Full-automatic dPCR system and use method thereof

Abstract

A fully automatic digital polymerase chain reaction (dPCR) system is provided. The target system includes: a sample preparation unit configured to receive a biological sample and extract nucleic acid therefrom; an analysis container; a separator configured to generate a plurality of sample partitions including the extracted nucleic acids and to distribute the generated sample partitions into the analysis container; a thermal cycler configured to adjust a temperature in the generated sample partition so as to amplify a target nucleic acid in the generated sample partition; and a sample reader configured to detect whether an amplified nucleic acid is present in each generated sample partition within the analysis container. The subject system also includes an anti-adhesion coating configured to at least reduce binding of the extracted nucleic acid to the dPCR system. Methods and kits for implementing the invention are also provided.

Description

Background Art

[0001] The medical diagnostics industry is an important part of today's healthcare infrastructure. However, current in vitro diagnostic assays, no matter how routine, have become a bottleneck in patient care. There are several reasons for this. First, many diagnostic assays can only be performed with very specialized equipment that is both expensive and can only be operated by trained clinicians. Such equipment exists in only a few locations, typically only one in any given urban area. This means that most hospitals need to send samples to these locations for analysis, resulting in transportation costs and delays, and even potential sample loss or mishandling. Second, the equipment in question is not typically "on-demand" but runs in batches, thus delaying the processing of many samples as they must wait for the machine to fill up before it can run. In addition, due to the long processing times, there may be long wait times for the machine to be available.

[0002] Polymerase chain reaction (PCR) is such a medical diagnostic method. PCR involves generating multiple copies of a nucleic acid sample (such as DNA) sufficient for analysis by exponential amplification of the sample. PCR typically includes a denaturation step of generating two single-stranded DNA molecules, an annealing step of attaching primers to the single-stranded DNA molecules, and an extension step of synthesizing new double-stranded DNA molecules. The PCR method typically involves exposing the sample to thermal cycling by using repeated cycles of higher and lower temperatures.

[0003] Digital polymerase chain reaction (dPCR) is a PCR method that allows for more precise quantification of nucleic acids. Compared to traditional PCR, dPCR involves performing the reaction in multiple partitions of the sample. In other words, the PCR reaction is performed in each sample partition. Subsequently, each sample portion can be analyzed for the presence of nucleic acids (e.g., using a fluorescent probe), thus improving the precision of quantification. Quantification is achieved by allowing the reaction to proceed for a fixed number of cycles sufficient to appropriately amplify 1 copy into 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., for diagnosing sepsis) has been proposed (see, for example, Abram et al. Lab on a Chip 20, no. 3 (2020): 477-489). It is believed that dPCR can reduce the time to detection (TTD) and improve the limit of detection compared to conventional blood culture. However, due to performance deficiencies, dPCR for sepsis detection has not been widely adopted. Summary of the Invention

[0004] The present inventors have recognized that performance deficiencies in certain technologies have hindered the widespread adoption of dPCR in applications where a small amount of analyte must be analyzed in a time-sensitive manner (e.g., sepsis diagnosis). In particular, it has been found that conventional dPCR systems suffer from the following problems: (1) sample volume and organism concentration issues, (2) long front-end preparation times, (3) adhesion of target analytes to inner surfaces, and (4) lack of automation. Regarding (1), it has been found that attempting to use conventional techniques with microfluidics / microdroplet technology to diagnose bacteremia is challenging because the concentration of organisms is extremely low. To achieve reasonable clinical sensitivity, an analytical sensitivity of approximately 1 organism / mL is required. Conventional blood culture systems use 10 mL of blood to overcome this problem, while typical microfluidics / microdroplet technology uses a sample input of 100 microliters (μL) to 1 mL. Regarding (2), most microdroplet systems operate in the range of approximately 10 μL / minute to approximately 50 μL / minute. Assuming a 10 mL input sample, this would correspond to droplet generation times of 200 minutes to 1000 minutes. In the extreme case of 1000 minutes, the time to generate droplets for a 10 mL sample is approximately 16.6 hours. Considering that the average time for a conventional blood culture to be positive is approximately 18 hours, no time-related benefits of dPCR in diagnostic applications (such as detecting bloodborne pathogens) have been realized. Regarding (3), the low concentration requirements of the target organism analyte per unit volume in the assay do not account for irreversible binding of the surface to the target organism analyte. However, currently available dPCR systems are affected by a non-negligible amount of analyte adhesion to the surface, thus further reducing the concentration of analyte available for measurement. Regarding (4), there is currently no fully automated dPCR system. This results in a significant amount of user involvement in sample handling using conventional means. At least considering the technical performance deficiencies (1) to (4), the present inventors have recognized the need for a fully automated and improved dPCR system. Embodiments of the present invention meet this need.

[0005] Aspects of the present invention include a fully automated digital polymerase chain reaction (dPCR) system. Systems of interest include: a sample preparation unit configured to receive a biological sample and extract nucleic acid therefrom; an analysis container; a divider configured to generate a plurality of sample partitions including the extracted nucleic acid, the sample partitions for distribution into the analysis container; a thermal cycler configured to regulate the temperature in the generated sample partitions to amplify a target nucleic acid in the generated sample partitions; and a sample reader configured to detect the presence or absence of the amplified nucleic acid in each of the generated sample partitions within the analysis container. Additionally, embodiments of the subject system 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 can include, for example, a hydrophilic component, a zwitterionic component, and / or an anti-fog agent. In an embodiment, at least one (and in some cases all) of the sample preparation unit, the analysis container, the divider, the thermal cycler, and the sample reader includes the anti-adhesion coating. The dPCR system can include a sample input block configured to receive a sample. In selected embodiments, the sample preparation unit includes a sample purifier that includes a magnet for extracting nucleic acid from a biological sample and / or a reagent reservoir, for example, by a magnetic bead capture oligonucleotide-mediated method. In some cases, the divider is a droplet generator. In some such cases, the droplet generator is configured to mix the sample with an immiscible liquid (e.g., oil), and the dPCR system includes a reservoir of the immiscible liquid. In certain embodiments, the analysis container can include a cuvette configured to rotate about an axis and translate along the axis. In a selected version, the sample reader is a three-dimensional (3D) particle counter. In additional embodiments, the divider is a liquid dispenser. In some such embodiments, the analysis container can be a microplate, and the sample reader is a microplate reader (e.g., a fluorescence microplate reader). In certain versions, the divider and the sample reader are configured to operate simultaneously. In some cases, the dPCR system further includes an automated liquid dispenser system (e.g., a pipette) operably connected to at least one of the sample preparation unit, the divider, the thermal cycler, and the sample reader. In a selected case, the subject dPCR system includes a robotic arm configured to transfer a sample from the sample preparation unit to the divider. The system can also include a waste reservoir for collecting used solids and / or liquids. Embodiments of the present invention also include a door (e.g., a sliding door) configured to enclose the system.

[0006] Aspects of the present invention also include methods of analyzing a sample. The methods of interest include introducing a biological sample into a dPCR system of the present invention (e.g., those as described herein) and analyzing the biological sample by dPCR. Embodiments of the method also include centrifuging the sample, e.g., prior to introducing it into the dPCR system. In certain cases, the method further includes dispensing the sample into a plurality of sub-batches and analyzing the plurality of sub-batches in parallel by the dPCR system. In certain cases, the method may involve pre-lysing the microorganisms in the sample; and analyzing high copy number targets (e.g., RNA, such as rRNA) in the sample.

[0007] Aspects of the present invention also include kits. The kits include one or more liquid containers configured for use in a dPCR system and including an anti-adhesion coating configured to at least reduce binding of extracted nucleic acids. The anti-adhesion coating for the subject liquid containers may include a hydrophilic component, a zwitterionic component, and / or an anti-fog agent. In some cases, the one or more liquid containers further include magnetic beads having an affinity for extracted nucleic acids. Embodiments of the containers also include optionally dried dPCR reagents (e.g., PCR premix) and / or buffers. The kits may also include one or more pipette tips including an anti-adhesion coating, one or more microplates including an anti-adhesion coating, and / or one or more cuvettes including an anti-adhesion coating. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The present invention may best be understood from the following detailed description when read in connection with the accompanying drawings. Included in the drawings are the following figures:

[0009] Figure 1 Shows a fully automated dPCR system according to certain embodiments of the present invention.

[0010] Figure 2 Shows components of a fully automated dPCR system including an anti-adhesion coating.

[0011] Figures 3A to 3B Shows embodiments of a dPCR system in which the divider is a droplet generator ( Figure 3A ) and embodiments of a dPCR system in which the divider is a dispenser ( Figure 3B ).

[0012] Figure 4 Shows a fully automated dPCR system according to certain embodiments of the present invention.

[0013] Figure 5 Shows a flow chart for practicing the method of the present invention according to certain embodiments. DETAILED DESCRIPTION

[0014] A fully automated digital polymerase chain reaction (dPCR) system is provided. The target system includes: a sample preparation unit configured to receive a biological sample and extract nucleic acid therefrom; an analysis container; a divider configured to generate a plurality of sample partitions including the extracted nucleic acid, the sample partitions being for distribution into the analysis container; a thermal cycler configured to regulate the temperature in the generated sample partitions to amplify a target nucleic acid in the generated sample partitions; and a sample reader configured to detect whether the amplified nucleic acid is present in each of the generated sample partitions within the analysis container. The system further includes 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 present invention are also provided.

[0015] Before describing the present invention in more detail, it is to be understood that the invention is not limited to the particular embodiments described, as these may vary. It is also to be understood that, since the scope of the invention will be limited only by the appended claims, the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0016] Where numerical ranges are provided, it is to be understood that, unless the context clearly dictates otherwise, to the tenth of the unit of the lower limit, the present invention includes each intermediate value between the upper and lower limits of such range and any other stated value or intermediate value within such stated range. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the present invention, subject to any specific excluded limitations within the stated range. When the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the present invention.

[0017] Certain ranges of values presented herein are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows as well as a number that is close to or approximates the number that follows. When determining whether a number is close to or approximates a specifically recited number, the unrecited number that is close to or approximates the specifically recited number may be a number that provides substantial equivalence to the specifically recited number in the context in which it occurs.

[0018] Unless otherwise defined, 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 described below.

[0019] All publications and patents cited in this specification are hereby incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are hereby incorporated by reference to disclose and describe the methods and / or materials associated with the cited publications. 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 dates, which may need to be independently confirmed.

[0020] Note that, as used herein and in the appended claims, the term "a" or "an" preceding an element does not exclude the presence of a plurality of such elements unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terms, such as "solely", "only", etc., in connection with the recitation of claim elements or the use of "negative" limitations.

[0021] After reading this disclosure, it will be apparent to those skilled in the art that the individual and separate embodiments described and shown herein have discrete components and features which can be readily separated from or combined with the features of any one of a number of other embodiments without departing from the scope or spirit of the present invention. Any of the methods recited may be carried out in the order of events listed or in any other order that is logically possible.

[0022] Although the systems and methods have been or will be described for purposes of grammatical fluidity and functional explanation, it should be clearly understood that, unless specifically provided otherwise in 35 U.S.C. § 112, the claims should not be construed as necessarily limited in any way by the use of the terms "means" or "step", but are to be given the full scope of the definition provided by the claims and equivalents thereof under the doctrine of judicial equivalents, and in the event 35 U.S.C. § 112 specifically provides for the claims, they are to be given their full statutory equivalents under 35 U.S.C. § 112.

[0023] Fully automated dPCR system

[0024] As discussed above, aspects of the present invention include a fully automated dPCR system. The system includes an anti-adhesion coating configured to at least reduce the binding of extracted nucleic acids to the dPCR system. The anti-adhesion coating described herein can reduce the binding of extracted nucleic acids to the dPCR system compared to the amount of binding that occurs in an actual or hypothetical conventional dPCR system lacking the anti-adhesion coating. For example, the anti-adhesion coating described herein can be configured to reduce the binding of extracted nucleic acids to the dPCR system by 10% or more than 10%, 15% or more than 15%, 20% or more than 20%, 25% or more than 25%, 30% or more than 30%, 35% or more than 35%, 40% or more than 40%, 50% or more than 50%, 55% or more than 55%, 60%, 65% or more than 65%, 70% or more than 70%, 75% or more than 75%, 80% or more than 80%, 85% or more than 85%, 90% or more than 90%, 95% or more than 95%, and up to including 100%. In some cases, it can be said that the anti-adhesion coating "prevents" the binding of extracted nucleic acids to the dPCR system. In some cases, reducing the binding of extracted nucleic acids to the surface of the dPCR system can increase the amount of target analyte available for analysis by dPCR by 10% or more than 10%, 15% or more than 15%, 20% or more than 20%, 25% or more than 25%, 30% or more than 30%, 35% or more than 35%, 40% or more than 40%, 50% or more than 50%, 55% or more than 55%, 60%, 65% or more than 65%, 70% or more than 70%, 75% or more than 75%, 80% or more than 80%, 85% or more than 85%, 90% or more than 90%, 95% or more than 95%, and up to including 100%. Any material suitable for coating a liquid container and capable of at least reducing (e.g., preventing) the binding of a bioanalyte (e.g., nucleic acid) thereto can be used. In some cases, the anti-adhesion coating can be a stable coating. In other words, subjecting the coating to one or more of autoclaving, washing with a detergent, or rinsing with a salt solution will not significantly alter the chemical properties of the coating.

[0025] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe any length polymer composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides), 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 10000 bases, greater than 100000 bases, greater than about 1000000 bases, up to about 10 10one or more bases, and the bases can be produced enzymatically or synthetically (e.g., peptide nucleic acids as described in U.S. Patent No. 5,948,902 and references cited therein), which can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to 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).

[0026] In some cases, the anti-adhesion coating includes a hydrophilic component. The hydrophilic component is described, for example, in U.S. Patent Application Publication 2006 / 0193894, the disclosure of which is incorporated herein by reference in its entirety. In some cases, a wetting agent, i.e., a reagent that reduces the total free energy of water and is capable of binding water, can be used to form the anti-adhesion coating. Suitable wetting agents include, for example, polymeric wetting agents and non-polymeric wetting agents. Exemplary polymeric wetting agents include, but are not limited to, 2-hydroxyethyl acrylate (HEA), 2-hydroxyethyl methacrylate (HEMA), dimethylacrylamide (DMA), polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol (PEG), di(ethylene glycol) vinyl ether (EO2V), cellulose derivatives, etc. and combinations thereof. Exemplary non-polymeric wetting agents include, but are not limited to, glycerol, urea, propylene glycol, non-polymeric diols, glycerin, etc.

[0027] In some embodiments, the anti-adhesion coating includes a zwitterionic component. The zwitterionic component is described, for example, in Baggerman et al. Langmuir 35, no. 5 (2019): 1072-1084; the disclosure of which is incorporated herein by reference in its entirety. In some cases, the zwitterionic component is a phosphocholine-containing polymer. In additional cases, the zwitterionic component includes acrylates and acrylamides, optionally having sulfobetaine and carboxybetaine moieties. The zwitterionic coating can be prepared by any convenient technique. In some cases, techniques including, but not limited to, atom transfer radical polymerization (ATRP), strain-promoted alkyne-azide cycloaddition (SPAAC), surface-initiated polymerization (e.g., surface-initiated atom transfer radical polymerization (SI-ATRP)), side-chain biofunctionalization using random copolymers, reversible addition-fragmentation chain transfer (RAFT) polymerization, combinations thereof, etc. are used to prepare the zwitterionic coating.

[0028] In some embodiments, the anti-adhesion coating is an anti-fogging agent. As discussed herein, "fogging" refers to the formation of small water droplets on a surface. The chemistry of the anti-fogging agent is based on the reaction of a polyol with a fatty acid such as stearic acid or lauric acid to form an ester. This forms a non-ionic surfactant that inhibits the formation of fog. The long carbon tail of the fatty acid is hydrophobic and remains entangled in the host polymer. The polyol with -OH groups is hydrophilic and is a non-ionic surfactant that tends to "frost" on the surface of the host polymer. Thus, the hydrophilicity of the host polymer is increased.

[0029] As described above, the target system includes a sample preparation unit, a separator, an analysis container, a thermal cycler, and a sample reader. Any one or all components of the subject dPCR system may include the anti-adhesion coating of the present invention. For example, at least one (including each) of the sample preparation unit, the analysis container, the separator, the thermal cycler, and the sample reader includes the anti-adhesion coating. In an alternative, each surface of the dPCR system configured to contact a portion of the sample includes the anti-adhesion coating.

[0030] Furthermore, the system of the present invention is fully automated. "Fully automated" means that the dPCR process can be performed by the system from start to finish without user interaction and intervention. Although the user may input the sample, provide reagents to the system, and remove waste, the dPCR process itself can be performed in an automated manner. In other words, sample lysis and DNA preparation are automatically performed by the robot and liquid handling components of the analyzer using protocols and reagents located within the system. Then, the system automatically mixes the sample and the PCR reagents and partitions the sample into multiple sample partitions. In addition, the sample reader automatically reads the sample. After completing the dPCR, the results will be automatically available and can also be automatically displayed. In an embodiment, the fully automated dPCR system can reduce the total time for dPCR by 10% or more than 10%, 15% or more than 15%, 20% or more than 20%, 25% or more than 25%, 30% or more than 30%, 35% or more than 35%, 40% or more than 40%, 50% or more than 50%, 55% or more than 55%, 60%, 65% or more than 65%, 70% or more than 70%, 75% or more than 75%, 80% or more than 80%, 85% or more than 85%, 90% or more than 90%, 95% or more than 95%, and reduction including 100% or more.

[0031] Sample preparation unit

[0032] The target sample preparation unit is configured to receive a biological sample and extract nucleic acids therefrom. In some cases, the sample preparation unit may include a sample input block configured to receive the sample. The sample input block can be any device configured to receive a biological sample, for example, in liquid form. For example, the sample input block can include a module having a plurality of grooves for receiving one or more than one sample. In some cases, the sample input block is configured such that the sample can be directly added to the grooves. In some such cases, the sample input block can be a microplate or the like. The sample input block can optionally be coated with the above anti-adhesion coating. In other cases, the sample input block is configured to receive one or more other liquid containers that themselves contain the sample (and can optionally be coated with an anti-adhesion coating). In this case, the sample can be contained in one or more sample tubes. Exemplary sample tubes include Eppendorf (e.g., 1.5 ml Eppendorf 2 ml Eppendorf ) and the like. In some cases, the sample input block is configured to be removed from the dPCR system (e.g., so that it can be loaded with samples, cleaned, etc.). In some such cases, the sample input block includes one or more than one handle.

[0033] The sample input block can be composed of any convenient material. In some cases, the sample input module includes one or more than one rigid plastic material, such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, and other polymeric plastic materials. In certain cases, the block includes a 3D printed polymer. Any convenient 3D printed polymer can be used, such as acrylonitrile butadiene styrene (ABS), acrylonitrile styrene acrylate (ASA), polyethylene terephthalate (PET), glycol-modified polyethylene terephthalate (PETG), polyaryletherketone (PAEK), polyetherimide (PEI), polycarbonate (PC), polypropylene (PP), nylon, and their composites and mixtures. In some cases, the sample input block includes one or more than one metal, including, for example, aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin, and their combinations and alloys.

[0034] The sample preparation unit of the present invention further includes a sample purifier. The disclosed sample purifier is configured to extract a target analyte (e.g., DNA, RNA) from a sample and prepare the extracted analyte for further analysis. In some embodiments, the sample purifier includes a plurality of recesses configured to receive a sample (e.g., from a sample input block). In some cases, the sample purifier is configured such that the sample can be directly added to the recesses. The recesses of the sample purifier may optionally be coated with the anti-adhesion coating described above. In other cases, the sample purifier is configured to receive one or more other liquid containers that themselves contain the sample to be purified (and may optionally be coated with an anti-adhesion coating). In such cases, the sample may be contained in one or more sample tubes. Exemplary sample tubes include Eppendorf (e.g., 1.5 mL Eppendorf 2 mL Eppendorf ) etc.

[0035] The target sample purifier further includes a plurality of synthetic particles located within the recesses, which can be used for sample purification. The synthetic particles can be, for example, beads. The beads can be, for example, silica beads, glass beads, magnetic beads, / beads, cellulose beads, polystyrene beads, or any combination thereof. The beads can include materials such as polydimethylsiloxane (PDMS), polystyrene glass, polypropylene, agarose, gelatin, hydrogel, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, cellulose, nylon, polysiloxane, or any combination thereof. The beads can include one or more types of solid, porous, or hollow spheres, balls, bearings, cylinders, or other similar configurations on which the analyte (e.g., nucleic acid) can be immobilized (e.g., covalently or non-covalently). The beads can be or include spherical (e.g., microspheres) or discrete particles having a non-spherical or irregular shape, such as cubes, cuboids, pyramids, cylinders, cones, ellipses, or discs, etc. In some embodiments, the beads can be non-spherical. In some cases, the beads are dried within the recesses of the sample purifier.

[0036] In addition, for the purposes of this disclosure, the particles can be magnetically responsive, e.g., by including one or more paramagnetic and / or superparamagnetic substances, such as magnetite. Such paramagnetic and / or superparamagnetic substances can be embedded in the matrix of the particles, and / or can be disposed on the outer surface and / or inner surface of the beads.

[0037] In certain embodiments, the particles are coated on their outer surfaces with a substance that non-specifically and reversibly binds nucleic acids (e.g., DNA). The beads can have any convenient surface chemistry configured to produce binding to the target nucleic acid. According to some embodiments, the substance comprises carboxyl groups that non-specifically and reversibly bind nucleic acids. Non-limiting examples of such a substance are succinic acid. In certain embodiments, the particle solid support is a solid phase reversible immobilization (SPRI) bead. Exemplary types of magnetic beads suitable for use in a 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, NeutAvidin TM -coated magnetic beads, and silica-coated magnetic beads, combinations thereof, and the like.

[0038] The size of the beads can vary. For example, the diameter of the beads can range from 0.1 micrometer to 50 micrometers. In some embodiments, the diameter of the beads can be or be approximately 0.1 micrometer, 0.5 micrometer, 1 micrometer, 2 micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers, 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, or a value or range between any two of these values. The diameter of the beads can be related to the diameter of the grooves of the sample purifier. In some embodiments, the diameter of the beads can be or be approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% longer or shorter than the diameter of the grooves, or a value or range between any two of these values. The diameter of the beads can be related to the diameter of the cells (e.g., a single cell captured by the pores of the matrix). In some embodiments, the diameter of the beads can be or be approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% longer or shorter than the cell diameter, or a number or range between any two of these values.

[0039] The sample purifier of the present invention further includes at least one magnet for extracting nucleic acids from a biological sample. The magnet of the sample purifier can be switched between an active position and an inactive position. In the active position, the magnet applies a magnetic force to the groove of the sample purifier. In the inactive position, the magnet does not apply a magnetic force to the groove of the sample purifier. For example, the sample purifier can include one or more magnets fixed to a support member; a motorized mechanism configured to move the support member in such a way that one or more magnets move back and forth along a fixed axis, and during at least a portion of the movement, one or more magnets remain in close proximity to one or more containers that contain magnetic particles in solution; and a control circuit for controlling the motorized mechanism. The motor can be controlled by a computer to operate at a specific speed; for example, at a rotational speed that causes the magnet to move vertically in the range of 1 mm / second to 20 mm / second. Thus, the magnetic separator can be configured to repeatedly move along the same axis, such as up and down, left and right, or back and forth several times. In some embodiments, the support member is located on one or more guiding members to ensure that the support member does not, for example, tilt, twist, or deflect, or undergo other internal movements (other than the controlled movement along the axis) and thereby reduce the efficacy of the separation.

[0040] For example, in some embodiments, the magnet can move between a first position or inactive position and a second position or active position, wherein in the first position or inactive position, the magnetic field generated by the magnet does not attract or applies a relatively weak attraction to the magnetic particles within the microarray, and wherein in the second position or active position, the magnetic field generated by the magnet can attract the magnetic particles. When the magnet is activated, the magnetic particles (optionally bound to the target analyte / nucleic acid) are immobilized within the groove while a washing step is performed. In an embodiment, when in the active position, the upper surface of the magnet can be parallel to the lower surface of the groove.

[0041] Embodiments of the sample purifier further include a stirrer. Any convenient device configured to stir or agitate the sample liquid during nucleic acid extraction can be used. In some cases, the stirrer is a vortexer (i.e., having a component configured to oscillate in a circular motion in a manner sufficient to generate a vortex). In some cases, the agitator is a rocker configured to tilt the purifier back and forth. In still other cases, the stirrer is a shaker.

[0042] The sample purifier can be composed of any convenient material. In some cases, the sample purifier includes one or more rigid plastic materials, such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, and other polymeric plastic materials. In certain cases, the block includes a 3D printed polymer. In some embodiments, the microplate is composed of polystyrene. In some cases, the sample purifier includes one or more metals, including, for example, aluminum, titanium, brass, iron, lead, nickel, steel (such as stainless steel), copper, tin, and combinations and alloys thereof.

[0043] The sample preparation unit can additionally provide reagent storage. For example, embodiments of the sample preparation unit include one or more reagent repositories. Any sealable container configured to hold dPCR reagents can be used. In certain cases, the reagents are located in tubes, bags, bottles, combinations thereof, etc. In some embodiments, the reagent repository includes a PCR premix. As is known in the art, a PCR premix contains precursors and enzymes for a PCR reaction (e.g., dNTPs, MgCl2, Taq polymerase, fluorescent dyes, etc.). Commercially available PCR premixes are produced by, for example, Sigma-Aldrich, Thermo Fisher, Bio-Rad, and Qiagen. In selected embodiments, the dPCR reagents are dried (e.g., lyophilized). For example, in one embodiment, one or more reagent repositories of the present invention include a dried premix. Then the nucleic acid extracted from the sample purifier can be placed into a container with the dried master mix and then agitated to obtain a solution containing the extracted nucleic acid and the necessary dPCR reagents.

[0044] The reagent repository can also include one or more dyes configured to stably bind to the target nucleic acid. In addition to the reagents already discussed and those known to persons skilled in the art of nucleic acid amplification and quantification, various detection reagents can also be included in the reagent repository, such as fluorescent dyes, fluorescent probes, and non-fluorescent dyes and non-fluorescent probes. For example, the system of the present invention can employ reagents suitable for TM TaqMan TMProbes; reagents suitable for SYBR green fluorescence detection; reagents suitable for molecular beacon reactions, such as molecular beacon probes; reagents suitable for scorpion reactions, such as scorpion probes; reagents suitable for fluorescent DNA-binding dye-type reactions, such as fluorescent probes; and / or reagents for LightUp assays, such as LightUp probes. In some embodiments, the reagent repository includes compositions for quantifying detectable signals (e.g., fluorescence) from partitions containing amplified nucleic acids (e.g., target amplicons, etc.). Such reagents can be used in the process of labeling amplified nucleic acids with detectable markers (e.g., during amplification, after amplification), exposing the partitions to a light source of a selected wavelength to cause the probe dyes bound to the amplicons to fluoresce, and detecting and / or measuring the resulting fluorescence. Fluorescence emitted from the partitions can be traced during the amplification reaction to allow monitoring of the reaction (e.g., using SYBR green-type compounds), or fluorescence can be measured after amplification.

[0045] In some embodiments, the present invention provides a system for detecting and / or quantifying the presence of a target nucleic acid in a partition by providing a probe specific for the target nucleic acid (e.g., TaqMan TM -type probe) in a partition amplification reaction and detecting / measuring the resulting fluorescence. In some embodiments, partitions containing amplified nucleic acids (e.g., target amplicons, etc.) will exhibit quantifiable post-amplification fluorescence. In some embodiments, detection of the fluorescence signal indicates the presence of template nucleic acid (e.g., target) in the partition.

[0046] In some embodiments, the system includes a reagent repository that contains containers (e.g., tubes, bags, bottles) that contain one or more buffers. Target buffers include, for example, rehydration buffer, lysis buffer, and elution buffer. For example, lysis buffer from the reagent repository can be used for nucleic acid extraction in a sample purifier. In an embodiment, after the nucleic acid has been extracted in the sample purifier, the system of the present invention is configured to elute the extracted nucleic acid using elution buffer. In some cases, the system of the present invention is configured to elute the nucleic acid in a volume less than the original sample volume. In some cases, the elution volume is less than 10% or more, less than 20% or more, less than 30% or more, less than 40% or more, less than 50% or more, less than 60% or more, less than 70% or more, less than 80% or more and including less than 90% or more. Rehydration buffer can be used to rehydrate dried dPCR materials (e.g., dried beads or dried premixes, etc.). Target buffers can include one or more of the following: guanidine, isopropanol, ethanol, Tris, and ethylenediaminetetraacetic acid (EDTA), etc.

[0047] A reagent tube containing a liquid or liquid reagent can be sealed with a laminated structure. The laminated structure typically has a heat-sealing layer, a plastic layer such as a polypropylene layer, and a metal layer such as an aluminum foil, where the heat-sealing layer is adjacent to one or more reagent tubes. An additional plastic film in the laminated material for a container containing a liquid reagent is typically for preventing the liquid from contacting the aluminum.

[0048] Separator

[0049] The system of the present invention further includes a separator. As used herein, the term "compartment" refers to a volume of fluid (e.g., liquid) that is a separate portion of the overall volume. A large-volume can be divided into any suitable number of smaller volumes (i.e., compartments). In an embodiment, the number of compartments can be from 10 2 to 10 7 . The compartments can be separated by a physical barrier or physical forces (e.g., surface tension, hydrophobic repulsion, etc.). The compartments generated from a larger volume can be substantially uniform in size (monodisperse), or can have non-uniform sizes (polydisperse). The compartments can be generated by any suitable means (e.g., emulsions, microfluidics, microspraying, etc.). Exemplary compartments are droplets.

[0050] The separator can be any device capable of generating a plurality of sample compartments containing the extracted nucleic acid. In some embodiments, the separator is a droplet generator. As used herein, the term "droplet" refers to a small amount of liquid that is immiscible with its surrounding environment (e.g., gas, liquid, surface, etc.). The droplet can reside on a surface and be encapsulated by a fluid immiscible with it (e.g., the continuous phase of an emulsion, a gas (e.g., air, nitrogen), or a combination thereof). Droplets are typically spherical or substantially spherical, but can also be non-spherical. The shape of other spherical or substantially spherical droplets can be changed by deposition onto a surface or shrinkage in a capillary channel of a smaller diameter. Droplets can be "simple droplets" or "composite droplets", where one droplet encapsulates one or more additional smaller droplets. Droplets generated by a droplet generator can have any convenient liquid volume, e.g., in the volume range of 0.1 μL to 1 μL. The diameter of the droplets provided herein and / or the average diameter of a set of droplets can vary and can be from 1 μm to 1000 μm, e.g., from 10 μm to 500 μm, e.g., from 50 μm to 200 μm.

[0051] When the separator is a droplet generator, in some cases, the dPCR system can be referred to as a droplet digital PCR system (ddPCR). ddPCR is described, for example, in Abram et al., Lab on a Chip 20, no. 3 (2020): 477-489, which is incorporated herein by reference in its entirety. In such an embodiment, the droplet generator can produce droplets by using two immiscible fluids: a dispersed phase and a continuous phase. In this case, the dispersed phase is a solution containing nucleic acids eluted from the sample preparation unit. In some cases, the dispersed phase is an aqueous liquid (e.g., the elution buffer that transfers the extracted nucleic acids is an aqueous liquid). The continuous phase is a liquid that is immiscible with the solution of the extracted nucleic acids (e.g., oil). Embodiments of the separator include a dispersed phase channel through which the dispersed phase flows, and one or more continuous phase channels through which the continuous phase flows. The dispersed phase channel and the continuous phase channels can be arranged in any suitable configuration. In some cases, the channels are arranged in a coaxial configuration, where the two phases flow in parallel. In other words, the dispersed phase channel is surrounded by the continuous phase channels such that the two channels are coaxial. The two phases can flow together in a manner that produces droplets. In additional embodiments, the continuous phase channel and the dispersed phase channel can be arranged in a T-junction configuration. In this configuration, the dispersed phase channel intersects the continuous phase channel at a 90-degree angle at the T-junction. In additional embodiments, the dispersed phase channel and the continuous phase channels are arranged in a flow focusing configuration. In this configuration, at least two continuous phase channels intersect the dispersed phase channel such that the dispersed phase is squeezed by two counter-flowing streams of the continuous phase. More details regarding droplet formation can be found in Baroud et al., Lab on a Chip 10, no. 16 (2010): 2032-2045, the content of which is incorporated herein by reference in its entirety.

[0052] Embodiments of systems where the separator is a droplet generator can additionally include immiscible liquid reservoirs (i.e., reservoirs). The immiscible liquid reservoirs can include any suitable one or more containers (e.g., tubes, bags, bottles) in which the aforementioned continuous phase can be stored and from which the same continuous phase can be withdrawn for the droplet generator. In some cases, the immiscible liquid is oil. Oils suitable for droplet formation are described, for example, in Baret, Jean-Cristophe, Lab on a Chip 12, no. 3 (2012): 422-433. Oils of interest can include, but are not limited to, silicone oils, hydrocarbon oils, and fluorinated oils. In selected embodiments, the immiscible liquid in the immiscible liquid reservoir includes a surfactant. Exemplary surfactants can include, for example, Triton X-100, ABIL EM90, PF-decanol, perfluorotetradecanoic acid (PFTD), PEG-capped perfluoropolyether (PFPE), FluoSurf TM and combinations thereof.

[0053] In an alternative embodiment where the separator is a droplet generator, the analysis vessel can be a cuvette. The term "cuvette" is used in its conventional sense to describe a tubular container having walls that are transparent to light for analyzing the contents of the analysis vessel. The cuvette can have any convenient cross-section. In some embodiments, the cuvette has a circular cross-section. In other embodiments, the cuvette has a square cross-section. The cuvette can be constructed of any suitable transparent material through which light can pass. In certain cases, the cuvette is made of plastic. In other embodiments, the cuvette is made of glass. In other embodiments, the cuvette is made of quartz (e.g., fused quartz). In certain cases, the cuvette is configured to move. In some such cases, the cuvette can be configured to rotate about a vertical axis and translate along the same vertical axis (i.e., move up and down). Any displacement scheme can be employed to move the cuvette, such as coupling to a movable support stage or directly to a motor-driven translation stage, a lead screw translation assembly, a gear-based translation device, such as using a stepper motor, a servo motor, a brushless motor, a brushed DC motor, a micro stepper drive motor, a high-resolution stepper motor, and other types of motors. The cuvette can be configured to move at multiple speeds. In some embodiments, the cuvette can be configured to translate at a speed of 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 of 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, the system includes multiple cuvettes, such as 2 or more cuvettes, 3 or more cuvettes, 4 or more cuvettes, and including 5 or more cuvettes.

[0054] Alternative embodiments of the separator described herein include a dispenser configured to dispense a small amount of liquid onto different parts of the analysis vessel, thereby creating sample partitions. In an embodiment, the dispenser can be configured to dispense a liquid volume in the range of 500 nL to 10,000 nL, such as 50 nL to 700 nL. Examples of automated dispensers suitable for use in the present system include, but are not limited to, those described in published PCT application publication numbers WO2009129397, WO2015192010, and WO2015192010; the disclosures of which are incorporated herein by reference.

[0055] In some cases, the analysis container to which the dispenser provides a liquid containing the target analyte is a microfluidic array dispensing (MAP) device. In such a case, the MAP device includes a series of microfluidic channels and microchambers. In an embodiment, the dispenser is configured to provide a sample liquid to an inlet hole. In such an embodiment, partitioning can occur by microfluidic transfer. In other words, the liquid can flow from the inlet hole through the channels and be collected in the microchambers. PCR reactions can occur in each microchamber. The number of microchambers within the MAP device can vary as needed. For example, in some embodiments, the number of microchambers can be from 500 to 50,000, such as from 1000 to 40,000, from 5000 to 30,000, and including from 15,000 to 25,000. In some embodiments, the MAP device including microchannels and microchambers includes the anti-adhesion coating described above. The MAP device is described, for example, in Dueck et al. Scientific reports 9, no. 1 (2019): 1-9; which is incorporated herein by reference in its entirety.

[0056] In some embodiments of a dPCR system in which the partitioner is a dispenser, the analysis container into which the dispenser disperses the liquid is a microplate. The microplate can have any convenient configuration. In some embodiments, the microplate is fabricated by inserting holes etched in the plate. The plate can be composed of any convenient material. In some cases, the microplate includes one or more rigid plastic materials, such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, and other polymeric plastic materials. In certain cases, the block includes a 3D printed polymer. Any convenient 3D printed polymer can be used, such as acrylonitrile butadiene styrene (ABS), acrylonitrile styrene acrylate (ASA), polyethylene terephthalate (PET), glycol-modified polyethylene terephthalate (PETG), polyaryletherketone (PAEK), polyetherimide (PEI), polycarbonate (PC), polypropylene (PP), nylon, and their composites and mixtures. In some cases, the sample input block includes one or more metals, including, for example, aluminum, titanium, brass, iron, lead, nickel, steel (such as stainless steel), copper, tin, and their combinations and alloys.

[0057] The microholes can be made in various shapes. Non-limiting examples of hole geometries can include cylindrical, conical, hemispherical, rectangular, or polyhedral (e.g., a three-dimensional geometry composed of several planes, such as a hexagonal column, octagonal column, inverted triangular pyramid, inverted square pyramid, inverted pentagonal pyramid, inverted hexagonal pyramid, or inverted truncated pyramid). The microholes can include shapes that combine two or more of these geometries. For example, the microhole can be partially cylindrical and the remainder conical.

[0058] The diameter of the micropores can be expressed in absolute dimensions. The diameter of the micropores can be from about 1 nanometer to about 1000 micrometers. In some embodiments, the diameter of the micropores can be or be about 1 micrometer, 2 micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers, 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, 200 micrometers, 300 micrometers, 400 micrometers, 500 micrometers, 600 micrometers, 700 micrometers, 800 micrometers, 900 micrometers, 1000 micrometers, or a value or range between any two of these values.

[0059] The depth of the micropores can vary, e.g., to provide effective trapping of droplets within the pores. The depth of the micropores can be determined based on their absolute dimensions. For example, the depth of the micropores can be from about 1 nanometer to about 1000 micrometers. In some embodiments, the depth of the micropores can be or be about 1 micrometer, 2 micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers, 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, 200 micrometers, 300 micrometers, 400 micrometers, 500 micrometers, 600 micrometers, 700 micrometers, 800 micrometers, 900 micrometers, 1000 micrometers, or a value or range between any two of these values.

[0060] The center-to-center distance or center-to-center spacing between the holes can be from about 1 micrometer to about 1000 micrometers. In some embodiments, the center-to-center distance between the holes can be or be approximately 1 micrometer, 2 micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers, 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, 200 micrometers, 300 micrometers, 400 micrometers, 500 micrometers, 600 micrometers, 700 micrometers, 800 micrometers, 900 micrometers, 1000 micrometers, or a value or range between any two of these values. The distance or spacing between the edges of the microholes can be from about 1 micrometer to about 1000 micrometers. In some embodiments, the distance between the edges of the microholes can be or be approximately 1 micrometer, 2 micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers, 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, 200 micrometers, 300 micrometers, 400 micrometers, 500 micrometers, 600 micrometers, 700 micrometers, 800 micrometers, 900 micrometers, 1000 micrometers, or a value or range between any two of these values. The microhole array can include microholes of different densities, such as from 100 microholes per square inch to 1000000 microholes per square inch. In some embodiments, the density of the microholes can be or be approximately 10 per cm 2 ² 2 、20 per cm 2 ² 2 、30 per cm 2 ² 2 、40 per cm 2 ² 2 、50 per cm 2 ² 2 、60 per cm 2 ² 2 、70 per cm 2 ² 2 、80 per cm 2 ² 2 、90 per cm 2 ² 2 、100 per cm 2 ² 2 、200 per cm 2 ² 2 、300 per cm 2 ²2 , 7,000 per cm 2 , 8,000 per cm 2 , 9,000 per cm 2 , 10,000 per cm 2 , 20,000 per cm 2 , 30,000 per cm 2 , 40,000 per cm 2 , 50,000 per cm 2 , 60,000 per cm 2 , 70,000 per cm 2 , 80,000 per cm 2 , 90,000 per cm 2 , 100,000 per cm 2 , or a value or range between any two of these values.

[0061] The total number of microholes on the substrate can vary depending on the pattern and spacing of the holes and the overall size of the array. The number of microholes in the array can vary, for example, from about 96 to about 1,000,000. In some embodiments, the number of microholes in the microhole array can be about 96. In some embodiments, the number of microholes can be about 150,000.

[0062] The microhole array can include surface features between the microholes that are designed to facilitate the introduction of liquid into the holes and / or prevent them from depositing on the surface between the holes. Non-limiting examples of suitable surface features include, but are not limited to, arched, ridged, or pointed surface features that surround the holes or span the surface between the holes.

[0063] Thermal cycler

[0064] The system also includes a thermal cycler configured to regulate the temperature in the generated sample partitions to amplify target nucleic acids in the generated sample partitions. The thermal cycler uses alternating cycles of heating and cooling (i.e., thermal cycling) to effect successive replication cycles. In some embodiments, the thermal cycler amplifies target nucleic acids (e.g., within the sample partitions). PCR can be performed by thermal cycling between two or more temperature set points, such as a higher denaturation temperature and a lower annealing / extension temperature, or by thermal cycling between three or more temperature set points, such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature, etc. PCR can be performed with reagents from the reagent repository, such as thermostable polymerases, such as Taq DNA polymerase (e.g., wild-type enzyme, Stoffel fragment, FastStart polymerase, etc.), Pfu DNA polymerase, S-Tbr polymerase, Tth polymerase, Vent polymerase, or combinations thereof, etc. Additional details regarding thermal cyclers can be found, for example, in 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; and U.S. Patent Application Publication Nos. 2006 / 0105433; 2008 / 0176292; 2017 / 0304828; and 2021 / 0291190; the contents of which are incorporated herein by reference.

[0065] Sample reader

[0066] Aspects of the system also include a sample reader. The sample reader can be configured to monitor fluorescence from a biochemical reaction. The reader can include, for example, a light source that selectively emits light in the absorption band of a fluorescent dye, a lens for focusing the light, and a light detector (e.g., a photodiode) that selectively detects light in the emission band of the fluorescent dye, where the fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof. Alternatively, the optical detector can include a bandpass-filtered diode (fluorescent probe) that selectively emits light in the absorption band of a fluorescent dye 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 multiple fluorescent dyes having different fluorescence emission spectra, where each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof. For example, the optical detector can be configured to independently detect multiple fluorescent dyes at multiple different locations, such as in a cuvette or a microplate, where each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof.

[0067] In an embodiment, the sample reader configured to detect the presence of amplified nucleic acid in each generated sample partition within an assay container is a particle counter, such as a two-dimensional (2D) particle counter or a three-dimensional (3D) particle counter. In some cases, the particle counter is a 3D particle counter. In an embodiment, the 3D particle counter includes a horizontal confocal microscope. In such an embodiment, the sample reader includes a light source. Any light source suitable for a horizontal confocal microscope may be employed. In some embodiments, the light source is a laser. In an embodiment, the laser can be any suitable laser, such as a continuous wave laser. For example, the laser can 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 can be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon fluoride (ArF) excimer laser, a krypton fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon fluoride (XeF) excimer laser or a combination thereof. In other cases, the subject flow cytometer includes a dye laser, such as a stilbene, coumarin, or rhodamine laser. In other cases, the target laser includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser and combinations thereof. In other cases, the subject flow cytometer includes a solid-state laser, such as a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser and combinations thereof.

[0068] The particle counter may additionally include one or more optical adjustment components for focusing the laser on an analysis container (e.g., a cuvette, a microplate). In certain embodiments, the optical adjustment element is located between the light source and the analysis container and may include any device capable of altering the spatial width of the illumination or some other characteristic of the illumination from the light source, such other characteristics being, for example, illumination direction, wavelength, beam width, beam intensity, and focus. The optical adjustment scheme may include any suitable device for adjusting one or more characteristics of the light source, including but not limited to lenses, mirrors, filters, optical fibers, wavelength splitters, pinholes, slits, collimation schemes, and combinations thereof. In certain embodiments, the target flow cytometer includes one or more focusing lenses. An example of a focusing lens can be a reducing lens. In other embodiments, the target flow cytometer includes an optical fiber. In certain cases, the optical adjustment component is an objective lens. The objective lens can have any suitable magnification, such as 10x, 20x, 50x, and 100x. In certain cases, the magnification of the objective lens is 20 times. The above rotation and translation of the cuvette transport the droplets through a Gaussian-shaped excitation volume.

[0069] The particle counter of the present invention may also include a dichroic mirror. After irradiation, after the sample emits a fluorescence signal, the emitted fluorescence can be collected by the above objective lens and directed to the dichroic mirror. Although the dichroic mirror is configured to pass the excitation light provided by the light source, it is configured to reflect light of the emission wavelength.

[0070] Embodiments of the particle counter also include a light detector configured to detect light emitted from the sample and reflected by the dichroic mirror. The target detector may include but is not limited to optical sensors or optical 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, as well as other detectors. In certain embodiments, a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS) image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor is used to measure the collected light. In certain embodiments, the detector is a photomultiplier tube, for example, the effective detection surface area of each region is 0.01 cm 2 to 10 cm 2 、for example 0.05 cm 2 to 9 cm 2 、for example 0.1 cm 2 to 8 cm2 , such as 0.5 cm 2 to 7 cm 2 and including 1 cm 2 to 5 cm 2 of the photomultiplier tube.

[0071] In the case where the separator is a dispenser configured to distribute the extracted nucleic acid droplets on, for example, a microplate, embodiments of the present invention include a microplate reader. The general meaning of the microplate reader discussed herein refers to an instrument configured to measure the properties of analytes within the wells of a microplate. In some cases, the microplate reader is a fluorescence microplate reader. In such a case, the microplate reader is configured to detect the presence or absence of fluorescence emission from each well of the microplate. In some cases, the microplate reader is a colorimetric microplate reader. In this case, the microplate reader is configured to detect the presence or absence of a colored compound in the solution within the wells of the microplate, where the presence of the colored compound may indicate the presence of the target nucleic acid. Additional details regarding the microplate reader 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 contents of which are incorporated herein by reference.

[0072] In some cases, each PCR reaction (i.e., in each droplet, well, or chamber, as appropriate) will generate a positive or negative result (i.e., the presence or absence of the target analyte). In some cases, the initial concentration of the target in the sample can be determined by counting the number of microchambers showing positive results and those showing negative results and applying Poisson statistics. In some cases, Poisson statistics can be used to provide a corrected concentration of the positive partition (i.e., containing the analyte), as follows:

[0073]

[0074] Delivery device

[0075] The system of the present invention may also include one or more than one delivery device. The delivery device of interest is configured to deliver a liquid (e.g., a liquid containing a sample, a reagent, etc.) from one part of the dPCR system to another part. Embodiments of the delivery device include an automated dispenser system. The automated dispenser system may be operably connected to at least one of the sample preparation unit, the partitioner, the thermal cycler, and the sample reader, and is configured to deliver a liquid between at least two of these components (e.g., at least 3 of these components and including all of these components) or their constituent elements. Suitable dispensers for use with the devices herein include one or more than one sensor; a manifold, one or more than one pump in fluid communication with the manifold; one or more than one dispensing head in fluid communication with the manifold; and an electrical connection that receives an electrical signal from an external controller, wherein the dispenser has no inlet or outlet for the fluid other than through one or more than one pump.

[0076] Embodiments of the automated dispenser system include an automated pipetting system. The pipette may be a single-channel pipette or a multi-channel pipette. In the case where the pipette is a multi-channel pipette, the number of channels may vary as needed. In some cases, the multi-channel pipette includes from 2 to 40 channels, such as from 5 to 20 channels. The pipette may be positioned on a robotic arm such that the pipette can automatically change its position within the dPCR system. For example, the pipette may be configured to aspirate a liquid inserted into the sample input block, change position, and transfer the liquid to the sample purifier. The pipette may also be configured to use disposable sterile tips (optionally coated with the anti-adhesion coating described herein). In some cases, the robotic arm will reposition the pipette to the tip storage area before each liquid delivery. In the tip storage area, the robotic arm may be configured to load a new tip for each pipette channel (e.g., by lowering the channel into the tip in a manner that holds the tip fixed to the channel). Additionally, after delivering the liquid, the robotic arm is configured to reposition the pipette to the waste repository where the used tips can be ejected and stored. Additional details regarding the automated dispenser system can be found in U.S. Patent Nos. 6,732,598; 7,429,360; 9,134,332; 9,335,336; and 10,704,039; the contents of which are incorporated herein by reference. In some embodiments, the system includes a plurality of automated pipetting systems, such as from 2 to 5 automated pipetting systems. In an alternative, the dPCR system of the present invention includes 2 automated pipetting systems. In some embodiments where the partitioner is a droplet generator, embodiments of the subject system include at least one pipetting system that is configured to obtain an aliquot of the amplified sample (e.g., from the thermal cycler) and transfer it to the sample reader.

[0077] The dispenser may also include a computer-controlled pump that is connected to the distribution manifold through associated computer-controlled valves. The distribution manifold may include a plurality of valves, such as solenoid valves configured to control the air flow through the pipette tips; in one exemplary embodiment, each pipettor has two valves, and there is an additional valve for venting the pump. Thus, for a dispenser with four pipette heads, it has nine valves. In another embodiment, each pipette has only one valve, and there is an additional valve for venting the pump. However, the distribution manifold is not limited to exactly including nine solenoid valves.

[0078] Embodiments of the dispenser include a pump for pumping air into and out of the distribution manifold. The distribution manifold includes a microfluidic network that evenly distributes air between one or more valves. Thus, by controlling the air flow through the manifold and various valves, the pressure above the pipette heads can be changed such that liquid is drawn into or expelled from the pipette tips attached to the respective pipette heads.

[0079] Embodiments of the dispenser may also operate in conjunction with a motorized plate that is configured to strip the pipette tips and align the pipettes during the process of dispensing fluid into the microfluidic cartridge, as further described herein. In such an embodiment, the pipette tips may all be aligned at the same spacing, above the respective sockets in the holder (above the pipette tip sheaths). A metal plate with elongated holes is located on the sockets. The pipette tips are partially inserted downward through the elongated holes into the sheaths, and the metal plate is moved forward in such a way that the pipette tips are clamped by the elongated portions of the holes. When the dispenser is moved upward, the pipette tips are separated from their respective heads. When the metal plate is subsequently moved back to its initial position, the pipette tips remain in their respective positions in the sockets.

[0080] In addition, or alternatively, the system may include one or more delivery devices that are configured to transport a container containing solid or liquid components (e.g., a liquid containing a sample, a reagent, etc.) from one part of the dPCR system to another part. In some such cases, one or more delivery devices may be robotic arms. The robotic arms may be configured for automatic control within a plurality of ranges of motion. For example, the arm may be configured to extend or retract in all directions along the X-axis and Y-axis. The arm is also capable of moving about its central point to allow for mechanical rotation of the arm. In some cases, the robotic arm is operably connected to a controller that is configured and / or trained to cause the robotic arm to retrieve a container of the system and reposition the container to another location within the system. Methods for programming a robotic arm to reposition an entity are described in U.S. Patent Application Publication No. 2006 / 0047363, the disclosure of which is incorporated herein by reference.

[0081] Figure 1 shows a fully automated dPCR system according to certain embodiments of the present invention. The fully automated dPCR system 100 includes a sample preparation unit 110, and the sample preparation unit 110 includes a sample input block 101, a sample purifier 102, and reagent repositories 103a and 103b. A user can place one or more samples into the sample input block 101. In some cases, this includes removing the sample input block 101, inputting the sample, and returning the module to the fully automated dPCR system 100. The sample from the sample input block 101 can be transferred to the sample purifier 102 (e.g., via a pipetting system 108). In the sample purifier 102, nucleic acids are extracted from the sample liquid. In Figure 1 one embodiment, the sample purifier 102 includes one or more magnets (not shown) configured to apply a magnetic force to magnetic beads (e.g., having the extracted nucleic acids stably bound thereto). Additionally, the sample purifier 102 can be equipped with a stirrer configured to break up the sample and facilitate nucleic acid extraction. In an embodiment, the pipetting system 108 is configured to supply reagents to the sample purifier for extracting and eluting nucleic acids from the sample (e.g., lysis buffer, elution buffer, etc.).

[0082] The reagent repository 103a includes one or more buffers required for sample preparation (e.g., rehydration buffer, elution buffer, etc.). In Figure 1 one embodiment, the reagent repository 103b includes a dry (e.g., lyophilized) PCR premix. The nucleic acids extracted in the elution buffer solution can be transferred from the sample purifier 102 into wells within the reagent repository 103b (e.g., via the pipetting system 108). The reagent repository 103b can be, for example, a microplate or a block including liquid containers such as tubes. Inserting the extracted nucleic acid solution into the reagent repository 103b causes the dry premix to enter the solution. This solution can then be transferred from the reagent repository 103b to a separator 104a. In some cases, the transfer is performed by the pipetting system 108. In other cases, the robotic arm 109 can be configured to transfer the container containing the solution (e.g., the reagent repository 103b) to the separator 104a. Used tips and liquid waste can be stored in the waste repository 107.

[0083] In Figure 1 one embodiment, the separator 104a is a droplet generator. However, other types of separators can also be employed, such as the dispenser discussed below with reference to Figure 3B The droplet generator is configured to divide the extracted nucleic acid solution into a plurality of droplets by combining the solution with an immiscible liquid. Figure 3AAn embodiment of the process is shown in more detail. After generating the droplets, the dispensed sample can then be transferred to a thermal cycler 105 where the extracted nucleic acids are amplified. This transfer can be performed, for example, by a robotic arm 109. After PCR, the partitioned samples can be read in a sample reader 106, which can be configured to determine positive or negative for the presence of the target analyte for each sample partition.

[0084] Figure 2 Components of a dPCR system with an anti - adhesion coating are shown. As Figure 2 shown, the container 201 includes wells 202. The wells 202 are coated with an anti - adhesion coating 203, as shown in the enlarged portion of the container 201. In Figure 2 the embodiment, the container 201 is similar to a microplate. However, it should be noted that any component of the dPCR system (e.g., Figure 1 any component shown in

[0085] Figure 3A An embodiment of the present invention is shown where the separator is a droplet generator. The droplet generator 301 is arranged in a flow - focusing configuration where a continuous phase (i.e., oil) causes the sample liquid 302 to break and form droplets 304. After an amplification step in a thermal cycler (not shown), the droplets 304 can be transferred to a cuvette 305. The droplets 304 can then be analyzed by a sample reader 310. In Figure 3A the embodiment, the sample reader 310 is a 3D particle counter. The sample reader 310 includes a light source 318, a dichroic mirror 319, an objective lens 315, and a detector 317. Light from the light source 318 passes through the dichroic mirror 319 and is focused by the objective lens 315 onto a portion of the cuvette 305 and illuminates the droplets 304. If the droplet contains nucleic acid, it will emit fluorescence, which propagates back through the objective lens 315, is reflected by the dichroic mirror 319, and is detected by the detector 317. The cuvette 305 is configured to rotate and translate as shown by the arrows, allowing different portions of the cuvette to be illuminated by the light source 318. In the embodiment, one or more than one of the microdroplet generator 301 and the cuvette 305 are coated with Figure 2 the anti - adhesion coating 203 shown in

[0086] Figure 3B is an embodiment of the present invention where the separator is a dispenser. As Figure 3BAs shown, the dispenser 310 is configured to partition sample liquid by dispensing a small amount of liquid into the wells of an assay container (i.e., microplate 311). Alternatively, the assay container 311 can be a MAP device. The microplate 311 and the sample partitioning therein can be subjected to thermal cycling (not shown) and then read by a sample reader 312. The sample reader 312 can be a fluorescence microplate reader and / or a colorimetric microplate reader, depending on the type of assay being run. In an embodiment, each of the dispenser 310 and the microplate 311 is coated with Figure 2 the anti-adhesion coating 203 shown in

[0087] In an embodiment, the fully automated dPCR system is a closed system. In other words, the components of the system are separated from the surrounding environment (e.g., by walls, doors, etc.). For example, in some cases, the dPCR system includes a workbench on which the functional components of the dPCR system are located (e.g., Figure 1 the components shown). The workbench can be surrounded by a wall that isolates the workbench from the surrounding environment. In some cases, the dPCR system includes a door configured to be a closed system. In some such cases, the door is a sliding door. In addition, embodiments of the system include a waste repository. The waste repository is optional. In embodiments where it is present, it is configured to receive used liquid reagents and / or used pipette tips. In other embodiments where it is absent, the used liquid reagents can be transferred to a location outside the holder and disposed of, e.g., the sample tube containing the original sample whose contents are being analyzed. In some embodiments, the waste repository is located under the workbench.

[0088] Figure 4 An embodiment of the present invention is shown, where the fully automated dPCR system is a closed system. The dPCR system 400 includes a workbench 401 on which the functional components of the dPCR system are located (see, for example Figure 1 ). The workbench and its components are enclosed by a wall and a sliding door 402. Below the workbench 401 is a compartment 403 where a waste reservoir is located for discarded liquid waste and / or pipette tips.

[0089] Computer control system

[0090] Aspects of the present disclosure further include a computer control system, where the system includes one or more than one computer implemented for full or partial automation. A processor, such as a microprocessor, is configured to control the functions of the various components of the system as shown above and thus communicate with each such component that requires control. In addition, the order in which the various functions are described is not limited to the order in which the processor executes instructions when the device is operating. It should also be understood that although a single processor is described as controlling all the operations of the dPCR system, for convenience, these operations can be distributed over more than one processor.

[0091] The processor can be configured to control various aspects of sample preparation and analysis. For example, the processor can be operably connected to each of a sample preparation unit (e.g., a sample purifier), a divider, a thermal cycler, and a sample reader, and initiate and control the activities of each of these elements, i.e., such a system is fully automated. Conveyor mechanisms, such as pipette systems and robotic arms, are likewise operably connected to the processor.

[0092] The system can include a display and an operator input device. For example, the operator input device can be a keyboard, a mouse, etc. The processor can be operably connected to the display, which can display the results of dPCR from the sample reader. The processing module includes a processor accessible to a memory, the memory having instructions stored thereon for performing the steps of the subject method. The processing module can include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input-output controller, a cache memory, a data backup unit, and many other devices. The processor can be an off-the-shelf processor, or some other processor available now or in the future. The processor executes the operating system, which is connected to the firmware and hardware in a known manner through an interface, and helps the processor coordinate and execute the functions of various computer programs that can be written in various programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages known in the art, and combinations thereof. The operating system typically cooperates with the processor to coordinate and execute the 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 based on known techniques. In some embodiments, the processor includes analog electronics that provide feedback control (e.g., negative feedback control).

[0093] The system memory can be any of a variety of known memory storage devices or future memory storage devices. Examples include any conventionally purchased random access memory (RAM), magnetic media such as a resident hard disk or tape, optical media such as a read-write optical disc, a flash device, or other memory storage devices. The memory storage device can be any of a variety of known devices or future devices, including a compact disc drive, a tape drive, or a floppy disk drive. Such a memory storage device reads from and / or writes to a program storage medium (not shown), such as a compact disc. Any of these program storage media, or other program storage media currently in use or likely to be developed in the future, can be regarded as a computer program product. It should be understood that these program storage media typically store computer software programs and / or data. The computer software programs, also known as computer control logic, are typically stored in the system memory and / or the program storage device used in conjunction with the memory storage device.

[0094] In some embodiments, the present invention describes a computer program product that includes a computer-usable medium having control logic (computer software program, including program code) stored therein. When executed by a processor and a computer, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. It will be apparent to those skilled in the art that implementing a hardware state machine can perform the functions described in the present invention.

[0095] The memory can be any suitable device for a processor to store and retrieve data, such as a magnetic storage device, an optical storage device, or a solid-state storage device (including magnetic disks, optical discs, magnetic tapes, or RAM, or any other suitable fixed or portable device). The processor can include a general-purpose digital microprocessor that is appropriately programmed by a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely through a communication channel, or pre-stored in a computer program product, such as a memory or any other portable or fixed computer-readable storage medium using any memory-related device. For example, a magnetic disk or an optical disc can carry the programming and can be read by a disk writer / reader. The system of the present invention also includes, for example, programming of an algorithm for practicing the above-described method in the form of a computer program product. The programming according to the present invention can be recorded on a computer-readable medium, for example, any medium directly readable and accessible 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 drives; and hybrid media of these categories, such as magnetic / optical storage media.

[0096] The processor can also access a communication channel to communicate with a user at a remote location. A remote location means that the user does not directly contact the system, and input information is relayed from an external device, such as a computer connected to a wide area network (“WAN”), a telephone network, a satellite network, or any other suitable communication channel including a mobile phone (i.e., a smart phone), to an input manager.

[0097] In some embodiments, the system according to the present disclosure can include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface can be used for wired or wireless communication, including but not limited to radio frequency (RF) communication (such as radio frequency identification (RFID)), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), communication protocols, and cellular communication, such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0098] In one embodiment, the communication interface may include one or more communication ports, such as physical ports or interfaces, such as USB ports, USB-C ports, RS-232 ports, or any other suitable electrical connection ports, to enable data communication between the subject system and other external devices such as computer terminals (e.g., in a doctor's office or hospital environment) for similar supplementary data communication.

[0099] In one embodiment, the communication interface is configured for infrared communication, communication, or any other suitable wireless communication protocol, enabling the subject system to communicate with computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, and other devices or any other communication devices available for user combination.

[0100] In one embodiment, the communication interface provides a connection for data transfer using the Internet Protocol (IP) via a cellular network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet via a Wi-Fi hotspot. In some embodiments, the communication interface provides a connection to a cloud-based platform on which dPCR data can be stored and / or accessed.

[0101] In one embodiment, the subject system communicates wirelessly with a server device via the communication interface, such as using common standards such as 802.11 or RF protocols, or the IrDA infrared protocol. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop computer; or a larger device, such as a desktop computer, household appliance, etc. In some embodiments, the server device has a display (such as a liquid crystal display (LCD)) and an input device (such as buttons, a keyboard, a mouse, or a touch screen).

[0102] In some embodiments, the communication interface is configured to communicate data stored in the subject system (e.g., in an optional data storage unit) with a network or server device automatically or semi-automatically using one or more of the above communication protocols and / or mechanisms.

[0103] The output controller may include a controller for any of a variety of known display devices for presenting information to a user (whether human or machine, whether local or remote). If one of the display devices provides visual information, this information can typically be organized logically and / or physically into a pixel array. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user and for processing user input information. The functional elements of a computer may communicate with each other via a system bus. In alternative embodiments, some of this communication may be accomplished using a network or other type of remote communication. The output manager may also provide information generated by the processing module to a user at a remote location according to known techniques, such as via the Internet, telephone, or satellite network. The presentation of data by the output manager may be based on a variety of known techniques. For example, the data may include SQL, HTML, or XML documents, email, or other files or other forms of data. The data may include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. Although they are typically part of the broad category of computers commonly referred to as servers, one or more platforms present in the subject system may generally be any type of known computer platform or a type to be developed in the future. However, they may also be mainframe computers, workstations, or other types of computers. They may be connected in a networked or other manner via any known or future type of wiring or other communication system (including wireless systems). They may be in the same location or physically separated. A variety of operating systems may be applied to any computer platform, most likely depending on the type and / or model of the computer platform selected. Suitable operating systems include XP, 7, 8, 10, OS / i5 / IBM Android TM , SGI Oracle etc.

[0104] Method for analyzing a sample

[0105] Aspects of the present invention also include methods of analyzing a sample. The subject methods include introducing a biological sample into the fully automated dPCR system of the present invention and analyzing the biological sample by dPCR. Embodiments of the subject methods also include overcoming sample volume and organism concentration issues prior to introducing the sample into the fully automated dPCR system. As discussed above in the Summary section, attempting to analyze samples using microfluidics / microdroplet technology is challenging because the concentration of organisms is extremely low. To achieve reasonable clinical sensitivity, an analytical sensitivity of approximately 1 organism / mL is required. Traditional blood culture systems use 10 mL of blood to overcome this problem, while typical microfluidics / microdroplet technology uses a sample input of 100 microliters (μL) to 1 mL.

[0106] In some embodiments, the method includes centrifuging the sample prior to introducing it into the dPCR system of the present invention. Such a method can include separating and concentrating the analyte in the sample. Centrifugation methods are described, for example, in Stevens et al. Critical reviews in microbiology 30, no. 1 (2004): 7-24; which is incorporated herein by reference in its entirety. Separation can be defined as the removal of a selected population from a complex mixture, while concentration is defined as a sample preparation process that reduces the sample volume while recovering all target analytes, such as cells. In some embodiments, the method includes centrifuging the sample by simple high-speed centrifugation (<60,000×g). This can be used to concentrate analytes (e.g., bacterial cells) from a microbial culture prior to nucleic acid extraction and detection by the fully automated dPCR system described herein. In additional embodiments, the method includes differential centrifugation. At each step of differential centrifugation, denser particles are separated from less dense particles. The centrifugation speed is increased until the target particles sediment, after which the final supernatant is removed and the pellet is resuspended for further analysis. In other embodiments, the method includes density gradient centrifugation. This technique relies on a suspension solution whose density decreases from the bottom (highest density) to the top (lowest density) of the tube. In some embodiments, the method includes enhancing centrifugation efficiency by coagulation and / or flocculation. Coagulation is facilitated by removing the electrostatic charge (e.g., typically by changing the pH value), which allows the particles to adhere to each other, thus facilitating sedimentation at a lower centrifugation speed. Flocculation is achieved by adding a small amount of a high molecular weight charged substance that bridges oppositely charged particles, producing loose aggregates that can be easily removed by centrifugation or filtration (see, e.g., Stevens et al.).

[0107] The method of the present invention may further include allocating a sample into a plurality of sub-batches and analyzing the plurality of sub-batches in parallel by a dPCR system. In such an embodiment, the method may include dividing the sample of interest into a plurality of sub-batches, for example, where the number of sub-batches is from 2 to 50, such as from 5 to 20. In some embodiments, the method includes dividing the sample into 10 or more sub-batches. The method according to this embodiment further includes processing the sub-batches in parallel. In other words, each sub-batch undergoes the same preparation procedure at the same time. The partitions created for the sub-batches may or may not be analyzed / read in parallel. In other words, the partitions may be read at the same or different times. This parallel processing of sub-batches is described in Gao et al., Analytica chimica acta 606, no. 1 (2008): 98-107; the entire content of which is incorporated herein by reference.

[0108] The method may also include pre-lysing the microorganisms in the sample and analyzing the high-copy number targets in the sample. For example, the method may include pre-lysing bacteria and analyzing high-copy number targets, such as analyzing rRNA present at >10,000 copies per bacterial cell. In an embodiment, this allows the user to obtain the most relevant portion of the entire sample for dPCR analysis. This may only require processing a portion of the original sample (e.g., blood). Any convenient lysis protocol may be used. In some embodiments, the method includes contacting the sample with a lysis buffer. The lysis buffer of interest may include, for example, NP-40 buffer, ammonium chloride-potassium buffer, SDS buffer, etc. A cell lysis protocol that may be used for the subject method is described, for example, in Hall et al., Micromachines 4, no. 3 (2013): 321-332, the content of which is incorporated herein by reference in its entirety. Any convenient assay may be used. In some cases, the assay is a TaqMan copy number assay, etc.

[0109] Desirable methods may additionally include reducing the time to detection (TTD). In other words, the methods according to embodiments of the present invention reduce the amount of time required to detect the presence of an analyte of interest by dPCR. Conventional dPCR systems typically require all sample partitions to be generated prior to analysis. However, the inventors have found that analysis of partitions (e.g., droplets) does not require all droplets to be generated. In other words, as new droplets are formed, intact partitions can be analyzed. Thus, the methods of the present invention include operating a partitioner and a sample reader simultaneously. Sample partitions can be evaluated while the partitioner is still generating more sample partitions. For example, assuming a single analyte of interest (e.g., an organism) in a 10 ml sample (e.g., blood) is randomly distributed, this means that in the worst case, the positive time for the microdroplet method will be close to 8 or 9 hours. Assuming a more typical case of 1 organism per ml and a more reasonable droplet generation rate of 20 microliters per minute, the average time to find a droplet containing the microbe is about 25 minutes, and the worst case is 50 minutes. Since the 18-hour TTD figure for blood cultures assumes all organism loads, it is reasonable to expect a droplet formation time of close to 25 minutes for a real system. In this way, simultaneous operation of the partitioner and the reader can significantly reduce the time required for diagnosis.

[0110] Figure 5 A flowchart showing the method of implementing the present invention according to certain embodiments is shown. Step 501 includes obtaining a biological sample for analysis. In an embodiment, the sample is a blood sample, although other samples can also be obtained as needed. In step 502, the biological sample obtained in step 501 is processed, wherein an analyte of interest (e.g., cells, nucleic acids) is extracted. Step 503 includes increasing the analyte concentration by one or more of the following prior to dPCR analysis: centrifuging the sample (503a); distributing the sample into multiple sub-batches and analyzing the multiple sub-batches in parallel by a dPCR system (503b); pre-lysing the microbes in the sample and analyzing high copy number targets in the sample (503c); and eluting the analyte extracted in step 502 into a small enough volume (503d). Step 504 includes introducing the sample into an automated digital polymerase chain reaction (dPCR) system, such as the system discussed above. Step 505 includes analyzing the biological sample by dPCR, which may include partitioning and reading simultaneously (505a).

[0111] In various embodiments, a sample is combined with a PCR reagent mixture comprising a polymerase and a plurality of nucleotides. The PCR reagent mixture can be in the form of one or more than one lyophilized pellet, and the step of preparing the PCR-ready sample can include contacting the PCR pellet with a liquid to produce a PCR reagent mixture solution. As described above, the system may already be pre-loaded with dried or lyophilized ASR reagents, so the user only needs to input the prepared polynucleotide sample into the dPCR system.

[0112] As described above, in some cases, the sample to be analyzed is a biological sample. The term "biological sample" in its conventional sense refers to a whole organism, plant, fungus, or a subset of animal tissues, cells, or components, which in some cases can be found in blood, mucus, lymph fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen, etc. In some embodiments, the biological sample is blood. Thus, a "biological sample" refers to a subset of a natural organism or its tissues, as well as homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, skin sections, respiratory sections, gastrointestinal sections, cardiovascular sections, and urogenital tracts, tears, saliva, milk, blood cells, tumors, organs. A biological sample can be any type of organism tissue, including healthy tissue and diseased tissue (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or its derivatives, e.g., plasma, tears, urine, semen, etc., where in some cases, the sample is a blood sample, which includes whole blood, such as blood obtained by venipuncture or finger prick (the blood does not necessarily need to be mixed with any reagents such as preservatives, anticoagulants, etc. before analysis).

[0113] In certain embodiments, the source of the sample is a "mammal" or "mammalian", where these terms are widely used to describe organisms within the class Mammalia, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The method can be used for samples from human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult), where in certain embodiments, the human subject is a juvenile, adolescent, or adult. Although the present invention is applicable to samples from human subjects, it should be understood that the method is also applicable to samples from other animal subjects (i.e., "non-human subjects"), such as but not limited to birds, mice, rats, dogs, cats, livestock, and horses.

[0114] In an embodiment, analyzing a biological sample by dPCR includes determining whether a certain analyte is present in the sample, i.e., whether the analyte is present in the sample. In some cases, the analyte is a microorganism, such as a virus, bacterium, or fungus. Optionally, a biological sample is analyzed by dPCR to determine the likelihood that the organism from which the sample was obtained has a disease. In some cases, the disease is sepsis or septicemia. Common bloodborne pathogens associated with these 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, etc.

[0115] In various embodiments, a probe selective for a polynucleotide sequence is added to the sample, wherein the step of preparing the PCR-ready sample includes contacting the neutralized polynucleotide sample or its PCR amplicon with the probe. The probe can be a fluorescent hybridization probe. The fluorescent hybridization probe can include a polynucleotide sequence conjugated to a fluorescent reporter dye and a fluorescent quencher dye. The PCR reagent mixture can further include a positive control plasmid and a plasmid fluorescent hybridization probe selective for at least a portion of the plasmid, and the microfluidic cartridge can be configured to permit independent optical detection of the fluorescent hybridization probe and the plasmid fluorescent hybridization probe.

[0116] In various embodiments, the probe can be selective for polynucleotide sequences characteristic of an organism, such as any organism employing 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, domestic animals, wild animals, extinct organisms, bacteria, fungi, viruses, plants, and the like. The probe can also be selective for organism components that use their own polynucleotides, such as mitochondria. In some embodiments, the probe is selective for microorganisms, such as organisms used in food production (e.g., yeast used in fermentation products, molds or bacteria used in cheese, etc.) or pathogens (e.g., humans, domestic animals or wild mammals, domestic birds or wild birds, etc.). In some embodiments, the probe is selective for organisms selected from Gram-positive bacteria, Gram-negative bacteria, yeast, fungi, protozoa, and viruses.

[0117] In various embodiments, the probe can be selective for polynucleotide sequences specific to organisms selected from: Staphylococcus spp., such as Staphylococcus epidermidis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus; Streptococcus (e.g., alpha-hemolytic, beta-hemolytic or gamma-hemolytic, group A, B, C, D or G), such as Streptococcus pyogenes, Streptococcus agalactiae; Enterococcus faecalis, Enterococcus durans, and Enterococcus faecium; nonenterococcal group D streptococci, such as Streptococcus bovis and Streptococcus equines, viridans Streptococci, such as Streptococcus mutans, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus mitior, Streptococcus milleri, Streptococcus constellatus, Streptococcus intermedius, and Streptococcus anginosus; Streptococcus iniae; Streptococcus pneumoniae; Neisseria, such as Neisseria meningitidis, Neisseria gonorrhoeae, saprophytic Neisseria spp.; Erysipelothrix, such as Erysipelothrix rhusiopathiae; Listeria spp., such as Listeria monocytogenes, the rare Listeria ivanovii, and Listeria seeligeri; Bacillus, such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus subtilus niger, Bacillus thuringiensis, Nocardia asteroides; Legionella, such as Legionella pneumophila (L.pneumonophilia), Pneumocystis, such as Pneumocystis carinii; Enterobacteriaceae, such as Salmonella, Shigella, Escherichia (such as Escherichia coli, Escherichia coli O157:H7); Klebsiella, Enterobacter, Serratia, Proteus, Morganella, Providencia, Yersinia, etc., such as Salmonella, such as Salmonella typhi, Salmonella paratyphi A, Salmonella schoumuelleri, Salmonella hirschfeldii, Salmonella dublin, Salmonella choleraesuis, Salmonella enteritidis, Salmonella typhimurium, Salmonella heidelberg, Salmonella newport, Salmonella infantis, Salmonella agona, and Salmonella saint-paul; Shigella, such as subgroup A, subgroup B, subgroup C, and subgroup D, such as Shigella flexneri, Shigella sonnei, Shigella boydii, Shigella dysenteriae, Proteus (Proteus mirabilis, Proteus vulgaris, and Proteus myxofaciens); Morganella (Morganella morganii); Providencia (Providencia rettgeri, Providencia alcalifaciens, and Providencia stuartii); Yersinia, such as Yersinia pestis, Yersinia enterocolitica Y.Yersinia enterocolitica); Haemophilus spp., such as Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus aphrophilus, Haemophilus ducreyi; Brucella spp., such as Brucella abortus, Brucella melitensis, Brucella suis, Brucella canis; Francisella spp., such as Francisella tularensis; Pseudomonas spp., such as Pseudomonas aeruginosa, Pseudomonas paucimobilis, Pseudomonas putida, Pseudomonas fluorescens; Burkholderia spp., Burkholderia (Pseudomonas) pseudomallei, Burkholderia mallei, Burkholderia cepacia, and Stenotrophomonas maltophilia; Campylobacter spp., such as Campylobacter fetus, Campylobacter jejuni; Helicobacter pylori (C. pylori, Helicobacter pylori); Vibrio spp., such as Vibrio cholerae, Vibrio parahaemolyticus, Vibrio mimicus, Vibrio alginolyticus, Vibrio hollisae, Vibrio vulnificus, and nonagglutinable vibrios; Clostridia spp., such as Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium botulinum; Actinomyces, such as Actinomyces israelii; Bacteroides spp., such as Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides distasonis;Distasonis), Bacteroides vulgatus, Bacteroides ovatus, and Bacteroides merdae; Prevotella, such as Prevotella melaninogenica; Fusobacterium; Treponema, such as Treponema pallidum subspecies endemicum, Treponema pallidum subspecies pertenue, Treponema carateum, and Treponema pallidum subspecies pallidum; Borrelia, such as Borrelia burgdorferi; Leptospira; Streptobacillus, such as Streptobacillus moniliformis; Spirillum, such as Spirillum minus; Mycobacterium, such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium fortuitum complex, Mycobacterium leprae, Mycobacterium asiaticum, Mycobacterium chelonae, subspecies abscessus, Mycobacterium fallax, Mycobacterium fortuitum, Mycobacterium malmoense, Mycobacterium shimoidei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium xenopi; Mycoplasma, such as Mycoplasma hominis, Mycoplasma orale, Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasma pneumoniae, Mycoplasma bovis, M.Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae; Mycoplasma, such as Mycoplasma genitalium; Ureaplasma, such as Ureaplasma urealyticum; Trichomonas, such as Trichomonas vaginalis; Cryptococcus, such as Cryptococcus neoformans; Histoplasma, such as Histoplasma capsulatum; Candida, such as Candida albicans; Aspergillus sp; Coccidioides, such as Coccidioides immitis; Blastomyces, such as Blastomyces dermatitidis; Paracoccidioides, such as Paracoccidioides brasiliensis; Penicillium, such as Penicillium marneffei; Sporothrix, such as Sporothrix schenckii; Rhizopus, Rhizomucor, Absidia, Basidiobolus; diseases caused by Bipolaris, Cladophialophora, Cladosporium, Drechslera, Exophiala, Phialophora, Xylohypha, Ochroconis, Rhinocladiella, Scolecobasidium, Wangiella; Trichosporon, such as Trichosporon beigelii; Blastoschizomyces, such as Blastoschizomyces capitatus; Plasmodium, such as Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariaePlasmodium malariae); Babesia sp; protozoa of the genus Trypanosoma, such as Trypanosoma cruzi; Leishmania, such as Leishmania donovani, Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania braziliensis, Leishmania viannia braziliensis; Toxoplasma, such as Toxoplasma gondii; Naegleria amoeba; Entamoeba histolytica; Giardia lamblia; Cryptosporidium, such as Cryptosporidium parvum; Isospora belli; Cyclospora cayetanensis, Ascaris lumbricoides; Trichuris trichiura; Ancylostoma duodenale or Necator americanus; Strongyloides stercoralis; Toxocara, such as Toxocara canis, Toxocara cati; Baylisascaris, such as Baylisascaris procyonis; Trichinella, such as Trichinella spiralis; Dracunculus, such as Dracunculus medinensis; Filarioidea; Wuchereria bancrofti, Brugia, such as Brugia malayi, Brugia timori; Onchocerca volvulus, Loa loa, Dirofilaria immitis; Schistosoma, such as Schistosoma japonicum, Schistosoma mansoni, Schistosoma mekongi, Schistosoma intercalatum, Schistosomahaematobium); Paragonimus spp., such as Paragonimus westermani, Paragonimus skrjabini; Clonorchis sinensis; Fasciola hepatica; Opisthorchis sp; Fasciolopsis buski; Diphyllobothrium latum; Taenia spp., such as Taenia saginata, Taenia solium; Echinococcus spp., such as Echinococcus granulosus, Echinococcus multilocularismultilocularis); Picornaviruses, rhinoviruses, echoviruses, coxsackieviruses, influenza virus; paramyxoviruses, such as types 1, 2, 3, and 4; adenoviruses; Herpesviruses, such as herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (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, [such as Venezuelan equine encephalitis, eastern equine encephalitis, western equine encephalitis]); Viral hemorrhagic fevers; (filoviruses, [such as Ebola, Marburg]); arenaviruses, [such as Lassa, Machupo]; Smallpox, variola; retroviruses, such as human immunodeficiency viruses 1 and 2; human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, and 35..

[0118] In various embodiments, the probe can be selective for polynucleotide sequences specific to the following organisms: Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella oxytoca, Klebsiella pneumoniae, Escherichia coli, Acinetobacter Baumannii, 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, Gardnerella vaginalis, Micrococcus spp., Haemophilus influenzae, Neisseria gonorrhoeae, Moraxella catarrhalis, Salmonella spp.) Chlamydia trachomatis, Peptostreptococcus productus, Peptostreptococcus, Anaerabius, Lactobacillus fermentum, Eubacterium lentum, Candida glabrata, Candida albicans, Chlamydia spp., Campylobacter spp., Salmonella spp., variola major, Yersinia pestis, Herpes Simplex Virus I (HSV I), Herpes Simplex Virus II (HSV II). In various embodiments, the probe can be selective for a polynucleotide sequence specific to Group B Streptococcus.

[0119] Performing dPCR on a sample can include heating a PCR reagent mixture and the polynucleotide sample under thermal cycling conditions suitable for generating PCR amplicons from the polynucleotide sample; contacting the polynucleotide sample or its PCR amplicon with at least one probe selective for a polynucleotide sequence, and independently contacting the polynucleotide sample and the negative control polynucleotide with the PCR reagent mixture under thermal cycling conditions suitable for independently generating the PCR amplicon of the polynucleotide sample and the PCR amplicon of the negative control polynucleotide; and / or contacting the polynucleotide sample or its PCR amplicon and the negative control polynucleotide or its PCR amplicon with at least one probe selective for a polynucleotide sequence.

[0120] In various embodiments, the method of performing 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 difference of about 20 kPa to 200 kPa, or in some embodiments about 70 kPa to 110 kPa, compared to ambient pressure.

[0121] In various embodiments, methods of using the devices described herein may further include one or more of the following steps: determining the presence of a polynucleotide sequence in a biological sample corresponding to a probe if the probe is detected in a neutralized polynucleotide sample or its PCR amplicon; determining that the result is contaminated if the probe is detected in a negative control polynucleotide or its PCR amplicon; and / or in some embodiments, where 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 includes determining that a PCR reaction has occurred if the plasmid probe is detected.

[0122] Kit

[0123] Aspects of the present disclosure also include kits, where the kits include one or more liquid containers configured for a digital polymerase chain reaction (dPCR) system. The one or more liquid containers include an anti-adhesion coating configured to at least reduce the binding of extracted nucleic acids to the dPCR system. The liquid containers in the subject kits can be any of the containers described herein. For example, the kits can include one or more microplates, tubes, pipette tips, cuvettes, etc. In some embodiments, the kits include containers from the reagent repositories suitable for the present invention. In such embodiments, the containers can contain one or more dPCR reagents or components. For example, the containers can contain one or more buffers (e.g., elution buffer, rehydration buffer, lysis buffer, etc.). In some cases, the containers contain immiscible liquids that can be used for droplet generation. In other cases, the containers of the subject kits are microplates, optionally with dried dPCR reagents (e.g., premixes) located therein. In some cases, the kits include one or more pipette tips comprising an anti-adhesion coating. In additional cases, the kits include one or more microplates comprising an anti-adhesion coating. In still additional embodiments, the kits include one or more cuvettes comprising an anti-adhesion coating. In some embodiments, the containers can be used in the sample purifier. In some such cases, the containers include magnetic beads suitable for nucleic acid extraction, such as those discussed above.

[0124] As described above, the anti-adhesion coating can be one of a plurality of coatings. Anti-adhesion coatings for the subject kits can include hydrophilic components, zwitterionic components, and / or anti-fogging agents.

[0125] In addition to the above components, the subject kits can also include (in some embodiments) instructions for practicing the subject methods. These instructions can be present in the kit in a variety of forms, and one or more than one form can be present in the kit. One form in which these instructions can be present is printed information on a suitable medium or substrate, such as one or more sheets of paper with printed information in the packaging of the kit, in a package insert, etc. Another form of these instructions is a computer-readable medium, such as a disk, a compact disc (CD), a portable flash drive, a hard drive, etc., on which information has been recorded. Another form in which these instructions can be present is a website address that can be used to access information on a removed site via the Internet.

[0126] Utility

[0127] The above systems, methods, and kits can be used for a variety of applications, including applications that require determining the presence or absence of an analyte in a biological sample. This analysis can be qualitative or quantitative. Similarly, in cases where the detection is qualitative, the method provides a reading or assessment of whether the target analyte is present in the sample being tested, e.g., an evaluation. In additional embodiments, the method provides a quantitative detection of whether the target analyte is present in the sample being analyzed, i.e., an assessment or evaluation of the actual amount of the target analyte in the sample being analyzed. In such embodiments, the quantitative detection can be absolute, or if the method is one for detecting two or more different target analytes in a sample, it can be relative. Thus, the term "quantitative", when used to quantify a target analyte in a sample, can refer to absolute quantification or relative quantification.

[0128] In certain embodiments, the present invention can be used to determine the presence or absence of bloodborne pathogens. For example, the systems and methods of the present invention can be used to detect sepsis and / or septicemia. The present invention is particularly suitable for situations where it is necessary to improve the efficiency of dPCR. Thus, the present invention can be used to improve (1) sample volume and organism concentration problems, (2) long front-end preparation times, (3) adhesion of the target analyte to the inner surface, and (4) lack of automation.

[0129] Samples (e.g., blood samples) can be obtained from any convenient source. In certain embodiments, a saliva sample is a sample derived from a "mammal" or "mammalian subject", where these terms are used broadly to describe organisms within the class Mammalia, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the subject is a human. The term "human" can include human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult), where in certain embodiments, the human subject is a juvenile, adolescent, or adult.

[0130] Although the foregoing invention has been described in detail for purposes of clarity of understanding by way of illustrations and examples, it will be apparent to those of ordinary skill in the art that, in light of the teachings of the present invention, some changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0131] Accordingly, the foregoing merely illustrates the principles of the present invention. It is to be understood 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 present invention and are included within the spirit and scope of the present invention. In addition, all of the examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventor to further the art and are to be construed as not being limited to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to cover both structural and functional equivalents thereof. In addition, regardless of their structure, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function. Furthermore, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0132] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are 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 a claim only when the exact phrase "means" or the exact phrase "step" is recited at the beginning of the limitation in the claim; if such exact phrase is not used in the limitation of the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

Claims

1. An automatic digital polymerase chain reaction (dPCR) system, comprising: A sample preparation unit configured to receive a biological sample and extract nucleic acid therefrom; An analysis container; A divider configured to generate a plurality of sample partitions including the extracted nucleic acid, the sample partitions being for distribution into the analysis container; And A thermal cycler configured to regulate the temperature in the generated sample partitions to amplify nucleic acid of interest in the generated sample partitions; And A sample reader configured to detect whether the amplified nucleic acid is present in each of the generated sample partitions within the analysis container; Wherein the dPCR system includes an anti-adhesion coating configured to at least reduce the binding of the extracted nucleic acid to the dPCR system.

2. The automatic dPCR system according to claim 1, wherein the anti-adhesion coating comprises a hydrophilic component.

3. The automatic dPCR system according to claim 1, wherein the anti-adhesion coating comprises an amphoteric ion component.

4. The automatic dPCR system according to claim 1, wherein the anti-adhesion coating is an anti-fogging agent.

5. The automatic dPCR system according to any one of the preceding claims, wherein at least one of the sample preparation unit, the analysis container, the divider, the thermal cycler, and the sample reader includes the anti-adhesion coating.

6. The automatic dPCR system according to claim 5, wherein each of the sample preparation unit, the analysis container, the divider, the thermal cycler, and the sample reader includes the anti-adhesion coating.

7. The automatic dPCR system according to claim 6, wherein each surface of the dPCR system configured to contact a part of the sample includes an anti-adhesion coating.

8. The automatic dPCR system according to any one of the preceding claims, wherein the sample preparation unit includes a sample input block configured to receive the sample.

9. The automatic dPCR system according to any one of the preceding claims, wherein the sample preparation unit includes a sample purifier, and the sample purifier includes a magnet for extracting nucleic acid from a biological sample.

10. The automatic dPCR system according to any one of the preceding claims, wherein the sample preparation unit includes a reagent repository.

11. The automatic dPCR system according to any one of the preceding claims, wherein the divider includes a droplet generator.

12. The automatic dPCR system according to claim 11, wherein the droplet generator is configured to mix the sample with an immiscible liquid.

13. The automatic dPCR system according to claim 12, further comprising an immiscible liquid repository.

14. The automatic dPCR system according to any one of claims 11 to 13, wherein the analysis container is a cuvette.

15. The automatic dPCR system according to claim 14, wherein the cuvette is configured to rotate about an axis and translate along the axis.

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

17. The fully automatic dPCR system according to any one of claims 1 to 10, wherein the separator is a liquid dispenser.

18. The fully automatic dPCR system according to claim 17, wherein the analysis container is a microplate.

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

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

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

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

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

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

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

26. A method for analyzing a sample, the method comprising: (a) introducing a biological sample into an automated digital polymerase chain reaction (dPCR) system, the system comprising: a sample preparation unit configured to receive a biological sample and extract nucleic acid therefrom; an analysis container; a separator configured to generate a plurality of sample partitions comprising the extracted nucleic acid for dispensing into the analysis container; and a thermal cycler configured to regulate the temperature in the generated sample partitions to amplify a target nucleic acid in the generated sample partitions; and a sample reader configured to detect the presence of the amplified nucleic acid in each of the generated sample partitions within the analysis container, 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 by dPCR.

27. The method according to claim 26, wherein the anti-adhesion coating comprises a hydrophilic component.

28. The method according to claim 26, wherein the anti-adhesion coating comprises a zwitterionic component.

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

30. The method according to any one of claims 26 to 29, wherein at least one of the sample preparation unit, the analysis container, the separator, the thermal cycler, and the sample reader comprises the anti-adhesion coating.

31. The method according to claim 30, wherein each of the sample preparation unit, the analysis container, the separator, the thermal cycler, and the 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 an anti-adhesion coating.

33. The method according to any one 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 one of claims 26 to 33, wherein the sample preparation unit comprises a sample purifier including a magnet for extracting nucleic acid from the biological sample.

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

36. The method according to any one of claims 26 to 35, wherein the divider comprises a droplet generator.

37. The method according to claim 36, wherein the droplet generator is configured to mix 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 one of claims 36 to 38, wherein the analysis container is a cuvette.

40. The method according to claim 39, wherein the cuvette is configured to rotate about an axis and translate along the 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 one of claims 26 to 35, wherein the divider is a liquid dispenser.

43. The method according to claim 42, wherein the analysis container is a microplate.

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

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

46. The method according to any one of claims 26 to 44, wherein the divider and the sample reader are configured to operate simultaneously.

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

48. The method according to any one 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 divider.

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

50. The method according to any one of claims 26 to 49, further comprising distributing the sample into a plurality of sub-batches and analyzing the plurality of sub-batches in parallel by the dPCR system.

51. The method according to any one of claims 26 to 50, further comprising: pre-lysing the microorganisms in the sample; and analyzing the high-copy-number targets of the sample.

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

53. A kit comprising: One or more liquid containers configured to be used in a digital polymerase chain reaction (dPCR) system and including 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 a hydrophilic component.

55. The kit according to claim 53, wherein the anti-adhesion coating comprises a zwitterionic component.

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

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

58. The kit according to any one of claims 53 to 57, wherein the one or more liquid containers further comprise dPCR reagents.

59. The kit according to claim 58, wherein the dPCR reagents are PCR premixes.

60. The kit according to claim 58 or 59, wherein the dPCR reagents are dry.

61. The kit according to any one of claims 53 to 60, wherein the one or more liquid containers 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 one of claims 53 to 63, wherein the kit comprises one or more pipette tips including an anti-adhesion coating.

65. The kit according to any one of claims 53 to 64, wherein the kit comprises one or more microplates including an anti-adhesion coating.

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

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