WO2018195594A1 - Extraction d'acide nucléique simple - Google Patents

Extraction d'acide nucléique simple Download PDF

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Publication number
WO2018195594A1
WO2018195594A1 PCT/AU2018/050374 AU2018050374W WO2018195594A1 WO 2018195594 A1 WO2018195594 A1 WO 2018195594A1 AU 2018050374 W AU2018050374 W AU 2018050374W WO 2018195594 A1 WO2018195594 A1 WO 2018195594A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
matrix
sample
dna
amplification
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PCT/AU2018/050374
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English (en)
Inventor
Michael Glenn MASON
Jose Ramon Botella
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The University Of Queensland
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2017901487A external-priority patent/AU2017901487A0/en
Application filed by The University Of Queensland filed Critical The University Of Queensland
Publication of WO2018195594A1 publication Critical patent/WO2018195594A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Definitions

  • THE present invention relates to nucleic acid isolation.
  • the invention relates to a nucleic acid-binding matrix for rapid and efficient nucleic acid isolation.
  • 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.
  • nucleic acid-based tests 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.
  • 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
  • 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 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.
  • isolation of the nucleic acid from the sample can be completed in less than about two (2) minutes.
  • the invention provides a method of extracting a nucleic acid from a sample, the method including the steps of:
  • the method of the first aspect consists of step (i) and step (ii).
  • 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).
  • 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.
  • extraction of the nucleic acid from the sample can be completed in less than about 2 minutes.
  • said extraction can be completed in less than about 1 minute.
  • said extraction can be completed in less than about 30 seconds.
  • the fibrous and/or porous matrix according to this aspect is a fibrous and/or porous membrane.
  • the fibrous and/or porous matrix according to this aspect is absorbent.
  • the matrix is hydrophilic.
  • the fibrous and/or porous matrix has a neutral or negative surface charge.
  • the surface charge is a negative surface charge.
  • the matrix according to this aspect is microporous.
  • the fibrous and/or porous matrix comprises cellulose, nylon, polyester, and/or polyvinyl or derivatives thereof.
  • the matrix comprises cellulose.
  • 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.
  • 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.
  • 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.
  • the nucleic acid remains in contact with an aqueous solution throughout the method.
  • 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.
  • the method of the second aspect consists of step (i) and step (ii).
  • the captured nucleic acid is eluted, enriched or purified prior to analysis.
  • the analysis is performed in situ on the fibrous and/or porous matrix.
  • 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.
  • 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.
  • 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.
  • the disease, disorder, or condition is caused by or associated with infection by a pathogen.
  • the pathogen is selected from the group consisting of a bacterium, a fungus, and a virus.
  • 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.
  • 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.
  • the nucleic acid and/or the sample according to the first to third aspects is of a plant.
  • the plant is a crop plant.
  • the nucleic acid and/or the sample according to the first to third aspects is of an animal.
  • the animal is a human.
  • a device for use according to the method of the first to third aspects 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.
  • said device consists of (a) and (b).
  • kits 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.
  • 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.
  • a protein includes one protein, one or more proteins or a plurality of proteins.
  • 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.
  • 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).
  • NTC no template control
  • Figure 2 sets forth capture and purification of nucleic acids using cellulosic paper.
  • Panel (A) ⁇ of purified DNA at different concentrations (0, 0.01, 0.1, 1 or lOng/ ⁇ ) was pipetted directly onto a Whatman No. l disc (3mm diameter) and then washed in 200 ⁇ 1 lOmM Tris (pH 8) before adding the disc to a PCR reaction. As a control ⁇ 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.
  • 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.
  • ⁇ 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, ⁇ of the raw lysate was added directly into a separate PCR reaction. No template controls (NTC) involved adding ⁇ of water instead of DNA template.
  • NTC template controls
  • Figure 4 sets forth DNA and RNA extraction from plant and animal pathogens using cellulosic paper.
  • 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 ⁇ 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/ ⁇ purified Arabidopsis genomic DNA solution for different amounts of time before washing for one minute and transferring 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/ ⁇ ) dissolved in water or in 150mM NaCl. DNA solution was removed from discs by centrifugation and the discs were added to a PCR amplification.
  • FIG. 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 ⁇ 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.
  • 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.
  • 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: 15- 16 Primers for detection of Actinobacillus pleuropneumoniae .
  • SEQ ID NOS: 17- 18 Primers for detection of Cucumber mosaic virus.
  • 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.
  • 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.
  • isolated material ⁇ e.g. a nucleic acid
  • 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.
  • 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.
  • nucleic acid designates single-or double-stranded
  • DNA and RNA 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 SequenaseTM.
  • a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
  • one aspect of the invention provides a method of extracting a nucleic acid from a sample, the method including the steps of:
  • nucleic acid 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.
  • 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.
  • the solution will be an aqueous or water-based solution.
  • 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.
  • the solvent of the sample in which the nucleic acid is in contact with consists essentially of, or consists of water.
  • other solvents may be used.
  • solvents comprising or consisting of an alcohol (e.g. ethanol) and/or a ketone (e.g. acetone).
  • the solvent will be or comprise a polar solvent.
  • step (ii) comprises elution of the nucleic acid from the fibrous and/or porous matrix.
  • the nucleic acid is eluted according to step (ii) by combining the matrix with a buffer.
  • the buffer comprises tris(hydroxymethyl)aminomethane (Tris), and optionally one or more metal salts.
  • the buffer is a phosphate buffer.
  • step (ii) comprises elution in a solution that contains one or more agents typically required for nucleic acid amplification (as herein described).
  • 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).
  • said solution may additionally or alternatively contain agents that have similar chemical structures or properties as the aforementioned agents for nucleic acid amplification reactions.
  • nucleic acid amplification agents 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- ⁇ 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.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • RCR rolling circle replication
  • NASBA nucleic acid sequence-based amplification
  • LCR ligase chain reaction
  • LAMP loop-mediated isothermal amplification of DNA
  • RPA recombinase polymerase amplification
  • the method of this aspect includes the further step of processing the sample, prior to step (i).
  • 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.
  • processing prior to step (i) comprises physical processing, such as grinding or crushing.
  • physical processing such as grinding or crushing.
  • 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.
  • 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.
  • processing prior to step (i) comprises chemical processing, such as chemical extraction or elution.
  • 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.
  • 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).
  • 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
  • PCR enhancer e.g. BSA
  • 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.
  • 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).
  • the purification according to step comprises washing the matrix in a wash solution or buffer.
  • the wash buffer comprises a pH buffering agent such as Tris.
  • the wash buffer may comprise one or more agents selected from the group consisting of a metallic salt, an alcohol, and a ketone.
  • the wash solution is substantially free of additional chaotropic agents.
  • the wash solution consists essentially of, or consist water and a pH buffering agent (e.g. Tris).
  • the wash solution consists essentially of, or consists of water.
  • nucleic acid of the sample is captured by the fibrous and/or porous matrix.
  • nucleic acid " ⁇ / 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • step (i) 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).
  • 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.
  • the extraction of the nucleic acid from the sample can be, or is, completed in less than about 2 minutes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method does not include a drying step.
  • the nucleic acid remains in contact with a solution throughout the method.
  • 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.
  • the matrix is a fibrous and/or porous membrane, although without limitation thereto.
  • a "membrane” will be understood to be a relatively thin, sheet-like structure.
  • the membrane may, but need not necessarily, be permeable to liquid.
  • the fibrous and/or porous matrix according to this aspect is absorbent.
  • "absorbent" matrices will have substantial capacity to take in and hold liquid.
  • the matrix is absorbent of water and aqueous solutions.
  • the fibrous and/or porous matrix according to this aspect is hydrophilic.
  • 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.
  • 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
  • hydrophobic matrices e.g. Immobilon-FL PVDF (catalogue number IPFL0010); and Hybond-C extra (catalogue number RPN203E) did not successfully capture nucleic acids.
  • the matrix according to this aspect is microporous.
  • a "microporous" matrix will be understood to be one which comprises pores below a certain micrometre size.
  • a microporous matrix according to this aspect comprises pores or openings with a diameter of less than: 200 ⁇ ; 175 ⁇ ; 150 ⁇ ; 100 ⁇ ; 75 ⁇ ; 50 ⁇ ; 25 ⁇ ; or ⁇ ⁇ ⁇ .
  • microporous matrices e.g. Whatman No. 1 : pore size - 11 ⁇ ; Whatman No. 4: pore size ⁇ 25 ⁇
  • the matrix according to this aspect has a neutral or negative surface charge.
  • surface charge can be measured by assessing zeta potential.
  • a net negative zeta potential corresponds to a negative surface charge of a matrix.
  • matrices with negative surface charge e.g. Whatman No. 1 and Whatman No. 2
  • matrices with neutral surface charge e.g. Amersham hybond-N (catalogue number RPN203N)
  • matrices with positive surface charge e.g. Amersham hybond-N+ (catalogue number RPN303B)
  • the matrix according to this aspect comprises cellulose.
  • the matrix consists of, or consists essentially of, cellulose-based paper.
  • 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.
  • sizing agents including rosin, gum, and starch
  • 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).
  • 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.
  • commercial photocopy paper did not successfully capture nucleic acids according to the method of this aspect.
  • 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.
  • the fibrous and/or porous matrix according to this aspect comprises nylon, polyester, and/or polyvinyl.
  • the matrix consists of, or consists essentially of, a nylon filter membrane.
  • 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.
  • the fibrous and/or porous matrix may consist of, or consists essentially of, a polyvinyl filter membrane.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • any agent contained within the sample itself will not be considered an additional nucleic acid binding agent or an additional chaotropic agent.
  • 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.
  • salts such as metal salts
  • 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.
  • no additional salt is used to facilitate nucleic acid capture according to the method of this aspect.
  • an additional nucleic acid binding agent such as a salt may improve or enhance nucleic acid capture by the fibrous and/or porous matrix.
  • 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.
  • nucleic acid binding agent 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.
  • the fibrous and/or porous matrix according to this aspect may be substantially free of other additional agents or reagents.
  • the fibrous and/or porous matrix according to this aspect is substantially free of additional agents which preserve nucleic acids.
  • additional agents are well known to the skilled person, and include those which decrease or remove nuclease activity (e.g. EDTA, guanidine thiocyanate etc.).
  • 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.
  • the fibrous and/or porous matrix according to this aspect is substantially free of additional agents which disrupt tissues and/or cells.
  • agents are well known to the skilled person, and include detergents such as Triton XI 00 and SDS.
  • solutions or buffers used according to the method of this aspect may be substantially free of additional agents.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sample according to the method of this aspect may be any suitable sample comprising a nucleic acid.
  • the nucleic acid of the sample is of, or derived from, a biological organism.
  • samples containing nucleic acids of other origin, e.g. synthetic nucleic acids, are also within the scope of this aspect.
  • 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.
  • 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.
  • the method of this aspect 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.
  • 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.
  • the biological organism is a plant, inclusive of any organism within the kingdom Plantae.
  • 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.
  • the plant species is selected from the group consisting of sugarcane, barley, wheat, sorghum, soybean, tomato, tobacco, mandarin, lime, lemon, and passionfruit.
  • 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.
  • the animal may be from any of the Ecdysozoa, Lophotrochozoa, Radiata, or Deuterostomia phyla.
  • the animal is selected from the group consisting of a mammal, a bird, a fish, a reptile, and an amphibian.
  • 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.
  • livestock e.g. horses, cattle and sheep
  • companion animals e.g. dogs and cats
  • laboratory animals e.g. mice, rats and guinea pigs
  • performance animals e.g. racehorses, greyhounds and camels
  • the animal is a human.
  • 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.
  • the biological organism may be a parasite or pathogen.
  • the pathogen is a microorganism selected from the group consisting of a virus, a bacteria, and a fungi.
  • 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.
  • 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.
  • 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.
  • the captured nucleic acid is eluted, enriched or purified prior to analysis.
  • the analysis is performed in situ on the matrix.
  • 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.
  • 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).
  • purification can be performed in embodiments wherein the analysis is performed in situ on the matrix.
  • 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.
  • 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.
  • the wash solution is substantially free of additional chaotropic agents.
  • 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.
  • analysis of the nucleic acid comprises nucleic acid sequence amplification.
  • 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.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • RCR rolling circle replication
  • RCR rolling circle replication
  • NASBA nucleic acid sequence-based amplification
  • next-Generation Sequencing' techniques 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.
  • 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.
  • 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.
  • POC point-of-care
  • analysis of the nucleic acid comprises nucleotide sequencing.
  • 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.
  • nucleic acid sequence amplification and nucleotide sequencing have vast application to nucleic acid analysis.
  • nucleic acid analysis using nucleic acid sequence amplification or nucleotide sequencing involves detection or identification of a nucleotide sequence of interest.
  • 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.
  • 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'.
  • 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.
  • 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).
  • the analysis is performed as point- of-care (POC) analysis.
  • POC point- of-care
  • 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.
  • the analysis according to this aspect comprises nucleic acid analysis by visual inspection or electronic determination.
  • 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.
  • 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.
  • the analysis involving visual inspection is as described in WO 2015/095929, incorporated herein by reference.
  • 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.
  • a solution comprising the particle may change colour.
  • the technique described in WO 2015/095929 generally does not require specialized (e.g. electronic) equipment such as spectrophotometers or thermal cyclers.
  • 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.
  • colorimetric or fluorescent dyes including, but not limited to, hydroxyl napthol blue, SYBR green, or SYTO 9.
  • 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.
  • 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.
  • 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.
  • the biological organism is a plant, inclusive of any organism within the kingdom Plantae.
  • 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.
  • the plant species is selected from the group consisting of sugarcane, barley, wheat, sorghum, soybean, tomato, tobacco, mandarin, lime, lemon, and passionfruit.
  • 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.
  • the animal may be from any of the Ecdysozoa, Lophotrochozoa, Radiata, or Deuterostomia phyla.
  • the animal is selected from the group consisting of a mammal, a bird, a fish, a reptile, and an amphibian.
  • 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.
  • livestock e.g. horses, cattle and sheep
  • companion animals e.g. dogs and cats
  • laboratory animals e.g. mice, rats and guinea pigs
  • performance animals e.g. racehorses, greyhounds and camels
  • the characteristic of interest may be any suitable characteristic of interest.
  • 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.
  • 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.
  • the sample is screened to determine the presence of infection or infestation with a pathogen or parasite.
  • 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.
  • 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).
  • a device for use, or when used, according to the method of the preceding aspect 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.
  • the device may be provided in a kit together with one or more reagents for amplifying, analyzing or detecting the nucleic acid.
  • reagents may include DNA polymerase enzymes, restriction endonucleases, probes and/or primers which in some embodiments may be labelled to facilitate detection.
  • 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.
  • 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.
  • handling portion 200 is formed from a waterproof coating overlying a region of the Whatman No. 1 filter paper extending from capture portion 100.
  • the waterproof coating is wax (Paraplast Plus, Fluka) however this can be varied as desired.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a user 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.
  • the user contacts capture portion 100 with the sample.
  • the user contacts capture portion 100 with a wash solution or wash buffer.
  • the user contacts capture portion 100 with an elution solution.
  • the waterproof coating of handling portion 200 prevents or at least constrains movement of liquid away from capture portion 100 into handling portion 200.
  • 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.
  • 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.
  • the total surface area of capture portion 100 of device 10 is about 5 mm 2 to about 50 mm 2 , including about: 10, 15, 20, 25, 30, 35, 40, and 45 mm 2 .
  • 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 (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 500 ⁇ 1 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.
  • extraction buffer 2% w/v CTAB, 1.42 M NaCl, 20mM EDTA, lOOmM Tris HCl pH8.0, 1% w/v PVP 40
  • DNA was suspended with ⁇ of H 2 0 and 50ug RNase A and followed by incubation at 37°C for 20 minutes to degrade RNA. ⁇ of 3M sodium acetate and ⁇ 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 50 ⁇ 1 of H 2 0 and quantified with NanoDrop ND- 1000 spectrophotometer.
  • 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). ⁇ of each solution was carefully added to two 70mm Whatman No.1 discs approximately 10mm from the centre of the disc.
  • 3mm diameter cellulose discs were cut from Whatman No. l using a hole puncher. ⁇ of lOng/ ⁇ , 1 ng/ ⁇ , O. lng/ ⁇ or 0.01 ng/ ⁇ purified DNA was loaded on cellulose discs respectively. Cellulose disc with identical DNA was then transferred into 200 ⁇ 1 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.
  • wash buffer lOmM Tris, 0.1% Tween-20
  • nucleic acid purification from plant tissues 5-10mg of leaf tissue was ground in 1.5mL tube with a plastic pestle in presence of 50 ⁇ 1 extraction buffer 1 (50mM Tris, 150mM NaCl, 2% PVP, 1% Tween-20) for approximately 30 seconds.
  • 50 ⁇ 1 extraction buffer 1 50mM Tris, 150mM NaCl, 2% PVP, 1% Tween-20
  • 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 200 ⁇ 1 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.
  • RNA purification from CMV-infected tomato leaves samples were prepared as described above with the exception that 50 ⁇ 1 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.
  • extraction buffer 2 800mM guanidine hydrochloride, 50mM Tris (pH 8), 0.5% Triton X100, 1% Tween- 20
  • LM-MEL-70 cells pellet previously stored at -20°C was lysed by adding 200 ⁇ 1 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 200 ⁇ 1 of wash buffer for one minute before transferring to PCR reaction mix.
  • 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 500 ⁇ 1 of extraction buffer 3 (1.5M guanidine hydrochloride, 50mM Tris (pH 8), lOOmM NaCl, 5mM EDTA, 1% Tween-20).
  • extraction buffer 3 1.5M guanidine hydrochloride, 50mM Tris (pH 8), lOOmM NaCl, 5mM EDTA, 1% Tween-20.
  • A. thaliana leaf tissue (approximately 30mm 2 ) was finely ground with plastic pestle in presence of 400 ⁇ 1 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 ⁇ of leaf tissue extract.
  • a 3mm diameter membrane discs was transferred into 25 ⁇ 1 of leaf tissue extract for 1 minute.
  • the membrane disc was then transferred to 200 ⁇ 1 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.
  • ⁇ of lOng/ ⁇ 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).
  • 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.
  • 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.
  • molten wax Paraplast Plus, Fluka
  • leaf tissue (approximately 200mm 2 ) was added to a 2mL tube containing 500 ⁇ 1 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.
  • 500 ⁇ 1 cell lysis buffer (20mM Tris, 25mM NaCl, 2.5mM EDTA, 0.05% SDS)
  • wash buffer lOmM Tris, 0.1% Tween-20
  • 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. 40 ⁇ 1 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 was performed by either polymerase chain reaction (PCR), Loop mediated isothermal amplification (LAMP), or Recombinase polymerase amplification (RPA).
  • PCR polymerase chain reaction
  • LAMP Loop mediated isothermal amplification
  • RPA Recombinase polymerase amplification
  • 15 ⁇ 1 reactions were performed using 7.5 ⁇ 1 of GoTaq Green Master Mix (Promega), 15 pmol of both forward and reverse primers (Table SI) and template DNA.
  • 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.
  • TwistAmp Basic RPA kit Twist DX
  • TwistAmp Basic RT-RPA kit Twist DX
  • Table SI both forward and reverse primer
  • cellulose discs containing extracted nucleic acids were added to 15 ⁇ 1 reactions containing: 20mM Tris (pH 8.8), lOmM (NH 4 )2S0 4 , 50mM KC1, 0.1% (v/v) Tween-20, 0.8M betaine, 8mM MgS0 4 , 1.2mM dNTPs, 4.8U Bst2.0 warmstart (NEB Biolabs, USA), 0.8 ⁇ of FIP and BIP primers and 0.2 ⁇ 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.
  • Whatman No.1 filter paper can entrap and retain DNA after washing
  • FIG. 2B a simple nucleic acid purification method
  • FIG. 2B A 7mm 2 (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 ⁇ of extract or a Whatman No.
  • 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).
  • Tomato plants infected with cucumber mosaic virus were tested using the filter paper DNA extraction method without any modifications.
  • RPA Recombinase Polymerase Amplification
  • 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).
  • LAMP Loop- mediated amplification
  • 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.
  • 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).
  • nucleic acids are able to rapidly bind to the cellulose fibres but are released at a much slower rate.
  • 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.
  • dipsticks made from Whatman No. l with a small 8 mm 2 (per face, approximately 16 mm 2 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.
  • 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.
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • lateral flow strips and cell culture/analysis
  • nucleic acid purification 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).
  • a small (7-8 mm 2 per face, or ⁇ 15 mm 2 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.
  • 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).
  • 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.
  • 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).
  • 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).
  • salts or dNTPs present in the amplification mix as has been reported for other systems (Tanaka et al. 2009).
  • 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).
  • 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.
  • thermocycler 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.
  • 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.
  • Extraction buffers assessed and found to be suitable for nucleic acid extraction include the following:
  • 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.
  • 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.
  • DNA and Whatman No. 1 were independently added ('Filter paper + DNA'), and filter paper or water only were added, to the PCR reaction.

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Abstract

L'invention concerne un procédé d'extraction d'un acide nucléique à partir d'un échantillon, le procédé comprenant les étapes consistant à combiner une matrice fibreuse et/ou poreuse avec un échantillon comprenant un acide nucléique, l'acide nucléique étant capturé par la matrice ; et à libérer l'acide nucléique de la matrice, afin d'extraire ainsi l'acide nucléique de l'échantillon. La matrice peut être une membrane. La matrice peut être microporeuse et/ou absorbante et/ou hydrophile. L'invention concerne également un dispositif destiné à être utilisé selon le procédé, le dispositif comprenant une partie capture et une partie manipulation.
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CN111549024A (zh) * 2020-05-09 2020-08-18 浙江省中药研究所有限公司 一种核酸提取试纸条及其使用方法
WO2021211849A1 (fr) * 2020-04-16 2021-10-21 Chhalliyil Pradheep Extraction et purification rapides d'arn
CN113528505A (zh) * 2021-06-22 2021-10-22 中国检验检疫科学研究院 一种基于膜吸附的黄瓜基因dna快速提取扩增方法
CN113755489A (zh) * 2021-10-13 2021-12-07 江苏溢纳生物科技有限公司 一种核酸提取的裂解结合液及其提取方法
WO2022232892A1 (fr) * 2021-05-06 2022-11-10 Fundação Oswaldo Cruz Cassette d'extraction d'acides nucléiques, méthode de diagnostic dans un échantillon biologique obtenu au moyen de ladite cassette d'extraction d'arn, trousse de diagnostic dans un échantillon biologique et méthode d'extraction de matériel génétique
WO2023049396A1 (fr) * 2021-09-23 2023-03-30 Ama Biotech Procédés et dispositifs d'échantillonnage de fluides corporels

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021211849A1 (fr) * 2020-04-16 2021-10-21 Chhalliyil Pradheep Extraction et purification rapides d'arn
CN111549024A (zh) * 2020-05-09 2020-08-18 浙江省中药研究所有限公司 一种核酸提取试纸条及其使用方法
WO2022232892A1 (fr) * 2021-05-06 2022-11-10 Fundação Oswaldo Cruz Cassette d'extraction d'acides nucléiques, méthode de diagnostic dans un échantillon biologique obtenu au moyen de ladite cassette d'extraction d'arn, trousse de diagnostic dans un échantillon biologique et méthode d'extraction de matériel génétique
CN113528505A (zh) * 2021-06-22 2021-10-22 中国检验检疫科学研究院 一种基于膜吸附的黄瓜基因dna快速提取扩增方法
WO2023049396A1 (fr) * 2021-09-23 2023-03-30 Ama Biotech Procédés et dispositifs d'échantillonnage de fluides corporels
CN113755489A (zh) * 2021-10-13 2021-12-07 江苏溢纳生物科技有限公司 一种核酸提取的裂解结合液及其提取方法

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