EP4277997A1 - Analyse d'amplification isotherme induite par boucle (lamp) pour cibles pathogènes - Google Patents

Analyse d'amplification isotherme induite par boucle (lamp) pour cibles pathogènes

Info

Publication number
EP4277997A1
EP4277997A1 EP22703179.6A EP22703179A EP4277997A1 EP 4277997 A1 EP4277997 A1 EP 4277997A1 EP 22703179 A EP22703179 A EP 22703179A EP 4277997 A1 EP4277997 A1 EP 4277997A1
Authority
EP
European Patent Office
Prior art keywords
lamp
saliva
composition
reaction
concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22703179.6A
Other languages
German (de)
English (en)
Inventor
Jordan SEVILLE
Darby MCCHESNEY
Jiangshan WANG
Murali Kannan MARUTHAMUTHU
Andres DEXTRE
Mohit Verma
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rtx Bbn Technologies Inc
Purdue Research Foundation
Original Assignee
Raytheon BBN Technologies Corp
Purdue Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Raytheon BBN Technologies Corp, Purdue Research Foundation filed Critical Raytheon BBN Technologies Corp
Publication of EP4277997A1 publication Critical patent/EP4277997A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/10—Nucleotidyl transfering
    • C12Q2521/101—DNA polymerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/10—Nucleotidyl transfering
    • C12Q2521/107—RNA dependent DNA polymerase,(i.e. reverse transcriptase)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/30—Oligonucleotides characterised by their secondary structure
    • C12Q2525/301—Hairpin oligonucleotides

Definitions

  • PCR Polymerase chain reaction
  • Quantitative PCR is an adaptation of PCR which allows monitoring of the amplification of a targeted nucleotide. Diagnostic qPCR has been applied to detect nucleotides that are indicative of infectious diseases, cancer, and genetic abnormalities.
  • Reverse transcription PCR is an adaptation of qPCR which allows detection of a target RNA nucleotides. Because of this ability, RT-PCR is well-suited for detecting virus pathogens. However, RT-PCR uses sizeable equipment which may not be available in certain point of care settings. Additionally, RT-PCR uses trained personnel, significant sample preparation, and time to perform and obtain results.
  • LAMP Loop-Mediated Isothermal Amplification
  • RT-LAMP Reverse-transcription LAMP
  • RT-LAMP Reverse-transcription LAMP
  • the present disclosure is drawn to technology (e.g., compositions, methods, systems, and assemblies) for use in detecting a target nucleotide using a LAMP analysis.
  • the target nucleotide can be known to reside in a pathogen of interest.
  • the LAMP analysis can be an RT-LAMP analysis.
  • methods of preparing saliva samples for loop-mediated isothermal amplification (LAMP) detection of a pathogen target are provided.
  • a method can include providing an amount of saliva from a test subject, and diluting the saliva in water to a degree that reduces a buffering capacity of the saliva while maintaining a sufficient concentration to allow for detection of the pathogen target.
  • the method can include reducing a viscosity of the sali va as compared to an original viscosity.
  • the viscosity can be reduced by one or more of dilution, filtering, or combinations thereof.
  • the viscosity can be reduced using filtering.
  • the viscosity can be reduced using a 10-micron filter.
  • the viscosity can be reduced to a degree that increases flowability through a solid phase medium as compared to an original viscosity.
  • the viscosity can be reduced to a range of from about 1 .0 centipoise (cP) to about 50 cP.
  • such a method can include filtering the saliva sample to a degree that adjusts a saliva sample pH to a test sample target range.
  • the test sample target range can be from about 7.2 to about 8.6.
  • the water can have a pH greater than 6.0 and can be substantially free of contaminants.
  • the saliva sample can consist essentially of saliva and water.
  • the saliva can be collected using sponge-based collection.
  • the saliva can be diluted in the water to a saliva to water ratio of about 1 : 1 to about 1:20. In another aspect, the saliva can be diluted in the water to a degree that provides the sample with an optical density at 600 nm (ODeoo) of less than 0.2. In a further aspect, the saliva has a volume from about 50 ( ul to about 100 pl. In yet another aspect, the saliva sample has a volume ranging from about 100 pl to about 1 nil. [0009] In an additional aspect, the pathogen target can comprise a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoa pathogen. In one aspect, the pathogen target can be a viral target.
  • the viral target can comprise a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a ds-DNA-RT virus.
  • the viral target can be H1N1, H2N2, H3N2, H 1 N 1 pdm09, or SARS-CoV-2.
  • the LAMP detection can comprise reverse transcription LAMP (RT- LAMP) detection.
  • RT- LAMP reverse transcription LAMP
  • test sample compositions for LAMP analysis can include: an amount of a test subject’s saliva that is sufficient to detect a pathogen target via a LAMP analysis in combination with an amount of water that reduces a buffering capacity of the saliva.
  • the composition can have a viscosity of from about 1.0 cP to about. 50 cP.
  • the composition can have a pH of from about 7.2 to about 8.6.
  • the composition can have a saliva to water ratio of about 1 : 1 to about 1 :20.
  • the composition can have an optical density at 600 nm (ODeoo) of less than 0.2.
  • the water can have a pH greater than 6,0 and can be substantially free of contaminants.
  • the composition can consist essentially of saliva and water.
  • the saliva can have a volume ranging from about 50 /.il to about 100 //I.
  • the saliva sample can have a volume of from about 100 ,ul to about 1 ml.
  • the pathogen target can comprise a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoa pathogen.
  • the pathogen target can be a viral target.
  • the viral target can comprise a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a ds-DNA-RT virus.
  • the viral target can comprise H1N1, H2.N2, H3N2, HlNlpdmO9, or SARS-CoV-2.
  • the buffering capacity of the composition can be less than 5 mM.
  • compositions for LAMP analysis on a solid phase medium can include one or more target primers, a DNA polymerase, and a re-solubilization agent.
  • a composition can be substantially free of non-pH sensitive agents capable of discoloring the solid phase medium.
  • the composition can include an antioxidant.
  • the composition can be substantially free of volatile agents.
  • the composition can be substantially free of hygroscopic agents.
  • the composition can further include reverse transcriptase,
  • the hygroscopic agents can absorb more than about 10 wt% when between about 40% and about 90% relative humidity (RH) at 25° C
  • the hygroscopic agents can include glycerol, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate, and combinations thereof.
  • the re-solubilization agent can be a surfactant.
  • the re-solubilization agent can comprise bovine serum albumin (BSA), casein, polysorbate 20, or combinations thereof.
  • the target primers can target a pathogen that can comprise a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoa pathogen.
  • the pathogen can be a viral pathogen.
  • the viral pathogen can comprise a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a ds-DNA-RT virus.
  • the viral pathogen can comprise H1N1, H2N2, H3N2, H1NlpdmO9, or SARS-CoV-2.
  • the composition can further comprise a non-discoloration additive.
  • the non-discoloration additive can comprise one or more of a sugar, a buffer, or combinations thereof.
  • the composition can further comprise an indicator.
  • a method for LAMP analysis on a solid phase medium can include providing an assembly of a solid phase medium and a composition as recited herein, depositing a biological sample onto the solid phase medium, and heating the assembly to an isothermal temperature sufficient to facilitate a LAMP reaction.
  • the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or combinations thereof.
  • the biological sample is saliva.
  • the LAMP analysis can be reverse transcriptase LAMP (RT-LAMP).
  • the method can further comprise detecting a viral pathogen.
  • a system for performing the LAMP analysis can comprise a composition as recited herein, and a solid phase medium on to which the composition is deposited.
  • compositions 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.
  • the LAMP analysis can be RT-LAMP.
  • the pH sensitive dye can be at least one of phenol red, phenolphthalein, azolitmin, bromothymol blue, naphtholphthalein, cresol red, or combinations thereof.
  • the plurality of non- interfering LAMP reagents can be substantially free of volatile reagents, pH- interfering reagents, magnesium-interfering reagents, or combinations thereof.
  • the plurality of non-interfering LAMP reagents can be substantially free of magnesium, ammonium sulfate, and ammonium carbonate.
  • the plurality of noninterfering LAMP reagents can comprise DNA polymerase, reverse transcriptase, target primers, or combinations thereof.
  • the composition can comprise an antioxidant.
  • the composition can further comprise carrier RNA, carrier DNA, RNase inhibitors, DNase inhibitors, guanidine hydrochloride, or combinations thereof.
  • the composition can further comprise a solid phase medium.
  • the composition can comprise a non-discoloration additive that can comprise a sugar, a buffer, a blocking agent, or combinations thereof.
  • a non-discoloration additive can comprise a sugar, a buffer, a blocking agent, or combinations thereof.
  • the sugar can comprise one or more of trehalose, glucose, sucrose, or combinations thereof.
  • the blocking agent can comprise bovine serum albumin, casein, or combinations thereof.
  • methods of performing a LAMP analysis with a pH- dependent output signal can include providing an assembly of a solid phase medium and a composition as recited herein, depositing a biological sample onto the solid phase medium, and heating the assembly to an isothermal temperature sufficient to facilitate a LAMP reaction.
  • the LAMP analysis can be RT-LAMP.
  • the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, and combinations thereof.
  • the biological sample can be saliva.
  • the method can further comprise detecting a viral pathogen.
  • methods of maximizing accuracy of an output signal in a pH-dependent LAMP analysis can comprise providing a reagent mixture that minimizes non-LAMP reaction produced discoloration from a signal output medium, and performing the LAMP reaction.
  • the method can comprise controlling production of protons from a non-LAMP reaction.
  • the method can comprise controlling oxidation from a non-LAMP reaction,
  • a method of maximizing accuracy of an output signal in a pH-dependent LAMP analysis can comprise substantially eliminating non-LAMP reaction produced discoloration from a signal output medium.
  • methods of maximizing a limit of detecti on (LOD) in a pH-dependent LAMP analysis can include substantially eliminating non-LAMP reaction produced discoloration from a signal output medium.
  • FIG. 1 depicts a method of preparing a saliva sample for loop-mediated isothermal amplification (LAMP) detection of a pathogen target in accordance with an example embodiment
  • FIG. 2 depicts a method for LAMP analysis m accordance with an example embodiment
  • FIG. 3 depicts a method of maximizing accuracy of an output signal in a pH-dependent LAMP analysis in accordance with an example embodiment
  • FIG. 4 illustrates that loop-mediate isothermal amplification (LAMP) can be obtained in a saliva sample in accordance with an example embodiment
  • FIG. 5 A illustrates a sponge-based collection device in accordance with an example embodiment
  • FIG. 5B illustrates a passive drool collection device in accordance with an example embodiment
  • FIG. 6A illustrates the limit of detection for various concentrations of sample and various collection devices in accordance with an example embodiment
  • FIG. 6B illustrates the effect of RNase inhibitors for various concentrations of template on the RT-LAMP colorimetric response in accordance with an example embodiment
  • FIG. 6C illustrates the effect of the saliva processing technique on the colorimetric LoD in accordance with an example embodiment
  • FIG. 6D illustrates the effect of carrier DNA concentration on colorimetric RT-LAMP response in accordance with an example embodiment
  • FIG. 6E illustrates the effect of Guanidine HCl on RT-LAMP colorimetric response in accordance with an example embodiment
  • FIG. 6F illustrates the effect of UDG on end-point RT-LAMP colorimetric response response in accordance with an example embodiment
  • FIG. 6G illustrates the effect of saliva processing on colorimetric response in accordance with an example embodiment
  • FIG. 7 is a chart illustrating the stability of frozen saliva samples in accordance with an example embodiment
  • FIG. 8 illustrates the limit of detection of fresh saliva in accordance with an example embodiment
  • FIG. 9 illustrates the limit of detection in a bovine nasal swab in accordance with an example embodiment
  • FIG. 10 illustrates the limit of detection on paper in accordance with an example embodiment
  • FIG. 11 illustrates the colonmetric transition for phenol red in accordance with an example embodiment
  • FIG. 12 illustrates buffer used for the paper-based assay in accordance with an example embodiment
  • FIG. 13 A illustrates paper LAMP validation in accordance with an example embodiment
  • FIG. 13B illustrates paper LAMP validation in accordance with an example embodiment
  • FIG. 14A illustrates low template concentration LAMP on paper at a 0-minute time point in accordance with an example embodiment
  • FIG. 14B illustrates low template concentration LAMP on paper at a 60-minute time point in accordance with an example embodiment
  • FIG. 15 illustrates whole untreated saliva with heat inactivated SARS-CoV-2 virus in accordance with an example embodiment
  • FIG. 16 illustrates a comparison of colorimetric and fluorometric RT-LAMP responses in accordance with an example embodiment
  • FIG. 17A illustrates use of calmagite as a LAMP colorimetric indicator in accordance with an example embodiment
  • FIG. 17B illustrates use of EBT as a LAMP indicator in accordance with an example embodiment
  • FIG. 17C illustrates LAMP on chromatography paper using EBT as a colorimetric reporter in accordance with an example embodiment
  • FIG. 17D illustrates colorimetric response of LAMP on various papers using EBT as an indicator in accordance with an example embodiment
  • FIG. 17E illustrates LAMP detection on biodyne A amphoteric paper using EBT as a colorimetric indicator in accordance with an example embodiment
  • FIG. 17F illustrates the effect of crystal violet concentration on LAMP colorimetric response in accordance with an example embodiment
  • FIG. 17G illustrates colorimetric LAMP using crystal violet at various concentration on paper in accordance with an example embodiment
  • FIG. 17H illustrates pH indicators as colorimetric reporters for RT-LAMP in accordance with an example embodiment
  • FIG. 171 illustrates the effect of cresol red concentration on colorimetric response of LAMP reaction in accordance with an example embodiment
  • FIG. 17 J the effect of concentration of various pH indicators on colorimetric response for RT- LAMP reaction in accordance with an example embodiment
  • FIG. 17L the effect of initial pH on RT-LAMP colorimetric response using Phenol red in accordance with an example embodiment
  • FIG. 18 illustrates the color stability of the drying process in accordance with an example embodiment
  • FIG. 19A illustrates the effect of elimination of single reactant on initial color of paper after drying in accordance with an example embodiment
  • FIG. 19B illustrates the effect of trehalose and Tween 20 on RT-LAMP colorimetric response in accordance with an example embodiment
  • formulation and “composition” are used interchangeably and refer to a mixture of two or more compounds, elements, or molecules. In some aspects, the terms “formulation” and “composition” may be used to refer to a mixture of one or more active agents with a carrier or other excipients.
  • soluble is a measure or characteristic of a substance or agent with regards to its ability to dissolve in a given solvent.
  • the solubility of a substance or agent in a particular component of the composition refers to the amount of the substance or agent dissolved to form a visibly clear solution at a specified temperature such as about 25°C or about 37°C.
  • lipophilic refers to compounds that are not freely soluble in water. Conversely, the term “hydrophilic” refers to compounds that are soluble in water.
  • a “subject” refers to an animal.
  • the animal may be a mammal.
  • the mammal may be a human.
  • non-liquid when used to refer to the state of a composition disclosed herein refers to the physical state of the composition as being a semi-solid or solid.
  • solid and “semi-solid” refers to the physical state of a composition that supports its own weight at standard temperature and pressure and has adequate viscosity or structure to not freely flow. Semi-solid materials may conform to the shape of a container under applied pressure.
  • a “solid phase medium,” “solid phase base” “solid phase substrate” “solid phase test substrate” “solid phase testing substrate,” and the like refer to a non-liquid medium, device, system, or environment.
  • the non-liquid medium may be substantially free of liquid or entirely free of liquid.
  • the non-liquid medium can comprise or be a porous material or a material with a porous surface.
  • the non-liquid medium can comprise or be a fibrous material or a material with a fibrous surface.
  • the non-liquid medium can be a paper.
  • a “non-discoloration additive” refers to an additive that minimizes or prevents a color change in the color of the solid phase medium from an original or starting color to a different color for reasons other than nucleotide amplification from a LAMP reaction taking place thereon or therein. For example, in one embodiment, such a color change can be minimized or reduced as compared to a color change that would take place without the nondiscoloration additive present.
  • non-LAMP reaction produced discoloration refers to any discoloration (e.g., change in color from an original color to another color) of the solid phase medium which is not the result of a nucleotide amplification from a LAMP reaction.
  • non- LAMP reaction produced discoloration can refer to discoloration of the solid phase medium resulting from one or more of: a volatile agent, a magnesium-interfering agent, an oxidizing agent, a pH change resulting from causes other than amplification from a LAMP reaction, drying, or combinations thereof.
  • a “volatile agent” refers to an agent that includes a composition that has a high vapor pressure or a low' boiling point.
  • ammonium sulfate can be a volatile agent because the ammonia can volatilize and leave behind sulfuric acid.
  • a composition, component, or element can have a high vapor pressure when the composition is in a gas phase at a temperature of more than about 30 °C.
  • a composition can have a low boiling point when the composition forms is in a gas phase at a temperature of less than about 80 °C
  • 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.
  • the ammonium ion can volatilize from ammonium sulfate, and the sulfate ion can react to form sulfuric acid and affect the pH of the reaction in the absence of amplification from the LAMP reaction.
  • comparative terms such as “increased,” “decreased,” “beter,” “worse,” “higher,” “lower,” “enhanced,” “maximized,” “minimized,” and the like refer to a property of a device, component, composition, or activity 7 that is measurably different from other devices, components, compositions or activities that are in a surrounding or adjacent area, that are similarly situated, that are in a single device or composition or in multiple comparable devices or compositions, that are in a group or class, that are in multiple groups or classes, or as compared to the known state of the art.
  • Coupled is defined as directly or indirectly connected in a chemical, mechanical, electrical or nonelectrical manner. Objects described herein as being “adjacent to” each other may be in physical contact with each other, in close proximity to each other, or in the same general region or area as each other, as appropriate for the context in which the phrase is used. Occurrences of the phrase “in one embodiment,” or “in one aspect,” herein do not necessarily all refer to the same embodiment or aspect.
  • the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result.
  • an object that is “substantially” enclosed would mean that the object is either completely enclosed or nearly completely enclosed.
  • the exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained.
  • the use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result.
  • compositions that is “substantially free of’ particles would either completely lack particles, or so nearly completely lack particles that the effect would be the same as if it completely lacked particles.
  • a composition that is “substantially free of’ an ingredient or element may still actually contain such item as long as there is no measurable effect thereof.
  • the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “a little above” or “a little below” the endpoint. Unless otherwise stated, use of the term “about” in accordance with a specific number or numerical range should also be understood to provide support for such numerical terms or range without the term “about”. For example, for the sake of convenience and brevity, a numerical range of “about 50 angstroms to about 80 angstroms” should also be understood to provide support for the range of “50 angstroms to 80 angstroms ” Furthermore, it is to be understood that in this specification support for actual numerical values is provided even when the term “about” is used therewith. For example, the recitation of “about” 30 should be construed as not only providing support for values a little above and a little below 30, but also for the actual numerical value of 30 as well.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • scalability the demand for testing is in the order of millions per week, but manufacturing new tests at that scale is difficult
  • sample processing many tests still use an extraction operation when using saliva
  • readability molecular tests often use fluorescence and thus, a fluorescence reader to report the results
  • RT-LAMP reverse-transcription loop-mediated isothermal amplification
  • SARS-CoV-2 a pathogen
  • diluted saliva e.g., 5 % v/v in water
  • RT-LAMP is a nucleic acid amplification technique conducted at a constant temperature with adequate diagnostic performance especially during the acute phase of infection. Since RT-LAMP can be conducted at a constant temperature, expensive thermal cycling equipment is not. used. Additionally, existing colorimetric reporters for LAMP products do not use fluorescence readers. Consequently, this test is suitable for use in point-of-care settings and is amenable to rapid development and scale-up, making it appropriate for use in public health emergencies.
  • RT-LAMP can be implemented on microfluidic paper-based analytical devices (pPADs) to detect various pathogens (e.g., SARS-CoV-2) where image analysis can be performed using a portable electronic device to distinguish between positive and negative responses.
  • pathogens e.g., SARS-CoV-2
  • a high-contrast RT-LAMP reaction on paper can provide a color change that can be visible to the naked eye.
  • polystyrene spacers can be used for preventing crosstalk between samples. The polystyrene spacers can be amenable to roll-to-roll fabrication for scale up of production.
  • Nucleic-acid-based COVID- 19 diagnosis methods use pre-processing to provide results.
  • on-paper colorimetric detection of SARS-CoV-2 can be performed with minimal pre-processing.
  • the device can have a sensitivity and specificity that can detect SARS- CoV-2 on paper without pre-amplification.
  • Other assays conducted in solution may not. be as scalable during manufacturing as paper-based assays.
  • the assay disclosed herein uses a dilution operation that can be completed in seconds, whereas other assays use various operations such as treatment with protease, heat-inactivation, and/or RNA extraction to detect SARS-CoV-2 (operations completed in at least 10 minutes and using additional equipment).
  • Saliva has various physical, chemical, and antibacterial properties that can cause difficulty in the context of a LAMP reaction.
  • saliva can dilute and remove organic acids from dental plaque that can interfere with a LAMP reaction.
  • the antibacterial agents in saliva e.g., mucins, amylases, lysozyme, and peroxidase enzyme, also present challenges.
  • peroxidase enzyme can form free radical compounds in bacterial cells that can cause them to undergo apoptosis-like death.
  • such a reaction can also provide an unstable redox environment that can complicate a LAMP reaction.
  • a method 100 of preparing a saliva sample for loop-mediated isothermal amplification (LAMP) detection of a pathogen target is provided.
  • LAMP loop-mediated isothermal amplification
  • One way of reducing the buffering capacity of saliva can include dilution.
  • such a method can comprise providing an amount of saliva from a test subject, as shown in block 110 and diluting the saliva in water to a degree that reduces a buffering capacity of the saliva while maintaining a sufficient concentration to allow for detection of the pathogen target, as shown in block 120.
  • the proteins present in saliva offer another challenge.
  • an excessively viscous sample can be difficult to test on a solid-based or solid-phase medium.
  • a slow flow rate in a solid-based medium can increase the reaction time, decrease uniformity of spreading, increase variability in results, and increase invalidity of results.
  • a viscous form of saliva does not spread evenly throughout a solid-based medium, a color-based indication can be difficult to read.
  • the decrease in uniform spreading can also increase the variability of results by adding uncertainty to the reading of results. Different technicians may interpret the results differently. In some cases, the results may be impracticable to read due to ambiguous or absent color changes. Therefore, controlling the viscosity of the saliva can prevent various complications that may occur.
  • the method can further comprise reducing a viscosity of the saliva as compared to an original viscosity.
  • the saliva can be reduced by one or more of dilution, filtering, the like, or combinations thereof.
  • the saliva when the viscosity of the saliva, is reduced using dilution, the saliva can be diluted in water to a saliva to water ratio of from about 1 : 1 to about 1 :20.
  • the viscosity of the saliva can be di luted in water to a saliva to water ratio of a bout 1 :1, 1 :2, 1 :4, 1:8, 1: 10, 1: 12, 1 :14, 1 :16, 1: 18, or 1 :20.
  • the saliva can be diluted in the water to a degree that provides the sample with an optical density at 600 nm (ODeoo) of less than about 0.2.
  • the saliva can have a volume range of from about 50 pl to about 100 pl.
  • the saliva sample can have a volume ranging from about 100 pl to about 1 ml.
  • diluting the saliva sample can reduce the impact of the effects arising from the buffering capacity and viscosity of the saliva.
  • Another way of reducing the impact of the viscosity of the saliva can include filtering.
  • the viscosity can be reduced using a filter having a rating between about 2 microns and 50 microns.
  • 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.
  • the filter rating can be an absolute micron rating in which the filter can remove at least about 98.7% of a specific particle size. Filtering the saliva, rather than dilution alone, can also remove saliva proteins (e.g., mucins, amylases, lysozyme, and peroxidase enzyme) that may interfere with a LAMP reaction.
  • saliva proteins e.g., mucins, amylases, lys
  • a viscosity of saliva can have a range of from about 1 centipoise (cP) and to about 100 cP before dilution or filtering.
  • the viscosity of the saliva can be reduced to a range of from about 1.0 cP to about 50 cP after dilution or filtering.
  • the viscosity of the saliva can be reduced to a range of from about. 1 .0 cP to about 10 cP after dilution or filtering.
  • Filtering the saliva can also adjust the pH range to a desirable level.
  • some pH indicators may display a color change within a specific pH range (e.g., 7.2 to about. 8.6). Therefore, the saliva can be filtered to a test sample target range depending on the type of pH indicator to be used. However, maintaining the test sample target range within physiological conditions can increase the uniformity of the LAMP reaction results.
  • the saliva can be filtered to a degree that adjusts the saliva sample pH to a test, sample target, range.
  • the test sample target range can include a pH range between about 7.2 to about 8.6.
  • the test sample target range can include a pH range between about 7.6 to about 8.2.
  • Adjusting the test sample target range to a desired level may not be sufficient to detect a pH change (or other colorimetric indication) in a LAMP reaction.
  • the saliva can be diluted with water to a degree that the buffering capacity of the composition is reduced relative to the buffering capacity before the dilution with water to allow a pH indication to be detected.
  • buffering capacity can be defined as the ability of a solution (e.g., the saliva, the water, or the saliva diluted with water) to resist changes in pH when acids or bases are added.
  • the buffering capacity can be defined as the amount of strong acid or strong base, grams equivalents, that is to be added to 1 liter of the solution to change the pH by one unit.
  • the buffering capacity of the saliva can be between 0.03 mg/ml to about 0.30 mg/ml before dilution with water, and the buffering capacity of the saliva diluted water can be between about 0.003 mg/ml to about 0.03 mg/ml after dilution with water. In another example, the buffering capacity of the saliva diluted water can be less than about 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM.
  • the water used to dilute the sample should be free of any contaminants or properties that might interfere with the LAMP reaction.
  • a pH that is too acidic can prevent the LAMP reaction from being detected if the pH prevents a pH-based indication change.
  • the saliva can be diluted in water, wherein the water can have a pH greater than about 6.0.
  • the water can have a pH less than about 8.0.
  • the water can be molecular grade water that is substantially free of contaminants, such as RNase and DNase. RNase can degrade the RNA in the saliva that is to be detected, and DNase can degrade the DNA formed during the LAMP reaction.
  • the saliva sample can consist essentially of saliva and water.
  • the saliva can be collected using one or more of a sponge-based collection method or a passive drool collection method.
  • Collecting saliva using a sponge-based collection method may provide the benefit of inherently filtering mucins and high molecular weight proteins out of the saliva as they will not be absorbed by the sponge, which can reduce the viscosity of the saliva and increase the rapidity, uniformity, and reliability of the saliva when used on a solid-based medium.
  • the saliva is collected using a drooling method, the unfiltered saliva can have a greater viscosity, and therefore a reduced absorption, and distribution on a solid-based medium.
  • the saliva when the saliva is collected via drooling, it can be subsequently filtered in order to remove mucins and other debris and reduce its viscosity.
  • a selected pathogen target can be detected from the saliva.
  • the pathogen target can be one or more of a viral pathogen, a bacterial pathogen, a fungal pathogen, a protozoa pathogen, the like, or combinations thereof.
  • the pathogen target in saliva can be detected when the nucleic acid from the pathogen target can be released from a cell wall, a cell membrane, a protein coat, or the like.
  • the pathogen target can be a viral target.
  • the viral target can be H1N1, H2N2, H3N2, HlNlpdmO9, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome (MERS), influenza, the like, or combinations thereof.
  • the viral target can be selected from a number of different viral species.
  • the viral target can be human coronavirus 229E, human coronavirus OC43, human coronavirus HKU1, human coronavirus NI..63, MERS-coronavirus, human respirovirus 1, human rubula virus 2, human respirovirus 3, human rubulavirus 4, human enterovirus, human respiratory virus, rhinovirus A, rhinovirus B, rhinovirus C, or combinations thereof.
  • the viral target can also be a form of influenza.
  • influenza can be any of Influenza A, Influenza B, Influenza C, or Influenza D.
  • the viral target can be a virus chosen from the order Nidovirale.
  • the viral target can be chosen from the alpha, beta, gamma or delta genera of the Nidovirale order.
  • the viral target can be a DNA virus selected from the group of families including: Adenoviridae, Papovaviridae, Parvoviridae, Herpesviridae, Poxviridae, Anelloviridae, Pleolipoviridae, the like, and combinations thereof.
  • the viral target can be an RNA virus selected from the group of families including: Reoviridae, Picornaviridae, Caliciviridae, Tbgaviridae, Arenaviridae, Flaviviridae, Orthomyxovindae, Paramyxoviridae, Bunyavindae, Rhabdoviridae, Filoviridae, Coronavindae, Astroviridae, Bomaviridae, the like and combinations thereof.
  • the viral target can be a reverse transcribing virus selected from the group of families including: Retroviridae, Caulimoviridae, Hepadnaviridae, the like, and combinations thereof.
  • the viral target can be a virus categorized by the Baltimore classification.
  • the viral target can be an RNA virus (e.g., Influenza A, Zika, Hepatitis C).
  • the viral target can be a DNA virus (e.g., Epstein Barr, Smallpox).
  • the viral target can be a positive sense RNA virus (e.g., Hepatitis A, rubella).
  • the viral target can be a negative sense RNA virus (e.g., Ebola, measles, mumps).
  • the viral target can be a dsDNA virus (e.g., chickenpox, herpes), an ssDNA virus, a dsRNA virus (e.g., a rotavirus), a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus (e.g., retroviruses), or a ds-DNA-RT virus (e.g., Hepatitis B).
  • the pathogen target can be a bacterial target.
  • the bacterial target can be selected from a genus including: Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, Yersinia, the like, and combinations thereof.
  • the bacterial target can be selected from a species including: Actinomyces israelii, Bacillus anthracis, Bordetella pertussis, B. abortus, B. cams, B. melitensis, B. suis, Corynebacterium diphlheriae, E. coll, Enterotoxigenic E. coli, Enteropathogenic E. coll, Enteroinvasive E.coli, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, M. tuberculosis, Mycoplasma pneumoniae, N meningitidis, S. typhi, S. sonnet, S.
  • the pathogen target can be selected from the species including: Chlamydia pneumoniae, Pneumocystis jirovecii, Candida albicans, Pseudomonas aeruginosa, Staphylococcus epidermis, Streptococcus salivarius, the like, and combinations thereof.
  • the pathogen target can also include various types of fungus.
  • the pathogen target can be a fungal target.
  • the fungal target can be selected from a genus including: Aspergillus, Histoplasma, Pneumocystis, Stachybotrys, the like, and combinations thereof.
  • the pathogen target can be a protist target.
  • the protist target can be selected from a genus including: plasmodium, trypanosomes, the like, and combinations thereof.
  • the LAMP detection can be reverse transcription LAMP (RT- LAMP).
  • cDNA can be generated from a target RNA with a reverse transcriptase enzyme. The cDNA can be amplified to a detectable amount.
  • LAMP can be used to amplify the DNA to a detectable amount without reversed transcribing the RNA to DNA.
  • the specific target nucleotide sequences to be detected can be target nucleotides corresponding to human biomarkers.
  • Any disease that has a target nucleotide corresponding to a human biomarker for a disease can be detected.
  • Various types of diseases can be detected including one or more of: breast cancer, pancreatic cancer, colorectal cancer, ovarian cancer, gastrointestinal cancer, cervix cancer, lung cancer, bladder cancer, many types of carcinomas, salivary gland cancer, kidney cancer, liver cancer, lymphoma, leukemia, melanoma, prostate cancer, thyroid cancer, stomach cancer, the like, or combinations thereof.
  • biomarkers for various types of diseases can be detected by detecting target nucleotides corresponding to one or more of: alpha fetoprotein, CAI 5-3 and CA27-29, CAI 9-9, Cl-125, calcitonin, calretinin, carcinoembryonic antigen, CD34, CD99MIC 2, CD117, chromogranin, chromosomes 3, 7, 17, and 9p21 , cytokeratin, cesmin, epithelial membrane antigen, factor VIII, CD31 FL1, glial fibrillary acidic protein, gross cystic disease fluid protein, hPG80, HMB-45, human chorionic gonadotropin, immunoglobulin, inhibin, keratin, lymphocyte marker, MART-1, Myo DI, muscle-specific actin, neurofilament, neuron-specific enolase, placental alkaline phosphatase, prostate-specific antigen, PTPRC, SI 00 protein, smooth muscle action, synaptophysm,
  • a test sample composition for loop-mediated isothermal amplification (LAM?) analysis can comprise an amount of a test subject’s saliva that is sufficient to detect a pathogen target via a LAMP analysis in combination with an amount of water that reduces a buffering capacity of the saliva.
  • the viscosity of the composition can be from about 1 .0 cP to about 50 cP.
  • the pH of the composition can be from about 7.2 to about 8.6. Selecting a viscosity and pH within these respective ranges can enhance the change in pH and therefore the color change resulting from a pH-based indicator.
  • the saliva can be diluted with water to place the viscosity and pH within the ranges enumerated above.
  • the saliva can be combined with the amount of water in a saliva to water ratio of from about 1 : 1 to about 1 :20.
  • 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.
  • the saliva can be combined with the amount of water to a degree that provides the sample with an optical density at 600 nm (ODeoo) of less than 0.2.
  • the amount of collected saliva can be higher than a threshold amount.
  • the saliva can have a volume ranging from about 50 pl to about 100 pl.
  • the saliva sample can have a volume ranging from about 100 pl to about 1 ml.
  • the saliva can also have various chemical properties (e.g., pH and buffering capacity) that can facilitate the LAMP reaction.
  • the water can have a pH greater than about 6.0 and can be substantially free of contaminants such as RNase and DNase.
  • the water can have a pH less than about 8.0 and can be substantially free of contaminants.
  • the composition can consist essentially of the saliva and the water.
  • the buffering capacity of the composition can be between about 0.003 mg/ml to about 0.03 mg/ml.
  • the buffering capacity of the composition can be less than about 5 mM, 4 mM, 3 inM, 2 mM, or 1 mM.
  • the pathogen target can comprise a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoa pathogen.
  • the pathogen target can be a viral target.
  • the viral target can comprise a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA- RT virus, or a ds-DNA-RT virus.
  • the viral target can comprise H 1 N 1, H2N2, H3N2, HlNlpdmO9, or SARS-CoV-2.
  • a variety of reagents can be used in a LAMP analysis depending on the testing medium, readout type, and overall environment of the designed system. Further, reaction components such as primers and enzymes can be selected in view of the specific target nucleotide sequences to be detected, organisms to be identified, etc. Additionally, the specifics of the test environment, such as liquid environment, anhydrous environment, housing, substrates, etc. can be taken into account as well as other needs such as storage on stability when selecting specific reagents to be involved in the reaction underlying the L A MP analysis.
  • a composition for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium can comprise one or more target primers, a DNA polymerase, and a re-solubilization agent.
  • the composition can be substantially free of non-pH sensitive agents capable of discoloring the solid phase medium.
  • the concentration of reagents can be increased when compared to a LAMP analysis in a liquid phase medium.
  • the concentration of DNA polymerase when used on the solid phase medium can be at least twice the concentration of DNA polymerase when used with a liquid medium.
  • the concentration of DNA polymerase when used on the solid phase medium can be at least three times the concentration of DNA polymerase when used with a liquid medium.
  • the concentration of DNA polymerase can be from about 300 U/mL to about 1000 U/mL when used on the solid-phase medium.
  • the concentration of DNA polymerase can be from about 600 U/mL to about 1000 U/mL when used on the solid phase medium.
  • the concentration of DNA polymerase can be from about 620 U/m to about 680 U/mL when used on the solid phase medium.
  • the composition can further comprise reverse transcriptase.
  • the reverse transcriptase can aid in the detection of RNA-based viruses.
  • the concentration of reverse transcriptase when used on the solid phase medium can be at least twice the concentration of reverse transcriptase when used with a liquid medium.
  • the concentration of reverse transcriptase can be least three times the concentration of reverse transcriptase when used with a liquid medium.
  • the concentration of reverse transcriptase can be from about 200 U/mL to about 600 U/mL when used on the solid-phase medium.
  • the concentration of reverse transcriptase can be from about 250 U/mL to about 500 U/mL when used on the solid phase medium. In yet another example the concentration of reverse transcriptase can be from about 290 U/mL to about 310 U/mL when used on the solid phase medium.
  • the composition can comprise a re-solubilization agent.
  • a re-solubilization agent can aid in the re-hydration of the LAMP reagents on the solid-based medium when a saliva sample is deposited on the solidbased medium.
  • the re-solubilization agent can be a surfactant.
  • the re-solubilization agent can comprise bovine serum albumin (BSA), casein, polysorbate 20, the like, or combinations thereof. BSA and casein can facilitate re-solubilization of the DNA polymerase, reverse transcriptase, and other related enzymes when the dried reagents are rehydrated.
  • BSA bovine serum albumin
  • casein can facilitate re-solubilization of the DNA polymerase, reverse transcriptase, and other related enzymes when the dried reagents are rehydrated.
  • Polysorbate 20 is a surfactant that can also aid in the re-solubilization of dried reagents.
  • the concentration of the re-solubilization agent can be from about 0,05 wt% to about 5 wt% when used on the solid-phase medium.
  • the concentrati on of the re-solubilization agent can be from about 0.5 wt% to about 3 wt%.
  • the concentration of the re-solubilization agent can be from about 0.5 wt.% to about 1.5 wt%.
  • the composition can further comprise an agent that can speed up the reaction, increase sensitivity, or a combination thereof.
  • an agent that can speed up the reaction can be included to speed up the reaction and increase sensitivity.
  • BSA can be included to speed up the reaction and increase sensitivity.
  • the inclusion of BSA can also introduce pH variations that can interfere with the readability of the results. Therefore, in some examples, the re-solubilization agent can include casein, polysorbate 20, the like, or combinations thereof.
  • Volatile agents can interfere with the LAMP reaction.
  • a volatile compound can ionize to a plurality of ions, and one of the ions can have a low boiling point. When the ion with the low boiling point evaporates, the remaining ion can further react. Some of the further reactions can include redox reactions, acid-base reactions, or other reactions that can affect the interpretation of a pH-based signal.
  • the composition can be substantially free of volatile agents.
  • the removal of volatile agents can increase the color contrast and decrease the reaction time of the solid-based medium when compared to the color contrast and reaction time when volatile agents are included.
  • 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 volatile agents.
  • Volatile agents can cause instability in the solid-based medium.
  • a LAMP reaction that contains a volatile compound, such as ammonium sulfate can cause instability in the solid-based medium when the ammonium ions partially convert the ammonium sulfate to ammonium which can volatilize and leave behind sulfate.
  • the sulfate can become sulfuric acid and reduce the pH which can affect the reading of the pH-based indicator (e.g., changing the phenol red indicator from red to yellow even when the LAMP reaction does not occur).
  • Replacing ammonium sulfate with betaine can prevent the non-LAMP reaction-based discoloration and stabilize the solid-based medium by preventing discoloration under storage.
  • the LAMP composition can comprise a non-volatile agent including a quaternary’ ammonium of low molecular weight of neutral charge, or an amide compound of low' molecular weight of neutral charge, the like, or combinations thereof.
  • the non-volatile agent can include, but is not limited to, N-Formylurea, Urea, L- Asparagine, Trimethylglycine (Betaine), 3-(Cyclohexylamino)-l -propanes ulphomc acid (CAPS), 3-(l-Pyridinio)-l- propanesulfonate (NDSB-201), N-Methylurea, Acetamide, Propionamide, Isobutyramide, Piracetam, 1,3-Dimethylurea, 1,1 -Dimethyl urea, Glycolamide, 2-Chloroacetamide, Succinimide, 2-Imidazolidone, Choline chloride.
  • Acetylcholine chloride Bethanechol chloride, L-Carnitine inner salt, O-Acetyl-L-carnitine hydrochloride, 4-(Cyclohexylamino)-l -butanesulfonic acid (CABS), Dimethylethylammoniumpropane sulfonate (NDSB-195), 3-(l-Methylpiperidinium)-l- propane sulfonate (NDSB-221), 3-(Benzyldimethylammonio)propanesulfonate (NDSB-256), and Dimethyl-2-hydroxyethylammonium-l -propane sulfonate (NDSB-211), the like, or combinations thereof.
  • CABS Cyclohexylamino)-l -butanesulfonic acid
  • NDSB-195 Dimethylethylammoniumpropane sulfonate
  • NDSB-221 3-(l-Methylpiperid
  • the concentration of the non-volatile agent including a quaternary ammonium of low molecular weight of neutral charge, or the amide compound of low molecular weight of neutral charge can be from about 1 mM to about 200 mM when used on the solidphase medium. In another example, the concentration of the non-volatile agent can be from about 10 mM to about 50 mM when used on the solid-phase medium. In yet another example, the concentration of the non-volatile agent can be from about 15 mM to about 25 mM when used on the solid-phase medium.
  • hygroscopic agents can interfere with the LAMP reaction.
  • a hygroscopic agent can retain an excessive amount of water and destabilize the reagents in the solid-based medium by slowing down or preventing drying.
  • the composition can be substantially free of hygroscopic agents.
  • a LAMP reaction that contains a hygroscopic agent, such as glycerol can contribute to the instability’ of reagents in the solid-based medium because the hygroscopic agent attract can attract water.
  • a hygroscopic agent can absorb more than about 10 wt % when between about 40% and about 90% relative humidity (RH) at 25° C.
  • a hygroscopic agent can include, but is not limited to, one or more of glycerol, ethanol, methanol, calcium chloride, potassium chloride, calcium sulfate, the like, or combinations thereof.
  • 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 agents.
  • Some additional agents can be included to prevent carryover contamination of previous LAMP reactions, primer dimerization, non-specific 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- stranded or double stranded DNA containing uracil.
  • dUTP deoxyuridine triphosphate
  • UDG uracil DNA glycosylase
  • the composition can further comprise an antioxidant.
  • the concentration of the antioxidant can be from about 0.1 mM to about 1 mM when used on the solid-phase medium.
  • the concentration of the antioxidant can be from about 0.2 mM to about 0.8 mM when used on the solid-phase medium.
  • the concentration of the antioxidant can be from about 0.2 mM to about 0.3 mM when used on the solid-phase medium. The antioxidant can stabilize the reagents on the solid-based medium by preventing oxidization-reduction reactions.
  • antioxidants can be used including, but not limited to: N-acetyl-cysteine, 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, zeaxanthm, apigenin, luteolin, tangeritin, isorhamnetin, kaempferol, myricetin, proanthocyamdins, quercetin, eriodyctiol, hesperetm, naringemn, catechin, gallocatechin, epicatechin, epigallocatechm, theaflavin, thearubigms, daidzein, genistein,
  • the composition can further comprise an indicator
  • the indicator can be a pH-based indicator, such as phenol red, when used with a solid-based medium. Phenol red has antioxidant properties that some other dyes do not have. The phenol red molecule is a conjugated bond system that might also contribute antioxidant properties.
  • the concentration of the indicator can be from about 0.1 mM to about 1 mM when used on the solid-phase medium. In another example, the concentration of the indicator can be from about 0.2 mM to about 0.8 mM when used on the solid-phase medium. In yet another example, the concentration of the indicator can be from about 0.2 mM to about 0.3 mM when used on the solid-phase medium.
  • the indicator can be one or more of a (i) magnesium colorimetric indicator, (ii) a pH colorimetric indicator, or (iii) a DNA intercalating colorimetric indicator.
  • the concentration of magnesium should be monitored to maintain the magnesium within a range of from about 0.01 mM to about 2 mM.
  • the concentration of magnesium should be monitored to prevent interference with DN A polymerase. Magnesium - a cofactor of DNA polymerase, can interfere with DNA polymerase when the magnesium concentration is outside a target range.
  • the LAMP reaction can also use various types of target primers.
  • Some target primers can include about 4 or 6 primers that can target 6 or 8 regions within a genome, respectively.
  • the concentration of the target primers can have a concentration from about 0.05 uM to about 5 ⁇ M when used on the solid-phase medium.
  • the concentration of the target primers can be from about 0.1 pM to about 3 pM when used on the solid-phase medium.
  • the concentration of the target primers can be from about 0.2 ⁇ M to about 1.6 pM when used on the solid- phase medium.
  • the target primers can be selected to target the genomes of various pathogens.
  • the target primers can target a pathogen that can comprise a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoa pathogen.
  • the pathogen target can be a viral target.
  • the viral target can comprise a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a ds-DNA-RT virus.
  • the viral target can comprise H1N1, H2N2, H3N2, HlNlpdmO9, or SARS-CoV-2.
  • the target primers can target nearly any pathogen target, in particular, those target pathogens as disclosed herein.
  • the composition can comprise a nondiscoloration additive.
  • the concentration of the non-discoloration additive can be from about 0.01 mM to about 1 M when used on the solid-phase medium.
  • the concentration of the non-discoloration additive can be from about 10 mMto about 500 mM when used on the solid-phase medium.
  • the concentration of the non- discoloration additive can be from about 200 mM to about 400 mM when used on the solidphase medium.
  • non-discoloration additives that can preserve the color of the solidbased medium in the absence of LAMP-reaction produced amplification and potentially increase the contrast when LAMP-reaction produced amplification occurs.
  • the non- discoloration additive can comprise one or more of a sugar, a buffer, the like, or combinations thereof.
  • a non-discoloration additive such as sugar can stabilize the solid-based medium and prevent discoloration under long-term storage conditions.
  • trehalose can preserve the stability of enzymes under freeze-drying conditions or when dried at ambient temperatures.
  • the sugar can comprise one or more of glucose, sucrose, trehalose, dextran, the like, or combinations thereof.
  • the concentration of the sugar can be from about 0.01 mM to about 1 M when used on the solid-phase medium.
  • the concentration of the sugar can be from about 10 mM to about 500 mM when used on the solid- phase medium.
  • the concentration of the sugar can be from about 200 mM to about 400 mM when used on the solid-phase medium.
  • the LAMP reaction can also include other reagents.
  • the composition can comprise one or more of an enzyme, a nucleic acid, or combinations thereof.
  • the enzyme can be an RNase inhibitor or a DNase inhibitor. Inclusion of an RNase inhibitor can slow the degradation of an RNA target to allow for an increased limit of detection. Inclusion of a DNase inhibitor can slow the degradation of a DNA target to also allow for an increased limit of detection.
  • the composition can comprise carrier DNA or carrier RNA.
  • the carrier DNA or carrier RNA can provide decoy substrate that sequesters the activity of DNase or RNase, respectively.
  • a selected amount of guanidine hydrochloride can stimulate the denaturing and exposing of RNA molecules which can further stabilize the LA1V1P reaction.
  • the concentration of the RNase or DNase inhibitor can be from about 0.01 ⁇ L per ml, of saliva sample to about 5 pl, per ml, of saliva sample when used on the solid-phase medium. In another example, the concentration of the RNase or DNase inhibitor can be from about 0.1 pl, per ml, of saliva sample to about 1 ⁇ L per ml, of saliva sample when used on the solid-phase medium. In yet another example, the concentration of the RNase or DNase inhibitor can be from about 0.5 ⁇ L per mL of saliva sample to about 1.5 ⁇ L per mL of saliva sample when used on the solid-phase medium.
  • the concentration of the carrier RNA or carrier DNA can be from about 0.01 ng/ ⁇ L to about 10 ng/ pl, when used on the solid-phase medium. In another example, the concentration of the carrier RNA or carrier DNA can be from about 0.1 ng/pl, to about 1 ng/pl, when used on the solid-phase medium. In yet another example, the concentration of the carrier RNA or carrier DNA can be from about 0.2 ng/ ⁇ L to about 0.4 ng/ ⁇ L when used on the solidphase medium.
  • the composition can further comprise a tonicity agent, a pH adjuster, a preservative, water, the like, or combinations thereof.
  • these ingredients/agents can be used to provide the composition with a range of specifically desired properties.
  • the tonicity of the composition can be from about 250 to about 350 milliosmoles/liter (mOsm/L).
  • the tonicity of the composition can be from about 270 to about 330 mOsm/L.
  • Tonicity agents can be present in the composition in various amounts.
  • the tonicity agent can have a concentration in the composition of from about 0.1 wl%, about 0.5 wt%, or about 1 wt% to about, 2 wt%, about 5 wl%, or about 10 wl%.
  • pH adjusters can be used to sel ect an initial pH of the compositi on before a LAMP reaction.
  • pH adjusters can also be used when the effects of the pH adjusters can be compensated for when interpreting results from the LAMP reaction.
  • Non-limiting examples of pH adjusters can include a number of acids, bases, and combinations thereof, such as hydrochloric acid, phosphoric acid, citric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, and the like.
  • the pH adjusters can be used to provide an appropriate pH for the composition.
  • the pH can be from about 5.5 to about 8.5.
  • the pH can be from about 5.8 to about 7.8.
  • the pH can be from about 6.5 to about 7.8.
  • the pH can be from about 7.0 to about 7.6. pH adjusters can be present in the composition in various amounts.
  • the pH adjuster can have a concentration in the composition of from about 0.01 wt%, about 0.05 wt%, about 0.1 wt%, or about 0.5 wt% to about 1 wt%, about 2 wt%, about 5 wt%, or about 10 wt%.
  • the shelf life of the composition can be enhanced by using preservatives.
  • preservatives can include benzalkonium chloride (BAK ), cetrimonium, sodium the like
  • Preservatives can be present in the composition in various amounts.
  • the preservative can have a concentration in the composition of from about 0.001 wt%, about 0.005 wt%, about 0.01 or about 0.05 wt% to about 0.1 wt%, about 0.25 wt%, about 0.5 wt%, or about 1 wt%.
  • a method 200 for LAMP analysis on a solid phase medium can comprise providing an assembly of a solid phase medium and a reaction composition in combination therewith, such as any of the ingredients or compositions as recited herein, as shown in block 210.
  • the method can comprise depositing a biological sample onto the solid phase medium, as shown in block 22.0.
  • the method can comprise heating the assembly to an isothermal temperature sufficient to facilitate a LAMP reaction, as shown in block 230.
  • the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, the like, or combinations thereof.
  • the biological sample can be saliva.
  • the method can comprise detecting a viral pathogen.
  • the viral pathogen can be a pathogen as disclosed herein.
  • the LAMP analysis can be reverse transcriptase LAMP (RT-LAMP).
  • the isothermal temperature sufficient to facilitate a LAMP reaction can be in a temperature range from about 50 °C to about 70 °C. In another aspect, the isothermal temperature sufficient to facilitate a LAMP reaction can be in a temperature range from about 60 °C to about 70 °C. In another aspect, the isothermal temperature sufficient to facilitate a LAMP reaction can be in a temperature range from about 60 °C to about 65 °C. The isothermal temperature can be selected based on one or more of the activity of the DNA polymerase, reverse transcriptase, or combinations thereof.
  • a temperature sufficient to facilitate a LAMP reaction can be in a temperature range of from about 60 °C to about 70 °C.
  • the isothermal temperature can be a temperature within a range that differs by less than 5 degrees Celsius.
  • a system for performing a LAMP analysis can comprise a composition as recited in this disclosure.
  • the system can comprise a solid phase medium on to which the composition is deposited.
  • magnesium colorimetric indicators When conducting a LAMP reaction, various indicators can be used to read the results of the reaction.
  • 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 should 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 because of the number of variables in play. Although all three indicators can be used in a LAMP Reaction, pH-based indicators may be subject to fewer variables.
  • a composition for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal can comprise a pH sensitive dye, and a plurality of non-interfering LAMP reagents.
  • the LAMP analysis can be reverse transcription LAMP (RT-LAMP).
  • the selection of pH-sensitive dye can depend on various factors, such as colorimetric range correlated to pH, degree of contrast between color changes, level of pH for a color change, uniformity of color change, reproducibility of color change, and the like.
  • phenol red can have a colorimetric range between a pH of about 6.8 and about 7.4. Below a pH of about 6.8, phenol red can turn yellow and above a pH of about 7,4, phenol red can turn red.
  • the degree of difference between yellow and red can be simple to read, and the pH change can occur at a pH level that mimics physiological conditions.
  • the pH sensitive dye can be a pH indicator with a color change around a pH of 6.5 to achieve a consistent and contrasting color change (e.g., Phenol red).
  • the pH sensitive dye can be at least one of phenol red, litmus, bromothymol blue, nitrazine yellow, cresol red, curcumin, brilliant yellow, in-cresol purple, a-naphtholphthalein, phenolphthalein, neutral red, acid fuchsin, azolitmin, the like, or combinations thereof.
  • the concentration of the pH sensitive dye can be from about 0.1 mMto about 1 mM when used on the solid-phase medium.
  • the concentration of the pH sensitive dye can be from about 0.2 mM to about 0.8 mM when used on the solid-phase medium. In yet another example, the concentration of the pH sensitive dye can be from about 0.2 mM to about 0.3 mM when used on the solid-phase medium.
  • the LAMP reaction should be substantially free of reagents that would introduce uncertainty into 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).
  • the plurality of non-interfering LAMP reagents can comprise DNA polymerase, reverse transcriptase, target primers, or combinations thereof.
  • the plurality of non-interfering LAMP reagents can be substantially free of volatile reagents, pH-interfering reagents, magnesium-interfering reagents, or combinations thereof.
  • the plurality of non- interfering LAMP reagents can be substantially free of magnesium, ammonium sulfate, or ammonium carbonate.
  • Magnesium, as a cofactor of DNA polymerase, should be tightly monitored to ensure that the LAMP reaction can proceed as designed.
  • Ammonium sulfate can ionize into the ammonium ion, which can leave behind a sulfate ion that can react to form sulfuric acid.
  • Ammonium carbonate can also ionize into an ammonium ion and leave behind a carbonate that can react to form carbonic acid. Therefore, the plurality of non-interfering LAMP reagents should be substantially free of these substances.
  • the plurality of non-interfering LAMP reagents can be substantially free of volatile reagents including, but not limited to: ammonium sulfate, and ammonium carbonate, the like, or combinations thereof.
  • 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 volatile reagents.
  • any pH- interfering reagents can interfere with the pH-dependent signal output when the pH-interfering reagents is not compensated for.
  • the plurality of non-interfering LAMP reagents can be substantially free of pH-mterfering reagents including, but not limited to a number of acids, bases, and combinations thereof.
  • 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 pH-interfering reagents.
  • the pH-dependent signal output can be negatively- affected when the LAMP reaction is interfered with.
  • magnesium as a cofactor of DNA polymerase, can interfere with the amplification from the LAMP reaction when the concentration is outside a selected range.
  • the plurality of non-interfering LAMP reagents can be substantially free of magnesium-interfering agents.
  • Magnesium-interfering agents can include magnesium-containing agents including, but not limited to: Mg 2 * Mg 1 *, magnesium carbonate, magnesium chloride, magnesium citrate, magnesium hydroxide, magnesium oxide, magnesium sulfate, magnesium sulfate heptahydrate, the like, or combinations thereof.
  • the composition can contain less than one or more of: 1.0 wt%, 0.5 wt%, 0.1 wt%, or 0.01 wt% of magnesium.
  • magnesium-interfering agents can include chelating agents that interfere with magnesium.
  • the composition can comprise a non-discoloration additive.
  • the non-discoloration additive can comprise one or more of a sugar, a buffer, a blocking agent, the like, or combinations thereof.
  • the sugar can stabilize the solid-based medium and prevent discoloration under long-term storage conditions.
  • the sugar can comprise one or more of: glucose, sucrose, trehalose, dextran, the like, or combinations thereof.
  • the concentration of the sugar can be from about 0.01 mM to about 1 M when used on the solid-phase medium. In another example, the concentration of the sugar can be from about 10 mM to about 500 mM when used on the solid- phase medium. In yet another example, the concentration of the sugar can be from about 200 mM to about 400 mM when used on the solid-phase medium.
  • a buffer can facilitate the stabilization of the LAMP reaction by removing the variability from the saliva sample.
  • a buffer can include one or more of phosphate-buffered saline (PBS), Dulbecco’s PBS, Alsever’s solution, Tris-buffered saline (TBS), HEPES, BICINE, water, balanced salt solutions (BSS), such as Hank’s BSS, Earle’s BSS, Grey’s BSS, Puck’s BSS, Simm’s BSS, Tyrode’s BSS, BSS Plus, Ringer’s lactate solution, normal saline (i.e. 0.9% saline), '/? normal saline, the like, or combinations thereof.
  • PBS phosphate-buffered saline
  • Dulbecco’s PBS Dulbecco’s PBS
  • Alsever Tris-buffered saline
  • HEPES Tris-buffered saline
  • BICINE
  • the concentration of the buffer can be from about 10 pM to about 20 mM when used on the solid-phase medium. In another example, the concentration of the buffer can be from about 100 pMto about 10 mM when used on the solid-phase medium. In yet another example, the concentration of the buffer can be from about 100 pM to about 500 pM when used on the solid-phase medium.
  • a blocking agent can decrease the amount of RNase-based degradation, DNase-based degradation, or other enzymatic degradations.
  • a blocking agent can include one or more of bovine serum albumin, casein, or combinations thereof.
  • the concentration of the blocking agent can be from about 0.01 wt% to about 5 wt% when used on the solid-phase medium.
  • the concentration of the blocking agent can be from about 0.01 wt% to about 1 wl% when used on the solid-phase medium.
  • the concentration of the blocking agent can be from about 0.02 wt% to about 0.06 wt% when used on the solid-phase medium.
  • composition can further comprise an antioxidant as disclosed herein.
  • composition can further comprise a solid phase medium.
  • the solid-phase medium can include, but is not limited to, one or more of: glass fiber, nylon, cellulose, polysulfone, polyethersulfone, cellulose acetate, nitrocellulose, polyester, hydrophilic polytetrafluoroethylene (PTFE ), or combinations thereof,
  • the composition can comprise one or more of an enzyme, a nucleic acid, or combinations thereof as disclosed herein.
  • the enzyme can be an RNase inhibitor or a DNase inhibitor.
  • the composition can comprise carrier DNA or carrier RNA.
  • the carrier DNA or earner RNA can provide decoy substrate that sequesters the activity of DNase or RNase, respectively.
  • a selected amount of guanidine hydrochloride can stimulate the denaturing and exposing of RNA molecules.
  • the concentration of the guanidine hydrochloride can be from about 1 mM to about 200 mM when used on the solid-phase medium.
  • the concentration of the guanidine hydrochloride can be from about 10 mM to about 100 mM when used on the solid-phase medium.
  • the concentration of the guanidine hydrochloride can be from about 20 mM to about 60 mM when used on the solid-phase medium.
  • a method of performing a LAMP analysis with a pH-dependent output signal can comprise providing an assembly of a solid phase medium and a composition as recited herein. The method can further comprise depositing a biological sample onto the solid phase medium. The method can further comprise heating the assembly to an isothermal temperature sufficient to facilitate a LAMP reaction.
  • the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, the like, or combinations thereof.
  • the biological sample can be saliva.
  • the method can comprise detecting a viral pathogen.
  • the viral pathogen can be a pathogen as otherwise disclosed herein.
  • a temperature sufficient to facilitate a LAMP reaction can be in a temperature range of from about 60 °C to about 70 °C.
  • the isothermal temperature can be a temperature within a range that differs by less than 5 degrees Celsius
  • the isothermal temperature sufficient to facilitate a LAMP reaction can be in a temperature range from about 50 °C to about 70 °C. In another aspect, the isothermal temperature sufficient to facilitate a LAMP reaction can be in a temperature range from about 60 °C to about 70 °C, In another aspect, the isothermal temperature sufficient to facilitate a L AMP reaction can be in a temperature range from about 60 °C to about 65 °C.
  • the isothermal temperature can be selected based on one or more of the activity of the DNA polymerase, reversed transcriptase, or combinations thereof
  • a method 300 of maximizing accuracy of an output, signal in a pH-dependent LAMP analysis can comprise providing a reagent mixture that minimizes non-LAMP reaction produced discoloration from a signal output medium as in block 310.
  • the method can further comprise performing the LAMP reaction, as shown in block 320.
  • the method can comprise controlling production of protons from a non- LAMP reaction.
  • the method can comprise controlling oxidation from a non- LAMP reaction.
  • a method of maximizing accuracy of an output signal in a pH-dependent LAMP analysis can comprise substantially eliminating non-LAMP reaction produced discoloration from a signal output medium.
  • a method of maximizing a level of detection (LOD) in a pH- dependent LAMP analysis can comprise substantially eliminating non-LAMP reaction produced discoloration from a signal output medium.
  • the color contrast can be enhanced and the sample variability can be mitigated without impacting the limit of detection by diluting the saliva to 5-10% with water.
  • the color contrast can be enhanced and the sample variability can be mitigated without impacting the limit of detection by filtering the saliva with a filter as otherwise disclosed herein.
  • DNase/RNase-free distilled water is prepared by filtration with 0.1 um membrane and tested for DNase and RNase activity. The DNase and RNase activity is tested in accordance with current U.S. Pharmacopeia (USP) monograph test standards for Water for Injection (WFI). Upon confirmation of no DNase, RNase, or protease activity the water is considered contaminant free and ready for use in preparing saliva samples.
  • USP U.S. Pharmacopeia
  • WFI Water for Injection
  • FIG. 4A illustrates flouorometric RT-qLAMP results for primer sets targeting RNaseP POP7 in 18% saliva spiked with 105 genome eq uivalents/r eaction of heat-inactivated SARS-CoV-2.
  • FIG. 4B illustrates flouorometric RT-qLAMP results for primer sets targeting RNaseP POP7 in water with 0.2 ng of synthetic RNaseP POP7 RNA.
  • the type of saliva collection device can facilitate a saliva sample in a LAMP reaction.
  • an operator may use protective equipment to protect from a pathogen that can be spread via airborne droplets (e.g., an aerosol virus). Therefore, operators can wear personal protective equipment to protect against the accidental contact with the aerosol virus.
  • the specific saliva collection device may be self-administered by the subject under the guidance of a healthcare professional.
  • the saliva collection device has a proven efficacy and can fall into two categories: sponge-based collection and passive drool collection as illustrated in FIGS. 5A and 5B.
  • a 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 sufficient volume has been collected. Once saturated, the sponge is inserted into a compression tube 506 and compressed against a filter which strains the saliva into a collection tube. A reason for this filtration operation is that it strains mucins and high molecular weight proteins out of the saliva and significantly reduces viscosity of the specimens. As a result, the solid phase medium can take-up and distribute the saliva in a more rapid, uniform, and reliable fashion.
  • the spongecollection 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 to compress the compression tube 506; and a sample volume adequacy indicator 512. to identify when sufficient saliva has been collected.
  • a passive drool device 500b can provide unfiltered saliva, with viscosity that slows absorption and distribution of the sample.
  • the passive drool device 500b can include: a collection funnel 522 for collecting saliva; an indicator line 528 for indicating when sufficient saliva has been collected; a collection tube 524 to collect the saliva; a tube cap 526; a volume indicator 530; and a tube cap storage 532.
  • the three devices were “Saliva SamplerTM” produced by StatSure Diagnostic Systems, Inc., “Pure*SALTM” produced by Oasis Diagnostics, and “Super* SALTM” also produced by Oasis Diagnostics.
  • the StatSure Saliva Sampler TM provides a tube containing a buffer (e.g., Buffer 2000) used to collect saliva from a patient.
  • Super* SAL uses a cylindrical absorbent pad and a collection tube to standardize the collection of saliva by removing any solid contaminants and mucinous material. Pure-SAL operates on a similar mechanism but includes an additional filter in the collection tube to remove contaminants.
  • FIG. 6A This data illustrates the LoD in saliva processed using different saliva collection devices (Pure-Sal, Super-Sal, Stat-sure).
  • the master-mix was treated with 0.6 microliter of HC1.
  • the Pure*SALTM and Super* SAL saliva collection devices illustrate a wider range of colorimetric response for a wider range of concentrations (1 to 10k genome equivalents per reaction of heat inactivated SARS-CoV-2).
  • a subject If a subject is self-testing, the subject collects a saliva specimen under the guidance of the healthcare professional into a specialized collection vessel which contains no additives and is thus safe for the subject to use in the collection.
  • the collected volume of saliva is approximately 100 ⁇ L.
  • the sponge sampler for example is inserted into the subject’s mouth and saliva is collected until the indicator on the sponge sampler changes color.
  • the sponge sampler is then inserted into a collection tube.
  • the sponge is then compressed to squeeze out the saliva (approximately 100 ⁇ L) into a collection tube containing an amount of water in it to dilute the saliva.
  • the saliva is diluted in the water to a saliva to water ratio of about 1 : 1 to about 1 :20.
  • the saliva is transferred from the collection tube to a test site.
  • RNAsecureTM AM7006, InvitrogenTM
  • IX RNAsecureTM was diluted from 25X stock using 1 ml of saliva.
  • the treated saliva was used as a matrix to spike heat-inactivated SARS-CoV-2 with a concentration range from 1000 copies to 62.5 copies/ react! on into the WarmstartTM colorimetric master mix along with 40mM of guanidine hydrochloride and 0.3ng/pl carrier DNA with a pH of 7.6.
  • the RNAsecureTM treated RT-LAMP was incubated at 65 °C to start the reaction. Fresh saliva under these conditions was tested without RNAsecureTM as a control.
  • RNAsecureTM did not show any significant increase in the Lol) of the reaction, as illustrated in FIG. 6B. That is, the addition of RNase inhibitor did not result in an appreciable increase in the measured parameters of the RT-LAMP reaction (e.g., the reaction speed, the falsepositive rate, or the limit of detection).
  • the pH of a frozen saliva sample can vary depending on the number of days at -20 degrees C before the saliva sample is thawed and tested.
  • the pH of the saliva sample from Donor 1 varied from a pH of 7.21 without any days between collection and testing to a pH of 7.46 after 6 days between collection/freezing and testing.
  • the pH of the saliva sample from Donor 2 varied from a pH of 7.00 without any days between collection and testing to a pH of 6.98 after 6 days between collection/freezing and testing.
  • the pH of the saliva sample from Donor 3 varied from a pH of 7.18 without any days between collection and testing to a pH of 7. 18 after 6 days between collection/freezing and testing.
  • the pH of the saliva sample from Donor 4 varied from a pH of 7.35 without any days between collection and testing to a pH of 7.47 after 6 days between collection/freezing and testing.
  • the pH of the saliva sample from Donor I varied from a pH of 7.22 without any days between collection and testing to a pH of 7.24 after 6 days between collection/freezing and testing.
  • FIG. 8 illustrates the limit of detection of fresh saliva.
  • Fresh saliva was collected using a drooling method and was diluted in water m a 1:3 ratio to obtain 25% saliva and 75% water.
  • Heat-inactivated SARS-CoV-2 was spiked into the 25% saliva with serial dilutions, as a control.
  • Dilution reduced the buffering capacity of saliva and decreased the concentration of inhibitory' components, both of which would delay colorimetric reporting. Dilution is less complex to the end-user compared to other pre-treatment operations found in a variety of studies, such as pre-treatment with proteases, Chelex® 100, or RNA extraction operations to inactivate inhibitory components of saliva.
  • the LoD of the colorimetric assay in 5% saliva that has been processed using Pure SALTM is 1000 copies/reaction (reaction volume 25 gL), which corresponds to 800 copies/gL of patient saliva after accounting for dilution (FIG. 6C).
  • reactions were taken using the flatbed scanner before and after the RT-LAMP reaction. Reactions consisted of 12.5 gL of NEB 2X colorimetric master mix, 2.5 gL of primer mix, 5 gL of water, and 5 gL of sample.
  • This LoD is several orders of magnitude higher than RT-PCR assays or other assays utilizing RNA extraction (on the order of 1 copy/reaction). However, these other assays were accompanied by pretreatment protocols and/or RNA extraction operations to achieve the reported LoD.
  • uracil-DNA glycosylase UDG
  • dUTP deoxy uridine triphosphate
  • FIG. 6F Colorimetric scans after 60 minutes of incubation at 65 °C for 25 ⁇ L reactions on the thermomixer and incubator with and without the addition of UDGs and dUTP.
  • the Primer set used was orflab.II.
  • the template was heat-inactivated virus at the indicated concentration.
  • For reactions with UDG, 1250 ⁇ L of NEB 2x colorimetric master mix was supplemented with 0.5 ⁇ L of Antarctic Thermolabile UDG, 3.5 ⁇ L of dUTP. For all other reactions NEB 2x colorimetric master mix was used.
  • the LoD of the RT-LAMP colorimetric assay in 5% processed saliva in solution increased to 250 copies/reaction (FIG. 6G).
  • FIG. 6G RT-LAMP colorimetric LoD using saliva processed with Pure- SALTM and saliva that was unprocessed. Plates were heated in an incubator set at 65 °C for 60 minutes. The Primer set used was orflab.II and the template was heat-inactivated virus at the indicated concentration (positive reactions) or nuclease-free water (negative reactions). Heating was conducted in an incubator (FisherbrandTM IsotempTM ) at 65 °C for 60 mins.
  • the colorimetric scans were taken using the flatbed scanner before and after the RT-LAMP reaction. 1250 iiL of NEB colorimetric master mix was supplemented with 0.5 jiL of Antarctic Thermolabile UDG, 3.5 ⁇ L of dUTP, carrier DNA (0.3 ng/ ⁇ L), and Guanidine HC1 (40 mM).
  • FIG. 9 illustrates the limit of detection in a bovine nasal swab that was re-suspended in about 1 mL water.
  • Heat-inactivated SARS-CoV-2 was spiked into water with the re-suspended background mucus and microbiome to obtain the same number of copies/reaction as in the previous example with saliva.
  • 5 p.L of sample was added to 20 ⁇ L of RT-LAMP. After incubating at 65 °C for about 1 hour, the color changed.
  • the LOD for the primer was about 250 copies/reaction in a volume of 25 ⁇ L, which is equivalent to about 5k copies/mL of nasal swab resuspension.
  • FIG. 10 illustrates the limit of detection on paper.
  • 20 ⁇ L of RT-LAMP reagents were added to Grade 1 chromatography paper.
  • Heat-inactivated SARS-CoV-2 was spiked into the 100% pooled saliva with serial dilutions of the virus.
  • 15 ⁇ L of about 100% saliva was added to each piece of paper. After incubating at 65 °C for 90 minutes the color changed.
  • the LOD for the primer was about 3k copies/reaction in a volume of 15 ⁇ L saliva, which is equivalent to about 20k copies/mL of saliva.
  • the reagents were included as shown in Table Al. In another example, the reagents were included as shown in Table A2. Table A-l
  • a buffer was used on the paperbased device to maintain a consistent starting pH
  • For phenol red a pH of 7.6 was a suitable starting point to enhance the colorimetric transition as illustrated in FIG. 11 , A few buffers having a pKa of about 8 were screened because the starting pH of 7.6 was close to the limits of the buffering range to allow a color change when amplification happens. 10 mM of BICINE buffer was used for the paper- based assay as illustrated in FIG. 12.
  • Colorimetric RT-LAMP master mix can be: KC1 (50 mM), MgSCk (8 mM), dNTP mixture (1.4 mM each dNTP), Bst 2.0 WarmStart® DNA Polymerase (0.32 U/gL), WarmStart® RTx Reverse Transcriptase (0.3 U/ ⁇ L), Phenol red (0.25 mM), dUTP (0.14 mM), Antarctic Thermolabile UDG (0.0004 U/ ⁇ L), Tween® 20 (1% v/v), betaine (20 mM), BSA (500 pg/niL), and trehalose (10% w/v).
  • D-(+)-trehalose dihydrate was titrated from 0% to 15% w/v using increments of 5% and lyophilized BSA was titrated from 0 to 1.25 mg/rnL using increments of 0.2 nig/mL.
  • concentrations for trehalose and BSA were 10% w/v and 0.626 mg/'niL, respectively.
  • Table 138-2 a Stocks of the LAMP primers can be made at a workable concentration in water for ease of setup.
  • a 10X Primer Mix containing all 6 LAMP primers, 10X Primer mix: 16 jiM FIP/BIP, 2 pM F3/B3, 4 ⁇ M Loop F/B can be made.
  • each reaction pad was transferred to a clean 1.5 mL microcentrifuge tube, 100 iiL Buffer EB was added to each tube. Reaction pads were submerged in Buffer EB overnight for eluting nucleic acids. Gel electrophoresis (2% agarose gel) was done with the eluent to verify the occurrence of LAMP amplification. A ladderlike pattern (typical LAMP product pattern) was shown in each positive pad lane while there was no obvious band in each negative lane (FIG. 13/X and 13B).
  • FIGS. 13 A and 13B paper LAMP validation was performed.
  • LAMP on paper with two conditions (with and without BSA in the reaction mix) was performed.
  • FIG. 13B associated gel electrophoresis (2% agarose) was performed.
  • the orf7ab.1 primer set targeting SARS-CoV-2 was used.
  • Negative reaction pads were reconstituted with 25 p.L nuclease free water.
  • Positive reaction pads were reconstituted with 25 ⁇ L 400 copies/ ⁇ L heat-inactivated SARS-CoV-2 virus. Heating was carried out in an incubator set at 65 °C and scanned in a flatbed scanner.
  • BSA is a reagent that can be used in the LAMP mix. Adding BSA can speed up reactions and increase sensitivity, as illustrated in FIG. 14A and FIG. 14B. In these reactions, a low template concentration LAMP on paper was performed subject to two conditions (with and without BSA m the reaction mix).
  • FIG. 14A shows a 0-minute time point.
  • FIG. B shows a 60- mmute time point.
  • Negative reaction pads were reconstituted with 25 ⁇ L nuclease free water.
  • Positive reaction pads were reconstituted with 25 fiL 8 copies/gL and 16 copies/ ⁇ L heat- inactivated SARS-CoV-2 virus (to reach a final concentration of 200 copies/reaction and 400 copies/reaction), respectively.
  • FIG. 13A and FIG. 14B show that after incubation (60 mm) negative paper pads containing BSA have a yellowish edge. After elution and running the eluent with gel electrophoresis, there was no DNA product visible on the gel, as shown in Figure 13B, indicating that the yellow color at the edge is not caused by off-target amplification or contamination. A heterogeneous distribution of BSA can lead to the yellow color at the edges upon application of heat.
  • Unusual pink color on paper pads During the process of preparing LAMP paper pads, there can be unusual pink spots different from the surrounding color, which can be caused by residual RNase AWAY either directly sprayed onto the pads and/or transferred via the forceps. RNase AWAY can degrade any RNA/DNA template added. If this occurs, thoroughly dry all equipment and surfaces, cut new 5x6 mm paper pads, and restart the ‘LAMP preparation’ section from Operation 5.
  • Overflowing of reagents after pad reconstitution During the sample loading operation, the pad can be unable to absorb the entire sample volume added to it for reconstitution. The template concentration may not be accurately represented by overflowed pads. Overflowing can be caused by insufficient drying of the pads. If this occurs: 1) dry for a longer time, 2) use an enhanced drying method such as heat drying (place on a clean microbiological incubator at 37°C; do NOT set the temperature higher than 45°C to prevent activation of the Bst 2.0 WarmStart® polymerase) or convective diving (use small fans to enhance airflow during drying), or 3) reduce reconstitution volume to 20 ⁇ L.
  • heat drying place on a clean microbiological incubator at 37°C; do NOT set the temperature higher than 45°C to prevent activation of the Bst 2.0 WarmStart® polymerase
  • convective diving use small fans to enhance airflow during drying
  • Negative controls exhibit color change: During the imaging and incubation operation, the negative pads can change at the same time or shortly after the sample- containing pads. This can be caused by either primer dimerization/non-specific amplification or carryover contaminants of previous LAMP reactions. To resolve, validate the primer in liquid-based LAMP prior to using them on paper. To control carryover contaminations, 1 ) implement dUTPs and UDG in all LAMP reactions, 2) maintain separate working stations for LAMP mixture preparation and sample addition, and 3) aliquot reagent stocks and use new aliquots if contamination is suspected to have occurred. Over-incubating the reaction can also induce nonspecific amplification. Do not exceed an incubation time of 75 minutes.
  • buffered salt solution e.g., transport media
  • EXAMPLE 14 Limit of Detection on Paper of untreated saliva with inactivated virus.
  • Fluorescent reporters would use an additional ultraviolet (UV) light source to be read without specialized instrumentation. However, a colorimetric assay using phenol red as an indicator would not use UV light, and can be interpreted by the naked eye. Polymerization of DNA produces protons and phenol red is responsive to pH. Diluted saliva (5% final concentration) was used to overcome the buffering capacity of saliva to measure changes in pH. Diluting saliva to a 5% final concentration also reduced the concentration of interferents (e.g., RNase).
  • interferents e.g., RNase
  • Fresh saliva (5%) with a pH of 6.5 was used to test the effect of carrier DN A along with guanidine chloride with the heat-inactivated SARS-CoV-2 at a concentration range of from 1000 copies to 62.5 copies/reaction (FIG. 6D).
  • Guanidine hydrochloride was reported to increase the sensitivity of LAMP. Its performance with our primer set was tested by adding 40mM of guanidine hydrochloride to the NEB WarmstartTM colorimetric master mix with a pH of 7.6. Pooled saliva (5%) with a pH of 6.5 was used to test the effect of guanidine chloride with the heat-inactivated SARS-CoV-2 with a concentration range of from 1000 copies to 62.5 copies/reaction. This same composition was also tested without adding guanidine chloride as a control. Guanidine chloride increased the replicate sensitivity and has a consistent amplification across the replicate (FIG. 6E).
  • Paper can be scaled up to millions of devices, but when RT-LAMP reagents were placed on paper, the paper changed color even when a negative control was used even though no amplification was occurring.
  • One possibility for this color change is oxidation of cellulose caused by the heat and the oxidizing nature 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. Eliminating ammonium sulfate maintained the color for a negative control. Increasing the concentration of phenol red, W'hich acts as an antioxidant, also maintained the color for the negative control as illustrated in FIG. 11.
  • EXAMPLE 18 Screening of Colorimetric Dyes
  • Three classes of colorimetric indicators were evaluated for a paper-based assay: (i) magnesium colorimetric indicators, (ii) pH colorimetric indicators, and (iii) DNA intercalating colorimetric indicators.
  • Bromothymol Blue (C AS# 76-59-5), Acid Fuchsin (CA S# 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), Phenol red sodium salt (CAS# 34487-61-1), Brilliant yellow (CAS# 3051-11-4), o-Cresolphthalein (CAS# 596-27-0), m-Cresol purple ((LAS# 2303-01-7), m-Cresol purple sodium salt (CAS# 62625-31-4 ), a- Naptholphthalein ((LAS# 596-01-0), and Neutral red ((LAS# 553-24-2) were screened.
  • Crystal violet (CAS# 548-62-9) was screened.
  • 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 whose concentration decreased throughout the RT-LAMP experiment due to the formation of magnesium pyrophosphate, a byproduct of the polymerase reaction.
  • FIG. 17A shows the colorimetric response of calmagite at varying concentrations throughout the LAMP reaction using genomic DNA as template.
  • LAMP detection was performed with increasing concentrations of calmagite (magnesium indicator).
  • the lolB.3 primer set targeting Histophilus somni genomic DNA was used.
  • Positive reactions were spiked with 5 ⁇ L of HS gDNA at a concentration of 0.2 ng/ ⁇ L.
  • Negative reactions used 5 ⁇ L of nuclease-free water. The total reaction volume was 25 ⁇ L.
  • Reactions were prepared using 12,5 ⁇ L of NEB Warmstart 2x master mix, 2.5 ⁇ L or primer mix, and 5 ⁇ L of either template (positive reactions) or water (negative) as above, and 5 ⁇ L of Calmagite prepared in water to produce a final concentration as indicated.
  • LAMP detection was performed with increasing concentrations of Eriochrome® Black T on chromatography paper in PCR tubes.
  • the lolB.3 primer set targeting Histophilus somni genomic DNA was used. Positive reactions were spiked with 5 ⁇ L of H. somni gDNA at a concentration of 0.2 ng/ ⁇ L. Negative reactions used 5 ⁇ L of nuclease-free water. Total reaction volume was 25 ⁇ L.
  • Reactions were prepared using 12.5 ⁇ L of NEB Warmstart 2x master mix, 2.5 ⁇ L or primer mix, and 5 ⁇ L of either template (positive reactions) or water (negative) as above, and 5 ⁇ L of EBT (300 pM) prepared in nuclease-free water.
  • EBT electrospray
  • the reaction consisted of 25 ⁇ L of EBT (300 pM) prepared in nuclease-free water.
  • LAMP detection was performed on multiple papers: chromatography grade 1 , anionic exchange nylon, cationic exchange nylon, poly ether sulfone membrane, asymmetric sub-micron polysulfone (BTS 0.8), asymmetric sub-micron polysulfone (BTS 100) and hydroxylated nylon 1.2.
  • the lolB.3 primer set targeting Histophilus somni (HS) genomic DNA was used. Positive reactions were spiked with 5 iiL of H.
  • Negative reactions used 5 iiL of nuclease-free water. Total reaction volume is 25 gL. Reactions were prepared using 12.5 ⁇ L of NEB Warmstart 2x master mix, 2.5 ⁇ L or primer mix, and 5 ⁇ L of either template (positive reactions) or water (negative) as above, and 5 ⁇ L of EBT (300 itM) prepared in nuclease-free water. Papers (as indicated) were placed in a a PCR tube containing 25 ⁇ L of reaction and was wicked by the paper over the course of reaction. After 60 minutes, the papers were removed and scanned. Gel was extracted using 30.
  • results were generated for overtime in a) PCR tubes, b) gel electrophoresis (2% agarose) of the extracted DNAfrom papers at 60 minutes, and c) scanned papers at 60 mintut.es.
  • the lolB.3 primer set targeting Histophilus somm (US) genomic DNA was used. Positive reactions were spiked with 5 ⁇ L of H. somni gDNA at a concentration of 0.2 ng/ ⁇ L. Negative reactions used 5 ⁇ L of nuclease-free water. Total reaction volume is 25 ⁇ L.
  • Reactions were prepared using 12.5 ⁇ L of NEB Warmstart 2x master mix, 2.5 ⁇ L or primer mix, and 5 ⁇ L of either template (positive reactions) or water (negative) as above, and 5 ⁇ L of EBT (300 pM) prepared in nuclease-free water.
  • Biodyne A amphoteric paper were placed in a PCR tube containing 25 ⁇ L of reaction and was wicked by the paper over the course of reaction. After 60 minutes, the papers were removed and scanned.
  • Reactions were prepared using 12.5 ⁇ L of NEB Warmstart 2x master mix, 2,5 ⁇ L or primer mix, and 5 ⁇ L of either template (positive reactions) or water (negative) as above, and 5 ⁇ L of Crystal violet prepared in nuclease- free water to result in a final concentration as indicated. Papers were placed in a PCR tube containing 25 ⁇ L of reaction and was wicked by the paper over the course of reaction. After 60 minutes, the papers were removed and scanned. Sodium sulfite and cyclodextrin were used to solubilize crystal violet.
  • the RT-LAMP solution was run in a 2% agarose gel, which showed that the positive reactions showed amplification at all tested concentrations of CV, while the negative reactions did not show amplification at any tested concentrations of CV.
  • the color change in the negative reactions was caused by the degradation of LC V to C V, not. because of binding of amplified DNA.
  • RT-LAMP detection was performed with increasing concentrations of cresol red sodium salt, Neutral red, Phenol red sodium salt, m-cresol purple, and m-cresol purple sodium salt in solution (pH indicator).
  • the N. 10 primer set targeting the N gene of SARS-CoV-2 was used. Positive reactions were spiked with 5 ⁇ L of in-vitro transcribed N gene RNA at a concentration of 0.2 ng/ ⁇ L. Negative reactions used 5 ⁇ L of nuclease-free water. Total reaction volume was 25 gL.
  • Reactions were prepared using 12.5 gL of NEB WarmstartTM 2x master mix, 2.5 ⁇ L or primer mix, and 5 ⁇ L of either template (positive reacti ons) or water (negative) as above, and 5 ⁇ L of the indicated pH indicator at the designated concentration in nuclease-free water. Reactions were carried out in an incubator and scanned every 20 minutes using a flatbed scanner.
  • RT-LAMP detection was performed with increasing concentrations of Cresol red, sodium salt, m-cresol purple, Bromothymol blue and Acid fuchsin in solution and b) associated gel electrophoresis (2% agarose) scan of products at 60 mins.
  • the N.10 primer set targeting the N gene of SARS-CoV-2 was used. Positive reactions were spiked with 5 ⁇ L of in-vitro transcribed N gene RNA at a concentration of 0.2 ng/ ⁇ L. Negative reactions used 5 ⁇ L of nuclease-free water.
  • Reactions were prepared using 12.5 ⁇ L of NEB Warmstart 2x master mix, 2.5 ⁇ L or primer mix, and 5 ⁇ L of either template (positive reactions) or water (negative) as above, and 5 ⁇ L of the indicated pH indicator to result in a final reaction concentration as indicated. Heating was carried out in an incubator set at 65 °C and scanned in a flatbed scanner every 20 minutes.
  • cresol red the pH indicator used in NEB’s colorimetric RT-LAMP kit
  • phenol red was also evaluated with respect to varying initial pH values resulting from the addition of HCl and KOH to the solution to provide the indicated initial pH value.
  • RT-LAMP detection with Phenol red at pH 8.1, 8.5, and 8.8 in solution was performed. Adjustments were made with HCl and KOH prior to the addition of template RNA.
  • the N. 10 primer set targeting the N gene of S ARS-CoV-2 was used. Positive reactions were spiked with 5 gL of in-vitro transcribed N gene RNA at a concentration of 0.2 ng/gL. Negative reactions used 5 gL, of nuclease-free water. Reactions consisted of 20 gL master mix and 5 gL of template as described above (positive) or nuclease-free water (negative).
  • pH indicators with a color change around pH 6.5 had the most consistent and the most contrasting color change (e.g., Phenol red).
  • the pH of the LAMP master mix was adjusted to 8.0, 8.5, or remained unadjusted (e.g., 7.6) and the water or synthetic RNA (N gene, 0.2 ng/gL) used for rehydration was also adjusted to 8.0, 8.5, or remained unadjusted (e.g., 5.5).
  • pH 7.6 is the unadjusted pH of the RT-LAMP reaction mixture.
  • Wet setup indicates 5 gL of synthetic RNA (N gene, 0.2 ng/gL, ‘+’ ) or water were added immediately after adding 20 gL of LAMP reaction master mix.
  • Dried setup indicates paper strips were left to dry for 30 minutes at room temperature after applying 20 gL LAMP master mix and then rehydrated with 25 gL synthetic RNA (‘v’) or water (‘-’).
  • FIG. 19B shows colorimetric RT-LAMP results with the inclusion of Trehalose or Tween 20 at the given concentration.
  • a composition for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal that comprises a pH sensitive dye; and a plurality of non-interfering LAMP reagents.
  • LAMP loop-mediated isothermal amplification
  • the pH sensitive dye can be at least one of phenol red, phenolphthalein, azolitmin, bromothymol blue, naphtholphthalein, cresol red, or combinations thereof.
  • the plurality of non-interfering LAMP reagents can be substantially free of volatile reagents, pFI-interfering reagents, magnesium-interfering reagents, or combinations thereof.
  • the plurality of non-interfering LAMP reagents can be substantially free of magnesium, ammonium sulfate, or ammonium carbonate.
  • the plurality of non-interfering LAMP reagents can comprise DNA polymerase, reverse transcriptase, target primers, or combinations thereof.
  • compositions for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal can further comprise an antioxidant.
  • compositions for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal can further comprise carrier RNA, carrier DNA, RNAase inhibitors, DNAase inhibitors, guanidine hydrochloride, or combinations thereof.
  • LAMP loop-mediated isothermal amplification
  • RT-LAMP reverse transcription LAMP
  • compositions for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal can further comprise a solid phase medium.
  • the composition can further comprise a nondiscoloration additive comprising a sugar, a buffer, a blocking agent, or combinations thereof.
  • compositions for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal can further comprise a sugar comprising one or more of trehalose, glucose, sucrose, dextran, or combinations thereof.
  • compositions for loop-mediated isothermal amplification (LAMP) analysis utilizing a pH-dependent output signal can further comprise a blocking agent comprising bovine serum albumin, casein, or combinations thereof.
  • a method of performing a LAMP analysis with a pH- dependent output signal that includes or comprises: providing an assembly of a solid phase medium and a composition as recited herein; depositing a biological sample onto the solid phase medium; and heating the assembly to an isothermal temperature sufficient to facilitate a LAMP reaction.
  • the biological sample can be one or more of saliva, mucus, blood, urine, feces, sweat, exhaled breath condensate, or combinations thereof.
  • the biological sample can be saliva
  • the method of performing a LAMP analysis with a pH-dependent output signal can further comprise detecting a viral pathogen.
  • the method of performing a LAMP analysis with a pH-dependent output signal can be reverse transcription LAMP (RT-LA.MP).
  • a method of maximizing accuracy of an output signal in a pH-dependent LAMP analysis that includes or comprises: providing a reagent mixture that minimizes non-LAMP reaction produced discoloration from a signal output medium; and performing the LAMP reaction.
  • a method of maximizing accuracy of an output signal in a pH-dependent LAMP analysis can comprise controlling production of protons from a non-LAMP reaction
  • a method of maximizing accuracy of an output signal in a pH-dependent LAMP analysis can comprise controlling oxidation from a non-LAMP reaction.
  • a method of maximizing accuracy of an output signal in a pH-dependent LAMP analysis that includes or comprises substantially eliminating non-LAMP reaction produced discoloration from a signal output medium.
  • a method of maximizing a level of detection (LOD) in a pH-dependent LAMP analysis that comprises substantially eliminating non-LAMP reaction produced discoloration from a signal output medium.

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Abstract

La présente divulgation concerne des compositions et des procédés d'analyse d'amplification isotherme induite par boucle (LAMP, « loop-mediated isothermal amplification ») utilisant un signal de sortie dépendant du pH. La composition peut comprendre un colorant sensible au pH et une pluralité de réactifs LAMP non interférents. Le procédé peut comprendre la fourniture d'un ensemble d'un milieu en phase solide et d'une composition, le dépôt d'un échantillon biologique sur le milieu en phase solide et le chauffage de l'ensemble à une température isotherme suffisante pour faciliter une réaction LAMP.
EP22703179.6A 2021-01-15 2022-01-15 Analyse d'amplification isotherme induite par boucle (lamp) pour cibles pathogènes Pending EP4277997A1 (fr)

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