Loop-mediated isothermal amplification (LAMP) on a solid-phase medium

Abstract

This disclosure relates to a loop-mediated isothermal amplification (LAMP) reaction assembly comprising a combination of a substantially hygroscopic-free LAMP reagent mixture and a solid-phase reaction medium. This disclosure also includes a system for colorimetric LAMP analysis comprising a substantially non-reactive solid-phase reaction medium and a non-interfering reagent mixture. This disclosure further 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 developing layer disposed on the reaction layer. This disclosure also includes a method for testing the presence of a target nucleotide sequence, comprising providing a biological sample and dispensing the sample into a test environment having a combination of a solid-phase reaction medium, a LAMP reagent mixture, and a pH-sensitive dye.

Description

Technical Field

[0001] [Related Applications]

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial Nos. 63 / 138,316 and 63 / 138,318, filed on January 15, 2021, each of which is incorporated herein by reference.

[0003] This invention relates to ring-mediated isothermal amplification (LAMP) in a solid medium. Prior Technology

[0004] [Background of the Invention]

[0005] Polymerase chain reaction (PCR) is a molecular biology technique that amplifies nucleotides for various analytical purposes. Quantitative PCR (qPCR) is an improvement on PCR, allowing for the monitoring of target nucleotide amplification. Diagnostic qPCR has been used to detect indicator nucleotides for infectious diseases, cancer, and gene abnormalities. Reverse transcription PCR (RT-PCR) is an improvement on qPCR, allowing for the detection of target RNA nucleotides. Because of this capability, RT-PCR is well-suited for detecting viral pathogens. However, RT-PCR requires substantial equipment, which may be unavailable in some on-site care settings. Furthermore, RT-PCR requires highly trained personnel, rigorous sample preparation, and a significant time commitment to execution and result acquisition.

[0006] Conversely, loop-mediated isothermal amplification (LAMP) is a simpler method for diagnostic identification of target nucleotides. Specifically, LAMP is a one-step nucleic acid amplification method for increasing specific nucleotide sequences. In addition to the isothermal heating process, LAMP can use simple visual output indicators, such as color changes, instead of the more complex fluorescent indicators used in PCR. To identify target nucleotides from RNA, reverse transcription LAMP (RT-LAMP), similar to RT-PCR, can be used, thus enabling diagnostic capabilities for identifying the presence of viral pathogens. Because LAMP is simpler and can be performed with less equipment and sample preparation, it is easier to use in on-site care settings such as clinics, emergency rooms, and even on mobile devices. Summary of the Invention

[0007] [Invention Summary]

[0008] This disclosure relates to a technique (e.g., method, system, and assembly) for detecting target nucleotides using loop-mediated isothermal amplification (LAMP) on a solid-phase medium. In some samples, it can be known that the target nucleotide is present in the pathogen of interest. In the case of a virus, the LAMP analysis can be a reverse transcription (RT) RT-LAMP analysis.

[0009] In some disclosed embodiments, a LAMP reaction assembly may comprise a combination of a substantially hygroscopic-free LAMP reagent mixture and a solid-phase reaction medium. In one embodiment, the solid-phase medium may be hydrophilic, absorbent, and porous.

[0010] In one state, the solid medium may be substantially free of magnesium interfering agents. In another state, the magnesium interfering agent may include magnesium-containing compounds and chelating agents that interfere with magnesium.

[0011] In another sample, the solid medium may be a cellulose-based medium. In one sample, the cellulose-based medium may have a surface area to thickness ratio between about 30 and about 600. In another sample, the cellulose-based medium may have a pore size greater than about 1 micrometer and less than about 100 micrometers. In one sample, the solid medium may comprise paper. In another sample, the solid medium may comprise glass fiber. In yet another sample, the solid medium may comprise nylon, polyurethane, polyether urethane, cellulose acetate, nitrocellulose, or hydrophilic polytetrafluoroethylene (PTFE), or combinations thereof.

[0012] In another embodiment, the LAMP reaction assembly may further comprise a binder that is substantially free of magnesium interfering agents and hygroscopic agents. In yet another embodiment, the LAMP reaction assembly may further comprise a spreading layer that is less hydrophilic than the solid-phase reaction medium.

[0013] In another disclosed embodiment, a method of manufacturing a LAMP reaction assembly as described above may include combining the substantially hygroscopic-free LAMP reagent mixture with the solid reaction medium such that the reagent mixture remains in contact with the solid reaction medium.

[0014] In one state, the method may include controlling discoloration using a non-discoloring additive. In another state, the non-discoloring additive may comprise a sugar, a buffer, an inhibitor, or a combination thereof. In yet another state, the non-discoloring additive may comprise a sugar comprising trehalose, glucose, sucrose, dextran, or a combination thereof. In yet another state, the non-discoloring additive may comprise an inhibitor comprising bovine serum albumin, casein, or a combination thereof.

[0015] In another disclosed embodiment, a method for performing 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 a LAMP reaction, and maintaining the temperature for a time sufficient to complete the LAMP reaction. In one sample, the biological sample may be one or more combinations of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or the like. In another sample, the biological sample may be saliva. In one sample, the method may further include detecting a viral pathogen. In another sample, the LAMP analysis may be reverse transcription LAMP (RT-LAMP).

[0016] In another disclosed embodiment, a system for colorimetric LAMP analysis may comprise a substantially non-reactive solid-phase reaction medium and a non-interfering reagent mixture. In one state, the substantially non-reactive solid-phase reaction medium may be hydrophilic, absorbent, and porous. In another state, the substantially non-reactive solid-phase reaction medium may be substantially free of oxidants, pH-disrupting agents, or combinations thereof.

[0017] In one state, the substantially non-reactive solid-phase reaction medium may have a buffering capacity from about 0.01 mM to about 5 mM. In another state, when combined with a pH-sensitive dye, the substantially non-reactive solid-phase reaction medium has a maximum absorption wavelength (λmax) within the test range. In another state, the substantially non-reactive solid-phase reaction medium may contain cellulose or glass fiber. In another state, the system may further contain a binder, a spreading layer, a spacer, a plastic carrier, or a combination thereof. In one state, each of the binder, spreading layer, spacer, or plastic carrier may be substantially free of oxidants and pH-disrupting agents. In another state, the non-interfering reagent mixture may further contain one or more target primers, DNA polymerases, or resolvants.

[0018] In another disclosed embodiment, a method for maximizing the accuracy of the chromatic output signal in a solid-phase pH-dependent LAMP analysis may include providing a solid-phase reaction medium that minimizes color changes caused by non-LAMP reactions, and performing the LAMP analysis on the solid-phase reaction medium. In one state, the method may include controlling non-LAMP-induced color changes caused by protons generated by non-LAMP reactions. In another state, the method may include using a color-fixing additive to control color changes caused by non-LAMP reactions. In one state, the color-fixing additive may comprise a sugar, a buffer, an inhibitor, or a combination thereof. In another state, the color-fixing additive may comprise a sugar comprising one or more of trehalose, glucose, sucrose, dextran, or combinations thereof. In yet another state, the color-fixing additive may comprise an inhibitor comprising bovine serum albumin, casein, or a combination thereof.

[0019] In yet another disclosed embodiment, a method for maximizing the detection level (LOD) in LAMP analysis may include providing a reaction environment and reagents that minimize non-LAMP reaction products.

[0020] In yet another disclosed embodiment, a system for colorimetric LAMP analysis may comprise a combination of a solid-phase reaction medium and a LAMP reagent, which, when stored at a selected temperature (e.g., room temperature of about 25°C), maintains the color of the solid-phase reaction medium having a hue within 10% of the initial hue of the solid-phase medium. In one state, the combination maintains color when stored for more than one or more of the following: 30 days, 90 days, 365 days, 2 years, or 5 years. In another state, the combination maintains color when stored at a relative humidity between about 40% and 90%. In yet another state, the selected temperature may be any temperature in the range between about –20°C and about 37°C.

[0021] In yet another disclosed embodiment, a method for manufacturing a colorimetric LAMP system as described above may include combining the 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 remain in contact. In one embodiment, the method may 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 embodiment, the coating may include dripping, spraying, impregnating, immersing, or atomizing the solution onto the substantially non-reactive solid-phase reaction medium. In yet another embodiment, a roll-to-roll (R2R) method may be used to combine the non-interfering reagent mixture with the substantially non-reactive solid-phase reaction medium.

[0022] In yet another disclosed embodiment, a solid-phase LAMP reaction medium may comprise 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 embodiment, the substrate may be an optically transparent material. In another embodiment, the adhesive layer may be substantially free of volatile agents. In yet another embodiment, the adhesive layer may be discontinuously disposed on the substrate. In yet another embodiment, the substrate may be an optically transparent plastic carrier.

[0023] In one sample, the solid-phase LAMP reaction medium may further include a test region. In one sample, the test region may be defined by at least two discontinuous adhesive layer segments. In another sample, the test region may be defined by at least three discontinuous adhesive layer segments. In yet another sample, the test region may be defined by at least four discontinuous adhesive layer segments.

[0024] In one state, the solid-phase LAMP reaction medium may further contain at least one substantially reagent-free fragment. In another state, the reaction layer may contain a reagent, including one or more target primers, a DNA polymerase, or a redissolving agent. In yet another state, the reagent may form a composition sufficient to carry out the LAMP reaction. In one state, the reaction layer may be discontinuous.

[0025] In another state, the solid-phase LAMP reaction medium may further comprise a developing layer comprising one or more of glass fiber, nylon, cellulose, polyurethane, polyetherurethane, cellulose acetate, nitrocellulose, polyester, hydrophilic polytetrafluoroethylene (PTFE), or combinations thereof. In one state, the developing layer may be optically transparent.

[0026] In another embodiment, the solid-phase LAMP reaction medium may include a spacer material. In one embodiment, the spacer material may comprise one or more of glass fiber, nylon, cellulose, polyurethane, polyetherurethane, cellulose acetate, nitrocellulose, polystyrene, polyester, hydrophilic polytetrafluoroethylene (PTFE), or combinations 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 from about 30 to about 600. In another embodiment, the reaction layer may have a thickness from about 0.05 mm to about 2 mm. In another embodiment, the reaction layer may have a width from about 4 mm to about 12 mm and a length from about 4 mm to about 25 mm. In another embodiment, the minimum space between segments of the reaction layer may be between about 1.8 mm and about 2.2 mm.

[0027] In another disclosed embodiment, a method for testing the presence of a viral pathogen may include providing a saliva sample from one of a subject and dispensing the sample into a test environment having a combination of a solid-phase reaction medium, a LAMP reagent mixture, and a pH-sensitive dye. In one embodiment, the method may include minimizing the amounts of volatile agents, hygroscopic agents, and non-pH-sensitive agents capable of discoloring the solid-phase medium. In another embodiment, the method may include providing one or more target primers, DNA polymerases, or redissolving agents in sufficient quantities to promote a LAMP reaction. In another embodiment, the method may include providing a reverse transcriptase in sufficient quantities to promote a reverse transcription LAMP (RT-LAMP) reaction. In another embodiment, the method may include providing one or more target primers in sufficient quantities to detect the viral pathogen. In yet another embodiment, the method may include generating a test result within one hour of dispensing the sample into the test environment.

[0028] In yet another disclosed embodiment, a method for confirming whether a saliva sample is suitable for testing using a solid-phase LAMP reaction may include providing a solid-phase reaction medium having at least one test site or test spot and a negative control site. In one embodiment, the at least one test site or test spot may include a combination of a LAMP reagent and a pH-sensitive dye. In another embodiment, the negative control site may include a pH-sensitive dye and exclude the LAMP reagent. In yet another embodiment, the method may include applying the saliva sample onto the solid-phase reaction medium. In yet another embodiment, the method may include confirming the activation of the pH-sensitive dye at the negative control site.

[0029] In one state, the pH-sensitive dye may be at least one of phenol red, phenolphthalein, litmus red, bromothymol blue, naphtholphthalein, cresol red, or combinations thereof. In another state, the LAMP reagent may be substantially free of volatile agents, pH-affecting agents, magnesium-containing agents, or combinations thereof. In yet another state, the LAMP reagent may contain non-interfering LAMP reagents, including DNA polymerase, reverse transcriptase, primers targeting specific regions, or combinations thereof.

[0030] In another embodiment, the method may further include providing a test location or test point defined by at least two discontinuous adhesive layer segments. In yet another embodiment, the method may include providing a test location or point defined by at least three discontinuous adhesive layer segments.

[0031] In yet another disclosed embodiment, a method for maximizing the accuracy of a positive test result from a solid-phase LAMP reaction may include providing a solid-phase reaction medium having at least three test sites or points, each comprising a common combination of a pH-sensitive dye and a LAMP reagent, wherein each site comprises a different primer sequence from a target pathogen. In one state, the method may include initiating a LAMP reaction. In another state, the method may include confirming a positive test result when at least two of the test sites or points activate the pH-sensitive dye and undergo a color change from a first color to a second color. In yet another state, the method may further include providing a sufficient amount of reverse transcriptase to promote a reverse transcription LAMP reaction.

[0032] In one state, the pH-sensitive dye may be at least one of phenol red, phenolphthalein, litmus red, bromothymol blue, naphtholphthalein, cresol red, or a combination thereof. In another state, the LAMP reagent may be substantially free of volatile agents, pH-affecting agents, magnesium-containing agents, or a combination thereof.

[0033] In another sample, the target pathogen may include a viral pathogen, bacterial pathogen, fungal pathogen, or protozoan pathogen. In one sample, the target pathogen may include a viral pathogen. In one sample, the viral pathogen may include dsDNA virus, ssDNA virus, dsRNA virus, positive-strand ssRNA virus, negative-strand ssRNA virus, ssRNA-RT virus, or ds-DNA-RT virus. In one sample, each primer sequence may be paired with a sequence from a viral target including H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.

[0034] In one disclosed embodiment, a method for testing the presence of a target nucleotide sequence may include providing a biological sample, dispensing the sample into a testing environment having a combination of a solid-phase reaction medium, a loop-mediated isothermal amplification (LAMP) reagent mixture, and a pH-sensitive dye.

[0035] In another state, the test environment may be substantially free of volatile agents, pH-affecting reagents, desiccants, or combinations thereof. In one state, the method may involve raising the temperature of the test environment at a rate of approximately 0.1 °C per second. In another state, the method may involve providing heating uniformity in the test environment with a variation of less than 1 °C. In another state, the method may involve providing a solid-phase reaction medium comprising either cellulose or glass fiber. In one state, the method may involve providing a quantity of reverse transcriptase sufficient to promote a reverse transcription LAMP (RT-LAMP) reaction.

[0036] In one state, the biological sample may be at least one of the following: saliva, mucus, blood, urine or feces, sweat, exhaled breath condensate, or a combination thereof. In one state, the method may 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 feces collection device.

[0037] In another sample, the target nucleotide sequence may originate from at least one of a viral pathogen, bacterial pathogen, fungal pathogen, or protozoan pathogen. In one sample, the target nucleotide sequence may originate from a viral pathogen. In one sample, the viral pathogen may be selected from the group consisting of: Coronaviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Adenoviridae, and Parvoviridae. In another sample, 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 sample, the target nucleotide sequence may originate from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pathogen.

[0038] In yet another disclosed embodiment, a biological sample testing device may include a substrate bonded to a combination of a solid-phase reaction medium, a dehydrated ring-mediated isothermal amplification (LAMP) reagent mixture, and a dehydrated pH-sensitive dye, wherein the device provides at least approximately 95% testing accuracy after being stored at room temperature for 6 months. In one embodiment, the device provides at least approximately 95% testing accuracy after being stored at room temperature for 12 months. In another embodiment, the device provides at least approximately 95% testing accuracy after being stored at room temperature for 2 years.

[0039] In yet another disclosed embodiment, a biological sample testing system may include a substrate bonded to a combination of a solid-phase reaction medium and a dehydrated ring-mediated isothermal amplification (LAMP) reagent mixture and a dehydrated pH-sensitive dye, the housing being operable to receive a biological sample. In one embodiment, the biological sample testing system may 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 embodiment, the biological sample testing system may contain a biological sample for a period of time for generating a test result using the pH-sensitive dye.

[0040] In one embodiment, the substrate may comprise an optically transparent material. In another embodiment, the substrate may be bonded to the solid-phase reaction medium using an adhesive. In yet another embodiment, the adhesive may be substantially optically transparent. In yet another embodiment, the substrate may comprise a portion of a housing.

[0041] In another embodiment, the biosample testing system may include an adhesive layer disposed on the substrate; a reaction layer disposed on the adhesive layer; and a spreader layer disposed on the reaction layer. In one embodiment, the biosample testing system may further include a spacer layer oriented in the same plane as the reaction layer. In another embodiment, the housing may be disposed abutted against the substrate. In another embodiment, the housing may be further disposed abutted against the spreader layer. In another embodiment, the housing may substantially enclose the substrate, adhesive layer, reaction layer, and spreader layer. Simple Explanation of the Diagram

[0042] The features and advantages of this disclosure will become apparent from the following detailed description, together with the accompanying drawings illustrating the features of this disclosure by way of example, wherein:

[0043] Figure 1 illustrates a method for performing LAMP analysis according to an exemplary embodiment;

[0044] Figure 2 illustrates a method for maximizing the accuracy of the chromaticity output signal in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis according to an exemplary embodiment;

[0045] Figure 3a illustrates a solid-phase LAMP reaction medium having three test sections according to an exemplary embodiment;

[0046] Figure 3b illustrates a solid-phase LAMP reaction medium with two test sections according to an exemplary embodiment;

[0047] Figure 3c illustrates a solid-phase LAMP reaction medium having four test sections according to an exemplary embodiment;

[0048] Figure 3d illustrates a solid-phase LAMP reaction medium with three test sections and no development layer according to an exemplary embodiment;

[0049] Figure 3e illustrates a solid-phase LAMP reaction medium with two test sections and no development layer according to an exemplary embodiment;

[0050] Figure 3f illustrates a solid-phase LAMP reaction medium with four test sections and no development layer according to an exemplary embodiment;

[0051] Figure 4 illustrates a method for testing the presence of viral pathogens according to an exemplary embodiment;

[0052] Figure 5 illustrates a method for determining whether a saliva sample is suitable for testing using a solid-phase LAMP reaction, according to an exemplary embodiment.

[0053] Figure 6 illustrates a method for maximizing the accuracy of positive test results from a solid-phase LAMP reaction according to an exemplary embodiment;

[0054] Figure 7 illustrates a method for testing the presence of a target nucleotide sequence according to an exemplary embodiment;

[0055] Figure 8a illustrates the operation of the biological testing kit according to an exemplary embodiment;

[0056] Figure 8b illustrates the color chart of the biological test kit according to an exemplary embodiment;

[0057] Figure 9 illustrates a paper LAMP assembly according to an exemplary embodiment;

[0058] Figure 10 illustrates the material screening of Grade 1 and Grade 222 chromatography papers according to an exemplary embodiment;

[0059] Figure 11 illustrates, according to an exemplary embodiment, the reaction of thermally deactivated SARS-CoV-2 in liquid using water incorporation;

[0060] Figure 12 illustrates the paper strip format according to an exemplary embodiment;

[0061] Figure 13 illustrates the solid-phase LAMP reaction medium according to an exemplary embodiment;

[0062] Figure 14 illustrates the solid-phase LAMP reaction medium according to an exemplary embodiment;

[0063] Figure 15A illustrates a comparison between Grade 1 chromatography paper and Grade 222 chromatography paper according to an exemplary embodiment;

[0064] Figure 15B illustrates the binding of dried RT-LAMP at different initial pH values ​​of the RT-LAMP reaction mixture according to an exemplary embodiment;

[0065] Figure 16 illustrates the test bar format according to an exemplary embodiment;

[0066] Figure 17 illustrates the test strip assembly process according to an exemplary embodiment; and

[0067] Figure 18A illustrates the operation of a biological testing system according to an exemplary embodiment.

[0068] Figure 18B illustrates the operation of the biological testing system according to an exemplary embodiment;

[0069] Figures 19A-19F illustrate schematic diagrams and colorimetric characterization analysis of the paper-based apparatus according to an exemplary embodiment;

[0070] Figures 20A-20C illustrate the digital analysis of paper-based colorimetric responses according to an exemplary embodiment;

[0071] Figure 21A illustrates the device efficacy under different concentrations of heat-deactivated SARS-CoV-2 according to an exemplary embodiment;

[0072] Figure 21B illustrates the phenol red color correction at various pH values ​​according to an exemplary embodiment;

[0073] Figure 21C illustrates the green channel color intensity of the RT-LAMP colorimetric response at different sample concentrations according to an exemplary embodiment;

[0074] Figure 21D illustrates a LAMP on chromatography paper using EBT as a colorimetric reporter reagent according to an exemplary embodiment;

[0075] Figure 21E illustrates the colorimetric response of LAMP on various types of paper using EBT as an indicator, according to an exemplary embodiment;

[0076] Figure 21F illustrates LAMP detection on biodyne A amphoteric paper using EBT as a colorimetric indicator according to an exemplary embodiment;

[0077] Figure 21G illustrates the elimination of the effect of a single reactant on the initial color of the dried paper according to an exemplary embodiment;

[0078] Figure 21H illustrates the effect of trehalose and Tween 20 on the RT-LAMP colorimetric response according to an exemplary embodiment;

[0079] Figure 21I illustrates the effect of saliva treatment on contrast color response according to an exemplary embodiment;

[0080] Figure 22A illustrates the effect of the disk on the RT-LAMP colorimetric response according to an exemplary embodiment;

[0081] Figure 22B illustrates the effect of the top cover on the RT-LAMP colorimetric response according to the exemplary embodiment;

[0082] Figure 22C illustrates the effect of the heating method according to the exemplary embodiment on the colorimetric response of RT-LAMP;

[0083] Figure 22D illustrates the effect of the heating rate on the RT-LAMP colorimetric response according to the exemplary embodiment; and

[0084] Figures 23A and 23B illustrate a colorimetric perception survey according to an exemplary embodiment.

[0085] Reference will now be made to the exemplary embodiments illustrated, and the same embodiments will be described using specific language. However, it should be understood that this is not intended to limit the scope of the art. Implementation

[0086] [Description of the Example]

[0087] Before describing the embodiments of the present invention, it should be understood that this disclosure is not limited to the specific structures, method steps, or materials disclosed herein, but extends to equivalents recognized by those skilled in the art. It should also be understood that the terminology used herein is for the purpose of describing particular paradigms or embodiments only and is not intended to be limiting. The same symbols in different figures represent the same elements. The numbers provided in the flowcharts and processes are provided to clearly illustrate the steps and operations and do not necessarily indicate a specific order or sequence.

[0088] Furthermore, the described features, structures, or properties can be combined in any suitable manner in one or more embodiments. Numerous specific details, such as examples of compositions, dosage forms, processes, etc., are provided in the following description to provide a thorough understanding of various embodiments of the invention. However, those skilled in the art will recognize that such detailed embodiments do not limit the overall inventive concept set forth herein, but are merely representative examples. [definition]

[0089] It should be noted that the singular forms “a,” “an,” and “the” used herein include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “a kind of excipient” includes reference to one or more such excipients, and reference to “the carrier” includes reference to one or more such carriers.

[0090] The terms “formulation” and “composition” as used herein are used interchangeably to refer to a mixture of two or more compounds, elements, or molecules. In some cases, the terms “formulation” and “composition” may refer to a mixture of one or more surfactants with a carrier or other excipients.

[0091] As used herein, the term "soluble" is a measure or characteristic of a substance or reagent in its ability to dissolve in a given solvent. The solubility of a substance or reagent in a particular component of a composition refers to the amount of the substance or reagent that dissolves at a specific temperature, such as about 25°C or about 37°C, to form a clearly transparent solution.

[0092] As used in this article, 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.

[0093] The term "subject" as used in this article refers to an animal. In one phenotype, the animal may be a mammal. In another phenotype, the mammal may be a human.

[0094] As used herein, the term “non-liquid” refers to the state of the composition disclosed herein as being in a semi-solid or solid physical state.

[0095] As used in this article, the terms "solid" and "semi-solid" refer to a physical state in which the constituents support their own weight and possess sufficient viscosity or structure to prevent free flow under standard temperature and pressure. Semi-solid materials can conform to the shape of a container under pressure.

[0096] As used herein, the term "solid medium" refers to a non-liquid medium. In one example, the non-liquid medium may be a material with a porous surface. In another example, the non-liquid medium may be a material with a fibrous surface. In yet another example, the non-liquid medium may be paper.

[0097] As used herein, the terms “solid medium,” “solid substrate,” “solid substrate,” “solid test substrate,” “solid test substrate,” etc., refer to a non-liquid medium, apparatus, system, or environment. In some cases, the non-liquid medium may be substantially free of liquid or completely free of liquid. In one example, the non-liquid medium may comprise or be a porous material or a material having a porous surface. In another example, the non-liquid medium may comprise or be a fibrous material or a material having a fibrous surface. In yet another example, the non-liquid medium may be paper.

[0098] As used herein, the term "color-reducing additive" means an additive that minimizes or prevents color changes in a solid-phase medium from its original or initial color to a different color due to reasons other than nucleotide amplification during or in the presence of a LAMP reaction. For example, in one embodiment, this color change can be minimized or reduced compared to the color change that would occur without the color-reducing additive.

[0099] As used herein, the term "non-LAMP reaction-induced discoloration" refers to any discoloration of a solid-phase medium that is not caused by nucleotide amplification via the LAMP reaction (e.g., a change in color from its original color to another). In some instances, non-LAMP reaction-induced discoloration may refer to a discoloration of a solid-phase medium caused by one or more of the following: volatile agents, magnesium interfering agents, oxidizing agents, pH changes caused by factors other than LAMP amplification, drying, or a combination thereof.

[0100] As used herein, the term "volatile agent" means an agent comprising a composition having a high vapor pressure or a low boiling point. In one example, ammonium sulfate may be a volatile agent because ammonia volatilizes, leaving sulfate ions. In one example, a composition may have a high vapor pressure when it is in the gas phase at a temperature above about 30°C. In one example, a composition may have a low boiling point when it is in the gas phase at a temperature below about 80°C.

[0101] The term "desiccant" as used in this article refers to a reagent that enhances the drying of a solid medium compared to the drying of a solid medium without the reagent.

[0102] As used in this article, the term "pH disruptor" refers to an agent that can affect the pH of a reaction, system, or environment due to factors other than LAMP amplification. In one example, ammonium ions can volatilize from ammonium sulfate, while sulfate ions can react to form sulfuric acid and affect the pH of the reaction in the absence of amplification from the LAMP reaction.

[0103] The term “non-pH-sensitive agent” as used in this article refers to a reagent that is substantially unaffected by pH changes.

[0104] In this disclosure, terms such as “comprising,” “including,” “containing,” and “having” may have the meanings they are given under U.S. Patent Law and may refer to “including,” “comprising,” etc., and are generally interpreted as open-ended terms. The terms “composed of” or “consisting of” are closed-ended terms that include only the components, structures, steps, etc., explicitly listed in conjunction with such terms, and the content that conforms to U.S. Patent Law. “Substantially composed of” or “substantially composed of” have the meanings they are generally given under U.S. Patent Law. In particular, these terms are generally closed-ended terms but allow for the inclusion of additional items, materials, components, steps, or elements that do not materially affect the essential and novel features or function of the relevant item. For example, if the language “substantially composed of” is used, the composition may contain trace elements that do not affect the properties or characteristics of the composition, even if such trace elements are not explicitly listed in the list of items following such terms. When open-ended terms such as “comprising” or “including” are used in a written description, it should be understood that they also directly support the language of “substantially composed of” and the language of “consisting of”, as explicitly stated, and vice versa.

[0105] The terms “first,” “second,” “third,” “fourth,” etc., used in the specification and claims of this invention, if applicable, are used to distinguish similar elements and are not necessarily used to describe a particular order or chronological sequence. It should be understood that any terms thus used are interchangeable where appropriate, allowing the embodiments described herein to be operated, for example, in order other than those shown or otherwise described herein. Similarly, if a method is described herein as comprising a series of steps, the order of these steps presented herein is not necessarily the only possible order in which they can be performed, and some of the stated steps may be omitted and / or other steps not described herein may be added to the method.

[0106] The terms used herein, such as “increased,” “decreased,” “better,” “worse,” “higher,” “lower,” “enhanced,” “maximized,” and “minimized,” are comparative terms meaning that the nature of an apparatus, component, composition, or activity is significantly different from that of other apparatuses, components, compositions, or activities in the following conditions: in the surrounding or adjacent area, in similar conditions, in a single apparatus or composition or in a number of comparable apparatuses or compositions, in a group or type, in multiple groups or types, or compared with the known state of the prior art.

[0107] As used herein, the term "coupling" is defined as a direct or indirect connection by chemical, mechanical, electrical, or non-electrical means. Objects described herein as "adjacent" to each other may be in actual contact, in close proximity, or located in the same general area or region, depending on the context in which the phrase is used. "Directly coupled" objects, structures, elements, or features are in contact with and attached to each other. Furthermore, as used in this written description, it should be understood that the use of the term "coupling" also supports "direct coupling," and vice versa.

[0108] As used herein, the term "substantially" refers to the complete or near-complete extent or degree of an effect, characteristic, property, state, structure, item, or result. For example, a "substantially" closed object means that the object is completely closed or almost completely closed. In some cases, the exact permissible deviation from absolute completeness may depend on the specific circumstances. However, in general, a degree of near-completeness will have the same overall result as achieving absolute completeness and fullness. When used in a negative sense, the use of "substantially" also applies to the complete or near-complete absence of an effect, characteristic, property, state, structure, item, or result. For example, a composition "substantially free of" particles can be completely free of particles or almost completely free of particles, such that the effect is the same as having no particles at all. In other words, a composition "substantially free of" a component or element may still actually contain that item, as long as it has no measurable effect.

[0109] The term “about” as used herein is used to provide flexibility for the endpoints of a numerical range by providing a given value that can be “slightly above” or “slightly below” the endpoints. Unless otherwise stated, the use of the term “about” for a particular number or range should also be understood as not supporting that numerical term or range without the term “about”. For example, for convenience and brevity, the numerical range of “about 50 angstroms to about 80 angstroms” should also be understood as supporting the range of “50 angstroms to 80 angstroms”. Furthermore, it should be understood that even when the term “about” is used in this specification, support for actual numerical values ​​is provided. For example, “about” 30 should be interpreted as supporting not only values ​​slightly above and slightly below 30, but also the actual value of 30.

[0110] The terms used herein, including multiple items, structural elements, constituent elements, and / or materials, may be presented in a common list for convenience. However, these lists should be interpreted as if each individual member of the list were individually identified as a separate and unique member. Therefore, no individual member in such a list should be construed as being substantially equivalent to any other member in the same list solely based on its presentation in a common group, without the indication to the contrary.

[0111] Concentration, quantity, level, and other numerical data may be expressed or presented in range format herein. It should be understood that this range format is used merely for convenience and brevity, and therefore should be interpreted flexibly to include not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges, or decimal units, covered within the range, as if each value and subrange were explicitly listed. For example, a numerical range of “about 1 to about 5” should be interpreted to include not only the explicitly listed values ​​of about 1 to about 5, but also the individual values ​​and subranges within that indicated range. Therefore, the numerical range includes individual values ​​such as 2, 3, and 4, and subranges such as from 1 to 3, from 2 to 4, and from 3 to 5, as well as individually 1, 2, 3, 4, and 5. The same principle applies to ranges that use only one value as a minimum or maximum value. Furthermore, this interpretation should apply regardless of the breadth of the range or the characteristics described.

[0112] Throughout this specification, the reference to "example" means that a particular feature, structure, or characteristic described in connection with that example is included in at least one embodiment. Therefore, the phrase "in an example" appearing in various places throughout this specification does not necessarily refer to the same embodiment. [Example]

[0113] Molecular testing for many pathogens (e.g., Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19) can be limited by laboratory conditions, resulting in significant delays (>24 hours) in providing results, hindering their adoption in designated care settings. Despite considerable efforts to develop designated care tests for SARS-CoV-2, several limitations remain: i) scalability (weekly testing demand is in the millions, but manufacturing new tests at this scale is difficult), ii) sample processing (many tests still use extraction procedures when saliva is used), and iii) readability (molecular tests often use fluorescence, thus requiring fluorescence readers to report results).

[0114] Current testing methods can be overcome by using paper-based devices and a site-specific care test using reverse transcription loop-mediated isothermal amplification (RT-LAMP). This test uses diluted saliva (e.g., 5% v / v in water) as a sample and reports a color change within 60 minutes in the presence of a pathogen (e.g., SARS-CoV-2). RT-LAMP is a nucleic acid amplification technique performed at an isothermal temperature, offering appropriate diagnostic performance, particularly during the acute phase of infection. Because RT-LAMP can be performed at an isothermal temperature, expensive thermal circulation equipment is unnecessary. Furthermore, existing colorimetric reporters for LAMP products do not use fluorescent readers. Therefore, this test is suitable for site-specific care settings and can be rapidly developed and scaled up, making it suitable for use in public health emergencies.

[0115] RT-LAMP can be performed on microfluidic paper-based analytical devices (μPADs) to detect various pathogens (e.g., SARS-CoV-2). Portable electronic devices can be used for image analysis to differentiate between positive and negative reactions. In one example, high-contrast RT-LAMP reactions on paper can provide visible color changes. Furthermore, to prevent crosstalk between samples, polystyrene spacers can be used instead of wax printing (which may have precisely aligned printing areas and dispense reagents). Polystyrene spacers are easy to manufacture roll-to-roll for scaled-up production.

[0116] Nucleic acid-based COVID-19 diagnostic methods utilize pretreatment to provide results. As disclosed herein, paper-based colorimetric detection of SARS-CoV-2 can be performed with minimal pretreatment. This device can provide the sensitivity and specificity for paper-based detection of SARS-CoV-2 without pre-amplification. Other analytical methods performed in solution may not be able to amplify during manufacturing as effectively as paper-based methods. Furthermore, the analytical method disclosed herein uses a dilution procedure that can be completed within seconds, whereas other analytical methods use various procedures such as protease treatment, heat deactivation, and / or RNA extraction to detect SARS-CoV-2 (procedures completed at least 10 minutes and using additional equipment). Material assembly for LAMP reaction in solid media

[0117] Performing LAMP reactions on solid media can be challenging due to performance requirements. For example, to maximize accuracy, the solid medium should support the LAMP reaction without interfering with the LAMP reaction or LAMP indicator. At a higher level, the solid medium is hydrated with the biological sample to allow the LAMP reaction to proceed, and then the results are read. However, a wide variety of reagents can interfere with the LAMP reaction or subsequent result readings. To minimize errors, care should be taken to reduce or avoid the challenges associated with providing the biological sample, hydrating the solid medium with the sample, integrating the LAMP reagent into the solid medium, performing the LAMP analysis, and obtaining a clear and legible test output signal.

[0118] Given the above background, in one disclosed embodiment, a loop-mediated isothermal amplification (LAMP) reaction assembly may comprise a combination of a substantially hygroscopic-free LAMP reagent mixture and a solid-phase reaction medium. When the LAMP reagent mixture is substantially hygroscopic-free, it minimizes or avoids difficulties that occur during the drying process of the solid-phase reaction medium. When hygroscopic agents are used, they, or the solid-phase reaction medium, may not dry completely, or may rehydrate to a certain extent during storage, and may interfere with the original color of the solid-phase reaction medium, potentially biasing the test results of the LAMP method.

[0119] In one embodiment, the hygroscopic agent may absorb more than about 10% by weight of the reagent at a relative humidity (RH) between about 40% and about 90% at 25°C. In one sample, the hygroscopic agent may comprise, but is not limited to, one or more of glycerol, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate, or combinations thereof. In some examples, LAMP reactions containing a hygroscopic agent (e.g., glycerol) can lead to instability of the reagent in the solid-phase medium because the hygroscopic agent attracts water. Therefore, an excess of hygroscopic agent in the solid-phase reaction medium should be avoided.

[0120] Furthermore, in another embodiment, the substantially hygroscopic-free LAMP reagent mixture may contain one or more of DNA polymerase, reverse transcriptase, target primer, or combinations thereof. When the LAMP reaction is a LAMP reaction or a reverse transcription LAMP (RT-LAMP) reaction, it may contain DNA polymerase. Additionally, when the LAMP reaction is an RT-LAMP reaction, the substantially hygroscopic-free LAMP reagent may further contain reverse transcriptase for reverse transcription of RNA into cDNA.

[0121] In one state, the solid-phase reaction medium may be substantially free of magnesium interfering agents. Magnesium can interfere with the LAMP reaction in several ways. First, magnesium can be a cofactor for DNA polymerase and should be closely monitored within a target concentration range to allow DNA polymerase to promote the LAMP reaction. Second, when magnesium-sensitive indicators are used in the LAMP reaction, magnesium interfering agents can invalidate the results or complicate the analysis of the results.

[0122] Therefore, in another state, the magnesium interfering agent may include magnesium-containing compounds and chelating agents that interfere with magnesium. In one state, the magnesium interfering agent may be substantially magnesium-free and includes, but is not limited to: Mg²⁺, Mg¹⁺, magnesium carbonate, magnesium chloride, magnesium citrate, magnesium hydroxide, magnesium oxide, magnesium sulfate, magnesium sulfate heptahydrate, etc., or combinations thereof. Because some solid-phase reaction media may have buffering capacity, residual ions, or chelating agents that interfere with magnesium, the concentration of magnesium (a cofactor for BST enzymes (e.g., DNA polymerase)) may be affected and interfere with the LAMP reaction. Therefore, the magnesium concentration should be controlled within a target magnesium range to promote the LAMP reaction. In one state, the composition may contain less than one or more of the following magnesium: 1.0 wt%, 0.5 wt%, 0.1 wt%, or 0.01 wt%.

[0123] The solid-phase reaction medium can possess various properties that promote the LAMP reaction. In a single state, the solid-phase reaction medium can be hydrophilic, absorbent, porous, or inert. The solid-phase reaction medium can be hydrophilic when the contact angle between the surface and edge of the droplet is less than about 90 degrees. The solid-phase reaction medium can be absorbent, as measured by the degree to which paper can absorb a certain amount of liquid. The solid-phase reaction medium can be porous when it has a pore size greater than at least 1 micrometer and less than or equal to one or more of about 100 micrometers, about 75 micrometers, about 50 micrometers, about 25 micrometers, about 10 micrometers, about 5 micrometers, or about 1 micrometer. The solid-phase reaction medium can be inert when it does not interfere with the LAMP reaction. The solid-phase reaction medium can also be inert when it does not interfere with the indications produced by the LAMP reaction.

[0124] The solid-phase reaction medium may contain or include various materials. In one state, the solid-phase reaction medium may contain one or more of nylon, polyurethane, polyether urethane, cellulose acetate, nitrocellulose, hydrophilic polytetrafluoroethylene (PTFE), etc., or combinations thereof. In another state, the solid-phase reaction medium may be a cellulose-based medium, such as Grade 1 chromatography paper, Grade 222 chromatography paper, etc., or combinations thereof.

[0125] In addition to the material type of the solid-phase reaction medium, the solid-phase reaction medium may also possess various physical properties that can adapt to or enhance the LAMP reaction and avoid interfering with the indicator or signal output generated by the LAMP reaction. In one sample, the solid-phase reaction medium may have a surface area to thickness ratio between about 30 and about 600. In another sample, the solid-phase reaction medium may have a surface area to thickness ratio between about 60 and about 400. In one sample, the solid-phase reaction medium may have a surface area to thickness ratio between about 100 and about 200. In another sample, the solid-phase reaction medium may be a cellulose-based medium having a surface area to thickness ratio between about 30 and about 600. In one sample, the cellulose-based medium may have a pore size greater than at least 1 micrometer and less than or equal to one or more of about 100 micrometers, about 75 micrometers, about 50 micrometers, about 25 micrometers, about 10 micrometers, about 5 micrometers, or about 1 micrometer.

[0126] The thickness of the solid-phase reaction medium can affect 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, and the concentration of reagents in the solid-phase reaction medium.

[0127] In one sample, the solid-phase reaction medium may comprise 222-grade chromatography paper, which provides increased homogeneity relative to 1-grade chromatography paper because 222-grade paper is thicker than 1-grade paper. Therefore, compared to 1-grade paper, 222-grade paper can concentrate reagents within a smaller surface area. In one sample, the chromatography paper may be 222-grade with a surface area of ​​approximately 5 mm × approximately 5 mm to provide the desired surface area to thickness ratio. In one example, 1-grade chromatography paper may have a cross-sectional dimension of 20 mm long and 5 mm wide and a thickness of 0.18 mm to provide a surface area to thickness ratio of approximately 555. In another example, 222-grade chromatography paper may have a cross-sectional dimension of 5 mm long and 5 mm wide and a thickness of 0.83 mm to provide a surface area to thickness ratio of approximately 30. Therefore, this example illustrates that a surface area to thickness ratio of about 30 (e.g., grade 222 chromatography paper) can provide increased uniformity compared to a surface area to thickness ratio of about 555 (e.g., grade 1 chromatography paper).

[0128] In addition to the materials disclosed herein, the solid-phase reaction medium may comprise one or more of paper or glass fibers. In one example, the paper may comprise one or more of α-cellulose, β-cellulose, γ-cellulose, or combinations thereof. In another example, the glass fibers may comprise one or more of A-glass, E-glass, S-glass, R-glass, C-glass, T-glass, D-glass, M-glass, ECR glass, or combinations thereof.

[0129] In some examples, the reaction assembly may include a binder. For instance, this binder can be used to bond the various fragments of the solid-phase reaction medium together. In one example, the reaction assembly may further include a binder that is substantially free of magnesium interfering agents, hygroscopic agents, or combinations thereof. This binder may be substantially free of magnesium interfering agents to avoid interference between magnesium and DNA polymerase, and to avoid complicating the reading of LAMP results when using magnesium-based indicators.

[0130] In some examples, the reaction assembly may also include a spreading layer. This spreading layer facilitates the uniform spreading of the biological sample across different sections of the solid-phase reaction medium. In another example, the spreading layer may have a lower hydrophilicity than the solid-phase reaction medium. A spreading layer with lower hydrophilicity than the solid-phase reaction medium promotes uniform spreading of the biological sample because the sample diffuses from the less hydrophilic spreading layer towards the more hydrophilic solid-phase reaction medium.

[0131] The configuration of the reagent mixture relative to the solid-phase reaction medium affects the LAMP reaction. In one embodiment, a method of manufacturing a LAMP reaction assembly as described herein may include combining the substantially hygroscopic-free LAMP reagent mixture with the solid-phase reaction medium such that the reagent mixture remains in contact with the solid-phase reaction medium. In one example, the reagent mixture may be in direct contact with the solid-phase reaction medium. In another example, the reagent mixture may be in indirect contact with the solid-phase reaction medium. When the reagent mixture remains in indirect contact with the solid-phase reaction medium, the intermediate material (e.g., an antioxidant) may enhance the LAMP reaction.

[0132] In some cases, the color of the solid-phase reaction medium can be affected by reagents not produced by the LAMP reaction. For example, volatile agents, such as ammonium sulfate, can form sulfate ions, which then react to form sulfuric acid. When using pH-based indicators to read the results of LAMP reactions, these non-LAMP reactions may hinder the correct reading of the results or may complicate the interpretation of the results.

[0133] In one embodiment, the method may include using a color-resistant additive to control color change. This color-resistant additive may comprise sugars, buffers, inhibitors, etc., or combinations thereof. In one example, the sugar may comprise trehalose, glucose, sucrose, dextran, etc., or combinations thereof. In another example, the inhibitor may comprise bovine serum albumin, casein, etc., or combinations thereof.

[0134] This sugar can prevent the effects of pH changes from non-LAMP reagents. This buffer can prevent the effects of pH changes caused by factors other than the LAMP reaction, thereby preventing interference with the LAMP reaction or LAMP results. This blocker can prevent the effects of factors other than the LAMP reaction by blocking the action of various enzymes such as RNase or DNase on the nucleic acid to be analyzed (e.g., RNA from viruses or DNA from pathogens).

[0135] In another embodiment, as shown in FIG1, method 100 for performing LAMP analysis may include providing a LAMP reaction assembly as described in this 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 a 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.

[0136] In one state, the biological sample may be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or combinations thereof. In another state, the biological sample may be saliva. In yet another state, the method may further include the detection of a viral pathogen. In yet another state, the LAMP analysis may be reverse transcription LAMP (RT-LAMP).

[0137] In another sample, the temperature sufficient to initiate the LAMP reaction is in the range of approximately 50°C to approximately 60°C. In another sample, the temperature sufficient to initiate the LAMP reaction is in the range of approximately 60°C to approximately 70°C. In another example, the isothermal temperature can be within a range differing by less than 5°C. In another sample, the temperature can be maintained at a level sufficient to complete the LAMP reaction for more than one or more of the following: 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, or 120 minutes. In another sample, the temperature can be maintained at a level sufficient to complete the LAMP reaction for less than one or more of the following: 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, or 120 minutes.

[0138] LAMP reaction assemblies can be manufactured to improve their uniformity, shelf life or storage life, and test effectiveness. In one embodiment, the LAMP reaction assembly can be manufactured using one or more of the following methods: slitting (cutting a roll into thinner rolls), monolithization (cutting into individual pieces by a cutter, rotary forging, or laser), reagent coating (reagent impregnation, spraying, or dispensing and drying), card lamination (applying a plastic backing to the roll), or combinations thereof. In one example, the LAMP reaction assembly can be manufactured using one or more of the following methods: slitting a roll into thinner rolls; cutting into individual pieces by a cutter or rotary forging; reagent coating using reagent impregnation; card lamination using a plastic backing applied to the roll, or combinations thereof. Materials for pH-based LAMP analysis in solid media

[0139] LAMP reactions can be challenging in solid-phase reaction media when hygroscopic agents and magnesium interfering agents are present. However, other factors can also interfere with the output and interpretation of LAMP results. When pH-based indicators are involved, the reagent mixture should be substantially free of interfering agents. In some cases, these interfering agents may include oxidants and pH-disrupting agents.

[0140] In one embodiment, a system for colorimetric ring-mediated isothermal amplification (LAMP) analysis may comprise a substantially non-reactive solid-phase reaction medium and a non-interfering reagent mixture. In one sample, the substantially non-reactive solid-phase reaction medium may be substantially free of oxidants and pH interfering agents. In one sample, the non-interfering reagent mixture may comprise one or more target primers, DNA polymerases, resolubilizing agents, or combinations thereof.

[0141] This oxidant interferes with the LAMP reaction. Therefore, the essentially non-reactive solid-phase reaction medium should not contain an oxidant. In one state, the oxidant may include, but is not limited to, one or more of the following: O₂, O₃, H₂O₂, F₂, Cl₂, halogens, HNO₃, nitrates, H₂SO₄, H₂S₂O₈, H₂SO₅, hypochlorite, chlorite, chlorate, perchlorate, chromium compounds, permanganate, sodium perborate, nitrous oxide, NO₂, N₂O₄, KNO₃, NaBiO₃, cerium compounds, lead dioxide, etc., or combinations thereof.

[0142] In some cases, simply avoiding the oxidant may not be sufficient to promote the LAMP reaction. In such cases, additional reagents may be included to prevent undesirable oxidation. In one state, the non-reactive solid-phase reaction medium may include one or more of the following: oxygen absorbers, desiccants, etc., or combinations thereof, to prevent oxidation of the non-reactive solid-phase reaction medium. In another state, to prevent oxidation of a non-reactive solid-phase reaction medium containing cellulose, the cellulose may be pretreated by thermal cycling to saturate the oxidation sites. In another example, an antioxidant may be added to prevent oxidation. In another state, a dye indicator (e.g., phenol red) may have antioxidant properties. In one state, the substantially non-reactive solid-phase reaction medium may contain less than one or more of the following oxidants: 1.0 wt%, 0.5 wt%, 0.1 wt%, or 0.01 wt%.

[0143] Besides oxidants, pH disruptors can hinder the correct interpretation of LAMP reaction results or further complicate the signal output of the LAMP reaction. In a single sample, pH disruptors may include, but are not limited to, one or more of the following: volatile agents, pH-affecting reagents, magnesium-containing reagents, or combinations thereof.

[0144] When reagents affecting pH are included, the resulting pH changes can complicate the analysis of pH-based results from LAMP reactions. In some cases, the pH-affecting reagents can be compensated for, and the analysis can be adjusted to allow for proper test interpretation. However, in other cases, pH-affecting reagents may make the interpretation of pH-based results ambiguous.

[0145] In one example, the substantially non-reactive solid-phase reaction medium may substantially be free of reagents that affect pH. In one sample, the reagents that affect pH may include acids, bases, or combinations thereof that interfere with pH sensitivity signals. In one sample, the substantially non-reactive solid-phase reaction medium may contain less than one or more of the following reagents that affect pH: 1.0 wt%, 0.5 wt%, 0.1 wt%, or 0.01 wt%.

[0146] When a volatile agent is included in the non-reactive solid-phase reaction medium, the volatile component may volatilize and leave behind components that can interfere with the pH-sensitive output signal or leave behind components that can further react to interfere with the pH-sensitive output signal. In one example, ammonium carbonate can form volatile ammonium ions and carbonate ions that can react to form carbonic acid. Carbonic acid may interfere with the pH-sensitive output signal by lowering the pH, even in the absence of a positive result from the LAMP reaction (e.g., the presence of a target pathogen and LAMP-induced amplification). Therefore, in one instance, the non-reactive solid-phase medium may be substantially free of volatile agents as defined herein.

[0147] When a magnesium-containing reagent is included in this non-reactive solid-phase reaction medium, it can interfere with the operation of DNA polymerase without close monitoring. Therefore, in one example, the substantially non-reactive solid-phase reaction medium may substantially be free of magnesium reagents as otherwise disclosed herein.

[0148] This substantially non-reactive solid-phase medium can be composed of various materials. In one state, the substantially non-reactive solid-phase reactive medium may include glass fiber, nylon, cellulose, polyurethane, polyetherurethane, cellulose acetate, nitrocellulose, hydrophilic PTFE, etc., or combinations thereof. In another state, the substantially non-reactive solid-phase reactive medium may be hydrophilic, absorbent, inert, and porous as disclosed herein.

[0149] The buffering capacity of the non-reactive solid-phase medium can affect the LAMP reaction. For example, strong buffers can prevent pH changes detected in the LAMP reaction. However, weak buffers can cause large pH fluctuations even in the absence of LAMP-induced amplification. In a single sample, the substantially non-reactive solid-phase reaction medium can have a buffering capacity from about 0.01 mM to about 5 mM. In one example, a detection limit of about 500 copies per 25 μl sample volume can be achieved by incorporating heat-deactivated virus into biobank saliva. However, the buffering capacity and pH of biobank saliva may differ from those of freshly collected saliva. Therefore, the detection limit (LOD) of freshly collected saliva can be adjusted based on the differences in buffering capacity and pH between biobank saliva and freshly collected saliva.

[0150] In some cases, technicians can interpret the pH-sensitive output signal from the non-reactive solid-phase reaction medium without specialized instruments. However, colorimetric readers offer a higher level of precision and accuracy. For example, in one instance, when combined with a pH-sensitive dye, the substantially non-reactive solid-phase reaction medium may have a maximum absorption wavelength (λmax) within the test range. In one example, when the pH-sensitive dye is phenol red, λmax is within the test range of approximately 443 nm to approximately 570 nm. Therefore, when the pH-sensitive dye is phenol red and the maximum absorption wavelength is within the detection range, technicians can use a colorimetric reader to detect positive or negative results.

[0151] In some samples, the system may further comprise components other than the non-reactive solid-phase reactive medium. In one sample, the system may further comprise an adhesive, a spreading layer, a spacer, a plastic carrier, or a combination thereof. In one sample, each of the adhesive, spreading layer, spacer, and plastic carrier may be substantially free of oxidants and pH-disrupting agents as disclosed herein.

[0152] In another embodiment, as shown in FIG2, a method 200 for maximizing the accuracy of the chromatic output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis may include: providing a solid-phase reaction medium, such as block 210, that minimizes color changes produced by non-LAMP reactions, and performing the LAMP analysis on the solid-phase reaction medium, as shown in block 220.

[0153] In one state, the method may further include controlling the non-LAMP-generated color change caused by protons produced by a non-LAMP reaction. In another state, the method may further include using a non-color-changing additive to control the non-LAMP-generated color change.

[0154] Color-changing additives prevent unwanted changes in colorimetric responses due to non-LAMP amplification. In a single sample, the color-changing additive may contain sugars, buffers, inhibitors, or combinations thereof.

[0155] In one example, the sugar may comprise one or more of trehalose, glucose, sucrose, dextran, or combinations thereof. In one sample, when used on the solid medium, the concentration of the sugar may range from about 0.01 mM to about 1 M. In another example, when used on the solid medium, the concentration of the sugar may range from about 10 mM to about 500 mM. In yet another example, when used on the solid medium, the concentration of the sugar may range from about 200 mM to about 400 mM.

[0156] In another example, the buffer may include phosphate-buffered saline (PBS), Dulbecco's PBS, Alsever's solution, Tris buffer (TBS), water, HEPES, BICINE, balanced salt solutions (BSS) such as Hank's BSS, Earle's BSS, Grey's BSS, Puck's BSS, Simm's BSS, Tyrode's BSS, BSS Plus, Ringer's lactate solution, physiological saline (i.e., 0.9% saline), 1 / 2 physiological saline, etc., or combinations thereof. In one instance, when used on a solid-phase medium, the concentration of the buffer may range from about 10 μM to about 20 mM. In another example, when used on a solid-phase medium, the concentration of the buffer may range from about 100 μM to about 10 mM. In yet another example, when used on a solid-phase medium, the concentration of the buffer may range from about 100 μM to about 500 μM.

[0157] In another example, the blocking agent may include one or more of bovine serum albumin, casein, or combinations thereof. In one embodiment, when used in a solid medium, the concentration of the blocking agent may be from about 0.01 wt% to about 5 wt%. In another example, when used in a solid medium, the concentration of the blocking agent may be from about 0.01 wt% to about 1 wt%. In yet another example, when used in a solid medium, the concentration of the blocking agent may be from about 0.02 wt% to about 0.06 wt%.

[0158] In another embodiment, a method for maximizing the detection level (LOD) in loop-mediated isothermal amplification (LAMP) analysis may include providing a reaction environment and reagents that minimize non-LAMP reaction products. In some examples, the reaction environment may be substantially free of one or more of the following: oxidants, pH interfering agents, hygroscopic agents, magnesium interfering agents, etc., or combinations thereof.

[0159] In another embodiment, a system for colorimetric ring-mediated isothermal amplification (LAMP) analysis may comprise a combination of a solid-phase reaction medium and a LAMP reagent, which, when stored at 25°C, maintains the color of the solid-phase reaction medium within 10% of its initial hue. In one state, the combination of the solid-phase medium and the LAMP reagent maintains color after storage for more than one or more of the following: 30 days, 90 days, 365 days, 2 years, 3 years, or 5 years. In another state, the combination of the solid-phase medium and the LAMP reagent maintains color when stored at a relative humidity between approximately 40% and 90%.

[0160] In another embodiment, a method for manufacturing a colorimetric LAMP system may include combining the 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 remain in contact.

[0161] In one example, the non-interfering reagent mixture can be kept in direct contact with the solid-phase reaction medium. In another example, the non-interfering reagent mixture can be kept in indirect contact with the solid-phase reaction medium. When the non-interfering reagent mixture is kept in indirect contact with the solid-phase reaction medium, the intermediate material (e.g., an antioxidant) can enhance the LAMP reaction.

[0162] In one embodiment, the method may 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 embodiment, the coating may include dripping, spraying, impregnating, soaking, or atomizing the solution onto the substantially non-reactive solid-phase reaction medium.

[0163] In some cases, the manufacturing process can affect the storage lifetime stability or uniformity of the LAMP analysis system. In one example, a roll-to-roll (R2R) method can be used to combine the non-interfering reagent mixture with the essentially non-reactive solid-phase reaction medium. Multiplex detection of selected targets using LAMP on a solid medium to test pathogens.

[0164] When using LAMP to detect pathogens on a solid-phase medium, there are several methods for multiplex detection of LAMP. First, the method can be multiplexed by including multiple control groups on the same solid-phase medium. For example, a positive control group can be used to verify the test's effectiveness. For instance, the LAMP reaction can test for salivary DNA or RNA biomarkers in a saliva sample to verify whether the LAMP reaction works as expected. In another example, a negative control group can be used to verify the test's effectiveness. For instance, the LAMP reaction can detect saliva samples that do not contain pathogens.

[0165] The second type of multiplex detection LAMP method involves including primers targeting multiple different pathogens on the same solid-phase medium. For example, the effectiveness of the test can be verified by testing viral and bacterial pathogens on the same solid-phase medium, thereby further characterizing biological samples.

[0166] A third approach to multiplex detection LAMP involves using different target primers to test the same pathogen. In one example, the first segment of the solid-phase medium can be used with a primer set to test for the first protein of the viral pathogen; the second segment of the solid-phase medium can be used with a second primer set to test for the second protein of the viral pathogen; and the third segment of the solid-phase medium can be used with a third primer set to test for the third protein of the viral pathogen. Each primer can target a different region of the viral pathogen genome to eliminate false negatives and false positives.

[0167] LAMP can be used for multiplex detection by including various components on a solid-phase LAMP reaction medium. In one embodiment, as shown in FIG3a, the solid-phase reaction medium 300a for performing LAMP analysis may include a substrate 302, an adhesive layer 304 disposed on the substrate 302, a reaction layer 306 disposed on the adhesive layer 304, which includes test points or reaction sites or fragments 305a, 305b, 305c and spacers 307a, 307b, 307c, and a spreading layer 308 disposed on the reaction layer 306. In one sample, the test points 305a, 305b and 305c may contain or otherwise hold reagents including one or more target primers, DNA polymerases, resolvants, etc. In one sample, the reagents can form a composition sufficient to carry out the LAMP reaction.

[0168] The spatially discontinuous reaction layer 306 can accommodate multiple control groups or multiple pathogen detection. For example, test point 305a can be a positive control group (e.g., testing for known saliva DNA or RNA biomarkers), test point 305b can be a negative control group (e.g., excluding colorimetric results testing of all reagents used in the LAMP reaction), and test point or reaction fragment 305c can test for the target pathogen.

[0169] Spatially discontinuous test sites or reaction locations 305a, 305b, and 305c also allow for multiple detection of various pathogens. For example, test site 305a can test for influenza, test site 305b can test for bacterial infections, and test site 305c can test for fungal infections.

[0170] The size of the reaction site or fragments 305a-305c may affect the multiplexing potential. In one sample, reaction fragments 305a-305c may have a thickness from about 0.05 mm to about 2 mm. In another sample, reaction fragments 305a-305c may have a width from about 4 mm to about 12 mm and a length from about 4 mm to about 25 mm. In one example, the reaction site 305a-305c may be spatially discontinuous. In another example, reaction fragments 305a-305c may have a surface area to thickness ratio from about 30 to about 600.

[0171] In one example, the solid-phase reaction medium 300a may be configured to receive a biofluid that can flow laterally across the spread layer 308 and migrate vertically downward to test points 305a-305c of the reaction layer 306. Test points 305a-305c may contain all components for the occurrence of RT-LAMP or LAMP reactions. In one example, test points 305a-305c may contain a redissolving agent (e.g., a surfactant), an enzyme (e.g., DNA polymerase, reverse transcriptase, DNase inhibitor, or RNase inhibitor), a stabilizer (e.g., an inhibitor, such as BSA or casein), a colorimetric indicator (e.g., a magnesium colorimetric indicator, a pH colorimetric indicator, or a DNA intercalation colorimetric indicator), and a buffer (e.g., 20 mM Tris).

[0172] The solid-phase reaction medium 300a can be configured to receive a reagent 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 introduce pH changes that may affect the readability of the results. Therefore, in some examples, the stabilizer can be casein, polysorbate 20, or a combination thereof.

[0173] The reaction fragments or points (e.g., test points) 305a-305c may comprise any suitable material disclosed herein. In one example, the reaction fragments 305a-305c may comprise one or more of glass fiber, nylon, cellulose, polyurethane, polyetherurethane, cellulose acetate, nitrocellulose, hydrophilic PTFE, etc., or combinations thereof. In one state, the pore size of the reaction fragments or points 305a-305c may range from about 1 to about 100 micrometers. The reaction fragments or points 305a-305c may be smooth and glossy in appearance.

[0174] In another configuration, reaction fragments 305a-305c provide a uniform final color in the readout area for accurate and precise signal output or detection. In one example, a biological sample can slowly migrate vertically downwards into reaction fragments 305a-305c. The final color intensity of reaction fragments 305a-305c can be measured by the user through optical observation and comparison with color charts or scales, or by using a handheld LED meter to measure percentage reflectance and converting it to copies per reaction of RNA or DNA using a set of curves calibrated against a laboratory reference instrument, or by obtaining an optical image of RGB values ​​or pixel counts, which can be calibrated against a laboratory reference instrument. The concentration of RNA or DNA can be determined by measuring the final color intensity or kinetic rate at a selected time.

[0175] In another state, the solid-phase LAMP reaction medium 300a may further comprise reaction fragments 305a-c. In one example, at least one segment may be substantially reagent-free (e.g., 305a may be substantially reagent-free). In another state, reaction fragments 305a-c may be defined by at least three discontinuous adhesive layer segments 304. The three reaction fragments 305a, 305b, and 305c may be specified for testing a pathogen, the same pathogen in multiple control groups, or different pathogens with or without specific positive or negative control groups. In short, reaction fragments 305a-c can be specified and configured for testing in virtually any array to provide any desired parameters or procedural criteria that produce specific results with a high degree of accuracy and reliability. Furthermore, the reaction layer 306 may be configured with a variety of numbers and arrangements of test points 305a-c. For example, the reaction layer may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other reasonable number of test points. Furthermore, such test points or reaction fragments can be arranged in a straight line, side by side, or almost any other desired spatial arrangement within the reaction layer 306.

[0176] In one state, as shown in Figure 3b, the solid-phase LAMP reaction medium 300b may contain reaction fragments 305a and 305b coupled between the binder layer 304 and the spread or distribution layer 308. In one state, at least one reaction fragment (e.g., 305a or 305b) may be a substantially reagent-free control segment or point. In another state, at least one of the reaction fragments (e.g., 305a or 305b) may be configured to test for a target pathogen (e.g., to contain or otherwise support reagents used for a selected LAMP reaction).

[0177] In another configuration, as shown in Figure 3c, the solid-phase LAMP reaction medium 300c may include a reaction layer 306 and reaction segments or points 305a, 305b, 305c, and 305d coupled between the binder layer 304 and the spreader layer 308. Each reaction segment may be separated from adjacent reaction segments or points by spacers (e.g., 307a, 307b, 307c, 307d, and 307e).

[0178] In another embodiment, as shown in Figure 3d, the solid-phase LAMP reaction medium 300d may include a substrate 302, an adhesive 304, and a reaction layer 306 having reaction segments or points 305a, 305b, and 305c, excluding the spread layer. In this example, the sample may be deposited individually or simultaneously on each segment of the reaction layers 305a, 305b, and 305c. Furthermore, in this embodiment, there are no spacers between adjacent reaction segments. However, in some embodiments, spacers may be included without the spread layer 308.

[0179] In yet another embodiment, as shown in Figure 3e, the solid-phase LAMP reaction medium 300e may comprise a substrate 302, an adhesive 304, and a reaction layer 306 having reaction segments or test points 305a and 305b, excluding a spread layer or any spacers. Similarly, in another embodiment, spacers may still be included, but a spread layer 308 may not be included. In this example, the sample may be deposited separately and / or collectively on each segment of the reaction layers 305a and 305b.

[0180] In yet another configuration, as shown in Figure 3f, the solid-phase reaction medium 300f suitable for LAMP analysis may include a substrate 302, an adhesive 304, and a reaction layer 306 having reaction segments 305a, 305b, 305c, and 305d, excluding a spread layer and without any spacers. Similarly, it should be understood that spacers may be included between adjacent reaction segments if desired, but without a spread layer. In this example, the sample may be deposited individually and / or collectively on each segment of the reaction layers 305a, 305b, 305c, and 305d.

[0181] It should also be understood that in other embodiments (not shown), a spread layer 308 may be present, having test points or reaction segments coupled between the spread layer and the adhesive 304, but without any spacers. In short, to provide the most accurate results, or to accommodate any manufacturing needs or obtain certain other benefits, the components of the solid-phase reaction medium may be selected according to the specific test to be performed.

[0182] In another embodiment, substrate 302 may be an optically transparent material. In another embodiment, substrate 302 may be an optically transparent plastic carrier. In another embodiment, adhesive layer 304 may be substantially free of volatile agents. In another embodiment, adhesive layer 304 may be substantially free of reagents that would interfere with the LAMP reaction. In another embodiment, adhesive layer 304 may be disposed continuously or discontinuously on the substrate.

[0183] The adhesive layer may comprise a variety of materials. In one example, the reaction layer 306 may comprise one or more of glass fiber, nylon, cellulose, polyurethane, polyetherurethane, cellulose acetate, nitrocellulose, hydrophilic PTFE, etc., or combinations thereof. In one example, the adhesive layer 304 may comprise an inert adhesive that does not affect the color change of the reaction fragments or test points in the reaction layer. In one example, the reaction layer 306 may be substantially free of chelating agents, magnesium, excessive buffering capacity, pH-affecting agents, or combinations thereof. In another example, the amount of adhesive may be minimized to prevent interference between the adhesive layer 304 and the reaction fragments 305a-c.

[0184] The spreading layer can be configured to enhance the distribution and uniformity of biological samples on reaction fragments 305a-305c. In another state, the spreading layer 308 can distribute saliva samples on the surface of the reaction layer 306 or measure saliva samples passing through the reaction layer. The spreading layer 308 can provide a uniform or substantially uniform concentration of saliva sample at 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 the same porosity, or an anisotropic layer with a porosity gradient, or combinations thereof. In one state, the anisotropic layer can have a pore size in the range of about 1 to about 100 micrometers. In one state, the spreading layer 308 can be hydrophilic enough to absorb and spread the sample. In another state, the hydrophilicity of the spreading layer 308 can be lower than that of the underlying reaction layer 306 to allow the sample to be extracted from the spreading layer 308 and enter the reaction layer 306.

[0185] When a homogeneous biological sample is provided, the precise permeability of the spreading layer 308 can be used to uniformly distribute the biological sample on the surface of the reaction layers 305a-c. In one state, the surface of the spreading layer 308 can be in direct contact with the reaction layers 305a-c to achieve uniform vertical transfer of the biological sample through lateral migration. In another state, the spreading layer 308 may comprise one or more of glass fiber, nylon, cellulose, polyurethane, polyetherurethane, cellulose acetate, nitrocellulose, polyester, hydrophilic polytetrafluoroethylene (PTFE), etc., or combinations thereof. In one example, the spreading layer 308 may be optically transparent. In one state, the material may be chemically treated to enhance the distribution of the biological sample on the multiple reaction segments 305a-305c.

[0186] Sufficient spacing between reaction segments or test points 305a-305c minimizes or eliminates the possibility of cross-contamination between reaction segments. For example, when the spacing between two segments allows reagent to flow from 305a to 305b, reaction segment 305a may contaminate adjacent segments, such as 305b. In another embodiment, the solid-phase LAMP reaction medium 300a may further include spacers 307a, 307b, 307c, and 307d, which can impose a minimum space of approximately 1.0 mm to approximately 3.0 mm (e.g., 2.5 mm) between reaction segments 305a-c.

[0187] In some embodiments, spacers 307a-307d may have a height greater than or equal to that of the reaction fragments or test points 305a-305d to create a physical barrier between them (e.g., when a sample is applied to each test point separately). In one example, the spacer height may be determined as a measurement of the distance between the substrate 302 and the top surface of the spacer. Similarly, the reaction fragment height may be determined as a measurement of the distance between the substrate 302 and the top surface of the reaction fragment. In some embodiments, the spacer may have a height that is 0 to 50% higher than the height of the reaction fragment.

[0188] In other embodiments, spacers 307a-307d may have a height less than or equal to that of the reaction fragments or test points 305a-305d to create a physical barrier between them (e.g., when a sample is applied to each test point separately). Similarly, the height of the reaction fragment may be determined as a measurement of the distance between the substrate 302 and the top surface of the reaction fragment. In some aspects, the spacer may have a height 0 to 50% lower than the height of the reaction fragment.

[0189] In one example, spacers 307a-d may include, but are not limited to, one or more of polyurethane, polyetherurethane, 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, glass fiber, nylon, cellulose, etc., or combinations thereof. In yet another example, spacers 307a-d may contain hydrophobic materials (e.g., polyurethane, polyetherurethane, cellulose acetate, nitrocellulose, polystyrene, polyester, hydrophilic polytetrafluoroethylene (PTFE), etc.), but not hydrophilic materials (glass fiber, nylon, cellulose, etc.). In one state, spacers 307a-d may be oriented in the same plane as and between reactive segments 305a-c.

[0190] In another example, as shown in Figure 3a, the solid-phase LAMP reaction medium may include a spreading layer 308 and a reaction layer 306 bonded by an adhesive layer 304. In one example, the material type of the spacers 307a-307d can enhance or otherwise positively influence the degree of spreading. For example, polystyrene spacers can produce a higher degree of spreading than other material types. At the expense of user convenience, a solid-phase reaction medium without a spreading layer 308 simplifies fabrication. In this case, the user can apply the saliva sample individually to each test point 305a-c. However, spacers made of materials that repel or minimally absorb the sample (such as polystyrene) can help the spreading layer, providing a uniform spreading result. Furthermore, spacers of such materials also facilitate sample spreading when the spreading layer 308 is not used.

[0191] Referring to Figure 4, a method 400 for testing the presence of a viral pathogen is shown, which may include providing a saliva sample from one of the subjects, as shown in block 410, and dispensing the sample into a test environment having a combination of a solid-phase reaction medium, a LAMP reagent mixture, and a pH-sensitive dye, as shown in block 420.

[0192] In one state, the method may include minimizing the amounts of volatile agents, hygroscopic agents, and non-pH-sensitive agents (capable of discoloring the solid-phase medium). In another state, the method may include providing one or more target primers, DNA polymerase, and redissolving agents in sufficient quantities to promote the LAMP reaction. In yet another state, the method may include providing a reverse transcriptase in sufficient quantities to promote the RT-LAMP reaction. In yet another state, the method may include providing one or more target primers in sufficient quantities to detect the viral pathogen. In another example, the method may include generating a test result within one hour of dispensing the sample to the test environment.

[0193] In yet another embodiment, as shown in FIG5, a method 500 for confirming whether a saliva sample is suitable for testing with a solid-phase LAMP reaction may include providing a solid-phase reaction medium having at least one test site or point, the at least one test site or point comprising a combination of a LAMP reagent and a pH-sensitive dye. In another embodiment, the method may further include providing a solid-phase reaction medium having at least one negative control site, the negative control site comprising a pH-sensitive dye but not the LAMP reagent, as shown in block 510. In another embodiment, the method may further include applying the saliva sample to the solid-phase reaction medium, as shown in block 520. In yet another embodiment, the method may further include confirming the activation of the pH-sensitive dye at the negative control site, as shown in block 530.

[0194] In one example, the pH-sensitive dye may be at least one of phenol red, phenolphthalein, litmus red, bromothymol blue, naphtholphthalein, cresol red, or combinations thereof. In another example, the LAMP reagent may be substantially free of volatile agents, pH-affecting agents, magnesium-containing agents, or combinations thereof. In yet another example, the LAMP reagent may contain non-interfering LAMP reagents, including DNA polymerase, reverse transcriptase, primers targeting specific regions, or combinations thereof.

[0195] In another embodiment, the method may further include providing a test location or point defined by at least two discontinuous adhesive layer segments. In yet another example, the method may further include providing a test location or point defined by at least three discontinuous adhesive layer segments.

[0196] In yet another embodiment, as shown in FIG6, a method 600 for maximizing the accuracy of a positive test result of a solid-phase LAMP reaction may include providing a solid-phase reaction medium having at least three test sites or points, each comprising a common combination of a pH-sensitive dye and a LAMP reagent, as shown in block 610. In one state, each site may include different primer sequences from a target pathogen. In another state, the method may include initiating a LAMP reaction, as shown in block 620. In yet another state, the method may include confirming a positive test result when at least two of the test sites or points activate the pH-sensitive dye and undergo a color change from a first color to a second color, as shown in block 630. In yet another state, the method may further include providing a sufficient amount of reverse transcriptase to promote the RT-LAMP reaction.

[0197] In one example, the pH-sensitive dye may be at least one of phenol red, phenolphthalein, litmus red, bromothymol blue, naphtholphthalein, cresol red, or a combination thereof. In another example, the LAMP reagent may be substantially free of volatile agents, pH-affecting agents, magnesium-containing agents, or a combination thereof.

[0198] In another sample, the target pathogen may include a viral pathogen, bacterial pathogen, fungal pathogen, or protozoan pathogen. In one sample, the target pathogen may include a viral pathogen. In another sample, the viral pathogen may include dsDNA virus, ssDNA virus, dsRNA virus, positive-strand ssRNA virus, negative-strand ssRNA virus, ssRNA-RT virus, or ds-DNA-RT virus. In another sample, the primer sequences may be paired with sequences from viral targets including H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.

[0199] In another state, the specific target nucleotide sequence to be detected may be a target nucleotide corresponding to a human biomarker. Any disease having a target nucleotide corresponding to a human biomarker of the disease to be detected can be detected. Various detectable disease types include one or more of the following: breast cancer, pancreatic cancer, colorectal cancer, ovarian cancer, gastrointestinal cancer, cervical cancer, lung cancer, bladder cancer, various epithelial cancers, salivary gland cancer, kidney cancer, liver cancer, lymphoma, leukemia, melanoma, prostate cancer, thyroid cancer, stomach cancer, etc., or combinations thereof. For example, biomarkers for various disease types can be detected by detecting one or more of the following target nucleotides: alpha fetal protein, CA15-3 and CA27-29, CA19-9, C125, calcitonin, calreticulin, carcinoembryonic antigen, CD34, CD99MIC 2, CD117, chromatin, chromosomes 3, 7, 17 and 9p21, cytokeratin, cesmin, epithelial membrane antigen, coagulation factor VIII, CD31 FL1, glial fibrillary acidic protein, macrocystic disease fluid protein, hPG80, HMB-45, human chorionic gonadotropin, immunoglobulins, inhibin, keratin, lymphocyte markers, MART-1, and Myo. D1, muscle-specific actin, neurofilaments, neuron-specific enolase, placental alkaline phosphatase, prostate-specific antigen, PTPRC, S100 protein, smooth muscle actin, synaptophysin, thymidine kinase, thyroglobulin, thyroid transcription factor-1, tumor M2-PK, vimentin, etc., or combinations thereof.

[0200] In one example, the positive control group may include a synthetic DNA target as one of the test segments to confirm that the DNA sample is stable. In another example, the negative control group may include: (a) an enzyme reagent without a primer, or (b) an enzyme reagent with a primer targeting a different virus.

[0201] In another example, three test segments can be used to enhance coverage of different viral strains (e.g., SARS-CoV-2). Detection time and limits can be selected for each primer. In one example, using the first primer achieves 97% viral (e.g., SARS-CoV-2) coverage. In another example, including two additional, slower and less sensitive primers compared to the first primer may not enhance viral coverage. In yet another example, multiple test segments can be used to achieve coverage of different viral pathogens (e.g., SARS-CoV-2, H1N1, H2N2, H3N2, H1N1pdm09, etc.). Solid-phase medium LAMP test procedure and method

[0202] LAMP assays on solid-phase media can be enhanced in several ways. First, the testing environment can be monitored to prevent changes that may affect the testing process. Second, the storage conditions of the biosample testing device affect its effectiveness. When these conditions are rigorously monitored, the biosample testing device can offer enhanced operability compared to liquid-based LAMP assays.

[0203] Various methods related to the testing environment can be used. In one embodiment, as shown in Figure 7, a method for testing the presence of a target nucleotide sequence may include: providing a biological sample, as shown in block 710, and dispensing the sample into a testing environment having a combination of a solid-phase reaction medium, a loop-mediated isothermal amplification (LAMP) reagent mixture, and a pH-sensitive dye, as shown in block 720. In one state, the method may include providing a sufficient amount of reverse transcriptase to facilitate the RT-LAMP reaction.

[0204] The type of biological sample can affect the testing environment. For example, saliva samples may have buffering properties, potentially reducing the contrast of the test output. In one example, the biological sample may be at least one of the following: saliva, mucus, blood, urine, sweat, exhaled breath condensate, or feces. In another example, the method may further include collecting the biological sample using one or more of the following: a saliva collection device, a nasal swab, a blood collection device, a urine collection device, a sweat collection device, an exhaled breath collection device, or a feces collection device.

[0205] The test environment can be controlled to provide consistent test outputs. In a single sample, the test environment may be substantially free of volatile agents, pH-affecting reagents, desiccants, or combinations thereof. Each of these reagents can potentially reduce the consistency of test results due to the introduction of variables (which may be compensated for through additional analysis).

[0206] Another environmental variable that can be monitored is the rate of temperature increase when heating the biological sample using a bioassay apparatus. In one case, the method may involve increasing the ambient temperature at a rate of approximately 0.1 °C / s. In one example, the ambient temperature may be increased at a rate of approximately 0.1 °C to approximately 0.2 °C / s. In one example, the reverse transcriptase may be activated at approximately 55 °C, while the DNA polymerase may be activated at approximately 65 °C. Therefore, a heating rate higher than approximately 0.2 °C / s can interfere with the coordinated action of reverse transcriptase and DNA polymerase in the LAMP reaction. In one example, the heating rate may be increased until the ambient temperature is in the range of approximately 60 °C to approximately 67 °C. In some cases, when the ambient temperature is increased to approximately 55 °C (i.e., the temperature at which reverse transcriptase can be activated), the bioassay apparatus may provide invalid results when increasing the temperature from 55 °C to approximately 65 °C at a rate of approximately 0.1 °C. Therefore, the heating rate should be monitored not only in the test environment temperature range of approximately 55°C to approximately 65°C, but also when the biological sample is heated to approximately 55°C.

[0207] Variations in the testing environment can also affect the results of biological sample testing devices. In a single sample, the method can include heating uniformity within the testing environment that provides a variation of less than 1°C. Spatial temperature variation around the test chamber should not exceed approximately 0.5°C to avoid interfering with the LAMP reaction.

[0208] In another embodiment, the method may include a saliva sample testing time of approximately 15 minutes to approximately 30 minutes. In another embodiment, the method may include a saliva sample testing time of approximately 30 minutes to approximately 45 minutes. In another embodiment, the method may include a saliva sample testing time of approximately 45 minutes to approximately 60 minutes. In another embodiment, the method may include a saliva sample testing time of approximately 60 minutes to approximately 90 minutes. In another embodiment, the method may include a nasopharyngeal sample testing time of approximately 20 minutes to approximately 30 minutes. In another embodiment, the method may include a nasopharyngeal sample testing time of approximately 30 minutes to approximately 40 minutes.

[0209] In another embodiment, the method may further comprise providing a solid-phase reaction medium comprising glass fiber, nylon, cellulose, polyurethane, polyetherurethane, cellulose acetate, nitrocellulose, hydrophilic PTFE, or combinations thereof. In yet another embodiment, the solid-phase reaction medium may comprise materials otherwise disclosed herein.

[0210] In one sample, the target nucleotide sequence may originate from at least one of a viral pathogen, bacterial pathogen, fungal pathogen, or protozoan pathogen. In another sample, the target nucleotide sequence may originate from a viral pathogen. In another sample, the viral pathogen may originate from the group consisting of: Coronaviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Adenoviridae, and Parvoviridae. In yet another sample, 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 sample, the target nucleotide sequence may originate from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pathogen.

[0211] The accuracy of this biological sample testing device may be affected by the length of storage time before testing and storage conditions (e.g., storage temperature, humidity, etc.). In one embodiment, a biological sample testing device may include a substrate that bonds a solid-phase reaction medium to a mixture of a dehydrated ring-mediated isothermal amplification (LAMP) reagent and a dehydrated pH-sensitive dye. In one state, the device provides at least about 95%, 96%, 97%, 98%, or 99% accuracy after being stored at a selected temperature (e.g., room temperature of about 25°C) for 6 months. In another state, the device provides at least about 95%, 96%, 97%, 98%, or 99% accuracy after being stored at a selected temperature for 12 months. In yet another state, the device provides at least about 95%, 96%, 97%, 98%, or 99% accuracy after being stored at a selected temperature for 2 years. In yet another state, the device provides at least about 95%, 96%, 97%, 98%, or 99% accuracy after being stored at a selected temperature for 3 years. In another case, the selected temperature can be any temperature in the range of approximately -20°C to approximately 37°C.

[0212] The biological sample testing system may include a housing. In yet another embodiment, the biological sample testing system may include a substrate bonded to a combination of a solid-phase reaction medium and a dehydrated ring-mediated isothermal amplification (LAMP) reagent mixture and a dehydrated pH-sensitive dye, the housing being operable to receive a biological sample. In one embodiment, 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 sufficient to produce a test result via the pH-sensitive dye.

[0213] In one embodiment, the substrate may comprise an optically transparent material. In another embodiment, the substrate may be bonded to the solid-phase reaction medium via an adhesive. In yet another embodiment, the adhesive may be substantially optically transparent. In yet another embodiment, the substrate may comprise a portion of a housing. In yet another embodiment, the biosample testing system may further comprise an adhesive layer disposed on the substrate; a reaction layer disposed on the adhesive layer; and a spread layer disposed on the reaction layer. In yet another embodiment, the biosample testing system may further comprise a spacer layer oriented in the same plane as the reaction layer. In yet another embodiment, the biosample testing system may further comprise a housing disposed against the substrate. In one example, the housing may further be disposed against the spread layer. In another example, the housing may substantially enclose the substrate, adhesive layer, reaction layer, and spread layer.

[0214] In one example, the operation of the biological testing system, as shown in Figure 8, may include: (a) collecting saliva using a sponge; (b) inserting the sponge into a collection tube; (c) diluting the collection tube with water to dilute the saliva sample to as low as 5% to stabilize the pH of the saliva and reduce its buffering capacity; (d) using a pipette to transfer the saliva sample onto a test strip in the test kit, thereby applying the saliva sample to a single location so that the capillary and mesh layer can spread the saliva sample onto individual segments of the reaction layer; (e) placing the cap on the test kit and locking the cap in place; (f) verifying that the pH of the saliva sample is within a test range by examining segments of the reaction layer; (g) inserting the test kit into a heater; and (h) verifying that the test kit is correctly positioned within the heater; (i) activating the heater; (j) reading the results approximately 30 minutes after the test is completed; (k) determining whether the result is valid; and (l) determining whether the result is positive or negative.

[0215] Procedure (a) can be performed by collecting saliva using a sponge collection device or a passive saliva collection device. When using a sponge collection device, the sponge is inserted into the collection tube to release saliva into the tube (procedure (b)). The collection tube can be diluted with water to reduce the buffering capacity of the saliva, decrease its viscosity, and improve sample homogeneity (procedure (c)). To transfer the saliva sample to the test strip, it can be applied individually to each segment of the test strip, or it can be applied to the center of the test strip and then spread out by the spreader to the entire test area of ​​the test strip (procedure (d)). The test strip can be covered to avoid contamination of the testing environment (procedure (e)). The pH of saliva samples can be tested using various methods, such as colorimetric instruments, fluorescence readers, or by simple comparison with the pH color chart of an in-use pH indicator (operation (f)). The test kit can be placed in a heater and started (operations (g), (h), and (i)) and the time recorded to measure the total reaction time and the time to reach a positive or negative result. The result can be read after 30 minutes (operation (j)) or immediately when a positive result is shown. The rate of positive result may be related to the concentration of the pathogen. The validity of the result can be determined based on the positive control group, negative control group, other pathogen results, or a combination thereof (operation (k)). When using a pH-based indicator, the result can be determined as positive or negative by comparing the color of the result with the color comparison chart of the pH-based indicator. The result can also be determined as positive or negative by measuring the wavelength of the absorbed color.

[0216] In another disclosed embodiment, a biological testing kit may include one or more of a saliva collection device, a collection tube, a test kit, a pipette, a heater, a colorimeter, or combinations thereof, as shown in Figures 8a and 8b. The saliva collection device may be a sponge collection device or a passive drooling device.

[0217] In one example, as shown in Operation 1 of Figure 8a, a user may collect saliva using one or more operations, including: (a) removing Pure-Sal™ from the pouch of the biotest kit, (b) placing the Pure-Sal™ sponge into the mouth to collect saliva until the indicator changes color or provides other indication that the saliva collection process is complete, (c) securing 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 (excluding some salivary degrading enzymes) into the compression tube, (f) closing the tube, and (g) mixing the tube by inverting it multiple times (e.g., 1 to 10 times).

[0218] After collecting and thoroughly mixing the saliva in the tube, the user can prepare the test kit as shown in operation 2 of Figure 8a. The user can (i) remove the test kit from the foil pouch, (ii) remove the protective sleeve from the test kit, and (c) place the test kit on a horizontal surface. The user can also transfer the sample as shown in operation 3 of Figure 8a, by filling the pipette to the black line with diluted saliva (e.g., diluted with water) and transferring it into the sample well. The user can also seal the test kit as shown in operation 4 of Figure 8a, by placing the protective sleeve on the test kit so that the fasteners are aligned and snapped together. The user can also heat the test kit as shown in operation 5 of Figure 8a, by placing the test kit in the auxiliary heater, closing the lid, and activating the auxiliary heater, which can be indicated by an LED when the reaction is complete.

[0219] After the reaction is complete, as shown in operation 6 of Figure 8a, the user can remove the test kit from the heater and compare it with the color chart, while ensuring that the orientation between the color chart and the test kit is correct. As further shown in Figure 8b, the color chart for the viral pathogen SARS-CoV-2 can contain 6 squares in 2 columns and 3 rows. The first row (-ve) indicates a negative result, the second row (+ve) indicates a positive result, and the third row (invalid test) indicates an invalid result.

[0220] For example, the first row could indicate a negative result because both the top and bottom squares are approximately reddish-orange, which can occur when no LAMP amplification of nucleotides has occurred in any of the squares. The second row could indicate a positive result because the top squares are approximately reddish-orange, which can occur when no LAMP amplification of nucleotides has occurred, but the bottom squares are approximately orange-yellow, which can occur when LAMP amplification of nucleotides has occurred, indicating the presence of a viral pathogen. The third row could indicate an invalid result because the top squares are approximately orange-yellow, which can occur when LAMP amplification of nucleotides has occurred, but the bottom squares are approximately orange-yellow, which can also occur when LAMP amplification of nucleotides has occurred. However, for the third row, the reason for the color change from reddish-orange to orange-yellow may be uncertain, as the negative control group (e.g., the top row) also showed a color change.

[0221] Therefore, users can compare the color chart with the tests in the test kit to determine the test results. The color chart shown in Figure 8b is an example. The color chart may include additional columns or rows to accommodate any number of tests for additional control groups, additional pathogens, or different primers for the same pathogen. Furthermore, the test can be performed by the subject themselves, or by a trained technician, nursing assistant, nurse, physician assistant, doctor, or any other person qualified to perform the test and interpret the results. [example]

[0222] The following examples are provided to facilitate a clearer understanding of certain embodiments of the invention and are in no way intended to limit it. Material assembly for paper-based LAMP reactions Example 1 – Paper LAMP Assembly

[0223] Regarding the paper-based LAMP assembly, several materials were screened to design a portable and compact assembly. For the development layer screening, two paper strips (one with a primer and one without) were placed together and saturated with 50 μL of RNA at a concentration of 0.2 ng / μL. Materials may not substantially affect the pH of the paper for several reasons: (a) the paper is too acidic to change color before incubation, or (b) the paper is too alkaline, which would prevent any color change in the reaction. Further testing using other equipment components was conducted to ensure materials prevented from interfering with each other between the two paper strips.

[0224] The paper LAMP assembly measures approximately 24 x 54 mm. The paper LAMP assembly comprises: (i) a readout layer, (ii) two reaction strips, and (iii) a developing layer. The readout layer includes a transparent 3 mm Melinex® backing for support. The Melinex® backing is attached to the two reaction strips, each formed from 5 mm x 20 mm chromatography paper (e.g., Whatman® Grade 1 chromatography paper) in contact with a double-sided adhesive (ArClean® 90178). The two test strips are separated by 2.5 x 20 mm 10 mm polystyrene spacers to prevent interference between the two test strips. The developing layer comprises a polyester mesh (Saaticare® PES 105 / 52). The sample is loaded onto the developing layer. Figure 9 shows an example of the assembly. Example 2 – Material Screening

[0225] The selection of paper material was based on five criteria: (a) the stability of the formulation when dried on the substrate, (b) the intensity of the color change upon sample rehydration, (c) the ability of the sample to be uniformly wicked throughout the paper substrate, (d) the ability of the material to remain inert throughout the reaction, and (e) the ability of the paper material to exhibit a color change during amplification. Whatman® Grade 1 chromatography paper was tested and used for optimization. When two test strips were assembled together, non-uniform fluid distribution was observed because the LAMP reaction used a large area of ​​Grade 1 chromatography. Selecting chromatography paper thicker than Grade 1 (e.g., Ahlstron 222 Grade 1 chromatography paper) allowed for a test strip surface area of ​​approximately 5 mm x 6 mm, while carrying the same amount of sample and reagents as Grade 1 chromatography paper, allowing for more uniform development during rehydration. Example 3 – Material Screening – Grade 1 and Grade 222 Chromatography Papers

[0226] In one example, as shown in Figure 10, these images illustrate examples of color contrasts that can be generated. In one example, Grade 1 chromatography paper was used. In another example, Grade 222 chromatography paper was used. In both cases, the RT-LAMP reagent was dried and rehydrated with 25 μL of 5% saliva and 95% water. Positive samples contained heat-deactivated SARS-CoV-2 incorporated at 10 k copies per reaction, and negative samples were 5% saliva (virus-free) and 95% water. Images were taken at time points after 90 minutes of incubation at 65°C.

[0227] As shown in Figure 10, the color contrast between negative and positive results on Grade 1 paper is not as pronounced as the color contrast between negative and positive results on Grade 222 paper. Therefore, thicker paper produces enhanced color contrast. Example 4 – Paper Assembly Procedure Guidelines

[0228] The paper assembly procedure may include: (1) cutting polyethylene terephthalate (PET) into rectangles of 80 mm x 7 mm, creating 2 for each device; (2) cutting chromatography paper into rectangles of 33 mm x 5 mm, creating 1 for each device; (3) cutting out a large piece of double-sided tape and attaching the PET rectangles to it, leaving a 2 cm gap between each PET rectangle; (4) cutting off all the rectangles, leaving excess tape around the edges; (5) peeling off the tape with PET on it and attaching the chromatography paper to the reverse side of the PET, leaving about 1 cm outside the tape; (6) using a single point near the inside of the device to add 25 μl of sample onto the chromatography paper; (7) attaching other PET strips to cover the sample (i.e., the tape side should contact the sample, and the PET side should be on the outside); (8) folding any tape stuck to the PET tape for further sealing; (9) adding 1 μl of PET to the bottom of a 15 ml tube. (10) Place the assembled paper apparatus into the tube so that the extended chromatography paper is in the solvent; (11) Close the tube; and (12) Incubate at 65°C for 1 hour. Materials for pH-based paper-based LAMP analysis Example 5 – Limit of Detection (LoD)

[0229] The LoD study used primer sets targeting various regions of SARS-CoV-2, and serially diluted 2-fold with heat-deactivated 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 a color change from red to yellow was observed in 4 / 4 replicates, thus confirming amplification. Example 6 – Limit of Detection (LoD)

[0230] The level of oscillation (LOD) for liquid-based LAMP reactions can be approximately 20 viral copies per μL sample volume. For solid-phase reaction media (e.g., chromatography paper), each reaction zone can hold approximately 25 μL of sample. Example 7 – The Effectiveness of LoD

[0231] After determining that the LoD in saliva was approximately 20 copies / μL, samples with the following relevant concentrations were prepared: (1) 20 copies / μL (1x LoD – 10 samples), (2) 40 copies / μL (2x 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 mixed saliva (30 aliquots). Results were also confirmed using image processing. Example 8 – LoD, Sensitivity, and Specificity

[0232] Multiple serial dilutions of heat-deactivated SARS-CoV-2 were performed in water (ranging from approximately 100 copies / reaction to 10⁵ copies / reaction). These serial dilutions served as templates for the liquid reaction, establishing a baseline LoD for potential primer candidates. Each viral concentration was reacted in triplicate on a white qPCR plate (e.g., Thermo Scientific® AB-0800W) and heated to 65°C for 60 minutes in a standard 75L bioincubator (e.g., Fisherbrand® Isotemp Microbiological Indicator, 15-103-0513). The color of the reaction mixture at different time points was recorded by scanning the plate with a desktop scanner (Epson® Perfection V800 Photo Color). The LoD of the primer set was determined by the lowest viral concentration that resulted in a strong color change in all three replicates. Any candidate capable of providing amplification at 10³ copies / reaction or less was then treated with the same viral dilution procedure (e.g., 2-fold dilution) and incorporated into a mixture of healthy saliva to examine matrix interference. Saliva LoD studies were then performed on a paper device to examine primer compatibility on a paper substrate.

[0233] As shown in Figure 11, the reaction on the right is carried out in a liquid environment, using a combination of heat-deactivated SARS-CoV-2 incorporated into water, and a colorimetric mixture with a fluorescent dye. The reaction volume is 25 μL. The black line represents the color change measured as an absorbance ratio (OD 430 / OD 560), and the light blue line represents the fluorescence change measured as a fluorescence intensity in units of 10³. Detection time may be faster when used with the built-in color / fluorescence reader.

[0234] For a viral concentration of approximately 1000 copies / reaction, the absorbance ratio and fluorescence activity peaked after approximately 17 minutes. For a viral concentration of approximately 500 copies / reaction, the absorbance ratio and fluorescence activity peaked after approximately 18 minutes. For a viral concentration of approximately 250 copies / reaction, the absorbance ratio and fluorescence activity peaked after approximately 19 minutes. For a viral concentration of approximately 125 copies / reaction, the absorbance ratio and fluorescence activity peaked after approximately 18 minutes. For a viral concentration of approximately 62.5 copies / reaction, the absorbance ratio and fluorescence activity peaked after approximately 17 minutes. For a viral concentration of approximately 31.25 copies / reaction, the absorbance ratio and fluorescence activity peaked after approximately 22 minutes. For the control group without a sample, the absorbance ratio and fluorescence activity did not peak as expected.

[0235] Sensitivity and specificity were determined using 30 human positive samples at 30 different LoD folds (1x, 2x, 4x, 40x, and 400x, repeated 10, 10, 4, 3, and 3 times, respectively) and 30 negative saliva samples from a plateless control group (NTC). The average green channel intensity of each reaction zone was captured using ImageJ to determine the colorimetric response intensity. Receiver operating characteristic (ROC) curves were generated by varying the threshold cutoff values ​​between positive and negative responses and calculating the sensitivity and specificity for each threshold. Sensitivity was calculated as the ratio of true positives to the total number of positives (including false positives). Specificity was calculated as the ratio of true negatives to the total number of negatives (including false negatives). Using paper LAMP for multiple target selection to test viral pathogens. Example 9 – Paper Slip Format

[0236] Figure 12 shows the strip format. The black line represents SAATICARE Hyphyl® polyester PES 105 / 52, with a thickness of 0.063 mm and a length of 50 mm. The red line represents Tekra Clear MELINEX® 454 polyester PET, with a thickness of 0.0762 mm and a length of 50 mm. The green line represents Adhesives Research ARclean® 90178 (AS-144), with a thickness of 0.038 mm and a length of 50 mm. The orange block represents Tekra Double White Opaque High Impact Polystyrene Litho Grade, with a thickness of 0.508 mm and a length of 2.5 mm. The blue block represents Ahistrom-Munksjö Cellulose Grade 222, with a thickness of 0.83 mm and a length of 5 mm. In this example, the strip has 5 spacers (orange) and 4 reactive layer sections (blue), as well as a spread layer (black), an adhesive layer (green), and a substrate (red). Example 10 – RT-LAMP Process

[0237] As shown in Figure 13, the solid-phase LAMP reaction medium may include: a 6 mm x 50 mm transparent 3 mm Melinex® 454 and Arcean® 90178; a reaction layer containing 5 mm Ahlstrom 222 reagent material (including 4 pink discontinuous reaction layer segments); spacers containing 2.5 mm 20 mil polystyrene (including 5 discontinuous spacers); a developing layer containing Saaticare® PES 105 / 52 hyphyl; and a substrate containing capillaries made of 14 mm Melinex, Arcean® 90178, and 3M 9962.

[0238] As shown in Figure 14, sufficient space between each reaction section or test area can prevent cross-contamination between reaction zones. In this example, the four orange reaction sections do not exhibit cross-contamination because the five spacers do not change color. Example 11-A – Paper Contrast

[0239] In another example, as shown in Figure 15, Grade 222 chromatography paper provides greater contrast than Grade 1 chromatography paper. The positive results (left side) of the Grade 222 paper show a greater contrast between the positive result (yellow-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 strip was tested with approximately 500 μM buffer solution, with a pH range from 8.0 for negative tests to 7.5 for positive tests. Example 11-B – The effect of phenol red concentration on paper

[0240] To distinguish between negative and positive results without inhibiting the reaction itself, phenol red concentrations were tested on Grade 1 and Grade 222 chromatography papers. For both types of papers, 250 μM phenol red per reaction showed consistent results. Lower dye concentrations showed a relatively pale and whitish color after 60 minutes of incubation, while for higher concentrations of phenol red, longer incubation times were used to differentiate between positive and negative results. Example 11-C – Effects of initial pH and drying on the response of paper-based colorimetric RT-LAMP using phenol red

[0241] Figure 15B illustrates the RT-LAMP reaction mixture under different initial pH conditions during drying. In this example, pH 7.6 is the unadjusted pH of the RT-LAMP reaction mixture. A wet configuration means adding 5 μL of synthetic RNA (N gene, 0.2 ng / μL, "+") or water ("-") immediately after adding 20 μL of the LAMP master mixture. A dry configuration means drying the strip at room temperature for 30 minutes after applying 20 μL of the LAMP master mixture, then rehydrating it with 25 μL of synthetic RNA ('+') or water ('-'). The LAMP reaction consisted of 12.5 μL of NEB 2x colorimetric master mixture, 2.5 μL of primer mixture, and 5 μL of phenol red, prepared in nuclease-free water (1 mM). The pH of the resulting mixture was adjusted with KOH. Grade 1 chromatography paper was used. The reaction was heated in an incubator set to 65°C for 120 minutes, and scanned using a flatbed scanner at time points of 45, 60, 90, and 120 minutes. Example 12 – Test Bar Format

[0242] As shown in Figure 16, the test strip format has a sample application side and a reading side (upper right of the figure). When a developing layer is present, the sample can be applied to a position on the sample application side. In other examples, without a developing layer, the sample can be applied to the sample application layer above the sections (orange) of the reaction layer. The reading side can be read without the need for specialized instruments, or using a colorimetric detector or a fluorescence detector.

[0243] As further shown in Figure 16, the test strip format also has four test reaction areas, an absorption / spreading layer, and a transparent single-sided adhesive that will conform to the shape and contact the absorption layer. Example 13 – Test Strip Assembly Process

[0244] Figure 17 illustrates the test strip assembly process, including cutting, where the reaction layer is coated with a relatively wide layer and cut to 5 mm, and then placed on a roll for lamination. The process also includes a first lamination step of laminating the reaction layer onto a clear single-sided adhesive. The process further includes a second lamination step performed online directly after the first lamination step, where a spread layer is laminated onto the reaction layer and the adhesive layer. This test strip assembly process may include materials such as: a polyurethane material coated with a pH reagent, a single-sided adhesive, and Grade 1 chromatography paper. Example 14 – RT-LAMP Process

[0245] A mixture of saliva and reagents was heated at 65°C to extract, reverse transcribe, and amplify SARS-CoV-2 viral RNA from the saliva in a one-pot mixture. Four primer sets were used for LAMP: 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 a final set targeting the human RNase P (RP) gene, serving as an on-board control. Each primer set consisted of six individual primers targeting specific regions of viral or human RNA that would be reverse transcribed and amplified during isothermal incubation using reverse transcriptase and strand displacement polymerase. Example 15 – Positive Control Group

[0246] The positive control reaction is included on the testing device as one of the three test strips used in conjunction with the viral RNA test strip. The positive control also serves as a positive sample control and an extraction control. If the control area of ​​the test strip shows a red-to-yellow color change, this indicates that the viral RNA, if present in the sample, has been successfully extracted from the virus, and that the reagents in the test have produced the expected amplified signal. If no color change occurs, the test should be considered invalid and repeated.

[0247] The positive control group was tested for human RNase P, a marker commonly found in human clinical specimens and a standard control in many RT-PCR kits. Amplification of this marker indicates successful lysis of human cells under these test conditions and can also be inferred as viral lysis.

[0248] The scientific basis for the control group is as follows. Unlike RT-PCR-based assays, our assay does not use chemical methods to extract viral RNA; heat treatment is sufficient. The polymerases in RT-PCR are sensitive to reaction inhibitors in biological sample matrices. Therefore, these assays typically involve RNA extraction and purification. Conversely, the Bst 2.0 polymerase used in the LAMP assay is highly stable in biological matrices, thus eliminating the need for extraction and purification. A temperature of 65°C is sufficient for the instrument to lyse viral particles, expose viral RNA, and support robust amplification. Example 16 – Negative Control Group

[0249] A negative, sample-free control test is performed using a test kit containing a blank test solution instead of a saliva sample. The blank test solution is a sterile pH buffer solution containing no RNA or DNA sample. If a color change is observed in this test, it indicates a possible false positive, and therefore any positive results obtained since the last control group run are invalid. Therefore, instructions to users are to perform this negative control group upon receiving a test kit using this representative test kit. This confirmation can also be repeated at defined time intervals.

[0250] The most likely cause of false positives in the negative control group is the retention effect of amplified DNA products from consecutive tests. The negative control group test is the last of a series of control measures, including manufacturing controls of test kits with strict sealing tolerances and routine disinfection and cleaning of the heater unit by the operator, to prevent false positives due to retention contamination. Example 17 – Internal Control Group

[0251] This testing assembly employs a unique control designed for use with saliva as the test matrix. Each test strip includes a pH indicator dye as an internal control to verify that the initial pH of the saliva sample is within the expected range. If the color changes from red to yellow on all four test strips upon sample application (before heating), the user should consider the sample invalid and discontinue testing. External factors such as eating, drinking, or using oral hygiene products before sampling can affect the initial pH of the sample. In the event of pH indicator failure, if the operator determines that one of these factors has affected the sample, the test can be repeated after 5 minutes to allow the patient's saliva pH to return to normal. Other external factors (e.g., certain diseases) can also systematically affect saliva pH; in such cases, alternative testing methods are required. Example 18 – Result Confirmation

[0252] The paper LAMP test is a qualitative test. It is based on visual color perception, which can be read by the operator using a color chart. A positive result produces a yellow color. All control groups should be examined before interpreting patient results. If the control groups are invalid, the test is invalid and patient results cannot be interpreted.

[0253] Because this is an isothermal RNA amplification, the presence of target SARS-CoV-2 RNA at the locus defined by the LoD assay will produce a positive result. In some cases, two positive results in the primer regions of the three target genes (Orf1ab, E gene, or RdRp gene) can confirm a positive test. The color of the test strip can be compared to the provided color interpretation table to confirm a positive result. A valid test requires the RNase P region of the test strip to turn yellow. If this region does not turn yellow, the test is invalid and should be repeated.

[0254] The first indicator of a reliable and legitimate test strip is that all four test strip areas do not turn yellow after saliva is applied and before the strip is heated. If this occurs, it may be due to recent food or fluid intake causing the saliva pH to be outside the acceptable range. The patient should rinse their mouth with water, wait at least 5 minutes, and then collect a new saliva sample.

[0255] Once the test reaction is completed in the heater unit, the on-board control group should show a positive yellow change in the RNase P area. Once this is confirmed, the color change of each bar area should be checked according to the provided color interpretation chart. Each bar area is categorized as positive (e.g., yellow change) or negative (e.g., pink). In one example, a positive result for both test areas indicates a confirmed diagnosis of SARS-CoV-2. Example 19 – Sources of False Positives

[0256] False positives are common in RT-LAMP analyses if appropriate measures are not taken to address them during the testing process. These false positives can originate from DNA aerosols formed during previous RT-LAMP reactions, which can linger in the environment and contaminate future experiments. These aerosols can contaminate individual reagents during reaction preparation or during transport, handling, sample loading, or incubation of the reaction mixture. Besides reducing the accuracy of RT-LAMP-related tests, aerosols can also introduce noise into experiments using specific sample concentrations (e.g., LoD studies). To address these various contamination points, all RT-LAMP-related procedures can be divided into several stages performed at different locations (i.e., reaction preparation, transport, loading / sealing, amplification incubation, and gel electrophoresis). By spatially separating these operations within the procedure, the possibility of introducing aerosols before reaction sealing can be minimized, or errors can be eliminated if false positives occur. Example 20 – Selection of discs, caps, sealants and tubes

[0257] Given the susceptibility of RT-LAMP to contamination and false positives, a comprehensive screening of 96-well qPCR discs, sealing methods, and PCR tubes was conducted. First, we screened Thermo Scientific® 96-well full-side edge PCR discs, specifically the clear (ThermoScientific® AB-0800) and white (AB-0800W) discs, as well as the FrameStar® 96-well full-side edge optical disc (Brooks Life Sciences® 4TI-0970). Clear discs are primarily used to aid in colorimetric analysis scanning. Among these clear discs, the white Thermo Scientific® 96-well full-side edge PCR disc demonstrated the best performance based on the average number of false positives.

[0258] Figure 22A shows colorimetric RT-LAMP scan images for the limit of detection (LoD) of orf1ab.II. The scan titles correspond to the catalog numbers of different disk types used to run the colorimetric LoD. Yellow wells indicate successful LAMP reactions, while red / orange wells indicate no or low-level amplification, respectively. 5 μL of heat-deactivated virus dilution at the labeled concentration was added to 20 μL of the reaction mixture. The master mixture consisted of 12.5 μL of NEB colorimetric 2x master mixture, 2.5 μL of primer mixture, and 5 μL of water. The endpoint image was captured after heating in an incubator set to 65°C for 60 minutes. Each primer set was repeated three times for each viral concentration.

[0259] For sealing methods, we investigated the following products: MicroAmp® Optical 8-tube cap strip (Thermo Fisher® 43-230-32), Thermo Scientific VersiCap Mat Cap Strip (Thermo Fisher® AB1820), Thermo Scientific Adhesive Plate Seals (Thermo Fisher® AB-0558), and MicroAmp Optical Adhesive Seal (Thermo Fisher® 43-119-71). Among these caps, the VersiCap Mat Cap Strip showed the best sealing performance when observing false positive rates; however, for colorimetric scanning, it was difficult to obtain an accurate image of the reaction process because the cap was not completely transparent. Therefore, adhesive seals were tested, and their performance was comparable after comparison. Furthermore, when pressure was applied simultaneously and uniformly on the disc, the adhesive seal performed better than the VersiCaps based on the false positive rate.

[0260] Figure 22B shows a colorimetric RT-LAMP scan image for the limit of detection (LoD) of orf1ab.II. The scan title corresponds to the catalog number of the different cap types used to run the colorimetric LoD. Yellow wells indicate a successful LAMP reaction, while red / orange wells indicate no or low-level amplification, respectively. 5 μL of heat-deactivated virus dilution in water was added to 20 μL of the reaction mixture to produce the final labeled concentration (positive reaction) or nuclease-free water (negative reaction). The master mixture consisted of 12.5 μL of NEB colorimetric 2x master mixture, 2.5 μL of primer mixture, and 5 μL of water. The plate was incubated at 65°C for 60 minutes, and the endpoint image was taken afterward. Each primer set was repeated three times for each viral concentration.

[0261] As alternatives to discs during imaging, we investigated the following PCR tube products: MicroAmp™ Optical 8-Tube Strip with optical cap (Thermo Fisher® A30588) and MicroAmp™ Optical 8-Tube Strip (Thermo Fisher® 4316567), using the same caps as those used for sealing discs. The tubes with optical caps performed best in terms of false positive rates.

[0262] A fully assembled quadruple test strip may include a spreading net, adhesive, and a transparent Melinexbacker. The test strip may also include a DNA sample in water at a concentration of 0.2 ng / μL, corresponding to approximately 10⁸ copies / μL. The sample volume can be 100 μL. The N gene primer can be used under alternating primer and primer-free conditions. The adhesive may have a buffering effect that inhibits the color response; therefore, the formulation should be compensated for. Paper LAMP Test Procedure and Method Example 21-A – Paper LAMP Testing Procedure

[0263] The paper LAMP test is designed for simple, on-site care use by trained healthcare professionals. The entire testing process is illustrated in Figure 18. The patient collects a saliva sample under the guidance of a healthcare professional. The saliva sample is collected in a specialized collection container, which is free of any additives and therefore safe for the patient to use during collection. The saliva volume is approximately 100 μL, which is easily collected by the patient. The test strip containing the specific RNA detection region described above is completely contained within a plastic strip cartridge. After collecting the saliva sample, the patient hands the collection container to the operator. The operator then applies the saliva sample to the indicated location on the test strip and closes the test cartridge casing. The test cartridge with the sample-containing strip is then placed in a heating device designed to securely hold the test cartridge in place to achieve the required heating to 65°C. Paper LAMP analysis is performed on the test strip at an isothermal temperature. During the LAMP reaction, nucleic acids in the saliva sample are identified, and the pH change caused by nucleic acid amplification at 65°C results in a color change on the strip.

[0264] The heater assembly is designed to provide uniform heating to the test strips. The heater assembly may feature (red-yellow-green) colored LEDs to indicate the heating progress to the user based on the incubation time and to alert the operator at the end of the test. During testing, the heater assembly may include a feature, such as a magnetic or mechanical lock, to ensure the test is not interrupted. The test reaction can be conducted at a constant temperature of 65°C for approximately 15 to 30 minutes. At the end of the analysis, the test carrier containing the test strips can be removed from the heater and visually read. Example 21-B – Paper LAMP Testing Procedure

[0265] Figure 18B illustrates the fabrication and use of a paper-based colorimetric molecular assay for SARS-CoV-2. The fabrication of the SARS-CoV-2 paper-based colorimetric molecular assay includes: (1a) creating a master mixture, (1b) transferring the mixture to a pad, (1c) quality checks, and (1d) air-drying the test apparatus. The use of the SARS-CoV-2 paper-based colorimetric molecular assay includes: (2a) sample collection, (2b) resuspending the sample in water, (2c) adding the sample to the pad, (2d) incubating at 65°C for approximately 60 minutes, and (2e) interpreting the results. Example 21-C – Paper LAMP Testing Procedure

[0266] The analytical workflow is shown in Figure 19A: saliva is collected, the sample is transferred to the paper apparatus, incubated at 65°C, and the results are read; a typical result is shown in Figure 19D. Figure 19C provides a schematic diagram of our paper apparatus structure. The paper apparatus comprises several 222-grade cellulose reaction pads separated by 20-mil polystyrene spacers to prevent cross-contamination between reaction zones. These components are attached to a transparent backing for structural support via double-sided adhesive. During manufacturing, the reagents for RT-LAMP are dried onto the reaction pads. These reagents are rehydrated when the user adds a sample to the reaction zone.

[0267] Figure 19 shows a schematic diagram of the paper apparatus and colorimetric characterization analysis. Figure 19A provides a schematic diagram of the workflow for using the apparatus. The control area represents the primer-free control group. Figure 19B provides a paper-based colorimetric LoD using heat-deactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at a specified concentration in 5% saliva. The negative replicate is an RT-LAMP reaction using nuclease-free water instead of heat-deactivated SARS-CoV-2. Data are taken from Figure 21A.

[0268] Figure 19C provides a schematic layout of the paper apparatus. Figure 19D provides typical colorimetric results for negative and positive runs. The control group is an RT-LAMP reaction without a LAMP primer. The positive reaction has 800 copies / μL incorporated into 5% saliva. Figure 19E provides the color gradient of possible results derived from the colorimetric results in cell B. Figure 19F provides a summary table of observations used to calculate the analytical sensitivity and specificity of the paper apparatus based on survey responses.

[0269] Each step of this analysis (Figure 19A) is designed to reduce complexity and mitigate user error in a place-based care setting. The sample collection step uses a sponge-based collection device that allows patients to collect the sample themselves, remove microparticles from the collected saliva, and minimize patient-to-patient outcome variability. The transfer step involves placing 25 μL of diluted saliva onto each of the two reaction zones on the device. For the incubation step, the paper device containing the sample is sealed in a resealable plastic bag and then placed in an incubator set at 65°C for 60 minutes. Finally, in the reading step, the user compares the colors of the control and reaction zones with the color bands in Figure 19C to determine the validity of the results and the presence of the pathogen of interest.

[0270] The platform comprises three main components: a primer set that adds specificity to the analysis, a paper apparatus containing two reaction zones (a control zone and a reaction zone), and a heat source for heating the paper apparatus to the reaction temperature. The primer set determines the pathogen targeted by the analysis. Therefore, the platform can be reconfigured to target different pathogens by redesigning the primer set, while keeping all other aspects of the apparatus and formulation the same. Furthermore, the paper apparatus can be configured to accommodate multiple reaction zones, allowing for the simultaneous detection of multiple targets.

[0271] The direct detection of SARS-CoV-2 in saliva is demonstrated through different colorimetric responses that can be read with the naked eye (Figure 19). This format is suitable for roll-to-roll manufacturing, with an estimated cost of approximately $10 per test. The limit of detection (LoD) of this test is 200 copies / μL of saliva. When measured using human samples (freshly collected saliva mixed with heat-deactivated SARS-CoV-2), the analytical sensitivity (positive predictive value) was 76%, the specificity (negative predictive value) was 100%, and the accuracy was approximately 91% (Figure 19F). Due to the subjectivity of color perception of the control pad, subjects incorrectly identified 20 out of 80 invalid devices, resulting in a false invalidity rate of 25%. When image processing was used to quantify color changes, the sensitivity increased to 97% and the accuracy to 98% (Figure 19C).

[0272] The device (Figure 19C) has dimensions of 6 mm x 20 mm and includes: a readout layer, two reaction strips, and spacers to prevent cross-contamination. The readout area includes an optically clear 3-mil MELINEX (Tekra MELINEX® 454 polyester (PET)) backing for support. It is attached to two 5 mm x 6 mm reaction strips (Ahlstrom-Munksjö Grade 222) using double-sided adhesive (Adhesives Research Acid-Free ARclean® 90178). These strips are separated by 2.5 x 6 mm 20-mil polystyrene spacers (Tekra Double White Opaque High Impact Polystyrene (HIPS) Litho Grade). 25 μL of sample is added during rehydration to saturate the strips.

[0273] A color band was created by averaging the RGB values ​​of phenol red on 222-stage chromatography paper over a range of pH. A linear gradient was then created using these averaged RGB values, and the optimal threshold determined from the ROC curve was annotated on this color band (Figure 19C).

[0274] After loading the sample onto the paper apparatus, the apparatus was placed in a 1" x 1" resealable plastic bag to prevent contamination during the RT-LAMP reaction. The plastic bag containing the paper apparatus was then placed in a 65°C incubator for 60 minutes. The bag was removed, scanned with a flatbed scanner (Fig. 19D), and compared with a colorimetric table (Fig. 19E) created by averaging the RGB values ​​of the phenol red response on a 222-level pad in buffer solutions with known pH ranges from 6 to 9 in the form of a known standard. The thresholds in the colorimetric table correspond to the thresholds determined from ROC analysis (Fig. 19B).

[0275] After determining the LoD in saliva to be 200 copies / μL (Fig. 19B), artificial samples with LoDs of 1x, 2x, 4x, 40x, and 400x were created. Thirty freshly collected saliva samples were used as negative samples (Fig. 21A). The results were quantified using image processing (Fig. 19A and Fig. 21C). A two-tailed t-test revealed a significant difference in the green channel intensity between the negative and positive colorimetric reaction pads (p < 0.001).

[0276] The image analysis showed 100% specificity, 97% sensitivity, and 98% accuracy (Figure 20C). Figure 20 shows the digital analysis of the colorimetric responses on paper. Figure 20A shows a box plot of the green channel intensity for 30 positive and 30 negative results of the paper-based RT-LAMP. Figure 20B shows the receiver operating characteristic (ROC) curves for 30 positive and 30 negative results of the paper-based RT-LAMP. Figure 20C shows a summary table of observations based on the image analysis.

[0277] In accordance with Purdue University IRB protocol #IRB-2021-375, colorimetric perception surveys were collected from participants (Figures 23A and 23B). Participants were given color bands with threshold annotations (Figure 19E). Participants were given multiple paper devices to scan and asked to classify the control group (left reaction area) as valid or invalid, and the SARS-CoV-2 reaction (right reaction area) as positive or negative. Observations classified as invalid were discarded from the analytical performance analysis, and the proportion of incorrectly identified invalid analyses was reported as error inefficiency.

[0278] Four participants were asked to use colorimetric strips (Figure 19) to classify 10 positive and 10 negative reactions shown in Figure 21A as valid or invalid (based on the left control area), and SARS-CoV-2 positive or negative (based on the right reaction area). Observations deemed invalid by the participants 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 valid using image analysis), where one observation was incorrectly classified as invalid. Therefore, our device calculated specificity and sensitivity of 100% and 76% respectively, considering colorimetric interpretation, with an accuracy of 91% (Figure 19F) and a false negative rate of 25%. Example 22 – Sample Stability

[0279] The standard time from saliva collection to test strip application is within 2 hours. Sample stability is allowed up to 24 hours from collection to testing, based on repeat testing of samples at 0-2 hours, 8-12 hours, and 20-24 hours (e.g., 15 positive and 15 negative). Samples collected rapidly from patients in a collection center or clinic setting can then be tested several hours later. One-site collection (e.g., in an outpatient clinic or drive-thru collection area) followed by transporting saliva samples to a second location can also be evaluated. Example 23 – Manufacturing

[0280] Dip coating and high-temperature drying do not appear to negatively impact the LAMP reaction. This has positive implications for manufacturing throughput and scalability. The test strip is designed to be mass-produced on existing paper processing equipment without significant tooling costs. The design is simple enough to provide a fast and easy path from prototyping to full-scale production, thus meeting the needs of emerging markets. Example 24 – Design of a Paper Device

[0281] Paper is widely used in pH indicators and urine test strips, primarily due to its low cost, low technical complexity, and ease of production using roll-to-roll manufacturing. In the device described herein, several types of paper and the use of selected chromatography paper were evaluated (Figures 21D-21F). Chromatography paper can be used in paper-based biosensors because it has a higher wicking capacity compared to other papers. Many paper-based devices use Grade 1 chromatography paper; however, due to the large area of ​​Grade 1 chromatography paper (5 mm x 20 mm), the solution distribution on the paper is uneven. Therefore, a different type of chromatography paper, Grade 222 (0.83 mm), was selected, which is approximately 4.6 times thicker than Grade 1 (0.18 mm). Due to its increased thickness, the same volume of liquid can be loaded into a reaction area that is 70% smaller due to the reduced device size (5 mm x 6 mm), thus achieving a more uniform distribution.

[0282] To reduce apparatus complexity, the components of the RT-LAMP reaction (without a sample) are dried on paper. Drying ensures stable distribution of the apparatus and ease of operation without affecting diagnostic performance; the user only needs to add the sample to rehydrate the reagents. After the reagents dry on the paper, over time, without any sample, the paper changes color from red to yellow, indicating a decrease in paper pH. Through a series of experiments, ammonium sulfate was identified as the cause of this change (Figure 21G). This color change may be due to cellulose oxidation caused by heating and the oxidative nature of ammonium sulfate, or acidification of the reagents due to ammonia released from the RT-LAMP mixture. To prevent color change without amplification, betaine was substituted for ammonium sulfate and the concentration of phenol red (as an antioxidant) was increased (Figure 21H).

[0283] The effectiveness of betaine in the LAMP reaction varies; however, betaine reduces oxidative damage and is therefore included. Trehalose and BSA were both added to the paper formulation (Fig. 21H).

[0284] In terms of the apparatus, two reaction zones were used: one targeting SARS-CoV-2 and one providing a primer-free control group to determine the stability of our reagents on paper (which should not react with any sample). A schematic diagram of the final apparatus is shown in Figure 19C, and results from the apparatus with and without heat-deactivated SARS-CoV-2 incorporated into saliva at a 5% reaction concentration are shown in Figure 19D. As shown in Figure 19B, the LoD (250 copies / reaction) analyzed on paper is comparable to the LoD observed in solution for the colorimetric RT-LAMP formulation (Figure 21I).

[0285] In terms of device construction, the Melinex® backing provides structural support. Two reaction pads are attached to the backing using double-sided adhesive without altering the pH. The number of reaction zones can be increased arbitrarily to enable multiple assays without changing the device design. A 20-mil polystyrene spacer is placed between the reaction pads to provide a physical barrier, preventing leakage from one reaction zone to an adjacent one, thus eliminating cross-contamination during reagent and sample addition. In some cases, a hydrophobic barrier created by wax printing can separate the reaction zones to prevent sample traversal; however, for roll-to-roll manufacturing, the use of wax is excluded, and spacers are used. Example 25 – Validity of Human Samples

[0286] To evaluate the analytical sensitivity and specificity of our paper-based assay, human samples were generated using heat-deactivated SARS-CoV-2 at a multiple of the LoD of orf7ab.I, and RT-LAMP analysis was run on paper. A total of 30 positive samples and 30 corresponding negative samples were used, which is the minimum number permitted by the U.S. Emergency Use Authorization (EUA). To determine the colorimetric threshold for distinguishing between positive and negative reactions, ROC curves were constructed by calculating sensitivity and specificity at different green channel intensity thresholds (Figure 20). At this threshold, the assay demonstrated the following analytical metrics: 97% sensitivity, 100% specificity, and 98% accuracy (Figures 20A and 20B). A significant difference was found between the positive and negative groups (p < 0.001). The paper strips were hand-cut, and slight variations in paper size could lead to differences in colorimetric responses. Therefore, large-scale manufacturing and quality control can further enhance the consistency between the two groups (positive and negative). This sensitivity is comparable to that of analytical methods using RNA extracts (sensitivity ~95%), and is better than reports of a significant decrease in sensitivity (approximately 80%) commonly observed for crude samples. Example 26 – Colorimetric Interpretation of Paper Devices

[0287] To observe the impact of color perception on the performance of our device, four participants were surveyed and asked to interpret the device's results. Each participant was provided with a color band, and after 60 minutes, the device was scanned (Figures 23A and 23B). Participants were asked to classify the results as valid or invalid (using a control pad) and positive or negative based on the thresholds marked on the color band. 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 participants identifying many positive reactions as invalid based on the control pad, resulting in a 25% false negative rate, which may be attributed to amplicon contamination of the control pad during the reaction. Furthermore, user interpretation of pads containing both yellow and red areas may introduce ambiguity, leading to an increased false positive rate or false negative rate. Recent findings suggest that this ambiguity is due to a third intermediate color cluster (and the positive / negative cluster), which is not adequately addressed in colorimetric analysis. Since invalid results are discarded from further analysis, this increased false negative rate may artificially affect the device's specificity and accuracy metrics. Example 27 – Effect of Exclusion of a Single Reactant on the Initial Color of Dried Paper

[0288] Figure 21G shows the basic formulation of 20 μL RT-LAMP master mix containing KCl (50 mM), MgSO₄ (8 mM), a molar mixture of dNTPs (1.4 mM each), 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), (NH₄)₂SO₄ (10 mM), and trehalose (10% w / v), along with 5 μL of nuclease-free water, added to Grade 1 chromatography paper and dried in a PCR preparation fume hood for 10 minutes. As shown, reactants were excluded from the basic formulation to determine the cause of the color change observed after drying. RT-LAMP primers and templates were not included in this study. Example 28 – Phenol Red Color Correction at Different pH Values

[0289] Figure 21B shows the correction of 250 μM phenol red at different pH values ​​on paper buffered with 20 mM Tris. The pH was adjusted using HCl or KOH. Images of the 222 grade chromatography paper (5 mm x 6 mm) were cropped into rectangles for easy comparison of colors between multiple test strips. Example 29 – Determination of the final device under different concentrations of thermally deactivated SARS-CoV-2

[0290] Figure 21A shows the final formulation of RT-LAMP using the orf7ab.I primer set and colorimetric RT-LAMP master mix. The left reaction zone on each device is the primer-free control group, in which all orf7ab.I primers are replaced with water and not included in the master mix. The right reaction zone contains heat-deactivated virus incorporated into treated saliva at a specified concentration in the case of a positive reaction, or contains treated saliva in the case of a negative reaction. The final reaction concentration of all treated saliva is 5%. The master mixture consisted of KCl (50 mM), MgSO₄ (8 mM), a molar mixture of dNTPs (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), betaine (20 mM), BSA (40 mg / mL), and trehalose (10% w / v). 222 grade chromatography paper was used. Example 30 – Green channel color intensity of RT-LAMP colorimetric responses at different concentrations

[0291] Figure 21C shows a scatter plot of the colorimetric response of paper pads at different concentrations. The green intensity threshold is shown by reference line 121. Example 31 – Effect of heating method on RT-LAMP colorimetric response

[0292] Figure 22C shows the colorimetric scan of the LAMP detection limit (LoD) of a 25 μL reaction after incubation at 65 °C for 60 min in different heating devices. The primer set used for these reactions was orf1ab.II. 5 μL of heat-deactivated virus dilution in water was added to a 20 μL reaction mixture to produce the final labeled concentration (positive reaction) or nuclease-free water (negative). The master mixture consisted of 12.5 μL of NEB Colorimetric 2x master mixture, 2.5 μL of primer mixture, and 5 μL of water. Example 32 – Effect of heating rate on RT-LAMP colorimetric response

[0293] Figure 22D shows the colorimetric scans after incubation of 25 μL of the reaction at 65 °C for 60 min at different heating rates on a qTower (96-well plate) and a thermal cycler (PCR tubes). The primer group used was orf1ab.II. 5 μL of heat-deactivated virus dilution was added to 20 μL of the reaction mixture to produce the final labeled concentration (positive reaction) or nuclease-free water (negative reaction). The master mixture consisted of 12.5 μL of NEB Colorimetric 2x master mixture, 2.5 μL of primer mixture, and 5 μL of water. Example 33 – Effects of Trehalose and Tween 20 on RT-LAMP Colorimetric Response

[0294] Figure 21H shows the colorimetric RT-LAMP results containing a given concentration of trehalose or Tween 20. The orf1ab.II primer set was used. Add 20 μL of a basic RT-LAMP master mix containing KCl (50 mM), MgSO4 (8 mM), a molar mixture of dNTPs (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, if indicated), betaine (20 mM), BSA (40 mg / mL), and trehalose (10% w / v, if indicated) onto grade 1 chromatography paper (5 mm x 20 mm) and allow to dry in a PCR preparation fume hood for 60 minutes. Add 25 μL of heat-deactivated SARS-CoV-2, in either 25% treated saliva (positive reaction) or nuclease-free water (negative reaction) to a final concentration of 1 x 10⁵ copies per reaction, to a dry reaction pad. Incubate the pad at 65°C for 60 minutes, then scan using a flatbed scanner.

[0295] The presence of ammonium sulfate causes the RT-LAMP reagent to change from red to yellow after drying, even without a sample. Increasing the concentration of phenol red and replacing ammonium sulfate with betaine can prevent this color change (Figure 21G). In addition, the addition of trehalose and bovine serum albumin (BSA) increases the reaction rate and improves the LoD (Figure 21H). Example 34 – Summary

[0296] A paper-based device for detecting pathogenic nucleic acids of interest in complex samples using loop-mediated isothermal amplification (LAMP) by generating a colorimetric response visible to the human eye. This device can detect SARS-CoV-2 in human saliva without pretreatment, demonstrating its practicality in emerging public health emergencies. The resulting device can detect the virus within 60 minutes and exhibits 97% analytical sensitivity and 100% specificity using image analysis, with a detection limit of 200 genome copies / μL in patient saliva. The device includes a configurable number of reaction zones constructed from 222-grade chromatography paper, separated by 20-mil polystyrene spacers, and attached to a Melinx® backing using ARclean® double-sided adhesive. The resulting device can detect multiple targets and various pathogens by changing the LAMP primer set.

[0297] The platform has the following characteristics: i) it uses saliva, ii) it requires minimal operator training, iii) it can be manufactured using a roll-to-roll method to achieve millions of tests, iv) it performs similarly to RT-qPCR in terms of analytical sensitivity and specificity, v) it provides a visible colorimetric response, vi) it is suitable for use in designated care settings, vii) it provides results in less than 60 minutes, and viiii) its estimated cost is ~ $10 per test.

[0298] Due to its simplicity and scalability, this test can be used in various settings, including home diagnostics. The platform can be easily reconfigured to target different pathogens by screening primer groups in solution. Multiplex detection can be achieved by adding additional reaction sites to the device. The platform's reconfigurable nature makes it suitable for detecting emerging pathogens in future public health emergencies. [Exemplary Example]

[0299] In one embodiment, a loop-mediated isothermal amplification (LAMP) reaction assembly is provided, which may comprise a combination of a substantially hygroscopic-free LAMP reagent mixture and a solid-phase reaction medium.

[0300] In one example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid-phase medium may be substantially free of magnesium interfering agents.

[0301] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the magnesium interfering agent may include a magnesium-containing compound and a chelating agent that interferes with magnesium.

[0302] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid medium can be hydrophilic, absorbent, and porous.

[0303] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid medium can be a cellulose-based medium.

[0304] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the cellulose-based medium may have a surface area to thickness ratio between about 30 and about 600.

[0305] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the cellulose-based medium may have a pore size of less than about 100 micrometers.

[0306] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid medium may comprise paper.

[0307] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the solid medium may comprise glass fibers.

[0308] In another example of a ring-mediated isothermal amplification (LAMP) reaction assembly, the solid medium may comprise nylon, polyurethane, polyetherurethane, cellulose acetate, nitrocellulose, or hydrophilic polytetrafluoroethylene (PTFE), or combinations thereof.

[0309] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the LAMP reaction medium may further contain a binder that is substantially free of magnesium interfering agents and hygroscopic agents.

[0310] In another example of a loop-mediated isothermal amplification (LAMP) reaction assembly, the LAMP reaction medium may further include an unfolding layer having lower hydrophilicity than the solid-phase reaction medium.

[0311] In one example, a method for manufacturing a LAMP reaction assembly as described herein is provided, the method comprising combining the substantially hygroscopic-free LAMP reagent mixture with the solid-phase reaction medium such that the reagent mixture remains in contact with the solid-phase reaction medium.

[0312] In one example, the method for manufacturing a LAMP reaction assembly as described herein may further include using a non-coloring additive to control color change.

[0313] In another example of a method for manufacturing a LAMP reaction assembly as described herein, the color-changing additive may comprise sugars, buffers, blockers, or combinations thereof.

[0314] In another example of a method for manufacturing a LAMP reaction assembly as described herein, the color-changing additive may comprise a sugar comprising trehalose, glucose, sucrose, dextran, or a combination thereof.

[0315] In another example of a method for manufacturing a LAMP reaction assembly as described herein, the color-changing additive may comprise a blocking agent comprising bovine serum albumin, casein, or a combination thereof.

[0316] In another example, a method for performing LAMP analysis is provided, which includes or comprises providing a LAMP reaction assembly as described herein; applying a biological sample to the reaction assembly; heating the assembly to a temperature sufficient to initiate a LAMP reaction; and maintaining the temperature for a period of time sufficient to complete the LAMP reaction.

[0317] In one example of a method for performing LAMP analysis, the biological sample may be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or combinations thereof.

[0318] In another example of the method for performing LAMP analysis, the biological sample could be saliva.

[0319] In another example of a method for performing LAMP analysis, the method may further include the detection of a viral pathogen.

[0320] In another example of a method for performing LAMP analysis, the LAMP analysis could be reverse transcription LAMP (RT-LAMP).

[0321] In one embodiment, a system for colorimetric ring-mediated isothermal amplification (LAMP) analysis is provided, comprising or including a substantially non-reactive solid-phase reaction medium and a non-interfering reagent mixture.

[0322] In one example of a system used for colorimetric ring-mediated isothermal amplification (LAMP) analysis, the substantially non-reactive solid-phase reaction medium may have a buffering capacity from about 0.01 mM to about 5 mM.

[0323] In another example of a system used for colorimetric ring-mediated isothermal amplification (LAMP) analysis, the substantially non-reactive solid-phase reaction medium may have a λ max range from about 443 nm to about 570 nm.

[0324] In another example of a system used for colorimetric ring-mediated isothermal amplification (LAMP) analysis, the substantially non-reactive solid-phase reaction medium may contain cellulose or glass fibers.

[0325] In another example of a system used for colorimetric ring-mediated isothermal amplification (LAMP) analysis, the substantially non-reactive solid-phase reaction medium can be hydrophilic, absorbent, and porous.

[0326] In another example of a system used for colorimetric ring-mediated isothermal amplification (LAMP) analysis, the substantially non-reactive solid-phase reaction medium may be substantially free of oxidants and pH-disrupting agents.

[0327] In another example of a system for colorimetric ring-mediated isothermal amplification (LAMP) analysis, the system may further include an adhesive; a developing layer; a spacer; and a plastic carrier, wherein each of the adhesive, developing layer, spacer, and plastic carrier is substantially free of oxidants and pH-disrupting agents.

[0328] In another example of a system used for colorimetric ring-mediated isothermal amplification (LAMP) analysis, the non-interfering reagent mixture may further contain one or more target primers, a DNA polymerase, and a redissolving agent.

[0329] In another example, a method is provided to maximize the accuracy of the chromatic output signal in a solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis, which may include: providing a solid-phase reaction medium that minimizes color changes produced by non-LAMP reactions; and performing the LAMP analysis on the solid-phase reaction medium.

[0330] In one example of a method to maximize the accuracy of the chromatic output signal in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis, the method may further include controlling non-LAMP-generated color changes caused by protons produced by non-LAMP reactions.

[0331] In another example of a method to maximize the accuracy of the chromatic output signal in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis, the method may further include the use of a non-color-changing additive to control the color change produced by non-LAMP reactions.

[0332] In another example of a method to maximize the accuracy of the chromatic output signal in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis, the color-changing additive may comprise sugars, buffers, blocking agents, or combinations thereof.

[0333] In another example of a method to maximize the accuracy of the chromatic output signal in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis, the color-changing additive may contain a sugar, which may include trehalose, glucose, sucrose, dextran, or combinations thereof.

[0334] In another example of a method to maximize the accuracy of the chromatic output signal in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis, the color-changing additive may contain a blocking agent comprising bovine serum albumin, casein, or a combination thereof.

[0335] In another example of a method to maximize the accuracy of the chromatic output signal in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis, a method to maximize the detection level (LOD) in loop-mediated isothermal amplification (LAMP) analysis may include providing a reaction environment and reagents that minimize non-LAMP reaction products.

[0336] In another example of a method to maximize the accuracy of the chromatic output signal in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis, a system for chromatic loop-mediated isothermal amplification (LAMP) analysis may comprise a combination of a solid-phase reaction medium and a LAMP reagent, which, when stored at 25°C, maintains the color of the solid-phase reaction medium within 10% of the initial hue of the solid-phase medium.

[0337] In one example of a method to maximize the accuracy of the chromaticity output signal in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis, the combination can maintain color for storage for more than one or more of the following: 30 days, 90 days, 365 days, 2 years, or 5 years.

[0338] In another example of a method to maximize the accuracy of chromatic output signals in solid-phase pH-dependent loop-mediated isothermal amplification (LAMP) analysis, this combination maintains color when stored at 25°C and relative humidity between approximately 40% and 90%.

[0339] In another example, a method for manufacturing a colorimetric LAMP system as described herein is provided, which may include combining the 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 remain in contact.

[0340] In one example of a method for manufacturing a colorimetric LAMP system as described herein, the manufacturing process may include: preparing a solution containing the non-interfering reagent mixture; and coating the reagent mixture onto the substantially non-reactive solid-phase reaction medium.

[0341] In another example of a method for manufacturing a colorimetric LAMP system as described herein, the coating may involve dripping, spraying, impregnating, soaking, or atomizing the solution onto the substantially non-reactive solid-phase reactive medium.

[0342] In another example of a method for manufacturing a colorimetric LAMP system as described herein, the non-interfering reagent mixture and the substantially non-reactive solid-phase reaction medium may be combined using a roll-to-roll (R2R) method.

[0343] It should be understood that the above methods are only used to illustrate some embodiments of the present invention. Many modifications and alternative arrangements can be devised by those skilled in the art without departing from the spirit and scope of the invention, and the appended claims are intended to cover these modifications and arrangements. Therefore, although the invention has been described in detail above in conjunction with what are now considered to be the most practical and preferred embodiments, it will be apparent to those skilled in the art that variations can be made without departing from the principles and concepts set forth herein.

[0344] 100, 200, 400, 500, 600, 700: Method 110, 120, 130, 140: Square 210, 220: Square 410, 420: Square 510, 520, 530: Square 610, 620, 630: Square 710, 720: Square 300a, 300b, 300c, 300d, 300e, 300f: Solid-phase reaction media 302:Substrate 304: Adhesive layer 306: Reaction Layer 305a, 305b, 305c, 305d: Test point, reaction location, reaction section 307a, 307b, 307c, 307d, 307e: Spacers 308: Expanded layer, distributed layer 121: Reference Line

Claims

1. A loop-mediated isothermal amplification (LAMP) reaction assembly comprising a solid-phase reaction medium, the solid-phase reaction medium comprising: 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; wherein the result of the loop-mediated isothermal amplification (LAMP) reaction is analyzed by color, wherein the analysis is performed by a pH-sensitive dye, wherein the pH-sensitive dye is included in the reaction layer along with the LAMP reagent mixture.

2. The LAMP reaction assembly as claimed in claim 1, wherein the solid-phase reaction medium is hydrophilic, absorbent and porous.

3. The LAMP reaction assembly of claim 1, wherein the solid-phase reaction medium is a cellulose-based medium.

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

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

6. The LAMP reaction assembly of claim 1, wherein the solid-phase reaction medium comprises paper.

7. The LAMP reaction assembly of claim 1, wherein the reaction layer comprises glass fiber.

8. The LAMP reaction assembly of claim 1, wherein the reaction layer comprises nylon, polyurethane, polyetherurethane, cellulose acetate, nitrocellulose, or hydrophilic polytetrafluoroethylene (PTFE), or combinations thereof.

9. The LAMP reaction assembly of claim 1, wherein the reaction layer is bonded to the substrate by an adhesive free of magnesium interfering agent and hygroscopic agent.

10. The LAMP reaction assembly of claim 1, further comprising a spread layer having a lower hydrophilicity than the reaction layer.

11. A method of manufacturing a LAMP reaction assembly as claimed in claim 1, comprising: bonding the LAMP reagent mixture to the reaction layer of the solid-phase reaction medium such that the LAMP reagent mixture and the reaction layer remain in contact.

12. The method of claim 11, wherein the solid-phase reaction medium is substantially free of magnesium interfering agents.

13. The method of claim 12, wherein the magnesium interfering agents include magnesium-containing compounds and chelating agents that interfere with magnesium.

14. The method of claim 11, further comprising: using a non-discoloring additive to control discoloration, wherein the non-discoloring additive comprises a sugar, a buffer, a blocker, or a combination thereof, the blocker comprising bovine serum albumin, casein, or a combination thereof.

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

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

17. A method for performing LAMP analysis, comprising: providing a LAMP reaction assembly as described in 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 the temperature for a period of time sufficient to complete the LAMP reaction.

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 combinations thereof.

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

20. The method of claim 17 further includes: 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, litmus red, bromothymol blue, naphtholphthalein, cresol red, or a combination thereof.

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

24. The LAMP reaction assembly of claim 22, wherein the solid-phase reaction medium has a λmax range from 443 nm to 570 nm.

25. The LAMP reaction assembly of claim 22, further comprising: an adhesive; a spreader; and a spacer; wherein the substrate is a plastic carrier, and wherein each of the adhesive, spreader, spacer and plastic carrier is free of oxidants and pH disruptors.

26. The LAMP reaction assembly of claim 22, comprising: a combination of the reaction layer and the LAMP reagent mixture, wherein, when stored at 25°C, the color of the solid reaction medium is maintained within 10% of the initial hue of the solid reaction medium.

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

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

29. The method of claim 11, wherein the method comprises: preparing a solution containing the LAMP reagent mixture; and applying the solution onto the reaction layer.

30. The method of claim 29, wherein the coating comprises dripping, spraying, impregnating, soaking or atomizing the solution onto the reactive layer.

31. The method of claim 29, wherein the LAMP reagent mixture is combined using a roll-to-roll (R2R) method.

Images