Loop-mediated isothermal amplification (LAMP) analysis for pathogenic targets

Abstract

The present disclosure relates to compositions, methods, and systems for loop-mediated isothermal amplification (LAMP) analysis on solid-phase media. The compositions can include one or more target primers, a DNA polymerase, and a resolubilizing agent. The compositions can be substantially free of non-pH sensitive agents that can discolor the solid-phase media. The methods can include providing an assembly of solid-phase media, depositing a biological sample on the solid-phase media, and heating the assembly to an isothermal temperature sufficient to promote a LAMP reaction. The systems can include the composition and the solid-phase media on which the composition is deposited.

Description

[Technical Field]

[0001] Related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 138,312, filed on 15 January 2021, which is incorporated herein by reference. [Background technology]

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

[0003] In contrast, loop-mediated isothermal amplification (LAMP) is a simpler approach to the diagnostic identification of target nucleotides. Specifically, LAMP is a one-step nucleic acid amplification method that amplifies a specific nucleotide sequence. In addition to using an isothermal heating process, LAMP allows the use of simpler visual output indicators, such as color changes, rather than the more complex fluorescent indicators used in PCR. Reverse transcription LAMP (RT-LAMP) can be used similarly to RT-PCR to identify target nucleotides from RNA and can be used for diagnostic purposes to distinguish the presence or absence of viral pathogens. Because LAMP is simpler, it can be performed with less equipment and sample preparation, thus facilitating its use in point-of-care settings such as clinics, emergency rooms, and even mobile bases. [Overview of the Initiative]

[0004] This disclosure relates to techniques (e.g., compositions, methods, systems, and assemblies) for use in detecting target nucleotides using LAMP analysis. In some embodiments, the target nucleotides may be known to be present in a pathogen of interest. If the pathogen is a virus, the LAMP analysis may be RT-LAMP analysis.

[0005] In some disclosed embodiments, a method is provided for preparing a saliva sample for loop-mediated isothermal amplification (LAMP) detection of a pathogen target. In one embodiment, such a method may include providing a certain amount of saliva from a test subject and diluting the saliva with water to a degree that reduces the buffering capacity of the saliva, while maintaining a concentration sufficient to enable detection of the pathogen target.

[0006] In one embodiment, the method may include reducing the viscosity of the saliva compared to its original viscosity. In another embodiment, the viscosity may be reduced by dilution, filtration, or a combination thereof. In yet another embodiment, the viscosity may be reduced using filtration. In yet another embodiment, the viscosity may be reduced using a 10-micron filter. In yet another embodiment, the viscosity may be reduced to the extent that the fluidity increases compared to the original viscosity, allowing it to pass through a solid phase medium. In yet another embodiment, the viscosity may be reduced to a range of about 1.0 centipoise (cP) to about 50 cP.

[0007] In one embodiment, such a method may include filtering the saliva sample until its pH is adjusted to the target range of the test sample. In another embodiment, the target range of the test sample may be approximately 7.2 to approximately 8.6. In yet another embodiment, the water may have a pH greater than 6.0 and may be substantially free of contaminants. In yet another embodiment, the saliva sample may essentially consist of saliva and water. In yet another embodiment, the saliva may be collected using a sponge-based collection method.

[0008] In one embodiment, saliva can be diluted with water until the saliva-to-water ratio is approximately 1:1 to approximately 1:20. In another embodiment, saliva can be diluted with water until the optical density (OD) of the sample at 600 nm is determined. 600 The solution can be diluted with water to a degree where the ratio is less than 0.2. In a further embodiment, the saliva has a volume of about 50 μl to about 100 μl. In yet another embodiment, the saliva sample has a volume in the range of about 100 μl to about 1 ml.

[0009] In additional embodiments, the pathogen target may include a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In one embodiment, the pathogen target may be a viral target. In another embodiment, the viral target may include a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a ds-DNA-RT virus. In yet another embodiment, the viral target may be H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.

[0010] In some embodiments, LAMP detection may include reverse transcription LAMP (RT-LAMP) detection.

[0011] In other disclosed embodiments, a test sample composition for LAMP analysis is disclosed, which may contain a sufficient amount of test control saliva to detect pathogen targets via LAMP analysis, combined with an amount of water to reduce the buffering capacity of the saliva.

[0012] In one embodiment, the composition may have a viscosity of about 1.0 cP to about 50 cP. In another embodiment, the composition may have a pH of about 7.2 to about 8.6. In yet another embodiment, the composition may have a saliva-to-water ratio of about 1:1 to about 1:20. In yet another embodiment, the composition may have an optical density (OD) at 600 nm. 600The pH may be less than 0.2. In another embodiment, the water may have a pH greater than 6.0 and may be substantially free of contaminants. In one embodiment, the composition may consist essentially of saliva and water. In another embodiment, the saliva may have a volume in the range of about 50 μl to about 100 μl. In yet another embodiment, the saliva sample may have a volume in the range of about 100 μl to about 1 ml.

[0013] In one embodiment, the pathogen target may include a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In another embodiment, the pathogen target may be a viral target. In yet another embodiment, the viral target may include a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a ds-DNA-RT virus. In yet another embodiment, the viral target may include H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2. In yet another embodiment, the buffering capacity of the composition may be less than 5 mM.

[0014] In further disclosed embodiments, a composition for LAMP analysis on a solid-phase medium may include one or more target primers, DNA polymerase, and a resolubilizer. In some embodiments, such a composition may be substantially free of non-pH-sensitive agents that can discolor the solid-phase medium. In one embodiment, the composition may include an antioxidant. In another embodiment, the composition may be substantially free of volatile agents. In yet another embodiment, the composition may be substantially free of hygroscopic agents. In yet another embodiment, the composition may further include a reverse transcriptase.

[0015] In one embodiment, the desiccant can absorb more than about 10% by weight when the relative humidity (RH) is between about 40% and about 90% at 25°C. In another embodiment, the desiccant may include glycerol, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate, and combinations thereof.

[0016] In another embodiment, the resolubilizer may be a surfactant. In another embodiment, the resolubilizer may include bovine serum albumin (BSA), casein, polysorbate 20, or a combination thereof.

[0017] In one embodiment, the target primer may target a pathogen that may include a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In one embodiment, the pathogen may be a viral pathogen. In another embodiment, the viral pathogen may include a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a ds-DNA-RT virus. In another embodiment, the viral pathogen may include H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.

[0018] In one embodiment, the composition may further include a non-coloring additive. The non-coloring additive may include one or more sugars, buffers, or combinations thereof. In another embodiment, the composition may further include an indicator.

[0019] In embodiments of other disclosures, a method for LAMP analysis on a solid-phase medium may include providing an assembly of the solid-phase medium and composition described herein, depositing a biological sample on the solid-phase medium, and heating the assembly to a temperature sufficient to facilitate the LAMP reaction. In one embodiment, the biological sample may be one or more of saliva, mucus, blood, urine, feces, sweat, breath condensate, or a combination thereof. In another embodiment, the biological sample is saliva. In one embodiment, the LAMP analysis may be reverse transcriptase LAMP (RT-LAMP). In another embodiment, the method may further include detecting viral pathogens.

[0020] In other disclosed embodiments, a system for performing LAMP analysis may comprise the compositions described herein and a solid-phase medium on which the compositions are deposited.

[0021] In a further disclosed embodiment, in a composition for loop-mediated isothermal amplification (LAMP) assay, a pH-dependent output signal comprising a pH-sensitive dye and a plurality of non-interfering LAMP reagents can be utilized. In one aspect, the LAMP assay can be RT-LAMP.

[0022] In one aspect, the pH-sensitive dye can be at least one of phenol red, phenolphthalein, azolitmin, bromothymol blue, naphthol phthalein, cresol red, or a combination thereof. In another aspect, the plurality of non-interfering LAMP reagents can be substantially free of volatile reagents, pH interfering reagents, magnesium interfering reagents, or a combination thereof.

[0023] In one aspect, the plurality of non-interfering LAMP reagents can be substantially free of magnesium, ammonium sulfate, and ammonium carbonate. In one aspect, the plurality of non-interfering LAMP reagents can include DNA polymerase, reverse transcriptase, target primers, or a combination thereof.

[0024] In another aspect, the composition can include an antioxidant. In another aspect, the composition can further include carrier RNA, carrier DNA, RNase inhibitor, DNase inhibitor, guanidine hydrochloride, or a combination thereof. In one aspect, the composition can further include a solid-phase medium.

[0025] In one aspect, the composition can include non-discoloring additives that can include sugars, buffers, blocking agents, or a combination thereof. In one aspect, the sugar can include one or more of trehalose, glucose, sucrose, or a combination thereof. In another aspect, the blocking agent can include bovine serum albumin, casein, or a combination thereof.

[0026] In other disclosed embodiments, a method of performing LAMP analysis using a pH-dependent output signal is provided, the method can include providing an assembly of a solid-phase medium and a composition as described herein, depositing a biological sample on the solid-phase medium, and heating the assembly to an isothermal temperature sufficient to promote the LAMP reaction. In one aspect, the LAMP analysis can be RT-LAMP. In one aspect, the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, breath condensate, and combinations thereof. In one aspect, the biological sample can be saliva. In another aspect, the method can further include detecting a viral pathogen.

[0027] In further disclosed embodiments, a method of maximizing the accuracy of the output signal in a pH-dependent LAMP analysis can include providing a reagent mixture that minimizes discoloration caused by non-LAMP reactions from the signal output medium, and performing the LAMP reaction. In one aspect, the method can include controlling the generation of protons from non-LAMP reactions. In another aspect, the method can include controlling the oxidation by non-LAMP reactions.

[0028] In other disclosed embodiments, a method of maximizing the accuracy of the output signal in a pH-dependent LAMP analysis can include substantially removing discoloration caused by non-LAMP reactions from the signal output medium.

[0029] In other disclosed embodiments, a method of maximizing the limit of detection (LOD) in a pH-dependent LAMP analysis can include substantially removing discoloration caused by non-LAMP reactions from the signal output medium.

[0030] The features and advantages of the present disclosure will become apparent from the following detailed description when taken in conjunction with the accompanying drawings which illustrate, by way of example, the features of the present disclosure. The accompanying drawings are as follows.

Brief Description of the Drawings

[0031] [Figure 1]A method for preparing a saliva sample for loop-mediated isothermal amplification (LAMP) detection of a pathogen target is shown according to an exemplary embodiment. [Figure 2] A method for LAMP analysis is shown according to an exemplary embodiment. [Figure 3] An exemplary embodiment illustrates a method for maximizing the accuracy of the output signal in pH-dependent LAMP analysis. [Figure 4] This example demonstrates that loop-mediated isothermal amplification (LAMP) can be obtained in a saliva sample. [Figure 5A] A sponge-based sampling device according to an exemplary embodiment is shown. [Figure 5B] A passive drool collection device according to an exemplary embodiment is shown. [Figure 6A] The detection limits of various sample concentrations and various sampling devices are shown according to exemplary embodiments. [Figure 6B] The effects of RNase inhibitors on templates of various concentrations on the RT-LAMP colorimetric response are shown according to exemplary embodiments. [Figure 6C] The effect of saliva treatment technology on colorimetric Level of Death (LOD) according to an exemplary embodiment is shown. [Figure 6D] The effect of carrier DNA concentration on the RT-LAMP colorimetric response, according to an exemplary embodiment, is shown. [Figure 6E] The effect of guanidine HCl on the RT-LAMP colorimetric response, according to an exemplary embodiment, is shown. [Figure 6F] The effect of UDG on the endpoint RT-LAMP colorimetric response, according to an exemplary embodiment, is shown. [Figure 6G] The effect of saliva treatment on the colorimetric response is shown according to an exemplary embodiment. [Figure 7] This chart shows the stability of a frozen saliva sample according to an exemplary embodiment. [Figure 8] The detection limits for untreated saliva are shown according to an exemplary embodiment. [Figure 9]The detection limits in a bovine nasal swab, according to an exemplary embodiment, are shown. [Figure 10] The detection limits on paper are shown according to an exemplary embodiment. [Figure 11] The colorimetric transition of phenol red according to an exemplary embodiment is shown. [Figure 12] The buffer used in a paper-based assay according to an exemplary embodiment is shown. [Figure 13A] A paper LAMP verification according to an exemplary embodiment is shown. [Figure 13B] A paper LAMP verification according to an exemplary embodiment is shown. [Figure 14A] The image shows a low template concentration LAMP on paper at time 0 minutes, according to an exemplary embodiment. [Figure 14B] The image shows a low template concentration LAMP on paper at 60 minutes, according to an exemplary embodiment. [Figure 15] The image shows an entire untreated saliva sample containing the thermoactivated SARS-CoV-2 virus, according to an exemplary embodiment. [Figure 16] A comparison of RT-LAMP colorimetric and fluorescence responses according to an exemplary embodiment is shown. [Figure 17A] The use of calmagite as a LAMP colorimetric indicator according to an exemplary embodiment is shown. [Figure 17B] The use of EBT as a LAMP indicator according to an exemplary embodiment is shown. [Figure 17C] A LAMP on chromatography paper using EBT as a colorimetric reporter, according to an exemplary embodiment, is shown. [Figure 17D] The colorimetric responses of LAMP on various papers, using EBT as an indicator, according to an exemplary embodiment, are shown. [Figure 17E] This exhibits LAMP detection on biodyne A amphoteric paper using EBT as a colorimetric indicator, according to an exemplary embodiment. [Figure 17F] The effect of crystal violet concentration on the LAMP colorimetric response, according to an exemplary embodiment, is shown. [Figure 17G]A colorimetric LAMP using various concentrations of crystal violet on paper, according to an exemplary embodiment, is shown. [Figure 17H] A pH indicator as a colorimetric reporter for RT-LAMP, according to an exemplary embodiment, is shown. [Figure 17I] The effect of cresol red concentration on the colorimetric response of the LAMP reaction, according to an exemplary embodiment, is shown. [Figure 17J] The effects of various pH indicator concentrations on the colorimetric response of an RT-LAMP reaction, according to an exemplary embodiment, are shown. [Figure 17K] A gel electrophoresis scan of RT-LAMP products using a pH indicator, according to an exemplary embodiment, is shown. [Figure 17L] The effect of initial pH on the RT-LAMP colorimetric response using phenol red, according to an exemplary embodiment, is shown. [Figure 18] This demonstrates the color stability of the drying process according to an exemplary embodiment. [Figure 19A] This illustrates the effect of removing a single reactant from the initial color of the paper after drying, according to an exemplary embodiment. [Figure 19B] The effects of trehalose and Tween20 on the RT-LAMP colorimetric response, according to an exemplary embodiment, are shown.

[0032] Herein, illustrative embodiments are used, and specific language is used to describe them. Nevertheless, it will be understood that the scope of the Art is not intended to be limited thereto. [Modes for carrying out the invention]

[0033] Before describing embodiments of the present invention, it should be understood that this disclosure is not limited to any specific structure, process step, or material disclosed herein, but extends to its equivalents as would be recognized by those skilled in the art. It should also be understood that the terms used herein are for illustrative purposes only and are not intended to limit any specific example or embodiment. The same reference numerals in different drawings represent the same component. Numbers provided in flowcharts and processes are provided for clarity to indicate steps and operations and do not necessarily indicate a specific order or sequence.

[0034] Furthermore, the described properties, structures, or features can be combined in any preferred manner in one or more embodiments. The following description provides many specific details, such as examples of compositions, dosage forms, treatments, etc., to provide a complete understanding of the various embodiments of the present invention. However, those skilled in the art will recognize that such detailed embodiments are not limiting to the overall inventive concept expressed herein, but merely representative of it.

[0035] definition As used herein, unless otherwise explicitly indicated by the context, the singular forms "a," "an," and "the" refer to multiple objects. For example, a reference to "excipient" refers to one or more such excipients, and a reference to "carrier" refers to one or more such carriers.

[0036] As used herein, the terms “compound” and “composition” are interchangeable and refer to a mixture of two or more compounds, elements, or molecules. In some embodiments, the terms “compound” and “composition” may be used to refer to a mixture of one or more activators with a carrier or other excipient.

[0037] As used herein, the term “solubility” is a measure or characteristic of a substance or drug relating to its ability to dissolve in a given solvent. The solubility of a substance or drug in a particular component of a composition refers to the amount of the substance or drug that dissolves at a specific temperature, such as about 25°C or about 37°C, to form a visibly clear solution.

[0038] As used herein, the term "lipophilic" refers to a compound that is poorly soluble in water. Conversely, the term "hydrophilic" refers to a compound that is soluble in water.

[0039] As used herein, “subject” refers to an animal. In one embodiment, the animal may be a mammal. In another embodiment, the mammal may be a human.

[0040] As used herein, “non-liquid” refers to the physical state of a composition as semi-solid or solid, when used to refer to the state of a composition disclosed herein.

[0041] As used herein, “solid” and “semi-solid” refer to the physical state of a composition having a suitable viscosity or structure that supports its own weight at standard temperature and pressure and does not flow freely. Semi-solid materials may conform to the shape of a container when pressure is applied.

[0042] As used herein, "solid-phase medium," "solid-phase base," "solid-phase substrate," "solid-phase test substrate," and "solid-phase inspection substrate" refer to a non-liquid medium, device, system, or environment. In some embodiments, the non-liquid medium may be substantially free of liquid or completely free of liquid. In one example, the non-liquid medium may include or be a porous material or a material having a porous surface. In another example, the non-liquid medium may include or be a fibrous material or a material having a fibrous surface. In yet another example, the non-liquid medium may be paper.

[0043] As used herein, “non-coloring additive” refers to an additive that minimizes or prevents a change in the color of a solid-phase medium from its original or starting color to a different color, for reasons other than nucleotide amplification from a LAMP reaction occurring on or within it. For example, in one embodiment, such a color change may be minimized or reduced compared to the color change that would occur in the absence of the non-coloring additive.

[0044] As used herein, “discoloration resulting from a non-LAMP reaction” refers to any discoloration of a solid-phase medium that is not a result of nucleotide amplification from a LAMP reaction (e.g., a change in color from the original color to another color). In some examples, discoloration resulting from a non-LAMP reaction may refer to discoloration of a solid-phase medium caused by one or more of the following: volatile agents, magnesium interferants, oxidizing agents, pH changes resulting from causes other than amplification by a LAMP reaction, drying, or a combination thereof.

[0045] As used herein, “volatile agent” refers to an agent comprising a composition having a high vapor pressure or a low boiling point. For example, ammonium sulfate can be a volatile agent because ammonia can volatilize and leave behind sulfuric acid. For example, a composition, component, or element may have a high vapor pressure if the composition exists in the gas phase at temperatures above about 30°C. For example, a composition may have a low boiling point if it exists in the gas phase at temperatures below about 80°C.

[0046] As used herein, a "pH interfering reagent" is a reagent that can affect the pH of a reaction, system, or environment for reasons other than amplification from a LAMP reaction. For example, ammonium ions may volatilize from ammonium sulfate, and sulfate ions may react to form sulfuric acid, both of which can affect the pH of the reaction in the absence of amplification from a LAMP reaction.

[0047] In this disclosure, “comprises,” “comprising,” “containing,” and “having,” etc., may have meanings attributed to them under U.S. patent law, and may mean “includes,” “including,” etc., and are generally interpreted as non-restrictive terms. The terms “consisting of” or “consists of” are restrictive terms and include only the components, structures, steps, etc., specifically enumerated in relation to such terms, as well as those subject to U.S. patent law. “Consisting essentially of” or “consists essentially of” have the meanings generally given to them under U.S. patent law. In particular, such terms are generally restrictive terms, except that they may include additional items, materials, components, steps, or components that do not substantially affect the basic and novel features or functions of the item(s) used in relation. For example, trace elements present in a composition but not affecting its properties or characteristics may be acceptable if they exist under the usage of "consisting essentially of," even if they are not explicitly listed in the list of items following such terminology. When non-restrictive terms such as "comprising" or "including" are used herein, the usages of "consisting essentially of" and "consisting of" should be given direct support as if they were explicitly stated, and vice versa.

[0048] The terms “first,” “second,” “third,” “fourth,” etc., in the specification and claims are used to distinguish between similar components and are not necessarily used to describe a specific sequential or chronological order. It should be understood that any such terms are interchangeable under appropriate circumstances so that the embodiments described herein may operate, for example, in an order other than those illustrated or otherwise described herein. Similarly, where a method is described herein as comprising a series of steps, the order of the steps presented herein is not necessarily the only order in which such steps may be performed, and certain steps among those described may be omitted, and / or certain other steps not described herein may be added to the method.

[0049] As used herein, comparative terms such as “increased,” “decreased,” “better,” “worsened,” “higher,” “lower,” “enhanced,” “maximized,” and “minimized” refer to characteristics of a device, component, composition, or activity that differ in a measurable manner from other devices, components, compositions, or activities in the surrounding or adjacent area, similarly located, within a single device or composition, or within multiple corresponding devices or compositions, within a group or class, or within multiple groups or classes, or in terms of known prior art.

[0050] As used herein, the term “coupled” is defined as being connected directly or indirectly in a chemical, mechanical, electrical, or non-electrical manner. Objects described herein as “adjacent” to each other may be in physical contact with each other, very close to each other, or in the same general area or scope, depending on the context in which the phrase is used. Where the phrases “in one embodiment” or “in one aspect” appear herein, they do not necessarily refer to the same embodiment or aspect.

[0051] As used herein, the term “substantially” refers to the complete or near-complete range or degree of an action, feature, characteristic, state, structure, item, or result. For example, an object that is “substantially” enclosed means that it is either completely enclosed or nearly completely enclosed. The exact acceptable degree of deviation from absolute completeness may, in some cases, depend on the specific circumstances. However, generally speaking, approaching completeness results in the same overall result as if absolute and total completeness were attained. The use of “substantially” is equally applicable when used in a negative sense to refer to the complete or near-complete absence of an action, feature, characteristic, state, structure, item, or result. For example, a composition that is “substantially” particle-free may either be completely particle-free or nearly particle-free to the extent that its effect is the same as if the particles were completely absent. In other words, a composition that is “substantially” particle-free may still contain such an item in practice, provided that its measurable effect is not present.

[0052] Where used herein, the term “about” is used to give flexibility to the endpoints of a numerical range by specifying that a given value may be “slightly above” or “slightly below” the endpoint. Unless otherwise specified, the use of the term “about” in reference to a particular number or numerical range should be understood as supporting such numerical term or numerical range without the term “about.” For example, for convenience and brevity, the numerical range “about 50 angstroms to about 80 angstroms” should also be understood as supporting the range “50 angstroms to 80 angstroms.” Furthermore, it should be understood herein that actual numerical values ​​are supported, even when the term “about” is used in conjunction with them. For example, the statement “about” 30 should be interpreted as supporting not only values ​​slightly above and slightly below 30, but also the actual numerical value of 30.

[0053] Where used herein, multiple items, structural elements, components, and / or materials may be presented in common lists for convenience. However, these lists should be interpreted as if each member of the list were individually identified as a separate and unique member. Therefore, no individual member of such a list should be interpreted as being substantially equivalent to any other member of the same list, based solely on its presentation within a common group, unless otherwise indicated.

[0054] Concentration, quantity, level, and other numerical data may be expressed or presented in range form as herein. Such range forms are used solely for convenience and brevity, and should therefore be interpreted flexibly to include not only the numbers explicitly listed as limits to the range, but also all individual numbers, subranges, or decimal units contained within that range, as if each number and subrange were explicitly listed. For example, the numerical range "approximately 1 to approximately 5" should be interpreted to include not only the explicitly listed values ​​of approximately 1 to approximately 5, but also the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 2, 3, and 4, as well as subranges such as 1 to 3, 2 to 4, and 3 to 5, and 1, 2, 3, 4, and 5 individually. The same principle applies to ranges that list only one number as the minimum or maximum value. Furthermore, such interpretations should apply regardless of the breadth or characteristics of the range described.

[0055] Throughout this specification, any reference to “an example” means that the particular characteristic, structure, or feature described in relation to 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.

[0056] Embodiment Many molecular tests for pathogens (e.g., Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19) can be limited to the laboratory, and therefore have a significant delay time (>24 hours) before results can be obtained, hindering their adoption in point-of-care settings. Several attempts have been made to develop point-of-care tests for SARS-CoV-2, but several limitations remain: i) scalability (the demand for tests is on the order of millions per week, but creating new tests at that scale is difficult), ii) sample handling (when using saliva, many tests still use extraction procedures), and iii) readability (because fluorescence is often used in molecular tests, a fluorescence reader is used to report the results).

[0057] Current testing methods can be overcome by using a point-of-care test that employs a paper-based device and reverse transcription loop-mediated isothermal amplification (RT-LAMP), which reports a color change in the presence of a pathogen (e.g., SARS-CoV-2) within 60 minutes using diluted saliva (e.g., 5% volume / volume in water) as a sample. RT-LAMP is a nucleic acid amplification technique that operates at a constant temperature, providing appropriate diagnostic performance, especially during the acute phase of infection. Because RT-LAMP can be operated at a constant temperature, it does not require expensive thermal cycling equipment. Furthermore, existing colorimetric reporters for LAMP products do not require a fluorescence reader. Therefore, this test is suitable for use in point-of-care settings and is suitable for use in public health emergencies due to its rapid development and scale-up capabilities.

[0058] RT-LAMP can be implemented in microfluidic paper-based analytical devices (μPADs) for detecting various pathogens (such as SARS-CoV-2), and image analysis can be performed using portable electronic devices to distinguish between positive and negative responses. In one example, high-contrast RT-LAMP responses on paper produce a visible color change. Furthermore, instead of using wax printing—which allows for precise alignment of the printing area and reagent distribution—polystyrene spacers can be used to prevent crosstalk between samples. Polystyrene spacers are suitable for roll-to-roll manufacturing to scale up production.

[0059] Nucleic acid-based COVID-19 diagnostic methods provide results using sample preparation. As disclosed herein, colorimetric detection of SARS-CoV-2 on paper can be performed with minimal sample preparation. This device can have the sensitivity and specificity to detect SARS-CoV-2 on paper without prior amplification. Other assays performed in solution may not be as scalable during preparation as paper-based assays. Furthermore, the assays disclosed herein use dilution steps that can be completed in seconds, while other assays use various steps to detect SARS-CoV-2, such as protease treatment, thermal inactivation, and / or RNA extraction (steps that take at least 10 minutes to complete and require additional equipment).

[0060] Sample collection and characterization for LAMP analysis Saliva possesses various physical, chemical, and antimicrobial properties that can create difficulties in relation to the LAMP reaction. For example, one physical property of saliva can dilute and remove organic acids from plaque, which can interfere with the LAMP reaction. Some chemical properties—electrolytes and buffering molecules that minimize pH changes—can also interfere with the LAMP reaction. Antimicrobial agents in saliva, such as mucin, amylase, lysozyme, and peroxidase enzymes, also present challenges. For example, peroxidase enzymes can form free radical compounds within bacterial cells, potentially inducing apoptosis-like death in these cells. However, such reactions can also create an unstable redox environment, potentially complicating the LAMP reaction.

[0061] With the above background in mind, a method 100 for preparing a saliva sample for loop-mediated isothermal amplification (LAMP) detection of a pathogen target is provided, as shown in the flowchart of Figure 1. Depending on the final signal output selected to indicate the test result, such as an optically detected pH-based color change, it may be desirable that the saliva in the sample does not cause a significant deviation of the overall sample pH from neutral. Various techniques and processes can be implemented to check or otherwise limit the buffering capacity or influence of saliva in the sample. Excessive buffering capacity may interfere with the pH variation used to detect pH-based color changes.

[0062] One method for reducing the buffering capacity of saliva is dilution. In one embodiment, such a method may include providing a certain amount of saliva from a test subject, as shown in block 110, and diluting the saliva with water to a degree that reduces the buffering capacity of the saliva while maintaining a concentration sufficient to enable the detection of the pathogen target, as shown in block 120.

[0063] The proteins present in saliva present additional challenges. For example, testing excessively viscous samples on a solid-based or solid-phase medium can be difficult. Slow flow rates in solid-based media can lead to longer reaction times, reduced uniformity of spreading, increased variability in results, and increased invalidity of results. For instance, if viscous saliva does not spread uniformly across a solid-based medium, color-based indications may be difficult to read. Reduced uniformity of spreading adds uncertainty to result interpretation and can increase variability. Interpretation of results may vary among technicians. In some cases, the color change may be ambiguous or nonexistent, making it impossible to interpret the results. Therefore, controlling the viscosity of saliva can prevent a variety of complex problems that may arise.

[0064] Therefore, in another embodiment, the method may further include reducing the viscosity of the saliva compared to its original viscosity. In one embodiment, the saliva can be reduced by dilution, filtration, or one or more of these in combination. In one embodiment, when reducing the viscosity of the saliva using dilution, the saliva can be diluted with water until the saliva-to-water ratio is about 1:1 to about 1:20. In another embodiment, the viscosity of the saliva can be diluted with water to a saliva-to-water ratio of about 1:1, 1:2, 1:4, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, or 1:20. In one embodiment, the saliva can be reduced by the optical density (OD) of the sample at 600 nm. 600 The solution can be diluted with water to a degree where the concentration is less than approximately 0.2. In one embodiment, the saliva may have a volume in the range of approximately 50 μl to approximately 100 μl. In another embodiment, the saliva sample may have a volume in the range of approximately 100 μl to approximately 1 ml.

[0065] In some cases, diluting the saliva sample can reduce the effects arising from the buffering capacity and viscosity of the saliva. Another method for reducing the effects of saliva viscosity involves filtration. In another embodiment, viscosity can be reduced using a filter with a rating between approximately 2 microns and 50 microns. In one example, the filter rating may be one or more of 2 microns, 5 microns, 8 microns, 10 microns, 15 microns, 20 microns, 25 microns, 40 microns, or 50 microns. In one embodiment, the filter rating may be an absolute micron rating, meaning the filter can remove at least about 98.7% of a given particle size. Filtration of saliva, as well as dilution, can also remove salivary proteins (e.g., mucin, amylase, lysozyme, and peroxidase enzymes) that may interfere with the LAMP reaction.

[0066] By dilution, filtration, or a combination of both, viscosity can be controlled to fall within a specific range. In yet another embodiment, viscosity can be reduced to such an extent that fluidity increases compared to the original viscosity, allowing it to pass through a solid-phase medium. In one example, the viscosity of saliva may range from about 1 centipoise (cP) to about 100 cP before dilution or filtration. In another example, the viscosity of saliva can be reduced to a range of about 1.0 cP to about 50 cP after dilution or filtration. In yet another example, the viscosity of saliva can be reduced to a range of about 1.0 cP to about 10 cP after dilution or filtration.

[0067] The pH range can also be adjusted to a desired level by filtering the saliva. For example, some pH indicators may display a color change within a specific pH range (e.g., 7.2 to about 8.6). Therefore, depending on the type of pH indicator used, the saliva can be filtered to the target range of the test sample. However, maintaining the target range of the test sample within physiological conditions can improve the uniformity of the LAMP reaction results. In one embodiment, the saliva can be filtered to the extent that the pH of the saliva sample is adjusted to the target range of the test sample. In one example, the target range of the test sample may include a pH range between about 7.2 and about 8.6. In another example, the target range of the test sample may include a pH range between about 7.6 and about 8.2.

[0068] When adjusting the target range of a test sample to a desired level, it may not be sufficient to detect the pH change (or other colorimetric indicator) in the LAMP reaction. In another example, the pH indicator can be made detectable by diluting saliva with water to such an extent that the buffering capacity of the composition decreases compared to the buffering capacity before dilution with water. In one example, buffering capacity can be defined as the ability of a solution (e.g., saliva, water, or saliva diluted with water) to resist a change in pH when an acid or base is added. In one example, buffering capacity can be defined as the amount of strong acid or strong base added to 1 liter of solution to change the pH by 1 unit, i.e., the gram equivalent. In one embodiment, the buffering capacity of saliva before dilution with water may be between 0.03 mg / ml and about 0.30 mg / ml, and the buffering capacity of the diluted saliva after dilution with water may be between about 0.003 mg / ml and about 0.03 mg / ml. In another example, the buffering capacity of the diluted saliva may be about 5 mM, 4 mM, 3 mM, 2 mM, or less than 1 mM.

[0069] The water used to dilute the sample must be free of any contaminants or properties that could interfere with the LAMP reaction. For example, if the pH is too acidic, the LAMP reaction may not be detectable if the pH-based indicator change is hindered. In one embodiment, saliva can be diluted with water, which may have a pH greater than approximately 6.0. In another embodiment, the water may have a pH less than approximately 8.0. In one example, the water may be molecular-grade water that is substantially free of contaminants such as RNase and DNase. RNase degrades RNA in the saliva to be detected, and DNase degrades DNA formed during the LAMP reaction. In another example, the saliva sample may essentially consist of saliva and water.

[0070] Minimizing the presence of undesirable salivary proteins can be achieved using specific saliva collection methods. In one embodiment, saliva can be collected using one or more of a sponge-based collection method or a sloshing collection method. When saliva is collected using a sponge-based collection method, there may be an advantage in that mucin and high molecular weight proteins are essentially filtered out of the saliva because they are not absorbed by the sponge. This can reduce the viscosity of the saliva when used on a solid-based medium, potentially improving the speed, uniformity, and reliability of the saliva. When saliva is collected using the sloshing method, unfiltered saliva may have a higher viscosity, leading to reduced absorption and distribution on a solid-based medium. As a result, in some embodiments, when saliva is collected via sloshing, it can be subsequently filtered to remove mucin and other debris and reduce its viscosity.

[0071] The selected pathogen target can be detected from saliva. In one embodiment, the pathogen target may be one or more of the following: viral pathogens, bacterial pathogens, fungal pathogens, protozoan pathogens, or combinations thereof. The pathogen target in saliva can be detected if nucleic acids from the pathogen target can be released from the cell wall, cell membrane, protein membrane, etc.

[0072] More specifically, in one embodiment, the pathogen target may be a viral target. In several embodiments, the viral target may be H1N1, H2N2, H3N2, H1N1pdm09, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome (MERS), influenza, or a combination thereof.

[0073] Viral targets can be selected from many different virus species. For example, viral targets may include human coronavirus 229E, human coronavirus OC43, human coronavirus HKU1, human coronavirus NL63, MERS coronavirus, human respirovirus 1, human rubravirus 2, human respirovirus 3, human rubravirus 4, human enterovirus, human respiratory virus, rhinovirus A, rhinovirus B, rhinovirus C, or combinations thereof.

[0074] The viral target may also be in the form of influenza. In one embodiment, the influenza may be influenza A, influenza B, influenza C, or influenza D. In one embodiment, the viral target may be a virus selected from the order Nidovirale. In one embodiment, the viral target may be selected from the genera Alpha, Beta, Gamma, or Delta of the order Nidovirale.

[0075] Various families of viruses can be detected. In one embodiment, the viral target may be a DNA virus selected from the group of families including Adenoviridae, Papovaviridae, Parvoviridae, Herpesviridae, Poxviridae, Anelloviridae, Pleolipoviridae, etc., and combinations thereof. In another embodiment, the viral target may be an RNA virus selected from the group of families including Reoviridae, Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Coronaviridae, Astroviridae, Bornaviridae, etc., and combinations thereof. In yet another embodiment, the viral target may be a reverse transcription virus selected from the group of families including Retroviridae, Caulimoviridae, Hepadnaviridae, etc., and combinations thereof.

[0076] More generally, the viral target may be a virus classified by the Baltimore classification. In one embodiment, the viral target may be an RNA virus (e.g., influenza A, Zika, hepatitis C). In one embodiment, the viral target may be a DNA virus (e.g., Epstein-Barr, smallpox). In one embodiment, the viral target may be a positive-sense RNA virus (e.g., hepatitis A, rubella). In one embodiment, the viral target may be a negative-sense RNA virus (e.g., Ebola hemorrhagic fever, measles, mumps). In another embodiment, the viral target may be a dsDNA virus (e.g., varicella, herpes), an ssDNA virus, a dsRNA virus (e.g., rotavirus), a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus (e.g., retrovirus), or a ds-DNA-RT virus (e.g., hepatitis B).

[0077] In addition to viral targets, in another embodiment, pathogen targets may be bacterial targets. In some examples, bacterial targets can be selected from genera including Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, Yersinia, and combinations thereof. In another example, bacterial targets can be selected from species including Actinomyces israelii, Bacillus anthracis, Bordetella pertussis, B. abortus, B. canis, B. melitensis, B. suis, Corynebacterium diphtheriae, E. coli, Enterotoxigenic E. coli, Enteropathogenic E. coli, Enteroinvasive E. coli, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, M. tuberculosis, Mycoplasma pneumoniae, N. meningitidis, S. typhi, S. sonnet, S. dysenteriae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Vibrio cholerae, Yersinia pestis, and combinations thereof.In another embodiment, the pathogen target can be selected from species including Chlamydia pneumoniae, Pneumocystis jirovecii, Candida albicans, Pseudomonas aeruginosa, Staphylococcus epidermis, Streptococcus salivarius, and combinations thereof.

[0078] Pathogen targets can include various types of fungi. In one embodiment, the pathogen target may be a fungal target. In some examples, the fungal target may be selected from genera including Aspergillus, Histoplasma, Pneumocystis, Stachybotrys, etc., and combinations thereof. In another embodiment, the pathogen target may be a protist target. In some examples, the protist target may be selected from genera including plasmodium, trypanosomes, etc., and combinations thereof.

[0079] If the pathogen target in saliva contains RNA, the RNA can be reverse transcribed. Therefore, in another embodiment, LAMP detection may be reverse transcribed LAMP (RT-LAMP). In this example, cDNA can be generated from the target RNA using reverse transcriptase. The cDNA can be amplified to a detectable level. If the pathogen target can be detected directly from DNA, LAMP can be used to amplify the DNA to a detectable level without reverse transcribing the RNA back into DNA.

[0080] In another embodiment, the specific target nucleotide sequence 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 can be detected. Various types of diseases can be detected, including breast cancer, pancreatic cancer, colorectal cancer, ovarian cancer, gastrointestinal cancer, cervical cancer, lung cancer, bladder cancer, many types of carcinoma, salivary gland cancer, kidney cancer, liver cancer, lymphoma, leukemia, melanoma, prostate cancer, thyroid cancer, stomach cancer, or one or more of these in combination. For example, biomarkers for various types of diseases include alpha-fetoprotein, CA15-3 and CA27-29, CA19-9, CI-125, calcitonin, calretinin, carcinoembryonic antigen, CD34, CD99MIC2, CD117, chromogranin, chromosomes 3, 7, 17, and 9p21, cytokeratin, cesmin, epithelial membrane antigen, factor VIII, CD31FL1, glial fibrillary acidic protein, and gross cystic disease fluid protein. This can be detected by detecting target nucleotides corresponding to one or more of the following: protein, hPG80, HMB-45, human chorionic gonadotropin, immunoglobulin, inhibin, keratin, lymphocyte markers, MART-1, MyoD1, muscle-specific actin, neuronal filaments, neuron-specific enolase, placental alkaline phosphatase, prostate-specific antigen, PTPRC, S100 protein, smooth muscle activity, synaptophysin, thymidine kinase, thyroglobulin, thyroid transcription factor-1, tumor M2-PK, vimentin, etc., or combinations thereof.

[0081] In another embodiment, the test sample composition for loop-mediated isothermal amplification (LAMP) analysis may contain a sufficient amount of test saliva to detect pathogen targets via LAMP analysis, combined with an amount of water that reduces the buffering capacity of the saliva. In one embodiment, the viscosity of the composition may be about 1.0 cP to about 50 cP. In another embodiment, the pH of the composition may be about 7.2 to about 8.6. Selecting viscosity and pH within these respective ranges facilitates pH changes and, therefore, color changes due to pH-based indicators.

[0082] Saliva can be diluted with water to bring its viscosity and pH within the ranges listed above. In one embodiment, saliva can be mixed with water in a saliva-to-water ratio of approximately 1:1 to approximately 1:20. In another embodiment, the saliva-to-water ratio may be approximately 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, or 1:20. In another embodiment, the saliva can be diluted with water to bring its viscosity and pH within the ranges listed above. 600 It can be combined with an amount of water up to such an extent that the ratio becomes less than 0.2.

[0083] To ensure that the amount of saliva contains a detectable amount of virus, the amount of saliva collected may be greater than the threshold amount. In one embodiment, the saliva may have a volume in the range of about 50 μl to about 100 μl. In another embodiment, the saliva sample may have a volume in the range of about 100 μl to about 1 ml.

[0084] Saliva may also possess various chemical properties (e.g., pH and buffering capacity) that can facilitate the LAMP reaction. In one embodiment, the water may have a pH greater than about 6.0 and may be substantially free of contaminants such as RNase and DNase. In another embodiment, the water may have a pH less than about 8.0 and may be substantially free of contaminants. In yet another embodiment, the composition may consist essentially of saliva and water. In one embodiment, the buffering capacity of the composition may be between about 0.003 mg / ml and about 0.03 mg / ml. In yet another example, the buffering capacity of the composition may be less than about 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM.

[0085] As previously stated, pathogen targets may include viral pathogens, bacterial pathogens, fungal pathogens, or protozoan pathogens. Pathogen targets may also be viral targets. Based on the Baltimore classification of viruses, in another embodiment, viral targets may include dsDNA viruses, ssDNA viruses, dsRNA viruses, positive-strand ssRNA viruses, negative-strand ssRNA viruses, ssRNA-RT viruses, or ds-DNA-RT viruses. In another embodiment, viral targets may include H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.

[0086] Reagent composition A variety of reagents can be used in LAMP analysis, depending on the test medium, readout type, and the overall environment of the designed system. Furthermore, reaction components such as primers and enzymes can be selected considering the specific target nucleotide sequence to be detected, the organism to be identified, etc. In addition, other needs such as the characteristics of the test environment (liquid environment, anhydrous environment, enclosure, substrate, etc.) and storage in a stable state can also be taken into consideration when selecting the specific reagents involved in the reaction that underlies LAMP analysis.

[0087] In one embodiment, a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid-phase medium may include one or more target primers, a DNA polymerase, and a resolubilizer. In one embodiment, the composition may be substantially free of non-pH-sensitive agents that can discolor the solid-phase medium.

[0088] When performing LAMP analysis on a solid-phase medium, the reagent concentration can be increased compared to LAMP analysis on a liquid-phase medium. In one embodiment, the concentration of DNA polymerase used on a solid-phase medium may be at least twice the concentration of DNA polymerase used on a liquid medium. In another embodiment, the concentration of DNA polymerase used on a solid-phase medium may be at least three times the concentration of DNA polymerase used on a liquid medium. In one example, the concentration of DNA polymerase used on a solid-phase medium may be about 300 U / mL to about 1000 U / mL. In another example, the concentration of DNA polymerase used on a solid-phase medium may be about 600 U / mL to about 1000 U / mL. In yet another example, the concentration of DNA polymerase used on a solid-phase medium may be about 620 U / mL to about 680 U / mL.

[0089] When LAMP analysis involves reverse transcriptase LAMP (RT-LAMP), the composition may further contain reverse transcriptase. Reverse transcriptase can be useful for detecting RNA-based viruses. In one embodiment, the concentration of reverse transcriptase when used on a solid-phase medium may be at least twice the concentration of reverse transcriptase when used on a liquid medium. In another embodiment, the concentration of reverse transcriptase may be at least three times the concentration of reverse transcriptase when used on a liquid medium. In one example, the concentration of reverse transcriptase when used on a solid-phase medium may be about 200 U / mL to about 600 U / mL. In another example, the concentration of reverse transcriptase when used on a solid-phase medium may be about 250 U / mL to about 500 U / mL. In yet another example, the concentration of reverse transcriptase when used on a solid-phase medium may be about 290 U / mL to about 310 U / mL.

[0090] In addition to target primers, DNA polymerase, and reverse transcriptase, the composition may include a resolubilizer. The resolubilizer may help rehydrate the LAMP reagent on a solid-based medium when a saliva sample is deposited on a solid-based medium. In one embodiment, the resolubilizer may be a surfactant. For example, the resolubilizer may include bovine serum albumin (BSA), casein, polysorbate 20, or a combination thereof. BSA and casein promote the resolubilization of DNA polymerase, reverse transcriptase, and other related enzymes when rehydrating the dried reagent. Polysorbate 20 is a surfactant that also helps resolubilize the dried reagent. In one example, the concentration of the resolubilizer may be about 0.05% to about 5% by weight when used on a solid-phase medium. In another example, the concentration of the resolubilizer may be about 0.5% to about 3% by weight. In yet another example, the concentration of the resolubilizer may be about 0.5% to about 1.5% by weight.

[0091] The composition may further include reaction-accelerating agents, sensitivity-enhancing agents, or combinations thereof. For example, BSA may be included to accelerate the reaction and enhance sensitivity. However, the inclusion of BSA can induce pH fluctuations, potentially impairing the readability of the results. Therefore, in some examples, the resolubilizing agent may include casein, polysorbate 20, or combinations thereof.

[0092] Volatile agents may interfere with the LAMP reaction. For example, volatile compounds may ionize into multiple ions, one of which may have a low boiling point. When the low boiling point ion evaporates, the remaining ions may react further. Some of these further reactions may include redox reactions, acid-base reactions, or other reactions that may affect the interpretation of the pH-based signal. In one embodiment, the composition may be substantially free of volatile agents. In one example, removing a volatile agent can increase the color contrast of the solid-based medium and shorten the reaction time compared to when the volatile agent is present. In one embodiment, the composition may contain less than one or more of the volatile agent in amounts of 1.0% by weight, 0.5% by weight, 0.1% by weight, or 0.01% by weight.

[0093] Volatile agents can cause instability in solid-based media. In some cases, in LAMP reactions involving volatile compounds such as ammonium sulfate, the solid-based media can become unstable if ammonium ions partially convert ammonium sulfate to ammonium, which then volatilizes, leaving behind sulfate ions. The sulfate ions become sulfuric acid, which can lower the pH and affect the reading of pH-based indicators (for example, phenol red indicator may change from red to yellow even if no LAMP reaction is occurring). Replacing ammonium sulfate with betaine can stabilize the solid-based media by preventing discoloration due to non-LAMP reactions and preventing discoloration during storage.

[0094] Therefore, by reducing the presence of volatile agents in the composition, the degree of interference between the LAMP reaction and its readings by pH-based indicators can be reduced. In one embodiment, the LAMP composition may include non-volatile agents, such as neutral-charged low-molecular-weight quaternary ammonium compounds, neutral-charged low-molecular-weight amide compounds, or combinations thereof. Examples of non-volatile agents include N-formylurea, urea, L-asparagine, trimethylglycine (betaine), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 3-(1-pyridinio)-1-propanesulfonate (NDSB-201), N-methylurea, acetamide, propionamide, isobutylamide, piracetam, 1,3-dimethylurea, 1,1-dimethylurea, glycolamide, 2-chloroacetamide, succinimide, 2-imidazolidone, choline chloride, acetylcholine chloride, betanethyl chloride, L-Cal Examples include, but are not limited to, intramolecular salts of nithine, O-acetyl-L-carnitine hydrochloride, 4-(cyclohexylamino)-1-butanesulfonic acid (CABS), dimethylethylammonium propanesulfonate (NDSB-195), 3-(1-methylpiperidinium)-1-propanesulfonate (NDSB-221), 3-(benzyldimethylammonio)propanesulfonate (NDSB-256), and dimethyl-2-hydroxyethylammonium-1-propanesulfonate (NDSB-211), or combinations thereof.

[0095] In one example, the concentration of a non-volatile agent, including a neutral-charged, low-molecular-weight quaternary ammonium or a neutral-charged, low-molecular-weight amide compound, may range from approximately 1 mM to approximately 200 mM when used in a solid-phase medium. In another example, the concentration of the non-volatile agent may range from approximately 10 mM to approximately 50 mM when used on a solid-phase medium. In yet another example, the concentration of the non-volatile agent may range from approximately 15 mM to approximately 25 mM when used on a solid-phase medium.

[0096] In addition to volatile agents, desiccants can also interfere with the LAMP reaction. Desiccants can destabilize reagents in solid-based media by retaining excess water, slowing or preventing drying. In one embodiment, the composition may be substantially desiccant-free. In some examples, LAMP reactions containing desiccants such as glycerol may contribute to the instability of reagents in solid-based media because the desiccant can attract water. In one example, a desiccant can absorb more than about 10% by weight at a relative humidity (RH) of about 40% to about 90% at 25°C. Examples of desiccants include, but are not limited to, one or more of glycerol, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate, or combinations thereof. In one embodiment, the composition may contain less than one or more of the desiccant in amounts of 1.0% by weight, 0.5% by weight, 0.1% by weight, or 0.01% by weight.

[0097] Several additional agents may be included to prevent carryover contamination of previous LAMP reaction products, primer dimerization, nonspecific amplification, or a combination thereof. Carryover contamination can be prevented by including deoxyuridine triphosphate (dUTP), uracil DNA glycosylase (UDG), or a combination thereof in the LAMP reaction product. These agents can catalyze the release of free uracil from uracil-containing single-stranded or double-stranded DNA.

[0098] Some pH-based indicators with antioxidant properties, such as phenol red, have been found to improve contrast and uniformity compared to other pH-based indicators with lower levels of antioxidant activity. In one embodiment, the composition may further contain an antioxidant. In one example, the concentration of the antioxidant may be about 0.1 mM to about 1 mM when used on a solid-phase medium. In another example, the concentration of the antioxidant may be about 0.2 mM to about 0.8 mM when used on a solid-phase medium. In yet another example, the concentration of the antioxidant may be about 0.2 mM to about 0.3 mM when used on a solid-phase medium. Antioxidants can stabilize reagents on solid-based media by preventing redox reactions.

[0099] N-acetylcysteine, hydroxytyrosol (HXT), superoxide dismutase (SOD), catalase, vitamin A, vitamin C, vitamin E, coenzyme Q10, manganese, iodide, melatonin, alpha-carotene, astaxanthin, beta-carotene, canthaxanthin, cryptoxanthin, lutein, lycopene, zeaxanthin, apigenin, luteolin, tangeretin, isorhamnetin, kaempferol, myricetin, proanthocyanidin, quercetin, eriodictiol, hesperetin, naringenin, catechin, gallocatechin, epicatechin, epigallocatechin A variety of antioxidants can be used, including but not limited to quinine, theaflavin, thearbigin, daidzein, genistein, glycitein, resveratrol, pterostilbene, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, chicoric acid, chlorogenic acid, cinnamic acid, ellagic acid, ellagitannin, gallic acid, gallotannin, rosmarinic acid, salicylic acid, curcumin, flavonolignan, xanthones, eugenol, capsaicin, bilirubin, citric acid, oxalic acid, phytic acid, R-alpha-lipoic acid, or combinations thereof.

[0100] While pH-based indicators have been discussed so far, other indicators can also be used. In one embodiment, the composition may further contain an indicator. In one example, the indicator may be a pH-based indicator such as phenol red when used with a solid-based medium. Phenol red has antioxidant properties not found in some other dyes. The phenol red molecule is a conjugated bond system, which may also contribute to its antioxidant properties. In one example, the concentration of the indicator may be about 0.1 mM to about 1 mM when used on a solid-phase medium. In another example, the concentration of the indicator may be about 0.2 mM to about 0.8 mM when used on a solid-phase medium. In yet another example, the concentration of the indicator may be about 0.2 mM to about 0.3 mM when used on a solid-phase medium.

[0101] Several other indicators can also provide appropriate colorimetric signals. In another example, the indicator may be one or more of (i) a magnesium colorimetric indicator, (ii) a pH colorimetric indicator, or (iii) a DNA intercalation colorimetric indicator. If the indicator is a magnesium colorimetric indicator, the magnesium concentration should be monitored to maintain it within the range of approximately 0.01 mM to approximately 2 mM. The magnesium concentration should also be monitored to prevent interference with DNA polymerase. Magnesium, a cofactor of DNA polymerase, can interfere with DNA polymerase if the magnesium concentration is outside the target range.

[0102] In the LAMP reaction, various types of target primers can also be used. Some target primers may include about four or six primers that can each target six or eight regions in the genome. In one embodiment, the concentration of the target primers may be about 0.05 μM to about 5 μM when used on a solid-phase medium. In another example, the concentration of the target primers may be about 0.1 μM to about 3 μM when used on a solid-phase medium. In yet another example, the concentration of the target primers may be about 0.2 μM to about 1.6 μM when used on a solid-phase medium.

[0103] Target primers can be selected to target the genomes of various pathogens. In one embodiment, target primers can target pathogens that may include viral pathogens, bacterial pathogens, fungal pathogens, or protozoan pathogens. In another embodiment, the pathogen target may be a viral target. In another embodiment, the viral target may include dsDNA viruses, ssDNA viruses, dsRNA viruses, positive-strand ssRNA viruses, negative-strand ssRNA viruses, ssRNA-RT viruses, or ds-DNA-RT viruses. In another embodiment, the viral target may include H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2. In short, target primers can target virtually any pathogen target, in particular the target pathogens disclosed herein.

[0104] If a solid-based medium contains an excess amount of volatile agents, oxidizing agents, pH interferants, magnesium interferants, or a combination thereof, the color of the solid-based medium may be affected in the absence of amplification by the LAMP reaction. To address this problem, in another embodiment, the composition may contain a non-coloring additive. In one embodiment, the concentration of the non-coloring additive may be about 0.01 mM to about 1 M when used on a solid-phase medium. In another example, the concentration of the non-coloring additive may be about 10 mM to about 500 mM when used on a solid-phase medium. In yet another example, the concentration of the non-coloring additive may be about 200 mM to about 400 mM when used on a solid-phase medium.

[0105] There are various non-coloring additives that can preserve the color of a solid-based medium when amplification by the LAMP reaction is absent, and potentially enhance contrast when amplification by the LAMP reaction occurs. For example, non-coloring additives may include one or more of the following: sugars, buffers, or combinations thereof.

[0106] In one example, non-coloring additives such as sugars can stabilize solid-based media and prevent discoloration under long-term storage conditions. For example, trehalose can maintain enzyme stability under freeze-drying conditions or when dried at ambient temperature. In one embodiment, the sugar may include one or more of glucose, sucrose, trehalose, dextran, or combinations thereof. In one embodiment, the sugar concentration may be about 0.01 mM to about 1 M when used on a solid-phase medium. In another example, the sugar concentration may be about 10 mM to about 500 mM when used on a solid-phase medium. In yet another example, the sugar concentration may be about 200 mM to about 400 mM when used on a solid-phase medium.

[0107] The LAMP reaction may also include other reagents. In one embodiment, the composition may include one or more enzymes, nucleic acids, or combinations thereof. In one example, the enzyme may be an RNase inhibitor or a DNase inhibitor. Including an RNase inhibitor can slow down the degradation of the RNA target and increase the detection limit. Including a DNase inhibitor can slow down the degradation of the DNA target and increase the detection limit. In one embodiment, the composition may include carrier DNA or carrier RNA. The carrier DNA or carrier RNA can provide a decoy substrate that sequesters the activity of DNase or RNase, respectively. In another example, a selected amount of guanidine hydrochloride can stimulate the denaturation and exposure of RNA molecules, thereby further stabilizing the LAMP reaction.

[0108] In one embodiment, when used on a solid-phase medium, the concentration of the RNase or DNase inhibitor may be approximately 0.01 μL to approximately 5 μL per 1 mL of saliva sample. In another embodiment, when used on a solid-phase medium, the concentration of the RNase or DNase inhibitor may be approximately 0.1 μL to approximately 1 μL per 1 mL of saliva sample. In yet another embodiment, when used on a solid-phase medium, the concentration of the RNase or DNase inhibitor may be approximately 0.5 μL to approximately 1.5 μL per 1 mL of saliva sample.

[0109] In one embodiment, the concentration of carrier RNA or carrier DNA may be about 0.01 ng / μL to about 10 ng / μL when used on a solid-phase medium. In another example, the concentration of carrier RNA or carrier DNA may be about 0.1 ng / μL to about 1 ng / μL when used on a solid-phase medium. In yet another example, the concentration of carrier RNA or carrier DNA may be about 0.2 ng / μL to about 0.4 ng / μL when used on a solid-phase medium.

[0110] In addition to the above, several other agents or components can be used in compositions suitable for carrying out the LAMP reaction described herein. For example, in another embodiment, the composition may further include isotonic agents, pH adjusters, preservatives, water, etc., or combinations thereof. Furthermore, these components / agents can be used to specifically provide the composition with a range of desired properties. In one embodiment, the tonicity of the composition may be about 250 to about 350 milliosmoles / liter (mOsm / L). In another embodiment, the tonicity of the composition may be about 270 to about 330 mOsm / L. The isotonic agent can be present in the composition in various amounts. In one embodiment, the isotonic agent may have a concentration of about 0.1% by weight, about 0.5% by weight, or about 1% to about 2% by weight, about 5% by weight, or about 10% by weight in the composition.

[0111] The composition should be substantially free of pH interfering reagents, but a pH adjuster can be used to select the initial pH of the composition before the LAMP reaction. Furthermore, a pH adjuster can also be used if its influence can be compensated for when interpreting the results of the LAMP reaction. Non-limiting examples of pH adjusters include numerous acids, bases, and combinations thereof, such as hydrochloric acid, phosphoric acid, citric acid, sodium hydroxide, potassium hydroxide, and calcium hydroxide. A pH adjuster can be used to provide the composition with an appropriate pH. In one embodiment, the pH may be about 5.5 to about 8.5. In another embodiment, the pH may be about 5.8 to about 7.8. In yet another embodiment, the pH may be about 6.5 to about 7.8. In yet another example, the pH may be about 7.0 to about 7.6. The pH adjuster can be present in the composition in various amounts. In one embodiment, the pH adjuster may have a concentration of about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, or about 0.5% to about 1% by weight, about 2% by weight, about 5% by weight, or about 10% by weight in the composition.

[0112] The shelf life of a composition can be extended by using preservatives. Non-limiting examples of preservatives include benzalkonium chloride (BAK), cetrimonium, sodium perborate, ethylenediaminetetraacetic acid (EDTA) and its various salt forms, and chlorobutanol. Preservatives can be present in the composition in various amounts. In one embodiment, the preservative may be present in the composition at concentrations of about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, or about 0.05% to about 0.1% by weight, about 0.25% by weight, about 0.5% by weight, or about 1% by weight.

[0113] In another embodiment, as shown in Figure 2, Method 200 for LAMP analysis on a solid-phase medium may include providing an assembly of a solid-phase medium and a reaction composition in combination with the solid-phase medium, such as any of the components or compositions listed herein, as shown in Block 210. In one embodiment, the Method may include depositing a biological sample on the solid-phase medium, as shown in Block 220. In another embodiment, the Method may include heating the assembly to a temperature sufficient to facilitate the LAMP reaction, as shown in Block 230.

[0114] In one embodiment, the biological sample may be one or more of the following: saliva, mucus, blood, urine, feces, sweat, breath condensate, etc., or a combination thereof. In another embodiment, the biological sample may be saliva. In one embodiment, the method may include the detection of a viral pathogen. In one embodiment, the viral pathogen may be a pathogen as disclosed herein. In another embodiment, the LAMP analysis may be reverse transcriptase LAMP (RT-LAMP).

[0115] In another embodiment, the isothermal temperature sufficient to promote the LAMP reaction may be in the temperature range of about 50°C to about 70°C. In another embodiment, the isothermal temperature sufficient to promote the LAMP reaction may be in the temperature range of about 60°C to about 70°C. In yet another embodiment, the isothermal temperature sufficient to promote the LAMP reaction may be in the temperature range of about 60°C to about 65°C. The isothermal temperature can be selected based on the activity of one or more of the DNA polymerase, reverse transcriptase, or a combination thereof.

[0116] In another example, the temperature sufficient to promote the LAMP reaction may be in the range of approximately 60°C to 70°C. In yet another example, isothermal temperatures may be within a range of less than 5 degrees Celsius.

[0117] In another embodiment, a system for performing LAMP analysis may have a configuration as described in this disclosure. In another embodiment, the system may include a solid-phase medium on which a composition is deposited.

[0118] Maximizing the output of the pH-sensitive signal. When performing a LAMP reaction, various indicators can be used to read the reaction results. Three types of colorimetric indicators are magnesium colorimetric indicators, pH colorimetric indicators, and DNA intercalate colorimetric indicators. Because magnesium can be a cofactor of DNA polymerase and its concentration must be strictly controlled, magnesium-based indicators may face various limitations when used in conjunction with LAMP reactions. DNA intercalate indicators may also face limitations depending on the number of variables involved. All three indicators can be used in LAMP reactions, but pH-based indicators may have fewer variables affected.

[0119] In one embodiment, a composition for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal may include a pH-sensitive dye and a plurality of non-interfering LAMP reagents. In one embodiment, the LAMP analysis may be reverse transcription LAMP (RT-LAMP).

[0120] The selection of pH-sensitive dyes can depend on various factors, including the colorimetric range correlated with pH, ​​the degree of contrast between color changes, the pH level at which color changes occur, the uniformity of color changes, and the reproducibility of color changes. For example, phenol red may have a colorimetric range between approximately 6.8 and 7.4 pH. Below approximately 6.8 pH, phenol red changes to yellow, and above approximately 7.4 pH, phenol red may change to red. The degree of difference between yellow and red is easily discernible, and pH changes can occur at pH levels that mimic physiological conditions.

[0121] In one embodiment, the pH-sensitive dye may be a pH indicator that exhibits a color change around pH 6.5 to achieve a consistent and contrasting color change (e.g., phenol red). In one embodiment, the pH-sensitive dye may be phenol red, litmus, bromothymol blue, nitrazine yellow, cresol red, curcumin, brilliant yellow, m-cresol purple, α-naphtholphthalein, phenolphthalein, neutral red, acid fuchsin, azolithomine, or a combination thereof. In one embodiment, the concentration of the pH-sensitive dye may be about 0.1 mM to about 1 mM when used on a solid-phase medium. In another example, the concentration of the pH-sensitive dye may be about 0.2 mM to about 0.8 mM when used on a solid-phase medium. In yet another example, the concentration of the pH-sensitive dye may be about 0.2 mM to about 0.3 mM when used on a solid-phase medium.

[0122] To maximize pH-sensitive signal output, the LAMP reaction must be substantially free of reagents that introduce uncertainty into the signal by interfering with the LAMP reaction (e.g., by interfering with DNA polymerase) or by interfering with the signal from the LAMP reaction (e.g., the pH signal). In one embodiment, the non-interfering LAMP reagents may include DNA polymerase, reverse transcriptase, target primers, or a combination thereof. In another embodiment, the non-interfering LAMP reagents may be substantially free of volatile reagents, pH interfering reagents, magnesium interfering reagents, or a combination thereof.

[0123] For example, several non-interfering LAMP reagents may be substantially free of magnesium, ammonium sulfate, or ammonium carbonate. Magnesium as a cofactor for DNA polymerase should be closely monitored to ensure that the LAMP reaction proceeds as designed. Ammonium sulfate is ionized to an ammonium ion, which may leave behind sulfate ions that can react to form sulfuric acid. Ammonium carbonate is also ionized to an ammonium ion, which may leave behind carbonate ions that can react to form carbonic acid. Therefore, several non-interfering LAMP reagents should be substantially free of these substances.

[0124] Because volatile agents may react and leave behind compositions that form acids or bases that may interfere with the pH-dependent signal from the LAMP reaction, volatile agents should be kept to a minimum. For example, several non-interfering LAMP reagents may substantially contain no volatile agents, including but not limited to ammonium sulfate, ammonium carbonate, or combinations thereof. In one embodiment, the composition may contain less than one or more of the following amounts of volatile agents: 1.0% by weight, 0.5% by weight, 0.1% by weight, or 0.01% by weight.

[0125] Furthermore, any pH interfering reagent may interfere with the pH-dependent signal output if those pH interfering reagents are not compensated for. For example, several non-interfering LAMP reagents may substantially not contain pH interfering reagents, including, but not limited to, a number of acids, bases, and combinations thereof. In one embodiment, the composition may contain less than one or more of the following amounts of pH interfering reagents: 1.0% by weight, 0.5% by weight, 0.1% by weight, or 0.01% by weight.

[0126] Even when pH is monitored, interference with the LAMP reaction can adversely affect pH-dependent signal output. For example, magnesium, as a cofactor for DNA polymerase, can interfere with amplification by the LAMP reaction if its concentration is outside the selected range. In one example, several non-interfering LAMP reagents may be substantially free of magnesium interferants. Magnesium interferants include Mg 2+ Mg 1+ Examples of magnesium-containing agents include, but are not limited to, magnesium carbonate, magnesium chloride, magnesium citrate, magnesium hydroxide, magnesium oxide, magnesium sulfate, magnesium sulfate heptahydrate, or combinations thereof. In one embodiment, the composition may contain less than one or more of the following amounts: 1.0% by weight, 0.5% by weight, 0.1% by weight, or 0.01% by weight. In another example, the magnesium interferant may include a chelating agent that interferes with magnesium.

[0127] Even when pH is monitored and the LAMP reaction is functioning correctly, discoloration of the solid-phase medium can occur due to other factors such as long-term storage. In one embodiment, the composition may include a non-discoloring additive. In one example, the non-discoloring additive may include one or more of the following: sugars, buffers, inhibitors, etc., or combinations thereof. In one example, sugars can stabilize the solid-based medium and prevent discoloration under long-term storage conditions. In one embodiment, the sugar may include one or more of the following: glucose, sucrose, trehalose, dextran, etc., or combinations thereof.

[0128] In one embodiment, the sugar concentration may be approximately 0.01 mM to approximately 1 M when used on a solid-phase medium. In another example, the sugar concentration may be approximately 10 mM to approximately 500 mM when used on a solid-phase medium. In yet another example, the sugar concentration may be approximately 200 mM to approximately 400 mM when used on a solid-phase medium.

[0129] Buffers can facilitate the stabilization of the LAMP reaction by removing variability from saliva samples. In one example, the buffer may include one or more of the following: phosphate-buffered saline (PBS), Dulbecco's PBS, Alceber's solution, Tris-buffered saline (TBS), HEPES, BICINE, water, buffered salt solutions (BSS), such as Hanks BSS, Earl's BSS, Grey's BSS, Puck's BSS, Simm's BSS, Tyrode's BSS, BSS Plus, Ringer's lactate solution, saline (i.e., 0.9% saline), 1 / 2 saline, or combinations thereof. In one embodiment, the concentration of the buffer may be about 10 μM to about 20 mM when used on a solid-phase medium. In another example, the concentration of the buffer may be about 100 μM to about 10 mM when used on a solid-phase medium. In yet another example, the concentration of the buffer solution can range from approximately 100 μM to approximately 500 μM when used on a solid-phase medium.

[0130] Blockers can reduce the amount of RNase-based degradation, DNase-based degradation, or other enzymatic degradation. In one example, the blocker may include one or more of bovine serum albumin, casein, or a combination thereof. In one embodiment, the concentration of the blocker may be about 0.01% to about 5% by weight when used on a solid-phase medium. In another example, the concentration of the blocker may be about 0.01% to about 1% by weight when used on a solid-phase medium. In yet another example, the concentration of the blocker may be about 0.02% to about 0.06% by weight when used on a solid-phase medium.

[0131] Antioxidants can enhance the uniformity and contrast of pH-dependent signals on a solid-phase medium by eliminating variables related to oxidation reactions. For example, a composition may further contain the antioxidants disclosed herein.

[0132] In another example, the composition may further include a solid-phase medium. Examples of solid-phase media include, but are not limited to, one or more of the following: glass fibers, nylon, cellulose, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, polyester, hydrophilic polytetrafluoroethylene (PTFE), or combinations thereof.

[0133] Certain additives can enhance the stability and uniformity of the LAMP reaction. In one embodiment, the composition may include one or more of the enzymes, nucleic acids, or combinations thereof disclosed herein. For example, the enzyme may be an RNase inhibitor or a DNase inhibitor. In another embodiment, the composition may include carrier DNA or carrier RNA. The carrier DNA or carrier RNA may provide a decoy substrate that sequesters the activity of DNase or RNase, respectively.

[0134] In another example, a selected amount of guanidine hydrochloride can stimulate the denaturation and exposure of RNA molecules. In one embodiment, the concentration of guanidine hydrochloride may be about 1 mM to about 200 mM when used on a solid-phase medium. In another example, the concentration of guanidine hydrochloride may be about 10 mM to about 100 mM when used on a solid-phase medium. In yet another example, the concentration of guanidine hydrochloride may be about 20 mM to about 60 mM when used on a solid-phase medium.

[0135] Maximizing the pH-dependent output signal can also be used in conjunction with other embodiments disclosed herein. In one embodiment, a method for performing LAMP analysis using the pH-dependent output signal may include providing an assembly of a solid-phase medium and composition as described herein. This method may further include depositing a biological sample onto the solid-phase medium. This method may further include heating the assembly to a temperature sufficient to facilitate the LAMP reaction.

[0136] As disclosed herein, in one embodiment, the biological sample may be one or more of the following: saliva, mucus, blood, urine, feces, sweat, breath condensate, etc., or a combination thereof. In another embodiment, the biological sample may be saliva. In one embodiment, the method may include the detection of a viral pathogen. In one embodiment, the viral pathogen may be a pathogen otherwise disclosed herein.

[0137] In one example, the temperature sufficient to promote the LAMP reaction may be in the range of approximately 60°C to 70°C. In another example, isothermal temperatures may be within a range of less than 5 degrees Celsius.

[0138] In another embodiment, the isothermal temperature sufficient to promote the LAMP reaction may be in the temperature range of about 50°C to about 70°C. In another embodiment, the isothermal temperature sufficient to promote the LAMP reaction may be in the temperature range of about 60°C to about 70°C. In yet another embodiment, the isothermal temperature sufficient to promote the LAMP reaction may be in the temperature range of about 60°C to about 65°C. The isothermal temperature can be selected based on the activity of one or more of the DNA polymerase, reverse transcriptase, or a combination thereof.

[0139] In another embodiment, as shown in Figure 3, method 300 for maximizing the accuracy of the output signal in pH-dependent LAMP analysis may include providing a reagent mixture that minimizes discoloration from the signal output medium due to non-LAMP reactions, as shown in block 310. This method may further include performing the LAMP reaction, as shown in block 320. In one embodiment, this method may include controlling the generation of protons due to non-LAMP reactions. In another embodiment, this method may include controlling oxidation due to non-LAMP reactions.

[0140] In another embodiment, a method for maximizing the accuracy of the output signal in pH-dependent LAMP analysis may include substantially removing discoloration caused by non-LAMP reactions from the signal output medium.

[0141] In another embodiment, a method for maximizing the level of detection (LOD) in pH-dependent LAMP analysis may include substantially removing discoloration resulting from non-LAMP reactions from the signal output medium. In one embodiment, diluting saliva with water to 5-10% can increase color contrast and reduce sample variability without affecting the detection limit. In another example, filtering saliva with a filter separately disclosed herein can increase color contrast and reduce sample variability without affecting the detection limit. [Examples]

[0142] The following examples are provided to facilitate a clearer understanding of certain embodiments of the present invention and are not intended to limit them in any way.

[0143] LAMP paper analysis for virus targeting in diluted saliva samples Example 1 - DNase / RNase-free distilled water DNase / RNase-free distilled water is prepared by filtering it through a 0.1 μm membrane and then tested for DNase and RNase activity. DNase and RNase activity is tested according to the current United States Pharmacopeia (USP) monograph testing standards for water for injection (WFI). Once the absence of DNase, RNase, or protease activity is confirmed, the water is considered contaminant-free and can be used to prepare saliva samples.

[0144] Example 2 - Amplification in saliva As shown in Figure 4, nucleic acid sequence primers were designed to target RNaseP in saliva as a positive control to confirm nucleotide amplification from saliva samples. Figure 4A shows the fluorescence quantification RT-qLAMP results for a primer set targeting RNaseP POP7 in 18% saliva spiked with 105 genome equivalent doses / reaction of heat-inactivated SARS-CoV-2. Figure 4B shows the fluorescence quantification RT-qLAMP results for a primer set targeting RNaseP POP7 in water containing 0.2 ng of synthetic RNaseP POP7 RNA.

[0145] As shown in Figure 4A, 18% saliva spiked with 105 genome equivalent amounts of heat-inactivated SARS-CoV-2 for each reaction was analyzed. In the left figure, primer (RNaseP.I), designed to target RNaseP using the mRNA sequence of the POP7 gene encoding the p20 subunit of RNaseP, did not adequately detect low levels of RNaseP, and therefore no amplification occurred. In the center figure, primer (RNaseP.II) was able to detect levels of RNaseP without amplifying the control without a template, resulting in amplification, as shown by the blue line without overlapping with the black line. In the right figure, primer (RNaseP.III) dimerized, resulting in amplification in both the black and blue lines (e.g., amplification is shown by the black line for the control without a template).

[0146] As shown in Figure 4B, water containing 0.2 ng of synthetic RNaseP POP7 RNA was analyzed. In the left figure, primer (RNaseP.I) dimerized, resulting in amplification as shown by the blue and black lines (e.g., amplified the control without a template). In the center figure, primer (RNaseP.II) detected the level of RNaseP without amplifying the control without a template, resulting in amplification as shown by the blue line without overlapping with the black line. In the right figure, primer (RNaseP.III) amplified RNase P without amplifying the control without a template, resulting in amplification as shown by the blue line, but no amplification as shown by the black line.

[0147] Example 3 - Saliva Collection Device Depending on the type of saliva collection device, saliva samples can be easily obtained in the LAMP reaction. In some cases, operators may use protective equipment to protect themselves from pathogens that may be spread via droplets (e.g., aerosol viruses). Therefore, operators may wear personal protective equipment to protect themselves from accidental contact with aerosol viruses. Certain saliva collection devices can be self-administered by subjects under the guidance of a healthcare professional. Saliva collection devices have been proven effective and can be classified into two categories: sponge-based collection and drooling collection, as shown in Figures 5A and 5B.

[0148] The sponge collection device 500a uses a sponge-like collection pad 504 to absorb saliva and includes a sample volume appropriate indicator 512 to indicate that a sufficient amount has been collected. Once saturated, the sponge is inserted into a compression tube 506 and compressed against a filter to filter the saliva into the collection tube. The reason for this filtration is to filter out mucin and high molecular weight proteins from the saliva, significantly reducing the viscosity of the sample. As a result, the solid phase medium can take in and disperse the saliva in a faster, more uniform, and reliable manner. The sponge collection device 500a may also include a compression seal 508 that forms a seal with the compression tube on the compression tube 506; a handle 510 for compressing the compression tube 506; and a sample volume appropriate indicator 512 for identifying that a sufficient amount of saliva has been collected.

[0149] The salivary device 500b can provide unfiltered saliva having a viscosity that slows the absorption and dispersion of the sample. The salivary device 500b may include a collection funnel 522 for collecting saliva; an indicator line 528 to indicate that sufficient saliva has been collected; a collection tube 524 for collecting saliva; a tube cap 526; a volume indicator 530; and a tube cap housing 532.

[0150] With either type of collection device, the residual risk of exposure to the operator is minimized. With sponge-based devices, there is a hypothetical risk of aerosol release during the compression process, especially if the user performs the compression process unintentionally. To control the risk of exposure, a medical operator may perform this operation. The collection device may have a compression seal to prevent aerosol backflow. With saliva collection, there is a slight risk of the outside of the device being contaminated with airborne saliva, which could cause secondary contamination of the operator if not handled properly. In both cases, the risk of exposure is reduced if the patient self-collects the saliva sample.

[0151] Three commercially available saliva collection devices were selected and their effects on the RT-LAMP response in saliva were evaluated. The three devices are the "Saliva Sampler®" from StatSure Diagnostic Systems, Inc., the "Pure·SAL®" from Oasis Diagnostics, and the "Super·SAL®" also from Oasis Diagnostics. The StatSure Saliva Sampler® provides a tube containing a buffer (e.g., Buffer 2000) used to collect saliva from patients. The Super·SAL standardizes saliva collection by removing solid and mucous contaminants using a cylindrical absorbent pad and collection tube. The Pure·SAL operates in a similar mechanism but includes an additional filter in the collection tube to remove contaminants.

[0152] Saliva pH was measured from treated saliva samples, and subsequent colorimetric analysis and fluorescence RT-LAMP LOD assays were performed using the treated saliva samples. This data is shown in Figure 6A. This data shows the LoD in saliva treated using various saliva collection devices (Pure-Sal, Super-Sal, Stat-sure). The master mix was treated with 0.6 microliters of HCl. Pure·SAL® and Super·SAL saliva collection devices show a wider range of colorimetric responses to a wider range of concentrations (1 to 10,000 genome equivalents per reaction of heat-inactivated SARS-CoV-2).

[0153] Example 4 - Saliva Collection Process If the subject performs the self-test, the subject will collect a saliva sample under the guidance of a medical professional in a dedicated collection container that does not contain additives and is therefore safe for the subject to use. The amount of saliva collected will be approximately 100 μL. For example, insert a sponge sampler into the subject's mouth and collect saliva until the color of the sponge sampler's indicator changes. Next, insert the sponge sampler into a collection tube. Then, compress the sponge to squeeze out the saliva (approximately 100 μL) into the collection tube containing an amount of water to dilute the saliva. Dilute the saliva with water until the saliva-to-water ratio is approximately 1:1 to approximately 1:20. Transfer the saliva from the collection tube to the test site.

[0154] Example 5 - Effects of RNase inhibitors on saliva The effect of adding an RNase inhibitor to the RT-LAMP reaction was evaluated by adding an RNase inhibitor to untreated saliva at a concentration of 1 μL per 1 mL of saliva.

[0155] The effect of RNAsecure® (AM7006, Invitrogen®) on newly collected saliva (5%) was tested to determine its suitability as a single-step process for the point-of-care RT-LAMP reaction. 1× RNAsecure® was diluted from 25× stock using 1 ml of saliva. Treated saliva was used as a matrix, and heat-inactivated SARS-CoV-2 was spiked into a pH 7.6 Warmstart® colorimetric master mix with 40 mM guanidine hydrochloride and 0.3 ng / μl carrier DNA in a concentration range of 1000 to 62.5 copies / reaction. The RNAsecure®-treated RT-LAMP was incubated at 65°C to initiate the reaction. Untreated saliva under these conditions was tested as a control without RNAsecure®.

[0156] 5 μL of thermo-inactivated virus was diluted with 5% treated saliva (i.e., final reaction concentration) and added to the RT-LAMP reaction to provide the indicated concentration with a final reaction volume of 25 μL. In case of a negative reaction, 5 μL of treated saliva (final reaction concentration 5%) was added instead of the diluted thermo-inactivated virus to provide the same reaction volume of 25 μL. Heating was performed in an incubator at 65°C for 60 minutes. Colorimetric scans were acquired using a flatbed scanner before and after the RT-LAMP reaction. 1250 μL of NEB colorimetric master mix was supplemented with 0.5 μL of Antarctic Thermolabile and 3.5 μL of dUTP 25×. An RNase inhibitor (RNASecure®) was diluted with total saliva to a 1× concentration, and then the saliva was diluted to 5% and added to the reactant.

[0157] As shown in Figure 6B, RNAsecure® did not show a significant increase in the reaction product's Level of Discharge (LoD). In other words, the addition of the RNase inhibitor did not significantly increase the measurement parameters of the RT-LAMP reaction (e.g., reaction rate, false positive rate, or detection limit).

[0158] Example 6 - Frozen saliva sample In some cases, due to logistical and transportation requirements, it may be desirable to freeze saliva samples for a certain period before analysis. Such situations may warrant special attention when performing the LAMP analysis described herein. As shown in Figure 7, the pH of frozen saliva samples may vary depending on the number of days at -20°C between thawing and testing. In one example, the pH of a saliva sample from donor 1 changed from pH 7.21 with no time between collection and testing to pH 7.46 after 6 days between collection / freezing and testing. In another example, the pH of a saliva sample from donor 2 changed from pH 7.00 with no time between collection and testing to pH 6.98 after 6 days between collection / freezing and testing. In one example, the pH of a saliva sample from donor 3 changed from pH 7.18 with no time between collection and testing to pH 7.18 after 6 days between collection / freezing and testing. In one example, the pH of the saliva sample from Donor 4 changed from pH 7.35 when there were no days between collection and testing to pH 7.47 after 6 days between collection / freezing and testing. In another example, the pH of the saliva sample from Donor 1 changed from pH 7.22 when there were no days between collection and testing to pH 7.24 after 6 days between collection / freezing and testing.

[0159] Example 7 - Detection limit in untreated saliva Figure 8 shows the detection limit of untreated saliva. Untreated saliva was collected using the drooling method and diluted with water in a 1:3 ratio to obtain 25% saliva and 75% water. As a control, heat-inactivated SARS-CoV-2 was serially diluted and spiked into 25% saliva. 5 μL of 25% saliva was added to 20 μL of RT-LAMP reagent to achieve a final saliva concentration of 5%. After incubation at 65°C for 1 hour, a color change occurred. The copy number on the y-axis represents the copy number at the original concentration of 100% saliva without dilution. The limit of detection (LOD) of the primer was 250 copies / reaction in a volume of 25 μL, which corresponds to approximately 200,000 copies / 1 mL of saliva.

[0160] Therefore, it was found that diluting saliva to 25% with nuclease-free water, and then further diluting it to a final saliva concentration of 5% before adding it to the RT-LAMP reaction, yielded results within 60 minutes. Dilution reduces the buffering capacity of the saliva and lowers the concentration of the inhibitory component, both of which delay the colorimetric reporting. Dilution is less complex for the end user compared to other pretreatment procedures found in various studies, such as pretreatment with proteases, Chelex® 100, or RNA extraction to inactivate the inhibitory component in saliva.

[0161] The Line of Dispersion (LoD) of a colorimetric assay in 5% saliva treated with Pure·SAL® was 1000 copies / reaction (reaction volume 25 μL), which corresponds to 1 μL of patient saliva after considering 800 copies / dilution (Figure 6C).

[0162] As shown in Figure 6C, the Level of Decomposition (LoD) may vary depending on the saliva collection device (Pure·SAL®, Super-Sal®, Stat-sure®). For all processing techniques, saliva diluted to 5% with water was tested. Primer set orflab.2 was used. 5 μL of thermo-inactivated virus diluted with 5% treated saliva (final reaction concentration) was added to the RT-LAMP reaction to obtain the indicated concentration and final reaction volume of 25 μL. In case of a negative reaction, 5 μL of treated saliva (final reaction concentration 5%) was added instead of the diluted thermo-inactivated virus to provide the same reaction volume of 25 μL. Heating was performed in an incubator at 65°C for 60 minutes. Colorimetric scans were acquired using a flatbed scanner before and after the RT-LAMP reaction. The reactant consisted of 12.5 μL of NEB 2× colorimetric master mix, 2.5 μL of primer mix, 5 μL of water, and 5 μL of sample.

[0163] This level of discharge (LoD) is several orders of magnitude higher than that of RT-PCR assays or other assays utilizing RNA extraction (approximately 1 copy / reaction). However, these other assays involved pretreatment protocols and / or RNA extraction procedures to achieve the reported LoD.

[0164] To increase this LoD, the inventors investigated the use of RNase inhibitors, guanidine HCl, and carrier DNA. The addition of RNase inhibitors decreased the LoD in 5% saliva (Figure 6B), which contradicts literature reports that utilizing RNase inhibitors in salivary RT-LAMP assays increased LoD; this discrepancy may be due to the type of RNase inhibitor used. Both guanidine HCl and carrier DNA increased LoD (Figures 6D and 6E), and these were added to the RT-LAMP reaction formulation for the colorimetric solution reaction. These components were not included because they would change color when dried on paper.

[0165] As shown in Figure 6D, 5 μL of thermo-inactivated virus diluted with 5% treated saliva (final reaction concentration) was added to the RT-LAMP reaction to obtain the indicated concentration and final reaction volume of 25 μL. In the case of a negative reaction, 5 μL of treated saliva (final reaction concentration 5%) was added instead of the diluted thermo-inactivated virus to obtain the same reaction volume of 25 μL. The primer set orflab.2 was used. Heating was performed in an incubator (Fisherbrand® Isotemp®) at 65°C for 60 minutes. Colorimetric scans were acquired using a flatbed scanner before and after the RT-LAMP reaction. 1250 μL of NEB colorimetric master mix was supplemented with 0.5 μL of Antarctic Thermolabile UDG, 3.5 μL of dUTP, and carrier DNA to obtain the indicated final reaction concentration.

[0166] As shown in Figure 6E, 5 μL of thermo-inactivated virus diluted with 5% treated saliva (final reaction concentration) was added to the RT-LAMP reaction to obtain the indicated concentration and final reaction volume of 25 μL. In the case of a negative reaction, 5 μL of treated saliva (final reaction concentration 5%) was added instead of the diluted thermo-inactivated virus to obtain the same reaction volume of 25 μL. The primer set orflab.2 was used. Heating was performed in an incubator (Fisherbrand® Isotemp®) at 65°C for 60 minutes. Colorimetric scans were acquired using a flatbed scanner before and after the RT-LAMP reaction. 1250 μL of NEB colorimetric master mix was supplemented with 0.5 μL of Antarctic Thermolabile UDG, 3.5 μL of dUTP, and guanidine HCl (40 mM).

[0167] Finally, to reduce carryover contamination, uracil-DNA glycosylase (UDG) and deoxyuridine triphosphate (dUTP) (Figure 6F) were included. Figure 6F shows colorimetric scans of 25 μL reactions on a thermomixer and incubator, with and without the addition of UDG and dUTP, after incubation at 65°C for 60 minutes. The primer set used was orflab.II. The template was thermo-inactivated virus at the indicated concentrations. For the reaction with UDG, 0.5 μL of Antarctic Thermolabile UDG and 3.5 μL of dUTP were added to 1250 μL of NEB 2× colorimetric master mix. For all other reactions, NEB 2× colorimetric master mix was used.

[0168] When guanidine HCl, carrier DNA, and UDG were included, the Level of Disability (LoD) of the RT-LAMP colorimetric assay in 5% treated saliva in solution increased to 250 copies / reaction (Figure 6G). Figure 6G shows the RT-LAMP colorimetric LoD using saliva treated with Pure·SAL® and untreated saliva. Plates were heated in an incubator set to 65°C for 60 minutes. The primer set used was orflab.II, and the template was specified concentrations of thermo-inactivated virus (positive reaction) or nuclease-free water (negative reaction). Heating was performed in an incubator (Fisherbrand® Isotemp®) at 65°C for 60 minutes. Colorimetric scans were acquired using a flatbed scanner before and after the RT-LAMP reaction. 1250 μL of NEB colorimetric master mix was supplemented with 0.5 μL of Antarctic Thermolabile UDG, 3.5 μL of dUTP, carrier DNA (0.3 ng / μL), and guanidine HCl (40 mM).

[0169] Example 8 - Detection limit in animal nasal swabs Figure 9 shows the detection limit in bovine nasal swabs resuspended in approximately 1 mL of water. Heat-inactivated SARS-CoV-2 was spiked into water along with resuspended background mucus and microbiome to obtain the same copy / reaction ratio as in the previous example using saliva. 5 μL of the sample was added to 20 μL of RT-LAMP. After incubation at 65°C for approximately 1 hour, a color change occurred. The LOD for the primer was approximately 250 copies / reaction in a 25 μL volume, which corresponds to approximately 5,000 copies / 1 mL of nasal swab resuspension.

[0170] Example 9 - Detection Limit on Paper Figure 10 shows the detection limits on paper. 20 μL of RT-LAMP reagent was added to Grade 1 chromatography paper. Heat-inactivated SARS-CoV-2 was spiked into 100% pooled saliva using serially diluted virus samples. 15 μL of approximately 100% saliva was added to each paper sample. After incubation at 65°C for 90 minutes, a color change occurred. The primer's LOD was approximately 3,000 copies / reaction in a volume of 15 μL, which corresponds to approximately 20,000 copies / 1 mL of saliva.

[0171] Reagent composition for facilitating LAMP analysis on paper Example 10 - Sample Reagent In one example, the reagents were included as shown in Table A1. In another example, the reagents were included as shown in Table A2. [Table 1] [Table 2]

[0172] Example 11 - Selection and concentration of buffer solution Because saliva pH can vary from sample to sample, a buffer was used in the paper-based device to maintain a consistent starting pH. For phenol red, pH 7.6 was a suitable starting point to promote the colorimetric transition, as shown in Figure 11. Since the starting pH of 7.6 was close to the limit of the buffer range that allows for a color change when amplification occurs, several buffers with a pKa of approximately 8 were screened. As shown in Figure 12, 10 mM BICINE buffer was used in the paper-based assay.

[0173] Example 12 - Effect of primer on reaction rate To increase the rate of the RT-LAMP reaction, we investigated including multiple primer sets in the RT-LAMP fluorescence reaction mixture. The investigation was performed in water using NEB LAMP fluorescent dye as the fluorescent indicator. Including multiple primer sets did not appear to significantly improve the reaction rate. Rather, the reaction proceeded mainly at the rate of the primer set that had the fastest reaction time when used individually.

[0174] Example 13 - Sample LAMP protocol, reagents, validation, and troubleshooting Sample Lamp protocol 13-A: Primer Mix 1. Obtain all six diluted primers from the freezer; 2. Mix 80 μl FIP, 80 μl BIP, 20 μl FB, 20 μl LB, 10 μl F3, and 10 μl B3 in a tube; 3. Add enough PCR-grade water until the total volume reaches 500 μl.

[0175] LAMP 1. Obtain the NEB Bst 2.0 Warmstart kit and primer mix; 2. While the reagents are thawing, spray with DNAway and wipe the surface with a Kimwipe after at least 5 minutes; 3. Label all required PCR tubes with the DNA samples and primers to be used. Be sure to add a negative control without DNA; 4. For each reaction, add 5 μl of PCR-grade water (or dye), 12.5 μl of the NEB Bst 2.0 Warmstart kit, and 2.5 μl of the primer mix. The master mix can be prepared according to the number of reactions to be performed; 5. If adding 5 μl of EBT dye, the concentration should be 1500 μM so that the final concentration is 300 μM; 6. For reactions that do not contain DNA, add an additional 5 μl of PCR-grade water and do not open again until it is ready to be filled into the gel; 7. When ready, place the PCR tubes in the PCR tray that was previously left in the pass-through chamber and transfer them to the BRK2037; 8. Once in the BRK2037, retrieve the sample DNA from the -20°C freezer; 9. Spray DNAway spray onto your hands and rub around the DNA sample tubes with your hands to coat them with the spray; 10. Add 5 μl of DNA sample as needed and close the tubes. Never open two DNA tubes at the same time or close the PCR tubes immediately after adding DNA; 11. Place the samples in a thermocycler set to 65°C for 1 hour and 80°C for 5 minutes (after this operation, the samples may be kept overnight at -20°C).

[0176] Sample reagent concentration 13-B: The colorimetric RT-LAMP master mix may consist of: KCl (50 mM), MgSO4 (8 mM), dNTP mixture (1.4 mM of each dNTP), Bst 2.0 WarmStart® DNA polymerase (0.32 U / μL), WarmStart® RTx reverse transcriptase (0.3 U / μL), phenol red (0.25 mM), dUTP (0.14 mM), Antarctic Thermolabile UDG (0.0004 U / μL), Tween® 20 (1% vol / volume), betaine (20 mM), BSA (500 μg / mL), and trehalose (10% wt / volume).

[0177] These components were titrated in a paper LAMP assay from a liquid concentration of 0.25× to over 5×. The concentration was determined by the rate of the LAMP reaction, the contrast between positive and negative LAMP results over a 60-minute reaction time, and the decrease in the amount of nonspecific amplification.

[0178] To determine the concentration of the protein stabilizing additive, D-(+)-trehalose dihydrate was titrated in 5% increments from 0% to 15% by weight / volume, and lyophilized BSA was titrated in 0.2 mg / mL increments from 0 to 1.25 mg / mL. The concentrations of trehalose and BSA were 10% by weight / volume and 0.626 mg / mL, respectively.

[0179] Sample Lamp protocol 13-C: reagent The reagents are shown in Table A-2 of Example 10.

[0180] device Tweezers, 0.5-10 μL pipettes, 2-20 μL pipettes, 20-200 μL pipettes, 100-1000 μL pipettes, Ahlstrom-Munksjo Grade 222, pH probe, heat source capable of reaching 65°C (e.g., incubator, water bath), PCR hood.

[0181] Disinfection: Spray RNase AWAY onto the pipette and all workbench (PCR hood) surfaces. Wipe thoroughly after applying RNase AWAY. Any remaining RNase AWAY may interfere with the reaction. To prevent cross-contamination, use separate rooms for preparing paper-based devices and filling samples. Pre-cut chromatography paper to 5mm x 6mm.

[0182] Preparation of LAMP: 1. Prepare the 2×LAMP mix in a PCR hood as shown in Table 13B-1. 2. Adjust the pH to approximately 7.5-8.0 (red but not pink) with 1M KOH (approximately 1-2 μL). Preciseness is not required. After pH adjustment, the 2×LAMP mix can be stored at -20°C. [Table 3]

[0183] 3. Prepare the master mix according to Table 13B-2. [Table 4]

[0184] 4. Adjust the pH to 8.0 with 0.1M KOH. Use a micro pH electrode. 5. Mix well. Place the paper pad on a clean surface inside the PCR hood. Add 30 μL of complete mix to the pre-cut Grade 222 paper pad. 6. Dry at room temperature for 60 minutes under the PCR hood. 7. After drying, collect the paper pad in a clean centrifuge tube or a clean resealable plastic bag.

[0185] Sample packing 1. Spray RNase AWAY onto the workbench and wipe clean with a wiper. 2. Remove the templates (DNA, RNA, thermoactivated virus) from the freezer. 3. Place the reaction pads on a clean surface. The pads can be placed on a new clear film and discarded after use. 4. Prepare the negative control pad first. Reconstitute the pad with 25 μL of non-template solvent (water, saliva). The reconstitution process should be gentle and avoid washing the reagents off the pad. 5. Using tweezers, place the negative control pad into a clean container (e.g., a 1-inch x 1-inch resealable plastic bag, centrifuge tube). 6. Dilute the template with solvent to the desired concentration. 7. Place additional reaction pads and reconstitute the pads with 25 μL of diluted template. 8. Using tweezers, place the positive pads into a clean container (e.g., a 1-inch x 1-inch resealable plastic bag, centrifuge tube). 9. Clean the workspace and bring the pads in for imaging and incubation.

[0186] Imaging and incubation: Note: Multiple imaging methods (e.g., time-lapse video, scanning) and heat sources (e.g., incubator, water bath) exist. This protocol can be used with a desktop scanner and a microbial incubator. 1. Place the pad on top of the scanner. Scan the pad before the reaction (0 minutes). 2. Preheat the incubator to 65°C. 3. Place the pad inside the incubator. Separate the pad. Uniform heating can affect the consistency of the results. 4. Remove the pad and repeat the scan at different time points (usually every 30 minutes). 5. After the final scan, dispose of the reaction pad in a biohazard waste container.

[0187] verification: To confirm the occurrence of LAMP amplification, each reaction pad was transferred to a clean 1.5 mL microcentrifuge tube. 100 μL of buffer EB was added to each tube. The reaction pads were immersed in buffer EB overnight to elute the nucleic acids. Gel electrophoresis (2% agarose gel) was performed using the eluate to confirm the occurrence of LAMP amplification. A ladder-like pattern (typical LAMP product pattern) was shown in each positive pad lane, but no clear bands were observed in each negative lane (Figures 13A and 13B).

[0188] As shown in Figures 13A and 13B, paper LAMP validation was performed. As shown in Figure 13A, LAMP on paper was performed under two conditions (with and without BSA in the reaction mixture). As shown in Figure 13B, associated gel electrophoresis (2% agarose) was performed. The orf7ab.1 primer set targeting SARS-CoV-2 was used. The negative reaction pad was reconstituted with 25 μL of nuclease-free water. The positive reaction pad was reconstituted with 25 μL of 400 copies / μL of heat-inactivated SARS-CoV-2 virus. Heating was performed in an incubator set to 65°C and scanned with a flatbed scanner.

[0189] BSA is a reagent that can be used in LAMP mixtures. As shown in Figures 14A and 14B, adding BSA can accelerate the reaction and improve sensitivity. In these reactions, low-template concentration LAMP on paper was performed under two conditions: with and without BSA in the reaction mixture. Figure 14A shows the time at 0 minutes. Figure B shows the time at 60 minutes. The orf7ab.1 primer was used in this experiment. The negative reaction pad was reconstituted with 25 μL of nuclease-free water. The positive reaction pad was reconstituted with 25 μL of heat-inactivated SARS-CoV-2 virus at concentrations of 8 copies / μL and 16 copies / μL, respectively (final concentrations were 200 copies / reaction and 400 copies / reaction).

[0190] However, BSA can also cause pH fluctuations in the device. Figures 13A and 14B show that after incubation (60 minutes), the negative paper pad containing BSA has yellowish edges. After elution, the eluate was run on gel electrophoresis, and as shown in Figure 13B, no DNA products were visible on the gel, indicating that the yellowing at the edges was not caused by off-target amplification or contamination. If BSA is heterogeneously distributed, the edges may turn yellow when heated.

[0191] troubleshooting: Unusual pink color on paper pad: During the process of preparing the LAMP paper pad, you may find unusual pink spots that are different in color from the surrounding area. This may be caused by residual RNase AWAY, either sprayed directly onto the pad and / or transferred via tweezers. RNase AWAY can degrade the added RNA / DNA template. If this occurs, completely dry all equipment and surfaces, cut out a new 5x6mm paper pad, and restart the "LAMP Preparation" section from operation 5.

[0192] Reagent overflow after pad reconstitution: During the sample filling procedure, the pad may not be able to absorb the total amount of sample added to it for reconstitution. The template concentration may not be accurately represented by the overflowed pad. Overflow may occur if the pad is not dried sufficiently. In such cases, 1) dry for a longer period of time, 2) use an improved drying method such as heat drying (place in a clean microbial incubator at 37°C; do not set the temperature above 45°C to prevent activation of Bst 2.0 WarmStart® polymerase) or convection drying (use a small fan to increase airflow during drying), or 3) reduce the reconstitution volume to 20 μL.

[0193] Negative control shows color change: During imaging and incubation procedures, the negative pad may change simultaneously with or immediately after the sample-containing pad. This can be caused by either primer dimerization / nonspecific amplification or carryover contaminants from previous LAMP reactants. To resolve this, validate primers with liquid-based LAMP before use on paper. To control carryover contamination, 1) introduce dUTP and UDG into all LAMP reactants, 2) maintain a separate workbench for LAMP mixture preparation and sample addition, and 3) portion reagent stocks and use new aliquots if contamination is suspected. Over-incubating reactants can induce nonspecific amplification. Incubation time should not exceed 75 minutes.

[0194] Sample pH and buffering capacity affect colorimetric readings: Since phenol red is a pH indicator, the sample pH and its buffering capacity can significantly affect the assay. The reagent compositions presented herein have been confirmed to work at saliva concentrations of 5–10% volume / volume (diluted with water). 5% saliva was chosen because it provides a fast response time and consistent results. Human saliva has a high buffering system, including bicarbonates, phosphates, and proteins, which prevents pH changes (and therefore color changes) at high saliva concentrations. Paper LAMP devices using nasal swabs resuspended in water were tested, and no inhibition by the sample matrix was observed. Colorimetric readings may also be interfered with by buffer salt solutions (such as transport media).

[0195] Example 14 - Detection limit on paper of untreated saliva containing inactivated virus. As shown in Figure 15, the detection limit of approximately 20 copies per μL of saliva was verified from all untreated saliva containing heat-inactivated SARS-CoV-2 virus. Various sample concentrations were prepared, including 20 copies / μL (1×LoD-10 sample), 40 copies / μL (2×LoD-10 sample), 100 copies / μL (2 samples), 1000 copies / μL (2 samples), 10,000 copies / μL (2 samples), 100,000 copies / μL (2 samples), and 1,000,000 copies / μL (2 samples). Aliquots of pooled saliva (30 aliquots) were used as negative samples. The results were confirmed using image processing.

[0196] Reagent composition that maximizes pH-sensitive signal output Example 15 - LAMP dye sample [Table 5]

[0197] Example 16- Fluorescent reporters may require an additional ultraviolet (UV) light source for reading without the use of specialized equipment. However, colorimetric assays using phenol red as an indicator do not use UV light and can be interpreted visually. Protons are generated by DNA polymerization, and phenol red responds to pH. Diluted saliva (final concentration 5%) was used to overcome the buffering capacity of saliva and measure the change in pH. Diluting saliva to a final concentration of 5% also reduced the concentration of interfering substances (e.g., RNase).

[0198] Incorporating carrier DNA and guanidine hydrochloride further enhanced the LoD, yielding comparable colorimetric reactions in water and saliva. The mechanism by which carrier DNA enhances LAMP results was unclear. This mechanism was investigated using a NEB 1kb DNA ladder (NEB-N3232L). The effect on LoD was studied using different concentrations of carrier DNA (0.3 ng / μl and 0.75 ng / μl). Guanidine chloride (40 mM) was also used. The pH of the complete master mix was maintained at 7.6, and the same conditions were tested even without carrier DNA. Using untreated saliva (5%) at pH 6.5, the effect of carrier DNA, along with guanidine chloride, was tested against heat-inactivated SARS-CoV-2 in the concentration range of 1000 copies to 62.5 copies / reaction (Figure 6D).

[0199] Guanidine hydrochloride has been reported to increase the sensitivity of LAMP. Performance with our primer set was tested by adding 40 mM guanidine hydrochloride to NEB Warmstart™ colorimetric master mix at pH 7.6. The effect of guanidine hydrochloride was tested against thermally inactivated SARS-CoV-2 in the concentration range of 1000 to 62.5 copies / reaction using pooled saliva (5%) at pH 6.5. This same composition was tested without guanidine hydrochloride as a control. Guanidine hydrochloride increased the sensitivity of replication and exhibited consistent amplification throughout replication (Figure 6E).

[0200] Because phenol red and fluorescent dyes report different mechanisms of LAMP-based nucleic acid amplification, these differences in signal measurement over time were investigated. The reactants were prepared on a FrameStar 96-well skirted optical bottom plate containing a combination of Warmstart® colorimetric LAMP 2× master mix and LAMP fluorescent dye, sealed with Thermo Scientific® Adhesive Plate Seals, and placed in a Clariostar® Plus microplate reader (BMG) for incubation and measurement of absorbance and fluorescence intensity. The color change over time was observed. 432nm / A 560nmwas expressed as a ratio. The absorbance values were A 432nm and A 560nm were subtracted by A 620nm to perform baseline correction.

[0201] Based on Figure 16, the color change in the reaction occurred later compared to the fluorescence change. The colorimetric analysis and fluorescence analysis data were collected using a BMG CLARIOstar (registered trademark) Plus plate reader. The reaction base mix was composed of NEB 2x Colorimetric LAMP Master Mix, 2.5 μL of primer mix, and 5 μL of 1:100 diluted NEB LAMP fluorescent dye (NEB B1700A). The chamber temperature of the plate reader was equilibrated to 65 °C before inserting the plate. 5 μL of heat-inactivated virus diluted with water was added to the reaction base mix to obtain the indicated final reaction concentration (positive reaction). For the NTC reaction, 5 μL of nuclease-free water was added to the reaction base mix. This difference in change suggests that the pH-based reporter responds slower to LAMP-based DNA amplification than the fluorescent reporter.

[0202] Example 17- Paper can be scaled up to millions of devices, but when the RT-LAMP reagent is placed on paper, the color of the paper changes even when amplification has not occurred or when a negative control is used. One possibility for this color change is the oxidation of cellulose caused by heat and the oxidizability of ammonium sulfate present in the RT-LAMP mixture. Another possibility for this color change is the acidification of the reagent due to the degassing of ammonia from the RT-LAMP mixture. When ammonium sulfate was removed, the color of the negative control was maintained. Even when the concentration of phenol red acting as an antioxidant was increased, the color of the negative control was maintained as shown in Figure 11.

[0203] Example 18 - Screening of Colorimetric Dyes Three classes of colorimetric indicators: (i) magnesium colorimetric indicators, (ii) pH colorimetric indicators, and (iii) DNA intercalating colorimetric indicators were evaluated for paper-based assays.

[0204] For magnesium indicators, we screened Calmagite (CAS#3147-14-6), Xylidly Blue-I (CAS#14936-97-1), Chlorophosphonazo III (CAS#1914-99-4), o-Cresolphthalein Complexone (CAS#2411-89-4), Eriochrome® Black T (EBT, CAS#1787-61-7), and Hydroxynaphthol Blue (HNB, CAS#63451-35-4).

[0205] pH indicators include bromothymol blue (CAS#76-59-5), acid fuchsin (CAS#3244-88-0), nitrazine yellow (CAS#5423-07-4), cresol red (CAS#1733-2-6), cresol red sodium salt (CAS#62625-29-0), curcumin (CAS#458-37-7), phenol red (CAS#143-74-8), and phenol red sodium salt. The following were screened: (CAS#34487-61-1), Brilliant Yellow (CAS#3051-11-4), o-Cresolphthalein (CAS#596-27-0), m-Cresol Purple (CAS#2303-01-7), m-Cresol Purple Sodium Salt (CAS#62625-31-4), α-Naphtholphthalein (CAS#596-01-0), and Neutral Red (CAS#553-24-2).

[0206] For DNA intercalation dyes, we screened for crystal violet (CAS#548-62-9).

[0207] Many magnesium indicators did not produce a consistent color change on paper. Metal ion indicators (calmagite and EBT) interacted with magnesium(II) ions in solution, and their concentration decreased throughout the RT-LAMP experiment due to the formation of magnesium pyrophosphate, a byproduct of the polymerase reaction.

[0208] Figure 17A shows the colorimetric response of karmagite at various concentrations throughout the LAMP reaction using genomic DNA as a template. LAMP detection was performed by increasing the concentration of karmagite (magnesium indicator). A lolB.3 primer set targeting Histophilus somni genomic DNA was used. For a positive reaction, 5 μL of HS gDNA was spiked at a concentration of 0.2 ng / μL. For a negative reaction, 5 μL of nuclease-free water was used. The total reaction volume was 25 μL. The reaction was prepared using 12.5 μL of NEB Warmstart 2× master mix, 2.5 μL of primer mix, and 5 μL of either the template (positive reaction) or water (negative reaction) as described above, and 5 μL of karmagite prepared with water to obtain the final concentrations shown.

[0209] At the concentrations tested, no visual changes were detected during the LAMP reaction. EBT showed a detectable color change from purple to dark blue between 0 and 60 minutes of the LAMP reaction; however, the color change was not clear, which may hinder clinical interpretation.

[0210] As shown in Figure 17B, LAMP detection was performed by increasing the concentration of Eriochrome® Black T (magnesium indicator). A lolB.3 primer set targeting Histophilus somni genomic DNA (gDNA) was used. For a positive reaction, 5 μL of HS gDNA was spiked at a concentration of 0.2 ng / μL. For a negative reaction, 5 μL of nuclease-free water was used. The total reaction volume was 25 μL. The reaction was prepared using 12.5 μL of NEB Warmstart 2× master mix, 2.5 μL of primer mix, and 5 μL of either the template (positive reaction) or water (negative), and 5 μL of EBT prepared with water to obtain the final concentrations shown.

[0211] Furthermore, no detectable color change occurred when LAMP was used on paper with EBT. As shown in Figure 17C, LAMP detection was performed on chromatography paper in PCR tubes by increasing the concentration of Eriochrome® Black T. The lolB.3 primer set targeting Histophilus somni genomic DNA was used. For the positive reaction, 5 μL of H. somni gDNA was spiked at a concentration of 0.2 ng / μL. For the negative reaction, 5 μL of nuclease-free water was used. The total reaction volume was 25 μL. The reaction was prepared using 12.5 μL of NEB Warmstart 2× master mix, 2.5 μL of primer mix, and either 5 μL of the above template (positive reaction) or water (negative reaction), and 5 μL of EBT (300 μM) prepared in nuclease-free water. In the case of the EBT reaction, the reaction consisted of 25 μL of EBT (300 μM) prepared in nuclease-free water.

[0212] By screening several different types of paper and confirming that amplification occurred with PES and polysulfone BTS 0.8 by gel electrophoresis, no colorimetric change was detected on any of the papers (Figures 17D and 17E).

[0213] As shown in Figure 17D, LAMP detection was performed on multiple paper substrates: chromatography grade 1, anion-exchange nylon, cation-exchange nylon, polyethersulfone membrane, asymmetric submicron polysulfone (BTS 0.8), asymmetric submicron polysulfone (BTS 100), and hydroxylated nylon 1.2. b) Endpoint scan of the paper in panel a at 60 minutes, and c) Gel electrophoresis (2% agarose) scan of the extracted LAMP product at 60 minutes. A lolB.3 primer set targeting Histophilus somni (HS) genomic DNA was used. For a positive reaction, 5 μL of H. somni gDNA was spiked at a concentration of 0.2 ng / μL. For a negative reaction, 5 μL of nuclease-free water was used. The total reaction volume was 25 μL. The reaction mixture was prepared using 12.5 μL of NEB Warmstart 2× master mix, 2.5 μL of primer mix, 5 μL of either the template (positive reaction) or water (negative reaction), and 5 μL of EBT (300 μM) prepared with nuclease-free water. Paper (as shown) was placed in a PCR tube containing 25 μL of reaction mixture and absorbed by the paper during the reaction. After 60 minutes, the paper was removed and scanned. The gel was extracted using 30.

[0214] As shown in Figure 17E, results were obtained over time for a) PCR tubes, b) gel electrophoresis (2% agarose) of DNA extracted from paper at 60 minutes, and c) a scan of paper at 60 minutes. A lolB.3 primer set targeting Histophilus somni (HS) genomic DNA was used. For a positive reaction, 5 μL of H. somni gDNA was spiked at a concentration of 0.2 ng / μL. For a negative reaction, 5 μL of nuclease-free water was used. The total reaction volume was 25 μL. The reaction mixture was prepared using 12.5 μL of NEB Warmstart 2× master mix, 2.5 μL of primer mix, 5 μL of either the template (positive reaction) or water (negative reaction), and 5 μL of EBT (300 μM) prepared with nuclease-free water. Biodyne A amphoteric paper was placed in PCR tubes containing 25 μL of reaction mixture and absorbed by the paper during the reaction. 60 minutes later, I took the paper out and scanned it.

[0215] Furthermore, stabilizing the crystal violet indicator in solution for the RT-LAMP reaction proved difficult. For leucocrystal violet (LCV), an unstable derivative of crystal violet, excess sodium sulfite was used to maintain colorless stability in solution. For water solubilization, sodium sulfite (SS) and beta-cyclodextrin (BCD) were used with LCV. Upon binding to dsDNA, LCV reverted to crystal violet (e.g., purple in solution). Consequently, it was expected that the solution color would change from colorless to purple throughout the RT-LAMP reaction as more dsDNA was generated as a result of amplification. However, when the LAMP reaction was performed with various concentrations of CV, color changes occurred in both positive and negative reactions.

[0216] As shown in Figure 17F, LAMP detection was performed using crystal violet, an intercalation dye in solution, and b) a scan of the product gel electrophoresis (2% agarose) at the relevant 60-minute time point was performed. A lolB.3 primer set targeting H. somni gDNA was used. For the positive reaction, 5 μL of H. somni gDNA was spiked at a concentration of 0.2 ng / μL. For the negative reaction, 5 μL of nuclease-free water was used. The total reaction volume was 25 μL. The reaction mixture was prepared using 12.5 μL of NEB Warmstart 2× master mix, 2.5 μL of primer mix, and 5 μL of either the template (positive reaction) or water (negative) described above, and 5 μL of crystal violet prepared with nuclease-free water to obtain the final concentration shown. Paper was placed in a PCR tube containing 25 μL of the reaction mixture and absorbed by the paper during the reaction. After 60 minutes, the paper was removed and scanned. Sodium sulfite and cyclodextrin were used to solubilize the crystal violet.

[0217] To confirm that amplification occurred in positive reactions but not in negative reactions, the RT-LAMP solution was run on a 2% agarose gel. The results showed amplification in positive reactions at all tested CV concentrations, while negative reactions did not show amplification at any tested CV concentration. Therefore, the color change in negative reactions was caused by the degradation of LCV to CV, and not by the binding of amplified DNA.

[0218] Further testing of CV on paper with different concentrations of CV, SS, and BCD yielded results that could not be distinguished between negative and positive reactions. As shown in Figure 17G, endpoint colorimetric scans were performed on paper for LAMP detection using the intercalating dye crystal violet (CV). CV was solubilized using sodium sulfite (SS) and beta-cyclodextrin (BCD). Paper was filled with 12.5 μL of NEB Warmstart® 2× master mix, 2.5 μL of primer mix, and 5 μL of either the template (positive reaction) or water (negative reaction) described above, and 5 μL of CV prepared in nuclease-free water at the indicated final concentrations.

[0219] Finally, several colorimetric pH indicators, encompassing a range of approximately 2 pH units with a color transition pH of approximately 7.0, were tested to see if they matched the expected pH range changes and transition points of the RT-LAMP reaction. These ranges were selected based on the initial starting pH of our LAMP colorimetric master mix and to overcome the buffering capacity of saliva. One exception in this selection process was acidic fuchsin, which encompasses a pH range of 3.0 and has a color transition pH of 5.0. Figures 17H–17K show the various concentrations of the selected pH indicators along with the corresponding gel electrophoresis results of the LAMP reaction at 60 minutes.

[0220] As shown in Figure 17H, RT-LAMP detection was performed by increasing the concentrations of cresol red sodium salt, neutral red, phenol red sodium salt, m-cresol purple, and m-cresol purple sodium salt (pH indicator) in solution. An N.10 primer set targeting the SARS-CoV-2 N gene was used. For a positive reaction, 5 μL of in vitro transcribed N gene RNA was spiked at a concentration of 0.2 ng / μL. For a negative reaction, 5 μL of nuclease-free water was used. The total reaction volume was 25 μL. The reaction mixture was prepared using 12.5 μL of NEB Warmstart® 2× master mix, 2.5 μL of primer mix, 5 μL of either the template (positive reaction) or water (negative reaction), and 5 μL of pH indicator at the specified concentration in nuclease-free water. The reaction was carried out in an incubator and scanned every 20 minutes using a flatbed scanner.

[0221] As shown in Figure 17I, LAMP detection was performed by increasing the concentration of cresol red (pH indicator). A lolB.3 primer set targeting H. somni gDNA was used. For a positive reaction, 5 μL of H. somni gDNA was spiked at a concentration of 0.2 ng / μL. For a negative reaction, 5 μL of nuclease-free water was used. The total reaction volume was 25 μL. The reaction mixture was prepared using 12.5 μL of NEB Warmstart® 2× master mix, 2.5 μL of primer mix, 5 μL of cresol red at the indicated final reaction concentration, and 5 μL of either the template (positive reaction) or water (negative reaction) described above.

[0222] As shown in Figure 17J, RT-LAMP detection was performed by increasing the concentrations of cresol red, sodium salt, m-cresol purple, bromothymol blue, and acidic fuchsin in solution, and b) the product was scanned by gel electrophoresis (2% agarose) at the relevant 60-minute time point. An N.10 primer set targeting the N gene of SARS-CoV-2 was used. For the positive reaction, 5 μL of in vitro transcribed N gene RNA was spiked at a concentration of 0.2 ng / μL. For the negative reaction, 5 μL of nuclease-free water was used. The reaction product was prepared using 12.5 μL of NEB Warmstart 2× master mix, 2.5 μL of primer mix, and either the template (positive reaction) or water (negative) described above, and 5 μL of the indicated pH indicator to obtain the indicated final reaction concentration. Heating was performed in an incubator set to 65°C, and scanned with a flatbed scanner every 20 minutes.

[0223] As shown in Figure 17K, an endpoint gel electrophoresis scan (60 minutes) of RT-LAMP products was performed on a 2% agarose gel.

[0224] As shown, one pH indicator that produced a clear colorimetric response between positive and negative reactions was cresol red. Furthermore, phenol red (the pH indicator used in NEB's colorimetric RT-LAMP kit) was also evaluated for the change in initial pH value resulting from the addition of HCl and KOH to the solution to provide the indicated initial pH value.

[0225] As shown in Figure 17L, RT-LAMP detection was performed using phenol red (pH indicator) at pH 8.1, 8.5, and 8.8 in solution. After preparation with HCl and KOH, template RNA was added. An N.10 primer set targeting the SARS-CoV-2 N gene was used. For a positive reaction, 5 μL of in vitro transcribed N gene RNA was spiked at a concentration of 0.2 ng / μL. For a negative reaction, 5 μL of nuclease-free water was used. The reaction consisted of 20 μL of master mix and 5 μL of the above template (positive) or nuclease-free water (negative). A 10 mL master mix was prepared using (NH4)2SO4 (20 mM), KCl (100 mM), MgSO4 (16 mM), dNTP mix (28 mM each of dNTPs), and tween20 (0.2% vol / vol). Therefore, phenol red showed a higher level of contrast between positive and negative reactions compared to cresol red.

[0226] As a result, pH indicators that exhibited a color change around pH 6.5 showed the most consistent and highest contrast color change (e.g., phenol red).

[0227] Example 19 - Effect of initial pH on paper To evaluate the color stability when drying is incorporated into the inventors' process, the pH of the LAMP master mix was adjusted to 8.0, 8.5, or left unadjusted (e.g., 7.6), and the water or synthetic RNA (N gene, 0.2 ng / μL) used for rehydration was also adjusted to 8.0, 8.5, or left unadjusted (e.g., 5.5).

[0228] As shown in Figure 18, pH 7.6 is the unadjusted pH of the RT-LAMP reaction mixture. The humid setting indicates that 5 μL of synthetic RNA (N gene, 0.2 ng / μL, "+") or water ("-") was added immediately after adding 20 μL of LAMP reaction master mix. The dry setting indicates that after applying 20 μL of LAMP master mix, the paper strip was air-dried at room temperature for 30 minutes, and then rehydrated with 25 μL of synthetic RNA ("+") or water ("-").

[0229] Adjusting the pH to 8.0 resulted in better color stability in the negative control, but pH 8.5 was too high, and no color change was discernible after 120 minutes of incubation under standard and dry settings. When the pH was left unadjusted, a color change occurred even when the control was packed.

[0230] Example 20 - Effects of trehalose and Tween20 on the colorimetric response of RT-LAMP Figure 19B shows the colorimetric RT-LAMP results with a given concentration of trehalose or Tween20. The orflab.II primer set was used. 20 μL of the RT-LAMP master mix, containing the basic formulation of KCl (50 mM), MgSO4 (8 mM), equimolar dNTP mixture (1.4 mM each of dNTPs), 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), Tween20 (1% vol / vol if indicated), betaine (20 mM), BSA (40 mg / mL), and trehalose (10% wt / vol if indicated), was added to Grade 1 chromatography paper (5 mm × 20 mm) and dried in a PCR preparation hood for 60 minutes. Final concentration per reaction: 1 × 10⁻¹⁶ in 25% treated saliva (positive reaction) or nuclease-free water (negative reaction) 5 25 μL of heat-inactivated copy of SARS-CoV-2 was added to a dry reaction pad. The pad was heated in an incubator set to 65°C for 60 minutes and scanned using a flatbed scanner.

[0231] When ammonium sulfate was included, the RT-LAMP reagent changed from red to yellow upon drying in the absence of a template. This color change was prevented by increasing the phenol red concentration and replacing ammonium sulfate with betaine (Figure 19A). Furthermore, the addition of trehalose and bovine serum albumin (BSA) increased the reaction rate and the level of discharge (LoD) (Figure 19B).

[0232] Exemplary Embodiments

[0233] In one example, a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid-phase medium is provided, comprising or comprising one or more target primers; DNA polymerase; and a resolubilizer; and substantially free of non-pH-sensitive agents that can discolor the solid-phase medium.

[0234] In one example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid-phase medium, the composition may further contain an antioxidant.

[0235] In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid-phase medium, the composition may be substantially free of volatile agents.

[0236] In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid-phase medium, the composition may be substantially desiccant-free.

[0237] In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid-phase medium, the desiccant can absorb more than about 10% by weight at relative humidity (RH) of about 40% to about 90% at 25°C.

[0238] In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium, examples of the desiccant can include glycerol, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate, and combinations thereof.

[0239] In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium, the resolubilizing agent can be a surfactant.

[0240] In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium, the resolubilizing agent can include bovine serum albumin (BSA), casein, polysorbate 20, and combinations thereof.

[0241] In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium, the target primer can target a pathogen including a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen.

[0242] In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium, the pathogen can be a viral pathogen.

[0243] In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium, the viral pathogen can include a dsDNA virus, a ssDNA virus, a dsRNA virus, a plus-strand ssRNA virus, a minus-strand ssRNA virus, a ssRNA-RT virus, or a ds-DNA-RT virus.

[0244] In another aspect, the viral pathogen can include H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.

[0245] In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium, the composition can further include reverse transcriptase.

[0246] In another example of a composition for loop-mediated isothermal amplification (LAMP) assays on a solid-phase medium, the composition can further comprise a non-discoloring additive.

[0247] In another example of a composition for loop-mediated isothermal amplification (LAMP) assays on a solid-phase medium, the non-discoloring additive comprises one or more of a sugar, a buffer, or a combination thereof.

[0248] In another example of a composition for loop-mediated isothermal amplification (LAMP) assays on a solid-phase medium, the composition can further comprise an indicator.

[0249] In one example, a method for LAMP assays on a solid-phase medium is provided, comprising providing an assembly of the solid-phase medium and the composition described herein, depositing a biological sample on the solid-phase medium, and heating the assembly to an isothermal temperature sufficient to promote a LAMP reaction.

[0250] In one example of a method for LAMP assays on a solid-phase medium, the biological sample is one or more of saliva, mucus, blood, urine, feces, sweat, breath condensate, and combinations thereof.

[0251] In another example of a method for LAMP assays on a solid-phase medium, the biological sample can be saliva.

[0252] In another example of a method for LAMP assays on a solid-phase medium, the method can further comprise detecting a viral pathogen.

[0253] In another example of a method for LAMP assays on a solid-phase medium, the LAMP assay can be reverse transcriptase LAMP (RT-LAMP).

[0254] In one example, a system for performing a LAMP assay is provided, comprising the composition described herein; and a solid-phase medium on which the composition is deposited, including or comprising.

[0255] It should be understood that the methods described above are merely illustrative of some embodiments of the present invention. Those skilled in the art will be able to devise many modifications and alternative configurations without departing from the spirit and scope of the invention, and the appended claims shall cover such modifications and configurations. Therefore, although the present invention has been described above in specific and detail in relation to what is currently considered the most practical and preferred embodiment of the invention, it will be apparent to those skilled in the art that variations may be made, including modifications, without departing from the principles and concepts set forth herein.

Claims

1. A composition for loop-mediated isothermal amplification (LAMP) analysis on a solid-phase medium, wherein the composition is One or more target primers; DNA polymerase; Reverse transcriptase; Solid-phase solubilizers, and Contains pH-sensitive dyes, The aforementioned composition does not contain ammonium sulfate, but contains solid-phase betaine. The solid phase medium is paper. The solubilizing agent, when directly depositing the biological sample onto the solid-phase medium, dissolves the biological sample at a specific temperature to form a liquid, and promotes the rehydration of the solid-phase betaine on the solid-phase medium. The composition comprises less than 1.0 weight percent (wt%) of a desiccant.

2. The composition according to claim 1, further comprising an antioxidant.

3. The composition according to claim 1, wherein it does not contain a volatile agent, and the volatile agent is ammonium sulfate.

4. The aforementioned desiccant includes glycerol, ethanol, methanol, calcium chloride, calcium sulfate, and combinations thereof. The composition according to claim 1, wherein the desiccant absorbs an amount exceeding 10% by weight when the relative humidity (RH) is 40% to 90% at 25°C.

5. The composition according to claim 1, wherein the solubilizing agent includes bovine serum albumin (BSA), casein, polysorbate 20, and combinations thereof.

6. The composition according to claim 1, wherein the target primer targets pathogens including viral pathogens, bacterial pathogens, fungal pathogens, or protozoan pathogens.

7. The composition according to claim 6, wherein the pathogen is a viral pathogen.

8. The composition according to claim 7, wherein the viral pathogen comprises a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a ds-DNA-RT virus.

9. The composition according to claim 7, wherein the viral pathogen comprises H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.

10. The composition according to claim 1, further comprising a non-discoloring additive.

11. The non-coloring additive is one or more of the following: sugar, buffer, or combination thereof. The composition according to claim 10, including

12. A method for LAMP analysis on a solid-phase medium, To provide a solid-phase medium and an assembly of the composition described in claim 1; Depositing a biological sample onto the solid phase medium; and The method comprising heating the assembly to a temperature sufficient to promote the LAMP reaction.

13. The method according to claim 12, wherein the biological sample is one or more of saliva, mucus, blood, urine, feces, and combinations thereof.

14. The method according to claim 12, wherein the biological sample is saliva.

15. The method according to claim 12, further comprising detecting a viral pathogen.

16. The method according to claim 12, wherein the LAMP analysis is performed using reverse transcriptase LAMP (RT-LAMP).

17. The composition according to claim 1; and The aforementioned composition is deposited on a solid-phase medium thereon. A system for performing LAMP analysis, including the above.

Images