Hand-held device for pre-symptomatic detection of infectious disease and methods of use thereof

Abstract

Described herein is a non-powered, handheld device for the amplification and detection of nucleic acids for early pathogen-agnostic detection of infectious disease.

Description

[0001] HAND-HELD DEVICE FOR PRE-SYMPTOMATIC DETECTION OF INFECTIOUS DISEASE AND METHODS OF USE THEREOF

[0002] STATEMENT OF GOVERNMENT INTEREST

[0003] This invention was made with government support under project / grant number MCDC20 16-004 awarded by the Defense Threat Reduction Agency (DTRA). The government has certain rights in the invention.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 687,021, filed August 26, 2024. The entire specification and figures of the above-referenced application are hereby incorporated, in their entirety by reference.

[0006] TECHNICAL FIELD

[0007] The present disclosure is directed to the field of nucleic amplification and detection, and in particular systems, methods, and apparatus directed to a non-powered, handheld device for amplification and detection of nucleic acids which can be used to for early pathogen-agnostic detection of infectious disease.

[0008] BACKGROUND

[0009] Early detection of pathogenic infection is vital for proper treatment and positive clinical outcomes. However, infected individuals may remain asymptomatic for several days postinfection while actively transmitting the pathogen to others. Traditional pathogen detection systems are often not effective at detecting the infection until after the onset of symptoms. Traditional pathogen testing includes serology or antibody-based tests, bacterial / viral / fungal growth cultures, and nucleic acid-based detection such as PCR (polymerase chain reaction). Such traditional tests are often time and labor intensive and are only effective after a patient has begun to show symptoms of the infection. Additionally, traditional diagnostic tests require clinical suspicion for a specific pathogen, expensive laboratory equipment, trained personnel, and have increased upstream and end-user costs.

[0010] For example, in a typical infection course exposure to an unknown pathogen occurs at day zero and then progresses through subsequent clinical stages of infection. As the pathogen replicates within the infected person, standard diagnostic tests are typically designed to work after the onset of symptoms, when people know there is something wrong and seek healthcare and diagnosis. However, at that point the person may have been contagious to others for days or even weeks. The opportunity to implement early quarantine and limit destructive downstream effects of unimpeded pathogen transmission has passed. This time delay to diagnosis can result in poorer patient outcomes and ongoing disease transmission before patients know they are contagious.

[0011] As opposed to the specialized, and later developing adaptive immune response, a host’s first line of defense against pathogenic microorganisms is the “innate immune” response. The body’s innate immunity is a self-amplifying and non-specific physiological response that occurs within hours of infection. As such, the ability to detect the presence of molecules produced by a host’s innate immune response may provide the ability to rapidly detect infection at the earliest stages while a patient is still asymptomatic. Such advancement would allow for more effective quarantine protocols, as well as improved treatment and clinical outcomes. As such, there exists a long-felt need for a diagnostic device for early pathogen-agnostic detection of infectious disease. As described below, the diagnostic device of the disclosure is adapted to detect RNA biomarkers that are upregulated in biological samples, such as saliva, upon exposure to a pathogen to provide early, pathogen-agnostic identification of infected individuals.

[0012] Additionally, detection of nucleic acids in other samples, such as environmental and pharmaceutical samples can have both clinical as well as industrial importance. However, again the time and expense required to retrieve, transport and process samples can be prohibitive. As a result, there is a need for a simple, inexpensive, portable, and technically reliable device for the amplification and detection and nucleic acids.

[0013] SUMMARY OF THE INVENTION

[0014] One aspect of the disclosure includes systems and methods for a nucleic acid amplification and detection device, also referred to herein as the assembly or device of the disclosure. In a preferred aspect, the present disclosure is directed to novel systems, methods, and apparatus for the amplification and detection of nucleic acids in a sample. In one preferred aspect, the present disclosure is embodied by novel systems, methods, and apparatus for early pathogen-agnostic detection of infectious disease.

[0015] The disclosure may further include systems, methods, and compositions for the amplification and detection of nucleic acids in a sample, such as an environmental, biological or pharmaceutical sample containing a quantity of nucleic acids, such as DNA or preferably RNA. The amplification and detection of nucleic acids includes nucleic acids containing somatic or genetic variant, nucleic acids that have been exposed to radiation and / or exhibit radiation induced mutations / darn age, detection of bacterial or viral pathogen nucleic acids in a sample, detection of nucleic acids that include genetic biomarkers, variants, or mutations indicative of a disease state or a predisposition to a disease state, detection of nucleic acids from a biological sample, such as preferably human or animal sample.

[0016] Other aspects of the present disclosure include a nucleic acid amplification and detection assembly adapted to amplify target nucleic acids in a sample, such as an environmental, biological, pharmaceutical, and / or diagnostic sample and the like. As described by Sawyer et al., in PCT / US2020 / 049290 (incorporated herein by reference), exemplary nucleotide transcript biomarkers, and preferably coding or non-coding RNA oligonucleotides, also referred to herein as RNA biomarkers, produced by a subject’s innate immune system in response to a pathogen or infection can be present in a biological sample, such as saliva. Notably, specific target RNA biomarkers produced by a patient's immune response (generally innate immune response or any other cellular pathway upregulated upon infection) are found in saliva may be indicative of early infection. As a result, one embodiment of the inventive technology includes systems, methods, and compositions for the amplification and detection of these target RNA biomarkers, which may act as indicators for early-infection in a subject. However, as noted above, target RNA transcript biomarkers present in a typical fluid sample provided by, in this embodiment a human subject, are generally present at low concentrations and require amplification to be detected. To overcome this physical limitation, the present disclosure includes systems, methods and apparatus for the isothermal amplification of the same.

[0017] In one preferred aspect, the present disclosure is directed to methods and apparatus for the sequential amplification and detection of one or more target nucleic acids in a sample. In this preferred aspect, a sample collector can be configured to collect a sample containing one or more nucleic acids to be amplified, which can be deposited into a reaction cartridge having a sample processing subassembly in fluid communication with an amplification subassembly. A heater subassembly can be positioned within the reaction cartridge and further be thermally coupled with the amplification subassembly. In this configuration, engagement of an injection assembly introduces a dilution solution into the sample processing subassembly and an activation solution to the heater subassembly, such introduction being sequential or simultaneous. The target nucleic acids are isolated and amplified within the amplification subassembly. The amplified nucleic acids, also referred to as the amplicon(s), are transported via a fluid transfer subassembly to a lateral flow assay adapted to receive and detect the amplified nucleic acids. In this manner, the collecting, isolation, processing, amplification, and detection of the target nucleic acids is performed by a hand-held and self-contained device.

[0018] In one aspect the disclosure describes a process to amplify and detect one or more nucleic acid oligonucleotides present in a sample. In a preferred aspect, a biological sample, such as a saliva sample, is collected by a sample collector and inserted into a reaction cartridge. The sample collector locks into place with the outer cover of the reaction cartridge to prevent users from removing the sample collector after insertion and to provide audible and tactile feedback to the user that the step is complete. Next, a user activates an injector by depressing a plunger responsive to a fluid reservoir having a first and second barrel. This plunger can also be locked to the outer cover of the reaction cartridge to prevent users from releasing the plunger. The engagement of the injector releases a sample dilution solution to flush the sample off the sample collector, dilute the sample with gentle detergents in preparation for the amplification reaction, and provide pressure to process the sample through a filter stack to remove cellular debris, remove amplification inhibitors, and other large particles. After filtration, a fractionator, shown in the figures as a fractionation pad, sequesters the initial fluid from the filter stack, which is generally low in sample concentration. The remaining sample is routed past the fractionator through an inline mixing channel to the amplification subassembly.

[0019] The action of depressing the plunger additionally releases a heater activation solution through a separate channel to activate an integrated chemical heater. In a preferred aspect, a sodium chloride activation solution saturates an exothermic fuel of magnesium-iron alloyed powder (Mg- Fe) initiating an exothermic reaction. A phase change material (PCM) that transitions from solid to liquid is positioned in between the exothermic fuel and one or more amplification chambers in the amplification subassembly so as to transmit and maintain a consistent and optimal temperature for the isothermal amplification reaction.

[0020] Upon delivery to the amplification subassembly, the diluted sample is routed to a plurality of separate amplification chambers containing lyophilized amplification reagents. One or more porous frits allow air to be flushed from the amplification chambers into vent channels but do not allow liquid to pass further. When the frit halt the flow of the diluted sample, a flow restrictor opens to force any excess diluted sample to a waste chamber, which preferably includes a waterproof vent to contain the unused sample. The integrated chemical heater subassembly provides thermal control for reverse transcription and isothermal amplification of one or more unique nucleic acid targets, and preferably RNA biomarkers, using reverse transcription recombinase polymerase amplification (RT-RPA) and 5’ modified primers. A control biomarker present at consistent levels in all samples is included as one of the targeted RNA biomarkers to monitor successful sample processing and amplification.

[0021] The user allows the amplification reaction to proceed for 15-20 minutes, then completes a simple user-initiated step to engage an amplicon transfer subassembly configured to puncture a barrier between the amplification chamber(s) and a lateral flow assay (LFA). The user next engages a fluid transfer subassembly via a push-button responsive to a fluid container causing the release of a transfer solution buffer to dilute the amplification reaction and deliver the amplified products for readout on the LFA.

[0022] The diluted amplification products are delivered to a LFA enclosed in the reaction cartridge. In a preferred embodiment, primers used in the amplification reaction, and preferably a RT-RPA reaction contain 5’ modifications for capture and detection of RNA biomarkers on the lateral flow strip. For example, a 5’ Fluorescein isothiocyanate (FITC) modification can be included on forward primers, a 5’ Biotin (Bio) is on the reverse primer of the control biomarker, and 5’ digoxigenin (Dig) can be incorporated on the reverse primer of a test biomarker. If the control and infection biomarkers were present in the original sample, gold-conjugated anti-FITC can bind the amplified products before being immobilized at the control (poly streptavidin) and test (anti-Dig) lines of the LFA for visual interpretation of results. Additionally, an excess gold line monitors the flow of gold on the strip and captures gold-conjugated goat anti-IgY with chicken IgY. The LFA is preferably allowed to resolve for 10-15 minutes prior to results readout.

[0023] Additional aspects of the invention may include one or more of the preferred embodiments set forth in the claims. Still further aspects of the invention may be evidenced from the specification, claims, and figures provided below

[0024] BRIEF DESCRIPTION OF THE FIGURES

[0025] Figure l is a front perspective view of a nucleic acid amplification and detection assembly in one embodiment thereof;

[0026] Figure 2 is a front view of a nucleic acid amplification and detection assembly with a sample collector positioned prior to insertion into a reaction cartridge in one embodiment thereof; Figure 3 is a front view of a nucleic acid amplification and detection assembly with a sample collector inserted into a reaction cartridge and an injection subassembly engaged in one embodiment thereof;

[0027] Figure 4A-B is a (A) front cross section view of a reaction cartridge having a fluid transfer subassembly and heater subassembly integrated into the cartridge and further responsive to an injection subassembly in one embodiment thereof; is a (B) cross section view of the back portion of a reaction cartridge having a fluid transfer subassembly and heater subassembly integrated into the cartridge and further responsive to an injection subassembly in one embodiment thereof;

[0028] Figure 5A-B is a (A) rear cross-section view of a reaction cartridge having a sample processing subassembly in communication with an amplification subassembly integrated into the cartridge and further responsive to an injection subassembly in one embodiment thereof; and is an alternative (B) rear cross-section view of a reaction cartridge having a sample processing subassembly in communication with an amplification subassembly integrated into the cartridge and further responsive to an injection subassembly in one embodiment thereof;

[0029] Figure 6 is a rear cross-section view of a reaction cartridge having a sample processing subassembly in communication with an amplification subassembly integrated into the cartridge and further responsive to an injection subassembly and a sample collector positioned within the cartridge in one embodiment thereof;

[0030] Figure 7 is an expanded view of an outer cover and reaction cartridge in relation to a fluid transfer subassembly having a spring-loaded push button configured to compress and rupture a fluid container containing a transfer solution in one embodiment thereof;

[0031] Figure 8 is an expanded view of an outer cover securing a reaction cartridge in relation to an injection subassembly configured to deliver a dilution and activation solution to the cartridge in one embodiment thereof;

[0032] Figure 9 is a front cross-section view of a reaction cartridge positioned in relation to a support film in one embodiment thereof;

[0033] Figure 10 is an expanded view of a fluid transfer subassembly positioned in relation to a reaction cartridge in one embodiment thereof;

[0034] Figure 11 is a detailed view of an amplification subassembly having a plurality of reaction chambers connected to a sample divider and venting paths in one embodiment thereof; Figure 12 is an expanded detailed view of an amplicon transfer subassembly in fluid communication with a LFA assay and fluid transfer subassembly in one embodiment thereof;

[0035] Figure 13 is an expanded view of a heater subassembly in relation to a reaction cartridge in one embodiment thereof;

[0036] Figure 14 is an expanded view of a heater subassembly in relation to a heating chamber integral with the reaction cartridge in one embodiment thereof;

[0037] Figure 15 is an expanded view of a lateral flow assay in relation to a reaction cartridge in one embodiment thereof;

[0038] Figure 16 is a perspective view of the rear surface of a reaction cartridge in relation to a support fdm forming the subassembly channels in one embodiment thereof;

[0039] Figure 17 is an expanded view of an amplicon transfer subassembly in relation to a reaction cartridge in one embodiment thereof;

[0040] Figure 18 is an expanded view of an amplicon transfer subassembly in relation to a reaction cartridge securing a heater subassembly in one embodiment thereof;

[0041] Figure 19 is a perspective view of a plurality of amplification reagents and venting frit in relation to the amplification chambers integrally positioned within the reaction cartridge in one embodiment thereof;

[0042] Figure 20 is an expanded view of a filter stack having a plurality of filters positioned adjacent to one another and a fractionator secured within the reaction cartridge in one embodiment thereof;

[0043] Figure 21 shows an expanded view of a lateral flow assay assembly in relation to a reaction cartridge in one embodiment thereof;

[0044] Figure 22 is an expanded view of an isolated sample collector positioned adjacent to a sample port of the reaction cartridge in one embodiment thereof;

[0045] Figure 23 is an expanded perspective view of an isolated injection assembly in one embodiment thereof;

[0046] Figure 24A-B shows a lateral flow strip design. (A) Schematic of isothermal amplification amplicons with Biotin, Dig, and FITC 5’ modifications for detection on the lateral flow strip; (B) shows a schematic of a lateral flow strip, with test, control, and excess gold binding sites labeled, along with conjugate and absorbent pads in one embodiment thereof. Figure 25A-B shows (A) a heater subassembly in relation to a reaction cartridge adjacent to (B) a cross-sectional view of a heater subassembly in one embodiment thereof.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] The present disclosure includes a nucleic acid amplification and detection assembly (100), (also generally referred to herein as the device or assembly (100), or device or assembly of the invention (100)), which is adapted to collect a sample, and preferably a saliva or other biological sample from a human subject, which may be further processed and filtered to release the nucleic acids from the sample, which then undergoes isothermal amplification followed by detection via a lateral flow assay.

[0049] The nucleic acid amplification and detection assembly (100) includes a sample collector (200) configured to collect a sample containing a quantity of nucleic acids. In the preferred embodiment shown in Figure 22, the sample collector (200) includes a collection handle (202) securing a sample swab (206), for example within a collector channel (not shown), adapted to allow a subject to self-collect a biological, environmental, or other specimen, such as preferably a saliva sample. The sample swab (206) of the invention includes an absorbent material adapted to collect a sample. For example, in one embodiment the sample swab (206) can be inserted into a subject’s mouth and capture a saliva sample sufficient for processing and later isothermal amplification and detection as further detailed below.

[0050] Specifically referring to Figure 22, the sample collector (200) can be inserted into the reaction cartridge (400) of the assembly (100) as detailed below and deposit a biological, or other sample therewith for initial processing. The sample collector (200) may include one or more extended surfaces securing one or more ring seals (210) that generate a hermetic seal when the sample collector (200) is inserted into the reaction cartridge (400) of the assembly (100) and further prevents the sample from flowing back out of the reaction cartridge (400). Moreover, the ring seals (210) further generate a pressure force from the downward movement of the sample collector (200) which can assist in the introduction of the sample into the device, and in particular the sample processing and amplification assemblies (402, 414) as described below.

[0051] As noted above, the sample collector (200) can be inserted into the reaction cartridge (400) of the assembly (100) via a sample port (218) positioned on the top surface of the cartridge (400). The sample collector (200) can be locked into position after insertion through the sample port (218) to prevent backflow of the sample and ensure sufficient sample material can be used for the subsequent processing and amplification steps. As shown in Figure 3, the sample collector (200) can include a twist lock mechanism including an extended tab (204) that can be secured by an outer cover (102) positioned over the reaction cartridge (400). In this preferred embodiment, the sample collector (200) is inserted into the reaction cartridge (400) via a sample port (218). In this initial configuration, the sample collector (200) can be twisted until the extended tab (204) engages the handle lock (110) thereby securing the sample collector (200) in place and preventing its removal from the reaction cartridge (400) until the extended tab (204) is disengaged from the handle lock (110).

[0052] The nucleic acid amplification and detection assembly (100) includes an injector assembly (300), also referred to as an injector (300). In a preferred embodiment, the injector assembly (300) is configured to introduce, preferably simultaneously, but in some embodiments sequentially a dilution solution (308) that is directed to the sample processing subassembly (402), and an activation solution (310) that is directed to a heater subassembly as described below (500). In a preferred embodiment, the dilution solution (308) can include one or more components for processing the sample containing the target nucleic acid(s). For example, a dilution solution (308) can include one or more buffers and detergents, as well as a lysis buffer solution configured to lyse cells present in the sample thereby releasing the nucleic acids into the solution for later processing. Additional embodiments can include a blocking solution to prevent generalized binding of the downstream lateral flow assay (800). The dilution solution (308) can further include a chelating agent, such as EGTA, and magnesium acetate (MgOAc). In this embodiment, the magnesium in the dilution solution (308) is taken up by the sample and such that the magnesium acetate prevents the inactivation of the downstream isothermal amplification reaction. Notably, descriptions and methods of performing isothermal amplification, including components necessary for isothermal amplification including various primers, and RNA biomarkers, and other isothermal amplification systems, generally referred to herein as amplification reagents, are described by Sawyer et al, in U.S. Patent Application No. 17 / 686,387 and are herein incorporated by reference.) Further example dilution solution (308) compositions are provided by Meyerson et al, in PCT / US2023 / 082792, which is incorporated herein by reference. In some embodiments, the term amplification reagents expressly includes primers for nucleic acid amplification.

[0053] Referring to Figure 23, the injector assembly (300) includes a fluid reservoir responsive to a barrel (306), which in a preferred embodiment includes two separate fluid compartments, specifically a first injection barrel (306A) and second first injection barrel (306B). Both the first and second injection barrels (306A-B) are responsive to a plunger (302) secured to a piston (304) that forms a seal between the barrel / plunger interface. Referring to Figure 23, the first and second injection barrels (306A-B) are in fluid communication with the reaction cartridge (400) however they are sealed at their terminal ends by a seal (314) that prevents the solution(s) from entering the reaction cartridge (400) until the injector assembly (300) is engaged. As shown in Figure 20, a puncture (316) is positioned below each of the first and second injection barrels (306A-B). In this embodiment, the punctures (316) are formed by lancet structures integrally formed by the reaction cartridge (400), however this is not limiting, as separable punctures secured to the reaction cartridge (400) or injector assembly (300) can further be encompassed by the current disclosure.

[0054] As noted above, once the sample collector (200) has been secured to the reaction cartridge (400) and the sample swab (206) positioned within a sample collection chamber (404) optionally having a pressure release vent (216) positioned above a pressure sensitive adhesive (216A), the sample collector (200) forming a seal with the reaction cartridge (400), the injector assembly (300) is preferably engaged. In this embodiment, the plunger (302) can be manually depressed causing the first and second injection barrels (306A-B) to traverse downward. This downward force overcomes the resistance of a compressible gasket (316) causing the seal (314) positioned at the terminal ends of the first and second injection barrels (306A-B) to be pierced by the punctures. This action allowing a controlled, and in this case simultaneous release of the dilution solution (308) from the first injection barrel (306A) and the activation solution (310) from the second injection barrel (306B). As described below, both solutions (308, 310) are not in fluid communication, but maintained separately such that they do not come into contact during the engagement of the injector assembly (300). While a preferred embodiment includes the simultaneous release of the both solutions (308, 310), in alternative embodiments, the sample collector (200) can include multiple independent plungers (302) responsive to the individual and injection barrels (306A-B).

[0055] As noted above, the reaction cartridge (400) of the disclosure preferably includes an enclosed system allowing the sequential processing and amplification of nucleic acids present in a sample. In the preferred embodiment shown in the figures, the reaction cartridge (400) includes a unitary plastic component, preferably formed through injection molding, 3D-printing, or other manufacturing process known in the art. As shown in Figures 4-5, the reaction cartridge (400) includes a front and rear surface. In this embodiment, the front surface includes integral positions to secure a heater subassembly (500), fluid transfer subassembly (600), amplicon transfer subassembly (700), and lateral flow assay (800). The rear surface of the reaction cartridge (400) includes integral positions to secure a sample processing subassembly (402) and amplification subassembly (414). As noted above, the various sub-assemblies can be in fluid communication one with another through one or more channels. In a preferred embodiment, the channels allowing fluid communication include microfluidic channels formed by integral channels incorporated into the body of the reaction cartridge (400) and sealed with a support film (118A, 118B) which can preferably be laser welded to the reaction cartridge (400).

[0056] The nucleic acid amplification and detection assembly (100) includes a reaction cartridge (400) housing a sample processing subassembly (402) configured to process and prepare nucleic acids in the sample for downstream amplification. In a preferred embodiment the sample processing subassembly (402) can include a sample collection chamber (404). Referring to Figure 5, the sample collection chamber (404) includes an integrally formed chamber formed by body of the reaction cartridge (400) and configured to secure the sample swab (206) for processing by a dilution solution(308).

[0057] As noted above, engagement of the injector assembly (300) causes a dilution solution (308) to be injected into the sample collection chamber (404) via a sample dilution channel (406). The dilution solution (308) can elute the sample positioned on the sample swab (206), and can further lyse cells present in the sample releasing the target nucleic acids to be amplified. The solution containing the eluted nucleic acids, sometimes referred to as the diluted sample, passes through a filter (408), and preferably a filter stack (408) having a plurality of differentially sized filters configured to remove amplification inhibitors, large particles, and cell by products, such as membranes, organelles, proteins, and glycoproteins.

[0058] The first fraction passing through the filter typically contains an insufficient quantity of the target nucleic acid for effective downstream amplification. As shown in Figure 5, a fractionator (410), provided in this example as a fractionator pad, is positioned within the reaction cassette (400) and in fluid communication with filter stack (408) such that the first fraction of the sample that passes through the filter is adsorbed by the fractionator (410) thereby allowing later fractions having higher concentrations of the target nucleic acid(s) to pass through for downstream amplification. The enriched fraction(s) containing the target nucleic acid(s) are mixed to ensure downstream amplification reactions receive approximately equivalent concentrations of the sample. Referring again to Figure 5, the enriched fraction(s) are introduced to a mixing channel (412) which is formed by a series of convoluted microfluidic pathways which allow the sample to be mixed for downstream amplification.

[0059] The nucleic acid amplification and detection assembly (100) includes a reaction cartridge (400) housing an amplification subassembly (412) in fluid communication with the sample processing subassembly (402) that is configured amplify the target nucleic acid(s), preferably via an isothermal amplification reaction. Referring to Figure 5, in a preferred embodiment the amplification subassembly (412) includes a sample divider (418) in fluid communication with the mixing channel (412). In this embodiment, the sample divider (418) is configured to divide and direct the processed sample to a plurality of amplification chambers (422) each containing amplification reagent(s) (420) necessary for nucleic acid amplification, as well as. In one embodiment, the amplified sample can pass from the amplification chamber (422) and be recombined at a reaction chamber outlet (430) for further processing as described herein. In a preferred embodiment, the amplification reagent(s) (420) include beads of lyophilized amplification reagent(s) that are hydrated and activated when they come into contact with the processed sample. Notably, while the preferred embodiment shown in the figures includes a sample divider and four separate amplification chambers (422) that can each amplify a different target nucleic acid, additional embodiments include a single amplification chamber (422) in fluid communication with the mixing channel (412). Moreover, in certain embodiments, the amplification reagent(s) (420) in each of the amplification chambers (422) are configured to amplify a single target nucleic acid, such as a single RNA biomarker, in alternative embodiments each of the amplification chambers (422) can contain amplification reagent(s) (420) configured to amplify one or more different target nucleic acids.

[0060] In certain embodiments, the amplification reagents (420) preferably includes reagents for an isothermal reaction selected from: Reverse-Transcription Recombinase Polymerase Amplification (RT-RPA); Recombinase polymerase amplification (RPA); nucleic acid sequencebased amplification (NASBA), loop-mediated isothermal amplification (LAMP); Reverse transcription loop-mediated isothermal amplification (RT-LAMP); strand displacement amplification (SDA); helicase-dependent amplification (HDA); nicking enzyme amplification reaction (NEAR); signal mediated amplification of RN A technology (SMART); rolling circle amplification (RCA); isothermal multiple displacement amplification (EVIDA); single primer isothermal amplification (SPIA); and polymerase spiral reaction (PSR).

[0061] In one embodiment the amplification reagent(s) (420) can be positioned within the amplification chamber(s) (422) and secured in place via an reaction chamber plug (432). As shown in Figure 19, in one embodiment the reaction chamber plug (432) can include multiple plus elements that can secure a plurality of amplification reagents (420) into their respective amplification chambers (422). In this configuration, the sensitive amplification reagent(s) (420), which generally require handling within a dry room, can be added to the amplification chamber(s) (422) later in the manufacturing process allowing the device to primarily be manufactured outside of a dry room environment.

[0062] The amplification chambers (422) are further configured to be in communication with one or more vents to allow air to pass through and out of the chamber as the processed sample displaces ambient air within the sample dilution channel (406) and amplification chambers (422) themselves. As generally shown in Figure 5, a porous venting frit (426) is positioned downstream from the reaction chamber outlet (430) and is capable of venting air from the amplification chambers (422). The venting frit (426) is composed of a hydrating polymer that expands when in contact with the processed sample which closes the air vent thereby preventing fluid from the processed sample from escaping the amplification chambers (422).

[0063] As noted above, the pressure exerted by the injector (300) provides additional forces to push the fluid through the sample processing subassembly (402) and downstream amplification subassembly (414) as described herein. However, once the flow of the processed sample stops, a flow restrictor (416) can be positioned above a pressure sensitive adhesive (416 A) and configured to be opened thereby providing excess fluid present in the sample dilution channel (406) an alternative pathway to a waste chamber (not shown) positioned within the reaction cartridge (400).

[0064] The nucleic acid amplification and detection assembly (100) includes a reaction cartridge (400) housing a heater subassembly (500) thermally coupled with the amplification subassembly (414). As described above, engagement of the injector (300) causes the introduction of an activation solution (310) into a heater activation channel (502). In the preferred embodiment shown in Figures 5 and 14, the heater activation channel (502) directs the activation solution (310) to an exothermal fuel (504) positioned within a heating chamber positioned adjacent to the amplification subassembly (414). As shown in Figure 14, the exothermal fuel (504) is positioned adjacent to a phase change material (512), and separated from the same by a heater seal (506). In one embodiment, a chamber insulator (516) is positioned above the exothermic fuel to improve performance and reliability of the exothermal reaction in varying external environment temperatures. When the exothermic fuel (504) comes into contact with an activation solution (310) an exothermic reaction is initiated, the heat from which causes a phase change material (512) positioned adjacent to the fuel (504) to reach a pre-determined temperature thereby heating the isothermal amplification of the amplification subassembly (414). More specifically, phase change material (512) is configured to provide a controlled, substantially constant temperature to the amplification chambers (422) for the amplification reaction by being at least partially converted from its solid form to its liquid form when heated by an exothermic chemical reaction generated by the exothermic fuel (414) and the activation solution (310).

[0065] In one embodiment, the exothermal fuel (504) can include a metal alloy and catalyst that can react with an activation solution (310). In this embodiment, an exothermic reaction occurs as the exothermic fuel (504) containing magnesium metal is converted to magnesium hydroxide in the presence of a metallic iron catalyst and an electrolyte activation solution (310), such as a sodium chloride solution. The phase change material (512) includes a quantity of a paraffin, while in other embodiments the phase change material (512) can be selected from a metal, an inorganic compound, an inorganic eutectic and an organic compound. In one embodiment shown in Figure 14, a chamber seal (518), which can include a foil seal is positioned below the phase change material to better regulate the controlled exothermic reaction and venting of the same. Additional phase change materials and exothermic fuels and reagents are described in U.S. Patent No. 8431387, which is incorporated herein by reference.

[0066] Referring to Figures 13-14, the exothermal fuel (504), heater seal (506) and phase change material (512) can be positioned within a heating chamber (514) formed by the reaction cartridge (400). These elements can further be encased by one or more insulation (508) elements as well as a vent (510), which can include a spacer formed by a non-insulative material. In this configuration, the insulation and vent (508, 510) elements direct heat from the phase change material (512) to the amplification subassembly (414) and allow that heat transfer to be maintained in different environmental conditions. The nucleic acid amplification and detection assembly (100) includes a reaction cartridge (400) housing a fluid transfer subassembly (600) in fluid communication with the amplification subassembly (414) and configured to convey the amplified nucleic acids or amplicons from the amplification chamber (422) to the lateral flow assay (800). As shown in Figure 10, the fluid transfer subassembly (600) includes a sealed fluid container (606) containing a transfer solution (608), such as a buffer positioned in the reaction cartridge (400). A spring (604) loaded push button (602), or other engagement device is positioned adjacent to the fluid container (606) with the button positioned in a button aperture (114) on the outer cover (102) so as to be manually accessible by a user. After the amplification reaction has been allowed to proceed, a subject can manually engage the push button (602) causing the fluid container (606) to rupture thereby releasing the transfer solution (608) into an amplicon transfer channel (702). In a preferred embodiment, the fluid container (606) includes a sealed blister pack containing the transfer solution (608). In a preferred embodiment the fluid container (606) comprises a cold-form foil packaging, which can preferably include aluminum foil is enclosed between layers of Nylon, PVC, or PVC / PVDC films capable of protecting the transferred solution (608) from moisture, oxygen, and light A lidding foil (614) is positioned on the underside of the fluid container (606) and secured to the reaction cartridge (400) via pressure sensitive adhesive (612).

[0067] Engagement of the spring (604) loaded push button (602) causes the fluid container (606) to be pierced by a puncture (616), shown in this embodiment as being positioned below the fluid container (606) and formed as an integral component of the reaction cartridge (400). In this configuration, the transfer solution (608) exits the ruptured fluid container (606) and is introduced to the amplicon transfer channel (702). The force applied to the push button (602) causes the transfer solution (608) to pass through the amplification chamber (422) containing the amplified nucleic acids or amplicons under pressure and carry the amplified nucleic acids through the amplicon transfer channel (702) for downstream detection by the lateral flow assay (800) as described below.

[0068] The nucleic acid amplification and detection assembly (100) includes a reaction cartridge (400) housing an amplicon transfer subassembly (700) in fluid communication with the fluid transfer subassembly (600). As noted above, the amplification of the target nucleic acid(s) can be allowed to proceed for a pre-determined length of time, preferably between 5-30 minutes prior to the amplicon’s transfer to the lateral flow assay (800). In a preferred embodiment, access to the lateral flow assay is blocked until engagement of an amplicon transfer subassembly (700). As shown in Figure 12, the access to the lateral flow assay (800) via the amplicon transfer channel (702) is initially blocked by a transfer seal (710). A piercer (706) is positioned adjacent to the transfer seal (710) and supported by a compressible gasket (708). The piercer (706) is further responsive to a switch (704) positioned on the external surface of the outer cover (102). In this configuration, a user can engage the switch (704) causing the piercer (706) to compress the gasket (708) and rupture the transfer seal (710) thereby establishing fluid communication between the amplicon transfer channel (702) and the lateral flow assay (800) such that amplified nucleic acid(s) can be transferred from the amplification chambers (422) to the lateral flow assay (800) for detection. In one embodiment, the switch (704) is positioned within a slide position capable of allowing the switch (704) to traverse forward engage the switch (704) causing the piercer (706) to compress the gasket (708) and rupture the transfer seal (710).

[0069] The nucleic acid amplification and detection assembly (100) includes a reaction cartridge (400) housing a lateral flow assay (800). As shown in Figure 21, a lateral flow assay (800) includes a retainer (818) configured to be secured within a cavity of the cartridge (400) and further secure a lateral flow strip (806), supported by a backing card (804) and in contact with a conjugate pad (808). An absorbent pad (814) is also positioned within the retainer (818) and in contact with the lateral flow strip (806) to facilitate the flow of the transfer fluid’s distribution actors the strip (806) via capillary action. The lateral flow strip (806) is further secured by one or more compression cleats (802) and support ribs (816) formed by the retainer (818). In one optional embodiment, a dam (not shown), which is preferably formed of silicone or other similar material is positioned at the proximal end of the lateral flow strip (806) to direct and facilitate distribution of the amplicon containing transfer fluid along the strip(806).

[0070] The lateral flow strip (806) can include an antibody-based capture mechanism. In this embodiment, the result of an isothermal RT-RPA reaction may include an amplicon that may act as a control biomarker, and another amplicon that may act as an infection biomarker. Once the amplification reaction is completed, the amplicon transfer subassembly (700) followed by the fluid transfer subassembly (600) can be engaged as described above allowing the amplified nucleic acid products to be introduced to the lateral flow strip (806). As shown in FIG. 31, these amplified nucleic acids can be introduced to one or more conjugated antibody reporter probes, which in a preferred embodiment may act as visual reporters at striping positions (812) by producing an observable indication of, for example the presence of a target RNA biomarker transcript in a sample.

[0071] In a preferred embodiment, the conjugate pad (808) preferably contains gold nanoparticles (GNPs) conjugated to anti-FITC antibodies that bind to a 5’ FITC modification on amplicons. The complexes then bind to striping positions (812) within a detection zone embedded on the nitrocellulose membrane, creating a visible line when amplicons are present. Exemplary methods, systems, and apparatus for the use and detection of amplified products on a lateral flow strip (806) resulting from an isothermal reaction such as a RT-RPA isothermal reaction is also described by Meyerson, et al., in PCT / US2023 / 082792, and are herein incorporated by reference.

[0072] Generally referring to Figures 7-8, the reaction cartridge (400) of the disclose can be positioned within the internal chamber (112) of an outer cover (102). As further shown in Figure 1, the outer cover (102) can include an aperture configured to allow the injector (300) to be positioned so as to be responsive with the reaction cartridge (400). The outer cover (102) can include a lateral flow readout (104) which can include a transparent viewing aperture configured to allow a user to view the results of the lateral flow assay (800). One or more diagnostic identifiers (106) can be positioned adjacent to the lateral flow readout (104) which can correspond to the striping position(s) (812) on the lateral flow strip (806) allowing a user to identify visually whether the test was positive or negative for the presence of the target nucleic acid as well as visual positive of negative control indications.

[0073] The terminology used herein is for describing embodiments and is not intended to be limiting. As used herein, the singular forms “a,” “and” and “the” include plural referents, unless the content and context clearly dictate otherwise. Thus, for example, a reference to “a biomarker” may include a combination of two or more such biomarkers. Unless defined otherwise, all scientific and technical terms are to be understood as having the same meaning as commonly used in the art to which they pertain. As used herein, “about” or “approximately” means within 10% of a stated concentration range or within 10%> of a stated time frame.

[0074] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0075] Nucleic acids and / or other moieties of the invention may be isolated or “extracted.” As used herein, “isolated” means separate from at least some of the components with which it is usually associated whether it is derived from a naturally occurring source or made synthetically, in whole or in part. Nucleic acids and / or other moieties of the invention may be purified. As used herein, purified means separate from the majority of other compounds or entities. A compound or moiety may be partially purified or substantially purified. Purity may be denoted by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.

[0076] The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (for example, a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.

[0077] A primer is preferably a single-stranded DNA. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 6 to about 225 nucleotides, including intermediate ranges, such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25 to 150 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein.

[0078] As used herein, a biological marker (“biomarker” or “marker”) is a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacological responses to therapeutic interventions, consistent with NIH Biomarker Definitions Working Group (1998). Markers can also include patterns or ensembles of characteristics indicative of particular biological processes. The biomarker measurement can increase or decrease to indicate a particular biological event or process. In addition, if the biomarker measurement typically changes in the absence of a particular biological process, a constant measurement can indicate occurrence of that process. In a preferred embodiment a biomarker includes one or more RNA transcripts that may be indicative of infection or other normal or abnormal physiological process.

[0079] As referred to herein, the terms “nucleic acid”, “nucleic acid molecules” “oligonucleotide”, “polynucleotide”, and “nucleotides” may interchangeably be used. The terms are directed to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof in the form of a separate fragment or as a component of a larger construct, linear or branched, single stranded, double stranded, triple stranded, or hybrids thereof. The term also encompasses RNA / DNA hybrids. The polynucleotides may include sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA. The DNA molecules may be, for example, but not limited to complementary DNA (cDNA), genomic DNA, synthesized DNA, recombinant DNA, or a hybrid thereof. The RNA molecules may be, for example, but not limited to: ssRNA or dsRNA and the like. The terms further include oligonucleotides composed of naturally occurring bases, sugars, and covalent intemucleoside linkages, as well as oligonucleotides having non-naturally occurring portions, which function similarly to respective naturally occurring portions. The terms “nucleic acid segment” and “nucleotide sequence segment,” or more generally “segment,” will be understood by those in the art as a functional term that includes both genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller engineered nucleotide sequences that are encoded or may be adapted to encode, peptides, polypeptides, or proteins. All nucleic acid primers, such as any SEQ IN NOs. incorporated by reference, are presented in the 5’ to 3’ prime direction unless otherwise noted.

[0080] As used herein, “complementary” refers to the ability of a single strand of a polynucleotide (or portion thereof) to hybridize to an anti-parallel polynucleotide strand (or portion thereof) by contiguous base-pairing between the nucleotides (that is not interrupted by any unpaired nucleotides) of the anti-parallel polynucleotide single strands, thereby forming a double-stranded polynucleotide between the complementary strands. A first polynucleotide is said to be “completely complementary” to a second polynucleotide strand if each and every nucleotide of the first polynucleotide forms base-paring with nucleotides within the complementary region of the second polynucleotide. A first polynucleotide is not completely complementary (i.e., partially complementary) to the second polynucleotide if one nucleotide in the first polynucleotide does not base pair with the corresponding nucleotide in the second polynucleotide. The degree of complementarity between polynucleotide strands has significant effects on the efficiency and strength of annealing or hybridization between polynucleotide strands. This is of particular importance in amplification reactions, which depend upon binding between polynucleotide strands. An oligonucleotide primer is “complementary” to a target polynucleotide if at least 50% (preferably, 60%, more preferably 70%, 80%, still more preferably 90% or more) nucleotides of the primer form base-pairs with nucleotides on the target polynucleotide.

[0081] As used herein, the term “detection” refers to the qualitative determination of the presence or absence of a microorganism in a sample. The term “detection” also includes the “identification” of a microorganism, i.e., determining the genus, species, or strain of a microorganism according to recognized taxonomy in the art and as described in the present specification. The term “detection” further includes the quantitation of a microorganism in a sample, e.g., the copy number of the microorganism in a microliter (or a milliliter or a liter) or a microgram (or a milligram or a gram or a kilogram) of a sample. The term “detection” also includes the identification of an infection in a subject or sample.

[0082] As used herein the term “pathogen” refers to an organism, including a microorganism, which causes disease in another organism (e.g., animals and plants) by directly infecting the other organism, or by producing agents that causes disease in another organism (e.g., bacteria that produce pathogenic toxins and the like). As used herein, pathogens include, but are not limited to bacteria, protozoa, fungi, nematodes, viroids and viruses, or any combination thereof, wherein each pathogen is capable, either by itself or in concert with another pathogen, of eliciting disease in vertebrates including but not limited to mammals, and including but not limited to humans. As used herein, the term “pathogen” also encompasses microorganisms which may not ordinarily be pathogenic in a non-immunocompromised host.

[0083] The term “infection,” or “infect” as used herein is directed to the presence of a microorganism within a subject body and / or a subject cell. For example, a virus may be infecting a subject cell. A parasite (such as, for example, a nematode) may be infecting a subject cell / body. In some embodiments, the microorganism may comprise a virus, a bacteria, a fungi, a parasite, or combinations thereof. According to some embodiments the microorganism is a virus, such as, for example, dsDNA viruses (such as, for example, Adenoviruses, Herpesviruses, Poxviruses), ssDNA viruses (such as, for example, Parvoviruses), dsRNA viruses (such as, for example, Reoviruses), (+) ssRNA viruses (+) sense RNA (such as, for example, Picomaviruses, Togaviruses), (-) ssRNA viruses (-) sense RNA (such as, for example, Orthomyxoviruses, Rhabdoviruses), ssRNA-RT viruses (+) sense RNA with DNA intermediate in life-cycle (such as, for example, Retroviruses), dsDNA-RT viruses (such as, for example, Hepadnaviruses). In some embodiments, the microorganism is a bacteria, such as, for example, a gram negative bacteria, a gram positive bacteria, and the like. In some embodiments, the microorganism is a fungi, such as yeast, mold, and the like. In some embodiments, the microorganism is a parasite, such as, for example, protozoa and helminths or the like. In some embodiments, the infection by the microorganism may inflict a disease and / or a clinically detectable symptom to the subject. In some embodiments, infection by the microorganism may not cause a clinically detectable symptom. In some embodiments, the microorganism is a symbiotic microorganism. In additional embodiments, the microorganism may comprise archaea, protists; microscopic plants (green algae), plankton, and the planarian. In some embodiments, the microorganism is unicellular (single-celled). In some embodiments, the microorganism is multicellular.

[0084] As used herein, the term “asymptomatic” refers to an individual who does not exhibit physical symptoms characteristic of being infected with a given pathogen, or a given combinations of pathogens.

[0085] Some embodiments of the invention comprise amplifying nucleic acids from a sample. As used herein, a “sample” can be any quantity of material that includes one or more nucleic acids, and preferably a liquid, semi-liquid or otherwise flowable sample that includes one or more nucleic acids. Examples include environmental samples, such as water, soil, and industrial samples as well as waste streams and the like. In additional embodiments, the sample may be a pharmaceutical sample containing an isolated or complex mixture of nucleic acids, and in particular therapeutic nucleic acids and the like. Further samples include biological samples, which may include prokaryotic as well as eukaryotic samples, as well as plant, fungi and / or isolated nucleic acids of the same.

[0086] As used herein, the term “biological sample” includes a sample from any bodily fluid or tissue. Biological samples or samples appropriate for use according to the methods provided herein include, without limitation, blood, serum, urine, saliva, tissues, cells, and organs, or portions thereof, as well as isolated nucleic acid samples derived from a subject, or other organism, such as a bacterium, plant, fungi or other cell. A “subject” is any organism of interest, generally a mammalian subject, and preferably a human subject. Some embodiments of the invention comprise detecting in a sample from a patient, a level of a biomarker, wherein the presence or expression levels of the biomarker are indicative of infection or possible infection by one or more pathogens.

[0087] Any isothermal amplification protocol can be used according to the methods provided herein. Exemplary types of isothermal amplification include, without limitation, Reverse- Transcription Recombinase Polymerase Amplification (RT-RPA); nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), Reverse transcription loop-mediated isothermal amplification (RT-LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HD A), nicking enzyme amplification reaction (NEAR), signal mediated amplification of RNA technology (SMART), rolling circle amplification (RCA), isothermal multiple displacement amplification (EVIDA), single primer isothermal amplification (SPIA), recombinase polymerase amplification (RPA), and polymerase spiral reaction (PSR), available at nature.com / articles / srepl2723 on the World Wide Web). In some cases, a forward primer is used to introduce a T7 promoter site into the resulting DNA template to enable transcription of amplified RNA products via T7 RNA polymerase. In other cases, a reverse primer is used to add a trigger sequence of a toehold sequence domain.

[0088] As used herein, the term “amplified” refers to polynucleotides that are copies of a particular polynucleotide, produced in an amplification reaction. An amplified product, according to the invention, may be DNA or RNA, and it may be double-stranded or single- stranded. An amplified product is also referred to herein as an “amplicon”. As used herein, the term “amplicon” refers to an amplification product from a nucleic acid amplification reaction. The term generally refers to an anticipated, specific amplification product of known size, generated using a given set of amplification primers.

[0089] As used herein, the term “lateral flow assay” means an assay where the sample flow takes place at least partly parallel to a surface through which the sample and / or chemical or physical phenomena contributed by the sample can be optically imaged. As used herein, “buffer” refers to a substance, which is typically a solution, that maintains a stable pH despite the addition of strong acids or bases and external influences of temperature, pressure, volume or redox potential. The buffer prevents changes in the concentration of additional chemicals, such as proton donor and acceptor systems, to prevent significant changes in hydrogen ion concentration (pH). The pH of all buffers is temperature and concentration dependent. The choice of buffer to be used to maintain the pH or pH range can be determined empirically by one skilled in the art based on the known buffering capacity of known buffers. Exemplary buffers include, but are not limited to: bicarbonate buffer, dimethylarsinate buffer, phosphate buffer or Tris buffer. For example, Tris buffer (tromethamine) is an amine-based buffer having a pKa of 8.06 and having an effective pH range of 7.9-9.2. For Tris buffer, the pH increased by about 0.03 units for every 1 °C decrease in temperature and decreased by 0.03-0.05 units for every 10-fold dilution.

[0090] As used herein “reagent” can refer broadly to any chemical or biochemical agent used in a reaction, including enzymes. A reagent can include a single agent which itself can be monitored or a mixture of two or more agents. A reagent may be living (e.g., a cell) or non-living. Exemplary reagents can include at least one of, but are not limited to, a lysis buffer, salt, a bead, a protease, an enzyme, a metal ion (for example magnesium salt), chelator, polymerase, primer, template, nucleotide triphosphate, label, dye, nuclease inhibitor, substrates, chromogens, cofactors, coupling enzymes, buffer, metal ions, inhibitors and activators, and the like.

[0091] As used herein, a system, apparatus, structure, article, element, or component that is “configured to” perform a particular function is in fact merely a function of that particular function after further modification, rather than having the potential to perform its particular function without any modification. In other words, any system, device, structure, article, element, or component is specifically selected, created, implemented, utilized, programmed, and / or designed for the purpose of performing a particular function, be done. As used herein, “configured to” means any system, device, structure, article, element, or element that actually enables the system, device, structure, article, element, or component to perform a specified function. , or that the property of the component is present. In this disclosure, a system, device, structure, article, element, or component that is said to be “configured to” perform a particular function can additionally or alternatively be “configured to” perform that function. It can be described as being “fitted to” and / or “operable to” perform.

Claims

CLAIMSWhat is claimed is:

1. A device for sequential nucleic acid amplification and detection comprising:- a sample collector configured to collect a sample containing one or more nucleic acid to be amplified, wherein the sample is deposited into a reaction cartridge having a sample processing subassembly in fluid communication with an amplification subassembly;- a heater subassembly thermally coupled with the amplification subassembly;- an injection assembly configured to introduce a dilution solution into the sample processing subassembly and an activation solution to the heater subassembly;- a lateral flow assay positioned within the reaction cartridge; and- fluid transfer subassembly configured to transfer amplified nucleic acids from the amplification subassembly to the lateral flow assay.

2. The device of claim 1, wherein said sample comprises a biological sample, a pharmaceutical sample, or an environmental sample.

3. The device of claim 1, wherein said nucleic acids present in said sample are selected from: RNA, DNA, one or more RNA biomarkers of infection, or a combination of the same.

4. The device of claim 1, wherein said sample collection assembly comprises a collection handle coupled with a sample collector configured to be inserted into a sample collection chamber of the reaction cartridge via a sample port.

5. The device of claim 4, wherein said handle comprises one or more ring seals.

6. The device of claim 5, wherein said sample collection assembly comprises a lock configured to secure the sample collector within the sample collection chamber.

7. The device of claim 6, wherein said lock comprises a tab configured to be rotatably secured with an outer cover of the reaction cartridge.

8. The device of claim 4, wherein said sample collection chamber comprises a waste chamber vent.

9. The device of claim 1, wherein said injection assembly comprises:- a first injection barrel containing a dilution solution;- a second injection barrel containing an activation solution; and- a plunger responsive to the first and second injection barrels, wherein the action of the plunger causes the dilution solution and the activation solution to be introduced into the reaction cartridge.

10. The device of claim 9, wherein said a plunger comprises a piston that is responsive to the first and second injection barrels.

11. The device of claim 10, wherein said piston comprises a plunger forming a seal when the plunger is activated.

12. The device of claim 10, wherein said injection assembly comprises a seal positioned at the terminal end of the first and second injection barrels.

13. The device of claim 12, wherein the action of the plunger causes the seal to be compressed into the puncture causing the dilution solution and the activation solution to be simultaneously introduced into the reaction cartridge.

14. The device of any of claims 9-13, wherein said sample processing subassembly comprises a sample dilution channel configured to direct the dilution solution to the sample collection chamber.

15. The device of claim 14, wherein said sample processing subassembly comprises a filter stack configured to receive and filter the diluted sample from the sample collection chamber.

16. The device of claim 15, wherein said filter stack comprises chelating agent.

17. The device of claim 15, wherein said sample processing subassembly comprises a fractionator in fluid communication with the filter stack configured to remove the first fraction of the solution passing through the filter stack that contains a small amount of the nucleic acids.

18. The device of claim 15, wherein said sample processing subassembly comprises a mixing channel in fluid communication with the filter stack.

19. The device of claim 18, wherein said amplification subassembly comprises one or more amplification chambers in fluid communication with the mixing channel.

20. The device of claim 19, wherein said one or more amplification chambers contain amplification reagents and primers necessary for amplification of the nucleic acids present in the sample, and wherein the reaction amplification reagents and primers are secured in the amplification chambers via a chamber plug.

21. The device of claim 20, wherein said amplification reagents comprises reagents for an isothermal reaction selected from: Reverse-Transcription Recombinase Polymerase Amplification (RT-RPA); Recombinase polymerase amplification (RPA); nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP); Reverse transcription loop-mediated isothermal amplification (RT-LAMP); strand displacement amplification (SDA); helicase-dependent amplification (HD A); nicking enzyme amplification reaction (NEAR); signal mediated amplification of RNA technology (SMART); rolling circle amplification (RCA); isothermal multiple displacement amplification (EVIDA); single primer isothermal amplification (SPIA); and polymerase spiral reaction (PSR).

22. The device of claim 18, wherein said amplification subassembly comprises a sample divider in fluid communication with the mixing channel and configured to direct the diluted sample to a plurality of amplification chambers each having amplification reagents and primers necessary for amplification of the nucleic acids present in the sample.

23. The device of any of claims 1-22, wherein said amplification subassembly comprises one or more venting frits.

24. The device of any of claims 1-23, wherein said wherein said amplification subassembly comprises a flow restrictor in fluid communication with the mixing channel.

25. The device of claim 1, wherein said heater subassembly comprises a heater activation channel configured to direct the activation solution to an exothermal fuel positioned within the reaction cartridge.

26. The device of claim 25, wherein said heater subassembly comprises a phase change material thermally coupled with the exothermic fuel such that activation of the fuel causes the phase change material to reach a pre-determined temperature thereby heating an isothermal amplification reaction within the amplification subassembly.

27. The device of claim 26, wherein said heater subassembly comprises an insulated portion and a vent portion configured to direct the thermal energy from the phase change material to the one or more amplification chambers within the reaction cartridge.

28. The device of claim 26, wherein said heater subassembly comprises a seal positioned between the phase change material and the exothermic fuel, and / or a seal positioned between the phase change material and a heating chamber.

29. The device of claim 26, wherein said phase change material comprises a paraffin.

30. The device of claim 26, wherein said phase change material is selected from the group consisting of: a metal, an inorganic compound, an inorganic eutectic, and an organic compound.

31. The device of claim 25, wherein said exothermal fuel comprises a Mg-Fe alloy32. The device of any of claims 1-31, wherein said fluid transfer subassembly comprises a fluid container containing a transfer solution.

33. The device of claim 32, wherein said fluid transfer subassembly comprises a push button positioned adjacent to the fluid container, wherein activation of the push button causes the transfer solution to be released from the fluid container and into an amplicon transfer channel where it passes through the one or more amplification chambers transferring the amplified nucleic acids to the lateral flow assay.

34. The device of claim 33, wherein said push button comprises a spring-loaded push button.

35. The device of claim 33, wherein activation of the push button causes a puncture to rupture the fluid container thereby releasing the transfer fluid into the amplicon transfer channel.

36. The device of any of claims 1-35, further comprising an amplicon transfer subassembly in fluid communication with the fluid transfer subassembly and configured to prevent the transfer of the amplified nucleic acids to the lateral flow away until activated.

37. The device of claim 36, wherein said amplicon transfer subassembly comprises a movable switch responsive to a piercer configured to puncture a transfer seal allowing the transfer fluid to enter the lateral flow assay.

38. The device of any of claims 1-37, wherein said lateral flow assay comprises a lateral flow strip adapted to receive and detect the amplified nucleic acids.

39. The device of claim 38, wherein said lateral flow strip comprises:- a backing card;- a membrane having one or more striping positions;- a conjugate pad; and- an absorbent pad.

40. The device of claim 39, wherein the lateral flow strip is secured by a retainer.

41. The device of claim 40, wherein the retainer comprises one or more compression cleats and one or more support ribs securing the lateral flow strip.

42. The device of claim 1, further comprising an outer cover positioned over the reaction cartridge.

43. A method of amplifying and detecting a nucleic acid, the method comprising:- inserting a sample collector containing a sample having one or more nucleic acids to be amplified and detected into a reaction cartridge;- activating an injection assembly thereby introducing a dilution solution to the sample and separately an activation solution to a heater subassembly;- filtering and transporting the diluted sample to an amplification subassembly containing reagents necessary for amplification of the nucleic acid;- amplifying the nucleic acid in the sample, wherein the heater subassembly is thermally coupled with the amplification chamber thereby heating the amplification reaction; and- engaging a fluid transfer subassembly thereby transferring the amplified nucleic acids from the amplification subassembly to a lateral flow assay.

44. The method of claim 43, wherein said sample comprises a biological sample, a pharmaceutical sample, or an environmental sample.

45. The method of any of claims 43-44, wherein said nucleic acids present in said sample are selected from: RNA, DNA, one or more RNA biomarkers of infection, or a combination of the same.

46. The method of claim 43, wherein the step of inserting comprises the step of inserting a sample collector into a sample collection chamber of the reaction cartridge via a sample port.

47. The method of claim 46, further comprising the step of locking the sample collector.

48. The method of claim 47, further comprising the step of forming a seal between the sample collector and the sample collection chamber.

49. The method of claim 43, wherein the step of activating an injection assembly comprises depressing a plunger responsive to a first and a second injection barrel, wherein the action of the plunger causes the dilution solution and the activation solution to be released into the reaction cartridge.

50. The method of claim 49, wherein the dilution solution is injected into a sample dilution channel configured to direct the dilution solution to the sample collection chamber.

51. The method of claim 50, further comprising the step of lysing the sample in the sample collection chamber.

52. The method of claim 43, wherein the step of filtering comprises the step of passing the diluted sample through a filter stack.

53. The method of claim 43, further comprising removing the first fraction of the solution passing through the filter stack that contains a small amount of nucleic acid.

54. The method of claim 43, wherein the step of transporting comprises the step of transporting the diluted sample through a mixing channel.

55. The method of claim 54, further comprising the step of engaging a flow restrictor in fluid communication with the mixing channel.

56. The method of claim 43, wherein the step of amplifying comprises contacting the sample with amplification reagents and primers necessary for amplification of the nucleic acids present in the sample within one or more amplification chambers.

57. The method of claim 43, further comprising the step of dividing the diluted sample into a plurality of amplification chambers each containing amplification reagents and primers necessary for amplification of the nucleic acids present in the sample.

58. The method of claim 43, wherein said amplification reagents comprises reagents for an isothermal reaction selected from: Reverse-Transcription Recombinase Polymerase Amplification (RT-RPA); Recombinase polymerase amplification (RPA); nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP); Reverse transcription loop-mediated isothermal amplification (RT-LAMP); strand displacement amplification (SDA); helicase-dependent amplification (HD A); nicking enzyme amplification reaction (NEAR); signal mediated amplification of RNA technology (SMART); rolling circle amplification (RCA); isothermal multiple displacement amplification (EVIDA); single primer isothermal amplification (SPIA); and polymerase spiral reaction (PSR).

59. The method of claim 56, further comprising the step of securing the amplification reagents within the amplification chambers via a reaction chamber plug.

60. The method of claim 49, wherein the activation solution is injected into a sample heater activation channel configured to direct the activation solution to an exothermal fuel positioned within the reaction cartridge and adjacent to a phase change material.

61. The method of claim 60, wherein the activation of the exothermic fuel causes the phase change material to reach a pre-determined temperature thereby heating an isothermal amplification reaction within the amplification subassembly.

62. The method of claim 43, wherein the step of engaging the fluid transfer subassembly comprises introducing a transfer solution into an amplicon transfer channel where it passes through the amplification subassembly transferring the amplified nucleic acids to the lateral flow assay.

63. The method of claim 62, wherein the step of introducing a transfer solution comprises puncturing a fluid container containing the transfer solution.

64. The method of claim 43, further comprising engaging an amplicon transfer subassembly in fluid communication with the fluid transfer subassembly, wherein the amplicon transfer subassembly prevents the transfer of the amplified nucleic acid to the lateral flow away until activated.

65. The method of claim 64, wherein the step of engaging an amplicon transfer subassembly comprises engaging a movable switch responsive to a piercer that when engaged causes the piercer to puncture a transfer seal allowing the transfer fluid to enter the lateral flow assay.

66. The method of claim 65, wherein the step of engaging an amplicon transfer subassembly occurs prior to engagement of the fluid transfer subassembly.

67. The method of claim 43-66, wherein said lateral flow assay comprises a lateral flow strip adapted to receive and detect the amplified nucleic acids.

68. The method of claim 67, wherein said lateral flow strip comprises:- a backing card;- a membrane having one or more striping positions;- a conjugate pad; andan absorbent pad.

69. The device of claim 68, wherein the lateral flow strip is secured by a retainer.

70. The device of claim 69, wherein the retainer comprises one or more compression cleats and one or more support ribs.

71. The device of claim 43, further comprising securing an outer cover over the reaction cartridge.

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