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WO2023056558A1 - Procédés et kits pour isoler des acides nucléiques - Google Patents

Procédés et kits pour isoler des acides nucléiques Download PDF

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Publication number
WO2023056558A1
WO2023056558A1 PCT/CA2022/051476 CA2022051476W WO2023056558A1 WO 2023056558 A1 WO2023056558 A1 WO 2023056558A1 CA 2022051476 W CA2022051476 W CA 2022051476W WO 2023056558 A1 WO2023056558 A1 WO 2023056558A1
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WIPO (PCT)
Prior art keywords
cartridge
sample
nucleic acids
fluid
nucleic acid
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CA2022/051476
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English (en)
Other versions
WO2023056558A9 (fr
WO2023056558A8 (fr
Inventor
Robert Matthew Mayall
Dawson Herman FOGEN
Dylan Matthew SILVER
Julia Cecile GUEGUENIAT
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Fredsense Technologies Corp
Original Assignee
Fredsense Technologies Corp
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Filing date
Publication date
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Publication of WO2023056558A1 publication Critical patent/WO2023056558A1/fr
Publication of WO2023056558A8 publication Critical patent/WO2023056558A8/fr
Publication of WO2023056558A9 publication Critical patent/WO2023056558A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • the present disclosure generally relates to methods for isolating nucleic acids, and, in particular, to methods and for isolating nucleic acids from samples containing microbial biological species, such as water samples.
  • nucleic acid assays involve the isolation of nucleic acid material from a sample obtained from a larger fluid source or fluid reservoir.
  • fluid contaminants such as minerals, suspended solids, debris or other contaminants that can be present in samples is often difficult to achieve.
  • the presence of contaminants can interfere with nucleic acid assays, including assays involving amplification of nucleic acid sequences, for example, polymerase chain reaction (PCR) based assays, and in particular when a target nucleic acid species is present in a fluid sample in very low amounts, for example, 50 copies or less.
  • PCR polymerase chain reaction
  • samples including in some cases water samples, may be classified as dangerous goods and shipment is subject to safety regulations, such as outlined in material data sheets.
  • safety regulations such as outlined in material data sheets.
  • an analytical laboratory receives samples containing live and potentially pathogenic microorganisms, and conversely, as the laboratory needs to dispose of such samples.
  • assays and techniques to analyze nucleic acid constituents which can be deployed at a sampling site are particularly desirable.
  • the present disclosure relates to the analysis of samples. [0011] In another broad aspect, the present disclosure related to methods for isolating microbial nucleic acid material from samples.
  • the present disclosure provides, in at least one embodiment, a method for isolating microbial nucleic acids from a sample comprising microbial biological species, the method comprising:
  • a cartridge comprising a reservoir containing a solid support material capable of binding nucleic acids, the cartridge further comprising a first opening to receive fluid and second opening to discharge fluid and a fluid flow path from the first opening through the reservoir to the second opening, to thereby allow the microbial nucleic acids to contact and bind to the solid support material;
  • washing the solid support material with a washing buffer by flowing the washing buffer through the cartridge, wherein the washing comprises fluidically coupling a vacuum aspirating pumping device to the first or the second opening of the cartridge and removing at least a portion of the washing buffer from the solid support material by vacuum aspirating the washing buffer from the solid support material;
  • the lysate can be filtered, and the filtered lysate can be mixed with the binding buffer.
  • the bindable mixture can be flowed along the fluid flow path through the cartridge by providing the bindable mixture in a fluid transfer device fluidically coupled to the first opening of the cartridge and transferring the bindable mixture from the fluid transfer device to the cartridge to thereby flow the bindable mixture through the cartridge.
  • the fluid transfer device can be a syringe comprising a piston, and the bindable mixture can be flowed through the cartridge by exerting downward pressure on the piston.
  • the second opening of the cartridge can be coupled to a vacuum aspirating pumping device, and the bindable mixture can be flowed through the cartridge by vacuum aspiration.
  • the washing buffer can be flowed through the cartridge by providing the washing buffer in a fluid transfer device fluidically coupled to the first opening of the cartridge and transferring the washing buffer from the fluid transfer device to the cartridge to flow the washing buffer along the fluid flow path through the cartridge.
  • the fluid transfer device can be a syringe comprising a piston, and the washing buffer can be flowed through the cartridge by exerting downward pressure on the piston, wherein the at least a portion of the washing buffer that is removed from the solid material is excess washing buffer not flowed through the cartridge by exertion of downward pressure on the piston.
  • the second opening of the cartridge can be coupled to a vacuum aspirating pumping device, wherein the at least a portion of the washing buffer that is removed from the solid material is all, or substantially all, of the washing buffer flowed through the cartridge.
  • the nucleic acids can be eluted by providing an elution buffer in a fluid transfer device fluidically coupled to the first opening of the cartridge and transferring the elution buffer from the fluid transfer device to the cartridge to flow the elution buffer along the fluid flow path through the cartridge.
  • the fluid transfer device can be a syringe.
  • the vacuum aspirating pumping device can be a pumping device operable at from about 5 pounds per square inch (psi) to about 50 psi.
  • the vacuum aspirating pumping device can be a battery operable device.
  • the lysis material can be particulate sodium chloride (NaCI) or potassium chloride (KCI), wherein upon mixing of the fluid sample and the lysis material a high salt concentration lysate containing at least about 3 M NaCI or 3 M KCI is formed.
  • NaCI sodium chloride
  • KCI potassium chloride
  • the lysis material can further include 2-amino-2-(hydroxymethyl)-1 ,3-propanediol (Tris) ethylenediaminetetraacetic acid (EDTA) (TE) to protect ribonucleic acids (RNA).
  • Tris 2-amino-2-(hydroxymethyl)-1 ,3-propanediol
  • EDTA ethylenediaminetetraacetic acid
  • RNA ribonucleic acids
  • the bindable mixture following mixing the lysate with the binding buffer to form the bindable mixture in step (b), the bindable mixture can be incubated for from about 10 minutes to about 60 minutes at room temperature prior to proceeding with step (c).
  • the binding buffer can be an ethanol solution comprising a sufficient concentration ethanol to upon mixing of the lysate therewith form a bindable mixture having a concentration ethanol of at least about 35% (v/v).
  • the washing buffer can be an ethanol-based washing buffer comprising about 100 m M NaCI, about 80% (v/v) ethanol having a pH of about 7.2.
  • the solid support material can be a silica mineral material.
  • the cartridge following the performance of step (d) and prior to the performance of step (e), the cartridge can be dried to ambient air for at least about two minutes.
  • the microbial nucleic acids in the eluate can be substantially pure, wherein the eluate exhibits an A260/A280 ratio of at least about 1.8.
  • the microbial nucleic acids in the eluate can be substantially pure, wherein in the eluate exhibits an A260/A280 ratio of from about 1.8 to about 2.2.
  • the method can further include a step (f) comprising obtaining an aliquot of the eluate, mixing the aliquot with a nucleic acid amplification mixture, wherein the nucleic acid amplification mixture comprises at least nucleic acid amplification primers, deoxynucleotides, a nucleic acid polymerase, and, optionally, a buffer and/or MgCh in concentrations sufficient to amplify the microbial nucleic acids in the eluate.
  • the nucleic acid amplification mixture can be a lyophilized nucleic acid amplification mixture.
  • the aliquot of the eluate can be mixed with the lyophilized nucleic acid amplification mixture without dilution of the aliquot.
  • the method can further include a step (f) comprising detecting the nucleic acids to thereby identify a microbial biological species present in the sample.
  • the method can further comprise a step (g) comprising amplifying the microbial nucleic acids and detecting the nucleic acids to thereby identify a microbial biological species present in the sample.
  • the microbial biological species can be a bacterial species.
  • the bacterial species can be Escherichia coli.
  • the microbial biological species can be a viral species.
  • the viral species can be a Severe Acute Respiratory Syndrome Coronavirus-2 virus (SARS-CoV-2) or a peppermint mild mottle virus (PMMoV).
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus-2 virus
  • PMMoV peppermint mild mottle virus
  • the method can be initiated from within from about 1 minute up to about 2 hours from the collection of the sample.
  • steps (a) - (e) of the method can be completed in about 30 minutes or less from initiation thereof.
  • the sample can be a fluid sample.
  • the fluid sample can be a water sample.
  • the water sample can be a waste water sample.
  • the water sample can be a drinking water sample.
  • kits for the extraction of nucleic acids from samples relate to kits for the extraction of nucleic acids from samples.
  • the present disclosure provides, in at least one embodiment, a kit for the extraction of nucleic acids from a sample, the kit comprising: (a) optionally at least one sample collection vessel;
  • a cartridge comprising a reservoir containing a solid support material capable of binding nucleic acids, the cartridge further comprising a first opening to receive fluid and second opening to discharge fluid and a fluid flow path from the first opening through the reservoir to the second opening;
  • kits can further contain a filter.
  • the fluid transfer device can be a syringe.
  • the kit can further comprise a vacuum aspirating pumping device.
  • the vacuum aspirating pumping device can be a pumping device operable at from about 5 pounds per square inch (psi) to about 50 psi.
  • the lysis material can be particulate sodium chloride (NaCI) or potassium chloride (KCI), wherein upon mixing of the fluid sample and the lysis material a high salt concentration lysate containing at least about 3 M NaCI or 3 M KCI is formed.
  • NaCI sodium chloride
  • KCI potassium chloride
  • the lysis material can further include 2-amino-2-(hydroxymethyl)-1 ,3-propanediol (Tris) ethylenediaminetetraacetic acid (EDTA) (TE) to protect ribonucleic acids (RNA).
  • Tris 2-amino-2-(hydroxymethyl)-1 ,3-propanediol
  • EDTA ethylenediaminetetraacetic acid
  • RNA ribonucleic acids
  • the binding buffer can be an ethanol solution comprising a sufficient concentration ethanol to upon mixing of the lysate therewith form a bindable mixture having a concentration ethanol of at least about 35% (v/v).
  • the washing buffer can be an ethanol-based washing buffer comprising about 100 mM NaCI, about 80% (v/v) ethanol having a pH of about 7.2.
  • the solid support material can be a silica mineral material.
  • kits for the extraction of nucleic acids from samples relate to uses of kits for the extraction of nucleic acids from samples.
  • the present disclosure provides, in at least one embodiment, a use of a kit comprising:
  • a cartridge comprising a reservoir containing a solid support material capable of binding nucleic acids, the cartridge further comprising a first opening to receive fluid and second opening to discharge fluid and a fluid flow path from the first opening through the reservoir to the second opening;
  • FIG. 1 shows a perspective view of an example syringe that can be used in accordance with an example embodiment of the present disclosure.
  • FIG. 2 shows a perspective view of an example cartridge that can be used in accordance with an example embodiment of the present disclosure.
  • FIG. 3 shows a perspective view of an example filter that can be used in accordance with an example embodiment of the present disclosure.
  • FIG. 4 shows an overview of an example step that can be performed in accordance with an example embodiment of the present disclosure, notably an example step to collect a fluid sample containing microbial biological species.
  • FIG. 5 shows an overview of an example step that can be performed in accordance with an example embodiment of the present disclosure, notably an example step involved in combining a fluid sample containing microbial biological species with a particulate lysis material to obtain a lysate containing nucleic acids.
  • FIGS. 6A - 6C show an overview of example steps that can be performed in accordance with an example embodiment of the present disclosure, notably example steps involved in filtering a lysate containing nucleic acids using a syringe and a filter to obtain a filtered lysate containing nucleic acids.
  • FIG. 7 shows an overview of an example step that can be performed in accordance with an example embodiment of the present disclosure, notably an example step involved in combining a filtered lysate containing nucleic acids with a binding fluid to obtain a bindable mixture containing nucleic acids.
  • FIGS. 8A - 8C show an overview of example steps that can be performed in accordance with an example embodiment of the present disclosure, notably example steps involved in collecting a bindable mixture containing nucleic acids in a syringe and coupling a cartridge including a solid support material capable of binding nucleic acids to the syringe.
  • FIGS. 9A - 9C show an overview of example steps that can be performed in accordance with an example embodiment of the present disclosure, notably example steps involved in flowing a bindable mixture containing nucleic acids through a cartridge including a solid support material capable of binding nucleic acids using a syringe to thereby bind the nucleic acids to the solid support material.
  • FIGS. 10A - 10D show an overview of example steps that can be performed in accordance with an example embodiment of the present disclosure, notably example steps involved in washing a cartridge including a solid support material with nucleic acids bound thereto using a washing buffer and flowing the washing buffer through the cartridge using a syringe (FIGS. 10A - 10B) and removing excess washing buffer by vacuum aspiration (FIG. 10C), and obtaining a cartridge containing washed solid support material with nucleic acids bound thereto (FIG. 10D).
  • FIGS. 10A - 10D show an overview of example steps that can be performed in accordance with an example embodiment of the present disclosure, notably example steps involved in washing a cartridge including a solid support material with nucleic acids bound thereto using a washing buffer and flowing the washing buffer through the cartridge using a syringe (FIGS. 10A - 10B) and removing excess washing buffer by vacuum aspiration (FIG. 10C), and obtaining a cartridge containing washed solid support
  • FIGS. 11A - 11C show an overview of example steps that can be performed in accordance with an example embodiment of the present disclosure, notably example steps involved in eluting nucleic acids from a solid support material contained in a cartridge using an eluent and flowing the eluent through the cartridge using a syringe, to obtain an eluent containing nucleic acids.
  • FIG. 12 depicts a graph obtained in the performance of certain experiments involving the isolation of nucleic acid material from a wastewater sample and the subsequent amplification of a SARS-CoV-2 S-gene.
  • the horizontal axis of the graph represents the number of PCR amplification cycles and the vertical axis represents the relative fluorescence units (RFUs), indicating the quantity of nucleic acid.
  • the experiments involved the comparison of four different conditions in a step in the isolation of nucleic acid material, notably the removal of washing buffer from a silicate column using: centrifugation (0); vacuum aspiration ( ⁇ ); air (A); and no removal (O).
  • the graph depicts the quantity of amplified nucleic acid material as a function of the number of PCR amplification cycles using nucleic acid material isolated under the noted conditions.
  • FIG. 13 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from wastewater samples obtained from three different sites and the subsequent PCR amplification of a SARS- CoV-2 S-gene.
  • the horizontal axis of the graph represents the number of amplification cycles and the vertical axis represents the relative fluorescence units (RFUs), indicating the quantity of nucleic acid.
  • the experiments involved the comparison of three samples collected at three different sampling sites: site 1 (A); site 2 ( ⁇ ); and site 3 (O).
  • the graph depicts the quantity of amplified nucleic acid material as a function of the number of PCR amplification cycles using nucleic acid material obtained from the three different samples.
  • FIG. 14 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample obtained and the subsequent PCR amplification of a SARS-CoV-2 S-gene.
  • the horizontal axis of the graph represents the number of amplification cycles and the vertical axis represents the relative fluorescence units (RFUs), indicating the quantity of nucleic acid.
  • the experiments involved the comparison of amplification using a sample containing SARS-CoV-2 viral nucleic acid material (O), and a control free of template nucleic acid material (A).
  • the graph depicts the quantity of amplified nucleic acid material as a function of the number of PCR amplification cycles under the noted conditions.
  • FIG. 15 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample and the subsequent amplification of a SARS-CoV-2 S-gene.
  • the horizontal axis of the graph represents the number of amplification cycles and the vertical axis represents the relative fluorescence units (RFUs), indicating the quantity of nucleic acid.
  • REUs relative fluorescence units
  • the experiments involved the comparison of four different conditions in a step in the isolation of nucleic acid material, notably 4 different lysis materials: (i) 4 M NaCI, 1 x TE (A); (ii) 2M NaCI, 1 x TE, 40% ethanol ( ⁇ ); (iii) 1 x TE (O); and (iv) 800 mM guanidine hydrochloride; 30 mM Tris*CI, pH 8.0; 30 mM EDTA, pH 8.0; 5% Tween®20; 0.5% Triton® X-100 centrifugation (0).
  • the graph depicts the quantity of amplified nucleic acid material as a function of the number of PCR amplification cycles using nucleic acid material isolated under the noted conditions.
  • FIG. 16 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of different conditions used to bind nucleic acid material to a silicate column, and in particular the percentage of ethanol used in a binding buffer.
  • the graph depicts the quantity of RNA recovered as a function of the percentage ethanol in the binding buffer.
  • FIG. 17 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect of lysis time duration used to lyse sample material.
  • the bar graph depicts nucleic amplification signal as a function of lysis time duration (10 minutes, 20 minutes, 30 minutes).
  • FIG. 18 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect of different filter materials to filter lysis sample material on amplification signal (AcQ).
  • the bar graph depicts nucleic amplification signal (AcQ) as a function of various filter materials polyethersulfone (PES) (B), or PVDF (C) (relative to nylon (A)).
  • FIG. 19 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect of different solid support materials, Zymo lll-P silica matrix material (A), Zymo V silica matrix material (B), and Zymo V-E silica matrix material (C) on the detected viral genomic copies per millilitre (cp/mL).
  • the bar graph depicts the detected viral genomic copies per millilitre (cp/mL) as a function of the different solid support materials.
  • FIG. 20 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of different volumes of sample material (10 ml and 50 ml) using Zymo lll-P silica matrix material (A), Zymo V silica matrix material (B), and Zymo V-E silica matrix material (C) on filtering time.
  • the bar graph depicts filtering time as a function of the different sample volumes (10 ml and 50ml).
  • FIG. 21 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect on filtering time of different vacuum aspiration conditions using a vacuum pump coupled to syringe containing solid support material while water is passed through the syringe, with the pump operating at 12 psi (5V), 27 psi (9V), 29 psi (12V), and 30 psi (16V), and using different volumes of water 10 ml, 25ml, and 50 ml.
  • FIG. 22 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect of different concentrations of dimethylsulfoxide (DMSO (0%, 5%(v/v), 10% (v/v)) in nuclease-free water eluent on the detected viral genomic copies per millilitre (cp/mL).
  • DMSO dimethylsulfoxide
  • the bar graph depicts the detected viral genomic copies per millilitre (cp/mL) as a function of the percentage of DMSO (0%, 5%(v/v), 10% (v/v)).
  • FIG. 23 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect of different eluents (Tris-EDTA buffer (TE 0.5X)) and nuclease-free water (NFW)) on the detected viral genomic copies per millilitre (cp/mL).
  • the bar graph depicts the detected viral genomic copies per millilitre (cp/mL) as a function of use of TE 0.5X)) and nuclease- free water (NFW).
  • a - F represent separately collected wastewater samples.
  • FIG. 24 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect of various volumes of eluent (Tris-EDTA buffer), 200 pl, 300 pl and 400 pl on the detected viral genomic copies per millilitre (cp/mL).
  • the bar graph depicts the detected viral genomic copies per millilitre (cp/mL) as a function of eluent volume (200 il, 300 pl and 400 pl).
  • FIG. 25 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect of detergents (0.05%, 0.1 % and 0.5% Tween®-80, 0.05% TX-100, 0.1 % TX-100) in lysis fluid on the detected viral genomic copies per millilitre (cp/mL).
  • FIG. 26 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect of the inclusion of detergent and glass beads in lysis fluid using two silica columns (Zymo P-lll (Column A) and Zymo V-E (Column B)) on viral target concentration.
  • the bar graph depicts the increase in viral yield (fold-change to Column A, cp/ml) as a function of the inclusion of detergent and/or glass beads.
  • FIG. 27 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect of using a 10 pl eluate in combination with a re-hydrated lyophilized RT-PCR mixture (10 pl), or a non-diluted eluate (20 pl) with a lyophilized RT-PCR mixture on the detected viral genomic copies per millilitre (cp/mL).
  • the bar graph depicts the detected viral genomic copies per millilitre (cp/mL) as a function of eluent dilution (undiluted: 20 pl; 2X diluted: 10 pl).
  • FIG. 28 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of different conditions in a step in the isolation of nucleic acid material from wastewater, notably an evaluation of the effect of diluting a fluid sample (no dilution; dilution 1/10) on the detected viral genomic copies per millilitre (cp/mL).
  • the bar graph depicts the detected viral genomic copies per millilitre (cp/mL) as a function of sample (No dilution; Dilution 1/10).
  • FIG. 29 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of the effect of total dissolve solids (TDS), ranging from 0 - 800 ppm, present in a fluid sample, on the quality of obtained nucleic acids, as determined by A260/A280.
  • TDS total dissolve solids
  • the graph shows the A260/A280 as a function of TDS in a fluid sample.
  • FIG. 30 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample.
  • the experiments involved an evaluation of the effect of storage time on detected viral genomic copies in fluid samples.
  • the bar graph depicts the detected viral genomic copies per millilitre as a function of storage time and shows detected viral genomic copies per millilitre (cp/mL) in fresh samples (Fresh) and samples stored or 24 hours at 4° C (24 h Storage).
  • a - G represent separately collected fluid samples.
  • FIG. 31 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample, notably the detection of pepper mild mottle (PMMoV) virus in wastewater samples obtained at different time points.
  • PMMoV pepper mild mottle
  • FIG. 32 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample, notably the detection of amplified E. coli nucleic acids
  • A growth medium
  • B non-lysed LB cultured E. coli cells used to isolate nucleic acids according to a method of the present disclosure
  • C lysed LB cultured E. coli cells used to isolate nucleic acids according to a method of the present disclosure
  • 1 no template DNA
  • 2 commercial RNA E. coli extraction kit
  • 3 LB cultured lysed E. coli cell mixture).
  • any range of values described herein is intended to specifically include the limiting values of the range, and any intermediate value or sub-range within the given range, and all such intermediate values and sub-ranges are individually and specifically disclosed (e.g., a range of 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5).
  • other terms of degree such as “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term, such as up to 15% for example, if this deviation would not negate the meaning of the term it modifies.
  • nucleic acid refers to of nucleoside or nucleotide polymer comprising nucleoside or nucleotide monomers, consisting of bases, sugars and intersugar (backbone) linkages.
  • the nucleic acid to which the present disclosure refers may be deoxyribonucleic nucleic acids (DNA) or ribonucleic acids (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil.
  • the nucleic acids may also contain modified bases.
  • modified bases include xanthine and hypoxanthine and aza and deaza analogs of nucleic acid constituents, and also modifications such as pseudouridylation, dihydrouridylation, and methylation of naturally occurring bases.
  • Nucleic acid polymers represent a sequence of nucleotide monomers.
  • microbial biological species can be said possess unique nucleic acid sequences which are distinct from nucleic acid sequences found in other microbial biological species.
  • microbial biological species and “microbial”, as used herein, refer to any type of microorganism, including, any bacteria, viruses, viroids, fungi, moulds, mycobacteria, protozoa, and the like, and further including any varieties, subtypes, or strains thereof. It is noted, in this respect, that, as used herein, the term “species” when contained within the term “microbial biological species”, is not intended to refer to the strict Linnean taxonomic meaning thereof, but rather has the more generic meaning of ‘kind’ or ‘sort’. Thus, a virus can be a microbial biological species, and two different viral strains, or two different bacterial strains, herein can be deemed to be two different microbial biological species.
  • an object material species e.g., nucleic acid material
  • an object material species such as nucleic acid material
  • an object material species is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, 95%, 96%, 97%, or 98%, and most preferably at least 99% of the macromolecular material species (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the object material species.
  • Purity can be measured by any appropriate method, e.g., by chromatography, gel electrophoresis, spectrophotometrically, absorbance or HPLC analysis.
  • detect and “detection”, as used herein, refer to the determination of the existence, presence or fact of a target, nucleic acid material, for example, or signal in a sample, or a reaction mixture, or the like. Detection is “quantitative” when it refers, relates to, or involves the measurement of a quantity or an amount of a molecule, such as a nucleic acid, or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the molecule or signal.
  • kits of the present disclosure can be used to isolate nucleic acids of a microbial biological species, for example, a bacterial species or a viral species, in a sample.
  • the methods disclosed herein involve, the isolation of nucleic acid material from microbial biological species present in a sample, and the separation thereof from contaminants in the sample, including non-nucleic acid material of the microbial biological species.
  • the herein disclosed methods can be rapid and easy to perform, and may be conducted in close proximity of, for example, a source fluid from which a fluid sample is obtained.
  • the present disclosure allows the isolation of a sample of nucleic acid material of which the composition can accurately reflect the composition of the nucleic acid constituents present in a larger body amount of fluid, such as a body of water, a river or lake, for example, from which the sample of is drawn.
  • the results obtained accurately correspond with the nucleic acid material present in the source fluid from which the sample is drawn, and permits an accurate qualitative and/or quantitative assessment of microbial biological species present in situ.
  • the methods of the present disclosure do not require the use of specialized laboratory equipment, such as centrifuges. Therefore, the methods of the present disclosure are particularly useful in settings where no laboratory or laboratory equipment is available for immediate analysis, such as, for example, a site at which a sample is collected.
  • the methods of the present disclosure can provide sufficiently pure nucleic acid material to allow amplification of specific nucleic acid species, so that it is possible to determine whether or not a specific microbial species is present in the sample, and, optionally, to determine what quantities of the microbial biological species are present in the sample.
  • the nucleic acid material obtained in accordance with the methods of the present disclosure can be sufficiently pure to allow for the detection of specific nucleic acid species present therein, even if such nucleic acid species are present in low copy numbers, for example, less than 50 copies in the sample.
  • nucleic acid material obtained in accordance with the methods of the present disclosure can be sufficiently pure to allow mixing of the undiluted nucleic acid material with commonly obtainable nucleic acid amplification ‘master mixtures’, for example, lyophilized ‘master mixtures’, and subsequent nucleic acid amplification.
  • the methods of the present disclosure can be performed without the use of hazardous chemicals, such as formaldehyde, formamide or guanidium thiocyanate (GITC), which are commonly used in methods known to the art for isolation of nucleic acids from samples and renders the methods impractical or unsuitable to use by non-qualified personnel.
  • hazardous chemicals such as formaldehyde, formamide or guanidium thiocyanate (GITC)
  • the methods of the present disclosure do not necessarily require the transportation of sample materials, thus the methods can be inexpensive and safe to conduct.
  • FIGS. 1 - 11 Example embodiments of methods to obtain nucleic acid material present in a fluid sample will in particular be discussed with reference to FIGS. 1 - 11.
  • FIGS. 1 - 11 represent example embodiments, which are not intended to be limiting. Other embodiments are discussed and may be understood by reference to the embodiments shown in FIGS. 1 - 11 , or may be implemented by those of skill in the art.
  • FIGS. 12 - FIGS. 32 certain results obtained in the performance of certain experiments to evaluate example embodiments and methods of the present disclosure are shown in FIGS. 12 - FIGS. 32.
  • FIGS. 1 - 11 illustrate example devices that may be used in accordance with the present disclosure to perform at least some of these multiple steps.
  • FIG. 4 illustrates an initial step of obtaining a fluid sample containing microbial biological species which can be used to extract nucleic acid material therefrom.
  • FIGS. 5 - 11 illustrate sequential further example steps of methods for treating the fluid sample to extract nucleic acid material.
  • FIGS. 1 - 3 depicted therein is syringe 10 (FIG. 1), cartridge unit 20 (FIG. 2), and filter unit 30 (FIG. 3). Further, depicted in FIGS. 1 - 3 are syringe and filter assembly 31 , and syringe and cartridge assembly 21 . Syringe and filter assembly 31 comprises syringe 10 and filter unit 30. Syringe and cartridge assembly 21 comprises syringe 10 and cartridge unit 20.
  • syringe 10 comprises syringe housing 18, having at its distal end aperture 17 through which a fluid may enter or exit syringe 10.
  • Syringe housing 18 further includes at its proximal end flange 11 providing a finger support and cooperating proximal handle 12 providing a further finger support.
  • Syringe 10 further comprises plunger unit 14 longitudinally movable within syringe housing 18, and piston 16 with seal 15 providing sealing against the inside wall of the syringe housing 18.
  • the diameter of distal aperture 17 is preferably substantially narrower than the diameter of syringe housing 18.
  • syringes comprising a seal are also known in the art as BD syringes. Syringes free of a seal are also known in the art as norm-ject syringes.
  • example embodiments herein involve the performance of multiple steps involving the transfer of multiple fluids using a syringe.
  • a single syringe may be used to practice the example steps illustrated in FIGS. 4 - 11 , most preferably after cleaning the syringe between contact with different fluids to prevent cross-contamination between different fluids.
  • different syringes may be used when different fluids are transferred.
  • FIGS. 4-10 For ease of illustration FIGS.
  • syringe 10 depicts a syringe referred to as “syringe 10”. It is to be understood, however, that in different embodiments, syringe 10 may represent multiple different syringes used to transfer fluids in multiple steps. It is further noted that in different embodiments, whether using a single syringe or using multiple syringes, in different steps, syringes capable of holding various fluid volumes may be used, ranging, for example, from about 250 pl to about 250 ml.
  • a syringe is a fluid transfer device.
  • the methods herein are illustrated in FIGS. 4, 6A - 6C, 8A - 8C, 9A - 9C, 10A - 10D, and 11A - 11 C using a syringe as a fluid transfer device to perform various steps of the example methods.
  • other fluid transfer devices for example, a pipette or a tube, may be used.
  • a pump for example a vacuum pump, may be used to aide in the transfer of fluids.
  • syringes and vacuum pumps are particularly convenient fluid transfer devices to conduct many steps of the herein disclosed methods.
  • cartridge unit 20 having cartridge housing 26, comprising an interior chamber containing therein a solid support material capable of binding nucleic acids (not visible).
  • Cartridge unit 20 further includes cartridge outlet 24 and cartridge inlet 22.
  • Cartridge inlet 22 is constructed to fit to syringe aperture 17 of syringe 10 in such a manner that when cartridge unit 20 and syringe 10 are reversibly coupled, cartridge inlet 22 and syringe aperture 17 form reversible joint 39 (see: e.g., FIG. 8C) through which fluid communication between fluid present in syringe housing 18 and the interior chamber (not visible) within cartridge housing 26 may readily be established.
  • Reversible joint 39 is constructed in such a manner that no fluid leakage occurs during fluid communication between syringe 10 and cartridge unit 20.
  • reversible joint 39 can be constructed using a screw thread structure.
  • filter unit 30 having filter housing 36, with a membrane filter contained in an interior chamber (not visible) in filter housing 36, and filter outlet 34 and filter inlet 32.
  • Filter inlet 32 is constructed to fit to syringe aperture 17 in such a manner that when filter unit 30 and syringe 10 are reversibly coupled via filter inlet 32 and syringe aperture 17, they together form reversible joint 38 (see: e.g., FIG. 6A) through which fluid communication between fluid present in syringe housing 18 and the interior chamber (not visible) within filter housing 36 may readily be established.
  • Reversible joint 38 is constructed in such a manner that no fluid leakage occurs during fluid communication between syringe 10 and filter unit 30.
  • reversible joint 38 can be constructed using a screw thread structure, a friction-fit coupling, or a Luer- lock coupling.
  • syringe 10 may initially be used to collect a fluid sample from a source fluid containing microbial biological species. Referring now to FIG. 4, syringe 10 may be used to draw fluid sample 40a comprising microbial biological species from vessel 41 containing source fluid 40 into syringe housing 18.
  • a fluid aliquot ranging, for example, from 1 ml to 250 ml
  • the fluid sample can be a water sample obtained from any source fluid, including a body of water, including by way of example, but not limitation, a naturally occurring body of water, such as an ocean, sea, bay, lake, river, stream, creek or channel, natural subterranean reservoir, or a man-made large body of water, such as a pond, pool, reservoir, man-made subterranean reservoir, canal, or ditch.
  • the body of water may be deemed potable, for example, water obtained from a municipal water drinking water system, or non-potable, for example, water obtained from a municipal sewage system, or industrial wastewater system.
  • the fluid sample can be obtained from a less voluminous quantity of the source fluid, for example, a fluid present in a vessel, container, tank, conduit, or receptacle used in an industrial, laboratory or domestic environment.
  • an obtained fluid sample may optionally be diluted, using an appropriate diluent, for example, water.
  • Dilution may, for example, be from about 2X to about 10X, for example, about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, or 10X. Dilution of the fluid sample may be desirable to dilute fluid contaminants which can impede analysis of the obtained microbial nucleic acids, as hereinafter further described.
  • a more or less solid sample material may be obtained from a more or less solid source material, for example, a mud or sludge sample. Such sample material may subsequently be contacted and mixed with a fluid, such as water, to obtain a liquid mixture, for example a liquid suspension or solution. The liquid mixture may then be used in the same manner as a liquid sample obtained from a source fluid, as described herein.
  • a fluid such as water
  • a fluid sample with solids suspended therein may be obtained, for example a wastewater sample, and the suspended solids may be isolated, for example by filtering, the wastewater sample, and recovering the filtered solids from the filter.
  • the solids may subsequently be resuspended in a fluid, for example, water.
  • the obtained liquid sample is subsequently considered a ‘fluid sample’, and used in accordance with the methods herein described.
  • a collected sample may be pre-treated in any desired manner, for example, a liquid sample may be heated or cooled (e.g., frozen and thawed) before being further processed in accordance with the methods of the present disclosure.
  • a syringe may conveniently be used to collect a fluid sample
  • any sampling device and technique may be used to collect a fluid sample in accordance herewith.
  • Liquid sampling devices that may be used include sample collection devices, such as sample containers, sample bottles, sample jugs, sample flasks, sample tubes, sampling taps or valves, syringes, pipettes etc., and further including electronically controlled automatic sampling devices (auto-samplers).
  • sample collection techniques may be varied, and selected as desired.
  • any receptacle capable of drawing fluid from the source fluid and containing an aliquot of the source fluid may be used, and such devices may generally be referred herein as “fluid transfer devices”.
  • the sampling device is contacted with the source fluid to draw and transfer an aliquot of the source fluid to the sampling device, and then the sampling device containing the fluid aliquot is separated from the source fluid to obtain a fluid sample.
  • the sampling technique and device provide a fluid sample which is substantially free of larger particulate matter and debris, for example, substantially free of particulate matter and debris larger in size than 100 pm, more preferably larger in size than about 10
  • the volume of the sample of fluid may vary, depending, for example, in part on the volume of the source fluid, and that fluid sampling techniques may vary depending on the source fluid, and may be adjusted as desirable.
  • the source fluid in accordance herewith, is further characterized in that it comprises microbial biological species.
  • the microbial biological species may be of a single taxonomic order, for example a single kingdom, phylum, class, order, family, genus, species, or strain.
  • the microbial biological species may represent a plurality of kingdoms, phyla, classes, orders, families, genera, species, or strains.
  • the kingdom, phylum, class, order, family, genus, species, or strains of microbial biological species may vary, and can, as will readily be appreciated by those of skill in the art, depend on the source of the fluid.
  • the microbial biological species may, for example, be pathogenic or represent other health or safety risks to humans or animals, or the microbial biological species may cause operational challenges in the performance of industrial processes.
  • the microbial biological species may be desirable, for example, certain microbial biological species performing certain catabolic or anabolic processes, for example, the degradation of waste products, including, for example, in wastewater treatment facilities.
  • any fluid sample containing nucleic acids, or suspected to contain nucleic acids may be collected, and used in accordance herewith.
  • the methods of the present disclosure are not intended to be limited with regards to the fluid source of the nucleic acid material.
  • RNA nucleic acids or DNA nucleic acids, or a mixture of RNA and DNA nucleic acids
  • methods of the present disclosure may be conducted using any collected fluid sample known to contain, or suspected to contain, any RNA or DNA nucleic acids, including, without limitation, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), plasmid DNA, microRNA (miRNA), or genomic DNA or RNA.
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • plasmid DNA plasmid DNA
  • microRNA microRNA
  • genomic DNA or RNA genomic DNA or RNA
  • Lysis material in this respect, is any material, i.e., substance, chemical compound or combination of chemical compounds, capable of lysing (i.e., breaking open) cellular membranes, and/or, in the case of viral microbial agents capable of substantially disrupting viral protein capsules. Lysis materials may be provided as solids, for example, solid particulates, or as liquids, for example, salts dissolved in water or buffer.
  • Lysis materials may contain, for example, ionic salts, including monovalent ionic salts, such as sodium chloride (NaCI), potassium chloride (KCI) or divalent ionic salts, such as ammonium sulfate (NH4)2SO4, for example, as well as buffering salts, such as Tris-HCl.
  • ionic salts including monovalent ionic salts, such as sodium chloride (NaCI), potassium chloride (KCI) or divalent ionic salts, such as ammonium sulfate (NH4)2SO4, for example, as well as buffering salts, such as Tris-HCl.
  • Lysis materials may further contain small amounts of detergents (e.g., from 0.01 % (v/v) to 1 % (v/v)), including, preferably, non-ionic detergents, such as Tween® 20, Tween® 80, Triton® X-100, or, optionally, ionic detergents, such as sodium dodecyl sulfate (SDS), for example.
  • detergents e.g., from 0.01 % (v/v) to 1 % (v/v)
  • non-ionic detergents such as Tween® 20, Tween® 80, Triton® X-100
  • ionic detergents such as sodium dodecyl sulfate (SDS), for example.
  • Lysis materials may further be fluids containing 2-amino-2-(hydroxymethyl)- 1 ,3-propanediol (Tris)-ethylenediaminetetraacetic acid (EDTA) (Tris-EDTA buffer, also referred to as TE buffer).
  • Tris-EDTA buffer also referred to as TE buffer.
  • [00130] Further optionally included in the lysis materials may be glass beads (such as 0.2 mm - 1 mm borosilicate beads), typically in volume less than 10% of the total volume (e.g., from 1 % to 5% of the total volume).
  • glass beads such as 0.2 mm - 1 mm borosilicate beads
  • TE buffer is preferably included in lysis fluids to prevent degradation of ribonucleic acids (RNA) by RNases.
  • Lysis material 52 and fluid sample 40 are mixed, for example by pouring a sample of fluid sample 40a present in first tube 50a into second tube 50b containing lysis material 52.
  • Combined fluid sample 40a and lysis material 52 are preferably thoroughly mixed, for example, by inverting tube 50b 10 - 15 times, or more, to thereby achieve lysis of the microbial biological species and obtain lysate 54.
  • such ionic salts can be included in particulate form in first tube 50a in amounts such that upon mixing between the lysis material and the fluid sample a high salt concentration lysate is obtained, e.g., a lysate having a relatively high ionic salt concentration, for example, preferably at least about 2 M KCI or NaCI, at least about 2.5 M KCI or NaCI, at least about 3 M KCI or NaCI, at least about 3.5 M KCI or NaCI, at least about 4 M KCI or NaCI, at least about 4.5 M KCI or NaCI, or at least about 5 M KCI or NaCI, ora lysate having a concentration of about 2.5 M KCI or NaCI to about 5 M KCI or NaCI, for example, about 2.5 M, 3 M, 3.5 M, 4 M,
  • lysis may be allowed to proceed for a brief period of time, for example, by incubating tube 50b for at least about 10 minutes, at least about 20 minutes, or at least about 30 minutes, or at least about 60 minutes, orfrom about 10 minutes to about 30 minutes, orfrom about 10 minutes to about 60 minutes, or for about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, preferably, at room temperature or ambient temperature.
  • lysate 54 is preferably filtered to remove insoluble materials that may be present in the lysate.
  • insoluble materials can include, debris, particulates, mineral aggregates, and the like, present in fluid sample 40a. This may be achieved by transferring lysate 54 to syringe 10 (thoroughly cleaned if syringe 10 was used to collect the fluid sample, or another clean syringe), and thereafter reversibly coupling filter unit 30 (shown in cross-section in FIGS. 6A - 6C) thereto and establishing joint 38.
  • Filter unit 30 comprises filter housing 36 and interior filter chamber 37 having an upper filter chamber portion 37a separated from a lower filter chamber portion 37b by membrane filter 35.
  • Membrane filter 35 has a pore size sufficiently large to allow the passage nucleic acid material through the membrane filter 35, but sufficiently small to prevent passage of larger insoluble particles, present in fluid sample 40a therethrough.
  • membrane filter 35 may have a pore size of ranging from about 1 pm to about 10 pm (e.g., about 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm) , or 1 pm to about 100 pm, or 5 pm to about 100 pm (e.g., about 5 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm), including for example, a polyethersulfone membrane filter, e.g., a 5 pm polyethersulfone (PES) membrane filter, a nylon membrane filter, or a polyvinylidene difluoride (PVDF) membrane filter.
  • PES polyethersulfone
  • PVDF polyvinylidene difluoride
  • Other filters that may be used are ceramic filters.
  • the filtering step may be performed once, or two or more times, using filters having membranes with different pore sizes, for example a first filtering step may be conducted using a membrane filter 35 may having a pore size ranging from 10 p,m to about 100pm, and a second filtering step using a membrane filter 35 may having a pore size ranging from 1 pm to about 10pm.
  • filter unit 30 coupled to syringe 10 through joint 38 formed by syringe aperture 17 of the syringe and filter inlet 24, and forming contiguous assembly 60.
  • Lysate 54 is filtered by moving plunger 14 downward (see: arrow d), thus moving piston 16 within syringe housing 18 downward from proximal portion 18b towards distal portion 18a of syringe housing 18, and thereby gradually emptying syringe housing 18 and pressing lysate 54 via joint 38 into upper filter chamber portion 37a, and filtering lysate 54, which upon passage through membrane filter 35 flows through bottom filter chamber portion 37b and exits filter unit 30 via filter outlet 26 to be collected in collection vessel 61.
  • Collection vessel 61 and contiguous assembly 60 can together be said to form filtering assembly 62.
  • membrane filter unit 30 contains insoluble particles, while filtered lysate 54b collected in collection vessel 61 contains nucleic acid material and other fluid constituents sufficiently small to traverse membrane filter 35.
  • Filtering assembly 62 may be disassembled by decoupling syringe 10 from filter unit 30 (see: arrow s1 ), and separating filter unit 30 from collection vessel 61 (see: arrow s2) containing filtered lysate 54b containing nucleic acids, in order to thereby obtain syringe 10, filter unit 30, and collection vessel 61c as shown in FIG. 6C.
  • FIGS. 6A - 6C are optional. It is generally desirable to conduct these steps, in particular, when fluid sample 40a contains substantial quantities of insoluble materials, as for example, may be the case when fluid sample 40a is a wastewater sample.
  • Second tube 70 contains binding fluid 71.
  • Suitable binding fluid is any fluid capable of promoting binding between nucleic acid material and a solid support material as hereinafter described, including, for example, an alcohol, such as a methanol, ethanol, propanol, or phenol solution.
  • Filtered lysate 54b containing nucleic acids and binding fluid 71 are mixed, for example, by pouring a volume of binding fluid 71 from tube 70 into an equal volume of filtered lysate 54b present in first tube 61.
  • the combined volumes are preferably thoroughly mixed, for example, by inverting tube 61 10 - 15 times, or more times, to thereby mix binding fluid 71 and filtered lysate 54b and obtain bindable mixture 73 containing nucleic acids.
  • the amount of an alcohol based binding fluid is selected so that upon mixing with the lysate (or filtered lysate, as the case may be), bindable mixture 73 includes about or at least about 35% (v/v), about or at least about 40% (v/v), about or at least about 45% (v/v), about or at least about 50% (v/v) of the alcohol.
  • a volume of an 80% (v/v) ethanol solution i.e., 80% (v/v) ethanol, 20% (v/v) water
  • bindable mixture 73 may be combined with an equal volume of lysate to obtain bindable mixture 73, wherein bindable mixture 73 then contains 40% (v/v) ethanol.
  • syringe 10 may be used to draw bindable mixture 73 containing nucleic acids from tube 61 into the syringe housing 18 by immersing aperture 17 of syringe 10 into bindable mixture 73, while having piston 16 positioned within distal portion 18a of syringe housing 18 and then moving plunger 14 upward (see: arrow u), thus moving piston 16 within syringe housing 18 upward from distal portion 18a towards proximal portion 18b of syringe housing 18, and thereby gradually filling syringe housing 18 with bindable mixture 73 containing nucleic acids.
  • cartridge unit 20 (shown in cross section) is provided, and reversibly coupled to syringe 10 via syringe aperture 17 of syringe 10 and cartridge inlet 22.
  • Cartridge unit 20 comprises cartridge housing 26 and an interior cartridge chamber 27 comprising solid support material 25.
  • Solid support material 25 can be any material capable of selectively binding nucleic acids present in a fluid contacted with the solid support material.
  • Solid support materials that can be used in this respect include, for example, solid support materials comprising or consisting of a mineral matrix, such as silica mineral matrix materials, including for example, a borosilicate glass fiber matrix material, or a Zymo lll-P silica matrix material, Zymo V silica matrix material, or Zymo V- E silica matrix material (Zymo Research Corp, Irvine CA); diatomaceous earth (see: e.g., U.S. Patent 5,075,430); a quaternary ammonium based anion exchange resin (Ferreira, G. N. et al., 2000, Biotechnol. Prog.
  • cartridge unit 20 coupled to syringe 10 through joint 39 formed by syringe aperture 17 of the syringe and cartridge inlet 22, together forming contiguous assembly 81.
  • cartridge unit 20 coupled to syringe 10 through joint 39 formed by syringe aperture 17 of syringe 10 and cartridge inlet 23, and forming contiguous assembly 81.
  • Bindable mixture 73 containing nucleic acids is flowed through cartridge unit 20 by moving plunger 14 downward (see: arrow d), thus moving piston 16 within syringe housing 18 downward from proximal portion 18b towards distal portion 18a of syringe housing 18, and thereby gradually emptying syringe housing 18 and pressing bindable mixture 73 containing nucleic acids via joint 39 through cartridge unit 20.
  • cartridge unit 20 comprises a fluid path therethrough from cartridge inlet 22 via cartridge chamber 27 to cartridge outlet 24.
  • bindable mixture 73 migrates through interior chamber 27 of cartridge unit 20 along the fluid path, nucleic acids present in bindable mixture 73 contact solid support material 25 and bind thereto.
  • bindable mixture 73b from which nucleic acids have been separated, due to the association thereof with solid support material 25, exits cartridge unit 20 via cartridge outlet 24 to be collected in tube 90.
  • T ube 90 and contiguous assembly 81 can together be said to form nucleic acid binding assembly 92.
  • cartridge unit 20 contains nucleic acid material bound to solid support 25 contained in interior chamber 27 of cartridge unit 20.
  • Nucleic acid binding assembly 92 may be disassembled by decoupling syringe 10 from cartridge unit 20 (see: arrow s1), and separating cartridge unit 20 from tube 90 (see: arrow s2) in order to obtain syringe 10, cartridge unit 20 containing nucleic acids and tube 90 as shown in FIG. 9C.
  • bindable mixture 73 may be transferred to cartridge unit 20 using a fluid transfer device, for example, by fluidically coupling syringe 10 to cartridge inlet 22 of cartridge 20, and instead of using plunger 14 to exert (downward) pressure on bindable mixture 73, a vacuum aspirating device may be fluidically coupled to cartridge outlet 24, and bindable mixture 73 may be flowed through cartridge 20 using the vacuum aspirating device.
  • a fluid transfer device for example, by fluidically coupling syringe 10 to cartridge inlet 22 of cartridge 20
  • a vacuum aspirating device may be fluidically coupled to cartridge outlet 24, and bindable mixture 73 may be flowed through cartridge 20 using the vacuum aspirating device.
  • Suitable vacuum aspirating devices and operating conditions are hereinafter described with reference to FIG. 10C.
  • cartridge unit 20 coupled to syringe 10 through joint 39 formed by syringe aperture 17 of the syringe and cartridge inlet 22, and forming a contiguous assembly 103.
  • Washing buffer 101 is flowed through cartridge unit 20 by moving plunger 14 downward (see: arrow d), thus moving piston 16 within syringe housing 18 downward from proximal portion 18b towards distal portion 18a of syringe housing 18, and thereby gradually emptying syringe housing 18 and pressing washing buffer 101 via joint 39 through cartridge unit 20 and washing solid support material 25 to which nucleic acids have been bound, in the process removing remnants of bindable mixture 73, without however removing nucleic acids from solid support material 25, and forming washed solid support 25c.
  • Washing buffer that may be used can be ethanol containing washing buffers, including alcohol based solutions, e.g., a 50% (w/w) to 95% (w/w) ethanol solution (Chen, C., and Thomas, C., 1980, Anal. Biochem. 101 : 339-341 ); Tris-NaCI based ethanol solutions, e.g., a 100 mM NaCI, 80% (v/v) ethanol, 10 mM Tris, pH 7.2 solution, or 1.5 M NaCI, 20% (v/v) ethanol, 10mM Tris, pH 7.2 solution (Whitney O.
  • alcohol based solutions e.g., a 50% (w/w) to 95% (w/w) ethanol solution (Chen, C., and Thomas, C., 1980, Anal. Biochem. 101 : 339-341 ); Tris-NaCI based ethanol solutions, e.g., a 100 mM NaCI, 80% (v/v)
  • a high concentration chaotropic agent based solution for example a 4 - 6 M sodium perchlorate based solution, e.g., a 10 mM Tris, 1 mM EDTA and 4 - 6 M NaCIO4 (Chen, C, and Thomas, C, 1980, Anal. Biochem. 101 : 339-341 ), pH 7.5 solution, or 50 mM Tris, 6 M NaCIC , pH 3 - 8 solution (Melzak, K., 1996, J. Colloid and Interface Science 181 , 635-644).
  • a 4 - 6 M sodium perchlorate based solution e.g., a 10 mM Tris, 1 mM EDTA and 4 - 6 M NaCIO4 (Chen, C, and Thomas, C, 1980, Anal. Biochem. 101 : 339-341 ), pH 7.5 solution, or 50 mM Tris, 6 M NaCIC , pH 3 - 8 solution (Melzak,
  • cartridge unit 20 Upon passage through cartridge unit 20, washing buffer containing remnants of bindable mixture 73 exits cartridge unit 20 via cartridge outlet 24 to be collected as spent washing buffer 101 b in collection vessel 102. Collection vessel 102 and contiguous assembly 103 can together be said to form washing assembly 105.
  • cartridge unit 20 Upon completion of flow through, cartridge unit 20 contains nucleic acid material bound to washed solid support 25c contained in interior chamber 27 of cartridge unit 20.
  • washing buffer 101 has passed through cartridge unit 20 and washing buffer 73b is collected as spent washing buffer 101 b in collection vessel 102.
  • washing buffer 101 may be transferred to cartridge unit 20 using a fluid transfer device, for example, by fluidically coupling syringe 10 to cartridge inlet 22 of cartridge 20, and instead of using plunger 14 to exert (downward) pressure on washing buffer 101 , a vacuum aspirating device may be fluidically coupled to cartridge outlet 24, and washing buffer 101 may be flowed through cartridge 20 using the vacuum aspirating device. Suitable vacuum aspirating devices and operating conditions are hereinafter described with reference to FIG. 10C.
  • multiple washes may be performed and the steps illustrated in FIGS. 10A and 10B may be iterated to more thoroughly remove final remnants of bindable mixture 73 from cartridge unit 20.
  • these steps may, for example, be conducted 2 or 3 times, each time using fresh washing buffer.
  • vacuum pump 110 is coupled to cartridge unit 20 via tubing 114 having distal end portion 114b coupled to vacuum pump 110, and proximal end portion 114a coupled to cartridge outlet 24 via coupling 112 formed by proximal end portion 114a of tubing 114 and cartridge outlet 24.
  • proximal end portion 114a of tubing 114 can also be coupled to cartridge inlet 22, after decoupling syringe 10 and cartridge unit 20.
  • vacuum pump 110 can preferably be a battery-operated vacuum pump, for example, a peristaltic pump, diaphragm pump, syringe pump, centrifugal pump (horizontal or vertical), rotary vane pump, axial flow pump, positive displacement pump, piston pump, progressive cavity pump, gear pump, lobe pump, or radial piston pump, for example.
  • a battery-operated vacuum pump for example, a peristaltic pump, diaphragm pump, syringe pump, centrifugal pump (horizontal or vertical), rotary vane pump, axial flow pump, positive displacement pump, piston pump, progressive cavity pump, gear pump, lobe pump, or radial piston pump, for example.
  • suitable pumps in accordance herewith deliver an operating vacuum pressure of from 5 pound per square inch (psi), or about 5 psi, to 50 psi, or about 50 psi, or from 12 psi, or about 12 psi, to 30 psi, or about 30 psi, for example, about 12 psi, about 13, psi, about 14 psi, about 15 psi, about 16 psi, about 17 psi, about 18 psi, about 19 psi, about 20 psi, about 21 psi, about 22 psi, about 23 psi, about 24 psi, about 25 psi, about 26 psi, about 27 psi, about 28 psi, about 29 psi, or about 30 psi, and preferably, from 25 psi, or about 25 psi, to 40 psi, or about 40 psi, including,
  • the operating pressure may be selected as function of the solid support material 25.
  • an operating pressure can be selected that maximizes flow through cartridge unit 20 without however substantially affecting or compromising the integrity of solid support material 25.
  • such operating pressure can be determined, by operating a selected pump at different operating pressures, and evaluate the flow of washing buffer (or bindable mixture, as the case may be) through cartridge unit 20 at different operating pressures.
  • battery operated diaphragm pumps are deemed particularly suitable, since they can provide an appropriate operating vacuum pressure and generally have modest power requirements.
  • Vacuum pump 110 is preferably run for at least about 1 minute, at least about 2 minutes, or at least about 5 minutes to thereby aspirate excess washing fluid and dry solid support material 25 to which nucleic acids have been bound.
  • vacuum pump 110 may be operated to aspirate fluids for a total time of, for example, example 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes or 50 minutes, where, as will be clear to those of skill in the art, larger fluid volumes generally will require longer run times.
  • Coupling of vacuum pump 110 to cartridge outlet 24 generally is preferably such that a fluidic coupling between vacuum pump 110 and cartridge outlet 24 is established via tubing 114.
  • Coupling 112 can conveniently be achieved using tubing (e.g., flexible silicone tubing or Tygon® tubing) or piping tightly coupled to cartridge outlet 24, including, for example, by slip coupling proximal end portion 114a of tubing 114 to cartridge outlet 24, and/or using adapter devices such as threaded tubes, connectors, clamps, clips, inserts, insert couplers, and the like, as necessary.
  • the length of tubing 114 may vary, but generally will be 1 m or less, e.g., for example, about 1 m, about 75 cm, or about 50 cm, as it generally will be possible to place cartridge unit 20 adjacent to vacuum pump 110 to perform the step illustrated in FIG. 10C. However, in circumstances where adjacent placement is not practical, longer tubing 114 may be used, as necessary. Distal end portion 114b and tubing 114 may be an integral part of vacuum pump 110, or distal end portion 114b may be releasably coupled to vacuum pump 110, again, by slip coupling and/or through an appropriate adapter device.
  • contiguous assembly 103 may be disassembled by decoupling tubing frontal portion 114a of tubing 114 from cartridge unit 20 and by decoupling syringe 10 from cartridge unit 20 (see: arrow s4) in order to obtain syringe 10 and cartridge unit 20 containing nucleic acids as shown in FIG. 10D.
  • cartridge unit 20 is left to dry following vacuum aspiration.
  • decoupled cartridge unit 20 is preferably left to dry, preferably to the ambient air, and preferably for at least 2 about minutes, at least about 3 minutes, at least about 5 minutes, or at least about 10 minutes, or from about 2 minutes to about 5 minutes or from about 1 minute to about 10 minutes, or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes, prior to proceeding with the elution steps illustrated in FIGS. 11A - 11C.
  • Such air drying of cartridge unit 20 is deemed beneficial, in particular, when the obtained eluate is subsequently used to detect nucleic acids therein, and when such detection involves the use of nucleic acid amplification.
  • a battery-operated vacuum pump for example, by using a diaphragm pump operated preferably at least at, for example, at about 5 psi, or from about 5 psi to about 50 psi ⁇ e.g., at about 12 psi, about 27 psi, about 29 psi, or about 30 psi) (see further Example 6)
  • the methods of the present disclosure can be conducted in close proximity of a fluid sampling site ⁇ e.g., as close as a few meters or tens of meters of a sampling site), and no transport of sample fluid to a laboratory is required.
  • the use of a battery-operated pump permits processing of the sample fluid immediately upon collecting the sample fluid.
  • the procedure may be initiated within 1 minute, within 5 minutes, within 10 minutes, within 30 minutes, within 1 hour or within 2 hours from collecting the sample fluid. In other embodiments, the procedure may be initiated within 2 hours to 24 hours from collecting the sample fluid. In such embodiments it may be beneficial to store the sample fluid at e.g., 4 °C prior to initiation of the procedure.
  • the method may be completed in about 30 minutes or less or about 45 minutes or less from its initiation.
  • the present disclosure allows the isolation of a sample of nucleic acid material of which the composition can accurately reflect the composition of the nucleic acid constituents present in the fluid sample, and thus the composition of the nucleic acid constituents present in the larger body of fluid from which the fluid sample of is drawn.
  • cartridge unit 20 coupled to syringe 10 through joint 39 formed by syringe aperture 17 of the syringe and cartridge inlet 22, and forming a contiguous assembly 105.
  • Eluent 152 is flowed through cartridge unit 20 by moving plunger 14 downward (see: arrow d), thus moving piston 16 within syringe housing 18 downward from proximal portion 18b towards distal portion 18a of syringe housing 18, and thereby gradually emptying syringe housing 18 and pressing eluent 152 via joint 39 through cartridge unit 20 and eluting nucleic acids from solid support material 25.
  • Eluent 152 is preferably a low ionic strength buffer containing no or low concentrations of alcohol.
  • the eluent may be water, and when RNA is isolated, is preferably RNase free water.
  • Further eluents may be a TE buffer containing e.g., from about 0.1 mM Tris to about 10 mM Tris, about 0.1 mM EDTA at about pH 7.5 to about pH 8.4 or 1 mM tricine at about pH 6.9 (Chen, C, and Thomas, C, 1980, Anal. Biochem. 101 : 339-341 ; Whitney O.
  • Eluents further may optionally include dimethylsulfoxide (DMSO), for example, up to about 10% (v/v) DMSO, e.g., about 1 % (v/v), about 2.5% (v/v), about 5% (v/v), about 7.5% (v/v), or 10% (v/v) DMSO.
  • Elution volumes may be as desired, and can range, for example, from about 200 p.l - 400 pl, when the initial fluid sample collected ranges from about 25 ml to 50 ml.
  • eluate 152b containing nucleic acids which have been removed from solid support material 25 exits cartridge unit 20 via cartridge outlet 24 to be collected in collection vessel 151. Collection vessel 151 and contiguous assembly 151 can together be said to form eluent assembly 155. Upon completion of flow through cartridge unit 20, eluent 152b contains nucleic acid material. [00156] Referring now to FIG. 11 B, once piston 16 reaches its most distal position within syringe housing 18 substantially all of eluent 152 will have passed through cartridge unit 20 and eluate 152b containing nucleic acids is collected in collection vessel 151 , and removed from solid support material 25.
  • Eluent assembly 155 may be disassembled by decoupling syringe 10 from cartridge unit 20 (see: arrow s1), and separating cartridge unit 20 from collection vessel 152b (see: arrow s2) in order to obtain syringe 10, cartridge unit 20 and collection vessel 152b containing nucleic acids as shown in FIG. 11C.
  • some or all of the steps depicted in FIGS. 4 - 11 may be conducted multiple times, including simultaneously, or more or less simultaneously.
  • multiple syringes 10 may be used to simultaneously, or more or less simultaneously, to obtain multiple samples 40a from different source fluids 40, or multiple samples 40a from the same source fluid 40 (e.g., for the purpose of obtaining duplicate results).
  • subsequent steps depicted in FIGS. 5 - 11 may be conducted simultaneously, or more or less simultaneously, using applicable multiple fluids (e.g., multiple fluids 54, 54b, 73, 73b).
  • a single volume of sample 40a, or other fluids may split in multiple smaller volumes of samples 40a (or 54, 54b, 73, 73b) and used to conduct applicable steps depicted in FIGS. 5 - 11.
  • multiple smaller volumes of sample 40a, or other fluids, e.g., 54, 54b, 73, 73b
  • manifolds may be used to couple multiple cartridge units 20 to a single vacuum pump 110, to simultaneously dry solid support material 25 in multiple cartridge units 20.
  • the obtained nucleic acids in eluate 152b can be substantially pure. Nucleic acid purity may be determined by any appropriate methodology, including, for example, spectrophotometrically, by determining the absorbance at 260 nm and 280 nm of a sample containing nucleic acid material.
  • a ratio of absorbance at 260 nm and 280 nm (A260/A280 ratio) of an eluate sample may be determined and may, for example, be at least about 1 .7, at least about 1 .8, at least about 1.9, or at least about 2.2, or, for example, from about 1 .7 to about 2.2 (e.g., about 1 .7, 1.8, 1 .9, 2.0, 2.1 , or 2.2). It is noted that the substantial nucleic acid purity of eluate 152b allows for the direct use of an eluate aliquot, for example, a 5 pl - 25 pl aliquot of eluate 152b in detection assays without further dilution of eluate 152b. However, eluate 152b may also optionally be diluted, for example, 2X, 5X, or 10X, using water or TE buffer, for example, prior to use in detection assays, for example, to conserve eluate material.
  • Obtained eluate 152b can be used to detect nucleic acids therein, using any suitable nucleic acid detection technology, including any qualitative or quantitative nucleic acid detection technology.
  • eluate obtained in accordance with the methods of the present disclosure is, in particular, suitable to be used in conjunction with techniques involving the amplification of a specific nucleic acid species, for example, a nucleic acid species having a nucleic acid sequence known to be uniquely representative of a particular bacterial species or strain or a particular viral species or strain.
  • nucleic acid species associated with spherical viruses include, for example, nucleic acid species associated with spherical viruses, a nucleic acid species associated with the SARS-CoV-2 virus (Severe Acute Respiratory Syndrome Coronavirus-2) (about 100 nm), including any variants thereof, such as the alpha-variant, beta-variant, gamma-variant and delta-variant, and other variants, which may evolve, or nucleic acid species associated with rod-shaped viruses, for example, a peppermint mild mottle virus (PMMoV) (about 20 nm x 300 nm).
  • SARS-CoV-2 virus severe Acute Respiratory Syndrome Coronavirus-2 virus
  • PMMoV peppermint mild mottle virus
  • Nucleic acid species associated with bacteria include nucleic acid species associated with Enterobacter, Klebsiella, Staphylococcus, Acinetobacter, Pseudomonas, Enterobacter, and further including, for example, nucleic acid species associated with Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae.
  • multiple species may be detected in a single sample, for example multiple viral strains (e.g., different SARS-CoV-2 virus strains or a SARS-CoV-2 and a PMMoV strain), or multiple bacterial strains, or one or more bacterial strains and one or more viral strains.
  • multiple viral strains e.g., different SARS-CoV-2 virus strains or a SARS-CoV-2 and a PMMoV strain
  • multiple bacterial strains e.g., different SARS-CoV-2 virus strains or a SARS-CoV-2 and a PMMoV strain
  • multiple bacterial strains e.g., different SARS-CoV-2 virus strains or a SARS-CoV-2 and a PMMoV strain
  • the eluate is suitable to amplify specific nucleic acid species therein, even when specific nucleic acid species are present in low copy numbers in the fluid sample, for example, when the fluid sample includes less than 50 copies, less than 25 copies, or even less than 10 copies of a specific nucleic acid species.
  • nucleic acid amplification techniques that may be used include nucleic acid amplification based technologies, such as polymerase chain reaction (PCR) based technologies and reverse transcriptase based technologies (RT- PCR), and further include isothermal amplification techniques such as nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (I), nicking enzyme amplification reaction (NEAR), signal mediated amplification of RNA technology (SMART), rolling circle amplification (RCA), isothermal multiple displacement amplification (IMDA), single primer isothermal amplification (SPIA), recombinase polymerase amplification (RPA), and polymerase spiral reaction (PSR), all of which can be used to amplify a specific nucleic acid species present in the eluent (see: for example: Fakrudin et al., 2013, J.
  • NASBA nucleic acid sequence
  • nucleic acid amplification mixtures are obtainable in what may be referred to as ‘amplification master mixtures’, e.g., ‘RT-PCR master mixtures’.
  • Such master mixtures may contain all, or substantially all, of the ingredients required for nucleic acid amplification, including, at least, (species specific) nucleic acid primers (for example, 25 pmol - 200 pmol), and deoxynucleotide triphosphates (dNTPs: dATPs, dGTPs, dTTPs, and dCTPs, for example, at a final concentration of 150 pM - 400 pM), and further preferably including a polymerizing enzyme ⁇ e.g., DNA polymerase, for example 2 - 10 enzyme units), MgCh (for example, at a final concentration of 0.5 mM - 1.
  • a polymerizing enzyme ⁇ e.g., DNA polymerase, for example 2 - 10 enzyme units
  • MgCh for example, at
  • Master mixtures are frequently provided in lyophilized form, as particulates, which prior to use are rehydrated using e.g., water or a buffer.
  • an aliquot of eluate may directly be added to a lyophilized ‘master mixture’, and nucleic acids present therein may directly be amplified.
  • the nucleic acid material in the eluate obtained in accordance herewith is substantially pure (e.g., exhibiting a A260/A280 ratio of at least 1.7), thus allowing for nucleic acid amplification and detection without dilution of the eluate, or without separate rehydration of an amplification ‘master mixture’.
  • eluate prepared in accordance with methods of the present disclosure may be used to amplify and detect one or more specific nucleic acid species present in the eluate.
  • a specific microbial biological species can be inferred to be present in the fluid sample, and by extension, in the source fluid from which the fluid sample was drawn.
  • kits to conduct the methods according to the present disclosure further includes a kit for the extraction of nucleic acids from a sample, the kit comprising
  • a cartridge comprising a reservoir containing a solid support material capable of binding nucleic acids, the cartridge further comprising a first opening to receive fluid and second opening to discharge fluid and a fluid flow path from the first opening through the reservoir to the second opening;
  • kit can further include a filter suitable to filter lysate.
  • the fluid transfer device can be a syringe, and in a further embodiment, the kit can include 2, 3, 4, 5 or 6 syringes.
  • the kit can further comprise a vacuum aspirating pumping device.
  • the vacuum aspirating pumping device can be a pumping device operable at from about 5 pounds per square inch (psi) to about 50 psi.
  • the pumping device can be a battery-operated device.
  • the lysis material in the kit can be particulate sodium chloride (NaCI) or potassium chloride (KCI), wherein upon mixing of the fluid sample and the lysis material a high salt concentration lysate containing at least about 3 M NaCI or 3 M KCI is formed.
  • NaCI sodium chloride
  • KCI potassium chloride
  • the lysis material can further include 2-amino-2- (hydroxymethyl)-1 ,3-propanediol (Tris) ethylenediaminetetraacetic acid (EDTA) (TE) to protect ribonucleic acids (RNA).
  • Tris 2-amino-2- (hydroxymethyl)-1 ,3-propanediol
  • EDTA ethylenediaminetetraacetic acid
  • RNA ribonucleic acids
  • the binding buffer in the kit can be an ethanol solution comprising a sufficient concentration ethanol to upon mixing of the lysate therewith form a bindable mixture having a concentration ethanol of at least about 35% (v/v).
  • the washing buffer in the kit can be an ethanol-based washing buffer comprising about 100 mM NaCI, about 80% (v/v) ethanol having a pH of about 7.2.
  • the solid support material in the cartridge in the kit can be a silica mineral material.
  • the kit can further include instructions for use of the kit to isolate nucleic acids.
  • the instructions may be included with the kit (e.g., printed on paper and directly included with the kit), or a reference may be provided for on-line access to the instructions, all of which are intended to be included herein.
  • the instructions additionally may include information regarding the components and use of the components included in the kit, for example, safety or storage information.
  • kits of the present disclosure can be used to isolate nucleic acid material of microbial biological species from a sample, such as a water sample.
  • the methods may be conducted directly at the site at which the sample is acquired in order to thereby evaluate the sample for the presence of nucleic acids of microbial biological species.
  • Example 1 Isolation of nucleic acids from a wastewater and amplification of SARS-CoV-2 S-gene.
  • Each of the four bindable mixtures were then passed through a separate silica column using another 60 mL syringe.
  • a separate syringe was then used to pass 2 mL wash buffer solution containing 100 mM NaCI, 80% Ethanol, and 10 mM Tris pH 7.2 through each of the four silica columns. Washing steps were repeated once more for each sample. Following washing the four silica columns were treated differently as follows. From a first column no excess wash buffer was removed. The second column was treated by pushing 100 ml of ambient air through the column using a 50 ml syringe.
  • a third column was coupled to a battery powered Walfront Mini Vacuum Pump, DC 12V 120kpa Vacuum Air Pump and the column was dried by running the vacuum pump at 65 kPa (9.4 psi) for 5 minutes.
  • a fourth column was inserted into an empty collection tube and centrifuged in an Eppendorf® Minispin® centrifuge at 13,400 rpm for 2 minutes. Following centrifugation, the collected tube was discarded. Nucleic acids were then eluted from each of the columns in 150 pL nuclease free water. The RNA samples were subsequently used to amplify SARS-CoV-2 RNA in technical duplicate using an S-gene specific primer set through RT-PCR.
  • FIG. 12 shows the results obtained.
  • RNA obtained from the first column (Centrifugation), the second column (Vacuum), the third column (Air), and the fourth column (No Buffer Removal) are indicated in the graph shown in FIG. 12 by open circles, triangles, squares, and diamonds, respectively.
  • a substantially lower concentration of amplified viral nucleic acid material was obtained when the remaining washing buffer was either not removed or removed by syringe drying the column than when remaining washing buffer was removed using a centrifuge or vacuum pump.
  • no removal of washing buffer or syringe drying results in an underestimation of the concentration of viral RNA actually present in the water sample.
  • no removal of washing buffer or syringe drying is likely to not result in any amplification of viral RNA. This would result in an erroneous assessment that no detectable viral RNA is present in the sample, when in fact viral RNA is present in the sample.
  • Example 2 Isolation of nucleic acids from multiple wastewater samples and amplification of SARS-CoV-2 S-gene.
  • Equal volumes of 80% (v/v) ethanol were added to each of the filtered lysates and thoroughly mixed to form a bindable mixture.
  • the bindable mixture was then passed through a silica column using another60 mL syringe. A separate syringe was then used to pass 2 mL wash buffer solution containing 100 mM NaCI, 80% Ethanol, and 10 mM Tris pH 7.2 through the silica column. Washing steps were repeated once more. Following washing the column was coupled to a battery powered Mini Vacuum Pump, DC 12V 120kpa Vacuum Air Pump and the column was dried by running the vacuum pump at 65 kPa (9.4 psi) for 5 minutes.
  • Nucleic acids were then eluted from the column in 150 pL nuclease free water. Concentrations of isolated RNA were measured via nanodrop, and the total amount of recovered RNA was determined to be 0.89, 11 .6, and 88.7 pg for site 1 , site 2, and site 3, respectively. The RNA samples were then used to amplify a SARS-CoV-2 RNA in technical duplicate using an S-gene specific primer set through RT-PCR. FIG. 13 shows the results obtained.
  • Example 3 Isolation of nucleic acids from another wastewater sample and amplification of SARS-CoV-2 S-gene.
  • a separate syringe was then used to pass 2 mL wash buffer solution containing 100 mM NaCI, 80% Ethanol, and 10 mM Tris pH 7.2 through the silica column. The washing steps was repeated once more. Following washing the column was coupled to a battery powered vacuum pump Mini Vacuum Pump, DC 12V 120kpa Vacuum Air Pump and the column was dried by running the vacuum pump at 65 kPa (9.4 psi) for 5 minutes. Nucleic acids were then eluted from the column in 150 pL nuclease free water. The RNA samples were then used to amplify SARS-CoV-2 RNA in technical duplicate using a SARS-CoV-2 S-gene specific primer set through RT-PCR.
  • FIG. 14 shows the results obtained. Data obtained from wastewater sample and no template control shown in open circles and triangles, respectively.
  • Example 4 Isolation of nucleic acids from a wastewater sample and amplification of SARS-CoV-2 S-gene using different lysis materials.
  • Solids captured by the filter were then mixed with 6 ml of four different lysis buffers as follows: (i) 4 M NaCI, 1 x TE; (ii) 2M NaCI, 1 x TE, 40% ethanol; (iii) 1 x TE; and (iv) 800 mM guanidine hydrochloride; 30 mM Tris-CI, pH 8.0; 30 mM EDTA, pH 8.0; 5% Tween® 20; 0.5% Triton® X-100. The samples were then thoroughly mixed and incubated at room temperature for a period of 10 minutes to form lysates.
  • a 60 mL syringe was used to pass the lysates through a 5 pm filter and the flow-through, i.e., filtered lysates, were collected in a 15 mL Falcon® tube.
  • the flow-through i.e., filtered lysates
  • an equal volume of various binding buffers was added to each sample. Specifically, 6 mL of 80% Ethanol was added to 6 mL of sample (i). Additionally, 6 mL of 40% Ethanol, 2M NaCI was added to 6 mL of sample (ii). Finally, 6 mL of 80% Ethanol, 4M NaCI was added to 6 mL sample (iii) and sample (iv).
  • bindable mixture was then passed through a silica column (Zymo-Spin V-E (Zymo Research Corp, Irvine CA, USA) using a 5 mL syringe. A separate syringe was then used to pass 2 mL Monarch® wash buffer solution through the silica column. The washing step was repeated once more. Following washing the remaining wash bufferwas removed by inserting the column into an empty collection tube and centrifugation in an Eppendorf® Minispin® centrifuge at 13400 rpm for 2 minutes.
  • RNA samples were then used to amplify SARS-CoV-2 RNA in technical duplicate using a SARS-CoV-2 S- gene specific primer set through RT-PCR. Recovered concentrations were measured via nanodrop and subsequent RT-PCR material was generated using Reliance mastermix (BioRad®, Hercules, California, USA) following the manufacturer’s protocol.
  • FIG. 15 shows the results obtained. Data obtained from lysis buffers (i), (ii), (iii), and (iv) are indicated by open triangles, squares, circles, and diamonds, respectively.
  • Example 5 Evaluation of binding buffers containing different concentrations of ethanol.
  • Binding buffers were made by mixing equal volumes lysing buffer (4M NaCI, 1 % TE) with ethanol at 1 1 different concentrations ranging from 25% (v/v) to 50% (v/v). Salt precipitation was assessed visually in each sample by turbidity and was found to occur when binding buffer contained >45% ethanol.
  • a total of 25 pg of purified SARS- CoV-2 S-gene RNA was added to each solution and passed through a Monarch® RNA clean up kit spin columns (New England Biolabs®, Ipswich, MA, USA) following the manufacturer's protocol. Washing columns were then washed a second time using the same washing conditions. Bound RNA was eluted from each column in 50 pL nuclease free water. The concentration of the eluted RNA was determined via nanodrop and the subsequent RNA recovery was calculated.
  • FIG. 16 shows the results obtained.
  • the bindable mixture was then passed through a Zymo lll-P silica column using the 60 mL syringe barrel as a reservoir.
  • a separate syringe was then used to pass 2 mL of wash buffer solution containing 100 mM NaCI, 80% (v/v) ethanol, and 10 mM Tris (pH 7.2) through the column.
  • the washing steps was repeated once more.
  • the silica column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)) and the column was air-dried for 5 minutes at 12 V.
  • Example 8 Evaluation of filter materials.
  • Volumes of 25 mL wastewater sample collected at a municipal wastewater treatment plant in a large city in Canada were mixed with 5.8 g NaCI and 1X Tris-EDTA buffer (pH 8.0). The samples were then thoroughly mixed and incubated at room temperature for 30 minutes to form a lysate. Following incubation of the lysate, a 60 mL syringe was used to pass the mixture through a 5 pm Nylon (A), polyethersulfone (PES) (B), or PVDF (C) filter and the flow-through, i.e., filtered lysate, was collected.
  • A Nylon
  • PES polyethersulfone
  • C PVDF
  • bindable mixture was then passed through a Zymo lll-P silica column using the 60 mL syringe barrel as a reservoir. A separate syringe was then used to pass 2 mL of wash buffer solution containing 100 mM NaCI, 80% (v/v) ethanol, and 10 mM Tris (pH 7.2) through the column. The washing steps was repeated once more.
  • the silica column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6))and the column was air-dried for 5 minutes at 12 V.
  • Nucleic acids were then eluted from the column in 200 pL nuclease-free water (VWR).
  • the bindable mixture was then passed through either a Zymo lll-P, V, or Zymo V-E silica column using the 60 mL syringe barrel as a reservoir (noted as A, B, and C, respectively).
  • a separate syringe was then used to pass 2 mL of wash buffer solution containing 100 mM NaCI, 80% (v/v) ethanol, and 10 mM Tris (pH 7.2) through the column. The washing steps was repeated once more.
  • the silica column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6))and the column was air-dried for 5 minutes at 12 V.
  • Nucleic acids were then eluted from the column in 200 pL nuclease-free water (VWR).
  • Example 10 Evaluation of filtered fluid volume.
  • volumes of either 10 or 50 mL of a wastewater sample collected at a municipal wastewater treatment plant in a large city in Canada were mixed with 5.8 g NaCI and 1x Tris-EDTA buffer (pH 8.0). The samples were then thoroughly mixed and incubated at room temperature for 30 minutes to form a lysate. Following incubation of the lysate, a 60 mL syringe was used to pass the mixture through a 5 pm Nylon filter and the flow-through, i.e., filtered lysate, was collected. An equal volume of 80% (v/v) ethanol was added to the filtered lysate and thoroughly mixed to form a bindable mixture.
  • Example 11 Evaluation of vacuum pump operating pressure.
  • Zymo V silica columns were attached to the bottom of a 60 mL syringe barrel and inserted into inlets on a vacuum manifold using a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump (as further referenced in Example 6)).
  • the bindable mixture was then passed through a Zymo V-E silica column using the 60 mL syringe barrel as a reservoir.
  • the silica column was washed with 10 mL of wash buffer containing 100 mM NaCI, 80% (v/v) reagent ethanol, and 10 mM Tris (pH 7.2), and then the column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6))and air-dried for 5 minutes at 16 V.
  • a battery powered vacuum pump Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)
  • Nucleic acids were then eluted from the column in 200 pL of nuclease-free water containing 0%, 5%, or 10% dimethylsulfoxide (DMSO).
  • DMSO dimethylsulfoxide
  • the nucleic acid samples were then used at 10 pL with 10 pL diluent to amplify SARS-CoV-2 RNA in technical duplicate using a SARS-CoV-2 N-gene specific primer set through RT-PCR. All samples were measured for viral genomic copies per millilitre (cp/mL) in the original wastewater.
  • Example 13 Evaluation of eluents.
  • Volumes of 25 mL wastewater samples ((A) - (F)) collected at various sites and/or times were mixed with 5.8 g NaCI and 1X Tris-EDTA buffer (pH 8.0). The samples were then thoroughly mixed and incubated at room temperature for 15 minutes to form a lysate. Following incubation of the lysate, a 60 mL syringe was used to pass the mixture through a 5 pm nylon filter and the flow-through, i.e., filtered lysate, was collected. An equal volume of 80% (v/v) ethanol was added to the filtered lysate and thoroughly mixed to form a bindable mixture.
  • the bindable mixture was then passed through a Zymo V-E silica column using the 60 mL syringe barrel as a reservoir.
  • the silica column was washed with 10 mL of wash buffer containing 100 mM NaCI, 80% (v/v) reagent ethanol, and 10 mM Tris (pH 7.2), and then the column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi) (as further referenced in Example 6)) and air-dried for 5 minutes at 16 V.
  • a battery powered vacuum pump Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi) (as further referenced in Example 6)
  • the bindable mixture was then passed through a Zymo V-E silica column using the 60 mL syringe barrel as a reservoir.
  • the silica column was washed with 10 mL of wash buffer containing 100 mM NaCI, 80% (v/v) reagent ethanol, and 10 mM Tris (pH 7.2), and then the column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa
  • Example 15 Evaluation of detergent in lysis fluid.
  • bindable mixture was then passed through a Zymo V-E silica column using the 60 mL syringe barrel as a reservoir.
  • the silica column was washed with 10 mL of wash buffer containing 100 mM NaCI, 80% (v/v) reagent ethanol, and 10 mM Tris (pH 7.2), and then the column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)) and air-dried for 5 minutes at 16 V. Nucleic acids were then eluted from the column in 200 pL of nuclease- free water.
  • a battery powered vacuum pump Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)
  • Example 16 Evaluation of detergent and beads in lysis fluid.
  • a 60 mL syringe was used to pass the mixture through a 5 pm nylon filter and the flow-through, i.e., filtered lysate, was collected.
  • An equal volume of 80% (v/v) ethanol was added to the filtered lysate and thoroughly mixed to form a bindable mixture.
  • the bindable mixture was then passed through a Zymo lll-P (Column A) or V-E (Column B) silica column using the 60 mL syringe barrel as a reservoir.
  • the silica column was washed with 10 mL of wash buffer containing 100 mM NaCI, 80% (v/v) reagent ethanol, and 10 mM Tris (pH 7.2), and then the column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)) and air-dried for 5 minutes. Nucleic acids were then eluted from the column in 200 pL of nuclease-free water.
  • a battery powered vacuum pump Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)
  • Example 17 Evaluation of use of undiluted eluate to amplify nucleic acid material.
  • the bindable mixture was then passed through a Zymo lll-P silica column using the 60 mL syringe barrel as a reservoir.
  • a separate syringe was then used to pass 2 mL of wash buffer solution containing 100 mM NaCI, 80% (v/v) ethanol, and 10 mM Tris (pH 7.2) through the column. This washing step was repeated once more.
  • the column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)) and air-dried for 5 minutes.
  • Nucleic acids were then eluted from the column in 200 pL of nuclease-free water.
  • the eluate may be used to rehydrate the lyophilized RT-qPCR reagent mixture.
  • the eluate may be used without dilution.
  • bindable mixture was then passed through a Zymo V-E silica column using the 60 mL syringe barrel as a reservoir.
  • the silica column was washed with 10 mL of wash buffer containing 100 mM NaCI, 80% (v/v) reagent ethanol, and 10 mM Tris (pH 7.2), and then the column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)) and air-dried for 5 minutes at 16 V. Nucleic acids were then eluted from the column in 200 pL of 0.5x Tris-EDTA buffer (pH 8.0).
  • Example 19 Evaluation of nucleic acid purity as a function of total dissolved solids (TDS) in sample fluid.
  • TDS values were measured using ZeroWater TDSmeter-20 ZT-2 Electronic Water Tester device directly in wastewater prior to lysis. Volumes of 25 ml_ wastewater sample collected at a municipal wastewater treatment plant in a large city in Canada were mixed with 5.8 g NaCI, 1x Tris-EDTA buffer (pH 8.0), and 0.5% (v/v) Tween®-80 detergent. The samples were then thoroughly mixed and incubated at room temperature for 15 minutes to form a lysate. Following incubation of the lysate, a 60 mL syringe was used to pass the mixture through a 5 pm nylon filter and the flow-through, /.e., filtered lysate, was collected.
  • bindable mixture was then passed through a Zymo V-E silica column using the 60 mL syringe barrel as a reservoir.
  • the silica column was washed with 10 mL of wash buffer containing 100 mM NaCI, 80% (v/v) reagent ethanol, and 10 mM Tris (pH 7.2), and then the column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)) and air-dried for 5 minutes at 16 V. Nucleic acids were then eluted from the column in 200 pL of nuclease- free water.
  • a battery powered vacuum pump Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)
  • Example 20 Evaluation of storage time of fluid samples.
  • the bindable mixture was then passed through a Zymo lll-P column using the 60 mL syringe barrel as a reservoir.
  • a separate syringe was then used to pass 2 mL of wash buffer solution containing 100 mM NaCI, 80% (v/v) ethanol, and 10 mM Tris (pH 7.2) through the column.
  • the washing steps was repeated once more.
  • the silica column was coupled to a battery-powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa, Vacuum Air Pump, running at 65 kPa (9.4 psi)) and the column was air-dried for 5 minutes at 12 V.
  • Nucleic acids were then eluted from the column in 200 pL nuclease-free water (VWR). The nucleic acid samples were then used at 10 pL with 10 pL diluent to amplify SARS-CoV-2 RNA in technical duplicate using a SARS-CoV-2 N-gene specific primer set through RT-PCR. All samples were measured for viral genomic copies per milliliter (cp/mL) in the original wastewater. The same experiment was repeated on the sample wastewater samples after 24 hours storage at 4°C. Each sample was tested at least two times (n > 2). Results are shown in FIG. 30.
  • Example 21 Detection of pepper mild mottle virus (PMMoV) in wastewater.
  • the silica column was washed with 10 mL of wash buffer containing 100 mM NaCI, 80% (v/v) reagent ethanol, and 10 mM Tris (pH 7.2), and then the column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)) and air-dried for 5 minutes. Nucleic acids were then eluted from the column in 200 pL of 0.5x Tris-EDTA buffer (pH 8.0).
  • the nucleic acid samples were then used at 20 pL to amplify PMMoV RNA in technical duplicate using a PMMoV specific primer set through RT-PCR.
  • Data shows the amplification signal (Cq) for each wastewater sample (in technical duplicate). Results are shown in FIG. 31.
  • Example 22 Detection of Escherichia coli in wastewater.
  • E. coli Escherichia coli
  • a 60 mL syringe was used to pass the mixture through a 5 pm nylon filter and the flow- through, i.e., filtered lysate, was collected.
  • a separate volume of E. coli bacteria was not lysed (Sample B) and added directly to the filter.
  • An equal volume of 80% (v/v) ethanol was added to the filtered samples and thoroughly mixed to form a bindable mixture.
  • the bindable mixture was then passed through a Zymo V-E silica column using the 60 mL syringe barrel as a reservoir.
  • the silica column was washed with 10 mL of wash buffer containing 100 mM NaCI, 80% (v/v) reagent ethanol, and 10 mM Tris (pH 7.2), and then the column was coupled to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)) and air-dried for 5 minutes at 16 V. Nucleic acids were then eluted from the column in 300 pL of 0.5x Tris-EDTA buffer (pH 8.0). The nucleic acid samples were then used at 10 pL with 10 pL diluent to amplify E.
  • a battery powered vacuum pump Mini Vacuum Pump, DC 12V, 120kPa Vacuum Air Pump, running at 65 kPa (9.4 psi)(as further referenced in Example 6)
  • Nucleic acids were then e
  • coli DNA/RNA in technical duplicate using a E. coli K12 rfb-50 specific primer set through RT-PCR.
  • Several controls were included in the RT-PCR: Sample 1 - RT-PCR reaction with all components except no DNA/RNA template was added; Sample 2 - template was purified RNA from E. coli bacteria using a commercial extraction kit; Sample 3- template was E. coli bacteria lysed by boiling at 95°C for 5 min. Reactions of 10 pL were run on 1.5% (w/v) agarose gel for 45 minutes @ 95V in cold buffer. DNA A ladder (NEB Quick-load 1 kb DNA Ladder) was included. Expected amplicon size is 969 bp.
  • FIG. 32 shows the amplification signal (Cq) for each wastewater sample (in technical duplicate), n.d., no amplification detected.

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Abstract

L'invention concerne des procédés d'isolement d'un matériau d'acide nucléique à partir d'un échantillon de fluide comprenant des espèces biologiques microbiennes. Les procédés impliquent la préparation d'un lysat et la liaison d'acides nucléiques à un matériau de support solide à l'aide d'un tampon pouvant se lier. Le matériau de support solide est lavé à l'aide d'un tampon de lavage et au moins une partie du tampon de lavage est retirée par aspiration sous vide à l'aide d'un dispositif de pompage à aspiration sous vide. Lors de l'élution à partir du matériau de support solide, un éluat contenant des acides nucléiques sensiblement purs peut être obtenu. L'éluat peut être utilisé pour détecter les acides nucléiques qu'il contient, y compris les espèces d'acides nucléiques spécifiques, afin d'identifier les espèces biologiques microbiennes présentes dans l'échantillon de fluide. L'invention concerne également des kits associés.
PCT/CA2022/051476 2021-10-06 2022-10-06 Procédés et kits pour isoler des acides nucléiques Ceased WO2023056558A1 (fr)

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