Loop-mediated isothermal amplification (LAMP) on solid-phase media

Abstract

The present disclosure relates to a loop-mediated isothermal amplification (LAMP) reaction assembly comprising a LAMP reagent mixture substantially free of hygroscopic agents in combination with a solid-phase reaction medium. The present disclosure also includes a system for chromatic LAMP analysis comprising a substantially non-reactive solid-phase reaction medium and a non-interfering reagent mixture. The present disclosure also includes a solid-phase LAMP reaction medium comprising a substrate, an adhesive layer disposed on the substrate, a reaction layer disposed on the adhesive layer, and a spreading layer disposed on the reaction layer. The present disclosure also includes a method of testing for the presence of a target nucleotide sequence, comprising providing a biological sample and dispensing the sample into a testing environment having a solid-phase reaction medium in combination with a LAMP reagent mixture and a pH-sensitive dye.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Applications Nos. 63 / 138,316 and 63 / 138,318, filed January 15, 2021, each of which is incorporated herein by reference. [Background technology]

[0002] Polymerase chain reaction (PCR) is a molecular biology technique that allows for the amplification of nucleotides for various analytical purposes. Quantitative PCR (qPCR) is an application of PCR that allows for the monitoring of the amplification of target nucleotides. Diagnostic qPCR has been applied to the detection of nucleotides indicative of infectious diseases, cancer, and genetic abnormalities. Reverse transcription PCR (RT-PCR) is an application of qPCR that allows for the detection of target RNA nucleotides. This capability makes RT-PCR suitable for the detection of viral pathogens. However, RT-PCR uses large equipment that may not be available in certain point-of-care settings. Furthermore, RT-PCR requires skilled personnel, significant sample preparation, and is time-consuming to perform and obtain results.

[0003] In contrast, loop-mediated isothermal amplification (LAMP) is a more simplified approach for diagnostically identifying target nucleotides. Specifically, LAMP is a single-step nucleic acid amplification method for amplifying specific nucleotide sequences. In addition to using an isothermal heating process, LAMP allows for the use of simple visual indicators, such as color changes, rather than the more complex fluorescent indicators used in PCR. Reverse transcription-LAMP (RT-LAMP) can be used similarly to RT-PCR to identify target nucleotides from RNA and thus can be used in a diagnostic capacity to identify the presence or absence of viral pathogens. Because LAMP is simpler, it requires less equipment and sample preparation, making it easier to use at the point of care, such as in clinics, emergency rooms, and even on a mobile basis. Summary of the Invention

[0004] The present disclosure relates to techniques (e.g., methods, systems, and assemblies) for use in detecting target nucleotides using loop-mediated isothermal amplification (LAMP) on solid-phase media. In some embodiments, the target nucleotide may be known to be present in a pathogen of interest. When the pathogen is a virus, the LAMP analysis can be a reverse transcription (RT) RT-LAMP analysis.

[0005] In some disclosed embodiments, the LAMP reaction assembly can include a LAMP reagent mixture that is substantially free of hygroscopic agents in combination with a solid-phase reaction medium. In one aspect, the solid-phase medium can be hydrophilic, absorbent, and porous.

[0006] In one aspect, the solid phase medium can be substantially free of magnesium-interfering agents, hi another aspect, the magnesium-interfering agents can include magnesium-containing compounds and chelating agents that interfere with magnesium.

[0007] In another embodiment, the solid phase medium may be a cellulose-based medium. In one embodiment, the cellulose-based medium may have a surface area-to-thickness ratio of about 30 to about 600. In another embodiment, the cellulose-based medium may have a pore size greater than about 1 micron and less than about 100 microns. In one embodiment, the solid phase medium may comprise paper. In another embodiment, the solid phase medium may comprise glass fiber. In yet another embodiment, the solid phase medium may comprise nylon, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, or hydrophilic polytetrafluoroethylene (PTFE), or a combination thereof.

[0008] In another embodiment, the LAMP reaction assembly may further include an adhesive that is substantially free of magnesium interfering agents and moisture absorbents. In another embodiment, the LAMP reaction assembly may further include a spreading layer that is less hydrophilic than the solid-phase reaction medium.

[0009] In another disclosed embodiment, a method of manufacturing the aforementioned LAMP reaction assembly may include combining a LAMP reagent mixture that is substantially free of moisture absorbents with a solid-phase reaction medium such that the reagent mixture is maintained in contact with the solid-phase reaction medium.

[0010] In one embodiment, the method can include controlling discoloration using a non-discoloring additive. In another embodiment, the non-discoloring additive can include a sugar, a buffer, a blocking agent, or a combination thereof. In another embodiment, the non-discoloring additive can include a sugar including one or more of trehalose, glucose, sucrose, dextran, or a combination thereof. In another embodiment, the non-discoloring additive can include a blocking agent including bovine serum albumin, casein, or a combination thereof.

[0011] In another disclosed embodiment, a method for performing a LAMP analysis may include providing a LAMP reaction assembly as described above, applying a biological sample to the reaction assembly, heating the assembly to a temperature sufficient to initiate the LAMP reaction, and maintaining the temperature for a time sufficient to complete the LAMP reaction. In one aspect, the biological sample may be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or a combination thereof. In another aspect, the biological sample may be saliva. In one aspect, the method may further include detecting a viral pathogen. In another aspect, the LAMP analysis may be reverse transcription-LAMP (RT-LAMP).

[0012] In another disclosed embodiment, a system for chromatic LAMP analysis can include a substantially non-reactive solid-phase reaction medium and a non-interfering reagent mixture. In one aspect, the substantially non-reactive solid-phase reaction medium can be hydrophilic, absorbent, and porous. In another aspect, the substantially non-reactive solid-phase reaction medium can be substantially free of oxidizing agents, pH-interfering agents, or combinations thereof.

[0013] In one embodiment, the substantially non-reactive solid phase reaction medium can have a buffer capacity of about 0.01 mM to about 5 mM. In another embodiment, the substantially non-reactive solid phase reaction medium, when combined with a pH-sensitive dye, has a maximum absorption wavelength (λ) within the test range. max ). In another embodiment, the substantially non-reactive solid phase reaction medium can include cellulose or glass fiber. In another embodiment, the system can further include an adhesive, a spreading layer, a spacer, a plastic carrier, or a combination thereof. In one embodiment, each of the adhesive, spreading layer, spacer, or plastic carrier can be substantially free of oxidizing agents and pH-interfering agents. In another embodiment, the non-interfering reagent mixture can further include one or more target primers, DNA polymerase, or resolubilizing agents.

[0014] In another disclosed embodiment, a method for maximizing the accuracy of a color output signal in a solid-phase pH-dependent LAMP assay may include providing a solid-phase reaction medium that minimizes color changes caused by events other than the LAMP reaction, and performing the LAMP assay on the solid-phase reaction medium. In one aspect, the method may include controlling color changes caused by events other than the LAMP reaction due to protons generated by events other than the LAMP reaction. In another aspect, the method may include using a non-color-changing additive to control color changes caused by events other than the LAMP reaction. In one aspect, the non-color-changing additive may include a sugar, a buffer, a blocking agent, or a combination thereof. In another aspect, the non-color-changing additive may include a sugar including one or more of trehalose, glucose, sucrose, dextran, or a combination thereof. In another aspect, the non-color-changing additive may include a blocking agent including bovine serum albumin, casein, or a combination thereof.

[0015] In yet another disclosed embodiment, a method for maximizing the level of detection (LOD) of a LAMP assay can include providing a reaction environment and reagents that minimize non-LAMP reaction products.

[0016] In yet another disclosed embodiment, a system for chromatic LAMP analysis can include a combination of a solid-phase reaction medium and a LAMP reagent that maintains the color of the solid-phase reaction medium with a color that is within 10% of the initial color of the solid-phase medium when stored at a selected temperature (e.g., room temperature of about 25°C). In one aspect, the combination can maintain the color when stored for more than one or more of 30 days, 90 days, 365 days, 2 years, or 5 years. In another aspect, the combination can maintain the color when stored at a relative humidity of about 40% to 90%. In another aspect, the selected temperature can be any temperature within the range of about -20°C to about 37°C.

[0017] In yet another disclosed embodiment, a method for producing the aforementioned chromatic LAMP system can include combining a non-interfering reagent mixture with a substantially non-reactive solid-phase reaction medium such that the non-interfering reagent mixture is maintained in contact with the substantially non-reactive solid-phase reaction medium. In one aspect, the method can include preparing a solution containing the non-interfering reagent mixture and coating the reagent mixture onto the substantially non-reactive solid-phase reaction medium. In another aspect, the coating can include dripping, spraying, impregnating, dipping, or misting the solution onto the substantially non-reactive solid-phase reaction medium. In another aspect, the non-interfering reagent mixture can be combined with the substantially non-reactive solid-phase reaction medium using a reel-to-reel (R2R) process.

[0018] In yet another disclosed embodiment, the solid-phase LAMP reaction medium may include a substrate, an adhesive layer disposed on the substrate, a reaction layer disposed on the adhesive layer, or a spreading layer disposed on the reaction layer. In one aspect, the substrate may be an optically transparent material. In another aspect, the adhesive layer may be substantially free of volatile agents. In another aspect, the adhesive layer may be discontinuously disposed on the substrate. In another aspect, the substrate may be an optically clear plastic carrier.

[0019] In one embodiment, the solid-phase LAMP reaction medium can further include a test zone. In one embodiment, the test zone can be defined by at least two segments of the discontinuous adhesive layer. In another embodiment, the test zone can be defined by at least three segments of the discontinuous adhesive layer. In another embodiment, the test zone can be defined by at least four segments of the discontinuous adhesive layer.

[0020] In one embodiment, the solid-phase LAMP reaction medium may further comprise at least one segment that is substantially free of reagents. In another embodiment, the reaction layer may comprise reagents including one or more target primers, DNA polymerase, or a resolubilizing agent. In another embodiment, the reagents may form a composition sufficient to carry out a LAMP reaction. In one embodiment, the reaction layer may be discontinuous.

[0021] In another embodiment, the solid-phase LAMP reaction medium may further comprise a spreading layer comprising one or more of glass fiber, nylon, cellulose, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, polyester, hydrophilic polytetrafluoroethylene (PTFE), or a combination thereof. In one embodiment, the spreading layer may be optically transparent.

[0022] In another embodiment, the solid-phase LAMP reaction medium may include a spacer material. In one embodiment, the spacer material may include one or more of glass fiber, nylon, cellulose, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, polystyrene, polyester, hydrophilic polytetrafluoroethylene (PTFE), etc., or a combination thereof. In another embodiment, the spacer material may be oriented in the same plane as the reaction layer and between segments of the reaction layer. In another embodiment, the reaction layer may have a surface area-to-thickness ratio of about 30 to about 600. In another embodiment, the reaction layer may have a thickness of about 0.05 mm to about 2 mm. In another embodiment, the reaction layer may have a width of about 4 mm to about 12 mm and a length of about 4 mm to about 25 mm. In another embodiment, the minimum space between segments of the reaction layer may be about 1.8 mm to about 2.2 mm.

[0023] In another disclosed embodiment, a method for testing for the presence of a viral pathogen may include providing a saliva sample from a subject and dispensing the sample into a test environment having a solid-phase reaction medium in combination with a LAMP reagent mixture and a pH-sensitive dye. In one aspect, the method may include minimizing the amount of volatile, hygroscopic, and non-pH-sensitive agents that may discolor the solid-phase medium. In another aspect, the method may include providing one or more target primers, a DNA polymerase, or a resolubilizing agent in an amount sufficient to promote the LAMP reaction. In another aspect, the method may include providing a reverse transcriptase in an amount sufficient to promote a reverse transcription-LAMP (RT-LAMP) reaction. In another aspect, the method may include providing one or more target primers in an amount sufficient to detect the viral pathogen. In another aspect, the method may include generating a test result in less than one hour after dispensing the sample into the test environment.

[0024] In yet another disclosed embodiment, a method for confirming the suitability of a saliva sample for testing by solid-phase LAMP reaction may include providing a solid-phase reaction medium with at least one test site or test spot and a negative control site. In one aspect, the at least one test site or test spot may include a combination of a LAMP reagent and a pH-sensitive dye. In another aspect, the negative control site may include a pH-sensitive dye and no LAMP reagent. In another aspect, the method may include applying the saliva sample to the solid-phase reaction medium. In another aspect, the method may include confirming activation of the pH-sensitive dye on the negative control site.

[0025] In one embodiment, the pH-sensitive dye may be at least one of phenol red, phenolphthalein, azolithin, bromothymol blue, naphtholphthalein, cresol red, or a combination thereof. In another embodiment, the LAMP reagent may be substantially free of volatile reagents, pH-affecting reagents, magnesium-containing reagents, or a combination thereof. In another embodiment, the LAMP reagent may include a non-interfering LAMP reagent including a DNA polymerase, a reverse transcriptase, a primer for a target region, or a combination thereof.

[0026] In another embodiment, the method can further include providing a test site or spot defined by at least two segments of the discontinuous adhesive layer. In another embodiment, the method can include providing a test site or spot defined by at least three segments of the discontinuous adhesive layer.

[0027] In yet another disclosed embodiment, a method for maximizing the accuracy of a positive test result from a solid-phase LAMP reaction can include providing a solid-phase reaction medium with at least three test sites or spots (each containing a common pH-sensitive dye and LAMP reagent combination), each containing a different primer sequence derived from a target pathogen. In one aspect, the method can include initiating the LAMP reaction. In another aspect, the method can include determining a positive test result when at least two of the test sites or spots activate the pH-sensitive dye to change from a first color to a second color. In another aspect, the method can further include providing a reverse transcriptase in an amount sufficient to promote the reverse transcription-LAMP reaction.

[0028] In one embodiment, the pH-sensitive dye can be at least one of phenol red, phenolphthalein, azolithin, bromothymol blue, naphtholphthalein, cresol red, or a combination thereof. In another embodiment, the LAMP reagents can be substantially free of volatile reagents, pH-affecting reagents, magnesium-containing reagents, or a combination thereof.

[0029] In another embodiment, the target pathogen may include a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In one embodiment, the target pathogen may include a viral pathogen. In one embodiment, the viral pathogen may include a dsDNA virus, a ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, a ssRNA-RT virus, or a ds-DNA-RT virus. In one embodiment, each primer sequence may match a sequence derived from a viral target including H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.

[0030] In one disclosed embodiment, a method for testing for the presence of a target nucleotide sequence may include providing a biological sample and dispensing the sample into a testing environment having a solid-phase reaction medium in combination with a loop-mediated isothermal amplification (LAMP) reagent mixture and a pH-sensitive dye.

[0031] In another embodiment, the test environment may be substantially free of volatile reagents, pH-affecting reagents, desiccants, or combinations thereof. In one embodiment, the method may include increasing the test environment temperature at a rate of about 0.1°C per second. In another embodiment, the method may include providing a heating uniformity of the test environment with a variation of less than 1°C. In another embodiment, the method may include providing a solid-phase reaction medium comprising cellulose or glass fiber. In one embodiment, the method may include providing a reverse transcriptase in an amount sufficient to promote a reverse transcription-LAMP (RT-LAMP) reaction.

[0032] In one embodiment, the biological sample can be at least one of saliva, mucus, blood, urine, or feces, sweat, exhaled breath condensate, or a combination thereof. In one embodiment, the method can include collecting the biological sample using one or more of a saliva collection device, a nasal swab, a blood collection device, a urine collection device, a sweat collection device, an exhaled breath condensate collection device, or a stool collection device.

[0033] In another embodiment, the target nucleotide sequence may be derived from at least one of a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In one embodiment, the target nucleotide sequence may be derived from a viral pathogen. In one embodiment, the viral pathogen is Coronoviridae , Orthomyxoviridae , Paramyxoviridae , Picornaviridae , Adenoviridae , and Parvoviridae In another aspect, the viral pathogen may be selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome (MERS), influenza, and H1N1. In one aspect, the target nucleotide sequence may be derived from a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen.

[0034] In yet another disclosed embodiment, a biological sample testing device can include a substrate that engages a solid-phase reaction medium in combination with a dehydrated reagent mixture and a dehydrated pH-sensitive dye for loop-mediated isothermal amplification (LAMP), the device providing a degree of testing accuracy of at least about 95% after six months of storage when stored at room temperature. In one aspect, the device can provide a degree of testing accuracy of at least about 95% after twelve months of storage when stored at room temperature. In another aspect, the device can provide a degree of testing accuracy of at least about 95% after two years of storage when stored at room temperature.

[0035] In yet another disclosed embodiment, a biological sample testing system can include a substrate that engages a solid-phase reaction medium in combination with a dehydrated reagent mixture and a dehydrated pH-sensitive dye for loop-mediated isothermal amplification (LAMP), the housing being operable to receive a biological sample. In one aspect, the biological sample testing system can include a heater configured to isothermally heat the container to an internal temperature sufficient to initiate and maintain a LAMP reaction between the LAMP reagent mixture. In another aspect, the biological sample testing system can include a biological sample for a period of time that is used to generate a test result via the pH-sensitive dye.

[0036] In one aspect, the substrate can comprise an optically transparent material. In another aspect, the substrate can engage the solid phase reaction medium via an adhesive. In another aspect, the adhesive can be substantially optically transparent. In another aspect, the substrate can form a portion of a housing.

[0037] In another aspect, the biological sample testing system may include an adhesive layer disposed on the substrate, a reaction layer disposed on the adhesive layer, and a spreading layer disposed on the reaction layer. In one aspect, the biological sample testing system may further include a spacer layer oriented in the same plane as the reaction layer. In another aspect, the housing may be disposed in contact with the substrate. In another aspect, the housing may further be disposed in contact with the spreading layer. In another aspect, the housing may substantially enclose the substrate, adhesive layer, reaction layer, and spreading layer.

[0038] The features and advantages of the present disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which together illustrate, by way of example, the features of the present disclosure. [Brief explanation of the drawings]

[0039] [Figure 1] 1 illustrates a method for performing a LAMP analysis according to an exemplary embodiment. [Figure 2] 1 illustrates a method for maximizing the accuracy of a color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay, according to an exemplary embodiment. [Figure 3a]1 illustrates a solid-phase LAMP reaction medium with three test sections, according to an exemplary embodiment. [Figure 3b] 1 illustrates a solid-phase LAMP reaction medium with two test sections, according to an exemplary embodiment. [Figure 3c] 1 illustrates a solid-phase LAMP reaction medium with four test sections, according to an exemplary embodiment. [Figure 3d] 1 illustrates a solid-phase LAMP reaction medium with three test sections and no spreading layer, according to an exemplary embodiment. [Figure 3e] 1 illustrates a solid-phase LAMP reaction medium with two test sections and no spreading layer, according to an exemplary embodiment. [Figure 3f] 1 illustrates a solid-phase LAMP reaction medium with four test sections and no spreading layer, according to an exemplary embodiment. [Figure 4] 1 illustrates a method for testing for the presence of a viral pathogen, according to an exemplary embodiment. [Figure 5] 1 illustrates a method for confirming the suitability of a saliva sample for testing by solid-phase LAMP reaction, according to an exemplary embodiment. [Figure 6] 1 illustrates a method for maximizing the accuracy of a positive test result from a solid-phase LAMP reaction, according to an exemplary embodiment. [Figure 7] 1 illustrates a method for testing for the presence of a target nucleotide sequence, according to an exemplary embodiment. [Figure 8a] 1 illustrates the operation of a biological test kit according to an exemplary embodiment. [Figure 8b] 1 illustrates a color chart for a biological test kit in accordance with an exemplary embodiment. [Figure 9] 1 illustrates a paper-based LAMP assembly according to an exemplary embodiment. [Figure 10] 1 illustrates material screening of Grade 1 and Grade 222 chromatography paper according to an exemplary embodiment. [Figure 11] 1 shows a reaction in a liquid using heat-inactivated SARS-CoV-2 added to water, according to an exemplary embodiment. [Figure 12] 1 illustrates the format of a paper strip according to an exemplary embodiment. [Figure 13] 1 illustrates a solid-phase LAMP reaction medium according to an exemplary embodiment. [Figure 14] 1 illustrates a solid-phase LAMP reaction medium according to an exemplary embodiment. [Figure 15A] 1 illustrates a comparison of Grade 1 and Grade 222 chromatography papers according to an exemplary embodiment. [Figure 15B] 1 illustrates RT-LAMP with the introduction of drying at different starting pH of the RT-LAMP reaction mixture, according to an exemplary embodiment. [Figure 16] 1 illustrates a test strip format according to an exemplary embodiment. [Figure 17] 1 illustrates an assembly process for a test strip according to an exemplary embodiment. [Figure 18A] 1 illustrates the operation of a biological testing system according to an exemplary embodiment. [Figure 18B] 1 illustrates the operation of a biological testing system according to an exemplary embodiment. [Figure 19] A illustrates a schematic diagram and colorimetric characteristics of a paper-based device according to an exemplary embodiment. B illustrates a schematic diagram and colorimetric characteristics of a paper-based device according to an exemplary embodiment. C illustrates a schematic diagram and colorimetric characteristics of a paper-based device according to an exemplary embodiment. D illustrates a schematic diagram and colorimetric characteristics of a paper-based device according to an exemplary embodiment. E illustrates a schematic diagram and colorimetric characteristics of a paper-based device according to an exemplary embodiment. F illustrates a schematic diagram and colorimetric characteristics of a paper-based device according to an exemplary embodiment. [Figure 20] A illustrates digital analysis of a colorimetric response on paper according to an exemplary embodiment. B illustrates digital analysis of a colorimetric response on paper according to an exemplary embodiment. C illustrates digital analysis of a colorimetric response on paper according to an exemplary embodiment. [Figure 21A] 1 illustrates validation of a device with various concentrations of heat-inactivated SARS-CoV-2, according to an exemplary embodiment. [Figure 21B] 1 illustrates color calibration of phenol red at various pH values, according to an exemplary embodiment. [Figure 21C] 1 illustrates the green channel color intensity of the RT-LAMP colorimetric reaction at various template concentrations, according to an exemplary embodiment. [Figure 21D] 1 illustrates LAMP on chromatography paper using EBT as a colorimetric reporter, according to an exemplary embodiment. [Figure 21E] 1 illustrates the colorimetric reaction of LAMP on various papers using EBT as an indicator, according to an exemplary embodiment. [Figure 21F] 1 illustrates LAMP detection on Biodyne A amphoteric paper using EBT as a colorimetric indicator, according to an exemplary embodiment. [Figure 21G] 10 illustrates the effect of removing a single reactant on the initial color of paper after drying, according to an exemplary embodiment. [Figure 21H] 1 illustrates the effect of trehalose and Tween 20 on the RT-LAMP colorimetric reaction, according to an exemplary embodiment. [Figure 21I] 1 illustrates the effect of saliva treatment on colorimetric responses, according to an exemplary embodiment. [Figure 22A] 1 illustrates the effect of a plate on a RT-LAMP colorimetric reaction, according to an exemplary embodiment. [Figure 22B] 1 illustrates the effect of a cap on a RT-LAMP colorimetric reaction, according to an exemplary embodiment. [Figure 22C] 1 illustrates the effect of heating methods on the RT-LAMP colorimetric reaction, according to an exemplary embodiment. [Figure 22D] 1 illustrates the effect of ramp rate on a RT-LAMP colorimetric reaction, according to an exemplary embodiment. [Figure 23A] 1 illustrates a colorimetric perception study in accordance with an exemplary embodiment; [Figure 23B] 1 illustrates a colorimetric perception study in accordance with an exemplary embodiment;

[0040] Reference will now be made to the illustrated exemplary embodiments, and specific language will be used herein to describe the same, it being understood nevertheless that no limitation of the scope of the technology is intended thereby. DETAILED DESCRIPTION OF THE INVENTION

[0041] Before describing embodiments of the present invention, it is to be understood that the present disclosure is not limited to the particular structures, process steps, or materials disclosed herein, but extends to equivalents thereof as would be recognized by one skilled in the relevant art. It should also be understood that the terminology used herein is used solely for the purpose of describing particular embodiment(s) and is not intended to be limiting. The same reference numbers in different drawings represent the same elements. Numbers provided in flowcharts and processes are provided to clearly illustrate steps and operations and do not necessarily indicate a particular order or sequence.

[0042] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details, such as examples of compositions, dosage forms, treatments, etc., are provided to provide a thorough understanding of various embodiments of the present invention. However, those skilled in the art will recognize that such specific embodiments are not limiting of the overall inventive concepts expressed herein, but are merely representative thereof.

[0043] definition It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an excipient" includes reference to one or more such excipients, and reference to "the carrier" includes reference to one or more such carriers.

[0044] As used herein, the terms "formulation" and "composition" are used interchangeably and refer to a mixture of two or more compounds, elements, or molecules. In some embodiments, the terms "formulation" and "composition" can be used to refer to a mixture of one or more active agents with a carrier or other excipient.

[0045] As used herein, the term "solubility" is a measure or characteristic of the ability of a substance or agent to dissolve in a given solvent. The solubility of a substance or agent in a particular component of a composition refers to the amount of the substance or agent that dissolves to form a visibly clear solution at a particular temperature, such as about 25°C or about 37°C.

[0046] As used herein, the term "lipophilic" refers to a compound that is not readily soluble in water. Conversely, the term "hydrophilic" refers to a compound that is soluble in water.

[0047] As used herein, "subject" refers to an animal. In one aspect, the animal can be a mammal. In another aspect, the mammal can be a human.

[0048] As used herein, "non-liquid," when used to refer to the state of the compositions disclosed herein, refers to the physical state of the composition as a semi-solid or solid.

[0049] As used herein, "solid" and "semi-solid" refer to the physical state of a composition that has the appropriate viscosity or structure to support its own weight at standard temperature and pressure and not flow freely. A semi-solid material can conform to the shape of a container under pressure.

[0050] As used herein, "solid phase medium" refers to a non-liquid medium. In one example, the non-liquid medium can be a material having a porous surface. In another example, the non-liquid medium can be a material having a fibrous surface. In yet another example, the non-liquid medium can be paper.

[0051] As used herein, "solid phase medium," "solid base," "solid phase substrate," "solid phase test substrate," "solid phase testing substrate," "solid phase reaction medium," and the like can be used interchangeably herein and refer to a non-liquid medium, device, system, or environment. In some aspects, the non-liquid medium may be substantially free of liquid or completely free of liquid. In one example, the non-liquid medium may include, or be a porous material or a material with a porous surface. In another example, the non-liquid medium may include, or be a fibrous material or a material with a fibrous surface. In yet another example, the non-liquid medium may be paper.

[0052] As used herein, a "non-color-changing additive" refers to an additive that minimizes or prevents the color of a solid-phase medium from changing from its original or initial color to a different color due to reasons other than nucleotide amplification by a LAMP reaction occurring on or in the solid-phase medium. For example, in one embodiment, such a color change can be minimized or reduced compared to the color change that would occur in the absence of the non-color-changing additive.

[0053] As used herein, "a discoloration occurring other than due to a LAMP reaction" refers to any discoloration of a solid-phase medium that is not the result of nucleotide amplification by a LAMP reaction (e.g., a change in color from its original color to another color). In some examples, a discoloration occurring other than due to a LAMP reaction may refer to a discoloration of a solid-phase medium caused by one or more of a volatile agent, a magnesium interfering agent, an oxidizing agent, a pH change due to a cause other than amplification by a LAMP reaction, drying, or a combination thereof.

[0054] As used herein, a "volatile agent" refers to an agent that comprises a composition having a high vapor pressure or a low boiling point. In one example, ammonium sulfate may be a volatile agent because the ammonium ion may volatilize, leaving sulfuric acid behind. In one example, a composition may have a high vapor pressure if it is in the gas phase at a temperature above about 30°C. In one example, a composition may have a low boiling point if it is in the gas phase at a temperature below about 80°C.

[0055] As used herein, "desiccant" refers to an agent that enhances the drying of a solid phase medium when compared to the drying of the solid phase medium without the desiccant.

[0056] As used herein, a "pH interfering reagent" is a reagent that can affect the pH of a reaction, system, or environment for reasons other than LAMP amplification. In one example, ammonium ions can volatilize from ammonium sulfate, and sulfate ions can react to form sulfuric acid, which can affect the pH of a reaction in the absence of LAMP amplification.

[0057] As used herein, a "non-pH sensitive agent" is a reagent that is substantially unaffected by changes in pH.

[0058] In this disclosure, terms such as "comprises," "comprising," "containing," and "having" can have the meaning ascribed to them in U.S. patent law and can mean "includes," "including," and the like, and are generally construed as open-ended terms. The terms "consisting of" or "consists of" are closed terms and include only those components, structures, steps, etc. specifically recited in conjunction with such term, in addition to those components, structures, steps, etc., that comply with U.S. patent law. "Consisting essentially of" or "consists essentially of" have the meaning generally ascribed to them by U.S. patent law. In particular, such terms are generally closed terms, except where permitted to include additional items, materials, components, steps, or elements that do not materially affect the basic and novel characteristics or function of the item(s) with which they are used. For example, trace elements that are present in a composition but do not affect the properties or characteristics of the composition are permissible when present under the term "consisting essentially of," even if they are not explicitly recited in the list of items following such terminology. When open-ended terms such as "comprising" or "including" are used in a written description, it is understood that the words "consisting of," as well as "consisting essentially of," should be directly supported as if explicitly recited, and vice versa.

[0059] When terms such as "first," "second," "third," and "fourth" appear in the specification and claims, they are used to distinguish between similar elements and not necessarily to describe a particular sequential or chronological order. All such terms used should be understood to be interchangeable, under appropriate circumstances, such that the embodiments described herein can operate in orders other than those illustrated or otherwise described herein. Similarly, when a method is described herein as including a series of steps, the order of such steps presented herein is not necessarily the only order in which such steps can be performed; in some cases, some of the described steps may be omitted, and / or certain other steps not described herein may be added to the method.

[0060] As used herein, comparative terms such as "increased," "decreased," "better," "worse," "higher," "lower," "enhanced," "maximized," "minimized," and the like refer to a property of a device, ingredient, composition, or activity that is measurably different from other devices, ingredients, compositions, or activities in surrounding or adjacent areas, similarly positioned, within a single device or composition, or within multiple comparable devices or compositions, within a group or class, or within multiple groups or classes, or compared to the known state of the art.

[0061] The term "coupled," as used herein, is defined as directly or indirectly connected in a chemical, mechanical, electrical, or non-electrical manner. Objects described herein as "adjacent" to one another may be in physical contact with one another, in close proximity to one another, or in the same general area or region as one another, depending on the context in which the phrase is used. "Directly coupled" objects, structures, elements, or features are in contact with and may be attached to one another. Furthermore, as used in this written description, it is understood that the use of the term "coupled" also supports "directly coupled," and vice versa.

[0062] As used herein, the term "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, a "substantially" enclosed object means that the object is either completely enclosed or nearly completely enclosed. The precise acceptable degree of deviation from absolute completeness may depend in some cases on the specific context. Generally speaking, however, approximating completeness results in an overall result similar to that obtained when absolute and total completeness were achieved. The use of "substantially" also applies when used in a negative sense to refer to the complete or nearly complete absence of an action, characteristic, property, state, structure, item, or result. For example, a composition "substantially free" of particles is either completely absent or nearly completely absent, such that the effect is the same as if the particles were completely absent. In other words, a composition that is "substantially free" of a component or element may still actually contain such an item as long as there is no measurable effect of that component or element.

[0063] As used herein, the term "about" is used to provide flexibility for the endpoints of numerical ranges by defining that a given value may be "slightly above" or "slightly below" the endpoint. Unless otherwise specified, the use of the term "about" in conjunction with a particular numerical value or range should also be understood to support such numerical term or range without the term "about." For example, for convenience and brevity, a numerical range of "about 50 angstroms to about 80 angstroms" should also be understood to support the range "50 angstroms to 80 angstroms." Furthermore, it should be understood that actual numerical values ​​are also supported herein, even when they are used with the term "about." For example, a statement of "about" 30 should be interpreted as supporting not only values ​​slightly above and slightly below 30, but also the actual numerical value of 30.

[0064] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in common lists for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Accordingly, no individual member of such a list should be construed as being de facto equivalent to any other member of the same list solely based on their presence within a common group, unless otherwise indicated.

[0065] Concentrations, amounts, levels, and other numerical data may be expressed or presented herein in a range format. It should be understood that such range format is used merely for convenience and brevity and should therefore be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limits, but also all individual numerical values, subranges, or fractional units subsumed within that range, as if each numerical value and subrange were explicitly recited. Illustratively, a numerical range of "about 1 to about 5" should be interpreted to include not only the explicitly recited values ​​of about 1 to about 5, but also each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 2, 3, and 4, as well as subranges such as 1 to 3, 2 to 4, and 3 to 5, individually, in addition to 1, 2, 3, 4, and 5. This same principle applies to ranges reciting only a single numerical value as a minimum or maximum value. Moreover, such interpretation should apply regardless of the width or characteristics of the range described.

[0066] Throughout this specification, a reference to "one example" means that the particular feature, structure, or characteristic described in connection with that example is included in at least one embodiment. Thus, the appearances of the phrase "in one example" in various places throughout this specification are not necessarily all referring to the same embodiment.

[0067] Embodiment Many molecular tests for pathogens (e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19) are limited to laboratories, which can have significant lag times (greater than 24 hours) before results are available, preventing their adoption in point-of-care settings. Although there have been several attempts to develop point-of-care tests for SARS-CoV-2, several limitations remain: i) scalability (demand for tests is in the millions per week, making it difficult to build new tests at that scale); ii) sample processing (when using saliva, many tests still require extraction procedures); and iii) readability (molecular tests often use fluorescence, requiring fluorescence readers to report results).

[0068] Current testing methods can be overcome with a point-of-care test using a paper-based device and reverse transcription loop-mediated isothermal amplification (RT-LAMP), which uses diluted saliva (e.g., 5% v / v in water) as a sample and reports a color change in the presence of pathogens (e.g., SARS-CoV-2) within 60 minutes. RT-LAMP is a nucleic acid amplification technique performed at a constant temperature, which has suitable diagnostic performance, especially during the acute phase of infection. Because RT-LAMP can be performed at a constant temperature, expensive thermal cycling equipment is not required. Furthermore, existing colorimetric reporters for LAMP products do not require fluorescent readers. As a result, this test is suitable for use in point-of-care settings and is amenable to rapid development and scale-up, making it suitable for use in public health emergencies.

[0069] RT-LAMP can be implemented in microfluidic paper-based analytical devices (μPADs) for detecting various pathogens (e.g., SARS-CoV-2), where image analysis can be performed using a portable electronic device to distinguish positive from negative reactions. In one example, a high-contrast RT-LAMP reaction on paper can result in a color change visible to the naked eye. Furthermore, instead of using wax printing (which involves precise alignment of the printing area and dispensing of reagents), polystyrene spacers can be used to prevent crosstalk between samples. Polystyrene spacers can be suitable for roll-to-roll fabrication for scale-up production.

[0070] Nucleic acid-based COVID-19 diagnostic methods require preprocessing to provide results. As disclosed herein, colorimetric detection of SARS-CoV-2 on paper can be performed with minimal preprocessing. The device may have the sensitivity and specificity to detect SARS-CoV-2 on paper without preamplification. Other assays performed in solution may not be as scalable during manufacturing as paper-based assays. Furthermore, while the assays disclosed herein use a dilution procedure that can be completed in seconds, other assays use various procedures, such as protease treatment, heat inactivation, and / or RNA extraction, to detect SARS-CoV-2 (procedures that take at least 10 minutes and require additional equipment).

[0071] Materials assembly for LAMP reaction on solid-phase media Performing a LAMP reaction on a solid-phase medium can be challenging due to performance requirements. For example, to maximize test accuracy, the solid-phase medium must support the LAMP reaction without interfering with the LAMP reaction or the LAMP reaction indicator. At a high level, the solid-phase medium is hydrated with the biological sample, allowing the LAMP reaction to proceed, and the results are then read. However, various reagents can interfere with the LAMP reaction or the subsequent reading of the results. To minimize errors, care can be taken to minimize or avoid potential issues with providing the biological sample, hydrating the solid-phase medium with the sample, incorporating the LAMP reagents into the solid-phase medium, performing the LAMP analysis, and obtaining a clear test output signal that is easily interpretable.

[0072] With the above background in mind, in one disclosed embodiment, a loop-mediated isothermal amplification (LAMP) reaction assembly may include a LAMP reagent mixture that is substantially free of hygroscopic agents in combination with a solid-phase reaction medium. If the LAMP reagent mixture is substantially free of hygroscopic agents, difficulties encountered during drying of the solid-phase reaction medium can be minimized or avoided. When hygroscopic agents are used, the hygroscopic agent or solid-phase reaction medium may not dry completely, or they may rehydrate to some extent during storage, and may interfere with the original color of the solid-phase reaction medium, which may distort the test results of the LAMP process.

[0073] In one example, the hygroscopic agent can be an agent that absorbs more than about 10% by weight at about 40% to about 90% relative humidity (RH) at 25°C. In one embodiment, the hygroscopic agent can include, but is not limited to, one or more of glycerol, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate, etc., or combinations thereof. In some examples, LAMP reactions containing hygroscopic agents such as glycerol can contribute to instability of reagents in the solid-phase medium because the hygroscopic agent may attract water. Therefore, excessive amounts of hygroscopic agents in the solid-phase reaction medium should be avoided.

[0074] Furthermore, in another embodiment, the LAMP reagent mixture substantially free of hygroscopic agents may include one or more of a DNA polymerase, a reverse transcriptase, a target primer, or a combination thereof. When the LAMP reaction is either a LAMP reaction or a reverse transcription-LAMP (RT-LAMP) reaction, a DNA polymerase may be included. Furthermore, when the LAMP reaction is an RT-LAMP reaction, the LAMP reagent substantially free of hygroscopic agents may further include a reverse transcriptase used to reverse transcribe RNA into cDNA.

[0075] In one embodiment, the solid-phase reaction medium may be substantially free of magnesium-interfering agents. Magnesium may interfere with the LAMP reaction in several ways. First, magnesium may be a cofactor for DNA polymerase, and it is necessary to closely monitor magnesium within a target concentration range so that the DNA polymerase can promote the LAMP reaction. Second, when a magnesium-sensitive indicator is used in the LAMP reaction, magnesium-interfering agents may invalidate the results of the LAMP reaction or complicate the analysis of the results.

[0076] Thus, in another aspect, magnesium interfering agents can include magnesium-containing compounds and chelating agents that interfere with magnesium. 2+ , Mg 1+The composition may be substantially free of magnesium, including, but not limited to, magnesium carbonate, magnesium chloride, magnesium citrate, magnesium hydroxide, magnesium oxide, magnesium sulfate, magnesium sulfate heptahydrate, etc., or combinations thereof. Some solid-phase reaction media may have some buffering capacity, residual ions, or chelating agents that can interfere with magnesium, which can affect the concentration of magnesium, a cofactor for BST enzymes (e.g., DNA polymerase), and this can interfere with the LAMP reaction. As a result, to facilitate the LAMP reaction, it is necessary to control the magnesium concentration within a target magnesium range. In one embodiment, the composition may contain one or more of less than 1.0 wt.%, less than 0.5 wt.%, less than 0.1 wt.%, or less than 0.01 wt.% magnesium.

[0077] The solid-phase reaction medium can have several characteristics to facilitate the LAMP reaction. In one embodiment, the solid-phase reaction medium can be hydrophilic, absorbent, porous, and inert. The solid-phase reaction medium can be hydrophilic if the contact angle between the surface and edge of the droplet is less than about 90 degrees. The solid-phase reaction medium can have absorbency measured by the degree to which paper can absorb a certain amount of liquid. The solid-phase reaction medium can be porous if the pore size of the solid-phase reaction medium is at least greater than 1 micron and is one or more of about 100 microns or less, about 75 microns or less, about 50 microns or less, about 25 microns or less, about 10 microns or less, about 5 microns or less, or about 1 micron or less. The solid-phase reaction medium can be inert if the medium does not interfere with the LAMP reaction. The solid-phase reaction medium can also be inert if the medium does not interfere with the indicator effect resulting from the LAMP reaction.

[0078] There are a variety of materials that the solid-phase reaction medium may comprise or include. In one embodiment, the solid-phase reaction medium may comprise one or more of nylon, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, hydrophilic polytetrafluoroethylene (PTFE), etc., or a combination thereof. In another embodiment, the solid-phase reaction medium may be a cellulose-based medium, such as Grade 1 chromatography paper, Grade 222 chromatography paper, etc., or a combination thereof.

[0079] In addition to the type of material of the solid-phase reaction medium, the solid-phase reaction medium may have several physical properties that can be compatible with or enhance the LAMP reaction and avoid interference with the indicating action or signal output resulting from the LAMP reaction. In one embodiment, the solid-phase reaction medium may have a surface area-to-thickness ratio of about 30 to about 600. In another embodiment, the solid-phase reaction medium may have a surface area-to-thickness ratio of about 60 to about 400. In one embodiment, the solid-phase reaction medium may have a surface area-to-thickness ratio of about 100 to about 200. In another embodiment, the solid-phase reaction medium may be a cellulose-based medium that may have a surface area-to-thickness ratio of about 30 to about 600. In one embodiment, the cellulose-based medium may have a pore size that is at least greater than 1 micron and one or more of about 100 microns or less, about 75 microns or less, about 50 microns or less, about 25 microns or less, about 10 microns or less, about 5 microns or less, or about 1 micron or less.

[0080] The thickness of the solid-phase reaction medium can contribute to the total reaction time of the LAMP reaction, the flow rate through the solid-phase reaction medium, the color contrast when using a colorimetric indicator, the uniformity of the colorimetric results, the concentration of reagents in the solid-phase reaction medium, etc.

[0081] In one embodiment, the solid-phase reaction medium can include Grade 222 chromatography paper, which is thicker than Grade 1 chromatography paper and therefore provides increased uniformity compared to Grade 1 chromatography paper. As a result, Grade 222 chromatography paper can concentrate reagents in a smaller surface area compared to Grade 1 chromatography paper. In one embodiment, the chromatography paper can be Grade 222, having a surface area of ​​about 5 mm by about 5 mm, to provide a desired surface area-to-thickness ratio. In one example, Grade 1 chromatography paper has cross-sectional dimensions of 20 mm long, 5 mm wide, and 0.18 mm thick, providing a surface area-to-thickness ratio of about 555. In another example, Grade 222 chromatography paper has cross-sectional dimensions of 5 mm long, 5 mm wide, and 0.83 mm thick, providing a surface area-to-thickness ratio of about 30. Thus, this example shows that a surface area to thickness ratio of about 30 (e.g., for grade 222 chromatography paper) can increase uniformity compared to a surface area to thickness ratio of about 555 (e.g., for grade 1 chromatography paper).

[0082] In addition to the materials disclosed herein, the solid-phase reaction medium may include one or more of paper or glass fiber. In one example, the paper may include one or more of alpha cellulose, beta cellulose, gamma cellulose, etc., or a combination thereof. In another example, the glass fiber may include one or more of A-glass, E-glass, S-glass, R-glass, C-glass, T-glass, D-glass, M-glass, ECR glass, etc., or a combination thereof.

[0083] In some examples, the reaction assembly may include an adhesive. For example, the adhesive may be used to adhere various segments of the solid-phase reaction medium to one another. In one example, the reaction assembly may further include an adhesive that is substantially free of magnesium-interfering agents, moisture-absorbing agents, or a combination thereof. The adhesive may be substantially free of magnesium-interfering agents when using a magnesium-based indicator to avoid interference between magnesium and DNA polymerase and avoid complications in reading the LAMP results.

[0084] In some examples, the reaction assembly may also include a spreading layer. The spreading layer may promote uniform spreading of the biological sample throughout various sections of the solid-phase reaction medium. In another example, the spreading layer may be less hydrophilic than the solid-phase reaction medium. Having a spreading layer that is less hydrophilic than the solid-phase reaction medium may promote uniform spreading of the biological sample as the biological sample diffuses from the less hydrophilic spreading layer into the more hydrophilic solid-phase reaction medium.

[0085] The arrangement of the reagent mixture relative to the solid-phase reaction medium can affect the LAMP reaction. In one embodiment, a method for producing a LAMP reaction assembly recited herein can include combining a LAMP reagent mixture substantially free of hygroscopic agents with a solid-phase reaction medium such that the reagent mixture is maintained in contact with the solid-phase reaction medium. In one example, the reagent mixture can be maintained in direct contact with the solid-phase reaction medium. In another example, the reagent mixture can be maintained in indirect contact with the solid-phase reaction medium. When the reagent mixture is maintained in indirect contact with the solid-phase reaction medium, an intervening material (e.g., an antioxidant) can enhance the LAMP reaction.

[0086] In some cases, the color of the solid-phase reaction medium can be affected by agents not originating from the LAMP reaction. For example, volatile agents such as ammonium sulfate can form sulfate ions, which can react to form sulfuric acid. When using pH-based indicators to read the results of a LAMP reaction, these non-LAMP reactions can interfere with the correct reading of the results or further complicate the interpretation of the results.

[0087] In one aspect, the method can include controlling discoloration using a non-discoloring additive. The non-discoloring additive can include a sugar, a buffer, a blocking agent, etc., or a combination thereof. In one example, the sugar can include one or more of trehalose, glucose, sucrose, dextran, etc., or a combination thereof. In another example, the blocking agent can include bovine serum albumin, casein, etc., or a combination thereof.

[0088] The sugar can prevent the influence of pH changes caused by factors other than the LAMP reagent. The buffer can prevent interference with the LAMP reaction or the results of the LAMP reaction by preventing the influence of pH changes caused by factors other than the LAMP reaction. The blocking agent can prevent the influence of factors other than the LAMP reaction by blocking the action of various enzymes such as RNase or DNase on the nucleic acid being analyzed (e.g., RNA from a virus or DNA from a pathogen).

[0089] 1 , a method 100 for performing a LAMP analysis may include providing a LAMP reaction assembly as enumerated in the present disclosure, as shown in block 110. The method may further include applying a biological sample to the reaction assembly, as shown in block 120. The method may further include heating the assembly to a temperature sufficient to initiate the LAMP reaction, as shown in block 130. The method may further include maintaining the temperature for a time sufficient to complete the LAMP reaction, as shown in block 140.

[0090] In one embodiment, the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or a combination thereof. In another embodiment, the biological sample can be saliva. In another embodiment, the method can further include detecting a viral pathogen. In another embodiment, the LAMP analysis can be reverse transcription-LAMP (RT-LAMP).

[0091] In another embodiment, the temperature sufficient to initiate the LAMP reaction can be in the range of about 50°C to about 60°C. In another embodiment, the temperature sufficient to initiate the LAMP reaction can be in the range of about 60°C to about 70°C. In another example, the isothermal temperatures can be within a range of temperatures that differ by less than 5°C. In another embodiment, the temperature can be maintained at a temperature sufficient to complete the LAMP reaction for one or more of more than 15 minutes, more than 30 minutes, more than 45 minutes, more than 60 minutes, more than 75 minutes, more than 90 minutes, more than 105 minutes, or more than 120 minutes. In another embodiment, the temperature can be maintained at a temperature sufficient to complete the LAMP reaction for one or more of less than 15 minutes, less than 30 minutes, less than 45 minutes, less than 60 minutes, less than 75 minutes, less than 90 minutes, less than 105 minutes, or less than 120 minutes.

[0092] LAMP reaction assemblies can be manufactured to enhance their uniformity, shelf life or shelf life, and testing effectiveness. In one aspect, LAMP reaction assemblies can be manufactured using one or more of the following techniques: slitting (dividing a roll into thinner rolls), singulation (cutting individual pieces with a guillotine, rotary die, or laser), reagent coating (impregnating, spraying, or dispensing reagents and drying), card lamination (adding a plastic backing to a reel), etc., or a combination thereof. In one example, LAMP reaction assemblies can be manufactured using one or more of the following techniques: dividing a roll into thinner rolls, cutting individual pieces with a guillotine or rotary die, reagent coating using reagent impregnation, card lamination using adding a plastic backing to a reel, etc., or a combination thereof.

[0093] Materials for pH-based LAMP analysis on solid-phase media Performing LAMP reactions on solid-phase reaction media can be difficult in the presence of moisture absorbers and magnesium interferents. However, there are other factors that can interfere with the output and interpretation of LAMP reaction results. When a pH-based indicator is required, the reagent mixture must be substantially free of interfering reagents. In some instances, interfering reagents may include oxidizing agents and pH interferents.

[0094] In one embodiment, a system for chromatic loop-mediated isothermal amplification (LAMP) analysis can include a substantially non-reactive solid-phase reaction medium and a non-interfering reagent mixture. In one aspect, the substantially non-reactive solid-phase reaction medium can be substantially free of oxidizing agents and pH-interfering agents. In one aspect, the non-interfering reagent mixture can include one or more target primers, a DNA polymerase, a resolubilizing agent, or a combination thereof.

[0095] Oxidizing agents may interfere with the LAMP reaction. Therefore, oxidizing agents should not be included in the substantially non-reactive solid-phase reaction medium. In one embodiment, the oxidizing agent may include, but is not limited to, one or more of O, O, H, O, F, Cl, halogens, HNO, nitrates, HSO, HSO, HSO, hypochlorite, chlorite, chlorate, perchlorate, chromium compounds, permanganate, sodium perborate, nitrous oxide, NO, NO, KNO, NaBiO, cerium compounds, lead dioxide, etc., or combinations thereof.

[0096] In some cases, avoiding oxidizing agents alone may not be sufficient to promote the LAMP reaction. In such cases, additional agents can be included to prevent undesired oxidation. In one embodiment, the non-reactive solid-phase reaction medium can include one or more of an oxygen absorber, a dehumidifier, etc., or a combination thereof, to prevent oxidation of the non-reactive solid-phase reaction medium. In another embodiment, to prevent oxidation of a non-reactive solid-phase reaction medium containing cellulose, the cellulose can be pre-treated by thermal cycling to saturate the oxidation sites. In another example, an antioxidant can be added to prevent oxidation. In another embodiment, a dye indicator (e.g., phenol red) can have antioxidant properties. In one embodiment, the substantially non-reactive solid-phase reaction medium can contain one or more of an oxidizing agent at less than 1.0 wt %, less than 0.5 wt %, less than 0.1 wt %, or less than 0.01 wt %.

[0097] In addition to oxidizing agents, pH interfering agents can prevent proper interpretation of the results of a LAMP reaction or further complicate the signal output of a LAMP reaction. In one embodiment, pH interfering agents can include, but are not limited to, one or more of a volatile reagent, a pH-affecting reagent, a magnesium-containing reagent, or a combination thereof.

[0098] When pH-affecting reagents are included, the resulting pH change can complicate the analysis of pH-based results from LAMP reactions. In some cases, the inclusion of pH-affecting reagents can be offset and the analysis adjusted to allow for proper interpretation of the test. However, in other cases, pH-affecting reagents can introduce uncertainty into the interpretation of pH-based results.

[0099] In one example, the substantially non-reactive solid phase reaction medium may be substantially free of pH-affecting reagents. In one embodiment, the pH-affecting reagents may include acids, bases, or combinations thereof that may interfere with pH-sensitive signals. In one embodiment, the substantially non-reactive solid phase reaction medium may contain less than 1.0 wt %, less than 0.5 wt %, less than 0.1 wt %, or less than 0.01 wt % of one or more pH-affecting reagents.

[0100] When a volatile reagent is included in the non-reactive solid phase reaction medium, the volatile component may volatilize, leaving behind a component that may interfere with the pH-sensitive output signal, or may further react to leave behind a component that interferes with the pH-sensitive output signal. In one example, ammonium carbonate may form ammonium ions, which volatilize, and carbonate ions, which react to form carbonic acid. Carbonic acid may interfere with the pH-sensitive output signal by lowering the pH in the absence of a positive result from the LAMP reaction (e.g., the presence of the target pathogen and LAMP-induced amplification). Thus, in one example, the non-reactive solid phase medium may be substantially free of volatile agents, as defined herein.

[0101] If a magnesium-containing reagent is included in the non-reactive solid-phase reaction medium, the magnesium-containing reagent may interfere with the operation of the DNA polymerase if not closely monitored. Thus, in one example, a substantially non-reactive solid-phase reaction medium may be substantially free of magnesium reagents, as otherwise disclosed herein.

[0102] The substantially non-reactive solid phase medium can be composed of a variety of materials. In one aspect, the substantially non-reactive solid phase reaction medium can include glass fiber, nylon, cellulose, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, hydrophilic PTFE, etc., or combinations thereof. In another aspect, the substantially non-reactive solid phase reaction medium can be hydrophilic, absorbent, inert, and porous, as disclosed herein.

[0103] The buffer capacity of the non-reactive solid-phase medium can affect the LAMP reaction. For example, a strong buffer can prevent the pH change detected from the LAMP reaction. However, a weak buffer can cause large pH fluctuations even in the absence of LAMP-induced amplification. In one embodiment, the substantially non-reactive solid-phase reaction medium can have a buffer capacity of about 0.01 mM to about 5 mM. In one example, heat-inactivated virus can be added to saliva stored in a biobank with a detection limit of about 500 copies per 25 μl sample volume. However, the buffer capacity and pH of saliva stored in a biobank may differ from those of freshly collected saliva. Therefore, the limit of detection (LOD) of freshly collected saliva can be adjusted based on the difference in buffer capacity and pH between the saliva stored in a biobank and the freshly collected saliva.

[0104] In some instances, the pH-sensitive output signal from a non-reactive solid-phase reaction medium can be interpreted by a technician without dedicated instrumentation. However, a colorimetric reader can provide an additional level of precision and accuracy. For example, in one instance, a substantially non-reactive solid-phase reaction medium, when combined with a pH-sensitive dye, exhibits a maximum absorption wavelength (λ ) within the test range. max In one example, if the pH-sensitive dye is phenol red, then λ max can be within the test range of about 443 nm to about 570 nm. Therefore, if the pH-sensitive dye is phenol red and the maximum absorption wavelength is within the test range, a technician can use a colorimetric reader to detect a positive or negative result.

[0105] In some embodiments, the system may further comprise components in addition to the non-reactive solid-phase reaction medium. In one embodiment, the system may further comprise an adhesive, a spreading layer, a spacer, a plastic carrier, or a combination thereof. In one embodiment, each of the adhesive, spreading layer, spacer, and plastic carrier may be substantially free of the oxidizing agents and pH interfering agents disclosed herein.

[0106] In another embodiment, as shown in FIG. 2 , a method 200 for maximizing the accuracy of a color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay may include providing a solid-phase reaction medium that minimizes color changes occurring outside of the LAMP reaction, as shown in block 210, and performing the LAMP assay on the solid-phase reaction medium, as shown in block 220.

[0107] In one embodiment, the method may further include controlling discoloration caused by protons generated outside of the LAMP reaction, hi another embodiment, the method may further include controlling discoloration caused outside of the LAMP reaction using a non-discoloring additive.

[0108] The non-color-changing additive can prevent undesired changes in the colorimetric reaction due to factors other than LAMP amplification. In one embodiment, the non-color-changing additive can include a sugar, a buffer, a blocking agent, etc., or a combination thereof.

[0109] In one example, the sugar may include one or more of trehalose, glucose, sucrose, dextran, etc., or a combination thereof. In one embodiment, the sugar concentration when used on a solid phase medium may be about 0.01 mM to about 1 M. In another example, the sugar concentration when used on a solid phase medium may be about 10 mM to about 500 mM. In yet another example, the sugar concentration when used on a solid phase medium may be about 200 mM to about 400 mM.

[0110] In another example, the buffer solution may include one or more of phosphate-buffered saline (PBS), Dulbecco's PBS, Alsever's solution, Tris-buffered saline (TBS), water, HEPES, BICINE, balanced salt solution (BSS) such as Hank's BSS, Earle's BSS, Gray's BSS, Puck's BSS, Simm's BSS, Tyrode's BSS, BSS Plus, lactated Ringer's solution, normal saline (i.e., 0.9% saline), half normal saline, etc., or combinations thereof. In one embodiment, the concentration of the buffer solution when used on a solid-phase medium may be about 10 μM to about 20 mM. In another example, the concentration of the buffer solution when used on a solid-phase medium may be about 100 μM to about 10 mM. In yet another example, the concentration of the buffer solution when used on a solid-phase medium may be about 100 μM to about 500 μM.

[0111] In another example, the blocking agent may include one or more of bovine serum albumin, casein, or a combination thereof. In one embodiment, the concentration of the blocking agent, when used on a solid phase medium, may be about 0.01% by weight to about 5% by weight. In another example, the concentration of the blocking agent, when used on a solid phase medium, may be about 0.01% by weight to about 1% by weight. In yet another example, the concentration of the blocking agent, when used on a solid phase medium, may be about 0.02% by weight to about 0.06% by weight.

[0112] In another embodiment, a method for maximizing the level of detection (LOD) of a loop-mediated isothermal amplification (LAMP) assay can include providing a reaction environment and reagents that minimize non-LAMP reaction products. In some examples, the reaction environment can be substantially free of one or more of oxidizing agents, pH interfering agents, hygroscopic reagents, magnesium interfering reagents, etc., or combinations thereof.

[0113] In another embodiment, a system for chromatic loop-mediated isothermal amplification (LAMP) analysis can include a combination of a solid-phase reaction medium and LAMP reagents that can maintain the color of the solid-phase reaction medium within 10% of the initial color of the solid-phase medium when stored at 25°C. In one aspect, the combination of the solid-phase medium and LAMP reagents can maintain the color when stored for more than one or more of 30 days, 90 days, 365 days, 2 years, 3 years, or 5 years. In another aspect, the combination of the solid-phase medium and LAMP reagents can maintain the color when stored at a relative humidity of about 40% to 90%.

[0114] In another embodiment, a method for producing a chromatic LAMP system can include combining a non-interfering reagent mixture with a substantially non-reactive solid phase reaction medium such that the non-interfering reagent mixture is maintained in contact with the substantially non-reactive solid phase reaction medium.

[0115] In one example, the non-interfering reagent mixture can be maintained in direct contact with the solid-phase reaction medium. In another example, the non-interfering reagent mixture can be maintained in indirect contact with the solid-phase reaction medium. When the non-interfering reagent mixture is maintained in indirect contact with the solid-phase reaction medium, an intervening material (e.g., an antioxidant) can enhance the LAMP reaction.

[0116] In one aspect, the method can include preparing a solution containing a non-interfering reagent mixture and coating the reagent mixture onto the substantially non-reactive solid phase reaction medium, hi another aspect, the coating can include dripping, spraying, impregnating, dipping, or misting the solution onto the substantially non-reactive solid phase reaction medium.

[0117] In some cases, the manufacturing process may affect the shelf-life stability or uniformity of the system for LAMP analysis. In one example, a non-interfering reagent mixture may be combined with a substantially non-reactive solid-phase reaction medium using a reel-to-reel (R2R) process.

[0118] Multiplexing selected targets for pathogen testing using LAMP on solid-phase media When using LAMP on a solid-phase medium to detect pathogens, there are various ways to multiplex LAMP. First, the process can be multiplexed by including multiple controls on the same solid-phase medium. For example, test validity can be verified using a positive control. For example, a LAMP reaction can test a saliva sample for saliva DNA or RNA biomarkers to verify that the LAMP reaction is functioning as expected. In another example, test validity can be verified using a negative control. For example, a LAMP reaction can test a saliva sample that does not contain pathogens.

[0119] A second way to multiplex LAMP can be by including primers targeting multiple different pathogens on the same solid-phase medium. For example, to further characterize a biological sample, one can verify test validity by testing for viral and bacterial pathogens on the same solid-phase medium.

[0120] A third method for multiplexing LAMP is to test the same pathogen using different target primers. In one example, a first section of the solid-phase medium can be tested using a primer set for a first protein of the viral pathogen, a second section of the solid-phase medium can be tested using a primer set for a second protein of the viral pathogen, and a third section of the solid-phase medium can be tested using a primer set for a third protein of the viral pathogen. Different regions of the viral pathogen's genome can be targeted by each primer to eliminate false negatives and false positives.

[0121] LAMP can be multiplexed by including various components on a solid-phase LAMP reaction medium. In one embodiment, as illustrated in FIG. 3 a, a solid-phase reaction medium 300 a for conducting a LAMP analysis can include a substrate 302, an adhesive layer 304 disposed on the substrate 302, a reaction layer 306 (including test spots or reaction locations or segments 305 a, 305 b, 305 c and spacers 307 a, 307 b, 307 c disposed on the adhesive layer 304), and a spreading layer 308 disposed on the reaction layer 306. In one aspect, the test spots 305 a, 305 b, and 305 c can contain or otherwise hold reagents, including one or more target primers, a DNA polymerase, a resolubilizing agent, etc. In one aspect, the reagents can form a composition sufficient to perform a LAMP reaction.

[0122] Spatially discontinuous reaction layer 306 may allow for multiplexing of multiple controls or multiple pathogens. For example, test spot 305a may be a positive control (e.g., testing for known saliva DNA or RNA biomarkers), test spot 305b may be a negative control (e.g., testing for a colorimetric result without all the reagents used in LAMP), and test spot or reaction segment 305c may test for a target pathogen.

[0123] Spatially discrete test spots or reaction locations 305a, 305b, and 305c may also allow for multiplexed detection of multiple pathogens, for example, test spot 305a may test for influenza, test spot 305b may test for bacterial infection, and test spot 305c may test for fungal infection.

[0124] The dimensions of the reaction locations or segments 305a-305c can affect the potential for multiplexing. In one embodiment, the reaction segments 305a-305c can have a thickness of about 0.05 mm to about 2 mm. In another embodiment, the reaction segments 305a-305c can have a width of about 4 mm to about 12 mm and a length of about 4 mm to about 25 mm. In one example, the reaction locations 305a-305c can be spatially discontinuous. In another example, the reaction segments 305a-305c can have a surface area-to-thickness ratio of about 30 to about 600.

[0125] In one example, the solid-phase reaction medium 300a can be configured to receive a biological fluid that can flow laterally across the spreading layer 308 and vertically downward into the test spots 305a-305c of the reaction layer 306. The test spots 305a-305c can contain all of the components used to generate an RT-LAMP or LAMP reaction. In one example, the test spots 305a-305c can contain a resolubilizing agent (e.g., a surfactant), an enzyme (e.g., a DNA polymerase, a reverse transcriptase, a DNase inhibitor, or an RNase inhibitor), a stabilizer (e.g., a blocking agent such as BSA or casein), a colorimetric indicator (e.g., a magnesium colorimetric indicator, a pH colorimetric indicator, or a DNA intercalating colorimetric indicator), and a buffer (e.g., 20 mM Tris).

[0126] The solid-phase reaction medium 300a can be configured to receive an agent that can accelerate the reaction, increase sensitivity, or a combination thereof. In one example, BSA can accelerate the reaction and increase sensitivity. However, the inclusion of BSA can also result in pH fluctuations that can impair the readability of the results. Thus, in some examples, the stabilizer can be casein, polysorbate 20, or the like, or a combination thereof.

[0127] The reaction segments or spots (e.g., test spots) 305a-305c can comprise any suitable material disclosed herein. In one example, the reaction segments 305a-305c can comprise one or more of fiberglass, nylon, cellulose, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, hydrophilic PTFE, etc., or combinations thereof. In one embodiment, the pore size of the reaction segments or spots 305a-305c can be about 1 to about 100 microns. The reaction segments or spots 305a-305c can have an optically clean and smooth appearance.

[0128] In another embodiment, the reaction segments 305a-305c can present a uniform final color within the read zone for accurate and precise signal output or detection. In one example, a biological sample can slowly move vertically downward into the reaction segments 305a-305c. The intensity of the final color of the reaction segments 305a-305c can be measured by the user by optical observation and comparison to a color chart or scale, or as percent reflectance units using a handheld LED meter, and can be converted to RNA or DNA copies per reaction using a curve set calibrated to a laboratory reference device, or as an optical image capturing RGB values ​​or pixel counts that can be calibrated to a laboratory reference device. The concentration of the RNA or DNA can be determined by measuring the final color intensity or kinetic rate at a selected time.

[0129] In another embodiment, the solid-phase LAMP reaction medium 300a may further include reaction segments 305a-c. In one example, at least one section may be substantially free of reagents (e.g., 305a may be substantially free of reagents). In another embodiment, the reaction segments 305a-c may be defined by at least three sections of the discontinuous adhesive layer 304. The three reaction segments 305a, 305b, and 305c may include designations for testing the same pathogen, one pathogen with multiple controls, or multiple different pathogens with or without specific positive or negative controls. In short, the reaction segments 305a-c may be designed and configured to test in nearly any sequence to provide any desired parameters or protocols that result in a specific test for a particular result with a high degree of accuracy and reliability. Furthermore, the reaction layer 306 may be configured with test spots 305a-c arranged in various numbers and positions. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other suitable number of test spots may be included in the reaction layer. Furthermore, such test spots or reaction segments may be arranged in a line, in parallel, or in nearly any other desired spatial arrangement within the reaction layer 306.

[0130] In one embodiment, as illustrated in FIG. 3b, a solid-phase LAMP reaction medium 300b can include reaction segments 305a and 305b coupled between an adhesive layer 304 and a spreading or distribution layer 308. In one embodiment, at least one reaction segment (e.g., 305a or 305b) can be a control section or spot that is substantially free of reagents. In another embodiment, at least one of the reaction segments (e.g., 305a or 305b) can be configured to test for a target pathogen (e.g., contain or otherwise support reagents for a selected LAMP reaction).

[0131] In yet another embodiment, as illustrated in Figure 3c, a solid-phase LAMP reaction medium 300c can include a reaction layer 306 with reaction segments or spots 305a, 305b, 305c, and 305d bonded between an adhesive layer 304 and a spreading layer 308. Each reaction segment can be separated from an adjacent reaction segment or spot by a spacer (e.g., 307a, 307b, 307c, 307d, and 307e).

[0132] In yet another embodiment, as illustrated in FIG. 3d, a solid-phase LAMP reaction medium 300d can include a substrate 302, an adhesive 304, and a reaction layer 306 (comprising reaction segments or spots 305a, 305b, and 305c) without a spreading layer. In this example, samples can be deposited onto each section of the reaction layer 305a, 305b, and 305c separately or simultaneously. Furthermore, in this embodiment, there are no spacers between adjacent reaction segments. However, in some embodiments, a spacer can be included without the presence of a spreading layer 308.

[0133] In yet another aspect, as illustrated in Figure 3e, a solid-phase LAMP reaction medium 300e can include a substrate 302, an adhesive 304, and a reaction layer 306 (with reaction segments or test spots 305a and 305b) without a spreading layer or any spacers. Again, in another embodiment, a spacer can still be included, without also including a spreader layer 308. In this example, the sample can be deposited separately and / or collectively onto each section of the reaction layers 305a and 305b.

[0134] In yet another embodiment, as shown in FIG. 3f, a solid-phase LAMP reaction medium 300f that can be used for LAMP analysis can include a substrate 302, an adhesive 304, and a reaction layer 306 (having reaction segments 305a, 305b, 305c, and 305d) without a spreading layer and without any spacers. Again, it should be understood that although no spreading layer is included, spacers can be included between adjacent reaction segments if desired. In this example, the sample can be deposited separately and / or collectively on each section of the reaction layer 305a, 305b, 305c, and 305d.

[0135] It should also be understood that in other embodiments (not shown), the spreading layer 308 may exist without any spacers, with the test spots or reaction segments bonded between the spreading layer and the adhesive 304. In short, depending on the particular test being performed, the components of the solid phase reaction medium may be selected to suit the needs of the test to provide the most accurate results, or to meet any manufacturing needs, or to obtain other particular benefits.

[0136] In another embodiment, substrate 302 can be an optically transparent material. In another embodiment, substrate 302 can be an optically clear plastic carrier. In another embodiment, adhesive layer 304 can be substantially free of volatile agents. In another embodiment, adhesive layer 304 can be substantially free of agents that may interfere with the LAMP reaction. In another embodiment, adhesive layer 304 can be disposed on the substrate either continuously or discontinuously.

[0137] The adhesive layer may include a variety of materials. In one example, the reactive layer 306 may include one or more of fiberglass, nylon, cellulose, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, hydrophilic PTFE, etc., or combinations thereof. In one example, the adhesive layer 304 may include an inert adhesive that does not affect the color change of the reactive segments or test spots within the reactive layer. In one example, the reactive layer 306 may be substantially free of chelating agents, magnesium, excess buffering capacity, reagents that affect pH, or combinations thereof. In another example, the amount of adhesive may be minimized to prevent interference between the adhesive layer 304 and the reactive segments 305a-c.

[0138] The spreading layer can be configured to enhance the distribution and uniformity of the biological sample across the reaction segments 305a-305c. In another embodiment, the spreading layer 308 can distribute or meter the saliva sample across the entire surface of the reaction layer 306. The spreading layer 308 can provide a uniform or substantially uniform concentration of the saliva sample between the interface between the spreading layer 308 and the reaction layer 306. The spreading layer 308 can be one or more of a mesh material, an isotropic porous material with uniform porosity throughout, an anisotropic layer with a gradient in porosity, or a combination thereof. In one embodiment, the anisotropic layer can have a pore size ranging from about 1 to about 100 microns. In one embodiment, the spreading layer 308 can be hydrophilic enough to absorb and spread the sample. In another embodiment, the spreading layer 308 can be less hydrophilic than the underlying reaction layer 306 so that the sample can be drawn out of the spreading layer 308 and into the reaction layer 306.

[0139] When a homogenous biological sample is provided, the precise transparency of the spreading layer 308 can be utilized to distribute the biological sample evenly and equally across the entire surface of the reaction layers 305a-c. In one embodiment, the surface of the spreading layer 308 can be in direct contact with the reaction layers 305a-c to uniformly move the biological sample vertically with horizontal movement of the biological sample. In another embodiment, the spreading layer 308 can comprise one or more of glass fiber, nylon, cellulose, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, polyester, hydrophilic polytetrafluoroethylene (PTFE), etc., or combinations thereof. In one example, the spreading layer 308 can be optically transparent. In one embodiment, the material can be chemically treated to enhance distribution of the biological sample across the multiple reaction segments 305a-c.

[0140] Appropriate spacing between reaction segments or test spots 305a-305c can minimize or eliminate the possibility of cross-contamination between each reaction segment. For example, reaction segment 305a may contaminate an adjacent segment, such as 305b, if the spacing between the two sections allows reagents to flow from 305a to 305b. In another embodiment, solid-phase LAMP reaction medium 300a can further include spacers 307a, 307b, 307c, and 307d, which can provide a minimum spacing of about 1.0 mm to about 3.0 mm (e.g., 2.5 mm) between reaction segments 305a-c.

[0141] In some embodiments, spacers 307a-307d may have a height equal to or greater than that of reaction segments or test spots 305a-305d to create a physical barrier between them (e.g., when samples are added separately to each test spot). In one example, the height of a spacer may be determined as a measure of the distance between substrate 302 and the top surface of the spacer(s). Similarly, the height of a reaction segment may be determined as a measure of the distance between substrate 302 and the top surface of a reaction segment. In some embodiments, a spacer may have a height that is 0-50% greater than the height of a reaction segment.

[0142] In other embodiments, spacers 307a-307d may have a height equal to or less than the reaction segments or test spots 305a-305d to create a physical barrier between them (e.g., when samples are added separately to each test spot). Similarly, the height of a reaction segment may be determined as a measure of the distance between substrate 302 and the top surface of the reaction segment. In some embodiments, the spacers may have a height that is 0-50% less than the height of the reaction segment.

[0143] In one example, spacers 307a-d may include, but are not limited to, one or more of polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, polystyrene, polyester, hydrophilic polytetrafluoroethylene (PTFE), etc., or combinations thereof. In another example, spacers 307a-d may include, but are not limited to, fiberglass, nylon, cellulose, etc., or combinations thereof. In another example, spacers 307a-d may include a hydrophobic material (e.g., polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, polystyrene, polyester, hydrophilic polytetrafluoroethylene (PTFE), etc.), but may not include a hydrophilic material (e.g., fiberglass, nylon, cellulose, etc.). In one embodiment, spacers 307a-d may be oriented in the same plane as and between reaction segments 305a-c.

[0144] In another example, as illustrated in FIG. 3a, a solid-phase LAMP reaction medium can include a spreading layer 308 and a reaction layer 306 joined by an adhesive layer 304. In one example, the material type of the spacers 307a-307d can enhance or otherwise positively affect the extent of spreading. For example, polystyrene spacers may allow for more spreading than other material types. A solid-phase reaction medium without the spreading layer 308 can simplify manufacturing at the expense of simplicity for the user. In this case, the user can apply the saliva sample to each of the test spots 305a-c individually. However, a spacer made of a material that repels or minimally absorbs the sample (e.g., polystyrene) can help result in uniform spreading by the spreading layer. Furthermore, such a spacer made of such a material can also help spread the sample when the spreading layer 308 is not used.

[0145] Referring to FIG. 4, a method 400 for testing for the presence of a viral pathogen is shown, which may include providing a saliva sample from a subject, as shown in block 410, and dispensing the sample into a testing environment having a solid-phase reaction medium in combination with a LAMP reagent mixture and a pH-sensitive dye, as shown in block 420.

[0146] In one aspect, the method may include minimizing the amount of volatile, hygroscopic, and non-pH-sensitive agents that may discolor the solid-phase medium. In another aspect, the method may include providing one or more target primers, a DNA polymerase, and a resolubilizing agent in amounts sufficient to facilitate a LAMP reaction. In another aspect, the method may include providing a reverse transcriptase in an amount sufficient to facilitate an RT-LAMP reaction. In another aspect, the method may include providing one or more target primers in an amount sufficient to detect a viral pathogen. In another example, the method may include generating a test result in less than one hour after dispensing the sample into a test environment.

[0147] In yet another embodiment, as shown in FIG. 5 , a method 500 for confirming the suitability of a saliva sample for testing by solid-phase LAMP reaction can include providing a solid-phase reaction medium with at least one test site or spot, wherein the at least one test site or spot includes a combination of a LAMP reagent and a pH-sensitive dye. In another aspect, the method can further include providing a solid-phase reaction medium with at least one negative control site, as shown in block 510, wherein the negative control site includes a pH-sensitive dye and no LAMP reagent. In another aspect, the method can further include applying the saliva sample to the solid-phase reaction medium, as shown in block 520. In another aspect, the method can further include confirming activation of the pH-sensitive dye on the negative control site, as shown in block 530.

[0148] In one example, the pH-sensitive dye may be at least one of phenol red, phenolphthalein, azolithin, bromothymol blue, naphtholphthalein, cresol red, or a combination thereof. In another example, the LAMP reagent may be substantially free of volatile reagents, pH-affecting reagents, magnesium-containing reagents, or a combination thereof. In another embodiment, the LAMP reagent may include a non-interfering LAMP reagent including a DNA polymerase, a reverse transcriptase, a primer for a target region, or a combination thereof.

[0149] In another embodiment, the method may further include providing a test site or spot defined by at least two sections of the discontinuous adhesive layer. In another example, the method may further include providing a test site or spot defined by at least three sections of the discontinuous adhesive layer.

[0150] In yet another embodiment, as shown in FIG. 6 , a method 600 for maximizing the accuracy of a positive test result from a solid-phase LAMP reaction may include providing a solid-phase reaction medium with at least three test sites or spots (each containing a common combination of pH-sensitive dye and LAMP reagents), as shown in block 610. In one aspect, each site may contain a different primer sequence derived from a target pathogen. In another aspect, the method may include initiating the LAMP reaction, as shown in block 620. In another aspect, the method may include determining a positive test result when at least two of the test sites or spots activate the pH-sensitive dye to change from a first color to a second color, as shown in block 630. In another aspect, the method may further include providing a reverse transcriptase in an amount sufficient to promote the RT-LAMP reaction.

[0151] In one example, the pH-sensitive dye can be at least one of phenol red, phenolphthalein, azolithin, bromothymol blue, naphtholphthalein, cresol red, or a combination thereof. In another embodiment, the LAMP reagents can be substantially free of volatile reagents, pH-affecting reagents, magnesium-containing reagents, or a combination thereof.

[0152] In another embodiment, the target pathogen may include a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In one embodiment, the target pathogen may include a viral pathogen. In another embodiment, the viral pathogen may include a dsDNA virus, a ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, a ssRNA-RT virus, or a ds-DNA-RT virus. In another embodiment, each primer sequence may match a sequence derived from a viral target including H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.

[0153] In another embodiment, the specific target nucleotide sequence to be detected can be a target nucleotide corresponding to a human biomarker. Any disease having a target nucleotide corresponding to a human biomarker for the disease can be detected. Various types of diseases can be detected, including one or more of breast cancer, pancreatic cancer, colorectal cancer, ovarian cancer, gastrointestinal cancer, cervical cancer, lung cancer, bladder cancer, many types of carcinoma, salivary gland cancer, renal cancer, liver cancer, lymphoma, leukemia, melanoma, prostate cancer, thyroid cancer, gastric cancer, etc., or combinations thereof. For example, biomarkers for various types of disease include alpha fetoprotein, CA15-3 and CA27-29, CA19-9, C125, calcitonin, calretinin, carcinoembryonic antigen, CD34, CD99MIC2, CD117, chromogranin, chromosomes 3, 7, 17, and 9p21, cytokeratin, cesmin, epithelial membrane antigen, factor VIII, CD31FL1, glial fibrillary acidic protein, total cystic disease fluid protein, hPG80, HMB-45, human chorionic gonadotropin, immunoglobulins, inhibin, keratin, lymphocyte markers, MART-1, Myoglobin, and IL-1. D1, muscle-specific actin, neurofilament, neuron-specific enolase, placental alkaline phosphatase, prostate-specific antigen, PTPRC, S100 proteins, smooth muscle action, synaptophysin, thymidine kinase, thyroglobulin, thyroid transcription factor-1, tumor M2-PK, vimentin, and the like, or combinations thereof.

[0154] In one example, a positive control may include a synthetic DNA target as one of the test sections to ensure that the DNA template is stable. In another embodiment, a negative control may include (a) an enzyme reagent without primers, or (b) an enzyme reagent with primers targeting a different virus.

[0155] In another example, three test sections can be used to enhance coverage of different viral strains (e.g., SARS-CoV-2). Detection timing and detection limits can be selected for each primer. In one example, 97% coverage of a virus (e.g., SARS-CoV-2) can be achieved using the first primer. In another example, including two additional primers that are slower and less sensitive than the first primer may not enhance viral coverage. In another example, multiple test sections can be used to achieve coverage of different viral pathogens (e.g., SARS-CoV-2, H1N1, H2N2, H3N2, H1N1pdm09, etc.).

[0156] Process and method of solid-phase media LAMP testing LAMP testing on solid-phase media can be enhanced in several ways. First, the testing environment can be monitored to prevent variations that may affect the testing process. Second, the storage conditions of the biological sample testing device can affect its effectiveness. Close monitoring of these conditions can enhance the operability of the biological sample testing device compared to liquid-based LAMP testing.

[0157] 7, a method for testing for the presence of a target nucleotide sequence may include providing a biological sample, as shown in block 710, and dispensing the sample into a test environment having a solid-phase reaction medium in combination with a loop-mediated isothermal amplification (LAMP) reagent mixture and a pH-sensitive dye, as shown in block 720. In one aspect, the method may include providing a reverse transcriptase in an amount sufficient to promote the RT-LAMP reaction.

[0158] The type of biological sample can affect the testing environment. For example, a saliva sample may have buffering capacity that can reduce the contrast of the test output. In one example, the biological sample can be at least one of saliva, mucus, blood, urine, sweat, breath condensate, or feces. In another example, the method can further include collecting the biological sample using one or more of a saliva collection device, a nasal swab, a blood collection device, a urine collection device, a sweat collection device, a breath collection device, or a stool collection device.

[0159] The test environment can be controlled to provide a consistent test output. In one embodiment, the test environment can be substantially free of volatile reagents, pH-affecting reagents, desiccants, or combinations thereof. Each of these reagents can reduce the consistency of test results by introducing variables that may be offset in further analysis.

[0160] Another test environment variable that can be monitored is the rate of temperature increase when heating a biological sample using a biological test instrument. In one embodiment, the method can include increasing the test environment temperature at a rate of about 0.1°C per second. In one example, the test environment temperature can be increased at a rate of about 0.1°C per second to about 0.2°C per second. In one example, reverse transcriptase can be activated at about 55°C, and DNA polymerase can be activated at about 65°C. As a result, a ramp rate greater than about 0.2°C per second can interfere with the coordinated action of reverse transcriptase and DNA polymerase in a LAMP reaction. In one example, the ramp rate can be increased until the test environment temperature is in the range of about 60°C to about 67°C. In some examples, if the test environment is increased to about 55°C (i.e., a temperature at which reverse transcriptase can be activated) at a ramp rate of about 0.1°C from 55°C to about 65°C, the biological sample test instrument may show invalid results. Therefore, the ramp rate needs to be monitored not only within the test environment temperature range of about 55°C to about 65°C, but also when the biological sample is heated to about 55°C.

[0161] Variations in the test environment can also affect the results from the biological sample testing instrument. In one embodiment, the method can include providing a heating uniformity in the test environment that varies by less than 1°C. To avoid interference with the LAMP reaction, the spatial variation in temperature around the test cartridge should not exceed about 0.5°C.

[0162] In another embodiment, the method may include a test time of about 15 minutes to about 30 minutes for a saliva sample. In another embodiment, the method may include a test time of about 30 minutes to about 45 minutes for a saliva sample. In another embodiment, the method may include a test time of about 45 minutes to about 60 minutes for a saliva sample. In another embodiment, the method may include a test time of about 60 minutes to about 90 minutes for a saliva sample. In another embodiment, the method may include a test time of about 20 minutes to about 30 minutes for a nasopharyngeal sample. In another embodiment, the method may include a test time of about 30 minutes to about 40 minutes for a nasopharyngeal sample.

[0163] In another embodiment, the method can further include providing a solid phase reaction medium comprising fiberglass, nylon, cellulose, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, hydrophilic PTFE, or the like, or a combination thereof. In another embodiment, the solid phase reaction medium can comprise a material disclosed elsewhere herein.

[0164] In one embodiment, the target nucleotide sequence may be derived from at least one of a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In one embodiment, the target nucleotide sequence may be derived from a viral pathogen. In another embodiment, the viral pathogen is Coronoviridae , Orthomyxoviridae , Paramyxoviridae , Picornaviridae , Adenoviridae , and ParvoviridaeIn another aspect, the viral pathogen may be selected from the group consisting of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-1), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS), influenza, and H1N1. In one aspect, the target nucleotide sequence may be derived from a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pathogen.

[0165] The testing accuracy of a biological sample testing device can be affected by the storage period and storage conditions (e.g., storage temperature, humidity, etc.) prior to testing. In one embodiment, the biological sample testing device can include a substrate that engages a solid-phase reaction medium in combination with a dehydrated reagent mixture and a dehydrated pH-sensitive dye for loop-mediated isothermal amplification (LAMP). In one aspect, the device can provide a degree of testing accuracy of at least about 95%, 96%, 97%, 98%, or 99% after six months of storage when stored at a selected temperature (e.g., room temperature of about 25°C). In one aspect, the device can provide a degree of testing accuracy of at least about 95%, 96%, 97%, 98%, or 99% after twelve months of storage when stored at a selected temperature. In another aspect, the device can provide a degree of testing accuracy of at least about 95%, 96%, 97%, 98%, or 99% after two years of storage when stored at a selected temperature. In another embodiment, the device may provide a degree of testing accuracy of at least about 95%, 96%, 97%, 98%, or 99% after 3 years of storage when stored at a selected temperature, hi another embodiment, the selected temperature may be any temperature within the range of about -20°C to about 37°C.

[0166] The biological sample testing system may include a housing. In yet another embodiment, the biological sample testing system may include a substrate that engages a solid-phase reaction medium in combination with a dehydrated reagent mixture and a dehydrated pH-sensitive dye of loop-mediated isothermal amplification (LAMP), the housing being operable to receive the biological sample. In one aspect, the biological sample testing system may further include a heater configured to isothermally heat the container to an internal temperature sufficient to initiate and maintain a LAMP reaction between the LAMP reagent mixture and the biological sample for a period of time used to generate a test result via the pH-sensitive dye.

[0167] In one aspect, the substrate may comprise an optically transparent material. In another aspect, the substrate may engage with the solid-phase reaction medium via an adhesive. In another aspect, the adhesive may be substantially optically transparent. In another aspect, the substrate may form a part of the housing. In another aspect, the biological sample testing system may further comprise an adhesive layer disposed on the substrate, a reaction layer disposed on the adhesive layer, and a spreading layer disposed on the reaction layer. In another aspect, the biological sample testing system may further comprise a spacer layer oriented in the same plane as the reaction layer. In another aspect, the biological sample testing system may further comprise a housing disposed in contact with the substrate. In one example, the housing may further be disposed in contact with the spreading layer. In another example, the housing may substantially surround the substrate, the adhesive layer, the reaction layer, and the spreading layer.

[0168] In one example, operation of the biological test system illustrated in FIG. 8 may include: (a) collecting saliva using a sponge; (b) pushing the sponge into the collection tube; (c) diluting the collection tube with water to dilute the saliva sample to 5% to stabilize the pH and reduce the buffering capacity of the saliva; (d) using a pipette to transfer the saliva sample to the test strip in the cartridge by applying the saliva sample to a single location, allowing the capillary and mesh layer to spread the saliva sample to individual segments of the reaction layer; (e) placing the cover on the cartridge and securing the cover in place; (f) verifying that the pH of the saliva sample is within the test range by examining the segments of the reaction layer; (g) inserting the cartridge into the heater and (h) verifying that the cartridge is properly positioned in the heater; (i) activating the heater; (j) reading the results approximately 30 minutes after the test is completed; (k) determining whether the results are valid; and (l) determining whether the results are positive or negative.

[0169] Operation (a) can be performed by collecting saliva via a sponge-based collection device or a passive salivation device. When using a sponge-based collection device, the sponge can be pressed into a collection tube, releasing the saliva into the collection tube (operation (b)). The collection tube can be diluted with water to reduce the buffering capacity of the saliva, reduce the viscosity of the saliva, and increase sample uniformity (operation (c)). When transferred to the test strip, the saliva sample can be applied individually to each section of the test strip, or it can be applied to the center of the test strip and a spreading layer can spread the sample across the entire test area of ​​the test strip (operation (d)). The test strip can be covered to avoid contamination from the testing environment (operation (e)). The pH of the saliva sample can be tested through various means, such as a colorimetric device, a fluorescence reader, or simply by comparing the pH indicator used to a pH color chart (operation (f)). The cartridge can be placed in a heater and run with time recording to measure the total reaction time and the time to reach a positive or negative result (operations (g), (h), and (i)). Results can be read after 30 minutes (operation (j)), or the results can be read immediately after the result indicates a positive result. The rate at which a positive result appears can be correlated with the concentration of the pathogen. Results can be determined to be valid based on the results of a positive control, a negative control, other pathogens, or a combination thereof (operation (k)). When a pH-based indicator is used, the color of the result can be compared to a color comparison chart for pH-based indicators to determine whether the result is positive or negative. The wavelength of the absorbed color can also be measured to determine whether the result is positive or negative.

[0170] In another disclosed embodiment, the biological test kit may include one or more of a saliva collection device, a collection tube, a cartridge, a pipette, a heater, a color chart, or combinations thereof, as illustrated in Figures 8a and 8b. The saliva collection device may be a sponge-based collection device or a passive salivation device.

[0171] In one example, as illustrated in operation 1 of FIG. 8 a, a user can collect saliva using one or more operations including: (a) removing the Pure-Sal™ from the pouch of the biological test kit; (b) placing the sponge in the Pure-Sal™ to collect saliva until the indicator changes color or some other indication that the saliva collection process is complete; (c) attaching the collection tube to the compression tube; (d) inserting the sponge sampler into the compression tube; (e) compressing the sponge to squeeze the collected saliva (free of some degradative salivary enzymes) into the compression tube; (f) closing the tube; and (g) mixing the tube by inverting it several times (e.g., 1-10 times).

[0172] After collecting saliva in the tube and properly mixing it, the user can prepare the cartridge, as shown in operation 2 of Figure 8a. The user can (i) remove the tester from the foil pouch, (ii) remove the sheath from the cartridge, and (c) place the cartridge on a horizontal surface. The user can also transfer the sample by filling the pipette with diluted saliva (e.g., diluted with water) up to the black line and transferring it to the sample well, as shown in operation 3 of Figure 8a. The user can also seal the cartridge by placing the sheath onto the cartridge so that the clips are aligned and interlocked, as shown in operation 4 of Figure 8a. The user can also heat the cartridge by placing the cartridge into the attached heater, closing the lid, and activating the attached heater (which can indicate when the reaction is complete with an LED), as shown in operation 5 of Figure 8a.

[0173] After the reaction is complete, the user can remove the cartridge from the heater and compare it to the color chart, while also ensuring the correct orientation of the color chart and cartridge, as shown in operation 6 of Figure 8a. As further shown in Figure 8b, the illustrated color chart for the viral pathogen SARS-CoV-2 can include two rows and three columns of six squares. The first column (-ve) can indicate a negative result, the second column (+ve) can indicate a positive result, and the third column (invalid test) can indicate an invalid result.

[0174] For example, the first column may indicate a negative result because both the top and bottom rows of squares are nearly red-orange. This could occur if nucleotide amplification of the LAMP reaction does not occur in either square. The second column may indicate a positive result because the top row of squares is nearly red-orange, which could occur if nucleotide amplification of the LAMP reaction does not occur, while the bottom square is nearly orange-yellow, which could occur if nucleotide amplification of the LAMP reaction occurs, indicating the presence of a viral pathogen. The third column may indicate an invalid result because the top row of squares is nearly orange-yellow, which could occur if nucleotide amplification of the LAMP reaction occurs, while the bottom square is nearly orange-yellow, which could occur if nucleotide amplification of the LAMP reaction occurs. However, in the third column, the reason for the color change from red-orange to orange-yellow may be uncertain because the negative control (e.g., the top row) also changes color.

[0175] Thus, the user can compare the color chart with the test in the cartridge to determine the test result. The color chart illustrated in Figure 8b is an example. Additional rows or columns can be included in the color chart to accommodate additional controls, additional pathogens, or any number of tests for different primers for the same pathogen. Furthermore, the test can be administered by the subject themselves, or by a trained technician, nursing assistant, nurse, physician assistant, doctor, or any other person qualified to administer the test and interpret the results. [Example]

[0176] The following examples are provided to facilitate a clearer understanding of certain embodiments of the present invention and are not meant to be limiting thereof in any way.

[0177] Materials assembly for paper-based LAMP reactions Example 1 - Paper-based LAMP assembly For paper-based LAMP assembly, several materials were screened to design a portable and compact assembly. For the spreading layer screening, two paper strips (one containing primers and the other not) were placed together, and 50 μL of RNA at a concentration of 0.2 ng / μL was loaded to impregnate both strips. Materials may not substantially affect the pH of the paper for several reasons: (a) the paper was too acidic, resulting in no color change before incubation, or (b) the paper was too basic, preventing the color change associated with the reaction. Materials that prevented crosstalk between the two paper strips, along with other device components, were further tested.

[0178] The dimensions of the paper-based LAMP assembly were approximately 24 x 54 mm. The paper-based LAMP assembly consisted of (i) a reading layer, (ii) two reaction strips, and (iii) a spreading layer. The reading layer consisted of a 3-millimeter transparent Melinex® support. Two reaction strips, each made from 5 mm x 20 mm chromatography paper (e.g., Whatman® Grade 1 chromatography paper), were attached to the Melinex® support with double-sided adhesive (ArClean® 90178). To prevent crosstalk between the two test strips, the two test strips were separated by a 2.5 x 20 mm, 10-millimeter polystyrene spacer. The spreading layer consisted of a polyester sulfone mesh (Saaticare® PES 105 / 52). The sample was loaded onto the spreading layer. Figure 9 shows an example of an assembly.

[0179] Example 2 - Materials Screening The paper-based material was selected based on five criteria: (a) formulation stability when dried on the substrate, (b) intensity of color change upon rehydration with sample, (c) ability of the sample to wick uniformly throughout the paper-based substrate, (d) ability of the material to remain inert throughout the reaction, and (e) ability of the paper-based material to exhibit a color change upon amplification. Whatman® Grade 1 chromatography paper was tested and used for optimization. When two test strips were assembled together, it was noted that the LAMP reaction utilized a larger area of ​​Grade 1 chromatography paper, resulting in uneven fluid distribution. By selecting a thicker chromatography paper than Grade 1 chromatography paper (e.g., Ahlstron Grade 222 chromatography paper), the surface area of ​​the test strip could be increased to approximately 5 mm × 6 mm while still retaining the same amount of sample and reagent as Grade 1 chromatography paper, allowing for more uniform spreading during rehydration.

[0180] Example 3 - Materials Screening - Grade 1 and Grade 222 Chromatography Paper In one example, as shown in Figure 10, images illustrate an example of the color contrast that can be produced. In one example, grade 1 chromatography paper was used. In another example, grade 222 chromatography paper was used. In each case, the RT-LAMP reagents were dried and rehydrated with 25 μL of 5% saliva and 95% water. Positive samples contained heat-inactivated SARS-CoV-2 spiked at 10,000 copies per reaction, while negative samples were 5% saliva (virus-free) and 95% water. Images were captured at 90 minutes of incubation at 65°C.

[0181] As shown in Figure 10, the color contrast between a negative result on Grade 1 paper and a positive result on Grade 1 paper is not as pronounced as the color contrast between a negative result on Grade 222 paper and a positive result on Grade 222 paper. Therefore, thicker paper enhances the color contrast.

[0182] Example 4 - Paper Assembly Protocol The paper assembly protocol included: (1) cutting polyethylene terephthalate (PET) into rectangles measuring 80 mm × 7 mm, two per device; (2) cutting chromatography paper into rectangles measuring 33 mm × 5 mm, one per device; (3) cutting a large sheet of double-sided tape and taping the PET rectangles onto it, leaving 2 cm between each PET rectangle; (4) cutting all rectangles so that excess tape remained on all edges; (5) peeling the tape off the PET and taping the chromatography paper to the other side of the PET, leaving approximately 1 cm outside the tape; and (6) removing the tape from the PET and taping the chromatography paper to the other side of the PET. (7) taping the other PET strip over the sample (i.e., the taped side touches the sample and the PET is on the outside); (8) folding the protruding tape over the PET tape to further seal; (9) adding 1 ml of solvent to the bottom of a 15 ml tube; (10) placing the assembled paper device into the tube so that the protruding chromatography paper is in the solvent; (11) closing the tube; and (12) incubating at 65°C for 1 hour.

[0183] pH-based paper-based material for LAMP analysis Example 5 - Limit of Detection (LoD) The LoD test used primer sets targeting various regions of SARS-CoV-2 and two-fold serial dilutions of heat-inactivated SARS-CoV-2 at concentrations of 2.5, 5, 10, and 20 copies / μL in a 25 μL reaction volume. Using a liquid-based colorimetric assay in water, the estimated LoD was approximately 20 copies / μL, and amplification was confirmed by a color change from red to yellow in four out of four replicates.

[0184] Example 6 - Limit of Detection (LoD) The LOD for liquid-based LAMP reactions can be a concentration of approximately 20 virus copies per μL of sample volume. For solid-phase reaction media (e.g., chromatography paper), each reaction zone can hold a sample volume of approximately 25 μL.

[0185] Example 7: Verification of LoD After determining the LoD to be approximately 20 copies / μL in saliva, the following samples were generated with relevant concentrations: (1) 20 copies / μL (1×LoD—10 samples), (2) 40 copies / μL (2×LoD—10 samples), (3) 100 copies / μL (2 samples), (4) 1000 copies / μL (2 samples), (5) 10,000 copies / μL (2 samples), (6) 100,000 copies / μL (2 samples), and (7) 1,000,000 copies / μL (2 samples). Negative samples were aliquots of pooled saliva (30 aliquots). Results were also confirmed using image processing.

[0186] Example 8 - LoD, Sensitivity, and Specificity Serial dilutions of heat-inactivated SARS-CoV-2 in water were prepared (approximately 100 copies / reaction ~10 5 These serial dilutions were used as templates in liquid reactions to establish a baseline LoD for potential candidate primer sets. Reactions were run in triplicate on white qPCR plates (e.g., Thermo Scientific® AB-0800W) for each virus concentration and heated to 65°C for 60 minutes in a standard 75L biological incubator (e.g., Fisherbrand® Isotemp Microbiological Indicator, 15-103-0513). The color of the reaction mixture at various time points was recorded by scanning the plate with a benchtop scanner (Epson® Perfection V800 Photo Color). The LoD of a primer set was determined by the lowest virus concentration that produced a strong color change in all three replicates. 10 3Any candidates that could produce amplification at or below 1 copy / reaction were then subjected to the same procedure using virus dilutions (e.g., 2-fold dilutions) and spiked into pooled normal saliva to check for matrix interference. Saliva LoD testing was then performed on a paper-based device to confirm primer compatibility on the paper substrate.

[0187] As illustrated in Figure 11, the reaction on the right was performed in liquid using a 25 μL reaction volume and heat-inactivated SARS-CoV-2 in water combined with a colorimetric mixture of fluorescent dyes. The black line represents the absorbance ratio (OD 430 / OD 560 ) and the light blue line indicates the color change measured at 10 3 The fluorescence change was measured in units of fluorescence intensity. When used in conjunction with the built-in color / fluorescence reader, detection times may be faster.

[0188] When the virus concentration is approximately 1000 copies / reaction, the absorbance ratio and fluorescence activity increase sharply after approximately 17 minutes. When the virus concentration is approximately 500 copies / reaction, the absorbance ratio and fluorescence activity increase sharply after approximately 18 minutes. When the virus concentration is approximately 250 copies / reaction, the absorbance ratio and fluorescence activity increase sharply after approximately 19 minutes. When the virus concentration is approximately 125 copies / reaction, the absorbance ratio and fluorescence activity increase sharply after approximately 18 minutes. When the virus concentration is approximately 62.5 copies / reaction, the absorbance ratio and fluorescence activity increase sharply after approximately 17 minutes. When the virus concentration is approximately 31.25 copies / reaction, the absorbance ratio and fluorescence activity increase sharply after approximately 22 minutes. For the no-template control, the absorbance ratio and fluorescence activity never increase sharply, as expected.

[0189] Sensitivity and specificity were determined using 30 artificially positive samples at various multiples of the LoD (1x, 2x, 4x, 40x, and 400x, with 10, 10, 4, 3, and 3 replicates, respectively) and 30 no-template control (NTC) negative saliva samples. Colorimetric reaction intensity was determined using ImageJ, and the mean intensity of the green channel for each reaction zone was extracted. Receiver operating characteristic (ROC) curves were generated by varying the threshold cutoff between positive and negative reactions and calculating the sensitivity and specificity at each threshold. Sensitivity was calculated as the ratio of true positives to total positives, including false positives. Specificity was calculated as the ratio of true negatives to total negatives, including false negatives.

[0190] Multiplexing selected targets for testing viral pathogens using paper LAMP Example 9 - Paper Strip Format Figure 12 illustrates the paper strip format. The black line represents SAATICARE Hyphyl® polyester PES 105 / 52, 0.063 mm thick and 50 mm long. The red line represents Tekra Clear MELINEX® 454 polyester PET, 0.0762 mm thick and 50 mm long. The green line represents Adhesives Research ARclean® 90178 (AS-144), 0.038 mm thick and 50 mm long. The orange block represents Tekra Double White Opaque High Impact Polystyrene Litho Grade, 0.508 mm thick and 2.5 mm long. The blue block represents Ahistrom-Munksjo Cellulose Grade 222, 0.83 mm thick and 5 mm long. In this example, the paper strip has a spreading layer (black), an adhesive layer (green), and a substrate (red), as well as five spacers (orange) and four reactive layer sections (blue).

[0191] Example 10 - RT-LAMP process As illustrated in FIG. 13, the solid-phase LAMP reaction medium can include a substrate comprised of a 6 mm x 50 mm, clear, 3 mm Melinex 454 with Arclean 90178; a reaction layer comprised of 5 mm Ahlstrom 222 reagent material (including four discontinuous pink reaction layer sections); a 2.5 mm spacer comprised of 20 mil polystyrene (including five discontinuous spacers); a spreading layer comprised of Saaticare PES 105 / 52 hyphyl; and a 14 mm capillary made of Melinex, Arclean 90178, and 3M 9962.

[0192] Crosstalk between reaction zones can be avoided by providing sufficient space between each reaction section or test area, as shown in Figure 14. In this example, the four orange reaction sections remain free of cross-contamination, as there is no color change in the five spacers.

[0193] Example 11-A - Paper Contrast In another example, as illustrated in Figure 15, Grade 222 chromatography paper may provide higher contrast than Grade 1 chromatography paper. The positive results (left side) on the 222 paper demonstrate higher contrast between the positive result (yellowish orange) on the top left and the negative result (dark orange) on the bottom left compared to the positive result (orange) on the top right and the negative result (dark orange) on the bottom right. Each paper strip was tested in approximately 500 µM buffer solutions at a pH range from 8.0 for negative tests to 7.5 for positive tests.

[0194] Example 11-B - Effect of Phenol Red Concentration on Paper To distinguish between negative and positive results without inhibiting the reaction itself, phenol red concentrations were tested on both Grade 1 and Grade 222 chromatography paper. Both types of chromatography paper showed consistent results at 250 μM phenol red per reaction. Lower dye concentrations showed a relatively faint, pale color after 60 minutes of incubation, while the higher phenol red paper pads required longer incubation times to distinguish between positive and negative results.

[0195] Example 11-C - Effect of initial pH and drying on colorimetric RT-LAMP reaction of paper using phenol red Figure 15B illustrates RT-LAMP with the introduction of drying at different starting pHs of the RT-LAMP reaction mixture. In this example, pH 7.6 is the unadjusted pH of the RT-LAMP reaction mixture. The wet setup indicated that 20 μL of LAMP reaction master mix was added, followed immediately by 5 μL of synthetic RNA (N gene, 0.2 ng / μL, "+") or water ("-"). The dry setup indicated that after applying 20 μL of LAMP master mix, the paper strip was allowed to dry at room temperature for 30 minutes and then rehydrated with 25 μL of synthetic RNA ("+") or water ("-"). The LAMP reaction included 12.5 μL of NEB 2x colorimetric master mix, 2.5 μL of primer mix, and 5 μL of phenol red, all prepared in nuclease-free water (1 mM). The pH of the resulting mixture was adjusted with KOH. Grade 1 chromatography paper was used. The mixture was heated in an incubator set at 65°C for 120 minutes, and scanned with a flatbed scanner at 45, 60, 90, and 120 minutes during the reaction.

[0196] Example 12 - Test Strip Format As shown in Figure 16, the test strip format has a sample application surface and a reading surface (top right of the figure). If a spreading layer is present, the sample can be applied to one location on the sample application surface. In other examples, the spreading layer is not used, and the sample can be applied to the sample application layer on each section of the reaction layer (orange). The reading surface can be read without a dedicated instrument or using a colorimetric or fluorescent detector.

[0197] As further illustrated in FIG. 16, the test strip format also has four test reaction areas, an absorbent / spreading layer, and a transparent single-sided adhesive that conforms to the shape and contacts the absorbent layer.

[0198] Example 13 - Test Strip Assembly Process 17 illustrates the test strip assembly process, which includes slitting (coating the reactive layer relatively wide, slitting it to 5 mm, and placing it on a reel for lamination). This process also includes a first lamination process of the reactive layer onto a transparent single-sided adhesive. This process also includes a second lamination process performed in-line immediately after the first lamination process, where a spreading layer is laminated to the reactive layer and adhesive layer. The test strip assembly process can include materials including a polysulfone material coated with a pH reagent, a single-sided adhesive, and chromatography paper.

[0199] Example 14 - RT-LAMP process RNA from SARS-CoV-2 virus in saliva was extracted, reverse transcribed, and amplified in a one-pot mixture by heating the saliva and reagent mixture at 65°C. The four primer sets used for LAMP included one targeting the SARS-CoV-2 RdRp gene, one targeting the SARS-CoV-2 envelope gene (E), one targeting the SARS-CoV-2 ORF1ab region, and finally one targeting the human RNase P (RP) gene (which served as an on-board control). Each primer set consisted of six individual primers targeting specific regions of viral or human RNA that were reverse transcribed and amplified during isothermal incubation using reverse transcriptase and strand-displacing polymerase.

[0200] Example 15 - Positive Control A positive control reaction is incorporated into the test device as one of the test areas run alongside the three test strips for viral RNA. The positive control simultaneously serves as a positive template control and an extraction control. If a color change from red to yellow occurs in the control area of ​​the test strip, this indicates that viral RNA, if present in the specimen, has been successfully extracted from the virus and that all reagents in the test are working as expected to generate an amplification signal. If a color change does not occur, the test is considered invalid and should be repeated.

[0201] The positive control detects human RNase P, a ubiquitous marker in human clinical specimens and a standard control in many RT-PCR kits. Amplification of this marker indicates that lysis of human cells occurs successfully under the test conditions, and it can be assumed that the virus is also lysed.

[0202] The scientific basis for the control is as follows: Unlike RT-PCR-based assays, our assay did not use chemical extraction of viral RNA; rather, heat treatment was sufficient. The polymerase in RT-PCR is highly sensitive to reaction inhibitors in biological sample matrices. Therefore, these tests typically involve RNA extraction and purification procedures. In contrast, the Bst 2.0 polymerase enzyme used in the LAMP assay is extremely robust in biological matrices, so extraction and purification are not used. The temperature of 65°C reached within the device is sufficient to lyse viral particles, expose viral RNA, and support robust amplification.

[0203] Example 16 - Negative Control A negative, no-template control test is performed using the test device with blank test solution in place of a saliva sample. The blank test solution is a sterile, pH-buffered solution that does not contain any RNA or DNA template. A color change in this test indicates a possible false positive and will invalidate any positive results obtained since the last control run. Therefore, the instructions to the user are to perform this negative control using one of these representative test kits upon receipt of the test kit. This check may be repeated at prescribed intervals.

[0204] The most likely cause of false positives in the negative control is carryover of amplified DNA products from successive tests. The negative control test is the last in a series of control measures to prevent false positives due to carryover contamination, including manufacturing controls for cartridges with strict seal tolerances and periodic wipe-cleaning and disinfection of heater fixtures by operators.

[0205] Example 17 - Internal Control The test assembly features a unique control designed for use with saliva as the test substrate. Each test strip contains a pH indicator dye that serves as an internal control to verify that the initial pH of the saliva sample is within the expected range. If a color change from red to yellow is observed on all four test strips upon sample application (before heating), the user should conclude that the sample is invalid and should not proceed with the test. External factors, such as eating, drinking water, or use of oral hygiene products prior to sample collection, can cause the sample's initial pH to be off. If the operator determines that one of these factors is affecting the sample in the event of a pH reading malfunction, the test can be retested after waiting five minutes for the patient's salivary pH to return to normal. Other external factors, such as certain illnesses, can also systemically affect salivary pH, necessitating the use of alternative testing methods.

[0206] Example 18 - Results confirmation The paper LAMP test is a qualitative test. It has a color-based visual result that the operator can read using a color chart for interpretation. A positive result will be yellow. All test controls must be examined before interpreting a patient's results. If the controls are not valid, the test is invalid and the patient's results cannot be interpreted.

[0207] Because this is an isothermal RNA amplification, the presence of target SARS CoV-2 RNA at levels defined by the LoD experiment will result in a positive test result. In some cases, a positive test interpretation can be confirmed by a positive result in two of the three target gene primer regions: Orf1ab, E gene, or RdRp gene. A positive result can be identified by comparing the color of the test strip to the provided color interpretation chart. The RNaseP region of the test strip should turn yellow, indicating a valid test. If the region does not turn yellow, the test is invalid and should be repeated.

[0208] The first indicator of a reliable and authentic test strip is that the four test strip areas do not turn yellow when saliva is applied before heating the test strip. If this occurs, the pH of the saliva is out of the acceptable range, possibly due to recent food or fluid intake. The patient should rinse their mouth with water, wait at least five minutes, and then re-collect saliva.

[0209] Upon completion of the test reaction within the heater fixture, the endogenous control should show a positive yellow color change in the RNaseP region. Once confirmed, the color change in each strip region should be examined by comparing it to the provided color interpretation chart. Each strip region is classified as positive (e.g., yellow change) or negative (e.g., pink color). In one example, a confirmed SARS CoV-2 infection may be indicated if two of the test regions are positive.

[0210] Example 19 - Sources of false positives False positives can often occur in RT-LAMP assays if proper care is not taken to address them during the assay procedure. These false positives may originate from DNA aerosols formed by previous RT-LAMP reactions, which persist in the environment for long periods and contaminate future experiments. These aerosols can contaminate individual reagents during reaction preparation or the reaction mixture when transferring, handling, loading samples into the reaction mixture, or incubating the reaction mixture. Apart from reducing the accuracy of RT-LAMP-related tests, aerosols can also add noise to experiments using specific template concentrations, such as LoD tests. To address these various points of contamination, all RT-LAMP-related procedures can be divided into stages (i.e., reaction preparation, transfer, loading / sealing, incubation for amplification, and gel electrophoresis) and performed in different locations. Spatial separation of operations within the protocol can minimize the possibility of aerosols being introduced before sealing the reaction or allow for problem resolution if a false positive occurs.

[0211] Example 20 - Screening of plates, caps, sealers, and tubes Considering the susceptibility of RT-LAMP to contamination and therefore false positives, we conducted an extensive screening of qPCR 96-well plates, sealing methods, and PCR tubes. First, we screened both the clear (ThermoScientific® AB-0800) and white (AB-0800W) Thermo Scientific® 96-well Full Skirted PCR Plates along with the FrameStar® 96-well Skirted Optical Bottom Plate (Brooks Life Sciences® 4TI-0970). The clear plates were primarily used to aid in scanning the colorimetric assay. Of these clear plates, the white-bottom Thermo Scientific® 96-well Full Skirted PCR Plate performed best based on the average number of false positives.

[0212] Figure 22A shows colorimetric RT-LAMP scan images for the limit of detection (LoD) of orf1ab.II. Scan titles are catalog numbers corresponding to the various plate types used to perform the colorimetric LoD. Yellow wells indicate successful LAMP reactions, while red / orange wells indicate no amplification or low levels of amplification, respectively. To 20 μL of reaction mixture, 5 μL of heat-inactivated virus water dilution was added at the indicated concentration. The reaction master mix consisted of 12.5 μL of NEB Colorimetric 2x Master Mix, 2.5 μL of primer mix, and 5 μL of water. Endpoint images were acquired after 60 minutes of heating in an incubator set at 65°C. Three replicates were performed for each primer set for each virus concentration.

[0213] The following sealing methods were investigated: MicroAmp® Optical 8 Cap Strips (Thermo Fisher® 43-230-32), Thermo Scientific VersiCap Mat Cap Strips (Thermo Fisher® AB1820), Thermo Scientific Adhesive Plate Seal (Thermo Fisher® AB-0558), and MicroAmp Optical Adhesive Seal (Thermo Fisher® 43-119-71). Based on the false positive rate, the VersiCap Mat Cap Strip provided the best seal. However, colorimetric scanning made it difficult to obtain an accurate image of the reaction progress because the caps were not completely transparent. Consequently, adhesive seals were tested and performed comparable to each other. Furthermore, when pressure was applied simultaneously and uniformly across the entire plate, the adhesive seal performed better than the VersiCap based on the false positive rate.

[0214] Figure 22B shows colorimetric RT-LAMP scan images for the limit of detection (LoD) of orf1ab.II. Scan titles are catalog numbers corresponding to the various cap types used to perform the colorimetric LoD. Yellow wells indicate successful LAMP reactions, while red / orange wells indicate no amplification or low levels of amplification, respectively. To 20 μL of reaction mixture, 5 μL of heat-inactivated virus water dilution was added to obtain the final indicated concentration (positive reaction) or nuclease-free water (negative reaction). The reaction master mix contained 12.5 μL of NEB Colorimetric 2x Master Mix, 2.5 μL of primer mix, and 5 μL of water. Endpoint images were taken after heating the plate at 65°C for 60 minutes. Three replicates were performed for each primer set for each virus concentration.

[0215] The following PCR tube products were investigated as alternatives to plates during imaging: MicroAmp™ Optical 8-Tube Strip with Attached Optical Cap (Thermo Fisher® A30588) and MicroAmp® Optical 8-Tube Strip (Thermo Fisher® 4316567) (the caps were the same as those used to seal the plates). Tubes with attached optical caps performed best in terms of false positive rate.

[0216] The fully assembled 4-plex test strip can include a spreading mesh, adhesive, and a see-through Melinex backing. 8 DNA template in water can also be included at a concentration of 0.2 ng / μL, equivalent to copies / μL. Sample volumes can be 100 μL. N gene primers can be used, alternating between primer and primer-free conditions. Adhesives may have a buffering effect that inhibits the color reaction, so this must be compensated for in the formulation.

[0217] Paper LAMP testing process and methods Example 21-A - Paper LAMP Test Process The paper LAMP test is designed for simple point-of-care use by trained healthcare professionals. The entire testing process is shown in Figure 18. Patients collect saliva samples under the guidance of healthcare professionals. The saliva sample is collected in a dedicated collection container containing no additives, making it safe for patients to use during collection. The saliva volume is approximately 100 μL, making collection easy for patients. The test strip, containing the specific RNA detection region described above, is housed entirely in a plastic strip holder. After collecting the saliva sample, the patient hands the collection container to the operator. The operator then applies the saliva sample to the designated location on the test strip and closes the cartridge housing. The cartridge with the sample-containing strip is then placed into a heater device designed to hold the cartridge securely in place and heat it to the desired temperature, up to 65°C. The paper LAMP assay proceeds isothermally on the test strip. During the LAMP reaction, nucleic acids are identified in the saliva sample, and at 65°C, a color change occurs on the strip due to a change in pH caused by nucleic acid amplification.

[0218] The heater device is designed to provide uniform heating across the entire test strip. The heater device can be equipped with (red-yellow-green) LED lights to indicate heating progress to the user regarding incubation time and to notify the operator when the test has reached completion. While the test is in progress, the heater device can include features, such as a magnetic or mechanical lock, to ensure the test is not interrupted. The test reaction is allowed to proceed for approximately 15-30 minutes at an isothermal temperature of 65°C. At the end of the assay, the test carrier with the test strip can be removed from the heater and visually read.

[0219] Example 21-B - Paper LAMP Test Process Figure 18B illustrates the fabrication and use of a paper-based colorimetric molecular test for SARS-CoV-2. Fabrication of a paper-based colorimetric molecular test for SARS-CoV-2 includes (1a) creating a master mix, (1b) transferring the mix to a pad, (1c) performing quality checks, and (1d) air-drying the test device. Use of a paper-based colorimetric molecular test for SARS-CoV-2 includes (2a) collecting the sample, (2b) resuspending it in water, (2c) adding the sample to the pad, (2d) incubating at 65°C for approximately 60 minutes, and (2e) interpreting the results.

[0220] Example 21-C - Paper LAMP Test Process The assay workflow (collecting saliva, transferring the sample to the paper-based device, incubating at 65°C, and reading the results) is illustrated in Figure 19A. Typical results are shown in Figure 19D. Figure 19C shows a schematic of the structure of our paper device. The paper device contained several grade 222 cellulose reaction pads separated by 20-mil polystyrene spacers to prevent crosstalk between reaction zones. These components were attached to a transparent backing via double-sided adhesive for structural support. During fabrication, the reagents for performing RT-LAMP were dried on the reaction pads. These reagents were rehydrated when the user added a sample to the reaction zone.

[0221] Figure 19 illustrates the schematic and colorimetric characteristics of the paper-based device. Figure 19A shows a schematic of the workflow for using the device. The control zone shows a no-primer control. Figure 19B shows the colorimetric LoD on paper using the indicated concentrations of heat-inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 5% saliva. Negative replicates are RT-LAMP reactions using nuclease-free water instead of heat-inactivated SARS-CoV-2. Data were taken from Figure 21A.

[0222] Figure 19C shows the schematic layout of the paper device. Figure 19D shows typical colorimetric results for negative and positive runs. The control is an RT-LAMP reaction without LAMP primers. Positive reactions include 800 copies / μL spiked into 5% saliva. Figure 19E shows the color gradient of possible results derived from the colorimetric results in panel B. Figure 19F shows a summary table of the observations used to calculate the analytical sensitivity and specificity of the paper device based on survey responses.

[0223] Each step in the assay (Figure 19A) was designed to reduce complexity and user error at the point-of-care site. The sample collection step uses a sponge-based collection device that allows patients to self-collect and removes particulates from the collected saliva, minimizing patient-to-patient variability. The transfer step involved placing 25 μL of diluted saliva in each of the two reaction zones on the device. The incubation step involved sealing the sample-loaded paper device in a resealable plastic bag and placing it in an incubator set at 65°C for 60 minutes. Finally, the read step involves the user comparing the color of the control and reaction zones to the color bar in Figure 19C to determine whether the results are valid and whether the target pathogen is present.

[0224] This platform consists of three main components: a primer set that confers specificity to the assay, a paper device containing two reaction zones (one control and one reaction), and a heating source used to heat the paper device to reaction temperature. The primer set determines which pathogen the assay targets. Therefore, the platform can be reconfigured to target different pathogens by redesigning the primer set while keeping all other aspects of the device and formulation identical. Furthermore, the paper-based device can be configured to accommodate multiple reaction zones, allowing for simultaneous detection of multiple targets.

[0225] Direct detection of SARS-CoV-2 in saliva is demonstrated via a clear colorimetric reaction that can be read using the naked eye (Figure 19). This format is amenable to roll-to-roll fabrication, with an expected cost per test of approximately $10. The limit of detection (LoD) of this test is 200 copies per 1 μL of saliva. When assessed visually, analytical sensitivity (positive predictive value) is 76%, specificity (negative predictive value) is 100%, and accuracy is approximately 91% when measured using an artificial sample (freshly collected saliva spiked with heat-inactivated SARS-CoV-2) (Figure 19F). Due to subjectivity in the perception of the control pad color, respondents incorrectly identified a total of 20 of the 80 devices presented as invalid, resulting in a false invalid rate of 25%. When image processing was used to quantify the color change, sensitivity increased to 97%, and accuracy was 98% (Figure 19C).

[0226] The device (Figure 19C) measured 6 mm x 20 mm and included a readout layer, two reaction strips, and a spacer to prevent crosstalk. The readout area contained a supporting, optically clear, 3 mil MELINEX (Tekra MELINEX® 454 polyester (PET)) backing. This was attached to two reaction strips of 5 mm x 6 mm chromatography paper (Ahlstrom-Munksjo Grade 222) using double-sided adhesive (Adhesives Research, acid-free ARclean® 90178). The strips were separated by a 2.5 x 6 mm 20 mil polystyrene spacer (Tekra Double White Opaque High Impact Polystyrene (HIPS) Litho Grade). Upon rehydration, 25 μL of sample was added to saturate the strips.

[0227] The color bar was created by averaging the RGB values ​​of phenol red on grade 222 chromatography paper over a range of pH values. These average RGB values ​​were used to create a linear gradient, and the optimal threshold determined from the ROC curve was annotated on the color bar (Figure 19C).

[0228] After loading the sample onto the paper-based device, the device was placed in a 1" x 1" resealable plastic bag to prevent contamination during the RT-LAMP reaction. The plastic bag containing the paper device was then placed in a 65°C incubator for 60 minutes. The bag was removed and scanned with a flatbed scanner (Figure 19D). The results were compared to a color chart (Figure 19E) created by averaging RGB values ​​from the phenol red reaction on a Grade 222 pad in buffers of known pH values ​​between 6 and 9 (Figure 21B). The threshold values ​​in the color chart correspond to the threshold values ​​determined from the ROC analysis (Figure 19B).

[0229] After determining the LoD of 200 copies / μL in saliva (FIG. 19B), artificial samples were created at 1x, 2x, 4x, 40x, and 400x the LoD. Thirty aliquots of freshly collected saliva were used as negative samples (FIG. 21A). Image processing was used to quantify the results (FIGS. 19A and 21C). Using a two-tailed Student's t-test, the difference in green channel intensity between the negative and positive colorimetric reaction pads was found to be significant (p<0.001).

[0230] The specificity using image analysis was 100%, the sensitivity was 97%, and the accuracy was 98% (Figure 20C). Figure 20 illustrates the digital analysis of the colorimetric reactions on paper. Figure 20A shows a boxplot of the green channel intensity of 30 positive and 30 negative RT-LAMP results on paper. Figure 20B shows the receiver operating characteristic (ROC) curve for 30 positive and 30 negative RT-LAMP results on paper. Figure 20C shows a summary table of the observations based on the image analysis.

[0231] Colorimetric perception surveys (Figures 23A and 23B) were collected from participants enrolled in the study in accordance with Purdue University IRB protocol number IRB-2021-375. Participants were given a color bar with annotated thresholds (Figure 19E). Participants were given multiple paper device scans and asked to classify the control pad (left reaction zone) as valid or invalid and the SARS-CoV-2 reaction (right reaction zone) as positive or negative. Observations classified as invalid were discarded from the assay performance analysis, and the proportion of assays incorrectly identified as invalid was reported as the false invalid rate.

[0232] Using the color bar (Figure 19), four participants were asked to classify the 10 positive and 10 negative reactions shown in Figure 21A as valid or invalid (according to the control zone on the left) and as positive or negative for SARS-CoV-2 (according to the reaction zone on the right). Observations that the participants considered invalid were discarded. Of the 40 true-positive observations (all valid using image analysis), participants incorrectly classified 19 as invalid. This contrasts with the 40 true-negative observations (36 of which were valid using image analysis) in which one observation was incorrectly classified as invalid. Thus, the specificity and sensitivity of our device, calculated taking colorimetric interpretation into account, were 100% and 76%, respectively, with an accuracy of 91% (Figure 19F), and a false-invalid rate of 25%.

[0233] Example 22 - Sample Stability The typical time from saliva collection to application to the test strip is within 2 hours. Sample stability, allowing testing up to 24 hours from sample collection, can be based on repeat testing of samples (e.g., 15 positive and 15 negative) at 0-2 hours, 8-12 hours, and 20-24 hours. Samples collected rapidly from patients at a collection center or clinical site can be subsequently tested several hours later. Single-site collection (e.g., collection at an outpatient facility or drive-through collection area, followed by transport of the saliva specimen to a second location, e.g., a site) can also be evaluated.

[0234] Example 23 - Preparation The dip coating and drying at elevated temperatures did not appear to adversely affect the LAMP reaction, which positively impacts manufacturing throughput and scalability. The test strips were designed to be manufactured at large scale using existing processing equipment without significant capital investment. The design is simple enough to achieve the goal of a fast and easy path from prototype to full-scale production to meet emerging market needs.

[0235] Example 24 - Paper-based device design Paper is widely used for pH indicators and urine strips, primarily due to its low cost, low technical complexity, and ease of production using roll-to-roll manufacturing. The use of several types of paper and selected chromatography paper was evaluated for the devices disclosed herein (Figures 21D-21F). Chromatography paper can be used for paper-based biosensors due to its enhanced capillary action capabilities compared to other papers. Many paper-based devices use Grade 1 chromatography paper. However, the large area of ​​Grade 1 chromatography paper (5 mm x 20 mm) results in uneven distribution of the solution across the paper. Therefore, we selected a different type of chromatography paper: Grade 222 (0.83 mm), which is approximately 4.6 times thicker than Grade 1 (0.18 mm). The increased thickness allows the device size to be reduced (5 mm x 6 mm), allowing the same volume of liquid to be loaded into a 70% smaller reaction area and distributed more evenly.

[0236] To reduce the complexity of the device, the components of the RT-LAMP reaction (without the template) were dried onto paper. Drying ensures stable distribution and ease of operation without compromising diagnostic performance. The user simply adds sample to rehydrate the reagents. When the reagents were dried onto the paper, the color of the paper changed from red to yellow over time in the absence of any template, indicating a decrease in the pH of the paper. Through a series of pull-out experiments, ammonium sulfate was found to be the cause of this change (Figure 21G). This color change could have been caused by oxidation of cellulose due to heating, the oxidizing properties of ammonium sulfate, or acidification of the reagents due to degassing of ammonia from the RT-LAMP mixture. To prevent color change in the absence of amplification, ammonium sulfate was replaced with betaine and the concentration of phenol red (which acts as an antioxidant) was increased (Figure 21H).

[0237] Although the effectiveness of betaine in LAMP reactions varies, it was included because it can reduce oxidative damage. Both trehalose and BSA were added to the formulation on paper (Figure 21H).

[0238] This device utilized two reaction zones: one targeting SARS-CoV-2 and one providing a no-primer control (which should not react with any sample) to determine the stability of our reagent on paper. A schematic of the final device is shown in Figure 19C, and results from devices with and without heat-inactivated SARS-CoV-2 spiked into saliva at a 5% reaction concentration are shown in Figure 19D. As shown in Figure 19B, the LoD of the assay on paper (250 copies / reaction) is comparable to that observed in solution of the colorimetric RT-LAMP formulation (Figure 21I).

[0239] For device construction, a Melinex® backing was used to provide structural support. Two reaction pads were attached to the backing using double-sided adhesive without altering their pH. The number of reaction zones can be optionally increased to allow for multiplexed detection without modifying the device design. 20-mil polystyrene spacers were added between the reaction pads to provide a physical barrier preventing leakage from one reaction zone to an adjacent one, thereby eliminating crosstalk during both reagent and sample addition. In some cases, reaction zones can be separated by a hydrophobic barrier created by wax printing, which prevents sample crossover. However, to enable roll-to-roll manufacturing, the use of wax was eliminated and spacers were used.

[0240] Example 25 - Validation of artificial samples To evaluate the analytical sensitivity and specificity of our assay on paper, we generated artificial samples using heat-inactivated SARS-CoV-2 at multiples of the LoD of orf7ab.I and performed the RT-LAMP assay on paper. A total of 30 positive samples and 30 corresponding negative samples were used, which is the minimum number required for Emergency Use Authorization (EUA) in the United States. To determine the colorimetric threshold for discriminating between positive and negative reactions, we constructed an ROC curve by calculating the sensitivity and specificity at various green channel intensity thresholds (Figure 20). At this threshold, the assay had the following analytical indices: sensitivity 97%, specificity 100%, and accuracy 98% (Figures 20A and 20B). The difference between the positive and negative groups was found to be significantly different (p<0.001). The paper strips were cut manually, and slight differences in paper size may result in differences in the colorimetric response. Therefore, large-scale production and quality control can further enhance the consistency within the two groups (positive and negative). This sensitivity is comparable to assays using RNA extracts (sensitivity of approximately 95%) and better than those reported with crude samples, where a significant loss of sensitivity (to approximately 80%) is common.

[0241] Example 26 - Colorimetric interpretation of paper-based devices To observe the impact of color perception on the performance of our device, we surveyed four participants and asked them to interpret the device results. Each participant was provided with a color bar and a 60-minute scan of the device (Figures 23A and 23B) and asked to classify the results as valid or invalid (using the control pad) and as positive or negative based on thresholds marked on the color bar. When user interpretation was introduced into the analysis, the device's sensitivity and accuracy decreased to 76% and 91%, respectively (Figure 19F). This low sensitivity stemmed from respondents identifying many positive reactions as invalid based on the control pad, resulting in a false invalid rate of 25%. This may be due to contamination of the control pad with amplicons in the reaction. Furthermore, user interpretation of pads with both yellow and red regions may create ambiguity, increasing the false positive or false invalid rate. Recent findings suggest that this ambiguity is due to a third, intermediate color cluster (in addition to the positive / negative cluster) that is not adequately addressed in colorimetric assays. This increase in false invalid rates may artificially affect the specificity and accuracy measures of the device, as invalid results were discarded from further analysis.

[0242] Example 27 - Effect of Removal of a Single Reactant on the Initial Color of Paper After Drying Figure 21G shows that 20 μL of the RT-LAMP master mix, containing a basic formulation containing KCl (50 mM), MgSO (8 mM), an equimolar dNTP mixture (1.4 mM for each dNTP), WarmStart BST 2.0 (0.32 U / μL), WarmStart RTx (0.3 U / μL), phenol red (0.25 mM), dUTP (0.14 mM), Antarctic UDG (0.0004 U / μL), Tween 20 (1% v / v), (NH)SO (10 mM), and trehalose (10% w / v), along with 5 μL of nuclease-free water, was added to grade 1 chromatography paper and allowed to dry for 10 minutes in a PCR preparation hood. To clarify the cause of the color change observed upon drying, reactants were omitted from the basic formulation as indicated. RT-LAMP primers and templates were not included in this study.

[0243] Example 28 - Color calibration of phenol red at various pH values Figure 21B shows the calibration of 250 μM phenol red at various pH values ​​on paper buffered with 20 mM Tris. The pH values ​​were adjusted using HCl or KOH. The images of the grade 222 chromatography paper (5 mm x 6 mm) were cropped to a rectangular shape to allow for easy color comparison across multiple strips.

[0244] Example 29 - Validation of the final device with various concentrations of heat-inactivated SARS-Cov-2 Figure 21A shows the final formulation of the RT-LAMP and colorimetric RT-LAMP master mixes using the orf7ab.I primer set. The left reaction zone of each device is a no-primer control, in which all orf7ab.I primers were replaced with water and not included in the master mix. The right reaction zone contains heat-inactivated virus spiked into treated saliva at the indicated concentrations for positive reactions or treated saliva for negative reactions. The final reaction concentration for all treated saliva was 5%. The master mix consisted of KCl (50 mM), MgSO (8 mM), an equimolar dNTP mixture (1.4 mM of each dNTP), WarmStart BST 2.0 (0.32 U / μL), WarmStart RTx (0.3 U / μL), phenol red (0.25 mM), dUTP (0.14 mM), Antarctic UDG (0.0004 U / μL), Tween 20 (1% v / v), betaine (20 mM), BSA (40 mg / mL), and trehalose (10% w / v). Grade 222 chromatography paper was used.

[0245] Example 30 - Color intensity of the green channel of the RT-LAMP colorimetric reaction at various template concentrations 21C shows a scatter plot of the colorimetric response of the paper pad at various concentrations, with the threshold for green color intensity indicated by the reference line 121.

[0246] Example 31 - Effect of heating method on RT-LAMP colorimetric reaction Figure 22C shows colorimetric scans for the limit of detection (LoD) of LAMP for 25 μL reactions between different heating devices after incubation at 65°C for 60 minutes. The primer set used for these reactions was orf1ab.II. To the 20 μL reaction mixture, 5 μL of heat-inactivated virus water dilution was added to obtain the final indicated concentration (positive reaction) or nuclease-free water (negative). The reaction master mix included 12.5 μL of NEB Colorimetric 2x Master Mix, 2.5 μL of primer mix, and 5 μL of water.

[0247] Example 32 - Effect on RT-LAMP colorimetric reaction or ramp rate on RT-LAMP colorimetric reaction Figure 22D shows colorimetric scans after incubating 25 μL reactions at 65°C for 60 minutes in a qTower (96-well plate) and a thermocycler (PCR tubes) at various ramp rates. The primer set used was orf1ab.II. To the 20 μL reaction mixture, 5 μL of heat-inactivated virus water dilution was added to obtain the final indicated concentration (positive reaction) or nuclease-free water (negative). The reaction master mix contained 12.5 μL of NEB Colorimetric 2x Master Mix, 2.5 μL of primer mix, and 5 μL of water.

[0248] Example 33 - Effect of trehalose and Tween 20 on RT-LAMP colorimetric reaction Figure 21H shows the results of colorimetric RT-LAMP with given concentrations of trehalose or Tween 20. The orf1ab.II primer set was used. Twenty microliters of the RT-LAMP master mix containing the basic formulation of KCl (50 mM), MgSO (8 mM), an equimolar dNTP mixture (1.4 mM for each dNTP), WarmStart BST 2.0 (0.32 U / μL), WarmStart RTx (0.3 U / μL), phenol red (0.25 mM), dUTP (0.14 mM), Antarctic UDG (0.0004 U / μL), Tween 20 (1% v / v where indicated), betaine (20 mM), BSA (40 mg / mL), and trehalose (10% w / v where indicated) was added to grade 1 chromatography paper (5 mm × 20 mm) and allowed to dry for 60 min in a PCR preparation hood. 1 × 10 nucleotides per reaction in 25% processed saliva were added to the paper and allowed to dry for 60 min in a PCR preparation hood. 5 Twenty-five microliters of heat-inactivated SARS-CoV-2 (positive reaction) or nuclease-free water (negative reaction) at a final concentration of 1 copy was added to the dry reaction pad. The pad was heated in an incubator set at 65°C for 60 minutes and then scanned using a flatbed scanner.

[0249] In the absence of template, the inclusion of ammonium sulfate caused the RT-LAMP reagent to change color from red to yellow upon drying. This color change was prevented by increasing the concentration of phenol red and replacing ammonium sulfate with betaine (Figure 21G). Furthermore, the addition of trehalose and bovine serum albumin (BSA) increased the reaction rate and improved the LoD (Figure 21H).

[0250] Example 34 - Summary A paper-based device for detecting nucleic acids of target pathogens in complex samples can use loop-mediated isothermal amplification (LAMP) by generating a colorimetric reaction visible to the human eye. To demonstrate the device's usefulness in emerging public health emergencies, it was used to detect SARS-CoV-2 in human saliva without pretreatment. The resulting device was capable of detecting the virus within 60 minutes, and using image analysis, demonstrated analytical sensitivity of 97% and specificity of 100%, with a detection limit of 200 genome copies per μL of patient saliva. The device contained a configurable number of reaction zones composed of grade 222 chromatography paper separated by 20-mil polystyrene spacers attached to a Melinex® backing with ARclean® double-sided adhesive. The resulting device can detect multiple targets and various pathogens by varying the LAMP primer set.

[0251] This platform has the following properties: i) uses saliva, ii) requires minimal operator training, iii) can be fabricated using roll-to-roll technology to enable millions of tests, iv) performs similarly to RT-qPCR assays in terms of analytical sensitivity and specificity, v) provides a colorimetric response that is visible to the naked eye, vi) is suitable for point-of-care use, vii) provides results in less than 60 minutes, and viii) is estimated to cost approximately $10 per test.

[0252] The test's simplicity and scalability allow for its use in a wide range of settings, potentially including in-home diagnostics. The platform can be easily reconfigured to target different pathogens by screening primer sets in solution. Multiplexing is possible by adding additional reaction sites to the device. Due to its reconfigurable nature, this platform can be used to detect emerging pathogens in future public health emergencies.

[0253] Exemplary Embodiments In one example, a loop-mediated isothermal amplification (LAMP) reaction assembly is provided that can include a LAMP reagent mixture that is substantially free of hygroscopic agents in combination with a solid-phase reaction medium.

[0254] In one example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid phase medium may be substantially free of magnesium interfering agents.

[0255] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the magnesium interfering agent can include a magnesium-containing compound and a chelating agent that interferes with magnesium.

[0256] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid phase medium can be hydrophilic, absorbent, and porous.

[0257] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid phase medium can be a cellulose-based medium.

[0258] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the cellulose-based medium can have a surface area-to-thickness ratio of about 30 to about 600.

[0259] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the cellulose-based medium can have a pore size of less than about 100 microns.

[0260] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid phase medium may include paper.

[0261] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid phase medium may include glass fibers.

[0262] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid phase medium may include nylon, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, or hydrophilic polytetrafluoroethylene (PTFE), or a combination thereof.

[0263] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the LAMP reaction medium can further include an adhesive that is substantially free of magnesium interfering agents and moisture absorbing agents.

[0264] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the LAMP reaction medium may further include a spreading layer that is less hydrophilic than the solid-phase reaction medium.

[0265] In one example, a method for producing a LAMP reaction assembly as recited herein is provided, which includes combining a LAMP reagent mixture that is substantially free of moisture absorbents with a solid-phase reaction medium such that the reagent mixture is maintained in contact with the solid-phase reaction medium.

[0266] In one example, the methods of making the LAMP reaction assemblies recited herein may further include controlling discoloration using a non-discoloring additive.

[0267] In another example of the method of making the LAMP reaction assembly recited herein, the non-color-changing additive may include a sugar, a buffer, a blocking agent, or a combination thereof.

[0268] In another example of the method of making the LAMP reaction assembly recited herein, the non-color-changing additive may comprise a sugar including one or more of trehalose, glucose, sucrose, dextran, or a combination thereof.

[0269] In another example of the method of making the LAMP reaction assembly recited herein, the non-color-changing additive may include a blocking agent comprising bovine serum albumin, casein, or a combination thereof.

[0270] In another example, a method of performing a LAMP analysis is provided that includes or comprises providing a LAMP reaction assembly as enumerated herein, applying a biological sample to the reaction assembly, heating the assembly to a temperature sufficient to initiate the LAMP reaction, and maintaining the temperature for a time sufficient to complete the LAMP reaction.

[0271] In one example method of performing a LAMP analysis, the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or a combination thereof.

[0272] In another example of a method for performing a LAMP analysis, the biological sample can be saliva.

[0273] In another example of a method of performing a LAMP analysis, the method may further include detecting a viral pathogen.

[0274] In another example of a method for performing a LAMP analysis, the LAMP analysis can be reverse transcription LAMP (RT-LAMP).

[0275] In one example, a system for chromatic loop-mediated isothermal amplification (LAMP) analysis is provided that includes or comprises a substantially non-reactive solid phase reaction medium and a non-interfering reagent mixture.

[0276] In one example of a system for chromatic loop-mediated isothermal amplification (LAMP) analysis, the substantially non-reactive solid phase reaction medium can have a buffer capacity of about 0.01 mM to about 5 mM.

[0277] In another example of a system for chromatic loop-mediated isothermal amplification (LAMP) assays, the substantially non-reactive solid-phase reaction medium has a λ range of about 443 nm to about 570 nm. max may have:

[0278] In another example of a system for chromatic loop-mediated isothermal amplification (LAMP) analysis, the substantially non-reactive solid phase reaction medium may comprise cellulose or glass fiber.

[0279] In another example of a system for chromatic loop-mediated isothermal amplification (LAMP) analysis, the substantially non-reactive solid phase reaction medium can be hydrophilic, absorbent, and porous.

[0280] In another example of a system for chromatic loop-mediated isothermal amplification (LAMP) analysis, the substantially non-reactive solid phase reaction medium may be substantially free of oxidizing agents and pH interfering agents.

[0281] In another example of a system for chromatic loop-mediated isothermal amplification (LAMP) analysis, the system can further include an adhesive, a spreading layer, a spacer, and a plastic carrier, each of which is substantially free of oxidizing agents and pH-interfering agents.

[0282] In another example of a system for chromatic loop-mediated isothermal amplification (LAMP) analysis, the non-interfering reagent mixture may further include one or more target primers, a DNA polymerase, and a resolubilizing agent.

[0283] In another example, a method is provided for maximizing the accuracy of a color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay, which may include providing a solid-phase reaction medium that minimizes color changes that occur outside of the LAMP reaction, and performing the LAMP assay on the solid-phase reaction medium.

[0284] In one example of a method for maximizing the accuracy of a color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay, the method may further include controlling color changes caused by protons generated outside of the LAMP reaction.

[0285] In another example of a method for maximizing the accuracy of a color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay, the method may further include using a non-color-changing additive to control color changes that occur outside of the LAMP reaction.

[0286] In another example of a method for maximizing the accuracy of the color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay, the non-color-changing additive may include a sugar, a buffer, a blocking agent, or a combination thereof.

[0287] In another example of a method for maximizing the accuracy of a color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay, the non-color-changing additive may comprise a sugar, including one or more of trehalose, glucose, sucrose, dextran, or combinations thereof.

[0288] In another example of a method for maximizing the accuracy of the color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay, the non-color-changing additive may include a blocking agent comprising bovine serum albumin, casein, or a combination thereof.

[0289] In another example of a method for maximizing the accuracy of a color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay, a method for maximizing the level of detection (LOD) of a loop-mediated isothermal amplification (LAMP) assay can include providing a reaction environment and reagents that minimize non-LAMP reaction products.

[0290] In another example of a method for maximizing the accuracy of a color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay, a system for a color loop-mediated isothermal amplification (LAMP) assay can include a combination of a solid-phase reaction medium and a LAMP reagent that maintains a color of the solid-phase reaction medium that is within 10% of the initial color of the solid-phase medium when stored at 25°C.

[0291] In one example of a method for maximizing the accuracy of a color output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assay, the combination can maintain color when stored for more than one or more of 30 days, 90 days, 365 days, 2 years, or 5 years.

[0292] In another example of a method for maximizing the accuracy of the color output signal in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) assays, this combination can maintain its color when stored at 25°C and approximately 40% to 90% relative humidity.

[0293] In another example, a method for producing a chromatic LAMP system as recited herein is provided, which may include combining a non-interfering reagent mixture with a substantially non-reactive solid phase reaction medium such that the non-interfering reagent mixture and the substantially non-reactive solid phase reaction medium are maintained in contact.

[0294] In one example of a method for manufacturing the chromatic LAMP system recited herein, the manufacturing process may include preparing a solution containing a non-interfering reagent mixture and coating the reagent mixture onto a substantially non-reactive solid-phase reaction medium.

[0295] In another example of a method for producing the color LAMP system recited herein, coating can include dripping, spraying, impregnating, dipping, or misting a solution onto a substantially non-reactive solid-phase reaction medium.

[0296] In another example of a method for producing the chromatic LAMP system recited herein, a non-interfering reagent mixture can be combined with a substantially non-reactive solid-phase reaction medium using a reel-to-reel (R2R) process.

[0297] It should be understood that the above-described methods are merely illustrative of some embodiments of the present invention. Numerous modifications and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the present invention, and the appended claims are intended to cover all such modifications and arrangements. Thus, while the present invention has been described above with specificity and detail in connection with what are presently considered to be the most practical and preferred embodiments of the invention, it will be apparent to those skilled in the art that variations, including modifications, can be made without departing from the principles and concepts described herein.

Claims

1. A loop-mediated isothermal amplification (LAMP) reaction assembly comprising a solid-phase reaction medium, wherein the solid-phase reaction medium comprises a substrate, a reaction layer disposed on the substrate, and a LAMP reagent mixture combined with the reaction layer, wherein the LAMP reagent mixture contains potassium chloride and does not contain glycerol, ethanol, methanol, calcium chloride, or calcium sulfate, a LAMP reaction assembly in which the result of the loop-mediated isothermal amplification (LAMP) reaction is analyzed by color, the analysis is performed by a pH-sensitive dye, and the pH-sensitive dye is included in the reaction layer together with the LAMP reagent mixture.

2. The LAMP reaction assembly according to claim 1, wherein the reaction layer is hydrophilic, absorbent, and porous.

3. The LAMP reaction assembly according to claim 1, wherein the reaction layer is a cellulose-based medium.

4. The LAMP reaction assembly according to claim 3, wherein the cellulose-based medium has a surface area to thickness ratio of about 30 to about 600.

5. The LAMP reaction assembly according to claim 3, wherein the cellulose-based medium has a pore size of less than about 100 microns.

6. The LAMP reaction assembly according to claim 1, wherein the reaction layer comprises paper.

7. The LAMP reaction assembly according to claim 1, wherein the reaction layer comprises glass fibers.

8. The LAMP reaction assembly according to claim 1, wherein the reaction layer comprises nylon, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, or hydrophilic polytetrafluoroethylene (PTFE), or a combination thereof.

9. The LAMP reaction assembly according to claim 1, wherein the reaction layer is adhered to the substrate by an adhesive that does not contain a magnesium interference agent and a moisture absorbent.

10. The LAMP reaction assembly according to claim 1, further comprising a developing layer having lower hydrophilicity than the reaction layer.

11. A method for manufacturing the LAMP reaction assembly according to claim 1, the method comprising combining the LAMP reagent mixture with the reaction layer of the solid-phase reaction medium so as to maintain contact between the LAMP reagent mixture and the solid-phase reaction medium.

12. The method according to claim 11, wherein the LAMP reagent mixture contains less than 1.0% by weight of a magnesium interference agent.

13. 13. The method of claim 12, wherein the magnesium interfering agent comprises a magnesium-containing compound and a chelating agent that interferes with magnesium.

14. further comprising using a non-discoloring additive to control discoloration; The method of claim 11 , wherein the non-color-changing additive comprises a sugar, a buffer, a blocking agent, or a combination thereof.

15. 15. The method of claim 14, wherein the non-discoloring additive comprises a sugar comprising one or more of trehalose, glucose, sucrose, dextran, or combinations thereof.

16. 15. The method of claim 14, wherein the non-discoloring additive comprises a blocking agent comprising bovine serum albumin, casein, or a combination thereof.

17. 1. A method for performing a LAMP analysis, comprising: Providing a LAMP reaction assembly according to claim 1; applying a biological sample to the LAMP reaction assembly; heating the LAMP reaction assembly to a temperature sufficient to initiate a LAMP reaction; and maintaining said temperature for a time sufficient to allow said LAMP reaction to complete; The method comprising:

18. 18. The method of claim 17, wherein the biological sample is one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or a combination thereof.

19. 18. The method of claim 17, wherein the biological sample is saliva.

20. 18. The method of claim 17, further comprising detecting a viral pathogen.

21. The method of claim 17, wherein the LAMP analysis is reverse transcription LAMP (RT-LAMP).

22. The LAMP reaction assembly of claim 1, wherein the pH-sensitive dye is selected from phenol red, phenolphthalein, azolithin, bromothymol blue, naphtholphthalein, cresol red, or a combination thereof.

23. 23. The LAMP reaction assembly of claim 22, wherein the solid phase reaction medium has a buffer capacity of about 0.01 mM to about 5 mM.

24. The solid-phase reaction medium has a λ range of about 443 nm to about 570 nm. ｍａｘ 23. The LAMP reaction assembly of claim 22, comprising:

25. glue, Deployment layer, a spacer; the substrate is a plastic carrier; 23. The LAMP reaction assembly of claim 22, wherein each of the adhesive, the spreading layer, the spacer, and the plastic carrier is free of oxidizing agents and pH interfering agents.

26. 23. A LAMP reaction assembly according to claim 22, comprising a combination of said solid phase reaction medium and LAMP reagents that maintains a color of said solid phase reaction medium that is within 10% of the initial color of said solid phase reaction medium when stored at 25°C.

27. 27. The LAMP reaction assembly of claim 26, wherein the combination maintains the color when stored for more than one or more of 30 days, 90 days, 365 days, 2 years, or 5 years.

28. 27. The LAMP reaction assembly of claim 26, wherein the combination maintains the color when stored at 25° C. and at a relative humidity of about 40% to 90%.

29. The manufacturing process comprises: preparing a solution containing the LAMP reagent mixture; and coating the LAMP reagent mixture onto the reaction layer; The method of claim 11 , comprising:

30. 30. The method of claim 29, wherein said coating comprises dripping, spraying, impregnating, dipping, or misting said solution onto said reaction layer.

31. 30. The method of claim 29, wherein the LAMP reagent mixture is combined with the reaction layer using a reel-to-reel (R2R) process.

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