Reaction cartridge assembly and methods of use thereof

Abstract

Among other things, the present application provides simplified reaction cartridge assemblies suitable for use in performing reactions, such as nucleic acid amplification reactions. In an embodiment, a reaction cartridge assembly comprises: an upper receiving reservoir; a reaction reservoir fixedly or removably attached to the upper receiving reservoir to provide fluid communication therebetween; and a closure cap, sized and configured to fit in the upper receiving reservoir, for sealing the reaction reservoir and / or for transferring materials into the reaction reservoir when the cap body is positioned in the upper receiving reservoir. Also provided are methods of performing a reaction, such as a nucleic acid amplification reaction, using a cartridge assembly that comprises an upper receiving reservoir, a reaction reservoir fixedly or removably attached to the upper receiving reservoir and a closure cap.

Description

REACTION CARTRIDGE ASSEMBLY AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application number 63 / 694,417 filed on September 13, 2024, the disclosure of which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0002] The present application pertains to the field of diagnostic assays, in particular, nucleic acid amplification-based assays and related reaction containers.BACKGROUND

[0003] Since the discovery of DNA, many nucleic acid amplification technologies have been developed to detect the presence, absence, or amount of specific DNA or RNA sequences. The Polymerase Chain Reaction (PCR) is a technique used to amplify and, thereby, detect or quantify a specific nucleic acid sequence of interest. This technique has formed the basis of numerous assays for detecting particular nucleic acid sequences. Multiplex PCR employs PCR to amplify and detect multiple target sequences simultaneously. While other nucleic acid amplification technologies have been developed, PCR remains the most commonly used in molecular diagnostic assays.

[0004] U.S. Patent 8,735,104 describes methods and an apparatus for nucleic acid analysis of swab samples without the need for purification. In one embodiment, a swab sample is collected using the cap of a reaction vessel. Insertion of the cap into the reaction vessel directly contacts the swab sample with a nucleic acid amplification reagent. One disadvantage of this method is that some samples contain high concentrations of nucleic acid amplification inhibitors, which can cause amplification to fail when directly contacted with a nucleic acid amplification reagent. Another disadvantage is that some samples contain cells that are difficult to lyse by direct contact with a nucleic acid amplification reagent and heating in a thermal cycler. Another disadvantage is that the apparatus requires that the nucleic acid amplification reagent be in liquid form so that it can be contacted withthe swab sample. However, nucleic acid amplification reagents in liquid form are typically not as stable as those in lyophilized or dried form. Another disadvantage is that the whole swab sample is contacted with the nucleic acid amplification reagent. In some cases, it may be advantageous to only contact a portion of the sample. However, one advantage of this method is that it does not require the use of a pipette or other liquid measuring device.

[0005] While the reaction vessel of U.S. Patent 8,735,104 has a single compartment, multicompartment cartridges have been disclosed before. For example, U.S. Patents 6,818,185; 8,703,476; and 9,789,483 disclose cartridges that have a reaction vessel and multiple compartments. A common disadvantage with these multi-compartment cartridges is they require valves, fluid channels, or other means for moving fluid from one compartment to another that are complicated to manufacture and are susceptible to failure in use.

[0006] U.S. Patent 11,185,864 discloses a single-compartment cartridge, whereby a pressing motion aliquots fluid containing a sample into a reaction vessel containing reaction chemicals via a chromatography element. This aliquoting is accomplished via a metering mechanism as shown in Figure 13. This metering mechanism can include a "fluid by-pass or pressure release channel" that "releases air under pressure from the lower portion of the metering to the upper portion of the chamber." There are many disadvantages to this cartridge, including the cost and difficulty of manufacturing the complicated metering mechanism; multiple points of failure in the metering mechanism; and the need to evacuate air so that air bubbles are not introduced into the chromatography element.

[0007] Thus, there remains a need for alternative reaction cartridges, particularly those that are suitable for use in performing nucleic acid amplification reactions, that overcome at least some of the drawbacks of previous reaction cartridges, tubes and vessels.

[0008] The above information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present disclosure.SUMMARY

[0009] An object of the present application is to provide a simplified reaction cartridge, for example, a simplified nucleic acid amplification cartridge, that is easier and / or less expensive to manufacture than cartridges currently in use.

[0010] Another object of one aspect of the present application is to provide a reaction cartridge that can enable a sample to be diluted before contacting the sample with a reagent, such as a nucleic acid amplification reagent. Another, related object is to provide a reaction cartridge that enables a sample to be aliquoted, without requiring the use of a metering mechanism, pipette, or other liquid measuring device and before contacting the sample aliquot with a nucleic acid amplification reagent.

[0011] Another object of one aspect of the present application is to provide a reaction cartridge that can facilitate or improve nucleic acid extraction of a sample before it is contacted with a nucleic acid amplification reagent.

[0012] In accordance with one aspect of the present application, there is provided a reaction cartridge assembly comprising: (a) an upper receiving reservoir defined by a housing wall having a top edge surrounding an upper opening and a bottom edge surrounding a lower opening; (b) a reaction reservoir comprising an upper open region that is fixedly or removably attached to the upper receiving reservoir at the lower opening to provide fluid communication between the upper receiving reservoir and the reaction reservoir; and (c) a closure cap comprising a top wall, a cap body depending therefrom that is sized and configured to fit in the upper receiving reservoir, and a cap component for sealing the reaction reservoir at the upper open region and / or for transferring materials into the reaction reservoir when the cap body is positioned in the upper receiving reservoir.

[0013] In some embodiments, the cap component is a hollow shaft extending through the cap body from the top wall and terminating at an aperture in a bottom surface of the cap body.

[0014] In some embodiments, the hollow shaft terminates at the aperture, which is positioned at a tip of the bottom surface that is configured to seal the upper open region of the reaction reservoir.

[0015] In some embodiments, one or more reagents are contained in the hollow shaft or affixed to an interior surface of the hollow shaft. Optionally, the one or more reagents are lyophilized, for example, the lyophilized reagents can be in the form of a lyophilized bead.

[0016] In some embodiments, the cap component further comprises a plunger that is displaceable within the hollow shaft. Optionally, the plunger is disposed entirely within, or disposable entirely into, the hollow shaft and below the top wall of the closure cap.

[0017] In some embodiments, the reaction cartridge comprises a temporary and removable barrier between the receiving reservoir and the reaction reservoir, optionally wherein the barrier is a plastic film.

[0018] In some embodiments, the cap component comprises a displaceable retaining member disposed within the hollow shaft. Optionally, a wall of the hollow shaft is configured to receivingly mate with the displaceable retaining member when the displaceable retaining member is displaced by the plunger.

[0019] In some embodiments, the cap component comprises a channel extending along a wall of the hollow shaft from the top wall of the closure cap and terminating at or before the aperture in the bottom surface of the cap body, optionally wherein the channel is a grooved channel.

[0020] In some embodiments, the reaction reservoir comprises: a bottom wall; and four planar side walls joined to the bottom portion. This configuration can be beneficial for insertion of the reaction reservoir in a thermal well of a thermal cycler and thus providing efficient heat transfer.

[0021] In some embodiments, the bottom wall comprises a closed, molded lens and one of the four side walls comprises or consists of a flat, optically transmissive (e.g., optically transparent) side wall. Optionally, the optically transmissive side wall is angularly offsetfrom the closed, molded lens by an angle of from approximately 90° to approximately 120°, preferably about 90°.

[0022] In some embodiments, the reaction reservoir is formed of a thermally stable plastic.

[0023] In some embodiments, the reaction cartridge assembly comprises one or more air escape vents formed when the cap body and the upper reservoir are assembled and wherein the one or more air escape vents are configured to allow air to escape from the reaction reservoir into the upper receiving reservoir.

[0024] In some embodiments, the reaction cartridge assembly comprises a closure cap insert comprising a top wall and an insert body depending therefrom, wherein the insert body is sized and configured to fit in the hollow shaft of the closure cap for depressing the plunger through the hollow shaft towards the reaction reservoir.

[0025] In some embodiments, the upper receiving reservoir of the reaction cartridge assembly comprises an interconnection mechanism to securely hold the closure cap in the upper receiving reservoir. Optionally, the interconnection mechanism comprises a protrusion from the housing wall of the upper receiving reservoir which is configured to secured ly mate with the closure cap.

[0026] In some embodiments, the reaction cartridge assembly comprises an identification tag, optionally, wherein the identification tag is configured to communicate wirelessly with a receiving device. The identification tag is optionally disposed on the closure cap, for example, on the top wall of the closure cap. The identification tag can be, for example, an RFID (radio frequency identification) tag, such as an NFC (near field communication) tag.

[0027] The present application further provides a method for performing a reaction, said method comprising the steps of: (i) providing a reaction cartridge comprising a receiving reservoir and a reaction reservoir; (ii) adding a fluid into the receiving reservoir of the reaction cartridge; (iii) combining a sample with the fluid in the receiving reservoir to form a fluid mixture; (iv) inserting a closure cap into the receiving reservoir to seal the reaction cartridge and to push the fluid mixture from the receiving reservoir into the reactionreservoir and to seal the reaction reservoir; and (v) allowing the reaction to proceed in the reaction reservoir.

[0028] In some embodiments the method further comprises introducing additional regents to the reaction reservoir prior to or during the step of allowing the reaction to proceed.

[0029] In some embodiments, the step of allowing the reaction to proceed comprises performing at least one thermal cycle by sequentially heating the reaction reservoir to a first temperature, maintaining the first temperature for a first preset time, cooling the reaction reservoir to a second temperature and maintaining the second temperature for a second preset time.

[0030] In some embodiments, the method comprises obtaining, by a collection device, the sample, optionally wherein the collection device is a swab.

[0031] In some embodiments, the sample is a sample swab and the step of combining sample with the fluid in the receiving reservoir comprises swirling the sample swab in the fluid for a sufficient time to release the sample components of interest from the swab into the fluid.

[0032] In some embodiments, the sample is or comprises tissue (such as obtained with a buccal swab), bodily fluid (such as sputum, semen, blood, urine, vaginal fluids and / or secretions, cerebrospinal fluid), faeces, wastewater, a forensic sample, or an environmental sample (e.g., pool water, water from a body of water such as a lake, an ocean, or a river, well water, etc.).

[0033] In some embodiments, the fluid in the receiving reservoir is an extraction reagent for lysing cells and / or extracting nucleic acid from the sample.

[0034] In some embodiments, the reaction is a nucleic acid amplification reaction, such as, for example, a non-isothermal amplification reaction.

[0035] In some embodiments, the method comprises identifying contents of the reaction cartridge and / or one or more reaction parameters using an identification tag. Optionally, the identification tag is disposed on the reaction cartridge or the closure cap.

[0036] In some embodiments, the method is performed using a reaction cartridge assembly as described and illustrated herein.

[0037] The present application further provides a kit comprising a reaction cartridge assembly, as described and illustrated herein, and, optionally, instructions for performing a reaction.

[0038] In some embodiments, the kit further comprises an instrument for detecting nucleic acids. Optionally, the instrument is for optically, electrochemically, or magnetically detecting nucleic acids.

[0039] In some embodiments, the kit further comprises a thermocycler, optionally, wherein the thermocycler comprises a thermoelectric heater / cooler. Optionally, the instrument for detecting nucleic acids is integrated with the thermocycler.

[0040] In some embodiments, the instrument or detecting nucleic acids comprises a sensor for detecting an identification tag disposed on a component of the reaction cartridge assembly.

[0041] In some embodiments, the kit further comprises a collection device (e.g., a sample collection device), optionally wherein the collection device is a swab.

[0042] In some embodiments, the kit further comprises one or more nucleic acid extraction reagents, wherein the one or more nucleic acid extraction reagents optionally comprise a nucleic acid extraction reagent is a detergent, a hydroxide, an alkaline solution, or a combination thereof.

[0043] In some embodiments, the kit further comprises a neutralizing fluid. Optionally, the neutralizing fluid contains a buffering agent which neutralizes a nucleic acid extraction reagent.

[0044] In some embodiments, the kit further comprises, or is for use with, software for analyzing the presence, absence, and / or amount of nucleic acids in a sample.

[0045] In some embodiments, the kit further comprises, or is for use with, software for analyzing the presence, absence, and / or amount of a pathogen in a sample.

[0046] The present application further provides a closure cap comprising: (i) a top wall, (ii) a cap body depending therefrom, and (iii) a cap component for sealing a reaction reservoir at an upper open region and / or for transferring materials into the reaction reservoir when the cap body is positioned in a receiving reservoir.

[0047] In some embodiments, the cap component is a hollow shaft extending through the cap body from the top wall and terminating at an aperture in a bottom surface of the cap body. Optionally, the hollow shaft terminates at the aperture, which is positioned at a tip of the bottom surface that is configured to seal the upper open region of the reaction reservoir. Further, in some embodiments, one or more reagents are contained in the hollow shaft or affixed to an interior surface of the hollow shaft. The one or more reagents are optionally lyophilized and may be, for example, in the form of a lyophilized bead.

[0048] In some embodiments, the cap component further comprises a plunger that is displaceable within the hollow shaft. Optionally, the plunger is disposed entirely within, or configured to be disposed entirely within, the hollow shaft and below the top wall of the closure cap.

[0049] In some embodiments of the closure cap, the cap component comprises a displaceable retaining member disposed within the hollow shaft. Optionally, the displaceable retaining member is hinged ly connected to an interior surface of the hollow shaft.

[0050] In some embodiments, the cap component comprises a channel extending along a wall of the hollow shaft from the top wall of the closure cap and terminating at or before the aperture in the bottom surface of the cap body, optionally wherein the channel is a grooved channel.

[0051] In some embodiments, the cap body is sized and configured to fit in a receiving reservoir, optionally wherein the receiving reservoir is an upper receiving reservoir of the reaction reservoir.

[0052] In some embodiments of the closure cap, the closure cap comprises an identification tag. Optionally, the identification tag is an RFID tag, for example, an NFC (near field communication) tag.

[0053] The present application further provides a reaction reservoir comprising: (a) bottom wall; and (b) four planar side walls joined to the bottom portion.

[0054] In some embodiments of the reaction reservoir, the bottom wall comprises a closed, molded lens and one of the four side walls comprises or consists of a flat, optically transmissive side wall. Optionally, the optically transmissive side wall is angularly offset from the closed, molded lens by an angle of from approximately 90° to approximately 120°, preferably about 90°.

[0055] In some embodiments the reaction reservoir is formed of a thermally stable plastic.

[0056] In some embodiments the reaction reservoir comprises an identification tag.BRIEF DESCRIPTION OF THE FIGURES

[0057] For a better understanding of the application as described herein, as well as other aspects and further features thereof, reference is made to the following description which is to be used in conjunction with the accompanying drawings, where:

[0058] Figure 1 is a top perspective view of a reaction cartridge according to one embodiment of the present application;

[0059] Figure 2 depicts a cross-section of the reaction tube depicted in Figure 1;

[0060] Figure 3 is a top perspective view of a closure cap according to one embodiment of the present application;

[0061] Figure 4 depicts a cross-section of the closure cap depicted in Figure 3;

[0062] Figure 5 is a top plan view of a reaction cartridge assembly according to one embodiment, comprising a reaction cartridge as shown in Figures 1 and 2 and a closure cap as shown in Figures 3 and 4;

[0063] Figure 6 depicts a cross-section of the reaction cartridge assembly depicted in Figure 5;

[0064] Figure 7 is a top plan view of a reaction cartridge assembly, according to one embodiment, seated in a thermal well of a thermal cycler; and

[0065] Figure 8 depicts a cross-section of the reaction cartridge assembly seated in a thermal well of a thermal cycler, as shown in Figure 7;

[0066] Figure 9A depicts a top perspective view of a closure cap according to an embodiment of the present application;

[0067] Figure 9B depicts a cross-section of the closure cap depicted in Figure 9A;

[0068] Figure 10 depicts a cross-section of a closure cap, according to one embodiment;

[0069] Figure 11 is a top plan view of a reaction cartridge assembly, according to one embodiment;

[0070] Figure 12 depicts a cross-section of the reaction cartridge assembly, as shown in Figure 11;

[0071] Figure 13 depicts a cross-section of the reaction cartridge assembly, as shown in Figure 11;

[0072] Figure 14 depicts a cross-section of the reaction cartridge assembly, as shown in Figure 11;

[0073] Figure 15 depicts a portion of the cross-section of the reaction cartridge assembly, as shown in Figure 11; and

[0074] Figure 16A depicts a cross-section of a closure cap insert, according to one embodiment; and

[0075] Figure 16B depicts a cross-section of a closure cap insert of Figure 16A and an exemplary embodiment of a closure cap insert inserted into a reaction cartridge assembly, according to one embodiment.DETAILED DESCRIPTION

[0076] Definitions

[0077] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which technologies disclosed herein belong.

[0078] As used in the specification and claims, the singular forms "a, ""an," and "the" include plural references unless the context clearly dictates otherwise.

[0079] The term "comprising," as used herein, will be understood to mean that the list following is non-exhaustive and may or may not include any other additional suitable items, for example one or more further feature(s), component(s), and / or ingredient(s) as appropriate.

[0080] The term "consisting of," as used herein, refers to compositions, methods, devices, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the present disclosure. Otherthan in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about."

[0081] Reference throughout this specification to "one embodiment," "an embodiment," "another embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "a further embodiment" or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0082] The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A," and "B".

[0083] The terms "about" and "approximately" are used herein as equivalents. Any numerals used in this application with or without about / approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0084] The term "amplification reaction" is used herein to refer to a reaction in which multiple copies of an original nucleic acid sequence are generated, typically by repeating an enzymatic duplication process for a number of cycles. When additional copies can be made from each of the duplicate copies made in an earlier cycle, the amplification process is said to be exponential with respect to the number of cycles. In certain embodiments, an amplification reaction is an isothermal amplification reaction. In certain embodiments, an amplification reaction is a non-isothermal amplification reaction.

[0085] Some amplification reactions, for example PCR and LCR, involve cycles of alternately high and low set temperatures, a process known as "thermal cycling." Amplification reactions which use thermal cycling are non-isothermal amplification reactions. PCR is an amplification reaction in which a polymerase enzyme, usually thermostable, generates multiple copies of the original sequence by extension of a primer using the original nucleic add as a template. PCR is described in more detail in U.S. Patent Nos 4,683,202 and 4,683,195 and elsewhere. LCR or "Ligase Chain Reaction" is a nucleic add amplification reaction in which a ligase enzyme, usually thermostable, generates multiple copies of the original sequence by ligating two or more oligonucleotide probes while they are hybridized to the target. LCR, and its variation, Gap LCR, are described in more detail in EP-A-320-308, EP-A-439-182 and WO 93 / 100447 and elsewhere.

[0086] Some amplification reactions, for example Loop-Mediated Isothermal Amplification (LAMP), Whole Genome Amplification (WGA), Strand Displacement Amplification (SDA), Helicase-Dependent Amplification (HDA), Recombinase Polymerase Amplification (RPA), Nucleic Acid Sequences Based Amplification (NASBA), and Transcription Mediated Amplification (TMA), do not involve cycling temperatures. In certain embodiments, an isothermal amplification reaction maintains a substantially constant or fixed temperature during amplification.

[0087] The term "amplicon" or "amplicon molecule," as used herein, refers to a target piece of nucleic acid (DNA or RNA), that is the source and / or product of nucleic acid amplification. In certain embodiments, an amplicon molecule is a nucleic acid molecule generated by transcription from a template nucleic acid molecule, or a nucleic acid molecule having a sequence complementary thereto, or a double-stranded nucleic acid including any such nucleic acid molecule. Transcription can be initiated from a primer.

[0088] As used herein, "diagnosis" refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have or develop a disease, disorder or condition, state, staging or characteristic of a disease, disorder or condition as manifested in the subject, information related to the nature or classification of a tumor, information related to prognosis and / or information useful in selecting an appropriate treatment. Selection of treatment may include the choice of a particular therapeutic agent, medicament, or other treatment modality such as surgery, radiation, etc., a choice about whether to withhold or deliver therapy, a choice relating to dosing regimen (e.g., frequency or level of one or more doses of a particular therapeutic agent or combination of therapeutic agents), etc.

[0089] The term "lens", as used herein, refers to a transparent substance with curved sides for concentrating or dispersing light rays. In some embodiments, a lens is transparent (e.g., transmissive) to light, for example, light having a visible, near infrared, infrared, and / or ultraviolet (UV) spectrum.

[0090] As used herein, the term "mutation" refers to a genetic variation in a biomolecule (e.g., a nucleic acid) as compared to a reference biomolecule. For example, a mutation in anucleic acid may, in some embodiments, comprise a nucleobase substitution, a deletion of one or more nucleobases, an insertion of one or more nucleobases, an inversion of two or more nucleobases, or a truncation, as compared to a reference nucleic acid molecule. Similarly, a mutation in a protein may comprise an amino acid substitution, insertion, inversion, or truncation, as compared to a reference polypeptide. Additional mutations, e.g., fusions and indels, are known to those of skill in the art. In some embodiments, a mutation comprises a genetic variant that is associated with a loss of function of a gene product. A loss of function may be a complete abolishment of function, e.g., an abolishment of the enzymatic activity of an enzyme, or a partial loss of function, e.g., a diminished enzymatic activity of an enzyme. In some embodiments, a mutant comprises a genetic variant that is associated with a gain of function, e.g., with a negative or undesirable alteration in a characteristic or activity in a gene product. In some embodiments, a mutant is characterized by a reduction or loss in a desirable level or activity as compared to a reference; in some embodiments, a mutant is characterized by an increase or gain of an undesirable level or activity as compared to a reference. In some embodiments, the reference biomolecule is a wild-type biomolecule.

[0091] As used herein, in its broadest sense, the term "nucleic acid" refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments e.g., as set forth herein, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments e.g., as set forth herein, the term nucleic acid refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside), and in some embodiments e.g., as set forth herein refers to a polynucleotide chain comprising a plurality of individual nucleic acid residues. A nucleic acid can be or include DNA, RNA, or a combination thereof. A nucleic acid can include natural nucleic acid residues, nucleic acid analogs, and / or synthetic residues. In some embodiments e.g., as set forth herein, a nucleic acid includes natural nucleotides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments e.g., as set forth herein, a nucleic acid is or includes of one or more nucleotide analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof).

[0092] In some embodiments e.g., as set forth herein, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments e.g., as set forth herein, a nucleic acid includes one or more introns. In some embodiments e.g., as set forth herein, a nucleic acid includes one or more genes. In some embodiments e.g., as set forth herein, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.

[0093] In some embodiments e.g., as set forth herein, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments e.g., as set forth herein, a nucleic acid can include one or more peptide nucleic acids, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone. Alternatively or additionally, in some embodiments e.g., as set forth herein, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments e.g., as set forth herein, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'- deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.

[0094] In some embodiments, e.g., as set forth herein, a nucleic acid is or includes at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues. In some embodiments, e.g., as set forth herein, a nucleic acid is partly or wholly single stranded, or partly or wholly double stranded.

[0095] As used herein the terms "sample" and "biological sample" means any sample, including, but not limited to cells, organisms, lysed cells, cellular extracts, nuclear extracts,components of cells or organisms, extracellular fluid, media in which cells are cultured, blood, plasma, serum, gastrointestinal secretions, tissues, homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears, vaginal fluids and / or secretions, and prostatic fluid. In addition, a sample can be a viral or bacterial sample, a sample obtained from an environmental source or a body of water (e.g., a lake, a reservoir, a well), a forensic sample, a food sample (e.g., a food source believed to be contaminated). In certain embodiments, a sample is obtained from a source (e.g., a subject, an environmental source, etc.) using a swab (e.g., a sample swab). As used herein, a "sample swab" may be used to refer to a sample taken with a collection device. In certain embodiments, a swab is a collection device which is made using a material (e.g., a fibrous material, a porous material, etc.) which allows for absorption and release of a sample from the device. In some embodiments, a sample is released from a swab upon contact with a fluid.

[0096] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.

[0097] The term "thermal cycler" or "thermocycler" is used herein to refer to a device used to heat, cool and / or hold a nucleic acid amplification reaction mixture between or at a set temperature for a set time duration over a set number of cycles. Thermocyclers used for real-time and / or quantitative PCR or LCR additionally comprise detection systems, typically optical detection systems, for monitoring reaction product generation. Real-time PCR detection systems are typically optical detection systems for measuring fluorescence signal generated during each amplification cycle as the fluorophore binds to the target sequence in amplicons.

[0098] As used herein, the term "single nucleotide polymorphism" or "SNP" refers to a particular base position in the genome where alternative bases are known to distinguish one allele from another. In some embodiments, one or a few SNPs and / or CNPs is / are sufficient to distinguish complex genetic variants from one another so that, for analytical purposes, one or a set of SNPs and / or CNPs may be considered to be characteristic of a particular variant, trait, cell type, individual, species, etc., or set thereof. In someembodiments, one or a set of SNPs and / or CNPs may be considered to define a particular variant, trait, cell type, individual, species, etc., or set thereof.

[0099] As used herein, the term "treatment" (also "treat" or "treating") refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result. In some embodiments, e.g., as set forth herein, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition using technologies described herein. In some embodiments, e.g., as set forth herein, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, or condition. In various examples, treatment is of an ailment caused by a pathogen (e.g., bacteria, virus), a condition relating to a genetic trait or disorder, or cancer.

[0100] Among other things, the present disclosure provides for a reaction cartridge and a reaction cartridge assembly that is suitable for performing reactions for detection of the presence or absence of a nucleic acid (DNA and / or RNA) and / or for quantification of an amount of nucleic acid in a sample. The detection of the presence, absence or amount of DNA and / or RNA is indicative, for instance, of the presence, absence or amount of a gene, an allele of a gene, a genetic trait or disorder, a polymorphism, a single nucleotide polymorphism (SNP) or of the presence of exogenous DNA or RNA in an organism, e.g., to determine the presence, absence, or amount of a pathogen (e.g., virus or bacteria) in an organism. Through the present disclosure, suitable remedies can be developed for the preparation of medicaments for the treatment of a so diagnosed ailment. For instance, the detection in a sample (e.g., blood) from an organism (e.g., a human) of a pathogen (e.g., a virus) can contribute to a diagnosis and determination of a corresponding treatment (e.g., an appropriate anti-viral or antibiotic).

[0101] Reaction Cartridge Assembly

[0102] The present application provides a reaction cartridge assembly comprising a reaction cartridge, useful for performing reactions, such as nucleic acid amplification reactions. The reaction cartridge comprises an upper portion having a receiving reservoir formed of a housing having an upper open end and a lower open end; a bottom portion comprising a bottom wall and side walls that form a reaction reservoir for accommodating a reaction mixture, wherein the lower open end of the housing of the upper portion is fixedly or removably attached to an upper opening of the reaction reservoir to thus provide fluid communication between the receiving reservoir and the reaction reservoir.

[0103] The present application further provides a reaction cartridge assembly comprising the reaction cartridge and a closure cap.

[0104] Closure Cap

[0105] The closure cap comprises a cap body adapted to fit within the upper open end of the receiving reservoir of the reaction cartridge. The closure cap further comprises a top wall with the cap body depending therefrom and being configured to be received within the housing of the receiving reservoir.

[0106] In some embodiments the cap body has a similar volume to that of the receiving reservoir and is configured to be matingly received in the receiving reservoir. In one example of this embodiment, if the housing forming the receiving reservoir is generally cylindrical, then the cap body can be similarly cylindrical and comprise a cylindrical wall that is sized to be flush against the internal surface of the cylindrical housing of the receiving reservoir when the cap body is positioned within the upper open end of the receiving reservoir of the reaction cartridge. Alternatively, if the housing is generally cuboid in shape, then the cap body can be similarly cuboid and also sized such that the walls of the cap body are flush against the internal surface of the cuboid housing of the receiving reservoir when the cap body is positioned within the upper open end of the receiving reservoir of the reaction cartridge.

[0107] In some embodiments, a cap body has a smaller volume than the volume of a receiving reservoir and is configured to be received in the receiving reservoir without contacting the internal surfaces of the reservoir housing. In this embodiment, the top wall of a closure cap is sized and configured to sealingly engage the housing of the receiving reservoir at the upper open end thereof.

[0108] A closure cap further includes a component for sealing a reaction reservoir at the upper opening thereof and / or for transferring materials, such as a fluid or reaction reagents, into the reaction reservoir. In one embodiment, this component comprises a hollow shaft extending from the top wall, through the cap body and terminating at the upper opening of the reaction reservoir. In some examples, the hollow shaft is cylindrical or tubular. In other examples, the hollow shaft tapers down from the top wall.

[0109] In some embodiments, the shaft terminates at a tip that is configured to seal the upper opening of the reaction reservoir so that the reaction reservoir is longer in fluid communication with the receiving reservoir. This allows a pre-determined volume of fluid to be sealed in the reaction reservoir, thus obviating the requirement for a metering mechanism, pipette, or other liquid measuring device. Optionally, the tip of the shaft extends into the reaction reservoir.

[0110] In some embodiments, the shaft is not hollow and contains reaction reagents held within or affixed to the tip or an interior surface thereof, such that the reaction reagents can be contacted with fluid in the reaction reservoir when the tip of the shaft seals the reaction reservoir.

[0111] In another embodiment, a closure cap further comprises a plunger that is movable (or displaceable) within the shaft, whereby depressing the plunger causes reaction reagents to be expelled from the shaft into the reaction reservoir. In another embodiment, the reagents are sealed within a soluble film that dissolves upon contact with a fluid.

[0112] In some embodiments, a reaction cartridge comprises a temporary and removable barrier between a receiving reservoir and a reaction reservoir. Disruption or puncture of the barrier will allow passage of one or more reagent into the reaction reservoir. In oneexample, a temporary and removable barrier is punctured when a plunger is depressed to expel reagents from within the shaft through the punctured barrier.

[0113] In some embodiments, a barrier is a plastic film.

[0114] In another embodiment, a reaction reservoir contains a lyophilized bead and a shaft terminates in a tip that punctures the barrier and allows the passage of one or more reagent into the reaction reservoir before it is sealed by the mating of the tip of the shaft with the reaction reservoir.

[0115] In some embodiments, reaction reagents within a shaft are in lyophilized form, for example, a lyophilized bead containing a mixture of reagents. The lyophilized reagents are then reconstituted when they contact fluid in the reaction reservoir. In certain embodiments, excipients are added to a lyophilized bead to increase the structural integrity and / or stability of the bead so that the bead does not immediately dissolve when contacted with fluid. Instead, a lyophilized bead can dissolve gradually during a reaction, for example, during an initial denaturation phase of a PCR. This is particularly useful if lyophilized reagents are affixed to the tip of the shaft and not within it.

[0116] In some embodiments, depressing a plunger pushes (either directly or indirectly) against a displaceable retaining member disposed within the shaft of a closure cap. A displaceable retaining member disposed within the shaft of a closure cap can serve to retain or hold reaction reagents within the shaft. In some embodiments, a depressing a plunger pushes a lyophilized bead through the shaft of a closure cap, causing the retaining member to be pushed out of the shaft and into an interior surface of the hollow shaft of a closure cap. In some embodiments, a retaining member is configured be hingedly connected to an interior surface of the shaft of a closure cap. In some embodiments, a retaining member is configured to hingedly connect to a shaft insert. In some embodiments, a retaining member is configured to hingedly connect to a shaft insert.

[0117] In some embodiments, depressing a plunger pushes a lyophilized bead against a rigid element at the tip of the shaft thereby fracturing the bead and causing it to be expelled and come into contact with fluid in the reaction reservoir.

[0118] In some embodiments, a plunger can be removed from the shaft after reagents have been expelled therefrom. Optionally, the plunger is replaced by a lid to close the shaft.

[0119] In some embodiments, a plunger is disposed completely within a closure cap. Disposing a plunger within a closure cap (for example during manufacturing and prior to shipment to a user) prevents the plunger from being depressed prior to use and releasing reaction reagents within the closure cap during, for example, shipment and user handling.

[0120] In certain embodiments, a plunger can be depressed using a closure cap insert as a lid. A closure cap insert is configured to depress the plunger. In certain embodiments, a closure cap insert comprises a top wall and an insert body depending therefrom, wherein the insert body is sized and configured to fit in a hollow shaft of a closure cap for depressing a plunger through the hollow shaft of the closure cap towards a reaction reservoir.

[0121] In certain embodiments, an interconnection mechanism securedly holds a closure cap in place in an upper receiving reservoir. In some embodiments, a closure cap securedly mates to an upper receiving reservoir of a reaction cartridge assembly when inserted into the supper receiving reservoir. In certain embodiments, an interconnection mechanism (e.g., a detent mechanism, a protrusion) from a housing wall of a receiving reservoir (e.g., an upper receiving reservoir) is configured to securedly mate with a closure cap. For example, a groove around a portion of or the whole closure cap can be configured to mate with a protrusion from a housing wall of a receiving reservoir. The groove of the closure cap and the protrusion from the housing wall of the receiving reservoir form an interconnection mechanism to securedly mate the components together.

[0122] In certain embodiments, an identification tag (e.g., as described herein) is disposed on a closure cap.

[0123] In certain embodiments, a shaft of a closure cap is configured to allow gas to escape as a plunger is depressed through the shaft. In some embodiments, a shaft comprises a channel extending along a wall of the shaft from a top wall of the closure cap and terminating at or before the aperture in the bottom surface of the cap body. In certain embodiments, the channel is a grooved channel.

[0124] Identification Tag

[0125] In some embodiments, a reaction cartridge assembly comprises an identification tag. An identification tag is useful for conveying information regarding, for example, a reaction taking place in a reaction cartridge (e.g., reaction temperature(s), reaction time(s), target nucleic acids), manufacturing information about a reaction cartridge or a component of the reaction cartridge (e.g., lot number, manufacturing date, reactants), or instructions on carrying out a reaction.

[0126] An identification tag is additionally useful in certain embodiments to avoid the need for additional user input when carrying out a reaction (e.g., as described herein). For example, an identification tag disposed on a component of a reaction cartridge assembly (e.g., on a closure cap, a reaction cartridge, or another component as described herein) may convey information about the component and / or information about reaction conditions.

[0127] In some embodiments, an identification tag is configured to communicate with an instrument (e.g., a thermocycler as described herein) or a device (e.g., a computer, a cellphone) used to control an instrument. For example, an instrument may be configured to communicate wirelessly with an identification tag on a component of a reaction cartridge assembly to determine which protocol to run and which target nucleic acid sequence is being identified.

[0128] In some embodiments, an identification tag is a tag which a user or device can observe visually including a string of alphanumeric characters or a QR (quick response) code.

[0129] In some embodiments, an identification tag is a tag which a device (e.g., a cellphone, a computer, etc.) can detect optically, magnetically, or using a radio frequency.

[0130] In some embodiments, an identification tag is a tag which is configured to communicate wirelessly (e.g., using a radio frequency) with a receiving device. In some embodiments, an identification tag is an RFID (radio frequency identification) tag. In some embodiments, an RFID tag operates in a low frequency band (30 KHz to 300 KHz range), in a high frequency band (3 MHz to 30 MHz range), or in a Ultra-High Frequency (UHF) band (300 MHz to 3 GHz range). In some embodiments, an RFID tag is an NFC (Near FieldCommunication) tag. An NFC tag operates at a frequency of about 13.56 MHz. In some embodiments, an NFC tag is compatible with or conforms to industry standards for NFC tags such as those established by ISO / IEC 14443 (ISO / IEC - International Organization for Standardization / International Electrotechnical Commission) and FeliCa.

[0131] In some embodiments, a user places an identification tag on a reaction cartridge assembly or a part thereof (e.g., a reaction cartridge, a closure cap) based on, for example, instructions provided in a kit.

[0132] Reaction Cartridge

[0133] As noted above, the reaction cartridge comprises an upper portion having a receiving reservoir and a bottom portion comprising a reaction reservoir.

[0134] In one embodiment, the upper element can be generally in the form of an open- ended funnel or housing that is fixedly or removably attached at an upper opening of the reaction reservoir to provide fluid communication between the receiving reservoir and the reaction reservoir. In some embodiments there is a temporary and removable barrier between the receiving reservoir and the reaction reservoir. Such a barrier can be disrupted to allow passage of a sample and / or one or more reagent from the receiving reservoir into the reaction reservoir

[0135] In one embodiment, the receiving reservoir can be pre-filled with a first fluid, such as a diluent or buffer for a reaction, where the first fluid optionally comprises additional reaction reagents. Alternatively, a user can add a first fluid to the receiving reservoir at the time of use.

[0136] In some embodiments, the first fluid in the receiving reservoir or added to the receiving reservoir contains a nucleic acid extraction reagent. In some embodiments, a nucleic acid extraction reagent is a detergent (e.g., Triton X-100, Trition X-114, Tween 20, Tween 80, NP-40, sodium dodecyl sulphate (SDS), cetyltrimethylammonium bromide (CTAB)), a hydroxide (e.g., sodium hydroxide), an alkaline solution (e.g., a solution with a pH greater than 7), or a combination thereof. Consequently, a nucleic acid extraction reagent can lyse cells and extract nucleic acids from a sample that is combined with the fluid in thereceiving reservoir. When a first fluid is added to a receiving reservoir at the time of use, a sample can be added to the receiving reservoir simultaneously with the first fluid or before or after addition of the first fluid.

[0137] In some embodiments, a second neutralizing fluid is added to a mixture of a first added fluid with a sample, following nucleic acid extraction. For example, if a first fluid contains sodium hydroxide, a second fluid may contain a buffering agent, such as Tris-HCI, to neutralize the hydroxide and thus lower the pH of the mixture.

[0138] The bottom portion of a reaction cartridge comprises a reaction reservoir for a reaction, such as a non-isothermal reaction (e.g., a nucleic acid amplification reaction). This reaction reservoir has a known volume.

[0139] In some embodiments, a reaction reservoir is for accommodating a reaction mixture, for example a nucleic acid amplification reaction mixture, and is sized to accommodate a reaction mixture having a volume in a range of: from about less than 1 nanoliter to about 1 milliliter; from about 1 nL to about 10 nL; from about 1 nL to about 1 pL; from about 1 pL to about 1 mL; from about 1 pL to about 10 pL; from about 1 nL to about 1.5 nL; from about 2 nL to about 2.5 nL; from about 3 nL to about 3.5 nL; from about 4 nL to about 4.5 nL; from about 5 nL to about 5.5 nL; from about 6 nL to about 6.5 nL; from about 7 nL to about 7.5 nL; from about 8 nL to about 8.5 nL; from about 9 nL to about 9.5 nL; from about 10 nL to about 20 nL; from about 30 nL to about 40 nL; from about 50 nL to about 60 nL; from about 70 nL to about 80 nL; from about 90 nL to about 100 nL; from about 200 nL to about 300 nL; from about 400 nL to about 500 nL; from about 600 nL to about 700 nL; from about 800 nL to about 900 nL; from about 1 pL to about 10 pL; from about 20 pL to about 30 pL; from about 40 pL to about 50 pL; from about 60 pL to about 70 pL; from about 80 pL to about 90 pL; from about 100 pL to about 200 pL; from about 300 pL to about 400 pL; from about 500 pL to about 600 pL; from about 700 pL to about 800 pL; or from about 900 pL to about 1 mL or more. In some embodiments, a reservoir for a reaction mixture is sized to contain a volume in the range of: from less than about 1 nanoliter to about 1 nL; from about 1.5 nL to about 2 nL; from about 2.5 nL to about 3 nL; from about 3.5 nL to about 4 nL; from about 4.5 nL to about 5 nL; from about 5.5 nL to about 6 nL; from about 6.5 nL to about 7 nL; from about 7.5 nL to about 8 nL; from about 8.5 nL to about 9 nL; from about 9.5 nL to about 10nL; from about 20 nL to about 30 nL; from about 40 nL to about 50 nL; from about 60 nL to about 70 nL; from about 80 nL to about 90 nL; from about 100 nL to about 200 nL; from about 300 nL to about 400 nL; from about 500 nL to about 600 nL; from about 700 nL to about 800 nL; from about 900 nL to about 1 pL; from about 10 pL to about 20 pL; from about 30 pL to about 40 pL; from about 50 pL to about 60 pL; from about 70 pL to about 80 pL; from about 90 pL it to about 100 pL; from about 200 pL to about 300 pL; from about 400 pL to about 500 pL; from about 600 pL to about 700 pL; from about 800 pL to about 900 pL; or from about 1 mL to more than about 1 mL.

[0140] In some embodiments, a reaction reservoir is formed from: a bottom end, distal from the upper opening, that comprises a closed, molded lens; and side walls connected to the bottom end, wherein at least one of the side walls comprises or consists of a flat, optically transmissive (e.g., optically transparent) material.

[0141] In accordance with some embodiments, a flat, optically transmissive surface on one side of a reaction reservoir is angularly offset at an angle of from about 90° to about 120° to the molded lens, or preferably about 90°.

[0142] In some embodiments, a reaction tube is in whole or in part made from plastic, glass, natural polymers, synthetic polymers, metal, or combinations thereof. In some embodiments, a reaction tube is made of any material suitable for conditions for nucleic acid amplification reactions, such as nucleic acid amplification reaction mixtures, chemical reagents and / or thermal cycling, such as, a thermally stable plastic. In some embodiments, a reaction tube is made, at least in part, of polypropylene.

[0143] In certain embodiments, a reaction cartridge is made of a polypropylene plastic for contact with one or more a thermal surface for heating and cooling the contents of the reaction reservoir. The polypropylene plastic is also optically transmissive to enable fluorescent detection of nucleic acid amplification, such as by real-time PCR.

[0144] Figures 1 to 15 depict illustrative, non-limiting examples of reaction cartridges of the present disclosure.

[0145] Figure 1 is a schematic showing a top perspective view of a reaction cartridge 10 according to an embodiment of the present disclosure. Figure 2 is a cross-section of reaction cartridge 10. As shown in Figures 1 and 2, reaction cartridge 10 includes a reaction reservoir 20 formed by four connecting, planar side walls 22 (not shown), 24, 26, and 28 and bottom wall 30. Reaction cartridge 10 further includes receiving reservoir 50 for receiving a sample and / or reagent(s). Receiving reservoir 50 is removably or fixedly attached to upper open end 40 of reaction reservoir 20. As shown in Figure 1, receiving reservoir 50 is formed from an open-ended housing 52, comprising a pair of opposing planar walls and a pair of opposing curved walls.

[0146] In the embodiments in which receiving reservoir 50 is removably attached to upper open end 40 of reaction reservoir 20, receiving reservoir 50 can be removed following addition of a sample and / or reagent(s) to allow a reaction mixture to be sealed within reaction reservoir 20 with a cap or lid (not shown).

[0147] In the embodiments in which receiving reservoir 50 is fixedly attached to upper open end 40 of reaction reservoir 20, a cap or a lid can be used to seal top, open end 55 of receiving reservoir 50. Alternatively, a plug-type element (not shown) can be matingly received in receiving reservoir 50 and thereby allow a reaction mixture to be sealed within reaction reservoir 20.

[0148] In some embodiments, reaction reservoir 20 has a rectangular pyramidal or square pyramidal shape truncated by bottom wall 30. All or a portion of bottom wall 30 forms a lens. Optionally, one of side walls 22, 24, 26, and 28 is formed of or comprises an optically transmissive (e.g., optically transparent) material.

[0149] Figure 3 is a schematic showing a top perspective view of a closure cap 100 according to one embodiment. Figure 4 is a cross-section of closure cap 100. As shown in Figures 3 and 4, closure cap 100 comprises a cap body 120, having an internal shaft 130, depending from top wall 140 having a cap body adapted to fit within the upper open end of the receiving reservoir of the reaction cartridge. The closure cap further comprises a top wall with the cap body depending therefrom and being configured to be received within thehousing of the receiving reservoir. Cap body 120 comprises air vents 122 and 124 into shaft 130.

[0150] Closure cap 100 further includes plunger 150, which is movable within shaft 130. Figures 3 and 4 illustrate plunger 150 fully depressed into shaft 130 toward dispensing hole 132 through which fluid or reagents found within shaft 130 can be expelled, for example, into the reaction reservoir of a reaction cartridge as described herein.

[0151] Figure 4 further illustrates the presence of lyophilized bead 134 adjacent dispensing hole 132. Lyophilized bead 134 can contain lyophilized reagents that can will be reconstituted when they contact fluid in the reaction reservoir.

[0152] Figure 5 is a top plan view of a reaction cartridge assembly 200 comprising reaction cartridge 10 and closure cap 100, according to one embodiment of the present application. Figure 6 is a cross-section of reaction cartridge assembly 200.

[0153] Figures 5 and 6 depict reaction cartridge assembly 200 with cap body 120 of closure cap 100 seated in receiving reservoir 50 of reaction cartridge 10. As shown Figures 5 and 6, when closure cap 100 is fully seated in receiving reservoir 50, the circumferential edges of top wall 140 engage with the upper edge of closure cap 100 and further includes plunger 150, which is movable within shaft 130. As illustrated in Figure 6, plunger 150 can comprise a curved or rounded end portion 155. Figures 5 and 6 illustrate plunger 150 fully depressed into shaft 130 toward dispensing hole 132 through which fluid or reagents found within shaft 130 can be expelled, for example, into reaction reservoir 20.

[0154] Figures 7 and 8 illustrate an embodiment in which a reaction cartridge assembly 201 with a cap body 121 of a closure cap 101 is seated in receiving reservoir 51 of reaction cartridge 11. As shown Figures 7 and 8, when closure cap 101 is fully seated in receiving reservoir 50, the circumferential edges of top wall 141 engage with the upper edge of closure cap 100 and further includes plunger 151, which is movable within shaft 130. As illustrated in Figure 8, plunger 151 can comprise a pointed or angled end portion 156. Figures 7 and 8 illustrate plunger 151 fully depressed into shaft 130 toward dispensing hole 132 through which fluid or reagents found within shaft 130 can be expelled, for example, into reaction reservoir 20.

[0155] Figures 6 and 8 further illustrate the presence of lyophilized bead 134 adjacent dispensing hole 132. Lyophilized bead 134 can contain lyophilized reagents that can be reconstituted when they contact fluid in reaction reservoir 20.

[0156] Reaction cartridge assembly 201 is configured for the bottom portion comprising reaction reservoir 20 to fit within a thermal well 300 of a thermal cycler assembly (not shown). As illustrated Figures 7 and 8, reaction reservoir 20 includes two opposing major planar walls 22 (not shown) and 24 spaced apart from each other by minor planar walls 26 and 28.

[0157] In some embodiments, reaction reservoir 20 has a rectangular pyramidal or square pyramidal shape truncated by bottom wall 30.

[0158] With this configuration of reaction reservoir 20, the reaction cartridge can be positioned with reaction reservoir 20 in the thermal well such that side walls 22, 24, 26, and 28 are flush with the side panels of thermal well 300. In certain embodiments, configuring side walls flush with side panels of a thermal well allows for more effective (e.g., efficient) heat transfer from the thermal well to a solution in a reaction reservoir.

[0159] Figure 9A is a schematic showing a top perspective view of a closure cap 900 according to an embodiment of the present application. Figure 9B depicts a cross-section of closure cap 900 depicted in Figure 9A. As shown in Figures 9A and 9B, closure cap 900 comprises cap body 902 depending from top wall 904. In certain embodiments, cap body 902 is adapted to fit within an upper open end of a receiving reservoir of a reaction cartridge. Closure cap 900 further comprises a top wall with cap body 902 depending therefrom and being configured to be received within the housing of the receiving reservoir. Cap body 902 comprises air vent 910. Air vent 910 is a grooved channel, which allows gases (e.g., air) to pass through the channel along a wall of shaft 912 and escape from a reaction cartridge assembly.

[0160] Closure cap 900 further includes plunger 914, which is movable within shaft 912. Depressing plunger 914 into shaft 912 toward dispensing hole 916 causes fluid or reagents found within shaft 912 to be expelled, for example, into a reaction reservoir of a reaction cartridge as described herein.

[0161] Closure cap 900 further illustrates lyophilized bead 918 being held in place by trap door 920. Trap door 920 is a displaceable retaining member disposed within shaft 912. Trap door 920 is hingedly connected to shaft insert 922. In some embodiments, trap door 920 is substantially perpendicular to shaft 912 when in a closed position. T rap door 920 forms an approximately 135° angle with shaft insert 922 when in a closed position, as shown. In some embodiments, trap door 920 forms an approximately 90° angle with shaft insert 922 when in a closed position. In some embodiments, trap door 920 forms an angle greater than 135° with shaft insert 922 when in a closed position. In some embodiments, trap door 920 forms an angle less than 90° with shaft insert 922 when in a closed position. Shaft insert 922 is inserted along a wall of shaft 912. Shaft insert 922 can be inserted into shaft 912 such that trap door 920 contacts shelf 924. Shelf 924 forms a protrusion from a wall of shaft 912. In some embodiments, the portion of shelf 924 in contact with trap door 920 is configured to allow to trap door 920 to slide past shelf 924 without catching as plunger 914 is depressed. A wall of shaft 912 is configured to receivingly mate with trap door 920 as plunger 914 is depressed. Depressing plunger 914 causes lyophilized bead 918 to move towards dispensing hole 916.

[0162] Figures 9A and 9B also depict cap groove 926 which traverses around cap side 928, which extends from top wall 904. In certain embodiments, cap groove 926 is configured to engage with a reaction cartridge. In certain embodiments, a cap groove can engage (e.g., matingly, sealingly) with a receiving reservoir of a reaction cartridge assembly to secure closure cap 900 into the receiving reservoir. In certain embodiments, once inserted into a receiving reservoir, cap groove 926 is configured to prevent or limit a user from removing closure cap 900 from the receiving reservoir. Identification tag 930 is disposed on top wall 904.

[0163] Figure 10 depicts a cross-section of a closure cap 1000, according to an illustrative embodiment. In certain embodiments, plunger 1002 is recessed into closure cap 1000. Recessing plunger 1002 in closure cap 1000 prevents unintentional depression plunger 1002 into shaft 1004, resulting in lyophilized bead 1004 being prematurely released through dispensing hole 1006. For example, a recessed plunger may be useful in preventing a user from unintentionally depressing a plunger as a closure cap is being handled by the user.Additionally, a recessed plunger may be useful in preventing unintentional release of, for example, a lyophilized bead during shipping.

[0164] Figures 11 and 12 depict reaction cartridge assembly 1100 according to an embodiment with cap body 902 seated in receiving reservoir 1102 of reaction cartridge 1104. As shown Figures 11 and 12, when closure cap 900 is fully seated in receiving reservoir 1102, wall 1108 engages with the upper edge of closure cap 900 and further includes plunger 914, which is movable within shaft 912. Cap body 902 is stabilized in receiving reservoir 1102 by ribs 1202 and 1204, which protrude from the interior surface of the receiving reservoir 1102. In certain embodiments, there are 2, 3, 4, 5, 6 or more ribs within the receiving reservoir. Additionally, when seated in receiving reservoir 1102, cap groove 926 engages matingly with side wall protrusion 1206 which traverses along inner wall 1208 of receiving reservoir 1102. In certain embodiments, cap groove 926 locks closure cap 900 into place such that dispensing hole 916 is aligned with an opening of reaction reservoir 1106.

[0165] As shown in Figure 12, reaction cartridge 1104 includes a reaction reservoir 1106 formed by four connecting, planar side walls 1210, 1212, 12014, and 1216 (not shown) and bottom wall 1218. Reaction cartridge 1104 further includes receiving reservoir 1106 for receiving a sample and / or reagent(s). Receiving reservoir 1102 is removably or fixedly attached to upper open end 1220 of reaction reservoir 1106. As shown in Figures 11 and 12, receiving reservoir 1102 is formed from an open-ended housing, comprising a pair of opposing planar walls and a pair of opposing curved walls.

[0166] In certain embodiments, rection reservoir 1106 is configured to allow light to pass through at least two side walls 1210, 1212, 12014, and 1216. The light shone through reaction reservoir 1106 passes through

[0167] Figure 13 illustrates plunger 914 fully depressed into shaft 912 toward dispensing hole 916 (not shown in Figure 13) through which fluid or reagents found within shaft 912 can be expelled into reaction reservoir 1106. In Figure 13, lyophilized bead 918 is ejected from shaft 912 into reaction reservoir 1106. In some embodiments, plunger 914 is depressed into shaft 912 by placing a closure cap insert (not pictured) into receivingreservoir 1102 of reaction cartridge 1104. In certain embodiments, a closure cap insert may have a protrusion which extends from a top wall of the closure cap which assists in depressing plunger 914 into shaft 912 to a desired depth. As plunger 914 is depressed into shaft 912, gas or air is allowed to escape from air vent 910 towards top wall 904. As plunger 914 is depressed into shaft 912, trap door 920, which is hinged ly attached to shaft insert 922, is displaced out of the hollow portion of shaft 912 and into a wall of shaft 912 such that is allows for plunger 914 to slide down towards dispensing hole 916 (not shown in Figure 13) and expel lyophilized bead 918 into reaction reservoir 1106. In certain embodiments, depressing plunger 914 also causes a portion of a sample to be ejected into reaction reservoir 1106 such that the sample and lyophilized bead 918 are placed in contact with one another.

[0168] Figure 14 depicts a cross-section of reaction cartridge assembly 1100, as shown in Figure 11. Figure 15 depicts an enlarged image of section G of the cross-section of reaction cartridge assembly 1100, shown in Figure 14. Figures 14 and 15 show plunger 914 completely depressed in reaction cartridge assembly 1100. Side air escape vents 1502, 1504 allow for air to escape from reaction reservoir 1106 as plunger 914 is depressed into shaft 912. In some embodiments, without sufficient venting, fluid would flow back into shaft 912 or receiving reservoir 1102 as plunger 914 is depressed.

[0169] In one embodiment, as illustrated in Figure 7, one of the side panels of thermal well 300 includes an opening 310. Opening 310 permits light to enter and exit the thermal well, for example, wherein the thermal well comprises a first opening 310 for excitation light from a light source to enter into the housing to excite a reaction mixture in the reaction cartridge, and a second opening (not shown) for light emitted from the reaction mixture to exit the housing for detection by the photodetector. Optionally, the second opening is at the bottom of thermal well 300.

[0170] In one embodiment, Figure 16A illustrates closure cap insert 1600. Figure 16B depicts a cross-section of closure cap insert 1600 of Figure 16A inserted into reaction cartridge assembly 1100. In Figure 16B, plunger 914 is depressed using closure cap insert 1600. Closure cap insert 1600 comprises top wall 1602 and insert body 1604 depending therefrom, wherein insert body 1604 is sized and configured to fit in a hollow shaft of aclosure cap of reaction assembly 1100 for depressing plunger 914 through a hollow shaft of the closure cap towards reaction reservoir 1106.

[0171] In some embodiments, the reaction cartridge is for use in a thermal cycler having a thermal well with openings for light transmission. In some embodiments, a reaction reservoir can comprise a flat, optically transmissive (e.g., optically transparent) side wall and a flat, optically transmissive bottom wall, or a molded lens in the bottom wall and a flat, optically transmissive side wall angularly offset from a closed, molded lens by an angle of from approximately 90° to approximately 120°, preferably 90°. In some embodiments, a thermal well configured to conform to a side wall of a reaction reservoir allows for a more efficient transfer of heat to the reaction reservoir.

[0172] Method for Performing a Non-isothermal Reaction

[0173] In another aspect of the present application, there is provided a method for performing a reaction (e.g., a non-isothermal reaction) using a reaction cartridge assembly of the present application. In some embodiments, a non-isothermal reaction (e.g., a nonisothermal amplification reaction) is performed using a reaction cartridge assembly of the present disclosure. In some embodiments, a non-isothermal reaction is for detection of the presence, absence, and / or amount of a target nucleic acid sequence in a sample comprising one or more nucleic acid sequences. In some embodiments, an amplification reaction can detect the presence, absence, and / or amount of two, three, four, five, six, or more target nucleic acid sequences.

[0174] Non-isothermal amplification reactions for detection of the presence, absence, and / or amount of a target nucleic acid are advantageous over isothermal amplification reactions as cycling temperatures in non-isothermal amplification reactions provide for predictable estimators of amplification progress. For non-isothermal reactions, each cycle of heating and cooling provides for a predictable degree or amount of nucleic acid amplification.

[0175] In an embodiment, a method comprises the steps of:- providing a reaction cartridge comprising a receiving reservoir and a reaction reservoir;- adding a fluid into the receiving reservoir of the reaction cartridge;- combining a sample with the fluid in the receiving reservoir;- inserting a closure cap into the receiving reservoir to seal the reaction cartridge and to push the fluid mixture from the receiving reservoir into the reaction reservoir and to seal the reaction reservoir using a shaft that is part of the cap;- optionally introducing additional regents to the reaction reservoir; and- optionally, performing at least one thermal cycle by sequentially heating the reaction reservoir to a first temperature, maintaining the first temperature for a first preset time, cooling the reaction reservoir to a second temperature and maintaining the second temperature for a second preset time.

[0176] In some embodiments, a sample is a sample swab and a step of combining sample with the fluid in the receiving reservoir comprises swirling the sample swab in the fluid for a sufficient time to release the sample components of interest from the swab into the fluid.

[0177] In some embodiments, a step of obtaining a sample is performed using a collecting device (e.g., a collection device as described herein). In some embodiments, a collection device is used to obtain a sample swab (e.g., a buccal swab, a swab from an environmental source, a swab from a forensic sample, etc.).

[0178] In some embodiments, a sample is a liquid sample that can be poured or aliquoted directly into the receiving reservoir.

[0179] In some embodiments, a step of performing at least one thermal cycle is for performing steps of amplification of one or more target nucleic acid sequences to thereby provide amplicons.

[0180] In some embodiments, a method additionally comprises a step of detecting the presence, absence and / or amount of amplicons produced by a reaction corresponding to a target nucleic acid sequence amongst nucleic acid sequences in a sample.

[0181] In certain embodiments, a method is performed in a reaction cartridge assembly as described herein.

[0182] In certain embodiments, a target nucleic sequence can be from DNA, genomic DNA, RNA, mRNA, cDNA, transgenic DNA, etc. In certain embodiments, a target nucleotide sequence is an exogenous sequence. In certain embodiments, a target nucleic sequence is a pathogen. In certain embodiments, a target nucleic sequence is derived from a pathogen.

[0183] In certain embodiments, a sample is from an organism. An organism can be, for example, a human, a non-human animal, a micro-organism, or a plant.

[0184] In certain non-limiting embodiments, a sample is or comprises tissue (such as tissue obtained using a buccal swab), bodily fluid (such as sputum, semen, blood, urine, vaginal fluids and / or secretions, cerebrospinal fluid), faeces, wastewater, a forensic sample, or an environmental sample (e.g., pool water, water from a body of water such as a lake, an ocean, or a river, well water, etc.). In certain embodiments, the sample may require or benefit from concentration (e.g., an environmental sample) and may be concentrated by pushing the sample across a filter using a syringe, and then the sample may be eluted off the filter by reversing the suction of the syringe.

[0185] In some embodiments, a receiving reservoir is pre-filled with fluid and, optionally, there is a temporary and removable barrier between a receiving reservoir and a reaction reservoir to retain the fluid in the receiving reservoir. In an embodiment, a step of inserting a closure cap into a receiving reservoir to push a fluid mixture into a reaction reservoir can comprise disrupting or puncturing a barrier. In one example, a temporary and removable barrier is punctured when a moveable plunger within a shaft in a closure cap body is depressed to puncture a barrier. Optionally, depressing a plunger additionally expels reagents from within a shaft into a reaction reservoir. Optionally, there is a lyophilized bead containing nucleic acid amplification reagents in a receiving reservoir that is sealed by a barrier. Disruption of a barrier allows fluid to fill a receiving reservoir. The receivingreservoir is then sealed to a pre-determined volume by the tip of the shaft mating with the top of the receiving reservoir.

[0186] In certain embodiments, a fluid poured into a reservoir contains a reagent for lysing cells in a sample. In certain embodiments, a lysing reagent is an acid or a base. In certain embodiments, a second fluid is poured into the reservoir after mixing of the first sample and a lysing fluid containing a lysing reagent in order to neutralize the lysing fluid.

[0187] Nucleic acid amplification methods typically employ two primers, deoxynucleotide triphosphates (dNTPs), and a (DNA) polymerase. A preferred method for amplification is Polymerase Chain Reaction, or "PCR". PCR protocols are well known in the art, and are described in standard laboratory textbooks e.g., Ausubel et al. (1995). Current Protocols in Molecular Biology, John Wiley & Sons, Inc. Other multiplex and / or isothermal amplification methods that may be applied include LCR, self-sustained sequence replication (3SR), Q-0- replicase mediated RNA amplification, rolling circle amplification (RCA), or strand displacement amplification (SDA).

[0188] Detection of the labelled amplicons can be performed by a detector to result in detection data. A detector is dependent on the general system with which the discrimination between amplicons of target sequences is performed but is also depending on the label that is present on a primer, such as a fluorescent or a phosphorescent label. In some embodiments, to discriminate between different target sequences in a sample, a difference in fluorescence spectrum of the respective corresponding amplicons is preferably used. In certain embodiments, at least one primer comprises a label, preferably a forward primer comprises a label. A label can be selected from a large group, amongst others comprising fluorescent and / or phosphorescent moieties such as dyes, chromophores, or enzymes, antigens, heavy metals, magnetic probes, phosphorescent moieties, radioactive labels, chemiluminescent moieties, or electrochemical detecting moieties. In certain embodiments, a label is a fluorescent or phosphorescent dye. Examples of such dyes include, but are not limited to, FAM, HEX, TET, JOE, NED, and (ET-) ROX.

[0189] By using different primer sets each containing a different label, the number of target sequences that can be discriminated in a sample and hence the number of target sequencesin a sample that can be detected can be increased by using additional labels. The maximum number of labels that can be used in one sample in a multiplex method is governed mostly by the limitations in the detection capabilities of the available detection platforms.

[0190] In certain embodiments, an amplification is performed using PCR with at least one forward and at least one reverse primer that are selective for a target sequence and not for any other sequence in the sample.

[0191] In certain embodiments, where a primer pair is used, at least one of either the forward or the reverse primer is labelled.

[0192] In certain embodiments, an amplification step is preceded or replaced by an assay for the detection of nucleic acids in samples.

[0193] In certain embodiments, amplicons are detected based on label, length, mobility, nucleotide sequence, mass, or a combination thereof.

[0194] In certain embodiments, amplicons are detected based on optical, electrochemical, or magnetic detection.

[0195] Reaction cartridge assemblies described herein provide at least some advantages over reaction cartridges and tubes typically used for reactions that require thermal cycling.

[0196] Using the present reaction cartridge assembly, a sample can be diluted to decrease the concentration of inhibitors or contaminants before being contacted with reaction reagents. Diluting inhibitors can help overcome the inhibitory effects of sample components that may be present along with nucleic acids. Further, a sample can be contacted with a nucleic acid lysis or extraction reagent before being contacted with the reaction reagents. Optionally, a sample can be contacted with a nucleic acid lysis or extraction reagent and then with a neutralization reagent before being contacted with the reaction reagents. The ability to perform these additional steps in the same assembly but before addition of reaction reagents is advantageous and further provides options for variation in pretreatment while using the same reaction cartridge. In contrast, a typical reaction vessel having fluids and reagents sealed into different compartments does not have this flexibility. Instead, all samples must go through the same workflow, whether or not they require all ofthe steps and fluids or reagents. In addition, the manufacturer may be required to hold inventory of reaction vessels with different configurations of fluids and reagents in their sealed compartments for multiple application - leading to increased costs.

[0197] The present reaction cartridge can retain a pre-determined, defined volume in the reaction reservoir after it is sealed with the shaft of the closure cap, such that an aliquot of the sample can be contacted with the reaction reagents without requiring the use of a metering device, pipette, or other liquid measuring device.

[0198] The reaction reagents can be conveniently stored in the closure cap in lyophilized form before being reconstituted by coming into contact with the sample in the reaction reservoir of the reaction cartridge.

[0199] Furthermore, embodiments of the present reaction cartridge assembly obviate the requirement for a multi-compartment cartridge that has valves, fluid channels, or other means for moving fluid from one compartment to another that are complicated to manufacture and are susceptible to breakage. Certain embodiments of present reaction cartridge assemblies described herein do not require a fluid by-pass or pressure release channel to release air under pressure, for example in a reaction reservoir.

[0200] In some embodiments, a step of identifying the contents of the reaction cartridge and / or one or more reaction parameters is performed. In certain embodiments, the step is performed using an identification tag. In some embodiments, an identification tag is disposed on a component of a reaction cartridge assembly (e.g., a reaction cartridge, a closure cap, or another component thereof).

[0201] Kits

[0202] The present disclosure includes, among other things, kits including one or more reaction cartridge assemblies for use in methods as provided herein, optionally in combination with instructions for use thereof in performing reactions (e.g., amplification reactions, e.g., isothermal amplification reactions, non-isothermal amplification reactions). In certain embodiments, a kit includes an instrument for detecting (e.g., optically, electrochemically, or magnetically detecting) the presence, absence, and / or amount ofamplicons using a reaction cartridge assembly (e.g., as described herein). In certain embodiments, a kit includes an instrument for detecting the presence, absence, or amount of a pathogen (e.g., a virus, a bacterium). In certain embodiments, a kit includes an instrument for detecting the presence, absence, or amount of a nucleic acid.

[0203] In some embodiments, an instrument for detecting nucleic acids includes a thermocycler to maintain and / or cycle temperature of a reaction cartridge assembly (e.g., as described herein). For example, a thermocycler can include a thermoelectric heater / cooler (e.g., a Peltier device) to heat and / or cool (e.g., rapidly heat and / or cool) a reaction cartridge to maintain and / or cycle the temperature at which an amplification reaction is taking place. In some embodiments, a thermoelectric heater / cooler can be used to cycle the temperature at which a reaction is taking place.

[0204] In certain embodiments, a kit includes one or more closure caps. In some embodiments, closure caps may contain reagents for performing a reaction (e.g., an amplification reaction) as described herein. In certain embodiments, closure caps contain reagents for amplification of one, two, three, four or more target sequences (e.g., DNA, RNA).

[0205] In certain embodiments, a kit includes a collection device for obtaining a sample comprising nucleic acids (e.g., DNA, RNA). In some embodiments, a collection device is used to obtain a sample swab (e.g., a buccal swab, a swab from an environmental source, a forensic sample, etc.). In some embodiments, a collection device is a sterile collection device (e.g., a sterile swab). In some embodiments, a sterile collection device is substantially free from nucleic acids (e.g., DNA, RNA) and / or other contaminants (e.g., pathogens, inhibitors, etc.) which interfere with amplification reactions as described herein. In some embodiments, a collection device can release a sample (e.g., nucleic acids from a sample, pathogens, etc.) into a reaction cartridge. In some embodiments, a collection device is contacted with a reagent (e.g., a liquid reagent) to release components of interest (e.g., nucleic acids, pathogens, cells, etc.) from the collection device.

[0206] In certain embodiments, a kit may include one or more reagents used in a reaction (e.g., an isothermal amplification reaction, a non-isothermal amplification reaction). Incertain embodiments, a kit includes one or more nucleic acid extraction reagents. In certain embodiments, a nucleic acid extraction reagent is a detergent (e.g., Triton X-100, Triton X- 114, Tween 20, Tween 80, NP-40, sodium dodecyl sulphate (SDS), cetyltrimethylammonium bromide (CTAB)), a hydroxide (e.g., sodium hydroxide), an alkaline solution, or a combination thereof.

[0207] In certain embodiments, a kit includes a neutralizing fluid. In certain embodiments, a neutralizing fluid contains a buffering agent which neutralizes a nucleic acid extraction reagent.

[0208] In certain embodiments, a kit includes lyophilized reagents for use in a reaction.

[0209] In certain embodiments, a kit includes software for analyzing the presence, absence, and / or amount of, for example, a pathogen, an amplicon, or nucleic acids in a sample.

[0210] ALTERNATIVE EMBODIMENTS

[0211] Embodiment 1. A reaction cartridge assembly comprising: a. an upper receiving reservoir defined by a housing wall having a top edge surrounding an upper opening and a bottom edge surrounding a lower opening; b. a reaction reservoir comprising an upper open region that is fixedly or removably attached to the upper receiving reservoir at the lower opening to provide fluid communication between the upper receiving reservoir and the reaction reservoir; and c. a closure cap comprising a top wall, a cap body depending therefrom that is sized and configured to fit in the upper receiving reservoir, and a cap component for sealing the reaction reservoir at the upper open region and / or for transferring materials into the reaction reservoir when the cap body is positioned in the upper receiving reservoir.

[0212] Embodiment 2. The reaction cartridge assembly of Embodiment 1, wherein the cap component is a hollow shaft extending through the cap body from the top wall and terminating at an aperture in a bottom surface of the cap body.

[0213] Embodiment 3. The reaction cartridge assembly of Embodiment 2, wherein the hollow shaft terminates at the aperture, which is positioned at a tip of the bottom surface that is configured to seal the upper open region of the reaction reservoir.

[0214] Embodiment 4. The reaction cartridge assembly of Embodiment 2 or 3, wherein one or more reagents are contained in the hollow shaft or affixed to an interior surface of the hollow shaft.

[0215] Embodiment 5. The reaction cartridge assembly of Embodiment 4, wherein the one or more reagents are lyophilized, optionally wherein the lyophilized reagents are in the form of a lyophilized bead.

[0216] Embodiment 6. The reaction cartridge assembly of any one of Embodiments 2 to 5, wherein the cap component further comprises a plunger that is displaceable within the hollow shaft.

[0217] Embodiment 7. The reaction cartridge assembly of any one of Embodiments 1 to 6, wherein the reaction cartridge comprises a temporary and removable barrier between the receiving reservoir and the reaction reservoir, optionally wherein the barrier is a plastic film.

[0218] Embodiment 8. The reaction cartridge assembly of any one of Embodiments 1 to 7, wherein the reaction reservoir comprises: a. a bottom wall; and b.four planar side walls joined to the bottom portion.

[0219] Embodiment 9. The reaction cartridge assembly of Embodiment 8, wherein the bottom wall comprises a closed, molded lens and one of the four side walls comprises or consists of a flat, optically transmissive side wall.

[0220] Embodiment 10. The reaction cartridge assembly of Embodiment 9, wherein the optically transmissive side wall is angularly offset from the closed, molded lens by an angle of from approximately 90° to approximately 120°, preferably about 90°.

[0221] Embodiment 11. The reaction cartridge assembly of any one of Embodiments 1 to 10, wherein the reaction reservoir is formed of a thermally stable plastic.

[0222] Embodiment 12. A method for performing a reaction, said method comprising the steps of: a. providing a reaction cartridge comprising a receiving reservoir and a reaction reservoir; b. adding a fluid into the receiving reservoir of the reaction cartridge; c. combining a sample with the fluid in the receiving reservoir to form a fluid mixture; d.inserting a cap closure into the receiving reservoir to seal the reaction cartridge and to push the fluid mixture from the receiving reservoir into the reaction reservoir and to seal the reaction reservoir; and e. allowing the reaction to proceed in the reaction reservoir.

[0223] Embodiment 13. The method of Embodiment 12, further comprising introducing additional regents to the reaction reservoir prior to or during the step of allowing the reaction to proceed.

[0224] Embodiment 14. The method of Embodiment 12 or 13, wherein the step of allowing the reaction to proceed comprises performing at least one thermal cycle by sequentially heating the reaction reservoir to a first temperature, maintaining the first temperature for a first preset time, cooling the reaction reservoir to a second temperature and maintaining the second temperature for a second preset time.

[0225] Embodiment 15. The method of any one of Embodiments 12 to 14, wherein the sample is a sample swab and the step of combining sample with the fluid in the receiving reservoir comprises swirling the swab in the fluid for a sufficient time to release the sample components of interest from the swab into the fluid.

[0226] Embodiment 16. The method of any one of Embodiments 12 to 15, wherein the sample is tissue, bodily fluid such as sputum, semen, blood, urine, and / or faeces, wastewater, a forensic sample or an environmental sample.

[0227] Embodiment 17. The method of any one of Embodiments 12 to 16, wherein the fluid in the receiving reservoir is an extraction reagent for lysing cells and / or extracting nucleic acid from the sample.

[0228] Embodiment 18. The method of any one of Embodiments 12 to 17, wherein the reaction is a nucleic acid amplification reaction.

[0229] Embodiment 19. The method of any one of Embodiments 12 to 17, wherein the method is performed using the reaction cartridge assembly of any one of Embodiments 1 to 11.

[0230] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill of those skilled in the art to which the disclosed technologies pertain and are herein incorporated by reference to the same extent as if each individual publication, patent, or patent applications was specifically and individually indicated to be incorporated by reference.

[0231] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

CLAIMSWE CLAIM:

1. A reaction cartridge assembly comprising: a. an upper receiving reservoir defined by a housing wall having a top edge surrounding an upper opening and a bottom edge surrounding a lower opening; b. a reaction reservoir comprising an upper open region that is fixedly or removably attached to the upper receiving reservoir at the lower opening to provide fluid communication between the upper receiving reservoir and the reaction reservoir; and c. a closure cap comprising a top wall, a cap body depending therefrom that is sized and configured to fit in the upper receiving reservoir, and a cap component for sealing the reaction reservoir at the upper open region and / or for transferring materials into the reaction reservoir when the cap body is positioned in the upper receiving reservoir.

2. The reaction cartridge assembly of claim 1, wherein the cap component is a hollow shaft extending through the cap body from the top wall and terminating at an aperture in a bottom surface of the cap body.

3. The reaction cartridge assembly of claim 2, wherein the hollow shaft terminates at the aperture, which is positioned at a tip of the bottom surface that is configured to seal the upper open region of the reaction reservoir.

4. The reaction cartridge assembly of claim 2 or 3, wherein one or more reagents are contained in the hollow shaft or affixed to an interior surface of the hollow shaft.

5. The reaction cartridge assembly of claim 4, wherein the one or more reagents are lyophilized, optionally wherein the lyophilized reagents are in the form of a lyophilized bead.

6. The reaction cartridge assembly of any one of claims 2 to 5, wherein the cap component further comprises a plunger that is displaceable within the hollow shaft.

7. The reaction cartridge assembly of claim 6, wherein the plunger is disposed entirely within the hollow shaft and below the top wall of the closure cap.

8. The reaction cartridge assembly of any one of claims 1 to 7, wherein the reaction cartridge comprises a temporary and removable barrier between the receiving reservoir and the reaction reservoir, optionally wherein the barrier is a plastic film.

9. The reaction cartridge assembly of any one of claims 2 to 8, wherein the cap component comprises a displaceable retaining member disposed within the hollow shaft.

10. The reaction cartridge assembly of claim 9, wherein a wall of the hollow shaft is configured to receivingly mate with the displaceable retaining member when the displaceable retaining member is displaced by the plunger.

11. The reaction cartridge assembly of any one of claims 2 to 10, wherein the cap component comprises a channel extending along a wall of the hollow shaft from the top wall of the closure cap and terminating at or before the aperture in the bottom surface of the cap body, optionally wherein the channel is a grooved channel.

12. The reaction cartridge assembly of any one of claims 1 to 11, wherein the reaction reservoir comprises: a. a bottom wall; and b. four planar side walls joined to the bottom portion.

13. The reaction cartridge assembly of claim 12, wherein the bottom wall comprises a closed, molded lens and one of the four side walls comprises or consists of a flat, optically transmissive side wall.

14. The reaction cartridge assembly of claim 13, wherein the optically transmissive side wall is angularly offset from the closed, molded lens by an angle of from approximately 90° to approximately 120°, preferably about 90°.

15. The reaction cartridge assembly of any one of claims 1 to 14, wherein the reaction reservoir is formed of a thermally stable plastic.

16. The reaction cartridge assembly of any one of claims 1 to 15, wherein the reaction cartridge assembly comprises one or more air escape vents formed when the cap body and the upper reservoir are assembled and wherein the one or more air escape vents are configured to allow air to escape from the reaction reservoir into the upper receiving reservoir.

17. The reaction cartridge assembly of any one of claims 6 to 16, wherein the reaction cartridge assembly comprises a closure cap insert comprising a top wall and an insert body depending therefrom, wherein the insert body is sized and configured to fit in the hollow shaft of the closure cap for depressing the plunger through the hollow shaft towards the reaction reservoir.

18. The reaction cartridge assembly of any one of claims 1 to 17, wherein the upper receiving reservoir comprises an interconnection mechanism to securely hold the closure cap in the upper receiving reservoir.

19. The reaction cartridge assembly of claim 18, wherein the interconnection mechanism comprises a protrusion from the housing wall of the upper receiving reservoir which is configured to securedly mate with the closure cap.

20. The reaction cartridge assembly of any one of claims 1 to 19, wherein the reaction cartridge assembly comprises an identification tag, optionally, wherein the identification tag is configured to communicate wirelessly with a receiving device.

21. The reaction cartridge assembly of claim 20, wherein the identification tag is disposed on the closure cap, optionally wherein the identification tag is disposed on the top wall of the closure cap.

22. The reaction cartridge assembly of claim 20 or 21, wherein the identification tag is an RFID (radio frequency identification) tag.

23. The reaction cartridge assembly of claim 22, wherein the RFID tag is an NFC (near field communication) tag.

24. A method for performing a reaction, said method comprising the steps of: a. providing a reaction cartridge comprising a receiving reservoir and a reaction reservoir; b. adding a fluid into the receiving reservoir of the reaction cartridge; c. combining a sample with the fluid in the receiving reservoir to form a fluid mixture; d. inserting a closure cap into the receiving reservoir to seal the reaction cartridge and to push the fluid mixture from the receiving reservoir into the reaction reservoir and to seal the reaction reservoir; and e. allowing the reaction to proceed in the reaction reservoir.

25. The method of claim 24, further comprising introducing additional regents to the reaction reservoir prior to or during the step of allowing the reaction to proceed.

26. The method of claim 24 or 25, wherein the step of allowing the reaction to proceed comprises performing at least one thermal cycle by sequentially heating the reaction reservoir to a first temperature, maintaining the first temperature for a first preset time, cooling the reaction reservoir to a second temperature and maintaining the second temperature for a second preset time.

27. The method of any one of claims 24 to 26, wherein the method comprises obtaining, by a collection device, the sample, optionally wherein the collection device is a swab.

28. The method of any one of claims 24 to 27, wherein the sample is a sample swab and the step of combining sample with the fluid in the receiving reservoir comprises swirling thesample swab in the fluid for a sufficient time to release the sample components of interest from the swab into the fluid.

29. The method of any one of claims 24 to 28, wherein the sample is tissue, bodily fluid, wastewater, a forensic sample, or an environmental sample.

30. The method of any one of claims 24 to 29, wherein the sample is a body fluid and wherein the body fluid is a member from the group consisting of sputum, semen, blood, urine, vaginal fluids and / or secretions, and faeces.

31. The method of any one of claims 24 to 30, wherein the fluid in the receiving reservoir is an extraction reagent for lysing cells and / or extracting nucleic acid from the sample.

32. The method of any one of claims 24 to 31, wherein the reaction is a nucleic acid amplification reaction.

33. The method of claim 32, wherein the nucleic acid amplification reaction is a nonisothermal amplification reaction.

34. The method of any one of claims 24 to 33, wherein the method comprises identifying contents of the reaction cartridge and / or one or more reaction parameters using an identification tag.

35. The method of claim 34, wherein the identification tag is disposed on the reaction cartridge or the closure cap.

36. The method of any one of claims 24 to 35, wherein the method is performed using the reaction cartridge assembly of any one of claim 1 to 23.

37. A kit comprising a reaction cartridge assembly (e.g., of any one of claims 1 to 23), optionally comprising instructions for performing a reaction.

38. The kit of claim 37, wherein the kit comprises an instrument for detecting nucleic acids.

39. The kit of claim 38, wherein the instrument optically, electrochemically, or magnetically detects nucleic acids.

40. The kit of claim 37-39, wherein the kit comprises a thermocycler, optionally, wherein the thermocycler comprises a thermoelectric heater / cooler.

41. The kit of claim 40, wherein the instrument for detecting nucleic acids is integrated with the thermocycler.

42. The kit of any one of claims 38-41, wherein the instrument comprises a sensor for detecting an identification tag disposed on a component of the reaction cartridge assembly.

43. The kit of claim 37-42, wherein the kit includes a collection device, optionally wherein the collection device is a swab.

44. The kit of claim 37-43, wherein the kit comprises one or more nucleic acid extraction reagents.

45. The kit of claim 44, wherein the one or more nucleic acid extraction reagents comprise a nucleic acid extraction reagent is a detergent, a hydroxide, an alkaline solution, or a combination thereof.

46. The kit of claim of any one of claims 37-45, wherein the kit comprises a neutralizing fluid.

47. The kit of claim 46, wherein the neutralizing fluid contains a buffering agent which neutralizes a nucleic acid extraction reagent.

48. The kit of claim of any one of claims 37-47, wherein the kit comprises software for analyzing the presence, absence, and / or amount of nucleic acids in a sample.

49. The kit of claim of any one of claims 37-48, wherein the kit comprises software for analyzing the presence, absence, and / or amount of a pathogen in a sample.

50. A closure cap comprising: a top wall, a cap body depending therefrom, and a cap component for sealing a reaction reservoir at an upper open region and / or for transferring materials into the reaction reservoir when the cap body is positioned in a receiving reservoir.

51. The closure cap of claim 50, wherein the cap component is a hollow shaft extending through the cap body from the top wall and terminating at an aperture in a bottom surface of the cap body.

52. The closure cap of claim 51, wherein the hollow shaft terminates at the aperture, which is positioned at a tip of the bottom surface that is configured to seal the upper open region of the reaction reservoir.

53. The closure cap of claim 51 or 52, wherein one or more reagents are contained in the hollow shaft or affixed to an interior surface of the hollow shaft.

54. The closure cap of claim 53, wherein the one or more reagents are lyophilized, optionally wherein the lyophilized reagents are in the form of a lyophilized bead.

55. The closure cap of any one of claims 51 to 54, wherein the cap component further comprises a plunger that is displaceable within the hollow shaft.

56. The closure cap of claim 55, wherein the plunger is disposed entirely within the hollow shaft and below the top wall of the closure cap.

57. The closure cap of any one of claims 51 to 56, wherein the cap component comprises a displaceable retaining member disposed within the hollow shaft.

58. The closure cap of claim 57, wherein the displaceable retaining member is hingedly connected to an interior surface of the hollow shaft.

59. The closure cap of any one of claims 51 to 58, wherein the cap component comprises a channel extending along a wall of the hollow shaft from the top wall of the closure cap and terminating at or before the aperture in the bottom surface of the cap body, optionally wherein the channel is a grooved channel.

60. The closure cap of any one of claims 51 to 59, wherein the cap body is sized and configured to fit in a receiving reservoir, optionally wherein the receiving reservoir is an upper receiving reservoir of the reaction reservoir.

61. The closure cap of any one of claim 51 to 60, wherein the closure cap comprises an identification tag.

62. The closure cap of claim 61, wherein the identification tag is an RFID tag, optionally, wherein the RFID (radio frequency identification) tag is an NFC (near field communication) tag.

63. A reaction reservoir comprising: a. a bottom wall; and b. four planar side walls joined to the bottom portion.

64. The reaction reservoir of claim 63, wherein the bottom wall comprises a closed, molded lens and one of the four side walls comprises or consists of a flat, optically transmissive side wall.

65. The reaction reservoir of claim 63 or 64, wherein the optically transmissive side wall is angularly offset from the closed, molded lens by an angle of from approximately 90° to approximately 120°, preferably about 90°.

66. The reaction reservoir of claims 63 to 65, wherein the reaction reservoir is formed of a thermally stable plastic.

67. The reaction reservoir of claims 63 to 66, wherein the reaction reservoir comprises an identification tag.

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