WO2018195594A1

WO2018195594A1 - Simple nucleic acid extraction

Info

Publication number: WO2018195594A1
Application number: PCT/AU2018/050374
Authority: WO
Inventors: Michael Glenn MASON; Jose Ramon Botella
Original assignee: University of Queensland UQ
Current assignee: University of Queensland UQ
Priority date: 2017-04-24
Filing date: 2018-04-24
Publication date: 2018-11-01
Anticipated expiration: 2019-10-24

Abstract

A method of extracting a nucleic acid from a sample is provided, the method including the steps of combining a fibrous and/or porous matrix with a sample comprising a nucleic acid whereby the nucleic acid is captured by the matrix; and releasing the nucleic acid from the matrix, to thereby extract the nucleic acid from the sample. The matrix may be a membrane. The matrix may be one or more of microporous, absorbent, and hydrophilic. Also provided is a device for use according to the method, the device comprising a capture portion and a handling portion.

Description

TITLE

SIMPLE NUCLEIC ACID EXTRACTION TECHNICAL FIELD THE present invention relates to nucleic acid isolation. In particular, the invention relates to a nucleic acid-binding matrix for rapid and efficient nucleic acid isolation.

BACKGROUND

The ability to amplify and detect specific nucleic acid sequences is a powerful tool routinely used for a wide variety of applications including disease diagnostics, qualitative trait loci (QTL) selection and mutant screening. In diagnostic applications, nucleic acid-based analysis has many advantages over more traditional methods such as enzyme or antibody-based assays offering increased sensitivity, faster sample-to- answer results and flexibility as it can be rapidly modified to meet new challenges as they arise.

New technologies are rapidly being developed allowing for nucleic acid-based tests to be performed in the field, and thus circumvent the need to transport samples to laboratories with sophisticated equipment. However, one of the major bottlenecks preventing the wide spread adoption of molecular diagnostics for field use is the requirement to purify nucleic acids from samples followed by the accurate transfer of a small volume of the purified nucleic acid into the amplification reaction Before analysis, nucleic acids must typically be first released from the sampled tissue and selectively retained while other compounds, especially those that interfere with the amplification process, including phenolics, polysaccharides and heme-containing compounds are removed. This is a complex task that has traditionally required trained technicians and involved many liquid handling steps.

The demand for simpler and more rapid nucleic acid purification methods has resulted in the expansion of commercially available solid-phase extraction kits. Many of these kits are based on the binding of nucleic acids to a solid silica support in the presence of a chaotropic salt; contaminants are then removed by a series of wash and centrifugation steps before finally eluting the nucleic acids from the silica in a low salt solution. Commercially available paramagnetic beads with a variety of different functionalised surface chemistries designed to capture and purify nucleic acids have also become available, removing the need for centrifugation. In these systems, a magnet is used to attract and hold the paramagnetic beads to the side of the tube to allow supernatant removal during the wash and elution steps. Nevertheless, while simpler and quicker than traditional nucleic acid extraction approaches, these techniques are still too complicated for effective incorporation into field-based point- of-need assays in many instances.

As such, there is presently a need for nucleic extraction techniques offering increased simplicity and/or speed. Techniques which are highly amenable to field- based point-of-need assays are particularly desirable.

SUMMARY

The present invention is broadly directed to extraction of nucleic acids using a nucleic acid-binding matrix that facilitates rapid and efficient isolation of nucleic acids from a sample. In some embodiments, isolation of the nucleic acid from the sample can be completed in less than about two (2) minutes.

In a first aspect the invention provides a method of extracting a nucleic acid from a sample, the method including the steps of:

(i) combining a fibrous and/or porous matrix with a sample comprising a nucleic acid whereby the nucleic acid is captured by the matrix; and

(ii) releasing the nucleic acid from the matrix;

to thereby extract the nucleic acid from the sample.

In some embodiments, the method of the first aspect consists of step (i) and step (ii).

In some embodiments, the method of this aspect includes the further step of enriching or purifying the nucleic acid captured by the fibrous and/or porous matrix, after step (i) and/or before step (ii).

Preferably, a substantial proportion of the nucleic acid of the sample is captured by the fibrous and/or porous matrix and/or released from the fibrous and/or porous matrix.

In preferred embodiments, extraction of the nucleic acid from the sample can be completed in less than about 2 minutes. Preferably, said extraction can be completed in less than about 1 minute. In a particularly preferred embodiment, said extraction can be completed in less than about 30 seconds.

Preferably, the fibrous and/or porous matrix according to this aspect is a fibrous and/or porous membrane.

Suitably, the fibrous and/or porous matrix according to this aspect is absorbent. In some preferred embodiments, the matrix is hydrophilic. In some preferred embodiments, the fibrous and/or porous matrix has a neutral or negative surface charge. Preferably, the surface charge is a negative surface charge.

In some preferred embodiments, the matrix according to this aspect is microporous.

In particularly preferred embodiments, the fibrous and/or porous matrix comprises cellulose, nylon, polyester, and/or polyvinyl or derivatives thereof. Preferably, the matrix comprises cellulose.

Preferably, the capture of the nucleic acid by the matrix according to the method of this aspect does not require additional nucleic acid binding agents to be added to the sample and/or the matrix. Preferably, the capture of the nucleic acid by the matrix does not require additional chaotropic agents to be added to the sample and/or the matrix.

In some preferred embodiments, the capture and/or retention of the nucleic acid by the matrix according to the method of this aspect does not require the addition of agents or reagents to the sample and/or matrix, other than water and a pH buffering agent. In some preferred embodiments, the capture and/or retention of the nucleic acid by the matrix does not require the addition of agents or reagents to the sample and/or matrix, other than water.

In some preferred embodiments of the method of this aspect, the nucleic acid remains in contact with an aqueous solution throughout the method. Preferably, the method of this aspect does not include a step of drying the matrix.

A second aspect of the invention provides a method of analysing a nucleic acid, the method including the steps of (a) capturing a nucleic acid according to step (i) of the first aspect; and (b) analysing the nucleic acid that is captured according to step (a), to thereby analyse the nucleic acid.

In some embodiments, the method of the second aspect consists of step (i) and step (ii).

In some embodiments of the second aspect, the captured nucleic acid is eluted, enriched or purified prior to analysis. In other embodiments of the second aspect, the analysis is performed in situ on the fibrous and/or porous matrix.

In some preferred embodiments, analysis of the nucleic acid according to the second aspect comprises nucleic acid sequence amplification. In some preferred embodiments, analysis of the nucleic acid according to the second aspect comprises nucleic acid sequencing. In particularly preferred embodiment, analysis of the nucleic acid according to the second aspect comprises analysis by visual inspection.

In a third aspect, the invention provides a method of screening a sample for a characteristic of interest, the method including the step of analysing an extracted nucleic acid from the sample according to the second aspect, and determining whether the sample has the characteristic of interest based on the results of the analysis of the nucleic acid, to thereby screen the sample for the characteristic of interest.

In certain embodiments the characteristic of interest that is screened for according to the method of the third aspect is the presence of a disease, disorder or condition. Preferably, the disease, disorder, or condition is caused by or associated with infection by a pathogen. In preferred embodiments, the pathogen is selected from the group consisting of a bacterium, a fungus, and a virus.

In one preferred embodiment, the nucleic acid according to the method of the first to third aspects is DNA. In another preferred embodiment, the nucleic acid according to the method of the first to third aspects is RNA.

Suitably, the nucleic acid and/or the sample according to the first to third aspects is of a biological organism. The biological organism may be a prokaryotic or eukaryotic organism.

In certain preferred embodiments, the nucleic acid and/or the sample according to the first to third aspects is of a plant. Preferably, the plant is a crop plant.

In certain preferred embodiments, the nucleic acid and/or the sample according to the first to third aspects is of an animal. Preferably, the animal is a human.

In a fourth aspect, there is provided a device for use according to the method of the first to third aspects, the device comprising: (a) a capture portion comprising a fibrous and/or porous matrix for combining with a nucleic acid whereby the nucleic acid is captured by the fibrous and/or porous matrix; and (b) a handling portion for a user. In a preferred embodiment, said device consists of (a) and (b).

In a fifth aspect there is provided a kit that comprises: a fibrous and/or porous matrix for use according to the method of the aforementioned aspects; or the device of the fourth aspect; optionally together with one or more reagents for amplifying, analysing or detecting the nucleic acid.

It will be appreciated that the indefinite articles "a" and "an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, "a" protein includes one protein, one or more proteins or a plurality of proteins.

As used herein, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE FIGURES

In order that the invention may be readily understood and put into practical effect, preferred embodiments will now be described by way of example with reference to the accompanying figures, wherein:

Figure 1 sets forth an assessment of untreated cellulosic paper and paper with the addition of DNA binding chemicals for nucleic acid isolation and amplification. Panel (A) Gel-red labelled salmon-sperm DNA in pH 5 (left image) or pH 8.5 (right image) buffer was added to the centre of a Whatman No.1 filter disc on which the chemicals: 1.25% chitosan (1), 2.5% dopamine (2), 2.5% spermine (3), 2.5% polyvinylpyriliodone (4), 1.25% polyethylenimine (5), and 3-Aminopropyl- trimethoxysilane (6) had been spotted. The filters were viewed under UV light before (upper images) and after (lower images) DNA and intercalating fluorescent dye (Gel red nucleic acid stain) addition. Panel (B) 3mm diameter discs of Whatman No. l paper that had been treated with or without 1.25% chitosan were incubated in Arabidopsis thaliana genomic DNA for 1 minute, then washed in pH 5 or pH 8.5 buffer for one minute and then transferred to a PCR mix for amplification, lul of water was used in place of the cellulose disc in the no template control (NTC).

Figure 2 sets forth capture and purification of nucleic acids using cellulosic paper. Panel (A) Ιμΐ of purified DNA at different concentrations (0, 0.01, 0.1, 1 or lOng/μΙ) was pipetted directly onto a Whatman No. l disc (3mm diameter) and then washed in 200μ1 lOmM Tris (pH 8) before adding the disc to a PCR reaction. As a control Ιμΐ of the same DNA solutions were directly added to the PCR reactions. Panel (B) An overview of the nucleic acid purification method using Whatman No. l discs. Tissue is ground in a 1.5ml eppendorf tube with a plastic pestle in the presence of extraction buffer. Nucleic acids are captured by a 3mm diameter Whatman No. l disc. The disc is then transferred to a tube containing wash buffer for one minute to remove contaminants present in the crude extract before transferring it to the tube containing the amplification reaction. The PCR reaction is performed without removing the disc from the tube. Panel (C) Whatman No.1 discs were immersed in an Arabidopsis thaliana leaf extract before being washed once, twice or not at all prior to amplification. As controls, Ιμΐ of crude extract or water (NTC) was added directly to the PCR reaction.

Figure 3 sets forth purification of nucleic acids using cellulosic paper from a range of plant and animal tissues. Panel (A) Genomic DNA from leaf tissues was extracted using the cellulose disc nucleic acid purification method. Universal primers designed against the 5.8S ribosomal RNA gene were used to amplify a product by PCR from each plant species with the exception of rice in which the betaine aldehyde dehydrogenase 2 (GenBank: KU308249.1) was amplified. Panel (B) Human whole blood was diluted 1 :5 in an extraction buffer containing proteinase K before using the cellulose disc method to purify genomic DNA in order to amplify a fragment of the BRAF gene (UniGene Hs.550061). Ιμΐ of each of the raw lysates was also added directly into separate PCR reactions. Purified Hela cells genomic DNA was used as a positive control. Panel (C) Genomic DNA purified from a human melanoma cell line (LM-MEL-70) using the cellulose disc method was used to amplify a fragment of the 28S ribosomal gene. As a control, Ιμΐ of the raw lysate was added directly into a separate PCR reaction. No template controls (NTC) involved adding Ιμΐ of water instead of DNA template.

Figure 4 sets forth DNA and RNA extraction from plant and animal pathogens using cellulosic paper. Panel (A) DNA was purified from Pseudomonas syringae infected Arabidopsis leaves at different stages of infection using cellulose discs and a fragment of the P. syringae genome amplified by PCR. Panel (B) A Whatman No. l disc was used to purify DNA from a lung swab of a pig infected with Actinobacillus pleuropneumoniae that had been placed in extraction buffer (see materials and methods). As a control, Ι μΐ of the raw lysate was added directly into a separate PCR reaction. Panel (C) Whatman No. l discs were used to purify nucleic acids from tomato plants infected with cucumber mosaic virus. The cellulose discs were added to RPA reactions with or without the presence of reverse transcriptase (RT). No template controls (NTC) involved adding Ιμΐ of water instead of DNA template. Panel (D) Cellulose discs were used to purify nucleic acids from tomato plants that were either healthy or infected with cucumber mosaic virus and subsequently amplify them in a LAMP isothermal reaction. Figure 5 sets forth an assessment of nucleic acid extraction using a variety of solid support matrices. Panel (A) Identical size fragments of a variety of sources were used to purify nucleic acids from an Arabidopsis leaf extract. The extracted nucleic acids were used for PCR amplification using primers designed for the G-protein gamma subunit 1 gene (AtAGGl). Panel (B) One, two or three discs (3mm diameter) of Whatman No. l, Hybond N or Scott-brand paper towel were incubated in purified Arabidopsis DNA, washed and then used in a PCR reaction using primers designed for the G-protein gamma subunit 1 gene.

Figure 6 sets forth binding and release of DNA from cellulosic paper. Panel (A) Whatman No. l discs were exposed to a lng/μΐ purified Arabidopsis genomic DNA solution for different amounts of time before washing for one minute and transferring to a PCR reaction. Panel (B) lOng purified Arabidopsis genomic DNA was pipetted onto Whatman No. l discs which were then washed in 10ml water with gentle agitation for different lengths of time prior to transferring the disc to a PCR reaction.

Figure 7 sets forth the use of salt to enhance DNA binding to cellulosic paper. Whatman No. l discs were incubated in purified Arabidopsis genomic DNA (lng/μΐ) dissolved in water or in 150mM NaCl. DNA solution was removed from discs by centrifugation and the discs were added to a PCR amplification.

Figure 8 sets forth dipstick based nucleic acid purification. Panel (A) The cellulose dipstick consists of a 2x40mm wax impregnated handle and a 2x4mm nucleic acid binding zone free of wax. Panel (B) An overview of the dipstick-based purification method in which tissue is homogenised by shaking it in a tube containing ball bearings and an appropriate extraction buffer. The dipstick is used to bind the nucleic acids by dipping it three times into the homogenate, washed by dipping it three times into a wash buffer and eluted by dipping it three times in the amplification reaction mix. Panel (C) Nucleic acids were purified using the cellulose dipstick method from Arabidopsis leaves infected with Fusarium oxysporum f.sp. conglutinans (upper image) or Pseudomonas syringae (lower image) and eluted into PCR reactions mixes containing pathogen specific primers. Panel (D) Nucleic acids were purified from tomato leaves infected with Cucumber mosaic virus using the cellulose dipstick method. The purified DNA was eluted directly into PCR amplification reaction mixes with (+RT) or without (-RT) AMV reverse transcriptase. No template controls (NTC) involved adding Ιμΐ of water instead of using dipstick- purified nucleic acids.

Figure 9 sets forth a comparison of cellulose dipsticks with a commercially available nucleic acid purification system. Panel (A) The time required, number of pipetting steps involved and the costs of all consumables, including tubes and pipette tips, were calculated for purification of nucleic acids from Arabidopsis leaf tissue using either the cellulose dipstick or Agencourt AMPure paramagnetic beads. All solutions that could be prepared in advance, including lysis and wash buffers were made and pre-aliquoted. The time and pipetting involved in the preparation of these solutions was not added to the tallies in the table. Panel (B) Purified Arabidopsis DNA at different concentrations was captured, washed and eluted using either the cellulose dipsticks or AMPure paramagnetic beads (Beckman Coulter). The eluted DNA was used in a PCR reaction with using primers designed for the G-protein gamma subunit 1 gene. Panel (C) Different volumes of an Arabidopsis leaf extract were captured, washed and eluted using either the cellulose dipsticks or AMPure paramagnetic beads and subsequently amplified in a PCR reaction as described above.

Figure 10 sets forth an assessment of various matrix types for use in DNA extraction and amplification.

Figure 11 sets forth a preferred embodiment of device 10 for use according to the methods of the invention. A preferred method is to create a dipstick that has an absorbent nucleic acid binding zone and a water repellent handle. The area of the nucleic acid binding zone can be altered to increase or decrease the amount of sample extract is used for nucleic acid purification.

Figure 12 sets forth as assessment of capture and release of nucleic acids using filter paper. Panel A illustrates band strength after amplification using limited PCR cycles of various concentrations of DNA added directly to the PCR reaction ('General PCR'); added directly to the PCR reaction in combination with the addition of filter paper to the reaction ('Filter paper + DNA'; included as a control); or added to filter, washed, then the filter paper subsequently added to the PCR reaction ('DNA in filter paper'). Filter paper, or water, added to the PCR reaction, were included as controls.

Figure 13 sets forth amplification of genomic DNA from filter paper after various periods of washing with water. 10 ng of Arabidopsis genomic DNA suspended in water was added onto 3.5mm diameter Whatman #1 discs. The discs were then placed into lOmL water for up to 24 hours with gentle rocking. Subsequently, each disc was transferred to a PCR reaction and the template was amplified. The 0 min sample was directly placed into the PCR reaction having never been incubated in the water. NoT = no template (negative) control, in which no template DNA was added to the PCR reaction.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOS: l-2 Primers for amplification 5.8S ribosomal RNA gene from Arabidopsis thaliana.

SEQ ID NOS:3-4 Primers for amplification of betaine aldehyde dehydrogenase 2 gene from rice.

SEQ ID NOS:5-6 Primers for amplification of 5.8S ribosomal RNA gene from tomato, sugarcane, sorghum, and soybean.

SEQ ID NOS:7-8 Primers for amplification of 5.8S ribosomal RNA gene from capsicum, tobacco, sweet potato, barely, wheat, mandarin, lime, lemon, orange, and passion fruit.

SEQ ID NOS:9-10 Cell line primers of Naito et al 1992 targeting melanoma line LM-MEL-70.

SEQ ID NOS: l 1-12 Primers for amplification of human BRAF gene

(UniGene Hs.550061).

SEQ ID NOS: 13-14 Primers for detection of Pseudomonas syringae.

SEQ ID NOS: 15- 16 Primers for detection of Actinobacillus pleuropneumoniae .

SEQ ID NOS: 17- 18 Primers for detection of Cucumber mosaic virus.

SEQ ID NOS: 19-20 Primers for detection of Fusarium oxysporum f. sp.

conglutinans .

SEQ ID NOS:21-24 Primers for detection of Cucumber mosaic virus using

LAMP amplification.

DETAILED DESCRIPTION

The present invention is at least partly predicated on the surprising discovery that certain matrices are highly amenable to capture, extraction, and/or purification of nucleic acids, without the need for additional chaotropic agents and/or additional nucleic acid binding agents. The invention is also at least partly predicated on the realisation that a property of certain matrices, particularly fibrous and/or porous matrices, makes them particularly useful for simple and/or rapid methods for isolation of nucleic acids. As used herein, a fibrous" matrix will be understood to comprise a plurality of fibres, threads, or filaments. A porous matrix will comprise a plurality of spaces or interstices (or 'pores'). Typically the pores of the porous matrix will be spread over a substantial proportion of the surface area of the matrix.

For the purposes of this invention, by "isolated" is meant material {e.g. a nucleic acid) that has been removed or extracted from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

By "enriched" or "purified" is meant isolated material having a higher incidence, representation or frequency in a particular state {e.g. an enriched or purified state) compared to a previous state prior to enrichment or purification.

The term "nucleic acid" as used herein designates single-or double-stranded

DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, microRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto.

A "polynucleotide " is a nucleic acid having eighty (80) or more contiguous nucleotides, while an "oligonucleotide " has less than eighty (80) contiguous nucleotides.

A "probe" may be a single or double-stranded oligonucleotide or polynucleotide, suitably labelled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

A "primer" is usually a single-stranded oligonucleotide, preferably having 15- 50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template" and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™.

Methods of extracting nucleic acids Accordingly, one aspect of the invention provides a method of extracting a nucleic acid from a sample, the method including the steps of:

(i) combining a fibrous and/or porous matrix with a sample comprising a nucleic acid whereby the nucleic acid is captured by the fibrous and/or porous matrix; and

(ii) releasing the nucleic acid from the fibrous and/or porous matrix, to thereby extract the nucleic acid from the sample.

In the context of interaction of a nucleic acid with a fibrous and/or porous matrix, as used herein "captured by" is meant that the nucleic acid is bound to, or held by or within the fibrous and/or porous matrix. Suitably, a substantial proportion of the nucleic acid of the sample that is absorbed by, or comes in contact with, the matrix, is captured by the matrix.

It will be appreciated that according to the method of this aspect, typically, although without limitation thereto, the fibrous and/or porous matrix will be combined with the nucleic acid of the sample wherein the nucleic acid is in solution in the sample. Preferably, the solution will be an aqueous or water-based solution. In particularly preferred embodiments, the solvent of the sample in which the nucleic acid is in contact with is substantial free of additional nucleic acid binding agents or additional chaotropic agents. In some preferred embodiments of this aspect, the solvent of the sample in which the nucleic acid is in contact with consists essentially of, or consists of water. However, in alternative embodiments, other solvents may be used. By way of non-limiting example, solvents comprising or consisting of an alcohol (e.g. ethanol) and/or a ketone (e.g. acetone). Typically, the solvent will be or comprise a polar solvent.

In a preferred embodiment of this aspect, step (ii) comprises elution of the nucleic acid from the fibrous and/or porous matrix. In certain preferred embodiments, the nucleic acid is eluted according to step (ii) by combining the matrix with a buffer. In a preferred embodiment, the buffer comprises tris(hydroxymethyl)aminomethane (Tris), and optionally one or more metal salts. In an embodiment, the buffer is a phosphate buffer.

In particularly preferred embodiments, step (ii) comprises elution in a solution that contains one or more agents typically required for nucleic acid amplification (as herein described). Typically, in these embodiments, said solution will comprise one or more agents including, but not limited to a pH buffer agent (e.g. Tris), dNTPs, a nucleic acid polymerase, and primers. Said solution may further comprises one or more of a metal and/or ammonium salt, polysorbate (e.g. Tween 20), and a zwitterion (e.g. an amino acid, preferably betaine). As will be readily appreciated by the skilled person, said solution may additionally or alternatively contain agents that have similar chemical structures or properties as the aforementioned agents for nucleic acid amplification reactions.

More generally, nucleic acid amplification agents, one or more of which can be suitable for inclusion in a solution in which the nucleic acid may be eluted according to step (ii) of the method of this aspect, include those used for techniques including, but not limited to, the polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA); ligase chain reaction (LCR); Q-β replicase amplification; loop-mediated isothermal amplification of DNA (LAMP); and recombinase polymerase amplification (RPA), as described in the corresponding citations hereinbelow provided and incorporated in full by reference.

In some embodiments, the method of this aspect includes the further step of processing the sample, prior to step (i). In embodiments which include this processing step, suitably, processing according to this step makes the nucleic acid more accessible for capture within the fibrous and/or porous matrix. Processing according to this step may comprise physical and/or chemical processing of the sample.

In certain preferred embodiments, processing prior to step (i) comprises physical processing, such as grinding or crushing. With reference to the Examples, in embodiments wherein the sample is a biological sample comprising tissues or cells, physical processing can be used to at least partially release the nucleic acid from the tissues or cells, such the nucleic acid it is more accessible to the fibrous and/or porous matrix. In a particularly preferred embodiment, the physical processing is grinding using beads such as ball-bearings or similar, as will be known to the skilled person. With reference to the Examples, it will be appreciated that such grinding offers efficiency of processing which can be advantageous in the context of the method of this aspect.

In certain preferred embodiments, processing prior to step (i) comprises chemical processing, such as chemical extraction or elution. With reference to the Examples, in embodiments wherein the sample is a biological sample comprising tissues or cells, chemical extraction using a suitable extraction solution or buffer can be used to at least partially release the nucleic acid from the tissues or cells, such that the nucleic acid is more accessible to the fibrous and/or porous matrix. In certain embodiments, the extraction buffer comprises one or more agents selected from the group consisting of Tris, one or more metallic salts (e.g. NaCl) and/or alkali compounds (e.g. NaOH), a polysorbate (e.g. Tween 20), a guanidine compound (e.g. guanidine hydrochloride), a surfactant and/or detergent (e.g. SDS; Triton XI 00), a chelating agent (e.g. EDTA), an antioxidant and/or protein denaturant (e.g. PVP), and a PCR enhancer (e.g. BSA).

In some particularly preferred embodiments, the agent that is added to the sample prior to step (i) for the purposes of chemical processing is not an additional nucleic acid binding agent, as hereinbelow defined. In some particularly preferred embodiment, the agent that is added to the sample prior to step (i) for the purposes of chemical processing is not an additional chaotropic agent, as hereinbelow defined.

In some embodiments, the method of this aspect includes a further step of purifying the nucleic acid contained by the fibrous and/or porous matrix, after step (i) and/or before step (ii). In certain preferred embodiments, the purification according to step comprises washing the matrix in a wash solution or buffer. In certain embodiments, the wash buffer comprises a pH buffering agent such as Tris. Additionally or alternatively, the wash buffer may comprise one or more agents selected from the group consisting of a metallic salt, an alcohol, and a ketone.

In preferred embodiments, the wash solution is substantially free of additional chaotropic agents. In a preferred embodiment, the wash solution consists essentially of, or consist water and a pH buffering agent (e.g. Tris). In a preferred embodiment, the wash solution consists essentially of, or consists of water.

It is preferred according to the method of this aspect that a substantial proportion or amount of the nucleic acid of the sample is captured by the fibrous and/or porous matrix. The skilled person will appreciate that, in this context, the nucleic acid "ø/ the sample" refers to nucleic acid of the sample which contacts the matrix. It will be readily understood by the skilled person that any nucleic acid that does not contact the matrix will not be captured by the matrix.

It will be further appreciated that, in this context, a "substantial portion, proportion or amount" will be at least partly related to the total amount or concentration of nucleic acid of the sample and/or the total capacity of the matrix to capture the nucleic acid. In embodiments, the amount or proportion of the nucleic acid that is extracted from the sample according to the method of this aspect is at least: 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total amount nucleic acid of the sample.

In embodiments, the amount or proportion of the nucleic acid that is extracted from the sample according to the method of this aspect is at least: 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total capacity of the fibrous and/or porous matrix in terms of the amount of captured nucleic acid.

Preferably, a substantial amount of the nucleic acid that is captured by the fibrous and/or porous matrix according to step (i) of the method of this aspect is released from the matrix according to step (ii) of the method of this aspect.

In embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total amount of the nucleic acid that is captured by the fibrous and/or porous matrix according to step (i) is released or eluted according to step (ii).

With reference to the Examples, it will be appreciated that a comparative assessment of band brightness after limited PCR amplification indicated that greater than 50% and up to about 100% of respective 0.1, 1, or 10 ng DNA samples was recovered from a preferred matrix of the invention. It will be appreciated that this indicates that a substantial portion of the DNA in the sample can be both captured and released from the matrix according to the method of this aspect.

Preferred embodiments of this aspect of the invention may offer benefits in relation to the speed with which the nucleic acid can be extracted from the sample.

In preferred embodiments of this aspect, the extraction of the nucleic acid from the sample can be, or is, completed in less than about 2 minutes.

Preferably, the isolation of the nucleic acid from the sample can be, or is, completed in less than about 1 minute. In a particularly preferred embodiment, the isolation of the nucleic acid from the sample can be, or is, completed in less than about 30 seconds.

It is particularly preferred according to this aspect that isolation of the nucleic acid including the processing the sample comprising the nucleic before step (i), and purifying the nucleic acid after step (i) and/or before step (ii) can be, or is, completed in less than about 30 seconds. In certain preferred embodiments, processing of the sample prior to step (i) can be, or is, completed in less than about 10 seconds, or more preferably about 8 seconds or less.

In certain preferred embodiments, combining the fibrous and/or porous matrix with the sample containing the nucleic acid whereby the nucleic acid is captured by the fibrous and/or porous matrix according to step (i) can be, or is, completed in less than about 5 seconds, or more preferably about 3 seconds or less.

In certain preferred embodiments, purifying the nucleic acid contained by the fibrous and/or porous matrix after step (i) and/or before step (ii) can be, or is, completed in less than about 5 seconds, or more preferably about 3 seconds or less.

In certain preferred embodiments, release of the nucleic acid according to step (ii) can be, or is, completed in less than about 5 seconds, or more preferably about 3 seconds or less.

As will be understood by the skilled person, existing methods for extracting nucleic acids using a matrix typically involve a drying step, wherein the nucleic acid is dried to the matrix to facilitate capture. Such drying steps often involve the addition of solutions comprising alcohol, and/or incubation or rest periods in which the drying and nucleic acid binding occurs. It will be appreciated that the inclusion of a drying step can add significantly to the time taken to perform such methods.

Advantageously, it has been discovered that the method of this aspect does not require a drying step. It has been surprisingly discovered that the nucleic acid can remain in contact with solution throughout the method, with suitable yields of nucleic acid obtained.

Accordingly, in certain preferred embodiments of the method of this aspect, the method does not include a drying step. Preferably, the nucleic acid remains in contact with a solution throughout the method. Preferably, the solution is an aqueous solution.

Fibrous and/or porous matrices with certain characteristics have been shown to be particular effective for the method of this aspect.

It is preferred that the matrix is a fibrous and/or porous membrane, although without limitation thereto. In this context, a "membrane" will be understood to be a relatively thin, sheet-like structure. The membrane may, but need not necessarily, be permeable to liquid. Suitably, the fibrous and/or porous matrix according to this aspect is absorbent. As used herein, "absorbent" matrices will have substantial capacity to take in and hold liquid. Preferably, the matrix is absorbent of water and aqueous solutions.

In some preferred embodiments, the fibrous and/or porous matrix according to this aspect is hydrophilic. As used herein, a "hydrophilic" matrix will be understood to be one which has a relatively strong binding affinity to water, as compared to, for example, non-polar solvents such as oils. With reference to the Examples and FIGS. 5 and 10, it will be appreciated that a variety of hydrophilic matrices e.g. Whatman No. l (catalogue number 1001055) and Whatman No.4 (catalogue number 1004042); Immobilon blotting filter paper (IBFP0785C); and Qiabrane nylon (60030) successfully captured nucleic acids according to the method of this aspect, whereas hydrophobic matrices, e.g. Immobilon-FL PVDF (catalogue number IPFL0010); and Hybond-C extra (catalogue number RPN203E) did not successfully capture nucleic acids.

In certain preferred embodiments, the matrix according to this aspect is microporous. As used herein, a "microporous" matrix will be understood to be one which comprises pores below a certain micrometre size. Preferably, a microporous matrix according to this aspect comprises pores or openings with a diameter of less than: 200 μπι; 175 μπι; 150μπι; 100 μπι; 75 μπι; 50 μπι; 25 μπι; or Ι ΐ μπι. With reference to the Examples and FIGS. 5 and 10, it will be appreciated that microporous matrices (e.g. Whatman No. 1 : pore size - 11 μπι; Whatman No. 4: pore size ~ 25 μπι) successfully captured nucleic acids according to the method of this aspect.

In some preferred embodiments, the matrix according to this aspect has a neutral or negative surface charge. The skilled person will appreciate that surface charge can be measured by assessing zeta potential. With reference to the Examples, a net negative zeta potential corresponds to a negative surface charge of a matrix. With reference to the Examples and FIGS. 5 and 10, it will be appreciated that matrices with negative surface charge, e.g. Whatman No. 1 and Whatman No. 2, and matrices with neutral surface charge, e.g. Amersham hybond-N (catalogue number RPN203N), successfully captured nucleic acids according to the method of this aspect, whereas matrices with positive surface charge, e.g. Amersham hybond-N+ (catalogue number RPN303B), were not suited for the rapid capture and elution of nucleic acids.

In certain preferred embodiments, the matrix according to this aspect comprises cellulose. In particularly preferred embodiments the matrix consists of, or consists essentially of, cellulose-based paper. As will be understood by the skilled person, cellulose-based paper comprises primarily cellulose, in addition to minor amounts of one or more other components such as sizing agents including rosin, gum, and starch; and fillers such as clay, chalk, and titanium oxide. Examples of cellulose- based paper matrices which are suitable for the method of this aspect include Whatman No. 1, Whatman No. 4, and 'Scott' brand Optimum towel' paper towel (catalogue number 4457).

It is preferred however, that in embodiments wherein the matrix comprises cellulose-based paper, the matrix does not comprise one or more additives or components typically present in commercial photocopy paper, preferably 'Australian' brand 80 gsm White Copy paper, but not present in filter paper, preferably Whatman filter paper, and/or paper towel, preferably Scott brand paper towel. With reference to the Examples, commercial photocopy paper did not successfully capture nucleic acids according to the method of this aspect. Without being bound by theory, it is considered that the presence of certain components or additives typically present in commercial photocopy paper, but not typically present in filter paper and/or paper towel, may prevent or constrain capture and/or release of nucleic acids according to the method of this aspect.

In certain preferred embodiments, the fibrous and/or porous matrix according to this aspect comprises nylon, polyester, and/or polyvinyl. In particularly preferred embodiments the matrix consists of, or consists essentially of, a nylon filter membrane. As will be understood by the skilled person, nylon filter membranes typically comprise nylon fibres which may be supported by polyester, and may also comprise one or more other minor components. Examples of nylon filter membranes which are suitable for the method of this aspect include Amersham hybond-N, and Qiabrane Nylon.

In some embodiments the fibrous and/or porous matrix may consist of, or consists essentially of, a polyvinyl filter membrane. As will be understood by the skilled person, polyvinyl filter membranes typically comprise polyvinyl fibres which may be supported by polyester, and may also comprise one or more other minor components.

Fibrous and/or porous matrices according to the method of this aspect may also consist of, consist essentially of, or comprise hybrid paper or membranes comprising one or more of cellulose, nylon, and/or polyester, and optionally one or more additional minor ingredients.

A particular benefit of preferred embodiments of the method of this aspect is that, as hereinabove described, the method does not necessarily require certain additional agents that are required for existing methods.

Thus, in some preferred embodiments no additional nucleic acid binding agents or reagents and/or no additional chaotropic agents or reagents are required to capture the nucleic acid using the fibrous and/or porous matrix according to the method of this aspect.

As used herein an "additional nucleic acid binding agent" will be understood to be an agent that is added or applied to the fibrous and/or porous matrix, and/or a solution containing or otherwise contacting the nucleic acid, which alters or chemically interacts with one or more of: the nucleic acid; the matrix; or a solvent containing the nucleic acid, and thereby substantially facilitates or enhances capture of the nucleic acid by the matrix. Non-limiting examples of nucleic acid binding agents include spermine; polyvinylyrilodone (PVP 40); polyethylenimine (PEI); dopamine, 3-aminopropyl trimethoxysilane (APTMS), and chitosan, which can themselves chemically bind or otherwise interact with nucleic acids, as will be appreciated by the skilled person.

As used herein an "additional chaotropic agenf will be understood to be an agent that is added or applied to the fibrous and/or porous matrix, and/or a solution containing or otherwise contacting the nucleic acid, which disrupts the structure and/or stability of the either the nucleic acid and/or the matrix and thereby substantially facilitates or enhances capture of the nucleic acid by the matrix. Non- limiting examples of chaotropic agents include guanidinium chloride, guanidinium thiocyanate, and alcohols such as ethanol, n-butanol and isopropanol, ketones such as acetone, which can themselves alter the structure and/or stability of nucleic acids in solution, as will be appreciated by the skilled person.

Furthermore, metal salts and similar agents, which can substantially enhance nucleic acid binding to a matrix by modifying or decreasing electrostatic repulsion between the nucleic acid and the matrix, are considered to fall within the scope of additional chaotropic agents, in the context of this aspect.

Additionally, agents such polyethylene glycol, and DNA compaction agents such as spermine, spermidine, and hexamminecobalt(III), which can similarly substantially enhance nucleic acid binding to a matrix by modifying interactions between nucleic acid molecules and/or the matrix, are considered to fall within the scope of nucleic acid binding agents and chaotropic agents.

It will nevertheless be understood that, for the purposes of this invention, any agent contained within the sample itself, will not be considered an additional nucleic acid binding agent or an additional chaotropic agent. Nor will derivatives of agents contained within the sample itself that may be produced upon combination of the sample with a solvent and/or the matrix be considered additional nucleic acid binding agents or additional chaotropic agents.

With reference to the Examples and Figures, it will be appreciated that, contrary to teachings in the prior art, it has been surprisingly found that certain matrices, including those comprising cellulose, nylon, and/or polyester as hereinabove described, can effectively capture nucleic acids in the absence of additional nucleic acid binding agents or additional chaotropic agents.

In particular regard to salts such as metal salts, with reference to FIG. 7, it has been surprisingly found that, although the addition of salt may enhance nucleic acid capture according to the method of this aspect, effective nucleic acid extraction can be performed in the absence of additional such salt.

Accordingly, in some preferred embodiments, no additional salt is used to facilitate nucleic acid capture according to the method of this aspect. In alternative embodiments, an additional nucleic acid binding agent such as a salt may improve or enhance nucleic acid capture by the fibrous and/or porous matrix.

In view of the surprising discovery set forth above, in certain particularly preferred embodiments, the fibrous and/or porous matrix is substantially free of additional nucleic acid binding agents. In certain particularly preferred embodiments, the fibrous and/or porous matrix is substantially free of additional chaotropic agents.

As used in the context of an additional agent according to the method of this aspect, such as a nucleic acid binding agent or chaotropic agent, "substantially free" will be understood to refer to the complete absence of the agent or the presence of only a quantity of such agents that does not have a significant effect on the sample, the nucleic acid, the matrix, and/or the interaction of the nucleic acid with the matrix.

Furthermore, in some embodiments, the fibrous and/or porous matrix according to this aspect may be substantially free of other additional agents or reagents. Preferably, the fibrous and/or porous matrix according to this aspect is substantially free of additional agents which preserve nucleic acids. Such agents are well known to the skilled person, and include those which decrease or remove nuclease activity (e.g. EDTA, guanidine thiocyanate etc.).

Preferably, the fibrous and/or porous matrix according to this aspect is substantially free of additional agents which degrade nucleic acids, such as DNase and RNase enzymes.

Preferably, the fibrous and/or porous matrix according to this aspect is substantially free of additional agents which disrupt tissues and/or cells. Such agents are well known to the skilled person, and include detergents such as Triton XI 00 and SDS.

It will be further understood that, in certain embodiments, solutions or buffers used according to the method of this aspect may be substantially free of additional agents.

In some embodiments, a solution (such as an extraction buffer, as hereinabove described) of the sample containing the nucleic acid according to the method of this aspect may additionally or alternatively be substantially free of one or more additional agents selected from the group consisting of additional nucleic acid binding agents, additional chaotropic agents, additional agents which preserve nucleic acids, additional agents which degrade nucleic acids, and additional agents which disrupt tissue and/or cells, as described above.

In some embodiments, the solution or buffer used for purification of the nucleic acid captured by the fibrous and/or porous matrix according to the method of this aspect may additionally or alternatively be substantially free of one or more additional agents selected from the group consisting of additional nucleic acid binding agents, additional chaotropic agents, additional agents which preserve nucleic acids, additional agents which degrade nucleic acids, and additional agents which disrupt tissue and/or cells, as described above.

In some embodiments, the solution or buffer used for elution of the nucleic acid captured by the fibrous and/or porous matrix according to the method of this aspect may additionally or alternatively be substantially free of one or more additional agents selected from the group consisting of additional nucleic acid binding agents, additional chaotropic agents additional agents which preserve nucleic acids, additional agents which degrade nucleic acids, and additional agents which disrupt tissue and/or cells, as described above.

It will be recognised by the skilled person that an advantage of preferred embodiments of the method of this aspect wherein additional chaotropic agents are not used for nucleic acid capture, e.g. by adding such agents to the sample and/or matrix, is that this can avoid the need for further chaotropic agents to be used in downstream steps, e.g. by including these in a solution for washing the nucleic acid.

Furthermore, by not including additional chaotropic agents, the need to remove such agents when eluting the nucleic acid can be obviated.

Accordingly, it will be appreciated that avoiding the use of additional chaotropic agents can contribute to the advantageous speed with which preferred embodiments of the method can be completed.

It will be appreciated that the sample according to the method of this aspect may be any suitable sample comprising a nucleic acid. Typically, the nucleic acid of the sample is of, or derived from, a biological organism. However, samples containing nucleic acids of other origin, e.g. synthetic nucleic acids, are also within the scope of this aspect.

As will be readily appreciated by the skilled person, single and double stranded RNA and DNA, as hereinabove described, share respective key chemical and structural characteristics regardless of origin. It is therefore expected that the method of this aspect can potentially be applied to single or double stranded RNA or DNA nucleic acids of any origin, based on the data presented in the Examples. It will be further appreciated that the method of this aspect is broadly applicable to a range of nucleic acid sizes.

In embodiments wherein the sample contains a nucleic acid of or derived from a biological organism, the sample may comprise cells or tissues of a biological organism, but need not necessarily do so, e.g. the sample may be an environmental sample in which nucleic acids of biological origin are present, but which does not comprise any substantial quantity of cells or tissue.

Based on the successful use of the method of this aspect for isolation of nucleic acids from a wide range of cells and tissues, it is expected that the method should be generally applicable to isolation of nucleic acids from samples comprising animal, plant, and/or microorganism cells or tissues. The skilled person will recognise that, in embodiments of the method including a processing step before step (i), the particular physical and/or chemical processing, and parameters thereof, that are used can be modified to suit specific cell or tissue types.

Suitably, in embodiments of this aspect wherein the nucleic acid of the sample is of or derived from a biological organism, the biological organism may be a prokaryotic organism or a eukaryotic organism. The biological organism may be a plant, an animal, a microorganism, or any other prokaryotic or eukaryotic organism inclusive of fungi and algae.

In certain embodiments, the biological organism is a plant, inclusive of any organism within the kingdom Plantae. Suitably, the plant may be any dicotyledon or monocotyledon, inclusive of crop plants such as legumes, cereals, and solanaceous plant species. The plant may be, for example, a grass species of the family Poaceae; a Saccharum species such as sugarcane; a cereal such as wheat, maize, sorghum, barley, and rice; a leguminous species such as beans and peanut; a solanaceous species such as tomato, tobacco, and potato; a tree species such as a fruit tree species; or a vine species such as a fruit or vegetable vine species. The plant may also be a model plant species such as the model dicotyledonous species Arabidopsis thaliana or the model monocotyledonous species Brachypodium distachyon.

In particularly preferred embodiments, the plant species is selected from the group consisting of sugarcane, barley, wheat, sorghum, soybean, tomato, tobacco, mandarin, lime, lemon, and passionfruit.

In some preferred embodiments, the biological organism is an animal, inclusive of any organism within the Animalia kingdom. The animal may be, for example an invertebrate such as an insect, nematode, mollusk, platyhelminth, or echinoderm, or a chordate inclusive vertebrates. Generally, the animal may be from any of the Ecdysozoa, Lophotrochozoa, Radiata, or Deuterostomia phyla.

In some preferred embodiments the animal is selected from the group consisting of a mammal, a bird, a fish, a reptile, and an amphibian. In embodiments wherein the animal is a mammal, the mammal may be a human or non-human mammal such as livestock (e.g. horses, cattle and sheep), companion animals (e.g. dogs and cats), laboratory animals (e.g. mice, rats and guinea pigs) and performance animals (e.g. racehorses, greyhounds and camels), although without limitation thereto. In one particularly preferred embodiment, the animal is a human. In embodiments wherein the biological organism is a microorganism, the microorganism may be selected from the group consisting of a virus, a bacteria, an archaea, a fungi or an algae.

In certain preferred embodiments, the biological organism may be a parasite or pathogen. In some preferred embodiments the pathogen is a microorganism selected from the group consisting of a virus, a bacteria, and a fungi.

It will be appreciated that in embodiments wherein the nucleic acid is of a parasite or pathogen, the sample according to the method of this aspect may comprise cells or tissue of another organism infected or infested with the parasite or pathogen. With reference to the Examples and FIGS. 4 and 8, it will be appreciated that samples containing plant tissue and cells or animal tissues and cells were used for isolation of pathogenic nucleic acids according to the method of this aspect.

Methods of analysing nucleic acids

A further aspect of the invention provides a method of analysing a nucleic acid, the method including the steps of (i) combining a fibrous and/or porous matrix with a sample comprising a nucleic acid whereby the nucleic acid is captured by the matrix; and (ii) analysing the nucleic acid that is captured according to step (i), to thereby analyse the nucleic acid.

In some embodiments of this aspect, the captured nucleic acid is eluted, enriched or purified prior to analysis.

In other embodiments of this aspect, the analysis is performed in situ on the matrix.

In certain preferred embodiments of this aspect the extracted nucleic acid is purified after step (i) and/or before step (ii) prior to analysis, as described in relation to the previous aspect. With reference the Examples, it has been determined that purification as hereinabove described can effectively remove other non-nucleic acid components from the fibrous and/or porous matrix, which may have an inhibitory effect on downstream analysis steps (e.g. nucleic acid amplification or sequencing as described below). It will be appreciated that purification can be performed in embodiments wherein the analysis is performed in situ on the matrix. In these embodiments, the fibrous and/or porous matrix may be washed using a wash solution or buffer as herein described, prior to performing analysis directly on the fibrous and/or porous matrix. In some embodiments, the wash solution is substantially free of one or more additional agents selected from the group consisting of additional nucleic acid binding agents, additional chaotropic agents, additional agents which preserve nucleic acids, additional agents which degrade nucleic acids, and additional agents which disrupt tissue and/or cells, as described above.

Preferably, the wash solution is substantially free of additional chaotropic agents.

In one particularly preferred embodiment, the wash solution consists essentially of, or consists of water and pH buffering agent. In one particularly preferred embodiment, the wash solution consists essentially of, or consists of, water.

In some preferred embodiments, analysis of the nucleic acid according to this aspect comprises nucleic acid sequence amplification. As used herein "nucleic acid sequence amplification" includes but is not limited to techniques such as polymerase chain reaction (PCR) as for example described in Chapter 15 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons NY USA 1995-2001) strand displacement amplification (SDA); rolling circle replication (RCR) as for example described in International Application WO 92/01813 and International Application WO 97/19193; nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et al. 1994, Biotechniques 17 1077; ligase chain reaction (LCR) as for example described in International Application WO89/09385 and Chapter 15 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY supra Q-β replicase amplification as for example described by Tyagi et al, 1996, Proc. Natl. Acad. Sci. USA 93 5395 and helicase- dependent amplification as for example described in International Publication WO 2004/02025; loop-mediated isothermal amplification of DNA (LAMP) as described by Notomi et a/., 2000, Nucleic Acids Res. 28(12): e63; and Recombinase Polymerase Amplification (RPA) as described by Piepenburg et a/., 2006, PLoS Biology 4(7): e204.

It will be further appreciated that 'Next-Generation Sequencing' techniques, such as hereinbelow described, frequently involve nucleic sequence amplification prior to sequencing. Particular sample preparation techniques applicable for various Next Generation sequencing approaches are known and have been extensively described, for example in manufacturer instructions for sample preparation kits available for proprietary sequencing technologies of Illumina (see, http://www.illumina.com/techniques/sequencing/ngs-library-prep.html); Pacific Biosystems (http://www.pacb.com/products-and-services/consumables/pacbio-rs-ii- consumables/sample-and-template-preparation-kits/); and Applied Biosystems (https://www.neb.com/applications/library-preparation-for-next-generation- sequencing/ion-torrent-dna-library-preparation). Such techniques are also within the scope of nucleic sequence amplification according to this aspect.

As set forth in the Examples, nucleic acid extracted from a sample according to a preferred embodiment of the above-described aspect including purification according after step (i) has been surprisingly and advantageously found to be highly amenable to amplification by multiple techniques including PCR, LAMP, and RPA. Generally, it is expected that such extracted nucleic acid will be broadly suitable for nucleic acid sequence amplification using a range of techniques. In certain particularly preferred embodiments nucleic acid sequence amplification performed as per analysis according to this aspect is by the use of a technique selected from the group consisting of HDA, LAMP, or RPA. It will be appreciated that these techniques do not require the use of a thermal cycler, and are therefore particularly amenable to nucleic acid amplification in the context of point-of-care (POC) analysis, as herein described.

In some preferred embodiments, analysis of the nucleic acid according to this aspect comprises nucleotide sequencing. As will be readily understood by the skilled person, a variety of techniques for nucleic acid sequencing exist. These include Sanger sequencing (Sanger et al. (1977) Proceedings of the National Academy of Sciences. 74(12) 5463-5467) and automated versions thereof, and newer technologies which are typically referred to as 'Next Generation' sequencing techniques (Mardis (2013) Annual Review of Analytical Chemistry. 6 287-303). Recently, nanopore sequence, particularly the Oxford Nanopore systems (including the 'MinlON') have seen substantial assessment and optimization for nucleotide sequencing. The skilled person is directed to Lu et al (2016) Genomics, Proteomics & Bioinformatics. 14(5) 265-279 for an overview of sequencing with the Oxford Nanopore MinlON system. It will be appreciated that portable nucleotide sequencers, such as the MinlON, are particular desirable for use in the context of point-of-care analysis, as herein described.

It will be readily appreciated by the skilled person that nucleic acid sequence amplification and nucleotide sequencing have vast application to nucleic acid analysis. Frequently, nucleic acid analysis using nucleic acid sequence amplification or nucleotide sequencing involves detection or identification of a nucleotide sequence of interest. Additionally or alternatively, nucleic acid analysis using nucleic acid sequence amplification or nucleotide sequencing may involve assessment of the degree or level of expression of a gene of interest. By way of non-limiting example, nucleic acid sequence amplification can be used to assess gene expression via techniques include real-time RT-PCR or qPCR; and next-generation sequencing has application to gene expression analysis via techniques such as cDNA sequencing or 'RNA-seq'. For gene expression analysis according to this method, RNA will typically be extracted and used for the production of corresponding cDNA, which is subsequently analysed e.g. using qPCR of by sequencing.

The analysis according to the method of this aspect can be any suitable analysis. By way of non-limiting example, the analysis may be a genetic marker analysis (e.g. detection of a gene or allele within the nucleic acid), a mutation analysis (e.g. detection of the presence or absence of a genetic mutation within the nucleic acid), an identification analysis (e.g. analysis of the origin of the nucleic acid), or a gene expression analysis (e.g. analysis of the degree of expression of the nucleic acid). In some particularly preferred embodiments, the analysis is performed as point- of-care (POC) analysis.

As will be readily understood by the skilled person, in a clinical context, POC analysis refers to analysis which can be performed at the time and place of care of a subject, e.g. at the bedside of a human patient. This is as compared to more traditional approaches to diagnostics which have typically required removal of a sample from the subject to a facility containing specialized equipment for testing, and which have generally required an extended period (e.g. hours to days) for the testing to be completed. More generally, as used herein, "POC analysis" will be understood to include and encompass any analysis which can be performed at or near the location of a sample in a relatively short time period. By way of non-limiting example, field testing of crops or livestock will be considered POC analysis for the purposes of this invention.

It will be appreciated that preferred embodiments of the method of nucleic acid extraction according to the aspect of the invention hereinabove described have advantages in the context of POC analysis. In particular, with reference to the Examples, it will be appreciated that preferred embodiments of this method can be performed with the use of minimal equipment, as compared to the substantial equipment typically required for existing methods of nucleic acid extraction (e.g. pipettes, water bath, timer, centrifuge, fume hood), and can be completed comparatively quickly. These characteristics make the method of nucleic acid extraction as herein described particularly desirable for combination with techniques for POC analysis, such as techniques involving nucleic acid amplification using HDA, LAMP, or RPA, or other similar techniques, or nucleic acid sequencing using portable nucleic acid sequencers.

In certain embodiments, the analysis according to this aspect comprises nucleic acid analysis by visual inspection or electronic determination. Without limitation, such analyses include those involving fluorescence, dye colour shifts, lateral flow devices, spectrometry such as Raman, and turbidity analysis, as will be known to the skilled person.

It will be appreciated that preferred embodiments of the method of nucleic acid extraction according to the aspect of the invention hereinabove described have advantages in the context of POC analysis as a component of diagnostic method involving diagnostic devices. Without limitation, such diagnostic devices involve those that make use of a detectable physical signal that includes, but is not limited to, changes in light intensity, absorbance, emission, wavelength, colour, electrical conduction, electrical resistance, or other electrical properties.

In one such embodiment that is particularly preferred in a POC context, the analysis involving visual inspection is as described in WO 2015/095929, incorporated herein by reference. It will be appreciated that for the analysis described in WO 2015/095929, generally a particle is combined with an amplified nucleic acid, wherein the characteristics of the particle are such that the nucleic acid forms a complex with the particle that can be observed by visual inspection. For example, when combined with a nucleic acid of interest, a solution comprising the particle may change colour. Advantageously, the technique described in WO 2015/095929 generally does not require specialized (e.g. electronic) equipment such as spectrophotometers or thermal cyclers.

In other particular embodiments that are preferred in POC contexts, the analysis according to this aspect comprises nucleic acid analysis by visual inspection or electronic determination when used in combination with colorimetric or fluorescent dyes including, but not limited to, hydroxyl napthol blue, SYBR green, or SYTO 9. When combined with nucleic acid extraction as hereinabove described, and nucleic acid amplification using HDA, LAMP, or RPA, or another similar technique, nucleic acid analysis by visual inspection, such as described above, is particularly beneficial for POC analysis according to this aspect. In this respect, it will be appreciated that it, at least in certain preferred embodiments (such as when performing analysis as described in WO 2015/095929) it may be possible to perform the entire process, from extraction to analysis, in the absence of specialized equipment or laboratory space, and rapidly (e.g. within minutes).

In particularly preferred embodiments, analysis of the nucleic acid according to this aspect can be performed in less than about 2 minutes, less than about 1 minute, or less than about 30 second.

Screening of samples

In a yet further aspect, the invention provides a method of screening a sample for a characteristic of interest, the method including the step of analysing a nucleic acid that is extracted from a sample according to the directly preceding aspect, and determining whether the sample has the characteristic of interest based on the results of the analysis, to thereby screen the sample for the characteristic of interest.

In preferred embodiments of this aspect, the sample is a sample comprising cells and/or tissue of a biological organism as hereinabove described. The biological organism may be a prokaryotic organism or a eukaryotic organism. The biological organism may be a plant, an animal, a microorganism, or any other prokaryotic or eukaryotic organism inclusive of fungi and algae.

In certain embodiments, the biological organism is a plant, inclusive of any organism within the kingdom Plantae. Suitably, the plant may be any dicotyledon or monocotyledon, inclusive of crop plants such as legumes, cereals, and solanaceous plant species. The plant may be, for example, a grass species of the family Poaceae; a Saccharum species such sugarcane; a cereal such as wheat, maize, sorghum, barley, and rice; a leguminous species such as beans and peanut; a solanaceous species such as tomato, tobacco, and potato; a tree species such as a fruit tree species; or a vine species such as a fruit or vegetable vine species. The plant may also be a model plant species including the model dicotyledonous species Arabidopsis or the model monocotyledonous species Brachypodium distachyon. In particularly preferred embodiments, the plant species is selected from the group consisting of sugarcane, barley, wheat, sorghum, soybean, tomato, tobacco, mandarin, lime, lemon, and passionfruit.

In some preferred embodiments, the biological organism is an animal, inclusive of any organism within the Animalia kingdom. The animal may be, for example an invertebrate such as an insect, nematode, mollusk, platyhelminth, or echinoderm, or a chordate inclusive of vertebrates. Generally, the animal may be from any of the Ecdysozoa, Lophotrochozoa, Radiata, or Deuterostomia phyla.

In some preferred embodiments the animal is selected from the group consisting of a mammal, a bird, a fish, a reptile, and an amphibian. In embodiments wherein the animal is a mammal, the mammal may be a human or non-human mammal such as livestock (e.g. horses, cattle and sheep), companion animals (e.g. dogs and cats), laboratory animals (e.g. mice, rats and guinea pigs) and performance animals (e.g. racehorses, greyhounds and camels), although without limitation thereto. In one particularly preferred embodiment, the animal is a human.

The characteristic of interest according to this aspect may be any suitable characteristic of interest. By way of non-limiting example, in embodiments wherein the sample is a plant sample, the characteristic of interest may be a characteristic of agricultural significance, such as seed, grain or other produce quality; stress tolerance, for example abiotic stress tolerance such as drought or salt resistance, and biotic stress resistance such as resistance to disease; produce yield; vigour; plant height; nutritional properties; and dormancy.

Similarly, non-human animal samples may be screened for physical characteristics, or characteristics associated with temperament, that may be interest in an agricultural or companion context. Human samples may also be screened for characteristics in a clinical context, such as developmental characteristics and genetic predispositions to genetic disorders, although without limitation thereto.

In some preferred embodiments of this aspect, the sample is screened to determine the presence of infection or infestation with a pathogen or parasite. Typically, such screening will involve the detection of a nucleic acid of a pathogen or parasite within a sample comprising cells and/or tissue of a biological organism that may be infected or infested with that pathogen or parasite. With reference to the Examples, it will be appreciated that the method of this aspect was used to detect infection of a plant (Arabidopsis thaliana) with bacterial {Pseudomonas syringae) and viral (Cucumber mosaic virus) pathogens, and to detect infection of an animal (pig) with a bacterial pathogen (Actinobacillus pleuropneumoniae).

Device and kit for nucleic acid capture and/or isolation

In another aspect, there is provided a device for use, or when used, according to the method of the preceding aspect, the device comprising: (a) a capture portion comprising a fibrous and/or porous matrix for combining with a nucleic acid whereby the nucleic acid is contained by the matrix; and (b) a handling portion for a user.

In some embodiments the device may be provided in a kit together with one or more reagents for amplifying, analyzing or detecting the nucleic acid. Such reagents may include DNA polymerase enzymes, restriction endonucleases, probes and/or primers which in some embodiments may be labelled to facilitate detection. In some embodiments the kit may further comprise a paramagnetic particle such as an SPRI particle.

FIG. 11 shows device 10, which is an embodiment of this aspect. Device 10 comprises capture portion 100; and handling portion 200. As depicted in FIG. 11, capture portion 100 is formed from Whatman No. 1 cellulose-based filter paper, however it will be appreciated that other suitable fibrous and/or porous matrices as herein described may alternatively be used.

As depicted in FIG. 11, handling portion 200 is formed from a waterproof coating overlying a region of the Whatman No. 1 filter paper extending from capture portion 100. As depicted in FIG. 11 the waterproof coating is wax (Paraplast Plus, Fluka) however this can be varied as desired. In FIG. 11, the region of the Whatman No. 1 filter paper which the waterproof coating overlies is depicted by dashed lines.

FIG. 8 A shows another embodiment of device 10. The embodiment depicted in FIG. 8 A is substantially as described above with reference to FIG. 11. However, this embodiment of device 10 has dimensions particularly adapted for use in isolation of nucleic acids according to the method hereinabove described, wherein the steps of the method can be performed using microcentrifuge tubes of a volume of 2 ml or less.

With reference to FIGS. 11 and 8, it will be readily appreciated that the particular shape and dimensions of device 10, including the shape and dimensions of each of capture portion 100 and handling portion 200, can be adjusted as desired. Adjustment may be performed, for example, in order to adapt device 10 to use according to the methods described in the preceding aspects with tubes or containers of various sizes, and/or to adapt device 10 to use according to the methods for capture and/or isolation of various nucleic acid types and/or quantities or concentrations and/or from various sample types.

In certain preferred embodiments, a length of the device is about 20 to about 100 mm, including about: 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mm. In certain preferred embodiments, a width of the device is about 0.5 to about 10 mm, including about: 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, and 9.5 mm.

In certain preferred embodiments, a proportion of a length of the capture portion of the device to a length of the handling portion of the device is about 0.05 to about 0.5, including about: 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, and 0.45.

With reference to FIG. 8B, when performing nucleic acid isolation according to the above described aspects using device 10, a user holds handling portion 200 of device 10, whereby capture portion 100 is not in direct contact with the user. In use, to perform step (i) of the method, the user contacts capture portion 100 with the sample. In use, for the purification step of the method after step (i) and/or before step (ii), the user contacts capture portion 100 with a wash solution or wash buffer. In use, to perform step (ii) of the method, the user contacts capture portion 100 with an elution solution. In use, the waterproof coating of handling portion 200 prevents or at least constrains movement of liquid away from capture portion 100 into handling portion 200.

With reference to the Examples, it has been realised that the binding capacity of capture portion 100 of the device of this aspect is at least partly determined by the volume and/or surface area of this portion. As such, advantageously, the size and/or shape of capture portion 100 may be adapted to adjust the amount of nucleic acid that is captured using device 10. This characteristic of device 10 can offer advantages for the flexibility of use of device 10 for methods involving various nucleic acids and/or samples types. Another advantage of this characteristic of device 10 is that it can facilitate repeatability of the concentration or amount of a nucleic acid that is captured and/or extracted using device 10.

In certain preferred embodiments, the total surface area of capture portion 100 of device 10 is about 5 mm² to about 50 mm², including about: 10, 15, 20, 25, 30, 35, 40, and 45 mm². In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES EXAMPLE 1; Nucleic acid purification from plants, animals and microbes

Materials and Methods

Plants, animals, and microbes

Plant materials used were Arabidopsis thaliana ecotype Columbia, capsicum {Capsicum annuum cv. warlock), tobacco (Nicotiana Benthamiana), tomato (Solanum lycopersicum cv. Micro Tom), sugarcane (Saccharum officinarum cv. Q208), sorghum (Sorghum biocolor cv. IS8525), soybean (Glycine max cv. Bunya), sweet potato (Ipomoea batatas cv. Northern star), rice (Oryza sativa cv. Topaz), barley (Hordeum vulgare line 2LZIB14), wheat (Triticum aestivum line S19-49), mandarin (Citrus reticulata), lime (Citrus aurantiifolia), lemon (Citrus limon), orange (Citrus sinensis), passion fruit (Passiflora edulis). Diseased plant materials included A. thaliana leaf tissue infected with Pseudomonas syringae pv tomato strain DC3000 or Fusarium oxysporum f.sp. conglutinans, and tomato leaf tissue infected with cucumber mosaic virus. Human samples included melanoma cell line LM-MEL-70 and blood. Diseased animal material was harvested by tissue swab from pig lung infected with Actinobacillus pleuropneumoniae .

Arabidopsis thaliana DNA purification

To obtain DNA for assessing nucleic acid-cellulose interaction, Arabidopsis thaliana (ecotype Columbia) DNA was extracted by modified CTAB DNA extraction (Doyle 1990). A. thaliana leaves were finely ground using liquid nitrogen and approximately lOOmg of leaf powder was mixed with 500μ1 of extraction buffer (2% w/v CTAB, 1.42 M NaCl, 20mM EDTA, lOOmM Tris HCl pH8.0, 1% w/v PVP 40) that was pre-heated 60°C. After 45 minutes at 60 °C, 500μ1 of chilled chloroform: isoamyl alcohol (24: 1, v/v) was added into the mixture and rocked gently at room temperature for 15 minutes, followed by centrifugation at 15,000g for 10 minutes. 200μ1 supernatant was transferred to a new tube and mixed gently with 400μ1 chilled ethanol. After incubation at -20 °C for 1 hour, the sample was centrifuged at 15,000g for lOmin to pellet the DNA.

The pellet was washed with 80% ethanol, followed by 100% ethanol. DNA was suspended with ΙΟΟμΙ of H₂0 and 50ug RNase A and followed by incubation at 37°C for 20 minutes to degrade RNA. ΙΟμΙ of 3M sodium acetate and ΙΟΟμΙ of isopropanol were added into sample and incubated at -20 °C for 10 minutes. DNA was pelleted by centrifugation at 15,000 g for 2 minutes and washed with 80% ethanol. After air drying, the DNA was suspended with 50μ1 of H₂0 and quantified with NanoDrop ND- 1000 spectrophotometer.

DNA binding to chemically modified cellulose

A number of chemicals with potential DNA binding capability were selected including spermine, polyvinylyrilodone (PVP 40) and the cationic polymers: polyethylenimine (PEI), dopamine, 3-aminopropyl trimethoxysilane (APTMS), and chitosan. Solutions were made containing the chemicals at either 1.25% (w/v) (Chitosan, APTMS, PEI) or 2.5% (w/v) (dopamine, spermine, PVP-40). Ιμΐ of each solution was carefully added to two 70mm Whatman No.1 discs approximately 10mm from the centre of the disc. The chemicals were allowed to fully dry onto the paper, before viewing the filter under UV light to assess the amount of fluorescence each chemical induces in the absence of DNA. 150μ1 of SOOng/μΙ salmon sperm DNA (Sigma) labelled with 0.5%(v/v) GelRed (Biotum) and buffered in either 50mM MES (pH 5) or 50mM Tris (pH 8.5) was added to the centre of each Whatman No. l disc. After approximately 5 minutes the movement of DNA by capillary action had stopped and the cellulose disc was viewed under UV light. DNA binding by the chemical was indicated by brighter fluorescence over the background.

DNA binding efficiency of cellulose

3mm diameter cellulose discs were cut from Whatman No. l using a hole puncher. Ιμΐ of lOng/μΙ, 1 ng/μΐ, O. lng/μΐ or 0.01 ng/μΐ purified DNA was loaded on cellulose discs respectively. Cellulose disc with identical DNA was then transferred into 200μ1 of wash buffer (lOmM Tris, 0.1% Tween-20) using a pipette tip and incubated in wash buffer for 1 minute, followed by disc transformation into a PCR amplification reaction. As control, the same amount of purified DNA was added directly into amplification reaction.

Cellulose disc nucleic acid purification

For nucleic acid purification from plant tissues, 5-10mg of leaf tissue was ground in 1.5mL tube with a plastic pestle in presence of 50μ1 extraction buffer 1 (50mM Tris, 150mM NaCl, 2% PVP, 1% Tween-20) for approximately 30 seconds. A 3mm diameter disc was cut from a piece of Whatman No. l using a hole puncher and transferred into the tissue extract for a minimum of three seconds. The disc was then transferred to 200μ1 of wash buffer (lOmM Tris, 0.1% Tween-20) using a pipette tip to remove contaminants including amplification inhibitors. After one minute, the disc containing nucleic acid was then transferred into an amplification reaction using a pipette tip. For RNA purification from CMV-infected tomato leaves, samples were prepared as described above with the exception that 50μ1 of extraction buffer 2 (800mM guanidine hydrochloride, 50mM Tris (pH 8), 0.5% Triton X100, 1% Tween- 20) was used to lyse the samples instead of extraction buffer 1.

For DNA purification from blood, samples were mixed with four volumes of extraction buffer with the addition of 40u of Proteinase K to aid DNA extraction. For DNA purification from human cell lines, LM-MEL-70 cells pellet previously stored at -20°C was lysed by adding 200μ1 extraction buffer 2 and vortexing for approximately 10 seconds. A 3mm Whatman No. l disc was incubated in lysate for one minute. The cellulose disc was transferred to 200μ1 of wash buffer for one minute before transferring to PCR reaction mix.

For DNA extraction from pig lung swabs, the outer lung tissue of the pig was surface sterilised by quartering with a hot spatula. An incision was made in the lung and a cotton swab rubbed against the inner lung tissue and then dropped into 500μ1 of extraction buffer 3 (1.5M guanidine hydrochloride, 50mM Tris (pH 8), lOOmM NaCl, 5mM EDTA, 1% Tween-20).

Assessing matrices suitable for nucleic acid purification

To determine which matrices have the capacity to purify nucleic acid, various matrices were investigated, including Whatman No. l (Whatman), Whatman No.4 (Whatman), DEAE-cellulose (Whatman), filter paper (Invitrogen), blotting filter paper (Immobilon), FL PVSF (Immobilon), hybond N (Amersham), hybond N+ (Amersham), Qiabrane nylon (Qiabrane), nylon plus (Qiabrane), hybond-C extra (Amersham), bleached photocopy paper, unbleached photocopy paper, and Scott paper towel (Scott). 3mm diameter discs were cut from either of membrane described above using a hole puncher for following analysis.

A. thaliana leaf tissue (approximately 30mm²) was finely ground with plastic pestle in presence of 400μ1 extraction buffer 1 (50mM Tris, 150mM NaCl, 2% PVP, 1%) Tween-20) for approximately 30 seconds. A. thaliana leaf tissue extract was aliquoted into 0.2mL tube after which each tube contained 25 μΐ of leaf tissue extract. A 3mm diameter membrane discs was transferred into 25μ1 of leaf tissue extract for 1 minute. The membrane disc was then transferred to 200μ1 of wash buffer (lOmM Tris, 0.1% Tween-20) using a pipette tip and briefly agitated by gently knocking the bottom of tube. After one minute incubation in wash buffer, the membrane disc was transferred into an amplification reaction using a pipette tip.

Assessing effect of the amount of membrane on nucleic acid purification

3mm diameter Whatman No.1, Hybond N or Scott paper towel discs were cut using a hole puncher. 20 μΐ of lng/μΐ purified A. thaliana DNA solution was added into 0.2mL tube containing either 1, 2 or 3 discs respectively and then removed out of tube after 1 minute. 200μ1 of wash buffer (lOmM Tris, 0.1% Tween-20) was added into the tube. After 1 minute incubation, PCR amplification reactions were transferred into 0.2mL tubes containing different amounts of discs, respectively.

Kinetics during DNA binding to and release from cellulose

3mm diameter Whatman No. l discs were obtained using a hole puncher. For DNA binding, 20μ1 of lng/μΐ purified A. thaliana DNA solution was added into 0.2mL tube containing 1 cellulose disc. After varying lengths of incubation time, DNA solution was removed from the tube. 200μ1 of wash buffer (lOmM Tris, 0.1% Tween-20) was transferred into the tube for 1 minute incubation and then removed, followed by transformation of a PCR reaction into the tube.

For DNA release, Ιμΐ of lOng/μΙ purified A. thaliana DNA solution was loaded on a Whatman No. l disc. The disc containing lOng purified DNA was then transferred into 15mL tube in presence of 10 mL of wash buffer (lOmM Tris, 0.1% Tween-20). 15mL tube was placed on mini rocker (Bio-Rad) and agitated gently for varying lengths of time. After agitation, the cellulose disc was transferred into a PCR amplification reaction.

DNA binding test with DNA buffered in 150mM ofNaCl

3mm diameter Whatman No. l discs were cut using a hole puncher. Cellulose discs were added into 20μ1 of lng/μΐ DNA buffered in water or 150mM of NaCl solution. After incubation for different lengths of time (1 or 60 minutes), DNA solution in 0.2mL tube with a hole at the bottom was removed from disc by spinning. Disc was then transferred into a PCR reaction.

Dipstick nucleic acid purification and subsequent amplification

Dipsticks were created by dipping half of a Whatman No. l filter into molten wax (Paraplast Plus, Fluka) to create a region that is impervious to water. After the wax had set, the partially wax-coated filter paper was cut into a 44mm wide rectangle of which approximately 40mm was coated in wax and 4mm was uncoated. This rectangle was then cut into approximately 2mm wide strips to create dipsticks with a 2x4mm nucleic acid binding area and a 2x40mm handle.

For nucleic acid purification using the dipsticks, leaf tissue (approximately 200mm²) was added to a 2mL tube containing 500μ1 cell lysis buffer (20mM Tris, 25mM NaCl, 2.5mM EDTA, 0.05% SDS) and two ball bearings. The plant tissue was macerated by shaking tube for approximately eight seconds. The dipstick was dipped into extract to bind nucleic acids then dipped into 1.75mL of wash buffer (lOmM Tris, 0.1% Tween-20) and then finally the bound nucleic acids were eluted by dipping the dipstick directly into amplification reaction. Each time the dipstick was dipped up and down in each solution three times and taking approximately three seconds. After elution, the dipstick was discarded and the DNA amplification reaction transferred to a thermocycler.

Magnetic beads nucleic acid extraction

Agencout AMPure XP PCR Purification kit (Beckman Coulter) was used to purify DNA following the manufacturer's recommendations. Briefly, one volume of sample was mixed with 1.8 volumes of paramagnetic particles. The mixture was incubated at room temperature for five minutes and then placed onto magnetic plate (Life technologies) for two minutes to pull down DNA bound paramagnetic particles. After supernatant was removed, paramagnetic particles were washed twice with 70% ethanol. After the 70% ethanol from the last wash was removed, the paramagnetic beads were air dried for five minutes. 40μ1 of water was added onto paramagnetic beads containing bound DNA and well mix by pipetting up and down for DNA elution before pulling the particles down to the bottom of the tube by magnet. The supernatant contained purified DNA was transferred to a new tube and was later used as template DNA in PCR amplifications.

Nucleic acid amplification

Nucleic acid amplification was performed by either polymerase chain reaction (PCR), Loop mediated isothermal amplification (LAMP), or Recombinase polymerase amplification (RPA). For PCR amplification, 15μ1 reactions were performed using 7.5μ1 of GoTaq Green Master Mix (Promega), 15 pmol of both forward and reverse primers (Table SI) and template DNA. Unless otherwise stated, PCR cycling parameters were as follows: 95°C for two minutes, 35 cycles of 95°C for 20 seconds, 55-60°C for 20 seconds, 72°C for 40 seconds, followed by final extension of 72°C for one minute. For reactions involving RPA, either TwistAmp Basic RPA kit (Twist DX) or TwistAmp Basic RT-RPA kit (Twist DX) was used as manufacturer's recommendations. Briefly, each RPA pellet was resuspended with 29.5μ1 of rehydration buffer and 0.48 μΜ of both forward and reverse primer (Table SI). The mix was the aliquoted evenly into four 0.2ml tubes, into which, the template DNA, water and 0.625 μΐ of 280mM magnesium acetate are added to make a final volume of 12.5μ1. RPA reactions or RT-RPA reaction were performed at 37°C or 42°C for 20 minutes respectively and the results visualised by agarose gel electrophoresis.

For reactions involving LAMP, cellulose discs containing extracted nucleic acids were added to 15μ1 reactions containing: 20mM Tris (pH 8.8), lOmM (NH₄)2S0₄, 50mM KC1, 0.1% (v/v) Tween-20, 0.8M betaine, 8mM MgS0₄, 1.2mM dNTPs, 4.8U Bst2.0 warmstart (NEB Biolabs, USA), 0.8μΜ of FIP and BIP primers and 0.2μΜ of F3 and B3 primers. Reactions were incubated at 63°C for 50 minutes followed by a five minute incubation at 80°C to denature the enzyme.

Results and Initial Discussion

Whatman No.1 filter paper can entrap and retain DNA after washing

To attempt to develop a simple nucleic acid purification method suitable for field-based (point-of-need, PON or point-of-care POC) diagnostics, we first investigated the ability of a number of cationic chemicals that could potentially help to capture anionic DNA and RNA by spotting them on to a piece of Whatman No. l paper (GE Healthcare, USA). We found that a number of compounds showed a strong ability to bind nucleic acids (FIG. 1 A) and these were further tested for their ability to capture genomic DNA that could be directly amplified from the modified cellulose in a PCR reaction. None of the chemicals examined produced reproducible amplification. However, surprisingly, we noted that the control, unmodified Whatman No. l paper, resulted in consistently strong amplification (FIG. IB).

The ability of cellulose-based paper to entrap or adsorb DNA under specific conditions has been reported but its use has been limited, to storage or transport and not for nucleic acid purification purposes under non-precipitating conditions (Alberts et al. 1968, Semancik 1986, Su et al. 1999, Nargessi 2005, Nargessi et al. 2010). We further examined the efficiency at which Whatman No.1 can capture DNA and retain DNA during a brief (1 minute) wash prior to DNA amplification directly from the paper. Our results show that the Whatman No. l has a relatively high efficiency as the amplification results were comparable to that observed when an identical amount of DNA template was added directly to the PCR reaction (FIG. 2A). These results suggest that cellulose can efficiently bind, or at least entrap, DNA and does not inhibit amplification reactions.

We then devised a simple nucleic acid purification method (FIG. 2B) to test whether it was possible to remove PCR-inhibiting chemical contaminants present in a plant crude extract while retaining enough DNA for amplification. A 7mm² (1.5mm diameter) disc of Whatman No. l paper was added to an Arabidopsis thaliana leaf extract for one minute before transferring it to a tube containing wash buffer for one minute and finally transferring it to the PCR reaction tube, where it remained for the entire PCR process. The primers used in the PCR were designed to amplify a 262bp fragment of the G protein gamma subunit gene (AtAGGl). No amplification occurred when either Ιμΐ of extract or a Whatman No. l filter soaked in extract was directly added to the amplification mix (FIG. 2C). However, briefly washing the extract soaked filter paper once prior to using the filter directly in a PCR reaction was sufficient to remove amplification inhibitors while retaining the captured plant DNA (FIG. 2C). Performing a second wash did not enhance or diminish the amplification efficiency (FIG. 2C).

Method evaluation and application to different systems

Having determined that the method was successfully applied to the model plant species Arabidopsis thaliana, its application to the development of nucleic acid- based diagnostics of commercially important crops was assessed. We successfully applied our cellulose-based method to number of agriculturally important species such as wheat, barely, rice, soybean, tomato, sugarcane, tobacco and sweet potato (FIG. 3 A). The method was also successfully used to produce PCR-ready DNA from mature leaves of a number of citrus tree species (mandarin, lime, lemon and orange) (FIG. 3 A), which are notoriously difficult due to their high levels of lignin, phenolics and polysaccharides (Cheng et al. 2003).

Furthermore, important human diseases such as HIV and hepatitis can be diagnosed using nucleic acid-based tests from blood samples, although it is essential to remove several inhibitory compounds prior to nucleic acid amplification (Akane et al. 1994). There are both commercial kits and published methods available that can extract DNA from blood but they require relatively extensive sample manipulation, which makes them suboptimal for PON applications (Ohhara et al. 1994, Rudbeck et al. 1998, Queipo-Ortuno et al. 1999). We therefore tested whether cellulose filter paper is capable of purifying DNA from whole blood samples in order to amplify a fragment of the human V-raf murine sarcoma viral oncogene homolog Bl (BRAF) gene by PCR (FIG. 3B).

Cell lysis was achieved by diluting the blood samples 1 :5 in an extraction buffer containing proteinase K. Direct addition of the sample to the PCR reaction did not result in detectable amplification. In contrast, immersing the filter paper in the sample followed by a one minute wash retained enough genomic DNA to allow amplification while removing inhibiting compounds from the sample, resulting in a clear amplification product. The cellulose disc method was also successfully used to amplify genomic DNA from melanoma cell line cultures while direct addition of lysate to the PCR reaction mix did not produce any amplicons (FIG. 3C).

One important application for the method described herein is as a first step in PON diagnostics, replacing the currently employed labour intensive procedures. Therefore, to test the ability of the method to detect plant pathogens, we infected Arabidopsis thaliana plants with the bacterial pathogen Pseudomonas syringae. Our method successfully extracted and amplified pathogenic DNA even before the symptoms were visible to the human eye (FIG. 4A). The cellulose disc method was also successfully used in the detection of animal pathogens including the bacteria Actinobacillus pleuropneumoniae in lung swabs from infected pigs (FIG. 4B).

Furthermore, we tested if the method could be used for the extraction of RNA.

Tomato plants infected with cucumber mosaic virus were tested using the filter paper DNA extraction method without any modifications. In this case we used Recombinase Polymerase Amplification (RPA) (isothermal) with the addition of reverse transcriptase to the reaction mix in order to perform reverse transcription and amplification simultaneously in a single tube. An amplification product was obtained in reactions containing reverse transcriptase while no amplification was observed on uninfected samples or reactions lacking reverse transcriptase (FIG. 4C). The cellulose disc method also works in conjunction with other isothermal methods including Loop- mediated amplification (LAMP) which detected the CMV RNA without requiring a reverse transcriptase due to the intrinsic reverse transcriptase activity of the Bst 2.0 enzyme (Shi et al. 2015) (FIG. 4D).

DNA capture and release

To explore the mechanism behind nucleic capture by Whatman No. l paper, we examined whether we could replace it in our simple extraction method with different types of solid supports. Our results show that other cellulose-based papers, including a common hand drying paper towel (Scott brand Optimum towel') can be used to purify crude plant extracts (FIG. 5A). However, not all cellulose papers can be used to purify nucleic acids as common photocopy paper, either bleached or unbleached, failed to amplify a product. Furthermore, we successfully used nylon membranes (Qiagen Qiabrane, Amersham Hybond-N) to purify DNA from the plant extract revealing that this method is not limited to cellulose-based supports. Positively charged supports failed to produce amplicons, independently of whether they were nylon- or cellulose-based (Amersham Hybond-N+, Qiabrane nylon plus, Hybond-C extra (nitrocellulose) and DEAE cellulose). This result indicates that, surprisingly, materials that are ideal for DNA capture are not necessarily ideal for use in DNA extraction. In general, the membranes which were found to be suitable for nucleic acid extraction were hydrophilic and microporous, and possessed neutral or negative surface charge (FIG. 10).

We further assessed whether the amount of the cellulose used in the DNA extraction has an effect on the amplification yield. After using 1, 2 or 3 discs in the extraction procedure, we observed an inverse relationship between the number of cellulose or nylon discs used and the amount of DNA amplified. Without being bound by theory, it is plausible that the more cellulose or nylon that is added to the amplification reaction results in a greater sequestration of primers, dNTPs or other reagents. Consistent with this, Scott paper towels which have 24% less cellulose by weight compared to Whatman No. l resulted in stronger amplification when an equal number of discs were used (FIG. 5B).

We hypothesised that the mechanism underlying our rapid purification method is based on differences in the kinetics of nucleic acid binding to and release from the cellulose. In our model, nucleic acids are able to rapidly bind to the cellulose fibres but are released at a much slower rate. Of note, other components present in the sample extracts, such as amplification inhibitors, either do not bind to the cellulose or are rapidly released and subsequently removed from the cellulose matrix during the brief washing step.

As predicted, we found that DNA binds to Whatman No. l and reaches an equilibrium with the surrounding liquid extremely fast (FIG. 6A). Binding for 7 seconds followed by a wash step resulted in strong amplification that could not be improved by longer binding times. In contrast, during the washing step, we found that DNA is released from the cellulose at a relatively slow rate (FIG. 6B and FIG. 13). Whatman No. l discs with 10 ng of purified genomic DNA directly added to them were washed for varying lengths of time by gently rocking in 10 ml of water before being dropped in the PCR amplification mix. As expected, the disc that was not washed and therefore contained the entire DNA sample gave the highest band intensity after amplification. Also, consistent with our hypothesis, Whatman No. l washed for up to 24 hours still retained enough DNA to give a positive amplification product. Most importantly, a brief one minute wash did not significantly affect the amount of DNA retained in the filter as can be seen by the almost identical intensity of the amplification bands when compared to the no wash control.

Previous studies have shown cellulose and nylon membranes carry a negative surface charges due to the presence of acidic groups, such as carboxyl and hydroxyl groups, on their surface (Dubitsky et al. 1995, Sood et al. 2010).

As DNA also carries a net negative charge, largely due to its electronegative phosphate backbone, the like charges between the DNA and the Whatman No. l surface will result in a repulsive force that will hinder DNA binding. We therefore predicted that the addition of salt could increase the binding of DNA to the Whatman No. l paper by counteracting the electrostatic repulsion. Our results confirmed the hypothesis as we observed significantly enhanced binding for DNA samples diluted in 150mM NaCl compared to those diluted in water (FIG. 7).

Pipette-free nucleic acid purification

We aimed to further simplify the nucleic acid extraction method by using a cellulose-based dipstick in order to streamline the handling and eliminate the need to transfer the cellulose disc between tubes. To this end, we designed dipsticks made from Whatman No. l with a small 8 mm² (per face, approximately 16 mm² total) DNA binding surface and a long water repellent handle by impregnating the filter paper with paraplast wax (FIG. 8 A). Using these dipsticks, we developed an improved method in which all reagents can be prepared in advance and stored for a long period of time at room temperature.

When needed, a nucleic acid extraction can be performed in less than 30 seconds without a pipette or any electrical device (FIG. 8B). Tissue is first homogenised in a tube containing the appropriate lysis buffer and ball bearings to help macerate the tissue. The cellulose dipstick is used to capture nucleic acids by dipping it into the lysate three times. Contaminants are removed from the dipstick by dipping it up and down in a wash solution three times. Finally, the bound nucleic acids are eluted from the cellulose by dipping the dipstick directly into the amplification mix three times. Using this method, we have successfully demonstrated that it is possible to rapidly purify DNA from plant leaves infected with the fungus Fusarium oxysporum or the bacteria Pseduomonas syringae (FIG. 8C). This method works equally well in extracting viral RNA from plant leaves that is suitable for use in reverse transcription PCR amplification (FIG. 8D).

To validate our newly developed nucleic acid purification method we compared it with a popular commercial rapid paramagnetic bead DNA extraction method (Beckman coulter, AMPure). We found that our method can purify amplifiable DNA significantly faster: under 30 seconds for our method versus 14.5 minutes for AMPure purification when following the manufacturers' recommended instructions (FIG. 9A). Importantly, the method achieves this speed and simplicity without the need for any pipetting. Our method is also significantly cheaper with consumables costing four times less than those required by the AMPure system and does not require the initial investment of USD $685-$876 for the specialised magnet plate. The sensitivity of our method was comparable to the commercial system as they could both extract amplifiable DNA from initial concentrations of O. lng/μΐ genomic DNA and above (FIG. 9B). In some experimental settings, there is a limited supply of sample tissue and it is therefore critical to be able to extract DNA from small volumes of tissue extract. We found that our method was again comparable the commercial system in its ability to purify nucleic acids from tissue extract volumes as low as 0.5μ1 (Figure 9C).

Further Discussion

New molecular technologies for point-of-need (PON) or point-of-care (POC) diagnostics are currently receiving substantial attention. Nucleic acid-based (molecular) assays offer greater sensitivity, specificity and speed over other technologies such as enzyme-linked immunosorbent assay (ELISA), lateral flow strips and cell culture/analysis (Dong et al. 2008, Liesenfeld et al. 2014). As such, molecular assays have the potential to revolutionize the early detection and continual monitoring of human, plant and animal diseases. However, a major bottleneck in molecular diagnostics is that they rely on nucleic acid purification, which is a relatively time consuming and laborious procedure that is not ideally suited to field- based testing (Mumford et al. 2006, Rahman et al. 2012, Thatcher 2015). We have determined that a small (7-8 mm² per face, or ~ 15 mm² total surface area) piece of cellulose-based paper is capable of purifying nucleic acids away from inhibitors in a wide range of plant, animal and microbial samples including whole blood and mature tree leaves. Furthermore, to optimized the method for field-based testing, we created a cellulose-paper-based dipstick that can bind, wash and elute purified nucleic acids in under 30 seconds without requiring any pipetting or electrical equipment. We found that, despite its speed, this method is comparable to a commercially available nucleic acid purification method in its ability to prepare amplification ready template nucleic acids (FIG. 9). The speed, simplicity and universality of this method makes it an attractive option for a broad range of diagnostic applications in both the field and laboratory settings.

A vast array of nucleic acid extraction procedures exist in the published literature however, the dipstick-purification system presented here has numerous advantages over existing methods. One key advantage is that the method is significantly faster and has fewer steps than common liquid-based DNA extraction methods (eg phenol/chloroform, CTAB or guanidinium salts etc.) or solid-phase DNA extraction methods involving silica or paramagnetic beads (FIG. 9). Furthermore, while a number of recently developed DNA extraction methods that utilise commercially available filters, including the silica-based Whatman Fusion 5 and the cellulose-based Whatman (Flinders Technology Associates (FTA) cards (Liu et al. 2011, Govindarajan et al. 2012, Gan et al. 2014, McFall et al. 2015), our method is much simpler and faster than any of the available membrane-based procedures. For example, FTA cards contain chemicals that lyse cells and protect the DNA from degradation and have been used for over a decade as a means to store and preserve DNA samples before processing (Gustavsson et al. 2009, Awad et al. 2014, Madhanmohan et al. 2015). These chemicals are inhibitory to DNA amplification and therefore must be removed through a number of washing and drying steps (Liu et al. 2011) before the DNA can be amplified from the FTA card. Additionally, unlike the 2 minute Fusion-5-based purification method, which can only capture DNA (Jangam et al. 2009, McFall et al. 2015), our method using Whatman No. l can also be used to extract RNA suitable for reverse-transcription and subsequent DNA amplification (FIG. 4 and FIG. 8).

In the context of POC field-based applications, an important issue with most extraction procedures is that, after purification, nucleic acids need to be accurately pipetted into the amplification mix. Adding too little or too much nucleic acids into an amplification reaction can result in a failure to amplify a product (Grunenwald 2003). A significant advantage of the method presented here is that the amount of nucleic acid transferred to the amplification reaction will be similar between samples of the same type because since the size of the DNA binding surface on the cellulose dipstick remains constant. Furthermore, the system can be fine-tuned by altering the size of the DNA binding surface in the dipstick thus optimising the amount of nucleic acid transferred for downstream applications. This is an important feature as it provides flexibility to adapt the method to different tissues (plant leaves, blood, saliva, etc.) depending on the intended application.

Overall cost, including the equipment required to perform the procedure, is a major determinant in the likelihood of broad scale adoption of diagnostic technologies (Yu et al. 2012, Haleyur Giri Setty et al. 2014). The cellulose dipstick purification method described here significantly increases the affordability of nucleic acid purification with the price per sample being $US 0.15 including plasticware and reagents at the time of writing, with the ball bearings being the major contributor to the final price. If the ball bearings used to homogenise the tissue are washed and reused, the cost can be further reduced to just $US 0.06 per sample. Whatman No. l paper is cheap and easy to obtain but not absolutely necessary as common paper towels proved to be as efficient, providing an even cheaper alternative.

Without being bound by theory, it is hypothesized that cellulose-based method for nucleic acid purification described herein takes advantage of four key cellulose characteristics. First, cellulose paper is capable of rapidly absorbing a relatively large amount of DNA/RNA relative to its mass through capillary action e.g. (Chen et al. 2015). Second, nucleic acids are either rapidly entrapped by, or bind to the cellulose fibres (FIG. 6). Third, a sufficient amount of nucleic acid is retained on the cellulose even after extended incubation in a large volume of water, while inhibitors including Proteinase K, cellulosic and phenolic compounds are rapidly eluted (FIGS. 2C, 3B, and 6B). Lastly, unlike positively charged membranes (FIG. 5), cellulose enables rapid elution of a sufficient quantity of bound nucleic acids into the amplification mix (FIGS. 6 A and 6B). This rapid elution from the cellulose may be catalysed by salts or dNTPs present in the amplification mix as has been reported for other systems (Tanaka et al. 2009). Collectively, it is hypothesized that these characteristics make cellulose an ideal material for a rapid and simple nucleic acid purification system that easily separates unwanted contaminants and inhibitors away from nucleic acids while also transferring a reproducible amount of nucleic acids into the amplification mix.

The use of cellulose for DNA purification under certain conditions is known and is claimed to have improved performance over silica-based DNA purification methods (Moeller et al. 2014, Promega 2016). Relevantly however, DNA purification using cellulose has been previously reported to be achieved by co-aggregating or adsorbing the DNA to the cellulose in the presence of various chemicals, including chaotropic salts (Linnes et al. 2014), ethanol (Su et al. 1999), and high salt and polyalkylene glycol concentrations (Nargessi 2005, Nargessi et al. 2007), which destabilise the DNA structure and facilitates its interaction with the cellulose fibres. Water or low salt solution is then used to elute the DNA from the cellulose.

However, in contrast to what has been reported in the literature, we have surprisingly found that cellulose can capture DNA in pure water (FIG. 6, 7, and 13) and moreover, retain the DNA in the presence of a large volume of water for over 24 hours (FIG. 6B). Furthermore, we have demonstrated that the method is not dependent on a specific buffer but can successfully purify nucleic acids from crude extracts with either guanidine hydrochloride-based (e.g. FIGS. 3B and 4B), SDS-based (FIGS. 8C and 8D), or Tween 20-based (e.g. FIG. 2C) extraction buffers. Although the exact mechanism of nucleic acid binding remains unclear, we observed that the amount of nucleic acid bound can be enhanced in the presence of salts (FIG. 7). This is likely due to the neutralisation of the negative charges on the surfaces of both the cellulose and nucleic acids thereby eliminating the repellent electrostatic forces between them.

As this study was focused on creating a pipette-free nucleic acid purification method and not a complete molecular diagnostic system, a mains powered thermocycler was used for most reactions. While this is suitable for laboratory-based research, it is obviously not ideal for field use. We have demonstrated that our method can be coupled with isothermal DNA amplification technology (Figures 4C, 4D), which have previously been successfully performed using simple portable devices that generate heat using either a small battery or chemical heat pad (Liu et al. 2011, Curtis et al. 2012, Almassian et al. 2013, Myers et al. 2013). A simple field-ready molecular diagnostic that requires minimal equipment and no pipetting should be achievable by coupling our dipstick nucleic acid purification system with isothermal DNA amplification and/or equipment-free naked eye visualisation methods (Hill et al. 2008, Goto et al. 2009, Rohrman et al. 2012, Rivas et al. 2014, Miyamoto et al. 2015, Tanner et al. 2015, Wee et al. 2015, Rodriguez et al. 2016). Such a system would be advantageous for a wide variety of applications including disease detection and monitoring, quarantine/border control, species identification, and quantitative trait loci screening.

We have described here a simple and rapid technique that allows researchers to obtain nucleic acid at a suitable purity and concentration for DNA amplification. We have reduced a complicated process to three simple steps that do not require any specialised equipment (e.g. pipettes or centrifuges) and takes less than 30 seconds to perform (FIG. 8). As such, the method is equally suited to both laboratory and field environments for a wide range of applications.

EXAMPLE 2: Extraction buffers

As set forth above, the skilled person will appreciate that the use of a particular extraction buffer can be optimized according to the characteristics of a sample containing the nucleic acid that is to be extraction, and/or the nucleic acid itself (e.g. DNA or RNA). Extraction buffers assessed and found to be suitable for nucleic acid extraction according to at least certain embodiments of the invention include the following:

- 50mM Tris (pH 8); 1.5M guanidine HC1; 5mM EDTA; and 1% Tween 20

- 50mM Tris (pH 8); 1.5M guanidine HC1; 5mM EDTA; 1% Tween 20; and 25% ethanol

- 50mM Tris (pH 8); 1.5M guanidine HC1; 5mM EDTA; 1% Tween 20; and 33% ethanol

- 800mM guanidine hydrochloride; 50mM Tris (pH 8); 0.5% Triton X100; and 1% Tween-20

- 20mM Tris (pH 8); 25mM NaCl; 2.5mM EDTA; and 0.05%SDS

- 50mM Tris (pH8); 150mM NaCl; 2% PVP; and 1% Tween-20

- 50mM Tris (pH8); 150mM NaCl; 1% BSA; 2% PVP; 1% Tween

- 25mM NaOH

- 50mM NaOH

EXAMPLE 3: Wash buffers

The following wash buffers have been tested and found to be suitable for nucleic acid extraction according to at least certain embodiments of the invention:

- Purified water

- lOmM Tris (pH 8) - lOmM Tris (pH 8)+0.1% Tween 20

- lOmM Tris (pH 8)+100mM NaCl

- 80% acetone

- 80% ethanol

- lOmM MES (pH 5)

- lOOmM Tris (pH 8.8)

- lOOmM Tris (pH 8.8) + 8mM MgS0₄

- 137mM NaCl, 3mM KC1, 10.6mM KH₂P0₄ (pH 7.4)

EXAMPLE 4: Quantification of nucleic acid recovered

For this example, 0.1, 1, or lOng of DNA was added to Whatman No. 1 cellulose paper, washed for 1 min in lOmM Tris and then added to a PCR reaction. For comparison 0.1, 1, or lOng of the DNA was added directly to another set of PCR reactions. PCR was then performed for a limited number of cycles, and corresponding samples of the reactions were visualized on a gel, with brightness of the bands compared (FIG. 12A). The experiment was performed in triplicate. For control purposes, DNA and Whatman No. 1 were independently added ('Filter paper + DNA'), and filter paper or water only were added, to the PCR reaction.

Quantification of band brightness on the gels indicated that between 58 to 100%) recovery of the DNA that was placed on the Whatman filters was achieved (Figure 13B). While the analysis is somewhat preliminary as there are a number of factors that could influence the brightness of bands on the gels, the similar band brightness achieved across the triplicates, and the consistent correlation with brightness and amount of input DNA in both the control and test samples, suggests that the data is a relatively reliable measure of the relative nucleic concentration. This example therefore provides an indication that the filter paper captures most of the DNA in the sample and releases most of the captured DNA upon elution.

Throughout the specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety. TABLES

Table 1. Listing of primers referred to in this application.

SEQ ID

Sequence (5'-3') NO Target species Primer source

GAGAGAGAGACTTCGACGACA 1 This

Arabidopsis thaliana application

G CTATCCACG AG AG ACCTACG 2

TTG TTTG GAGCTTGCTGATG 3 Bradbury et

Rice al. 2005

CATAG G AG C AG CTG AAATATATACC 4

YG ACTCTCG G CAACG G ATA 5 Tomato, sugarcane, sorghum, Cheng et al.

soybean 2016

RGTTTC I 1 1 I CCTCCGCTTA 6

YG ACTCTCG G CAACG G ATA 7 Capsicum, tobacco, sweet

potato, barely, wheat, Cheng et al. mandarin, lime, lemon, orange, 2016

GCGTTCAAAGAYTCGATG RTTC 8 passion fruit

AAACTCTGGTGGAGGTCCGT 9 Naito et al.

Cell line 1992

CTTACCAAAAGTGGCCCACTA 10

ATAG GTG ATTTTG GTCTAG CTACTGT 11 This

Human blood application

AGTAACTCAG CAG CATCTC AG G 12

AAAGCCGCATATCCCCCA 13 This

Pseudomonas syringae application

TCAG ATACCG TCTCCTCAC AC 14

AAGGTTGATATGTCCGCACC 15 Actinobacillus Gram et al.

pleuropneumoniae 1998

CACCG ATTACG CCTTG CCA 16

AG I I AA I CC I 1 I GCCGAAA I 1 I GA I I C I AC 17 Wee et al.

Cucumber mosaic virus 2015

GTGCTCGATGTCAACATGAAGTACTAGCTC 18

TCTTATCCCATCCCC AG CAT 19 Fusarium oxysporum f.sp This

conglutinans (PCR application

CAACTCCTGTACGG ATTG CG 20 amplification)

GGATACATGAGTGTCCCTCAAGTG 21

Cucumber mosaic virus (LAMP This

ACAACAGCAAAACACCGCTT 22

amplification) application

CTTGTCG CCTAG ATCAG CTAAG TATCG AAC AGTTTCTACCG ATG CTG AAG G 23

AG CAGTG CGTCACATTACATAACCTGTCTC C ATG G G ACAATC ATAC G 24

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Claims

1. A method of extracting a nucleic acid from a sample, the method including the steps of:

(ii) releasing the nucleic acid from the matrix,

to thereby extract the nucleic acid from the sample.

2. The method of claim 1, wherein a substantial proportion of the nucleic acid of the sample is captured by the matrix and/or released from the matrix.

3. A method of analysing a nucleic acid, the method including the steps of:

(ii) analysing the nucleic acid that is captured according to step (i),

to thereby analyse the nucleic acid, wherein the nucleic acid is optionally released from the matrix before step (ii).

4. The method of any one of claims 1-3, wherein the method consists of step (i) and step (ii).

5. The method of any preceding claim, wherein the method includes the further step of processing the sample, prior to step (i).

6. The method of claim 5, wherein processing the sample comprises physical processing.

7. The method of claim 5 or claim 6, wherein processing the sample comprises chemical processing.

8. The method of any preceding claim, wherein capture of the nucleic acid by the matrix does not require additional nucleic acid binding agents to be added to the sample and/or the matrix.

9. The method of any preceding claim, wherein capture of the nucleic acid by the matrix does not require additional chaotropic agents to be added to the sample and/or the matrix.

10. The method of any preceding claim, wherein the retention of the nucleic acid by the matrix does not require additional nucleic acid binding agents and/or additional chaotropic agents to be added to the sample and/or the matrix.

11. The method of any preceding claim, wherein capture and/or retention of the nucleic acid by the matrix does not require the addition of agents or reagents to the sample and/or matrix, other than water and optionally a pH buffering agent.

12. The method of any preceding claim, including the further step of purifying the nucleic acid captured by the fibrous and/or porous matrix, after step (i) and/or before step (ii).

13. The method of claim 12, wherein the step of purifying the nucleic acid comprises, or consists of, adding water to the fibrous and/or porous matrix, optionally wherein the water includes a pH buffering agent.

14. The method of any one of claims 1-3, including the step of adding a solution comprising one or more additional agents to the sample and/or matrix.

15. The method of claim 14, wherein the solution comprises salt.

16. The method of any preceding claim, wherein the nucleic acid remains in contact with an aqueous solution throughout the method.

17. The method of any preceding claim, wherein extraction or analysis of the nucleic acid can be completed in less than about 2 minutes; less than about 1 minute; or less than about 30 seconds.

18. The method of any preceding claim, wherein the matrix is a membrane.

19. The method of any preceding claim, wherein the matrix is microporous.

20. The method of any preceding claim, wherein the matrix is absorbent.

21. The method of any preceding claim, wherein the matrix is hydrophilic.

22. The method of any preceding claim, wherein the matrix has a neutral or negative surface charge.

23. The method of any preceding claim, wherein the matrix comprises cellulose, nylon, and/or polyester.

24. The method of claim 20, wherein the matrix comprises, consists essentially of, or consists of, cellulose paper.

25. The method of any preceding claim, wherein the matrix is substantially free of additional nucleic acid binding agents.

26. The method of any one of claims 3-25, wherein analysis of the nucleic acid comprises nucleic acid amplification.

27. The method of claim 26, wherein analysis of the nucleic acid comprises visualization of the nucleic acid by binding the nucleic acid to a particle and/or dye.

28. The method of any one of claims 3-27, including the step of determining whether the sample has a characteristic of interest based on the results of the analysis.

29. The method of any preceding claim, wherein the nucleic acid and/or the sample is of a plant.

30. The method of any preceding claim, wherein the nucleic acid and/or the sample is of an animal.

31. The method of any preceding claim, wherein the nucleic acid and/or the sample is of a microorganism.

32. The method of any preceding claim, wherein the nucleic acid is DNA.

33. The method of any one of claims 1-29, wherein the nucleic acid is RNA.

34. The method of any preceding claim, including the step of selecting the surface area of the matrix based on the amount of nucleic acid to be captured by the matrix.

35. A device for use according to the method of any preceding claim, the device comprising, or consisting of: (a) a capture portion comprising a fibrous and/or porous matrix for combining with a nucleic acid whereby the nucleic acid is captured by the matrix; and (b) a handling portion for handling by a user, whereby the user is not in direct contact with the capture portion.

36. A kit that comprises: a fibrous and/or porous matrix for use according to the method of any one of claims 1-34; or the device of claim 35; optionally together with one or more reagents for amplifying, analysing or detecting the nucleic acid.