TW202242131A - Loop-mediated isothermal amplification (lamp) analysis for pathogenic targets - Google Patents

Abstract

The present disclosure is drawn to compositions, methods, and systems for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium. The composition can comprise one or more target primers, a DNA polymerase, and a re-solubilization agent. The composition can be substantially free of non-pH sensitive agents capable of discoloring the solid phase medium. The method can comprise providing an assembly of a solid phase medium, depositing a biological sample onto the solid phase medium, and heating the assembly to an isothermal temperature sufficient to facilitate a LAMP reaction. The system can comprise a composition and a solid phase medium on to which the composition is deposited.

Description

Loop-Mediated Isothermal Amplification (LAMP) Analysis for Pathogenic Targets

Related applications

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/138,312, filed January 15, 2021, which is hereby incorporated by reference.

The present invention relates to loop-mediated isothermal amplification (LAMP) assays for pathogenic targets.

Background of the invention

Polymerase chain reaction (PCR) is a molecular biology technique that amplifies nucleotides for various analytical purposes. Quantitative PCR (qPCR) is a modification of PCR that allows monitoring the amplification of target nucleotides. Diagnostic qPCR has been applied to detect indicative nucleotides for infectious diseases, cancer, and genetic abnormalities. Reverse transcription PCR (RT-PCR) is a modification of qPCR that allows detection of target RNA nucleotides. Because of this capability, RT-PCR is well suited for the detection of viral pathogens. However, RT-PCR requires considerable equipment, which may not be available in some point-of-care settings. Furthermore, RT-PCR requires well-trained personnel, rigorous sample preparation, and time-to-result.

In contrast, loop-mediated isothermal amplification (LAMP) is a simpler method for the diagnostic identification of target nucleotides. In particular, LAMP is a one-operation nucleic acid amplification method for increasing specific nucleotide sequences. In addition to using a constant temperature heating process, LAMP can also test indicators using simple visual output, such as a color change, rather than the more complex fluorescent indicators used in PCR. In order to identify target nucleotides from RNA, RT-PCR-like reverse transcription LAMP (RT-LAMP) can be used, which can be used in the diagnostic ability to identify the presence or absence of viral pathogens. Because LAMP is simpler and can be performed with less equipment and sample preparation, it is easier to use in point-of-care settings, such as clinics, emergency rooms, or even on a mobile basis.

Summary of the invention

The present disclosure relates to techniques (eg, compositions, methods, systems, and assemblies) for detecting target nucleotides using LAMP analysis. In some aspects, the target nucleotide may be known to be present in the pathogen of interest. In cases where the pathogen is a virus, the LAMP assay may be a RT-LAMP assay.

In some disclosed embodiments, a method of preparing a saliva sample for loop-mediated isothermal amplification (LAMP) detection of a pathogenic target is provided. In one aspect, such a method may include providing an amount of saliva from a test subject, and diluting the saliva in water to reduce the buffering capacity of the saliva while maintaining a sufficient concentration to allow detection of the pathogenic target Degree.

In one aspect, the method can include reducing the viscosity of the saliva compared to an original viscosity. In another aspect, the viscosity can be reduced by one or more of dilution, filtration, or a combination thereof. In another aspect, filtration can be used to reduce viscosity. In another aspect, a 10 micron filter can be used to reduce viscosity. In yet another aspect, the viscosity can be reduced to such an extent that fluidity through the solid medium is increased as compared to the original viscosity. In yet another aspect, the viscosity can be reduced to a range of about 1.0 centipoise (cP) to about 50 cP.

In one aspect, such a method can include filtering the saliva sample to the extent that the pH of the saliva sample is adjusted to a test sample target range. In another aspect, the test sample target range can be from about 7.2 to about 8.6. In another aspect, the water can have a pH greater than 6.0 and can be substantially free of contaminants. In yet another aspect, the saliva sample can consist essentially of saliva and water. In yet further aspects, saliva may be collected using sponge-based collection.

In one aspect, the saliva can be diluted in water to a saliva to water ratio of about 1:1 to about 1:20. In another aspect, the saliva can be diluted in water to an extent that provides a sample with an optical density at 600 nm ( _OD6oo ) of less than 0.2. In other aspects, the saliva has a volume of from about 50 μl to about 100 μl. In yet another aspect, the saliva sample has a volume of from about 100 μl to about 1 ml.

In another aspect, the pathogenic target can comprise a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In one aspect, the pathogenic target can be a viral target. In another aspect, the viral target can include a dsDNA virus, ssDNA virus, dsRNA virus, sense ssRNA virus, reverse strand ssRNA virus, ssRNA-RT virus, or ds-DNA-RT virus. In another aspect, the virus target can be H1N1, H2N2, H3N2, H1N1pdm09 or SARS-CoV-2.

In some aspects, the LAMP detection can comprise a reverse transcription LAMP (RT-LAMP) detection.

In other disclosed embodiments, a test sample composition for LAMP analysis is disclosed, and may include: an amount of saliva of a test subject sufficient to detect a pathogenic target by LAMP analysis, and an agent that reduces the buffering capacity of the saliva water volume.

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

In one aspect, the pathogenic target can comprise a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In another aspect, the pathogenic target can be a viral target. In another aspect, the viral target can comprise a dsDNA virus, ssDNA virus, dsRNA virus, sense ssRNA virus, reverse strand ssRNA virus, ssRNA-RT virus, or ds-DNA-RT virus. In yet another aspect, the viral target may comprise H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2. In yet another aspect, the buffering capacity of the composition may be less than 5 mM.

In yet other disclosed embodiments, a composition for performing LAMP assays on a solid medium can include one or more primers of interest, a DNA polymerase, and a resolubilizing agent. In some aspects, such a composition may be substantially free of non-pH sensitive agents capable of discoloring the solid phase medium. In one aspect, the composition can include an antioxidant. In another aspect, the composition can be substantially free of volatile agents. In yet another aspect, the composition can be substantially free of hygroscopic agents. In one other aspect, the composition may further include reverse transcriptase.

In one aspect, the hygroscopic agent can absorb more than about 10% by weight at 25°C between about 40% and about 90% relative humidity (RH). In another aspect, the moisture absorbent may include glycerin, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate, and combinations thereof.

In another aspect, the redissolving agent can be a surfactant. In another aspect, the resolubilizing agent may comprise bovine serum albumin (BSA), casein, polysorbate 20, or a combination thereof.

In one aspect, the target primer can target a pathogen, which can include a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In one aspect, the pathogen can be a viral pathogen. In another aspect, the viral pathogen may comprise dsDNA virus, ssDNA virus, dsRNA virus, positive strand ssRNA virus, reverse strand ssRNA virus, ssRNA-RT virus or ds-DNA-RT virus. In another aspect, the viral pathogen may comprise H1N1, H2N2, H3N2, H1N1pdm09 or SARS-CoV-2.

In one aspect, the composition may further include a color-changing additive. The non-discoloration additive may comprise one or more of sugars, buffers or combinations thereof. In another aspect, the composition may further include an indicator.

In other disclosed embodiments, methods for performing LAMP analysis on solid media may include providing an assembly having a solid media and a composition as described herein; depositing a biological sample onto on the solid medium; and heating the assembly to a constant temperature sufficient to promote the LAMP reaction. In one aspect, the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or combinations thereof. In another aspect, the biological sample is saliva. In one aspect, the LAMP assay can be reverse transcriptase LAMP (RT-LAMP). In another aspect, the method may further comprise detecting a viral pathogen.

In other disclosed embodiments, a system for performing LAMP analysis may comprise a composition as described herein, and a solid medium on which the composition is deposited.

In yet further disclosed embodiments, compositions for loop-mediated isothermal amplification (LAMP) assays can utilize a pH-dependent output signal, which can include pH-sensitive dyes and various non-interfering LAMP reagents. In one aspect, the LAMP assay can be RT-LAMP.

In one aspect, the pH-sensitive dye can be at least one of phenol red, phenolphthalein, litmus red, bromevanillol blue, naphtholphthalein, cresol red, or a combination thereof. In another aspect, the plurality of non-interfering LAMP reagents can be substantially free of volatile agents, pH disruptors, magnesium disruptors, or combinations thereof.

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 comprise DNA polymerase, reverse transcriptase, target primers, or a combination thereof.

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

In one aspect, the composition may include a non-discoloration additive, which may include a sugar, a buffer, a blocking agent, or a combination thereof. In one aspect, the sugar may comprise one or more of trehalose, glucose, sucrose, or a combination thereof. In another aspect, the blocking agent may comprise bovine serum albumin, casein, or a combination thereof.

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

In further disclosed embodiments, a method of maximizing the accuracy of an output signal in a pH-dependent LAMP assay may comprise providing a reagent mixture that minimizes discoloration from non-LAMP reactions of signal output mediators and performing the LAMP reaction. In one aspect, the method can comprise controlling the production of protons from non-LAMP reactions. In another aspect, the method can comprise controlling oxidation from non-LAMP reactions.

In other disclosed embodiments, a method of maximizing the accuracy of output signals in pH-dependent LAMP assays can include substantially removing discoloration from non-LAMP reactions of a signal output medium.

In other disclosed embodiments, a method of maximizing the limit of detection (LOD) in a pH-dependent LAMP assay can include substantially removing discoloration from non-LAMP reactions from a signal output mediator.

Description of the embodiment

Before describing the embodiments of the present invention, it should be understood that this disclosure is not limited to the specific structures, method steps or materials disclosed herein, but extends to equivalents recognized by those of ordinary skill in the related art. It is also to be understood that terminology used herein is for the purpose of describing particular examples or embodiments only and is not intended to be limiting. The same symbols in different drawings represent the same elements. Numbers provided in flowcharts and procedures are provided to clearly illustrate steps and operations and do not necessarily imply a particular order or sequence.

Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as examples of compositions, dosage forms, treatments, etc., to provide a thorough understanding of various inventive embodiments. Those skilled in the relevant art will recognize, however, that such detailed embodiments do not limit, but are merely representative of, the general inventive concepts set forth herein. definition

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

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

As used herein, the term "solubility" is a measure or property of a substance or agent in terms of its ability to dissolve in a given solvent. The solubility of a substance or reagent in a specific component of a composition means the amount of the substance or reagent that dissolves at a specific temperature such as about 25°C or about 37°C to form an apparently transparent solution.

As used herein, the term "lipophilic" means a compound that is not readily soluble in water. Conversely, the term "hydrophilic" means a compound that is soluble in water.

The term "subject" as used herein means an animal. In one aspect, the animal can be a mammal. In another aspect, the mammal can be a human.

As used herein, the term "non-liquid" when used to refer to the state of the compositions disclosed herein means that the physical state of the composition is semi-solid or solid.

The terms "solid" and "semi-solid" as used herein mean a physical state in which a composition supports its own weight at standard temperature and pressure and has sufficient viscosity or structure not to flow freely. Semi-solid materials can conform to the shape of a container under applied pressure.

As used herein, the terms "solid medium", "solid substrate", "solid substrate", "solid test substrate", "solid test substrate" and the like mean a non-liquid medium, device, system or environment. In some aspects, the non-liquid medium can be substantially or completely free of liquid. In one example, the non-liquid medium may comprise or be a porous material or a material having a porous surface. In another example, the non-liquid medium may comprise or be a fibrous material or a material having a fibrous surface. In yet another example, the non-liquid medium can be paper.

As used herein, the term "non-discoloration additive" means one that minimizes or prevents the color of the solid phase medium from changing from the original or starting color due to reasons other than nucleotide amplification on or in which the LAMP reaction occurs. Additives for color change to different colors. For example, in one embodiment, this color change can be minimized or reduced compared to what would occur without the non-color changing additive.

As used herein, the term "non-LAMP reaction-induced discoloration" means any discoloration of the solid phase medium (eg, a color change from an original color to another color) that is not caused by nucleotide amplification by a LAMP reaction. In some instances, non-LAMP reaction-induced discoloration may mean discoloration of the solid phase medium caused by one or more of the following: volatile agents, magnesium interfering agents, oxidizing agents, pH changes due to causes other than amplification of the LAMP reaction , drying or a combination thereof.

The term "volatile agent" as used herein means an agent including a composition having a high vapor pressure or a low boiling point. In one example, ammonium sulfate can be a volatile agent because ammonia evaporates leaving sulfuric acid. In one example, the composition, component or element can have a high vapor pressure when the composition is in the gas phase at a temperature above about 30°C. In one example, the composition can have a low boiling point when the composition is in the gas phase at a temperature below about 80°C.

As used herein, the term "pH disruptor" is an agent that can affect the pH of a reaction, system or environment for reasons other than amplification of a LAMP reaction. In one example, ammonium ions will volatilize from ammonium sulfate, while sulfate ions can react to form sulfuric acid and affect the pH of the reaction without amplification from the LAMP reaction.

In this disclosure, "comprising", "comprising", "comprising", and "having", etc. may have the meanings assigned to them in U.S. patent law, and may mean "including", "comprising", etc., and are generally interpreted as open formula term. The term "consisting of" or "consisting of" is a closed term, which only includes the components, structures, steps, etc. clearly listed in combination with such term, and the contents complying with the US patent law. "Consisting essentially of" or "consisting essentially of" has the meaning generally assigned to them under US patent law. In particular, these terms are largely closed terms, but allow for the inclusion of additional items, materials, components, steps or elements that do not materially affect the basic and novel characteristics or functions of the item concerned. For example, if present in the language "consisting essentially of", the composition is permitted to contain trace elements that do not affect the properties or characteristics of the composition, even if such trace elements are not expressly listed in the list of items following such term . When an open-ended term such as "comprises" or "comprises" is used in a written description, it should be understood that it also directly supports language "consisting essentially of" as well as language "consisting of", as expressly As stated, and vice versa.

The terms "first", "second", "third", "fourth", etc., in the specification and claims of the invention, if any, are used to distinguish similar elements and not necessarily to describe specific sort or chronological order. It is to be understood that any terms so used are interchangeable under appropriate circumstances such that the embodiments described herein are, for example, capable of operation in other orderings than those illustrated or otherwise described herein. Similarly, if a method is described herein as comprising a series of steps, the order in which the steps are presented is not necessarily the only order in which the steps may be performed, and some of the described steps may be omitted from the method and /or possibly adding some other steps not described here.

Terms used herein such as "increased", "decreased", "better", "worse", "higher", "lower", "enhanced", "maximized ", "minimized" and other comparative terms mean the property of a device, component, composition or activity that is significantly different from other devices, components, compositions or activities in the surrounding or adjacent In an area, in a similar state, in a single device or composition or among comparable devices or compositions, in a group or type, in groups or types or compared to the known state of the art .

As used herein, the term "coupled" is defined as directly or indirectly connected chemically, mechanically, electrically or non-electrically. Items referred to herein as being "adjacent" to each other may be in physical contact with each other, in close proximity to each other, or in the same general area or area as each other, depending on the context in which the phrase is used. Appearances of the phrase "in one embodiment" or "in one aspect" herein are not necessarily all referring to the same embodiment or aspect.

The term "substantially" as used herein means the complete or nearly complete range or degree of an action, feature, property, state, structure, item or result. For example, an article that is "substantially" enclosed means that the article is completely enclosed, or nearly completely enclosed. In some cases, the exact permissible degree of deviation from absolute perfection may depend on the circumstances. In general, however, the closeness of completeness will have the same overall result as the attainment of absolute completeness and completeness. The use of "substantially" when used in a negative connotation applies equally to meaning a complete or almost complete lack of an action, characteristic, property, state, structure, item or result. For example, a composition that is "substantially free" of particles may be completely free of particles, or nearly completely free of particles, so that the effect is the same as having no particles at all. In other words, a composition that is "substantially free" of an ingredient or element may actually contain that item as long as it has no measurable effect.

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

As used herein, the terms multiple items, structural elements, constituent elements and/or materials, may be presented in a common list for convenience. However, these lists should be construed as if the individual members of the list are individually identified as a single and unique member. Accordingly, no individual member of such list should be construed as de facto equivalent to any other member of the same list solely based on their presentation in a common group without an indication to the contrary.

Concentrations, amounts, levels, and other numerical data may be expressed or presented herein in a range format. It should be understood that this range format is used merely for convenience and brevity, and should therefore be construed flexibly to include not only the values expressly recited as range limits, but also all individual values or subranges, or decimal units, encompassed within that range, with respect to It is as if each value and subrange were explicitly enumerated. By way of example, a numerical range of "about 1 to about 5" should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also individual values and subranges within the indicated range. Accordingly, this numerical range includes individual values such as 2, 3, and 4 and subranges such as from 1-3, from 2-4, and from 3-5, etc., as well as 1, 2, 3, 4, and 5 individually. The same principle applies to ranges that have only one value as the minimum or maximum value. Moreover, such an interpretation should apply regardless of the breadth of the scope or the characteristics described.

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

Molecular testing for many pathogens (e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19) may be limited to the laboratory and therefore have significant delays in providing results (> 24 hours), hindering its adoption in point-of-care settings. Although there have been many attempts to develop point-of-care tests for SARS-CoV-2, there are still some limitations: i) Scalability (the weekly test demand is in the millions, but manufacturing new tests at this scale difficult), ii) sample handling (when saliva is used, many tests still use an extraction procedure), and iii) readability (molecular tests often use fluorescence, so results are reported using a fluorescent reader).

Current testing methods can be overcome by point-of-care testing using paper-based devices and reverse transcription loop-mediated constant temperature amplification (RT-LAMP), which can use diluted saliva (e.g., 5% v/v in water) as a sample, Within 60 minutes, report a color change in the presence of a pathogen (eg, SARS-CoV-2). RT-LAMP is a nucleic acid amplification technique that is performed at constant temperature and has suitable diagnostic properties especially during the acute phase of infection. Because RT-LAMP can be performed at a constant temperature, it is not necessary to use expensive thermal cycling equipment. In addition, existing colorimetric reporters for LAMP products do not use fluorescent readers. Therefore, this test is suitable for use in point-of-care settings and can be rapidly developed and scaled up, making it suitable for use in public health emergencies.

RT-LAMP can be implemented on microfluidic paper-based analysis devices (μPADs) to detect various pathogens (eg, SARS-CoV-2), where image analysis can be performed using a portable electronic device to distinguish positive from negative reactions. In one example, a high-contrast RT-LAMP reaction on paper provides a color change visible to the naked eye. Furthermore, to prevent crosstalk between samples, polystyrene spacers can be used instead of wax printing (which may have precisely aligned printed areas and dispense reagents). Polystyrene spacers are easily manufactured roll-to-roll for scale-up.

Nucleic acid-based diagnostic methods for COVID-19 use preprocessing to deliver results. As disclosed herein, colorimetric detection of SARS-CoV-2 on paper can be performed with minimal preprocessing. The device can have the sensitivity and specificity to detect SARS-CoV-2 on paper without pre-amplification. Other assays performed in solution may not scale as well during manufacturing as paper-based assays. In addition, the assay disclosed herein uses a dilution operation that can be completed in seconds, while other assays use various operations such as protease treatment, heat deactivation, and/or RNA extraction to detect SARS-CoV-2 (operation completed at least 10 minutes and use additional equipment). Sample Collection and Characterization for LAMP Analysis

Saliva has various physical, chemical and antibacterial properties which may cause difficulties in the case of LAMP reactions. For example, in one physical property, saliva dilutes and removes organic acids from dental plaque, which may interfere with the LAMP reaction. Some chemical properties—electrolytes and buffer molecules that minimize pH changes—may also interfere with the LAMP reaction. Antibacterial agents in saliva, such as mucin, amylase, lysozyme and peroxidase enzymes also present challenges. For example, peroxidase enzymes can form free radical compounds in bacterial cells, which can cause them to undergo apoptosis-like death. However, this reaction may also provide an unstable redox environment, complicating the LAMP reaction.

With the above background in mind, as shown in the flowchart in FIG. 1 , a method 100 for preparing a saliva sample for loop-mediated isothermal amplification (LAMP) detection of pathogenic targets is provided. Depending on the signal output ultimately chosen to display the test result, eg optical detection of a pH-based color change, it is desirable to ensure that saliva in the sample does not shift the pH of the entire sample substantially from neutral. A variety of techniques and methods can be implemented to detect or limit the buffering capacity or influence of saliva in a sample. Excessive buffering capacity may prevent fluctuations in pH for detecting pH-based color changes.

One method of reducing the buffering capacity of saliva may include dilution. In one embodiment, such a method may comprise providing an amount of saliva from a test subject, as shown at block 110, and diluting the saliva in water to reduce the buffering capacity of the saliva while maintaining a sufficient concentration to The extent to which detection of the pathogenic target is permitted is indicated by block 120 .

The proteins present in saliva present another challenge. For example, a sample that is too viscous may be difficult to test in a solid base or medium. Slow flow rates in solid-based media may increase reaction time, reduce development uniformity, increase result variability, and increase result ineffectiveness. For example, color-based indications can be difficult to read when viscous saliva does not spread evenly throughout the solid-based medium. Reduced spread uniformity may also increase result variability by adding read uncertainty. Different techniques may interpret different results. In some cases, results may not be readable due to unclear or no color changes. Therefore, controlling the viscosity of saliva prevents various complicating factors that may occur.

Thus, in another embodiment, the method may further comprise reducing the viscosity of the saliva compared to the original viscosity. In one aspect, the saliva can be reduced by one or more of dilution, filtration, etc., or a combination thereof. In one aspect, when dilution is used to reduce the viscosity of saliva, the saliva can be diluted in water to a saliva to water ratio of from about 1:1 to about 1:20. In another aspect, the viscosity of the saliva can be diluted in 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 aspect, the saliva can be diluted in water to an extent that provides the sample with an optical density at 600 nm ( _OD6oo ) of less than about 0.2. In one aspect, the saliva can have a volume ranging from about 50 μl to about 100 μl. In another aspect, the saliva sample may have a volume ranging from about 100 μl to about 1 ml.

In some cases, diluting a saliva sample can reduce the shock due to the buffering capacity and viscosity of saliva. Other methods of reducing the impact of saliva viscosity may include filtration. In another aspect, a filter having a rating between about 2 microns and 50 microns can be used to reduce viscosity. In one example, the filter grade can 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 aspect, the filter rating can be an absolute micron rating, wherein the filter can remove at least about 98.7% of a specified particle size. Filtering saliva, rather than diluting it separately, also removes salivary proteins (eg, mucin, amylase, lysozyme, and peroxidase enzymes) that may interfere with the LAMP reaction.

Viscosity can be controlled to fall within a specified range by a combination of dilution, filtration, or both. In yet another aspect, the viscosity can be reduced to such an extent that the flow through the solid medium is increased compared to the original viscosity. In one example, the viscosity of saliva may range from about 1 centipoise (cP) to about 100 cP prior to dilution or filtration. In one example, after dilution or filtration, the viscosity of the saliva can be reduced to a range from about 1.0 cP to about 50 cP. In another example, after dilution or filtration, the viscosity of the saliva can be reduced to a range from about 1.0 cP to about 10 cP.

Filtering saliva can also adjust the pH range to a desired level. For example, certain pH indicators can exhibit a color change over a characteristic pH range (eg, 7.2 to about 8.6). Thus, depending on the type of pH indicator used, the saliva is filtered to the target range of the test sample. However, maintaining the test sample target range under physiological conditions can increase the uniformity of LAMP response results. In one aspect, the saliva can be filtered to the extent that the pH of the saliva sample is adjusted to a test sample target range. In one example, the test sample target range can include a pH range between about 7.2 and about 8.6. In another example, the test sample target range can include a pH range between about 7.6 and about 8.2.

Adjusting the test sample target range to a desired level may not be sufficient to detect pH changes (or other colorimetric indications) in the LAMP reaction. In another example, the saliva may be diluted with water to such an extent that the buffering capacity of the composition is reduced to allow detection of an indication of pH relative to the buffering capacity prior to dilution with water. In one example, buffering capacity can be defined as the ability of a solution (eg, saliva, water, or saliva diluted with water) to resist changes in pH when an acid or base is added. In one example, the buffering capacity can be defined as the amount, in gram equivalents, of a strong acid or base added to 1 liter of solution to change the pH by one unit. In one aspect, the buffering capacity of the saliva can be between 0.03 mg/ml and about 0.30 mg/ml before dilution with water, and the buffering capacity of the saliva can be between about 0.003 mg/ml and about 0.03 mg/ml after dilution with water. Between mg/ml. In another example, the buffering capacity of water-diluted saliva can be less than about 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM.

Water used to dilute samples should be free of any contaminants or properties that may interfere with the LAMP reaction. For example, a pH that is too acidic may prevent the detection of a LAMP reaction if the pH would prevent a change based on the pH indication. In one aspect, the saliva can be diluted in water, wherein the water can have a pH greater than about 6.0. In another aspect, the water can have a pH of less than about 8.0. In one example, the water can be molecular grade water that is substantially free of contaminants such as RNase and DNase. RNase degrades the RNA to be detected in saliva, while DNase degrades DNA formed during the LAMP reaction. In another example, the saliva sample can consist essentially of saliva and water.

Minimizing the presence of unwanted salivary proteins can be done using specific saliva collection methods. In one aspect, saliva may be collected using one or more of a sponge-based collection method or a passive drool collection method. Collection of saliva using a sponge-based collection method provides the benefit of naturally filtering out mucin and high molecular weight proteins from the saliva as they cannot be absorbed by the sponge, which reduces the viscosity of the saliva when used on a solid-based medium And improve rapidity, uniformity and reliability. Unfiltered saliva may be more viscous when collected using the spit method, thereby reducing absorption and distribution on solid-based media. As a result, in some embodiments, when saliva is collected by spit, it can be filtered afterwards to remove mucin and other debris and reduce its stickiness.

Selected pathogenic targets can be detected from saliva. In one aspect, the pathogenic target can be one or more of viral pathogens, bacterial pathogens, fungal pathogens, protozoan pathogens, etc. or a combination thereof. Pathogenic targets can be detected in saliva when nucleic acids from the pathogenic targets can be released from cell walls, cell membranes, protein coats, etc.

More specifically, in one aspect, the pathogenic target can be a viral target. In some aspects, the viral target can 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, etc. or a combination thereof.

The viral target can be selected from many different viral species. In one example, the viral target can be human coronavirus 229E, human coronavirus OC43, human coronavirus HKU1, human coronavirus NL63, MERS-coronavirus, human respiratory virus 1, human rubulavirus 2 (human rubulavirus 2) , human respiratory virus 3, human mumps virus 4, human enterovirus, human respiratory virus, rhinovirus A, rhinovirus B, rhinovirus C, or combinations thereof.

The viral target may also be in the form of influenza. In one aspect, the influenza can be any of influenza A, influenza B, influenza C, or influenza D. In one aspect, the viral target may be a virus selected from the order Nidovirale . In one aspect, the viral target may be selected from the alpha, beta, gamma, delta genera of the order Reticuloviridae.

Detectable viruses are of various virus families. In one aspect, the viral target may be a DNA virus selected from the group consisting of the following families: Adenoviridae , Papovaviridae , Parvoviridae , Herpesviridae ( Herpesviridae ), poxviridae ( Poxviridae ), anelloviridae ( Anelloviridae ), halophilic bacteria polymorphic virus family ( Pleolipoviridae ) , etc. and combinations thereof. In another aspect, the viral target may be an RNA virus selected from the group comprising the following families: Reoviridae , Picornaviridae , Caliciviridae , Togaviridae , Arenaviridae , Flaviviridae , Orthomyxoviridae , Paramyxoviridae , Bunyaviridae , Baculoviridae Rhabdoviridae , Filoviridae , Coronaviridae , Astroviridae , Bornaviridae , etc. and combinations thereof. In another aspect, the viral target may be a retrovirus selected from the family group comprising: Retroviridae , Caulimoviridae , Hepadnaviridae , etc. and etc. combination.

More generally, the viral target may be a virus classified according to the Baltimore classification. In one aspect, the viral target can be an RNA virus (eg, influenza A, Zika, hepatitis C). In one aspect, the viral target can be a DNA virus (eg, Epstein Barr, smallpox). In one aspect, the viral target can be a positive-sense RNA virus (eg, hepatitis A, rubella). In one aspect, the viral target can be an anti-sense RNA virus (eg, Ebola, measles, mumps). In another aspect, the viral target can be a dsDNA virus (e.g., chickenpox, herpes), ssDNA virus, dsRNA virus (e.g., rotavirus), positive-strand ssRNA virus, reverse-strand ssRNA virus, ssRNA-RT virus ( eg, retroviruses) or ds-DNA-RT viruses (eg, hepatitis B).

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

The pathogenic targets may also include various types of fungi. In one aspect, the pathogenic target can be a fungal target. In some examples, the fungal target can be selected from genera including: Aspergillus , Histoplasma , Pneumocystis , Stachybotrys , etc. and combinations thereof. In another aspect, the pathogenic target can be a protozoan target. In some examples, the protozoan target can be selected from a genus comprising: plasmodium , trypanosomes , and the like, and combinations thereof.

When the pathogenic target in saliva includes RNA, the RNA can be reverse transcribed. Thus, in another aspect, the LAMP detection can be a reverse transcription LAMP (RT-LAMP). In this example, reverse transcriptase enzymes are used to generate cDNA from target RNA. The cDNA can be amplified to a detectable amount. When the pathogenic target can be detected directly from DNA, then the DNA can be amplified to a detectable amount using LAMP, without the need for reverse transcription of RNA to DNA.

In another aspect, the specific target nucleotide sequence to be detected may be a target nucleotide sequence corresponding to a human biomarker. Any disease having a target nucleotide corresponding to a human biomarker for the disease being detected can be detected. Various detectable disease types include one or more of the following: breast cancer, pancreatic cancer, colorectal cancer, ovarian cancer, gastrointestinal cancer, cervical cancer, lung cancer, bladder cancer, multiple epithelial cancers, salivary gland cancer, kidney cancer, liver cancer , lymphoma, leukemia, melanoma, prostate cancer, thyroid cancer, gastric cancer, etc. or a combination thereof. For example, biomarkers of various disease types can be detected by detecting target nucleotides corresponding to one or more of the following: alpha-fetoprotein, CA15-3 and CA27-29, CA19-9, C!-125, calcitonin , calretinin, carcinoembryonic antigen, CD34, CD99MIC 2, CD117, chromosomal granulin, chromosome 3, 7, 17 and 9p21, cytokeratin, cesmin, epithelial membrane antigen, coagulation factor VIII, CD31 FL1, glial fibrils Acidic protein, macrocystic disease liquid protein, hPG80, HMB-45, human chorionic gonadotropin, immunoglobulin, inhibin, keratin, lymphocyte markers, MART-1, Myo D1, muscle-specific actin, Neurofilament, neuron-specific enolase, placental alkaline phosphatase, prostate-specific antigen, PTPRC, S100 protein, smooth muscle actin, synaptophysin, thymidine kinase, thyroglobulin, thyroid transcription factor-1, tumor M2-PK, vimentin, etc. or a combination thereof.

In another embodiment, the test sample composition of the loop-mediated constant temperature amplification (LAMP) assay can include an amount of saliva of a test subject sufficient to detect a pathogenic target by the LAMP assay, and can reduce the amount of saliva of the test subject. A combination of water volumes for buffer capacity. In one aspect, the viscosity of the composition can be from about 1.0 cP to about 50 cP. In another aspect, the pH of the composition may be from about 7.2 to about 8.6. Selecting the viscosity and pH within these respective ranges can enhance the change in pH and thus the color change produced by the pH-based indicator.

The saliva can be diluted with water to bring the viscosity and pH within the above range. In one aspect, the saliva can be combined with an amount of water in a saliva to water ratio of from about 1:1 to about 1:20. In another aspect, the ratio of saliva to water may be about 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:1 16. 1:18 or 1:20. In another aspect, the saliva can be combined with the amount of water to provide the sample with an optical density at 600 nm (OD ₆₀₀ ) of less than 0.2.

To ensure that the amount of saliva contains a detectable amount of virus, the amount of saliva collected may be above a threshold amount. In one aspect, the saliva can have a volume ranging from about 50 μl to about 100 μl. In another aspect, the saliva sample may have a volume ranging from about 100 μl to about 1 ml.

Saliva may also have various chemical properties (eg, pH and buffering capacity) that promote LAMP reactions. In one aspect, the water can have a pH greater than about 6.0 and be substantially free of contaminants such as RNase and DNase. In another aspect, the water can have a pH of less than about 8.0 and be substantially free of contaminants. In another aspect, the composition may consist essentially of the saliva and the water. In one aspect, the composition may have a buffer capacity between about 0.003 mg/ml and about 0.03 mg/ml. In another example, the composition may have a buffer capacity of less than about 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM.

As previously mentioned, the pathogenic target may comprise a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. The pathogenic target may be a viral target. In another aspect, the viral target can comprise a dsDNA virus, ssDNA virus, dsRNA virus, sense ssRNA virus, reverse strand ssRNA virus, ssRNA-RT virus, or ds-DNA-RT virus, according to the Baltimore Classification of Viruses. In another aspect, the viral target may comprise H1N1, H2N2, H3N2, H1N1pdm09 or SARS-CoV-2. Reagent composition

A variety of different reagents can be used in LAMP assays depending on the test medium, the type of readout, and the overall environment for which the system is designed. Furthermore, reaction components such as primers and enzymes can be selected according to the specific target nucleotide sequence to be detected, the organism to be identified, and the like. Furthermore, details of the test environment, such as liquid environment, anhydrous environment, housing, substrate, etc., as well as other requirements, such as storage stability, can be considered when selecting specific reagents to participate in reactions under LAMP analysis.

In one embodiment, a composition for a loop-mediated isothermal amplification (LAMP) assay on a solid medium may comprise one or more primers of interest, a DNA polymerase, and a resolubilizing agent. In one aspect, the composition may be substantially free of non-pH sensitive agents capable of discoloring the solid phase medium.

Compared with LAMP analysis in liquid medium, when performing LAMP analysis on solid phase medium, the concentration of reagents can be increased. In one aspect, the concentration of the DNA polymerase when used on a solid medium can be at least twice the concentration of the DNA polymerase used with a liquid medium. In another aspect, the concentration of the DNA polymerase when used on a solid medium can be at least three times the concentration of the DNA polymerase used with a liquid medium. In one example, the concentration of DNA polymerase can be from about 300 U/mL to about 1000 U/mL when used on a solid medium. In another example, the concentration of DNA polymerase can be from about 600 U/mL to about 1000 U/mL when used on a solid medium. In yet another example, the concentration of DNA polymerase can be from about 620 U/mL to about 680 U/mL when used on a solid medium.

When the LAMP assay involves reverse transcriptase LAMP (RT-LAMP), the composition may further comprise reverse transcriptase. The reverse transcriptase can aid in the detection of RNA-based viruses. In one aspect, when used on a solid medium, the concentration of reverse transcriptase can be at least twice the concentration of reverse transcriptase used with liquid medium. In another aspect, when used on a solid medium, the concentration of reverse transcriptase may be at least three times the concentration of reverse transcriptase used with liquid medium. In one example, the concentration of reverse transcriptase can be from about 200 U/mL to about 600 U/mL when used on a solid medium. In another example, the concentration of reverse transcriptase can be from about 250 U/mL to about 500 U/mL when used on a solid medium. In yet another example, the concentration of reverse transcriptase can range from about 290 U/mL to about 310 U/mL when used on a solid medium.

In addition to the target primer, DNA polymerase and reverse transcriptase, the composition may also include a resolubilizing agent. When the saliva sample is deposited on the solid-based medium, the resolubilizing agent helps to rehydrate the LAMP reagent on the solid-based medium. In one aspect, the redissolving agent can be a surfactant. For example, the resolubilizing agent may comprise bovine serum albumin (BSA), casein, polysorbate 20, etc. or combinations thereof. BSA and casein can facilitate the resolubilization of DNA polymerase, reverse transcriptase and other related enzymes when the dried reagents are rehydrated. Polysorbate 20 is a surfactant that also aids redissolution of dried reagents. In one example, when used on a solid medium, the concentration of the redissolving agent can be from about 0.05% to about 5% by weight. In another example, the concentration of the redissolving agent may be from about 0.5% to about 3% by weight. In yet another example, the concentration of the redissolving agent may be from about 0.5% to about 1.5% by weight.

The composition may further include agents that can accelerate the reaction, increase the sensitivity, or a combination thereof. In one example, BSA can be included to speed up the reaction and increase sensitivity. However, the inclusion of BSA may also cause pH changes which may interfere with the readability of the results. Thus, in some examples, the resolubilizing agent may include casein, polysorbate 20, etc., or combinations thereof.

Volatile agents may interfere with the LAMP reaction. For example, a volatile compound may ionize into multiple ions, one of which may have a low boiling point. When the low-boiling ions evaporate, the remaining ions are available for further reactions. Some further reactions may include redox reactions, acid-base reactions, or other reactions that may affect the interpretation of the pH-based signal. In one aspect, the composition can be substantially free of volatile agents. In one example, the removal of the volatile agent increases the color contrast and decreases the reaction time of the solid-based medium compared to the color contrast and reaction time when the volatile agent is included. In one aspect, the composition may contain less than one or more of the following volatile agents: 1.0%, 0.5%, 0.1%, or 0.01% by weight.

Volatile agents may cause instability of solid-based media. In some instances, LAMP reactions containing volatile compounds such as ammonium sulfate can lead to instability of solid-based media when ammonium ions partially convert ammonium sulfate to ammonium, which volatilizes leaving sulfate. Sulfate can change to sulfuric acid, lowering the pH, which can affect the interpretation of pH-based indicators (eg, turning a phenol red indicator from red to yellow even though the LAMP reaction has not occurred). Replacing ammonium sulfate with betaine prevents discoloration not based on the LAMP reaction and stabilizes the solid-based medium by preventing discoloration upon storage.

Thus, reducing the presence of volatile agents in the composition reduces the degree of interference with the LAMP reaction and its interpretation by pH-based indicators. In one aspect, the LAMP composition may include a non-volatile agent, including a neutrally charged low molecular weight quaternary ammonium or a neutrally charged low molecular weight amide compound, or a combination thereof. In one example, the non-volatile agent may include, but is not limited to, N-formylurea, urea, L-asparagine, trimethylglycine (betaine), 3-(cyclohexylamino )-1-propanesulfonic acid (CAPS), 3-(1-pyridinyl)-1-propanesulfonate (NDSB-201), N-methylurea, acetamide, propionamide, isobutyramide Amine, Piracetam, 1,3-dimethylurea, 1,1-dimethylurea, glycolamide, 2-chloroacetamide, succinimide, 2-tetrahydroimidazolone , choline chloride, acetylcholine chloride, bethanechol chloride, L-carnitine inner salt, O-acetyl-L-carnitine hydrochloride, 4-(cyclohexyl Amino)-1-butanesulfonic acid (CABS), ammonium dimethylethylpropanesulfonate (NDSB-195), 3-(1-methylpiperidinium)-1-propanesulfonate (NDSB-221 ), 3-(benzyldimethylammonium)propanesulfonate (NDSB-256) and dimethyl-2-hydroxyethylammonium-1-propanesulfonate (NDSB-211), etc., or etc. combination.

In one example, when used on the solid medium, the non-volatile agent, comprising a neutrally charged low molecular weight quaternary ammonium or a neutrally charged low molecular weight amide compound, can be present at a concentration of from about 1 mM to about 200 mM . In another example, when used on the solid medium, the concentration of the non-volatile agent can be from about 10 mM to about 50 mM. In yet another example, when used on the solid medium, the concentration of the non-volatile agent can be from about 15 mM to about 25 mM.

In addition to volatile agents, hygroscopic agents can also interfere with the LAMP reaction. Hygroscopic agents retain excess water and destabilize reagents in solid-based media by slowing or preventing drying. In one aspect, the composition may be substantially free of hygroscopic agents. In some instances, LAMP reactions containing hygroscopic agents, such as glycerol, can lead to instability of the reagents in solid media due to the hygroscopic agent absorbing water. In one example, the hygroscopic agent can absorb more than about 10% by weight between about 40% and about 90% relative humidity (RH) at 25°C. In one example, the hygroscopic agent may include, but not limited to, one or more of the following: glycerin, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate, etc., or a combination thereof. In one aspect, the composition may contain less than one or more of the following moisture absorbents: 1.0%, 0.5%, 0.1%, or 0.01% by weight.

Additional reagents may be included to prevent carryover from previous LAMP reactions, primer dimerization, non-specific amplification carryover. Inclusion of deoxyuridine triphosphate (dUTP), uracil DNA glycosidase (UDG), or a combination thereof in the LAMP reaction can prevent carryover carry-over contamination. These agents catalyze the release of free uracil from uracil-containing single- or double-stranded DNA.

Certain pH-based indicators with antioxidant activity, such as phenol red, have been found to increase contrast and uniformity compared to other pH-based indicators with a reduced degree of antioxidant activity. In one aspect, the composition may further include an antioxidant. In one example, when used on the solid-based medium, the concentration of the antioxidant can be from about 0.1 mM to about 1 mM. In another example, when used on the solid-based medium, the concentration of the antioxidant can be from about 0.2 mM to about 0.8 mM. In yet another example, when used on the solid-based medium, the concentration of the antioxidant may be from about 0.2 mM to 0.3 mM. The antioxidant can stabilize the reagents on the solid-based medium by preventing redox reactions.

Various antioxidants that can be used include, but are not limited to: N-acetyl-cysteine, hydroxytyrosol (HXT), superoxide dismutase (SOD), catalase, vitamin A, vitamin C, Vitamin E, coenzyme Q10, manganese, iodide, melatonin, α-carotene, astaxanthin, β-carotene, canthaxanthin, cryptoxanthin, lutein, lycopene, zeaxanthin, carotene Coriander yellow, luteolin, tangerine, isorhamnetin, kaempferol, myricetin, proanthocyanidins, quercetin, eriodyctiol, hesperidin, naringin Ligand, catechin, gallocatechin, epicatechin, epigallocatechin, theaflavins, thearubigins, daidzein, genistein, soybean Glycitein, resveratrol, pterostilbene, cyanine, delphinidin, mallowin, geranioside, paeoniflorin, petunia, cichoric acid, chlorogenic acid, cinnamic acid, Turkish tannin Acid, Gallotannin, Gallic Acid, Tannin, Rosmarinic Acid, Salicylic Acid, Curcumin, Flavonoid Lignan, Oxanthones, Eugenol, Capsaicin, Bilirubin, Lemon acid, oxalic acid, phytic acid, R-alpha-lipoic acid, etc., or combinations thereof.

Although pH-based indicators have been discussed so far, other indicators can also be used. In one aspect, the composition may further include an indicator. In one example, when used with a solid-based medium, the indicator may be a pH-based indicator, such as phenol red. Phenol red has some antioxidant properties not found in other dyes. Phenol red molecules are conjugated bond systems that may also contribute to antioxidant properties. In one example, when used on the solid medium, the concentration of the indicator can be from about 0.1 mM to about 1 mM. In another example, when used on the solid medium, the concentration of the indicator can be from about 0.2 mM to about 0.8 mM. In yet another example, when used on the solid medium, the concentration of the indicator can be from about 0.2 mM to about 0.3 mM.

Some other indicators may also provide appropriate colorimetric signals. In another example, the indicator can be one or more of: (i) a magnesium colorimetric indicator, (ii) a pH colorimetric indicator, or (iii) a DNA intercalation colorimetric indicator. When the indicator is a magnesium colorimetric indicator, the concentration of magnesium should be monitored so as to maintain the magnesium in the range of from about 0.01 mM to about 2 mM. Also, the concentration of magnesium should be monitored to prevent interference with DNA polymerase. Magnesium – a cofactor for DNA polymerase, interferes with DNA polymerase when magnesium concentration exceeds a target range.

LAMP reactions can also use various types of target primers. Some target primers may include about 4 or 6 primers, which may target 6 or 8 regions within the genome, respectively. In one aspect, the target primer may have a concentration of from about 0.05 μM to about 5 μM when used on a solid medium. In another example, when used on a solid medium, the target primer may be present at a concentration of from about 0.1 μM to about 3 μM. In yet another example, when used on a solid medium, the target primer may be present at a concentration of from about 0.2 μM to about 1.6 μM.

Primers of interest can be selected to target the genome of various pathogens. In one aspect, the target primer can target a pathogen, which can comprise a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. In another aspect, the pathogenic target can be a viral pathogen. In another aspect, the viral target can comprise a dsDNA virus, ssDNA virus, dsRNA virus, sense ssRNA virus, reverse strand ssRNA virus, ssRNA-RT virus, or ds-DNA-RT virus. In another aspect, the viral target may comprise H1N1, H2N2, H3N2, H1N1pdm09 or SARS-CoV-2. In summary, this target primer can target almost any pathogenic target, especially those disclosed herein.

When the solid medium contains excessive volatile agents, oxidants, pH interfering agents, magnesium interfering agents, etc. or a combination thereof, the color of the solid medium without amplification of the LAMP reaction will be affected. To solve this problem, in another aspect, the composition may contain a color-changing additive. In one aspect, the non-discoloration additive may be present at a concentration of from about 0.01 mM to about 1M when used on a solid medium. In another example, when used on a solid medium, the non-discoloration additive may be present at a concentration of from about 10 mM to about 500 mM. In yet another example, when used on a solid medium, the non-discoloration additive may be present at a concentration of from about 200 mM to about 400 mM.

There are various color-changing additives that maintain the color of the solid-based medium in the absence of amplification by the LAMP reaction and potentially increase contrast when amplification by the LAMP reaction occurs. In one embodiment, the color-changing additive may include one or more of sugars, buffers, etc., or a combination thereof.

In one example, a non-discoloration additive, such as sugar, stabilizes the solid-based medium and prevents discoloration under long-term storage conditions. For example, trehalose can maintain the stability of the enzyme under freeze-drying conditions or when dried at ambient temperature. In one aspect, the sugar may comprise one or more of the following: glucose, sucrose, trehalose, dextran, etc., or a combination thereof. In one aspect, the sugar may be present at a concentration of from about 0.01 mM to about 1M when used on a solid medium. In another example, the sugar may be present at a concentration of from about 10 mM to about 500 mM when used on a solid medium. In yet another example, when used on a solid medium, the sugar may be present at a concentration of from about 200 mM to about 400 mM.

The LAMP reaction may also include other reagents. In one aspect, the composition may comprise one or more enzymes, nucleic acids, or a combination thereof. In one example, the enzyme can be an RNase inhibitor or a DNase inhibitor. The inclusion of an RNase inhibitor slows the degradation of the RNA target, allowing for improved detection limits. The inclusion of a DNase inhibitor slows down the degradation of DNA targets, allowing for increased detection limits. In one aspect, the composition may comprise carrier DNA or carrier RNA. The carrier DNA or carrier RNA can provide a bait matrix for sequestering DNase or RNase activity, respectively. In another example, a defined amount of guanidine hydrochloride stimulates the denaturation and exposure of RNA molecules, which further stabilizes the LAMP response.

In one aspect, when used on a solid medium, the RNase or DNase inhibitor may be present at a concentration of about 0.01 μL per mL of saliva sample to about 5 μL per mL of saliva sample. In another example, when used on a solid medium, the RNase or DNase inhibitor may be present at a concentration of about 0.1 μL per mL of saliva sample to about 1 μL per mL of saliva sample. In yet another example, when used on a solid medium, the RNase or DNase inhibitor may be present at a concentration of about 0.5 μL per mL of saliva sample to about 1.5 μL per mL of saliva sample.

In one aspect, the carrier RNA or carrier DNA may be present at a concentration of from about 0.01 ng/μL to about 10 ng/μL when used on a solid medium. In another example, when used on a solid medium, the carrier RNA or carrier DNA may be present at a concentration of from about 0.1 ng/μL to about 1 ng/μL. In yet another example, when used on a solid medium, the carrier RNA or carrier DNA may be present at a concentration of from about 0.2 ng/μL to about 0.4 ng/μL.

In addition to the foregoing, many other agents or ingredients may be used in compositions suitable for carrying out the LAMP reactions described herein. For example, in another aspect, the composition may further include a tonicity agent, a pH regulator, a preservative, water, etc. or a combination thereof. Additionally, these ingredients/agents can be used to provide the composition with a range of particularly desirable properties. In one aspect, the composition may have a tonicity of from about 250 to about 350 milliosmoles/liter (mOsm/L). In another aspect, the composition may have a tonicity of from about 270 to about 330 mOsm/L. Tonicity agents can be present in the composition in various amounts. In one aspect, the concentration of the tonicity agent in the composition may be from about 0.1%, about 0.5% or about 1% by weight to about 2%, about 5% or about 10% by weight.

Although the composition should be substantially free of pH interfering agents, pH adjusters can be used to select the initial pH of the composition prior to the LAMP reaction. In addition, when the effect of the pH adjuster can be compensated when interpreting the results of the LAMP reaction, the pH adjuster can also be used. Non-limiting examples of pH adjusters may include combinations of various acids, bases, and the like, such as hydrochloric acid, phosphoric acid, citric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, and the like. The pH adjuster can be used to provide a suitable pH for the composition. In one aspect, the pH can be from about 5.5 to about 8.5. In one aspect, the pH can be from about 5.8 to about 7.8. In another example, the pH can be from about 6.5 to about 7.8. In yet other examples, the pH can be from about 7.0 to about 7.6. The pH adjuster can be present in the composition in various amounts. In one aspect, the pH adjuster may have a concentration in the composition from about 0.01% by weight, about 0.05% by weight, about 0.1% by weight or about 0.5% by weight to about 1% by weight, about 2% by weight, about 5% by weight or about 10% by weight.

The use of preservatives increases the shelf life of the composition. Non-limiting examples of preservatives may include alkyldimethylbenzylammonium chloride (BAK), cetrimonium, sodium perborate, ethylenediaminetetraacetic acid (EDTA) and various salt forms thereof, chloroprene Alcohol and so on. Preservatives can be present in the composition in various amounts. In one aspect, the concentration of the preservative in the composition can be from about 0.001 wt%, about 0.005 wt%, about 0.01 wt% or about 0.05 wt% to about 0.1 wt%, about 0.25 wt%, about 0.5 wt% % by weight or about 1% by weight.

In another embodiment, as shown in FIG. 2, a method 200 for LAMP analysis on a solid medium can include providing an assembly having a solid medium and a reaction composition (as described herein) combination of any of the ingredients or compositions described above), as shown in block 210. In one aspect, the method can include depositing a biological sample onto the solid medium, as represented by block 220 . In another aspect, the method can include heating the assembly to a constant temperature sufficient to promote the LAMP reaction, as represented by block 230 .

In one aspect, the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, etc., or a combination thereof. In another aspect, the biological sample can be saliva. In one aspect, the method can comprise detecting a viral pathogen. In one aspect, the viral pathogen can be a pathogen as disclosed herein. In another aspect, the LAMP assay can be reverse transcriptase LAMP (RT-LAMP).

In another aspect, the isothermal temperature sufficient to promote the LAMP reaction may range from about 50°C to about 70°C. In another aspect, the isothermal temperature sufficient to promote the LAMP reaction may range from about 60°C to about 70°C. In another aspect, the isothermal temperature sufficient to promote the LAMP reaction may range from about 60°C to about 65°C. The thermostatic temperature can be selected based on one or more of the activities of DNA polymerase, reverse transcriptase, or combinations thereof.

In another example, the temperature sufficient to promote the LAMP reaction may be in the range of about 60°C to about 70°C. In another example, the thermostatic temperature may be a temperature within a range that differs by less than 5°C.

In another embodiment, a system for performing LAMP analysis may comprise the compositions described in this disclosure. In another aspect, the system can include a solid medium on which the composition is deposited. Maximizes pH-sensitive signal output

When performing a LAMP reaction, various indicators can be used to read the reaction result. Colorimetric indicators include magnesium colorimetric indicator, pH colorimetric indicator and DNA intercalation colorimetric indicator. Because magnesium acts as a cofactor for DNA polymerase and its concentration should be tightly controlled, magnesium-based indicators may face various limitations when used in LAMP reactions. DNA intercalation indicators may also face limitations due to the high variability in their use. Although all three indicators can be used in LAMP reactions, the pH-based indicators may be subject to fewer variables.

In one embodiment, a composition for a loop-mediated isothermal amplification (LAMP) assay utilizing a pH-dependent output signal may comprise a pH-sensitive dye and non-interfering LAMP reagents. In one aspect, the LAMP assay can be reverse transcription LAMP (RT-LAMP).

The choice of pH sensitive dye may depend on various factors such as colorimetric range in relation to pH, degree of contrast between color changes, pH level of color change, uniformity of color change, reproducibility of color change, and the like. For example, phenol red can have a colorimetric range of pH between about 6.8 and about 7.4. Below a pH of about 6.8, phenol red will turn yellow, and above a pH of about 7.4, phenol red will turn red. The degree of difference between yellow and red is easy to read, and changes in pH can occur at pH levels that mimic physiological conditions.

In one aspect, the pH sensitive dye can be a pH indicator (eg, phenol red) that has a color change around pH 6.5 to achieve a consistent and contrasting color change. In one aspect, the pH-sensitive dye can be at least one of the following: phenol red, litmus, brometrivanol blue, nitrazine yellow, cresol red, curcumin, brilliant yellow, m-cresol violet, α-naphtholphthalein, phenolphthalein, neutral red, acid fuchsin, litmus, etc. or combinations thereof. In one aspect, the pH sensitive dye may be present at a concentration of from about 0.1 mM to about 1 mM when used on a solid medium. In another example, the pH sensitive dye may be present at a concentration of from about 0.2 mM to about 0.8 mM when used on a solid medium. In yet another example, the pH sensitive dye may be present at a concentration of from about 0.2 mM to about 0.3 mM when used on a solid medium.

In order to maximize the pH-sensitive signal output, the LAMP reaction should be substantially free of compounds that would be indeterminate by interfering with the LAMP reaction (e.g., interfering with DNA polymerase) or interfering with the signal (e.g., pH signal) from the LAMP reaction. Reagents that specifically introduce this signal. In one aspect, the plurality of non-interfering LAMP reagents can comprise DNA polymerase, reverse transcriptase, target primers, or a combination thereof. In another aspect, the plurality of non-interfering LAMP reagents can be substantially free of volatile agents, pH disruptors, magnesium disruptors, or combinations thereof.

In one example, the plurality of non-interfering LAMP reagents can be substantially free of magnesium, ammonium sulfate, or ammonium carbonate. Magnesium acts as a cofactor for DNA polymerase and should be closely monitored to ensure that the LAMP reaction will perform as designed. Ammonium sulfate can ionize to ammonium ions, which will leave sulfate ions, which can react to form sulfuric acid. Ammonium carbonate can also ionize to ammonium ions and leave carbonate groups that can react to form carbonic acid. Therefore, the plurality of non-interfering LAMP reagents should be substantially free of such substances.

Volatile agents should be kept to a minimum because they leave behind species that can react to form acids or bases, which interfere with the pH-dependent signal from the LAMP reaction. In one example, the plurality of non-interfering LAMP reagents can be substantially free of volatile agents, including but not limited to: ammonium sulfate, ammonium carbonate, and the like, or combinations thereof. In one aspect, the composition may contain less than one or more of the following volatile agents: 1.0%, 0.5%, 0.1%, or 0.01% by weight.

Furthermore, any pH disturber will interfere with the pH-dependent signal output when the pH disturber is not compensated. In one example, the plurality of non-interfering LAMP reagents can be substantially free of pH interfering agents, including but not limited to combinations of acids, bases, and the like. In one aspect, the composition may contain less than one or more of the following pH disruptors: 1.0%, 0.5%, 0.1%, or 0.01% by weight.

Even when pH has been monitored, pH-dependent signal output may be negatively affected when the LAMP reaction is perturbed. For example, magnesium acts as a cofactor for DNA polymerase and interferes with the amplification of LAMP reactions when concentrations are outside the selected range. In one example, the plurality of non-interfering LAMP agents can be substantially free of magnesium interfering agents. Magnesium interfering agents may include magnesium-containing agents, including but not limited to: Mg ²⁺ , Mg ¹⁺ , magnesium carbonate, magnesium chloride, magnesium citrate, magnesium hydroxide, magnesium oxide, magnesium sulfate, magnesium sulfate heptahydrate, and the like, or combinations thereof. In one aspect, the composition may contain less than one or more of the following magnesium: 1.0%, 0.5%, 0.1%, or 0.01% by weight. In another example, the magnesium interferer can include a magnesium interferent chelator.

Even if the pH has been monitored and the LAMP reaction is functioning properly, discoloration of the solid phase media may be caused by other factors, such as long-term storage. In one aspect, the composition may include a non-discoloration additive. In one example, the color-changing additive may include one or more of sugars, buffers, blocking agents, etc., or combinations thereof. In one example, the sugar stabilizes the solid-based medium and prevents discoloration under long-term storage conditions. In one aspect, the sugar may include one or more of the following: glucose, sucrose, trehalose, dextran, etc., or a combination thereof.

In one aspect, the sugar may be present at a concentration of from about 0.01 mM to about 1M when used on a solid medium. In another example, the sugar may be present at a concentration of from about 10 mM to about 500 mM when used on a solid medium. In yet another example, the sugar may be present at a concentration of from about 200 mM to about 400 mM when used on a solid medium.

Buffers facilitate the stabilization of LAMP reactions by eliminating variability from saliva samples. In one example, the buffering agent may include one or more of the following: phosphate buffered saline (PBS), Dulbecco's PBS, Alsever's solution, Tris buffered solution (TBS), HEPES, BICINE, water, balanced salt solution (BSS ), such as Hank's BSS, Earle's BSS, Gray's BSS, Puck's BSS, Simm's BSS, Tyrode's BSS, BSS Plus, Ringer's lactic acid solution, normal saline (ie 0.9% saline), 1/2 physiological Salt water, etc. or a combination thereof. In one aspect, when used on a solid medium, the buffer may be present at a concentration of from about 10 μM to about 20 mM. In another example, when used on a solid medium, the buffer may be present at a concentration of from about 100 μM to about 10 mM. In yet another example, when used on a solid medium, the buffer may be present at a concentration of from about 100 μM to about 500 μM.

A blocking agent can reduce the amount of RNase-based degradation, DNase-based degradation, or other enzymatic degradation. In one example, the blocking agent may include one or more of bovine serum albumin, casein, or a combination thereof. In one aspect, when used on a solid medium, the concentration of the blocking agent may be from about 0.01% to about 5% by weight. In another example, when used on a solid medium, the concentration of the blocking agent may be from about 0.01% to about 1% by weight. In yet another example, when used on a solid medium, the concentration of the blocking agent may be from about 0.02% to about 0.06% by weight.

Antioxidants can increase the uniformity and contrast of pH-dependent signals on solid media by eliminating the variables associated with oxidation reactions. In one example, the composition further comprises an antioxidant as disclosed herein.

In another example, the composition may further include a solid medium. The solid phase medium may include, but is not limited to, one or more of the following: glass fiber, nylon, cellulose, polyethylene, polyethersulfone, cellulose acetate, nitrocellulose, polyester, hydrophilic polytetrafluoroethylene (PTFE) or a combination thereof.

Certain additives increase the stability and uniformity of the LAMP reaction. In one aspect, the composition may comprise a combination of one or more enzymes, nucleic acids, or the like as disclosed herein. In one example, the enzyme can be an RNase inhibitor or a DNase inhibitor. In another aspect, the composition may comprise carrier DNA or carrier RNA. The carrier DNA or carrier RNA can provide a bait matrix that sequesters DNase or RNase activity, respectively.

In another example, a defined amount of guanidine hydrochloride stimulates denaturation and exposure of RNA molecules. In one aspect, the guanidine hydrochloride may be present at a concentration of from about 1 mM to about 200 mM when used on a solid medium. In another example, when used on a solid medium, the guanidine hydrochloride may be present at a concentration of from about 10 mM to about 100 mM. In yet another example, when used on a solid medium, the concentration of the guanidine hydrochloride can be from about 20 mM to about 60 mM.

Maximization of the pH-dependent output signal can also be used in conjunction with other embodiments disclosed herein. In one embodiment, a method of LAMP analysis using a pH-dependent output signal can include providing an assembly having a solid phase medium and compositions described herein. The method can further comprise depositing a biological sample onto the solid medium. The method can further comprise heating the assembly to a constant temperature sufficient to promote the LAMP reaction.

As disclosed herein, in one aspect, the biological sample can be one or more of: saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, etc. or combinations thereof. In another aspect, the biological sample can be saliva. In one aspect, the method can comprise detecting a viral pathogen. In one aspect, the viral pathogen can be a pathogen as otherwise disclosed herein.

In one example, the temperature sufficient to promote the LAMP reaction may be in the range of about 60°C to about 70°C. In another example, the thermostatic temperature may be a temperature within a range that differs by less than 5°C.

In another aspect, 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 aspect, the isothermal temperature sufficient to promote the LAMP reaction may be in the temperature range of about 60°C to about 70°C. In another aspect, the isothermal temperature sufficient to promote the LAMP reaction may be in the temperature range of about 60°C to about 65°C. The thermostatic temperature can be selected based on one or more of the activities of DNA polymerase, reverse transcriptase, or combinations thereof.

In another embodiment, as shown in FIG. 3, a method 300 for maximizing the accuracy of an output signal in a pH-dependent LAMP assay may include providing a reagent mixture that reacts non-LAMP from a signal output medium. The resulting discoloration is minimized, as in block 310 . The method may further comprise performing a LAMP reaction, as represented by block 320 . In one aspect, the method can comprise controlling the production of protons from non-LAMP reactions. In another aspect, the method can comprise controlling oxidation from non-LAMP reactions.

In another embodiment, a method of maximizing the accuracy of output signals in pH-dependent LAMP assays may comprise substantially removing discoloration from non-LAMP reactions of a signal output medium.

In another embodiment, a method of maximizing the level of detection (LOD) in a pH-dependent LAMP assay may comprise substantially removing discoloration from non-LAMP reactions of a signal output mediator. In one aspect, by diluting the saliva with water to 5-10%, the color contrast can be enhanced and sample variability can be reduced without affecting the detection limit. In another example, by filtering the saliva using a filter as otherwise disclosed herein, color contrast can be enhanced and sample variability can be reduced without affecting detection limits. example

The following examples are provided to facilitate a clearer understanding of certain embodiments of the invention and are by no means meant to limit them. Paper-based LAMP analysis of viral targets in diluted saliva samples Example 1 - DNase/RNase Free Distilled Water

Use 0.1μm membrane filtration to prepare DNase/RNase-free distilled water, and test the DNase and RNase activity. DNase and RNase activity were tested according to current United States Pharmacopeia (USP) Water for Injection (WFI) monograph test standards. After confirming the absence of DNase, RNase, or protease activity, the water was considered contamination-free for the preparation of saliva samples. Example 2 - Amplification in saliva

A nucleic acid sequence primer targeting RnaseP in saliva was designed as a positive control to confirm the nucleotide amplification from the saliva sample, as shown in FIG. 4 . Figure 4A illustrates fluorescent RT-qLAMP results for a primer set targeting RNaseP POP7 in 18% saliva spiked with 105 genome equivalents/reaction of heat-inactivated SARS-CoV-2. Figure 4B illustrates the fluorescent RT-qLAMP results of primer sets targeting RNaseP POP7 in water with 0.2 ng of synthetic RNaseP POP7 RNA.

As shown in Figure 4A, 18% saliva that had been spiked with 105 genome equivalents per reaction of heat-inactivated SARS-CoV-2 was analyzed. In the left panel, no amplification occurred because the primer targeting RNaseP (RNaseP.I) was designed using the mRNA sequence of the POP7 gene encoding the p20 subunit of RNaseP, and low-level RNaseP was not sufficiently detected. In the middle panel, amplification occurs, as shown by the blue line, which does not overlap with the black line, because the primer (RNaseP.II) was able to detect the level of RNaseP without amplifying the no-sample control. In the right panel, both black and blue lines are amplified due to dimerization of the primer (RNaseP.III) (eg, amplification is shown in the no-sample control black line).

As shown in Figure 4B, water containing 0.2 ng of synthetic RNaseP POP7 RNA was analyzed. In the left panel, amplification occurs in the blue and black lines due to dimerization of the primer (RNaseP.I) (eg, amplification of the no-plate control). In the middle panel, amplification occurred, as shown by the blue line, which did not overlap with the black line, because the primer (RNaseP.II) was able to detect the level of RNaseP without amplifying the no-sample control. In the right panel, the blue line was amplified, but the black line was not, because the primer (RNaseP.III) amplified RNase P in the absence of the no-sample control. Example 3 - Saliva Collection Device

The type of saliva collection device that facilitates the collection of saliva samples for LAMP reactions. In some cases, operators may use protective equipment to prevent pathogen transmission through airborne droplets (eg, aerosolized viruses). Therefore, operators may wear personal protective equipment to prevent accidental exposure to aerosol viruses. Specific saliva collection devices can be self-collected by subjects under the guidance of health care professionals. Saliva collection devices have proven efficacy and can be divided into two categories: sponge-based collection and passive drool collection, as shown in Figures 5A and 5B.

The sponge collection device 500a uses a sponge-like collection pad 504 to absorb saliva, and includes a sample volume sufficiency indicator 512 to indicate when a sufficient volume has been collected. Once saturated, the sponge is inserted into the compression tube 506 and compressed against the filter, which will filter the saliva into the collection tube. The principle of this filtration operation is to remove mucin and high molecular weight proteins from saliva and significantly reduce the viscosity of the sample. As a result, the solid phase medium can absorb and distribute saliva in a more rapid, uniform and reliable manner. The sponge collection device 500a may also include: a compression seal 508 on the compression tube 506 to form a seal with the compression tube; a handle 510 for compressing the compression tube 506; and a sample volume sufficiency indicator for identifying when enough saliva has been collected device 512.

The passive drool device 500b can provide unfiltered saliva with a viscosity that slows sample absorption and distribution. The passive drooling device 500b may include: a collection funnel 522 for collecting saliva; an indicator line 528 for indicating when sufficient saliva has been collected; a collection tube 524 for collecting saliva; a cap 526; a volume indicator 530; And cap memory 532.

With both types of collection devices, the residual risk of operator exposure is minimized. For sponge-based devices, there is a postulated risk of aerosol release during compression operations, especially if the user is particularly rough during the compression process. Health care operators can do this to control exposure risk. The collection device may include a compression seal to prevent aerosol backflow. In terms of passive drool collection, there may be some risk of contaminating the exterior of the device with stray saliva which, if not handled properly, could transmit cross-contamination to the operator. In both cases, self-collection of saliva samples by patients reduced the risk of exposure.

Three commercial saliva collection devices were selected to evaluate their effect on RT-LAMP responses in saliva. These three devices are "Saliva Sampler™" manufactured by StatSure Diagnostic Systems, Inc., "Pure•SAL™" manufactured by Oasis Diagnostics, and "Super•SAL™" manufactured by Oasis Diagnostics. The StatSure Saliva Sampler™ is provided in a tube containing a buffer (eg, Buffer 2000) for collecting saliva from patients. Super•SAL standardizes saliva collection by removing any solid contaminants and mucous material using cylindrical absorbent pads and collection tubes. Pure•SAL works by a similar mechanism, but includes an additional filter in the collection tube to remove contaminants.

Saliva pH was measured from treated saliva samples, which were then used for colorimetric and fluorescent RT-LAMP LOD analysis. This data is presented in Figure 6A. The data illustrate the LoD in saliva treated with different saliva collection devices (Pure-Sal, Super-Sal, Stat-sure). Treat the master mix with 0.6 μl of HCl. The Pure•SAL™ and Super•SAL saliva collection devices demonstrate a broader colorimetric response to a wider range of concentrations (1 to 10k genome equivalents of thermally deactivated SARS-CoV-2 per reaction). Example 4 - Saliva Collection Process

If the subject conducts the self-test, the subject will collect the saliva sample into a special collection container without any additives under the guidance of health care professionals, so it is safe for the subject to use when collecting. The volume of saliva collected was approximately 100 μL. For example, insert the sponge sampler into the subject's mouth and collect saliva until the indicator on the sponge sampler changes color. Then insert the sponge sampler into the collection tube. The sponge is then compressed, and the saliva (approximately 100 μL) is squeezed into a collection tube filled with a certain amount of water to dilute the saliva. Saliva is diluted in water to a saliva to water ratio of about 1:1 to about 1:20. Transfer saliva from the collection tube to the testing site. Example 5 – Effects of RNase Inhibitors on Saliva

RNase inhibitors were added to untreated saliva at a concentration of 1 μL per ml of saliva to determine the effect of adding RNase inhibitors on the RT-LAMP response.

The effect of RNAsecure™ (AM7006, Invitrogen™) on freshly collected saliva (5%) was tested for its suitability as a single protocol for point-of-care RT-LAMP reactions. Dilute 25X RNAsecure™ stock solution to 1X with 1 ml of saliva. Treated saliva was used as a substrate to add heat-inactivated SARS-CoV-2 at concentrations ranging from 1000 copies to 62.5 copies/reaction to Warmstart along with 40 mM Guanidine HCl and 0.3 ng/μl carrier DNA ™ Colorimetric Master Mix, pH 7.6. Incubate RNAsecure™-treated RT-LAMP at 65°C to initiate the reaction. Fresh saliva under these conditions was tested without RNAsecure™ as a control.

5 µL of heat-inactivated virus was diluted in 5% treated saliva (i.e., final reaction concentration) and added to the RT-LAMP reaction to give the indicated concentrations in a final reaction volume of 25 µL. For negative reactions, add 5 µL of treated saliva (5% final reaction concentration) instead of diluted heat-inactivated virus to give the same 25 µL reaction volume. Heating was carried out in an incubator at 65°C for 60 minutes. Colorimetric scanning was performed using a flatbed scanner before and after the RT-LAMP reaction. Add 0.5 μL of Antarctic Thermolabile and 3.5 μL of dUTP to 1250 μL of NEB colorimetric master mix. 25X RNase Inhibitor (RNASecure™) was diluted in whole saliva to generate a 1X concentration and then added to the reaction diluted to 5% saliva.

RNAsecure™ did not show any significant increase in the LoD of the response, as shown in Figure 6B. That is, the addition of an RNase inhibitor did not result in a significant increase in measured parameters (eg, reaction rate, false positive rate, or limit of detection) of the RT-LAMP reaction. Example 6 – Freezing a saliva sample

In some cases, it may be necessary to freeze saliva samples for a period of time prior to analysis due to logistics, shipping needs, etc. Such situations may require special care when performing the LAMP analysis described here. As shown in Figure 7, the pH of frozen saliva samples varied with the number of days at -20°C before the saliva samples were thawed and tested. In one example, the pH of the saliva sample from Donor 1 varied from 7.21 without any days between collection and testing, to pH 7.46 after a 6-day interval between collection/freezing and testing. In another example, the pH of the saliva sample from Donor 2 varied from 7.00 without any days between collection and testing, to pH 6.98 after a 6-day interval between collection/freezing and testing. In one example, the pH of the saliva sample from Donor 3 varied from 7.18 without any days between collection and testing, to pH 7.18 after a 6-day interval between collection/freezing and testing. In one example, the pH of the saliva sample from Donor 4 varied from 7.35 without any days between collection and testing, to pH 7.47 after a 6-day interval between collection/freezing and testing. In one example, the pH of the saliva sample from Donor 1 varied from 7.22 without any days between collection and testing, to pH 7.24 after a 6-day interval between collection/freezing and testing. Example 7 - Limit of detection in fresh saliva

Figure 8 illustrates the detection limit for fresh saliva. Fresh saliva was collected using the drool method and diluted in water at a ratio of 1:3 to obtain 25% saliva and 75% water. Heat-inactivated SARS-CoV-2 was added to 25% saliva by serial dilution as a control group. Add 5 µL of 25% saliva to 20 µL of RT-LAMP reagent to make a final concentration of 5% saliva. After incubation for 1 hour at 65°C, the color changed. Copy numbers on the y-axis represent the original concentration in 100% saliva without dilution. The limit of detection (LOD) of the primers was 250 copies/reaction in a volume of 25 μL, corresponding to approximately 200k copies/mL saliva.

Therefore, it was determined that saliva diluted to 25% with nuclease-free water and further diluted to a final concentration of 5% after addition to the RT-LAMP reaction gave results within 60 min. Dilution reduces the buffering capacity of saliva and reduces the concentration of inhibitory components, both of which delay colorimetric reporting. Compared to other pretreatment procedures found in various studies, such as pretreatment with protease, Chelex® 100 or RNA extraction procedures to deactivate inhibitory components in saliva, dilution is less complicated for the end user.

In 5% saliva that had been treated with Pure SAL ^™ , the LoD of the colorimetric assay was 1000 copies/reaction (reaction volume 25 μL), corresponding to 800 copies/μL of patient saliva after dilution (Figure 6C).

As shown in Figure 6C, different saliva collection devices (Pure·SAL ^™ , Super-Sal ^™ , Stat-sure ^™ ) can result in different LoDs. Tested on 5% saliva diluted in water for all treatment techniques. Use the primer set orf1ab.2. 5 μL of heat-inactivated virus diluted in 5% treated saliva (final reaction concentration) was added to the RT-LAMP reaction resulting in the indicated concentrations and a final reaction volume of 25 μL. For negative reactions, add 5 μL of treated saliva (5% final reaction concentration) instead of diluted heat-inactivated virus, providing the same 25 μl reaction volume. Heat in a 65°C incubator for 60 minutes. Colorimetric scanning was performed using a flatbed scanner before and after the RT-LAMP reaction. The reaction contained 12.5 μL NEB 2X colorimetric master mix, 2.5 μL primer mix, 5 μL water, and 5 μL sample.

This LoD is several orders of magnitude higher (approximately 1 copy number/reaction) than RT-PCR assays or other assays utilizing RNA extraction. However, these other analyzes were accompanied by preprocessing procedures and/or RNA extraction operations to achieve the reported LoD.

To enhance this LoD, we investigated the use of RNase inhibitors, guanidine hydrochloride and carrier DNA. Addition of RNase inhibitor decreased the LoD in 5% of saliva (Fig. 6B), which contradicts reports in the literature that RT-LAMP analysis in saliva treated with RNase inhibitor increased LoD; this difference may be due to the due to the type of RNase inhibitor. Both guanidine hydrochloride and carrier DNA increased the LoD (Figure 6D and Figure 6E) and were added to our RT-LAMP reaction recipe for colorimetric solution reactions. None of these ingredients are included because of the color change that occurs when drying on paper.

As shown in Figure 6D, 5 µL of heat-inactivated virus diluted in 5% treated saliva (final reaction concentration) was added to the RT-LAMP reaction, yielding the indicated concentrations and a final reaction volume of 25 µL. For negative reactions, 5 μL of treated saliva (5% final reaction concentration) was added instead of diluted heat-inactivated virus, resulting in the same 25 μl reaction volume. The primer set orf1ab.2 was used. Heat at 65° C. for 60 minutes in an incubator (Fisherbrand ^™ Isotemp ^™ ). Colorimetric scanning was performed using a flatbed scanner before and after the RT-LAMP reaction. 0.5 μL of Antarctic Thermolabile UDG, 3.5 μL of dUTP and carrier DNA were supplemented to 1250 μL of NEB Colorimetric Master Mix to produce the indicated final reaction concentrations.

As shown in Figure 6E, 5 µL of heat-inactivated virus diluted in 5% treated saliva (final reaction concentration) was added to the RT-LAMP reaction, yielding the indicated concentrations and a final reaction volume of 25 µL. For negative reactions, 5 μL of treated saliva (5% final reaction concentration) was added instead of diluted heat-inactivated virus, resulting in the same 25 μl reaction volume. Use the primer set orf1ab.2. Heat at 65° C. for 60 minutes in an incubator (Fisherbrand ^™ Isotemp ^™ ). Colorimetric scanning was performed using a flatbed scanner before and after the RT-LAMP reaction. Add 0.5 μL of Antarctic Thermolabile UDG, 3.5 μL of dUTP and guanidine hydrochloride (40 mM) to 1250 μL of NEB colorimetric master mix.

Finally, uracil-DNA glycosylase (UDG) and deoxyuridine triphosphate (dUTP) were included (Figure 6F) to reduce carryover contamination. As shown in FIG. 6F , a colorimetric scan was performed on 25 μL of the reaction incubated at 65° C. for 60 minutes in an incubator and incubator with and without the addition of UDG and dUTP. The primer set used was orf1ab.II. The samples are heat-inactivated virus at the indicated concentrations. For reactions with UDG, 1250 μL of NEB 2x Colorimetric Master Mix was supplemented with 0.5 μL of Antarctic Thermolabile UDG, 3.5 μL of dUTP. For all other reactions, use NEB 2x Colorimetric Master Mix.

The LoD of the RT-LAMP colorimetric assay in 5% treated saliva solution increased to 250 copies/reaction when guanidine hydrochloride, carrier DNA, and UDG were included (Fig. 6G). RT-LAMP colorimetric LoD using Pure·SAL ^™ treated saliva and untreated saliva as shown in Figure 6G. Plates were heated for 60 minutes in an incubator set at 65°C. The primer set used was orf1ab.II and the template was the indicated concentration of heat-inactivated virus (positive response) or nuclease-free water (negative response). Heating was performed at 65°C for 60 minutes in an incubator (Fisherbrand ^™ Isotemp ^™ ). Colorimetric scanning was performed using a flatbed scanner before and after the RT-LAMP reaction. Add 0.5 μL of Antarctic Thermolabile UDG, 3.5 μL of dUTP, carrier DNA (0.3 ng/μL) and guanidine hydrochloride (40 mM) to 1250 μL of NEB colorimetric master mix. Example 8 – Limits of detection for animal nasal swabs

Figure 9 illustrates the limit of detection of bovine nasal swabs resuspended in approximately 1 mL of water. Spiking heat-inactivated SARS-CoV-2 into water with resuspended background mucus and microbial communities resulted in the same number of copies/reactions as in the previous saliva paradigm. Add 5 μL of sample to 20 μL RT-LAMP. After about 1 hour of incubation at 65°C, the color changes. The LOD of this primer in a volume of 25 μL was about 250 copies/reaction, corresponding to about 5k copies/mL nasal swab resuspension. Example 9 - Detection Limits on Paper

Figure 10 illustrates the detection limit on paper. Add 20 µL of RT-LAMP reagent to grade 1 chromatography paper. Heat-inactivated SARS-CoV-2 was spiked into 100% pooled saliva and the virus was serially diluted. Add 15 µL of approximately 100% saliva to each sheet. After incubation at 65°C for 90 minutes, the color changed. The LOD of this primer in 15 μL of saliva is about 3k copies/reaction, corresponding to about 20k copies/mL of saliva. Example 10 of Reagent Composition to Facilitate On-Paper LAMP Analysis - Sample Reagents

In one example, the reagents include those shown in Table A1. In another example, the reagents include those shown in Table A2. Table A-1 Reagent Deoxynucleotide (dNTP) solution group (dATP, dCTP, dGTP and dTTP each 100 mM) Deoxynucleotide (dNTP) solution mixture TWEEN® 20 Bst 2.0 WarmStart® DNA Polymerase Bst 3.0 DNA polymerase WarmStart® RTx Reverse Transcriptase Tris-HCl Primer group Target DNA for QA testing Table A-2 Reagent Potassium Chloride ( magnesium sulfate Deoxynucleotide triphosphate (dNTP) Bst 2.0 DNA polymerase WarmStart® RTx Reverse Transcriptase Phenol red Antarctic Thermolabile Uracil DNA Glycosylase (UDG) Polysorbate 20 betaine bovine serum albumin Trehalose nuclease free water RNase AWAY Example 11 - Buffer selection and concentration

Since the pH of saliva can vary from sample to sample, use buffers on paper devices to maintain a consistent starting pH. For phenol red, pH 7.6 is a suitable starting point for enhanced colorimetric transition, as shown in Figure 11. Some buffers with pKa around 8 were screened because the starting pH 7.6 is near the limit of the buffer range for color change when amplification occurs. As shown in Figure 12, 10 mM BICINE buffer was used for paper assay. Example 12 - Influence of Primers on Reaction Rate

In order to increase the rate of the RT-LAMP reaction, studies including multiple primer sets in the fluorescent RT-LAMP reaction mixture were performed. The study was performed in water using NEB LAMP fluorescent dye as a fluorescent indicator. Inclusion of multiple primer sets did not appear to significantly increase the reaction rate. Instead, the reaction proceeds primarily at the rate of the primer set with the fastest reaction time when used alone. Example 13 - Sample LAMP Protocol, Reagents, Validation, and Troubleshooting Sample LAMP Protocol 13-A: Primer Mix

1. Remove all 6 diluted primers from refrigerator; 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 to 500 μl. LAMP

1. Obtain the NEB Bst 2.0 Warmstart kit and the primer mixture; 2. After the reagent is thawed and sprayed with the DNAway for at least 5 minutes, wipe the surface with a Kimwipe; 3. Label all PCR tubes that require DNA samples and primers to be used. Be sure to add a negative control group without adding DNA; 4. Add 5μl PCR grade water (or dye), 12.5μl NEB Bst 2.0 Warmstart reagent set and 2.5μl primer mix to each reaction. A master mix can be made for several reactions to be performed; 5. If 5 μl of EBT dye is added, the concentration should be 1500 μM, so the final concentration is 300 μM; 6. Reactions without DNA should add an additional 5 μl of PCR grade water and wait until necessary Load onto the gel to open again; 7. When ready, the PCR tubes should be placed in the PCR tray previously left in the transfer chamber and transferred to the BRK 2037; 8. After entering the BRK 2037, remove from the -20°C freezer 9. Spray your hands with DNAway spray and rub your hands around the DNA sample tube so that it is also covered by the spray; 10. Add 5 μl of DNA sample where appropriate and close the tube. Do not open the two DNA tubes at the same time, and close the PCR tube immediately after adding DNA; 11. Put the sample into a thermal cycler set at 65°C for 1 hour and 80°C for 5 minutes (after this operation, the sample can be stored at -20°C C overnight). Sample Reagent Concentration 13-B:

Colorimetric RT-LAMP Master Mix can be: KCl (50 mM), MgSO ₄ (8 mM), dNTP Mix (1.4 mM 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% v/v), Betaine (20 mM) , BSA (500μg/mL) and trehalose (10% w/v).

These components were each titrated from 0.25X liquid concentration to 5X or higher for paper LAMP analysis. The concentration depends on the rate of the LAMP reaction, the contrast between positive and negative LAMP results at 60 minutes reaction time, and the reduction in non-specific amplification.

To determine the concentration of protein stabilizing additives, D-(+)-trehalose dihydrate was increased from 0% to 15% w/v in 5% increments, and lyophilized BSA increased from 0 to 1.25 mg/mL. The concentrations of trehalose and BSA were 10% w/v and 0.626 mg/mL, respectively. Sample Lamp Experiment Procedure 13-C: Reagents

Reagents are shown in Table A-2 of Example 10. equipment

Tweezers, 0.5-10 μL pipette, 2-20 μL pipette, 20-200 μL pipette, 100-1000 μL pipette, Ahlstrom-Munksjö Grade 222, pH probe, heat source up to 65°C (eg, incubation box, water bath), PCR fume hood. disinfect:

Spray pipettes and all bench (PCR fume hood) surfaces with RNase AWAY. Wipe thoroughly after applying RNase AWAY. If any remains, RNase AWAY will interfere with the reaction. Use separate rooms for paper setup and sample loading to help prevent cross-contamination. Pre-cut 5 mm x 6 mm chromatography paper. LAMP preparation:

1. Prepare the 2X LAMP mix shown in Table 13B-1 in a PCR fume hood. 2. Adjust the pH to pH ~7.5–8.0 (red but not pink) with 1M KOH (about 1-2 μL). It doesn't need to be precise. After adjusting the pH, the 2X LAMP mix can be stored at -20 °C. Table 13B-1 2X LAMP mix Component volume unit stock solution concentration unit final concentration unit KCL 100 µL 1000 mM 100 mM _MgSO4 160 µL 100 mM 16 mM dNTPs 280 µL 10 mM 2.8 mM Bst 2.0 DNA polymerase 10.8 µL 120 U/μL 1.296 U/μL RTx reverse transcriptase 40 µL 15 U/μL 0.6 U/μL Phenol red 20 µL 25 mM 0.2 mM dUTPs 2.8 µL 100 mM 0.28 mM Antarctic Thermolabile UDG 0.4 µL 1 U/μL 0.0004 U/μL Polysorbate 20 100 µL 20 % 2 % nuclease free water 286 µL - - - - total 1000 µL

3. Prepare a master mix according to Table 13B-2. Table 13B-2 COMPLETE MIXTURE FOR PAPER LAMP Component volume unit stock solution concentration unit final concentration unit 2X LAMP mix 125 µL - - - - 10X Primer Mix ^a 25 µL - - - - Betaine 1 µL 5 m 20 mM bovine serum albumin 3.13 µL 40 mg/mL 0.626 mg/mL Trehalose 36 µL 689 mg/mL - - water 9.2 µL total 200 µL ^a The stock solution of LAMP primer can be made in water at a concentration that can be used for easy setup. 10X primer mix containing all 6 LAMP primers. 10X Primer Mix: 16 μM FIP/BIP, 2 μM F3/B3, 4 μM Ring F/B can be prepared.

4. Adjust the pH to 8.0 with 0.1M KOH. Use a micro pH electrode. 5. Mix thoroughly. Place the paper pads on a clean surface inside the PCR fume hood. Add 30 µL of the complete mixture onto pre-cut 222 grade paper pads. 6. Dry in PCR fume hood for 60 minutes at room temperature. 7. Once dry, collect the paper pads into a clean centrifuge tube or a clean resealable plastic bag. sample loading

1. Spray the workbench with RNase AWAY and clean with a rag. 2. Remove the sample plate (DNA, RNA, heat-inactivated virus) from the refrigerator. 3. Place the reaction pad on a clean surface. You can place the pad on a new clear film and discard after use. 4. Prepare the negative control pad first. The pad was reconstituted with 25 μL of non-sampling solvent (water, saliva). The recovery process should be gentle to avoid washing reagents from the pad. 5. Use tweezers to place the negative control pad into a clean container (eg, 1” x 1” resealable plastic bag, centrifuge tube). 6. Dilute the sample to the desired concentration with solvent. 7. Deploy more reaction pads and reconstitute the pads with 25 μL of the diluted sample. 8. Using tweezers, place the positive pad into a clean container (eg, 1” x 1” resealable plastic bag, centrifuge tube). 9. Clear work area and bring the pad for imaging and incubation. Imaging and incubation:

Note: There are various imaging methods (eg, time-lapse video, scanning) and heat sources (eg, incubator, water bath). For this protocol, a desktop scanner and a microbe incubator can be used. 1. Place the pads on top of the scanner. The pads were scanned prior to reaction (0 min). 2. Preheat the incubator to 65°C. 3. Place the pads in the incubator. Separate the pads. Uniformity of heating can affect the consistency of results. 4. Remove the pads and repeat the scan at different time points (usually every 30 minutes). 5. After the final scan, dispose of the reaction pad in biohazard waste. Effective:

To verify that LAMP amplification occurs, transfer each reaction pad to a clean 1.5 mL microcentrifuge tube. Add 100 µL of Buffer EB to each tube. Soak the reaction pad in Buffer EB overnight for nucleic acid extraction. Gel electrophoresis (2% agarose gel) was performed on the eluate to verify the occurrence of LAMP amplification. A ladder-like banding pattern (typical of LAMP product pattern) was shown in each positive pad lane, while no distinct bands were present in each negative lane (FIGS. 13A and 13B).

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, the relevant gel electrophoresis (2% agarose) was performed. The orf7ab.1 primer set targeting SARS-CoV-2 was used. Reconstitute the negative reaction pad with 25 µL of nuclease-free water. Recover the positive reaction pad with 25 µL of 400 copies/µL heat-inactivated SARS-CoV-2 virus. Heat in an incubator set to 65°C and scan in a flatbed scanner.

BSA is a reagent that can be used in LAMP mixtures. Adding BSA can speed up the reaction and increase the sensitivity, as shown in Figure 14A and Figure 14B. In these reactions, low sample concentration LAMP on paper was run under two conditions (with and without BSA in the reaction mixture). Figure 14A shows the 0 minute time point. Panel B shows the 60 min time point. This experiment uses the orf7ab.1 primer. Reconstitute the negative reaction pad with 25 µL of nuclease-free water. Reconstitute the positive reaction pads with 25 μL of heat-inactivated SARS-CoV-2 virus of 8 copies/μL and 16 copies/μL (to achieve a final concentration of 200 copies/reaction and 400 copies/reaction).

However, BSA may also introduce pH changes to the device. Figures 13A and 14B show that after incubation (60 minutes), negative paper pads containing BSA had a yellowish edge. After elution, the eluate was run with gel electrophoresis, and no DNA product was visible on the gel, as shown in Figure 13B, indicating that the yellow color at the edge was not caused by off-target amplification or contamination. The uneven distribution of BSA can lead to a yellowish edge when heated. Troubleshooting:

Unusual pink color on paper pads: During the preparation of LAMP paper pads, unusual pink spots may appear that are different in color from the surrounding area, this may be due to residual RNase AWAY sprayed directly on the pad and/or transferred by tweezers Caused. RNase AWAY degrades any added RNA/DNA samples. If this happens, dry all equipment and surfaces thoroughly, cut a new 5x6 mm paper pad, and restart the "LAMP preparation" section from step 5.

Reagent spillage after pad reconstitution: During a sample loading operation, the pad may not be able to absorb the full sample volume added to it for reconstitution. Spilled pads may not be an accurate representation of sample concentration. Spills can be caused by insufficient pad drying. If this happens: 1) dry longer, 2) use enhanced drying methods such as heat drying (place in a clean microbiological incubator at 37°C; do not set the temperature above 45°C to prevent activation of Bst 2.0 WarmStart® polymerase) or convective drying (use a small fan to enhance airflow during drying), or 3) reduce the reconstituted volume to 20 μL.

Negative controls show color changes: Negative pads may change at the same time as, or shortly after, pads containing samples during imaging and incubation procedures. This could be caused by primer dimerization/nonspecific amplification or carryover contamination from previous LAMP reactions. To solve this problem, validate the primers in liquid-based LAMP before using them on paper. To control carryover contamination, 1) implement dUTP and UDG in all LAMP reactions, 2) maintain separate workstations for LAMP mix preparation and sample addition, and 3) aliquot reagent stock solutions and use new ones if contamination is suspected Aliquot. Over-incubation of the reaction may also induce non-specific amplification. Do not incubate for more than 75 minutes.

Sample pH and buffering capacity affect colorimetric readings: Since phenol red is a pH indicator, the pH of the sample and its buffering capacity can have a significant impact on the analysis. Saliva concentrations of 5-10% v/v (diluted with water) have been shown to work with the reagent compositions presented here. 5% saliva was chosen because this concentration has a faster response time and produces consistent results. Human saliva has a complex buffer system that includes bicarbonate, phosphate, and proteins that prevent pH changes (and thus color changes) at high saliva concentrations. Paper LAMP devices with nasal swabs resuspended in water were tested and did not show any inhibition by the sample matrix. Colorimetric readings may also be affected by buffered saline solutions (eg, transport media). Example 14 - On-paper detection limit of untreated saliva with deactivated virus.

As shown in Figure 15, whole untreated saliva with heat-inactivated SARS-CoV-2 virus validated a detection limit of approximately 20 copies per μL of saliva. Using aliquots of pooled saliva (30 aliquots) as negative samples, make various sample concentrations including 20 copies/μL (1x LoD – 10 samples), 40 copies/μL (2x LoD – 10 samples), 100 copies/μL (2 samples), 1000 copies/μL (2 samples), 10,000 copies/μL (2 samples), 100,000 copies/μL (2 samples) sample), 1,000,000 copies/μL (2 samples). Confirm the results using image processing. Example of Reagent Composition to Maximize pH Sensitive Signal Output 15 - Sample LAMP Dye Table B dye Phenol red solution litmus Bromevanillol Blue Nitrazine Yellow Cresyl red, sodium salt curcumin bright yellow m-cresyl violet α-Naphtholphthalein neutral red acid fuchsin Acid fuchsin, calcium salt Example 16 -

Fluorescent reporters are read using an additional ultraviolet (UV) light source without the need for specialized equipment. However, colorimetric assays using phenol red as an indicator do not use UV light and can be read by the naked eye. Polymerization of DNA generates protons and phenol red is pH responsive. Diluted saliva (5% final concentration) was used to overcome the buffering capacity of saliva to measure changes in pH. Diluting saliva to a final concentration of 5% also reduced the concentration of interferents (eg, RNase).

Binding of carrier DNA and guanidine hydrochloride also enhanced the LoD and provided comparable colorimetric responses in water and saliva. The mechanism by which carrier DNA increases LAMP outcomes is unclear. This mechanism was explored by using the NEB 1 kb DNA ladder (NEB-N3232L). Different concentrations of carrier DNA (0.3ng/μl and 0.75ng/μl) were used to study the effect on LoD. Guanidine chloride (40 mM) was also used. The pH of the complete master mix was maintained at 7.6, and the same conditions were also tested without carrier DNA. The effect of carrier DNA was tested using fresh saliva (5%) at pH 6.5 together with guanidine chloride and heat-inactivated SARS-CoV-2 at concentrations ranging from 1000 copies to 62.5 copies/reaction (Fig. 6D).

Guanidine hydrochloride has been reported to increase the sensitivity of LAMP. Performance with our primer set was tested by adding 40 mM Guanidine HCl to NEB Warmstart™ Colorimetric Master Mix at pH 7.6. The effect of guanidine chloride was tested using pooled saliva (5%) at pH 6.5 and heat-inactivated SARS-CoV-2 at concentrations ranging from 1000 copies to 62.5 copies/reaction. The same composition was also tested without the addition of guanidine chloride as a control. Guanidine chloride increased replication sensitivity and had consistent amplification across replicates (Fig. 6E).

Due to the different mechanisms by which phenol red and fluorescent dyes report on LAMP-based nucleic acid amplification, differences in these time-dependent signal measurements were investigated. To prepare reactions using the combination of Warmstart™ Colorimetric LAMP 2x Master Mix and LAMP Fluorescent Dye on FrameStar 96-well Lined Optical Bottom Plates, seal with Thermo Scientific™ Adhesive Sealing Film, and read in Clariostar™ Plus Microplates It is used to incubate and measure the absorbance and fluorescence intensity in the detector (BMG). The color change over time is expressed as the ratio _A432nm / _A560nm . Absorbance values were baseline corrected by subtracting A ₆₂₀ nm from A _{432 nm} and A _{560 nm} .

According to Figure 16, the color change in the reaction occurs later than the fluorescence change. Colorimetric and fluorometric data were collected on a BMG CLARIOstar® Plus plate reader. The reaction base mix consisted of NEB 2x colorimetric LAMP master mix, 2.5 μL primer mix and 5 μL 1:100 dilution of NEB LAMP fluorescent dye (NEB B1700A). The chamber temperature of the disc reader was allowed to equilibrate to 65°C before inserting the disc. 5 μL of heat-inactivated virus diluted in water was added to the reaction base mixture to produce the indicated final reaction concentrations (positive reactions). For NTC reactions, add 5 µL of nuclease-free water to the reaction base mix. This difference in variation suggests that the pH-based reporter responds more slowly to LAMP-based DNA amplification than the fluorescent reporter. Example 17 –

Paper can be scaled up to millions of devices, but when RT-LAMP reagents are placed on the paper, even with a negative control, the paper changes color even though no amplification occurs. One possibility for this color change is thermally induced oxidation of the cellulose and the oxidative nature of the ammonium sulfate present in the RT-LAMP mixture. Another possibility for this color change is acidification of the reagents due to outgassing of the ammonia in the RT-LAMP mixture. Removal of ammonium sulfate preserves the color of the negative control. Increasing the concentration of phenol red as an antioxidant also maintained the color of the negative control, as shown in FIG. 11 . Example 18 - Screening of Colorimetric Dyes

Three types of colorimetric indicators were evaluated for paper analysis: (i) magnesium colorimetric indicators, (ii) pH colorimetric indicators, and (iii) DNA intercalation colorimetric indicators.

In terms of magnesium indicator, calcium magnesium indicator (CAS# 3147-14-6), xylidine blue I (CAS# 14936-97-1), chlorophosphine azo III (CAS# 1914-99-4) , o-cresolphthalein complexing agent (CAS# 2411-89)-4), Eriochrome® Black T (EBT, CAS# 1787-61-7) and hydroxynaphthol blue (HNB, CAS# 63451-35-4) for screening .

In terms of pH indicators, p-Brodivanillol Blue (CAS# 76-59-5), Acid Fuchsin (CAS# 3244-88-0), Nitrazine Yellow (CAS# 5423-07-4), Cresol Red (CAS# 1733-2-6 ), Cresyl Red Sodium Salt (CAS# 62625-29-0), Curcumin (CAS# 458-37-7), Phenol Red (CAS# 143-74-8), Phenol red sodium salt (CAS# 34487-61-1), brilliant yellow (CAS# 3051-11-4), o-cresolphthalein (CAS# 596-27-0), m-cresol violet (CAS# 2303-01- 7), m-cresyl violet sodium salt (CAS #62625-31-4), α-naphtholphthalein (CAS # 596-01-0) and neutral red (CAS # 553-24-2) for screening.

For DNA intercalating dyes, crystal violet (CAS# 548-62-9) was screened.

Many magnesium indicators do not produce a consistent color change on paper. Metal ion indicators (calcium magnesium indicator and EBT) will interact with magnesium(II) ions in solution, and the concentration of magnesium ions decreases throughout the RT-LAMP experiment due to the formation of magnesium pyrophosphate, a by-product of the polymerase reaction. .

Figure 17A shows the colorimetric response of different concentrations of calcium and magnesium indicators throughout a LAMP reaction using genomic DNA as a template. The LAMP assay is performed with increasing concentrations of a calcium and magnesium indicator (magnesium indicator). The loIB.3 primer set targeting Histophilus somni genomic DNA was used. Positive reactions were spiked with 5 μL of HS gDNA at a concentration of 0.2 ng/μL. Negative reactions use 5 µL of nuclease-free water. The total reaction volume was 25 μL. Reactions were prepared using 12.5 μL of NEB Warmstart 2x Master Mix, 2.5 μL of Primer Mix, and 5 μL of the above sample (positive reaction) or water (negative), and 5 μL of calcium and magnesium indicator prepared in water to produce the indicated final concentration.

At the concentrations tested, no visual changes were detected during the LAMP reaction. Between the 0-minute and 60-minute time points of the LAMP response, the EBT showed a detectable color change from purple to dark blue; however, the color change was insignificant, which may interfere with interpretation in a clinical setting.

As shown in Figure 17B), LAMP detection was performed at increasing concentrations of Eriochrome® Black T (magnesium indicator). The loIB.3 primer set targeting H. somniophilus genomic DNA (gDNA) was used. Positive reactions were spiked with 5 μL of HS gDNA at a concentration of 0.2 ng/μL. Negative reactions use 5 µL of nuclease-free water. The total reaction volume was 25 μL. Reactions were prepared using 12.5 μL of NEB Warmstart 2x master mix, 2.5 μL of primer mix, and 5 μL of the template as above (positive reactions) or water (negative), and 5 μL of EBT prepared in water to yield the indicated final concentrations.

Furthermore, the use of LAMP on paper using EBT produced no detectable color change. As shown in Figure 17C, LAMP detection was performed with increasing concentrations of Eriochrome® Black T on chromatography paper in PCR tubes. The lolB.3 primer set targeting the H. somniophilus genomic DNA was used. Positive reactions were spiked with 5 μL of H. somnus genomic DNA at a concentration of 0.2 ng/μL. Negative reactions use 5 µL of nuclease-free water. The total reaction volume was 25 μL. Reactions were prepared using 12.5 μL of NEB Warmstart 2x Master Mix, 2.5 μL of Primer Mix, and 5 μL of the plate as above (positive reaction) or water (negative), and 5 μL of EBT (300 μM) prepared in nuclease-free water. For the EBT reaction, the reaction consisted of 25 μL of EBT (300 μM) prepared in nuclease-free water.

After screening several different types of paper and confirming the amplification on PES and polyblast BTS 0.8 by gel electrophoresis, no colorimetric change could be detected on any paper (Figure 17D and Figure 17E).

As shown in Figure 17D, LAMP assays were performed on various papers: chromatography grade 1, anion-exchange nylon, cation-exchange nylon, polyether membrane, asymmetric submicron poly(BTS 0.8), asymmetric submicron poly(BTS 100) and hydroxylated nylon 1.2. b) endpoint scan of the paper in panel a at 60 min and c) gel electrophoresis (2% agarose) scan of the extracted LAMP product at 60 min. The loIB.3 primer set targeting H. somniophilus (HS) genomic DNA was used. Positive reactions were spiked with 5 μL of H. somnus genomic DNA at a concentration of 0.2 ng/μL. Negative reactions use 5 µL of nuclease-free water. The total reaction volume was 25 μL. Reactions were prepared using 12.5 μL of NEB Warmstart 2x Master Mix, 2.5 μL of Primer Mix, and 5 μL of the plate as above (positive reaction) or water (negative), and 5 μL of EBT (300 μM) prepared in nuclease-free water. Paper (as indicated) was placed in a PCR tube, which contained 25 μL of the reaction and was wicked by the paper during the reaction. After 60 minutes, remove the paper and scan it. Use 30 to extract the gel.

As shown in Figure 17E, results generated from a) time-lapse in PCR tubes, b) gel electrophoresis (2% agarose) of DNA extracted from paper at 60 minutes and c) scanning of paper at 60 minutes . The loIB.3 primer set targeting H. somniophilus (HS) genomic DNA was used. Positive reactions were spiked with 5 μL of H. somnus genomic DNA at a concentration of 0.2 ng/μL. Negative reactions use 5 µL of nuclease-free water. The total reaction volume was 25 μL. Reactions were prepared using 12.5 μL of NEB Warmstart 2x Master Mix, 2.5 μL of Primer Mix, and 5 μL of the plate as above (positive reaction) or water (negative), and 5 μL of EBT (300 μM) prepared in nuclease-free water. Biodyne A amphoteric paper was placed in a PCR tube, which contained 25 μL of the reaction and was wicked by the paper during the reaction. After 60 minutes, remove the paper and scan it.

In addition, the crystal violet indicator in the solution of RT-LAMP reaction is difficult to stabilize. For leuco crystal violet (LCV), an unstable crystal violet derivative, an excess of sodium sulfite is used to maintain the colorless stability in solution. For dissolution in water, LCV uses sodium sulfite (SS) and β-cyclodextrin (BCD). After binding to dsDNA, LCV converts back to crystal violet (eg, purple in solution). Therefore, throughout the RT-LAMP reaction, the color of the solution is expected to change from colorless to purple due to the amplification of more dsDNA. However, when LAMP reactions were performed at different concentrations of CV, color changes occurred in both positive and negative reactions.

LAMP analysis was performed using an intercalating dye, crystal violet solution, and b) correlated gel electrophoresis (2% agarose) scan of the product at 60 minutes as shown in Figure 17F. The lolB.3 primer set targeting the H. somniophilus genomic DNA was used. Positive reactions were spiked with 5 μL of H. somnus genomic DNA at a concentration of 0.2 ng/μL. Negative reactions use 5 µL of nuclease-free water. The total reaction volume was 25 μL. Reactions were prepared using 12.5 μL of NEB Warmstart 2x Master Mix, 2.5 μL of Primer Mix, and 5 μL of the template (positive reaction) or water (negative) as above, and 5 μL of crystal violet prepared in nuclease-free water to produce the indicated final concentration. Place the test paper into the PCR tube, which contains 25 μL of the reaction and is wicked by the paper during the reaction. After 60 minutes, remove the paper and scan. Sodium sulfite and cyclodextrin are used to dissolve crystal violet.

To confirm that amplification occurred in positive reactions but not in negative reactions, the RT-LAMP solution was electrophoresed on a 2% agarose gel, which showed that positive reactions showed amplification, while negative reactions showed amplification in All CV concentrations tested showed no amplification. Therefore, the color change in the negative reaction was caused by the degradation of LCV to CV and not due to the incorporation of amplified DNA.

Further testing of CV on paper at different concentrations of CV, SS and BCD provided results that could not distinguish between negative and positive reactions. Endpoint colorimetric scans were performed for LAMP detection on paper using the intercalating dye crystal violet (CV) as shown in Figure 17G. Sodium sulfite (SS) and β-cyclodextrin (BCD) were used to dissolve CV. Load 25 μL of the LAMP reaction onto the paper using 12.5 μL of NEB Warmstart™ 2x Master Mix, 2.5 μL of Primer Mix, and 5 μL of the above sample (positive reaction) or water (negative), and 5 μL of CV preparations were prepared in nuclease-free water at the indicated concentrations.

Finally, several colorimetric pH indicators were tested covering a range of about 2 pH units with a color transition pH of about 7.0 to match the expected pH range change and transition point of the RT-LAMP reaction. These ranges were chosen based on the initial pH of our LAMP Colorimetric Master Mix and also to overcome the buffering capacity of saliva. An exception to this selection process is acid fuchsin, which covers a 3.0 pH range and has a color conversion pH of 5.0. Figures 17H-17K show various concentrations of selected pH indicators and the associated gel electrophoresis results at 60 minutes for the LAMP reaction.

As shown in FIG. 17H , RT-LAMP detection was performed with increasing concentrations of cresyl red sodium salt, neutral red, phenol red sodium salt, m-cresyl violet, and m-cresyl violet sodium salt (pH indicator). The N.10 primer set targeting the N gene of SARS-CoV-2 was used. A positive reaction was spiked with 5 μL of in vitro transcribed N gene RNA at a concentration of 0.2 ng/μL. Negative reactions use 5 µL of nuclease-free water. The total reaction volume was 25 μL. Prepared using 12.5 μL NEB Warmstart™ 2x Master Mix, 2.5 μL Primer Mix, and 5 μL of the above sample (positive reaction) or water (negative), and 5 μL of the indicated pH indicator at the indicated concentration in nuclease-free water Reactant. Reactions were performed in an incubator and scanned every 20 minutes using a flatbed scanner.

As shown in Figure 17I, LAMP detection was performed with increasing concentrations of cresyl red (pH indicator). The lolB.3 primer set targeting the H. somniophilus genomic DNA was used. Positive reactions were spiked with 5 μL of H. somnus genomic DNA at a concentration of 0.2 ng/μL. Negative reactions use 5 µL of nuclease-free water. The total reaction volume was 25 μL. Reactions were prepared using 12.5 μL of NEB Warmstart™ 2x master mix, 2.5 μL of primer mix, 5 μL of cresyl red to give the indicated final reaction concentration, and 5 μL of the template as above (positive reaction) or water (negative).

As shown in Figure 17J, RT-LAMP detection was performed with increasing concentrations of cresyl red, sodium salt, m-cresyl violet, bromevanillol blue, and acid fuchsin, and b) the product was correlated at 60 minutes Gel electrophoresis (2% agarose) scanning. The N.10 primer set targeting the N gene of SARS-CoV-2 was used. A positive reaction was spiked with 5 μL of in vitro transcribed N gene RNA at a concentration of 0.2 ng/μL. Negative reactions use 5 µL of nuclease-free water. Reactions were prepared using the following: 12.5 μL of NEB Warmstart 2x Master Mix, 2.5 μL of Primer Mix, 5 μL of Plate as above (positive reaction) or water (negative), and 5 μL of the indicated pH indicator to produce the final indicated reaction concentration. Warming was performed in an incubator set at 65°C and scanning was performed every 20 minutes in the flatbed scanner.

An endpoint gel electrophoresis scan (60 min) of RT-LAMP products was performed on a 2% agarose gel as shown in Figure 17K.

As shown, one pH indicator that produces a distinct colorimetric reaction between positive and negative reactions is cresyl red. In addition, phenol red (the pH indicator used in NEB's colorimetric RT-LAMP kit) was also evaluated for different initial pHs resulting from the addition of HCl and KOH to the solution to provide the specified initial pH.

As shown in Figure 17L, RT-LAMP detection was performed with phenol red solutions (pH indicators) at pH 8.1, 8.5 and 8.8. Adjust with HCl and KOH before adding sample RNA. The N.10 primer set targeting the N gene of SARS-CoV-2 was used. In the positive reaction, 5 μL of in vitro transcribed N gene RNA at a concentration of 0.2 ng/μL was added. Negative reactions use 5 µL of nuclease-free water. Reactions consisted of 20 μL of master mix with 5 μL of the above-mentioned template (positive reaction) or nuclease-free water (negative). Make 10 mL of master with (NH ₄ ) ₂ SO ₄ (20 mM), KCl (100 mM), MgSO ₄ (16 mM), dNTP mix (28 mM each dNTP), tween 20 (0.2% v/v) Mixture. Therefore, phenol red produces a higher level of contrast between positive and negative reactions than cresyl red.

Thus, pH indicators that change color around pH 6.5 have the most consistent and largest contrast color change (eg, phenol red). Example 19 - Effect of initial pH on paper

To assess color stability with the addition of dryness in our method, the pH of the LAMP master mix was adjusted to 8.0, 8.5 or left unadjusted (eg, 7.6), water or synthetic RNA (N gene , 0.2 ng/μL) adjusted to 8.0, 8.5 or left unadjusted (eg, 5.5).

As shown in Figure 18, pH 7.6 is the unadjusted pH of the RT-LAMP reaction mixture. The wet setting refers to adding 5 μL of synthetic RNA (N gene, 0.2 ng/μL, “+”) or water (“-”) immediately after adding 20 μL of LAMP reaction master mix. The dry setting refers to the strips being left to dry at room temperature for 30 min after addition of 20 μL of LAMP master mix, and then rehydrated with 25 μL of synthetic RNA (‘+’) or water (‘-’).

Adjusting the pH to 8.0 in the negative control produced better color stability, while pH 8.5 was too high to obtain a noticeable color change for the standard and dry settings after 120 minutes of incubation. When the pH was not adjusted, the color changed even when the control was loaded. Example 20 - Effect of Trehalose and Tween 20 on the Colorimetric Response of RT-LAMP

Figure 19B shows the results of colorimetric RT-LAMP containing given concentrations of trehalose or Tween20. Use the orf1ab.II primer set. Dispense 20 µL containing KCl (50 mM), MgSO ₄ (8 mM), equimolar dNTP mix (1.4 mM each dNTP), WarmStart BST 2.0 (0.32 U/µL), WarmStart RTx (0.3 U/µL), phenol red (0.25 mM), dUTP (0.14 mM), Antarctic UDG (0.0004 U/μL), Tween 20 (1% v/v if specified), Betaine (20 mM), BSA (40 mg/mL) and seaweed Sugar (10% w/v, if specified) in RT-LAMP master mix was added to grade 1 chromatography paper (5 mm x 20 mm) and allowed to dry in a PCR prep hood for 60 minutes. Add 25 µL of heat-inactivated SARS-CoV- ² at a final concentration of 1x105 copies per reaction in 25% treated saliva (positive reaction) or nuclease-free water (negative reaction), to the dry reaction pad middle. The pads were heated for 60 min in an incubator set at 65 °C and then scanned using a flatbed scanner.

Inclusion of ammonium sulfate causes a color change from red to yellow after drying of the RT-LAMP reagent when no template is present. This color change was prevented by increasing the concentration of phenol red and replacing ammonium sulfate with betaine (Figure 19A). Furthermore, the addition of trehalose and bovine serum albumin (BSA) increased the reaction rate and increased the LoD ( FIG. 19B ). exemplary embodiment

In one example, there is provided a composition for performing loop-mediated constant temperature amplification (LAMP) analysis on a solid medium, which comprises or comprises one or more target primers; a DNA polymerase; and a redissolving agent; wherein The composition is substantially free of non-pH sensitive agents capable of discoloring the solid phase medium.

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

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

In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid medium, the composition can be substantially free of hygroscopic agents.

In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid medium, the hygroscopic agent is between about 40% and about 90% relative humidity (RH) at 25°C. Can absorb more than about 10% by weight.

In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid medium, the hygroscopic agent may include glycerol, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate and combinations thereof.

In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid medium, the redissolving agent may be a surfactant.

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

In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid medium, the target primer can target a pathogen comprising a viral pathogen, a bacterial pathogen, a fungal pathogen, or a native animal pathogen.

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

In another example of a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid medium, the viral agent may comprise dsDNA virus, ssDNA virus, dsRNA virus, sense ssRNA virus, anti-sense ssRNA virus, ssRNA-RT virus or ds-DNA-RT virus.

In another aspect, the viral pathogen may comprise H1N1, H2N2, H3N2, H1N1pdm09 or SARS-CoV-2.

In another example of the composition for loop-mediated isothermal amplification (LAMP) analysis on a solid medium, the composition may further comprise a reverse transcriptase.

In another example of the composition for loop-mediated isothermal amplification (LAMP) analysis on a solid medium, the composition may further include a discoloration additive.

In another example of the composition for loop-mediated isothermal amplification (LAMP) analysis on a solid medium, the non-discoloration additive comprises one or more of sugars, buffers, or combinations thereof.

In another example of the composition for loop-mediated isothermal amplification (LAMP) analysis on a solid medium, the composition may further comprise an indicator.

In one example, a method for performing LAMP analysis on a solid medium is provided, comprising providing an assembly having a solid medium and a composition as described herein; depositing a biological sample onto the on a solid medium; and heating the assembly to a constant temperature sufficient to promote the LAMP reaction.

In one example of a method for LAMP analysis on a solid medium, the biological sample is one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or combinations thereof.

In another example of the method for LAMP analysis on a solid medium, the biological sample can be saliva.

In another example of the method for performing LAMP analysis on a solid medium, the method may further comprise detecting a viral pathogen.

In another example of a method for performing a LAMP assay on a solid medium, the LAMP assay may be reverse transcriptase LAMP (RT-LAMP).

In one example, there is provided a system for performing LAMP analysis comprising or comprising a composition as described herein; and a solid medium on which the composition is deposited.

It should be understood that the above methods are only used to illustrate some embodiments of the present invention. Numerous modifications and alternative arrangements can be devised by those skilled in the art without departing from the spirit and scope of the invention, and the appended patent application is intended to cover such modifications and arrangements. Thus, while the invention has been described with particularity and detail in connection with what is presently considered to be the most practical and preferred embodiment of the invention, it will be apparent to those of ordinary skill in the art that the invention can be practiced without departing from the principles and principles described herein. Making changes in the case of concepts.

100: method 110,120: block 200: method 210,220,230: blocks 300: method 310,320: block 500a: Sponge collection device 504: Sponge collection pad 506: Compression tube 508: Compression seals 510: handle 512: Sample volume sufficiency indicator 500b: Passive drooling device 522: Collection funnel 524: collection tube 526: pipe cap 528: indicator line 530: volume indicator 532: cap memory

Features and advantages of the present disclosure will become apparent by reference to the following detailed description, taken in conjunction with the accompanying drawings illustrating by way of example features of the disclosure, in which:

1 depicts a method for preparing a saliva sample for loop-mediated constant temperature amplification (LAMP) detection of pathogenic targets according to an exemplary embodiment;

Figure 2 depicts a method for LAMP analysis according to an exemplary embodiment;

3 depicts a method of maximizing the accuracy of an output signal in a pH-dependent LAMP assay, according to an exemplary embodiment;

Figure 4 illustrates that loop-mediated isothermal amplification (LAMP) can be obtained in saliva samples according to an exemplary embodiment;

Figure 5A illustrates a sponge-based collection device in accordance with an exemplary embodiment;

Figure 5B illustrates a passive drool collection device in accordance with an exemplary embodiment;

Figure 6A illustrates various sample concentrations and detection limits for various collection devices in accordance with an exemplary embodiment;

Figure 6B illustrates the effect of RNase inhibitors on the colorimetric response of RT-LAMP for various concentrations of samples according to exemplary embodiments;

FIG. 6C illustrates the effect of saliva treatment techniques on contrast color LoD in accordance with an exemplary embodiment;

Figure 6D illustrates the effect of vector DNA concentration versus color RT-LAMP response according to an exemplary embodiment;

Figure 6E illustrates the effect of guanidine hydrochloride on the colorimetric response of RT-LAMP according to exemplary embodiments;

Figure 6F illustrates the effect of UDG on the colorimetric response of endpoint RT-LAMP according to an exemplary embodiment;

Figure 6G illustrates the effect of saliva treatment on contrast color response in accordance with an exemplary embodiment;

Figure 7 is a graph illustrating the stability of frozen saliva samples according to an exemplary embodiment;

Figure 8 illustrates the detection limit of fresh saliva according to an exemplary embodiment;

Figure 9 illustrates the detection limit of a bovine nose swab according to an exemplary embodiment;

Figure 10 illustrates the detection limit on paper according to an exemplary embodiment;

Figure 11 illustrates the colorimetric transition of phenol red in accordance with exemplary embodiments;

Figure 12 illustrates buffers for paper-based assays according to an exemplary embodiment;

Figure 13A illustrates the effectiveness of paper LAMP in accordance with exemplary embodiments;

Figure 13B illustrates the effectiveness of paper LAMP in accordance with exemplary embodiments;

Figure 14A illustrates low template concentration LAMP at the 0 minute time point on paper according to an exemplary embodiment;

Figure 14B illustrates low template concentration LAMP at the 60 minute time point on paper according to an exemplary embodiment;

Figure 15 illustrates whole unprocessed saliva with heat-inactivated SARS-CoV-2 virus, according to an exemplary embodiment;

Figure 16 illustrates a comparison of colorimetric and fluorescent RT-LAMP responses in accordance with exemplary embodiments;

17A illustrates the use of wortensite as a LAMP colorimetric indicator, according to an exemplary embodiment;

Figure 17B illustrates the use of EBT as a LAMP indicator according to an exemplary embodiment;

Figure 17C illustrates LAMP on chromatography paper using EBT as a colorimetric reporter, according to an exemplary embodiment;

Figure 17D illustrates the colorimetric response of LAMP on various papers using EBT as an indicator, according to an exemplary embodiment;

Figure 17E illustrates LAMP detection on Biodyne A amphoteric paper using EBT as a colorimetric indicator, according to an exemplary embodiment;

Figure 17F illustrates the effect of crystal violet concentration on the colorimetric response of LAMP, according to an exemplary embodiment;

17G illustrates a colorimetric LAMP using different concentrations of crystal violet on paper, according to an exemplary embodiment;

Figure 17H illustrates a pH indicator as a colorimetric reporter for RT-LAMP according to an exemplary embodiment;

Figure 17I illustrates the effect of cresyl red concentration on the colorimetric response of a LAMP reaction according to an exemplary embodiment;

FIG. 17J , the effect of the concentration of various pH indicators on the colorimetric response of the RT-LAMP reaction according to exemplary embodiments;

Figure 17K, Gel electrophoresis scan of RT-LAMP products using a pH indicator according to an exemplary embodiment;

Figure 17L, Effect of initial pH on colorimetric response of RT-LAMP using phenol red, according to exemplary embodiments;

Figure 18 illustrates the color stability of the drying process according to an exemplary embodiment;

Figure 19A illustrates the effect of eliminating a single reactant on the initial color of paper after drying according to an exemplary embodiment; and

Figure 19B illustrates the effect of trehalose and Tween 20 on the colorimetric response of RT-LAMP according to exemplary embodiments.

Reference will now be made to the exemplary embodiments described, and specific language will be used herein to describe the same. It should be understood, however, that no limitation of the scope of the present technology is intended.

(none)

Claims (22)

A composition for performing loop-mediated constant temperature amplification (LAMP) analysis on a solid medium, comprising: one or more target primers; a DNA polymerase; and re-dissolving agent; Wherein the composition does not substantially contain non-pH sensitive agents capable of discoloring the solid phase medium. The composition according to claim 1, further comprising an antioxidant. The composition according to claim 1, wherein the composition substantially does not contain volatile agents. The composition according to claim 1, wherein the composition substantially does not contain a hygroscopic agent. The composition of claim 4, wherein the hygroscopic agent absorbs more than about 10% by weight between about 40% and about 90% relative humidity (RH) at 25°C. The composition according to claim 4, wherein the hygroscopic agent comprises glycerin, ethanol, methanol, calcium chloride, calcium sulfate and combinations thereof. The composition according to claim 1, wherein the redissolving agent is a surfactant. The composition according to claim 1, wherein the redissolving agent comprises bovine serum albumin (BSA), casein, polysorbate 20 and combinations thereof. The composition according to claim 1, wherein the target primer targets a pathogen, and the pathogen includes a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen. The composition according to claim 9, wherein the pathogen is a viral pathogen. The composition according to claim 10, wherein the viral pathogen comprises dsDNA virus, ssDNA virus, dsRNA virus, positive strand ssRNA virus, reverse strand ssRNA virus, ssRNA-RT virus or ds-DNA-RT virus. The composition of claim 10, wherein the viral pathogen comprises H1N1, H2N2, H3N2, H1N1pdm09 or SARS-CoV-2. The composition according to claim 1, further comprising a reverse transcriptase. The composition according to claim 1, further comprising a color-changing additive. The composition according to claim 14, wherein the non-discoloration additive comprises one or more of sugars, buffers or combinations thereof. The composition according to claim 1, further comprising an indicator. A method for performing LAMP analysis on a solid medium comprising: Provide a solid phase medium and an assembly of the composition as described in Claim 1, depositing a biological sample onto the solid medium; and The assembly is heated to a constant temperature sufficient to promote the LAMP reaction. The method according to claim 17, wherein the biological sample is one or more of saliva, mucus, blood, urine, feces and combinations thereof. The method of claim 17, wherein the biological sample is saliva. The method as claimed in item 17, further comprising: A viral pathogen is detected. The method of claim 17, wherein the LAMP assay is reverse transcriptase LAMP (RT-LAMP). A system for performing LAMP analysis comprising: The composition as described in claim 1; and A solid medium on which the composition is deposited.

Images