by gentle vortexing, and incubated at 75 C for 10 minutes without shaking. 10 pL of a cell lysis reagent, such as FASTBREAK™ (Promega), is then added and the contents mixed by gentle vortexing and then incubated for 15 minutes at room temperature while shaking at 300 rpm to provide a solution comprising the final treated fraction.

[0023] The solution comprising the final treated fraction may be transferred into a pathogen lysis tube containing various sized homogenizer beads, where the beads may be of the same or different diameters and made from zirconium, silica, glass, steel, ceramic, or combinations thereof and labelled Pathogen Lysis Tube L. Pathogen Lysis Tube L is then placed in a bead beater or tissue homogenizer for 5 minutes at 50 Hz with the beater held so it remains secure. After 5 minutes, the beater and tube are tightened and then beat for five more minutes at 50 Hz with the beater held so that it remains secure. Pathogen Lysis Tube L is then centrifuged at 10,000 xg for 1 minute to reduce foam. The supernatant from the final treated fraction is then moved to a fresh microcentrifuge tube labeled BF2 DNase and saved for extraction.

[0024] The eukaryotic fraction may be captured, and any residual bacteria removed, by removing or cutting the filters of Fl and F2. The filters may be cut to increase surfacearea, or to help the filters fit in a tube, and then added to a tube labelled HF. If using device 10, filter manifolds 16 may be separated from device 10 and filters 32 removed from filter manifolds 16. 400 pL PBS and 40 pL of a Fastbreak ™ (Promega) prokaryotic lysis solution are added to HF followed by gentle vortexing. HF is incubated for 15 minutes at room temperature and then shaken at 300 rpm. HF is then centrifuged at 10,000 xg for 10 minutes. The supernatant may be removed and saved, leaving about 50 pL and the filter pieces. 1 pL DNase, 10 pL DNase buffer and 39 pL PCR grade H2O are added to the pellet on ice, mixed by gentle vortexing, and then incubated at 37 C for 10 minutes without shaking. The results may then be marked as the eukaryotic fraction (HF) for subsequent extraction.

[0025] The isolated eukaryotic cells and prokaryotic cells may now be processed in the conventional manner, e.g., the pellets may then be treated with Dnase and inactivated prior to DNA extraction, and then DNA may be extracted, assessed and interpreted for the particular application.

Claims

CLAIMSWhat is claimed is:

1. A device for differential cell separation, comprising: a filter manifold having an inner surface that defines a central cavity and a filter support positioned in the central cavity; a filter suspended in the central cavity by the filter support; a syringe interconnected to a first side of the filter manifold to dispense a fluid onto the filter; and a recovery tube interconnected to a second side of the filter manifold and in communication with the cavity of the filter manifold to receive the fluid dispensed from the syringe after the fluid has passed through the filter.

2. The device of claim 1, further comprising an adaptor interconnecting the syringe to the first side of the filter manifold.

3. The device of claim 2, wherein the adaptor includes a first set of threads that can engage a corresponding second set of threads of the first side of the filter manifold.

4. The device of claim 3, wherein the syringe is connected to the adaptor by a luer lock.

5. The device of claim 1, further comprising an end cap interconnecting the recovery tube to the second side of the filter manifold.

6. The device of claim 5, wherein the end cap includes a first set of threads that engage a corresponding second set of threads of the filter manifold.

7. The device of claim 6, wherein the end cap include a tube fitting that frictionally engages an opening of the recovery tube.

8. The device of claim 1, further comprising a second filter manifold having a second central cavity and a second filter support positioned in the second central cavity, wherein the second filter manifold is coupled between the filter manifold and the recovery tube with the second central cavity is in communication with the first filter cavity and the second filter cavity is in communication with the recovery tube.

9. The device of claim 1, wherein the filter support extends circumferentially around an inside of the filter manifold that forms the central cavity.

10. The device of claim 9, wherein the filter is secured in a filter cup having an upper rim that engages the fdter support.

11. The device of claim 10, wherein the upper rim of the filter cup includes a series of opening that align with an receive a corresponding series of posts extending from the fdter support.

12. A method of separating any prokaryotic cells from any eukaryotic cells in a sample, comprising the steps of: providing a first filter manifold with a first central cavity and a first filter suspended in the first central cavity and a first recovery tube connected to the first filter manifold and in communication with the first central cavity downstream of the first filter; suspending a first filter in the first filter manifold; applying an unknown sample to the first filter; pumping air onto the first filter using a syringe coupled to the first filter manifold; washing the first filter with a first solution; collecting the first solution in the first recovery tube as a first fraction; providing a second filter manifold with a second central cavity and a second filter suspended in the second central cavity and a second recovery tube connected to the second filter manifold and in communication with the second central cavity downstream of the second fdter; suspending a second filter in the second filter manifold; applying the first fraction to the second filter; pumping air onto the second fdter using the syringe coupled to the second fdter manifold; washing the second filter with a second solution; and collecting the second solution in the second recovery tube as a second fraction.

13. The method of claim 12, wherein any of the amount of prokaryotic cells in the second fraction may be removed by adding a buffer to the second fraction, incubating the second fraction after adding the buffer and centrifuging the second fraction after incubating to provide a first supernatant and a first pellet containing the amount of prokaryotic cells.

14. The method of claim 13, further comprising the steps of: cutting the first filter and the second filter into pieces; placing the pieces into a test tube; adding an amount of buffer and an amount of a prokaryotic lysis reagent; mixing the pieces with the amount of buffer and the amount of the prokaryotic lysis reagent; andcentrifuging the pieces with the amount of buffer and the amount of the prokaryotic lysis reagent to form a second supernatant and a second pellet containing the amount of eukayotic cells.

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