WO2023056558A1

WO2023056558A1 - Methods and kits for isolating nucleic acids

Info

Publication number: WO2023056558A1
Application number: PCT/CA2022/051476
Authority: WO
Inventors: Robert Matthew Mayall; Dawson Herman FOGEN; Dylan Matthew SILVER; Julia Cecile GUEGUENIAT
Original assignee: Fredsense Technologies Corp.
Priority date: 2021-10-06
Filing date: 2022-10-06
Publication date: 2023-04-13
Also published as: WO2023056558A9; WO2023056558A8

Abstract

Methods for isolating nucleic acid material from a fluid sample comprising microbial biological species are provided. The methods involve preparing a lysate and binding nucleic acids to a solid support material using a bindable buffer. The solid support material is washed using a washing buffer and at least a portion of the washing buffer is removed by vacuum aspiration using a vacuum aspirating pumping device. Upon elution from the solid support material an eluate containing nucleic acids that are substantially pure can be obtained. The eluate may be used to detect nucleic acids therein, including specific nucleic acid species, to thereby identify microbial biological species present in the fluid sample. Related kits are also provided.

Description

METHODS AND KITS FOR ISOLATING NUCLEIC ACIDS

RELATED APPLICATION

[0001] This application claims the benefit of priority to United States Provisional Patent Application No. 63/252,668 filed October 6, 2021 ; the entire contents of United States Provisional Patent Application No. 63/252,668 are hereby incorporated by reference.

FIELD OF THE DISCLOSURE

[0002] 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.

BACKGROUND

[0003] The following paragraphs are provided by way of background to the present disclosure. They are not however an admission that anything discussed therein is prior art or part of the knowledge of persons skilled in the art.

[0004] Techniques to assay the nucleic acid constituents present in samples, such as fluid samples are highly desirable, since such techniques allow for the qualitative and quantitative assessment of living material, such as microbial biological species present in the samples. Thus, for example, bodies of water, such as rivers, lakes, or wastewater effluents, are commonly monitored for the presence of microbial biological species, including, for example, water borne pathogenic bacteria and viruses, to mitigate health and safety risks.

[0005] Some known techniques to assay nucleic acids in samples involve the isolation of nucleic acid material from a sample obtained from a larger fluid source or fluid reservoir. However, such isolation is frequently challenging since separation of nucleic acid material from 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. Thus, techniques that permit the isolation of high-quality nucleic acid materials substantially free of contaminants, are particularly desirable.

[0006] Furthermore, many techniques known to the art for isolating nucleic acids involve the use of laboratory equipment, such as centrifuges, for example, to separate nucleic acids from contaminating materials. However, such techniques are generally not practical to implement at remotely located sample collection sites.

[0007] Other shortcomings associated with known assays to evaluate nucleic acid materials in samples are associated with the handling of samples, including fluid samples. In many instances, samples are transported from a sampling site to a laboratory to perform a nucleic acid analysis. However, alterations in microbial constituency can occur when samples are obtained from locations where there is no access to near-by analytical laboratory facilities and storage and transport of the sample is required. By way of example, temperature fluctuations can result in a changes in microbial and nucleic acid constituents in a fluid sample. Thus, a laboratory nucleic acid characterization may not be an accurate reflection of the in situ constituent nucleic acids present in the fluid reservoir from which the fluid sample was collected. Furthermore, 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. Yet further safety and health complications may arise when an analytical laboratory receives samples containing live and potentially pathogenic microorganisms, and conversely, as the laboratory needs to dispose of such samples. For at least these reasons, assays and techniques to analyze nucleic acid constituents which can be deployed at a sampling site are particularly desirable.

[0008] There remains, therefore, an ongoing need in the art for improved methods and systems for isolating nucleic acid material from samples, and in particular there is a need for rapid and easy to operate methods that permit the isolation of nucleic acids at sampling sites.

SUMMARY

[0009] The following paragraphs are intended to introduce the reader to the more detailed description that follows and not to define or limit the claimed subject matter of the present disclosure.

[0010] In one broad aspect, 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.

[0012] Accordingly, in one aspect, in accordance with the teachings herein, 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) mixing the sample with a lysis material to lyse the microbial biological species and form a lysate comprising microbial nucleic acids;

(b) mixing the lysate with a binding buffer to form a bindable mixture comprising microbial nucleic acids in solution;

(c) flowing the bindable mixture through 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;

(d) 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; and

(e) eluting the microbial nucleic acids from the solid support material to obtain an eluate comprising the microbial nucleic acids in solution.

[0013] In at least one embodiment, in an aspect, the lysate can be filtered, and the filtered lysate can be mixed with the binding buffer.

[0014] In at least one embodiment, in an aspect, 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. [0015] In at least one embodiment, in an aspect, 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.

[0016] In at least one embodiment, in an aspect, 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.

[0017] In at least one embodiment, in an aspect, 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.

[0018] In at least one embodiment, in an aspect, 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.

[0019] In at least one embodiment, in an aspect, 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.

[0020] In at least one embodiment, in an aspect, 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.

[0021] In at least one embodiment, in an aspect, the fluid transfer device can be a syringe.

[0022] In at least one embodiment, in an aspect, the vacuum aspirating pumping device can be a pumping device operable at from about 5 pounds per square inch (psi) to about 50 psi.

[0023] In at least one embodiment, in an aspect, the vacuum aspirating pumping device can be a battery operable device.

[0024] In at least one embodiment, in an aspect, 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.

[0025] In at least one embodiment, in an aspect, the lysis material can further include 2-amino-2-(hydroxymethyl)-1 ,3-propanediol (Tris) ethylenediaminetetraacetic acid (EDTA) (TE) to protect ribonucleic acids (RNA).

[0026] In at least one embodiment, in an aspect, 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).

[0027] In at least one embodiment, in an aspect, 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).

[0028] In at least one embodiment, in an aspect, 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.

[0029] In at least one embodiment, in an aspect, the solid support material can be a silica mineral material.

[0030] In at least one embodiment, in an aspect, 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.

[0031] In at least one embodiment, in an aspect, 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.

[0032] In at least one embodiment, in an aspect, 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.

[0033] In at least one embodiment, in an aspect, 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. [0034] In at least one embodiment, in an aspect, the nucleic acid amplification mixture can be a lyophilized nucleic acid amplification mixture.

[0035] In at least one embodiment, in an aspect, the aliquot of the eluate can be mixed with the lyophilized nucleic acid amplification mixture without dilution of the aliquot.

[0036] In at least one embodiment, in an aspect, the method can further include a step (f) comprising detecting the nucleic acids to thereby identify a microbial biological species present in the sample.

[0037] In at least one embodiment, in an aspect, 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.

[0038] In at least one embodiment, in an aspect, the microbial biological species can be a bacterial species.

[0039] In at least one embodiment, in an aspect, the bacterial species can be Escherichia coli.

[0040] In at least one embodiment, in an aspect, the microbial biological species can be a viral species.

[0041] In at least one embodiment, in an aspect the viral species can be a Severe Acute Respiratory Syndrome Coronavirus-2 virus (SARS-CoV-2) or a peppermint mild mottle virus (PMMoV).

[0042] In at least one embodiment, in an aspect, the method can be initiated from within from about 1 minute up to about 2 hours from the collection of the sample.

[0043] In at least one embodiment, in an aspect, steps (a) - (e) of the method can be completed in about 30 minutes or less from initiation thereof.

[0044] In at least one embodiment, in an aspect, the sample can be a fluid sample.

[0045] In at least one embodiment, in an aspect, the fluid sample can be a water sample.

[0046] In at least one embodiment, in an aspect, the water sample can be a waste water sample.

[0047] In at least one embodiment, in an aspect, the water sample can be a drinking water sample.

[0048] In another aspect, the present disclosure relates to kits for the the extraction of nucleic acids from samples. Thus, in an aspect, 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;

(b) a vessel containing lysis material;

(c) a vessel containing binding buffer;

(d) 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;

(e) a vessel containing washing buffer;

(f) a vessel containing eluent;

(g) optionally at least one fluid transfer device; and

(h) optionally at least one eluent collection vessel, together with instructions to perform the methods of the present disclosure. [0049] In at least one embodiment, in an aspect, the kit can further contain a filter.

[0050] In at least one embodiment, in an aspect, the fluid transfer device can be a syringe.

[0051] In at least one embodiment, in an aspect, the kit can further comprise a vacuum aspirating pumping device.

[0052] In at least one embodiment, in an aspect, the vacuum aspirating pumping device can be a pumping device operable at from about 5 pounds per square inch (psi) to about 50 psi.

[0053] In at least one embodiment, in an aspect, 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.

[0054] In at least one embodiment, in an aspect, the lysis material can further include 2-amino-2-(hydroxymethyl)-1 ,3-propanediol (Tris) ethylenediaminetetraacetic acid (EDTA) (TE) to protect ribonucleic acids (RNA).

[0055] In at least one embodiment, in an aspect, 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).

[0056] In at least one embodiment, in an aspect, 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. [0057] In at least one embodiment, in an aspect, the solid support material can be a silica mineral material.

[0058] In another aspect, the present disclosure relates to uses of kits for the extraction of nucleic acids from samples. Thus, in an aspect, the present disclosure provides, in at least one embodiment, a use of a kit comprising:

(a) optionally at least one sample collection vessel;

(b) a vessel containing lysis material;

(c) a vessel containing binding buffer;

(e) a vessel containing washing buffer;

(f) a vessel containing eluent;

(g) optionally at least one fluid transfer device; and

(h) optionally at least one eluent collection vessel, together with instructions to perform the methods of the present disclosure, to isolate microbial nucleic acids.

[0059] Otherfeatures and advantages or the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating preferred implementations of the present disclosure, is given by way of illustration only, since various changes and modification within the spirit and scope of the disclosure will become apparent to those of skill in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The disclosure is in the hereinafter provided paragraphs described, by way of example, in relation to the attached figures. The figures provided herein are provided for a better understanding of the example embodiments and to show more clearly how the various embodiments may be carried into effect.

[0061] FIG. 1 shows a perspective view of an example syringe that can be used in accordance with an example embodiment of the present disclosure.

[0062] FIG. 2 shows a perspective view of an example cartridge that can be used in accordance with an example embodiment of the present disclosure. [0063] FIG. 3 shows a perspective view of an example filter that can be used in accordance with an example embodiment of the present disclosure.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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).

[0071] 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.

[0072] 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. In an aspect, 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.

[0073] 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. In an aspect, 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.

[0074] 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. In an aspect, 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.

[0075] 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. In an aspect, 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.

[0076] FIG. 16 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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.

[0077] FIG. 17 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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). [0078] FIG. 18 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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)).

[0079] FIG. 19 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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.

[0080] FIG. 20 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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).

[0081] FIG. 21 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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. The bar graph depicts filtering time as a function of the different sample volumes (10 ml, 25 ml, and 50ml), and different pump operating voltages (12 psi (5V), 27 psi (9V), 29 psi (12V), and 30 psi (16V)). [0082] FIG. 22 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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). 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)).

[0083] FIG. 23 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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.

[0084] FIG. 24 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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).

[0085] FIG. 25 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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). The bar graph depicts the detected viral genomic copies per millilitre (cp/mL) as a function of various included detergents (0.05%, 0.1% and 0.5% Tween®-80, 0.05% TX-100, 0.1 % TX-100). [0086] FIG. 26 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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.

[0087] FIG. 27 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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).

[0088] FIG. 28 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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).

[0089] FIG. 29 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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. The graph shows the A260/A280 as a function of TDS in a fluid sample.

[0090] FIG. 30 depicts a graph obtained in the performance of certain further experiments involving the isolation of nucleic acid material from a wastewater sample. In an aspect, 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.

[0091] 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.

[0092] 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).

[0093] The figures together with the following detailed description make apparent to those skilled in the art how the disclosure may be implemented in practice.

DETAILED DESCRIPTION

[0094] Various methods, compositions and systems will be described below to provide at least one example of at least one embodiment of the claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover methods, compositions and systems that differ from those described below. The claimed subject matter is not limited to any method, composition or system having all of the features of methods, compositions or systems described below, or to features common to multiple methods, compositions or systems described below. It is possible that a method, composition, or system described below is not an embodiment of any claimed subject matter. Any subject matter disclosed in methods, compositions or systems described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicants, inventors or owners do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.

[0095] As used herein and in the claims, the singular forms, such as “a”, “an” and “the” include the plural reference and vice versa unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, the terms “comprise,” “comprises” and “comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers. The term “or” is inclusive unless modified, for example, by “either”. The term “and/or” is intended to represent an inclusive or. That is “X and/or Y” is intended to mean X or Y or both, for example. As a further example, X, Y and/or Z is intended to mean X or Y or Z or any combination thereof.

[0096] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as being modified in all instances by the term “about.” The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1 % and 15% of the stated number or numerical range, as will be readily recognized by the context. Furthermore, 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). Similarly, 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.

[0097] Unless otherwise defined, scientific and technical terms used in connection with the formulations described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0098] All publications, patents, and patent applications referred herein are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically indicated to be incorporated by reference in its entirety.

Terms and definitions

[0099] The term “nucleic acid”, as used herein, refer 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. Examples of such 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. In this respect, microbial biological species can be said possess unique nucleic acid sequences which are distinct from nucleic acid sequences found in other microbial biological species. Reference is further made herein to a “species of nucleic acids”, by which is meant a polymeric nucleic acid molecule having a particular specific nucleic acid sequence by which it can be distinguished from other nucleic acid species having other nucleic acid sequences.

[00100] The terms “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. [00101] The terms “substantially pure” and “isolated”, as used herein, as may be used interchangeably herein to describe an object material species, e.g., nucleic acid material, which has been separated from components that naturally accompany it. Typically, an object material species, such as nucleic acid material, 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.

[00102] The terms “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.

General implementation

[00103] In general, the methods and 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.

[00104] In broad terms, 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. Thus, 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. Thus, when the nucleic acid material is subsequently used for qualitative and/or quantitative characterization, 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.

[00105] Furthermore, 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.

[00106] Furthermore, 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. Furthermore, the 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.

[00107] Furthermore, 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.

[00108] In addition, 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.

[00109] In what follows selected embodiments are described with reference to the drawings. 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. However, it is to be clearly understood that the embodiments illustrated in 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. Furthermore, 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.

[00110] The example embodiments hereinafter discussed with respect to FIGS. 1 - 11 involve, in an aspect, the sequential performance of multiple steps. In this respect, FIGS. 1 - 3 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.

[00111] Referring initially to 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.

[00112] Referring further now to FIG. 1, 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. Thus, when downward pressure is applied to fluid filled syringe housing 18 using plunger unit 14, a substantial fluid pressure in narrow distal aperture 17 is achieved. Such pressure is deemed beneficial as it can weaken the integrity of the cellular membranes when the syringe is used to obtain a fluid sample as hereinafter described. It is noted that in other embodiments no seal is included. 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.

[00113] As will become clear, in an aspect, example embodiments herein involve the performance of multiple steps involving the transfer of multiple fluids using a syringe. It is noted that hereinafter in FIGS. 4, 6A - 6C, 8A - 8C, 9A - 9C, 10A - 10D, and 11 A - 11C reference is made to syringe 10. In this respect, 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. Alternatively, different syringes may be used when different fluids are transferred. For ease of illustration FIGS. 4, 6A - 6C, 8A - 8C, 9A - 9C, 10A - 10D, and 11 A - 11 C, depict 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.

[00114] It is further also noted that a syringe is a fluid transfer device. In an aspect, 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. In alternate embodiments, in one or more steps, instead of a syringe, other fluid transfer devices, for example, a pipette or a tube, may be used. In some embodiments, a pump, for example a vacuum pump, may be used to aide in the transfer of fluids. In general, the inventors believe that syringes and vacuum pumps are particularly convenient fluid transfer devices to conduct many steps of the herein disclosed methods.

[00115] Depicted in FIG. 2 is 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. Thus, in an example embodiment, reversible joint 39 can be constructed using a screw thread structure.

[00116] Depicted in FIG. 3 is 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. Thus, in an example embodiment, reversible joint 38 can be constructed using a screw thread structure, a friction-fit coupling, or a Luer- lock coupling.

[00117] In an aspect, 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. This may be achieved by immersing aperture 17 of syringe 10 into source fluid 40, 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 a fluid aliquot (ranging, for example, from 1 ml to 250 ml) of source fluid 40 to obtain fluid sample 40a of source fluid 40 contained within syringe 10.

[00118] It is noted that in accordance herewith, any natural or artificial source of source fluid may be used. In certain embodiments, 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. In other embodiments, 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.

[00119] Furthermore, it is noted that 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.

[00120] Furthermore, it is noted that in some embodiments, 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.

[00121] Furthermore, it is noted that in some embodiments, 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.

[00122] Furthermore, it is noted that 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.

[00123] Although, as hereinbefore described with reference to FIG. 4, 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). As will be understood by those of skill in the art, depending on the source from which the sample material is collected, sample collection techniques may be varied, and selected as desired. Thus, 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”. In general terms, 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. In certain embodiments, 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 |im. The skilled artisan will readily recognize that 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.

[00124] The source fluid, in accordance herewith, is further characterized in that it comprises microbial biological species. In some embodiments, the microbial biological species may be of a single taxonomic order, for example a single kingdom, phylum, class, order, family, genus, species, or strain. In other embodiments, 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. In other embodiments, 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.

[00125] Thus, it is to be clearly understood that, in accordance herewith, any fluid sample containing nucleic acids, or suspected to contain nucleic acids, may be collected, and used in accordance herewith. Thus, the methods of the present disclosure are not intended to be limited with regards to the fluid source of the nucleic acid material.

[00126] Furthermore, it is noted that a fluid sample containing any nucleic acid material can be used. In this respect, it is to be understood that included herein are embodiments comprising RNA nucleic acids, or DNA nucleic acids, or a mixture of RNA and DNA nucleic acids, and thus, it is to be understood that the 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. [00127] Referring now to FIG. 5, shown therein is tube 50a (e.g., a 50 ml Falcon® tube) in which fluid sample 40a containing microbial biological species has been collected. Second tube 50b contains lysis material 52. 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.

[00128] 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.

[00129] 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).

[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).

[00131] 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. In embodiments hereof where ionic salts are used as lysis materials, 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,

4.5 M or 5 M KCI or NaCI. In example embodiments, a lysis material that may be used is an NaCI or KCI salt, and 1 x TE buffer (10mM Tris-HCI containing 1 mM EDTA«Na2, pH = 7.0 - 9.0), wherein the obtained lysate has an ionic salt 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, 4.5 M or 5 M KCI or NaCI, and 1x TE.

[00132] Subsequent to mixing of lysis material 52 and fluid sample 40, 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.

[00133] Referring next to FIGS. 6A - 6C, lysate 54 is preferably filtered to remove insoluble materials that may be present in the lysate. Such 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. Thus, 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. Other filters that may be used are ceramic filters. Furthermore, 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.

[00134] Continuing to refer to FIG. 6A, shown therein is 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. Upon completion of filtering, 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.

[00135] Referring now to FIG. 6B, once piston 16 reaches its most distal position within syringe housing 18, generally all of lysate 54 has passed through filter unit 30 and has been collected in collection vessel 61. 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.

[00136] It is noted that the steps illustrated in 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.

[00137] Referring next to FIG. 7, shown therein is collection vessel 61 (depicted with lid 76) containing filtered lysate 54b. Second tube 70 contains binding fluid 71. Suitable binding fluid, in this respect, 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. Preferably, 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. Thus, it will be clear to a person of skill in the art that, for example, a volume of an 80% (v/v) ethanol solution (i.e., 80% (v/v) ethanol, 20% (v/v) water) may be combined with an equal volume of lysate to obtain bindable mixture 73, wherein bindable mixture 73 then contains 40% (v/v) ethanol.

[00138] Referring next to FIG. 8A, 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.

[00139] Referring now to FIG. 8B, once bindable mixture 73 containing nucleic acids has been collected in syringe 10, 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. 16 (3) 416-424); or a metal oxide-based material, such as titanium dioxide, for example (Patel, S. et al., 2016, J. Mol. Liquids (213) 304- 311 ), all of which can selectively bind nucleic acids. [00140] Referring now to FIG. 8C, shown therein is 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.

[00141] Referring next to FIG. 9A, shown therein is 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. As will be clear, cartridge unit 20 comprises a fluid path therethrough from cartridge inlet 22 via cartridge chamber 27 to cartridge outlet 24. As 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. Upon passage through cartridge unit 20, 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. Upon completion of flow through cartridge unit 20, cartridge unit 20 contains nucleic acid material bound to solid support 25 contained in interior chamber 27 of cartridge unit 20.

[00142] Referring now to FIG. 9B, once piston 16 reaches its most distal position within syringe housing 18 bindable mixture 73 has passed through cartridge unit 20 and bindable mixture 73b from which nucleic acids have been separated, is collected in tube 90. 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.

[00143] In an alternative embodiment, instead of using syringe 10 to exert (downward) pressure on bindable mixture 73 to pass bindable mixture 73 through cartridge unit 20, 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. Suitable vacuum aspirating devices and operating conditions, in this respect, are hereinafter described with reference to FIG. 10C.

[00144] Referring next to FIG. 10A, shown therein is 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. et al., 2020, dx.doi.org/10.17504/protocols.io.bpdfmi3n), or 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). 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. 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.

[00145] Referring now to FIG. 10B, once piston 16 reaches its most distal position within syringe housing 18 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.

[00146] In an alternative embodiment, instead of using syringe 10 to exert (downward) pressure on washing buffer 101 to pass washing buffer 101 through cartridge unit 20, 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.

[00147] In some embodiments, 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. Thus, these steps may, for example, be conducted 2 or 3 times, each time using fresh washing buffer.

[00148] Referring next to FIG. 10C, in orderto remove excess washing buffer 101 from solid support material 25 within chamber 27 of the cartridge unit 20, 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. In alternate embodiments, proximal end portion 114a of tubing 114 can also be coupled to cartridge inlet 22, after decoupling syringe 10 and cartridge unit 20.

[00149] It is noted that in the hereinbefore described alternative embodiment in which a vacuum aspirating device is fluidically coupled to cartridge unit 22 to flow washing buffer through cartridge unit 22, all, or substantially all, of washing buffer 101 can be flowed through cartridge unit 22 by means of vacuum pump 110. Thus, it is to be understood that vacuum pump 110 can be used to remove at least a portion of washing buffer 101 from cartridge unit 20.

[00150] For field operations, 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. Example diaphragm pumps that may be used in accordance herewith include, for example, Joto Fluid, P-G3053-4 (see: https://www.jotofluid.com/product/GED1224120P402-42min-120kPa-Mini-Air-Pump-12- Volt-Electrical-Diaphragm-Pump-Air-Compressors.html), Topsflo, TM30A-D (see: http://www.topsflo.com/mini-diaphragm-pump/tm30a-d.html), and Vikeye, Vikyeqg2kszd47h (see: https://www.amazon.ca/Negative-Pressure-Analysis-Sampling- lnstrument/dp/B07RL7F736/ref=rtpb_2/137-8158367-

6402815?pd_rd_w=bHcMo&pf_rd_p=20523d9a-69e9-4cf2-81 bb- 78c83b713159&pf_rd_r=693XJQESNEMSK6QMYWVR&pd_rd_r=27cf4268-ac69-4c6c- 824b-6a0212b81 a9b&pd_rd_wg=7lean&pd_rd_i=B07RL7F736&psc=1). In general, 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, for example, about 25 psi, about 30 psi, about 35 psi, or about 40 psi. Furthermore, it is noted that the operating pressure may be selected as function of the solid support material 25. In this respect, 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. As will be understood by those of skill in the art, 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. In this respect, 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. In embodiments where vacuum pump 110 is used to aspirate bindable mixture 73 and all of substantially all of washing buffer 101 , depending on the fluid volumes used, 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.

[00151] Upon completion of vacuum aspiration, 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.

[00152] It is generally desirable that cartridge unit 20 is left to dry following vacuum aspiration. Thus, 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.

[00153] It is noted that by using 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. Furthermore, the use of a battery-operated pump permits processing of the sample fluid immediately upon collecting the sample fluid. For example, 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. Thus, 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.

[00154] Referring next to FIG. 11 A, shown therein is 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. Thus, for example, 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. et al., 2020, dx.doi.org/10.17504/protocols.io.bpdfmi3n; and Ali, N. et al., 2017, Biomed. Res. Int. Art ID NO: 9306564). 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.

[00155] Upon passage through cartridge unit 20, 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.

[00157] It is noted that in some embodiments, in accordance herewith, some or all of the steps depicted in FIGS. 4 - 11 may be conducted multiple times, including simultaneously, or more or less simultaneously. Thus, referring to FIG. 4, for example, 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). Similarly, 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). Furthermore, a single volume of sample 40a, or other fluids, (e.g., 54, 54b, 73, 73b) 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. Conversely, multiple smaller volumes of sample 40a, or other fluids, (e.g., 54, 54b, 73, 73b) may be combined to obtain a single larger volume of samples 40a (or 54, 54b, 73, 73b) and used to conduct applicable steps depicted in FIGS. 5 - 11. Furthermore, it is noted that in the performance of the step depicted in FIG. 10C, 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.

[00158] 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. Thus, 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.

[00159] 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. In this respect, 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. This includes, 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). 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. Furthermore, it is noted that 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.

[00160] In view of the purity of the nucleic acid material in the eluate that can be obtained in accordance with the methods of present disclosure, 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. In this respect, 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. Pharm. Bioallied Sci. 5(4): 245-252; NASBA: see: Morabito K., et al., 2013, Molecular Diagnosis and Therapy 17: 185-192; LAMP: see: Becherer L. et al., 2020, Analytical Methods 12: 717-746; SDA: see: Walker G.T. et al., 1992. Nucl. Acids. Res. 20(7) 1691-1696; HDA: see: Xu, V. M. et al., 2004, EMBO Rep. 5(8) 795- 800; NEAR: see: Qian C., et al., 2019, Analytica Chimica Acta 1050: 1-15; SMART: see: Wharam S.D. et al., 2001 , Nucleic Acids Res. 29(11): E54-4; RCA: see: U.S. Patent Application have publication no 20080227160; IMDA: see: Dean F. B. et al., 2002, Proc. Natl. Acad. Sci. (USA), 99(8) 5261-5266; SPIA: see: United States Patent 8,034,568; RPA: see: Lobato M. and O’Sullivan C.K., 2018, Trends in Anal. Chem. 98: 19-35; PSR: see: Liu W. et al., 2015, Sci. Rep. 5, 12723). As will be known to those of skill in the art, these techniques may be used to achieve qualitative or quantitative detection of a specific nucleic acid species.

[00161] As is further known to those of skill in the art, 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. 5 mM), and buffer {e.g., a 100-150 mM Tris-CI, 20 - 80 mM (NH₄)₂SO4, 0.01 % - 0.04% (v/v) Tween®-20 buffer, pH 8.0 - 9.5). Master mixtures are frequently provided in lyophilized form, as particulates, which prior to use are rehydrated using e.g., water or a buffer. In accordance herewith, 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’.

[00162] Thus, it will be clear that 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. Upon such amplification, 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.

[00163] In another aspect, the present disclosure provides kits to conduct the methods according to the present disclosure. Accordingly, the present disclosure, in an aspect, further includes a kit for the extraction of nucleic acids from a sample, the kit comprising

(a) optionally at least one sample collection vessel;

(b) a vessel containing lysis material;

(c) a vessel containing binding buffer;

(e) a vessel containing washing buffer;

(f) a vessel containing eluent;

(g) optionally at least one fluid transfer device; and

(h) optionally at least one eluent collection vessel, together with instructions to perform the methods of the present disclosure. [00164] In one embodiment, the kit can further include a filter suitable to filter lysate.

[00165] In one embodiment, the fluid transfer device can be a syringe, and in a further embodiment, the kit can include 2, 3, 4, 5 or 6 syringes.

[00166] In one embodiment, the kit can further comprise a vacuum aspirating pumping device.

[00167] In one embodiment, the vacuum aspirating pumping device can be a pumping device operable at from about 5 pounds per square inch (psi) to about 50 psi. In one embodiment, the pumping device can be a battery-operated device.

[00168] In one embodiment, , 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.

[00169] In one embodiment, the lysis material can further include 2-amino-2- (hydroxymethyl)-1 ,3-propanediol (Tris) ethylenediaminetetraacetic acid (EDTA) (TE) to protect ribonucleic acids (RNA).

[00170] In one embodiment, 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).

[00171] In one embodiment, 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.

[00172] In one embodiment, the solid support material in the cartridge in the kit can be a silica mineral material.

[00173] 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.

[00174] As can now be appreciated, the methods and 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.

[00175] Of course, the above-described example embodiments of the present disclosure are intended to be illustrative only and in no way limiting. The described embodiments are susceptible to many modifications of composition, details, and order of operation. The invention, rather, is intended to encompass all such modifications within its scope, as defined by the claims, which should be given a broad interpretation consistent with the description as a whole.

EXAMPLES [00176] Hereinafter are provided examples of further specific embodiments for performing the methods of the present disclosure, as well as embodiments representing the kits of the present disclosure. It is noted that the examples are provided in further reference to using the example embodiments shown in FIGS. 1 - 11 , and the results are shown in FIGS. 12 - 32.

Example 1 - Isolation of nucleic acids from a wastewater and amplification of SARS-CoV-2 S-gene.

[00177] For each of four methods tested (as described below) a total volume of 25 mL wastewater sample collected at municipal wastewater treatment plant located in a large Canadian city (population > 1 million) was mixed with 5.8 g NaCI and 1 x TE. The samples were then thoroughly mixed and incubated at room temperature for a period of 10 minutes to form lysates. Following incubation of the lysates, a 60 mL syringe was used to pass each of the four lysates through a separate 5 m filter and the four flow-throughs, i.e., filtered lysates, were collected. Equal volumes of 80% (v/v) ethanol were added to each of the four filtered lysates and thoroughly mixed to form bindable mixtures. 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. Data from 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.

[00178] Referring further to FIG. 12, 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. Thus, no removal of washing buffer or syringe drying results in an underestimation of the concentration of viral RNA actually present in the water sample. In particular, in instances when the water sample contains even lower concentrations of viral RNA, 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.

[00179] A total volume of 25 mL each of wastewater samples collected at three different Canadian municipal wastewater treatment plants (site 1 = a large city with a population > 1 million; site 2 = a small town with a population < 10,000 people; and site 3 = a medium-size city with a population <100,000 people) were mixed with 5.8 g NaCI and 1 x TE. The samples were then thoroughly mixed and incubated at room temperature for a period of 10 minutes to form a lysate. Following incubation of the lysate, a 60 mL syringe was used to pass the mixtures through a 5 p.m filter and the flow-through, i.e., filtered lysate was collected. 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.

[00180] A total volume of 25 mL a wastewater sample collected at a municipal wastewater treatment plant in a large city in Canada was mixed with 5.8 g NaCI and 1 x TE. The samples were then thoroughly mixed and incubated at room temperature for a period of 10 minutes to form a lysate. Following incubation of the lysate, a 60 mL syringe was used to pass the mixture through a 5 pm filter and the flow-through, i.e., filtered lysate, was collected. An equal volume of 80% ethanol was added to the filtered lysate and thoroughly mixed to form a bindable mixture. The bindable mixture was then passed through a 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 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. A control sample which contains all components of the RT-PCR reactions with the exception that no RNA template was added was also used for amplification using the same S-gene primer set. 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. [00181] A total volume of 20 mL of four wastewater samples collected at a municipal waste water treatment plant located in a small town in Canada (population < 10,000) and passed through a 5 pm filter. 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. Following incubation of the 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. In order for each sample to have the same or similar final binding solution 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). Mixtures were thoroughly mixed to form a bindable mixture. The 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. 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. 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.

[00182] 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.

Example 6 - Evaluation of solid support drying time.

[00183] Zymo V-E silica columns were attached to the bottom of a 60 ml_ syringe barrel and inserted into inlets on a vacuum manifold connected to a battery powered vacuum pump (Mini Vacuum Pump, DC 12V 120kpa Vacuum Air Pump, running at 65 kPa (9.4 psi) (see: https://www.amazon.ca/Negative-Pressure-Analysis-Sampling- lnstrument/dp/B07RL7F736/ref=rtpb_2/137-8158367-

6402815?pd_rd_w=bHcMo&pf_rd_p=20523d9a-69e9-4cf2-81 bb-

78c83b713159&pf_rd_r=693XJQESNEMSK6QMYWVR&pd_rd_r=27cf4268-ac69-4c6c- 824b-6a0212b81 a9b&pd_rd_wg=7lean&pd_rd_i=B07RL7F736&psc=1). Using the syringe barrel as a reservoir, 10 mL of wash buffer containing 100 mM NaCI, 80% (v/v) reagent ethanol, and 10 mM Tris (pH 7.2) was completely passed through the silica column at constant vacuum. Each column was left to vacuum dry for 2, 3, or 5 min, and measured after each timepoint. Residual liquid remaining in the silica fret was collected by centrifugation at 12,000 x g for 1 min into a 1.5 mL tube and weighed (with the conversion of 1 mg = 1 pL at ~0.83 mg/pL density of 80% (v/v) ethanol). Table 1 shows the results.

Table 1 : Solid support drying times

[00184] It is noted that insufficient removal of washing buffer can impede nucleic acid amplification.

Example 7 - Evaluation of lysis time.

[00185] 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 either 10, 20, or 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 polyethersulfone (PES) 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 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. Following the washing, 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 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 compared relative to the average amplification signal (Cq) of wastewater samples lysed for 10 min. Each condition was tested at least twice (n = >2). The results are shown in FIG. 17.

Example 8 - Evaluation of filter materials. [00186] 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. 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 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. Following the washing, 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 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 compared relative to the average amplification signal (Cq) of wastewater samples filtered thru nylon material. Each condition was tested at least twice (n = >2). The results are shown in FIG. 18.

Example 9 - Evaluation of solid support materials.

[00187] 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 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 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. Following the washing, 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 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. Each set of columns was tested six times (n = 6). The results are shown in FIG. 19.

Example 10 -Evaluation of filtered fluid volume.

[00188] 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. The bindable mixture was then passed through either a Zymo lll-P, V, or V-E silica column using the 60 mL syringe barrel as a reservoir (noted as A, B, and C, respectively) and the total time to filter was recorded. Each set of columns was tested once (n = 1 ). The results are shown in FIG. 20. It is noted that nucleic acid yield and filtration time may be adjusted and optimized, as desired, by selection of specific solid support materials. In this respect, it is noted that, for example, column material B (Zymo V) yielded more nucleic acid material (see: Example 9), but more time is required to yield the nucleic acid material (see: Example 10).

Example 11 - Evaluation of vacuum pump operating pressure. [00189] 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)). Using the syringe barrel as a reservoir, a volume of either 10, 25 or 50 mL of purified water was completely passed through the silica column, at either 12 psi (operating voltage 5V), 27 psi (operating voltage 9V), 29 psi (operating voltage 12V), or 30 psi (operating voltage 16V) vacuum strength and the filtering time was measured. Each set of volumes was tested six times (n = 6). The results are shown in FIG. 21.

Example 12 - Evaluation of concentration DMSO in eluent.

[00190] 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, 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. Nucleic acids were then eluted from the column in 200 pL of nuclease-free water containing 0%, 5%, or 10% dimethylsulfoxide (DMSO). 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. Each set of DMSO concentrations was tested thrice (n = 3 to 16). The results are shown in FIG. 22.

Example 13 - Evaluation of eluents. [00191] 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. Nucleic acids were then eluted from the column in 200 pL of either nuclease-free water (NFW) or 0.5X Tris- EDTA buffer (pH 8.0). 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. Each set of columns was tested three times (n = 3). The results are shown in FIG. 23.

Example 14 - Evaluation of eluent volume.

[00192] 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 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

*+o 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 either 200, 300, or 400 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 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. Each set of columns was tested 2 times (n = 2). The results are shown in FIG. 24. It is noted that a larger eluent volume may be preferrable, since trace amounts of substances present in the initial fluid sample carried over into the eluate may inhibit nucleic acid amplification, and such inhibiting substances may be diluted when a larger volume of eluent is used.

Example 15 - Evaluation of detergent in lysis fluid.

[00193] 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 varying concentrations of Tween®-80 or Triton® X-100 (TX-100) 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, 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. Nucleic acids were then eluted from the column in 200 pL of nuclease- free water. 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. Each set of columns was tested 2 times (n = 2). Results are shown in FIG. 25. Example 16 - Evaluation of detergent and beads in lysis fluid.

[00194] 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), with or without 2% (v/v) beads (VWR beads 0.4 mm, cat number 30623-120, Chemglass, VWR) and/or 0.5% (v/v) Tween®-80 detergent. The samples were then thoroughly mixed and incubated at room temperature for 15 minutes or 30 minutes (for beads only) 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 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. 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 compared relative to the average SARS- CoV-2 detection of wastewater samples extracted with lll-P column and no added beads or detergent. Each set of columns was tested 3 times (n = 3). Results are shown in FIG. 26.

Example 17 - Evaluation of use of undiluted eluate to amplify nucleic acid material.

[00195] 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 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. 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. Following washing, 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 nucleic acid samples were then used at 20 pL or 10 pL with 10 pL diluent to rehydrate a lyophilized RT-qPCR reagent mixture 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. Each set of elution volumes was tested seventeen times (n = 17). The results are shown in FIG. 27. It is noted that it is not necessary to rehydrate the lyophilized RT-qPCR reagent mixture, e.g., with water or a buffer, and that, instead, the eluate may be used to rehydrate the lyophilized RT-qPCR reagent mixture. Thus, the eluate may be used without dilution.

Example 18 - Evaluation of dilution of fluid sample.

[00196] Volumes of 25 mL of undiluted and 10-fold diluted 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, 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. 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 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. Four samples were tested for dilution (n = 4). The results are shown in FIG. 28. It is noted that a diluted fluid sample may be preferrable, since trace amounts of substances present in the initial fluid sample carried over into the eluate may inhibit nucleic acid amplification, and such inhibiting substances may be diluted when an undiluted fluid sample is used.

Example 19 - Evaluation of nucleic acid purity as a function of total dissolved solids (TDS) in sample fluid.

[00197] 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. 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. Nucleic acids were then eluted from the column in 200 pL of nuclease- free water. The nucleic acid samples were measured for purity using 2 pL on a NanoDrop ND-1000 spectrophotometer at absorbance 260 and 280 nm. Measurements were referenced against nuclease-free water. Each sample was measured (n = 50). Results are shown in FIG. 29. It is noted that the method yields nucleic acid material exhibiting an A260/A280 ratio ranging from about 1 .8 to 2.2, regardless of whether the starting fluid material has a low TDS content (100 ppm) or a high TDS (800 ppm). Thus, the results in this Example indicate that the methods of the present disclosure can yield substantially pure nucleic acid material from starting fluid samples that contain a wide range of totally dissolved solids.

Example 20 - Evaluation of storage time of fluid samples.

[00198] Volumes of 25 mL wastewater samples (A) - (G) collected at a municipal waste water 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 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 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. Following the washing, 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.

[00199] Volumes of 25 mL of wastewater sample collected at a municipal wastewater treatment plant in a large city in Canada were mixed with 5.8 g NaCI, Ix Tris-

04 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, 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. 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.

[00200] Subculture of Escherichia coli (E. coli) bacteria was inoculated by 100-fold dilution in LB growth media from an overnight culture incubated at 37°C at 300 rpm. This subculture was grown to mid-log phase (ODeoo = 0.5), and then diluted 5-fold with LB growth media prior to extraction. A contamination control with only LB growth media was also included in the extraction. Volumes of 25 mL of either LB only (Sample A) or E.coli bacteria (Sample C) were mixed with 5.8 g NaCI, 1x Tris-EDTA buffer (pH 8.0), and 0.5% (v/v) Tween®-80 detergent. These 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. In addition, 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. 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. In addition, reactions from Samples 2, A, B, and C were analyzed on gel electrophoresis prior to RT-PCR amplification. The amplicon was detected by visual inspection of the gel following gel electrophoresis in control Samples 2 and 3, as well as in test samples B and C, following RT-PCR amplification. The amplicon was not detected on the gel in control Samples 1 or A, or in any sample prior to RT-PCR amplification.

[00201] Using the same RT-PCR amplification reactions of the nucleic acid samples prepared for the gel electrophoresis experiment, FIG. 32 shows the amplification signal (Cq) for each wastewater sample (in technical duplicate), n.d., no amplification detected.

Claims

1. A method for isolating microbial nucleic acids from a sample comprising microbial biological species, the method comprising:

2. A method according to claim 1 , wherein the lysate is filtered, and the filtered lysate is mixed with the binding buffer.

3. A method according to claim 1 , wherein the bindable mixture is 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.

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4. A method according to claim 3, wherein the fluid transfer device is a syringe comprising a piston, and the bindable mixture is flowed through the cartridge by exerting downward pressure on the piston.

5. A method according to claim 3, wherein the second opening of the cartridge is coupled to a vacuum aspirating pumping device, and the bindable mixture is flowed through the cartridge by vacuum aspiration.

6. A method according to claim 1 , wherein the washing buffer is 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.

7. A method according to claim 6, wherein the fluid transfer device is a syringe comprising a piston, and the washing buffer is flowed through the cartridge by exerting downward pressure on the piston, wherein the at least a portion of the washing buffer removed from the solid material is excess washing buffer not flowed through the cartridge by exertion of downward pressure on the piston.

8. A method according to claim 6, wherein the second opening of the cartridge is coupled to a vacuum aspirating pumping device, and 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.

9. A method according to any one of claims 1 to 8, wherein the nucleic acids are 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.

10. A method according to claim 9, wherein the fluid transfer device is a syringe.

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11. A method according to any one of claims 1 , 5, or 8, wherein the vacuum aspirating pumping device is a pumping device operable at from about 5 pounds per square inch (psi) to about 50 psi.

12. A method according to claim 11 , wherein the vacuum aspirating pumping device is a battery operable device.

13. A method according to claim 1 , wherein the lysis material is 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.

14. A method according to claim 13, wherein the lysis material furthers include 2- amino-2-(hydroxymethyl)-1 ,3-propanediol (Tris) ethylenediaminetetraacetic acid (EDTA) (TE) to protect ribonucleic acids (RNA).

15. A method according to claim 1 , wherein following mixing the lysate with the binding buffer to form the bindable mixture in step (b), the bindable mixture is incubated for from about 10 minutes to about 60 minutes at room temperature prior to proceeding with step (c).

16. A method according to claim 1 , wherein the binding buffer is 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).

17. A method according to claim 1 , wherein the washing buffer is an ethanol-based washing buffer comprising about 100 mM NaCI, about 80% (v/v) ethanol having a pH of about 7.2.

18. A method according to claim 1 , wherein the solid support material is a silica mineral material.

19. A method according to claim 1 , wherein following the performance of step (d) and prior to the performance of step (e), the cartridge is dried to ambient air for at least about two minutes.

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20. A method according to any one of claims 1 to 19, wherein the microbial nucleic acids in the eluate are substantially pure, wherein the eluate exhibits an A260/A280 ratio of at least about 1 .8.

21. A method according to any one of claims 1 to 19, wherein the microbial nucleic acids in the eluate are substantially pure, wherein in the eluate exhibits an A260/A280 ratio of from about 1 .8 to about 2.2.

22. A method according to claim 1 , wherein the method further includes 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 MgCb in concentrations sufficient to amplify of the microbial nucleic acids in the eluate.

23. A method according to claim 22, wherein the nucleic acid amplification mixture is a lyophilized nucleic acid amplification mixture.

24. A method according to claim 23, wherein the aliquot of the eluate is mixed with the lyophilized nucleic acid amplification mixture without dilution of the aliquot.

25. A method according to claim 1 , wherein the method further includes a step (f) comprising detecting the nucleic acids to thereby identify a microbial biological species present in the sample.

26. A method according to any one of claims 22 to 24, wherein the method further comprises 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.

27. A method according to claims 25 or 26, wherein the microbial biological species is a bacterial species.

28. A method according to claim 27, wherein the bacterial species is Escherichia coli.

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29. A method according to claim 26, wherein the microbial biological species is a viral species.

30. A method according to claim 29, wherein the viral species is a Severe Acute Respiratory Syndrome Coronavirus-2 virus (SARS-CoV-2) or a peppermint mild mottle virus (PMMoV).

31. A method according to any one of claims 1 to 30, wherein the method is initiated from within from about 1 minute up to about 2 hours from the collection of the sample.

32. A method according to any one of claims 1 to 31 , wherein steps (a) - (e) of the method are completed in about 30 minutes or less from initiation thereof.

33. A method according to any one of claims 1 to 32, wherein the sample is a fluid sample.

34. A method according to claim 33, wherein the fluid sample is a water sample.

35. A method according to claim 34, wherein the water sample is a wastewater sample.

36. A method according to claim 34, wherein the water sample is a drinking water sample.

37. A kit for the extraction of nucleic acids from a sample, the kit comprising

(a) optionally at least one sample collection vessel;

(b) a vessel containing lysis material;

(c) a vessel containing binding buffer;

(e) a vessel containing washing buffer;

61 (f) a vessel containing eluent;

(g) optionally at least one fluid transfer device; and

(h) optionally at least one eluent collection vessel, together with instructions to perform the methods of the present disclosure.

38. A kit according to claim 37, wherein the kit further contains a filter.

39. A kit according to claim 37, wherein the fluid transfer device is a syringe.

40. A kit according to claim 37, wherein the kit further comprises a vacuum aspirating pumping device.

41. A kit according to claim 40, wherein the vacuum aspirating pumping device is a pumping device operable at from about 5 pounds per square inch (psi) to about 50 psi.

42. A kit according to claim 40, wherein the lysis material is 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.

43. A kit according to claim 42, wherein the lysis material further includes 2-amino-2- (hydroxymethyl)-1 ,3-propanediol (Tris) ethylenediaminetetraacetic acid (EDTA) (TE) to protect ribonucleic acids (RNA).

44. A kit according to claim 40, wherein the binding buffer is 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).

45. A kit according to claim 40, wherein the washing buffer is an ethanol-based washing buffer comprising about 100 mM NaCI, about 80% (v/v) ethanol having a pH of about 7.2.

46. A kit according to claim 40, wherein the solid support material is a silica mineral material.

47. A use of a kit according to claim 40 to isolate microbial nucleic acids.