WO2024158712A1

WO2024158712A1 - Method and device for preparing and extracting an agricultural biomolecule

Info

Publication number: WO2024158712A1
Application number: PCT/US2024/012439
Authority: WO
Inventors: Jonathon DIVER; Blake FREML; Hena GUO; Pa Kou LOR; Mark Nicolas; Justin Andrew SCHARES; Robert Shartle; Huyen Tran; Ming X. Tan
Original assignee: Pioneer Hi-Bred International, Inc.
Priority date: 2023-01-23
Filing date: 2024-01-22
Publication date: 2024-08-02

Abstract

The present invention provides an improved method and device for preparing, extracting, separating and/or purifying an agricultural biomolecule, for example, nucleic acid from seed samples, from leaf samples, from fungi, from insects or from soil microbes. In one embodiment the device is an extraction tube with breakable ampoules comprising the extraction buffers, and a removable filter that remains separated from the reaction until the lysate is ready to be filtered upon dispensing. The other end of the extraction tube may comprise a removable endcap with an integrated measuring scoop for measuring ground samples and inserting the measured samples into the extraction tube or an integrated microneedle patch for sample collection.

Description

METHOD AND DEVICE FOR PREPARING AND EXTRACTING AN AGRICULTURAL BIOMOLECULE

Field

[0001] The present invention provides an improved method and device for preparing, extracting, separating and/or purifying an agricultural biomolecule, for example, nucleic acid or proteins from seed samples, from leaf samples, from fungi, from insects and/or from soil microbes.

BACKGROU N D

[0002] Nucleic acid-based sequencing procedures typically require nucleic acid extractions from biological substances. Nucleic acid extraction, whether using polymerase chain reaction (PCR) or PCR free sequencing, is an important first step in this process, and requires clean purified nucleic acids free of cell lysate. This extraction and purification can be particularly problematic for plant cell extracts, which comprise high amounts of structural molecules such as lignin, cellulose (including cellulose microfibrils), hemicellulose, pectin and cross-linking glycans. Further, portable devices have been developed that allow for in-field nucleic acid based sequencing, and associated portable extraction devices and methods are needed that allow for in-field extraction and purification of nucleic acids from plants as well as other agricultural samples such as fungi, insects and soil microbes.

[0003] It is an object of the present invention to provide an in-field method and device for extracting and purifying an agricultural biomolecule, such as a nucleic acid from a plant cell.

SU M MARY

[0004] One embodiment relates to a method for preparing a biomolecule-containing composition, the method comprising the steps of (a) providing a device comprising a body, the body defining an inner chamber comprising at least one breakable ampoule, and wherein the inner chamber has a volume sufficient to receive a sample comprising a biomolecule, a first opening located at one end of the device comprising a removable scoop endcap, said removable scoop endcap comprising an inner cavity into which a sample may be inserted into the inner cavity and said removable scoop endcap forms a watertight seal with the inner chamber upon sample insertion, a second opening located at or towards the opposing end of the device comprising a watertight puncturable seal, an attachable filter housing capable of puncturing the opposing end watertight puncturable seal, said attachable filter housing comprising at least one filter and further comprising a nozzle end; and (b) adding a sample comprising a biomolecule to the inner chamber of said device by addition of the sample to the inner cavity of the scoop endcap and attachment of the scoop endcap with the inner chamber, wherein the inner chamber comprises one or more reagents in the one or more breakable ampoules, and wherein at least one of the sample and the one or more reagents comprises a liquid; and (c) breaking one or more ampoules and shaking the device to create a mixture comprising extracted biomolecules, and (d) affixing the attachable filter housing to the end of the device in a manner that punctures the puncturable seal and allows the liquid to pass through the filter and through the nozzle, and e) thereby recovering the biomolecule-containing composition.

[0005] One embodiment relates to a method for preparing a biomolecule-containing composition, the method comprising the steps of (a) providing a device comprising a body, the body defining an inner chamber comprising at least one breakable ampoule, and wherein the inner chamber has a volume sufficient to receive a sample comprising a biomolecule, a first opening located at one end of the device comprising a removable endcap, said removable endcap comprising a microneedle patch, wherein said removable endcap forms a watertight seal with the inner chamber upon sample insertion, a second opening located at or towards the opposing end of the device comprising a watertight puncturable seal, an attachable filter housing capable of puncturing the opposing end watertight puncturable seal, said attachable filter housing comprising at least one filter and further comprising a nozzle end; and (b) removing the endcap from the tube and pressing the microneedle patch surface into plant tissue, then reinserting the endcap into the tube by attachment of the endcap back into the inner chamber of the extraction tube, wherein the inner chamber comprises one or more reagents in the one or more breakable ampoules, and wherein at least one of the sample and the one or more reagents comprises a liquid; and (c) breaking one or more ampoules and shaking the device to create a mixture comprising extracted biomolecules, and (d) affixing the attachable filter housing to the end of the device in a manner that punctures the puncturable seal and allows the liquid to pass through the filter and through the nozzle, and e) thereby recovering the biomolecule-containing composition.

[0006] Another embodiment relates to a method for preparing a biomolecule-containing composition, the method comprising the steps of (a) providing a device comprising a body, the body defining an inner chamber comprising at least one breakable ampoule, and wherein the inner chamber has a volume sufficient to receive a sample comprising a biomolecule, a first opening located at one end of the device comprising a removable scoop endcap, said removable scoop endcap comprising an inner cavity into which a sample may be inserted into the inner cavity and said removable scoop endcap forms a watertight seal with the inner chamber upon sample insertion, a second opening located at or towards the opposing end of the device comprising a capable filter housing, said capable filter housing comprising at least one filter and further comprising a nozzle end with a removable cap over the nozzle end; and (b) adding a sample comprising a biomolecule to the inner chamber of said device by addition of the sample to the inner cavity of the scoop endcap and attachment of the scoop endcap with the inner chamber, wherein the inner chamber comprises one or more reagents in the one or more breakable ampoules, and wherein at least one of the sample and the one or more reagents comprises a liquid; and (c) breaking one or more ampoules and shaking the device to create a mixture comprising extracted biomolecules, and (d) removing the removable cap from the capable filter housing to allow liquid to pass through the filter and through the nozzle, and e) thereby recovering the biomolecule-containing composition.

[0007] Another embodiment relates to a method for preparing a biomolecule-containing composition, the method comprising the steps of (a) providing a device comprising a body, the body defining an inner chamber comprising at least one breakable ampoule, and wherein the inner chamber has a volume sufficient to receive a sample comprising a biomolecule, a first opening located at one end of the device comprising a removable endcap that comprises a microneedle patch, said removable endcap forming a watertight seal with the inner chamber upon sample insertion, a second opening located at or towards the opposing end of the device comprising a capable filter housing, said capable filter housing comprising at least one filter and further comprising a nozzle end with a removable cap over the nozzle end; and (b) removing the endcap from the tube and pressing the microneedle patch surface into plant tissue, then reinserting the endcap into the tube by attachment of the endcap back into the inner chamber of the extraction tube, wherein the inner chamber comprises one or more reagents in the one or more breakable ampoules, and wherein at least one of the sample and the one or more reagents comprises a liquid; and (c) breaking one or more ampoules and shaking the device to create a mixture comprising extracted biomolecules, and (d) removing the removable cap from the capable filter housing to allow liquid to pass through the filter and through the nozzle, and e) thereby recovering the biomolecule-containing composition. [0008] In some embodiments, the reagents comprise one or more of a lysis buffer, a precipitation buffer, and alkaline proteinase or a cell wall degrading enzyme, or a cellulase, hemicellulase, pectinase, glucouronidase, glucanase, chitinase, laminarinase, lyticase, lysozyme, subtilisin, proteinase K, trypsin, caldolysin, or a combination of any two or more thereof.

[0009] In some embodiments the method uses two or more ampoules that can be broken in sequence, as a way of sequencing the timing of the reaction.

[0010] In some embodiments the sample tested is ground seed, such as soybean seed. In some embodiments the sample tested is leaf or other plant tissue, such as stalk, meristem or root tissue.

[0011] The biomolecule being extracted may be a nucleic acid. In some embodiments the nucleic acid is DNA. In some embodiments the nucleic acid is RNA. In some embodiments the nucleic acid is a mixture of DNA and RNA. In some embodiments, a first fluorescent probe identifies a first transgenic trait. In some embodiments a second fluorescent probe identifies a wild type or non-transgenic sample. In some embodiments, the relative proportion of transgenic and wild type varieties in the sample may be measured, thereby quantifying the amount of the transgenic trait present in the overall sample. In other embodiments, two or more fluorescent probes may be used that identify two or more transgenic traits, thereby quantifying the relative proportions of the two or more transgenic traits present in the overall sample. In some embodiments, quantitative PCR may be used to identify the fluorescence at different cycle times, and to measure the relative proportion of the sequence detected by the fluorescent probe at each cycle.

[0012] In some embodiments the nucleic acid is DNA from a plant tissue. In other embodiments, the nucleic acid is DNA from a pathogen endogenous to the plant tissue.

[0013] Another embodiment provides a device for preparing a biomolecule-containing composition from a sample, the device comprising a body, the body defining an inner chamber comprising at least two breakable ampoules, and wherein the inner chamber has a volume sufficient to receive a sample comprising a nucleic acid, a first opening located at one end of the device comprising a removable endcap, said removable endcap comprising an integrated sample collection and measurement system that may be inserted into the inner cavity and said removable endcap forms a watertight seal with the inner chamber upon sample insertion, a second opening located at or towards the opposing end of the device comprising a watertight seal, and an attachable filter housing capable of replacing or puncturing the opposing end watertight seal, said attachable filter housing comprising at least one filter and further comprising a nozzle end.

[0014] Another embodiment provides a device for preparing a biomolecule-containing composition from a sample, the device comprising a body, the body defining an inner chamber comprising at least two breakable ampoules, and wherein the inner chamber has a volume sufficient to receive a sample comprising a nucleic acid, a first opening located at one end of the device comprising a removable endcap, said removable endcap comprising an integrated sample collection and measurement system that may be inserted into the inner cavity and said removable endcap forms a watertight seal with the inner chamber upon sample insertion, a second opening located at or towards the opposing end of the device comprising a capable filter housing comprising a nozzle end with a removable top cap, said removable top cap capable of forming a watertight seal between the nozzle tip and the inside of the top cap, said capable filter housing comprising at least one filter.

[0015] In some embodiments the device comprises two or more ampoules that can be broken in sequence, as a way of sequencing the timing of the reaction. In some embodiments, the reagents in the ampoules may comprise one or more of a lysis buffer, a precipitation buffer, and alkaline proteinase or a cell wall degrading enzyme, or a cellulase, hemicellulase, pectinase, glucouronidase, glucanase, chitinase, laminarinase, lyticase, lysozyme, subtilisin, proteinase K, trypsin, caldolysin, or a combination of any two or more thereof. In some embodiments, the first ampoule comprises a lysis buffer, and the second ampoule comprises a precipitation buffer.

[0016] In some embodiments the endcap comprises a grip edge that extends beyond the body of the device, which grip may be sized for fingers in order to allow for easy removal and reattachment of the endcap. In some embodiments, the removeable and re-attachable endcap comprises an integrated sample collection system that collects a known quantity of sample. In some embodiments, the endcap comprises an internally rounded scoop that eliminates or minimizes the amount of edges in which a sample might agglomerate in a way that prevents contact with the reagents. In some embodiments, a leaf punch or microneedle patch can be added to the internal recessed portion of the end cap instead of the scoop. In other embodiments, the microneedle patch can be integrated with the end cap, so that it is flush or raised from the internal portion, so that the endcap can be removed, the microneedle patch portion pressed against sample tissue and reinserted into the extraction tube, where the sample tissue captured by the microneedle patch portion will be able to come in contact with the biomolecule extraction reagents.

[0017] In some embodiments, the device comprises a means for conducting the reactions apart from a filter, wherein the filter is utilized in the final step of expelling the final product of the reaction into an analytic device. In some embodiments the means for separating the filter from the reaction is a puncturable seal, such as a foil seal, waver seal, silicone seal, polyurethane seal, or the like. In some embodiments the means for separating the filter from the reaction may be a removable cap that may be replaced with a housing comprising one or more filters. In some embodiments, both may be used, and the filter housing may comprise an edge or other feature that may be used to puncture the puncturable seal when the filter housing is inserted on or into the extraction device.

[0018] In some embodiments the analytic device is a PCR machine. In some embodiments, the analytic device may be a sequencer or an ELISA assay.

BRI EF DESCRI PTION OF DRAWINGS

[0019] The invention will now be described by way of example only and with reference to the drawings in which:

[0020] FIG. 1 depicts a top angled view of the extraction and purification device, with the filter and application tip in position for extrusion of the purified nucleic acids, with the exterior of the reaction chamber transparent. For nucleic acid extraction, the top breakable ampoule holds the precipitation buffer, while the bottom ampoule holds the lysis buffer.

[0021] FIG. 2 depicts a top view of the extraction device, showing the filter and top cap system in open positions. The removeable and re-attachable endcap is engaged in the bottom portion of the extraction tube, and a puncturable seal may be engaged at the top portion of the tube.

[0022] FIG. 3 depicts a top view of the endcap when removed from the extraction tube. This embodiment shows a measuring scoop integrated into the endcap.

[0023] FIG. 4 depicts a partial cut-out side view of the scoop endcap, showing the rounded bottom in the measuring portion in the interior of the scoop endcap.

[0024] Fig. 5 depicts a partial cut-out side view of the nozzle, including the nozzle tip, showing the proportions and dimensions of one embodiment with a 0.7mm diameter center at the constriction point. [0025] Fig. 6 depicts a top side view of a lacerating leaf punch that may be used in the endcap to obtain leaf punch tissue samples for analysis.

[0026] Fig. 7 depicts a cross section view of the removable endcap to illustrate the location of the second filter that can be laced with magnetic beads to facilitate subsequent DNA analysis.

DETAI LED DESCRI PTION OF TH E I NVENTION

[0027] The present invention relates to a device and method for preparing, separating, extracting and/or purifying a biomolecule from a sample. The device comprises an extraction tube defining an inner chamber, which inner chamber houses the biomolecule extraction. In one embodiment, the inner chamber comprises two ampoules. The first ampoule comprises a lysis buffer to lyse the cell walls and release the nucleic acids and/or other biomolecules to be prepared, separated, extracted and/or purified. The second ampoule comprises a buffer or other purifying reagent to isolate and prepare the biomolecule for assay. In the case where the biomolecule is a nucleic acid, the second ampoule may comprise a precipitation buffer.

[0028] A removable endcap with an integrated sample collection and measurement system serves to seal the bottom end of the extraction tube. The outer perimeter of the removable endcap forms a tight seal with the inner perimeter of the extraction tube and prevents the aqueous material from leaking from the extraction tube. In the embodiment shown, the removable endcap comprises an inner cavity preformed into a measuring scoop calibrated to hold a measured amount of powdered plant (or other) material. In the embodiment shown in Fig. 1, the removeable endcap is premeasured to hold 0.10 grams of soy powder obtained from ground soybean seeds, which sample size amount has been optimized for use with 1.5 mL lysis buffer and 1.5 mL precipitation buffer. However, in other embodiments the scoop size may be optimized in size to account for the different sample size of different types of samples or different sample preparation techniques, such as different grinding size or particle grinding, crushing, leaf punches, etc. and may be a volume anywhere between 2 cubic centimeters and 30 cubic centimeters, or any integer or fractional volume in between. Each ampoule may be adjusted in volume, and may range from .5 mL of volume to 30 mL of volume, or any integer or fractional volume in between. The size of the extraction tube may be adjusted accordingly to accommodate the sample size and ampoule volumes. The ampoules may be made of a glass capsule, a ceramic capsule, plastic or any other easily hand or tool breakable material. The composition of the extraction tube is flexible enough to allow the user to break the ampoule by hand or with a hand operated clamp. Liquid in the ampoules may have a layer of inert gas above the liquid to prevent oxidation or pH changes.

[0029]

[0030] 1. Definitions

[0031] The term "and/or" can mean "and" or "or".

[0032] The term "collection and measurement system" refers to a removable endcap design that can be used to collect a known quantity of a sample. Non-limiting examples of such a system include a measuring scoop formed into the internal portion of the endcap, a recessed portion of the endcap adapted to collect and hold a sample of known size such as a leaf punch, a microneedle patch that when pressed into tissue will collect a consistent quantity of such tissue. Such term also includes an associated device, such as leaf punch, that can deposit a leaf disc of known size into the endcap. Such term does not include a swab that collects an unknown quantity of sample.

[0033] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

[0034] The term "nucleic acid" as used in this specification refers to a single- or doublestranded polymer of deoxyribonucleotides (DNA), ribonucleotide bases (RNA) or known analogues of natural nucleotides, or mixtures thereof. The term includes reference to synthetic, modified or tagged nucleotides.

[0035] The term "(s)" following a noun contemplates the singular or plural form, or both.

[0036] The term "sample" as used in this specification refers to any material from which a biomolecule is to be prepared, extracted, purified and/or separated. The sample may comprise a natural or biological sample, for example, ground seed, ground leaf tissue, leaf punches, insects, fungi fruiting bodies (ground or cut), and/or soil containing microorganisms, such as nematodes in a soil sample or already purified out of a soil sample. The sample may be obtained from the plant or insect using a leaf punch or microneedle patch or other sample-holding matrix. In some cases the sample-holding matrix will have been used to obtain the sample from a source and is able to be added directly to the device for extraction, purification, separation or preparation of the biomolecule from the sample. One such example would be a removeable endcap with an integrated swab with absorbent material for liquid sample collection.

[0037]

[0038] 2. Device and Method of the Invention

[0039] The extraction device 100 of the present invention as shown in FIG. 1 comprises an extraction tube 110 defining an inner chamber housed within an outer body. While in the embodiment shown the extraction device is in the form of a tube, it is hereby noted that other shapes and sealing mechanisms may be used, so long as it remains possible to break the ampoules in sequence. Both ampoules are sealed, and the reagents inside are not released until the ampoules are broken. A hand operated clamp (not shown) has been designed that grips the tube and can be squeezed by hand to create pressure that breaks an individual ampoule, and then can slide up the extraction tube 110 in a position where it is used to break the subsequent ampoule.

[0040] The extraction device 100 and all associated components, other than the ampoules, may be entirely constructed of polypropylene, although polyethylene, polyvinyl alcohol, polyvinyl chloride, or other material readily apparent to those of ordinary skill in the art may be used, in whole or in part. In one embodiment, the extraction tube 110 is constructed of polypropylene and the removable endcap is constructed of low density polyethylene. In this embodiment, the polypropylene should allow for enough flexibility so that the user can break the one or more ampoules. In another embodiment, the removable endcap is constructed of polyvinyl alcohol with an integrated microneedle patch. The microneedle patch may be fabricated through a vacuum-based micromolding procedure. In one embodiment, the microneedle patch is a 15 by 15 microneedle array, with each needle about 800 pm in height, 150 pm in base radius, and 5 pm in tip radius, with a fracture force strong enough to insert into plant (or skin) tissues without breaking. Microneedle patches may comprise arrays of any configuration or shape, as well as various ranges of needle sizes.

[0041] As can be seen in FIG. 2, the extraction tube 110 comprises an opening at each end. At the top end of the extraction tube 110 a puncturable seal 120 may be affixed during assembly of the extraction device 100. In one embodiment, the puncturable seal 120 is made of a laminate foil or film, such as heat staked foil coated with low density polyethylene. In another embodiment, the puncturable seal 120 may be an end of the extraction tube 110 that can be punctured or cut, such as a one-way pressure valve, a pressure burst valve, a burst disc, a rupture disk or other breakable or removable barrier. The valve, disc or other breakable or removable barrier, all of which are included within the definition of a puncturable seal, may be injection molded as part of the extraction device. In another embodiment, a removeable water-tight cap, or top cap 180 may be used by affixing the filter housing to the extraction tube 110 with the top cap 180 forming an airtight seal over the nozzle tip 175, so that the top cap 180 creates an air pressure block in the filter housing 160 that prevents liquid from passing through the filter and into the filter housing 160 during the mixing stage of the reaction. In certain embodiments the volume of air space in the nozzle 170 and filter housing 160 is minimized to allow the air pressure block to form, and the total volume of space in the nozzle 170 is less than 400 uL, and the total volume of space in the filter housing 160 is less than 2000 uL. In the embodiment shown in Fig. 5, the nozzle 170 (inclusive of the nozzle tip 175) is 318 uL and the filter housing is 1864 uL.

[0042] At the bottom end of the extraction tube 100, there is an endcap 200 that may be removed and reattached to the tube. In other embodiments (not shown in the figures), the scoop portion of the endcap may be replaced with a recessed portion sized to accommodate a leaf disc or microneedle patch, or an integrated leaf punch or microneedle patch constructed as part of the endcap. Interior to the extraction tube are at least two breakable ampoules. The first breakable ampoule 130 comprises a lysis buffer to lyse the cell walls and release the nucleic acids and/or other biomolecule to be prepared, separated, extracted and/or purified. The second breakable ampoule 140 comprises a buffer or other purifying reagent to isolate and prepare the biomolecule for assay. In the case where the biomolecule is a nucleic acid, the second ampoule may comprise a precipitation buffer. The lysis buffer may be any solution known to those of skill in the art, but in one embodiment described in more detail below, the lysis buffer comprises 2% sodium dodecyl sulfate (SDS) when the volume of the lysis buffer is 2.0 mL or less and .10 g of powdered seed is used. In another embodiment, the lysis buffer is an alkaline DNA extraction solution with from 1.5% to 2.0% SDS. In one embodiment, the lysis buffer may be a hotshot lysis buffer. In another embodiment, sodium lauryl sulfate (SLS) or another anionic surfactant may be used instead of SDS. The purifying reagent may be any reagent capable of purifying the biomolecule of interest. For any antigen, the purifying reagent may be beads with conjugated antibodies, designed to capture a target cell or protein for enrichment or evaluation. Beads capable of binding DNA may also be used, or the DNA and other nucleic acids may be precipitated with a precipitation buffer for enrichment or evaluation, including subsequent attachment to magnetic beads or other types of beads. In one embodiment, the nucleic acid is precipitated with a precipitation buffer. The precipitation buffer may be any such buffer known to one of ordinary skill in the art, including but not limited to alcohol (e.g., isopropanol or ethanol) and salt (e.g. sodium acetate, ammonium acetate, sodium chloride or lithium chloride). In one embodiment, the precipitation buffer comprises a potassium acetate solution.

[0043] To perform the extraction with the device of the embodiment shown in the figures, the endcap 200 is removed from the device, the internal cavity 210 of the endcap 200 forms a scoop that is filled with a sample, such as powder produced by grinding soybean seed (or soil or other types of agricultural seeds to be tested), and reinserted into the extraction tube 110. To minimize sampling area, the endcap 200 comprises an inner cavity 210 calibrated in volume to serve as a measuring device, while the outer surface 220 of the endcap 200 is sized to form a friction fit with the extraction tube 110. In one embodiment, the endcap 200 comprises a grip portion 230 to facilitate removal and reattachment to the extraction tube 110. In another embodiment, the internal cavity 210 of the endcap 200 comprises a rounded bottom 240 to facilitate dispersion of the sample when the sample filled endcap 200 is inserted into the extraction tube 110 and shaken to mix the sample with the lysis buffer released after breaking the first breakable ampoule 130. In some embodiments the curved bottom 240 at the base of the inner cavity 210 of the scoop endcap 200 may be in the shape of a parabola or hemisphere. In one embodiment, the inner cavity 210 of the endcap 200 is sized to accommodate .10 grams of soybean seed ground to a fine powder.

[0044] In an alternative embodiment, the removable endcap 200 will not comprises a scooped inner cavity 210, and instead will comprise an integrated microneedle patch. One such microneedle patch for use in such an embodiment has been described in Extraction of Plant DNA by Microneedle Patch for Rapid Detection of Plant Diseases, by Paul, R. et al, ACS nano, 2019, published by the American Chemical Society, DOI: 10.1021/acsnano.9b00193, which is incorporated by reference herein. Such a microneedle patch can be integrated into the surface of the removable endcap that is exposed in the interior of extraction tube 110 when the endcap is inserted. For example, in one such embodiment (not shown), the integrated microneedle patch would be flush with or slightly raised from the inner surface of the endcap. The endcap could be removed from the tube and the microneedle patch pressed onto the surface of the leaf, stem, plant, insect or other tissue (or even skin) to collect the sample and then replaced into the extraction tube 110, so that when the endcap is replaced in the extraction tube 110 the surface of the microneedle patch comprising the tissue with the DNA (or RNA, polysaccharide or other biomolecule) will be exposed to the reagents released upon the breaking of the one or more ampoules. In this alternative embodiment, an additional ampoule comprising a buffer solution that washes the genetic material off of the microneedles and directly into the interior of the extraction tube 110 may be used.

[0045] In another alternative embodiment, a lacerating leaf punch may be used. As shown in the representative embodiment illustrated in Fig. 6, the leaf punch simultaneously extracts a leaf disk and has a lacerating effect on the leaf disk. Several leaf punch designs were tested, with various reagent concentrations as described in Example 5 below. Fig. 6 shows the removable endcap base 500, a raised cylinder 505 similar in diameter to the inner surface of the extraction tube 110, such that a watertight seal or air block is formed, a frustoconical portion 515, and a lacerating cone 520 that comes to a point at the top to provide the initial puncture of the leaf. Other designs (not shown) comprised a cylinder raised above the removable endcap base, similar to that of the embodiment shown in Fig. 6, and with a raised cutting pedestal for leaf cutting also similar to that of the embodiment shown in Fig. 6, except with the base of the cylinder being slightly recessed within the top portion of the raised cylinder. An additional design (not shown) comprised a single raised cylinder that, at the top portion, terminated in a hyperbolic paraboloid surface that served as cutting surface. Yet a further design (not shown) comprised a raised cylinder that, at the top portion, comprised a depression surrounded by a raised scallop wall that served as the cutting surface. All designs successfully cut out leaf disks from which DNA could be extracted. The latter two designs were satisfactory, but in some cases the leaf disk remained stuck in or onto the raised cylinder, thereby making DNA extraction less predictable. The advantage of the design shown in Fig. 6 is that the leaf disk easily detached from the raised cylinder portion, and would have a laceration through the center of the leaf disk that further facilitated DNA extraction. Therefore, this design was especially beneficial when the laceration leaf punch was be repeatedly used to obtain multiple leaf punches for the DNA extraction, as described below in Example 5.

[0046] When conducting the extraction reaction, the first ampoule with the lysis buffer is broken, thereby releasing the lysis buffer into the interior of the extraction tube 110. The extraction tube is shaken to mix the lysis buffer and the sample, resulting in DNA (or RNA or other biomolecule) lysis from the cells or microbes. After lysis is complete, the second ampoule with the precipitation buffer (or other purifying reagent) is broken, thereby releasing the precipitation buffer into the interior of the extraction tube 110. Once the precipitation reaction is complete, the attachable filter housing is inserted through the puncturable seal 120, with the exterior of the attachable filter housing 160 forming a seal with the interior of the extraction tube 110. The integrated seal puncturing edge 165 of the attachable filter housing 160 can be used to pierce the puncturable seal 120. A double "O" ring connector 190 with an attached top cap 180 helps to secure the seal to prevent leakage, while also serving to facilitate storage and attachment of the attachable filter housing 160 with nozzle 170 and the top cap 180.

[0047] The attachable filter housing 160 comprises at least one filter. In one embodiment, two filters are used, with the first filter heat staked or otherwise attached to match the slant of the seal puncturing edge 165 of the attachable filter housing 160, and the second positioned inside the attachable filter housing. In one embodiment, the position of the first filter is as shown in Fig. 2, with the first filter 310 at the base of the filter housing 160, and the second filter 320 at the entrance to the nozzle 170 as shown in Fig. 5. In some embodiments, the filter is heat staked to match the slant of the seal puncturing edge 165 has a larger pore size than the second filter 320. Filters of several pore sizes and materials all functioned well. The one or more filters may comprise or be operably engaged with a purifying material that can bind or retain substances that are deleterious to downstream applications of the biomolecule. For example, the purifying material may bind or retain salts including potassium, calcium, magnesium or sodium salts, detergents including sodium dodecyl sulfate, peptides or peptide complexes. Purifying materials may include but are not limited to activated charcoal, a chelating resin, an ion exchange resin, a desalting resin, a gel, a clay, a concentrating agent, a water absorption material, a cross-linked dextran gel, agarose, polyacrylamide, silica, silicon dioxide, zeolite, diatomaceous earth, coal-derived activated charcoal, plant-derived activated charcoal and/or paramagnetic silica.

[0048] Pressure may be applied to the extraction tube 110 to expel the precipitated DNA (or RNA or other purified biomolecule) through the one or more filters and out the nozzle tip 175 into any suitable enrichment or evaluation device. The nozzle 170 and nozzle tip 175 may be sized appropriately for the intended assay device, such as a microfluidic device or gene chip. This can be done immediately, such as in the field, at a grain elevator or other point of grain delivery, or the nozzle 170 may be capped with a removable seal at the nozzle tip 175 with top cap 180 for shipment and analysis at another location, such as when DNA (or another biomolecule) is extracted from a leaf, insect, fungal or soil sample taken in a field.

[0049] The attachable filter housing 160, top cap 180 and or removeable end cap 200 may be configured to engage with the extraction tube, or with each other, by friction fit or by a threading arrangement.

[0050] In one embodiment, the nozzle 170 and nozzle tip 175 is sized to produce droplets that dispense droplets of a known size and shape, so that the droplet will comprise a known amount of the biomolecule of interest in a known volume of solution. For example, the nozzle 170 of the embodiment shown in Fig. 5 comprises a constriction point (300) of 0.7mm, which widens out slightly to the end point of the tip to produce a droplet with an average volume of 22 microliters. Given the known volume, viscosity and surface tension dynamics produced with the known reagents and quantities used in the extraction device 100, the amount of isolated DNA obtained from a level scoop end cap 200 of ground soybean seed would be in the range of 3.3 to 11.2 nanograms per microliter. The droplet can be sized accordingly for the different quantity of isolated DNA (or RNA or other biomolecule) that would be obtained by using a microneedle patch or leaf punch of known sample size.

[0051] The device, and particularly the nozzle 170 may be configured to engage with many standard laboratory collection tubes, tube strips or plates, for example, mini-prep tubes, PCR tubes, or custom vessels for specific diagnostic apparatus. These include but are not limited to chip, microfluidic and flow cell based diagnostic apparatus. An increasing number of such devices are being developed at a portable scale, which can perform PCR, detect DNA (including in real time) and/or sequence DNA (or RNA). Non-limiting examples of these devices include the MobiNAAT, described in Mobile nucleic acid amplification testing (mobiNAAT) for Chlamydia trachomatis screening in hospital emergency department settings, Shin et al, Scientific Reports, 4495 (2017), the MinlON device produced by Oxford Nanopore Technologies, the nanofluidic Processor developed by miDiagnostics, the Accula™ system produced by Thermofisher, and the be.well ™ device being developed by Alveo Technologies. Some of these chip, microfluidic and flow cell based devices are designed to work with swabs as versus liquid drops, in which case the nozzle or the top cap may be adapted to comprise a swab (not shown). The swab may be a removeable swab positioned partially within the nozzle housing, with a portion that binds or absorbs the purified DNA or other biomolecule positioned past the filter or filters, which could then be removed in order to insert the portion of the swab comprising the purified DNA or other biomolecule into the diagnostic device. Alternatively, in an embodiment not shown in the figures, the swab could be integrated into the nozzle tip 175 or top cap 180, such that the end of the nozzle tip 175 is a swab that has bound or absorbed the purified DNA or other biomolecule and could be inserted directly into the diagnostic device. The swab could further comprise hollow tubes or channels that would direct the purified DNA or other biomolecule into the binding or absorbent portion of the swab.

[0052]

[0053] 3. Applications of the Device and Method

[0054] The device and methods are suitable for preparing, extracting, purifying or separating biomolecules from a wide range of samples.

[0055] In one embodiment the sample is derived from plant tissue. In various embodiments the sample is derived from the leaves, stems, roots, flowers, seeds, sap, bark, pollen or nectar of a plant. One embodiment involves collection of DNA from the epidermis and mesophyll layer of a leaf, and may also comprise collection of an endogenous pathogen from the infected mesophyll layer of the plant tissue.

[0056] In one embodiment the sample is ground seed. One embodiment involves testing ground grain for the presence and identification of specific transgenic traits. One embodiment specifically involves testing ground soybean seeds for the identification of two or more transgenic traits present in the DNA of the seeds. In one embodiment the sample is obtained from a microorganism. In various embodiments the sample comprises bacteria, yeast, fungi, endophytes or spores.

[0057] In one embodiment the sample is a crude or unprocessed sample. For example, the sample may be a crude sample obtained from a subject or source and applied directly to the device without any processing or purification steps undertaken.

[0058] In one embodiment the sample comprises a partially purified preparation comprising a biomolecule. For example, in various embodiments the sample comprises a cell lysate, partially degraded tissue, or a sample that has undergone one or more grinding and/or partial purification steps. In one embodiment the method of the invention is used to remove one or more residual contaminants or undesirable substances from a sample comprising a biomolecule to obtain a composition comprising a substantially pure biomolecule.

[0059] In various embodiments the one or more reagents comprises an enzyme. In various embodiments the enzyme is selected from the group comprising a thermostable enzyme, a thermophilic enzyme, a mesophilic enzyme, a proteolytic enzyme, an alkaline proteinase, a serine protease, a metalloproteinase, a neutral proteinase, a threonine proteinase, an aspartate proteinase, a cysteine proteinase, a cell-wall degrading enzyme, and a combination of any two or more thereof. In various embodiments the one or more reagents comprises cellulase, hemicellulase, pectinase, glucouronidase, glucanase, chitinase, laminarinase, lyticase, lysozyme, subtilisin, proteinase K, trypsin, Bacillus sp. EA1 proteinase, thermolysin, caldolysin, a pectate lyase, polygalacturonase, lysozyme, a lysin, a lytic enzyme, a Thermus proteinase, or a combination of any two or more thereof.

[0060] In one embodiment the one or more reagents comprises one or more non-enzymatic reagents. In one embodiment the one or more reagents comprises one or more cations selected from the group comprising potassium, sodium, magnesium and calcium ions. In one embodiment the one or more reagents comprises one or more non-ionic surfactants selected from the group comprising a polyethylene oxide, a co-polymer of ethylene oxide and propylene oxide, a fluorosurfactant, a polysorbate, or a combination of any two or more thereof.

[0061] It will be appreciated by those skilled in the art that the device and methods of the invention are suitable for the preparation, extraction, separation, or purification of various types of biomolecules from a range of sample types for many medical, laboratory, horticultural, veterinary, agricultural, environmental, forensic or diagnostic applications.

[0062] The method and device of the invention are useful for applications where the sample comprises minute quantities of the biomolecule, where the biomolecule is of relatively poor quality, or where it is critical that the composition comprising the biomolecule comprises low or no contaminants.

[0063] The method and device of the invention are particularly useful for extracting or purifying nucleic acids, such as deoxyribose nucleic acid (DNA) or ribonucleic acid (RNA) for a variety of molecular biology applications. For example, the method and device of the invention may be used to produce a composition comprising nucleic acid extracted from a sample that is suitable for immediate use for a polymerase chain reaction (PCR), reverse transcriptase PCR (RT- PCR), quantitative PCR (qPCR or qRT-PCR), forensic DNA fingerprinting, fluorescence-based detection, chip-based hybridization detection, evaporation enrichment, DNA sequencing, RNA sequencing, molecular beacons, electrophoresis, direct electronic detection or nanopore analysis. [0064] The method and device of the invention are suitable for the preparation of nucleic acids for applications where the concentration of nucleic acid in the sample may be very low and where contamination may lead to an incorrect analysis of the nucleic acid.

[0065] An advantage of the invention is that the device is portable and the method may be carried out using simple equipment. Therefore, the method and device of the invention are particularly suited to point-of-use applications. For example, the device may be used in the field to obtain rapid extraction or purification of biomolecules from sample to reduce the potential for contamination or degradation of the biomolecule.

[0066] The invention consists in the foregoing and also envisages constructions of which the following gives examples only and in no way limit the scope thereof. By non-limiting example, one embodiment of this invention may comprises an extraction tube having an opening at only one end, the sample can be loaded at the open end, the removable cap can form a seal for the reaction, which can then be removed and replaced with the filter housing and/or nozzle for dispersing the biomolecule.

[0067] EXAMPLES

[0068] Example 1 - Test to determine the importance of preventing liquid flow through the filters during the lysis and precipitation reaction.

[0069] 1. Extraction

[0070] A device as shown in FIG. 1 and described above was used.

[0071] Example 1A - failed experiment

[0072] .10 g of soybean seed ground to a powder was loaded into a test squeeze tube capped with a filtered dispense tip that was not protected from the reaction by a puncturable seal 120 or other method. This served as a proxy for the device described above. 1.5 mL of hotshot lysis buffer was added and the sample and lysis buffer mixture was shaken by hand for 5 seconds. Next, 1.5 mL of precipitation buffer was added and the sample and lysis buffer mixture was inverted and shaken by hand for 5 seconds. Two reps of 3 drops of liquid for each rep (approximately 70 to 100 uL) were expelled through the filtered dispense tip onto a PCR MobiNAAT device, as described in US20210114036, run with an assay to detect a molecular stacked transgenic trait that confers the soybean plant with resistance to 2,4-D choline, glyphosate and glufosinate. A qualitative real time polymerase chain reaction (qPCR) assay was run with a fluorescent probe designed to bind to a DNA locus indicative of the presence or absence of a transgenic trait in order to test for the quality of the DNA extraction. Various filter pore sizes were used ranging from 7 microns to 56 microns. All samples failed regardless of the filter pore sizes or filter combinations, and the transgenic trait failed to be detected. It is believed that this experiment failed because lysis and/or precipitation buffer flowed past the first filter prior to completion of the reaction.

[0073] Example IB - Successful experiment

[0074] Example 1A was re-run with exactly the same protocol, except that after mixing with the precipitation buffer a fresh unused filter housing was placed onto the squeeze tube prior to expelling the two reps of 3 drops of liquid each into the PCR MobiNAAT device. A 7 micron filter cap was used. After replacement with the fresh unused filter cap, all PCR tests were successfully run, and the molecular stacked transgenic trait was successfully detected.

[0075] These experiments indicated the need to keep the filters and attachable filter housing 160 separate from the extraction reaction as the reaction occurred, and led to the development of the puncturable seal 120 in combination with the seal puncturing edge 165 of the attachable filter housing 160. As an alternative to a puncturable seal 120, a seal between the nozzle tip 175 and the bottom edge of the top cap 180 can be used to create air pressure in the filter housing 160 that prevented lysis buffer and/or precipitation buffer from flowing past the first filter prior to completion of the reaction. In one successful test of this alternative embodiment, dual 5 micron filters were used and the internal volume of the filter housing 160 was 1,864 uL.

[0076] Example 2

[0077] This example investigates optimal reaction volumes for extraction of nucleic acid from a sample of soybean seed ground to a powder using a device and method of the invention.

[0078] 2A. Ground Seed to Lysis Buffer Ratio

[0079] The extraction method was tested with a range of ground soybean seed in amounts of ,10g and ,25g, and with various amounts of hotshot lysis buffer, as shown in table 2A. A control sample of 0.25 g ground soybean powder and 4.00 mL lysis buffer was used. [0080] Table 2A - Ground Seed to Lysis Buffer Ratios

[0081] A benchtop method was used, in which the ground soybean seed was added to a 5 mL tube containing the hotshot lysis buffer and shaken by hand for 5 seconds. Next, an equal volume of precipitation buffer was added to the lysate and briefly mixed. After precipitation, the liquid lysate was transferred to a syringe with a 5 um syringe filter attached. The lysate was forced through the filter. 100 uL filtered lysate was loaded onto a PCR device. If the device were used, the ground soybean seed would be added to the extraction tube 110 via the scoop endcap 200. The lysis buffer would be added at the distal end to simulate the release of lysis buffer that would occur upon breaking of an ampoule of lysis buffer, in the amounts shown in Table 2A, and the distal end would be sealed with a removable seal to simulate a puncturable seal 120. The mixture would be shaken by hand for 5 seconds. Next, 1.5 mL of precipitation buffer would be added and the sample and lysis buffer mixture would be inverted and shaken by hand for 5 seconds.

[0082] Two reps of 3 drops of lysate for each rep (approximately 70-100uL) was expelled through a filter and into the PCR device. [0083] A qualitative real time polymerase chain reaction (qPCR) assay was run with FAM and VIC fluorescent probes. The FAM fluorescent probe was designed to bind to a DNA locus indicative of the presence of the molecular stacked transgenic trait that confers the soybean plant with resistance to 2,4-D choline, glyphosate and glufosinate, in order to test for the quality of the DNA extraction. The VIC fluorescent probe indicates wildtype and that the trait is not present. The results show that at a g/mL ratio of .250 g/mL or above, there was a reduction in PCR performance or consistency. Results at a g/mL ration of .100 g/mL were very favorable, with early cycle time detection, and very consistent results that matched the control sample. A g/mL range between and including .05 to .200 is preferred.

[0084] 2B. SDS percentage in Lysis Buffer

[0085] The extraction method was tested with a range of sodium dodecyl sulfate (SDS) at the optimal .10/2.0mL ratio determined in Example 2A above. While one would have expected a reduced amount of SDS in the hotshot lysis buffer as compared with the 2% amount contained in the .25g/4.0mL ratio, an unexpectedly early response was shown in cycle time 1 using a scaled down range of .10/2.0mL while maintained, and not similarly scaling down, the SDS percentage, as shown in Table 2B.

[0086] The qPCR protocol described in Example 2A was used, with a second assay run using two fluorescent probes (FAM and VIC) in order to detect the relative quantities of the transgenic trait as versus wildtype.

Table 2B

[0087] With powder from ground seeds, the tests unexpectedly indicate maintaining 2% SDS in the lysis buffer, at decreased quantities of lysis buffer, achieves resolution of FAM and FAM + VIC at an earlier stage of amplification.

[0088] 2C. Precipitation Buffer Amount

[0089] The extraction method was tested with a range of precipitation buffer amounts.

Surprisingly, when tested on powder from ground seeds, lower amounts of precipitation buffer gave increased performance with earlier cycle time amplification and detection with little adverse impact on pH.

Table 2C

[0090] The qPCR protocol described in Example 2A was used, with a second assay run using two fluorescent probes (FAM and VIC) in order to detect the relative quantities of the transgenic trait as versus wildtype.

[0091] Accordingly, with powder from ground seeds, the precipitation buffer showed resolution of FAM and FAM + VIC at earlier cycle times, while not otherwise impacting pH, in amounts ranging from .5 mL to 2.0 mL.

[0092] Example 3 - Filters

[0093] This example investigates filter sizes and filter size combinations to determine the optimal filter configuration for extraction of nucleic acid from a sample of soybean seed ground to a powder using a device and method of the invention.

[0094] Five filter assemblies were tested against two material types. The first material (Type 1) was a finely ground soybean seed. The second material (Type 2) was a finely ground high oil soybean seed, which high oil can cause difficulties during extraction. The lysis extraction and nucleic acid purification were performed as described in Example 2. Five filter combinations were tested, an initial filter of 7, 10, 15, 31, or 56 microns, and a second filter of 7 microns. The first, or initial filter, was angled to provide additional surface area for the liquid to pass through and to prevent precipitate from clogging of the filter housing 160. The second filter was positioned on the interior of the nozzle 170 at second filter location 320, which is positioned between the filter housing 160 and the nozzle 170. The first filter was positioned on the incoming edge of the attachable filter housing 160 so that the nucleic acids and extraction mixture would pass through the first filter prior to reaching the second filter. A 5 micron filter inserted at the end of a syringe and extruded with pressure was used as a control. [0095] All samples dispensed easily regardless of pore size, without clogging. Four dispenses were made per extraction, and qPCR was run for both FAM and FAM + VIC as described in prior examples. In all cases, the nucleic acid extraction with the lysis and precipitation buffers occurred prior to contact with the filters as described in Example IB.

[0096] QPCR of the results showed good results with FAM and with FAM + VIC in all samples, with a combination of two sequential 7 um filters giving the most consistent performance in terms of both amplification and cycle time values. Larger pore sizes did allow for successful amplification, but resulted in more cases of either failed or delayed amplification.

[0097] Example 4 - DNA Coated Magnetic Beads

[0098] Following DNA extraction, in some cases it is desirable to attach the extracted DNA to magnetic beads, such as carboxylate-modified magnetic beads, for further purification and/or DNA analysis as already described herein. Various methods of attaching DNA to magnetic beads were tested in conjunction with the extraction of DNA from soybean seed ground to a powder. An optimal and efficient configuration that was identified was to dry the beads onto the internal side of the second filter 320 positioned within removable endcap 160 downstream of the first filter 310, as shown in Fig. 7. The filter must be of a porosity large enough to permit the beads with attached DNA to pass through. For example, magnetic bead sizes in the range of .5 to 10 microns can be used with a filter with a porosity of 1, 5, 10 or 20 microns as appropriate. As noted in Example 3 above, 7 micron filters provided consistent performance, so beads smaller than 7 microns would be optimal for use with these filters.

[0099] Example 5 - Leaf extraction

[0100] Leaf DNA was extracted using the laceration leaf punch described herein and shown in Fig. 6. Both maize and soy leaf tissue was tested and successful DNA extractions were obtained from plant leaf tissue from each of these species. The optimal DNA yield range of about 2.0 to 3.5 ng/uL was obtained from mature maize leaf tissue by using 1 ml of hotshot alkaline lysis extraction buffer and 1 ml of a Tris-EDTA buffer, although 1.5 ml of lysis buffer was also successfully used. DNA yields ranged from about .5 ng/uL to 3.5 ng/uL. In all cases, the DNA yield obtained from using the laceration leaf punch exceeded the amount of the DNA yield obtained by using a traditional circulatory laboratory leaf punch to create leaf discs that were then loaded into the extraction tube device described herein for DNA extraction. The DNA extraction yield also tended to be greater the greater the number of leaf disks used in the extraction.

[0101] Table 5

[0102] INDUSTRIAL APPLICATION

[0103] The methods and devices of the invention have utility for a wide range of agricultural and related environmental applications, including the extraction, separation or purification of biomolecules such as nucleic acids from samples for amplification, identification, analysis and diagnostics. In particular, the methods and devices of the invention have utility at point of seed or grain deliveries to test for the presence or absence of one or more transgenic traits in the same rapid assay. The present invention also provides an improved method and device for preparing, extracting, separating and/or purifying an agricultural biomolecule, for example, nucleic acid from seed samples, from leaf samples, from fungi, from insects or from soil microbes. Soil microbes may include, but are not limited to, bacteria, actinomycetes, fungi, protozoa and nematodes. When nucleic acids are extracted, the methods and devices of the invention include immediately testing the nucleic acids or capping the ends of the tube and storing the extracted nucleic acids in the extraction tube for delivery to a laboratory.

Claims

Claims The invention claimed is:

1. A method for preparing a biomolecule-containing composition, the method comprising the steps of a) providing a device comprising a body, the body defining

1. an inner chamber comprising at least one breakable ampoule, and wherein the inner chamber has a volume sufficient to receive a sample comprising a biomolecule, ii. a first opening located at one end of the device comprising a removable endcap, said removable endcap comprising an integrated sample collection and measurement system, and said removable endcap forms a watertight seal with the inner chamber upon sample insertion, iii. a second opening located at or towards the opposing end of the device comprising a watertight seal, iv. an attachable filter housing capable of releasing the watertight seal, said attachable filter housing comprising at least one filter and further comprising a nozzle end; b) adding a sample comprising a biomolecule to the inner chamber of said device by reattachment of the removable endcap comprising the integrated sample collection and measurement system into the inner chamber, wherein the inner chamber comprises one or more reagents in the one or more breakable ampoules, and wherein at least one of the sample and the one or more reagents comprises a liquid; c) breaking the one or more ampoules and shaking the device to create a mixture comprising extracted biomolecules, d) affixing the attachable filter housing to the end of the device in a manner that releases the watertight seal and allows the liquid to pass through the filter and through the nozzle, and e) thereby recovering the biomolecule-containing composition.

2. The method according to claim 1, wherein the one or more reagents comprises: a) a lysis buffer, b) a precipitation buffer, c) an alkaline proteinase or a cell-wall degrading enzyme, or d) cellulase, hemicellulase, pectinase, glucouronidase, glucanase, chitinase, laminarinase, lyticase, lysozyme, subtilisin, proteinase K, trypsin, caldolysin, or a combination of any two or more thereof.

3. The method according to claim 1, wherein the device comprises two ampoules, and the first ampoule comprises a lysis buffer, and the second ampoule comprises a precipitation buffer.

4. The method of claim 3, wherein the first ampoule is broken, the device is shaken, and then the second ampoule is broken and the device is shaken for a second time.

5. The method according to claim 1, wherein the internal portion of the removable endcap comprises a scoop for collecting and measuring a sample of ground seed.

6. The method according to claim 5, wherein the ground seed is soybean seed.

7. The method according to claim 6, wherein the biomolecule is a nucleic acid that is bound to a magnetic bead within the attachable filter housing.

8. The method according to claim 7, wherein the nucleic acid comprises at least two fluorescent probes, wherein a first fluorescent probe identifies a transgenic trait, and a second fluorescent probe identifies the non-transgenic wild type.

9. The method according to claim 8, wherein quantitative PCR or quantitative reverse transcriptase PCR is used to detect the sequences identified by the at least two fluorescent probes.

10. The method according to claim 9, wherein the amount of sample and reagents comprise from .10g to .25g of sample, from ImL to 4mL of lysis buffer in the first ampoule, said lysis buffer comprising 2% sodium dodecyl sulfate, and from ,5mL to 2mL of precipitation buffer in the second ampoule.

11. The method according to claim 1, wherein the internal portion of the removable endcap comprises a scoop for collecting and measuring a soil sample.

12. The method according to claim 11, wherein the sample comprises a nematode.

13. The method according to claim 1, wherein the removable endcap comprises a microneedle patch or leaf punch for collecting a known quantity of tissue.

14. The method according to claim 13, wherein the tissue is diseased plant leaf tissue.

15. The method of claim 13, wherein the leaf punch creates a leaf tissue disk that also comprises lacerations internal to the perimeter of the leaf tissue disk.

16. The method of claim 1, wherein the watertight seal is formed by a puncturable seal at the second opening, and the puncturable seal is punctured upon the insertion of the filter housing into the second opening.

17. The method of claim 16, wherein the puncturable seal is comprised of a laminate foil or film.

18. The method of claim 1, wherein the watertight seal is formed by an air pressure block in the attachable filter housing, and wherein the air pressure block is released with the removal of a top cap from the nozzle tip end of the attachable filter housing.

19. A device for extracting a nucleic acid, the device comprising i. a body, the body defining an inner chamber comprising at least two breakable ampoules, and wherein the inner chamber has a volume sufficient to receive a sample comprising a nucleic acid, ii. a first opening located at one end of the device comprising a removable endcap, said removable endcap comprising an integrated sample collection and measurement system insertable into the inner cavity, wherein said removable endcap forms a watertight seal with the inner chamber upon sample insertion, iii. a second opening located at or towards the opposing end of the device comprising a watertight seal, and iv. an attachable filter housing capable of replacing or puncturing the opposing end watertight seal, said attachable filter housing comprising at least one filter and further comprising a nozzle end.

20. The device of claim 19 comprising two ampoules, wherein the first ampoule comprises a lysis buffer, and the second ampoule comprises a precipitation buffer.

21. The device of claim 19, wherein the watertight seal at the opposing end of the device is a puncturable seal, and the attachable filter housing punctures the puncturable seal when the filter housing is placed in or onto the body of the device.

22. The device of claim 19, wherein the watertight seal at the opposing end of the device is formed by an air pressure block, which air pressure block is released by the removal of a top cap from the nozzle tip end of the attachable filter housing.

23. The device of claim 19, wherein the attachable filter housing comprises at least one filter comprising magnetic beads that bind DNA.

24. The device of claim 19, wherein the removable endcap comprises a curved inner cavity forming a measuring scoop, and wherein one level scoop endcap of ground soybean seed input into the device will dispense in the range of 3 to 12 nanograms per microliter of DNA, in an average droplet size of 22 microliters.

25. The device of claim 19, wherein the removable endcap comprises a microneedle patch or a leaf punch.

26. The device of claim 25, wherein the removable endcap comprises a leaf disk punch comprising at least one lacerating point that creates lacerations internal to the perimeter of the leaf tissue disk.