LARGE- VOLUME SAMPLES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/272,557, filed December 29, 2015, the disclosure of which is incorporated by reference in its entirety herein.

BACKGROUND

[0002] Testing aqueous samples for the presence of microorganisms (e.g., bacteria, viruses, fungi, spores, etc.) and/or other analytes of interest (e.g., toxins, allergens, hormones, etc.) can be important in a variety of applications, including food and water safety, infectious disease diagnostics, and environmental surveillance. For example, comestible samples, such as beverages, process water, and/or public water consumed by the general population may contain or acquire microorganisms that can flourish or grow as a function of the environment in which they are located. This growth may lead to the proliferation of pathogenic organisms, which may produce toxins or multiply to infective doses. By way of further example, a variety of analytical methods can be performed on samples of non-comestible samples (e.g., groundwater, urine, etc.) to determine if a sample contains a particular analyte. For example, groundwater can be tested for a microorganism or a chemical toxin; and urine can be tested for a variety of diagnostic indicators to enable a diagnosis (e.g., diabetes, pregnancy, etc.).

[0003] Microbiological quality of water supplies and recreational water is typically assessed by detection and enumeration of coliforms, fecal coliforms, and Escherichia coli. The traditional methods use most-probable-number (MPN) using serial dilutions within replicate tubes incubated with selective media or a membrane filtration method where a known amount of water is filtered through a membrane and the membrane is placed on media selective for the bacterial group of interest. The regulations for drinking water require detection of 1 cfu/100 ml and the samples are typically brought to the lab and processed. Although large samples of water are typically available for processing, it is difficult to transport large volumes of water to the lab and process them. It can be difficult to detect 1 cfu rapidly without any enrichment.

SUMMARY

[0004] The present disclosure generally relates to a system and device for detecting target microorganisms. In addition, the present disclosure relates to a method for detecting target microorganisms in a sample. In particular, the present disclosure relates to detecting target microorganisms present in a relatively large sample volume. [0005] In one aspect, the present disclosure provides a system for detecting microorganisms. The system can comprise a lysate collection vessel, a plurality of cell-disrupting particles, a filter unit, and a closure element. The lysate collection vessel can comprise an open end configured to receive a sample, a closed end, and a cavity that tapers from a wider portion proximate the open end to a narrower portion proximate the closed end. The filter unit can comprise a housing having a first end, a second end comprising an outlet, and a fluid pathway extending from the first end to the outlet; and a membrane filter disposed in the fluid pathway. The closure element can form a barrier to prevent passage of a microorganism into the filter unit through the outlet. The filter unit can be configured so that liquid passing through the fluid pathway from the first end to the outlet passes through the membrane filter. In any

embodiment of the system, the lysate collection vessel can be operatively coupled to the filter unit, wherein operatively coupling the lysate collection vessel to the filter unit can comprise coupling the open end of the lysate collection vessel to the first end of the filter unit. In any embodiment, the cell-disrupting particles can be fabricated from a material selected from the group consisting of silica, zirconium, silicon carbide, garnet, steel, and combinations thereof.

[0006] In any of the above embodiments, the system further can comprise a cell disruption buffer. In any of the above embodiments, the means for operatively coupling the open end of the lysate collection vessel to the first end of the filter unit can include a first engagement structure that is an integral part of the lysate collection vessel and a complementary second engagement structure that is an integral part of the filter unit. In any of the above

embodiments, the cell-disrupting particles can have a density of about 2.5 g/cc to about 7.9 g/cc. In any of the above embodiments, the cell-disrupting particles can have a mean particle diameter of about 0.1 μπι to about 2 μπι.

[0007] In another aspect, the present disclosure provides a container. The container can comprise a filter unit comprising a housing with a first end, a second end with an outlet, a fluid flow path extending from the first end to the outlet, and a filter disposed in the flow path; a closure element that forms a barrier to prevent passage of a microorganism into the filter unit through the outlet; and a lysate collection vessel sealingly coupled to the filter unit. The filter unit can be configured to receive a liquid sample and retain microorganisms therefrom on the filter. The container can define an interior volume with a perimeter. The filter defines a portion of the perimeter. The filter can have a first side that faces the interior volume. The first side can have a filtrand disposed thereon. A plurality of cell-disrupting particles can be disposed in the interior volume.

[0008] In any of the above embodiments of the container, the lysate collection vessel can be removably coupled to the filter unit. In any of the above embodiments of the container, the filter unit can be configured so that a liquid sample can be urged through the liquid flow path and thereby causing the microorganisms to be retained on the filter. In any of the above embodiments of the container, the container can be configured to be placed into a centrifuge and subjected to a force of about 500 ^χ g to about 10,000 ^χ g, inclusive.

[0009] In yet another aspect, the present disclosure provides a method. The method can comprise providing any of the above embodiments of the system, moving a liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter, coupling the open end of the lysate collection vessel to the filter unit to form a cell-disruption container containing the plurality of cell-disrupting particles disposed therein, agitating the cell- disruption container under conditions that cause the cell -disrupting particles to disrupt microbial cells disposed on the membrane filter to form a liquid lysate, moving a portion of the liquid lysate to a predefined location in the lysate collection vessel, and analyzing the portion of the liquid lysate to detect an analyte associated with a target microorganism.

[0010] In any embodiment of the method, moving a liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter can comprise operatively connecting the second end of the filter unit to a source of negative pressure. In any of the above embodiments of the method, analyzing the liquid lysate can comprise detecting a polynucleotide that indicates a presence of the target microorganism or analyzing the liquid lysate can comprise detecting an antigen that indicates a presence of the target microorganism.

[0011] In yet another aspect, the present disclosure provides a kit. The kit can comprise a lysate collection vessel, a plurality of cell -disrupting particles, a filter unit, and a membrane filter. The lysate collection vessel can comprise an open end configured to receive a sample, a closed end, and a cavity that tapers from a wider portion proximate the open end to a narrower portion proximate the closed end. The filter unit can comprise a housing having a first end, a second end comprising an outlet, and a fluid pathway extending from the first end to the outlet. The membrane filter can be configured to be positioned in the fluid pathway so that liquid passing through the fluid pathway from the first end to the outlet passes through the membrane filter. In any embodiment, the kit further can comprise a closure element that forms a barrier to prevent passage of a microorganism into the filter unit through the outlet.

[0012] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0013] As used herein, "a," "an," "the," "at least one," and "one or more" are used interchangeably. Thus, for example, an apparatus comprising "a" filter can be interpreted to mean that the culture device can comprise "one or more" filters. [0014] The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0015] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0016] The features and advantages of the present invention will be understood upon consideration of the detailed description of the preferred embodiment as well as the appended claims. These and other features and advantages of the invention may be described below in connection with various illustrative embodiments of the invention.

[0017] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The figures and the detailed description which follow more particularly exemplify illustrative embodiments. Other features, objects and advantages will become apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

[0018] FIG. 1 is an exploded perspective view of one embodiment of a container assembly of a system for detecting a microorganism according to the present disclosure.

[0019] FIG. 2 is an assembled side cross-sectional view of the container assembly of FIG. 1.

[0020] FIG. 3 is an exploded perspective view of one embodiment of a filtration assembly used in a method of detecting a microorganism according to the present disclosure, the filtration assembly comprising a receptacle portion, a connector subassembly, and a filter unit.

[0021] FIG. 4 is an assembled perspective view of the filtration assembly of FIG. 3.

[0022] FIG. 5 is a block diagram of one embodiment of a method of detecting a target microorganism according to the present disclosure.

DETAILED DESCRIPTION

[0023] Before any embodiments of the present disclosure are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms "connected" and "coupled" and variations thereof are used broadly and encompass both direct and indirect connections and couplings. Further, "connected" and "coupled" are not restricted to physical or mechanical connections or couplings. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure. Furthermore, terms such as "front," "rear," "top," "bottom," and the like are only used to describe elements as they relate to one another, but are in no way meant to recite specific orientations of the apparatus, to indicate or imply necessary or required orientations of the apparatus, or to specify how the invention described herein will be used, mounted, displayed, or positioned in use.

[0024] In a variety of samples that are desired to be tested for an analyte of interest, the analyte can be present in the sample at a low concentration. For example, regulations for water safety testing can require that testing devices be able to detect 1 colony-forming unit (cfu) of a bacterium of interest in 100 mL of water. Such a low concentration can be difficult or impossible to detect in a reasonable amount of time, much less in a "rapid" time frame, which is described in greater detail below. In order to decrease detection time, in some cases, the sample may need to be concentrated into a smaller volume. That is, in some cases, in order to reach an appropriate concentration of an analyte of interest so as to achieve a detection threshold of an analytical technique in a shorter amount of time, the sample may need to be concentrated by several orders of magnitude.

[0025] The present disclosure generally relates to systems and methods for detecting the presence or absence of an analyte of interest in a sample, particularly, in liquid samples, and more particularly, in dilute aqueous samples. Furthermore, the present disclosure generally relates to systems and methods for rapidly detecting the analyte. In some embodiments, the analyte is selected for detecting (e.g., the presence or absence of) Escherichia coli or other coliforms, for example, in a water sample. Detection of microorganisms (or other analytes) of interest in a water sample can be difficult, because of the low concentration of these microorganisms. As a result of the low concentration, detection in existing systems and methods can be very slow, because the microorganism(s) need to be grown (or the analyte concentration needs to be increased) to a detectable level, which can take time. The present inventors, however, have invented systems and methods for decreasing the time needed to detect a target microorganism in a water sample, and particularly, a dilute water sample.

[0026] Systems and methods of the present disclosure employ a lysate collection vessel that is adapted to contain a sample during a cell-disruption process and, subsequently, move (e.g., by centrifugation) the sample containing lysed cells to a predefined location. The lysate collection vessel can include a wall (or sidewall or slanted wall) that extends to (e.g., tapers toward) a closed end, which can serve as a convenient predefined location to which the sample of lysed cells can be moved for convenient access. [0027] Methods of the present disclosure generally include providing any embodiment of the system described herein, moving a liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter; coupling the open end of the lysate collection vessel to the filter unit to form a cell-disruption container containing the plurality of cell-disrupting particles disposed therein; agitating the cell-disruption assembly under conditions that disrupt microbial cells disposed on and in the membrane filter to form a liquid lysate; moving a portion of the liquid lysate to a predetermined location in the lysate collection vessel; and analyzing the liquid lysate to detect an analyte associated with a target microorganism.

[0028] Samples comprising larger volumes of liquid can be difficult to analyze because any target microorganisms present in this larger volume may not be easily or accurately detected (e.g., by imaging or optically interrogating) at least partly because the microorganism(s), if present, may exist in a relatively low concentration in these larger volumes, and/or because the larger volume may not be suitably positioned (e.g., in an analytical instrument) for detecting the microorganisms or an analyte associated therewith.

[0029] As for retrieving a concentrated sample (filtrand) from a filter, in the systems and methods of the present disclosure, the lysate collection vessel can be used in concert with the filter unit and cell-disruption particles to disintegrate cells present in the filtrand. The disintegrated sample (liquid lysate) then can be moved to a predefined location in the lysate collection vessel where a portion of it can be recovered and analyzed. That is, in some embodiments, systems and methods of the present disclosure can employ a sequential combination of filtration, cell disruption, and centrifugation in order to isolate and detect an analyte indicative of a target microorganism, if present in a sample. Advantageously, the filtrand does not need to be eluted from the filter before any microorganisms present in the filtrand are subjected to cell disruption. This enhances detection of analytes because the method does not require elution of the microorganisms from the filter in order to detect the microorganisms.

[0030] For example, a large dilute aqueous sample can be filtered to retain on the filter of the filter unit the microorganisms, if present, by size, charge and/or affinity. The

microorganisms can be retained on a first side of a filter, which can then be oriented to face a lysate collection vessel during subsequent cell disruption and centrifugation processes.

Optionally, a diluent (e.g., a buffer) can be contacted with the filter, and microorganisms, if present in the sample. In such embodiments, the filtrand on the filter plus any additional diluent that is added can form the "sample concentrate", cells (e.g., microorganisms) in the sample concentrate can be disrupted (e.g., by "bead-beating"), and the resulting cell lysate can be moved (e.g., by centrifugation) to a predefined location in the lysate collection vessel, where a portion of the lysate can be removed for analysis. The analysis can detect the presence/absence of an analyte that is associated with (e.g., uniquely associated with) the target microorganism.

[0031] Systems of the present disclosure can include parts of a container assembly, the parts being configured to facilitate the processes of filtering and centrifuging, as well as facilitating a mechanical cell disruption process. In addition, the systems and methods of the present disclosure allow for concentration of a large volume sample down to a very small volume, for example, from about 1 L down to about 250 to 1000 microliters. After disruption of the cells, the cell lysate further can be concentrated using the commercially available devices such as Centricon (EMD Millipore, Billerica, MA) centrifugal devices.

[0032] Particularly, in any embodiment, the systems and methods of the present disclosure can include performing a concentration step comprising filtering an original sample using a filter that is configured to retain one or more target microorganisms to form a filtrand on one side of the filter; optionally adding one or more diluents to the filtrand and using the filtrand and any added diluents to form a sample concentrate; using particles to disrupt cells, if present, in the sample concentrate to form a liquid cell lysate; moving the resulting liquid cell lysate (e.g., by centrifugation) to a predefined location in the lysate collection vessel; and analyzing a portion of the liquid lysate to detect an analyte. The analyte may be associated with (e.g., uniquely associated with) a target microorganism.

[0033] In some existing systems and methods, portions of the sample can become irreversibly trapped in the filter during filtration. Trapping can be overcome using isoporous filters, however, filtration through isoporous filters can be slow, and the pores of the isoporous filter can be easily and rapidly plugged during filtration. Other existing systems and methods rely on the elution of analytes from the filter and subsequent detection of the eluted analytes. However, an advantage of methods of the present disclosure is that elution of target microorganisms from the filter membrane is not necessary in order to detect the analytes associated with the target microorganism because the disruption process disintegrates cells that are washed off the membrane filter and dislodges and/or disintegrates the membrane filter and cells that remain trapped on or in the membrane filter.

[0034] In some embodiments, rapid detection can refer to detection in no greater than 8 hours; in some embodiments, no greater than 6 hours; in some embodiments, no greater than 5 hours; in some embodiments, no greater than 4 hours; in some embodiments, no greater than 3 hours; in some embodiments, no greater than 2 hours; and, in some embodiments, no greater than 1 hour.. The detection time, however, can be dependent upon the type and quantity of microorganisms being detected and/or on the detection technology used in the method.

[0035] Samples to be analyzed for a target microorganism can be obtained in a variety of ways. For example, in any embodiment, the sample to be analyzed itself may be a liquid sample, such as a dilute liquid sample and/or a dilute aqueous sample. In some embodiments, the sample can include the liquid resulting from washing or rinsing a source of interest (e.g., a surface, fomite, etc.) with an appropriate suspending medium (e.g., a buffer solution). In any embodiment, a liquid sample can be prefiltered (e.g., to remove relatively large (e.g., >50 micron) particles) before using the prefiltered sample in the method and device of the present disclosure.

[0036] The term "source" can be used to refer to a substance or environmental surface desired to be tested for the presence of target microorganisms. The source can be a solid, a liquid, a semi-solid, a gelatinous material, and combinations thereof. In some embodiments, the source can be interrogated by a substrate (e.g., a swab or a wipe) that was used, for example, to collect residual matter from a surface of interest. In some embodiments, a liquid substance can include a solid substrate, which can be further broken apart (e.g., during an agitation or dissolution process) to enhance retrieval of the source and any target microorganism associated therewith. A surface of interest can include at least a portion of a variety of surfaces, including, but not limited to, walls (including doors), floors, ceilings, drains, refrigeration systems, ducts (e.g., air ducts), vents, toilet seats, handles, doorknobs, handrails, bedrails (e.g., in a hospital), countertops, tabletops, eating surfaces (e.g., trays, dishes, etc.), working surfaces, equipment surfaces, clothing, etc., and combinations thereof. All or a portion of the source can be used to obtain a sample that is to be analyzed using the methods of the present disclosure. For example, a "source" can be a water supply (e.g., a potable water supply) or water (e.g., potable water) moving through a pipeline, and a relatively large volume sample can be taken from that source to form a sample that will be tested with the systems and methods of the present disclosure. Therefore, the "sample" can also be from any of the above-described sources.

[0037] The term "fomite" is generally used to refer to an inanimate object or substrate capable of carrying and/or transferring potentially-infectious microorganisms. Fomites can include, but are not limited to, cloths, mop heads, towels, sponges, wipes, eating utensils, coins, paper money, cell phones, clothing (including shoes), doorknobs, feminine products, diapers, etc., portions thereof, and combinations thereof.

[0038] The term "analyte" is generally used to refer to a substance or biomolecule to be detected (e.g., by a laboratory or field test). A sample can be tested for the presence and/or quantity of particular analytes that are associated with (e.g., uniquely associated with) a target microorganism or group of microorganisms. Examples of analytes can include, but are not limited to, biomolecules (e.g., polypeptides, polynucleotides, polysaccharides, and the like) or small molecules associated with a target microorganism.

[0039] A variety of testing methods can be used to identify or quantitate an analyte of interest, including, but not limited to, biochemical assays (e.g. immunoassay, nucleic acid amplification), or a combination thereof. In some embodiments, analytes of interest can be detected genetically; immunologically; colorimetrically; fluorimetrically; lumimetrically; by detecting, for example, a polynucleotide or antigen released from a cell in the sample; by detecting light that is indicative of the analyte associated with the target microorganism; by detecting light by absorbance, reflectance, fluorescence, or combinations thereof; or combinations thereof.

[0040] Specific examples of testing methods that can be used include, but are not limited to, enzyme assays, antigen-antibody interactions, molecular sensors (affinity binding), thermal analysis, spectroscopy (e.g., mass spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, infrared (IR) spectroscopy, x-ray spectroscopy, attenuated total reflectance spectroscopy, Fourier transform spectroscopy, gamma-ray spectroscopy, etc.), spectrophotometry (e.g., absorbance, reflectance, fluorescence, luminescence (e.g., detection of ATP bioluminescence), colorimetric detection etc.), electrochemical analysis, genetic techniques (e.g., polymerase chain reaction (PCR), transcription mediated amplification (TMA), hybridization protection assay (HP A), DNA or RNA molecular recognition assays, etc.), adenosine triphosphate (ATP) detection assays, immunological assays (e.g., enzyme- linked immunosorbent assay (ELISA)), or a combination thereof.

[0041] The term "microorganism" is generally used to refer to any prokaryotic or eukaryotic microscopic organism that can be captured by a filter, including without limitation, one or more of bacteria (e.g., motile or vegetative, Gram positive or Gram negative), viruses (e.g.,

Norovirus, Norwalk virus, Rotavirus, Adenovirus, DNA viruses, RNA viruses, enveloped, non- enveloped, human immunodeficiency virus (HIV), human Papillomavirus (HPV), etc.), bacterial spores or endospores, algae, fungi (e.g., yeast, filamentous fungi, fungal spores), prions, mycoplasmas, and protozoa. In some cases, the microorganisms of particular interest are those that are pathogenic, and the term "pathogen" is used to refer to any pathogenic microorganism. Examples of pathogens can include, but are not limited to, members of the family Enter obacteriaceae, or members of the family Micrococaceae, or the genera

Staphylococcus spp., Streptococcus, spp., Pseudomonas spp., Enterococcus spp., Salmonella spp., Legionella spp., Shigella spp., Yersinia spp., Enterobacter spp., Escherichia spp., Bacillus spp., Listeria spp., Campylobacter spp., Acinetobacter spp., Vibrio spp., Clostridium spp., and Corynebacterium spp. Particular examples of pathogens can include, but are not limited to, Escherichia coli including enterohemorrhagic E. coli e.g., serotype 0157:H7, 0129:H11;

Pseudomonas aeruginosa; Bacillus cereus; Bacillus anthracis; Salmonella enteritidis;

Salmonella enterica serotype Typhimurium; Listeria monocytogenes; Clostridium botulinum; Clostridium perfringens; Staphylococcus aureus; methicillin-resistant Staphylococcus aureus; Campylobacter jejuni; Yersinia enterocolitica; Vibrio vulnificus; Clostridium difficile; vancomycin-resistant Enter ococcus; Cronobacter sakazakii; and coliforms. Environmental factors that may affect the growth of a microorganism can include the presence or absence of nutrients, pH, moisture content, oxidation-reduction potential, antimicrobial compounds, temperature, atmospheric gas composition and biological structures or barriers.

[0042] The term "biomolecule" is generally used to refer to a molecule, or a derivative thereof, that occurs in or is formed by an organism. For example, a biomolecule can include, but is not limited to, at least one of an amino acid, a nucleic acid, a polypeptide, a protein, a polynucleotide, a lipid, a phospholipid, a saccharide, a polysaccharide, and combinations thereof. Specific examples of biomolecules can include, but are not limited to, a metabolite (e.g., staphylococcal enterotoxin), an allergen (e.g., peanut allergen(s ), egg allergen(s ), pollens, dust mites, molds, danders, or proteins inherent therein, etc.), a hormone, a toxin (e.g., Bacillus diarrheal toxin, aflatoxin, Clostridium difficile toxin etc.), RNA (e.g., mR A, total RNA, tR A, etc.), DNA (e.g., plasmid DNA, plant DNA, etc.), a tagged protein, an antibody, an antigen, ATP, and combinations thereof.

[0043] The term "diluent" is generally used to refer to a liquid added to a source material to disperse, dissolve, suspend, emulsify, wash and/or rinse the source. A diluent can be used in forming a liquid composition, from which a sample to be analyzed using the methods of the present disclosure can be obtained. In some embodiments, the diluent is a sterile liquid. In some embodiments, the diluent can include a variety of additives, including, but not limited to, surfactants, or other suitable additives that aid in dispersing, dissolving, suspending or emulsifying the source for subsequent analyte testing; rheological agents; antimicrobial neutralizers (e.g., that neutralize preservatives or other antimicrobial agents); pH buffering agents; enzymes; an agent to neutralize sanitizers (e.g., sodium thiosulfate neutralization of chlorine); stabilizing agents (e.g., that stabilize the analyte(s) of interest, including solutes, such as sodium chloride, sucrose, etc.); or a combination thereof. In some embodiments, the diluent can include sterile water (e.g., sterile double-distilled water (ddttO)); one or more organic solvents to selectively dissolve, disperse, suspend, or emulsify the source; aqueous organic solvents, or a combination thereof. In some embodiments, the diluent is a sterile buffered solution (e.g., Butterfield's Buffer, available from Edge Biological, Memphis TN).

[0044] The term "agitate" and derivatives thereof is generally used to describe the process of giving motion to a liquid composition, for example, to mix or blend the contents of such liquid composition. A variety of agitation methods can be used, including, but not limited to, manual shaking, mechanical shaking, ultrasonic vibration, vortex stirring, manual stirring, mechanical stirring (e.g., by a mechanical propeller, a magnetic stir bar, or another agitating aid, such as ball bearings), manual beating, mechanical beating, blending, kneading, and combinations thereof. [0045] The term "filtering" is generally used to refer to the process of separating matter by size, charge and/or function. For example, filtering can include separating soluble matter and a solvent (e.g., diluent) from insoluble matter, or filtering can include separating soluble matter, a solvent and relatively small insoluble matter from relatively large insoluble matter. As a result, a liquid composition can be "pre-filtered" to obtain a sample that is to be analyzed using the methods of the present disclosure. A variety of filtration methods can be used, including, but not limited to, passing the liquid composition (e.g., comprising a source of interest, from which a sample to concentrated can be obtained) through a filter, other suitable filtration methods, and combinations thereof.

[0046] A "filter" is generally used to describe a device used to separate the soluble matter (or soluble matter and relatively small (e.g., <10 μπι diameter, <5 μπι diameter, <1 μπι diameter, <0.5 μπι diameter, <0.2 μπι diameter) insoluble matter) and solvent from the insoluble matter (or relatively large insoluble matter) in a liquid composition and/or to filter a sample during sample concentration. Examples of filters can include, but are not limited to, a woven or non- woven mesh (e.g., a wire mesh, a cloth mesh, a plastic mesh, etc.), a woven or non-woven polymeric web (e.g., comprising polymeric fibers laid down in a uniform or nonuniform process, which can be calendered), a surface filter, a membrane (e.g., a ceramic membrane (e.g., ceramic aluminum oxide membrane filters available under the trade designation

ANOPORE from Whatman Inc., Florham Park, NJ), a polycarbonate membrane (e.g., track- etched polycarbonate membrane filters available under the trade designation NUCLEOPORE from Whatman, Inc.)), a polyester membrane (e.g., comprising track-etched polyester, etc.), a sieve, a frit, filter paper, foam, etc., and combinations thereof.

[0047] In any embodiment, the filter can be configured to separate a target microorganism from a sample, for example, by size, charge, and/or affinity. For example, in some

embodiments, the filter can be configured to retain a target microorganism, such that a filtrand retained on the filter comprises the target microorganism.

[0048] In any embodiment, the filter can be configured to retain at least 30% of the target microorganisms in a sample; in any embodiment, at least 50%; in any embodiment, at least 80%; in any embodiment, at least 85%; in any embodiment, at least 90%; and in any embodiment, at least 95% of the target microorganisms in the sample.

[0049] Additional examples of suitable filters are described in co-pending PCT Publication No. W02011/156251 (Rajagopal, et al.), which claims priority to US Patent Application No. 61/352,229; PCT Publication No. W02011/156258 (Mach et al), which claims priority to US Patent Application No. 61/352,205; PCT Publication No. W02011/152967 (Zhou), which claims priority to US Patent Application Nos. 61/350,147 and 61/351,441; and PCT Publication No. W02011/153085 (Zhou), which claims priority to US Patent Application Nos. 61/350, 154 and 61/351,447, all of which are incorporated herein by reference in their entirety.

[0050] In some embodiments, the term "filtrand" is generally used to describe the solid remaining after a liquid source (e.g., water to be tested) has been filtered to separate insoluble matter from soluble matter. Such a filtrand can be diluted or resuspended to form a sample concentrate to be further processed in a method of the present disclosure. The filtrand may be present on one surface or side of the filter, and/or may have penetrated at least partially into the depth of the filter.

[0051] "Bead beating" is a term used herein to describe a method of extracting

polynucleotides (e.g., DNA) from cells (e.g., microbial cells). A method of obtaining microbial DNA from soil samples is described by Yeates et al. ("Methods for microbial extraction from soil for PCR amplification"; 1998; C. Yeates, M R. Gillings, A D. Davidson, N. Altavilla, and D.A. Veal; Biological Procedures Online; Vol. 1, pp. 40-47) and is incorporated herein by reference in its entirety. In addition, Lavender et al. ("A cross comparison of QPCR to agar- based or defined substrate test methods for the determination of Escherichia coli and enterococci in municipal water quality monitoring programs"; 2009; J.S. Lavender and J.L. Kinzelman; Water Research; Vol. 43, pp 4967-4979) and Haugland et al. ("Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis"; 2005; R.A. Haugland, S.C. Siefring, L.J. Wymer, K.P. Brenner, and A. P. Dufour; Water Research; Vol. 39, pp 559-568), both of which are incorporated herein by reference in their entireties, describe methods of extracting microbial DNA from microbes captured on membrane filters.

[0052] Bead beating methods typically include placing cells (e.g., microorganism cells) into a container with a buffer and a plurality of particles (e.g., glass beads) and subjecting the container to agitation forces (e.g., in a bead beater such as, for example, a Mini-Beadbeater-16 available from BioSpec Products, Bartlesville, OK) sufficient to cause disruption of the cells in the container.

[0053] In some embodiments, the g-force exerted on the liquid cell lysate in the subsequent centrifugation step can aid in moving the liquid lysate to a predefined location and aid in separating the liquid lysate from the particulate cell-lysing agent and from fragments of the filter, if present in the liquid cell lysate. In any embodiment, the centrifugation step will cause the liquid cell lysate to move toward the closed end of the lysate collection vessel. In addition, cell -disrupting particles and/or fragments of the filter may sediment to the closed end of the lysate collection vessel where the liquid lysate typically forms a discrete layer on top of the particles and the fragments. [0054] The phase "substantially transparent" is generally used to refer to a body or substrate that transmits at least 50% of electromagnetic radiation having wavelengths at a selected wavelength or within a selected range of wavelengths in the ultraviolet to infrared spectrum (e.g., from about 200 nm to about 1400 nm; "UV-IR"), in some embodiments, at least about 75% of a selected wavelength (or range) in the UV-IR spectrum, and in some embodiments, at least about 90% of a selected wavelength (or range) in the UV-IR spectrum.

[0055] The phrase "substantially non-transparent" is generally used to refer to a body or substrate that transmits less than 50% of electromagnetic radiation having wavelengths at a selected wavelength or within a selected range of wavelengths in the ultraviolet to infrared spectrum (e.g., from about 200 nm to about 1400 nm; "UV-IR"), in some embodiments, less than 25% of a selected wavelength (or range) in the UV-IR spectrum, and in some

embodiments, less than 10% of a selected wavelength (or range) in the UV-IR spectrum.

[0056] Various details of "substantially transparent" and "substantially non-transparent" materials are described in PCT Patent Publication No. WO 2011/063332, which is incorporated herein by reference in its entirety.

[0057] The terms "hydrophobic" and "hydrophilic" are generally used as commonly understood in the art. Thus, a "hydrophobic" material has relatively little or no affinity for water or aqueous media, while a "hydrophilic" material has relatively strong affinity for water or aqueous media. The required levels of hydrophobicity or hydrophilicity may vary depending on the nature of the sample, but may be readily adjusted based on simple empirical

observations of a liquid sample when applied to various hydrophobic or hydrophilic surfaces.

[0058] FIGS. 1-4 illustrate components of two sample detection systems according to the present disclosure, wherein like numerals represent like elements. FIG. 1 illustrates a sample detection system 100 according to one embodiment of the present disclosure. In any embodiment, the sample detection system 100 can be used to concentrate a sample and to form a cell lysate (e.g., in a collection vessel) in order to rapidly detect an analyte that is associated with a target cell (e.g., a microorganism), if present, in the sample.

[0059] As shown in FIG. 1, in some embodiments, the system 100 can include a lysate collection vessel 102. The lysate collection vessel 102 can be configured to be coupled with a filter unit 108, such that the lysate collection vessel 102 and the filter unit 108 can be removably or permanently coupled together to form a cell disruption container 500. Preferably, the lysate collection vessel 102 and/or the filter unit 108 is adapted to form a liquid-tight seal when the lysate collection vessel and the filter unit are coupled together. The system further comprises a plurality of cell-disrupting particles 126.

[0060] The lysate collection vessel 102 can be adapted to contain material that is to be analyzed, for example, to detect an analyte that indicates a presence of one or more target microorganisms in a sample. The material generally comprises a filtrand collected by the filter unit and a relatively small volume of liquid. The lysate collection vessel 102 can be sized and shaped, as desired, to accommodate the material to be analyzed, and the shape and

configuration of the lysate collection vessel 102 and the filter unit 108 is shown by way of example only.

[0061] As shown in FIG. 1, the lysate collection vessel 102 can be an elongated tube having a closed end 118 and an open end 116. The lysate collection vessel 102 has an interior cavity 105 that tapers from a wider cross-sectional area proximate the open end 116 to a narrower cross-sectional area proximate the closed end 118.

[0062] The filter unit 108 comprises a filter housing 132 that can include a first end 144 and a second end 145. The second end 145 can be opposite the first end 144. The first end 144 of the filter housing 132 can have an opening (e.g., to receive a sample into the filter unit 108 as described herein) and can be configured to be operatively coupled to the lysate collection vessel 102, and particularly, the open end 116 of the lysate collection vessel 102, such that coupling the filter unit 108 and the lysate collection vessel 102 together joins the open end 116 of the lysate collection vessel 102 to the opening at the first end 144 of the filter housing 132.

[0063] The second end 145 of the filter housing 132 comprises an outlet 139. A fluid pathway extends from the opening at the first end 144 to the outlet 139. A membrane filter 112 is disposed in the fluid pathway. The filter unit 108 is configured so that liquid passing through the fluid pathway from the first end 144 to the outlet 139 passes through the membrane filter 112.

[0064] It should be understood that a variety of shapes and dimensions of the lysate collection vessel 102 and the filter unit 108 can be used. In addition, a variety of coupling means can be employed to removably and/or permanently couple the lysate collection vessel 102 and the filter unit 108, including, but not limited to, screw threads (as shown or otherwise), a clamp (e.g., a spring-loaded clamp, a snap-type clamp, etc.); a clip (e.g., a spring-loaded clip, etc.); ties (e.g., wire ties); one or more magnets; tape; an adhesive; a cohesive; snap-fit engagement (e.g., wherein the filter unit 108 functions as a flip-top cap); press-fit engagement (also sometimes referred to as "friction-fit engagement" or "interference-fit engagement"); thermal bonding (e.g., heat and/or pressure applied to one or both of the components to be coupled); welding (e.g., sonic (e.g., ultrasonic) welding); other suitable coupling means; and combinations thereof.

[0065] The open end 116 of the lysate collection vessel 102 is configured to be coupled to the first end 144 of the filter unit 108. As shown in FIG. 1, in some embodiments, the open end 116 of the lysate collection vessel 102 can be dimensioned to be received in the filter unit 108 (i.e., the filter housing 132), and the threads 123 of the lysate collection vessel can be configured to cooperate and engage with the protrusions 129 (see FIG. 1) of the filter unit 108. Such engagement can allow the lysate collection vessel 102 and the filter unit 108 to be screwed together, and particularly, to be removably coupled together. The filter unit 108 and the lysate collection vessel 102 can be removably coupled together by other means (e.g., via any of the removable coupling means described herein. In some embodiments, the lysate collection vessel 102 can instead be dimensioned to receive at least a portion of the filter housing 132.

[0066] As shown in FIG. 2, the lysate collection vessel 102 can further include a wall 138 (i.e., an internal wall) that defines at least a portion of an inner surface of the lysate collection vessel 102. The wall 138 extends from the open end 116 to the closed end 118.

[0067] By way of example only, the lysate collection vessel 102 is illustrated as being the larger portion of the cell disruption container 500, such that the lysate collection vessel 102 acts as the tube or reservoir of the cell disruption container 500, and the filter unit 108 acts as the cap or cover of the cell disruption container 500. However, it should be understood that the sizes, shapes, and relative sizes of the components of the cell disruption container 500 can be adjusted to suit a particular sample or situation.

[0068] The filter housing 132 is configured to house and retain a filter 112. By way of example only, as shown in FIG. 1, the filter 112 can be positioned against a filter support 131 disposed in the filter housing 132. As further shown in FIG. 1, the filter support 131 can include or at least partially define a plurality of apertures 137 that are disposed in the fluid pathway that extends through the filter housing 132 to the outlet 139 (see FIGS. 1-2). Such an outlet 139 can function as the outlet of the filter unit 108, and can be coupled to a suction source (not shown) to perform a filtration step that moves a liquid sample through the filter unit 108.

[0069] In some embodiments, a portion of the filter 112 (e.g., a periphery thereof) can be ultrasonically welded to the filter housing 132 (e.g., a ledge or flange, not shown, within the housing 132 on which the periphery of the filter 112 will sit). In any embodiment, a porous layer (e.g., a nonwoven fabric, not shown) can be provided to help support the filter 112 during filtration and to enhance the integrity of the ultrasonic seal. Such an ultrasonic weld can provide a hermetic seal, in addition to providing means for coupling the filter 112 to the filter housing 132. Still, in some embodiments, the filter 112 can be integrally formed with the filter housing 132 or sandwiched between two mating parts.

[0070] The filter unit 108 (or the filter housing 132) can include a first end 144 and a second end 145. The filter 112 is disposed in the fluid pathway such that a fluid sample moving through the fluid pathway passes through the filter 112. A sample filtrate (not shown) resulting from a liquid passing through the filter 112 exits the filter unit 108 at the outlet 139 at the second end 145. The second end 145 is configured to be coupled (e.g., by inserting the second end 145 of the filter housing 132 into a vacuum flask or by attaching a vacuum hose to the outlet 139) to a source of negative pressure.

[0071] As shown in FIGS. 1 and 2, in some embodiments, the outlet 139 of the filter unit 108 that is used in the filtration step of any embodiment of the method of the present disclosure can be sealed or closed, for example, by a closure element 117. For example, the closure element 117 can be coupled to the second end 145 (e.g., coupled to the outlet 139) following the filtration step, and particularly when the filter unit 108 and the lysate collection vessel 102 are coupled together to form the cell disruption container 500.

[0072] When coupled to the second end, the closure element 117 forms a barrier to prevent passage of a microorganism into the filter unit through the outlet. Optionally, the closure element 117 also seals the second end 145 of the filter unit 108 so that liquid cannot enter or escape the filter unit through the outlet 139. The closure element 117 can be fabricated from a variety of materials including, but not limited to a fibrous material (e.g., a woven material, a nonwoven material) configured (e.g., via porosity) to prevent passage of microorganisms (e.g., bacteria, yeast, mold, spores) therethrough, rubber, plastic, metal, glass, or combinations thereof. In any embodiment, the closure element can be fabricated from material that can be sterilized using suitable sterilization processes that are known in the art. Optionally, the closure element 117 can be fabricated from a material that also prevents passage of a liquid (e.g., an aqueous liquid) therethrough. In any embodiment, the closure element 117 may be a plastic or metal film that can be attached (e.g., via a pressure sensitive adhesive) to seal the second end 145 (e.g., to seal the outlet 139 at the second end).

[0073] The closure element 117 can be coupled to the outlet 139 via friction fit, for example. Alternatively, the closure element 117 can be coupled to the outlet 139 or second end 145 of the filter housing using an adhesive (e.g., a pressure-sensitive adhesive) or other suitable secural means.

[0074] The filter 112 can be configured to retain a target microorganism from the sample, if present. The filter 112 can be configured either by effective pore size, charge, affinity, or other suitable means for retaining the target microorganism.

[0075] Particularly, exemplary filters 112 can be made by, for example, TIPS (thermally induced phase separation) process, SIPS (solvent induced phase separation) process, VIPS (vapor induced phase separation) process, stretching process, track-etching, or electrospinning (e.g., PAN fiber membranes). Suitable membrane materials include, for example, polyolefins (e.g., polyethylene and/or polypropylene), ethylene-chlorotrifluoroethylene copolymer, poly aery lonitrile, polycarbonate, polyester, polyamide, polysulfone, polyethersulfone, polyvinylidene fluoride (PVDF), cellulose ester, and/or combinations thereof. [0076] Suitable membranes may be characterized as porous membranes or as nanofiber membranes. Nanofiber filter membranes can have the fiber diameter less than 5 μιη such as, for example, less than 1 μιη. Nanofiber membranes may be prepared from, for example, polyacrylonitrile, polyvinylidene fluoride, a cellulose ester, polyvinyl acetate, polyvinyl alcohol, polyvinyl butyral, and/or combinations thereof.

[0077] Certain TIPS polyolefin membranes can be prepared so that they possess a single, homogeneous zone of membrane structure, each zone having a different pore microstructure. In other cases, a TIPS membrane may be prepared as a multi-zone membrane that includes two or more zones, each zone having a different pore microstructure. A multi-zone TIPS membrane may contain distinct zones or, alternatively, may possess a transition zone between two otherwise distinct zones.

[0078] Exemplary filter membranes include membranes that are described in, for example, in U.S. Patent No. 4,539,256; U.S. Patent No. 4,726,989; U.S. Patent No. 4,867,881; U.S. Patent No. 5,220,594; U.S. Patent No. 5,260,360; PCT Publication No. WO2010/078234; PCT Publication No. WO2010/071764; PCT Publication No. WO2011/152967; and PCT Publication

No. WO2011/151085.

[0079] The filter 112 can include a first side 113 that is oriented toward the first end 144 (i.e., the first side faces the source of liquid sample during the filtration step of the method of the present disclosure and which faces the lysate collection vessel 102 during the cell disruption step of the method of the present disclosure) and a second side 116 that is oriented toward the second end 145 (e.g., the second side faces the outlet 139). During the filtration step of the method of the present disclosure, the sample is separated into a filtrand 151 that is retained on the first side 113 of the filter 112 and a filtrate (not shown) that passes through the filter. The filtrate can either be discarded or can be re-run through the filter, or otherwise processed.

[0080] While the filtrand may be described as being retained on the first side 113 of the filter 112, this does not necessarily mean that filtrand is not present in any of the depth of the filter 112 (as may be the case for a porous polymer film with a wide distribution of pore sizes). Rather, this means that the sample was filtered through the filter 112 from the first side 113 to a second (opposite) side, and that the first side 113 is the side of the filter 112 that a sample contacts as it is passing through the filter during filtration and the first side 113 is the side of the filter 112 that faces the lysate collection vessel 102 when the cell disruption container 500 of the present disclosure is formed. It is possible that at least some of the filtrand may be present below the surface of the first side 113 of the filter 112, that is, at least partially into the depth of the filter 112. However, particular filters or types of filters (e.g., multi-zone filters, isoporous filters) can be employed to inhibit the sample from moving so far into the depth of the filter 112 that extraordinary time and/or efforts are required to lyse the microorganisms retained by the filter 1 12.

[0081] Returning to FIG. 1, by way of further example, the filter unit 108 includes an inner surface that includes one or more protrusions 129, and the lysate collection vessel 102 includes an outer surface 122 that includes one or more tracks or threads 123 adjacent the open end 1 16. The protrusions 129 of the filter unit 108 are configured to cooperate and engage with the threads 123 of the lysate collection vessel 102, such that the filter unit 108 and the lysate collection vessel 102 can be coupled together.

[0082] The specific style of protrusions 129 and threads 123 shown in FIG. 1 includes a series of circumferentially-spaced protrusions 129 and threads 123 so that any protrusion 129 on the filter unit 108 can be coupled to any thread 123 on the lysate collection vessel 102 and turned from an unlocked to a locked position by rotating the filter unit 108 and the lysate collection vessel 102 relative to one another (e.g., 90 degrees, if 4 sets of protrusions

129/threads 123 are employed and evenly spaced about the inner surface 120 of the filter unit 108 and the outer surface 122 of the lysate collection vessel 102). The illustrated coupling mechanism between the lysate collection vessel 102 and the filter unit 108 is shown by way of example only as an efficient means for closing the lysate collection vessel 102.

[0083] Thus, in the illustrated embodiment of the container 500 of FIGS. 1-2, the means for operatively coupling the open end of the lysate collection vessel to the first end of the filter unit include a first engagement structure (i.e., the threads 123) that is an integral part of the lysate collection vessel and a complementary second engagement structure (i.e., the protrusions 129) that is an integral part of the filter unit.

[0084] In some embodiments, the lysate collection vessel 102, the filter unit 108, and the closure element 1 17 can be coupled together in such a way that the interior of the cell disruption container 500 is sealed from ambience (e.g., forming a liquid-tight seal, a hermetic seal, or a combination thereof). For example, in some embodiments, one or more seals (e.g., O- rings) can be employed between the lysate collection vessel 102 and the filter unit 108, or one or both of the lysate collection vessel 102 and the filter unit 108 can include one or more seals (e.g., O-rings).

[0085] The lysate collection vessel 102 and the filter unit 108 can be formed of a variety of materials, including, but not limited to, polymeric materials, metals (e.g., aluminum, stainless steel, etc.), ceramics, glasses, and combinations thereof. Examples of polymeric materials can include, but are not limited to, polyolefins (e.g., polyethylene, polypropylene, combinations thereof, etc.), polycarbonate, acrylics, polystyrene, high density polyethylene (HDPE), polypropylene, other suitable polymeric materials capable of forming a self-supporting container, or a combination thereof. The term "self-supporting" is generally used to refer to an object that does not collapse or deform under its own weight. For example, a bag is not "self- supporting" in that it does not maintain its shape, but rather collapses or distorts, under its own weight. The lysate collection vessel 102 and the filter unit 108 can be formed of the same or different materials.

[0086] The lysate collection vessel 102 and the filter unit 108, or a portion thereof, can be substantially transparent, opaque (i.e., substantially non-transparent), or somewhere in between (e.g., translucent), and can be any suitable size, depending on the type, amount and/or size of sample to be analyzed, and the type, amount and/or size of concentrate to be collected and interrogated. In some embodiments, the lysate collection vessel 102 can have a capacity of at least about 1 mL, at least about 5 mL, at least about 10 mL, at least about 25 mL, or at least about 50 mL. That is, in some embodiments, the capacity, or volume, of the lysate collection vessel 102 can range from about 1 mL to about 50 mL, and in some embodiments, can range from about 1 mL to about 10 mL.

[0087] In addition, as shown in FIG. 1, in some embodiments, a filter unit gasket (e.g., an O- ring) 135 can be employed in the cell disruption container 500 between the filter housing 132 (i.e., between the filter 112) and the lysate collection vessel 102. Such a filter unit gasket 135 can enhance the seal between the filter housing 132 and the lysate collection vessel 102.

[0088] A system of the present disclosure further comprises a plurality of particles 126. The particles 126 are adapted (e.g., compositionally and/or sized and or shaped) to disrupt cells (e.g., microbiological cells) when placed in a suitable container that is subjected to suitable external agitational or vibrational force. The particles 126 can have a mean particle diameter of about 0.1 mm to about 2.0 mm, for example. Preferably, the particles 126 have a mean particle diameter of about 0.1 mm to about 0.5 mm. In any embodiment, the particles 126 can have a density of about 2.5 g/cc to about 7.9 g/cc. The particles 126 can be made from any suitable material including, but not limited to, silica, zirconium, silicon carbide, garnet, steel, and combinations thereof.

[0089] In any embodiment, the system of the present disclosure optionally may comprise a cell disruption buffer (not shown). The cell disruption buffer may be disposed in the lysate collection vessel, for example. Alternatively, the cell disruption buffer may be provided in a separate container (not shown) and can be deposited into the container 500 of FIGS. 1 and 2 prior to performing the agitation step of the method of the present disclosure. The cell disruption buffer may comprise one or more compounds that buffer the cell lysate within a predefined pH range (e.g., 6.5 to 8.5, inclusive). The cell disruption buffer optionally can comprise one or more compounds (e.g., betaine, trehalose, serum albumin, and/or a surfactant) for stabilizing biomolecules released from cells. [0090] In some embodiments, a filter unit 108 of the present disclosure can be a part of a filtration assembly that is used in a system and a method of the present disclosure. FIGS. 3and 4 show various views of one embodiment of a filtration assembly 450 used to move a liquid sample through the filter unit in order to capture a target microorganism from the sample onto and/or in the membrane filter. The filtration assembly 450 can be used in a system according to the present disclosure. Thus, in any embodiment of a system of the present disclosure, the filter unit is a part of the filtration assembly described herein.

[0091] As shown in FIGS. 3 and 4, filtration assembly 450 comprises a receptacle portion 306 coupled to the filter unit 108 via a connector subassembly 220. In some embodiments, the connector subassembly 220 or parts thereof can be coupled (e.g., removably or permanently) to the receptacle portion 306, and in some embodiments, the connector subassembly 220 or parts thereof can be integrally formed with the receptacle portion 306. By way of example only, the connector subassembly 220 is illustrated in FIGS. 3 and 4 as being adapted to be coupled between the receptacle portion 306 and the filter unit 108.

[0092] The receptacle portion 306 can include a first end 354 that at least partially defines a reservoir 358, and a second end 356 configured to be coupled to the filter unit 108 (i.e., either directly or indirectly). The first end 354 can either be an open end or a closed end. By way of example only, in FIGS. 3 and 4, the first end 354 is closed and the second end 356 is open, such that a sample can be added to the reservoir 358 via the second end 356, e.g., prior to coupling the receptacle portion 306 to the filter unit 108. However, in some embodiments, the first end 354 can be open to allow a sample to be added to the reservoir 358 via the first end 354, and the first end 354 can remain open, or it can be closed by a cover or lid.

[0093] In some embodiments, the receptacle portion 306 of FIGS. 3 and 4 can have a capacity of at least about 1 mL, at least about 5 mL, at least about 10 mL, at least about 25 mL, at least about 50 mL, at least about 100 mL, at least about 250 mL, or at least about 1 liter. That is, in some embodiments, the capacity, or volume, of the receptacle portion 306 can range from about 1 mL to about 1000 mL, and in some embodiments, can range from about 10 mL to about 250 mL. In any embodiment, the capacity, or volume, of the receptacle portion 306 can be no more than about 25 mL; and in any embodiment, can be no more than about 50 mL; and in any embodiment, can be no more than about 100 mL; and in any embodiment, can be no more than about 250 mL; and in any embodiment, can be no more than about 500 mL; and in any embodiment, can be no more than about 1000 mL.

[0094] The filtration assembly 450, and particularly, the receptacle portion 306, can be adapted to contain a sample that is to be analyzed, for example, for one or more analytes of interest. The filtration assembly 450 (or the receptacle portion 306) can be sized and shaped, as desired, to accommodate the sample to be analyzed, and the shape and configuration of the receptacle portion 306 and the filter unit 108 is shown by way of example only.

[0095] As shown in FIG. 3, in some embodiments, the connector subassembly 220 can include a connector 222 and a connector subassembly gasket 224. The connector subassembly gasket 224 can be configured to be coupled between the connector 222 and the receptacle portion 306. In some embodiments, as shown, the connector subassembly gasket 224 can be dimensioned to be received within the second end 356 of the receptacle portion 306 and can further include a bore or an aperture 226 dimensioned to receive a first end 223 of the connector 222. For example, aperture 226 can be shaped and dimensioned to receive a connection tube 221 of the connector 222. Such a configuration can aid in creating a seal (e.g., a liquid-tight seal, a hermetic seal, or a combination thereof) between the receptacle portion 306 and the filter unit 108, while maintaining fluid communication between the receptacle portion 306 and the filter unit 108 via the aperture 226 formed therein. The shape and configuration of the connector subassembly gasket 224, the connector 222, and the second end 356 of the receptacle portion 306 are shown by way of example only; however, it should be understood that any shapes and configurations and relative structures of these elements can be employed to achieve the same function.

[0096] As mentioned above, the connector 222 can include a first end 223 configured to be coupled to the receptacle portion 306 and a second end 225 configured to be coupled to the filter unit 108. A variety of coupling means can be employed to removably and/or permanently couple the receptacle portion 306 and the connector 222, as well as the connector 222 and the filter unit 108, including, but not limited to, screw threads (as shown), a clamp (e.g., a spring- loaded clamp, a snap-type clamp, etc.); a clip (e.g., a spring-loaded clip, etc.); ties (e.g., wire ties); one or more magnets; tape; an adhesive; a cohesive; a hook-and-loop fastener; snap-fit engagement (e.g., wherein the filter housing 132 functions as a flip-top cap); press-fit engagement (also sometimes referred to as "friction-fit engagement" or "interference-fit engagement"); thermal bonding (e.g., heat and/or pressure applied to one or both of the components to be coupled); welding (e.g., sonic (e.g., ultrasonic) welding); other suitable coupling means; and combinations thereof.

[0097] The threads 227 on the connector 222 cooperate and engage with protrusions 129 (see FIG. 1) of the filter unit 108 to allow the filter unit 108 to be screwed onto the connector subassembly 220 for coupling to the connector subassembly 220 and the receptacle portion 306. In some embodiments, the receptacle portion 306 or the filter unit 108 can itself include all of the features of the connector subassembly 220. Alternatively, in some embodiments, the receptacle portion 306 and the filter unit 108 can be configured to be coupled directly to one another. [0098] As shown in FIGS. 3 and 4, the filtration assembly 450 can include a receptacle portion 306 that is adapted to contain a sample (e.g., a large volume aqueous sample) and a filter unit 108 that includes a filter 112 that can be configured to retain a microorganism (e.g., a target microorganism), if present, from the sample. The filter unit 108 and the receptacle portion 306 can be configured to be removably coupled together to form the filtration assembly 450.

[0099] The threaded connection between the connector 222 and the filter unit 108 is shown by way of example only; however, in some embodiments, manufacturability may be enhanced by employing a friction-fit (e.g., press-fit) or snap-fit-type coupling means (as shown for the coupling between the connector 222 and the connector subassembly gasket 224) between these components.

[00100] As shown in FIGS. 3 and 4, the connector subassembly 220 (e.g., the connector 222) can further include a vent port 228 and a filter plug 230 that can serve as an air inlet during the filtration process, particularly, in embodiments in which the first end 354 of the receptacle portion 306 is closed. The filter plug 230 can be used to filter inlet air as it enters the filtration assembly 450 during a filtration process. In some embodiments, the filter plug 230 can be formed of a hydrophobic material (e.g., polypropylene, polyethylene, polytetrafluoroethylene (PTFE), or a combination thereof) to inhibit sample leakage out of the vent port 228 and contamination into the vent port 228.

[00101] The filter unit 108 can include a filter housing 132 configured to house and retain the filter 112, as discussed herein. By way of example only, as shown in FIG. 1, the filter 112 can be positioned against a filter support 131 disposed in the filter housing 132. In any

embodiment, a porous layer (e.g., a nonwoven fabric, not shown) can be disposed between the filter 112 and the filter support 131. The porous layer can provide additional support to keep the membrane intact during the filtration process. As further shown in FIG. 1, the filter support 131 can include or at least partially define a plurality of apertures 137 that are in fluid communication with an outlet 139 (see FIGS. 3 and 4). Such an outlet 139 can function as the outlet of the filtration assembly 450 and/or of the filter unit 108, and can be coupled to a source of negative pressure (not shown) to facilitate a filtration step that moves the sample through the receptacle portion 306, the connector subassembly 220 (if employed), and the filter unit 108.

[00102] In some embodiments, the filter 112 (e.g., a periphery thereof) can be ultrasonically welded to the filter housing 132 (e.g., a ledge or flange within the housing 132 on which the periphery of the filter 112 will sit). Such an ultrasonic weld can provide a hermetic seal, in addition to providing means for coupling the filter 112 to the filter housing 132. Still, in some embodiments, the filter 112 can be integrally formed with the filter housing 132 or sandwiched between two mating parts. [00103] The filter unit 108 (or the filter housing 132) can include a first end 144 and a second end 145. The filter 1 12 is disposed in the fluid pathway such that a fluid sample moving through the fluid pathway passes through the filter 1 12. A sample filtrate (not shown) resulting from a liquid passing through the filter 1 12 exits the filter unit 108 at an outlet 139 at the second end 145. The second end 145 is configured to be coupled (e.g., by inserting the second end 145 of the filter housing 132 into a vacuum flask or by attaching a vacuum hose to the outlet 139) to a source of negative pressure.

[00104] In addition to being adapted for coupling the filter unit 108 to the receptacle portion (e.g., via the connector subassembly 220), the first end 144 of the filter unit 108 is adapted for coupling the filter unit 108 to the lysate collection vessel 102, as shown in FIG. 1. For example, the protrusions 129 of the filter unit 108 are configured for coupling with the threads 227 of the connector subassembly 220 (or of the receptacle portion 306 if the connector subassembly 220 is not employed) and the threads 123 of the lysate collection vessel 102 as shown in FIG. 2. In addition, by way of example only, in some embodiments, the first end 144 can be dimensioned to receive the second end 225 of the connector subassembly 220 (or vice versa). Alternatively, if the connector subassembly 220 is not employed, the first end 144 can be configured to be coupled directly to the receptacle portion 306.

[00105] In addition, as shown in FIGS. 1 and 3, in some embodiments, a filter unit gasket (e.g., an O-ring) 135 can be employed between the filter housing 132 (i.e., between the filter 1 12) and the connector subassembly 220 (or the receptacle portion 306 if the connector subassembly 220 is not employed) and/or between the filter housing 132 (i.e., between the filter 1 12) and the lysate collection vessel 102. Such a filter unit gasket 135 can enhance the seal between the receptacle portion 306 (e.g., or the connector subassembly 220) and the filter unit 108. By way of example only, the same filter unit gasket 135 is shown as being employed in the filtration assembly 450 (FIG. 3) and the cell disruption container 500 (FIG. 1); however, this need not be the case.

[00106] In another aspect, the present disclosure provides a method of detecting a target microorganism in a sample (e.g., an aqueous sample that is characterized by having a relatively large volume). The method can comprise providing any embodiment of the system of the present disclosure, moving a liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter, moving a liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter, agitating the cell-disruption container under conditions that cause the cell -disrupting particles to disrupt microbial cells disposed on the membrane filter to form a liquid lysate, moving a portion of the liquid lysate to a predefined location (e.g., proximate the closed end) in the lysate collection vessel, and analyzing the liquid lysate to detect an analyte associated with a target microorganism. [00107] In any embodiment of the method, moving a liquid sample through the filter unit can comprise coupling the filter unit to a structure (e.g., a reservoir such as, for example, the receptacle portion of the filtration assembly disclosed herein; a conduit such as, for example, a faucet or a water line) that is configured to deliver the liquid sample to the filter unit.

[00108] With reference to FIG. 5, a sample detection method 600 will now be described, with continued reference to the sample detection system 100 of FIGS. 1-2 and the filtration assembly 45 O of FIGS. 3-4.

[00109] The method 600 comprises the step 670 of providing a microbial detection system according to any embodiment of the present disclosure. In the illustrated embodiment of FIG. 5, the system comprises the components of the filtration assembly 450 shown in FIGS 3-4 and the lysate collection vessel 102 of FIGS. 1-2.

[00110] The method 600 further comprises the step 672 of moving a liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter. In the illustrated embodiment of FIG. 5, the filter unit 108 is coupled to the receptacle portion 306 via the connector subassembly 220. The portion can be any part (e.g., up to 5%, up to 10%, up to

20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 100%) of the liquid sample. In any embodiment, the portion can be a predefined volume (e.g., about 1 mL, 2 mL, 5 mL, 25 mL, 50 mL, 100 mL, 250 mL, 500, mL, 1 L, 2 L, 5 L, 10 L, or more than 10 L).

[00111] In any embodiment, moving a liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter can comprise operatively connecting the second end of the filter unit to a source of negative pressure. Connecting the second end of the filter unit to a source of negative pressure can be done, for example, by attaching a vacuum hose to the outlet 139 or by inserting the second end into a vacuum flask (not shown) that is connected to a vacuum source.

[00112] In any embodiment of the method 600 wherein a filtration assembly (e.g., filtration assembly 450) is used, the filtration assembly can be placed in an "upright" orientation (as shown in FIG. 4) wherein the reservoir 358 is superior to the filter unit 108 such that a liquid sample (not shown) can be deposited into the reservoir 358. At least a portion or all of the liquid sample can move therefrom (e.g., via positive pressure applied to the reservoir, via gravity flow, and/or via negative pressure applied to the outlet 139) in a direction from the reservoir 358, through the connector subassembly 220 and the filter 112, and exit the filtration assembly 450 via the outlet 139. As the liquid sample passes through the filter 112, microorganisms (including target microorganisms), if present, are retained by the filter.

[00113] After moving the liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter, the method further comprises the step 674 of closing the outlet of the filter unit with the closure element. Closing the outlet serves at least two functions: i) it keeps material (e.g., filter fragments, intact cells, cell lysate) from

unintentionally exiting the cell disruption container and ii) it prevents the ingress of target microorganisms into the filter unit from sources other than the liquid sample.

[00114] The method 600 further comprises the step 676 of coupling the open end of the lysate collection vessel to the filter unit to form a cell-disruption container containing the plurality of cell-disrupting particles disposed therein. Referring back to FIGS. 1-4, after the portion of the sample is moved through the filter unit, the first end of the filter unit 108 is detached from the receptacle portion 306 and/or connector subassembly 220 and the closure element 1 17 is used to seal the outlet 139 of the filter unit. The filter unit 108 is then coupled (e.g., reversibly coupled) to the lysate collection vessel 102 (e.g., by coupling the first end of the filter unit to the open end of the lysate collection vessel) to form a cell disruption container 500. A plurality of cell disruption particles are disposed in the lysate collection vessel 102 or, optionally, can be added to the lysate collection vessel or the filter unit 108 (e.g., between the filter 112 and the first end 144 of the filter unit) before forming the cell disruption container 500. Optionally, a volume of liquid suspending medium (e.g., sterile water, a sterile buffer; not shown) may be deposited (e.g., either separately or with the cell disruption particles suspended therein) into the lysate collection vessel or the filter unit (e.g., between the filter and the first end of the filter unit) before forming the cell disruption container. The suspending medium can facilitate the bead beating process and can serve as the carrier solvent in which the biomolecules from ruptured cells are suspended.

[00115] It is contemplated that the step 674 of closing the outlet of the filter unit with the closure element may be performed either before or after the step 676 of coupling the open end of the lysate collection vessel to the filter unit to form a cell-disruption container containing the plurality of cell-disrupting particles disposed therein.

[00116] After the cell disruption container is formed, the method 600 further comprises the step 678 of agitating the cell -disruption container under conditions that cause the cell- disrupting particles to disrupt microbial cells disposed on the membrane filter to form a liquid lysate. The liquid lysate may comprise liquid from the liquid sample that remained in the filter or in the second end of the filter unit after the moving step 672, liquid from the optional liquid suspending medium, liquid from any liquid in which the plurality of cell-disruption particles were suspended prior to adding the particles to the cell disruption container, liquid from the cells present in the filtrand, and liquid from combinations of any two or more of the foregoing liquids.

[00117] The cell disruption container is agitated under conditions that cause cells disposed therein to be disrupted. "Cell disruption", as used herein, refers to a bead beating process that releases from an intact cell, or otherwise makes accessible within a ruptured cell, biological molecules (e.g., polynucleotides, polypeptides) that are normally enveloped by an intact cell membrane. Cell disruption processes such as bead beating are well known in the art, as discussed herein. Without limitation, the cell disruption container is agitated at about 2000 oscillations per minute using, for example, a vortex mixing machine. The agitation causes high-speed collisions between the particles and the microbial cells, typically causing the cells to rupture, which may cause release of the cellular contents. Rupturing the cells provides access to polynucleotides associated with the cell. Certain polynucleotides are uniquely associated with certain target microorganisms. Thus, disrupting the cells can make it possible to detect polynucleotides associated with target microorganisms using nucleic acid amplification techniques (e.g., PCR), for example.

[00118] After agitating the cell disruption container under conditions that cause cells disposed therein to be disrupted, the method 600 further comprises the step 680 of moving a portion of the liquid lysate to a predetermined location. In any embodiment, the predetermined location can be a predetermined location (e.g., proximate the closed end) in the lysate collection vessel. In any embodiment, the portion of the liquid lysate can be moved to the predetermined location, for example, by placing the cell disruption container into an appropriate (e.g., similarly- dimensioned) centrifuge rotor or carrier and subjecting the cell disruption container to a centrifugal force sufficient to move the liquid lysate to the predefined location (e.g., proximate the closed end of the lysate collection vessel). Once the portion of the liquid cell lysate is moved to the predefined location, a part or all of the portion can be analyzed.

[00119] If centrifugation is used to move the portion of the liquid lysate to the predefined location, the g-force in the centrifugation step can be at least about 500 ^« g (e.g., 500 * 9.8 m/s ² on earth, at sea level), in some embodiments, at least about 1000 ^« g, and in some embodiments, at least about 5000 ^« g. In some embodiments, the g-force in the centrifugation step can be up to about 10,000 ^« g, inclusive.

[00120] If centrifugation is used to move the portion of the liquid lysate to the predefined location, the duration of the centrifugation, can be at least about 1 minute; in some

embodiments, at least about 5 minutes; and in some embodiments, at least about 10 minutes. In some embodiments, the duration of the centrifugation step, if used, can be up to about 20 minutes, inclusive.

[00121] Referring back to FIG. 5, the method 600 of the present disclosure further comprises the step 682 of analyzing the liquid lysate to detect the target microorganism. Detecting the target microorganism comprises detecting an analyte associated with (e.g., uniquely associated with) the target microorganisms. Suitable analytes include, but are not limited to,

polynucleotides (e.g., DNA, RNA), proteins, and cell wall components (e.g., glycans, lipopolysaccharides) that can be detected using detection methods that are known in the art. Suitable detection methods include rapid detection methods. Rapid detection methods generally provide a result in 2 hours or less; preferably, in 90 minutes or less; more preferably, in 60 minutes or less.

[00122] Suitable detection methods include, without limitation, genetic methods (e.g., nucleic acid hybridization and/or nucleic acid amplification (e.g., PCR, rtPCR, LCR, NASBA, 3SR, RCA, and LAMP)) and immunological methods (e.g., ELISA, lateral flow assays, TRF assays, and IMS-ECL assays).

[00123] Genetic methods that include nucleic acid hybridization and/or amplification can comprise detecting a polynucleotide that indicates the presence of the target microorganism. In any embodiment, the polynucleotide may be unique to the target microorganism. In any embodiment, the polynucleotide may be unique to the genus or the species of target microorganisms and a limited number (e.g., one, two, or three) genera or species of non-target microorganisms.

[00124] A method of the present disclosure that uses nucleic acid amplification to detect target microorganisms can be adapted to selectively detect live target microorganisms (i.e., acellular nucleic acid and/or nucleic acid from dead microorganisms is not detected in the method). Fittipaldi et al (Journal of Microbiological Methods, 2012, vol. 91, pp. 276-289; which is incorporated herein by reference in its entirety) describe the use of light-reactive viability dyes to suppress amplification of polynucleotides from nonviable microorganisms. Thus, a method of the present disclosure optionally can comprise, before detecting the target microorganisms by nucleic acid amplification, i) contacting the sample filtrand with a light- reactive viability dye capable of suppressing amplification of an acellular polynucleotide or a polynucleotide present in a nonviable microorganism and ii) exposing the contacted filtrand to a suitable source and quantity of electromagnetic energy to suppress amplification of a viability dye -bound polynucleotide.

[00125] The filtrand can be contacted with the viability dye, for example, by adding the dye to the sample before it is passed through the membrane filter or, alternatively, by contacting a small amount of the day with the filter after the filtrand from the sample is collected on the filter, as described in Example 3 hereinbelow. Advantageously, the latter method can be performed using a system or container of the present disclosure wherein the viability dye is disposed; optionally, with the cell-disrupting particles; in the lysate collection vessel.

[00126] Advantageously, exposing the contacted filtrand to a suitable source and quantity of electromagnetic energy to suppress amplification of a viability dye-bound polynucleotide can be performed while the filter membrane is disposed in the filter unit, as described in Example 3. Thus, the filter does not need to be removed or otherwise handled prior to exposing the filtrand to the electromagnetic energy.

[00127] Immunological methods can comprise detecting an antigen that indicates a presence of the target microorganism. In any embodiment, detecting the antigen can comprise detecting an antigen that is unique to the target microorganism. In any embodiment, the antigen may be unique to the genus or the species of target microorganisms and a limited number (e.g., one, two, or three) genera or species of non-target microorganisms. In any embodiment, detecting the antigen can comprise detecting an antigen that is normally intracellular or otherwise is not detectable in a suspension of intact target microorganisms.

[00128] In yet another aspect, the present disclosure provides a container. The container comprises a lysate collection vessel, a filter unit, and a closure element. One embodiment of the cell disruption container 500 is depicted in FIGS. 1 and 2 and described herein.

[00129] The lysate collection vessel of the container can be, for example, any embodiment of the lysate collection vessel 102 shown in FIGS. 1 and 2 and described herein. The filter unit of the container can be, for example, any embodiment of the filter unit 108 shown in FIGS. 1 and 2 and described herein. The filter unit 108 can comprise a filter housing 132 with a first end, 144, a second end 145, a fluid flow path extending from the first end to the second end, and a filter 112 disposed in the flow path. The filter unit 108 is sealingly coupled to the lysate collection vessel 102 such that liquid and/or microorganisms cannot leak from the cell disruption container 500 at the location where the filter unit and lysate collection vessel are coupled.

[00130] The closure element can be, for example, and embodiment of the closure element 117 shown in FIGS. 1 and 2 and described herein. The closure element 117 forms a barrier preventing the passage of microorganisms into the filter unit 108 through the second end 145. The filter unit of the container is configured to receive a liquid sample and retain

microorganisms therefrom on the filter.

[00131] The cell disruption container 500 defines an interior volume. The interior volume of the cell disruption container 500 has a perimeter (not shown), portions of which are defined by the filter 112 and interior surface of the wall(s) of the lysate collection vessel 102. The filter 112 has a first side 113 that faces the interior volume of the cell disruption container 500. In any embodiment of the container, the first side can have a filtrand disposed thereon (i.e., the filter unit is used for filter a sample before it is used to form the container). The container further comprises a plurality of cell-disrupting particles disposed therein (i.e., the particles are disposed in the interior volume of the container).

[00132] In any embodiment of the container, the lysate collection vessel can be removably coupled to the filter unit. In any embodiment of the container, the lysate collection vessel is coupled to the filter unit such that the first side of the filter is oriented toward the cavity of the lysate collection vessel (e.g., as shown in the illustrated embodiment of FIGS. 1 and 2 and described herein).

[00133] In any embodiment, the container of the present disclosure is configured to be placed into a centrifuge and subjected to a force of about 500 ^x g to about 10,000 ^χ g, inclusive. For example, the materials used for the components of the container and the thicknesses of the materials are sufficient to withstand, without substantial deformation and/or structural disintegration, the centrifugal forces within the aforementioned range. In any embodiment, the assembled container is dimensioned to fit in a standard centrifuge rotor (e.g., a rotor that holds 50-mL centrifuge tubes) such that the walls of the rotor substantially support the container during centrifugation.

[00134] In any embodiment, a method according to the present disclosure comprises:

providing any embodiment of the system disclosed herein, moving a liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter, coupling the open end of the lysate collection vessel to the filter unit to form a cell-disruption container containing the plurality of cell-disrupting particles disposed therein, agitating the cell-disruption container under conditions that cause the cell -disrupting particles to disrupt microbial cells disposed on the membrane filter to form a liquid lysate, moving a portion of the liquid lysate at a predefined location in the lysate collection vessel, and analyzing the portion of the liquid lysate to detect an analyte associated with a target microorganism; each of the steps as disclosed hereinabove.

[00135] In any embodiment, a method according to the present disclosure consists essentially of: providing any embodiment of the system disclosed herein, moving a liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter, coupling the open end of the lysate collection vessel to the filter unit to form a cell-disruption container containing the plurality of cell-disrupting particles disposed therein, agitating the cell- disruption container under conditions that cause the cell-disrupting particles to disrupt microbial cells disposed on the membrane filter to form a liquid lysate, moving a portion of the liquid lysate at a predefined location in the lysate collection vessel, and analyzing the portion of the liquid lysate to detect an analyte associated with a target microorganism; each of the steps as disclosed hereinabove.

[00136] In yet another aspect, the present disclosure provides a kit. The kit can comprise a lysate collection vessel, a plurality of cell -disrupting particles, a filter unit, and a membrane filter, each according to any of the embodiments described herein. The membrane filter is configured to be positioned in the fluid pathway so that liquid passing through the fluid pathway from the first end to the outlet passes through the membrane filter. Optionally, the membrane filter may be provided separate from the filter unit and may be placed into the liquid pathway of the filter unit (as with currently-available reusable filter units) prior to use. In any embodiment of the kit, the plurality of cell-disrupting particles may be provided in a container or they may be provided in the lysate collection vessel. In any embodiment, the kit further can comprise a closure element that forms a barrier to prevent passage of a microorganism into the filter unit through the outlet, as described herein.

[00137] The embodiments described above and illustrated in the figures are presented by way of example only and are not intended as a limitation upon the concepts and principles of the present disclosure. As such, it will be appreciated by one having ordinary skill in the art that various changes in the elements and their configuration and arrangement are possible without departing from the spirit and scope of the present disclosure.

[00138] All references and publications cited herein are expressly incorporated herein by reference in their entirety into this disclosure.

[00139] The following embodiments are intended to be illustrative of the present disclosure and not limiting.

Exemplary Embodiments

[00140] Embodiment A is a system for preparing a sample to detect a microorganism, the system comprising:

a lysate collection vessel comprising:

an open end configured to receive a sample;

a closed end;

a cavity that tapers from a wider portion proximate the open end to a narrower portion proximate the closed end;

a plurality of cell-disrupting particles;

a filter unit comprising:

a housing having a first end, a second end comprising an outlet, and a fluid pathway extending from the first end to the outlet;

a membrane filter disposed in the fluid pathway; and

a closure element that forms a barrier to prevent passage of a microorganism into the filter unit through the outlet;

wherein the membrane filter has a first side that is oriented toward the first end and a second side that is oriented toward the second end;

wherein the filter unit is configured so that liquid passing through the fluid pathway from the first end to the outlet passes through the membrane filter. [00141] Embodiment B is the system of Embodiment A, wherein the cell-disrupting particles are fabricated from a material selected from the group consisting of silica, zirconium, silicon carbide, garnet, steel, and combinations thereof.

[00142] Embodiment C is the system of any one of the preceding Embodiments, further comprising a cell disruption buffer.

[00143] Embodiment D is the system of any one of the preceding Embodiments, wherein the means for operatively coupling the open end of the lysate collection vessel to the first end of the filter unit include a first engagement structure that is an integral part of the lysate collection vessel and a complementary second engagement structure that is an integral part of the filter unit.

[00144] Embodiment E is the system of any one of the preceding Embodiments, wherein the cell-disrupting particles have a density of about 2.5 g/cc to about 7.9 g/cc.

[00145] Embodiment F is the system of any one of the preceding Embodiments, wherein the cell -disrupting particles have a mean particle diameter of about 0.1 μπι to about 2 μπι.

[00146] Embodiment G is the system of any one of the preceding Embodiments, further comprising a receptacle portion adapted to couple to the filter unit, wherein the receptacle portion comprises a reservoir configured to hold the sample.

[00147] Embodiment H is the system of Embodiment G, further comprising a connector having a first end adapted to couple to the receptacle portion and a second end adapted to couple to the filter unit.

[00148] Embodiment I is the system of any one of the preceding Embodiments, wherein the lysate collection vessel is operatively coupled to the filter unit, wherein operatively coupling the lysate collection vessel to the filter unit comprises coupling the open end of the lysate collection vessel to the first end of the filter unit.

[00149] Embodiment J is a method for detecting an analyte of interest in a sample, if present, the method comprising:

providing the system of any one of Embodiments A through I;

moving a liquid sample through the filter unit so that a portion of the liquid passes through the membrane filter;

coupling the open end of the lysate collection vessel to the filter unit to form a cell- disruption container containing the plurality of cell-disrupting particles disposed therein; agitating the cell-disruption container under conditions that cause the cell-disrupting particles to disrupt microbial cells disposed on the membrane filter to form a liquid lysate; moving a portion of the liquid lysate at a predefined location in the lysate collection vessel; and analyzing the portion of the liquid lysate to detect an analyte associated with a target microorganism.

[00150] Embodiment K is the method of Embodiment J, wherein contacting a liquid sample with the membrane filter under conditions that cause a portion of the liquid move from the first end through the membrane filter toward the second end comprises operatively connecting the second end of the filter unit to a source of negative pressure.

[00151] Embodiment L is the method of Embodiment J or Embodiment K, wherein analyzing the liquid lysate comprises detecting a polynucleotide that indicates a presence of the target microorganism.

[00152] Embodiment M is the method of Embodiment J or Embodiment K, wherein analyzing the liquid lysate comprises detecting an antigen that indicates a presence of the target microorganism.

[00153] Embodiment N is the method of any one of Embodiments J through L:

wherein analyzing the portion of the liquid lysate to detect an analyte associated with a target microorganism comprises amplifying a polynucleotide associated with the target microorganism;

wherein, before amplifying a polynucleotide associated with the target microorganism, the method further comprises:

contacting the sample filtrand with a light-reactive viability dye capable of suppressing amplification of an acellular polynucleotide or a polynucleotide present in a nonviable microorganism; and

exposing the contacted filtrand to a suitable source and quantity of electromagnetic energy to suppress amplification of a viability dye-bound

polynucleotide.

[00154] Embodiment O is a container, comprising:

a filter unit comprising a housing with a first end, a second end with an outlet, a fluid flow path extending from the first end to the outlet, and a filter disposed in the flow path; a closure element that forms a barrier to prevent passage of a microorganism into the filter unit through the outlet; and

a lysate collection vessel sealingly coupled to the filter unit;

wherein the filter unit is configured to receive a liquid sample and retain

microorganisms therefrom on the filter;

wherein the container defines an interior volume with a perimeter;

wherein the filter defines a portion of the perimeter;

wherein the filter has a first side that faces the interior volume;

wherein the first side has a filtrand disposed thereon; wherein a plurality of cell-disrupting particles is disposed in the interior volume.

[00155] Embodiment P is the container of Embodiment O, wherein the lysate collection vessel is removably coupled to the filter unit.

[00156] Embodiment Q is the container of Embodiment O or Embodiment P, wherein the filter unit is configured so that a liquid sample can be urged through the liquid flow path thereby causing the microorganisms to be retained on the filter.

[00157] Embodiment R is the container of any one of Embodiments O through Q, wherein the lysate collection vessel is coupled to the filter unit such that the first side of the filter is oriented toward the cavity of the lysate collection vessel.

[00158] Embodiment S is the container of any one of Embodiments O through R, wherein the container is configured to be placed into a centrifuge and subjected to a force of about 500 ^χ g to about 10,000 ^χ g, inclusive.

[00159] Embodiment T is a kit, comprising:

a lysate collection vessel comprising:

an open end configured to receive a sample;

a closed end;

a cavity that tapers from a wider portion proximate the open end to a narrower portion proximate the closed end;

a plurality of cell-disrupting particles;

a filter unit comprising:

a housing having a first end, a second end comprising an outlet, and a fluid pathway extending from the first end to the outlet; and

a membrane filter;

wherein the membrane filter is configured to be positioned in the fluid pathway so that liquid passing through the fluid pathway from the first end to the outlet passes through the membrane filter.

[00160] Embodiment U is the kit of Embodiment T, wherein the membrane filter is disposed in the fluid pathway in the filter housing.

[00161] Embodiment V is the kit of Embodiment T or Embodiment U, wherein the plurality of cell-disrupting particles is disposed in the lysate collection vessel.

[00162] Embodiment W is the kit of any one of Embodiments T through V, further comprising a closure element that forms a barrier to prevent passage of a microorganism into the filter unit through the outlet.

[00163] Embodiment X is the kit of any one of Embodiments T through W, further comprising a light-reactive viability dye. EXAMPLES

[00164] Preparative Example 1. Bacteria used in examples.

[00165] The various bacteria used in the examples (Table 1) were obtained from ATCC (Manassas, VA).

[00166] Table 1. Bacteria used in examples.

[00167] Pure cultures of the bacterial strains were inoculated into Tryptic Soy Broth (TSB, Becton-Dickinson; Franklin Lakes, NJ) and were grown overnight at 37°C. The cultures were diluted serially in Butterfield phosphate buffer (Whatman, Piscataway, NJ) to obtain a desired concentration of colony forming units (cfu) per ml for spiking into water samples. The bacteria were quantified by inoculating one milliliter of appropriate dilutions of the diluted bacterial suspensions into 3M™ Petrifilm™ E.coii/Coliform Count Plates (3M Company, St. Paul, MN) according to manufacturer's instruction. The plates were incubated overnight at 37°C and the resulting colonies were read using 3M™ Petrifilm™ Plate Reader (3M Company) to determine the concentration of colony forming units (cfu) in each original bacterial suspension.

[00168] Preparative Example 2. Preparation of TIPS (R1901-11) membranes.

[00169] A multi-zone microporous polypropylene membrane (designated herein as R1901- 11) was prepared as described in International Patent Publication No. WO2010/078234 using both a 40 mm twin screw extruder and a 25 mm twin screw extruder. Melt streams from the two extruders were cast into a single sheet through a multi -manifold die.

[00170] Melt stream 1.

[00171] Polypropylene (PP) resin pellets (F008F from Sunoco Chemicals, Philadelphia, PA) and a nucleating agent (MILLAD 3988, Milliken Chemical, Spartanburg, SC) were introduced into a 40 mm twin screw extruder which was maintained at a screw speed of 250 rpm. The mineral oil diluent (Mineral Oil SUPERLA White 31, Chevron Corp., San Ramon, CA) was fed separately from the reservoir into the extruder. The weight ratio of PP/diluent/nucleating agent was 29.25%/70.7%/0.05%. The total extrusion rate was about 30 lb/hr (13.6 kg/hr) and the extruder's eight zones were set to provide a decreasing temperature profile from 271° C to 177° C.

[00172] Melt stream 2.

[00173] PP resin pellets and MILLAD 3988 were introduced into a 25 mm twin screw extruder which was maintained at a screw speed of 125 rpm. The mineral oil diluent was fed separately from the reservoir into the extruder. The weight ratio of PP/diluent/nucleating agent was 29.14%/70.7%/0.16%. The total extrusion rate was about 6 lb/hr (2.72 kg/hr) and the extruder's eight zones were set to provide a decreasing temperature profile from 271° C to 177° C.

[00174] The multi-zone film was cast from the multi -manifold die maintained at 177° C onto a patterned casting wheel. The temperature of casting wheel was maintained at 60° C and the casting speed was 3.35 m/min (1 1 ft/min). The resulting film was washed in-line in a solvent to remove mineral oil in the film and then air dried. The washed film was sequentially oriented in the length and cross direction 1.8 x 2.80 at 99°C and 154°C, respectively.

[00175] Surface modification of TIPS membranes.

[00176] The TIPS membranes were coated with polyethylene glycol (PEG) as described in PCT Publication No. WO2011/153085.

[00177] A 5-wt% EVAL stock solution was made by dissolving an ethylene -vinyl alcohol copolymer (EVAL) with 44 mole% ethylene content (EVAL44, Sigma- Aldrich Co., St Louis, MO, USA) in an ethanol (AAPER Alcohol and Chemical Co. Shelbyville, KY)/water solvent mixture (70 vol% ethanol) in a water bath at temperature 70-80° C. From the above stock solution, a solution was made containing 1 -wt% EVAL44, 2- wt% SR@610 (Sartomer, Warrington, PA), 1 wt% reactive photoinitiator VAZPIA ( 2-[4-(2- hydroxy-2- methylpropanoyl)phenoxy] ethyl-2-methyl-2-N-propenoylamino propanoate, as disclosed in U.S. Patent No. 5,506,279) in ethanol/water mixture solvent (70 vol% ethanol).

[00178] A TIPS microporous membrane was saturated with the coating solution above in a heavy weight PE bag. Effort was made to remove the excessive surface solution by paper towel wiping after the saturated membrane was removed from the PE bag. The membrane was allowed to dry by solvent evaporation at room temperature for 10-12 hours. Then, the dry membrane was saturated with a 20-wt% NaCl aqueous solution. After that, the membrane went through a nitrogen inert Fusion UV system with H-bulb on a conveying belt. The speed of the belt was 20 feet per minute (fpm). The membrane was sent through the UV system again in the same speed with the opposite membrane side facing the light source. The cured membrane sample was washed in excessive deionized water and dried at 90°C for 1 to 2 hours until completely dry. The dried membranes were stored in a PE bag at room temperature.

[00179] Example 1. Preparation of molded filtration devices

[00180] CAD designs were made to make a filter unit, lysate collection vessel, connector subassembly, and cap similar to the filter unit 108, lysate collection vessel 102, connector subassembly 220, and closure element 117 shown in FIGS. 1-4.

[00181] The lysate collection vessel had an interior volume of approximately 15 mL. The interior volume has a tapered closed end. The filter unit and lysate-collection vessel were dimensioned so that, when coupled together, the resulting container could fit into a standard centrifuge carrier for 50-mL centrifuge tubes.

[00182] The filter unit was dimensioned to hold a 32 mm membrane filter. A filter unit gasket was made to provide a tight seal between the membrane filter and the lysate collection vessel and a connector subassembly gasket was made to provide a liquid-tight seal between the connector subassembly and a sample bottle. The filter unit and the lysate collection vessel were made by injection molding using polypropylene, while the gaskets were made using injection-molded medical grade SANTOPRENE™ thermoplastic material (ExxonMobil Chemical; Spring, TX). The connector subassembly had an opening to fit a sintered polypropylene to allow air movement during vacuum filtration. The parts were used to assemble a filtration assembly as shown in Fig. 3. The membrane filter was either

ultrasonically sealed to the filter support of the filter unit or used without any sealing.

[00183] Example 2. Use of an integrated filtering/bead beating assembly for rapid detection of microorganisms in a water sample.

[00184] One liter of tap water sample was collected in a sterile Nalgene high density polyethylene bottle (Thermo Fisher Scientific, Rochester, NY) containing 1 gram of reagent- grade sodium thiosulfate. E. coli was grown in TSB and serially diluted in Butterfield phosphate buffer to obtain approximately 10-1000 colony forming units/ml. 1 ml of the serially diluted bacteria was added to 1 liter of tap water and mixed thoroughly. 32 mm-diameter circular filter membranes were cut (if necessary) from the 0.4μ 3M multizone membrane described in Preparative Example 2 or a track etched 0.4μ polycarbonate Isopore™ Membrane Filter (cat# HTTP04700; EMD Millipore Corporation; Billerica, MA) and placed onto the filter support of the filter unit. The filter assembly was assembled and the filter assembly connector was inserted into the bottle and the filter unit was connected to a 1 liter vacuum filtering flask.

[00185] The water sample spike with bacteria was filtered through the membranes at a vacuum pressure of about 20 inches of mercury using an Air Cadet Vacuum/Pressure Station (model No. 420-3901, Barnant Company; Barrington, IL). The filter assembly was removed from the vacuum flask, the filter unit was disconnected from the connector subassembly and coupled to the lysate-collection vessel with the membrane filter facing the interior of the lysate collection vessel to form the container of FIGS. 1-2. Prior to forming the container, 300 mg of 0.1 mm Zirconia/silica beads (Cat # 1 lG79101z, Biospec Products, Bartiesville, OK) in 500 μΐ of AE buffer (Qiagen, Valencia, CA) was placed into the lysate collection vessel. The container was vortexed in a vortex mixer (Fisher Vortex Genie 2, Cat#12-812, Fisher

Scientific, Pittsburg, PA) for 2 min. The container was removed from the vortex mixer, centrifuged in a in a multipurpose centrifuge (Model 5804, Eppendorf) with a swinging bucket centrifuge rotor (A-4-44) and for 5 min at 5000 RPM (4500 g), and the resulting supernatant was removed from the container and transferred to a 1.5 mi microfuge tube (Plastibrand microtubes).

[00186] qPCR Analyses.

[00187] Bacterial DNA was detected in the supernatants using qPCR. A 3M Integrated Cycler (Catalog # MOL 1001; available from Focus Diagnostics, Cypress, CA) was used for

DNA. amplification procedures. The amplification reagents (described below) were loaded into a Direct Amplification Disc ("DAD"; which can be obtained from Focus Diagnostics in Catalog # MOL2650 SIMPLEX A™ FluA/B & RSV Direct Kit) was used with the 3M integrated Cycler for amplification procedures.

[00188] Sample Ready™ - Custom Manufactured Reagents for detecting E. coli and Total coliforms was obtained from BioGX (Birmingham, AL) and used as directed by the manufacturer to detect bacterial DNA in the supernatant from the bead beating process. The lyophilized reagents were reconstituted with molecular grade water to obtain 1.25x concentration of reagents. 50 μΐ of the reagent was added into the reagent chamber and 50 μΐ of the bead beaten sample was added to the sample chamber of Direct Amplification Disc. The wells were sealed with the peeled adhesive foil. After loading all of the wells in the disc, the disc was loaded onto the Integrated Cycler. Thermal cycling was carried out with the following conditions: 5 min at 99 ^° C for denaturation followed by 45 cycles of 10 sec at 99 ^° C and 43 sec at 62 ^° C. The results indicated that with integrated filtration device, 10 to 100 cells could be detected by qPCR after filtration and extraction of DNA from membrane by coupling the centrifuge tube with beads and vortexing to extract DNA (Table 2).

[00189] Table 2. Detection of E. coli by qPCR using integrated filtration and bead beating device. Primers used in the TC analysis detect total coliforms in the sample. Primers used in the EC analysis detect total Escherichia coli in the sample. Primers used in the IPC analysis detect internal positive control (for PCR verification) in the sample. Sample A had approximately 1000 cfu/mL, Sample B had approximately 100 cfu/mL, and Sample C had approximately 10 cfu/mL, as determined by plate counts.

Sample ID TC (FAM) C _t EC (JOE) Ct IPC (Q670) Ct

A 25.8 27.8 32.2

A 26.3 27.6 32.8

B 30.6 30.7 32.1

B 30.8 30.5 32.3

C 0 33.8 31.9

C 0 34 31.5

No Template Control (NTC) 0 0 31.4 [00190] Example 3. Use of an integrated filtering/bead beating assembly for rapid detection of live/dead microorganisms in a water sample.

[00191] a) Heat killed cells: E. coli was grown in Tryptic Soy Broth for about 16 h at 37 ^° C (about 10 ⁹ cfu/ml) and 1 ml of cells was placed in a water batch (90 ^° C) for about 1 min to obtain mixture of live and dead cells. The control and heat killed cells were serially diluted in

Butterfield phosphate buffer to determine colony forming units/ml of sample. One milliliter of appropriate dilutions of the diluted bacterial suspensions was inoculated into 3M™ Petrifilm™ E.coli/Coliform Count Plates (3M Company) according to manufacturer's instruction. The plates were incubated overnight at 37° C and the resulting colonies were read using

3M™ Petrifilm™ Plate Reader (3M Company) to determine the concentration of colony forming units (cfu) in each original bacterial suspension. The heat treatment typically resulted in about 2 log reduction of viable cells.

[00192] An aliquot (0.1 ml) of the control (not heat treated) and heat killed cells was added to 1 liter of sterile deionized water and mixed thoroughly. 32 mm-diameter circular filter membranes were cut (if necessary) from 0.4μ track etched polycarbonate Isopore™ Membrane

Filter (cat# HTTP04700; EMD Millipore Corporation) and placed onto the filter support of the filter unit. The filter assembly was assembled and the filter assembly connector was inserted into the bottle and the filter unit was connected to a 1 liter vacuum filtering flask.

[00193] The water samples spiked with bacteria were filtered through the membranes at a vacuum pressure of about 15 inches of mercury using an Air Cadet Vacuum/Pressure Station

(model No. 420-3901, Barnant Company). After filtration, the samples were processed as described below.

[00194] After filtration, the filter assembly was removed from the vacuum flask, the filter unit was disconnected from the connector subassembly and coupled to the lysate-collection vessel with the membrane filter facing the interior of the lysate collection vessel to form the container of FIGS. 1-2. The lysate vessel contained 300 mg of 0.1 mm Zirconia/silica beads (Cat # 1107910 lz, Biospec Products,) in 500 μΐ of AE buffer (Qiagen) with 50 μΜ of propidium monoazide (PMA, Biotium, Inc., Hayward, CA).

[00195] The containers were incubated in dark for 10 min at room temperature and then exposed to blue LED (460 nm) in a dark chamber for 15 min. All the containers were vortexed in a vortex mixer (Fisher Vortex Genie 2, Cat#12-812) for 2 min. The container was removed from the vortex mixer, centrifuged in a in a multipurpose centrifuge (Model 5804, Eppendorf) with a swinging bucket centrifuge rotor (A-4-44) and for 5 min at 5000 RPM (4500 g), and the resulting supernatant was removed from the container and transferred to a 1.5 ml sterile microfuge tube (Plastibrand microtubes). A known aliquot of the sample (1 μΐ) was used for [00196] E. coli (uidA) PMA real time PCR kit for detecting E. coli was obtained from Biotium and used as directed by the manufacturer to detect bacterial DNA in the supernatant from the bead beating process. 1 μΐ of DNA sample was added to 96-well PCR plate containing 19 μΐ of reaction mix (primers, probes, and enzyme mix). Thermal cycling was carried out using Applied Biosystems® 7500 Real-Time PCR. System (Thermo Fisher Scientific} with the following conditions: 5 minutes at 95° C for denaturation followed by 40 cycles of: 5 seconds at 95° C. and 30 seconds at 64° C.

[00197] As seen in Table 3, the treatment of membranes with PM A in the integrated device was able to differentiate live/dead cells. The Ct values were higher for heat killed cells than for control cells (not heat treated). The addition of bead beating beads to the membrane along with buffer containing PMA did not adversely affect the ability of the PMA to inhibit amplification of free DNA and/or DNA present in dead cells. The data indicate that the integrated device can be used in conjunction with PMA to differentially detect live cells.

[00198] Table 3. Detection of live/dead E. coli by qPCR using integrated filtration and bead beating device.

[00199] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[00200] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[00201] Various modifications may be made without departing from the spirit and scope of the invention. These and other embodiments are within the scope of the following claims.