EP4630582A1

EP4630582A1 - Fully automated dpcr systems and methods of use thereof

Info

Publication number: EP4630582A1
Application number: EP22968045.9A
Authority: EP
Inventors: Rich MOORE; James Price; Jim PETISCE; Courtney PASSOW
Original assignee: Becton Dickinson and Co
Current assignee: Becton Dickinson and Co
Priority date: 2022-12-08
Filing date: 2022-12-08
Publication date: 2025-10-15
Also published as: JP2026501127A; WO2024123340A1; CN120322564A

Abstract

Fully automated digital polymerase chain reaction (dPCR) systems are provided. Systems of interest include a sample preparation unit configured to receive a biological sample and extract nucleic acids therefrom, an analysis vessel, a partitioner configured to generate a plurality of sample partitions comprising the extracted nucleic acids and distribute the generated sample partitions into the analysis vessel, a thermocycler configured to modulate temperature in the generated sample partitions so as to amplify nucleic acids of interest in the generated sample partitions, and a sample reader configured to detect whether amplified nucleic acid is present in each of the generated sample partitions within the analysis vessel. The subject systems also include an anti-adhesion coating configured to at least reduce the binding of the extracted nucleic acid to the dPCR system. Methods and kits for practicing the invention are also provided.

Description

FULLY AUTOMATED dPCR SYSTEMS AND METHODS OF USE THEREOF

INTRODUCTION

The medical diagnostics industry is a critical element of today's healthcare infrastructure. At present, however, in vitro diagnostic analyses no matter how routine have become a bottleneck in patient care. There are several reasons for this. First, many diagnostic analyses can only be done with highly specialist equipment that is both expensive and only operable by trained clinicians. Such equipment is found in only a few locations often just one in any given urban area. This means that most hospitals are required to send out samples for analyses to these locations, thereby incurring shipping costs and transportation delays, and possibly even sample loss or mishandling. Second, the equipment in question is typically not available “on-demand” but instead runs in batches, thereby delaying the processing time for many samples because they must wait for a machine to fill up before they can be run. In addition, a long wait time may exist for a machine to become available due to lengthy processing times.

Polymerase chain reaction (PCR) is one such medical diagnostic method. PCR involves generating a number of copies of a nucleic acid sample (e.g., DNA) sufficient for analysis by exponentially amplifying the sample. PCR generally involves a denaturation step that yields two single-stranded DNA molecules, an annealing step in which primers attach to the single-stranded DNA molecules, and an elongation step synthesizing a new double-stranded DNA molecule. Methods of PCR generally involve exposing samples to thermal cycling by using repeated cycles of higher and lower temperatures.

Digital polymerase chain reaction (dPCR) is a method of PCR allowing for more precise quantitation of the nucleic acids. In contrast to traditional PCR, dPCR involves carrying out a reaction in a plurality of partitioned samples. In other words, a PCR reaction is carried out in each of the sample partitions. Each sample partition may subsequently be analyzed for the presence or absence of the nucleic acid (e.g., using fluorescent probes), thereby increasing the precision of quantitation. Quantitation is achieved by running the reaction through a fixed number of cycles, sufficient to suitably amplify 1 copy to a detectable response and then counting the number of reactive and non-reactive subvolumes. The use of dPCR in a clinical setting to detect bloodborne pathogens (e.g., to diagnose sepsis) has been proposed (see, e.g., Abram et al. Lab on a Chip 20, no. 3 (2020): 477-489). It is believed that dPCR may be able to reduce time to detection (TTD) and improve limits of detection as compared to conventional whole blood cultures. However, dPCR for sepsis detection has yet to reach widespread adoption due to performance deficiencies.

SUMMARY

The present inventors have realized that certain technical performance deficiencies have prevented dPCR from widespread adoption in applications where a small amount of analyte must be analyzed in a time-sensitive manner (e.g., sepsis diagnosis). In particular, it was found that conventional dPCR systems suffer from (1 ) specimen volume and organism concentration issues, (2) long front-end preparation time, (3) target analyte adhesion to interior surfaces, and (4) lack of automation. With respect to (1 ), it was found that attempting to use microfluidic/microdroplet technologies to diagnose bacteremia using conventional techniques is challenging because the concentration of organism is extremely low. To achieve reasonable clinical sensitivity an analytical sensitivity of ~1 organism/ml is required. Classical blood culture systems use 10 ml of blood to overcome this issue and typical microfluidic/microdroplet techniques use 100’s of microliters (pL) to 1 ml of sample input. Regarding (2), most microdroplet systems operate in a range from ~10 pL/min to ~50 pL/min. Assuming a 10ml input sample, that would correspond to 200 to 1000 minutes of droplet generation time. At the extreme of 1000 minutes time to generate the droplets would be -16.6hrs for 10 ml sample. Given that the average time to positivity for a conventional blood culture is ~18 hours, any timing-related benefits of dPCR in diagnostic applications such as the detection of bloodborne pathogens are currently unrealized. With respect to (3), low concentration of target organism analytes per unit volume within an assay requires that none of the assay surfaces irreversibly bind the target organism analytes. However, currently available dPCR systems suffer from non-negligible amounts of analyte adhering to surfaces and therefore further lowering the concentration of analyte that is available to by assayed. With respect to (4), there are currently no fully automated systems for dPCR. This results in a considerable amount of user involvement in the processing of samples using conventional means. At least in view of technical performance deficiencies (1 )-(4), the inventors have realized fully automated and improved dPCR systems are needed. Embodiments of the present invention satisfy this need.

Aspects of the invention include fully automated digital polymerase chain reaction systems. Systems of interest include a sample preparation unit configured to receive a biological sample and extract nucleic acids therefrom, an analysis vessel, a partitioner configured to generate a plurality of sample partitions comprising the extracted nucleic acids for distribution into the analysis vessel, a thermocycler configured to modulate temperature in the generated sample partitions so as to amplify nucleic acids of interest in the generated sample partitions, and a sample reader configured to detect whether amplified nucleic acid is present in each of the generated sample partitions within the analysis vessel. In addition, embodiments of the subject systems include an anti- adhesion coating configured to at least reduce the binding of the extracted nucleic acid to the dPCR system. The anti-adhesion coating may include, for example, hydrophilic components, zwitterionic components, and/or an anti-fogging agent. In embodiments, at least one — and in certain cases, all — of the sample preparation unit, analysis vessel, partitioner, thermocycler and sample reader comprises the anti-adhesion coating. The dPCR systems may include a sample input block configured to receive the sample. In select embodiments, the sample preparation unit comprises a sample purifier comprising a magnet for extracting nucleic acids from the biological sample and/or a reagent repository, e.g., via a magnetic bead capture oligonucleotide mediate protocol. In some cases, the partitioner is a droplet generator. In some such cases, the droplet generator is configured to combine the sample with an immiscible liquid (e.g., oil), and the dPCR system includes a repository for the immiscible liquid. The analysis vessel may, in certain embodiments, include a cuvette that is configured to rotate around and translate along an axis. In select versions, the sample reader is a three-dimensional (3D) particle counter. In additional embodiments, the partitioner is a liquid dispenser. In some such embodiments, the analysis vessel may be a microwell plate, and the sample reader is a microplate reader (e.g., a microplate fluorescence reader). In certain versions, the partitioner and sample reader are configured to operate simultaneously. In some instances, the subject dPCR systems additionally include an automated liquid dispenser system (e.g., pipettor) operably connected to at least one of the sample preparation unit, the partitioner, the thermocycler, and the sample reader. In select cases, the subject dPCR systems include a robotic arm configured to transfer the sample from the sample preparation unit to the partitioner. Systems may further include a waste repository for collecting used solids and/or liquids. Embodiments of the invention further include a door (e.g., a sliding door) configured to enclose the system.

Aspects of the invention also include methods of analyzing a sample. Methods of interest include introducing a biological sample into a dPCR system of the invention (e.g., such as those described herein), and analyzing the biological sample via dPCR. Embodiments of the subject methods also include centrifuging the sample, e.g., prior to its introduction into the dPCR system. In certain instances, methods also include apportioning the sample into a plurality of sub-lots, and analyzing the plurality of sublots via the dPCR system in parallel. Methods may, in some cases, involve pre-lysing micro- organisms in the sample; and assaying the sample for a high copy number target (e.g., an RNA, such as an rRNA).

Aspects of the invention further include kits. The subject kits include one or more liquid vessels configured for use in a dPCR system and comprising an anti-adhesion coating configured to at least reduce the binding of extracted nucleic acids. The anti- adhesion coating for use in the subject liquid vessels may include hydrophilic components, zwitterionic components, and/or anti-fogging agents. In some cases, the one or more liquid vessels further comprise magnetic beads having an affinity for the extracted nucleic acids. Embodiments of the vessels also include an, optionally dried, dPCR reagent (e.g., PCR master mix) and/or a buffer. Kits may also include one or more pipettor tips comprising the anti-adhesion coating, one or more microwell plates comprising the anti-adhesion coating, and/or one or more cuvettes comprising the anti- adhesion coating. BRIEF DESCRIPTION OF THE FIGURES

The invention may be best understood from the following detailed description when read in conjunction with the accompanying drawings. Included in the drawings are the following figures:

FIG. 1 depicts a fully automated dPOR system according to certain embodiments of the invention.

FIG. 2 depicts a component of the fully automated dPCR system comprising the anti-adhesion coating.

FIG. 3A-B depict an embodiment of the dPCR system where the partitioner is a droplet generator (FIG. 3A) and an embodiment of the dPCR system where the partitioner is a liquid dispenser (FIG. 3B).

FIG. 4 depicts a fully automated dPCR system according to certain embodiments of the invention.

FIG. 5 depicts a flowchart for practicing methods of the invention according to certain embodiments.

DETAILED DESCRIPTION

Fully automated digital polymerase chain reaction (dPCR) systems are provided. Systems of interest include a sample preparation unit configured to receive a biological sample and extract nucleic acids therefrom, an analysis vessel, a partitioner configured to generate a plurality of sample partitions comprising the extracted nucleic acids for distribution into the analysis vessel, a thermocycler configured to modulate temperature in the generated sample partitions so as to amplify nucleic acids of interest in the generated sample partitions, and a sample reader configured to detect whether amplified nucleic acid is present in each of the generated sample partitions within the analysis vessel. The subject systems also include an anti-adhesion coating configured to at least reduce the binding of the extracted nucleic acid to the dPCR system. Methods and kits for practicing the invention are also provided.

Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

While the system and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of “means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

FULLY AUTOMATED dPCR SYSTEMS

As discussed above, aspects of the invention include fully automated dPCR systems. The subject systems include an anti-adhesion coating configured to at least reduce the binding of an extracted nucleic acid to the dPCR system. Anti-adhesion coatings as described herein may reduce binding of the extracted nucleic acid to the dPCR system as compared to the amount of binding that would occur in an actual or hypothetical conventional dPCR system lacking an anti-adhesion coating. For example, the anti-adhesion coating described herein may be configured to reduce the binding of the extracted nucleic acid to the dPCR system by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 50% or more, 55% or more, 60%, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, and including by 100%. In certain cases, the anti-adhesion coating may be said to “prevent” the binding of the extracted nucleic acid to the dPCR system. In some instances, reduction of the binding of extracted nucleic acid to the surfaces of the dPCR system may increase the amount of target analyte that is available to be assayed via dPCR by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 50% or more, 55% or more, 60%, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, and including by 100%. Any material that is suitable for being coated on a liquid vessel and is capable of at least reducing (e.g., preventing) the binding of biological analytes (e.g., nucleic acids) thereto may be employed. The anti-adhesion coating may, in some cases, be a stable coating. In other words, subjecting the coating to one or more of autoclaving, washing with a cleaning agent, or rinsing with a saline solution does not substantially alter the chemical properties of the coating.

The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, greater than 10,000 bases, greater than 100,000 bases, greater than about 1 ,000,000, up to about 10¹⁰ or more bases composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may be produced enzymatically or synthetically (e.g., peptide nucleic acid as described in U.S. Patent No. 5,948,902 and the references cited therein) which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Naturally- occurring nucleotides include guanine, cytosine, adenine, thymine and uracil (G, C, A, T and U respectively).

In certain cases, the anti-adhesion coating comprises hydrophilic components. Hydrophilic components are described in, e.g., U.S. Patent Application Publication 2006/0193894, the disclosure of which is incorporated by reference herein in its entirety. In certain cases, the anti-adhesion coating may be formed using a humectant, i.e., an agent that lowers the total free energy of water and is capable of binding water. Suitable humectants include, e.g., polymeric humectants and non-polymeric humectants. Exemplary polymeric humectants include, but are not limited to, hydroxyethyl acrylate (HEA), 2-hydoxyethyl methacrylate (HEMA), dimethacrylamide (DMA), polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol (PEG), di(ethylene glycol)vinyl ether (EO2V), cellulose derivatives, and the like and combinations thereof. Exemplary non-polymeric humectants include, but are not limited to, glycerin, urea, propylene glycol, non- polymeric diols, glycerols, and the like.

In some embodiments, the anti-adhesion coating comprises zwitterionic components. Zwitterionic components described in, e.g., Baggerman et al. Langmuir35, no. 5 (2019): 1072-1084; incorporated by reference herein in its entirety. In some instances, the zwitterionic components are phosphoryl choline-containing polymers. In additional cases, the zwitterionic components include acrylates and acrylamides, optionally with sulfobetaine and carboxybetaine moieties. Zwitterionic coatings may be prepared via any convenient technique. In some cases, zwitterionic coatings are prepared using techniques that include, but are not limited to, atom- transfer radical polymerization (ATRP), strain promoted alkyne-azide cycloaddition (SPAAC), surface-initiated polymerization (e.g., SI-ATRP), biofunctionalization of side chains using random copolymers, reversible addition-fragmentation chain-transfer (RAFT) polymerization, combinations thereof, and the like.

In some embodiments, the anti-adhesion coating is an anti-fogging agent. As discussed herein, “fogging” refers to formation of small water droplets on a surface. The chemistry of anti-fogging agents are based on a polyalcohol being reacted with a fatty acid like stearic or lauric acid to form an ester. This forms a non-ionic surfactant which acts to inhibit fog formation. The long carbon tail of the fatty acid is hydrophobic and stays entangled in the host polymer. The polyalcohol with its -OH groups is hydrophilic and a non-ionic surfactant which prefers to “bloom” to the host polymer surface. Consequently, the hydrophilicity of the host polymer is increased.

As discussed above, systems of interest include a sample preparation unit, a partitioner, an analysis vessel, a thermocycler, and a sample reader. Any one, or all, of the components of the subject dPCR systems may include an anti-adhesion coating of the invention. For example, at least one (including each) of the sample preparation unit, analysis vessel, partitioner, thermocycler and sample reader comprises the anti- adhesion coating. In select cases, each surface of the dPCR system configured to contact a portion of the sample comprises the anti-adhesion coating.

In addition, systems of the invention are fully automated. By “fully automated”, it is meant that the dPCR process, from start to finish, may be performed by the system without user interaction and intervention. While the user may input sample, supply the system with reagents, and remove waste, the dPCR process itself may be executed in an automated fashion. In other words, sample lysis and DNA preparation is automatically performed by the analyzer's robotic and liquid handling components using protocols and reagents located within the system. The system then automatically mixes the sample and PCR reagents, and partitions the sample into multiple sample partitions. In addition, sample is automatically read by the sample reader. Results, which will be automatically available upon completion of dPCR, may also be automatically displayed. In embodiments, the subject fully automated dPCR systems may reduce total time for dPCR by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 50% or more, 55% or more, 60%, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, and including by 100% or more.

Sample Preparation Units

Sample preparation units of interest are configured to receive a biological sample and extract nucleic acids therefrom. In some instances, sample preparation units include a sample input block configured to receive the sample. The sample input block may be any device configured to receive a biological sample, e.g., in a liquid form. For example, the sample input block may comprise a block having a plurality of recessed wells for receiving sample(s). In some instances, the sample input block is configured such that sample may be added directly to the recessed wells. In some such instances, the sample input block may be a microwell plate, or the like. The sample input block may optionally be coated with the anti-adhesion coating discussed above. In other instances, the sample input block is configured to receive one or more other liquid vessels which, themselves, contain the sample (and may be optionally coated with the anti-adhesion coating). In such instances, the sample may be contained in one or more sample tubes. Exemplary sample tubes include Eppendorf Tubes® (e.g., 1 .5 mL, 2mL Eppendorf Tubes®), and the like. In some cases, the sample input block is configured to be removed from the dPCR system (e.g., so it can be loaded with sample, cleaned, etc.). In some such cases, the sample input block includes one or more handles.

The sample input block may be comprised of any convenient material. In some cases, the sample input block includes one or more rigid plastic materials such as, for example, polycarbonates, polyvinyl chloride (PVC), polyurethanes, polyethers, polyamides, polyimides, among other polymeric plastic materials. In certain cases, the block includes a 3D printed polymer. Any convenient 3D printed polymer may be employed, such as, for example, acrylonitrile butadiene styrene (ABS), acrylic styrene acrylonitrile (ASA), polyethylene terephthalate (PET), glycol-modified polyethylene terephthalate (PETG), polyaryletherketones (PAEK), polyetherimides (PEI), polycarbonate (PC), polypropylene, (PP), nylon as well as composites and hybrids thereof. In some cases, the sample input block includes one or more metals including, for example, aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin as well as combinations and alloys thereof.

Sample preparation units of the invention also include a sample purifier. The disclosed sample purifier is configured to extract an analyte (e.g., DNA, RNA) of interest from the sample and prepare the extracted analyte for further analysis. In some embodiments, the sample purifier comprises a plurality of recessed wells configured to receive sample (e.g., from the sample input block). In some instances, the sample purifier is configured such that sample may be added directly to the recessed wells. The recessed wells of the sample purifier may be coated with the anti-adhesion coating discussed above. In other cases, the sample purifier is configured to receive one or more other liquid vessels which, themselves, contain the sample to be purified (and may be optionally coated with the anti-adhesion coating). In such instances, the sample may be contained in one or more sample tubes. Exemplary sample tubes include Eppendorf Tubes® (e.g., 1 .5 mL, 2mL Eppendorf Tubes®), and the like. Sample purifiers of interest additionally include a plurality of synthetic particles located within the recesses that may be employed for sample purification. The synthetic particles can be, for example, beads. The bead can be, for example, a silica gel bead, a glass bead, a magnetic bead, a Dynabead®, a Sephadex®/ Sepharose® bead, a cellulose bead, a polystyrene bead, or any combination thereof. The bead can comprise a material such as polydimethylsiloxane (PDMS), polystyrene glass, polypropylene, agarose, gelatin, hydrogel, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, cellulose, nylon, silicone, or any combination thereof. The bead can comprise one or more types of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration on which an analyte (e.g., nucleic acid) can be immobilized (e.g., covalently or non-covalently). A bead can be, or comprise, a discrete particle that is spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like. In some embodiments, a bead can be non-spherical in shape. In some cases, the beads are dried within the recesses of the sample purifier.

Additionally, and for purposes herein, the particles may be magnetically responsive, e.g., by virtue of comprising one or more paramagnetic and/or superparamagnetic substances, such as for example, magnetite. Such paramagnetic and/or superparamagnetic substances may be embedded within the matrix of the particles, and/or may be disposed on an external and/or internal surface of the bead.

In certain embodiments, the particles are coated with a substance on their external surface that binds nucleic acids (e.g., DNA) non-specifically and reversibly. The beads may have any convenient surface chemistry that is configured to create binding to a target nucleic acid. According to some embodiments, the substance comprises carboxyl groups that non-specifically and reversibly bind nucleic acids. A non-limiting example of such a substance is succinic acid. In certain embodiments, the particulate solid supports are solid phase reversible immobilization (SPRI) beads. Exemplary types of beads that may be adapted for use in the sample purifier include, but are not limited to, carboxylate-modified magnetic beads, amine-blocked magnetic beads, oligo(dT)- coated magnetic beads, streptavidin-coated magnetic beads, streptavidin-blocked magnetic beads, NeutrAvidin™-coated magnetic beads, and silica-coated magnetic beads, combinations thereof, and the like.

The size of the beads can vary. For example, the diameter of the bead can range from 0.1 micrometer to 50 micrometers. In some embodiments, the diameters of beads can be, or be about, 0.1 , 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 micrometers, or a number or a range between any two of these values. The diameters of the bead can be related to the diameter of the recesses of the sample purifier. In some embodiments, the diameters of the bead can be, or be about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or a range between any two of these values, longer or shorter than the diameter of the recess. The diameter of the beads can be related to the diameter of a cell (e.g., a single cell entrapped by a well of the substrate). In some embodiments, the diameters of the beads can be, or be about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or a number or a range between any two of these values, longer or shorter than the diameter of the cell.

Sample purifiers of the invention also include at least one magnet for extracting nucleic acids from the biological sample. The magnet of the sample purifier is switchable between an active position and an inactive position. In the active position, the magnet applies a magnetic force to the recesses of the sample purifier. In the inactive position, the magnet does not apply a magnetic force to the recesses of the sample purifier. For example the sample purifier may include one or more magnets affixed to a supporting member; a motorized mechanism configured to move the supporting member in such a manner that the one or more magnets move backwards and forwards along a fixed axis, and during at least a portion of the motion, the one or more magnets maintain close proximity to one or more receptacles which contain the magnetic particles in solution; and control circuitry to control the motorized mechanism. The motor can be computer controlled to run at a particular speed; for example at a rotational speed that leads to vertical motion of the magnet in the range 1 -20 mm/s. The magnetic separator can thus be configured to move repetitively, e.g., up and down, from side to side, or backwards and forwards, along the same axis several times. In some embodiments, the supporting member rides on one more guiding members to ensure that the supporting member does not, for example, tip, twist, or yaw, or undergo other internal motions while moving (other than that of controlled motion along the axis) and thereby reduce efficacy of the separation.

For example, in some embodiments the magnet can be movable between a first position or inactive position in which a magnetic field produced by the magnet does not attract magnetic particles within the microwell array or exerts a relatively weak attractive force on magnetic particles, and a second position or active position in which the magnetic field produced by the magnet can attract magnetic particles. While the magnet is active, magnetic particles (optionally bound to target analyte/nucleic acid) are immobilized within the recesses while wash steps are performed. In embodiments, when in the active position, a superior surface of the magnet can be in parallel with an inferior surface of the recesses.

Embodiments of the sample purifier also include an agitator. Any convenient device configured to stir or disturb sample liquid during nucleic acid extraction may be employed. In certain cases, the agitator is a vortexer (i.e. having a component configured to oscillate in a circular motion in a manner sufficient to create a vortex). In certain cases, the agitator is a rocker configured to tilt the purifier back and forth. In still further cases, the agitator is a shaker.

The sample purifier may be comprised of any convenient material. In some cases, the sample purifier includes one or more rigid plastic materials such as, for example, polycarbonates, polyvinyl chloride (PVC), polyurethanes, polyethers, polyamides, polyimides, among other polymeric plastic materials. In certain cases, the block includes a 3D printed polymer. In some embodiments, the microplate is comprised of polystyrene. In some cases, the sample purifier includes one or more metals including, for example, aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin as well as combinations and alloys thereof.

The sample preparation unit may additionally provide for reagent storage. For example, embodiments of the sample preparation unit include one or more reagent repositories. Any sealable vessel configured to contain dPCR reagents may be employed. In certain cases, the reagents are positioned within tubes, bags, bottles, combinations thereof, and the like. In some embodiments, the reagent repository comprises a PCR master mix. As is known in the art, PCR master mix comprises precursors and enzymes for use in a PCR reaction (e.g., dNTPs, MgCl2, Taq polymerase, fluorochromes, etc.). Commercially available PCR master mixes are produced by, e.g., Sigma-Aldrich, Thermo Fisher, Bio-Rad and Qiagen. In select embodiments, the dPCR reagents are dried (e.g., lyophilized). For example, in one embodiment, one or more reagent repositories of the invention include dried master mix. Extracted nucleic acid from the sample purifier may then be placed in a container having the dried master mix and then agitated, resulting in a solution comprising both the extracted nucleic acids and necessary dPCR reagents.

Reagent repositories may also include one or more dyes configured to stably associate with target nucleic acids. In addition to the reagents already discussed, and those known to those of skill in the art of nucleic acid amplification and quantification, various detection reagents, such as fluorescent and non-fluorescent dyes and probes may be included in the reagent repository. For example, systems of the invention may employ reagents suitable for use in a TaqMan™ reaction, such as a TaqMan™ probe; reagents suitable for use in a SYBR Green fluorescence detection; reagents suitable for use in a molecular beacon reaction, such as molecular beacon probes; reagents suitable for use in a scorpion reaction, such as a scorpion probe; reagents suitable for use in a fluorescent DNA-binding dye-type reaction, such as a fluorescent probe; and/or reagents for use in a LightUp protocol, such as a LightUp probe. In some embodiments, the reagent repository includes compositions for quantifying a detectable signal (e.g. fluorescence) from partitions containing amplified nucleic acid (e.g. target amplicons, etc.). Such reagents may be employed during labeling of (e.g. during amplification, post- amplification) amplified nucleic acids with a detectable label, exposing partitions to a light source at a wavelength selected to cause the amplicon bound probe dye to fluoresce, and detecting and/or measuring the resulting fluorescence. Fluorescence emitted from the partitions can be tracked during amplification reaction to permit monitoring of the reaction (e.g., using a SYBR Green-type compound), or fluorescence can be measured post-amplification.

In some embodiments, the present invention provides systems for detecting and/or quantifying the presence of a target nucleic acid in partitions by providing a probe with specificity for a target nucleic acid (e.g., a TaqMan™-type probe) in partitioned amplification reactions, and detecting/measuring the resulting fluorescence. In some embodiments, partitions containing amplified nucleic acid (e.g., target amplicons, etc.) will exhibit quantifiable post-amplification fluorescence. In some embodiments, detection of a fluorescent signal is indicative of the presence of the template nucleic acid (e.g., target) in the partition.

In some embodiments, systems include a reagent repository comprising vessels (e.g., tubes, bags, bottles) including one or more buffers. Buffers of interest include, e.g., rehydration buffers, lysis buffers and elution buffers. For example, lysis buffer from the reagent repository may be employed in nucleic acid extraction in the sample purifier. In embodiments, after nucleic acid has been extracted in the sample purifier, systems of the invention are configured to elute the extracted nucleic acids using the elution buffer. In some cases, systems of the invention are configured to elute the nucleic acids in a volume that is smaller than the original sample volume. In some cases, the eluted volume is 10% smaller or more, 20% smaller or more, 30% smaller or more, 40% smaller or more, 50% smaller or more, 60% smaller or more, 70% smaller or more, 80% smaller or more and including 90% smaller or more. Rehydration buffers may be employed to rehydrate dried dPCR materials (e.g., such as dried beads or dried master mix, etc.). Buffers of interest may include one or more of the following: guanidine, isopropanol, ethanol, Tris, and ethylenediaminetetraacetic acid (EDTA), and the like.

Reagent tubes that hold liquids or liquid reagents can be sealed with a laminate structure. The laminate structure typically has a heat seal layer, a plastic layer such as a layer of polypropylene, and a layer of metal such as aluminum foil, wherein the heat seal layer is adjacent the one or more reagent tubes. The additional plastic film that is used in a laminate for receptacles that contain liquid reagents is typically to prevent liquid from contacting the aluminum.

Partitioners

Systems of the invention additionally include a partitioner. As used herein, the term “partition” refers to a volume of fluid (e.g. liquid) that is a separated portion of a bulk volume. A bulk volume may be partitioned into any suitable number of smaller volumes (i.e. partitions). In embodiments, the number of partitions may range from 10² to 10⁷. Partitions may be separated by a physical barrier or by physical forces (e.g. surface tension, hydrophobic repulsion, etc.). Partitions generated from the larger volume may be substantially uniform in size (monodisperse) or may have non-uniform sizes (polydisperse). Partitions may be produced by any suitable manner (e.g. emulsion, microfluidics, microspray, etc.). Exemplary partitions are droplets.

The partitioner may be any device capable of generating a plurality of sample partitions comprising the extracted nucleic acids. In some embodiments, the partitioner is a droplet generator. As used herein, the term “droplet” refers to a small volume of liquid that is immiscible with its surroundings (e.g. gases, liquids, surfaces, etc.). A droplet may reside upon a surface, be encapsulated by a fluid with which it is immiscible (e.g. the continuous phase of an emulsion, a gas (e.g. air, nitrogen)), or a combination thereof. A droplet is typically spherical or substantially spherical in shape, but may be non-spherical. The shape of an otherwise spherical or substantially spherical droplet may be altered by deposition onto a surface or constriction in a capillary channel of smaller diameter. A droplet may be a “simple droplet” or a “compound droplet,” wherein one droplet encapsulates one or more additional smaller droplets. The droplets created by the droplet generator may have any convenient volume of liquid, such as where the volume ranges from 0.1 pL to 1 pL. The diameter of a droplet and/or the average diameter of a set of droplets provided herein may vary, and can range from 1 pm to 1000 pm, such as 10 pm to 500 pm, such as 50 pm to 200 pm.

Where the partitioner is a droplet generator, the dPCR system may, in certain cases, be referred to as a droplet digital PCR system (ddPCR). ddPCR is described in, e.g., Abram et al. Lab on a Chip 20, no. 3 (2020): 477-489; herein incorporated by reference in its entirety. In such embodiments, the droplet generator may generate droplets by employing two immiscible fluids: a dispersed phase and a continuous phase. In this instance, the dispersed phase is a solution comprising the eluted nucleic acids from the sample preparation unit. In certain cases, the dispersed phase is an aqueous liquid (e.g., the elution buffer in which the extracted nucleic acids are transported is an aqueous liquid). The continuous phase is a liquid (e.g., an oil), that is immiscible with the solution of extracted nucleic acids. Embodiments of the partitioner include a dispersed phase channel through which the dispersed phase is flowed, and one or more continuous phase channels through which the continuous phase is flowed. The dispersed phase channel and continuous phase channel(s) may be arranged in any suitable configuration. In certain cases, the channels are arranged in a co-axial configuration in which the two phases are flowed in parallel. In other words, the dispersed phase channel is surrounded by the continuous phase channel such that the two channels are co-axial. The two phases may be co-flowed in such a manner that droplets are produced. In additional embodiments, a continuous phase channel and a dispersed phase channel may be arranged in a T-junction configuration. In such a configuration, the dispersed phase channel meets the continuous phase channel at 90 degrees in a T-shaped junction. In still other embodiments, a dispersed phase channel and a continuous phase channel are arranged in a flow-focusing configuration. In such a configuration, at least two continuous phase channels meet a dispersed phase channel such that the dispersed phase is squeezed by two counterflowing streams of the continuous phase. Additional details regarding droplet formation may be found in Baroud et al. Lab on a Chip 10, no. 16 (2010): 2032-2045, herein incorporated by reference in its entirety.

Embodiments of the system where the partitioner is a droplet generator may additionally include a immiscible liquid repository (i.e., reservoir). The immiscible liquid repository may comprise any suitable vessel(s) (e.g., tubes, bags, bottles) in which the aforementioned continuous phase may be stored and from which the same continuous phase may be drawn for use in the droplet generator. In some cases, the immiscible liquid is an oil. Oils suitable for use in droplet formation are described in, e.g., Baret, Jean-Cristophe. Lab on a Chip 12, no. 3 (2012): 422-433. Oils of interest may include, but are not limited to, silicon oils, hydrocarbon oils, and fluorinated oils. In select embodiments, the immiscible liquid in the immiscible liquid repository comprises a surfactant. Exemplary surfactants may include, e.g., Triton X-100, ABIL EM90, PF- decanol, perfluorotetradecanoic acid (PFTD), PEG end-capped perfluoropolyether (PFPE), FluoSurf™, or the like, and combinations thereof.

In select embodiments where the partitioner is a droplet generator, the analysis vessel may be a cuvette. The term “cuvette” is used in its conventional sense to describe a tube-like vessel having light-accessible walls for the analysis of the contents of the vessel. The cuvette may have any convenient cross-section. In some embodiments, the cuvette possesses a circular cross-section. In other embodiments, the cuvette possesses a square cross-section. The cuvette may be constructed from any suitable transparent material through which light may be transmitted. In certain cases, the cuvette is constructed from plastic. In other embodiments, the cuvette is constructed from glass. In still other embodiments, the cuvette is constructed from quartz (e.g., fused quartz). In certain cases, the cuvette is configured to move. In some such cases, the cuvette may be configured to rotate around a vertical axis, and translate (i.e., move up and down) along the same vertical axis. Any displacement protocol may be employed to move the cuvette, such as a coupling to a moveable support stage or directly with a motor actuated translation stage, leadscrew translation assembly, geared translation device, such as those employing a stepper motor, servo motor, brushless electric motor, brushed DC motor, micro-step drive motor, high resolution stepper motor, among other types of motors. The cuvette may be configured to move at multiple speeds. In some embodiments, the cuvette may be configured to translate at a speed ranging from 1 mm/s to 20 mm/s, such as 2 mm/s to 10 mm/s, and including 3 mm/s to 7 mm/s. In some cases, the cuvette is configured to translate at a speed ranging from 100 rpm to 300 rpm, such as 125 rpm to 275 rpm, such as 150 rpm to 250 rpm, and including 175 rpm to 225 rpm. In some embodiments, systems include a plurality of cuvettes such as 2 or more cuvettes, 3 or more cuvettes, 4 or more cuvettes and including 5 or more cuvettes.

Alternative embodiments of the partitioner described herein include a liquid dispenser configured to dispense small amounts of liquid onto different portions of an analysis vessel and thereby generate sample partitions. In embodiments, the liquid dispenser may be configured to dispense volumes of liquid ranging from 500 nL to 10,000 nL, such as 50 nL to 700 nL. Examples of automated liquid dispensers include that may be adapted for use in embodiments of the present systems include, but are not limited to, those described in published PCT application publication nos.

W02009129397, WO2015192010 and WO2015192010; the disclosures of which are herein incorporated by reference. In some cases, the analysis vessel into which the liquid dispenser provides liquid comprising the target analyte is a microfluidic array partitioning (MAP) device. In such cases, the MAP device includes a series of microfluidic channels and microchambers. In embodiments, the liquid dispenser is configured to provide sample liquid to an inlet well. In such embodiments, the partitioning may occur via microfluidic transfer. In other words, liquid may flow through the channels from the inlet well and be collected into the microchambers. A PCR reaction may take place within each of the microchambers. The number of microchambers within the MAP device may vary, as desired. For example, the number of microchambers may range in some embodiments from 500 to 50,000, such as 1 ,000 to 40,000, from 5,000 to 30,000 and including 15,000 to 25,000. In some embodiments, the MAP device — including the microchannels and microchambers — includes the anti-adhesion coating discussed above. MAP devices are described in, e.g., Dueck et al. Scientific reports 9, no. 1 (2019): 1 -9; incorporated by reference herein.

In some embodiments of the dPCR system where the partitioner is a liquid dispenser, the analysis vessel into which the partitioner disperses liquid is a microwell plate. Microwell plates may have any convenient configuration. In some embodiments, microwell plates are fabricated by inserting wells in plates using lithography. The plates may be comprised of any convenient material. In some cases, the microwell plates include one or more rigid plastic materials such as, for example, polycarbonates, polyvinyl chloride (PVC), polyurethanes, polyethers, polyamides, polyimides, among other polymeric plastic materials. In certain cases, the block includes a 3D printed polymer. Any convenient 3D printed polymer may be employed, such as, for example, acrylonitrile butadiene styrene (ABS), acrylic styrene acrylonitrile (ASA), polyethylene terephthalate (PET), glycol-modified polyethylene terephthalate (PETG), polyaryletherketones (PAEK), polyetherimides (PEI), polycarbonate (PC), polypropylene, (PP), nylon as well as composites and hybrids thereof. In some cases, the sample input block includes one or more metals including, for example, aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin as well as combinations and alloys thereof. Microwells can be fabricated in a variety of shapes. Non-limiting exemplary well geometries can include cylindrical, conical, hemispherical, rectangular, or polyhedral (e.g., three dimensional geometries comprised of several planar faces, for example, hexagonal columns, octagonal columns, inverted triangular pyramids, inverted square pyramids, inverted pentagonal pyramids, inverted hexagonal pyramids, or inverted truncated pyramids). The microwells can comprise a shape that combines two or more of these geometries. For example, a microwell can be partly cylindrical, with the remainder having the shape of an inverted cone.

The diameter of a microwell can be specified in terms of absolute dimensions. The diameter of a microwell can range from about 1 nanometer to about 1000 micrometers. In some embodiments, the microwell diameter can be, or be about, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrometers, or a number or a range between any two of these values.

The depth of the microwell can vary, for example, to provide efficient trapping of droplets within the wells. The depth of a microwell can be specified in terms of its absolute dimension. For example, the depth of a microwell can range from about 1 nanometer to about 1000 micrometers. In some embodiments, the microwell depth can be, or be about, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrometers, or a number or a range between any two of these values.

The center-to-center distance or the center-to-center spacing between wells can vary from about 1 micrometer to about 1000 micrometers. In some embodiments, the center-to-center distance between wells can be, or be about, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrometers, or a number or a range between any two of these values. The distance or the spacing between the edges of the microwells can vary from about 1 micrometer to about 1000 micrometers. In some embodiments, the distance between the edges of the wells can be, or be about, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrometers, or a number or a range between any two of these values. A microwell array can comprise microwells at varying densities, for example ranging from 100 microwells per inch² to 1000000 microwells per inch². In some embodiments, the density of the microwell array can be, or be about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, or a number or a range between any two of these values, microwells per cm²

The total number of microwells on a substrate can vary based on the pattern and the spacing of the wells and the overall dimensions of the array. The number of microwells in the array can vary, for example, ranging from about 96 to about 1000000. In some embodiments, the number of microwells in the microwell array can be about 96. In some embodiments, the number of microwells can be about 150000.

A microwell array can comprise surface features between the microwells that are designed to help guide liquid into the wells and/or to prevent them from settling on the surfaces between wells. Non-limiting examples of suitable surface features include, but are not limited to, domed, ridged, or peaked surface features that encircle the wells or straddle the surface between wells.

Thermocvclers

The subject systems also include a thermocycler configured to modulate temperature in the generated sample partitions so as to amplify nucleic acids of interest in the generated sample partitions. Thermocyclers employ alternating cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. In some embodiments, thermocyclers amplify target nucleic acids (e.g. within sample partitions). PCR may be performed by thermal cycling between two or more temperature set points, such as a higher melting (denaturation) temperature and a lower annealing/extension temperature, or among three or more temperature set points, such as a higher melting temperature, a lower annealing temperature, and an intermediate extension temperature, among others. PCR may be performed with reagents from the reagent repository, e.g., a thermostable polymerase, such as Taq DNA polymerase (e.g., wild- type enzyme, a Stoffel fragment, FastStart polymerase, etc.), Pfu DNA polymerase, S- Tbr polymerase, Tth polymerase, Vent polymerase, or a combination thereof, among others. Additional details regarding thermocyclers may be found in, e.g., U.S. Patent Nos 5,716.842; 6,153,426; 6,556,940; 7,030,340; 7,939,312; RE39,566; 8,940,524; 9,034,635; 9,352.322; 9,475,053; 10,406,527; 10,632,466 and 10,960,399; as well as U.S. Patent Application Publications Nos 2006/0105433; 2008/0176292; 2017/0304828; and 2021/0291190; the disclosures of which are incorporated by reference herein.

Sample Reader

Aspects of the subject systems also include a sample reader. The sample reader may be configured to monitor fluorescence from biochemical reactions. The reader can include, for example, a light source that selectively emits light in an absorption band of a fluorescent dye, lenses for focusing the light, and a light detector (for example a photodiode) that selectively detects light in an emission band of the fluorescent dye, wherein the fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof. Alternatively, the optical detector can include a band pass-filtered diode that selectively emits light in the absorption band of the fluorescent dye (a fluorogenic probe) and a bandpass filtered photodiode that selectively detects light in the emission band of the fluorescent dye. For example, the optical detector can be configured to independently detect a plurality of fluorescent dyes having different fluorescent emission spectra, wherein each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof. For example, the optical detector can be configured to independently detect a plurality of fluorescent dyes at a plurality of different locations of, for example, a cuvette or microwell plate, wherein each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof.

In embodiments, the sample reader configured to detect whether amplified nucleic acid is present in each of the generated sample partitions within the analysis vessel is a particle counter, such as a two-dimensional (2D) or three-dimensional (3D) particle counter. In certain cases, the particle counter is a 3D particle counter. In embodiments, the 3D particle counter includes a horizontal-geometry confocal microscope. In such embodiments, the sample reader includes a light source. Any light source suitable for use in horizontal-geometry confocal microscopy may be employed. In some embodiments, the light source is a laser. In embodiments, the laser may be any convenient laser, such as a continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser and a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some instances, the laser is a gas laser, such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon chlorine (XeCI) excimer laser or xenon-fluorine (XeF) excimer laser or a combination thereof. In other instances, the subject flow cytometers include a dye laser, such as a stilbene, coumarin or rhodamine laser. In yet other instances, lasers of interest include a metal-vapor laser, such as a helium-cadmium (HeCd) laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser, helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser, copper laser or gold laser and combinations thereof. In still other instances, the subject flow cytometers include a solid-state laser, such as a ruby laser, an Nd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser, Nd:YVO₄ laser, Nd:YCa4O(BO₃)₃ laser, Nd:YCOB laser, titanium sapphire laser, thulim YAG laser, ytterbium YAG laser, ytterbiunWs laser or cerium doped lasers and combinations thereof.

The particle counter may additionally include one or more optical adjustment components for focusing laser light on the analysis vessel (e.g., cuvette, microwell plate). In certain embodiments, the optical adjustment component is located between the light source and the analysis vessel, and may include any device that is capable of changing the spatial width of irradiation or some other characteristic of irradiation from the light source, such as for example, irradiation direction, wavelength, beam width, beam intensity and focal spot. Optical adjustment protocols may include any convenient device which adjusts one or more characteristics of the light source, including but not limited to lenses, mirrors, filters, fiber optics, wavelength separators, pinholes, slits, collimating protocols and combinations thereof. In certain embodiments, particle counters of interest include one or more focusing lenses. The focusing lens, in one example, may be a de-magnifying lens. In certain cases, the optical adjustment component is an objective lens. The objective lens may have any suitable magnification, such as 10x, 20x, 50x and 100x. In some cases, the objective lens has a magnification of 20x. The above-described rotation and translation of the cuvette transports droplets across the Gaussian-shaped excitation volume.

Particle counters of the invention may additionally include a dichroic mirror. After fluorescent signal is emitted in the sample following irradiation, the emitted fluorescent light may be collected by the objective lens described above, and directed to the dichroic mirror. While the dichroic mirror is configured to pass the excitation light provided by the light source, it is configured to reflect emission wavelength light.

Embodiments of the particle counters also include a light detector configured to detect the light emitted from the sample and reflected by the dichroic mirror. Detectors of interest may include, but are not limited to, optical sensors or detectors, such as active-pixel sensors (APSs), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, among other detectors. In certain embodiments, the collected light is measured with a charge-coupled device (CCD), semiconductor charge-coupled devices (CCD), active pixel sensors (APS), complementary metal-oxide semiconductor (CMOS) image sensors or N-type metal- oxide semiconductor (NMOS) image sensors. In certain embodiments, the detector is a photomultiplier tube, such as a photomultiplier tube having an active detecting surface area of each region that ranges from 0.01 cm² to 10 cm², such as from 0.05 cm² to 9 cm², such as from 0.1 cm² to 8 cm², such as from 0.5 cm² to 7 cm² and including from 1 cm² to 5 cm².

Where the partitioner is a liquid dispenser configured to distribute extracted nucleic acid droplets on, e.g., a microwell plate, embodiments of the invention include a microplate reader. Microplate readers are discussed herein in their conventional sense to refer to instruments configured to measure properties of analytes within the well of microplates. In some cases, the microplate reader is a microplate fluorescence reader. In such cases, the microplate reader is configured to detect the presence or absence of fluorescent emission from each well of the microwell plate. In some cases, the microplate reader is a microplate colorimetric reader. In such cases, the microplate reader is configured to detect the presence or absence of colored compounds in solutions within the wells of the microwell plate, where the presence of the colored compounds may indicate the presence of the target nucleic acid. Additional details regarding microplate readers can be found in U.S. Patent Nos. 5,784,152; 9,029,101 ; 9,733,124; 9,964,556; 9,994,889; 10,072,982; 10,180,441 ; 10,527,550; the disclosures of which are incorporated by reference herein.

In certain cases, each PGR reaction (i.e., within each droplet, well, or chamber, as applicable) will generate a positive or negative result (i.e., presence or absence of target analyte). In some cases, the original concentration of target in the sample can be determined by counting the number of microchambers exhibiting positive versus negative results and applying Poisson statistics. In some cases, Poisson statistics may be employed to provide a corrected concentration of positive partitions (i.e., comprising the analyte), as follows:

Conveyance Devices

Systems of the invention may additionally include one or more conveyance devices. Conveyance devices of interest are configured to convey liquid (e.g., sample- containing liquid, reagents, etc.) from one portion of the dPCR system to another. Embodiments of the conveyance device include an automated liquid dispenser system. The automated liquid dispenser system may be operably connected to at least one of the sample preparation unit, the partitioner, the thermocycler, and the sample reader, and be configured to convey liquid between at least two of these components (such as at least 3 of these components, and including all of these components), or their constituent elements. A suitable liquid dispenser for use with the apparatus herein comprises one or more sensors; a manifold, one or more pumps in fluid communication with the manifold; one or more dispense heads in fluid communication with the manifold; and electrical connections that accept electrical signals from an external controller, wherein the liquid dispenser has no inlet or outlet for fluids, other than through the one or more pumps.

Embodiments of the automated liquid dispenser system include an automatic pipettor system. The pipettor may be a single-channel pipettor or a multi-channel pipettor. Where the pipettor is a multi-channel pipettor, the number of channels may vary, as desired. In some cases, the multi-channel pipettor includes a number of channels ranging from 2 to 40, such as 5 to 20. The pipettor may be positioned on a robotic arm, such that the pipettor may automatically change location within the dPCR system. For example, the automatic pipettor may be configured to draw in liquid inserted into the sample input block, change location, and transfer the liquid to the sample purifier. Pipettors may additionally be configured to use disposable, sterile, tips (optionally coated with the anti-adhesion coating described herein). In some cases, the robotic arm relocates the pipettor to a tip storage area before each conveyance of liquid. At the tip storage area, the robotic arm may be configured to load a new tip for each pipettor channel (e.g., by lowering the channel into the tip in such a manner that the tip remains fixed to the channel). In addition, following the conveyance of liquid, the robotic arm is configured to relocate the pipettor to a waste repository where the used tip(s) may be discharged and stored. Further details regarding automatic liquid dispenser systems may be found in U.S. Patent Nos. 6,732,598; 7.429,360; 9,134,332 9,335,336; and 10,704,039, the disclosures of which are incorporated by reference herein. In some embodiments, systems include a plurality of automatic pipettor systems, such as where the number of automatic pipettor systems ranges from 2 to 5. In select cases, dPCR systems of the inventions include 2 automatic pipettor systems. In some embodiments where the partitioner is a droplet generator, embodiments of the subject systems include at least one pipettor system configured to take an aliquot of amplified sample (e.g., from the thermocycler) and transfer it to the sample reader.

The liquid dispenser can further comprise a computer-controlled pump connected to a distribution manifold with related computer controlled valving. The distribution manifold can comprise a number of valves, such as solenoid valves configured to control the flow of air through the pipette tips; in an exemplary embodiment, there are two valves for each pipette, and one additional valve to vent the pump. Thus, for a liquid dispenser having four pipette heads, there are nine valves. In another embodiment there is only one valve for each pipette, and one additional valve to vent the pump. However, the distribution manifold is not limited so comprising exactly nine solenoid valves.

Embodiments of the liquid dispenser include a pump for pumping air in and out of the distribution manifold. The distribution manifold comprises a microfluidic network that distributes air evenly amongst the one or more valves. Thus, by controlling flow of air through the manifold and various valves, pressure above the pipette heads can be varied so that liquid is drawn up into or expelled from a pipette tip attached to the respective pipette heads.

Embodiments of the liquid dispenser can also operate in conjunction with a motorized plate configured to strip the pipette tips and align the pipettes during dispensing of fluid into a micro fluidic cartridge, as further described herein. In such embodiments, the pipette tips may be aligned, all at the same pitch, above respective sockets (over a pipette tip sheath) in a holder. A metal plate having elongated holes lies over the sockets. The pipette tips are inserted part way down into the sheath through the elongated holes, and the metal plate is moved along in such a manner that the pipette tips are clamped by the elongated portion of the holes. When the liquid dispenser is moved up, the pipette tips become detached from their respective heads. When the metal plate is subsequently moved back to its initial position, the pipette tips remain in place in their respective sockets.

In addition, or alternatively, systems may include one or more conveyance devices configured to convey vessels containing solid or liquid components (e.g., sample-containing liquid, reagents, etc.) from one portion of the dPCR system to another. In some such cases, the one or more conveyance devices may be robotic arms. The robotic arm may be configured for automatic control in a plurality of ranges of motion. For example, the arm may be configured to extend or telescope in all directions along the X and Y axes. The arm may additionally be capable of movement around its central point to allow for rotation of the arm mechanism. In some cases, the robotic arm is operably connected to a controller that has been configured and/or trained to cause the robotic arm to obtain a vessel of the system, and relocate said vessel to another location within the system. Methods for programming a robotic arm to relocate entities are described in U.S. Patent Application Publication No. 2006/0047363, the disclosure of which is incorporated by reference herein.

FUG. 1 depicts a fully automated dPCR system according to certain embodiments of the invention. Fully automated dPCR system 100 includes a sample preparation unit 1 10 comprising a sample input block 101 , a sample purifier 102, and reagent repositories 103a and 103b. A user may place one or more aliquots of sample into the sample input block 101. In some cases, this includes removing the sample input block 101 , inputting the sample, and returning the block to the fully automated dPCR system 100. Sample from the sample input block 101 may be transferred to the sample purifier 102 (e.g., via pipettor system 108). In the sample purifier 102, nucleic acid is extracted from the sample liquid. In the example of FIG. 1 , sample purifier 102 includes one or more magnets (not shown) configured to apply a magnetic force to magnetic beads (e.g., having extracted nucleic acid stably associated therewith). In addition, sample purifier 102 may be equipped with an agitator configured to break-up the sample and facilitate nucleic acid extraction. In embodiments, pipettor system 108 is configured to supply reagents to the sample purifier for the extraction and elution of nucleic acids form the sample (e.g., lysis buffers, elution buffers, etc.).

Reagent repository 103a includes one or more buffers needed for sample preparation (e.g., rehydration buffers, elution buffers, etc.). In the example of FIG. 1 , reagent repository 103b includes dried down (e.g., lyophilized) PCR master mix. Extracted nucleic acid in an elution buffer solution may be transferred from the sample purifier 102 to wells within reagent repository 103b (e.g., via automatic pipettor system 108). Reagent repository 103b may be, e.g., a microwell plate or a block comprising liquid vessels such as tubes. Insertion of the extracted nucleic acid solution to reagent repository 103b causes the dried down master mix to enter solution. This solution may then be transferred from reagent repository 103b to the partitioner 104a. In some cases, the transfer is carried out by the pipettor system 108. In other cases, a robotic arm 109 may be configured to transfer the vessel containing the solution (e.g., reagent repository 103b) to the partitioner 104a. Spent tips and liquid waste may be deposited in waste repository 107. In the embodiment of FIG. 1, partitioner 104a is a droplet generator. However, other types of partitioners may also be employed, such as the liquid dispenser discussed below with respect to FIG. 3B. The droplet generator is configured to partition the extracted nucleic acid solution into a plurality of droplets by combining the solution with an immiscible liquid. One embodiment of this process is shown in greater detail in FIG. 3A. Following droplet generation, the partitioned sample may then be transferred to the thermocycler 105 where the extracted nucleic acid is amplified. This transfer may be carried out, e.g., by robotic arm 109. After PGR, the partitioned sample may be read in sample reader 106, which may be configured to determine the positivity or negativity of each sample partition with respect to the presence of the analyte of interest.

FIG. 2 depicts a component of the dPCR system having an anti-adhesion coating. As shown in FIG. 2 vessel 201 comprises wells 202. Wells 202 are coated with anti-adhesion coating 203, as shown in the magnified portion of vessel 201 . In the example of FIG. 2, vessel 201 resembles a microwell plate. However, it should be noted that any component of the dPCR system (such as any of the components shown in FIG. 1) may be coated with the anti-adhesion coating in the same fashion. In some cases, the entirety of the surface of vessel 201 is coated with anti-adhesion coating 203 (i.e., not just the wells).

FIG. 3A presents an embodiment of the invention in which the partitioner is a droplet generator. Droplet generator 301 is arranged in a flow focusing configuration in which a continuous phase (i.e., oil) causes sample liquid 302 to break off and form droplets 304. Following an amplification step in the thermocycler (not shown), droplets 304 may be transferred to cuvette 305. Droplets 304 may subsequently be analyzed by sample reader 310. In the embodiment of FIG. 3A, sample reader 310 is a 3D particle counter. Sample reader 310 includes light source 318, dichroic mirror 319, objective lens 315, and detector 317. Light from light source 318 passes through dichroic mirror 319 and is focused by objective lens 315 onto a certain portion of the cuvette 305 and irradiates droplets 304. If a droplet contains nucleic acid, it will emit fluorescent light which is back-propagated through objective lens 315, reflected by dichroic mirror 319, and detected by detector 317. Cuvette 305 is configured to rotate and translate as shown by arrows, allowing different portions of the cuvette to be illuminated by light source 318. In embodiments, one or more of the droplet generator 301 and the cuvete 305 is coated with anti-adhesion coating 203 shown in FIG. 2.

FIG. 3B presents an embodiment of the invention where the partitioner is a liquid dispenser. As shown in FIG. 3B, liquid dispenser 310 is configured to partition sample liquid by dispensing small amounts into the wells of an analysis vessel (i.e. , microplate 311 ). Alternatively, the analysis vessel 311 could be a MAP device. The microplate 311 and sample partitions therein may be subjected to thermocycling (not shown), and then read by sample reader 312. The sample reader 312 may be a microplate fluorescence reader and/or a microplate colorimetric reader depending on the type of assay being run. In embodiments, each of liquid dispenser 310 and microplate 311 are coated with anti-adhesion coating 203 shown in FIG. 2.

In embodiments, the fully automated dPCR system is an enclosed system. In other words, components of the system are separated from the ambient environment (e.g., by walls, doors, etc.). For example, in some cases, the dPCR system includes a deck where the functional components of the dPCR system are located (e.g., the components shown in FIG. 1). The deck may be surrounded by walls enclosing the deck from the ambient environment. In some cases, the dPCR system includes a door configured to enclose the system. In some such cases, the door is a sliding door. In addition, the embodiments of the system include a waste repository. The waste repository is optional. In embodiments where it is present, it is configured to receive spent liquid reagents and/or spent pipettor tips. In other embodiments, where it is not present, spent liquid reagents can be transferred to and disposed of at a location outside of the holder, such as, for example, a sample tube that contained the original sample whose contents are being analyzed. In some embodiments, the waste repository is located under the deck.

FIG. 4 presents an embodiment of the invention in which the fully automated dPCR system is an enclosed system. dPCR system 400 includes deck 401 where the functional components of the dPCR system are located (see, e.g., FIG. 1). The deck and its components are enclosed by walls and sliding door 402. Below deck 401 is a compartment 403 in which a waste reservoir is located for discarded liquid waste and/or pipettor tips. COMPUTER-CONTROLLED SYSTEMS

Aspects of the present disclosure further include computer-controlled systems, where the systems include one or more computers for complete automation. A processor, such as a microprocessor, is configured to control functions of various components of the system as shown above, and is thereby in communication with each such component requiring control. Furthermore, the order in which the various functions are described, is not limiting upon the order in which the processor executes instructions when the apparatus is operating. It is also to be understood that, although a single processor is described as controlling all operations of the dPCR system, such operations may be distributed, as convenient, over more than one processor.

The processor can be configured to control various aspects of sample preparation and analysis. For example the processor may be operably connected to each of the sample preparation unit (e.g., the sample purifier), the partitioner, the thermocycler, and the sample reader, and initiate and control the activity of each of these elements, i.e., such the system is fully automated. Conveyance mechanisms, such as the automated liquid dispenser system and the robotic arms, are likewise operably connected to the processor.

Systems may include a display and operator input device. Operator input devices may, for example, be a keyboard, mouse, or the like. The processor may be operably connected to the display, which display may show the results of the dPCR from the sample reader. The processing module includes a processor which has access to a memory having instructions stored thereon for performing the steps of the subject methods. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, memory storage devices, and input-output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or it may be one of other processors that are or will become available. The processor executes the operating system and the operating system interfaces with firmware and hardware in a well-known manner, and facilitates the processor in coordinating and executing the functions of various computer programs that may be written in a variety of programming languages, such as Java, Perl, C++, Python, other high level or low level languages, as well as combinations thereof, as is known in the art. The operating system, typically in cooperation with the processor, coordinates and executes functions of the other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. In some embodiments, the processor includes analog electronics which provide feedback control, such as for example negative feedback control.

The system memory may be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic medium such as a resident hard disk or tape, an optical medium such as a read and write compact disc, flash memory devices, or other memory storage device. The memory storage device may be any of a variety of known or future devices, including a compact disk drive, a tape drive, or a diskette drive. Such types of memory storage devices typically read from, and/or write to, a program storage medium (not shown) such as a compact disk. Any of these program storage media, or others now in use or that may later be developed, may be considered a computer program product. As will be appreciated, these program storage media typically store a computer software program and/or data. Computer software programs, also called computer control logic, typically are stored in system memory and/or the program storage device used in conjunction with the memory storage device.

In some embodiments, a computer program product is described comprising a computer usable medium having control logic (computer software program, including program code) stored therein. The control logic, when executed by the processor the computer, causes the processor to perform functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementation of the hardware state machine so as to perform the functions described herein will be apparent to those skilled in the relevant arts.

Memory may be any suitable device in which the processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including magnetic or optical disks or tape or RAM, or any other suitable device, either fixed or portable). The processor may include a general-purpose digital microprocessor suitably programmed from a computer readable medium carrying necessary program code. Programming can be provided remotely to processor through a communication channel, or previously saved in a computer program product such as memory or some other portable or fixed computer readable storage medium using any of those devices in connection with memory. For example, a magnetic or optical disk may carry the programming, and can be read by a disk writer/reader. Systems of the invention also include programming, e.g., in the form of computer program products, algorithms for use in practicing the methods as described above. Programming according to the present invention can be recorded on computer readable media, e.g., any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; portable flash drive; and hybrids of these categories such as magnetic/optical storage media.

The processor may also have access to a communication channel to communicate with a user at a remote location. By remote location is meant the user is not directly in contact with the system and relays input information to an input manager from an external device, such as a computer connected to a Wide Area Network (“WAN”), telephone network, satellite network, or any other suitable communication channel, including a mobile telephone (i.e., smartphone).

In some embodiments, systems according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and/or transmitter for communicating with a network and/or another device. The communication interface can be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., Radio-Frequency Identification (RFID), Zigbee communication protocols, Wi-Fi, infrared, wireless Universal Serial Bus (USB), Ultra-Wide Band (UWB), Bluetooth® communication protocols, and cellular communication, such as code division multiple access (CDMA) or Global System for Mobile communications (GSM). In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port to allow data communication between the subject systems and other external devices such as a computer terminal (for example, at a physician’s office or in hospital environment) that is configured for similar complementary data communication.

In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the subject systems to communicate with other devices such as computer terminals and/or networks, communication enabled mobile telephones, personal digital assistants, or any other communication devices which the user may use in conjunction.

In one embodiment, the communication interface is configured to provide a connection for data transfer utilizing Internet Protocol (IP) through a cell phone network, Short Message Service (SMS), wireless connection to a personal computer (PC) on a Local Area Network (LAN) which is connected to the internet, or Wi-Fi connection to the internet at a Wi-Fi hotspot. In some embodiments, the communication interface provides connection to a cloud-based platform where dPCR data may be stored and/or accessed.

In one embodiment, the subject systems are configured to wirelessly communicate with a server device via the communication interface, e.g., using a common standard such as 802.1 1 or Bluetooth® RF protocol, or an IrDA infrared protocol. The server device may be another portable device, such as a smart phone, Personal Digital Assistant (PDA) or notebook computer; or a larger device such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), as well as an input device, such as buttons, a keyboard, mouse or touch-screen.

In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the subject systems, e.g., in an optional data storage unit, with a network or server device using one or more of the communication protocols and/or mechanisms described above. Output controllers may include controllers for any of a variety of known display devices for presenting information to a user, whether a human or a machine, whether local or remote. If one of the display devices provides visual information, this information typically may be logically and/or physically organized as an array of picture elements. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing graphical input and output interfaces between the system and a user, and for processing user inputs. The functional elements of the computer may communicate with each other via system bus. Some of these communications may be accomplished in alternative embodiments using network or other types of remote communications. The output manager may also provide information generated by the processing module to a user at a remote location, e.g., over the Internet, phone or satellite network, in accordance with known techniques. The presentation of data by the output manager may be implemented in accordance with a variety of known techniques. As some examples, data may include SQL, HTML or XML documents, email or other files, or data in other forms. The data may include Internet URL addresses so that a user may retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The one or more platforms present in the subject systems may be any type of known computer platform or a type to be developed in the future, although they typically will be of a class of computer commonly referred to as servers. However, they may also be a main-frame computer, a workstation, or other computer type. They may be connected via any known or future type of cabling or other communication system including wireless systems, either networked or otherwise. They may be co-located or they may be physically separated. Various operating systems may be employed on any of the computer platforms, possibly depending on the type and/or make of computer platform chosen. Appropriate operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, QS/400®, i5/OS®, IBM i®, Android™, SGI IRIX®, Oracle Solaris® and others. METHODS OF ANALYZING A SAMPLE

Aspects of the invention also include methods of analyzing a sample. Methods of interest include introducing a biological sample into a fully automated dPCR system of the invention, and analyzing the biological sample via dPCR. Embodiments of the subject methods also include overcoming specimen volume and organism concentration issues prior to the introduction of the sample into the fully automated dPCR system. As discussed above in the Summary section, attempting to use microfluidic/microdroplet technologies to analyze samples is challenging because the concentration of organism is extremely low. To achieve reasonable clinical sensitivity an analytical sensitivity of ~1 organism/ml is required. Classical blood culture systems use 10 ml of blood to overcome this issue and typical microfluidic/microdroplet techniques use 100’s of microliters (pL) to 1 ml of sample input.

In some embodiments, methods include centrifuging the sample prior to introduction into the dPCR systems of the invention. Such methods may include separating and concentrating analytes in the sample. Methods of centrifugation are described in, e.g., Stevens et al. Critical reviews in microbiology 30, no. 1 (2004): 7-24, herein incorporated by reference in its entirety. Separation can be defined as the removal of a select population from a complex mixture, while concentration is defined as a sample preparation process that seeks to reduce sample volume while simultaneously recovering all of the analytes (e.g., cells) of interest. In some embodiments, methods include centrifuging the sample via simple high-speed centrifugation (<60,000 x g). Such may be used to concentrate analytes (e.g., bacterial cells) from microbiological media before extracting nucleic acids and detecting by the fully automated dPCR systems described herein. In additional embodiments, methods include differential centrifugation. At each step of differential centrifugation, the particles of higher density are separated from those that are less dense. The speed of centrifugation is increased until the target particle settles, after which the final supernatant is removed and the pellet is resuspended for further assay. In further embodiments, methods include density gradient centrifugation. Such techniques rely upon a suspending solution that decreases in density from the bottom (highest density) to the top (lowest density) of the tube. In some embodiments, methods include increasing the efficiency of centrifugation via coagulation and/or flocculation. Coagulation is facilitated by the removal of electrostatic charges (e.g., usually by pH change), which allows particles to adhere to one another, thereby facilitating sedimentation by lower centrifugation speeds. Flocculation is achieved by adding small amounts of high molecular weight, charged materials which bridge oppositely charged particles to produce a loose aggregate which may be readily removed by centrifugation or filtration (see, e.g., Stevens et al.).

Methods of the invention may additionally include apportioning the sample into a plurality of sub-lots, and analyzing the plurality of sublots via the dPCR system in parallel. In such embodiments, methods may include dividing a sample of interest into multiple sub-lots, such as where the number of sublots ranges from 2 to 50, such as 5 to 20. In some embodiments, methods include dividing the sample into 10 or more sub- lots. Methods according to this embodiment also include processing the sub-lots in parallel. In other words, each sub-lot is subjected to the same preparation procedure at the same time. The partitions created with respect to the sub-lots may or may not be analyzed/read in parallel. In other words, the partitions may be read at the same or different times. Such parallel processing of sub-lots is described in Gao et al. Analytica chimica acta 606, no. 1 (2008): 98-107; incorporated by reference herein in its entirety.

Methods may additionally include pre-lysing micro-organisms in the sample and assaying the sample for a high copy number target. For example, methods may include pre-lysing bacteria and assaying for a high copy number target such as rRNA which is present at >10,000 copies per bacterial cell. In embodiments, such allows a user to obtain the most relevant portion of the whole sample for dPCR analysis. This may require processing only a portion of the original sample (e.g., blood). Any convenient lysis protocol may be employed. In some embodiments, methods include contacting a lysis buffer with the sample. Lysis buffers of interest may include, for example, NP-40 buffer, Ammonium-Chloride-Potassium buffer, SDS buffer, and the like. Cell lysis protocols that may be adapted for use in the subject methods are described in, e.g., Hall et al. Micromachines 4, no. 3 (2013): 321 -332; herein incorporated by reference in its entirety. Any convenient assay may be employed. In some cases, the assay is a TaqMan copy number assay, or the like. Methods of interest may additionally involve reducing time to detection (TTD). In other words, methods according to embodiments of the invention reduce the amount of time required for the presence of an analyte of interest to be detected via dPCR. Conventional dPCR systems generally require all sample partitions to be produced prior to analysis. However, the present inventors have discovered that analysis of the partitions (e.g., droplets) does not require that all droplets be produced. In other words, completed partitions can be analyzed while new droplets are being formed. As such, methods of the invention involve operating the partitioner and sample reader simultaneously. Sample partitions can be evaluated while the partitioner is still generating more sample partitions. For example, assuming a random distribution of a single analyte of interest (e.g., organism) in a 10 ml sample (e.g., blood) would mean that the time to positivity of a microdroplet approach would be close to 8 or 9 hours in the worst case. Assuming a more typical case of 1 organism per ml and a more reasonable droplet creation rate of 20 pL/min, the mean time to discovery of a droplet containing microorganisms is ~25min and the worst case is 50 minutes. Since the 18 hr TTD figure for blood culture assumes all organism loads it is reasonable to expect a real system to have a droplet formation time of close to 25 minutes. As such, simultaneous operation of the partitioner and reader can substantially reduce time required for a diagnosis.

FIG. 5 presents a flowchart for practicing methods of the invention according to certain embodiments. Step 501 includes obtaining a biological sample for analysis. In embodiments, the sample is a blood sample, although other samples may also be obtained, as desired. In step 502, the biological sample obtained in step 501 is subjected to an process in which the analyte of interest (e.g., cells, nucleic acids) are extracted. Step 503 includes increasing analyte concentration prior to dPCR analysis by one or more of: centrifuging the sample (503a); apportioning the sample into a plurality of sub-lots, and analyzing the plurality of sublots via the dPCR system in parallel (503b); pre-lysing micro-organisms in the sample, and assaying the sample for a high copy number target (503c); and eluting the analyte extracted in step 502 into a sufficiently smaller volume (503d). Step 504 includes introducing the sample into an automated digital polymerase chain reaction (dPCR) system, such as the one discussed above. Step 505 includes analyzing the biological sample via dPCR, which may include simultaneous partitioning and reading (505a).

In various embodiments, the sample is combined with a PCR reagent mixture comprising a polymerase enzyme and a plurality of nucleotides The PCR reagent mixture can be in the form of one or more lyophilized pellets and the steps by which the PCR-ready sample is prepared can involve contacting the PCR pellet with liquid to create a PCR reagent mixture solution. As discussed above, systems may have dried down or lyophilized ASR reagents preloaded such that the user only needs to input prepared polynucleotide sample into the dPCR system.

As discussed above, in some instances, the sample analyzed is a biological sample. The term “biological sample” is used in its conventional sense to refer to a whole organism, plant, fungi or a subset of animal tissues, cells or component parts which may in certain instances be found in blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen, or the like. In some embodiments, the biological sample is blood. As such, a “biological sample” refers to both the native organism or a subset of its tissues as well as to a homogenate, lysate or extract prepared from the organism or a subset of its tissues, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, sections of the skin, respiratory, gastrointestinal, cardiovascular, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. Biological samples may be any type of organismic tissue, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or derivative thereof, e.g., plasma, tears, urine, semen, etc., where in some instances the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or fingerstick (where the blood may or may not be combined with any reagents prior to assay, such as preservatives, anticoagulants, etc.).

In certain embodiments the source of the sample is a “mammal” or “mammalian”, where these terms are used broadly to describe organisms which are within the class Mammalia, including the orders carnivore (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subjects are humans. The methods may be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., neonates, infant, juvenile, adolescent, adult), where in certain embodiments the human subject is a juvenile, adolescent or adult. While the present invention may be applied to samples from a human subject, it is to be understood that the methods may also be carried-out on samples from other animal subjects (that is, in “non-human subjects”) such as, but not limited to, birds, mice, rats, dogs, cats, livestock and horses.

In embodiments, analyzing the biological sample via dPCR includes determining the presence or absence of a certain analyte in a sample, i.e., whether the analyte is present in the same or not present in the sample. In certain cases, the analyte is a microorganism, such as a virus, bacterium, or fungus. In select cases, the biological sample is analyzed via dPCR to determine the likelihood that the organism from which the sample was obtained suffers from a disorder. In some cases, the disorder is sepsis or septicemia. Common bloodborne pathogens associated with such conditions include, but are not limited to, Escherichia coli, Acinetobacter baumanii, Salmonella enterica, Shigella dysenteriae, Pseudomonas aeruginosa, Proteus mirabilis, Serratia marcescens, Neisseria meningitides, Klebsiella pneumonia, Streptococcus pneumonia, Staphylococcus aureus, Haemophilus influenzae, Legionella sp., Chlamydia pneumoniae, Listeria monocytogenes, Klebsiella sp., Enterobacter sp., Bacteroides fragilis, and the like.

In various embodiments, a probe is added to the sample that can be selective for a polynucleotide sequence, wherein the steps by which the PCR-ready sample is prepared involve contacting the neutralized polynucleotide sample or a PCR amplicon thereof with the probe. The probe can be a fluorogenic hybridization probe. The fluorogenic hybridization probe can include a polynucleotide sequence coupled to a fluorescent reporter dye and a fluorescence quencher dye. The PCR reagent mixture can further include a positive control plasmid and a plasmid fluorogenic hybridization probe selective for at least a portion of the plasmid and the microfluidic cartridge can be configured to allow independent optical detection of the fluorogenic hybridization probe and the plasmid fluorogenic hybridization probe. In various embodiments, the probe can be selective for a polynucleotide sequence that is characteristic of an organism, for example any organism that employs deoxyribonucleic acid or ribonucleic acid polynucleotides. Thus, the probe can be selective for any organism. Suitable organisms include mammals (including humans), birds, reptiles, amphibians, fish, domesticated animals, wild animals, extinct organisms, bacteria, fungi, viruses, plants, and the like. The probe can also be selective for components of organisms that employ their own polynucleotides, for example mitochondria. In some embodiments, the probe is selective for microorganisms, for example, organisms used in food production (for example, yeasts employed in fermented products, molds or bacteria employed in cheeses, and the like) or pathogens (e.g., of humans, domesticated or wild mammals, domesticated or wild birds, and the like). In some embodiments, the probe is selective for organisms selected from the group consisting of gram positive bacteria, gram negative bacteria, yeast, fungi, protozoa, and viruses.

In various embodiments, the probe can be selective for a polynucleotide sequence that is characteristic of an organism selected from the group consisting of Staphylococcus spp., e.g., S. epidermidis, S. aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Staphylococcus; Streptococcus(e.g., a, p or y-hemolytic, Group A, B, C, D or G) such as S. pyogenes, S. agalactiae; E. faecalis, E. durans, and E. faecium; nonenterococcal group D. streptococci, e.g., S. bovis and S. equines, Streptococci viridans, e.g., S. mutans, S. sanguinis, S. salivarius, S. mitior, A. milleri, S. constellatus, S. intermedius, and S. anginosus; S. iniae; S. pneumoniae; Neisseria, e.g., N. meningitidis, N. gonorrhoeae, saprophytic Neisseria spp.; Erysipelothrix, e.g., E. rhusiopathiae; Listeria spp., e.g., L. monocytogenes, rarely L. ivanovii and L. seeligeri; Bacillus, e.g., B. amhracis, B. cercus, B, sabtilis, B. subtilus niger, B. thuringiensis, Nocardia asteroides; Legionella, e.g., L. pneumonophilia, Pneumocystis, e.g., P. carinii; Enterobacteriaceae such as Salmonella, Shigella, Escherichia (e.g., E. coli, E. coli0157:H7); Klebsiella, Enterobacter, Serratia, Proteus, Morganella, Providencia, Yersinia, and the like, e g., Salmonella, e.g., S. typhi S. paratyphi A, B (S. schoumuelleri), and C (S. hirschfeldii), S. dublin, S. choleraesuis, S. enteritidis, S. typhimurium, S. heidelberg, S. newport, S. infantis, S. agona, and S. saint-paul; Shigella e.g., subgroups: A, 8, C, and D, such as S. flexneri, S. sonnei, S. boydii, S. dysenteriae, Proteus (P. mirabilis, P. vulgaris, and P. myxofaciens), Morganella (M. morganii); Providencia (P. rettgeri, P. alcalifaciens, and P. stuartii); Yersinia, e.g., Y. pestis, Y. enterocolitica; Haemophilus, e.g., H. influenzae, H. parainfluenzae, H. aphrophilus, H. ducreyi; Brucella, e.g., B. abortus, B. melitensis, B. suis, B. canis; Francisella, e.g., F. tularensis, Pseudomonas, e.g., P. aeruginosa, P. paucimobilis, P. putida, P. fluorescens, Burkholderia (Pseudomonas) pseudomallei, Burkhoideria mallei, Burkholderia cepacia and Stenotrophomonas maltophilia, Campylobacter, e.g., ; C. fetus, C. jejuni. C. pylori (Helicobacter pylori); Vibrio, e.g., V. cholerae, V. parahaemolyticus, V. mimicus, V. alginolyticus, V. hollisae, V. vulnificus, and the nonagglutinable vibrios; Clostridia, e.g., C. perfringens, C. tetani, C. difficile, C. botulinum; Actinomyces, e.g., A. israelii; Bacteroides, e.g., B. fragilis, B. thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus, , and B. merdae; Prevotella, e.g., P. melaninogenica; genus Fusobacterium; Treponema, e.g., T. pallidum subspecies endemicum, T, pallidum subspecies pertenue, T. carateum, and T. pallidum, subspecies pallidum; genus Borrelia, e.g., B. burgdorferi; genus Leptospira;

Streptobacillus, e.g., S. moniliformis; Spirillum, e.g., S. minus; Mycobacterium, e.g., M. tuberculosis, M. bovis, M. africanum, M. avium, M. intracellulare, M. kansasii, M. xenopi, M. marinum, M. ulcerans, the M. fortuirum complex (M. foruitum), M. leprae, M. asiaticum, M. chelonae, subsp. abscessus, M. fallax, M. fortuitum, M. malmoense, M. shimoidei, M. simiae, M. szulgai, M. xenopi; Mycoplasma, e.g., M. hominis, M. orale, M. salivarium, M. fermentans, M. pneumoniae, M. bovis, M. tuberculosis, M. avium, M. leprae; Mycoplasma, e.g., M. genitalium; Ureaplasma, e.g., U. urealyticum;

Trichomonas, e.g., T. vaginalis; Cryptococcus, e.g., C. neoformans; Histoplasma, e.g., H. capsulatum; Candida, e.g., C. albicans; Aspergillus sp; Coccidioides, e.g., C. immitis; Blastomyces, e.g., B. dermatitidis; Paracoccidioides, e.g., P. brasiliensis; Penicillium, e.g., P. mameffei; Sporcthrix, e.g., S. schenckii; Rhizopus, Rhizomucor, Absidia, and Bastdiobolus; diseases caused by Bipolaris, Cladophialophora, Cladosporium, Drechslera, Exophiala, Phialophora, Xylohypha, Ochroconis, Rhinocladiella, Scolecobasidium, and Wangiella; Trichosporon, e.g., T, beigelii; Blastoschizomyces, e.g., B. capitatus; Plasmodium, e.g., P. falciparum, P. vivax, P. ovale, and P. malariae; Babesia sp, protozoa of the genus Trypanosoma, e.g., T. cruzi, Leishmania, e.g., L. donovani, L. major, L. tropica, L. mexicana, L. braziliensis, L. viannia braziliensis; Toxoplasma, e.g., T. gondii; Amoebas of the genera Naegleria; Entamoeba histolytica,; Giardia lamblia; genus Cryptosporidium, e.g., C. parvum; Isospora belli; Cyclospora cayetanensis, Ascaris lumbricoides; Trichuris trichiura; Ancylostoma duodenale or Necator americanus; Strongyloides stercoralis Toxocara, e.g., T. canis, T. cati; Baylisascaris, e.g., B. procyonis; Triehinella, e g., T. spiralis; Dracunculus, e.g., D. medinensis; genus Filarioidea; Wuchereria bancrofti, Brugia, e.g., B. malayi, B. timori; Onchocerca volvulus; Loa loa; Dirofilaria immitis; genus Schistosoma, e.g., S. japenicum, S. mansoni, S. mekongi, S. intercalatum , S. haematobium; Paragonimus, e.g., P. Westermani, P. skrjabini ; Clonorchis sinensis; Fasciola hepatica; Opisthorchis sp; Fasciolopsis buski; Diphyllocularis latum, Taenia, e.g., T. saginata, T. solium, Echinococcus, e.g., E. granulosus, E. multilocularis; Picornavirus, rhinoviruses echoviruses, coxsackieviruses, influenza virus; paramyxoviruses, e.g., types 1 , 2, 3, and 4; adnoviruses; Herpesviruses, e.g., HSV-1 and HSV-2; varicella-zoster virus; human T- lymphotrophic virus type I and type II), Arboviruses and Arenaviruses; Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae; Flavivirus; Hantavirus; Viral encephalitis (alphaviruses [e.g., Venezuelan equine encephalitis, eastern equine encephalitis, western equine encephalitis]); Viral hemorrhagic fevers (filoviruses, [e.g., Ebola, Marburg] and arenaviruses (e.g., Lassa, Machupo); Smallpox (variola); retroviruses e.g., human immunodeficiency viruses 1 and 2; human papillomavirus (HPV) types 6, 1 1 , 16, 18, 31 , 33, and 35.

In various embodiments, the probe can be selective for a polynucleotide sequence that is characteristic of an organism selected from the group consisting of Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella oxytoca, Klebsiella pneumoniae, Escherichia coli, Acinetobacter Baumunnii, Serratia marcescens, Enterobacter aerogenes, Enterococcus faecium, vancomycin-resistant enterococcus (VRE), Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus viridans, Listeria monocytogenes, Streptococcus Group B, Streptococcus Group C, (Streptococcus Group G, Streptococcus Group F, Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus epidermidis, Gardenetella vaginalis, Micrococcus spp., Haemophilus influenzae, Neisseria gonorrhoeae, Moraxella catarrhalis, Salmonella spp., Chlamydia trachomatis, Peptostreptococcus productus, Peptostreptococcus, uanaerabius, Lactobacillus fermentum, Eubacterium lentum, Candida glabrata, Candida albicans, Chlamydia spp., Camplobacter spp., Salmonella spp., smallpox (variola major), Yersina Pestis, Herpes Simplex Virus I (HSV I), and Herpes Simplex Virus II (HSV II). In various embodiments, the probe can be selective for a polynucleotide sequence that is characteristic of Group B Streptococcus.

Carrying out dPCR on a sample can include heating the PCR reagent mixture and the polynucleotide sample under thermal cycling conditions suitable for creating PCR amplicons from the polynucleotide sample; contacting the polynucleotide sample or a PCR amplicon thereof with at least one probe that is selective for a polynucleotide sequence, independently contacting each of the polynucleotide sample and a negative control polynucleotide with the PCR reagent mixture under thermal cycling conditions suitable for independently creating PCR amplicons of the polynucleotide sample and PCR amplicons of the negative control polynucleotide; and/or contacting the polynucleotide sample or a PCR amplicon thereof and the negative control polynucleotide or a PCR amplicon thereof with at least one probe that is selective for a polynucleotide sequence.

In various embodiments, a method of carrying out PCR on a sample can further include one or more of the following steps: heating the biological sample, pressurizing the biological sample at a pressure differential compared to ambient pressure of between about 20 kilopascals and 200 kilopascals, or in some embodiments between about 70 kilopascals and 110 kilopascals.

In various embodiments, a method of using the apparatus described herein can further include one or more of the following steps: determining the presence of a polynucleotide sequence in the biological sample, the polynucleotide sequence corresponding to the probe, if the probe is detected in the neutralized polynucleotide sample or a PCR amplicon thereof; determining a contaminated result if the probe is detected in the negative control polynucleotide or a PCR amplicon thereof; and/or in some embodiments, wherein the PCR reagent mixture further comprises a positive control plasmid and a plasmid probe selective for at least a portion of the plasmid, the method further including determining a PCR reaction has occurred if the plasmid probe is detected.

KITS

Aspects of the present disclosure further include kits, where kits include one or more liquid vessels configured for use in a digital polymerase chain reaction (dPCR) system. The one or more liquid vessels include an anti-adhesion coating configured to at least reduce the binding of extracted nucleic acids. The liquid vessel(s) in the subject kits may be any of the vessels described herein. For example, the kits may include one or more microwell plates, tubes, pipette tips, cuvettes, and the like. In some embodiments, kits include vessels suitable for use in a reagent repository of the invention. In such embodiments, the vessels may contain one or more dPCR reagents or components. For example, the vessels may contain one or more buffers (e.g., elution buffers, rehydration buffers, lysis buffers, etc.). In some cases, vessels contain a immiscible liquid that can be employed in droplet creation. In still other cases, vessels of the subject kits are microwell plates, optionally having dried dPCR reagents positioned herein (e.g., master mix). In some cases, the kit comprises one or more pipettor tips comprising the anti-adhesion coating. In additional cases, the kit comprises one or more microwell plates comprising the anti-adhesion coating. In still further embodiments, the kit comprises one or more cuvettes comprising the anti-adhesion coating. In some embodiments, the vessel may be employed in the subject sample purifier. In some such cases, the vessel comprises magnetic beads suitable for nucleic acid extraction, e.g., such as those discussed above.

As discussed above, the anti-adhesion coating may be one of multiple coatings. For example, coatings for use in the subject kits may include hydrophilic components, zwitterionic components, and/or anti-fogging agents.

In addition to the above components, the subject kits may further include (in some embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), portable flash drive, Hard Drive etc., on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

UTILITY

The above-described systems, methods and kits find use in a variety of applications, including applications where it is desired to determine the presence or absence of an analyte in a biological sample. The analysis may be qualitative or quantitative. As such, where detection is qualitative, the methods provide a reading or evaluation, e.g., assessment, of whether or not a target analyte is present in the sample being assayed. In yet other embodiments, the methods provide a quantitative detection of whether the target analyte is present in the sample being assayed, i.e. , an evaluation or assessment of the actual amount of the target analyte in the sample being assayed. In such embodiments, the quantitative detection may be absolute or, if the method is a method of detecting two or more different target analytes in a sample, relative. As such, the term "quantifying" when used in the context of quantifying a target analyte(s) in a sample can refer to absolute or to relative quantification.

In particular embodiments, the present invention may be employed to determine the presence or absence of bloodborne pathogens. For example, the systems and methods of the invention may be employed to detect sepsis and/or septicemia. The invention particularly finds use where it is desirable to improve the efficiency of dPCR. As such, the invention may be utilized to improve (1 ) specimen volume and organism concentration issues, (2) long front-end preparation time, (3) target analyte adhesion to interior surfaces, and (4) lack of automation.

Samples (e.g., blood samples) may be obtained from any convenient source. In certain embodiments, the saliva sample is one that is obtained from a "mammal" or "mammalian subject", where these terms are used broadly to describe organisms which are within the class Mammalia, including the orders carnivore (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, subjects are humans. The term "humans" may include human subjects of both genders and at any stage of development (e.g., fetal, neonates, infant, juvenile, adolescent, adult), where in certain embodiments the human subject is a juvenile, adolescent or adult.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that some changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

What is claimed is:

1 A fully automated digital polymerase chain reaction (dPCR) system comprising: a sample preparation unit configured to receive a biological sample and extract nucleic acids therefrom; an analysis vessel; a partitioner configured to generate a plurality of sample partitions comprising the extracted nucleic acids for distribution into the analysis vessel; and a thermocycler configured to modulate temperature in the generated sample partitions so as to amplify nucleic acids of interest in the generated sample partitions; and a sample reader configured to detect whether amplified nucleic acid is present in each of the generated sample partitions within the analysis vessel; wherein the dPCR system comprises an anti-adhesion coating configured to at least reduce the binding of the extracted nucleic acid to the dPCR system.

2. The fully automated dPCR system according to Claim 1 , wherein the anti- adhesion coating comprises hydrophilic components.

3. The fully automated dPCR system according to Claim 1 , wherein the anti- adhesion coating comprises zwitterionic components.

4. The fully automated dPCR system according to Claim 1 , wherein the anti- adhesion coating is an anti-fogging agent.

5. The fully automated dPCR system according to any of the preceding claims, wherein at least one of the sample preparation unit, analysis vessel, partitioner, thermocycler and sample reader comprises the anti-adhesion coating.

6. The fully automated dPCR system according to Claim 5, wherein each of the sample preparation unit, analysis vessel, partitioner, thermocycler and sample reader comprises the anti-adhesion coating.

7. The fully automated dPCR system according to Claim 6, wherein each surface of the dPCR system configured to contact a portion of the sample comprises the anti- adhesion coating.

8. The fully automated dPCR system according to any of the preceding claims, wherein the sample preparation unit comprises a sample input block configured to receive the sample.

9. The fully automated dPCR system according to any of the preceding claims, wherein the sample preparation unit comprises a sample purifier comprising a magnet for extracting nucleic acids from the biological sample.

10. The fully automated dPCR system according to any of the preceding claims, wherein the sample preparation unit comprises a reagent repository.

1 1 . The fully automated dPCR system according to any of the preceding claims, wherein the partitioner comprises a droplet generator.

12. The fully automated dPCR system according to Claim 11 , wherein the droplet generator is configured to combine the sample with an immiscible liquid.

13. The fully automated dPCR system according to Claim 12, further comprising an immiscible liquid repository.

14. The fully automated dPCR system according any of Claims 11 to 13, wherein the analysis vessel is a cuvette.

15. The fully automated dPCR system according to Claim 14, wherein the cuvette is configured to rotate around and translate along an axis.

16. The fully automated dPCR system according to Claim 14 or 15, wherein the sample reader is a 3D particle counter.

17. The fully automated dPCR system according to any of Claims 1 to 10, wherein the partitioner is a liquid dispenser.

18. The fully automated dPCR system according to Claim 17, wherein the analysis vessel is a microwell plate.

19. The fully automated dPCR system according to Claim 18, wherein the sample reader is a microplate fluorescence reader.

20. The fully automated dPCR system according to Claim 18, wherein the sample reader is a microplate colorimetric reader.

21 . The fully automated dPCR system according to any of the preceding claims, wherein the partitioner and sample reader are configured to operate simultaneously.

22. The fully automated dPCR system according to any of the preceding claims, further comprising an automated liquid dispenser system operably connected to at least one of the sample preparation unit, the partitioner, the thermocycler, and the sample reader.

23. The fully automated dPCR system according to any of the preceding claims, further comprising a robotic arm configured to transfer the sample from the sample preparation unit to the partitioner.

24. The fully automated dPCR system according to any of the preceding claims, further comprising a waste repository.

25. The fully automated dPCR system according to any of the preceding claims, further comprising a door configured to enclose the system.

26. A method of analyzing a sample, the method comprising:

(a) introducing a biological sample into an automated digital polymerase chain reaction (dPCR) system comprising: a sample preparation unit configured to receive a biological sample and extract nucleic acids therefrom; an analysis vessel; a partitioner configured to generate a plurality of sample partitions comprising the extracted nucleic acids for distribution into the analysis vessel; and a thermocycler configured to modulate temperature in the generated sample partitions so as to amplify the nucleic acids of interest in the generated sample partitions; and a sample reader configured to detect whether amplified nucleic acid is present in each of the generated sample partitions within the analysis vessel, wherein the dPCR system comprises an anti-adhesion coating configured to at least reduce the binding of the extracted nucleic acid to the dPCR system; and

(b) analyzing the biological sample via dPCR.

27. The method according to Claim 26, wherein the anti-adhesion coating comprises hydrophilic components.

28. The method according to Claim 26, wherein the anti-adhesion coating comprises zwitterionic components.

29. The method according to Claim 26, wherein the anti-adhesion coating is an anti- fogging agent.

30. The method according to any of Claims 26 to 29, wherein at least one of the sample preparation unit, analysis vessel, partitioner, thermocycler and sample reader comprises the anti-adhesion coating.

31 . The method according to Claim 30, wherein each of the sample preparation unit, analysis vessel, partitioner, thermocycler and sample reader comprises the anti- adhesion coating.

32. The method according to Claim 31 , wherein each surface of the dPCR system configured to contact a portion of the sample comprises the anti-adhesion coating.

33. The method according to any of Claims 26 to 32, wherein the sample preparation unit comprises a sample input block configured to receive the sample.

34. The method according to any of Claims 26 to 33, wherein the sample preparation unit comprises a sample purifier comprising a magnet for extracting nucleic acids from the biological sample.

35. The method according to any of Claims 26 to 34, wherein the sample preparation unit comprises a reagent repository.

36. The method according to any of Claims 26 to 35, wherein the partitioner comprises a droplet generator.

37. The method according to Claim 36, wherein the droplet generator is configured to combine the sample with an immiscible liquid.

38. The method according to Claim 37, wherein the dPCR system further comprises an immiscible liquid repository.

39. The method according to any of Claims 36 to 38, wherein the analysis vessel is a cuvette.

40. The method according to Claim 39, wherein the cuvette is configured to rotate around and translate along an axis.

41 . The method according to Claim 39 or 40, wherein the sample reader is a 3D particle counter.

42. The method according to any of Claims 26 to 35, wherein the partitioner is a liquid dispenser.

43. The method according to Claim 42, wherein the analysis vessel is a microwell plate.

44. The method according to Claim 43, wherein the sample reader is a microplate fluorescence reader.

45. The method according to Claim 40, wherein the sample reader is a microplate colorimetric reader.

46. The method according to any of Claims 26 to 44 wherein the partitioner and sample reader are configured to operate simultaneously.

47. The method according to any of Claims 26 to 46, wherein the fully automated dPCR system further comprises an automated liquid dispenser system operably connected to at least one of the sample preparation unit, the partitioner, the thermocycler, and the sample reader.

48. The method according to any of Claims 26 to 47, wherein the fully automated dPCR system further comprises a robotic arm configured to transfer the sample from the sample preparation unit to the partitioner.

49. The method according to any of Claims 26 to 48, further comprising centrifuging the sample.

50. The method according to any of Claims 26 to 49, further comprising apportioning the sample into a plurality of sub-lots, and analyzing the plurality of sublots via the dPCR system in parallel.

51 . The method according to any of Claims 26 to 50, further comprising: pre-lysing micro-organisms in the sample; and assaying the sample for a high copy number target.

52. The method according to Claim 51 , wherein the high copy number target is an RNA.

53. A kit comprising: one or more liquid vessels configured for use in a digital polymerase chain reaction (dPCR) system and comprising an anti-adhesion coating configured to at least reduce the binding of extracted nucleic acids.

54. The kit according to Claim 53, wherein the anti-adhesion coating comprises hydrophilic components.

55. The kit according to Claim 53, wherein the anti-adhesion coating comprises zwitterionic components.

56. The kit according to Claim 53, wherein the anti-adhesion coating is an anti- fogging agent.

57. The kit according to any of Claims 53 to 56, wherein the one or more liquid vessels further comprise magnetic beads having an affinity for the extracted nucleic acids.

58. The kit according to any of Claims 53 to 57, wherein the one or more liquid vessels further comprise a dPCR reagent.

59. The kit according to Claim 58, wherein the dPCR reagent is a PCR master mix.

60. The kit according to Claim 58 or 59, wherein the dPCR reagent is dried.

61 . The kit according to any of Claims 53 to 60 wherein the one or more liquid vessels further comprise a buffer.

62. The kit according to Claim 61 , wherein the buffer is a rehydration buffer.

63. The kit according to Claim 61 , wherein the buffer is an elution buffer.

64. The kit according to any of Claims 53 to 63, wherein the kit comprises one or more pipettor tips comprising the anti-adhesion coating.

65. The kit according to any of Claims 53 to 64, wherein the kit comprises one or more microwell plates comprising the anti-adhesion coating.

66. The kit according to any of Claims 53 to 65, wherein the kit comprises one or more cuvettes comprising the anti-adhesion coating.