Systems and devices for portable nucleic acid amplification and detection

Abstract

The present invention is directed to novel systems, methods, and apparatus for power-less, field-ready diagnostic devices for the early detection and amplification of nucleic acids in a sample.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international PCT application claims the benefit of and priority to U.S. Provisional Application No. 63 / 430,631, filed December 6, 2022, the specification, claims and drawings of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to the field of diagnostic devices, and in particular to systems, methods, and apparatus directed to novel, non-powered, portable devices for the amplification and detection of nucleic acids. In a preferred embodiment, the present invention includes systems, methods, and apparatus directed to novel, non-powered, portable devices for early, pathogen-nonspecific detection of infectious diseases. [Background technology]

[0003] Early detection of pathogen infection is essential for appropriate treatment and favorable clinical outcomes. However, infected individuals may remain asymptomatic for several days after infection while actively transmitting the pathogen to others. Traditional pathogen detection systems are often ineffective at detecting 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 once patients begin to show symptoms of infection. Furthermore, traditional diagnostic tests require clinical suspicion for a specific pathogen, expensive laboratory equipment, and trained personnel, increasing upstream and end-user costs.

[0004] For example, in a typical infection course, exposure to an unknown pathogen occurs on day 0, followed by progression to subsequent clinical stages of infection. As the pathogen replicates within an infected person, standard diagnostic tests are typically designed to function after the onset of symptoms, when people realize something is wrong and seek medical attention. However, at that point, the person may have been infectious to others for days or even weeks. The opportunity to implement early isolation and limit the devastating downstream effects of unimpeded pathogen transmission has passed. This time delay to diagnosis can worsen patient outcomes and result in ongoing disease transmission before the patient knows they are infectious.

[0005] In contrast to the specific, slower-developing adaptive immune response, the host's first line of defense against pathogenic microorganisms is the "innate immune" response. The body's innate immunity is a self-amplifying, nonspecific physiological response that occurs within hours of infection. Therefore, the ability to detect the presence of molecules produced by the host's innate immune response could potentially lead to the rapid detection of infections at their earliest stages, when patients are still asymptomatic. Such advances would enable more effective isolation protocols, as well as improved treatment and clinical outcomes. Therefore, there is a long-felt need for a diagnostic device for early, nonspecific pathogen detection of infectious diseases. As described below, the diagnostic device of the present invention is adapted to detect RNA biomarkers that are upregulated in biological samples such as saliva upon exposure to pathogens, allowing for early, nonspecific pathogen identification of infected individuals.

[0006] Additionally, the detection of nucleic acids in other samples, such as environmental and pharmaceutical samples, can be of both clinical and industrial importance. However, again, the time and expense required to collect, transport, and process the samples can be prohibitive. As a result, there is a need for simple, inexpensive, portable, and technically reliable devices for the amplification and detection of nucleic acids. Summary of the Invention

[0007] The inventive technology described herein may include systems, methods, and compositions for amplifying and detecting nucleic acids in a sample. In a preferred embodiment, the sample of the present invention may include an environmental sample, a biological sample, or a pharmaceutical sample containing a predetermined amount of nucleic acid. In another preferred embodiment, the present invention includes novel systems, methods, and compositions for amplifying and detecting nucleic acids in a sample using an amplification pad embedded with reagents, preferably lyophilized reagents, necessary for amplifying nucleic acids such as DNA or RNA.

[0008] In other embodiments, detecting nucleic acids includes detecting nucleic acids containing somatic or genetic mutations, detecting nucleic acids that have been exposed to radiation and / or exhibit radiation-induced mutations / damage, detecting bacterial or viral pathogen nucleic acids in a sample, detecting nucleic acids containing genetic biomarkers, variants, or mutations that indicate a disease state or predisposition to a disease state, detecting nucleic acids from environmental samples, and detecting nucleic acids from agricultural or plant samples.

[0009] In a preferred aspect, the amplification products produced by the amplification pad of the present invention may be in fluid communication with an assay, such as a lateral strip assay. In this embodiment, the sample may comprise a liquid sample that provides sufficient capillary action through the device and amplification pad to transfer the amplification products produced within the pad to the assay portion of the device.

[0010] In a preferred embodiment, the present invention includes a nucleic acid amplification device. In a preferred embodiment, the device includes a collection handle configured to collect a sample containing nucleic acids, a sample processing assembly in fluid communication with the amplification assembly, and a heater assembly thermally coupled to the amplification assembly and responsive to a heater actuation assembly. In another preferred embodiment, the device may further include a lateral flow assay configured to receive the amplification product from the amplification assembly.

[0011] In another specific embodiment, the technology of the present invention may include systems, methods, and compositions for efficiently lysing cells that may be present in a sample, such as a biological sample. In this embodiment, a lysis pad embedded with buffer and / or reagents sufficient to lyse the cells in the sample, thereby releasing nucleic acids, may be placed within the device. In another specific embodiment, the technology of the present invention may include systems, methods, and compositions for efficiently filtering the lysed cells. In this embodiment, filters, and preferably filter stacks described herein, may filter the liquid sample while preventing undesirable inhibitors of subsequent nucleic acid amplification, for example, through chelating materials.

[0012] In another specific aspect, the present technology may include systems, methods, and compositions for early detection of pathogens and / or infections in asymptomatic subjects through a novel diagnostic device. In a preferred embodiment, the diagnostic device may include a lateral flow assay configured to detect one or more oligonucleotide transcript biomarkers produced by a subject's innate immune system in response to a pathogen or infection and present in saliva.

[0013] Additional aspects of the present invention may include one or more of the preferred embodiments set forth in the claims. Further aspects of the present invention will become apparent from the specification, claims, and drawings provided below. [Brief explanation of the drawings]

[0014] [Figure 1A] (A) An exemplary model of an assembled diagnostic device in one embodiment of the present invention. [Figure 1B] (B) Exploded model of an assembled diagnostic device, in one embodiment. [Figure 1C] (C) Cross-sectional view of an assembled diagnostic device in one embodiment of the present invention. [Figure 2]A-B are the absorption volumes and dimensions of the tested collection materials. The values ​​presented were calculated by subtracting the pre-collection mass from the post-collection mass. Mass was measured to approximate sample volume. (A) Maximum collection capacity of collection pads soaked in PBS (30 x 8 mm Porex collection pads n = 11, all other pad sizes n = 1). Collection pads were attached to the collection handle of the diagnostic device and soaked in PBS until fully saturated. (B) Average maximum oral collection volume of saliva (Porex n = 8; SalivaBio n = 4). Collection pads were attached to the collection handle of the diagnostic device and placed in the oral cavity until the pad reached saturation. [Figure 3] A-D show the optimization and measurement of RNase inhibition by mouse RNase inhibitor (mRI). (A) Diagram of the RNase Alert assay. A quenched fluorescent ssRNA probe reports RNase activity upon physical separation of the quencher and fluorescent dye by RNase degradation. Data are collected immediately after saliva is added to the assay, and RNase activity is calculated from the linear slope during the first 90 seconds of measurement. (B) RNase activity in five individual saliva samples with or without the addition of 4 U / μl mRI. (C) RT-qPCR data for a panel of 12 biomarkers, showing the relative abundance of each biomarker at the indicated times after lysis. Each time point is from a pooled saliva sample run through a separate device. ΔCt values ​​were normalized to the 0 second time point. Horizontal bars represent the mean ± standard deviation. (D) RNase activity in four saliva samples before and after rehydration of an amplification pad containing 4 U / μl lyophilized mRI. The saliva in the rehydrated pads underwent a 20-minute RPA incubation at 39°C before being added to the RNase Alert assay, demonstrating long-lasting RNase inhibition. Ct = cycle threshold. [Figure 4]A–D are optimizations of saliva treatment with Chelex-100. (A) Inhibition of RPA by saliva. Titrations of pooled saliva were used to reconstitute lyophilized RPA reactions in the presence or absence of cDNA template to identify inhibition of RPA by saliva above 25% (v / v). (B) Removal of saliva inhibitors by Chelex-100. Pooled saliva was treated with 20%, 5%, and 0% (w / v) Chelex-100 in liquid form and then added to RPA with cDNA template (+) or no template (-) at a final concentration of 95% (v / v). (C) Comparison of 30-minute in-solution treatment with direct column Chelex-100 treatment. Saliva was treated in liquid form at 0% and 5% (w / v) or in column form at 5% and 20% (w / v) and then added to RPA with cDNA template (+) or no template (-) at a final concentration of 95% (v / v). (D) Identification of persistent RT-specific inhibitors in saliva after treatment with Chelex-100. Saliva was treated with 20% (w / v) Chelex-100 for 1 minute and then added to RT-RPA with cell line cDNA, RNA, or no template (-). All reactions contained 4 U / μl RNase inhibitor and 0.0625 U / μl Transcriptor RT. [Figure 5]A-D are optimizations of filter stack height, diameter, and material composition. Mass was measured to approximate saliva volume. (A) Squeezed mass of the collection pad and retained mass of the dissolving pad. A 35.5mm x 10mm Porex collection pad was used with a 10mm diameter dissolving pad of various heights. (B) Sample mass squeezed downstream of the dissolving pad. A 35.5mm x 8mm Porex collection pad was used with a 10mm diameter dissolving pad of various heights. (C) Squeezed mass of the collection pad and retained mass of the dissolving pad. A 30mm x 10mm SalivaBio collection pad was used with a dissolving pad of various dimensions or material density. (D) Sample mass squeezed downstream of the dissolving pad. A 30mm x 10mm SalivaBio collection pad was used with a dissolving pad of various dimensions or material density. (A / C) Squeezed mass of the collection pad was calculated by subtracting the pre-collection mass from the post-collection mass. The retained mass of the dissolving pad was calculated by subtracting the dry dissolving pad mass from the wet pad mass. (B / D) The downstream sample squeezed mass was calculated by subtracting the retained mass of the dissolving pad from the squeezed mass of the collection pad. [Figure 6] Tracking the dye front through the diagnostic device. Xylene cyanol dye was dried onto an 11x10mm dissolving pad before assembly into the device. A SalivaBio 30x10mm collection pad was saturated with 1.2mL of PBS and squeezed through the device. The sample flows through the dissolving pad, reconstituting and washing away the xylene cyanol dye, then saturating the transfer and amplification pads. The device (top), transfer pad, and amplification pad (bottom) were imaged before (left) and after (right) adding PBS to the device. [Figure 7]Evaluation of optimized sample processing and lysis buffer components. (A) RT-RPA efficiency in the presence of Buffer 20 and Buffer 21. Cell line RNA was used as the (+) template and (-) no-template control. (B) Optimized initial / pre-infection diagnostic sample processing and lysis tested in five individuals. -RT = no reverse transcriptase was included in the RT-RPA reaction; +RT = Superscript IV was added to the RPA reaction at a final concentration of 1 U / μl. [Figure 8] Comparison of RT resistance to saliva inhibition. 50 μl RT-RPA reactions were prepared using SuperScript IV (1 U / μl), NxtScript (1 U / μl), NxtScript 2G (1 U / μl), or MMLVHP (5 U / μl) and then lyophilized onto amplification pads. MMLVHP was tested at 5 U / μl, which is cost-equivalent to the 1 U / μl of other RT enzymes. Water and saliva samples were processed in a diagnostic device equipped with a collection pad, Buffer 21 lysis pad, 10 μm filter, 75 mg of Chelex-100, and a 1 μm filter. Samples were combined with cell line RNA (+) or water (-) and used to reconstitute RT-RPA amplification pads. The LFA signal in reactions without cell line RNA (-) indicates amplification of endogenous CXCL8 RNA in saliva (red box). "Saliva without Chelex" was processed through the diagnostic device without Chelex-100. [Figure 9] Comparison of the effect of pad density on reaction efficiency. (A) Pad-driven RPA reactions were performed on three densities of amplification pad material. The reactions were then diluted 10-fold and developed on lateral flow strips: high density = 0.25 g / cc, medium density = 0.16 g / cc, low density = 0.07 g / cc. [Figure 10]A-B: Limit of detection (LoD) study of host biomarkers in RPA. (A) Heat map of CALR LoD across various temperature ranges (X-axis). A dilution series of starting template concentrations (Y-axis) was assayed by RPA at temperatures ranging from 29°C to 43°C for 20 minutes. Results were measured by amplicon fluorescence intensity (AU = arbitrary units) from agarose gel electrophoresis. (B) A dilution series of double-stranded CALR biomarker PCR products was performed in liquid RPA reactions using the TwistDx TwistAmp Basic RPA kit. Reactions were incubated at 37°C for 20 minutes. (C) A dilution series of double-stranded CXCL8 biomarker PCR products was performed in pad-driven RPA reactions. Reactions were incubated at 44°C for 20 minutes. [Figure 11]Integration of RT-RPA amplification. (A) Integration of pad material into the amplification device (also called the amplification assembly). T1 and T2 = Transfer pad material, Porex HRM PP / PE blend, 0.16 g / cc, blocked with 1% PEG20K and 0.2% Tween-20. The Amp pad is the same HRM PP / PE blend material, with RT-RPA reagents lyophilized with 0.1% Tween-20, 2% sucrose, and 1% mannitol. (B) Amplification performance using the configuration shown in A. Template wicked through the pad material, and a total of 10 copies of in vitro transcribed CXCL8 RNA reached the amplification pad. The amplification assembly was incubated in an incubator set at 44°C for 20 minutes, and then amplicons were visualized on a lateral flow strip. (C) Integration of pad material into the complete diagnostic device. (D) Amplification of the entire device using the setup shown in C. Template was absorbed into the collection pad and then squeezed out within the device. The sample was passed through a blank lysis pad (without lysis buffer), then through a 10 μm filter, 75 mg of Chelex-100, and a 1 μm filter, then wicked through the pad material toward the amplification pad. Double-stranded DNA template was added, delivering 10 copies of template to the amplification pad. The complete diagnostic device was incubated for 40 minutes in an incubator set at 44°C, and then amplicons were visualized on the lateral flow strip. [Figure 12] Vertical strip embodiment of a diagnostic device. Additional subcomponents were integrated into the final design, including a collection pad, filtration stack, filter housing, amplification pad, and lateral flow strip. An incubation film is shown that inhibits the flow of amplification products onto the lateral flow strip. Once the reaction is complete, the incubation film can be easily peeled back down to the point of contact between the amplification pad and the conjugate pad of the lateral flow strip. [Figure 13]Alternative designs for the diagnostic device. CAD models of the collection handle and changes made to the assembled SLA device from version 1.0 (left / green) to version 1.4 (right / blue). The model was modified to accommodate design constraints for low-volume production molding. Assembled SLA device shown with and without ghosted housing and strip cover. [Figure 14] Amplifier assembly and base plate for relocating pressure relief holes. The original injection molded design (left) had three pressure relief holes on the front of the amplifier assembly. Alternative embodiments included a redesigned amplifier assembly and base plate, shown in exploded view (center) or mated view (right). Orange circles highlight the pressure relief holes. [Figure 15] Time course study of CALR amplification in RPA. A dilution series from 10 to 10 input copies (Y-axis) was incubated in RPA reactions at 34 °C for the indicated amount of time (X-axis), and the reactions were visualized by agarose gel (A) and lateral flow strips (B). The expected size of the CALR amplicon is shown to the left of the gel in panel A. [Figure 16] A-B: Characterization of CXCL8 (A) and DDX58 (B) biomarker amplification across temperature (X-axis) and template concentration (Y-axis). Relative band intensities (arbitrary units) of amplicons were determined by quantifying bands on an agarose gel (left). Selected reactions were also assayed on lateral flow strips (right). [Figure 17] A-B, Time course study of CXCL8 (A) and DDX58 (B) amplification in RPA. A dilution series of 10 to 10 input copies was incubated in RPA reactions at 34 °C for the indicated amount of time, and the reactions were visualized by agarose gel and lateral flow strips. The expected size of the target amplicon is indicated to the left of the gel. [Figure 18]A-B: Preliminary LoD study with RT-RPA at various temperatures. RT-RPA reactions using IVT template dilutions of CALR (A) and CXCL8 (B) were incubated for 15 min at the indicated temperatures and then visualized on an agarose gel. The expected sizes of the CALR and CXCL8 amplicons are shown to the left of the gel in panels A and B, respectively. Results were also visualized using lateral flow strips (C). [Figure 19] A-B: Comparison of the LoDs of the reference (A) and infected (B) test lines on lateral flow strips. Dilutions of mimetic concentrations ranging from 5 ng to 0.1 ng were developed on the lateral flow strips for 15 minutes at room temperature and then imaged. [Figure 20] Exemplary oligo modifications for RT-RPA amplicon detection on lateral flow strips. Reference / control biomarkers are amplified with FITC-modified forward primers and biotin-modified reverse primers for detection with polystreptavidin on lateral flow strips. Infection biomarkers are amplified with FITC-modified forward primers and DIG-modified reverse primers for detection with anti-DIG on lateral flow strips. Gold-conjugated anti-FITC binds to the amplicons and then immobilizes them in the detection zone on the strip, generating a signal at the infection or reference line. An excess gold wire monitors the flow of gold on the strip and detects gold-conjugated anti-IgY (made in rabbits) using an anti-rabbit antibody. Anti-DIG and anti-FITC are mouse monoclonal antibodies. [Figure 21] Example of early wet-lab primer screening. Eight primer sets for the same target (RACK1) were screened by RPA using cell line cDNA (+) as template or a no-template control (-). 20 μl TwistDx Basic Kit reactions were incubated at 39°C for 20 minutes. Reactions were analyzed on a 2% agarose gel. [Figure 22]Multiplexing with control and reference biomarkers in RT-RPA. The reference biomarker (CALR) and infection biomarker (IFIT2) were assayed individually or together in multiplexed RT-RPA reactions using RNA isolated from a human lung cell line (+) or a no-template control (-). Each oligo was added at a final concentration of 240 nM, and Transcriptor Reverse Transcriptase (3531287001; MilliporeSigma) was added at a final concentration of 0.0625 U / μL. Reactions were incubated at 39°C for 15 minutes and analyzed by lateral flow strips. [Figure 23] A-B show examples of 4-plex and 5-plex RT-RPA reactions. (A) Multiplexed RT-RPA reactions were performed on RNA isolated from a human lung cell line (+) or a no-template control (-) at 39°C for 15 min and analyzed on a 4% agarose gel. Each oligo was at a final concentration of 180 nM. The predicted amplicon size for each target is shown to the right of the gel. Note that DDX58 and CALR have very similar amplicon sizes and are indistinguishable on the gel. (B) Multiplexed RT-RPA reactions with and without RNA template were analyzed on a lateral flow strip. [Figure 24] Effect of oligo concentration on lateral flow strips. Multiplexed RT-RPA reactions were performed on RNA isolated from human lung cell lines (+) or no-template controls (-) at 39°C for 15 minutes and analyzed on lateral flow strips. The relative and absolute concentrations of primers were varied as indicated on the strips. Infecting primers: CXCL8, DDX58, OAS2, IFIT2. [Figure 25]Close-up view of the saliva processing subcomponents for infection diagnostics. Saliva is collected using a collection pad and transferred to the device. The collection pad also reduces sample viscosity and removes large particles from the saliva. The lysis pad contains lyophilized buffer for lysis of human cells and inactivation of salivary RNases. The debris filter removes cellular debris and remaining large salivary particles prior to biomarker amplification. The RT-RPA amplification pad contains lyophilized reaction buffer and enzymes for amplification of human biomarkers of infection. [Figure 26] Interference of saliva in RT-RPA amplification. cDNA and 500 or 50 pg of RNA were used as templates in RT-RPA reactions targeting the CXCL8 biomarker. Saliva (0 μl, 5 μl, 1 μl, or 0.1 μl) was added to the reactions to test for interference in RT-RPA. Reactions labeled in red do not contain reverse transcriptase. The expected size of the CXCL8 amplicon is shown on the left side of the gel. [Figure 27] Example of a salivary RNase activity assay performed on TapeStation High Sensitivity RNA ScreenTapes (5067; Agilent). Results from three independent assays are shown. Lanes 1–3 (left to right) are experimental controls containing 500 pg of RNA in RNase-free water, untreated saliva without added cell line RNA, and untreated saliva incubated with cell line RNA. The lane labeled "Incomplete RNase Inactivation" contains no ribosomal RNA band, indicating the presence of RNase activity. The lane labeled "Sufficient RNase Inactivation" contains a sharp ribosomal band (similar to RNA in water), indicating that RNase was inactivated. gDNA = genomic DNA. [Figure 28]Testing for Reagent Interference in RT-RPA. Lysis and RNase Inhibitor Reagents were tested in RT-RPA using cDNA and RNA templates. RT-RPA reactions were prepared using various concentrations of labeled reagents as described above (final concentrations in a 20 μl RT-RPA reaction are reported). Amplification products were run on a 2% agarose gel, and band intensities were compared to control (no added reagent) reactions. Reactions labeled red lacked reverse transcriptase. The expected size of the CXCL8 amplicon is indicated on the left side of the gel. GuHCl = guanidine hydrochloride; Triton = Triton X-100. [Figure 29] Crude saliva extracts amplified by RT-RPA. Saliva was collected from seven individuals using selected saliva collection pads. Saliva was extracted from the pads using a 3 ml syringe and treated with an optimized lysis buffer. Five μl of the treated saliva was used as a template for an RT-RPA reaction to amplify the human RNA biomarker CXCL8. The reaction products were run on lateral flow strips for infection diagnosis (see above). Five of the seven extracts were successfully amplified, as indicated by the line on the lateral flow strip for the CXCL8 amplicon. Samples 1 and 6 failed to produce an amplification product. [Figure 30] Pad-driven amplification—wet reagent. A porous 3mm x 3.5mm x 12mm pad was fully saturated with the RPA liquid reaction. After incubation, the reaction was squeezed from the pad using a syringe and diluted 50-fold with PBS before being applied to a lateral flow strip. NTC = no template control. [Figure 31] A-B, Lyophilization of RPA reactions with and without glycine and trehalose. (A) Lyophilization of the original RPA components and the associated lateral flow signal after rehydration and amplification. (B) Lyophilization of the reformulated RPA components with added glycine and trehalose and the associated lateral flow signal after rehydration and amplification. [Figure 32]Amplification of NCL (reference biomarker) and OAS2 (infection biomarker) primers by full-pad RPA. Pads were fully saturated with RPA reaction reagents and lyophilized under our optimized conditions. The lyophilized pads were rehydrated with water containing cDNA template and incubated at 39°C for 20 minutes before application to the lateral flow strip. Experiments were performed in duplicate. NCL and OAS2 amplicons bind to distinct locations on the lateral flow strip. NTC = no template control. [Figure 33] In one embodiment, RACK1 (reference biomarker) primers were amplified with the current diagnostic device. Pad-driven amplification was performed using a diagnostic device with all subcomponents integrated. In this preliminary experiment, a solution containing cDNA template was added to the device, which was then incubated at 39°C for 20 minutes. After incubation, the pull tab was removed and the amplified product was run onto a lateral flow strip. RACK1 amplicons were detectable on the lateral flow strip, albeit at low levels. This integrated prototype will be used in subsequent experiments to further optimize other aspects of the device, from saliva collection to amplification efficiency. [Figure 34] 10 is a cross-sectional view of an alternative diagnostic device, according to one embodiment. [Figure 35]Amplification in a fully integrated device. (A) Amplification of RNA template diluted in water. 105 copies / μl of RNA template in water or water alone (no template) was absorbed onto a collection swab attached to the collection handle, and then the sample was expressed into the reaction cartridge by inserting the collection handle. The device was placed in a 45°C incubator for 20 minutes. After 20 minutes, the pull tab was removed to release the reaction onto the lateral flow strip. Results were read through the strip cover window 15 minutes after removing the pull tab. A device with two lines (control and test) indicates a positive result, while a device with one line (control only) indicates a negative result. Three replicates are shown for each template amount. (B) Amplification of DNA template diluted in saliva. Saliva was collected from three individuals, and DNA template was then spiked into each saliva sample to a final concentration of 105 copies / μl. The DNA template-spiked saliva samples were assayed in the reaction cartridge as described in A. Three replicates are shown for each saliva sample. (C) Amplification of endogenous RNA from saliva samples. Saliva samples were collected from individual donors by holding the collection swab in the mouth. After absorption, the sample was expressed into the reaction cartridge by inserting the collection handle, and the sample was then assayed as described in A. RNA templates present in saliva could be detected in four of the six samples shown. [Figure 36] Schematic of a reaction cartridge, with visible components of the reaction cartridge annotated. [Figure 37] Cross-sectional view of the reaction cartridge showing subassemblies of the device. [Figure 38] Exploded view of the reaction cartridge. [Figure 39] FIG. 1 is an exploded view of the heater actuation assembly in the context of the reaction cartridge. [Figure 40] FIG. 10 is an exploded view of the heater subassembly in the context of the reaction cartridge. [Figure 41] FIG. 10 is an exploded view of the saliva processing subassembly in the context of the reaction cartridge. [Figure 42]Exploded view of the lateral flow assay subassembly in the context of the reaction cartridge. [Figure 43] A cross-sectional schematic of the reaction cartridge, with all components within each subassembly annotated. [Figure 44] Schematic of the saliva collection device, with key features of the device design annotated. [Figure 45] Schematic cross-section of a saliva collection device, with key features of the device design annotated. [Figure 46] An exploded view of the saliva collection device, with key features of the device design annotated. [Figure 47] Implementation of the saliva sufficiency indicator for interpersonal normalized sample volume. (A) Schematic of the saliva sufficiency indicator. The sufficiency indicator display window appears white before sample collection and turns dark blue once sufficient sample is collected. (B) Sample collection volume from 1 minute of saliva collection across individuals using various saliva collection swab materials. Each swab material differs in density, diameter, length, and maximum sample collection volume. The blue line represents the minimum sample volume required to activate the device. (C) Sample collection volume (left, n=31) and time (right, n=31) using the saliva sufficiency indicator across individuals. The blue line represents the minimum sample volume required to activate the device. [Figure 48] Thermal profiles of various chemical reactions investigated for use in the device. A) Galvanic corrosion between magnesium-iron alloy powder and sodium chloride solution. B) Calcium oxide dissolution reaction in water. C) Copper sulfate hydration reaction with water. D) Iron powder oxidation reaction. E) Sodium acetate crystallization reaction. Temperature probes were placed inside the reactions carried out in PCR tubes. [Figure 49]Modification of the magnesium-iron fuel source reaction to achieve isothermal conditions. A) Thermal effect of varying the total volume of 1% NaCl solution used to rehydrate a fixed amount of magnesium-iron powder. B) Thermal effect of varying the concentration of NaCl solution used to rehydrate a fixed amount of magnesium-iron powder. C) Thermal profile of a phase change material that melts at 42°C heated by a magnesium-iron fuel source. D) Thermal profile of a custom chemical heater consisting of three annular cylinders. The outermost cylinder contains the fuel (magnesium-iron mixed with sand), the middle cylinder contains the phase change material (melting at 42°C), and the innermost cylinder is the liquid amplification reaction chamber. Thermal profiles of four different temperature probes are shown: two in different regions of the fuel source, one in the phase change material, and one in the amplification reaction chamber. [Figure 50]Chemical heater design and functional testing. A) Diagram of the concentric ring design used to house three separate chambers: 1. chemical fuel and filler, 2. phase change material (PCM), and 3. amplification reaction. B) Image of a proof-of-concept prototype chemical heater. A 3D-printed housing was used to separate the chambers: 1. fuel (38 mg Mg-Fe alloy) and filler (100 mg vermiculite), 2. PCM (800 μL Rubitherm RT44HC), and 3. a reaction chamber containing 100 μL of water. Thermal probes (yellow) were inserted to monitor the temperature at each location. C) Example thermal profiles of the chemical fuel, PCM, and reaction chamber upon activation with 300 μL of 10% sodium chloride, recorded at the indicated locations by the thermocouple probes. The green area of ​​the graph represents the temperature range in which RT-RPA amplification can occur. D) Comparison of the thermal profiles of the reaction chambers of three independently prepared chemical heaters with that of a BioRad T100 thermal cycler (TC) set at 42 °C. The green area of ​​the graph represents the temperature range in which RT-RPA amplification can occur. E) LFA results of RT-RPA amplification of the indicated template concentrations in 20 μL reactions using either a T100 thermal cycler (TC) or a chemical heater as the heat source. The presence of a red band at the location labeled "Amplicon Detected" indicates successful amplification of the template. NTC = No Template Control. [Figure 51] Integration of heater elements into the current platform prototype. A) Schematic of the heater subassembly arrangement in the overall device. Shown is the fluid capsule holding the chemical fuel, phase change material, and activator solution delivered to the heater assembly containing the isothermal amplification reaction. B) Comparison of the thermal profiles of three devices with integrated heaters with that of a thermal cycler (TC). C) Detection of RNA-templated multiplexed amplification (control and test biomarkers) on a lateral flow strip. The amplification reaction was performed in a fully integrated device containing an active heater element. [Figure 52]Isothermal amplification and detection of RNA from capillary blood. A) 200 microliters of capillary blood was collected using a fingerstick lancet. RNA from the sample was purified using a column purification kit and eluted in 50 microliters, yielding approximately 1.5 micrograms of RNA in total. The indicated nanogram amounts were added to a 20-minute RT-RPA reaction, and amplicons of a host biomarker (CXCL8) were separated on a lateral flow strip. The upper red indicator line is the lateral flow control line. B) RT-RPA reactions using 2.8 ng of RNA were performed as in A), with increasing hemoglobin concentrations, and amplification products were separated on a lateral flow strip. NTC = no template control. [Figure 53] Isothermal amplification and detection of endogenous RNA from crude blood samples. A) 50 microliters of capillary blood was collected using a TAP Microselect device and diluted to various concentrations with DEPC-treated water. 1 μL of each dilution was then added to a 20 μL RT-RPA reaction with and without a 10 ng RNA template spike to monitor potential inhibition from the blood. No inhibition was observed in any reactions with spiked RNA template. Endogenous amplification of a host biomarker (RACK1) was observed on a lateral flow strip at a 10% blood dilution in water. The upper red indicator line is the lateral flow control line. B) Lyophilized reagents for the RT-RPA reaction were resuspended in a 10% blood diluent so that the final reaction consisted of 50% or 100% diluted blood without an additional template spike. No inhibition was observed with increasing concentrations of blood. Instead, stronger endogenous amplification of host biomarkers (CXCL8 and RACK1) was observed on later flow strips. NTC = no template control. [Figure 54]Comparison of RNA expression in venous and capillary blood. A) Equal amounts of RNA purified from venous or capillary blood from six donors were subjected to an RT-qPCR panel to measure the indicated human RNA biomarkers. B) RNA from 50 microliters of capillary blood from three donors was prepared using either a crude extraction method (10-fold dilution in water, centrifugation to collect the cell pellet, and resuspending the pellet in 20 microliters of water) or spin-column purification. The RNA samples were subjected to an RT-qPCR panel to measure the indicated human biomarkers. Data are presented in boxplot format. [Figure 55] Prototyping and conceptualization of the capillary blood collection handle. A) Capillary tube used to collect prepared capillary blood samples (water containing blue dye). The indicator line (black) on the capillary tube represents the sample sufficiency line at 20 microliters. B) The capillary tube containing the prepared sample is attached to a syringe containing lysis buffer, which mixes with the sample as it is expressed into the device. C) Description of the capillary blood collection handle and its compatibility with the current device prototype. [Figure 56] FIG. 1 shows sequential two-step sample amplification and delivery to a lateral flow assay in one embodiment. [Figure 57] (A) Shown is a fluid injection assembly having an injector configured to pass a fluid, such as a buffer, through a collection pad and into a sample processing assembly; (B) Shown is a fluid injection assembly having a fluid, such as a buffer, disposed in a reservoir and responsive to a collection handle and configured to inject the fluid into the sample processing assembly in response to a first movement of the collection handle. [Figure 58] 1 shows a cross-sectional view of a check valve positioned between a sample processing assembly and an amplification assembly in one embodiment. [Figure 59]Magnesium transfer pad integration. A) Cross-sectional schematic of the amplifier subassembly showing the location of the magnesium transfer pad located above the amplification pad. B) Water samples spiked (+) or unspiked (-) with nucleic acid template with 14 mM magnesium (liquid, blank transfer pad) or without (magnesium in the transfer pad). The samples were processed through the device, and the amplification reaction was carried out within the device. Two amplification products (healthy line and infected line) were separated on a lateral flow strip. C) Experiment performed similar to B), except the sample used was pooled, boiled saliva. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates to novel systems, methods, and devices for amplifying and detecting nucleic acids in a sample. In a preferred embodiment, the present invention is embodied by novel systems, methods, and devices for early, pathogen-nonspecific detection of infectious diseases. In a preferred embodiment, the present invention includes a diagnostic device (100) adapted to amplify nucleic acids in a sample, such as an environmental sample, a biological sample, a pharmaceutical sample, and / or a diagnostic sample. As described in PCT / US2020 / 049290 by Sawyer et al. (incorporated herein by reference), exemplary nucleotide transcript biomarkers, preferably coding or encoded RNA oligonucleotides 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. It should be noted that specific target RNA transcripts or biomarkers produced by a patient's immune response (generally the innate immune response or any other cellular pathway upregulated during infection) can be found in saliva and provide an indication of early infection. As a result, one embodiment of the present technology includes systems, methods, and compositions for detecting these target oligonucleotides, e.g., target RNA transcripts, which can serve as biomarkers for early infection in a subject. However, as described above, in this embodiment, the target RNA transcript biomarkers present in a typical liquid sample provided from a human subject are generally present at low concentrations and require amplification for detection. To overcome this physical limitation, the present invention includes systems, methods, and devices for isothermal amplification of target RNA transcript biomarkers.

[0016] In a preferred embodiment, the diagnostic device (100) of the present invention, also generally referred to herein as device (100) or device of the present invention (100), is adapted to collect an environmental or biological sample, preferably a sample such as a saliva sample from a subject, which is further processed and filtered to release nucleic acids from the sample, followed by isothermal amplification and subsequent detection on a lateral flow strip.

[0017] Generally, with reference to the preferred embodiment shown in FIG. 1 , the diagnostic device (100) of the present invention includes a collection handle (102). In this preferred embodiment, the collection handle (102) may include a handle portion for securing a collection pad (108) adapted to allow a subject to self-collect a biological specimen, preferably a saliva sample. As described below, the collection pad (108) of the present invention includes an absorbent material adapted to be inserted into a subject's oral cavity and capture a sufficient saliva sample for processing and subsequent isothermal amplification and detection by a lateral flow strip (109), as described in further detail below. Specifically, with reference to FIG. 1C , in this embodiment, the collection handle (102) of the present invention is configured to form a plunger for insertion into the interior compartment (119) of the device (100) so that a biological or other sample can be deposited therein for initial processing. 1C, the collection handle (102) may include one or more sealing positions (118) that can form an airtight seal when the handle is inserted, further preventing the backflow of the sample from the internal compartment (119) of the device (100). In yet other embodiments, the collection handle (102) may be secured to the device (100) of the present invention such that the biological specimen is secured within the internal compartment (119). Examples of locks may include a slide lock position, a twist lock position, a slotted lock position, a snap lock position, or a quick release lock position.

[0018] The diagnostic device (100) of the present invention includes a barrel (105). As shown in the embodiment highlighted in FIG. 1, the barrel (105) of the present invention is configured to receive a sample, for example, via a collection pad (108) or by depositing the sample directly thereon. As shown in FIG. 1A, the barrel (105) can be disposed within a housing (103) that forms an interior compartment (119). In this embodiment, the collection handle (102) and collection pad (108) can be disposed within the barrel (105) and / or housing and sealed to prevent contamination prior to use. Furthermore, the collection handle (102) and collection pad (108) can be removed from the barrel (105) and / or housing (103) assembly and used to collect a sample as described above. When reinserted into the device, they create a seal such that downward movement of the collection handle (102) acts as a plunger, generating an internal pressure differential to force the biological specimen through the lysis pad (110) and filter stack (111), as described below. It should be noted that while this embodiment depicts barrel (105) and housing (103) as separable components, in certain alternative embodiments, barrel (105) and housing (103) may comprise integral components that, together with other elements described in more detail below, form an integrated or semi-integrated device.

[0019] The diagnostic device (100) of the present invention includes a lysis pad (110). As shown in the embodiment highlighted in FIG. 1, the lysis pad (110) of the present invention is disposed within the barrel (103) and can thereby be in fluid communication with a biological specimen delivered from the collection pad (108) or deposited directly in the internal compartment (119). In this embodiment, the lysis pad (110) of the present invention contains a lyophilized buffer adapted to cause lysis of cells in the sample and, optionally, inactivation of RNases. In a preferred embodiment, the lyophilized buffer of the present invention may include a lysis buffer formulation identified as Buffer 21 (5 mM TCEP-HCl, 7.5% Tween-20, 0.00167% digitonin), and optionally, a portion of lyophilized mouse RNase inhibitor (mRI). Exemplary alternative lysis buffer formulations are provided below in Table 10.

[0020] As described in more detail in the Examples below, the dissolving pad (110) is preferably adapted to lyse only a portion of the biological sample, preferably only the first approximately 0.5 mL of saliva expressed from the collection pad (108). In this configuration, the lysed sample is followed by additional unlysed saliva, creating a "front" of lysed sample that can be maintained until it reaches the amplification assembly. As described in more detail below, in an alternative embodiment, inactivation of RNases in the biological sample can occur after cell lysis as part of the present invention.

[0021] The diagnostic device (100) of the present invention includes an amplification assembly (106). As shown in the embodiment highlighted in Figure 1, the amplification assembly (106) of the present invention can be configured to receive and be in fluid communication with a barrel (105), thereby allowing a sample to pass from a lysing pad (110) to the amplification assembly (106), for example, via a channel (115). Referring again to Figure 1, the amplification assembly (106) of the present invention can be adapted to accommodate a filter stack (111), a transfer channel (115) in fluid communication with a reservoir (116) formed by a base plate (107), one or more transfer pads (112, 113), and at least one amplification pad (114), each of which is further described below.

[0022] As described above, the diagnostic device (100) of the present invention includes a filter stack (111) that can be disposed within the amplification assembly (106). As shown in the embodiment highlighted in FIG. 1, the filter stack (111) of the present invention can include one or more filters disposed adjacent to a column portion (111c) containing a chelating agent configured to prevent positively charged molecules, such as divalent cations and positively charged macromolecules, in the sample from inhibiting the DNA polymerase and reverse transcriptase used in the subsequent isothermal amplification step. As described further below, flow of the sample, particularly a biological saliva sample, through the filter stack (111) of the present invention reduces viscosity and improves the overall flow of the sample through the device (100).

[0023] Referring again to FIG. 1 , the filter stack (111) of the present invention may include first and second filters (111a, 111b) sandwiched between a column section (111c) containing an agent, such as a chelating agent, configured to suppress divalent cations and positively charged molecules in the sample from inhibiting downstream isothermal amplification. In a preferred embodiment, the column section (111c) may include a predetermined amount of Chelex-100, which may further be in the form of a resin. As used herein, Chelex-100 is a chelating material used to purify other compounds by ion exchange. It is notable for its ability to bind transition metal ions. It is a styrene-divinylbenzene copolymer containing iminodiacetic acid groups.

[0024] As shown in the preferred embodiment of FIG. 1C, the first and second filters (111a, 111b) of the filter stack (111) may contain different filtration sizes, while the column section (111c) may contain a predetermined amount of Chelex-100. In this preferred embodiment, the first upper filter (111a) may contain a larger filter size than the lower second filter (111b). For example, in the preferred embodiment shown in the drawing, the first upper filter (111a) may contain a filter size of approximately 10 μm, while the lower second filter (111b) may contain a filter size of approximately 1 μm. This configuration prevents the Chelex-100 resin from passing through the second filter (111b), thereby preventing the Chelex-100 resin or other chelating agents from interacting with magnesium, which is required for downstream isothermal amplification, preferably by RT-RPA. Additionally, the column section (111c) containing a chelating agent such as Chelex-100 can be dried before being packed into the filter stack (111). This step minimizes the occurrence of air pockets within the filter stack (111).

[0025] In yet a further embodiment, a third filter (111d) may be included in the filter stack (111). In this embodiment, the third filter (111d) of the present invention may be positioned below the second filter (111c) and may have a smaller filter size than the second filter (111c). In a preferred embodiment, the third filter (111d) of the present invention may have a filter size of approximately 0.4 μm or less. By positioning the third filter (111d) below the second filter (111c), trapped air can be squeezed out through the dry column section (111c), preferably containing a chelating agent such as Chelex-100, before the sample flows, further promoting more efficient rehydration of the resin.

[0026] The diagnostic device (100) of the present invention includes an amplification assembly (106) disposed below a filter stack (111) forming a transfer channel (115) adapted to convey a processed and filtered sample (in this case, a saliva sample) to a reservoir (116) formed by a base plate (107). In this embodiment, the sample passing through the collection pad (108), dissolving pad (110), and filter stack (111) enters the base plate (107), where it is deposited in the reservoir (116), is picked up by one or more transfer pads (112 or 113), and is transferred to an amplification pad (114) disposed within the amplification assembly (106). This amplification pad contains lyophilized reagents necessary for isothermal amplification of the sample, as described below. As will be described below, the reservoir (116) allows excess sample to be collected, allowing the lysed sample "front" to be more efficiently wicked up by the unprocessed sample into the transfer pads (112, 113), then into the amplification pad, and ultimately onto the lateral flow strip (109). In this way, the excess sample collected in the reservoir provides sufficient liquid volume to drive fluid transport through the device, in effect driving capillary action of the amplified sample across the lateral flow strip (109).

[0027] 14, to prevent excessive pressure from being created within the amplification assembly (106) due to compression of the collection pad (110), the base plate (107) of the present invention may include one or more pressure relief locations (121). Again, while the base plate (107) is shown as a separable part of the device (100), in alternative embodiments, the base plate (107) may be integral with one or more components of the device (100) described herein.

[0028] As described above, the diagnostic device (100) of the present invention includes one or more transfer pads (112 or 113) adapted to wick the sample from the base plate (107) toward the amplification pad (114) containing embedded isothermal amplification reagents. In this embodiment, the transfer pad (112 or 113) and amplification pad (114) are formed from a material sufficiently porous to rapidly absorb the sample, prevent mixing of lysed and unlysed samples, and maintain a "leading edge" of the lysed sample along the processing path toward the lateral flow strip (109). In a preferred embodiment, the amplification pad (114) of the present invention contains lyophilized reaction buffer and enzymes for amplifying human infection biomarkers using RT-RPA or other isothermal amplification methods described herein. Note that descriptions and methods for performing RT-RPA, including the necessary components for RT-RPA, including various primers and RNA biomarkers, as well as other isothermal amplification systems, are described in U.S. Patent Application No. 17 / 686,387 to Sawyer et al., which is incorporated herein by reference. The components required to perform RT-RPA or other isothermal amplification methods can be further lyophilized at the distal end of the amplification pad (114) to reduce the amount of reagents required and generate a sample flow reserve after the reaction, allowing dilution for flow onto the lateral flow strip (109). In other embodiments, the amplification pad (114) can be formed from a porous material embedded and dried with RT-RPA reagents and optimized excipients, such as 0.1% Tween-20, 2% sucrose, 1% mannitol, and optionally an RNase inhibitor, such as mRI.

[0029] As described above, the diagnostic device (100) of the present invention includes a lateral flow strip (109) that can be secured within a strip cover (104). In this embodiment, the strip cover (104) of the present invention secures the lateral flow strip (109) in place, protects it from environmental exposure, and includes a transparent window for visually observing test results. Additionally, the strip cover (104) creates a pressure gradient to ensure uniform flow of amplification products from the sample across the lateral flow strip (109), regardless of orientation. The lateral flow strip (109) of the present invention can be further protected by a removable barrier, such as a pull tab (117) that contains the sample within the amplification pad (114) during incubation, and the pull tab can be removed to allow readout of amplification products from the sample transferred from the amplification pad (114) to the lateral flow strip (109).

[0030] Exemplary methods, systems, and devices for the use and detection of amplification products on a lateral flow strip (109) resulting from an isothermal reaction, such as RT-RPA, are also described in U.S. Patent Application No. 17 / 686,387 to Sawyer et al., incorporated herein by reference. As shown in FIG. 20 , the present invention may include a lateral flow strip (109) with an antibody-based capture mechanism. In this embodiment, the result of an isothermal RT-RPA reaction may include an amplified RPA product that can act as a control biomarker and another amplified RPA product that can act as an infection biomarker. Upon completion of the RT-RPA reaction in the amplification pad (114), the pull tab (117) can be removed, allowing fluid communication from the pad to the strip, thereby introducing the amplified products to one or more conjugated antibody reporter probes. In a preferred embodiment, this may act as a visual reporter, for example, by generating an observable indication of the presence of target RNA biomarker transcripts in the sample. More specifically, as shown in Figure 20, an isothermal RT-RPA reaction can generate at least two amplified RPA products, or amplicons, a control biomarker and an infection biomarker, each with a modified 5' ssDNA overhang region forming the probe capture region and the target capture region, respectively. In this embodiment, the control biomarker can include a dsDNA transcript region bound to a 5' FITC forward ssDNA oligo (green) and a 5' biotin reverse ssDNA oligo (orange). The infection biomarker in this embodiment can include a dsDNA transcript region bound to a 5' FITC forward ssDNA oligo (green and pink) and a 5' DIG ssDNA reverse oligo (blue). As further shown in Figure 20, the GNPs can be conjugated to an anti-FITC (fluorescein isothiocyanate) antibody, preferably an anti-FITC antibody produced in rabbits. As shown in Figure 20, streptavidin may also be stripped onto a lateral flow strip (109) membrane to capture control biomarker amplicons present in the amplified RPA product.In this embodiment, an anti-DIG (digoxigenin) antibody, preferably an anti-DIG antibody produced in a mouse, may also be stripped onto a lateral flow strip (109) membrane to capture infection biomarker amplicons present in the amplified RPA product.

[0031] As further shown in FIG. 20, the hybrid dsDNA control amplicon probe and infection amplicon probe generated in the amplification reaction within the amplification pad (114) may be combined with an anti-FITC antibody-conjugated GNP reporter probe. In this embodiment, the anti-FITC antibody may bind to the 5' FITC forward oligos of the control biomarker and infection biomarker to form agglutination complexes. In this embodiment, the agglutination complexes may be further introduced into the lateral flow strip (109) of the present invention. The amplification products driven by saliva present in the reservoir (116) flow by capillary action through the lateral flow strip (109) membrane, such as a nitrocellulose fiber membrane, toward an absorbent pad region on the lateral flow strip (109). This absorbent pad region may include a detection zone with one or more capture probes embedded on the surface of the lateral flow strip, preferably the nitrocellulose membrane of the lateral flow strip (109). The position and orientation of the capture probes embedded in the lateral flow strip (109) may be adjusted to optimize signal generation or sample-probe interaction.

[0032] As described above, the capture probe may comprise an immobilized streptavidin base tetramer embedded in the nitrocellulose surface of the lateral flow strip (109). This immobilized streptavidin base may be conjugated to a biotin-TEG linker, which may further be conjugated to a ssDNA target capture probe sequence that may be complementary to the target capture region on the hybrid dsDNA probe, preferably the 5' biotin-reverse oligo. Furthermore, the capture probe may comprise an immobilized anti-DIG antibody that may be configured to bind to the 5' DIG-reverse oligo. In this configuration, the control biomarker and infection biomarker amplicons may be bound to their respective locations by their respective capture probes. As described above, the GNP reporter probe of the present invention generates a red signal when immobilized in solution or on a lateral flow strip. Therefore, a visible signal may be generated in the detection zone when a certain concentration of complex aggregates are captured in close proximity to each other. A visible signal within this detection zone may indicate a positive result indicating the presence of the target pathogen, or an early sign of infection in the subject. Of course, the above are merely exemplary embodiments, and many uses and configurations of lateral flow assays can be adapted to the present invention.

[0033] In another preferred embodiment, the present invention includes a nucleic acid amplification device (200), also referred to generally herein as an amplification device (200), or the device of the present invention (200), configured to process a sample containing a predetermined amount of nucleic acid that can be further amplified and detected, for example, by a lateral flow assay. Generally, with reference to the preferred embodiment shown in Figures 44-46, the amplification device (200) of the present invention includes a collection handle (238) configured to collect a sample containing a predetermined amount of nucleic acid. In the embodiment shown in Figure 44, the collection handle (238) includes an extended collection arm (242) that secures a collection pad (239) adapted to allow a subject to self-collect a biological or environmental sample, such as, preferably, a saliva sample.

[0034] As discussed above, the collection pad (239) of the present invention comprises an absorbent material adapted to collect a biological or environmental sample. For example, in one embodiment, the collection pad (239) can be inserted into a subject's mouth and capture a saliva sample sufficient for processing and subsequent detection by isothermal amplification and lateral flow assay (225), as described in further detail below. With specific reference to FIG. 44, in this embodiment, the collection handle (238) of the present invention is configured to form a plunger that can be inserted into the processing chamber (204) of the device (200) to deposit a biological or other sample therein for initial processing. 44-45, the collection handle (238) may include one or more extended surfaces, which in this embodiment have one or more O-rings (241) secured to them, forming a seal (240) that creates an airtight seal when the collection handle (238) is inserted into the processing chamber (204) and further prevents sample from backflowing from the processing chamber (204) of the amplification device (200). Additionally, the seal (240) further generates pressure from the downward movement of the collection handle (238), which can aid in the introduction of the sample into the device, particularly into the sample processing and amplification assemblies (250, 251), as highlighted below.

[0035] 44-46, the collection handle (238) may further include one or more secondary arms (242), which in this preferred embodiment are disposed adjacent to and generally parallel to the collection arm (242). The secondary arms (242) of the present invention may further include one or more docking locations (244), which in this preferred embodiment may include a plurality of raised surfaces that may mate with corresponding recesses, for example, in the reagent syringe (206) of the heater actuation assembly (252), as described below.

[0036] The collection handle (238) of the present invention may further include a sample sufficiency indicator (245). Referring to the embodiment shown in Figures 45-46, the sample sufficiency indicator (245) may include one or more strips of material configured to provide a visual indication, such as the release of a dye or other chemical indicator, in response to the presence of a sample, such as a saliva sample. In this embodiment, a user may contact the collection pad (239) with a source of sample, for example, by inserting it into the user's oral cavity. As the collection pad (239) becomes saturated with sample, a portion of it may come into contact with the sample sufficiency indicator (245), causing a corresponding color change.

[0037] In this example, the sample sufficiency indicator (245) can be calibrated so that the time it takes for the sample to pass through the sample sufficiency indicator (245), for example, by capillary action, corresponds to the sufficient time required to provide a sufficient volume of sample, such as a saliva sample or other environmental or biological sample. Referring again to FIG. 46, the sample sufficiency indicator (245) can be calibrated by being positioned under a cover (246) having a viewing opening (247). In this embodiment, the viewing opening (247) is positioned at a distal point of the collection pad (239), such that the length of time it takes for the color change to reach the viewing opening (247) and become visible to the user is calibrated to the amount of time required to collect an appropriately sized sample for processing and subsequent amplification, as described below. Of course, the type of sample being collected, as well as characteristics such as viscosity and nucleic acid concentration, can be taken into account when calibrating the position of the sample sufficiency indicator (245). In another embodiment, the sample sufficiency indicator (245) of the present invention can be separated from the collection pad (239) by a sample contact interface (248) that can facilitate the flow of sample to the sample sufficiency indicator (245).

[0038] The amplification device (200) of the present invention further includes a collection handle (238) having a capillary collection assembly (300). As shown in FIG. 55, in a preferred embodiment, the capillary collection assembly (300) of the present invention includes a capillary collection channel (301) in fluid communication with one or more vent holes (302) configured to define the volume of sample captured within the channel (301). In this configuration, a fluid sample, preferably a blood sample, can be collected by contacting the end of the capillary collection channel (301) with a predetermined volume of blood or other fluid and drawing the blood into the channel by capillary action. As further shown in FIG. 55, the capillary collection assembly (300) can further include a solution, such as a buffer solution, in response to a plunger (303) configured to pass the solution through the channel and transfer a fluid sample containing a predetermined amount of nucleic acids to the processing chamber (204) of the sample processing assembly (250).

[0039] The amplification device (200) of the present invention further includes a collection handle (238) having a fluid injection assembly (400). As shown in Figure 57, in a preferred embodiment, the fluid injection assembly (400) can include a collection handle (238) having a fluid reservoir (401) that preferably contains a buffer solution (402) in response to an injector (403). In this configuration, a predetermined amount of a sample containing nucleic acids can be deposited onto a collection pad (239). The injector (403) can then be actuated to squeeze the buffer solution (402) from the reservoir and pass through the collection pad (239), transporting the sample in solution to the processing chamber (204) of the sample processing assembly (250).

[0040] In another embodiment, the amplification device (200) of the present invention further includes a fluid injection assembly (400) configured to deliver a fluid, preferably a buffer solution (402), in response to a first actuation of the collection handle (238), as described below. As shown in Figure 57, in this preferred embodiment, the buffer solution (402) or other fluid is disposed within a fluid reservoir (401) and separated from the processing chamber (204) by a fluid seal (405). A lancet (407) is disposed adjacent to the fluid seal (405) and, when punctured, allows the buffer solution (402) to be delivered to the processing chamber (204) and mixed with the sample prior to delivery to the reaction chamber (213). The fluid reservoir (401) of the present invention further includes a buffer plug (404) disposed above the buffer solution (402), which is configured to prevent fluid from leaking from the reservoir (401), for example, when the amplification device (200) is inverted or otherwise moved from a substantially upright position. Additionally, in some embodiments, a fluid plunger (not shown) can be disposed above the buffer plug (404).

[0041] As further shown in FIG. 57, the fluid reservoir (401) of the present invention is coupled with a gasket (406), which is preferably made from a compressible material that allows the fluid reservoir (401) to be depressed in response to a first movement of the collection handle (238), as described herein. In this embodiment, the secondary arm (243) of the collection handle (238) is positioned to be inserted into the fluid reservoir (401) such that the plug (207), and optionally the plunger (not shown), are depressed as a result of this first movement of the collection handle (238). This first movement applies a downward force to the fluid reservoir (401), thereby compressing the gasket (406), such that the fluid seal (405) is punctured by a lancet (407) positioned below the seal, allowing a buffer solution (402) or other solution to be delivered to the processing chamber (204) and mixed with the sample prior to delivery to the reaction chamber (213).

[0042] The amplification device (200) of the present invention includes a processing chamber (204). As shown in the embodiment highlighted in FIG. 37, the processing chamber (204) of the present invention is configured to accept a sample, for example, via a collection handle (238) or by directly depositing the sample therein. As shown in FIG. 1A, the processing chamber (204) is disposed within a housing (201) that forms an internal compartment. In this embodiment, the collection handle (238) is housed within the processing chamber (204) and can be sealed as described above to prevent contamination prior to use. Additionally, the collection handle (238) can be removed from the processing chamber (204) and used to collect a sample, for example, via a collection pad (239) as described above, and, when reinserted into the device, forms a seal such that the downward movement of the collection handle (238) and seal (240) acts as a plunger, creating an internal pressure differential to force the sample through the processing assembly (250) and into the amplification assembly (251), as described below. It should be noted that while the present embodiment depicts the processing chamber (204) and housing (201) as separable components, in certain alternative embodiments, the processing chamber (204) and housing (201), among other elements described herein, may comprise integral components that form an integrated or semi-integrated device.

[0043] In a preferred embodiment, the amplification device (200) of the present invention includes a lysis pad (202). As shown in FIG. 43, the lysis pad (202) of the present invention is disposed within the processing chamber (204), thereby allowing fluid communication with a sample delivered from the collection pad (239) or a sample directly deposited in the internal compartment of the processing chamber (204). In this embodiment, the lysis pad (202) of the present invention includes a lyophilized buffer adapted to cause cell lysis and, optionally, inactivation of RNases in the sample. In a preferred embodiment, the lyophilized buffer of the present invention may include a lysis buffer formulation identified as Buffer 21 (5 mM TCEP-HCl, 7.5% Tween-20, 0.00167% Digitonin), and optionally, a portion or lyophilized mouse RNase inhibitor (mRI). In a preferred embodiment, the lysis buffer may include a solution containing 1.5% Tween-20 and 3.0% sucrose. Additional exemplary alternative lysis buffer formulations are provided in Table 10 below. It should be noted that for samples that do not involve or require cell lysis, the lysis pad of the present invention can be omitted. Furthermore, lysis buffers for lysis of various cells, whether eukaryotic, prokaryotic, fungal, or plant cells, will be known to those skilled in the art.

[0044] The amplification device (200) of the present invention includes one or more filters (203a, 203b, 203c) that can be positioned within the processing chamber (204) adjacent to the dissolving pad (202). As shown in the embodiment highlighted in Figure 41, the filter (111) of the present invention can include one or more filters positioned adjacent to a column section (not shown), commonly referred to as a filter stack, that contains a chelating agent configured to prevent positively charged molecules in the sample, such as divalent cations and positively charged macromolecules, from inhibiting the DNA polymerase and reverse transcriptase used in the subsequent isothermal amplification step. As explained further below, flow of the sample, particularly a saliva sample, through the filter (203) of the present invention reduces viscosity and improves the overall flow of the sample through the device (100).

[0045] Referring again to Figure 41, the filter (203) of the present invention may include a first filter and a second filter (203a, 203b), which are sandwiched between column sections (not shown) containing an agent, e.g., a chelating agent, configured to prevent divalent cations and positively charged molecules in the sample from inhibiting downstream isothermal amplification. In a preferred embodiment, the column section (not shown) may further include a predetermined amount of Chelex-100, which may be in the form of a resin. As used herein, Chelex-100 is a chelating material used to purify other compounds via ion exchange. It is notable for its ability to bind transition metal ions. It is a styrene-divinylbenzene copolymer containing iminodiacetic acid groups.

[0046] As further shown in the preferred embodiment of Figure 47, the first and second filters (203a, 203b) of the filter stack can have different filtering sizes. In this preferred embodiment, the first upper filter (203a) can have a larger filtering size than the lower second filter (203b). For example, in the preferred embodiment shown in the figure, the first upper filter (203a) can have a filtering size of approximately 10 μm, while the lower second filter (203b) can have a filtering size of approximately 1 μm. In this configuration, the Chelex-100 resin placed between the two filters (203a, 203b) is prevented from passing through the second filter (203c), thereby preventing the resin or other chelating agents from interacting with magnesium, preferably required for downstream isothermal amplification by RT-RPA or RPA. Additionally, the column section (not shown) containing the chelating agent, such as Chelex-100, can be dried before loading into the filter stack. This process uses a filter stack to minimize the creation of air pockets.

[0047] In yet a further embodiment, a third filter (203c) may be included in the filter stack. In this embodiment, the third filter (203c) of the present invention may be positioned below the second filter (203b) and may have a smaller filter size than the second filter (203b). In a preferred embodiment, the third filter (203c) of the present invention may have a filter size of approximately 0.4 μm or less. Placing the third filter (203c) below the second filter (203b) allows trapped air to be squeezed through the dried column section prior to sample flow, further promoting more efficient rehydration of the resin.

[0048] The amplification device (200) of the present invention includes an amplification assembly (251). In a preferred embodiment, a transfer channel disposed below the filter (203) is adapted to deliver the processed and filtered sample to a reaction housing (205) having a reaction chamber (213) including a transfer pad (215) disposed adjacent to an amplification pad (216). In this embodiment, the sample passes from the collection pad (239), through the lysing pad (202) and filter (203), and into the reaction chamber (213), where it is delivered to the transfer pad (215), for example, by wicking action. The sample is then transferred to an amplification pad (216) disposed adjacent to and in fluid communication with the transfer pad (215). As shown in the figure, in this configuration, gravity and the internal pressure generated by the collection handle (238) collect the sample, thereby allowing the lysed sample "front" to be more efficiently wicked up by the unprocessed sample into the transfer pad(s) (215), then pushed forward onto the amplification pad (216), and ultimately into the lateral flow assay (225), as shown below. In one embodiment, the reaction housing (205) can include an overflow reservoir (205a) configured to allow excess sample or other fluids, such as buffer, to flow into the overpressure created within the amplification assay (251).

[0049] As described above, the amplification device (200) of the present invention includes a transfer pad (215) adapted to receive a sample from the sample processing assembly (250) and deliver it to an amplification pad (216) embedded with amplification reagents, preferably lyophilized isothermal amplification reagents. In this embodiment, the transfer pad (215) and amplification pad (216) are formed from a material that is sufficiently porous to rapidly absorb the sample, prevent mixing of lysed and unlysed samples, and maintain a "leading edge" of the lysed sample along a processing path toward the lateral flow assay (225), described below. In a preferred embodiment, the amplification pad (215) of the present invention contains lyophilized reaction buffer and enzymes for amplifying nucleic acids, such as human biomarkers of infection, using RT-RPA or other isothermal amplification methods, such as RPA, as described herein.

[0050] In a preferred embodiment, the transfer pad (215) of the present invention further includes an embedded amount of magnesium, preferably in the form of magnesium acetate (MgOAc). This magnesium is taken up by the sample, thereby preventing the magnesium acetate from inactivating the isothermal amplification reaction. Additionally, descriptions and methods for performing isothermal amplification, including the components necessary for isothermal amplification, including various primers and RNA biomarkers, as well as other isothermal amplification systems, are described in U.S. Patent Application No. 17 / 686,387 to Sawyer et al., which is incorporated herein by reference. The components necessary for performing RT-RPA, RPA, or other isothermal amplification methods can be further lyophilized at the distal end of the amplification pad (216), thereby reducing the required reagent volume and creating a sample flow reserve after the reaction, allowing for dilution for flow to the lateral flow assay (225).

[0051] In other embodiments, the amplification pad (216) can be formed of a porous material that has been impregnated and dried with amplification reagents and optimized excipients, such as 0.1% Tween-20, 2% sucrose, 1% mannitol, one or more primers for amplification of control and target nucleic acids, and optionally an RNase inhibitor, such as mRI.

[0052] In another embodiment, the amplification pad (216) can be pretreated to prevent nonspecific or undesired binding. In this preferred embodiment, the amplification pad (216) can be treated with a blocking solution to inhibit nonspecific binding prior to the addition of nucleic acid amplification reagents and excipients, as well as control and target primers. In a preferred embodiment, the blocking solution (not shown) of the present invention comprises a solution containing predetermined amounts of bovine serum albumin (BSA), Tween-20, and Tris-HCl. In a specific embodiment, the blocking solution (not shown) of the present invention comprises a solution containing 0.2% BSA, 0.1% Tween-20, and 100 mM Tris-HCl (pH 8.3).

[0053] Referring again to FIG. 42, the amplification device (200) of the present invention includes a lateral flow assay (225) that can be placed in fluid communication with an amplification assembly (251) in response to a second actuation of the collection handle (238), as described above. The isothermal amplification reaction in the amplification pad (216) may require a specified reaction time to generate sufficiently amplified nucleic acid (also called an amplification product or amplicon) for subsequent detection. As shown in the preferred embodiment of FIG. 43, depression of the collection handle (238) is calibrated so that the first actuation is a sufficient travel length to deposit the sample into the sample processing and amplification assembly (250, 251). As described above, the first actuation of the collection handle (238) is depression, with its travel blocked by the pull tab (224). In a preferred embodiment, the pull tab (224) of the present invention is positioned below the collection handle (238), has an extended lip (224a), and is secured to the processing chamber (204), thereby blocking downward movement of the collection handle (238) (also referred to as the first movement of the collection handle (238)). In this manner, the sample of the present invention is transferred from the sample processing and amplification assembly (250, 251) by the first movement of the collection handle (238), where the isothermal amplification reaction can proceed until the second movement of the collection handle (238).

[0054] As shown in Figure 47, an amplicon seal (222) is positioned between the reaction chamber (213) and the lateral flow assay (225), thereby physically isolating the amplification products generated in the amplification pad (216) from contacting the lateral flow assay (225) until a second actuation of the collection handle (238) is performed. In this embodiment, the pull tab (224) of the present invention is separated or otherwise removed, thereby allowing further depression of the collection handle (238). This further depression of the collection handle (238) pushes the sample processing and amplification assembly (250, 251) downward, causing the lancet (235) to engage and pierce the amplicon seal (222), thereby transferring the amplification products to the baseplate reservoir (223), where they can contact the assay transfer pad (226). Note that in one embodiment, a compression foot (249) may be positioned adjacent to the reaction chamber (213) to provide a backstop for compression of the transfer and amplification pads (215, 216).

[0055] Referring again to Figure 42, the assay transfer pad (226) of the present invention can be formed of glass fiber or other material that allows the amplification products released by the lancet (235) to be wicked up and transferred from the amplification pad (216). In this preferred embodiment, the assay transfer pad (226) of the present invention can be positioned such that a portion of the pad is disposed on the bottom surface of the baseplate reservoir (223). In this configuration, the assay transfer pad (226) is in fluid communication with the lateral flow assay (225), thereby transferring the amplification products from the assay transfer pad (226) to the lateral flow assay (225) by capillary action. In a preferred embodiment, the lateral flow assay (225) includes a conjugate pad in fluid communication with a membrane (228), which is further in fluid communication with an absorbent pad (229), all of which can be immobilized on a backing (230) support. In this embodiment, the cover (230) secures all or part of the components of the lateral flow assay (225) in place, protecting them from environmental exposure, and may include a transparent window (232) for visual observation of test results. Additionally, the cover (230) may generate a pressure gradient that allows amplification products to flow uniformly from the sample across the lateral flow assay (225) regardless of orientation. In another embodiment, the lateral flow assay (225) of the present invention may be mounted in a housing (201) so that the assay, including the membrane (228) that displays the assay result, may be removed for further processing or recording.

[0056] Exemplary methods, systems, and devices for the use and detection of lateral flow assays (225) derived from isothermal reactions such as RT-RPA or RPA are described above and further in U.S. Patent Application No. 17 / 686,387 to Sawyer et al., which is incorporated herein by reference.

[0057] The amplification device (200) of the present invention includes a heater assembly (253) thermally coupled to an amplification assembly (251). In one embodiment, the heater assembly (253) of the present invention generates heat at a constant temperature as a result of the liquid form of the exothermic phase change material (218) being in equilibrium with the solid form of the exothermic phase change material (218). In this embodiment, a predetermined amount of phase change material (218) and an exothermic fuel (220) are disposed in the reaction chamber (213) such that the amount is thermally coupled to the amplification pad (216). As shown in FIG. 43, the transfer pad and amplification pads (215, 216) are positioned in a central portion of the reaction chamber (213). A predetermined amount of phase change material (218), such as Rubitherm RT44HC, is disposed adjacent to the transfer pad and amplification pads (215, 216) such that the amount is thermally coupled. A quantity of exothermic fuel (220), such as a dry magnesium-iron (Mg-Fe) alloy, is supported by a cap (221) and is disposed adjacent to and similarly thermally coupled to the phase change material (218). In a preferred embodiment, a quantity of filler (219), such as sand, vermiculite, or other similar compound, is disposed adjacent to or mixed with the exothermic fuel (220) to generate a fuel component for an exothermic reagent (209), such as a salt solution, as described below.

[0058] When the exothermic fuel (220) contacts the exothermic reagent (209), an exothermic reaction is initiated, causing the phase change material (218) to reach a predetermined temperature, thereby heating the isothermal amplification of the amplification pad (216). More specifically, the phase change material (218) is configured to at least partially convert from its solid form to a liquid form upon heating by the exothermic chemical reaction generated by the exothermic fuel (220) and the exothermic reagent (209), thereby providing a controlled, substantially constant temperature for the amplification reaction in the reaction chamber (213). In one embodiment, the phase change material (218) includes a predetermined amount of paraffin, while in other embodiments, the phase change material (218) can be selected from metals, inorganic compounds, inorganic eutectics, and organic compounds. Additional phase change materials, exothermic fuels, and exothermic reagents are described in U.S. Patent No. 8,431,387, which is incorporated herein by reference.

[0059] The heater assembly (212) of the present invention is responsive to a heater actuation assembly (252). In a preferred embodiment, the heater actuation assembly (252) of the present invention includes an exothermic reagent (209) disposed within the reagent syringe (206) and separated from the heater assembly by a reagent seal (210) that, when punctured, allows the exothermic reagent (209) to be transferred to the heater assembly (212), where it contacts the exothermic fuel (220) and initiates an exothermic reaction. In a preferred embodiment, the exothermic reagent (209) is released and transferred to the heater assembly (212) in response to a first actuation of the collection handle (238). As shown in FIGS. 42-42, the reagent syringe (206) of the present invention includes an exothermic reagent (209), such as a sodium chloride solution, secured within the syringe body by the reagent seal (210), which prevents the exothermic reagent (209) from being transferred to the heater assembly (212). The reagent syringe (206) of the present invention further includes a plug (207) disposed above the exothermic reagent (209), the plug (207) being configured to prevent the exothermic reagent (209) from leaking from the syringe, for example, when the amplification device (200) is inverted or otherwise moved from a substantially upright position. Additionally, in some embodiments, a plunger (208) can be disposed above the plug (207).

[0060] As further shown in Figure 42, the reagent syringe (206) of the present invention is coupled with a gasket (211), which is preferably made of a compressible material and allows the reagent syringe (206) to be depressed in response to a first movement of the collection handle (238). In this embodiment, the secondary arm (243) of the collection handle (238) is positioned to be inserted into the reagent syringe (206), thereby depressing the plug (207) and optionally the plunger (208) as a result of the first movement of the collection handle (238). This first movement applies a downward force to the reagent syringe (206), thereby compressing the gasket (211), thereby allowing the reagent seal (210) to be punctured by the lancet (212) positioned below the seal and the transfer of the exothermic reagent (209) to the heater assembly (253).

[0061] As further shown in Figure 42, the amplification device (200) of the present invention includes an absorbent collar (236) positioned adjacent to the reaction housing (205) and configured to absorb exothermic reagents that may leak from the heater assembly (253). Also shown in Figure 42, the amplification device (200) of the present invention includes a frit (237) positioned above the heater assembly (253), particularly above the exothermic fuel (220), preferably adjacent to the filler (219), to contain the components of the heater assembly (253) necessary to generate the exothermic reaction, such as sand or magnesium iron, while allowing the exothermic reagent (209) (sodium chloride solution) to enter the assembly and, conversely, allow gases generated by the exothermic reaction to escape.

[0062] In particular, as shown in FIG. 36, the pull tab (224) of the present invention includes an opening configured to allow the secondary arm (243) of the collection handle (238) to be inserted through the tab and into the reaction syringe, thereby completing the first sequential depression stroke of the collection handle (238) as generally described herein.

[0063] As shown in Figure 58, the amplification device (200) of the present invention includes a check valve (409). In this embodiment, the check valve (409) is positioned between the filter (203) and the reaction chamber (213) containing the transfer pad and amplification pads (215, 216). In this configuration, the check valve (409) of the present invention contains the liquid in the sample after it passes through the filter and into the reaction chamber (213). This allows the amplification device (200) to tip or tilt during use without causing leakage, loss of material, or invalidating the assay due to backflow contamination of the sample.

[0064] The terminology used herein is for the purpose of 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 otherwise defined, all scientific and technical terms should be understood to have the same meaning as commonly used in the relevant technical field. As used herein, "about" or "approximately" means within 10% of a stated concentration range or within 10% of a stated time frame.

[0065] The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements, whether related or unrelated to those elements specifically identified by the "and / or" clause, may optionally be present. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may 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.

[0066] Nucleic acids and / or other moieties of the present invention may be isolated or "extracted." As used herein, "isolated" means separated from at least some of the components with which it is normally associated, whether naturally occurring or synthetically produced in whole or in part. Nucleic acids and / or other moieties of the present invention may be purified. As used herein, purified means separated from a majority of other compounds or entities. Compounds or moieties may be partially purified or substantially purified. Purity may be indicated by gravimetric measurements or determined using various analytical techniques, including, but not limited to, mass spectrometry, HPLC, etc.

[0067] The term "primer" as used herein refers to an oligonucleotide that can serve as a starting point for DNA synthesis under appropriate conditions. Such conditions include conditions in which the synthesis of primer extension products complementary to a nucleic acid strand is induced in the presence of four nucleoside triphosphates and an agent for extension (e.g., DNA polymerase or reverse transcriptase) in an appropriate buffer and at an appropriate temperature.

[0068] The primer is preferably 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 15 to 35 nucleotides, 18 to 75 nucleotides, and 25 to 150 nucleotides. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. The 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 appropriate primers for amplification of a given target sequence is well known in the art and described in the references cited herein.

[0069] As used herein, a biological marker ("biomarker" or "marker") is a characteristic that is objectively measured and evaluated as an indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic intervention, consistent with the NIH Biomarker Definitions Working Group (1998). Markers can also include patterns or ensembles of characteristics that indicate a particular biological process. Biomarker measurements can increase or decrease to indicate a particular biological event or process. In addition, if a biomarker measurement typically changes in the absence of a particular biological process, a constant measurement can indicate the occurrence of that process. In a preferred embodiment, a biomarker comprises one or more RNA transcripts that can indicate infection or other normal or abnormal physiological processes.

[0070] As referred to herein, the terms "nucleic acid," "nucleic acid molecule," "oligonucleotide," "polynucleotide," and "nucleotide" may be used interchangeably. These terms refer to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof, linear or branched, single-stranded, double-stranded, triple-stranded, or hybrids thereof, either in the form of separate fragments or as components of larger constructs. The term also encompasses RNA / DNA hybrids. Polynucleotides can include sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA. DNA molecules can be, for example, but are not limited to, complementary DNA (cDNA), genomic DNA, synthetic DNA, recombinant DNA, or hybrids thereof. RNA molecules can be, for example, but are not limited to, ssRNA or dsRNA. These terms further include oligonucleotides composed of naturally occurring bases, sugars, and covalent internucleoside linkages, as well as oligonucleotides containing non-naturally occurring portions that function similarly to their respective naturally occurring portions. The terms "nucleic acid segment" and "nucleotide sequence segment," or more generally, "segment," will be understood by those skilled in the art as functional terms that include both genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller engineered nucleotide sequences that encode or can be adapted to encode peptides, polypeptides, or proteins. All nucleic acid primers, e.g., SEQ ID NOs: 445-468, are presented in the 5' to 3' prime direction unless otherwise specified.

[0071] As used herein, "complementary" refers to the ability of a single strand of a polynucleotide (or a portion thereof) to hybridize to an antiparallel polynucleotide strand (or a portion thereof) by adjacent base pairing between nucleotides of the antiparallel polynucleotide single strand (which is uninterrupted by unpaired nucleotides), thereby forming a double-stranded polynucleotide between the complementary strands. A first polynucleotide is said to be "fully complementary" to a second polynucleotide strand if each and every nucleotide of the first polynucleotide base pairs with a nucleotide in the complementary region of the second polynucleotide. A first polynucleotide is not fully complementary to a second polynucleotide (i.e., is partially complementary) if a nucleotide in the first polynucleotide does not base pair with the corresponding nucleotide in the second polynucleotide. The degree of complementarity between polynucleotide strands significantly affects the efficiency and strength of annealing or hybridization between polynucleotide strands. This is particularly important in amplification reactions, which rely on binding between polynucleotide strands. An oligonucleotide primer is "complementary" to a target polynucleotide if at least 50% (preferably 60%, more preferably 70%, 80%, and even more preferably 90% or more) of the nucleotides of the primer base pair with nucleotides on the target polynucleotide.

[0072] 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 art-accepted taxonomy and as described herein. The term "detection" further includes quantification of the copy number of a microorganism in a sample, for example, in a microliter (or milliliter or liter) or microgram (or milligram or gram or kilogram) sample. The term "detection" also includes the identification of an infection in a subject or sample.

[0073] As used herein, the term "pathogen" refers to an organism (including a microorganism) that causes disease by directly infecting other organisms (e.g., animals and plants) or by producing an agent (e.g., a bacterium that produces a pathogenic toxin) that causes disease in other organisms. As used herein, pathogens include, but are not limited to, bacteria, protozoa, fungi, nematodes, viroids, and viruses, or any combination thereof, each of which is capable of inducing disease by itself or in association with another pathogen in vertebrates, including, but not limited to, mammals, and in humans, including, but not limited to, humans. As used herein, the term "pathogen" also encompasses microorganisms that would not normally be pathogenic in a non-immunocompromised host.

[0074] As used herein, the term "infection" or "infected" refers to the presence of a microorganism in a subject's body and / or cells. For example, a virus may infect a subject's cells. A parasite (e.g., nematode, etc.) may infect a subject's cells / body. In some embodiments, the microorganism may include a virus, a bacterium, a fungus, a parasite, or a combination thereof. According to some embodiments, the microorganism is a virus, such as a dsDNA virus (e.g., adenovirus, herpesvirus, poxvirus, etc.), a ssDNA virus (e.g., parvovirus, etc.), a dsRNA virus (e.g., reovirus, etc.), a (+)ssRNA virus (+)sense RNA (e.g., picornavirus, togavirus, etc.), a (-)ssRNA virus (-)sense RNA (e.g., orthomyxovirus, rhabdovirus, etc.), a ssRNA-RT virus (+)sense RNA with a DNA intermediate in its life cycle (e.g., retrovirus, etc.), a dsDNA-RT virus (e.g., hepadnavirus, etc.), etc. In some embodiments, the microorganism is a bacterium, such as a gram-negative bacterium, a gram-positive bacterium, etc. In some embodiments, the microorganism is a fungus, such as a yeast, a mold, etc. In some embodiments, the microorganism is a parasite, such as a protozoan or a helminth, etc. In some embodiments, infection with the microorganism can result in disease and / or clinically detectable symptoms in a subject. In some embodiments, infection with the microorganism may not cause clinically detectable symptoms. In some embodiments, the microorganism is a symbiotic microorganism. In additional embodiments, the microorganism can include archaea, protists; microalgae (green algae), plankton, and planaria. In some embodiments, the microorganism is unicellular (single cell). In some embodiments, the microorganism is multicellular.

[0075] 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 combination of pathogens.

[0076] Some embodiments of the present invention involve amplifying nucleic acids from a sample. As used herein, a "sample" can be any volume of material, including one or more nucleic acids, preferably a liquid, semi-liquid, or other fluid sample containing one or more nucleic acids. Examples include environmental samples such as water, soil, and industrial samples, as well as waste streams. In additional embodiments, the sample can be a pharmaceutical sample containing isolated or complex mixtures of nucleic acids, particularly therapeutic nucleic acids. Additional samples include prokaryotic and eukaryotic samples, as well as biological samples containing plants, fungi, and / or nucleic acids isolated therefrom.

[0077] As used herein, the term "biological sample" includes samples from any bodily fluid or tissue. Biological samples or samples suitable for use in accordance with the methods provided herein include, but are not limited to, blood, serum, urine, saliva, tissues, cells, and organs, or portions thereof, as well as isolated nucleic acid samples from a subject or other organisms, such as bacteria, plants, fungi, or other cells. A "subject" is any organism of interest, generally a mammalian subject, preferably a human subject. Some embodiments of the present invention include detecting the level of a biomarker in a sample from a patient, wherein the presence or expression level of the biomarker indicates infection or possible infection by one or more pathogens.

[0078] Any isothermal amplification protocol can be used in accordance with the methods provided herein. Exemplary types of isothermal amplification include, but are not limited to, 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 (HDA), nicking enzyme amplification reaction (NEAR), signal-mediated amplification of RNA (SMART), rolling circle amplification (RCA), isothermal multiple displacement amplification (IMDA), single primer isothermal amplification (SPIA), recombinase polymerase amplification (RPA), and polymerase spiral reaction (PSR) (available on the World Wide Web at nature.com / articles / srepl2723). In some cases, a forward primer is used to introduce a T7 promoter site into the resulting DNA template, allowing transcription of the amplified RNA product by T7 RNA polymerase. In other cases, a reverse primer is used to add the trigger sequence of the toehold sequence domain.

[0079] As used herein, the term "amplified" refers to a polynucleotide that is a copy of a specific polynucleotide produced in an amplification reaction. According to the present invention, the amplification product may be DNA or RNA, and may be double-stranded or single-stranded. The amplification 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. This term generally refers to an expected specific amplification product of known size generated using a given set of amplification primers.

[0080] As used herein, the term "lateral flow assay" refers to an assay in which sample flow occurs at least partially parallel to a surface that can optically image the sample and / or chemical or physical phenomena caused by the sample.

[0081] The invention generally described herein will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects of embodiments of the invention. These examples are not intended to limit the invention, and one of ordinary skill in the art will appreciate from the above teachings and the following examples that other techniques and methods may be employed to satisfy the claims and without departing from the scope of the claimed invention. Indeed, while the invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the scope of the invention as encompassed by the appended claims. [Example]

[0082] Example 1: Overview of the diagnostic device As mentioned above, in a preferred embodiment, the diagnostic device of the present invention describes a self-contained device that uses a minimal approach to processing saliva for biomarker amplification, as well as a single low-temperature (near body temperature) amplification method and an integrated lateral flow assay for amplification readout. In a preferred embodiment, the diagnostic device of the present invention may include multiple injection-molded parts, a porous pad material embedded with lyophilized sample processing reagents, a filter stack, and a lateral flow assay (LFA) (Figures 1B and 1C).

[0083] Example 2: Saliva sample collection and initial processing As depicted in Figure 1 , in a preferred embodiment, a biological sample, preferably a saliva sample, is collected by holding an absorbent material in a subject's oral cavity for a specified amount of time and then inserting and compressing the material into the device of the present invention to express the biological sample. Initial evaluation determined that the saliva collection volume requirement was at least 1.0 mL of saliva. A sample collection of less than 1.0 mL would not saturate the amplification pad material and therefore would not provide the volume required to activate the lateral flow strip. The inventors initially selected a 30 mm x 8 mm collection pad manufactured by Porex, Inc., made from a porous high-relief medium (HRM) polypropylene / polyethylene (PP / PE) blend. This material was tested by direct oral saliva collection (holding a swab in the oral cavity and measuring the mass every minute after collection to calculate the absorption volume). This material was only able to collect less than 1.0 mL of saliva across multiple individuals. Therefore, it was determined that further optimization of the saliva collection pad's material and dimensions was necessary to accommodate the volume requirements of the device.

[0084] Seven additional collection pad materials, varying in dimensions, density, and composition, were screened to ensure direct oral saliva collection of volumes greater than 1.0 mL (Table 1). The maximum collection volume for each material was determined by immersing the collection material in phosphate-buffered saline (PBS) and measuring the volume absorbed (Figure 2A). Furthermore, the maximum oral collection volume across individuals was tested by holding the swab directly in the oral cavity until fully saturated and measuring the volume absorbed (Figure 2B).

[0085] Of the materials tested, Salimetrics' SalivaBio Oral Swab (SOS) material demonstrated the highest volume collection capability, with a maximum absorption volume of 1.6 mL. Furthermore, this material consistently absorbed greater than 1.0 mL during direct oral saliva collection. The 35.5 mm x 8 mm Porex HRM fiber media collected sufficient volumes in PBS immersion tests, but was unable to reliably collect more than 1.0 mL during direct oral collection. Smaller dimensions of the same material were not tested because the 35.5 mm x 8 mm size did not absorb enough saliva. Of the seven materials screened, the 30 mm x 10 mm Salimetrics SOS met all requirements and was therefore selected for integration into the initial embodiment of the diagnostic device of the present invention.

[0086] Example 3: Monitoring RNase activity and optimizing inhibition We monitored RNase activity in representative saliva samples and sought to optimize RNase inhibition. Saliva contains abundant RNases, and limiting their activity is crucial because these enzymes degrade the RNA template targeted by our device. Limiting RNase activity allows time for reverse transcriptase to synthesize complementary DNA (cDNA) from the RNA, thereby providing a template for the RPA reaction. While all saliva samples contain abundant RNases, significant variability in RNase activity exists between individuals. To measure RNase activity in saliva samples, we used the Invitrogen RNase Alert Assay (catalog number AMI964). This assay contains a short single-stranded RNA (ssRNA) probe with a fluorescent dye at one end and a quencher at the other. RNase degrades the ssRNA probe, releasing the quencher and allowing the fluorescent dye to fluoresce (Figure 3A).

[0087] To determine the variability of RNase activity between individuals, we tested saliva samples from five donors with and without the addition of 4 U / μL mouse RNase inhibitor (mRI, NEB catalog no. M0314) in the RNase Alert Assay (Figure 3B). While there was certainly variability in RNase activity among the five saliva samples, the difference between the highest and lowest activity samples was less than two-fold. Importantly, the addition of 4 U / μL mRI reduced RNase activity to negligible amounts in all five samples, providing an approach to protect salivary RNA within the device.

[0088] In one embodiment, mRIs would be introduced into the saliva sample prior to lysis to inhibit RNases prior to releasing RNA from cells. In an alternative configuration, mRIs could be lyophilized within the amplification pad, concentrating the mRI treatment only in the 100 μL lysis tip used for amplification; however, this approach leaves the RNA unprotected for the short period between lysis and rehydration of the amplification pad. To determine whether the RNA can remain unprotected during this time, we performed reverse transcription quantitative polymerase chain reaction (RT-qPCR) on a panel of relevant biomarkers to measure RNA degradation immediately after lysis (Figure 3C). Each time point used the same pooled saliva sample but was passed through separate inventive devices, resulting in some variability between time points. A slight increase of 1–2 cycle threshold (Ct) values ​​was observed within a few minutes after lysis, indicating some RNA degradation. However, this represents a relatively small amount of RNA loss and should not affect the ability to amplify target biomarkers.

[0089] To confirm that the lyophilized mRI within the amplifier pads could rehydrate and inhibit salivary RNase, four amplifier pads were rehydrated with saliva and the contents of the rehydrated pads were measured using the RNase Alert Assay (Figure 3D). Near-complete inhibition of RNase activity was observed in the saliva-rehydrated amplifier pads, demonstrating an efficient approach for inhibiting salivary RNase within the device.

[0090] Example 4: Addressing additional amplification inhibitors in saliva In a preferred embodiment, collected saliva samples provide all of the liquid required for reconstitution of reaction components. To identify challenges in reconstituting RPA reagents using saliva, we monitored RPA efficiency while increasing the concentration of processed saliva. Saliva was squeezed from the collection pad, treated with lyophilized lysis buffer, and then passed through 10 μm and 1 μm filter stacks. To eliminate the compounding effects of RNase activity and focus on amplification inhibitors, we monitored RPA efficiency using a cDNA template. RPA efficiency was maintained up to 25% (v / v) of processed saliva in the reaction; however, above 25%, the RPA reaction was completely inhibited by saliva (Figure 4A).

[0091] To identify amplification inhibitors in saliva, we first eliminated protein-based inhibitors by heat-treating saliva samples before adding them to the RPA reaction, and observed similar inhibition even with increasing saliva concentrations. We further ruled out the possibility that the high abundance of nucleic acids in saliva was the cause of inhibition by demonstrating that RPA was not inhibited even with the addition of 3 μg of cell line DNA. After eliminating protein and nucleic acid inhibitors, we conducted a literature search for additional compounds that inhibit the amplification reaction in saliva. We found reports that positively charged molecules in saliva, such as divalent cations and positively charged macromolecules, can be inhibitory to DNA polymerase and reverse transcriptase, which are necessary for amplification in our device. Previous reports have identified Chelex-100 (BioRad catalog number 1421253) as an effective agent for removing positively charged inhibitors from saliva and obtaining samples suitable for amplification. Chelex-100 is a styrene-divinylbenzene copolymer resin (wet bead size 75-150 μm) modified with paired iminodiacetate ions to chelate positively charged molecules.

[0092] We tested Chelex-100 for its ability to remove inhibitors of RPA in saliva. When saliva was treated with 5% or 20% Chelex-100 (w / v) solutions for 30 min, efficient amplification was maintained in RPA reactions containing 95% saliva, whereas amplification was inhibited in untreated saliva (Figure 4B). Chelex-100 maintained its effectiveness even after short-term direct treatment, in which saliva was passed directly through a Chelex-100-packed column (Figure 4C). For direct treatment of saliva, 20% Chelex-100 (w / v) was more advantageous than 5%. We further investigated the efficiency of reverse transcription-RPA (RT-RPA) in saliva treated with 20% Chelex-100. We found that our current reverse transcriptase of choice remained susceptible to inhibitors, even after treatment with Chelex-100 (Figure 4D). Increasing the concentration of reverse transcriptase showed promise in overcoming this problem, and we further addressed it by screening for more inhibitor-resistant reverse transcriptases, as described in more detail below.

[0093] To incorporate a Chelex-100 column into our device, we packed beads between the 10 μm and 1 μm filters in the filter stack. Due to the resin's large size, Chelex-100 cannot pass through the 1 μm filter, which is necessary to prevent Chelex-100 from interacting with the magnesium required for the RT-RPA reaction downstream of the device. In a preferred embodiment, the diagnostic device can be packaged dry with a desiccant for maximum stability, but Chelex-100 is supplied in a hydrated form. If the resin is allowed to dry within the device, air pockets will form, and these must be expelled to maintain efficacy. This problem can be addressed in two ways: 1) Drying the Chelex-100 before packing it into the filter stack minimizes the formation of air pockets. 2) Adding a 0.4 μm filter after the 1 μm filter facilitates the passage of trapped air through the dried Chelex-100 prior to the saliva flow, facilitating more efficient rehydration of the resin by saliva.

[0094] Example 5: Identification and optimization of dissolving pad materials In one embodiment, the lysis buffer is lyophilized into a PP / PE blend fiber pad. When the saliva collection pad is inserted into the device, the collection pad and dissolution pad come into contact, allowing saliva to pass through and rehydrate the lysis buffer (Figure 1). To reduce the volume requirements of the device, multiple dissolution pad sizes and fiber densities were tested. Sample retention tests were performed on dissolution pads with a 10 mm diameter and heights of 6, 8, 10, and 12 mm. While shorter dissolution pads retained less volume than taller pads, the overall compression of the collection pad and the amount of sample squeezed out decreased with height (Figure 5A). Due to these two opposing effects, changing the height of the dissolution pad did not change the sample volume flowing to downstream components of the device (Figure 5B).

[0095] Volume retention tests were also performed across pad densities (0.07 g / cc vs. 0.16 g / cc) and wider diameters (Figures 5C and 5D). As observed when adjusting pad height, lower density materials retained less volume, but also reduced sample squeeze-out from the collection pad. Increasing the diameter to 11 mm reduced sample retention and increased sample squeeze-out from the collection pad, ultimately optimizing delivery to the remainder of the device. The 11 mm diameter also prevents sample from flowing around the dissolving pad and ensures that all sample contacts the dissolving pad as it passes through the device. Therefore, the 11 mm x 10 mm, 0.16 g / cc density dissolving pad was selected for incorporation into the device because it exhibited optimal squeeze-out and retention characteristics while ensuring sample passage through the material.

[0096] Example 6: Maintaining the leading edge of a lysed sample To minimize the volume of pad material used in the device and the overall device dimensions, the dissolving pad is designed to dissolve only the first 0.5 mL of saliva expressed from the collection pad. After the lysed sample, additional undissolved saliva is injected, forming a "front" of lysed sample that must be maintained until it reaches the amplification reaction within the device. To determine the feasibility of maintaining a dissolving front within the device, we dried xylene cyanol dye into the dissolving pad and monitored its position after sample processing (Figure 6). Using this surrogate indicator of the sample dissolving front, we demonstrated that sample expressed through the dissolving pad reconstitutes the reagents dried within the pad, which are then extruded downstream of the device along with additional sample from the collection pad. The transfer and amplification pads rapidly absorb the sample, preventing mixing of the lysed and undissolved samples and maintaining the lysed sample front. This is indicated by the higher concentration of dye absorbed by the amplification pad compared to the transfer pad (Figure 6).

[0097] Example 7: Optimization of lysis buffer and incorporation of optimized sample processing The device of the present invention can employ a minimal approach to saliva processing for biomarker amplification and detection. For example, in one embodiment, a lysis buffer formulation containing Buffer 20 (100 mM guanidine hydrochloride, 5 mM TECEP-HCl, 7.5% Tween-20, 0.00167% digitonin, and 4 U / μl RNase inhibitor) can be used to achieve three key functions: 1) inactivation of salivary RNases, 2) compatibility with RT-RPA, and 3) release of RNA biomarkers from cells in saliva without the use of heat.

[0098] In continued testing, we found that 100 mM guanidine reduced the overall efficiency of the RT-RPA reaction (Figure 7A). Furthermore, as mentioned above, we found that by transferring the RNase inhibitor from the lysis buffer to the amplification reaction, we could reduce the amount of RNase inhibitor used and reduce costs. Therefore, we modified the lysis buffer formulation to Buffer 21 (5 mM TCEP-HCl, 7.5% Tween-20, 0.00167% digitonin) and removed guanidine hydrochloride and the RNase inhibitor. This optimized formulation improved the overall reaction efficiency (Figure 7A). We lyophilized Buffer 21 into selected lysis pad materials and processed saliva from five donors through the device (Figure 7B). This process involves squeezing the sample from the collection pad, which forces the sample through a lysis pad embedded with Buffer 21 and 4 U / μl mRI, followed by a 10 μm filter, 100 mg of Chelex-100, and a 1 μm filter. The resulting sample was used to reconstitute the RPA reagents (Twist rehydration buffer and Twist enzyme), and reverse transcriptase and modified RPA primers were added to the reaction.

[0099] Example 8: Optimization and integration of pad-driven RT-RPA amplification Saliva processed through the filter and buffer of the device of the present invention was shown to remove amplification inhibitors and reduce viscosity, enabling efficient RPA amplification using cDNA templates. While cDNA-templated reactions amplified robustly across samples, in a preferred embodiment, the device of the present invention will incorporate reverse transcriptase (RT) to first convert salivary RNA biomarkers to cDNA. In initial RT-RPA experiments using 0.0625 U / μl of Transcriptor RT, we observed persistent RT-specific inhibition by saliva. To mitigate this issue, we screened eight RT enzymes at multiple concentrations, focusing on their resistance to inhibitors found in saliva (Table 2).

[0100] Initial RT screening was performed to determine compatibility with RPA reaction conditions, including low-temperature incubation and buffer conditions different from PCR. Furthermore, primer-dimer formation was monitored in the presence of each RT. Four of the most promising RT enzymes were ultimately selected based on their ability to overcome saliva inhibition across samples and maintain high amplification efficiency for exogenous RNA (A549 cell line RNA) and endogenous RNA (saliva-derived CXCL8 RNA) even in high saliva concentrations. The RT enzymes, along with the RPA reagents, were lyophilized onto amplification pad material, and RT performance was evaluated using water, 96.6% saliva, 48.3% saliva, and saliva without Chelex-100 treatment. All conditions were tested with and without spike-in of additional RNA template (Figure 8). In RNA spike-in reactions, all RTs showed LFA-positive results in water and 48.3% saliva, and no primer-dimer formation was observed. None of the RT enzymes were able to amplify RNA in the presence of saliva without Chelex-100 treatment. The difference in amplification efficiency between the RT enzymes was most pronounced in the 96.6% saliva, where MMLV HP produced the strongest LFA signal and was the only RT in this experiment to amplify endogenous salivary RNA in high saliva concentrations. The conditions shown in Figure 8 were repeated in multiple experiments and are used as representative data. All four RTs were able to amplify endogenous RNA to varying degrees.

[0101] Example 9: Optimization of pad materials and excipients Initial attempts at pad-driven amplification demonstrated inefficiencies and indicated that further optimization was required for sufficient amplification. Amplification pad material was explored as one avenue for optimizing performance. Twelve materials were evaluated, and three were selected for wet-lab testing (Figure 9). All three materials were HRM PP / PE blends manufactured by Porex, ranging in density from 0.25 g / cc (highest density) to 0.07 g / cc (lowest density). The high-density material produced the lowest amplification signal, while the lowest-density material produced the highest signal, though slightly stronger than the medium-density material. The low-density material produced stronger amplification but proved difficult to integrate into device fabrication due to its lack of rigidity. The medium-density pad was selected for further optimization due to its ease of use.

[0102] The medium-density pad offered the best balance of amplification efficiency and rigidity, but the reaction efficiency was significantly reduced compared to liquid reactions. To improve performance, we conducted a screen to optimize the lyophilization excipients as a means of improving reaction efficiency (Table 3). We found that adding 0.1% Tween-20 to the 2% sucrose and 1% mannitol already used for lyophilization significantly improved pad amplification efficiency. Henceforth, all amplification pads were dried with 0.1% Tween-20 added.

[0103] To further improve reaction efficiency and prevent template loss in the pad materials upstream of amplification, we blocked these materials with 0.2% Tween-20 and 1% PEG20K. The combined effect of adding excipients and blocking the upstream materials significantly improved reaction efficiency in pad-driven amplification.

[0104] Example 10: Integration of pad-driven RT-RPA amplification Previous data showed that the limit of detection (LoD) in liquid RPA reactions for reference biomarkers was 10 copies of template (Figure 10A). These data were measured using a dilution series of double-stranded DNA product spanning the desired amplification region of the biomarker of interest, with the template being approximately 10 copies per reaction. 6 In this liquid RPA LoD study, CALR DNA was used as the reference biomarker, but similar LoDs have been observed when CXCL8 RNA was used as the reference.

[0105] We replicated the LoD study with pad-driven amplification, under the assumption that amplification efficiency would likely be reduced. A new dilution series of double-stranded DNA product was generated, again at approximately 10 per reaction. 6 This dilution series was run through a pad-driven RPA, resulting in a template LoD of approximately 10 in a 100-μl reaction. 3 Preliminary experiments using RNA templates suggested an additional 10- to 100-fold decrease in efficiency in RT-RPA, with an LoD of 10 in a 100-μl pad-driven RT-RPA reaction. 4 ~10 3 After incorporating the improvements described above into pad-driven amplification, we integrated an amplification pad into our device. The sample passed through the collection pad, dissolution pad, and filter stack within the device before entering the base plate, where it was wicked up by a transfer pad (HRM PP / PE blend fiber from Porex with a density of 0.16 g / cc). From the transfer pad, the sample passed through a second transfer pad of the same material and then onto the same pad material containing lyophilized RT-RPA reagents.

[0106] Amplification assemblies were generated on a blocked transfer pad (1% PEG20K, 0.2% Tween-20) followed by an amplification pad embedded with lyophilized RT-RPA reagents and optimized excipients (0.1% Tween-20, 2% sucrose, 1% mannitol) (Figure 11A). Template (in vitro transcribed CXCL8 RNA) was pipetted onto the baseplate and allowed to passively wick through the material toward the amplification pad. Magnesium was supplied with the template to activate the RT-RPA enzyme, but for this purpose the magnesium eventually dried into the transfer pad material. Sufficient RNA template was obtained by 10 7 The copies were added to the base plate so that they reached the amplification pad. The device was then incubated in an incubator set at 44°C for 20 minutes, after which amplification in this setup was successfully observed on the lateral flow strip (Figure 11B).

[0107] Next, we extended this to integration into the entire device. In this setup, the template was absorbed into the collection pad and then squeezed into the device, allowing the template to pass through the dissolution pad, the filter / Chelex stack, and onto the baseplate (Figure 11C). The template was then wicked up through the pad material toward the amplification pad, similar to the previous setup. After incubating the device in an incubator, only 10% of the template delivered to the amplification pad was absorbed. 4 Although the lateral flow strips were replicated, successful amplification was observed (Figure 11D). In this experiment, double-stranded DNA covering the amplified region of CXCL8 was used as the template, but subsequent experiments demonstrated that RNA templates could also be successfully amplified in the device of the present invention.

[0108] In a preferred embodiment, the amplification assembly and base plate include separate compartments to contain excess saliva and eliminate alternative routes to the LFA other than via the amplification pad. Furthermore, compression of the sample pad was found to create excessive pressure in the amplification assembly. This pressure buildup led to pressure venting through the amplification pad, causing the sample to exit the distal end of the amplification pad, resulting in reagent loss and premature flow to the LFA. This problem was resolved by adding a pressure relief location, which in this embodiment included three 1 mm diameter pressure relief holes on the front of the amplification assembly. Additionally, to better regulate the flow front within the device, the amplification assembly device is configured to first direct the sample to the base plate, from which the lysed sample front is wicked up into the transfer pad and then pushed forward to the amplification pad.

[0109] Example 11: Optimization of sample volume tolerance During functional testing, we evaluated the range of volumes acceptable to the device. The minimum required volume for our device is defined by the retention volume of the pad material placed throughout the device and the volume required to activate the LFA. When we tested a range of volumes collected between individuals using our selected collection pad material, we observed variability in collected volumes between users, ranging from 1.0 mL to 1.5 mL. When testing this volume range, we found that the device could only accept sample volumes between 1.0 and 1.1 mL. Volumes greater than 1.1 mL caused flooding of the device, with excess sample exiting the pressure relief hole in the front of the amplification assembly and resulting in premature flow to the LFA.

[0110] To expand the device's volume tolerance, we improved the mechanism by which excess sample is processed. First, we relocated the pressure relief hole to the side of the amplifier assembly by redesigning the mating surface between the amplifier assembly and base plate (Figure 14). Second, we added absorbent material near the pressure relief hole to capture expelled sample and reduce the possibility of premature LFA activation. These improvements to the amplifier assembly and base plate successfully expanded the device's volume tolerance. Functional testing with both PBS and saliva samples using the revised injection-molded parts demonstrated that a wide range of volumes was tolerated, achieving a 100% (24 / 24) success rate.

[0111] Example 12: Evaluation of isothermal chemical methods In total, over 15 isothermal amplification techniques were evaluated for suitability in the device of the present invention. From this preliminary list, four isothermal chemistries were selected for in-house testing: helicase-dependent amplification (HDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), and recombinase polymerase amplification (RPA).

[0112] All four amplification chemistries were performed as endpoint reactions using primers specifically designed for each chemistry, followed by visualization using agarose gel electrophoresis. Each isothermal chemistry showed varying degrees of success, but all were inferior to RPA. Both HDA and NASBA resulted in high levels of nonspecific amplification, with little target product amplified using either reaction chemistry. SDA failed to achieve any target amplification, even after multiple attempts at primer and enzyme optimization. Because HDA and NASBA amplified the intended target, albeit minimally, real-time quantification protocols were established for each isothermal chemistry. Both chemistries yielded remarkably similar Ct values ​​between templated and nontemplated reactions. The high level of nonspecific amplification indicated by Ct values ​​can make it difficult to distinguish between positive and negative samples.

[0113] Example 13: Recombinase Polymerase Amplification (RPA) Reaction Conditions To evaluate recombinase polymerase amplification (RPA) reaction conditions that allow discrimination between biomarker concentrations, double-stranded DNA products were generated for the three biomarkers of interest, spanning the desired amplification region. These products were assayed by qPCR as a dilution series targeting a Ct range of 17-35, corresponding to approximately 10 copies to approximately 10 copies per reaction. These dilution series were then used as starting templates for RPA reactions at temperatures near 37 °C as a means of correlating Ct values ​​from qPCR with RPA readouts on an LFA.

[0114] To determine the expected baseline for template studies at various temperatures, we first performed a dilution series using one of the reference biomarkers, CALR, as the template. The CALR template copy numbers tested ranged from 17.53 to 35.0 Ct (approximately 10 per reaction). 6 The dilution series corresponded to 10 copies to 10 copies. This dilution series was run using the TwistAmp® Basic Kit (TABAS03KIT; TwistDx) at temperatures ranging from 29 to 43°C for 20 minutes, followed by visualization of the amplification products on an agarose gel. Band intensities from the gel were quantified using ImageJ and normalized between experiments (Figure 10A). All concentrations of CALR template tested (including as few as 10 copies) were detectable at incubation temperatures of 37°C or higher. The reactions showing the two lowest amplicon intensities at each temperature were also assayed on lateral flow strips (Figure 10B). If positive, reactions using higher copy numbers of template incubated at the same temperature were presumed positive but were not performed on lateral flow strips to conserve reagents. The limit of detection (LoD) increased as the reaction temperature decreased. When the incubation temperature was 31°C, the LoD of the assay was 10 copies per reaction, and at 29°C, it was 10 copies per reaction. 6 None of the CALR inputs tested, including the highest copy input, was detected.

[0115] After determining that standard RPA reactions could detect as few as 10 copies of template when incubated near body temperature, we determined whether the reaction time could be shortened. Using the same template and dilution series as above, identical reactions were incubated at 34°C for 5, 10, 15, or 20 minutes (Figure 15). After a 5-minute incubation period, no detectable amplification was observed at any template concentration tested. After 10 minutes, detectable amplification occurred for templates up to 104, which increased to 103 after 15 minutes of incubation. No difference was observed between the 15- and 20-minute time points for detectable amplification on the lateral flow strips. Therefore, it was determined that a 15-minute incubation period was sufficient for amplification, and that shorter incubations could potentially be used to differentiate between biomarker copy numbers.

[0116] Example 14: Consistency of LoD across biomarkers After establishing a baseline LoD across time and temperature using the exemplary CALR reference biomarker, the LoD study was replicated using two infectious disease biomarkers, CXCL8 and DDX58. Because all CALR dilutions were detectable above 37°C, a dilution series was generated for each biomarker as with CALR and then used as reaction templates at temperatures ranging from 29°C to 37°C. The LoD study for these two biomarkers uses a 15-minute incubation time. When comparing the LoDs between biomarkers, some differences were observed. These differences included a 10-minute incubation time at 29°C. 5 At 37°C, the LoD of both infectious disease biomarkers increased (10 copies), whereas at 37°C the LoD of CALR was 10 copies. 3These detection differences may be due to reduced sensitivity of the infection line on the lateral flow strip, differences in incubation parameters between the reference and infectious disease biomarkers, and primer efficiency for each amplification target.

[0117] Example 15: Extended Time Course Study To further characterize the RPA reaction, a time course evaluation at 34°C was also performed for the exemplary infectious disease biomarkers. Because the CALR template showed no detectable amplification after 5 minutes, the time course experiment was modified to incubation times of 7, 11, and 15 minutes for the two infectious disease biomarkers. Both biomarkers produced visible bands after 7 minutes when visualized on an agarose gel, but amplification did not reach detectable levels on lateral flow strips (Figure 17). For the DDX58 biomarker, 10 amplifications were observed by 11 minutes. 5 Copy template was detectable on the lateral flow strip. Between the 7 and 11 minute time points, the detection limit on the agarose gel was 10 per reaction. 3 Although the number of copies of the template improved, the amount of amplicon generated was insufficient for detection on the lateral flow strip. Between the 11 and 15 minute time points, the LoD on the lateral flow strip did not improve, and after 15 minutes it was 10 5 Although previous experiments showed that the LoD of the DDX58-initiated template was 10 when incubated at 33.4°C, 3 For CXCL8, the agarose gel showed 10 copies after 11 minutes. 3 On lateral flow strips, CXCL8 was detected at 10 min after 11 min and did not improve at 15 min. 5 Copy the template until it was detected, and at 15 minutes, 10 3 The differences in detection limits between biomarkers indicate that amplification rates are likely biomarker and primer dependent.

[0118] Example 16: Reverse transcription recombinase polymerase amplification (RT-RPA) and integrated device After establishing a baseline LoD for RPA at various time and temperature points using DNA templates, preliminary RT-RPA studies were conducted using RNA in vitro transcripts (IVT) generated for each of the previously characterized biomarkers. A dilution series of IVT specifically synthesized for the three biomarkers was run in RT-qPCR, again targeting a Ct range of 17–35. The DDX58 biomarker dilution series was excluded from the initial RT-RPA study because it was not within the target Ct range. For CALR and CXCL8, 15-minute RT-RPA reactions were performed at incubation temperatures of 31°C, 37°C, and 43°C, followed by visualization using agarose gel electrophoresis (Figure 18). RT-RPA does not appear to significantly affect reaction efficiency in most cases. Even when reaction efficiency decreases, the LoD does not decrease by more than one order of magnitude compared to RPA using DNA templates. Furthermore, the decrease in LoD is template dependent.

[0119] At incubation temperatures of 37°C and 43°C, all dilutions of CALR IVT were detectable on the lateral flow strip. At these temperatures, all dilutions of CALR DNA were also detectable. Surprisingly, all dilutions of CXCL8 IVT were also detectable at 43°C, with down to 100 copies of starting template detectable at 37°C. This was an improvement over the LoD of 1,000 copies observed when using CXCL8 DNA product as a template at 37°C. Because CXCL8 DNA template RPA was not performed at 43°C, we cannot determine how the efficiency of the reverse transcriptase reaction at this temperature is affected. At lower incubation temperatures, a decrease in RT-RPA efficiency begins to be observed. At 31°C, the CALR DNA template was detected at 10 copies. 4 Although RT-RPA using an IVT template was able to detect up to 10 copies, 5The transcription efficiency at low temperatures may be due to the temperature sensitivity of the reverse transcriptase used in RT-RPA. Furthermore, the difference in LoD between the biomarkers may be due to differences in the secondary structure of each RNA template.

[0120] Example 17: Detection limits of isothermal amplification products on lateral flow strips To determine the LoD of each band on the strip, modified double-stranded DNA products (called mimics) for reading on the lateral flow strip were generated for both the reference and infected lines. The concentrations of both mimics were quantified, and a dilution series was generated for each mimic. Lateral flow strips were run with 5 ng, 1 ng, 0.5 ng, 0.25 ng, or 0.1 ng of either the infected or reference mimic in a final volume of 80 μl PBS (Figure 19). After running the strips for 15 minutes, an LoD of 0.5 ng was observed for the reference line, and an LoD of 1 ng was observed for the infected line.

[0121] Example 18: Conditions for multiplexed isothermal amplification reactions We developed a protocol for designing and selecting primers for use in RT-RPA that amplifies mRNA while excluding genomic DNA (gDNA) or off-target DNA / RNA and limits primer-dimer formation. RPA primers were synthesized with 5'-end modifications to enable detection on lateral flow strips. The forward primer was 5' fluorescein (FITC)-modified, the reverse primer for the reference biomarker was 5' biotin (Bio)-modified, and the reverse primer for the infection biomarker was 5' digoxigenin (Dig)-modified (Figure 20). Therefore, amplification of the reference (control) biomarker within the device was detected at the reference control line of the LFA in all samples, while amplification of the infection biomarker was detected at the infection line only in infected individuals.

[0122] To design primers for RT-RPA, we used NCBI primer design software, following the parameter guidelines in the TwistDx assay design manual and parameters optimized based on our own empirical data. The key parameters for enabling selective and efficient amplification of target templates are as follows:

[0123] Maximum product size: 200bp Primer size: 27-36bp Primer GC content (%): 35-70 Primer melting temperature: 59-70℃, maximum Tm difference 10℃ Primers must be separated by at least one intron (length >800 bp) in the corresponding genomic DNA. From the output generated using the above search parameters, primers were screened for potential off-target amplification using the following references: refseq mRNA (H. sapiens) and refseq representative genomes (H. sapiens and bacteria). Primers with mismatches in the 3' region and / or at least seven mismatches distributed throughout the entire length of the primer were selected for off-target amplification. Primers that met the above selection criteria were subjected to thermodynamic analysis to select primers that are unlikely to form primer dimers and lead to nonspecific readouts on LFA. The predicted interactions between the forward and reverse primers were evaluated using IDT's Oligo Analyzer Tool (heterodimer analysis). From this output, primers predicted to interact with a delta G value close to 0 (low probability) or at the internal 5' end (interactions that cannot be extended by polymerase) were selected.

[0124] After the selected primers arrived in the laboratory, they were screened by RPA using cell line cDNA as template and a no-template control to assess the level of primer noise. (Figure 21) shows an example of such a screen. In this example, two RACK1 primer sets, RPA2 and RPA6, were selected for further use based on their strong signal in the template reaction and minimal primer noise in the no-template control.

[0125] In total, we conducted wet-lab screening of over 130 primer sets for 25 targets using the above method. Nearly all of these tested primer sets were able to efficiently amplify target cDNA or RNA. Primers that met the selection criteria of absence of primer noise in no-template control reactions were ordered with LFA-compatible 5'-end modifications. The modified primers were evaluated for false-positive results on lateral flow strips in no-template control reactions. Only primer dimers containing both a forward and a reverse primer generated false-positive signals on lateral flow strips.

[0126] Example 19: Multiplexing of RT-RPA primers After an extensive primer design and screening process, specific primer pairs were used in multiplexed RT-RPA reactions. Figure 22 shows an example of multiplexing two exemplary primer sets, namely CALR and IFIT2, on a lateral flow strip. After identifying optimal conditions for multiplexing two targets, we explored higher multiplexing in a single RT-RPA reaction. Our selected primer design and the robustness of the RPA allowed us to observe the amplification of five products on a gel (Figure 23). The ability to multiplex up to five targets allows us to strategically detect biomarkers within the device in a way that improves sensitivity and specificity for pre-symptomatic infection. Next, we explored how primer concentration can be used to modulate the signal from multiple biomarkers (Figure 24). We found that the total primer concentration and the primer concentration ratio for the reference biomarker and the infection biomarker can affect the specificity and intensity of the readout results.

[0127] One of the challenges of multiplex RT-RPA is the increased tendency for primer dimer formation, which can affect the readout results on lateral flow strips. The inventors investigated the use of betaine as an additive to reduce the occurrence of primer dimers. Betaine effectively lowers the melting temperature of oligonucleotides, thereby preventing the formation of primer dimers during isothermal amplification. In preliminary experiments, we found that betaine can reduce primer noise in multiplex RT-RPA reactions, but some primer sets lose efficiency in the presence of high concentrations of betaine. Therefore, in specific embodiments, betaine can be an effective additive for reducing primer dimers in RT-RPA reactions.

[0128] Example 20: Buffers and filters for minimal processing of saliva for compatibility with multiplex isothermal amplification reactions In one embodiment, saliva processing using the device of the present invention can incorporate three physical filters (a collection pad, a lysis pad, and a debris filter) and a lyophilized lysis buffer to release and protect RNA molecules for amplification by RT-RPA (Figure 25). The collection pad is required to reduce the viscosity of the sample, collect and release a sufficient amount of sample required for the device, and remove large particles such as food and mucin (a high-molecular-weight glycoprotein present in saliva). The lysis pad is embedded with a lyophilized lysis buffer to release and protect intracellular RNA biomarkers. Finally, the debris filter serves to remove post-lysis cellular debris and remaining large inhibitory particles before the sample flows into the RT-RPA amplification pad. The selection of the collection pad, lysis pad, and debris filter is discussed in further detail below.

[0129] As described above, the device of the present invention is designed to provide the entire volume of liquid required for the saliva sample being tested to reconstitute all lyophilized reagents (including the lysis buffer and RT-RPA reagents) and allow them to flow throughout the device. While this approach reduces the complexity, cost, and ease of use of the device, it creates the challenge that the upstream lysis buffer must be fully compatible with the downstream reaction without dilution. Additionally, because the device is non-electrically powered, the simplest approach to processing saliva samples involves a buffer that does not require heat to lyse the sample and inactivate salivary RNases. Therefore, the lysis buffer must fulfill three functions: 1) inactivate salivary RNases, 2) maintain compatibility with RT-RPA, and 3) lyse cells in the saliva to release RNA targets without heating.

[0130] RNases are abundant and potent RNA-degrading enzymes readily found in human saliva. RNases must be inactivated to prevent immediate degradation of the RNA biomarkers targeted by the device. To demonstrate the effect of salivary RNases on RT-RPA reactions, untreated saliva was spiked into RPA reactions containing either cDNA or RNA templates derived from a human lung cell line. Reactions containing more than 0.1 μl of saliva showed interference with the RNA target but not with the cDNA template (Figure 2.5.2). This indicates that RNases found in saliva degrade the RNA template prior to amplification, but that saliva does not otherwise inhibit the RPA reaction.

[0131] To screen chemical and protein-based reagents for salivary RNase inhibition, we developed an RNase activity assay in which purified cell line RNA was treated with potential RNase inhibitors and then incubated with saliva samples at 37°C for 30 minutes (Figure 2.5.3). After incubation, the RNA was purified and analyzed using an Agilent TapeStation. To determine RNase activity in the presence of various inhibitors, we compared the brightness of eukaryotic ribosomal RNA bands (28S and 18S). Furthermore, the Agilent TapeStation calculates an RNA Integrity Score (RIN) as a measure of RNA quality (RIN 10 = fully intact RNA, RIN 1 = fully degraded RNA). However, contaminating nucleic acids (e.g., bacterial 16S and 23S rRNA) can interfere with this calculation.

[0132] To screen chemical and protein-based reagents that inhibit salivary RNase, we developed an RNase activity assay. In this assay, purified cell line RNA was treated with potential RNase inhibitors and then incubated with saliva samples at 37°C for 30 minutes (Figure 2.5.3). After incubation, the RNA was purified and analyzed using an Agilent TapeStation. To determine RNase activity in the presence of various inhibitors, we compared the brightness of eukaryotic ribosomal RNA bands (28S and 18S). Furthermore, the Agilent TapeStation calculates an RNA Integrity Score (RIN) as a measure of RNA quality (RIN 10 = fully intact RNA, RIN 1 = fully degraded RNA), but contaminating nucleic acids (e.g., bacterial 16S and 23S rRNA) can interfere with this calculation. In total, 54 unique reagents, concentrations, and buffer compositions were tested for their ability to inactivate RNase. Table 6 is a non-exhaustive list of reagents that have been tested for RNase inactivation.

[0133] Reagents under consideration for this device were tested for compatibility with RT-RPA at concentrations expected to be required for lysis. To test for interference, lysis or RNase inhibitor reagents were spiked into RT-RPA reactions using purified cell line RNA or cDNA as template. Reactions were analyzed on an agarose gel to confirm the presence or absence of amplification products. An example of an RT-RPA interference assay is shown in Figure 28. In total, over 20 lysis buffers and 17 lysis and RNase inhibitor reagents were tested at various concentrations for interference in RT-RPA. Table 7 provides a non-exhaustive list of the reagents tested. Expected results of RT-RPA compatibility testing are shown below.

[0134] · Complete interference: no amplification bands.

[0135] Partial interference: Band intensity is reduced compared to the control reaction. The tested reagent reduces the overall reaction efficiency.

[0136] No interference: amplified bands have the same intensity as the control reaction.

[0137] Reverse transcriptase interference or degradation of the RNA target: band present in the cDNA reaction and absent in the RNA template reaction.

[0138] We further screened various concentrations of lysis reagents for their ability to lyse cells present in saliva. Commonly used lysis reagents include chaotropic salts (e.g., guanidine hydrochloride), anionic detergents (e.g., sodium dodecyl sulfate), and nonionic detergents (e.g., Triton X-100). After confirming that the selected lysis buffer did not interfere with RT-RPA and was effective in inhibiting RNase activity, we determined the lysis efficiency by directly testing crude saliva extracts in RT-RPA. Initial screening of candidate lysis buffers was performed by treating saliva samples with the selected buffer and then adding the saliva directly to the RT-RPA reaction. Lysis efficiency was determined by comparing the amplified band intensity on agarose gels and lateral flow strips with a positive control (purified cell line RNA). Three candidate buffers passed this screening. The following buffer (Buffer #20) was selected for further evaluation: 100 mM guanidine hydrochloride, 5 mM TCEP-HCl, 7.5% tween-20, 0.0016% digitonin, and 4 U / μl RNase inhibitor.

[0139] To determine the robustness of this lysis buffer, seven raw saliva samples were collected using selected collection pads and expelled from the collection pads using a 3 ml syringe. The saliva samples were treated with Buffer #20, after which 5 μl of treated saliva was used as template for a 20 μl RT-RPA reaction. The RT-RPA reaction was incubated at 42°C for 20 minutes, heat-inactivated at 95°C for 10 minutes, and analyzed on a lateral flow strip. Amplification of the CXCL8 biomarker (a transcript abundant in healthy volunteer saliva) was observed in five of the seven saliva samples tested (Figure 29).

[0140] Example 21: Hydrodynamic characterization Characterization of the fluid dynamics of human saliva was essential to ensure that appropriate mechanical processes and sample handling were included in the device design. This ensured the collection, transport, processing, mixing, and eventual amplification of biomarkers from the saliva sample. In this embodiment, simulated sample collection using a sublingually inserted pad improved the saliva collection experience for the user compared to passively dripping saliva into a vial. It also served as an initial metering measure to ensure sufficient sample volume was collected. An example of this is shown in Table 8. For both saliva and water, the coefficient of variation (CV) for the total saturated volume across three replicate measurements was less than 3.5%. Furthermore, expression of solution from the saturated collection pad was sufficient for the product, with CVs for the remaining sample volume of water less than 3% and saliva less than 10% (Table 8). The greater variability observed in saliva expression is likely due to variability in mucin concentration between samples. The final collection volume required for amplification within the device must take into account the variability observed in saliva expression from the collection pad.

[0141] Alternative embodiments were constructed and tested, ultimately resulting in an exemplary device integrating a 3 mL syringe with several subcomponents. We verified successful delivery of saliva samples through a sealed device that integrates several sample processing steps, including sample collection, reagent mixing, incubation, and delivery to a lateral flow strip (Figure 12).

[0142] Example 22: Optimization of filtration compression force Due to limitations inherent in lateral flow technology, saliva filtration is necessary to reduce and normalize the viscosity of saliva samples. A series of experiments was conducted to identify the filtration requirements necessary to mitigate signal inhibition and flow irregularities caused by salivary mucins. Specifically, to determine the effect of mucins, saliva was collected from three subjects at three time points, yielding samples with various mucin concentrations. Each sample was then tested under four filtration conditions: 1) no filtration, 2) filtration through a 10 μm filter, 3) filtration through a gradient stack containing a 10 μm filter, a 1 μm filter, and a 0.45 μm filter, and 4) absorption and squeezing from the collection pad before passing through the gradient filter stack (Table 9). Sample viscosity was measured by adding blue dye to each sample at the time of collection and recording two visual measurements from the samples run on the test strip: 1) the time required for the sample to wick up to the control line, and 2) the percentage of solution absorbed by the absorbent pad.

[0143] Saliva was found to significantly reduce the flow rate and sample volume through lateral flow test strips (Table 9). Passing the sample through a 10 μm filter significantly improved these problems, with additional 1 μm and 0.45 μm filters providing slight additional improvement. Thus, in preferred embodiments, the device can incorporate a method of compressive force filtration through a membrane having a porosity of at least 10 μm prior to sample delivery to the test strip.

[0144] Example 22: Evaluation of pad-driven amplification Pad-driven amplification uses a porous material that is impregnated with RPA reagents and then dried. Preliminary studies showed lower than desired amplification, necessitating additional work to understand the efficiency of liquid reactions in vials compared to pad materials. As shown in Figure 30, pad materials fully saturated with wet RPA reagents maintain comparable amplification efficiency compared to vial reactions.

[0145] Because amplification failed with pads containing lyophilized RPA reagent but not with the liquid reagent, optimization efforts focused on the lyophilization conditions. To improve the efficiency of the RPA reaction after lyophilization, the RPA mixture was reformulated by incorporating additional excipients before lyophilization. Several excipients, including trehalose, glycine, mannitol, sorbitol, and sucrose, were added to the RPA formulation. While the lyophilized cake in the original RPA tube was very poor (Figure 31), the addition of trehalose and glycine prevented the cake from collapsing and maintained its structure after drying (Figure 31B). Dried reactions from both conditions were used for amplification. There was a significant difference in amplification efficiency between the two formulations: there was no detectable amplification with the original RPA formulation, whereas the reformulated reaction resulted in successful amplification. A final formulation containing 2% sucrose and 1% mannitol was selected, and subsequent formulation and drying parameters were developed and tested. Further optimization of the freeze-drying procedure will increase the primary and secondary drying cycle times by approximately 25% and reduce the initial heat treatment before evacuation to -44°C.

[0146] Because the reconstituted RPA mixture was successfully amplified, additional primers were tested using pad amplification. Figure 32 illustrates the drying method for reagents onto the pad and demonstrates successful amplification of both the NCL (reference biomarker) and OAS2 (infection biomarker) primer sets. In a preferred embodiment, a portion of the RPA mixture is lyophilized at the distal end of the pad, reducing the amount of reagent required and providing additional sample flow after the reaction, allowing for dilution for flow to the lateral flow strip. The feasibility of this approach is demonstrated in Figure 33. Here, amplification is performed within one embodiment of the present invention, in which all subcomponents are customized and integrated into a single device. The device was assembled with an amplification pad cut to one-quarter the length of the original amplification pad. This pad was saturated with primers and RPA reagents and dried, then supported within the device by a blank amplification pad three-quarters the original length. A solution containing cDNA template and magnesium acetate was added to the test device and allowed to incubate at 39°C for 20 minutes. Amplification occurred within the prototype device. Thus, we have demonstrated that amplification using reduced amounts of RPA reagent within the distal end of the pad is feasible. Table 1. Evaluation of saliva collection pads for diagnostic devices [Table 1] Table 2. Reverse transcriptases screened for RT-RPA [Table 2] Table 3. Excipients tested for improving pad-driven amplification [Table 3] Table 4. Device subcomponents and their functions in one embodiment [Table 4] Table 6. Reagents used in saliva RNase inactivation screening. Results were compiled from multiple RNase inactivation screens as shown in Figure 2.5.3. A pass (green) indicates sufficient RNase inactivation, a partial pass (yellow) indicates partial RNase inactivation, and a fail (red) indicates incomplete RNase inactivation. [Table 6] Table 7. Reagents used for compatibility screening in RT-RPA. Pass (green) indicates that the reagent did not interfere with RT-RPA. Partial pass (yellow) indicates that the reagent partially interfered with the RT-RPA reaction. Fail (red) indicates that the reagent completely interfered with the RT-RPA reaction. Reagent concentrations are the final concentrations in the RT-RPA reaction. [Table 7-1] [Table 7-2] Table 8. Collection pad absorption and squeeze-out volume evaluation [Table 8] Table 9. Evaluation of flow characteristics of lateral flow test strips using raw and filtered saliva. [Table 9-1] [Table 9-2] [Table 9-3] Table 10: Example formulation of lysis buffer [Table 10-1] [Table 10-2] [Table 10-3]

[0147] Preserved embodiment The present invention further includes the following additional conserved embodiments.

[0148] 1. A nucleic acid amplification device comprising: a collection pad adapted to collect the sample; a lysis pad disposed in fluid communication with the collection pad containing a predetermined amount of lyophilized buffer adapted to lyse cells in the sample; a filter stack in fluid communication with the dissolving pad; a reservoir for receiving a sample from the filter stack; one or more transfer pads adapted to transfer the sample to an amplification pad containing lyophilized reagents necessary for the isothermal amplification of nucleic acids present in said sample; a lateral flow strip adapted to receive the amplification product from the amplification pad.

[0149] 2. The device of embodiment 1, wherein the sample comprises a biological sample.

[0150] 3. The device of embodiment 2, wherein the biological sample is a saliva sample.

[0151] 4. The device of embodiment 3, wherein the collection pad is secured to a collection handle.

[0152] 5. The device of embodiment 1, wherein the dissolving pad is secured within a barrel and in fluid communication with an amplification assembly that secures the one or more transfer pads and the amplification pad.

[0153] 6. The device of embodiment 1, wherein the filter stack comprises a column portion containing an agent, such as a chelating agent, configured to inhibit divalent cations and positively charged molecules, disposed between a first filter and a second filter, and optionally a third filter disposed below the second filter.

[0154] 7. The device of embodiment 6, wherein the second filter has a smaller filter size than the first filter, and optionally the third filter has a smaller filter size than the second filter.

[0155] 8. The device of embodiment 6, wherein the chelating agent comprises Chelex-100.

[0156] 9. The device of embodiment 1, wherein the reservoir is disposed within a base plate.

[0157] 10. The device of embodiment 9, wherein the base plate further comprises one or more pressure relief locations.

[0158] 11. The device of embodiment 1, wherein lyophilized reaction buffer and enzymes for isothermal amplification are disposed in the distal portion of the amplification pad.

[0159] 12. The device of embodiment 1, wherein the one or more transfer pads and the amplification pad are sufficiently porous to form a lysed sample front along a processing path toward a lateral flow strip.

[0160] 13. The device of embodiment 1, wherein the sample in the reservoir generates sufficient capillary action to transfer amplification products from the amplification pad to the lateral flow strip.

[0161] 14. The device of embodiment 1, further comprising a strip cover that secures the lateral flow strip.

[0162] 15. The device of embodiment 14, wherein the strip cover is fixed to a housing, the housing being optionally positioned over the barrel and adapted to receive the collection handle.

[0163] 16. The device of embodiment 15, wherein the housing includes a locking position.

[0164] 17. The device of embodiment 1, wherein the lyophilized reagents comprise reagents for RT-RPA.

[0165] 18. The device of embodiment 17, wherein the reagents for RT-RPA comprise a reagent having one or more additional excipients and / or RNase inhibitors.

[0166] 19. The device of embodiment 1, wherein the nucleic acids present in the sample are selected from RNA, DNA, and / or one or more RNA biomarkers of infection.

[0167] 20. A filter for a diagnostic device, comprising: A nucleic acid amplification device comprising a filter stack, the filter stack further comprising a column portion containing a chelating agent configured to inhibit divalent cations and positively charged molecules, disposed between a first filter and a second filter, and optionally a third filter disposed below the second filter; A filter for a diagnostic device, wherein a sample is passed through the filter stack prior to amplification of nucleic acids present in the sample.

[0168] 21. The filter for the device of embodiment 20, wherein the sample comprises a biological sample.

[0169] 22. A filter for a device according to embodiment 20, wherein the sample comprises a biological sample having cells that are lysed before passing through the filter stack.

[0170] 23. A filter for a device as described in embodiment 20, wherein the second filter has a smaller filter size than the first filter, and the optional third filter has a smaller filter size than the second filter.

[0171] 24. A filter for the device of embodiment 20, wherein the chelating agent comprises Chelex-100.

[0172] 25. A filter for a device according to embodiment 20, wherein the first filter has a filter size of about 10 μM.

[0173] 26. A filter for a device according to embodiment 20, wherein the second filter has a filter size of about 1 μM.

[0174] 27. A filter for a device according to embodiment 20, wherein the nucleic acids present in the sample are selected from RNA, DNA, and / or one or more RNA biomarkers of infection.

[0175] 28. A filter for a device according to embodiment 20, wherein the optional third filter has a filter size of about 0.4 μM.

[0176] 29. A filter for a device according to embodiment 20, wherein the isothermal amplification comprises RT-RPA.

[0177] 30. A filter for a device according to embodiment 20, wherein the filter stack is in fluid communication with an amplification pad containing lyophilized reagents necessary for oligonucleotide amplification.

[0178] 31. A filter for a device according to embodiment 30, wherein the lyophilized reaction buffer and enzyme for oligonucleotide amplification are located in the distal portion of the amplification pad.

[0179] 32. A filter for a device according to embodiment 30, wherein the filter stack is in fluid communication with an amplification pad via one or more transfer pads.

[0180] 33. A filter for a device according to embodiment 30, wherein the amplification pad is in fluid communication with a lateral flow strip adapted to receive amplification products from the amplification pad.

[0181] 34. A filter for a device according to embodiment 30, wherein the sample passes through a dissolving pad before contacting the filter stack.

[0182] 35. An isothermal amplification device comprising: an amplification pad adapted to receive the sample, containing lyophilized reagents necessary for the isothermal amplification of one or more nucleic acids present in the sample; a lateral flow strip adapted to receive the amplification product from the amplification pad.

[0183] 36. The device of embodiment 35, wherein the sample comprises a biological sample.

[0184] 37. A device according to embodiment 35, wherein lyophilized reaction buffers and enzymes for isothermal amplification are disposed in the distal part of the amplification pad.

[0185] 38. The device of embodiment 35, wherein the isothermal amplification comprises RT-RPA.

[0186] 39. The device of embodiment 35, further comprising one or more transfer pads adapted to transfer the sample to an amplification pad containing lyophilized reagents necessary for the isothermal amplification of one or more biomarkers.

[0187] 40. The device of embodiment 39, wherein the one or more transfer pads and the amplification pad are sufficiently porous to form a front of the sample to be processed along a processing path toward the lateral flow strip.

[0188] 41. The device of embodiment 35, further comprising a reservoir for receiving the sample, the reservoir creating capillary action sufficient to transfer amplification products from the amplification pad to the lateral flow strip.

[0189] 42. The device of embodiment 35, wherein the nucleic acids present in the sample are selected from RNA, DNA, and RNA biomarkers of infection.

[0190] 43. The device of embodiment 35, further comprising a lysis pad containing a predetermined amount of lyophilized buffer adapted to lyse cells in the sample.

[0191] 44. The device of embodiment 35, further comprising a filter stack in fluid communication with the dissolving pad.

[0192] 45. The device described in embodiment 44, wherein the filter stack includes a column portion containing a chelating agent configured to inhibit divalent cations and positively charged molecules, disposed between a first filter and a second filter, and optionally a third filter.

[0193] 46. ​​A device according to embodiment 45, wherein cells in the sample are lysed before passing through the filter stack.

[0194] 47. The device of embodiment 45, wherein the second filter has a smaller filter size than the first filter, and the optional third filter has a smaller filter size than the second filter.

[0195] 48. The device of embodiment 45, wherein the chelating agent comprises Chelex-100.

[0196] 49. The device of embodiment 45, wherein the first filter has a filter size of about 10 μM.

[0197] 50. The device of embodiment 45, wherein the second filter has a filter size of about 1 μM.

[0198] 51. The device of embodiment 45, wherein the optional third filter has a filter size of about 0.4 μM.

[0199] 52. A system for nucleic acid amplification, comprising: a sample containing a predetermined amount of nucleic acid; A filter, a reaction chamber and / or amplification pad containing reagents necessary for the amplification of the nucleic acids present in the sample; a lateral flow strip adapted to receive the amplification product from the amplification pad.

[0200] 53. The system of embodiment 52, wherein the sample comprises a biological sample.

[0201] 54. The system of embodiment 53, wherein the biological sample is a saliva sample.

[0202] 55. The system of embodiment 54, wherein the filter comprises a filter stack having a column portion containing an agent, such as a chelating agent, configured to inhibit divalent cations and positively charged molecules, disposed between a first filter and a second filter, and optionally a third filter disposed below the second filter.

[0203] 56. The system of embodiment 55, wherein the second filter has a smaller filter size than the first filter, and optionally the third filter has a smaller filter size than the second filter.

[0204] 57. The system of embodiment 55, further comprising a third filter positioned below the second filter.

[0205] 58. The system of embodiment 55, wherein the chelating agent comprises Chelex-100.

[0206] 59. A reservoir for receiving a sample, the system according to embodiment 52.

[0207] 60. The system of embodiment 59, wherein the reservoir is responsive to one or more pressure release positions.

[0208] 61. The system of embodiment 60, wherein the reagents include reagents necessary for isothermal amplification.

[0209] 62. The system of embodiment 61, wherein the reagents necessary for isothermal amplification are lyophilized.

[0210] 63. The system of embodiment 62, wherein the lyophilized reagents for isothermal amplification are located in the distal portion of the amplification pad.

[0211] 64. The system of embodiment 53, further comprising a lysis pad containing a predetermined amount of lyophilized buffer adapted to lyse cells in the biological sample.

[0212] 65. The system of embodiment 64, wherein the buffer is lyophilized.

[0213] 66. The system of embodiment 52, further comprising one or more transfer pads adapted to transfer the sample to the reaction chamber and / or amplification pad.

[0214] 67. The system of embodiment 64, wherein the one or more transfer pads and amplification pads have sufficient porosity to form a front of the dissolved sample along a processing path toward the lateral flow strip.

[0215] 68. The system of embodiment 59, wherein the sample in the reservoir generates capillary action sufficient to transfer amplification products from the amplification pad to the lateral flow strip.

[0216] 69. The system of embodiment 52, further comprising a strip cover that secures the lateral flow strip.

[0217] 70. The system of embodiment 52, wherein the strip cover is fixed to the housing.

[0218] 71. The system of embodiment 52, wherein the reagents include reagents for RT-RPA.

[0219] 72. The system of embodiment 71, wherein the reagents for RT-RPA comprise lyophilized reagents for RT-RPA.

[0220] 73. The system of embodiment 72, wherein the lyophilized reagent for RT-RPA comprises a lyophilized reagent having one or more additional excipients and / or RNase inhibitors.

[0221] 74. The system of any of embodiments 1, 35, or 52, further comprising a removable barrier between the reaction chamber and / or amplification pad and the lateral flow strip.

[0222] 75. The system of embodiment 74, wherein the removable barrier includes a pull tab configured to separate the reaction chamber and / or amplification pad from the lateral flow strip, and removing the pull tab allows amplification products to move from the reaction chamber and / or amplification pad to the lateral flow strip.

Claims

1. A nucleic acid amplification device, comprising: a collection handle configured to collect a sample containing nucleic acids; a sample processing assembly in fluid communication with the amplification assembly; a heater assembly thermally coupled to the amplification assembly and responsive to a heater actuation assembly.

2. The device of claim 1 , further comprising a lateral flow assay configured to receive the amplification product from the amplification assembly.

3. The device of claim 1 , wherein the sample comprises a biological sample or an environmental sample.

4. The device of claim 3 , wherein the biological sample comprises a saliva sample.

5. 10. The device of claim 1, wherein the sample processing assembly includes a processing chamber that receives the collection handle and further secures one or both of a lysing pad and one or more filters.

6. 10. The device of claim 1, wherein the amplification assembly includes a transfer pad adapted to transfer the sample to an amplification pad containing lyophilized reagents necessary for amplification of nucleic acids present in the sample.

7. 7. The device of claim 6, wherein the amplification pad is treated with a blocking solution before being embedded with the lyophilized reagents necessary for amplification of nucleic acids present in the sample.

8. 7. The device of claim 6, wherein the lyophilized reagents comprise reagents for an amplification 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 (HDA), nicking enzyme amplification reaction (NEAR), signal-mediated amplification of RNA technology (SMART), rolling circle amplification (RCA), isothermal multiple strand displacement amplification (IMDA), single primer isothermal amplification (SPIA), and polymerase spiral reaction (PSR).

9. The device of claim 8 , wherein the reagent comprises a reagent having one or more additional excipients, RNase inhibitors, or DNase inhibitors.

10. 7. The device of claim 6, wherein the nucleic acid present in the sample is selected from RNA, DNA, one or more RNA biomarkers of infection, or a combination thereof.

11. 7. The device of claim 6, wherein the transfer pad is embedded with a predetermined amount of magnesium acetate (MgOAc) or magnesium (Mg).

12. The device of claim 1 , wherein the heater assembly includes an inner housing containing a phase change material that is responsive to an exothermic fuel.

13. The device of claim 12 , wherein the exothermic fuel comprises a Mg—Fe alloy.

14. 13. The device of claim 12, wherein the heater actuation assembly includes an exothermic reagent configured to actuate the exothermic fuel to cause the phase change material to reach a predetermined temperature, thereby heating the amplifier pad.

15. The device of claim 14 , wherein the phase change material comprises paraffin.

16. 15. The device of claim 14, wherein the phase change material is selected from the group consisting of metals, inorganic compounds, inorganic eutectics, and organic compounds.

17. 15. The device of claim 14, wherein the exothermic reagent is disposed in a reagent syringe and separated from the heater assembly by an exothermic reagent seal.

18. The device of claim 14 , wherein the pyrogenic reagent comprises a salt solution.

19. The device of claim 2 , further comprising an amplicon seal separating the amplification assembly and the lateral flow assay.

20. 20. The device of claim 19, wherein the assay transfer pad is in fluid communication with the lateral flow strip such that the amplification products are transferred from the assay transfer pad to the lateral flow strip by capillary action.

21. 6. The device of claim 5, wherein the filter comprises a column portion containing a chelating agent configured to inhibit divalent cations and positively charged molecules disposed between a first filter and a second filter, and optionally a third filter disposed below the second filter.

22. The device of claim 1 , wherein the nucleic acid sample is introduced into the sample processing assembly and the amplification assembly by a first movement of the collection handle.

23. The device of claim 1 , wherein the heater actuation assembly is responsive to a first movement of the collection handle to activate the heater assembly.

24. The device of claim 2 , wherein the lateral flow assay receives the amplification product from the amplification assembly in response to a second movement of a collection handle.

25. 1. A nucleic acid amplification and detection device comprising: a collection handle configured to collect the nucleic acid sample; a sample processing assembly in fluid communication with an amplification assembly, the nucleic acid sample being introduced into the sample processing assembly and the amplification assembly by a first movement of the collection handle; a heater assembly thermally coupled to the amplifier assembly and responsive to a heater actuation assembly, the heater actuation assembly activating the heater assembly in response to the first movement of the collection handle; a lateral flow assay configured to receive the amplification product from the amplification assembly in response to a second movement of the collection handle.

26. 26. The device of claim 25, wherein the collection handle includes a collection pad secured to the collection handle.

27. 26. The device of claim 25, wherein the sample is a biological sample or an environmental sample.

28. 28. The device of claim 27, wherein the biological sample is a saliva sample.

29. 26. The device of claim 25, wherein the sample processing assembly includes a processing chamber adapted to receive the collection handle and further secures one or both of a lysis pad containing a predetermined amount of lyophilized buffer adapted to lyse cells present in the sample, and a filter adapted to filter the sample.

30. 26. The device of claim 25, wherein the amplification assembly includes a transfer pad adapted to transfer the sample to an amplification pad containing lyophilized reagents necessary for amplification of nucleic acids present in the sample.

31. 31. The device of claim 30, wherein lyophilized reaction buffers and enzymes for amplification are disposed in a distal portion of the amplification pad.

32. 31. The device of claim 30, wherein the lyophilized reagents comprise 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 (HDA), nicking enzyme amplification reaction (NEAR), signal-mediated amplification of RNA technology (SMART), rolling circle amplification (RCA), isothermal multiple displacement amplification (IMDA), single primer isothermal amplification (SPIA), and polymerase spiral reaction (PSR).

33. 33. The device of claim 32, wherein the reagent comprises a reagent having one or more additional excipients, RNase inhibitors, or DNase inhibitors.

34. 31. The device of claim 30, wherein the nucleic acid present in the sample is selected from RNA, DNA, one or more RNA biomarkers of infection, or a combination thereof.

35. 31. The device of claim 30, wherein the transfer pad is embedded with a predetermined amount of magnesium acetate (MgOAc) or magnesium (Mg).

36. 26. The device of claim 25, wherein the heater assembly includes an inner housing containing a phase change material that is responsive to an exothermic fuel.

37. 37. The device of claim 36, wherein the exothermic fuel comprises a Mg-Fe alloy.

38. 37. The device of claim 36, wherein the heater actuation assembly includes an exothermic reagent configured to actuate the exothermic fuel to cause the phase change material to reach a predetermined temperature, thereby heating an isothermal amplification reaction within the amplification pad.

39. 39. The device of claim 38, wherein the phase change material comprises paraffin.

40. 39. The device of claim 38, wherein the phase change material is selected from the group consisting of metals, inorganic compounds, inorganic eutectics, and organic compounds.

41. 39. The device of claim 38, wherein the exothermic reagent is disposed within a reagent syringe, separated from the heater assembly by an exothermic reagent seal, and is released in response to the first movement of the collection handle.

42. 42. The device of claim 41, wherein the first movement of the collection handle engages a lancet with the reagent seal and brings the exothermic reagent in the reagent syringe into contact with the exothermic fuel of the heater assembly.

43. 42. The device of claim 41, wherein the pyrogenic reagent comprises a salt solution.

44. 26. The device of claim 25, wherein the second movement of the collection handle engages a lancet with an amplicon seal that separates the amplification assembly and the lateral flow assay, thereby bringing the amplification product into contact with an assay transfer pad.

45. 26. The device of claim 25, wherein the second movement of the collection handle compresses the amplification pad and the transfer pad, thereby bringing the amplification product into contact with an assay transfer pad.

46. 46. ​​The device of claim 45, wherein the assay transfer pad is in fluid communication with the lateral flow strip such that the amplification products are transferred from the assay transfer pad to the lateral flow strip by capillary action.

47. 30. The device of claim 29, wherein the filter comprises a column portion containing a chelating agent configured to inhibit divalent cations and positively charged molecules disposed between a first filter and a second filter, and optionally a third filter disposed below the second filter.

48. 48. The device of claim 47, wherein the second filter has a smaller filter size than the first filter, and optionally the third filter has a smaller filter size than the second filter.

49. 48. The device of claim 47, wherein the chelating agent comprises Chelex-100.

50. 26. The device of claim 25, further comprising a strip cover that secures the lateral flow strip.

51. 51. The device of claim 50, wherein the strip cover is optionally positioned over a processing chamber and secured to a housing adapted to receive the collection handle.

52. 26. The device of claim 25, further comprising a pull tab removably coupled to the collection handle, the pull tab configured to allow the first movement of the collection handle and to prevent the second movement of the collection handle until removed.

53. 26. The device of claim 25, further comprising a check valve disposed between the sample processing assembly and the amplification assembly.

54. 1. A device for the continuous amplification and detection of nucleic acids, comprising: a collection handle containing a nucleic acid sample, wherein a first actuation of the handle delivers the sample to a processing chamber containing one or both of a lysing pad and one or more filters; a transfer pad in fluid communication with the processing chamber for transferring the sample to an amplification pad containing lyophilized reagents necessary for amplification of nucleic acids present in the sample; a reaction housing including a heater assembly thermally coupled to the amplifier pad; a heater actuation assembly including a pyrogenic reagent disposed within the reagent syringe and separated from the heater assembly by a reagent seal, the pyrogenic reagent being released in response to the first actuation of the collection handle, thereby actuating the heater assembly; a lateral flow assay separated from the amplification pad by an amplicon seal and adapted to receive amplification products from the amplification pad in response to a second movement of the collection handle.

55. 55. The device of claim 54, wherein the sample is a biological sample or an environmental sample.

56. 45. The device of claim 44, wherein the biological sample is a saliva sample.

57. 55. The device of claim 54, further comprising a pull tab removably coupled to the collection handle, the pull tab configured to allow the first movement of the collection handle and to prevent the second movement of the collection handle until removed.

58. The collection handle a collection arm that secures a collection pad configured to be inserted into the processing chamber; a secondary collection arm configured to be inserted into the reagent syringe of the heater actuation assembly.

59. 55. The device of claim 54, wherein the collection handle includes a seal.

60. 60. The device of claim 59, wherein the seal comprises one or more O-rings.

61. 55. The device of claim 54, wherein the collection handle includes a sample sufficiency indicator in fluid communication with the collection pad.

62. 62. The device of claim 61, wherein the sample sufficiency indicator is located below a cover having a viewing opening.

63. 55. The device of claim 54, wherein the lyophilized reagents comprise lyophilized reaction buffers and enzymes for isothermal amplification disposed in a distal portion of the amplification pad.

64. 55. The device of claim 54, wherein the lyophilized reagents comprise 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 (HDA), nicking enzyme amplification reaction (NEAR), signal-mediated amplification of RNA technology (SMART), rolling circle amplification (RCA), isothermal multiple displacement amplification (IMDA), single primer isothermal amplification (SPIA), and polymerase spiral reaction (PSR).

65. 65. The device of claim 64, wherein the reagent comprises a reagent having one or more additional excipients, RNase inhibitors, or DNase inhibitors.

66. 55. The device of claim 54, wherein the nucleic acids present in the sample are selected from RNA, DNA, and / or one or more RNA biomarkers of infection.

67. 55. The device of claim 54, wherein the lysis pad comprises a predetermined amount of lyophilized buffer adapted to lyse cells present in the sample.

68. 55. The device of claim 54, wherein the one or more filters include a plurality of filters, each filter having a smaller size than the preceding filter.

69. 55. The device of claim 54, wherein the one or more filters comprise: a column portion comprising a chelating agent configured to inhibit divalent cations and positively charged molecules disposed between a first filter and a second filter, and optionally a third filter disposed below the second filter.

70. 55. The device of claim 54, wherein the transfer pad and the amplification pad have sufficient porosity to form a lysed sample front along a processing path toward a lateral flow strip.

71. 55. The device of claim 54, wherein the transfer pad is embedded with a predetermined amount of magnesium acetate (MgOAc) or magnesium (Mg).

72. 55. The device of claim 54, wherein the heater actuation assembly includes a plunger and / or plug that forms a seal in response to the secondary arm of the collection handle.

73. 55. The device of claim 54, wherein the heater actuation assembly includes a lancet positioned below the reagent seal positioned above a gasket, and wherein the first movement of the collection handle causes downward movement of the secondary arm, thereby generating internal pressure and allowing the lancet to puncture the reagent seal and transfer the exothermic reagent to a heater element.

74. 55. The device of claim 54, wherein the transfer pad and / or the amplification pad are disposed within a reaction chamber thermally coupled to the heater assembly.

75. 55. The device of claim 54, wherein the heater assembly includes a phase change material disposed between the reaction chamber and an exothermic fuel, the quantity of the phase change material being configured to at least partially convert from its solid form to a liquid form when heated by an exothermic chemical reaction produced by the exothermic fuel and the exothermic reagent, thereby providing a controlled, substantially constant temperature in the reaction chamber for an amplification reaction.

76. 76. The device of claim 75, wherein the pyrogenic reagent comprises a salt solution.

77. 76. The device of claim 75, wherein the exothermic fuel comprises a Mg-Fe alloy.

78. 76. The device of claim 75, wherein the phase change material comprises paraffin.

79. 76. The device of claim 75, wherein the phase change material is selected from the group consisting of metals, inorganic compounds, inorganic eutectics, and organic compounds.

80. 76. The device of claim 75, wherein the heater assembly includes a quantity of filler material disposed adjacent the heat-generating fuel.

81. 76. The device of claim 75, wherein the heater assembly includes a cap positioned adjacent the exothermic fuel.

82. 76. The device of claim 75, further comprising an absorbent collar disposed adjacent a top surface of the heater element.

83. 76. The device of claim 75, further comprising a frit positioned above and adjacent to the heater element, the frit configured to allow an exothermic reagent solution to flow into the heater element and to allow gases produced by an exothermic reaction to escape from the heater element.

84. 55. The device of claim 54, wherein the second movement of the collection handle engages a lancet with an amplicon seal that separates the amplification pad and the lateral flow assay, thereby contacting the amplification product with the assay transfer pad.

85. 55. The device of claim 54, wherein the second movement of the collection handle compresses the amplification pad and the transfer pad, thereby causing the amplification product to contact an assay transfer pad.

86. The lateral flow assay comprises: Conjugate pad and A membrane and 55. The device of claim 54, further comprising an absorbent pad.

87. The lateral flow assay comprises: Backing and 87. The device of claim 86, further comprising a strip cover that secures the lateral flow strip.

88. 55. The device of claim 54, further comprising a check valve disposed between the filter and the transfer pad.

89. 55. The device of claim 54, wherein the collection handle containing the nucleic acid sample comprises a capillary collection assembly.

90. The capillary collection assembly includes: a capillary collection channel; one or more vents in fluid communication with the channel; 90. The device of claim 89, comprising: a plunger-responsive solution configured to transport the nucleic acid sample through the channel and into the processing chamber.

91. 91. The device of claim 90, wherein the nucleic acid sample comprises a blood sample.

92. 55. The device of claim 54, further comprising a fluid injection assembly.

93. The fluid injection assembly includes: a fluid reservoir in communication with the processing chamber; an injector responsive to said collection handle; a plug, and optionally a plunger, disposed within the fluid reservoir; a fluid seal disposed above the gasket.

94. 94. The device of claim 93, wherein the injector includes an arm on the collection handle that responds to a plunger and / or the plug, and wherein the first movement of the collection handle causes downward movement of the arm, thereby generating internal pressure and allowing a lancet to puncture the fluid seal and transfer the fluid to the processing chamber.

95. The fluid injection assembly includes: a fluid reservoir disposed on the collection handle; an injector configured to inject fluid into the processing chamber through a pad containing the nucleic acid sample.

96. The device of any one of claims 93 to 95, wherein the fluid comprises a buffer solution.

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