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

Abstract

The present disclosure relates to compositions and methods for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal. The composition can include a pH-sensitive dye and a plurality of non-interfering LAMP reagents. The method can include providing an assembly of a solid phase medium and the composition, depositing a biological sample on the solid phase medium, and heating the assembly to an isothermal temperature sufficient to promote a LAMP reaction.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 138,314, filed January 15, 2021, which is incorporated herein by reference. [Background technology]

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

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

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

[0005] In some disclosed embodiments, methods for preparing a saliva sample for loop-mediated isothermal amplification (LAMP) detection of a pathogen target are provided. In one aspect, such methods can include providing a volume of saliva from a test subject and diluting the saliva with water to a level that reduces the buffering capacity of the saliva while maintaining a concentration sufficient to allow detection of the pathogen target.

[0006] In one embodiment, the method can include reducing the viscosity of the saliva compared to its original viscosity. In another embodiment, the viscosity can be reduced by one or more of dilution, filtration, or a combination thereof. In another embodiment, filtration can be used to reduce the viscosity. In a further embodiment, a 10 micron filter can be used to reduce the viscosity. In yet another embodiment, the viscosity can be reduced to a level that increases the fluidity through a solid phase medium compared to the original viscosity. In yet another embodiment, the viscosity can be reduced to a range of about 1.0 centipoise (cP) to about 50 cP.

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

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

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

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

[0011] In other disclosed embodiments, a test sample composition for a LAMP assay is disclosed, which can include a sufficient amount of test control saliva to detect a pathogen target via a LAMP assay, in combination with an amount of water to reduce the buffering capacity of the saliva.

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

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

[0014] In yet other disclosed embodiments, a composition for LAMP analysis on a solid-phase medium can include one or more target primers, a DNA polymerase, and a resolubilizing agent. In some aspects, such compositions can be substantially free of non-pH-sensitive agents that can discolor 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 another aspect, the composition can further include a reverse transcriptase.

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

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

[0017] In one embodiment, 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 embodiment, the pathogen can be a viral pathogen. In another embodiment, the viral pathogen can include a dsDNA virus, a ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, a ssRNA-RT virus, or a ds-DNA-RT virus. In another embodiment, the viral pathogen can include H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.

[0018] In one embodiment, the composition can further comprise a non-color-changing additive. The non-color-changing additive can comprise one or more of a sugar, a buffer, or a combination thereof. In another embodiment, the composition can further comprise an indicator.

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

[0020] In other disclosed embodiments, a system for performing a LAMP assay can include a composition described herein and a solid phase medium on which the composition is deposited.

[0021] In yet further disclosed embodiments, a composition for loop-mediated isothermal amplification (LAMP) analysis can utilize a pH-dependent output signal that can include a pH-sensitive dye and a plurality of non-interfering LAMP reagents. In one aspect, the LAMP analysis can be RT-LAMP.

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

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

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

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

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

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

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

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

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

[0031] [Figure 1]According to an exemplary embodiment, a method for preparing a saliva sample for loop-mediated isothermal amplification (LAMP) detection of a pathogen target is presented. [Figure 2] 1 illustrates a method for LAMP analysis according to an exemplary embodiment. [Figure 3] According to an exemplary embodiment, a method for maximizing the accuracy of the output signal in a pH-dependent LAMP assay is shown. [Figure 4] In accordance with an exemplary embodiment, it is shown that loop-mediated isothermal amplification (LAMP) can be obtained in saliva samples. [Figure 5A] 1 shows a sponge-based collection device, according to an exemplary embodiment. [Figure 5B] 1 illustrates a passive drool collection device according to an exemplary embodiment. [Figure 6A] 1 illustrates the detection limits of various concentrations of samples and various collection devices, according to an exemplary embodiment. [Figure 6B] 1 shows the effect of an RNase inhibitor on various concentrations of template on the RT-LAMP colorimetric response, according to an exemplary embodiment. [Figure 6C] 10 illustrates the effect of saliva processing techniques on colorimetric LoD, according to an exemplary embodiment. [Figure 6D] 1 shows the effect of carrier DNA concentration on RT-LAMP colorimetric response, according to an exemplary embodiment. [Figure 6E] 1 shows the effect of guanidine HCl on RT-LAMP colorimetric response, according to an exemplary embodiment. [Figure 6F] 1 shows the effect of UDG on end-point RT-LAMP colorimetric response, according to an exemplary embodiment. [Figure 6G] 10 shows the effect of saliva treatment on colorimetric response, according to an exemplary embodiment. [Figure 7] 1 is a chart showing the stability of frozen saliva samples, according to an exemplary embodiment. [Figure 8] 10 illustrates the detection limit for untreated saliva, according to an exemplary embodiment. [Figure 9]1 shows the limit of detection in bovine nasal swabs, according to an exemplary embodiment. [Figure 10] 1 illustrates detection limits on paper according to an exemplary embodiment. [Figure 11] 1 shows the colorimetric transition of phenol red, according to an exemplary embodiment. [Figure 12] 1 shows buffers used in a paper-based assay, according to an exemplary embodiment. [Figure 13A] 1 illustrates a paper LAMP verification according to an exemplary embodiment. [Figure 13B] 1 illustrates a paper LAMP verification according to an exemplary embodiment. [Figure 14A] 1 shows low template concentration LAMP on paper at 0 minutes, according to an exemplary embodiment. [Figure 14B] 1 shows low template concentration LAMP on paper at 60 minutes, according to an exemplary embodiment. [Figure 15] 1 shows unprocessed whole saliva containing heat-inactivated SARS-CoV-2 virus, according to an exemplary embodiment. [Figure 16] 1 shows a comparison of RT-LAMP colorimetric and fluorescent responses, according to an exemplary embodiment. [Figure 17A] 1 illustrates the use of calmagite as a LAMP colorimetric indicator, according to an exemplary embodiment. [Figure 17B] 1 illustrates the use of EBT as a LAMP indicator, according to an exemplary embodiment. [Figure 17C] 1 shows LAMP on chromatography paper using EBT as a colorimetric reporter, according to an exemplary embodiment. [Figure 17D] 1 shows the colorimetric response of LAMP on various papers using EBT as an indicator, according to an exemplary embodiment. [Figure 17E] 1 shows LAMP detection on biodyne A amphoteric paper using EBT as a colorimetric indicator, according to an exemplary embodiment. [Figure 17F] 1 shows the effect of crystal violet concentration on LAMP colorimetric response, according to an exemplary embodiment. [Figure 17G]1 shows colorimetric LAMP using various concentrations of crystal violet on paper, according to an exemplary embodiment. [Figure 17H] 1 shows a pH indicator as a colorimetric reporter for RT-LAMP, according to an exemplary embodiment. [Figure 17I] 1 shows the effect of cresol red concentration on the colorimetric response of a LAMP reaction, according to an exemplary embodiment. [Figure 17J] 1 shows the effect of various pH indicator concentrations on the colorimetric response of the RT-LAMP reaction, according to an exemplary embodiment. [Figure 17K] 1 shows a 17K gel electrophoresis scan of RT-LAMP products using a pH indicator, according to an exemplary embodiment. [Figure 17L] 1 shows the effect of initial pH on RT-LAMP colorimetric response using phenol red, according to an exemplary embodiment. [Figure 18] 10 illustrates color stability of the drying process, according to an exemplary embodiment. [Figure 19A] 10 illustrates the effect of removing a single reactant on the initial color of paper after drying, according to an exemplary embodiment. [Figure 19B] 1 shows the effect of trehalose and Tween 20 on RT-LAMP colorimetric response, according to an exemplary embodiment.

[0032] Reference will now be made to exemplary embodiments and specific language will be used herein to describe the same, it being understood that no limitation on the scope of the technology is intended thereby. DETAILED DESCRIPTION OF THE INVENTION

[0033] Before describing embodiments of the present invention, it is to be understood that the present disclosure is not limited to the particular structures, process steps, or materials disclosed herein, but extends to equivalents thereof as would be recognized by one skilled in the relevant art. It should also be understood that the terminology used herein is used only for the purpose of describing specific examples or embodiments, and is not intended to be limiting. The same reference numbers in different drawings represent the same components. Numbers provided in flowcharts and processes are provided for clarity in indicating steps and operations, and do not necessarily indicate a particular order or sequence.

[0034] 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 embodiments of the present invention. However, those skilled in the art will recognize that such specific embodiments are not limiting of the overall inventive concepts set forth herein, but are merely representative thereof.

[0035] definition 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 a "carrier" includes reference to one or more such carriers.

[0036] As used herein, the terms "formulation" and "composition" are used interchangeably and refer to a mixture of two or more compounds, elements, or molecules. In some embodiments, the terms "formulation" and "composition" can be used to refer to a mixture of one or more active agents with a carrier or other excipient.

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

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

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

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

[0041] As used herein, "solid" and "semi-solid" refer to the physical state of a composition that has a suitable viscosity or structure to support its own weight at standard temperature and pressure and is not free-flowing. A semi-solid material may conform to the shape of a container upon application of pressure.

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

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

[0044] As used herein, "non-LAMP reaction-induced discoloration" refers to any discoloration of a solid-phase medium (e.g., a change in color from an original color to another color) that is not the result of nucleotide amplification from a LAMP reaction. In some examples, non-LAMP reaction-induced discoloration may refer to discoloration of a solid-phase medium due to one or more of a volatile agent, a magnesium interfering agent, an oxidizing agent, a pH change resulting from a cause other than amplification by a LAMP reaction, drying, or a combination thereof.

[0045] As used herein, a "volatile agent" refers to an agent comprising a composition having a high vapor pressure or a low boiling point. In one example, ammonium sulfate may be a volatile agent because ammonia may volatilize, leaving sulfuric acid. In one example, a composition, component, or element may have a high vapor pressure if the composition exists in the gas phase at a temperature above about 30°C. In one example, a composition may have a low boiling point if the composition exists in the gas phase at a temperature below about 80°C.

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

[0047] In this disclosure, terms such as "comprises," "comprising," "containing," and "having" can have the meaning ascribed to them in U.S. patent law and can mean "includes," "including," etc., and are generally construed as open-ended terms. The terms "consisting of" or "consists of" are closed terms and include only those components, structures, steps, etc. that are specifically recited in association with such terms, as well as those pursuant to U.S. patent law. "Consisting essentially of" or "consists essentially of" have the meaning generally given them by U.S. patent law. In particular, such terms are generally closed terms, except that they may include additional items, materials, ingredients, steps, or components that do not materially affect the basic and novel characteristics or function of the item(s) with which they are used. For example, trace elements that are present in a composition but do not affect the properties or characteristics of the composition would be permissible if present under the phrase "consisting essentially of," even if they are not explicitly set forth in the list of items following such terminology. When open-ended terms such as "comprising" or "including" are used herein, it is understood that the phrases "consisting essentially of" as well as "consisting of" should be given direct support as if explicitly set forth, and vice versa.

[0048] Terms such as "first," "second," "third," and "fourth" in the specification and claims are used to distinguish between similar elements and are not necessarily used to describe a particular sequential or chronological order. It should be understood that any such terms used are interchangeable under appropriate circumstances, such that the embodiments described herein can, for example, operate in orders other than those illustrated or otherwise described herein. Similarly, when a method is described herein as including a series of steps, the order of the steps presented herein is not necessarily the only order in which such steps may be performed; certain of the described steps may be omitted and / or certain other steps not described herein may be added to the method.

[0049] As used herein, comparative terms such as "increased," "decreased," "better," "worse," "higher," "lower," "enhanced," "maximized," "minimized," and the like refer to the properties of a device, component, composition, or activity that are measurably different from other devices, components, compositions, or activities in the surrounding or adjacent area, similarly located, within a single device or composition or multiple comparable devices or compositions, within a group or class, within multiple groups or classes, or that are measurably different compared to known prior art.

[0050] The term "coupled," as used herein, is defined as directly or indirectly connected in a chemical, mechanical, electrical, or non-electrical manner. Objects described herein as "adjacent" to one another may be in physical contact with one another, in close proximity to one another, or in the same general area or region of one another, depending on the context in which the phrase is used. Appearances of the phrases "in one embodiment" or "in one aspect" herein do not necessarily all refer to the same embodiment or aspect.

[0051] As used herein, the term "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is "substantially" encapsulated means that the object is either completely encapsulated or nearly completely encapsulated. The exact acceptable degree of deviation from absolute perfection may depend on the specific situation. Generally speaking, however, approximating perfection results in the same overall result as if absolute and total perfection were achieved. The use of "substantially" is equally applicable when used in a negative sense to refer to the complete or nearly complete absence of an action, characteristic, property, state, structure, item, or result. For example, a composition that is "substantially free" of particles will either be completely devoid of particles or nearly completely devoid of particles to the extent that the effect is the same as if the particles were completely absent. In other words, a composition that is "substantially free" of a component or element may still actually contain such an item so long as there is no measurable effect of that component.

[0052] As used herein, the term "about" is used to provide flexibility to the endpoints of numerical ranges by specifying that a given value may be "slightly above" or "slightly below" the endpoints. Unless otherwise specified, the use of the term "about" in conjunction with a particular number or numerical range should be understood to support such numerical term or range without the term "about." For example, for convenience and brevity, a numerical range of "about 50 angstroms to about 80 angstroms" should also be understood to support the range "50 angstroms to 80 angstroms." Furthermore, it should be understood that, in this specification, actual numerical values ​​are supported even when the term "about" is used in conjunction with them. For example, a statement of "about" 30 should be interpreted as supporting the actual numerical value of 30, as well as supporting values ​​slightly above and slightly below 30.

[0053] As used herein, a plurality of items, structures, components, and / or materials may be presented in common lists for convenience; however, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such a list should be construed as being effectively equivalent to any other member of the same list solely based on presentation within a common group, absent a contrary indication.

[0054] Concentrations, amounts, levels, and other numerical data may be expressed or presented herein in a range format. It should be understood that such range formats are used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limits, but also all individual numerical values, subranges, or fractional units subsumed within that range, as if each numerical value and subrange were explicitly recited. By way of illustration, a numerical range of "about 1 to about 5" should be interpreted to include not only the explicitly recited values ​​of about 1 to about 5, but also each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 2, 3, and 4, as well as subranges such as 1 to 3, 2 to 4, and 3 to 5, and the values ​​1, 2, 3, 4, and 5 individually. This same principle applies to ranges reciting only a single numerical value as the minimum or maximum value. Furthermore, such interpretation should apply regardless of the breadth or character of the range described.

[0055] References throughout this specification to "an example" mean that the particular feature, structure, or characteristic described in connection with that example is included in at least one embodiment. Thus, the appearances of the phrase "in one example" in various places throughout this specification do not necessarily all refer to the same embodiment.

[0056] Embodiment Many molecular tests for pathogens (e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19) can be confined to laboratories and therefore have significant delays (>24 hours) before results are available, hindering their adoption in point-of-care settings. While there have been several attempts to develop point-of-care tests for SARS-CoV-2, several limitations remain: i) scalability (demand for testing is on the order of millions per week, making it difficult to create new tests at that scale); ii) sample processing (when using saliva, many tests still use extraction procedures); and iii) readability (molecular tests often use fluorescence, requiring fluorescence readers to report results).

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

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

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

[0060] Sampling and characterization for LAMP analysis Saliva has various physical, chemical, and antibacterial properties that can pose challenges in the LAMP reaction. For example, one physical property of saliva is that it dilutes and removes organic acids from dental plaque, which can hinder the LAMP reaction. Some chemical properties, such as electrolytes and buffer molecules that minimize pH changes, can also hinder the LAMP reaction. Antibacterial agents in saliva, such as mucin, amylase, lysozyme, and peroxidase enzymes, also pose challenges. For example, peroxidase enzymes can form free radical compounds within bacterial cells, causing apoptosis-like death in the cells. However, such reactions can also provide an unstable redox environment, complicating the LAMP reaction.

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

[0062] One method for reducing the buffering capacity of saliva can be dilution, and in one embodiment, such a method can include providing a volume of saliva from a test subject, as shown in block 110, and diluting the saliva with water to a level that reduces the buffering capacity of the saliva while maintaining a concentration sufficient to allow detection of the pathogen target, as shown in block 120.

[0063] Proteins present in saliva present additional challenges. For example, overly viscous samples can be difficult to test with solid-based or solid-phase media. Slow flow rates in solid-based media can increase reaction times, reduce uniformity of spreading, and increase result variability and invalidity. For example, if saliva in its viscous form does not spread evenly throughout the solid-based media, color-based displays can be difficult to read. Reduced uniform spreading can add uncertainty to result reading and increase result variability. Different technicians may interpret results differently. In some cases, the color change may be ambiguous or absent, making it impractical to read the results. Therefore, controlling the viscosity of saliva can prevent various potential complications.

[0064] Thus, in another embodiment, the method can further include reducing the viscosity of the saliva compared to its original viscosity. In one aspect, the viscosity of 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 the saliva, the saliva can be diluted with water to a saliva-to-water ratio of about 1:1 to about 1:20. In another aspect, the viscosity of the saliva can be reduced by diluting with water to a saliva-to-water ratio of about 1:1, 1:2, 1:4, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, or 1:20. In one aspect, the viscosity of the saliva can be reduced by measuring the optical density at 600 nm (OD ) of the sample. 600 ) is less than about 0.2. In one embodiment, the saliva may have a volume ranging from about 50 μl to about 100 μl. In another embodiment, the saliva sample may have a volume ranging from about 100 μl to about 1 ml.

[0065] In some cases, diluting a saliva sample can reduce the effects of saliva's buffering capacity and viscosity. Another method for reducing the effects of saliva viscosity includes filtration. In another embodiment, viscosity can be reduced using a filter rated between about 2 microns and 50 microns. In one example, the filter rating 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 embodiment, the filter rating can be an absolute micron rating, which allows the filter to remove at least about 98.7% of a specific particle size. In addition to dilution, filtering saliva can also remove saliva proteins (e.g., mucin, amylase, lysozyme, and peroxidase enzymes) that may interfere with the LAMP reaction.

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

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

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

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

[0070] Minimizing the presence of undesirable saliva proteins can be achieved using certain saliva collection methods. In one aspect, saliva can be collected using one or more of a sponge-based collection method or a saliva-flow collection method. Collecting saliva using a sponge-based collection method may have the advantage that mucins and high-molecular-weight proteins are not absorbed by the sponge and are therefore essentially filtered out of the saliva, which may reduce the viscosity of the saliva when used on a solid-based medium, making the saliva more rapid, uniform, and reliable. When saliva is collected using a saliva-flow method, unfiltered saliva may be more viscous, which may reduce absorption and distribution on a solid-based medium. As a result, in some embodiments, when saliva is collected via saliva-flow, it can be subsequently filtered to remove mucins and other debris and reduce its viscosity.

[0071] The selected pathogen target can be detected in saliva.In one embodiment, the pathogen target can be one or more of viral pathogens, bacterial pathogens, fungal pathogens, protozoan pathogens, etc., or a combination thereof.The pathogen target in saliva can be detected when the nucleic acid from the pathogen target can be released from cell wall, cell membrane, protein membrane, etc.

[0072] More specifically, in one embodiment, the pathogen target can be a viral target. In some embodiments, 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.

[0073] 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 respirovirus 1, human rubulavirus 2, human respirovirus 3, human rubulavirus 4, human enterovirus, human respiratory virus, rhinovirus A, rhinovirus B, rhinovirus C, or a combination thereof.

[0074] The viral target may also be a form of influenza. In one embodiment, the influenza may be influenza A, influenza B, influenza C, or influenza D. In one embodiment, the viral target may be a virus selected from the order Nidovirales. In one embodiment, the viral target may be selected from the alpha, beta, gamma, or delta genera of the order Nidovirales.

[0075] There are various families of viruses that can be detected. In one aspect, the viral target is: Adenoviridae , Papovaviridae , Parvoviridae , Herpesviridae , Poxviridae , Anelloviridae In another embodiment, the viral target may be a DNA virus selected from the group of families including: Reoviridae , Picornaviridae , Caliciviridae , Togaviridae , Arenaviridae , Flaviviridae , Orthomyxoviridae , Paramyxoviridae , Bunyaviridae , Rhabdoviridae , Filoviridae , Coronaviridae , Astroviridae , Bornaviridae In another embodiment, the viral target may be an RNA virus selected from the group consisting of: Retroviridae , Caulimoviridae , Hepadnaviridae The virus may be a reverse transcribing virus selected from the group consisting of:

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

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

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

[0079] If the pathogen target in saliva contains RNA, the RNA can be reverse transcribed. Thus, in another embodiment, LAMP detection can be reverse transcription-LAMP (RT-LAMP). In this example, reverse transcriptase can be used to generate cDNA from the target RNA. The cDNA can be amplified to a detectable amount. If the pathogen target can be detected directly from DNA, LAMP can be used to amplify the DNA to a detectable amount without reverse transcribing the RNA to DNA.

[0080] In another embodiment, the specific target nucleotide sequence to be detected can be a target nucleotide corresponding to a human biomarker. Any disease having a target nucleotide corresponding to a human biomarker for the disease can be detected. Various types of diseases can be detected, including one or more of breast cancer, pancreatic cancer, colon cancer, ovarian cancer, gastrointestinal cancer, cervical cancer, lung cancer, bladder cancer, many types of carcinoma, salivary gland cancer, kidney cancer, liver cancer, lymphoma, leukemia, melanoma, prostate cancer, thyroid cancer, gastric cancer, etc., or combinations thereof. For example, biomarkers for various types of disease include alpha fetoprotein, CA15-3 and CA27-29, CA19-9, CI-125, calcitonin, calretinin, carcinoembryonic antigen, CD34, CD99MIC2, CD117, chromogranin, chromosomes 3, 7, 17, and 9p21, cytokeratin, cesmin, epithelial membrane antigen, factor VIII, CD31FL1, glial fibrillary acidic protein, gross cystic disease fluid protein, and cystic fibrosis protein. The detection can be by detecting target nucleotides corresponding to one or more of: human chorionic gonadotropin, human PG80, HMB-45, human chorionic gonadotropin, immunoglobulin, inhibin, keratin, lymphocyte marker, MART-1, MyoD1, muscle-specific actin, neurofilament, neuron-specific enolase, placental alkaline phosphatase, prostate-specific antigen, PTPRC, S100 protein, smooth muscle activity, synaptophysin, thymidine kinase, thyroglobulin, thyroid transcription factor-1, tumor M2-PK, vimentin, and the like, or a combination thereof.

[0081] In another embodiment, a test sample composition for loop-mediated isothermal amplification (LAMP) analysis can include a sufficient amount of saliva from a test subject to detect a pathogen target via LAMP analysis, combined with an amount of water to reduce the buffering capacity of the saliva. 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 can be from about 7.2 to about 8.6. Selecting a viscosity and pH within these respective ranges facilitates the pH change and, therefore, the color change resulting from the pH-based indicator.

[0082] The saliva can be diluted with water to achieve a viscosity and pH within the ranges listed above. In one embodiment, the saliva can be mixed with water at a saliva-to-water ratio of about 1:1 to about 1:20. In another embodiment, the saliva-to-water ratio can be about 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, or 1:20. In another embodiment, the saliva can be diluted to achieve a saliva-to-water ratio of about 1:1 to about 1:20. In another embodiment, the saliva can be diluted to achieve a saliva-to-water ratio of ... to about 1:20. In another embodiment, the saliva can be diluted to achieve a saliva-to-water ratio of about 1:1 to about 1:20. In another embodiment, the saliva can be diluted to achieve a saliva-to-water ratio of about 1:1 to about 1:20. In another embodiment, the saliva can be diluted to achieve a saliva-to-water ratio of about 1:1 to about 1:20. In another embodiment, the saliva can be diluted to achieve a saliva-to-water ratio of about 1:1 to about 1:20. In another embodiment, the saliva can be diluted to achieve a saliva-to-water ratio of about 1:1 to about 1:20. In another embodiment, the saliva can be diluted to achieve a saliva-to-water ratio of about 600 ) can be combined with an amount of water to such an extent that the .gtoreq.(.times. ...

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

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

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

[0086] Reagent Composition Depending on the test medium, readout type, and overall environment of the designed system, various reagents can be used in LAMP analysis. Furthermore, reaction components such as primers and enzymes can be selected taking into consideration the specific target nucleotide sequence to be detected, the organism to be identified, etc. Furthermore, characteristics of the test environment, such as a liquid environment, an anhydrous environment, housing, substrate, etc., as well as other needs such as storage stability, can also be taken into consideration when selecting the specific reagents involved in the reaction underlying the LAMP analysis.

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

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

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

[0090] In addition to the target primer, DNA polymerase, and reverse transcriptase, the composition can include a resolubilizing agent. The resolubilizing agent may aid in the rehydration of the LAMP reagents on the solid-based medium when the saliva sample is deposited on the solid-based medium. In one embodiment, the resolubilizing agent can be a surfactant. For example, the resolubilizing agent can include bovine serum albumin (BSA), casein, polysorbate 20, etc., or a combination thereof. BSA and casein promote the resolubilization of the DNA polymerase, reverse transcriptase, and other related enzymes when the dried reagents are rehydrated. Polysorbate 20 is a surfactant that also aids in the resolubilization of the dried reagents. In one example, the concentration of the resolubilizing agent when used on a solid-phase medium can be about 0.05% to about 5% by weight. In another example, the concentration of the resolubilizing agent can be about 0.5% to about 3% by weight. In yet another example, the concentration of the resolubilizing agent can be about 0.5% to about 1.5% by weight.

[0091] The composition may further include an agent that accelerates the reaction, an agent that enhances sensitivity, or a combination thereof. In one example, BSA may be included to accelerate the reaction and enhance sensitivity. However, the inclusion of BSA may induce pH fluctuations, which may impair the readability of the results. Therefore, in some examples, the resolubilizing agent may include casein, polysorbate 20, etc., or a combination thereof.

[0092] Volatile agents can 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 ion evaporates, the remaining ion may undergo further reactions. Some of these further reactions may include oxidation-reduction reactions, acid-base reactions, or other reactions that may affect the interpretation of pH-based signals. In one embodiment, the composition may be substantially free of volatile agents. In one example, removing a volatile agent can increase the color contrast of the solid-based medium and shorten the reaction time compared to the color contrast and reaction time when a volatile agent is present. In one embodiment, the composition may contain less than one or more of 1.0 wt %, 0.5 wt %, 0.1 wt %, or 0.01 wt % of a volatile agent.

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

[0094] Therefore, reducing the presence of volatile agents in the composition can reduce the degree of interference with the LAMP reaction and its readout by the pH-based indicator. In one embodiment, the LAMP composition can include a non-volatile agent, such as a neutrally charged, low-molecular-weight quaternary ammonium salt, a neutrally charged, low-molecular-weight amide compound, or a combination thereof. In one example, the non-volatile agent includes N-formyl urea, urea, L-asparagine, trimethylglycine (betaine), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 3-(1-pyridinio)-1-propanesulfonate (NDSB-201), N-methyl urea, acetamide, propionamide, isobutyramide, piracetam, 1,3-dimethyl urea, 1,1-dimethyl urea, glycolamide, 2-chloroacetamide, succinimide, 2-imidazolidone, choline chloride, acetylcholine chloride, bethanechol chloride, L-calcium phosphate, and the like. Examples of suitable amines include, but are not limited to, carnitine inner salt, O-acetyl-L-carnitine hydrochloride, 4-(cyclohexylamino)-1-butanesulfonic acid (CABS), dimethylethylammonium propanesulfonate (NDSB-195), 3-(1-methylpiperidinium)-1-propanesulfonate (NDSB-221), 3-(benzyldimethylammonio)propanesulfonate (NDSB-256), and dimethyl-2-hydroxyethylammonium-1-propanesulfonate (NDSB-211), or combinations thereof.

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

[0096] In addition to volatile agents, hygroscopic agents can also interfere with LAMP reactions. Hygroscopic agents can retain excessive amounts of water, slowing or preventing drying and thereby destabilizing reagents in solid-based media. In one embodiment, the composition can be substantially free of hygroscopic agents. In some examples, LAMP reactions involving hygroscopic agents, such as glycerol, can contribute to reagent instability in solid-based media because the hygroscopic agent can attract water. In one example, the hygroscopic agent can absorb more than about 10% by weight at 25°C and about 40% to about 90% relative humidity (RH). In one example, the hygroscopic agent can include, but is not limited to, one or more of glycerol, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate, and the like, or combinations thereof. In one embodiment, the composition can contain less than one or more of 1.0 wt%, 0.5 wt%, 0.1 wt%, or 0.01 wt% of the hygroscopic agent.

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

[0098] It has been discovered that some pH-based indicators with antioxidant activity, such as phenol red, can provide improved contrast and uniformity compared to other pH-based indicators with less antioxidant activity. In one embodiment, the composition can further comprise an antioxidant. In one example, the concentration of the antioxidant when used on a solid-phase medium can be about 0.1 mM to about 1 mM. In another example, the concentration of the antioxidant when used on a solid-phase medium can be about 0.2 mM to about 0.8 mM. In yet another example, the concentration of the antioxidant when used on a solid-phase medium can be about 0.2 mM to about 0.3 mM. The antioxidant can stabilize the reagent on the solid-based medium by preventing redox reactions.

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

[0100] Although pH-based indicators have been discussed above, other indicators can also be used. In one embodiment, the composition can further comprise an indicator. In one example, the indicator can be a pH-based indicator such as phenol red when used with a solid-phase medium. Phenol red has antioxidant properties that some other dyes lack. The phenol red molecule is a conjugated bond system, which may also contribute to its antioxidant properties. In one example, the concentration of the indicator can be about 0.1 mM to about 1 mM when used with a solid-phase medium. In another example, the concentration of the indicator can be about 0.2 mM to about 0.8 mM when used with a solid-phase medium. In yet another example, the concentration of the indicator can be about 0.2 mM to about 0.3 mM when used with a solid-phase medium.

[0101] Some other indicators can also provide an appropriate colorimetric signal. 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 intercalating colorimetric indicator. If the indicator is a magnesium colorimetric indicator, the magnesium concentration should be monitored to maintain it within a range of about 0.01 mM to about 2 mM. The magnesium concentration should also be monitored to prevent interference with DNA polymerase. Magnesium, a cofactor for DNA polymerase, can interfere with DNA polymerase if the magnesium concentration is outside the target range.

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

[0103] The target primers can be selected to target the genomes of various pathogens. In one embodiment, the target primers can target pathogens, which can include viral pathogens, bacterial pathogens, fungal pathogens, or protozoan pathogens. In another embodiment, the pathogen target can be a viral target. In another embodiment, the viral target can include a dsDNA virus, a ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, a ssRNA-RT virus, or a ds-DNA-RT virus. In another embodiment, the viral target can include H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2. In short, the target primers can target almost any pathogen target, particularly a target pathogen as disclosed herein.

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

[0105] A variety of non-color-changing additives exist that can preserve the color of the solid-based medium in the absence of LAMP amplification and potentially enhance contrast when LAMP amplification occurs. In one example, the non-color-changing additive can include one or more of a sugar, a buffer, etc., or a combination thereof.

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

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

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

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

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

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

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

[0113] 2, a method 200 for LAMP analysis on a solid phase medium can include providing an assembly of a solid phase medium and a reaction composition, such as any of the components or compositions listed herein, in combination with the solid phase medium, as shown in block 210. In one aspect, the method can include depositing a biological sample on the solid phase medium, as shown in block 220. In another aspect, the method can include heating the assembly to an isothermal temperature sufficient to promote the LAMP reaction, as shown in block 230.

[0114] In one embodiment, 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 embodiment, the biological sample can be saliva. In one embodiment, the method can include detecting a viral pathogen. In one embodiment, the viral pathogen can be a pathogen as disclosed herein. In another embodiment, the LAMP analysis can be reverse transcriptase LAMP (RT-LAMP).

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

[0116] In another example, a temperature sufficient to promote a LAMP reaction can be in the range of about 60° C. to about 70° C. In another example, an isothermal temperature can be within a range of temperatures that differ by less than 5 degrees Celsius.

[0117] In another embodiment, a system for performing a LAMP analysis can include a configuration as described in the present disclosure. In another aspect, the system can include a solid phase medium onto which the composition is deposited.

[0118] Maximizing pH-sensitive signal output When performing a LAMP reaction, various indicators can be used to read the reaction results. Three types of colorimetric indicators include magnesium colorimetric indicators, pH colorimetric indicators, and DNA intercalating colorimetric indicators. Because magnesium can be a cofactor for DNA polymerase and its concentration must be tightly controlled, magnesium-based indicators can face various limitations when used in the context of a LAMP reaction. DNA intercalating indicators can also face limitations due to the number of variables involved. All three indicators can be used in a LAMP reaction, although pH-based indicators may be affected by fewer variables.

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

[0120] The choice of pH-sensitive dye can depend on various factors, such as the colorimetric range relative to pH, the degree of contrast between the color changes, the pH level at which the color changes, the uniformity of the color change, and the reproducibility of the color change. For example, phenol red can have a colorimetric range between about pH 6.8 and about pH 7.4. Below a pH of about 6.8, phenol red can change to yellow, and above a pH of about 7.4, phenol red can change to red. The degree of difference between the yellow and red colors can be easily read, and pH changes can occur at pH levels that mimic physiological conditions.

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

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

[0123] In one example, the non-interfering LAMP reagents may be substantially free of magnesium, ammonium sulfate, or ammonium carbonate. Magnesium, as a cofactor for DNA polymerase, should be closely monitored to ensure that the LAMP reaction can proceed as designed. Ammonium sulfate can be ionized to ammonium ions, leaving sulfate ions that can react to form sulfuric acid. Ammonium carbonate can also be ionized to ammonium ions, leaving carbonate ions that can react to form carbonic acid. Therefore, the non-interfering LAMP reagents must be substantially free of these substances.

[0124] Volatile agents should be minimized because they can leave compositions that can react to form acids or bases that can interfere with the pH-dependent signal from the LAMP reaction. In one example, the non-interfering LAMP reagents can be substantially free of volatile reagents, including, but not limited to, ammonium sulfate, ammonium carbonate, and the like, or combinations thereof. In one aspect, the composition can contain less than one or more of the following amounts of volatile reagents: 1.0 wt %, 0.5 wt %, 0.1 wt %, or 0.01 wt %.

[0125] Furthermore, any pH-interfering reagents may interfere with the pH-dependent signal output if they are not compensated for. In one example, the plurality of non-interfering LAMP reagents may be substantially free of pH-interfering reagents, including, but not limited to, numerous acids, bases, and combinations thereof. In one aspect, the composition may contain less than one or more of 1.0 wt %, 0.5 wt %, 0.1 wt %, or 0.01 wt % of pH-interfering reagents.

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

[0127] Even if the pH is monitored and the LAMP reaction is functioning properly, discoloration of the solid-phase medium may occur due to other factors, such as long-term storage. In one embodiment, the composition may include a non-discoloring additive. In one example, the non-discoloring additive may include one or more of a sugar, a buffer, a blocking agent, etc., or a combination thereof. In one example, the sugar may stabilize the solid-based medium and prevent discoloration under long-term storage conditions. In one embodiment, the sugar may include one or more of glucose, sucrose, trehalose, dextran, etc., or a combination thereof.

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

[0129] The buffer can promote stabilization of the LAMP reaction by removing variability from the saliva sample. In one example, the buffer can include one or more of phosphate-buffered saline (PBS), Dulbecco's PBS, Alsever's solution, Tris-buffered saline (TBS), HEPES, BICINE, water, buffered saline (BSS), such as Hank's BSS, Earle's BSS, Gray's BSS, Puck's BSS, Simm's BSS, Tyrode's BSS, BSS Plus, lactated Ringer's solution, saline (i.e., 0.9% saline), half-normal saline, or a combination thereof. In one embodiment, the buffer concentration can be about 10 μM to about 20 mM when used on a solid-phase medium. In another example, the buffer concentration can be about 100 μM to about 10 mM when used on a solid-phase medium. In yet another example, the concentration of the buffer can be from about 100 μM to about 500 μM when used on a solid phase medium.

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

[0131] Antioxidants can enhance the uniformity and contrast of pH-dependent signals on solid phase media by eliminating variables associated with oxidation reactions. In one example, the composition can further comprise an antioxidant as disclosed herein.

[0132] In another example, the composition can further comprise a solid phase medium, including, but not limited to, one or more of glass fiber, nylon, cellulose, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, polyester, hydrophilic polytetrafluoroethylene (PTFE), or combinations thereof.

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

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

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

[0136] 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 a combination thereof. In another embodiment, the biological sample can be saliva. In one aspect, the method can include detecting a viral pathogen. In one aspect, the viral pathogen can be a pathogen otherwise disclosed herein.

[0137] In one example, a temperature sufficient to promote a LAMP reaction can be in the range of about 60° C. to about 70° C. In another example, an isothermal temperature can be within a range that differs by less than 5 degrees Celsius.

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

[0139] 3, a method 300 for maximizing accuracy of an output signal in a pH-dependent LAMP analysis can include providing a reagent mixture that minimizes discoloration from the signal output medium due to non-LAMP reactions, as shown in block 310. The method can further include performing a LAMP reaction, as shown in block 320. In one aspect, the method can include controlling proton production due to the non-LAMP reaction. In another aspect, the method can include controlling oxidation due to the non-LAMP reaction.

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

[0141] In another embodiment, a method for maximizing the level of detection (LOD) in a pH-dependent LAMP assay can include substantially eliminating discoloration caused by non-LAMP reactions from the signal output medium. In one aspect, diluting saliva with 5-10% water can enhance color contrast and reduce sample variability without affecting the detection limit. In another example, filtering saliva with a filter as disclosed elsewhere herein can enhance color contrast and reduce sample variability without affecting the detection limit. [Example]

[0142] The following examples are provided to facilitate a clearer understanding of certain embodiments of the present invention and are not meant to be limiting thereof in any way.

[0143] Paper LAMP assay for viral targets in diluted saliva samples Example 1 - DNase / RNase-free distilled water DNase / RNase-free distilled water is prepared by filtering through a 0.1 μm membrane and tested for DNase and RNase activity. DNase and RNase activity are tested according to the current United States Pharmacopeia (USP) monograph test criteria for Water for Injection (WFI). If no DNase, RNase, or protease activity is found, the water is considered contaminant-free and is used to prepare saliva samples.

[0144] Example 2 - Amplification in saliva As shown in Figure 4, nucleic acid sequence primers were designed to target RNaseP in saliva as a positive control to confirm nucleotide amplification from saliva samples. Figure 4A shows the results of fluorometric RT-qLAMP of a primer set targeting RNaseP POP7 in 18% saliva spiked with 10 genome equivalents / reaction of heat-inactivated SARS-CoV-2. Figure 4B shows the results of fluorometric RT-qLAMP of a primer set targeting RNaseP POP7 in water containing 0.2 ng of synthetic RNaseP POP7 RNA.

[0145] As shown in Figure 4A, 18% saliva spiked with 10 genome-equivalents of heat-inactivated SARS-CoV-2 per reaction was analyzed. In the left panel, primers (RNaseP.I) designed to target RNaseP using the mRNA sequence of the POP7 gene, encoding the p20 subunit of RNaseP, did not adequately detect low levels of RNaseP, resulting in no amplification. In the middle panel, primers (RNaseP.II) were able to detect RNaseP levels without amplifying the no-template control, resulting in amplification, as indicated by the blue line without overlapping with the black line. In the right panel, primers (RNaseP.III) dimerized, resulting in amplification in both the black and blue lines (e.g., amplification was indicated by the black line in the no-template control).

[0146] As shown in Figure 4B, water containing 0.2 ng of synthetic RNaseP POP7 RNA was analyzed. In the left panel, primer (RNaseP.I) dimerized, resulting in amplification at the blue and black lines (e.g., amplifying the no-template control). In the middle panel, primer (RNaseP.II) was able to detect RNase P levels without amplifying the no-template control, resulting in amplification as shown by the blue line without overlapping with the black line. In the right panel, primer (RNaseP.III) amplified RNase P without amplifying the no-template control, resulting in amplification at the blue line but not the black line.

[0147] Example 3 - Saliva collection device Depending on the type of saliva collection device, saliva samples can be easily obtained for LAMP reactions. In some cases, operators may use protective equipment to protect against pathogens (e.g., aerosol viruses) that may be spread through droplets. Therefore, operators can wear personal protective equipment to protect against accidental contact with aerosol viruses. Certain saliva collection devices can be self-administered by subjects under the guidance of a medical professional. Saliva collection devices have proven their effectiveness and can be divided into two categories: sponge-based collection and saliva collection, as shown in Figures 5A and 5B.

[0148] The sponge collection device 500a uses a sponge-like collection pad 504 to absorb saliva and includes a sample-volume adequacy indicator 512 to indicate when a sufficient amount has been collected. Once saturated, the sponge is inserted into a compression tube 506 and compressed against a filter, straining the saliva into the collection tube. This filtering step is performed to filter out mucins and high-molecular-weight proteins from the saliva and significantly reduce the viscosity of the sample. As a result, the solid phase medium can capture and distribute the saliva more quickly, uniformly, and reliably. The sponge collection device 500a can 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 adequacy indicator 512 to identify when sufficient saliva has been collected.

[0149] The saliva collection device 500b can provide unfiltered saliva with a viscosity that slows the absorption and dispersion of the sample. The saliva collection device 500b can include a collection funnel 522 for collecting saliva, an indicator line 528 that indicates when sufficient saliva has been collected, a collection tube 524 for collecting saliva, a tube cap 526, a volume indicator 530, and a tube cap holder 532.

[0150] With both types of collection devices, the residual risk of exposure to the operator is minimal. With sponge-based devices, there is a hypothetical risk of aerosols being released during the compression maneuver, especially if the user inadvertently performs the compression process. To control the risk of exposure, this maneuver may be performed by the medical operator. The collection device may have a compression seal to prevent backflow of aerosols. With saliva collection, there is a slight risk of the outside of the device becoming contaminated with airborne saliva, which may cause cross-contamination for the operator if not handled properly. In both cases, exposure risk is reduced by the patient self-collecting the saliva sample.

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

[0152] Saliva pH was measured from the processed saliva samples, and subsequent colorimetric and fluorometric RT-LAMP LOD assays were performed using the processed saliva samples. The data are shown in Figure 6A. The data demonstrate the LOD in saliva processed using various saliva collection devices (Pure-Sal, Super-Sal, and Stat-sure). The master mix was treated with 0.6 microliters of HCl. The Pure·SAL™ and Super·SAL saliva collection devices exhibit a broader colorimetric response across a wider range of concentrations (1 to 10,000 genome equivalents per reaction of heat-inactivated SARS-CoV-2).

[0153] Example 4 - Saliva Collection Process When a subject self-tests, under the guidance of a medical professional, the subject collects a saliva specimen in a dedicated collection container that contains no additives and is therefore safe for the subject to use for collection. The amount of saliva collected is approximately 100 μL. For example, a sponge sampler is inserted into the subject's mouth, and saliva is collected until the sponge sampler's indicator changes color. The sponge sampler is then inserted into a collection tube. The sponge is then compressed to squeeze out saliva (approximately 100 μL) into a collection tube containing a quantity of water to dilute the saliva. The saliva is diluted with water until the saliva-to-water ratio is approximately 1:1 to approximately 1:20. The saliva is then transferred from the collection tube to the testing site.

[0154] Example 5 - Effect 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 reaction.

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

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

[0157] As shown in Figure 6B, RNAsecure™ did not significantly increase the LoD of the reaction, i.e., the addition of an RNase inhibitor did not significantly increase the measured parameters of the RT-LAMP reaction (e.g., reaction rate, false positive rate, or detection limit).

[0158] Example 6 - Frozen saliva samples In some cases, due to logistics, transportation needs, etc., it may be desirable to freeze saliva samples for a period of time prior to analysis. Such situations may merit special attention when performing the LAMP assay described herein. As shown in Figure 7, the pH of frozen saliva samples can vary depending on the number of days at -20°C before the saliva sample is thawed and tested. In one example, the pH of saliva samples from Donor 1 varied from pH 7.21 without any time between collection and testing to pH 7.46 six days after collection / freezing and testing. In another example, the pH of saliva samples from Donor 2 varied from pH 7.00 without any time between collection and testing to pH 6.98 six days after collection / freezing and testing. In one example, the pH of saliva samples from Donor 3 varied from pH 7.18 without any time between collection and testing to pH 7.18 six days after collection / freezing and testing. In one example, the pH of saliva samples from donor 4 varied from pH 7.35 with no days between collection and testing to pH 7.47 after 6 days between collection / freezing and testing. In one example, the pH of saliva samples from donor 1 varied from pH 7.22 with no days between collection and testing to pH 7.24 after 6 days between collection / freezing and testing.

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

[0160] Therefore, we found that diluting saliva to 25% with nuclease-free water and further diluting it to a final concentration of 5% saliva upon addition to the RT-LAMP reaction yielded results within 60 minutes. Dilution reduces the buffering capacity of saliva and reduces the concentration of inhibitory components, both of which delay the colorimetric report. Dilution is less complicated for end users than other pretreatment procedures found in various studies to inactivate inhibitory components in saliva, such as pretreatment with proteases, Chelex® 100, or RNA extraction procedures.

[0161] The LoD of the colorimetric assay in 5% saliva processed using Pure·SAL™ was 1000 copies / reaction (25 μL reaction volume), which corresponds to 800 copies / μL of patient saliva after accounting for dilution (Figure 6C).

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

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

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

[0165] 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 to obtain 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 the diluted heat-inactivated virus to obtain the same 25 μL reaction volume. Primer set orflab.2 was used. Heating was performed at 65°C for 60 minutes in an incubator (Fisherbrand™ Isotemp™). Colorimetric scans were acquired using a flatbed scanner before and after the RT-LAMP reaction. 1250 μL of NEB colorimetric master mix was supplemented with 0.5 μL of Antarctic Thermolabile UDG, 3.5 μL of dUTP, and carrier DNA to obtain the indicated final reaction concentrations.

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

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

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

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

[0170] Example 9 - Detection limits on paper Figure 10 shows the limit of detection on paper. 20 μL of RT-LAMP reagents were added to Grade 1 chromatography paper. Heat-inactivated SARS-CoV-2 was spiked into 100% pooled saliva using serial dilutions of the virus. 15 μL of approximately 100% saliva was added to each paper strip. After 90 minutes of incubation at 65°C, a color change occurred. The LOD of the primers was approximately 3,000 copies / reaction in a 15 μL volume, which corresponds to approximately 20,000 copies / mL of saliva.

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

[0172] Example 11 - Buffer Selection and Concentration Because saliva pH can vary from sample to sample, a buffer solution was used in the paper-based device to maintain a consistent starting pH. For phenol red, pH 7.6 was a good starting point to promote the colorimetric transition, as shown in Figure 11. Because the starting pH of 7.6 was near the end of the buffer range where a color change was possible when amplification occurred, several buffers with pKa values ​​of approximately 8 were screened. As shown in Figure 12, a 10 mM BICINE buffer was used for the paper-based assay.

[0173] Example 12 - Effect of primers on reaction rate To increase the rate of the RT-LAMP reaction, we investigated the inclusion of multiple primer sets in the RT-LAMP fluorescent reaction mixture. The study was conducted in water using NEB LAMP fluorescent dye as the fluorescent indicator. The inclusion of multiple primer sets did not appear to significantly improve the reaction rate. Rather, the reaction proceeded primarily at the rate of the primer set with the fastest reaction time when used alone.

[0174] Example 13 - Sample LAMP Protocol, Reagents, Validation, and Troubleshooting Sample Lamp Protocol 13-A: Primer Mix 1. Get all six diluted primers from the freezer; 2. Mix 80 μl FIP, 80 μl BIP, 20 μl FB, 20 μl LB, 10 μl F3, and 10 μl B3 in a tube; 3. Add enough PCR-grade water to make 500 μl.

[0175] LAMP 1. Obtain the NEB Bst 2.0 Warmstart Kit and Primer Mix; 2. While the reagents are thawing, spray with DNAway for at least 5 minutes, then wipe the surface with a Kimwipe; 3. Label all required PCR tubes with the DNA sample and primers you will be using. Be sure to include a negative control with no DNA added; 4. Add 5 μl of PCR-grade water (or dye), 12.5 μl of the NEB Bst 2.0 Warmstart Kit, and 2.5 μl of the Primer Mix per reaction. Master mixes can be created depending on the number of reactions to be performed; 5. If adding 5 μl of EBT dye, the concentration should be adjusted to 1500 μM, for a final concentration of 300 μM; 6. For reactions without DNA, add an additional 5 μl of PCR-grade water and do not reopen until needed to load the gel; 7. When ready, place the PCR tubes in the PCR tray previously left in the pass-through chamber and transfer to BRK2037; 8. Once in BRK2037, obtain the sample DNA from the -20°C freezer; 9. Spray your hands with DNAway spray and rub your hands around the DNA sample tubes to ensure they are also covered; 10. Add 5 μl of DNA sample if necessary and close the tubes. Never open two DNA tubes at the same time or close the PCR tubes immediately after adding the DNA; 11. Place the samples in a thermocycler set to 65°C for 1 hour and 80°C for 5 minutes (after this procedure, samples can be kept at -20°C overnight).

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

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

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

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

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

[0181] Disinfection: Spray RNase AWAY on pipettes and all work surface (PCR hood) surfaces. Wipe off thoroughly after applying RNase AWAY. Residual RNase AWAY may interfere with the reaction. To prevent cross-contamination, use separate rooms for manufacturing paper-based devices and loading samples. Pre-cut chromatography paper to 5mm x 6mm.

[0182] Preparation of LAMP: 1. In a PCR hood, prepare the 2x LAMP mix as shown in Table 13B-1. 2. Adjust the pH to approximately 7.5-8.0 (red, but not pink) with 1M KOH (approximately 1-2 μL). It does not need to be exact. After adjusting the pH, the 2x LAMP mix can be stored at -20°C. [Table 3]

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

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

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

[0186] Imaging and incubation: NOTE: Multiple imaging methods (e.g., time-lapse video, scanning) and heat sources (e.g., incubators, water baths) exist. A benchtop scanner and a microbial incubator can be used in this protocol. 1. Place the pad on top of the scanner. Scan the pad before the reaction (0 min). 2. Preheat the incubator to 65 °C. 3. Place the pad in the incubator. Separate the pad. Uniformity of heating can affect the consistency of results. 4. Remove the pad and repeat the scan at different time points (typically every 30 min). 5. After the final scan, discard the reaction pad in a biohazard waste container.

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

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

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

[0190] However, BSA may also cause pH fluctuations in the device. Figures 13A and 14B show that after incubation (60 min), the negative paper pad containing BSA has yellowish edges. After elution, the eluate was run on gel electrophoresis, and no DNA product was visible on the gel, as shown in Figure 13B, indicating that the yellow color at the edges is not due to off-target amplification or contamination. If BSA is distributed unevenly, applying heat may cause the edges to turn yellow.

[0191] troubleshooting: Abnormal pink color on the paper pad: During the process of preparing the LAMP paper pad, abnormal pink spots different from the surrounding color may be present. This may be caused by residual RNase AWAY, either sprayed directly onto the pad and / or transferred via tweezers. RNase AWAY can degrade added RNA / DNA templates. If this occurs, thoroughly dry all instruments and surfaces, cut a new 5 x 6 mm paper pad, and resume the "LAMP Preparation" section from step 5.

[0192] Reagent overflow after pad reconstitution: During the sample loading procedure, the pad may not be able to absorb the entire sample volume added to it for reconstitution. The template concentration may not be accurately represented by an overflowing pad. Insufficient pad drying can cause overflow. In such cases, try the following: 1) drying for a longer period of time; 2) using improved drying methods such as heat drying (place in a clean microbial incubator at 37°C; do not set the temperature higher than 45°C to prevent activation of Bst 2.0 WarmStart® Polymerase) or convection drying (use a small fan to increase airflow during drying); or 3) reducing the reconstitution volume to 20 μL.

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

[0194] Sample pH and buffer capacity affect colorimetric readings: Because phenol red is a pH indicator, sample pH and its buffer capacity can significantly affect the assay. The reagent composition presented herein has been confirmed to work at saliva concentrations of 5–10% volume / volume (diluted with water). 5% saliva was chosen because it provides fast response times and consistent results. Human saliva contains a sophisticated buffering system, including bicarbonate, phosphate, and proteins, which prevents pH changes (and therefore color changes) at high saliva concentrations. Testing of a paper LAMP device using nasal swabs resuspended in water showed no inhibition by the sample matrix. Colorimetric readings can also be hindered by buffered salt solutions (e.g., transport media).

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

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

[0197] Example 16 Fluorescent reporters require an additional ultraviolet (UV) light source for reading without specialized equipment. However, colorimetric assays using phenol red as an indicator do not use UV light and can be read with the naked eye. DNA polymerization generates protons, and phenol red responds to pH. Diluted saliva (5% final concentration) was used to measure changes in pH by overcoming the buffering capacity of saliva. Diluting saliva to a final concentration of 5% also reduced the concentration of interfering substances (e.g., RNase).

[0198] Incorporation of carrier DNA and guanidine hydrochloride also enhanced the LoD, resulting in comparable colorimetric responses in water and saliva. The mechanism by which carrier DNA enhanced LAMP results was unclear. This mechanism was investigated using the NEB 1 kb DNA ladder (NEB-N3232L). Different concentrations of carrier DNA (0.3 ng / μl and 0.75 ng / μl) were used to study their effect on the 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 tested without carrier DNA. The effect of carrier DNA, along with guanidine chloride, on heat-inactivated SARS-CoV-2 at concentrations ranging from 1,000 copies to 62.5 copies per reaction was tested using untreated saliva (5%) at pH 6.5 (Figure 6D).

[0199] Guanidine hydrochloride has been reported to increase the sensitivity of LAMP. Performance using our primer set was tested using NEB Warmstart™ colorimetric master mix at pH 7.6 with 40 mM guanidine hydrochloride. The effect of guanidine chloride was tested on heat-inactivated SARS-CoV-2 at concentrations ranging from 1,000 copies to 62.5 copies per reaction using pooled saliva (5%) at pH 6.5. This same composition was tested without guanidine chloride as a control. Guanidine chloride increased the sensitivity of replication, with consistent amplification across replicates (Figure 6E).

[0200] Because phenol red and fluorescent dyes have different mechanisms for reporting LAMP-based nucleic acid amplification, these differences in signal measurement over time were investigated. Reactions were prepared in FrameStar 96-well skirted optical bottom plates containing Warmstart™ colorimetric LAMP 2x master mix and a combination of LAMP fluorescent dyes, sealed with Thermo Scientific™ Adhesive Plate Seals, and placed in a Clariostar™ Plus microplate reader (BMG) for incubation and measurement of absorbance and fluorescence intensity. Color change over time was measured using A 432nm / A 560nmThe absorbance values ​​were expressed as a ratio of A 432nm and A 560nm A 620nm Baseline correction was performed by subtracting

[0201] Based on Figure 16, the color change in the reaction occurred later than the fluorescence change. Colorimetric and fluorometric data were collected using a BMG CLARIOstar® Plus plate reader. The reaction base mix consisted of NEB 2x colorimetric LAMP master mix, 2.5 μL of primer mix, and 5 μL of a 1:100 dilution of NEB LAMP fluorescent dye (NEB B1700A). The plate reader chamber temperature was equilibrated to 65°C before inserting the plate. Five μL of heat-inactivated virus diluted in water was added to the reaction base mix to obtain the final reaction concentrations shown (positive reaction). For NTC reactions, 5 μL of nuclease-free water was added to the reaction base mix. This difference in change suggests that the pH-based reporter responds more slowly to LAMP-based DNA amplification than the fluorescent reporter.

[0202] Example 17 Although the paper can be scaled up to millions of devices, placing the RT-LAMP reagents on the paper changed color even when no amplification occurred and a negative control was used. One possibility for this color change is heat-induced oxidation of the cellulose and the oxidizing properties of ammonium sulfate present in the RT-LAMP mixture. Another possibility for this color change is acidification of the reagents due to degassing of ammonia from the RT-LAMP mixture. Removing ammonium sulfate maintained the color of the negative control. Even with increasing concentrations of phenol red, which acts as an antioxidant, the color of the negative control was maintained, as shown in Figure 11.

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

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

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

[0206] For DNA intercalating dyes, crystal violet (CAS#548-62-9) was screened.

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

[0208] Figure 17A shows the colorimetric response of calmagite at various concentrations across LAMP reactions using genomic DNA as a template. LAMP detection was performed with increasing concentrations of calmagite (a magnesium indicator). The lolB.3 primer set targeting Histophilus somni genomic DNA was used. For positive reactions, 5 μL of HS gDNA was spiked at a concentration of 0.2 ng / μL. For negative reactions, 5 μL of nuclease-free water was used. 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 either the above template (positive reaction) or water (negative reaction), and 5 μL of calmagite prepared with water to achieve the final concentrations shown.

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

[0210] LAMP detection was performed with increasing concentrations of Eriochrome™ Black T (magnesium indicator) as shown in Figure 17B. The lolB.3 primer set targeting Histophilus somni genomic DNA (gDNA) was used. For positive reactions, 5 μL of HS gDNA was spiked at a concentration of 0.2 ng / μL. For negative reactions, 5 μL of nuclease-free water was used. 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 either the template (positive reaction) or water (negative reaction), as described above, and 5 μL of EBT adjusted with water to the final concentrations shown.

[0211] Furthermore, LAMP on paper using EBT did not produce a detectable color change. As shown in Figure 17C, LAMP detection was performed on chromatography paper in PCR tubes using increasing concentrations of Eriochrome™ Black T. The lolB.3 primer set targeting Histophilus somni genomic DNA was used. For positive reactions, 5 μL of H. somni gDNA was spiked at a concentration of 0.2 ng / μL. For negative reactions, 5 μL of nuclease-free water was used. 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 either the template (positive reaction) or water (negative reaction), as described above, and 5 μL of EBT (300 μM) prepared in nuclease-free water. For EBT reactions, the reaction consisted of 25 μL of EBT (300 μM) prepared in nuclease-free water.

[0212] By screening several different types of paper and confirming by gel electrophoresis that amplification was occurring on PES and polysulfone BTS 0.8, we were unable to detect a colorimetric change on any of the papers (Figures 17D and 17E).

[0213] As shown in Figure 17D, LAMP detection was performed on several papers: chromatography grade 1, anion-exchange nylon, cation-exchange nylon, polyethersulfone membrane, asymmetric submicron polysulfone (BTS 0.8), asymmetric submicron polysulfone (BTS 100), and hydroxylated nylon 1.2. b) Endpoint scan of the paper in panel a at 60 min, and c) gel electrophoresis (2% agarose) scan of the extracted LAMP products at 60 min. The lolB.3 primer set targeting Histophilus somni (HS) genomic DNA was used. For positive reactions, 5 μL of H. somni gDNA was spiked at a concentration of 0.2 ng / μL. For negative reactions, 5 μL of nuclease-free water was used. 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 either 5 μL of template (positive reaction) or water (negative reaction), and 5 μL of EBT (300 μM) prepared in nuclease-free water. Paper (as indicated) was placed in a PCR tube containing 25 μL of reaction, and the reaction was wicked up by the paper over the course of the reaction. After 60 minutes, the paper was removed and scanned. The gel was extracted using 30.

[0214] As shown in Figure 17E, results were obtained over time for a) PCR tubes, b) gel electrophoresis (2% agarose) of DNA extracted from the paper at 60 min, and c) scanning of the paper at 60 min. The lolB.3 primer set targeting Histophilus somni (HS) genomic DNA was used. For positive reactions, 5 μL of H. somni gDNA was spiked at a concentration of 0.2 ng / μL. For negative reactions, 5 μL of nuclease-free water was used. 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 either the template (positive reaction) or water (negative reaction), as described above, and 5 μL of EBT (300 μM) prepared in nuclease-free water. Biodyne A amphoteric paper was placed in the PCR tube containing the 25 μL reaction mixture, and the paper wicked up the reaction mixture throughout the course of the reaction. After 60 minutes, I took the paper out and scanned it.

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

[0216] As shown in Figure 17F, LAMP detection was performed using crystal violet, an intercalating dye, in solution, and a) gel electrophoresis (2% agarose) scans of the products at the relevant 60-minute time point. The lolB.3 primer set targeting H. somni gDNA was used. For positive reactions, 5 μL of H. somni gDNA was spiked at a concentration of 0.2 ng / μL. For negative reactions, 5 μL of nuclease-free water was used. 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 either the template (positive reaction) or water (negative reaction), as described above, and 5 μL of crystal violet prepared in nuclease-free water to achieve the indicated final concentrations. Paper was placed in a PCR tube containing 25 μL of the reaction mixture and wicked up over the course of the reaction. After 60 minutes, the paper was removed and scanned. Sodium sulfite and cyclodextrin were used to solubilize the crystal violet.

[0217] To confirm that amplification was occurring in the positive but not the negative reactions, the RT-LAMP solution was run in a 2% agarose gel. This showed that the positive reactions showed amplification at all CV concentrations tested, while the negative reactions showed no amplification at any CV concentration tested. Therefore, the color change in the negative reactions was caused by degradation of the LCV to the CV and not by binding of amplified DNA.

[0218] Further testing of the CV on paper with different concentrations of CV, SS, and BCD yielded results that were indistinguishable between negative and positive reactions. As shown in Figure 17G, endpoint colorimetric scanning was performed on paper for LAMP detection using the intercalating dye crystal violet (CV). The CV was solubilized using sodium sulfite (SS) and beta-cyclodextrin (BCD). The paper was loaded with 12.5 μL of NEB Warmstart™ 2x Master Mix, 2.5 μL of primer mix, and 5 μL of either the template (positive reaction) or water (negative reaction) described above, as well as 5 μL of CV prepared with nuclease-free water at the final concentrations indicated.

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

[0220] As shown in Figure 17H, RT-LAMP detection was performed using increasing concentrations of cresol red sodium salt, neutral red, phenol red sodium salt, m-cresol purple, and m-cresol purple sodium salt (pH indicator) in solution. The N.10 primer set targeting the SARS-CoV-2 N gene was used. For positive reactions, 5 μL of in vitro transcribed N gene RNA was spiked at a concentration of 0.2 ng / μL. For negative reactions, 5 μL of nuclease-free water was used. 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 either the above template (positive reaction) or water (negative reaction), and 5 μL of the indicated pH indicator at the indicated concentration in nuclease-free water. Reactions were performed in an incubator and scanned every 20 minutes using a flatbed scanner.

[0221] LAMP detection was performed with increasing concentrations of cresol red (a pH indicator) as shown in Figure 17I. The lolB.3 primer set targeting H. somni gDNA was used. For positive reactions, 5 μL of H. somni gDNA was spiked at a concentration of 0.2 ng / μL. For negative reactions, 5 μL of nuclease-free water was used. 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 cresol red for the final reaction concentrations shown, and 5 μL of either the template (positive reaction) or water (negative).

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

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

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

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

[0226] As a result, pH indicators with a color change around pH 6.5 showed the most consistent and most contrasting color changes (e.g., phenol red).

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

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

[0229] Adjusting the pH to 8.0 gave better color stability in the negative control, but a pH of 8.5 was too high for the standard and dry settings to produce a discernible color change after 120 minutes of incubation. If the pH was left unadjusted, the color changed even when the control was filled.

[0230] Example 20 - Effect of Trehalose and Tween 20 on RT-LAMP Colorimetric Response Figure 19B shows colorimetric RT-LAMP results with the inclusion of trehalose or Tween 20 at the indicated concentrations. The orflab.II primer set was used. Twenty microliters of RT-LAMP master mix containing the following basic formula was added to Grade 1 chromatography paper (5 mm × 20 mm): KCl (50 mM), MgSO (8 mM), equimolar dNTP mixture (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, where indicated), betaine (20 mM), BSA (40 mg / mL), and trehalose (10% w / v, where indicated). The mixture was then dried for 60 minutes in a PCR preparation hood. Final concentration of 1 x 10 per reaction in 25% treated saliva (positive reaction) or nuclease-free water (negative reaction) 5 A 25 μL copy of heat-inactivated SARS-CoV-2 was added to a dry reaction pad, which was heated in an incubator set at 65°C for 60 minutes and scanned using a flatbed scanner.

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

[0232] Exemplary Embodiments In one embodiment, a composition for loop-mediated isothermal amplification (LAMP) assays utilizing a pH-dependent output signal is provided, comprising a pH-sensitive dye and a plurality of non-interfering LAMP reagents.

[0233] In one example of a composition for a loop-mediated isothermal amplification (LAMP) assay utilizing a pH-dependent output signal, the pH-sensitive dye can be at least one of phenol red, phenolphthalein, azolithin, bromothymol blue, naphtholphthalein, cresol red, or a combination thereof.

[0234] In one example of a composition for a loop-mediated isothermal amplification (LAMP) assay utilizing a pH-dependent output signal, the plurality of non-interfering LAMP reagents can be substantially free of volatile reagents, pH-interfering reagents, magnesium-interfering reagents, or combinations thereof.

[0235] In one example of a composition for a loop-mediated isothermal amplification (LAMP) assay that utilizes a pH-dependent output signal, the plurality of non-interfering LAMP reagents can be substantially free of magnesium, ammonium sulfate, or ammonium carbonate.

[0236] In one example of a composition for a loop-mediated isothermal amplification (LAMP) assay that utilizes a pH-dependent output signal, the plurality of non-interfering LAMP reagents can include a DNA polymerase, a reverse transcriptase, a target primer, or a combination thereof.

[0237] In one example of a composition for use in a loop-mediated isothermal amplification (LAMP) assay that utilizes a pH-dependent output signal, the composition can further include an antioxidant.

[0238] In one example of a composition for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal, the composition can further include carrier RNA, carrier DNA, an RNAase inhibitor, a DNAase inhibitor, guanidine hydrochloride, or a combination thereof.

[0239] In one example of a composition for a loop-mediated isothermal amplification (LAMP) assay that utilizes a pH-dependent output signal, the LAMP assay can be a reverse transcription LAMP (RT-LAMP).

[0240] In one example of a composition for loop-mediated isothermal amplification (LAMP) analysis that utilizes a pH-dependent output signal, the composition can further include a solid phase medium.

[0241] In one example of a composition for a loop-mediated isothermal amplification (LAMP) assay that utilizes a pH-dependent output signal, the composition can further include a non-color changing additive including a sugar, a buffer, a blocking agent, or a combination thereof.

[0242] In one example of a composition for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal, the composition can further include sugars including one or more of trehalose, glucose, sucrose, dextran, or combinations thereof.

[0243] In one example of a composition for a loop-mediated isothermal amplification (LAMP) assay that utilizes a pH-dependent output signal, the composition can further include a blocking agent comprising bovine serum albumin, casein, or a combination thereof.

[0244] In one example, a method is provided for performing a LAMP assay using a pH-dependent output signal, the method including or comprising providing an assembly of a solid phase medium and a composition described herein; depositing a biological sample on the solid phase medium; and heating the assembly to an isothermal temperature sufficient to promote the LAMP reaction.

[0245] In one example, a method of performing a LAMP analysis using a pH-dependent output signal, wherein the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or a combination thereof.

[0246] In one example, a method for performing a LAMP assay with a pH-dependent output signal, wherein the biological sample can be saliva.

[0247] In one example, a method of performing a LAMP assay using a pH-dependent output signal, the method can further include detecting a viral pathogen.

[0248] In one example, a method for performing a LAMP assay using a pH-dependent output signal, wherein the LAMP assay can be a reverse transcription LAMP (RT-LAMP).

[0249] In one example, a method is provided for maximizing the accuracy of an output signal in a pH-dependent LAMP assay, the method including or comprising providing a reagent mixture that minimizes discoloration caused by non-LAMP reactions from the signal output medium and performing a LAMP reaction.

[0250] In another example, a method for maximizing the accuracy of an output signal in a pH-dependent LAMP assay can include controlling the production of protons by non-LAMP reactions.

[0251] In one example, a method for maximizing the accuracy of an output signal in a pH-dependent LAMP assay, the method can include controlling oxidation due to non-LAMP reactions.

[0252] In one example, a method is provided for maximizing the accuracy of an output signal in a pH-dependent LAMP assay, the method including or comprising substantially removing discoloration caused by non-LAMP reactions from the signal output medium.

[0253] In one example, a method is provided for maximizing the level of detection (LOD) in a pH-dependent LAMP assay, which comprises substantially removing discoloration caused by non-LAMP reactions from a signal output medium.

[0254] It should be understood that the above methods are merely illustrative of some embodiments of the present invention. Numerous modifications and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the present invention, and the appended claims are intended to cover all such modifications and arrangements. Thus, while the present invention has been described with specificity and detail in connection with what are presently considered to be the most practical and preferred embodiments thereof, it will be apparent to those skilled in the art that variations can be incorporated and made without departing from the principles and concepts set forth herein.

Claims

1. 1. An assembly for a loop-mediated isothermal amplification (LAMP) assay utilizing a pH-dependent output signal for analyzing a biological sample selected from saliva or a nasal swab, comprising: Assemble a solid phase medium and a composition for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal; The composition comprises: pH-sensitive dyes, and Multiple non-interfering LAMP reagents Including, The plurality of non-interfering LAMP reagents at least one of a DNA polymerase, a reverse transcriptase, and a target primer; The magnesium interfering reagent contains less than 1.0% by weight of the composition in terms of magnesium content, and the magnesium interfering reagent contains Mg ２＋ , Mg １＋ , magnesium carbonate, magnesium chloride, magnesium citrate, magnesium hydroxide, magnesium oxide, magnesium sulfate, magnesium sulfate heptahydrate, or a combination thereof; and is free of volatile reagents selected from the group consisting of ammonium sulfate, ammonium carbonate, or combinations thereof; the composition further comprises a sugar comprising one or more of trehalose, glucose, sucrose, dextran, or a combination thereof; The assembly, wherein the pH-sensitive dye is at least one of phenol red, phenolphthalein, azolithmine, bromothymol blue, naphtholphthalein, cresol red, or a combination thereof.

2. The assembly of claim 1 further comprising an antioxidant.

3. 10. The assembly of claim 1, further comprising a carrier RNA, a carrier DNA, an RNAase inhibitor, a DNAase inhibitor, guanidine hydrochloride, or a combination thereof.

4. The assembly of claim 1, wherein the LAMP analysis is reverse transcription LAMP (RT-LAMP).

5. 10. The assembly of claim 1, further comprising a non-discoloring additive comprising a buffer, a blocking agent, or a combination thereof.

6. 10. The assembly of claim 1, further comprising a blocking agent comprising bovine serum albumin, casein, or a combination thereof.

7. 1. A method for performing a LAMP assay with a pH dependent output signal, comprising: providing the assembly of claim 1, comprising a solid phase medium and a composition for loop-mediated isothermal amplification (LAMP) analysis; depositing a biological sample onto the solid phase medium; and heating the assembly to an isothermal temperature sufficient to promote the LAMP reaction; The method comprising:

8. The method of claim 7 , wherein the biological sample is saliva.

9. 8. The method of claim 7, further comprising detecting a viral pathogen.

10. The method of claim 7, wherein the LAMP analysis is reverse transcription LAMP (RT-LAMP).

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