EP4110945A2 - Schneller diagnosetest für lampe - Google Patents

Schneller diagnosetest für lampe

Info

Publication number
EP4110945A2
EP4110945A2 EP21772871.6A EP21772871A EP4110945A2 EP 4110945 A2 EP4110945 A2 EP 4110945A2 EP 21772871 A EP21772871 A EP 21772871A EP 4110945 A2 EP4110945 A2 EP 4110945A2
Authority
EP
European Patent Office
Prior art keywords
lamp
nucleic acid
amplification
sample
target nucleic
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
EP21772871.6A
Other languages
English (en)
French (fr)
Inventor
Nathan TANNER
Yinhua Zhang
Eric Hunt
Gregory Patton
Guoping REN
Zhiru LI
Andrew Barry
Nicole Nichols
Catherine B. POOLE
Harriet M. STRIMPEL
Ivan R. CORREA, Jr.
Clotilde Carlow
Esta Slayton
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.)
New England Biolabs Inc
Original Assignee
New England Biolabs Inc
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 New England Biolabs Inc filed Critical New England Biolabs Inc
Publication of EP4110945A2 publication Critical patent/EP4110945A2/de
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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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

Definitions

  • mNGS is restricted by throughput, turnover time, high costs and requirement for high technical expertise.
  • RT-qPCR requires multiple steps and expensive laboratory instruments and is difficult to utilize outside of well-equipped facilities.
  • a rapid, specific, and sensitive diagnostic single test for one or several pathogens would be desirable to identify and track infected humans, animals or plants in a widespread epidemic that might threaten health and well-being.
  • Target pathogens and particularly RNA viruses, naturally display mutations and changes in their genomic sequences that can impact the sensitivity and accuracy of the molecular diagnostic test when the mutations occur in the regions targeted by the oligonucleotide primers and/or probes.
  • the ongoing SARS-CoV-2 pandemic has seen the emergence of numerous viral variants from different regions of the world, with prominent effects on detection using molecular assays.
  • the B.1.1.7 "alpha" variant features a 6-base deletion (removing 2 amino acids of the spike protein, ⁇ 69-70) which causes a failure of the S Gene assay in the widely used TaqPathTM COVID-19 multiplex RT-qPCR test (Vogels et al.
  • TaqMan® probes rely on specific hybridization to targeted sequences and may display greater sensitivity to mutations than when they occur in primer binding regions; the ⁇ 69-70 deletion occurs in the TaqPath probe region and completely prevents detection of RNA with that deletion.
  • a master mix comprising: a DNA polymerase suitable for isothermal amplification of DNA; dNTPs (dATP, dGTP, dCTP, and dTTP); and at least one dye that changes color or fluorescence in response to DNA amplification.
  • the master mix may be dried or may be in a weakly buffered solution.
  • the master mix is a LAMP master mix.
  • the parameters tested herein may be applicable to master mixes for non-LAMP isothermal amplification reactions.
  • the DNA polymerase in the master mix may be any polymerase suitable for use in an isothermal amplification reaction (e.g., in a Loop-Mediated Isothermal Amplification (LAMP) reaction).
  • Suitable DNA polymerases are known in the art and include strand displacing DNA polymerases preferably mesophilic DNA polymerases such as Bst polymerase or variants thereof or Bsu DNA polymerase or variants thereof.
  • a reversible inhibitor of the DNA polymerase may be included in the master mix.
  • the reversible inhibitor may inhibit the polymerase at temperatures below 50°C.
  • examples of reversible inhibitors include aptamers and antibodies.
  • the at least one dye may be a colored dye detectable in visible light, or may be a fluorescent dye, so long as the dye provides a change in signal (e.g., a change in color, or a change in color intensity or fluorescence intensity) in response to an amplification reaction.
  • a change in signal e.g., a change in color, or a change in color intensity or fluorescence intensity
  • the at least one colorimetric or fluorescent dye is or includes a dye that is pH sensitive.
  • the master mix containing the dye is used in an amplification reaction that alters the pH of the reaction mix, the spectral or fluorescent properties of the dye change (e.g. the dye changes color), which provides confirmation that amplification has occurred.
  • pH sensitive dyes examples include colorimetric dyes such as phenol red, cresol red, m-cresol purple, bromocresol purple, neutral red, phenolphthalein, naphtholphthein, and thymol blue; and fluorescent dyes such as 2',7'-Bis-(2- Carboxyethyl)-5-(and-6)-Carboxyfluorescein or a carboxyl seminaphthorhodafluor (e.g. SNARF-1).
  • the at least one pH sensitive dye is phenol red.
  • the at least one dye is or includes a dye that is not pH sensitive, such as a metallochromic indicator.
  • a metallochromic indicator When the master mx containing the metallochromic indicator dye is used in an amplification reaction that alters the availability of one or more metal ions in the reaction mix, the spectral or fluorescent properties of the dye change (e.g. the dye changes color), which provides confirmation that amplification has occurred.
  • the metallochromic indicator dye is 4-(2-pyridylazo) resorcinol (PAR). If PAR is used, the master mix may additionally comprise manganese ions such that the PAR in the master mix is complexed with Mn ions to form a PAR-Mn complex.
  • a metallochromic dye is hydroxynaphthol blue (Wastling et al. (2010) PLoS Negl Trap Dis 4(11): e865. doi: 10.1371 /journal .pntd.0000865).
  • the visually detectable dye in the reaction mix is in the range of 50 ⁇ M -200 ⁇ M.
  • the master mix for LAMP may optionally further comprise at least one set of primers (e.g., two, three, four, or five sets of primers) having specificity for a target nucleic acid. If multiple primer sets are used, each primer set may target a different nucleic acid sequence within the target nucleic acid (e.g., two or more different viral gene sequences).
  • the master mix may comprise at least two sets of primers, each specific for a different SARS-CoV-2 target sequence (e.g., in the ORF la gene and/or Gene N of SARS-CoV-2). Frequently it is preferred that the primers are provided separately from the master mix.
  • the master mix may comprise a reverse transcriptase, such as an HIV derived reverse transcriptase, an intron encoded reverse transcriptase, or a reverse transcriptase variant of Moloney murine leukemia virus.
  • the master mix may comprise a reversible inhibitor for the reverse transcriptase that inhibits activity of the enzyme at or below 40°C.
  • reversible inhibitors include one or more inhibitory oligonucleotides or antibodies.
  • One or more inhibitors of RNase activity that are not reversible may be included in the master mix for inhibiting RNase activity.
  • the master mix may include an RNAse inhibitor that is not an aptamer.
  • the master mix may comprise dUTP and/or a uracil DNA glycosylase (UDG), such as a thermolabile UDG.
  • UDG uracil DNA glycosylase
  • the master mix optionally comprises a molecule comprising C- (NH 2 ) 2 NH; such as guanidine hydrochloride, guanidine thiocyanate, guanidine chloride, guanidine sulfate, or arginine.
  • the master mix comprises guanidine hydrochloride.
  • the C- (NHz) 2 NH containing molecule can be present in the master mix at a concentration in the range of up to 60 mM, such as in the range of 20 mM-40 mM (e.g., about 20 mM, 30 mM or 40 mM).
  • the master mix does not contain a molecule comprising C- (NHz) 2 NH.
  • a composition comprising C-(NH 2 ) 2 NH; such as guanidine hydrochloride, guanidine thiocyanate, guanidine chloride, guanidine sulfate, or arginine is not included in the master mix may be provided separately such as contained in a lysis buffer or in a mixture containing primer sets for adding to a LAMP reaction mixture.
  • a concentration of guanidine salt in the LAMP reaction mixture is preferably 40mM guanidine salt.
  • the C-(NH 2 ) 2 NH containing molecule can be present in the composition at a concentration in the range of up to 60 mM, such as in the range of 20 mM-40 mM (e.g., about 20 mM, 30 mM or 40 mM).
  • the master mix contains primers that are specific for a target polynucleotide. In other embodiments, the master mix does not contain primers where these are added separately.
  • the master mix may be freeze dried, air dried, or lyophilized.
  • the master mix may be immobilized, for example on paper, or on a natural or synthetic polymer.
  • the dried master mix is reconstituted prior to use in an amplification reaction.
  • the master mix is in solution (e.g., following reconstitution)
  • the master mix is in a weakly buffered solution, such as in a Tris buffer.
  • the weakly buffered solution preferably has a concentration less than 5 mM, such as less than 5 mM Tris or equivalent buffer.
  • the weakly buffered solution is in the range of 0.5 mM to 5 mM, such as 0.5 mM to 5 mM Tris or equivalent buffer.
  • the pH of the master mix may be buffered in the range of pH 7.5-pH 9.0; such as in the range of pH 7.8- pH 8.5, or pH 8.1- pH 8.5.
  • the liquid form of the master mix may be in any suitable reaction container.
  • kits comprising a master mix as described herein.
  • the kit may optionally further comprise a heating block or water bath suitable for heating a reaction tube, plate, or paper, or a plurality of the same to a temperature suitable for isothermal amplification.
  • a method for determining whether a target nucleic acid is present in a sample comprising: bringing an aliquot of the sample into contact with a master mix as described herein to form a reaction mixture wherein the reaction mix additionally includes primers that are specific for the target nucleic acid.
  • the sample may be in an aqueous solution or absorbed to a test matrix such as paper.
  • the master mix may include additional reagents to enhance sensitivity of the assay such as a guanidine salt, or this could be added separately to the reaction.
  • one primer set or a plurality of primer sets may be included in the master mix or added separately to the reaction mix. Not all target specific primer sets work equally well.
  • Determination as to whether the target nucleic acid is present in the sample may then proceed by detecting a change in the spectral properties, color, or fluorescence of the reaction mixture.
  • the method involves isothermal amplification of the target nucleic acid in a LAMP reaction or a helicase-dependent amplification reaction (HDA).
  • the method involves colorimetric LAMP, which may be pH sensitive or may be pH insensitive (e.g., using PAR).
  • the method uses two, three, four, or five sets of target-specific primers in a multiplexed reaction (e.g., multiplexed LAMP); wherein the primers are added to the reaction mixture in the master mix or are added separately.
  • the sample may be a clinical sample, such as a sample of a body fluid (e.g., blood, sputum, saliva, mucous, lymph, sweat, urine, feces, etc.) or a sample taken from a swab such as a nasal, oral, or buccal swab, which may be from a human or other mammalian subject.
  • the sample may alternatively be an environmental sample.
  • the method is performed directly on the sample (e.g., crude tissue or cell lysate, or whole blood) without a step of purifying target nucleic acid from the sample.
  • the sample may be added to a lysis buffer such as exemplified herein for saliva that may nonetheless be suitable for any body fluid.
  • the sample may alternatively be a sample of purified nucleic acid.
  • an aqueous solution may include sterile water or a weak buffer (e.g., 0.5 mM to 5 mM, such as 0.5 mM to 5 mM Tris or equivalent buffer) where the aqueous solution optionally contains a nuclease inhibitor such as an RNase inhibitor or a DNase inhibitors or both.
  • a weak buffer e.g., 0.5 mM to 5 mM, such as 0.5 mM to 5 mM Tris or equivalent buffer
  • a nuclease inhibitor such as an RNase inhibitor or a DNase inhibitors or both.
  • the target nucleic acid may be any DNA or RNA of interest.
  • the nucleic acid may be associated with a pathogen or a diagnostic target for pathogenesis.
  • the target nucleic acid is RNA or DNA of a target pathogen.
  • the target nucleic acid is from a bacterium.
  • the target nucleic acid is from a multi-cellular parasite, such as a parasitic nematode.
  • the pathogen is a virus; for example, an RNA virus, such as a coronavirus.
  • the pathogen may be SARS-CoV-2.
  • the target SARS-CoV-2 RNA sequence is the ORFla gene and/ or Gene N or portion thereof.
  • a method for determining whether a SARS-CoV-2 nucleic acid is present in a sample comprising: bringing an aliquot of the sample in an aqueous solution into contact with a master mix as described herein to form a reaction mixture, wherein the master mix or reaction mixture comprises at least one set of primers specific for a target SARS-CoV-2 nucleic acid; and determining whether the target SARS-CoV-2 nucleic acid is present in the sample by detecting a change in the color or fluorescence of the mixture.
  • the nucleic acid is associated with gene expression, or may be an indicator of a metabolic response to a pharmaceutical preparation or allergen.
  • the method may be for determining a gene expression profile in response to an environmental or metabolic event, or in response to a therapeutic treatment.
  • the target nucleic acid is DNA, and the method is for determining one or more genetic loci correlated to a phenotype.
  • the genetic loci may be selected from the group consisting of a single nucleotide polymorphism (SNP) in a genome, an exon, or a gene in a genome.
  • the method uses a pH-sensitive dye.
  • a pH-sensitive dye During nucleic acid amplification, hydrogen ions accumulate in the reaction mixture so that the mixture becomes increasingly acidic with increasing amplification. pH sensitive dyes change their color, color intensity, or fluorescent intensity, in response to the change in pH in the reaction mixture.
  • the method uses PAR as the dye, and the master mix or the reaction mixture further comprises manganese ions (e.g., about 0.4 mM Mn ions per 100 ⁇ M PAR).
  • the complex of PAR with manganese ions is in a red-colored state.
  • Pyrophosphate produced during the nucleic acid amplification process, as a byproduct of primer nucleic acid polymerization sequesters manganese with a higher affinity than does PAR, thereby removing the manganese from solution and returning PAR to a non-complexed, yellow-colored state.
  • the reaction mixture further comprises a non-ionic detergent such as Triton X-100 (e.g. at about l%-4%, such as about 1% or 2%), which is shown herein to further enhance the color-change observed when using PAR.
  • the concentration of the visually detectable dye in the reaction mix may be in the range of 50 ⁇ M -250 ⁇ M; such as at 50 ⁇ M -150 ⁇ M, for example at about 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, or 150 ⁇ M.
  • the reaction mixture further comprises a molecule comprising C-(NH 2 ) 2 NH; such as guanidine hydrochloride, guanidine thiocyanate, guanidine chloride, guanidine sulfate, or arginine.
  • the molecule is guanidine hydrochloride.
  • the C-(NH 2 ) 2 NH containing molecule can be added to the reaction at a concentration of up to 60 mM, such as in the range of 20 mM -40 mM (e.g., about 20 mM, 30 mM, or 40 mM).
  • the sodium chloride concentration in the reaction mixture is preferably less than 40 mM; for example, the reaction mixture may contain NaCI at a concentration of about 20 mM or about 10 mM.
  • the reaction mixture may alternatively, or additionally, comprise KCI at a concentration of less than 100 mM (e.g., less than 40 mM).
  • the method may comprise the step of combining one or more RNase inhibitors and/or thermolabile Proteinase K with the sample, prior to combining the sample with the master mix to form the reaction mixture.
  • one or more RNase inhibitors and/or thermolabile Proteinase K can be added to the reaction mixture together with the sample, in which case the thermolabile Proteinase K should be inactivated prior to adding the master mix.
  • the method may comprise adding a reverse transcriptase to the reaction mixture (either via the master mix, or separately) if the target nucleic acid is RNA, such as viral RNA.
  • the reaction mixture may further comprise dUTP and UDG (e.g., thermolabile UDG), which may be added to the reaction mixture from the master mix. Alternatively, the dUTP may be added to the reaction mixture from the master mix, and the UDG may be added separately to the reaction mixture.
  • the method comprises analyzing multiple samples.
  • the method may use a reaction container that has multiple compartments each for analyzing a separate sample.
  • the master mix is dried and immobilized onto e.g., paper, and an aliquot (e.g., droplet) of the sample is added to the paper, followed by a heating step, resulting in amplification of target nucleic acid in the sample.
  • an aliquot e.g., droplet
  • the change in the spectral or fluorescent properties of the dye can be detected by eye or using a spectrophotometer or fluorimeter or recorded by means of a camera or other color sensitive recording device.
  • the method involves comparing the spectral or fluorescent properties of the dye before and after amplification has occurred.
  • the change in spectral properties or fluorescence of the mixture can be recorded by a spectrophotometer having dual wavelength capabilities, digitized, and stored by a computer.
  • composition comprising: one or more primer sets suitable for amplification, such as for an isothermal amplification reaction such LAMP, the primer sets having specificity for a single target nucleic acid of interest; and a buffer containing a molecule comprising C- (NH 2 ) 2 NH.
  • the composition comprises two, three, four, or five primer sets, each having specificity for a single target nucleic acid of interest; such as a viral RNA sequence (e.g., SARS- CoV-2 RNA).
  • the composition comprises C-(NH 2 ) 2 NH such as selected from guanidine hydrochloride, guanidine thiocyanate, guanidine chloride, guanidine sulfate, or arginine.
  • the molecule is present in the composition at a concentration in the range of up to 60 mM, such as in the range of 20 mM-40 mM (e.g., about 20 mM, 30 mM, or 40 mM).
  • the composition may further comprise one or more reagents selected from a DNA polymerase, such as Bst polymerase; a reverse transcriptase; and an RNAse inhibitor.
  • the composition may comprise dNTPs, which may optionally include dUTP; and/or may comprise a thermolabile UDG (also referred to herein as uracil deglycosylase).
  • the composition may further comprise a reporter molecule for detecting amplification in the presence of a target nucleic acid; for example, a colorimetric or fluorescent dye as described herein (e.g., PAR).
  • a method of isothermal amplification comprising: (a) adding any embodiment of the master mix described herein that contains a suitable polymerase, and dNTPs to a sample comprising a target nucleic acid to form a reaction mixture, and in the presence of suitable primers allowing amplification to occur; and (b) detecting whether the target nucleic acid is present in the sample.
  • the target nucleic acid may be as described above.
  • the target nucleic acid is a viral nucleic acid such as viral RNA.
  • the target nucleic acid may be a SARS-CoV-2 RNA.
  • the target SARS-CoV-2 RNA sequence is located within the ORFla gene and/or Gene N.
  • the molecule comprising C-(NH 2 ) 2 NH is present in the reaction mixture at a concentration of up to 60 mM, such as in the range of 20 mM-40 mM (e.g. about 20 mM, 30 mM or 40 mM).
  • the reaction mixture may further comprise NaCI at a concentration of less than 40 mM; such as at a concentration of about 20 mM.
  • the reaction mixture may alternatively, or additionally, comprise KCI at a concentration of less than 100 mM (e.g., less than 40 mM).
  • a method for detecting amplification of a target nucleic acid comprising: providing an amplification reaction mixture containing a target nucleic acid and a master mix or composition as defined herein; and detecting a change in the spectral or fluorescent properties of the dye resulting from amplification of the target nucleic acid.
  • the target nucleic acid may be as described above.
  • the target nucleic acid is a viral nucleic acid such as viral RNA.
  • the target nucleic acid may be a SARS-CoV-2 RNA.
  • Embodiments describe the use of immobilized reagent and lyophilized reagents in receiving vessels for saliva and other body fluids to assist in streamlining workflows and improving sensitivity of assays.
  • a kit is provided for Loop-Mediated Isothermal Amplification (LAMP), that includes either separately or combined in a mixture: (a) a strand displacing polymerase, for example, a Family A DNA polymerase, for example a mesophilic bacterial strand displacing polymerase, for example a bacillus family A strand displacing DNA polymerase capable of copying DNA at a temperature in the range of 50°C-68°C; (b) a reversible inhibitor of the polymerase, for example, an oligonucleotide reversible inhibitor also referred to as an aptamer that binds and inactivates the polymerase at temperatures at or below 50°C but is released from the polymerase at temperatures above 50°C, permitting specific amplification with reduced background; (c) a thermolabile UDG that is inactivated at a temperature of above 50°C, a temperature that is preferably lower than the temperature at which the polymerase is active and isothermal
  • the kit may comprise in addition to the above components, a reverse transcriptase for example a virus derived reverse transcriptase, or an intron expressed reverse transcriptase and a reversible inhibitor of the reverse transcriptase that permits the reverse transcriptase to be active above 50°C but not below 50°C so as to reduce background and enhance sensitivity of the LAMP amplification.
  • a reverse transcriptase for example a virus derived reverse transcriptase, or an intron expressed reverse transcriptase
  • a reversible inhibitor of the reverse transcriptase that permits the reverse transcriptase to be active above 50°C but not below 50°C so as to reduce background and enhance sensitivity of the LAMP amplification.
  • the reversible inhibitor of the reverse transcriptase and the strand displacing polymerase should not adversely affect either the effectiveness of each inhibitor or the desired activity of either enzyme at the desired temperature.
  • the kit may additionally contain a receiving container for a biological sample.
  • the receiving container may be a vessel with a lid or a paper, a microfluidic device or a polymer surface to which the reagents and/or sample are attached and suitable for absorbing reagents or sample in a liquid form.
  • the receiving container may be a vessel for combining reagents in a liquid form with samples in a liquid form.
  • the kit may also contain lysis reagents for combining with the biological sample prior to performing LAMP with the kit reagents. These lysis reagents are for adding to or contained in the receiving container for lysing a biological sample to release any target nucleic acids therein for amplification by LAMP.
  • the lysis reagents may include a reducing agent such as Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and a metal chelator such as EDTA.
  • the lysis mixture may further include a salt of C-(NH 2 ) 2 NH + .
  • the lysis mixture may further include a poloxamer. Additionally, the kit may contain at least one set of LAMP primers.
  • the kit may contain a plurality of primer sets for amplifying a plurality of target nucleic acids in the biological sample such as for example different respiratory RNA viruses.
  • the kit may contain a plurality of primer sets for amplifying a single nucleic acid target such as a viral genome such as Gene N and Gene E in SARS-CoV-2 in the biological sample.
  • the kit may contain a plurality of primer sets for amplifying a single or multiple target nucleic acids from single or multiple samples. Pooling samples is one form of high throughput analysis of populations having a low infection rate with a pathogen.
  • any of the amplification reagents or the reagents in the lysis mix may be freeze dried or lyophilized preferably excluding salts of C-(NH 2 ) 2 NH + .
  • any of the specified enzyme and/or oligonucleotide reagents may be immobilized or incorporated on a matrix. If any of the reagents are supplied in a buffer for use with pH dependent colorimetric LAMP, then the buffering capacity should be no greater than equivalent to 5 mM Tris.
  • the kit provided herein may include instructions for use with a biological sample where the biological sample is selected from a body fluid or tissue, an agricultural sample, a food sample, a waste product, and a pathogen for example, the biological sample is a body fluid or tissue selected from the group consisting of mucous, urine, lymph, blood, saliva, feces, sputum, sweat, semen and biopsy, for example a saliva or a nasal swab.
  • the instructions provide how to test for a target RNA genome in a single viral strain or in multiple viral strains or from multiple samples from a population of subjects.
  • the kit may contain a receiving container for a biological sample, that includes a vessel with a lid, the lid containing a solution of indicator reagent for release into the reaction vessel after lysis of the biological sample or after LAMP.
  • Instructions for use of the kit may include a method for high sample throughput workflow enabled by the kit that is partially or completely automated and further comprises a recording device for storing and/or reporting positive sample data after detection of a change in color or fluorescence of the indicator resulting from amplification of the target nucleic acid.
  • a reaction mixture in one aspect, includes a thermolabile UDG, a strand displacing polymerase, a reversible inhibitor of the polymerase, and a salt of C-(NH 2 ) 2 NH + .
  • a lysis mixture for releasing an RNA from a biological sample for detection by amplification and/or sequencing, comprising a poloxamer, a reducing agent and a metal chelating agent.
  • the lysis mixture may further include a salt of C-(NH 2 ) 2 NH +
  • a master mix in another aspect, includes a thermolabile UDG, a strand displacing polymerase, a reversible inhibitor of the polymerase, a reverse transcriptase and a reversible inhibitor of the reverse transcriptase.
  • a method for amplifying any target nucleic acid in a biological sample by LAMP includes: (a) combining the biological sample with a lysis reagent to form a lysis mix; (b) incubating the lysis mix at a temperature of at least 60°C for a period of time in the range of 3 minutes to 45 minutes; (c) combining an aliquot of the heat treated mix after step (b) with amplification reagents that include a strand displacing polymerase, a reversible inhibitor of the polymerase, a thermolabile UDG, nucleoside triphosphates, and at least one set of LAMP primers that hybridize to the target nucleic acid, to produce a reaction mix; and (d) incubating the reaction mix under amplification conditions for LAMP to permit inactivation of the thermolabile UDG and amplification of the target nucleic acid.
  • the amplification reagents may further include a reverse transcriptase and a re
  • the lysis reagent in (a) may include at least one of a salt of C-(NH 2 ) 2 NH + and a poloxamer to produce a lysis mix.
  • the lysis reagent may also include a reducing agent and a metal chelating reagent.
  • the lysis mix may be incubated at 95°C for 5 minutes to break open the sample and release target nucleic acid.
  • any of the reagents in the lysis mix may be immobilized and/or lyophilized or freeze dried. Prior to the addition of the biological sample.
  • the method may comprise a further step of determining if amplification has occurred by measuring fluorescence and/or color changes of one or more indicators in the amplification reagent mix.
  • the biological sample may be saliva
  • the target nucleic acid may be an RNA virus such as a coronavirus
  • the amplification reagents may include a reverse transcriptase and a reverse transcriptase reversible inhibitor single or multiple sets of LAMP primers for amplifying at least two sequences in a target DNA or a cDNA of an RNA target such as Gene E and Gene N sequences in SARS-CoV-2, and/or for amplifying multiple target nucleic acids from different viruses and/or for amplifying target nucleic acids from different biological samples or from different animal subjects.
  • a method for analyzing a biological sample to determine the presence of a target nucleic acid includes: (a) combining the biological sample with a lysis reagent comprising a reducing agent, a metal chelator and at least one of a guanidine salt and a poloxamer to produce a lysis mix; and (b) determining the presence of the target nucleic acid by selectively amplifying, by means of, for example, LAMP, the target nucleic acid in a reaction mix that comprises an aliquot of the lysis mix.
  • the reaction mix in (b) may include an indicator reagent that changes color or provides fluorescence if amplification occurs, and the method may include the additional step of determining whether the reaction mix contains the target nucleic acid based on a change in color or fluorescence.
  • a method for detecting an RNA virus in saliva or a nasal swab of a subject includes (a) collecting saliva in a receiving container that comprises: (i) substrate immobilized oligonucleotides for binding viral RNA and a lysis reagent mix comprising two or more reagents (for example, 3 or more reagents) selected from a guanidinium salt, a poloxamer, a reducing agent, a DNase inhibitor, an RNase inhibitor, a detergent, a metal chelator and a proteolytic agent; or (ii) lyophilized substrate immobilized oligonucleotides for binding viral RNA, and/or one or more lyophilized reagents contained in a lysis reagent mix, wherein the lysis reagent mix comprises two or more reagents (for example, 3 or more reagents) selected from a poloxamer, a reducing agent, DNase inhibitor, an RNase inhibitor,
  • the receiving container may be selected from a paper substrate, a microfluidic device or a polymer surface.
  • the reaction mix of (d) in this method may further include an indicator reagent that changes color or provides fluorescence if amplification occurs, and wherein the method further comprises detecting a change in a signal that indicates the presence of coronavirus in the saliva of the subject.
  • the method may include amplification reagents for LAMP as specified in the kit above and single or multiple primer sets as specified above also.
  • the amplification reagents may include LAMP primer sets targeting a second viral genome that is not a coronavirus wherein the LAMP primer sets are combined in a single reaction mix and wherein the LAMP primer set for the coronavirus is linked to a colorimetric indicator that changes color after amplification that is detectable at one wavelength and a second LAMP primer set for amplifying a second non coronavirus nucleic acid, having a colorimetric or fluorescent indicator that changes color after amplification that is detectable at a second wavelength.
  • One embodiment provides for a method for amplifying a target nucleic acid by LAMP, that includes: (a) combining in a mixture, a biological sample from a mammalian subject with a buffer comprising a poloxamer; (b) heating the mixture to a temperature of at least 65°C for an effective time to denature proteins in the biological sample; (c) allowing the sample to cool; and (d) amplifying one or more nucleic acids from the mix by LAMP where the biological sample may be saliva, a nasal swab or a buccal swab.
  • a kit for use in diagnostic detection of a target nucleic acid and variants thereof having undefined mutations, obtained from a cell or virus in a biological sample.
  • the kit may include (a) a lyophilized mixture of a strand displacing polymerase and an indicator reagent and optionally a lyophilized reverse transcriptase, wherein (i) the indicator reagent is characterized by its ability to change color or provide fluorescence in a nucleic acid amplification reaction; and (ii) the strand displacing polymerase when rehydrated is capable of amplifying DNA at a temperature in the range of 50°C-68°C; and (b) a universal primer set suitable for loop mediated amplification (LAMP) of the target nucleic acids and variants thereof containing undefined mutations within one or more of the primer binding sites.
  • Any of the reagents in the kit may be combined in a mixture in a single container or provided in separate containers.
  • the universal primer set as defined below is preferably suitable for LAMP and is capable of hybridizing to the target DNA in the presence of a plurality of undefined mutations to provide a positive result for the target DNA in a predetermined assay time period otherwise determined for a positive sample of a target nucleic acid having a known sequence.
  • the universal primer set may be similarly diagnostic for the target nucleic acids and variants thereof where deletions and additions in the BIF and FIP primer binding sites of the variants do not exceed 6-9 nucleotides.
  • the target DNA is the reverse transcription product of an RNA virus, for example, a coronavirus.
  • the indicator reagent is a molecular beacon.
  • the kit may include (c) lysis reagent in a container for receiving the biological sample, wherein the lysis reagents comprise a reducing agent and a metal chelator.
  • the lysis reagent may include the reducing agent is Tris (2-carboxyethyl) phosphine hydrochloride (TCEP).
  • TCEP Tris (2-carboxyethyl) phosphine hydrochloride
  • the lysis reagents may include at least one of a salt of C-(NH 2 ) 2 NH + and a poloxamer.
  • the kit may include one or more of components in (a)-(b) are immobilized on a substrate.
  • the lyophilized indicator reagent may be a metallochromic dye or a molecular beacon.
  • the reverse transcriptase may be a virus encoded reverse transcriptase, or a bacteria encoded intron II reverse transcriptase.
  • a method for detecting a target nucleic acid or unknown variant thereof in a biological sample by LAMP that includes combining the biological sample with a lysis reagent to form a lysis mix; incubating the lysis mix at a temperature of at least 60°C for a period of time in the range of 2 minutes to 45 minutes; combining in a reaction mix, an aliquot of the heat treated lysis mix with amplification reagents comprising a strand displacing polymerase, a reversible inhibitor of the polymerase, nucleoside triphosphates, and at least one set of LAMP primers that is capable of hybridizing to the target nucleic acid and to undefined variants of the target nucleic acid; and incubating the reaction mix for a reaction positive period of time under amplification conditions for LAMP to detect the presence of the target nucleic acid or undefined variants thereof in the sample.
  • the lysis reagent in the method may include a reducing agent and a metal chelating reagent. Once combined with the biological sample, the lysis mix may be heated to 95°C for 5 minutes.
  • the method may include a reverse transcriptase and a reversible inhibitor of the reverse transcriptase in the amplification reagents.
  • Any of the amplification reagents and/or lysis reagents may be lyophilized prior to combining with the biological sample.
  • Any of the amplification reagents and/or lysis reagents may be immobilized on a matrix prior to or during the method.
  • An example of a biological sample is saliva for detecting a target nucleic acid such as an RNA virus for example, a coronavirus is saliva.
  • FIGs. 11A, 13A-13D, and 14 describe color changes from metallochromic dyes where colors change from brown/orange to yellow. These colors are represented by dots that vary in density according to whether the reaction is positive (high density) or negative (1 or 2 dots) or somewhere in between.
  • FIG. 1 shows detection sensitivity of synthetic SARS-CoV-2 RNA amplicons by a pH dependent colorimetric LAMP assay using phenol red as the indicator that changed color from red to yellow when amplification of a target nucleic acid occurred, causing the pH of the reaction mixture to decrease.
  • 5 sets of LAMP primers (1a-A, 1a-B, N-A, N-B, and la-C) were tested with templates ranging from 120x10 s to 120 copies of viral RNA (Twist Biosciences), or a no-template control (NTC). Yellow, positive amplification; pink, no amplification.
  • FIG. 2A - FIG. 2B shows that RNA can be detected as efficiently as DNA using pH dependent colorimetric LAMP (New England Biolabs, Ipswich, MA (M1800)) using the primers described in Example 1 that targeted two different SARS-CoV-2 sequences.
  • pH dependent colorimetric LAMP New England Biolabs, Ipswich, MA (M1800)
  • RNA and gBIock double-stranded DNA (dsDNA) templates in LAMP amplification is shown using real time amplification curves.
  • Two primer sets (ORFla-A in FIG. 2A and Gene N-A in FIG. 2B) were used to amplify either RNA (green curves, dilutions from 120x10 s to 120 copies) or gDNA (blue curves, 60x10 s to 60 copies).
  • RNA green curves, dilutions from 120x10 s to 120 copies
  • gDNA blue curves, 60x10 s to 60 copies.
  • For ORFla-A primer set the gBIock is faster than RNA template; for Gene N-A primer set, the RNA is slightly faster.
  • Each "cycle" represents 20 seconds, with 30 minute timepoint noted by dashed line.
  • 3B shows that pH dependent colorimetric LAMP can detect viral genomes in total cell lysates and whole blood without requiring a purification step to remove total RNA.
  • the primer sets described in Example 1 were used here. Lysis was performed using a cell lysis reagent (Luna® Cell Ready (New England Biolabs, Ipswich, MA)).
  • FIG. 3A shows direct RNA LAMP detection using total cell lysate. The approximate maximum number of copies of synthetic RNA added to each LAMP reaction is shown.
  • the viral RNA was spiked into a Hela cell lysate. For 4800 copies of viral RNA, there were about 200 Hela cells present. NC, no cell, and no template control.
  • FIG. 3B shows colorimetric LAMP detection of various amounts of target RNA spiked in whole blood. The number of copies of the target RNA that could be detected are shown. In a control, 5 ng of Jurkat total RNA was added, which is similar to the total RNA present in the reaction with blood samples.
  • FIG. 4A - FIG. 4B shows the detection of various closely related nematode parasites was achieved for as little as 0.01 pg of nematode DNA in an environmental sample using the colorimetric LAMP described in Example 1.
  • the control was a reagent mix absent nematode sample.
  • Example 1 describes the test for an RNA virus, the same methodology applies to detecting the DNA from the various nematodes using an appropriate set of primers. As indicated in the figure, the assay could detect 0.01 pg-0.1 pg of nematode parasite DNA.
  • FIG. 5A- FIG. 5B shows that pH colorimetric LAMP is a useful diagnostic tool for detecting tick borne pathogens as it is both sensitive and specific for the target.
  • FIG. 5A shows detection of 1.28 fg target DNA from a specific target tick borne pathogen, with negative results (pink) from samples of non-target tick borne pathogens and hosts.
  • FIG. 5B shows specificity for the target DNA with negative (pink) results for non-target DNA.
  • no template DNA control 2) pathogen target DNA; 3) DNA from other tick-borne pathogen 1 (negative control); 4) DNA from other tick-borne pathogen 2 (negative control); 5) DNA from other tick-borne pathogen 3 (negative control); 6) tick DNA; 7) mosquito DNA; 8) DNA from other tick-borne pathogen 4 (negative control).
  • FIG. 6A shows that pH-dependent colorimetric LAMP sensitivity is unaffected by the presence of dUTP and UDG.
  • Two CP-LAMP MM contained a 50/50 mixture of dUTP/dTTP replacing dTTP.
  • One CP- LAMP MM did not contain UDG (no UDG).
  • One CP-LAMP MM (with UDG) includes 0.02 U/ ⁇ L thermolabile uracil DNA glycosylase (UDG) (New England Biolabs, Ipswich, MA).
  • a third LAMP MM contained neither UDG nor dUTP. In all other respects the same protocols were followed as described in Example 1 and in the figures above.
  • FIG. 6B shows that carryover of polynucleotide substrate is effectively prevented by including dUTP in the LAMP reaction prevented over 10 fold dilutions of the dll template from a first sample to a second master mix that does not contain any target nucleic acid. Each sample from left to right is a 10 fold dilution of the previous sample. At 50 fold -60 fold dilution, the carryover material was destroyed by the thermolabile UDG (0.02U/ ⁇ l). Carryover prevention (CP) was determined in CP-LAMP MM.
  • FIG. 7 shows that lyophilized pH-dependent LAMP MM is equally effective when compared to non-lyophilized LAMP MM stored at -20°C.
  • the starting pH of the lyophilized LAMP when reconstituted was reduced by 0.25 units in this example.
  • FIG. 8A-FIG. 8C shows real time detection of target RNA (Jurkat total RNA) using a lyophilized LAMP MM and an HMBS2 primer set and a -20°C storage preparation of LAMP MM.
  • the LAMP MM contained the fluorescent dye (Syto-9) for following amplification.
  • FIG. 8D provides a comparison of the rate of LAMP using a previously lyophilized LAMP MM or a MM stored at -20°C.
  • FIG. 9A-FIG. 9C shows that no difference in sensitivity of the LAMP reaction was observed using a LAMP MM versus -20°C stored LAMP MM for DNA and RNA analyses, where RNA analysis additionally required a reverse transcriptase in the MM.
  • Fig. 9A and 9B showed a color change with phenol red while FIG. 9C used a fluorescent dye to detect amplification.
  • WarmStart® LAMP Kit (DNA & RNA) is provided New England Biolabs.
  • FIG. 10 shows the mechanism of the colorimetric response for 4-(2-pyridylazo) resorcinol (PAR).
  • PAR is a known metallochromic indicator which, in the absence of metals in solution, exhibits a yellow color. When complexed with manganese in solution PAR produces a red color.
  • pyrophosphate is produced as a by-product of primer nucleic acid polymerization.
  • the pyrophosphate by-product sequesters manganese with a higher affinity than does PAR, resulting in the dissociation of Mn from PAR and thereby returning PAR to a yellow-colored state.
  • FIG. 11A and FIG. 11B shows that because pyrophosphate exhibits a higher affinity for manganese ions than PAR, this property can be used to detect amplification of nucleic acids using LAMP. Pyrophosphate generated during nucleic acid polymerization precipitates manganese from solution, thus disrupting the PAR:Mn complex and restoring the yellow color. This is demonstrated spectroscopically by spiking in pyrophosphate to restore the yellow color (bottom row) (FIG. 11A). The color change can be further enhanced by the addition of Triton X-100 (FIG. 11B).
  • FIG. 12 shows that PAR has been demonstrated to be compatible with use in microfluidic paper- based analytical devices ( ⁇ PADs) (Meredith, et al. Anal. Methods (2017) 9, 534-540).
  • FIG. 12B shows the results of a paper-based spot test showing metal-PAR reactivity (orange and red color formation) for a number of transition, alkali, and alkaline earth metals.
  • FIG. 13 shows that PAR provides the means of a colorimetric endpoint for LAMP in a LAMP MM.
  • a color change is observed when precipitation of manganese occurs that is caused by the release of pyrophosphates in a LAMP reaction.
  • the enzymes added to perform LAMP in the presence of resorcinol and conditions of the reaction are as follows:
  • PAR concentrations left to right 150 ⁇ M, 100 ⁇ M, 75 ⁇ M, 50 ⁇ M;
  • Mn 2+ concentration 0.5mM MnCl 2 ;
  • Target polynucleotide is the BRCA gene in Hela cell genomic DNA.
  • the non-template control retains the red color of PAR bound to manganese ions while the positive sample turned yellow corresponding to the reaction of manganese with pyrophosphate.
  • FIG. 14 shows the strong color reaction of a PAR-based LAMP in the presence of 2% Triton X-100 and 0.5 mM MnCl 2 , Bst 2.0 polymerase, IX Isothermal Amplification Buffer, 200 ⁇ M PAR and the BRCA2b primer set using 1 ⁇ l Hela cells.
  • the non-template control retains the red color of PAR bound to manganese ions while the positive sample turned yellow corresponding to the reaction of manganese with pyrophosphate.
  • FIG. 15 shows examples of 4 different guanidine salts (also called guanidinium salts) for enhancing the LAMP reaction.
  • FIG. 16 shows that guanidine hydrochloride (GuCI) increases the reaction speed of a LAMP amplification reaction. Detection of two different genes-BRACA and CFTR were achieved using LAMP MM and increasing concentrations of GuCI.
  • FIG. 17A-17C shows that GuHcl was effective in increasing the rate of isothermal amplification reactions and this effect was enhanced by selecting a range of concentrations for NaCI or KCI in the reaction buffer.
  • FIG. 17A shows the results of standard HDA reactions in IsoAmp II kit (H0110) and 0.1ng plasmid, with 10mM NaCI versus 40mM NaCI, in which guanidinium hydrochloride was added at a final concentration of 0 mM-60 mM.
  • the reactions were performed at 65°C and EvaGreen® dye (Biotium, Inc., Hayward, CA) was included to monitor the progression of amplification.
  • the effect of reducing NaCI concentrations was most noticeable at higher concentrations of GuCI (30 mM-60 mM guanidine hydrochloride) resulting in a reduction of Time to threshold (Tt) of 35 minutes (40mM NaCI) to 12.3 minutes (10 mM NaCI).
  • FIG. 17B shows an increase in the rate of amplification using the LAMP assay described in Example 1 and Bst 2.0 DNA polymerase with a Iambda2 primer set and 0.5 ng lambda DNA in ThermoPol buffer containing lOmM KCI plus or minus 30 mM guanidine hydrochloride.
  • the addition of guanidine stimulated the LAMP amplification rate significantly at the lower end of the KCI concentration (less than 40 mM KCI).
  • FIG. 17C shows an increase in the rate of amplification using the LAMP assay described in Example 1 and Bst 3.0 DNA polymerase (also referred to as BTB3) with a Iambda2 primer set and 0.5 ng lambda DNA in an isothermal amplification buffer containing 50 mM KCL plus or minus 30 mM guanidine hydrochloride.
  • BTB3 Bst 3.0 DNA polymerase
  • the addition of guanidine stimulated the LAMP amplification rate significantly at the lower end of the KCI concentration (less than 100 mM KCI).
  • FIG. 18A-18D shows that guanidine hydrochloride not only increases LAMP reaction speed but also improves the limit of detection sensitivity.
  • FIG. 18A shows that colorimetric LAMP could detect 100 copies of synthetic SARS-CoV-2 RNA with 40mM guanidine hydrochloride using primer set 1. The color change from pink to yellow indicates a positive detection. "None” denotes no guanidine hydrochloride. In the presence of 40 mM guanidine hydrochloride, 8/8 positive reactions were detected, whereas 5/8 positive reactions were detected without it.
  • FIG. 18B shows the results of real time colorimetric LAMP using primer set 1 in the presence and absence of 40 mM Guanidine HCI.
  • the reaction also contains 1 ⁇ M dsDNA binding dye Syto-9 for monitoring the real time progression of the amplification.
  • FIG. 18C shows the results with 4 different primer sets. In all cases, sensitivity was increased in the presence of guanidine hydrochloride. The percentage of positive reactions for detecting 100 copies of SARS-CoV-2 RNA is shown in the table. The table shows that guanidine improves the detection sensitivity of all primer sets.
  • FIG. 18D shows a diagram of SARS-CoV-2 with the location of 2 template sequences (E and N) in the target nucleic acid.
  • FIG. 19A-19C shows that guanidine allows efficient multiplexing of LAMP amplifications with multiple LAMP primer sets in the same reaction without adverse effects on the rate of amplification while significant improvements in sensitivity were observed.
  • FIG. 19A shows sensitivity of pH colorimetric LAMP by the percentage of positive samples detected using single sets of primers (identified as sets 3 and 4) and when sets 3 and 4 are combined in the presence or absence of 40 mM guanidine hydrochloride in a 40 minute incubation.
  • the tables shows that 92.2% positives were detected for known test samples containing 50 copies synthetic SARS-CoV-2 RNA using a combined set of primers 3 and 4 with guanidine, compared with 28% for single sets of primers in the absence of guanidine.
  • FIG. 19B shows the sensitivity of pH colorimetric LAMP for detecting 12.5 copies of synthetic SARS-CoV-2 RNA in the presence of guanidinium hydrochloride and a plurality of primer sets.
  • the results shows an increase of detection rate with any combinations of 2 primer sets (3+4, 3+5, 4+5).
  • the reactions including all 3 primer sets (3+4+5) also showed further increase of detection rate over any 2 primer sets, providing detection of 57% of all positives in a 40 minute incubation.
  • the reactions without template remained negative and showed no sign of amplification signal, indicating robust specific amplification.
  • FIG. 19C shows that real time amplification with guanidine hydrochloride resulted in an expected rate of amplification with a combination of primer sets 3 and 4 and 50 copies of template CV- 19 RNA where the combination of primer sets did not adversely affect the rate of amplification.
  • FIG. 20 shows an example of the use of lysis buffer for SARS-CoV-2 detection in saliva
  • 900 ⁇ l of saliva from a patient is added to 100 ⁇ l of 10X lysis buffer (1), mixed (2) and then heated to 95°C for 5 minutes (3).
  • 2 ⁇ l of the sample is then added to 18 ⁇ l of pH colorimetric LAMP Master mix either containing primers to SARS-CoV-2 (target) or primers for actin (control) (4). After an incubation for 45 minutes at 65°C, the test tubes were examined for a color change from pink to yellow indicative of the presence of SARS-CoV-2 (5).
  • FIG. 21A-21B shows that various ratios of the reagents in the lysis buffer spiked with synthetic SARS-CoV-2 RNA were tested to determine which combination if any interfered with pH colorimetric LAMP and if not which conditions provided the greatest sensitivity for detecting 40 copies of the virus genome.
  • the results did not suggest any interference and the saliva lysis mixture containing 4 mM TCEP (reducing agent) and 75 mM LiCI at pH 8.0 with 400 mM guanidine hydrochloride (GnHCL) gave the best results.
  • FIG. 21A shows 6 different conditions for the saliva lysis buffer.
  • FIG. 21B shows the color changes indicative of a positive result under the 6 different test conditions where 4 mM TCEP (reducing agent) and 75 mM LiCI at pH 8.0 with 400 mM guanidine hydrochloride (GnHCL) resulted in 100% detection of 40 copies of viral genome.
  • FIG. 22 shows that 8 mM TCEP in addition to 75 mM LiCI and 400 mM GnHCL performed similarly to 4 mM TCEP in the saliva lysis buffer when 5 ⁇ l 10X lysis buffer was added to 45 ⁇ l saliva sample containing inactive virus particles (SeraCare) and heated for 5 minutes at 95°C. 2 ⁇ l of this sample was then added to 18 ⁇ l LAMP master mix and incubated for 35 minutes at 65°C.
  • inactive virus particles SeraCare
  • FIG. 23A-23D shows the effect of varying the LAMP assay time after the 5 minute saliva lysis reactions on saliva spiked with 10,000 cps/ml synthetic SARS-CoV-2 RNA (20 copies/2 pl) using a saliva lysis buffer containing 8 mM TCEP, 0 mM LiCI/75 mM LiCI and 400 mM GnCL. Increased sensitivity was observed over time with the presence of LiCI consistently contributing to increased sensitivity as the time of incubation increased beyond 35 minutes. Saliva not containing RNA and H 2 O were used as negative controls.
  • FIG. 24 shows that when a saliva sample spiked with a known copy number of synthetic SARS- CoV-2 input (Twist) is treated with saliva lysis buffer (8 mM TCEP, 75 mM LiCI and 400 mM GnCI), followed by RT-qPCR, the lysis buffer was shown to have minimal or no adverse effect on the RT-qPCR reaction.
  • FIG. 25 shows that the lysis buffer described in FIG. 23A-23D and FIG. 24 can provide similar sensitivity of virus detection as that reported by others after purification of the viral RNA from nasopharyngeal swabs and saliva (Wyllie et al. MedRxiv April 22, 2020: https://doi.org/10.1101/2020.04.16.20067835).
  • FIG. 26 shows that endpoint absorbance ratio (432 nm/560 nm wavelengths) at a range of pH from pH 4 - pH 11 can be measured by a colorimeter.
  • the highest positive signal at 560 nm is between pH 9 - pH 11 and the highest negative signals at 432 nm occurs at pH 4- pH 6.
  • the 432/560 nm signal ratio can be used to determine positive and negative samples in pH-dependent colorimetric LAMP.
  • FIG. 27 shows the absorbance results after pH dependent colorimetric LAMP was performed on samples containing synthetic SARS-CoV-2 RNA using dual primer sets of N2 and El after no incubation at 65°C and after 20 minutes incubation at 65°C. The samples were allowed to cool to room temperature before the color was measured in the SpectraMax® (Molecular Devices, San Jose, CA). Data is provided from a SpectraMax readout that provided 100% detection of 20 copies of SARS-CoV-2 RNA and 62% detection of 10 copies of SARS-CoV-2 RNA using a sample spiked with 20 copies of SARS-CoV-2 RNA.
  • FIG. 28A-28F shows an example of an automated workflow that permits 100,000 reactions in about 20 hours. This is calculated from a batch size of 5,760 reactions (15 x 384 well plates or 60 x 96 tube racks) with a process time of 40 minutes/sample and 100 minutes/batch.
  • FIG. 28A shows how an individual saliva sample from a collection tube might be placed in a tube containing saliva lysis buffer in a 96 tube rack.
  • FIG. 28B shows a robot that can transfer samples (for example 3 ⁇ l) from individually 2D barcoded sample collection tubes or batches of 4 x 96 tube racks to 384 well plates with a linear barcode to associate each sample to a discrete well location in 4 minutes.
  • FIG. 28C shows a robot liquid handler that can add for example 17 ⁇ l of reaction mix (e.g. 2 ⁇ L lOx primer mix, 10 ⁇ L WarmStart Colorimetric Lamp 2X Master Mix (M1800), 5 ⁇ L of DNAse, RNAse free H2 O) into the 384 well plate within about 1 minute.
  • reaction mix e.g. 2 ⁇ L lOx primer mix, 10 ⁇ L WarmStart Colorimetric Lamp 2X Master Mix (M1800), 5 ⁇ L of DNAse, RNAse free H2 O
  • FIG. 28D shows a stack of plates each with plastic seal ready for the LAMP reaction.
  • FIG. 28E shows two devices for performing LAMP that requires incubation at 65°C for a period of time such as 30 minutes. This may be achieved by means of a horizontal conveyor belt that sends each plate through a heated chamber so that the residence time in the chamber is the desired incubation time. Alternatively, this may be achieved by stacking plates in a tower incubator where heating occurs for the programmed time.
  • FIG. 28F shows a robotic plate handler that takes the 384 plates from the incubator and places them in sequence in a SpectraMax or other spectrophotometer (absorbance reader) that records the 2D barcode on the plate and the color of each well at specific wavelengths.
  • FIG. 29 shows a schematic for handling large numbers of patients at a pop-up laboratory using embodiments of the rapid LAMP method for detecting SARS-CoV-2.
  • FIG. 30 (a)-(c) shows a workflow for a LAMP reaction using each of 3 different types of sample collection. These are: FIG. 30 (a) collect patient saliva sample into an empty tube, inactivation of any infectious virus in the absence of a buffer using a temperature of 65°C for 30 minutes, 75°C for 15 minutes or 95°C for 5 minutes and then transfer for example, 1 volume of the sample into 1 volume of 2X buffer or an equivalent ratio using 4X buffer or 10X buffer resulting in a IX mixture;
  • FIG. 30 (b) incorporating a volume of a concentrated viral inactivation buffer (for example 2X, 5X or 10X) into a compartment of a saliva collection tube prior to collection of saliva from the patient in the same tube. After collecting the saliva and closing the lid of the tube, the sample is mixed with viral inactivation buffer released from the compartment;
  • a concentrated viral inactivation buffer for example 2X, 5X or 10X
  • FIG. 30 (c) a volume of viral inactivation buffer is present in a sample tube for receiving a nasal or oral swab from a patient where the contents of the swab are directly released into the buffer.
  • the viral inactivation buffer contains a poloxamer surfactant such as PF68 suitable for reducing RNase activity in addition to a reducing agent (e.g., TCEP) and a metal chelator (e.g., EDTA).
  • a heating step at 95°C for 5 minutes is performed that is generally expected to break up the cells and any viruses releasing RNA while inactivating nucleases.
  • An aliquot of the patient sample is then transferred into standard LAMP master mix containing a DNA polymerase, and reversible inhibitor, a reverse transcriptase with reversible inhibitor, primer sets and nucleoside triphosphates for fluorescent LAMP (e.g., fluorescent LAMP using an intercalating dye or DARQ LAMP for multiplexing) or colorimetric LAMP.
  • fluorescent LAMP e.g., fluorescent LAMP using an intercalating dye or DARQ LAMP for multiplexing
  • colorimetric LAMP e.g., colorimetric LAMP.
  • an aliquot can be used for RT-qPCR or for any other diagnostic assay including sequencing.
  • FIG. 31A-31E show minimal interference between primer sets in a multiplex reaction.
  • 24 samples were tested for SARS-CoV-2 and 8 samples were tested for the actin control.
  • Rox Integrated DNA technologies (IDT) Wisconsin
  • IDT Integrated DNA technologies
  • FIG. 31A shows DARQ.
  • FIG. 31B shows DARQ.
  • LAMP with El, ACTB and EIA primer sets (all with Q-FIP:Fd) in the presence of SARS-CoV-2 target RNA and ACTB RNA.
  • FIG. 31C shows DARQ LAMP with El, ACTB , EIA and EIB primer sets (all with Q-FIP:Fd) in the presence of SARS-CoV-2 target RNA and ACTB RNA.
  • FIG. 31D shows DARQ LAMP with El, ACTB, EIA and EIB primer sets (all with Q-FIP:Fd) in the presence of ACTB RNA.
  • FIG. 31E provides a summary table of results provided by 31A-31D.
  • FIG. 32A-32C shows that SARS-CoV-2 and Influenza strain A or B can be detected in a single sample in a multiplex reaction.
  • FIG. 32A DARQ. LAMP detection of SARS-CoV-2 (labeled with JOE) and Flu A (labeled with Cy5) provides similar results to the detection of Flu A and SARS-CoV-2 alone.
  • FIG. 32B DARQ LAMP detection of SARS-CoV-2 (labeled with JOE) and Flu B (labeled with FAM) provides similar results to the detection of Flu B and SARS-CoV-2 alone.
  • FIG. 32C DARQ LAMP detection of Flu A (Cy5) and Flu B (FAM) provides similar results to the detection of Flu A and Flu B alone.
  • FIG. 33 shows a supported lipid bilayer (SLB) 96 well plate with numbered wells containing serial dilutions of virus spiked into negative saliva samples.
  • SLB supported lipid bilayer
  • FIG. 34 shows the results of a colorimetric LAMP assay using inactivated SARS-Cov-2 virus spiked negative saliva.
  • the dilution series shows samples where the limit of detection (LOD) is 40cps/uL.
  • FIG. 35 shows that all samples were positive that contained 40 copies of inactivated virus into saliva with LAMP reactions for each sample.
  • FIG. 36 A-36F shows that mutation position effects on RT-LAMP amplification does not affect sensitivity but can make small changes in time to endpoint. Plots of the effects of change relative to the WT primer set for three genes from SARS Covid 2- Asle (circle), El (square), N2 (triangle) are provided for each primer.
  • FIG. 37A-37B shows that a molecular beacon assay is an effective end point for a universal LAMP assay.
  • FIG. 37A describes the design of SGF LAMP primers and beacons.
  • Upper panel shows the locations of various LAMP primers and molecular beacons.
  • the lower panel compares the sequences for wt, SGF deletion, SGFwt-MB and SGFdel-MB. Dashes: bases deleted in SGF deletion; Bold: LNA base; Underlined: stem region; Italics, non-target sequence, attached fluorophores and quenchers.
  • FIG 37B shows comparable sensitivity of detection of SARS-CoV-2 sequences including wild type (SGFwt) and variant RNA (SGFdel) by LAMP using molecular beacons.
  • SGFwt wild type
  • SGFdel variant RNA
  • results of LAMP reactions with either WT RNA (left panels) or B.1.1.7 RNA (right) in the presence of SYTO-9, SGFdel-MB, or SGFwt-MB are shown.
  • the primer set amplifies both the wt and B.1.1.7 RNA with similar efficiency as detected with SYTO-9 (top).
  • MB beacon was added as a reporter, both SGFdel-MB (middle) and SGFwt-MB (lower) showed only with their intended template RNAs from 50-10,000 copies.
  • Embodiments utilize isothermal amplification, such as LAMP, and simple visual detection of amplification in a liquid or on a reaction matrix for potential use in rapid, field applications.
  • LAMP is an isothermal amplification protocol first developed by the Eiken Chemical Co. in Japan (see for example, Notomi, et al. Nucleic Acid Research (2000) 28, E63). LAMP is also described in detail in US 6,410,278 and Mori, et al. (J. Infect. Chemother. 2009 15: 62-9).
  • four primers recognize six unique target sequences on the template strand. Two of the primers are designated as “inner primers" (FIP and BIP) and two are designated “outer primers" (F3 and B3).
  • the inner primers In addition to containing a sequence that is complementary to a target sequence at their 3' ends, the inner primers also contain a tail that comprises a sequence that is downstream of the 3' end of primers in the template.
  • extension of an inner primer results in a product that has a self-complementary sequence at the 5' end.
  • Displacement of this product by an outer primer generates a product that has a loop at the 5' end.
  • the primer sets used in LAMP typically contain four, five or six template-complementary sequences, where four sequences are found at the 3' ends of the primers and two of the sequences are found at the 5' ends of two of the primers.
  • the initial reaction in LAMP results in a DNA product that has a dumbbell-like structure.
  • the ends form stem loops and the single stranded region in between the stem loops is copied from the template.
  • This product self-primes its own amplification to amplify the template sequence.
  • LAMP uses a strand-displacing polymerase, and is isothermal, that is it does not require heating and cooling cycles.
  • Primer sets for LAMP as used herein refer to four to six primers that includes optionally loop forward and/or backward primers (LF and LB) in addition to forward internal primers (FIP) and backward internal primers (BIP) and forward primer (F3) and backward primer (B3).
  • LF and LB loop forward and/or backward primers
  • F3 forward internal primers
  • BIP backward internal primers
  • F3 forward primer
  • B3 forward primer
  • LAMP assays are described herein that enable rapid and sensitive detection of target nucleic acids; such as a nucleic acid of or associated with a pathogen, such as a virus, in a human or animal population. These assays are simple and portable while retaining sensitivity and minimizing false positives and negatives.
  • the LAMP assays described herein rely on detection of a change in some aspect, such as fluorescence, or color of a dye in a reaction mix due to a change in pH or metal ions. Turbidity may also be used as an end point in some LAMP assays.
  • the LAMP assays described herein were tested for their ability to detect not only the target nucleic acid but also variants thereof.
  • the test sample was SARS-Cov-2 which has been able to evolve by mutating its RNA to escape the host immune system and to be more infectious.
  • SARS-CoV- 2 variant strains B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta) and C.37 (lambda) emerged in different regions of the world, and rapidly became the most frequently isolated strains worldwide. They have numerous and distinctive missense mutations, most prominently located in the spike protein. While only alpha carries the ⁇ 69-70 deletion, all but delta carry a 9-base, 3-amino acid deletion at positions 3675-3677 in the Orfla sequence (termed the "SGF" deletion).
  • the virus can be sequenced and the mutations defined. After the mutations have been discovered, new primer pairs can be used to detect viral infections with confidence by means of RT-PCR amplification tests.
  • references herein to embodiments that relate to "colorimetric tests” refer to detection of a target nucleic acid (e.g., a target pathogen nucleic acid) by a change in color of a dye in a reaction mix due to a change in pH or metal ion binding or dissociation.
  • target nucleic acid refers to any nucleic acid that is targeted by primers to determine its presence in a biological sample.
  • the diagnostic LAMP methods described herein may be used for detecting pathogens, including any of: prokaryotes such as bacteria, eukaryotic pathogens such as multicellular parasites, fungi, single cell pathogens such as trypanosomes or yeasts, and mycoplasma; as well as for use in genetic tests, such as for genetic diseases, and in personalized medicine, which may require SNP detection or gene analysis of the genome of a subject or RNA analysis to determine gene expression profiles in response to an environmental or metabolic event.
  • Other diagnostic uses include testing food for undesirable biological entities and monitoring environmental samples which may include biological material and are here referred to as biological samples also.
  • Many of the examples provided herein are directed to the SARS- CoV-2 RNA virus because of a sensitive diagnostic test is of paramount importance. However, the improvements in sensitivity described here may be applied to any colorimetric or fluorescent LAMP based assay to detect a target nucleic acid and indeed may find applications in non-LAMP assays also.
  • Described herein are modifications to isothermal amplification diagnostic assays (e.g. LAMP) to improve their sensitivity.
  • LAMP isothermal amplification diagnostic assays
  • nuclease inhibitors e.g., RNase and/or DNase inhibitors
  • thermolabile Proteinase K added to the sample (e.g., into the sample tube) to facilitate the efficiency of the LAMP reaction by removing unwanted protein, followed by heat inactivation of the Proteinase K prior to subsequent isothermal amplification (e.g., LAMP or RT-LAMP) reactions;
  • thermolabile UDG e.g., thermolabile UDG
  • Bst polymerase refers to any of Bst large fragment or mutant of the Bst polymerase or Bst large fragment. Examples of mutants of Bst polymerase are described in US 9,157,073, US 9,993,298, and US 9,127,258.
  • master mix refers to a combination of reagents which can be added to a sample to execute a reaction in an assay where the combination enhances the efficiency and speed of performing the assay.
  • the master mixes described herein include a mesophilic strand displacing DNA polymerase and may additionally include other enzymes such as a reverse transcriptase, uracil deglycosylase (also referred to herein as uracil DNA glycosylase) for example, a thermolabile UDG which becomes inactivated in the temperature range of 50°C-60°C or at 65°C; and a thermolabile Proteinase K such as a thermolabile Proteinase K.
  • the master mix may also include a reversible inhibitor of DNA polymerase activity.
  • a reversible inhibitor is an oligonucleotide known as an aptamer that binds to the DNA polymerase and blocks its activity below a selected temperature (for example 50°C, 55°C or 60°C) but above that temperature, the oligonucleotide is disassociated from the enzyme, permitting the reverse transcriptase to become active.
  • the master mix includes a reverse transcriptase and a reversible inhibitor of reverse transcriptase activity for inhibiting the activity of these enzymes below 40°C or below 45°C or below 50°C.
  • a reversible inhibitor is an oligonucleotide known as an aptamer that binds to the reverse transcriptase and blocks its activity below a selected temperature (for example 40°C) but above that temperature, the oligonucleotide is disassociated from the enzyme, permitting the reverse transcriptase to become active. This permits setting up a reaction at room temperature while avoiding nonspecific amplification.
  • the master mix may also contain inhibitors of nucleases such as RNase inhibitors and/or DNAse inhibitors. These inhibitors may be chemical reagents such as poloxamers, and/or aptamers.
  • nucleases may also be inactivated by submitting the sample combined with a lysis buffer to a high temperature for an effective time such as
  • the master mixed may also include dNTPs such as dTTP, dATP, dGTP and dCTP as well as dUTP for carryover prevention.
  • dNTPs such as dTTP, dATP, dGTP and dCTP
  • a 2X master mix may contain the dNTPs in equal quantities except the dUTP at 50% concentration of the other dNTPs.
  • the master mix may include single strand binding proteins and/or helicases to reduce nonspecific amplification.
  • the master mix may include a pH-sensitive dye or a metallochromic dye.
  • the master mix may be lyophilized or freeze dried.
  • RNA may be the template nucleic acid. It is not required for DNA.
  • the master mix for use in pH colorimetric LAMP may have a low buffer concentration such as 5 mM Tris or less. A low buffer concentration is not required if a metallochromic dye of fluorescent dye is used to detect amplification.
  • the master mix may be prepared in a 2X, 3X, 4X, 5X, 10X or any suitable concentration. The master mix once diluted by the sample will result in a IX concentration.
  • the master mix may contain primers or primers are not contained in the master mix.
  • Kits refer to a combination of materials that are needed to perform a reaction.
  • a kit may contain multiple tubes or a single tube.
  • the kit may include a mixture of lyophilized reagents and reagents in a storage buffer.
  • the kits described herein contain multiple tubes wherein the master mix is contained in one tube, guanidine salt in a second tube and oligonucleotide primers in a third tube.
  • the oligonucleotide primers in the third tube comprise a plurality of sets of LAMP primers, for example two sets of primers consisting of 8-12 primers where each set has 4-6 primers and wherein both sets of primers target different sequences in a single nucleic acid or different nucleic acid targets.
  • the third tube may contain more than 2 sets of LAMP primers where a plurality of sets may target a single nucleic acid or multiple different nucleic acid sequences.
  • a plurality of sets of LAMP primers in the third tube may target the genome of SARS-CoV-2 and a different set or plurality of sets of LAMP primers in the third tube may target the genome of influenza virus.
  • the plurality of LAMP primers in the third tube may additionally contain a sample identification sequence if multiple samples are pooled for combining with the kit.
  • the kit contains a 2X master mix, a 5X-25X guanidinium salts, and 5X-25X of LAMP primer sets.
  • purified nucleic acid such as RNA or DNA
  • direct tissue or cell lysate can be analyzed in a colorimetric LAMP such as a pH dependent colorimetric LAMP assay, a fluorescent LAMP assay or a metallochromic based LAMP assay.
  • a colorimetric LAMP such as a pH dependent colorimetric LAMP assay, a fluorescent LAMP assay or a metallochromic based LAMP assay.
  • Either purified or lysate can be used in a simple, rapid method for SARS-CoV-2 RNA detection.
  • a sample is obtained from the nasal passages of a patient (human or mammal) by nasal swab or from the buccal cavity.
  • the biological sample may be one or more of the following body fluids: saliva, sputum, mucous, blood, semen, urine, sweat, lymph fluid, feces.
  • the biological sample may alternatively be a tissue sample.
  • the biological sample may include pathogens such as parasites such as viruses, bacteria, archaea, worms, ticks , single cell organisms, and fungi, Samples may also be obtained from an environmental source such as a food, plant, sewage, water, dust, or surface swab of an object.
  • Samples can be placed into a small volume of water saline, TE, or suitable transport medium (for example, a universal transport medium for viruses) or directly into a lysis buffer that inactivates and breaks open the pathogen if that is the diagnostic target while protecting the released target nucleic acids from nuclease digestion .
  • the samples may be further purified from lysis buffer or added without further purification to a LAMP amplification master mix for testing for the presence of a pathogen, gene, SNP, mutation or other nucleic acid target.
  • the pH of the sample may determine the type of assay detection that is preferably used.
  • urine has an acid pH so a metallochromic based assay that is not pH sensitive might be used, instead of a pH endpoint.
  • the nucleic acids in saliva may come from cheek cells and tongue cells as well as any typical oral microbial species.
  • Saliva can be collected from a human subject using a commercial collection device such as provided by for example any of Boca Scientific Inc. (Westwood, MA), Salimetrics LLC (Carlsbad, CA), Mantacc (Shenzhen, China), Greiner Bio-One BD Sputum Collection (Monroe, NC).
  • Saliva has some challenges as a body fluid for nucleic acid analysis. Its pH and composition may vary according to the biology of the individual and also according to the recent intake of food and/or liquid into the mouth. For example, it is well known that a glass of water containing the juice of a squeezed lemon will cause the saliva to become acidic for a short time after intake.
  • the pH can be normalized to the extent necessary with respect to subsequent dilution before initiation of the amplification reaction so that the concentration of buffer in the final reaction mixture is less than a corresponding amount of 5 mM Tris while optimizing the pH to be preferably within the range of pH 7.9- pH 8.3.
  • the subject providing the saliva might avoid ingestion of a particular food or drink for a predetermined number of minutes (such as 30 minutes) before providing the saliva sample.
  • a detergent may be added to the collection tube if storage before analysis is intended.
  • Triton X-100 in relatively high concentrations has been shown not to interfere with a subsequent colorimetric LAMP reaction (see for example, FIG. 11B).
  • the lysis buffer as described below will inactivate the virus and release nucleic acid for amplification or for sequencing.
  • the lysis buffer described in Example 9 is suited for amplification generally as illustrated for RT-QPCR and for LAMP.
  • the saliva lysis buffer may also be applied to other body fluids for a similar purpose.
  • the lysis buffer may be used for obtaining nucleic acid samples suitable for direct sequencing such as by Oxford Nanopore.
  • the sensitivity of the methods described herein was tested in samples of human SARS-CoV-2 negative saliva spiked with either SARS-CoV-2 viral RNA (Twist Synthetic SARS-CoV-2 RNA Control 2 (MN908947.3) (Twist Biosciences, San Francisco, CA) or virus particles provided by SeraCare (Milford, MA).
  • SARS-CoV-2 viral RNA Twist Synthetic SARS-CoV-2 RNA Control 2 (MN908947.3) (Twist Biosciences, San Francisco, CA) or virus particles provided by SeraCare (Milford, MA).
  • An aliquot of the spiked saliva samples was added to the lysis buffer that was then analyzed using a pH colorimetric LAMP assay as shown in FIG. 20.
  • Using the methods described herein it was possible to detect less than a 100 copies of viral RNA (80 copies of virus) derived from a saliva sample, more particularly less than 80 particles, more particularly, 40 particles or less with up to 100% efficacy.
  • a microfuge tube receives the sample, for example a swab, in a suitable buffer.
  • a reaction platform such as a 96 well dish, 384 well dish or other multi-well dish may be used for multiple sample analysis.
  • the sample may be spotted onto a paper, plastic, or glass surface.
  • the sample may also be introduced into a microfluidic device, such as a lab-on-a-chip.
  • an Echo® Liquid Handler (Labcyte Inc., San Jose, CA) may be used to handle fluid samples. Because the one tube reaction is simple, any automated liquid handler of device may be used for analysis of multiple samples.
  • a master mix can include or be combined with oligonucleotide probes or primers.
  • a master mix can be added directly to the sample, or alternatively, a portion of the sample can be added to the master mix.
  • the primers and/or probes can be added to the master mix prior to adding the mixture to the sample or the primers and/or probes can be added to the sample prior to or after addition of the master mix.
  • the LAMP primers and/or probes may be incorporated into the LAMP master mix so that all that is required is to add an aliquot of sample (purified nucleic acid or lysed cell or fluid sample) to the master mix and to raise the temperature to 60°C-65°C for a 15 minute, 30 minute, 45 minute or 60 minute incubation time for amplification to occur, to detect a change in color or fluorescence that defines the presence of the target nucleic acid.
  • the master mix will contain a DNA polymerase dNTPs and a suitable buffer or water.
  • the master mix may additionally include one or more of the following: probes or primers, a plurality of sets of primers, reverse transcriptase, dUTP and UDG, a helicase, a single strand binding protein, a carboxamide, an RNase inhibitor (e.g., murine RNase inhibitor or an aptamer), a pH-sensitive dye or a metallochromic dye such as PAR, and/or manganese ions, and/or guanidine salts.
  • the master mix may have been previously stored at -20°C , prepared in a liquid formulation that is stable at room temperature or lyophilized.
  • the second sample is then exposed to UDG prior to amplification and this enzyme creates an abasic site at the incorporated uracil. Consequently, it is not possible to amplify sample 1 amplified DNA in sample 2 which would result in a false positive.
  • the UDG is temperature inactivated or it would adversely affect the desired amplification. It is desirable that the diagnostic test described herein is simple to perform, and also rapid and sensitive. Hence a thermolabile UDG is incorporated into the master mix along with dUTP so that once the temperature is raised to the temperature required for amplification, namely 55°C-75°C, the UDG is inactivated.
  • a preferred feature of embodiments of the diagnostic test described herein is inhibition of carryover contamination.
  • a 2X LAMP master mix containing a buffer concentration of less than 8 mM TRIS buffer and the pH sensitive dye was tested with and without dUTP and Antarctic UDG. It was found that master mixes containing dUTP and Antarctic UDG yielded substantially the same speed, sensitivity and specificity observed in the absence of UDG (see Examples 3 and 4 and FIG. 6A-FIG. 6B). Because prevention of carryover is important for high throughput screening, preferably, UDG and dUTP should be included in master mixes for pH colorimetric LAMP detection of pathogens.
  • Thermolabile Proteinase K (see for example, US patent application, serial no. 16/719,097) and/or a proteinase inhibitor such as murine proteinase K inhibitor and/or an RNase inhibitor (see for example US provisional application serial no. 62/992,921) and/or a DNase inhibitor can be added to the sample or the reaction tube into which the sample is added. Where thermolabile Proteinase K is used, it may be inactivated before addition of the master mix. Thermolabile Proteinase K is available from New England Biolabs, Inc, Ipswich, MA.
  • LAMP master mix containing enzymes, dNTPs, buffer, and pH sensitive dyes (e.g. colorimetric or fluorometric dyes), and/or other dyes that bind metals (e.g. PAR), may be freeze dried or lyophilized and stored at room temperature as a master mix, for example a 2X master mix or 5X master mix or a 10X master mix until needed.
  • the master mix may then be added to a solution containing primers and/or sample to be tested for target nucleic acid in a reaction container, a microfluidic device, a lab on a chip device or a matrix such as paper, plastic, or glass.
  • the pH and buffer concentration of the master mix is not relevant until the dried master mix is added to the sample or the master mix is rehydrated. It was here shown that the pH of the buffer of rehydrated master mix is slightly changed when the master mix is rehydrated so this is taken into account during formulation.
  • pH dependent colorimetric LAMP pH dependent colorimetric LAMP is shown here to be quick, easy, reliable, and suitable for scale-up in molecular diagnosis of viruses and multicellular parasites and their pathogens.
  • a desirable format for measuring a color change is a strip test such as routinely used for pregnancy tests that rely on antibodies (CVS) or the Quickvue® in-line StrepA test (Quidel Corporation, San Diego, CA) or equivalent.
  • a liquid test may be convenient in which the sample is added to a first solution (e.g., a lysis buffer) from which an aliquot is removed to a second solution containing the master mix that is then heated on a small pad provided with the test kit (this could be an equivalent of an activated handwarmer or directions for heating a small amount of water in a kettle or a heat block).
  • a pH change will result in a color change denoting a positive result.
  • a metallochromic dye such as PAR has been found to be useful in a colorimetric LAMP assay and provides an alternative should body fluid to be tested be acidic such as urine.
  • the PAR based assay is described in the figures and examples.
  • Manganese used in the PAR colorimetric endpoint LAMP assay is a suitable ion as it does not negatively affect the activity of the polymerase or the reverse transcriptase in a LAMP reaction.
  • PAR it is not pH sensitive, it has advantages in a variety of situations in which pH colorimetric LAMP is not best suited as enumerated herein.
  • Either pH colorimetric LAMP or PAR may be used in conjunction with Calcein which also binds to manganese to give a fluorescent signal and/or with the fluorescent dye, Syto-9.
  • Guanidine salts may also be used in conjunction with colorimetric LAMP to enhance the sensitivity of the assay.
  • a master mix may contain a pH sensitive dye, fluorescent dye or PAR, a polymerase, plus optionally a reverse transcriptase for RNA detection (such as for an RNA virus), reversible inhibitors for the polymerase and reverse transcriptase to reduce unwanted background noise and increase sensitivity, primers and dNTPs form a reaction mix when combined with a sample for determining the presence of a target nucleic acid.
  • the master mix is preferably in a concentrated form, for example a 2X, 3X, 5X, 7X or 10X master mix where the final amount of master mix after combination with a sample is IX.
  • Some of the considerations in forming the master mix beyond those established in commercial master mixes may include one or more of the following parameters: (1) the concentration of dye; and (2) the amount of buffer; (3) the pH; (4) dilution of the master mix in the sample; and (5) the proposed incubation time of the reaction mix to which the master mix is added.
  • the concentration of dye in the master mix should be sufficient such that when diluted with sample, it enables unambiguous visual detection of a positive sample.
  • the concentration of dye must not be so high as to adversely affect the activity of the polymerase and/or reverse transcriptase.
  • the visually detectable dye in the reaction mix (IX master mix plus sample) is in the range of 50 ⁇ M -200 ⁇ M.
  • the amount of buffer in a liquid concentrated master mix should be sufficient to maintain the enzymes and other reagents in a stable condition but suitable for dilution to detect a color change if amplification of a target nucleic acid has occurred.
  • buffer concentrations of greater than 5 mM e.g., 5 mM Tris
  • the buffer concentration was formulated to meet the IX amount of master mix to have an equivalence to 0.5 mM Tris-5 mM Tris.
  • PAR or fluorescence based LAMP do not require this limit on buffer concentration.
  • LAMP master mixes may be prepared as 2X, 3X, 5X, 7X or 10X although 2X, 5X or 10X are the usually preferred concentrations for easy dilution into a sample.
  • enzyme inhibitors such as blood, sputum, and urine but not necessarily nasal or buccal swabs
  • adjustment of the acidic pH of urine or the alkali pH of sodium hydroxide treated sputum may be desirable to ensure that the pH of the sample does not inhibit the detection of the amplified target nucleic acid in the sample.
  • Buccal and nasal swabs can be placed in water or transport media and added in a 1:1 ratio to a volume of master mix resulting in a pH and buffer concentration as described above.
  • it may be desirable to extract the nucleic acid for example, using Monarch® (New England Biolabs, Ipswich, MA), Qiagen or Roche purification kit prior to testing.
  • the samples can be used in higher volumes especially if eluted from extraction matrices in diluted buffer or water. Lysis buffers are discussed below.
  • FIG. 2A-FIG. 2B shows the sensitivity of assays described herein for nematode parasites as little as 0.01-0.1 picograms nematode DNA can be detected.
  • Example 3B shows detection of 1.28 femtograms of DNA of a tick-borne pathogen from whole ticks.
  • Embodiments of the test described herein for a target RNA of a virus in a nasal or oral swab can be performed within 15 minutes.
  • Example 1 provides an example of pH dependent colorimetric LAMP as used for detection of viral pathogens such as RNA or DNA viruses for example, SARS- CoV-2 RNA.
  • the results for SARS-CoV-2 RNA in FIG. 21-FIG. 27 show that the virus can be reliably detected at between 10-40 copies in under 60 minutes.
  • Biological material that may contain one or more pathogens can be sampled for one or more target nucleic acids in parallel to obtain distinctive results for each or together for purposes of efficiency initially but that might result in secondary tests.
  • a sample is taken from a subject to test for SARS-CoV-2 and influenza, it might be desirable to perform tests in parallel using one or more primer sets that are specific for each target nucleic acid. Samples may be obtained from any biological source.
  • samples may be derived from blood (e.g., from a venous draw), serum, plasma, urine, feces, sputum, hair follicles, lavage, nasal, oral or buccal swabs, and/or saliva may be used for detecting the pathogen using pH dependent color LAMP or PAR dependent color LAMP that is not pH dependent.
  • the samples may be dried, placed in an aqueous solution that may be selected from water or stored in a transport medium, for example saline, TE or a transport medium available from Copan (Murrieta, CA).
  • a transport medium for example saline, TE or a transport medium available from Copan (Murrieta, CA).
  • Such products include Sputum dipperTM, SnotBusterTM, UriSpongeTM and UTM® Universal Transport SystemTM.
  • RNAses that might be contained in the biological material. These RNAses are particular concern if they are released when a diagnostic test requires testing for RNA such as the viral RNA diagnostics for SARS-CoV-2 are performed.
  • the RNases that are released from the sample can digest the viral RNA from disrupted virus of interest reducing the sensitivity of the test.
  • Detergents are routinely used to disrupt biological material. Examples of such detergents include Tween and Triton X. These detergents appear to contribute to the observed loss of viral RNA (within 5 minutes at room temperature) once the biological sample is exposed to these detergents. Not wishing to be limited by theory, it is hypothesized either that these detergents either activate cell RNases or cause the viruses to break open releasing the viral RNA that is quickly enzymatically digested by RNAses.
  • One way to prevent RNase digestion is to heat the sample to 95°C for at least 5 minutes immediately after adding buffer containing Tween or Triton X to the samples. This heat inactivates the majority of RNAses. However, without some alternative intervention, the availability of intact sample RNA can be significantly reduced.
  • a family of surfactants also called an antifoaming agents specifically poloxamers, that can inactivate the virus particles and either inhibit RNases or do not break open the virus particles, so that substantially all or a significant portion of the RNA remains intact for at least 2 hours, 3 hours, 4 hours, 5 hours and up to at least 6 hours at room temperature after addition of the surfactant prior to a heat step at 95°C to release viral RNA from virus particles and inactivate the RNase.
  • the observed advantage of the poloxamer PF68 is described in Examples 13-15. It is expected that other related poloxamer surfactants will be similarly effective. Examples of other members of this group of surfactants are provided below.
  • RNA from a sample Since the improvement observed relates to the preparation of RNA from a sample, it is believed that this group of surfactants can be effective in the purification of RNA and in improving the sensitivity of colorimetric and fluorescent LAMP reactions as well as RT-qPCR reactions and also in the preparation of samples for nucleic acid sequencing.
  • the improvements relate to obtaining RNA from any biological source including any diagnostic test known in the art for a virus from any biological material.
  • RNA virus is described in FIG. 30 in 3 different initial workflows where saliva or other body fluid or buccal or nasal swab is collected in a tube, or in a microwell in a 96 well dish or a 384 well dish that may be empty or may contain a buffer with the foaming agent or may contain buffer in a compartment in the tube or well that is released on introduction of sample. If the collection tube receives the sample in the absence of buffer, an aliquot can then be transferred to receiving compartment (tube or a multi-sample microwell dish) where each well or tube contains buffer.
  • the tubes or wells containing sample in buffer containing the poloxamer is then heated to 95°C for 5 minutes and an aliquot transferred into a fluorescent LAMP, DARQ LAMP, colorimetric LAMP (cLAMP) or RT-qPCR reaction tube to determine the presence of the virus or viruses.
  • a fluorescent LAMP, DARQ LAMP, colorimetric LAMP (cLAMP) or RT-qPCR reaction tube to determine the presence of the virus or viruses.
  • the poloxamer foaming agent may be best suited for adding to the sample at the earliest convenience. It may also be used for DNA viruses or other samples. There was no adverse effect of the carryover of the poloxamer from sample tubes to the amplification reaction.
  • the poloxamer agent is important for improved results while the buffer used along with the poloxamer does not appear to be of critical importance.
  • a buffer containing TCEP and EDTA may be adequate (for example, 100X buffer containing 0.5 MTCEP (2.5 ml)) .5 M EDTA (1 ml) and 1.1 N NaoH (1.5 mis) described in Rabe and Cepko (2020) PNAS vol 117, pp24450-24458) can be used without LiCI.
  • Guanidinium chloride has proved useful for colorimetric LAMP, it is not required in a fluorescent or DARQ LAMP reaction.
  • the volume of buffer in the tubes or wells is preferably 2X the volume in a 364 well dish (12.5 p).
  • SARS-CoV-2 LAMP tests were analyzed using a Cq value for real time analysis to detect virus and also endpoint +/- analysis.
  • SARS-CoV-2 it is important to understand the amount of virus in a patient as determined by a nasal or buccal swab or saliva that corresponds to active infection. It has been observed that titers of the order of 2xl0 4 virus particles/ml (20 virus particles/pl) may be detected at the onset of infection or at the end of a recovery phase of infection whereby virus titers in the region of 10 9 -10 11 signal an infectious dose.
  • colorimetric LAMP test can readily be performed on multiple samples simultaneously as needed, for example in 96 well or 384 well plates with instantaneous detection of positive samples by eye or by spectrophotometer to detect a color change.
  • colorimetric LAMP can consistently detect 100% of samples containing 50-80 particles/ ⁇ l (5-8xl0 4 particles/ml) and is described herein, can detect RNA corresponding to as few as 25 viruses with 90% success rate. These detection ranges of 25-50 virus particles are below the concentration required to detect virus load of an active spreader using saliva, nasal or buccal swabs.
  • Embodiments are provided for lysis buffers suitable for LAMP.
  • the lysis buffer was prepared as a 2X mix but could also be prepared as a 4X, 5X or 10X mix or indeed any concentrated form limited only by possible undesirable precipitation of individual reagents at high concentrations.
  • the 2X lysis mix described herein comprises 800 mM guanidine HCI, 4% Triton X -100, 80 mM TCEP and 150 mM LiCI at pH 8.0. This 2X lysis mix can be combined with an equal volume of saliva resulting in a final concentration of 400 mM guanidine HCI, 2% Triton X-100, 40 mM TCEP and 75 mM LiCI at pH 8.0.
  • Triton X or Tween could be replaced with a non- ionic detergent having improved properties with respect to RNases in saliva.
  • Triton X and Tween 20 were very effective at lysing virus particles at room temperature, these detergents appeared to have the effect of exacerbating RNAse activity with an adverse effect on the sensitivity of an isothermal amplification performed on saliva.
  • an alkoxylated alcohol which is an amphiphilic water soluble polymorphic block copolymer of poly(ethylene oxide) (PEO) and polypropylene oxide) (PPO), did not have the undesirable effect on RNAse activity observed for the other detergents.
  • a preferable composition would be an ABA triblock copolymer, wherein A is the hydrophilic block PEO and B is the hydrophobic block PPO.
  • Preferable copolymers have molar mass ratio between the PEO and PPO blocks from 1:9 to 8:2. Examples of such block copolymers are Pluronic detergents and poloxamers.
  • Pluronic F68 CAS [691397-13-4]
  • Thermo Fisher, Waltham MA The structure is provided below.
  • Detergents of this class may also be referred as Epoxyethane-epoxypropane copolymer, Ethoxylated propoxylated propylene glycol, Ethylene glycol-propylene glycol copolymer, Ethylene glycol- propylene glycol polymer, Ethylene oxide-l,2-propylene oxide copolymer, Ethylene oxide-propylene oxide copolymer, Ethylene oxide-propylene oxide copolymer ethylene glycol ether, Ethylene oxide- propylene oxide copolymer, Ethylene-propylene glycol copolymer, Methyloxirane-oxirane copolymer, Oxirane-methyloxirane copolymer, Oxirane-propylene oxide copolymer, Oxyethylene-oxypropylene copolymer, Oxypropylene-oxyethylene copolymer, Oxypropylene-oxypropylene copolymer, Poly(oxyethylene) poly(oxy
  • PEO-PPO-PEO triblock copolymers are also referred as Ethene oxide-propene oxide triblock copolymer, Ethylene glycol-propylene glycol copolymer triblock, Ethylene glycol-propylene glycol triblock copolymer, Ethylene oxide-Excenol 1020-propylene oxide triblock copolymer, Ethylene oxide- propylene oxide triblock copolymer, Ethylene oxide-propylene oxide-ethylene oxide triblock copolymer, Oxirane-methyloxirane triblock copolymer, Oxirane-oxypropylene triblock copolymer, Oxirane- propylene oxide triblock copolymer, Poly(ethylene oxide)-block-poly(propylene oxide)-block- poly(ethylene oxide), Polyethylene oxide polypropylene oxide polyethylene oxide triblock copolymer, Polypropylene-polyethylene-polypropylene triblock copolymer, Propylene glycol-ethylene glyco
  • Most preferable detergents are Pluronic F68 and Poloxamer 188.
  • Other detergents of interest are Pluronic L31, Pluronic L61, Pluronic L81, Pluronic L101, Pluronic L121, Pluronic L42, Pluronic L62, Pluronic L72, Pluronic L92, Pluronic L122, Pluronic L43, Pluronic L63, Pluronic L44, Pluronic L64, Pluronic P84, Pluronic P103, Pluronic P123, Pluronic P65, Pluronic P75, Pluronic P85, Pluronic P105, Pluronic F77, Pluronic F87, Pluronic F127, Pluronic F38, Pluronic F88, Pluronic F98, and Pluronic F108.
  • Poloxamer 10R5 Poloxamer 1100, Poloxamer 122, Poloxamer 123, Poloxamer 127, Poloxamer 17R4, Poloxamer 181, Poloxamer 183, Poloxamer 202, Poloxamer 237, Poloxamer 272, Poloxamer 317, Poloxamer 333, Poloxamer 334, Poloxamer 407, Poloxamer 68, Poloxamer F 98, Poloxamer P 188, and Poloxamer P 407.
  • amphiphilic block copolymers of interest include combination of one or more of the selected polymers polyethylene glycol (PEG), poly(D,L-lactide-co-glycolide) (PLGA), poly(lactic acid) (PLA), poly(glutamic acid) (PGA), poly(caprolactone) (PCL), N-(2-hydroxypropyl)-methacrylate (HPMA) copolymers, and poly(amino acids).
  • PEG polyethylene glycol
  • PLA poly(D,L-lactide-co-glycolide)
  • PLA poly(lactic acid)
  • PGA poly(glutamic acid)
  • PCL poly(caprolactone)
  • HPMA N-(2-hydroxypropyl)-methacrylate
  • Immobilized reagents can improve a diagnostic workflow.
  • Examples of immobilized reagents that may be used for enrichment of a target nucleic acid such as a target RNA contained in a target pathogen or exosome in a sample may include one or more of: an oligonucleotide for hybridizing to the target nucleic acid, a poly-dT oligonucleotide, an oligonucleotide that serves as primer or probe for reverse transcription and/or replication of a target nucleic acid; a nucleic acid binding protein such as pl9 (see for example US 8,753,809), and where the target nucleic acid is DNA, an oligonucleotide reagent that hybridizes top the target DNA, a transcriptional activator protein that is sequence specific for the target DNA such as described in (see for example 9,963,687), or a nonspecific DNA binding protein such as a repair protein moiety or a polymerase moiety (such as SS07); a
  • Immobilized reagents may include a combination so that the pathogen or exosome may be immobilized and on release of the nucleic acid from pathogen and exosome possibly in the same receiving tube, immobilized oligonucleotides can bind target nucleic acid for further characterization involving amplification to generate a detectable signal with high sensitivity.
  • Reagents for LAMP may be immobilized on a substrate instead of the primers or target nucleic acid.
  • LAMP reagents such as any or all of the strand displacing polymerase, reverse transcriptase, uracil deglycosylase, nuclease inhibitors and stabilizing reagents may be immobilized through the use of linkers and/or tags as discussed below.
  • a mixture of immobilized reagents may be utilized in a composition where a portion of the immobilized reagents bind to and enrich for one or more pathogens or exosomes or their nucleic acids in a sample where the one or more pathogens and/or exosomes may be different targets from a single subject (for example, SARS-CoV-2 and Influenza viruses) or the same or different targets from multiple subjects.
  • a single subject for example, SARS-CoV-2 and Influenza viruses
  • Embodiments of the method described herein include washing and replacement of the biological fluid with a suitable RNAse or DNAse free buffer containing a lysis reagent, where a second portion of immobilized reagents captures and enriches the nucleic acid from the lysed particles.
  • a nucleic acid from a biological sample including environmental samples can be detected after enrichment in a workflow that involves a minimum number of steps to produce a sensitive and specific result.
  • a reagent is added to an environmental sample or a body fluid or a solution containing the contents of a swab (see above) to releases nucleic acid from a pathogen or exosome contained in the sample.
  • the target nucleic acid hybridizes to an immobilized oligonucleotide on a substrate, such as a bead, so that separation of the immobilized nucleic acid from the rest of the solution can occur and a new solution can be added that has the necessary reagents for reverse transcription if the nucleic acid is RNA and for amplification, for example, using a polymerase chain reaction (PCR) or by isothermal amplification, for example, LAMP.
  • PCR polymerase chain reaction
  • LAMP isothermal amplification
  • the amplification event can then be detected using fluorescence or color by eye or by means of a simple device.
  • Example 14 describes this approach for a coronavirus.
  • the hybridized target nucleic acid can be released from the substrate after enrichment or the immobilized oligonucleotide can act as a primer or probe so as to function both to enrich the target nucleic acid and to initiate cDNA synthesis and/or replication.
  • an immobilized reagent is intended to enhance efficiency of the workflow and potentially also enhance sensitivity of an assay for detecting the pathogen.
  • Others have used immobilization for enrichment of exons in a genomic DNA sample (see for example US 9,708,658, US 9,567,632, US 10,087,481 and US 10,246702).
  • Embodiments described herein differ from the approaches described in these references because they are applicable to detecting a pathogen in a biological fluid and because the immobilized reagents may preferably serve as primers or probes to enrich and reverse transcribe and/or amplify the nucleic acid obtained from the biological sample in a single step.
  • immobilized reagents may be provided in a lyophilized state and hydrated by the addition of a body fluid sample or sample containing a swab.
  • the rehydrated reagent may include RNase inhibitors, DNase inhibitors, a detergent and/or surfactant, salts, reducing agents such as DTT or TCEP and proteolytic agents such as Proteinase K (such as a heat labile Proteinase K) and salts at a suitable concentration for aiding hybridization that is well understood in the art and/or destabilizing protein.
  • a detergent such as a non-ionic detergent includes an amphiphilic water soluble polymorphic block copolymer of poly(ethylene oxide) (PEO) and polypropylene oxide) (PPO), Tween 80 or Triton X suitable for disrupting a virus or exosome to release the nucleic acid.
  • the type of detergent and/or surfactant may be selected depending on what proteolytic agents and reducing agents are included to aid in the release of nucleic acid from pathogens or exosomes. If the target nucleic acid is RNA, the use of reducing agents such as DTT, TCEP are desirable for stabilizing the RNA.
  • the receiving tube may include in addition to lyophilized reagents or buffer containing reagents, a concentration of chaotropic salts for denaturing proteins.
  • a heating step may be included where heat can cause denaturing of both proteins and nucleic acids and can be used to (a) inactive proteins (e.g., DNases and RNases) in the absence of proteinase K and (b) to melt nucleic acid secondary and/or intermolecular hybridization structures.
  • a heat-denaturing step can be damaging to nucleic acids, particularly in the presence of divalent metals,
  • substrates as used with reference to enrichment refer to immobilization substrates.
  • Substrates may be biological, non-biological, organic, inorganic or a combination thereof, and may be in the form of solid or porous particles, strands, precipitates, gels, sheets, tubings, spheres, containers, capillaries, pads, slices, films, plates such as microplates, slides, and have any convenient shape, including flat, disc, sphere, circle, etc.
  • a substrate as used herein is meant to comprise any material (porous or non-porous, flexible or rigid) material onto which it is desired to capture and immobilize a reagent oligonucleotide.
  • the substrate may form a colloidal solution from microbeads.
  • the substrate may be beads that sediment in a solution.
  • the substrate may be magnetic beads that can be concentrated in one part of a tube in response to an external magnet.
  • the substrate may have any desired shape. A spherical shape is convenient because of the maximal surface area to volume.
  • the substrate may include any desirable material known in the art suitable for binding oligonucleotides including magnetic, paramagnetic or non-magnetic beads.
  • the substrate for immobilizing reagents may be beads.
  • suitable beads include magnetic or paramagnetic beads are coated with negatively charged molecules (e.g., carboxyl- containing molecules) which reversibly bind viral nucleic acids in the presence of a crowding agent (e.g., polyethylene glycol, such as 10-50% PEG) and salt (e.g., NaCI, such as 1-4 M NaCI).
  • a crowding agent e.g., polyethylene glycol, such as 10-50% PEG
  • salt e.g., NaCI, such as 1-4 M NaCI.
  • SPRI® and AMPure® beads Beckman Coulter, Brea, CA.
  • Electrostatic reversible binding of nucleic acids to magnetic or paramagnetic beads may be improved in the presence of chaotropic salts (e.g., guanidine hydrochloride, guanidinium thiocyanate, etc.).
  • chaotropic salts e.g., guanidine hydrochloride, guanidinium thiocyanate, etc.
  • magnetic or paramagnetic beads are coated with positively charged molecules (e.g., amino-containing molecules) which reversibly bind viral nucleic acids.
  • magnetic or paramagnetic beads are coated with silica and reversibly binds nucleic acids based on salt concentration.
  • Substrate may be modified with secondary molecules to provide surfaces that facilitate coating of the substrate with a reagent. Examples are provided below.
  • the surface of the substrate may be composed of a variety of materials, for example, polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, membranes, etc., provided that the surface may support functional groups.
  • Convenient substrates include: glass such as glass slides or glass beads; plastic such as in microtiter plates; functionalized polymers such as polymer beads.
  • the surface of the substrates may be chemically modified for example by oxides such as silicon dioxide, tantalum pentoxide or titanium dioxide, or by metals, such as noble metal surfaces such as gold or silver, copper or aluminum surfaces.
  • the substrate surface may be magnetized for example by Fe, Mn, Ni, Co, and their oxides.
  • the substrate may be nanoparticles made of lll-V semiconductor material (quantum dots) using for example GaN, GaP, GaAs, InP, or InAs) or ll-VI semiconductor material using for example ZnO, ZnS, CdS, CdSe, or CdT or Ln-doped fluoride nanocrystals, rare earth-doped oxidic nanomaterials.
  • lll-V semiconductor material quantum dots
  • GaN GaN
  • GaP GaAs, InP, or InAs
  • ll-VI semiconductor material using for example ZnO, ZnS, CdS, CdSe, or CdT or Ln-doped fluoride nanocrystals, rare earth-doped oxidic nanomaterials.
  • the surface of the substrate may inherently contain a linker, cross linker or spacer, a binder, a functional group, or electrostatic charge to prepare the substrate for a coating with oligonucleotide reagent.
  • the substrate surface for binding an oligonucleotide can be added or activated in a coating (such as a polymer coating) of the substrate using a suitable material.
  • Activation as used herein means a modification of a functional group on the surface of the substrate to enable coupling of a binding agent to the surface.
  • polymer coatings include any suitable class of compounds, for example, polyethylene glycols, polyethylene imides, polysaccharides, polypeptides, or polynucleotides.
  • Attachment of the polymers to the substrate surface may be achieved by a variety of methods which are readily apparent to a person skilled in the art. For example, polymers having trichlorosilyl or trisalkoxy groups may be reacted with hydroxyl groups on the substrate surface to form siloxane bonds. Attachment to a gold or silver surface may take place via thiol groups on the polymer. Alternatively, the polymer may be attached via an intermediate species, such as a self-assembled monolayer of alkanethiols.
  • the type of polymers selected, and the method selected for attaching the polymers to the surface will thus depend on the polymer having suitable reactivity for being attached to the substrate surface, and on the properties of the polymers regarding non-specific adsorption to, especially, DNA and RNA.
  • the functional groups may be present on the polymer or may be added to the polymer by the addition of single or multiple functional groups.
  • a spacer arm can be used to provide flexibility to the binding oligonucleotide allowing it to interact with its environment in a way which minimizes steric hindrance with the solid support.
  • the substrate can be modified with a binder, e.g., an antibody (or antibody fragment) or another affinity binder, e.g.
  • streptavidin suitable for binding an oligonucleotide molecule that has been modified with the corresponding affinity ligand, e.g. biotin, and another affinity binder, e.g. an antibody recognizing part of the sequence of a biomolecule.
  • affinity ligand e.g. biotin
  • another affinity binder e.g. an antibody recognizing part of the sequence of a biomolecule.
  • a binder as used herein means any agent that is a member of a specific binding pair.
  • binders include agonists and antagonists for cell membranes, toxins and venoms, antigenic determinants, hormones and hormone receptors, steroids, peptides, enzymes, substrates, cofactors, drugs, lectins, sugars, oligonucleotides, oligosaccharides, proteins, glycoproteins, cells, cellular membranes, organelles, cellular receptors, vitamins, viral epitopes, and immunoglobulins, e.g., monoclonal and polyclonal antibodies.
  • binding pairs include biotin-steptavidin/avidin, hapten/antigen-antibody, carbohydrate-lectin, or others known to those skilled in the art.
  • SNAP-tag/AGT and benzylguanine derivatives U.S. Patent Nos. 7,939,284; 8,367,361; 7,799,524; 7,888,090; and 8,163,479) or pyrimidine derivatives (U.S. Patent No. 8,178,314), CLIP-tag/ACT and benzylcytosine derivatives (U.S. Patent No. 8,227,602), HaloTag and chloroalkane derivatives (Los, et al.
  • oligonucleotides can be functionalized with benzylguanine, pyrimidine, benzylcytosine, chloroalkane, or beta-lactam derivatives respectively, and subsequently be captured in a solid support modified with SNAP-tag/AGT, CLIP-tag/ACT, HaloTag or serine-beta-lactamases.
  • Coating of the surface of the substrate with an oligonucleotide reagent by means of a binder may be achieved using an affinity binding pair such as biotinylated oligonucleotides for binding streptavidin-functionalized beads.
  • affinity binding pair such as biotinylated oligonucleotides for binding streptavidin-functionalized beads.
  • Other affinity groups on oligonucleotide reagents include desthiobiotin, avidin, NeutrAvidin, protein A, protein B, maltose-binding protein, chitin, poly-histidine, HA-tag, c-myc tag, FLAG-tag, SNAP-tag, S-tag and glutathione-S-transferase (GST).
  • magnetic or paramagnetic beads are coated with a poly dT sequence (e.g., 10-25 dT oligonucleotide) which is then used as for the direct binding of poly(A)+ viral RNA.
  • a poly dT sequence e.g., 10-25 dT oligonucleotide
  • Cap-binding protein attached to an immobilization substrate can serve as reagents to directly bind target nucleic acid such as m7G(5')ppp(5')N (where N is any canonical nucleotide, including nucleotides with 2' O-methylation) capped viral RNA (see for example PCT/US2020/031653 for modified caps).
  • the oligonucleotide for hybridizing target nucleic acid should be at least 6 nucleotides long and may include one or more modified nucleotides.
  • the oligonucleotide may be designed to function as a probe or primer for reverse transcription and/or amplification.
  • the oligonucleotide may be any of an RNA, DNA, peptide nucleic acid (PNA), a lock nucleic acid (LNA), an unlock nucleic acid (UNA), a triazole nucleic acid, a phosphorothioate oligonucleotide, or a combination thereof.
  • Modified nucleotides may improve oligonucleotide chemical stability (e.g., resistance to degradation at basic or acidic pH, at high temperatures, upon UV radiation, etc.), mechanical stability (e.g., resistance to degradation following mechanical manipulation such as sonication, acoustic shearing, etc.), and/or enzymatic stability (e.g., resistance to degradation in the presence of a nuclease such as a restriction enzyme, a DNase, a RNase, etc.). Modified nucleotides may also improve oligonucleotide ability to hybridize (e.g., through base pairing) with a fully or partially complementary nucleic acid targeted sequence.
  • chemical stability e.g., resistance to degradation at basic or acidic pH, at high temperatures, upon UV radiation, etc.
  • mechanical stability e.g., resistance to degradation following mechanical manipulation such as sonication, acoustic shearing, etc.
  • enzymatic stability e.g., resistance to degradation in
  • base modifications include 2-aminopurine, 2,6-diaminopurine, 5-iodouracil, 5- bromouracil, 5-fluorouracil, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil, 5-proprynyluracil, 5- methylcytosine, 5-hydromethylcytosine, 5-formylcytosine, 5-carboxycytosine, 5-iodocytosine, 5- bromocytosine, 5-fluorocytosine, 5-proprynylcytosine, 4-ethylcytosine, 5-methylisocytosine, 5- hydroxycytosine, 4-methylthymine, thymine glycol, ferrocene thymine, pyrrolo cytosine, inosine, 1- methyl-inosine, 2-methylinosine, 5-hydroxybutynl-2'-deoxyuracil (Super T), 8-aza-7-deazaguanine (Super G), 5-nitroindole
  • sugar modifications include but are not limited to those found in dideoxynucleotides (e.g., ddGTP, ddATP, ddTTP, and ddCTP), 2'- or 3'-O-alkyl-nucleotides (e.g., 2'-O-methyl-nucleotides and 3'-O-methyl-nucleotides), 2'- or 3'-O-methoxyethyl-nucleotides (MOE), 2'- or 3' -fluoro-nucleotides, 2'- or 3'-O-allyl-nucleotides, 2'- or 3'-O-propargyl-nucleotides, 2'- or 3'-amine-nucleotides (e.g., 3'-deoxy-3'-amine-nucleotides), 2'- or 3'-O-alkylamine-nucleotides (e.g.,
  • sugar modifications include those found in the monomers that comprise the backbone of synthetic nucleic acids such as 2'-O,4'-C-methylene- ⁇ -D-ribonucleic acids or locked nucleic acids (LNAs), methylene-cLNA, 2',4'-(N-methoxy)aminomethylene bridged nucleic acids (N-MeO-amino BNA), 2',4'-aminooxymethylene bridged nucleic acids (N-Me-aminooxy BNA), 2'-O,4'-C- aminomethylene bridged nucleic acids (2',4'-BNA(NC)), 2'4'-C-(N-methylaminomethylene) bridged nucleic acids (2',4'-BNA(NC)[NMe]), peptide nucleic acids (PNA), triazole nucleic acids, morpholine nucleic acids, amide-linked nucleic acids, 1,5-anhydrohexitol nucleic acids (HNA), tri
  • Oligonucleotides as described herein may be attached to a substrate via a linker, a functional group, by cross linking, or by electrostatic charge that enables coating of the substrate with the reagent oligonucleotide but avoids non-specific binding of the target oligonucleotide.
  • the activated functional groups on the surface may be present on the predefined regions only, or alternatively on the entire surface, are reacted selectively with the functional groups present in the oligonucleotide molecules.
  • the necessary reaction conditions including time, temperature, pH, solvent(s), additives, etc. will depend on inter alia the particular species used and appropriate conditions for each particular situation will readily be apparent to the skilled person.
  • the oligonucleotide reagent may be attached to the substrate through the oligonucleotide 5' end, 3' end or through an internal nucleotide.
  • the oligonucleotide may be attached to the solid support through a linear or branched linker.
  • the linker separating the oligonucleotide from the substrate may serve as steric spacer and does not necessarily have to be of defined length. Examples of suitable linkers may be selected from any of the hetero-bifunctional cross-linking molecules described by Hermanson, Bioconjugate Techniques, 2nd Ed; Academic Press: London, Bioconjugate Reagents, pp 276-335 (2008), incorporated by reference.
  • the linker may be a flexible linker connecting the solid support to one or a plurality of same or different oligonucleotides.
  • (b) Functional Group or Cross Linking A variety of methods are known for attaching an oligonucleotide to a substrate, including covalent bonding to the support surface and non-covalent interaction (binding by adsorption, e. g. cationic surfaces) of the oligonucleotide with the surface. Typically, covalent immobilization involves the reaction of an active functional group on the oligonucleotide with an activated functional group on the solid surface.
  • reactive functional groups include amines, hydroxylamines, hydrazines, hydrazides, thiols, phosphines, isothiocyanates, isocyanates, N-hydroxysuccinimide (NHS) esters, carbodiimides, thioesters, haloacetyl derivatives, sulfonyl chlorides, nitro- and dinitrophenyl esters, tosylates, mesylates, triflates, maleimides, disulfides, carboxyl groups, hydroxyl groups, carbonyldiimidazoles, epoxides, aldehydes, acyl-aldehydes, ketones, azides, alkynes, alkenes, nitrones, tetrazines, isonitriles, tetrazoles, and boronates.
  • Examples of such reactions include the reaction between an amine and an activated carboxy group forming an amide, between a thiol and a maleimide forming a thioether bond, between an azide and an alkyne derivative undergoing a 1,3-dipolar cycloaddition reaction, between an amine and an epoxy group, between an amine and another amine functional group reacting with an added bifunctional linker reagent of the type of activated bis-dicarboxylic acid derivative giving rise to two amide bonds, or other combinations known in the art.
  • Other reactions, such as UV-mediated cross-linking can be used for covalent attachment of oligonucleotide to solid supports. Functionalization of surfaces with biological materials can also be used for attaching oligonucleotides to solid supports.
  • oligonucleotides can be specifically or nonspecifically attached to SNAP-tag/AGT, CLIP-tag/ACT, HaloTag or serine-beta-lactamases and subsequently be captured in a solid support functionalized with benzylguanine, pyrimidine, benzylcytosine, chloroalkane, or beta-lactam derivatives, respectively.
  • the activated surface functional groups may be present in predefined regions of the substrate only, or alternatively on the entire surface of the substrate, and can be reacted selectively with functional groups present in the oligonucleotide molecules.
  • the necessary reaction conditions including time, temperature, pH, solvent(s), additives, etc. will depend on inter alia the particular species used and appropriate conditions for each particular situation will readily be apparent to the skilled person.
  • Oligonucleotides can be synthesized to incorporate a desired functional group. Individual nucleotides can be modified either chemically or enzymatically with any type of functional group in order to provide the desired reactivity. This chemical or enzymatic functionalization can be extended to DNA and RNA molecules. For example, oligonucleotides can be specifically or nonspecifically attached to SNAP-tag/AGT,
  • CLIP-tag/ACT HaloTag or serine-beta-lactamases and subsequently be captured on a substrate functionalized with benzylguanine, pyrimidine, benzylcytosine, chloroalkane, or beta-lactam derivatives, respectively.
  • acyl carrier proteins and modifications thereof binding proteins
  • binding protein which are coupled to a phosphopantheteine subunit from Coenzyme A (binder substrate) by a synthase protein
  • proteins or fragments thereof allowing convenient binding of DNA to a solid support are e.g. chitin binding domain (CBD) (see for example, US 584,247 and US 7,732,565 and New England Biolabs) maltose binding protein (MBP) (See for example US 5,643,758 and US 8,623,615 and NEBExpress® MBP Fusion system (New England Biolabs, Ipswich, MA) omitting TEV cleavage), glycoproteins, transglutaminases, dihydrofolate reductases, glutathione-S-transferase al (GST), FLAG tags, S-tags, His-tags, and others known to those skilled in the art.
  • an oligonucleotide is modified with a molecule which is one part of a specific binding pair and capable of specifically binding to a partner covalently or noncovalently attached to a solid support.
  • Coating of magnetic or paramagnetic beads with oligonucleotide primers may be achieved using an affinity binding pair, such as in streptavidin-functionalized beads and biotinylated oligonucleotides.
  • affinity binding pair such as in streptavidin-functionalized beads and biotinylated oligonucleotides.
  • Other preferred affinity groups include desthiobiotin, avidin, NeutrAvidin, protein A, protein B, maltose- binding protein, chitin, poly-histidine, HA-tag, c-myc tag, FLAG-tag, SNAP-tag, S-tag and glutathione-S- transferase (GST).
  • magnetic or paramagnetic beads are coated with a poly dT sequence (e.g.
  • RNA 10-25 dT oligonucleotide
  • a target nucleic acid having a poly A tail such as a poly(A)+ viral RNA
  • Further preferred embodiments include magnetic or paramagnetic beads coated with a cap-binding protein (an example of such a protein is the translation initiation factor 4E, elF4E) which can be used for the direct binding of m7G(5')ppp(5')N (where N is any canonical nucleotide, including nucleotides with 2' O-methylation) capped viral nucleic acid (see for example PCT/US2020/031653).
  • a cap-binding protein an example of such a protein is the translation initiation factor 4E, elF4E
  • m7G(5')ppp(5')N where N is any canonical nucleotide, including nucleotides with 2' O-methylation
  • a further consideration is the use of immobilized enzymes to improve a coronavirus testing workflow that utilizes saliva (or other bodily fluid) as a starting material to test for the presence of virus and uses LAMP to amplify nucleic acids derived from any SARS-CoV-2 virus present in the saliva to determine whether an individual is infected.
  • the enzymes involved in reverse transcription and LAMP are stably and efficiently immobilized on a microchip or any column reactor or fluid channel network (including low-cost polymers, such as elastomer, and paper).
  • the enzymes may be irreversibly adsorbed or covalently linked to the solid surface using any one of the methods described in this invention.
  • the saliva sample and any other buffers and reagents are flowed through the microreactor by a peristaltic pump. Any unwanted biological material may be separated from the desired viral genome by means of an integrated in-line microchannel containing an appropriate separation material.
  • the separation material may be irreversibly adsorbed or covalently linked to channel to permit separation through one or more of the following techniques: size-selection, ion exchange, size-exclusion, affinity selection, hybridization, and liquid chromatography.
  • the saliva nucleic acid is then flowed through one or more channel(s) where the enzymes involved in reverse transcription and LAMP are immobilized. A readout of the LAMP reaction will indicate or not the presence of viral genome.
  • FIG. 28A-FIG. 28F An example of an automated workflow from sample to collection to output was envisaged in FIG. 28A-FIG. 28F. Although there are many possibilities in the uses of robotic devices for individual steps, the exemplified workflow is predicted to have a capability to perform 100,000 reactions in 20 hours.
  • LAMP sensitivity has been improved by reducing background and enhancing signal and these improvements can be followed through to the present assay. See for example: US 9,121,046, US 9,546,358, US 9,074,249, US 9,074,243, US 9,157,073, and US 9,127,258 in addition to US 9,580,748, US 9,034,606, and US 10,597,647 all incorporated in entirety by reference. These modifications can be incorporated into the colorimetric LAMP assay described herein to improve the detection of pathogens even further. US 10,253,357 is also incorporated by reference.
  • the diagnostic test of whether a pathogen is present in a sample can be scaled up without any difficulty so that any of l-1000s of reactions can be performed at the same time. If the reactions are performed in 96 well dishes or in 1000 well dishes, a robot liquid handler can add master mix to each well and then the swab sample and a computer system can record the color changes and the location of the well testing positive. For individual or small numbers of samples, the reactions might be performed in microfuge or PCR tubes. The entire diagnostic test can be completed within 4 hours, for example in less than 3 hours, for example less than 2 hours, for example less than 1 hour from the time of taking the sample to obtaining a result. The diagnostic test can be performed in a doctor's office or even at home.
  • the master mix including enzymes, dyes, primers and nucleotides may be dried onto a solid matrix such as paper so that addition of a measured droplet of the sample onto the paper and a heating step even on a surface heated by a homemade water bath, or small heating block that can be transported in a backpack for environmental use results in a color change to indicate a result.
  • the master mix may be freeze dried or lyophilized and contained in a tube ready for addition of a sample in the home or clinic.
  • Embodiments include incorporating amplification reagents in a facemask possibly immobilized on beads such that when droplets of saliva containing virus contact the beads, fluorescence results from an isothermal amplification reaction.
  • LAMP as described in embodiments herein requires a 65°C temperature step, this requirement may be circumvented in the future for LAMP or by use of other isothermal amplification methods.
  • the combination of a saliva droplet containing a virus particle interacting with immobilized regent on a bead might trigger an exothermic chemical reaction. The higher the virus load, the stronger the signal that would result from amplification.
  • the signal from the production of hydrogen ions or change in flow of electrons (that result from amplification as pyrophosphates are released when dNTPs are incorporated during amplification reaction) that generates a visual signal might in turn trigger a sound wave that is amplified resulting in an audible sound.
  • Such microelectronic technology already exists in a different formats and could be constructed using synthetic biology constructed circuits. An audible sound could alert the wearer of the mask of 3 rd party released virus without the need to remove the mask while a visual signal would alert others of the wearer of the mask being infected.
  • a discrete portion of a face mask may contain a chamber containing lysis reagent in a dried or liquid form so that incoming saliva droplets will be lysed and the polynucleotides released.
  • Embodiments describing improvements in the LAMP reaction that include carryover prevention, RNase inhibition, enhancement of sensitivity and rate of the LAMP reaction by the use of guanidine salts and/or reduced NaCI or KCI in the buffer, selecting primer sets and multiplexing primer sets, lyophilization of reagents can be combined in any combination for purposes of automation of large numbers of assays for genomic studies, gene expression studies or epidemiology analysis as well as point of care and miniaturization of tests to act as environmental sensors of pathogens.
  • Multiplexing as discussed herein can be achieved by pooling patient samples or purified nucleic acids from different sources and testing for the presence of a single species of target sequence such as contained in a single infectious agent such as SARS-CoV-2.
  • the samples in the pool can be differentiated by the use of random sequence identifiers referred to in the art as "unique identifiers "(U IDs), "unique molecular identifiers” (UMIs) or “degenerate base regions” (DBRs) etc. pH or fluorescent colorimetric LAMP as discussed herein is well suited for analyzing pooled samples.
  • Multiplexing may also be used on a single vertebrate subject sample, to determine the presence any one of several different target nucleic agents such as genes, or infectious agents such as viruses, bacterial or fungi such as respiratory viruses such a coronavirus, a rhinovirus, a respiratory syncytial virus, an Influenza virus, a parainfluenza virus, a metapneumovirus, an adenovirus and a bocavirus.
  • target nucleic agents such as genes, or infectious agents such as viruses, bacterial or fungi such as respiratory viruses such a coronavirus, a rhinovirus, a respiratory syncytial virus, an Influenza virus, a parainfluenza virus, a metapneumovirus, an adenovirus and a bocavirus.
  • a multiplex test would include Influenza A and B and SARS-CoV-2 that may occur together or separately in humans in winter seasons in northern and temperate zones.
  • Other vertebrates may include mammals such as wild or domestic mammals
  • Multiplex LAMP for multiple targets is preferably performed using fluorescent colorimetric LAMP for example using "Detection of Amplification by Releasing of Quenching” (DARQ) that supplements a standard LAMP primer set with a pair of oligonucleotides in duplex form for detection (see for example, US patent 9,074,243).
  • the DARQ oligonucleotide duplex consists of a 5'-modified version of the FIP primer (Q-FIP) annealed to a 3'-modified oligonucleotide represent the Fl region (Fd), complementary to the FIP in its 5' section.
  • the fluorescent dye and quencher can be present either on the 5' or complementary 3' oligonucleotide of the duplex.
  • the two modifications are selected to be dark quencher- fluorophore pairs, and when the quenched FIP is incorporated into the LAMP product, subsequent amplification displaces the Fd oligo, releasing quenching and producing fluorescent signal specific to the label and target of interest.
  • a list of some fluorescent dyes and quenchers are provided in US 9,074,243.
  • Example 17 describe a diagnostic assay in which multiple LAMP primer sets were used in a single tube and together were capable of detecting the two most common influenza strains and SARS-CoV-2 (SARS-CoV-2, Influenza A, Influenza B, and an internal control).
  • the diagnostic assay also conformed to the useful properties of speed, sensitivity and non-interference thereby expanding the utility of the widely- used LAMP chemistry to a multiplex diagnostic setting (see for example, FIGs 31A-31E and 32A- 32C).
  • the selection of appropriate targets, confirmation of minimum interference between primer sets, and choice of a suitable internal control to verify LAMP was achieved to enable a successful multiplex reaction to be performed.
  • an internal control a cellular RNA such as actin (ACTB) could be effectively incorporated into a multiplex DARQ LAMP reaction without interference providing that the concentration of the primer set for detecting the control was lower than the concentration used to detect respiratory pathogens in the sample.
  • the concentration of the control primer set should preferably be between 25% and 80% of the concentration of the primer sets for pathogen targets. For example, the preferred range was 50%-80% or 50%-75%.
  • Criteria were identified to establish the number and type of fluorescent dyes including wavelength emission spectrum, sensitivity of emitted signal from the unquenched sample and suitable multichannel devices for reading endpoint or continuous signals from multiple different fluorescent labels from a single reaction vessel.
  • End point fluorescence measurements could be reliably used to distinguish between positive and negative samples using a threshold value that was set based on negative (background) values. Positives were matched to those called during real time monitoring.
  • the end point result demonstrates compatibility of endpoint, plate-reader measurements with multiplex DARQ LAMP, enabling use on a wider range of instrument types and increasing potential test throughput compared with real time detection.
  • a limitation of real time detection is that it relies on certain detection instruments that are tied up during the entire incubation time of samples thereby negatively impacting sample throughput.
  • DARQ. LAMP could be used for detecting 5 different targets using 5 different sets of
  • LAMP. primers The limitation of 5 was set by the number of channels in a BioTek reader (BioTek, Winooski, VT). BioTek Synergy Neo2 microplate reader was used detecting a fluorescence signal for 5-FAM (Excitation, 484/20, Emission 530/25, Signal Gain 75), HEX (524/20, 565/20, 75), 5-ROX (569/20, 615/25, 85) and Cy5 (640/20, 682/20, 75). Indeed, there are as many as a hundred different fluorescent dyes that have different wavelengths across the 480 nm-640 nm spectrum.
  • a reader might accommodate primer sets and targets that exceed 5 targets in a single multiplex reaction.
  • Primer sets against two different Influenza RNA targets and one SARS-CoV-2 RNA target and a single internal control were used to establish the feasibility of multiplexing.
  • Sources of commonly understood terms and symbols may include: standard treatises and texts such as Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994), and Hale & Markham, the Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) and the like.
  • Numeric ranges are inclusive of the numbers defining the range. All numbers should be understood to encompass the midpoint of the integer above and below the integer i.e. the number 2 encompasses 1.5-2.5. The number 2.5 encompasses 2.45-2.55 etc.
  • sample numerical values may represent, unless specified otherwise, an intermediate value in a range of values. If specified, an individual numerical value may represent an extreme point in a range. Where a plurality of numerical values are provided these may represent the extremes of a range unless specified. If specified, these values may represent intermediate values in a range.
  • non-naturally occurring refers to a composition that does not exist in nature.
  • a “non-naturally occurring” protein may have an amino acid sequence that is different from a naturally occurring amino acid sequence for example, one or more amino acid substitutions, deletions or insertions at the N-terminus, the C-terminus and/or between the N- and C-termini of the protein.
  • the non-naturally occurring protein may have less than 100% sequence identity to the amino acid sequence of a naturally occurring protein although it may have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 98.5% or at least 99% identity to the naturally occurring amino acid sequence.
  • a non-naturally occurring protein may include a protein that has a post-translational modification pattern that is different from the protein in its natural state for example, an N-terminal methionine or may lack one or more post-translational modifications (e.g., glycosylation, 5 phosphorylation, etc.) if it is produced by a different (e.g., bacterial) cell.
  • a post-translational modification pattern that is different from the protein in its natural state for example, an N-terminal methionine or may lack one or more post-translational modifications (e.g., glycosylation, 5 phosphorylation, etc.) if it is produced by a different (e.g., bacterial) cell.
  • non-naturally occurring refers to a nucleic acid that contains: a) a sequence of nucleotides that is different from a nucleic acid in its natural state (i.e., having less than 100% sequence identity to a naturally occurring nucleic acid sequence); b) one or more non-natural nucleotide monomers (which may result in a non-natural backbone or sugar that is not G, A, T or C); and/or c) may contain one or more other modifications (e.g., an added label or other moiety) to the 5'- end, the 3' end, and/or between the 5'- and 3' -ends of the nucleic acid.
  • modifications e.g., an added label or other moiety
  • non-naturally occurring refers to: (a) a combination of components that are not combined by nature, e.g., because they are at different locations, in different cells or different cell compartments; (b) a combination of components that have relative concentrations that are not found in nature; (c) a combination that lacks something that is usually associated with one of the components in nature; (d) a combination that is in a form that is not found in nature, e.g., dried, freeze dried, crystalline, aqueous; and/or (e) a combination that contains a component that is not found in nature.
  • a composition may contain a "non-naturally occurring" buffering agent (e.g., Tris, HEPES, TAPS, MOPS, tricine or MES), a detergent, a dye, a reaction enhancer or inhibitor, an oxidizing agent, a reducing agent, a solvent or a preservative that is not found in nature.
  • a non-naturally occurring buffering agent e.g., Tris, HEPES, TAPS, MOPS, tricine or MES
  • the non-naturally occurring polymerase may be purified so that it does not contain DNases, RNases or other proteins with undesirable enzyme activity or undesirable small molecules that could adversely affect the sample substrate or reaction kinetics.
  • a master mix comprising: a strand displacing polymerase suitable for Loop- Mediated Isothermal Amplification (LAMP) of DNA; dATP, dGTP, dCTP, and dTTP; at least one reagent that changes color or provides fluorescence if amplification occurs; wherein the master mix is either dried or in a weakly buffered solution at a starting pH which is measurably altered during amplification.
  • LAMP Loop- Mediated Isothermal Amplification
  • master mix according to any of 1 through 6, wherein the master mix further comprises an aptamer for regulating the activity of the strand displacing polymerase.
  • RNase inhibitors comprise an aptamer for inhibiting RNase A, an aptamer for inhibiting RNase I and/or a protein inhibitor of RNAse.
  • the master mix according to 1 through 9 further comprising at least one set of primers having specificity for a target nucleic acid.
  • the master mix according to 1 through 10 further comprising a plurality of sets of primers having specificity for a target nucleic acid.
  • the at least one reagent includes a dye that is pH sensitive and changes color after an amplification reaction in which the pH is altered.
  • the master mix according to 1 through 12 further comprising dUTP.
  • the master mix according to 1 through 15 wherein the metallochromic indicator is 4-(2-pyridylazo) resorcinol (PAR). 17. The master mix according to 1 through 16, wherein the master mix is freeze dried, air dried, or lyophilized.
  • a method for determining whether a target nucleic acid is present in a sample comprising:
  • sample is a clinical sample, such as a sample of a body fluid or a sample taken from a swab, an environmental sample, or a sample of purified nucleic acid.
  • target nucleic acid is: associated with a pathogen or is a diagnostic target for pathogenesis; associated with gene expression; or an indicator of a metabolic response to a pharmaceutical preparation or allergen.
  • a composition comprising: one or more primer sets suitable for amplification, the primer sets having specificity for a single template nucleic acid of interest; and a buffer containing a molecule comprising C-(NH 2 ) 2 NH + .
  • composition according to 32, wherein the molecule comprising C-(NH 2 ) 2 NH + is selected from guanidine hydrochloride, guanidine thiocyanate, arginine, and guanidine sulfate.
  • composition according to 32 or 33, wherein the one or more primer sets are primer sets for Loop-Mediated Isothermal Amplification (LAMP).
  • LAMP Loop-Mediated Isothermal Amplification
  • composition according to any of 32 through 36 further comprising one or more reagents selected from an RNAse inhibitor, a reverse transcriptase, a thermolabile RNAse inhibitor, a reverse transcriptase, a thermolabile RNAse inhibitor, a reverse transcriptase, a thermolabile RNAse inhibitor, a reverse transcriptase, a thermolabile RNAse inhibitor, a reverse transcriptase, a thermolabile
  • Proteinase K and a polymerase.
  • composition according to any of 32 through 37 further comprising dNTPs; and optionally further comprising a reporter molecule for detecting amplification in the presence of a target nucleic acid.
  • composition according to 37, wherein the reporter molecule comprises a metallochromic dye.
  • composition according to 38, wherein the metallochromic indicator is 4-(2- pyridylazo) resorcinol (PAR).
  • a method of isothermal amplification comprising: (a) adding the composition according to any of 1 through 20, and 32 through 38, to a sample comprising a target nucleic acid; and (b) detecting whether the target nucleic acid is present in the sample.
  • LAMP isothermal amplification
  • a method for detecting a target nucleic acid in a biological sample comprising: (a) treating the biological sample with a lysis mixture comprising a C-(NH 2 ) 2 NH + salt, and a reducing agent; at an elevated temperature for an effective period of time;
  • (a) is 95°C and the effective time is 5 minutes.
  • a composition comprising: a guanidine salt, a reducing agent and detergent.
  • composition according to 51 further comprising LiCI.
  • guanidine salt is guanidine hydrochloride (Guanidine MCI).
  • composition according to any of 51 through 53, wherein the reducing agent is
  • composition according to any of 51 through 54, wherein the reducing agent is
  • TEP Tris(2-carboxyethyl)phosphine hydrochloride
  • composition according to any of 51 through 55 further comprising a biological sample.
  • a method for preparing a biological sample for an amplification reaction comprising:
  • a 2X lysis mixture comprises guanidine salt in the range of 300 mM-1.5 M guanidine hydrochloride, TritonX in the range of 1%-6%, and
  • TCEP in the range of 40 mM-120 mM.
  • step of increasing the temperature further comprises raising the temperature in the range of 65°C-95°C wherein the time of incubation at the raised temperature is inversely proportional to the temperature.
  • the method of 70 further comprises incubating the sample at room temperature for
  • step of amplifying further comprises amplifying the target polynucleotide by LAMP.
  • sequencing further comprises adding sample barcodes for large scale multiplexing of samples, wherein the bar code is added in the primer where amplification precedes sequencing or by ligation when the target polynucleotides are sequenced directly.
  • kits comprising the master mix of any of 1 through 20, and/or the compositions of 32 through 39, and 51 through 62, wherein the kit optionally further comprises a heating block suitable for heating a reaction tube, plate, or paper, or a plurality of the same.
  • a kit for performing multiplex loop mediated amplification (LAMP) reaction comprising:
  • kit according to 79 wherein the stand-displacing DNA polymerase of (b) is contained in a master mix that further comprises: deoxynucleotide triphosphates including dUTP; a reversible inhibitor of polymerase activity that blocks polymerase activity at a temperature below 40°C; and at least one dye or fluorescent indicator for detecting an amplification product by a change in color or fluorescence.
  • kit according to 80 further comprising instructions to combine the reagents of (a)-(c) with a target DNA in order to amplify the different template sequences by LAMP.
  • kit according to 81 further comprising a reducing agent.
  • TCEP Tris(2-carboxyethyl)phosphine hydrochloride
  • each set of oligonucleotide primers comprises 4, 5 or 6 primers.
  • kit according to 79 wherein the target nucleic acid is derived from a coronavirus RNA and each set of primers hybridizes to a different template sequence in the coronavirus RNA genome, or cDNA copy thereof.
  • oligonucleotide primers comprise a mixture of 12 different primers.
  • the oligonucleotide primers target multiple target nucleic acids with a plurality of sets of oligonucleotide primers for each target.
  • a method comprising:
  • composition further comprises a reverse transcriptase if the target nucleic acid is RNA, and the method comprises reverse transcribing the RNA into DNA.
  • composition of claim 79 further comprises at least one additional reagent selected from the group consisting of: reverse transcriptase,
  • UDG UDG
  • dUTP dATP
  • dTTP dGTP
  • dCTP a color or fluorescent indicator that changes color or fluorescence when amplification has occurred
  • a reducing agent a detergent
  • each set of oligonucleotide primers comprises 4 or 6 primers.
  • the plurality of sets of primers for the target nucleic acid comprise at least 2 sets of primers, each set of primers hybridizing to a different target sequence in the coronavirus genome, or cDNA copy thereof.
  • the plurality of sets of primers for the target nucleic acid comprise at least 2 sets of primers and (a) further comprises including multiple different target nucleic acids wherein a plurality of sets of primers hybridize to a different target sequence in each target nucleic acid.
  • a method for detecting SARS-CoV-2 virions in saliva of a subject comprising:
  • oligonucleotides are characterized by a single sequence capable of binding to either end of the virion RNA or to an internal sequence in the virion RNA.
  • oligonucleotides are characterized by a plurality of different sequences wherein each sequence targets a different region of the viral genome.
  • oligonucleotides are characterized by one or a plurality of sequences capable of acting as a primer to initiate reverse transcription.
  • oligonucleotides are characterized by a plurality of sequences capable of targeting different viral RNA genomes such as SARS-
  • proteolytic reagents are selected from Proteinase K, thermolabile Proteinase K, salt, detergent, and reducing agent.
  • RNAse inhibitors are aptamers or antibodies.
  • a composition comprising a receiving vessel containing reagents comprising (i) oligonucleotides immobilized on a substrate in a diluting volume of buffer containing
  • oligonucleotide immobilized on a substrate DNase inhibitors, RNase inhibitors and proteolytic agents or (ii) lyophilized oligonucleotide immobilized on a substrate, DNase inhibitors, RNase inhibitors and one or more proteolytic agents for rehydration in contact with the saliva; wherein the oligonucleotides are characterized by one or more sequences, wherein at least one sequence is complementary to a portion of SARS-CoV-2 RNA.
  • composition according to 119 wherein the oligonucleotides are capable of hybridizing to at least one of the polyA tail of the viral RNA, the capped end of the RNA or to an internal target sequence.
  • the reagents further comprise one or more of the following: Proteinase K, thermolabile Proteinase K, aptamer inhibitors of RNAses and/or DNases, antibody inhibitors of RNAses and/or DNases, salts to facilitate hybridization and/or protein denaturation, detergents, surfactants and/or reducing agents.
  • a kit comprising the compositions of 119 through 124.
  • a composition comprising a mixture of LAMP primer sets wherein each of a plurality of different primer sets in the mixture have a specificity for a different respiratory virus.
  • a composition according to 126 further comprising a primer set in the mixture having a specificity for a cellular non-viral target.
  • target pathogens selected from a coronavirus, a rhinovirus, a respiratory syncytial virus, an influenza virus, a parainfluenza virus, a metapneumovirus, an adenovirus and a bocavirus.
  • Q.-FIP FIR primer
  • composition according to any of 126 through 130, wherein the concentration of the primer set in the mixture that is specific for the cellular non-viral target has a concentration in the range of 25%-80% for example 50-75% of the primer sets for respiratory viruses.
  • a kit comprising:
  • a composition comprising a biological sample combined with a buffer containing an antifoaming agent, wherein the antifoaming agent is a poloxamer.
  • composition according to 134 further comprising one or more LAMP primer sets.
  • a kit comprising, a reaction container for receiving a swab containing a biological sample or a liquid biological fluid, the reaction container optionally containing a buffer containing a poloxamer or wherein the buffer containing the poloxamer is contained in a distinct compartment in the reaction container or is provided in a separate tube for receiving an aliquot of the biological sample.
  • a method comprising:
  • each primer set comprises a quenched fluorescent dye in a duplex oligonucleotide.
  • the plurality of LAMP primer sets are specific for one or more virus nucleic acids selected from the group consisting of a coronavirus, a rhinovirus, a respiratory syncytial virus, an influenza virus, a parainfluenza virus, a metapneumovirus, an adenovirus and a bocavirus.
  • a kit for Loop-Mediated Isothermal Amplification comprising:
  • thermolabile uracil DNA glycosylase UDG
  • nucleoside triphosphates comprising dATP, dGTP, dCTP, and dTTP; and dUTP; and at least one indicator reagent that changes color or provides fluorescence if amplification occurs; wherein any of (a) to (e) are separate or combined into one or more mixtures in the kit.
  • the kit according to 148 wherein the strand displacing polymerase is a mesophilic bacterial strand displacing polymerase and the temperature for copying the DNA is 65°C for less than 1 hour.
  • kit according to 148 or 149, wherein the reversible inhibitor is an oligonucleotide.
  • kit according to any of the preceding embodiments, further comprising a receiving container for a biological sample.
  • kit further comprising lysis reagents for adding to or contained in the receiving container for lysing a biological sample to release any target nucleic acids therein for amplification by Loop-
  • lysis reagents comprise a reducing agent and a metal chelator.
  • lysis reagents comprise a salt of C-(NH 2 ) 2 NH + .
  • kit according to any of the preceding claims, further comprising: in a separate container, at least one set of LAMP primers.
  • Isothermal Amplification (LAMP) primers further comprise a plurality of primer sets for amplifying a plurality of target nucleic acids in the biological sample.
  • Isothermal Amplification (LAMP) primers further comprise a plurality of primer sets for amplifying a nucleic acid target from a single virus strain in the biological sample.
  • kits according to any of claims 148-151 and 155-159, wherein any or all of the lysis reagents and/or reagents for Loop-Mediated Isothermal Amplification (LAMP) amplification except for a salt of C-(NH 2 ) 2 NH + , are lyophilized, freeze dried or in a solution.
  • LAMP Loop-Mediated Isothermal Amplification
  • kit according to any of 148-151 and 155-160, further comprising a buffer with a buffering concentration of no more than equivalent to 5 mM Tris buffer for use with the kit components if the indicator in (e) is a pH dependent colorimetric dye.
  • the kit according to 152 further comprising instructions for use with a biological sample wherein the biological sample is selected from a body fluid or tissue, an agricultural sample, a food sample, a waste product, and a pathogen. 163.
  • the biological sample is a body fluid or tissue selected from the group consisting of mucous, urine, lymph, blood, saliva, feces, sputum, sweat, semen and biopsy.
  • the kit according to any of the previous claims were in the enzymes and/or oligonucleotides and/or Loop-Mediated Isothermal Amplification (LAMP) primer sets are immobilized on a substrate for reacting with a nucleic acid in the biological sample.
  • LAMP Loop-Mediated Isothermal Amplification
  • kit according to any of 148-161 and 163-165, wherein the indicator reagent is a metallochromic dye.
  • the reverse transcriptase is selected from a virus derived reverse transcriptase, or from a Group II intron reverse transcriptase.
  • kit according to 168-169 further comprising a reversible inhibitor of the reverse transcriptase.
  • kits according to 152 wherein the biological sample is saliva or a nasal swab and the target nucleic acid is a single target RNA virus genome.
  • the receiving container for the biological sample is a vessel with a lid, the lid containing a solution of indicator reagent for release into the reaction vessel after lysis of the biological sample or after Loop-Mediated Isothermal Amplification (LAMP).
  • LAMP Loop-Mediated Isothermal Amplification
  • kit according to any of 148-172 suitable for use in a high sample throughput workflow that is partially or completely automated and further comprises a recording device for storing and/or reporting positive sample data after detection of a change in color or fluorescence of the indicator resulting from amplification of the target nucleic acid.
  • the receiving container is selected from paper, a microfluidic device and a polymer surface.
  • a reaction mixture comprising: a thermolabile uracil DNA glycosylase (UDG), a strand displacing polymerase, a reversible inhibitor of the polymerase, and a salt of
  • a lysis mixture for releasing an RNA from a biological sample for detection by amplification and/or sequencing comprising a poloxamer, a reducing agent and a metal chelating agent.
  • a master mix comprising: a thermolabile uracil DNA glycosylase (UDG), a strand displacing polymerase, a reversible inhibitor of the polymerase, a reverse transcriptase and a reversible inhibitor of the reverse transcriptase.
  • UDG thermolabile uracil DNA glycosylase
  • a strand displacing polymerase a strand displacing polymerase
  • a reversible inhibitor of the polymerase a reverse transcriptase and a reversible inhibitor of the reverse transcriptase.
  • LAMP Mediated Isothermal Amplification
  • step (c) combining an aliquot of the heat treated mix after step (b) with amplification reagents comprising a strand displacing polymerase, a reversible inhibitor of the polymerase, a thermolabile uracil DNA glycosylase (UDG), nucleoside triphosphates, and at least one set of LAMP primers that hybridize to the target nucleic acid, to produce a reaction mix; and
  • the lysis reagent in (a) comprises at least one of a salt of C-(NH 2 ) 2 NH + and a poloxamer to produce a lysis mix.
  • lysis reagent comprises a reducing agent and a metal chelating reagent.
  • any of the reagents in the lysis mix and any of the amplification reagents may be lyophilized prior to combining with the biological sample.
  • the method according to 186 further comprising (e) detecting a change in color or fluorescence of the indicator reagent corresponding to the amplification of the target nucleic acid.
  • RNA virus and the amplification reagents include a reverse transcriptase and a reverse transcriptase reversible inhibitor.
  • RNA virus is a coronavirus and the amplification reagents include multiple sets of primers.
  • the multiple sets of primers include a plurality of primers sets for amplifying at least two different sequences in the coronavirus genome or cDNA copy of the coronavirus genome.
  • a method for analyzing a biological sample to determine the presence of a target nucleic acid comprising:
  • a lysis reagent comprising a reducing agent, a metal chelator and at least one of a guanidine salt and a poloxamer to produce a lysis mix
  • reaction mix comprises an indicator reagent that changes color or provides fluorescence if amplification occurs, and the method further comprises determining whether the reaction mix contains the target nucleic acid based on a change in color or fluorescence.
  • a method for detecting an RNA virus in saliva or a nasal swab of a subject comprising:
  • substrate immobilized oligonucleotides for binding viral RNA and a lysis reagent mix comprising two or more reagents selected from a guanidinium salt, a poloxamer, a reducing agent, a DNase inhibitor, an RNase inhibitor, a detergent, a metal chelator and a proteolytic agent; or
  • lyophilized substrate immobilized oligonucleotides for binding viral RNA and/or one or more lyophilized reagents contained in a lysis reagent mix, wherein the lysis reagent mix comprises two or more reagents selected from a poloxamer, a reducing agent, DNase inhibitor, an RNase inhibitor, a detergent, a metal chelator and a proteolytic agent, wherein the lyophilized reagents become rehydrated when contacted by the collected saliva;
  • step (b) incubating the receiving container after step (a) at an effective temperature and time to release nucleic acid from any coronaviruses in the saliva for binding to the immobilized oligonucleotides;
  • step (d) adding amplification reagents to the substrate after step (c), wherein amplification reagents comprise reverse transcription reagents and DNA amplification reagents, to make a reaction mix; and
  • the receiving container is selected from a paper substrate, a microfluidic device or a polymer surface.
  • the lysis reagent mix in (i) comprises three or more reagents selected from a guanidinium salt, a poloxamer, a reducing agent, a
  • DNase inhibitor, an RNase inhibitor, a detergent, a metal chelator and a proteolytic agent or in (ii) comprises three or more reagents where at least one reagent is lyophilized that is selected from a poloxamer, a reducing agent, DNase inhibitor, an RNase inhibitor, a detergent, a metal chelator and a proteolytic agent.
  • reaction mix of (d) further comprises an indicator reagent that changes color or provides fluorescence if amplification occurs, and wherein the method further comprises detecting a change in a signal that indicates the presence of coronavirus in the saliva of the subject.
  • amplification reagents further comprise at least two sets of Loop-Mediated Isothermal Amplification (LAMP) primers that target different regions of a coronavirus genome.
  • LAMP Loop-Mediated Isothermal Amplification
  • LAMP Loop-Mediated Isothermal Amplification
  • LAMP Amplification
  • the method of 206, wherein the biological sample is saliva, a nasal swab or a buccal swab.
  • a kit for use in diagnostic detection of a target nucleic acid and variants thereof having undefined mutations, obtained from a cell or virus in a biological sample comprising:
  • the indicator reagent is characterized by its ability to change color or provide fluorescence in a nucleic acid amplification reaction
  • the strand displacing polymerase when rehydrated is capable of amplifying DNA at a temperature in the range of 50°C-68°C
  • a universal primer set suitable for loop mediated amplification (LAMP) of the target nucleic acids and variants thereof having undefined mutations wherein any of the reagents in the kit may be combined in a mixture in a single container or provided in separate containers.
  • LAMP loop mediated amplification
  • the kit according to 208 wherein the target nucleic acid is a target DNA that is the reverse transcription product of an RNA virus.
  • lysis reagents comprise at least one of a salt of C-(NH 2 ) 2 NH + and a poloxamer.
  • kit according to any of 208-216 comprising the reverse transcriptase, wherein the reverse transcriptase is a virus encoded reverse transcriptase, or a bacteria encoded intron II reverse transcriptase.
  • step (c) combining in a reaction mix, an aliquot of the heat treated lysis mix of step (b) with amplification reagents comprising a strand displacing polymerase, a reversible inhibitor of the polymerase, nucleoside triphosphates, and at least one set of LAMP primers that is capable of hybridizing to the target nucleic acid and to undefined variants thereof; and
  • amplification reagents include a reverse transcriptase and a reversible inhibitor of the reverse transcriptase.
  • any of the lysis reagents and any of the amplification reagents may be lyophilized prior to combining with the biological sample.
  • 225 The method according to any of 218-223, wherein the target nucleic acid is an RNA genome from a virus.
  • SARS-CoV-2 virus RNA is analyzed directly from nasal swabs using a visual, colorimetric detection.
  • This simple and sensitive method provides an opportunity to facilitate virus detection in the field without a requirement for complex diagnostic infrastructure.
  • the general features of the method were reported in Tanner, et al. BioTechniques 58:59-68 (2015) and reagents for conducting the method are provided by New England Biolabs (M1800).
  • the sensitivity of the method was tested for the coronavirus described as SARS-CoV-2.
  • LAMP oligonucleotide primers targeting two fragments were designed using the online software Primer Explorer V5 (available for free use at: https://primerexplorer.jp/e/). The two fragments corresponded to the 5' region of the ORFla gene and Gene N. Three sets of primers were designed to target ORF1 and two for GeneN. Each set of primers was tested with synthetic DNA substrates (gBIocks®, Integrated DNA Technologies, Coralville, Iowa) and RNA (in vitro transcribed RNA from that DNA) prior to clinical use.
  • synthetic DNA substrates gBIocks®, Integrated DNA Technologies, Coralville, Iowa
  • RNA in vitro transcribed RNA from that DNA
  • DNA fragments containing two SARS-CoV-2 sequences were synthesized as gBIocks.
  • T7 RNA polymerase promoter sequences were added by PCR (M0493) (numbers indicate New England Biolabs, Inc. catalog ID numbers unless otherwise noted).
  • the PCR reaction utilized primer pairs where one primer of the pair containing the T7 RNA promoter sequence.
  • the PCR amplicon was then transcribed by in vitro transcription (E2050) to produce RNA with sequences that mimicked the selected portions of the SARS-CoV-2 virus. This RNA was purified using RNA clean up columns (T2040).
  • the resulting RNAs as well as the corresponding gBIocks DNA were serially diluted in 10-fold increments using 0.1X TE buffer containing 0.01% Tween 20.
  • All LAMP assays were performed in a 20 ⁇ l reaction mixture containing 2 ⁇ L of 10X primer mix of 16 ⁇ M (each) of Forward Inner Primer (FIP) and Backward Inner Primer (BIP), 2 ⁇ M (each) of F3 and B3 primers, 4 ⁇ M (each) of Forward Loop (LF) and Backward Loop (LB) primers, 10 ⁇ L of WarmStart Colorimetric Lamp 2X master mix (M1800) 5 ⁇ L of DNAse, RNase free water and 3 ⁇ l of RNA template. Individual primer pair sets that were optimal (where one set includes 6 primers) were selected for 0RF1 and Gene N.
  • test samples were prepared as follows: synthetic viral RNAs were spiked into Hela cells, which were then diluted and lysed using Luna Cell Ready Lysis Module (E3032). Each lysate was then diluted 10X with O.lx TE +0.01% Tween 20 and 1 ⁇ L was added to standard colorimetric LAMP reactions.
  • synthetic RNA described above was spiked into 200 ⁇ L whole human blood (Quadrant Health Strategies, Beverly, MA) and then purified the total blood RNA using Monarch Total RNA Miniprep Kit (T2010).
  • pH dependent detection sensitivity assays showed that all five primer sets provided similar detection sensitivity and could consistently detect as low as 120 copies of the viral RNA (or 4.8 copies/ ⁇ L) as determined by serial dilution of ⁇ 120 million copies down to ⁇ 120 copies (per 25 ⁇ L reaction) at 10-fold intervals in the colorimetric LAMP reactions.
  • the relative efficiency of pH dependent colorimetric LAMP using RNA or DNA templates was determined from the real time LAMP signal using synthetic RNA with similarly diluted gBIock dsDNA.
  • Example 2 Analysis of total RNA from crude lysates for identification of SARS-CoV-2 (COVID-19) virus RNA
  • thermolabile UDG a thermolabile UDG
  • 50:50 dTTP: dUTP a 50:50 dTTP: dUTP in addition to dCTP, dGTP and dATP was demonstrated.
  • FIG. 6A shows that this change in reagents did not affect the sensitivity or the specificity of the assay. Moreover, these additions to the master mix were effective in removing carryover nucleic acids from one sample to the next.
  • Each RT-LAMP reaction contained IX CP-LAMP MM (no UDG or with UDG), IX LAMP primers, genomic RNA, and IX LAMP fluorescent dye in a reaction volume of 25 ⁇ L.
  • the plate was incubated at 65°C for 75 minutes in a qPCR instrument, then imaged in a flatbed fluorescence scanner. The results are shown in FIG. 6A.
  • a control was added as shown which had neither dUTP or UDG. No difference was observed between control and samples with the carryover prevention additives.
  • Each RT-LAMP reaction contains IX CP-LAMP MM (with UDG), IX LAMP primers, genomic RNA, and IX LAMP fluorescent dye in a reaction volume of 25 ⁇ L. Both rows are identical replicates.
  • the first well in each row contains approximately 1 ng (1000 pg) of genomic RNA. From the second well onwards, 10-fold dilutions were completed, with the last well in each row serving as a no-template (NT) reaction with no RNA.
  • the plate was incubated at 65°C for 75 minutes in a qPCR instrument, then imaged in a flatbed fluorescence scanner. No amplified product was observed in amounts where carryover occurs.
  • Example 4 Nasal sampling for detecting SARS-CoV-2
  • Nasal samples are collected by swab and placed in sterile water in a microfuge tube. An aliquot of the sample is then combined with a master mix prepared as described above (see Example 3). Thermolabile UDG (New England Biolabs, Ipswich, MA) is added according to the manufacturer's instructions.
  • Four primer sets from Table 1 described in Example 2 can be used here although a single set of primers for each of 0RF1 and GeneN is sufficient. Modifications of the primers described in Table 1 can also be utilized. Other regions in the virus may be additionally or alternatively utilized.
  • the reaction mixture is then heated to a temperature of about 65°C using a temperature block for 15-60 minutes at which time the amplification is complete.
  • Example 5 Colorimetric LAMP using lyophilized colorimetric LAMP mix
  • 2X master mix (2X MM) for colorimetric LAMP was prepared using standard concentrations of LAMP reaction components described above (Bst 2.0 and RTx enzymes, aptamers to both, nucleotides, pH dependent dye, salt, detergent in a weakly buffered solution) together with 150 mM Trehalose, glycerol-free WarmStart RTx and high concentrations of WarmStart Bst 2.0. Extra KOH was added to half of the mix to increase the pH of the 2X MM to pH 8.2 from pH 8.0.
  • the reconstituted 2X MM and the 2X MM previously stored at -20°C were then added to 12.5 ⁇ l of primer/template mix and 1 ⁇ M dsDNA binding fluorescent dye (Syto- 9).
  • the primer set for HMBS2 was used for RT-LAMP and the template was Jurkat total RNA at 10 ng or 0.3 ng.
  • the reaction was incubated in a Bio-Rad IQTM5 Real Time PCR machine (Bio-Rad, Hercules, CA) to monitor the speed of the reaction and colorimetric or fluorometric detection of amplicons at the end of a 45 minute incubation at 65°C.
  • Example 6 PAR-based Colorimetric Detection of Nucleic Acid Amplification is an effective alternative to pH dependent colorimetric LAMP
  • a standard 2X LAMP master mix was prepared (see Example 1) and added to DNA so that the reaction mix contained using the following DNA polymerases in separate reactions: Bst LF, Bst 3.0, Bst 2.0 or WarmStart Bst 2.0 (all products from New England Biolabs, Ipswich, MA) in standard amplification buffer containing Tris-HCI, pH 8.8 at 25°C; (NH 4 ) 2 SO 4 ; KCI; MgSO 4 . The buffer was varied from 0-4% Triton X-100. PAR concentration was varied from 250 ⁇ M to 50 ⁇ M PAR with results shown for 150 ⁇ M, 100 ⁇ M, 75 ⁇ M and 50 ⁇ M (FIG. 13).
  • MnCl 2 was used throughout at concentrations in the range of 0.4 mM -1.6 mM. In FIG. 13, the reactions shown contained 0.5 ⁇ M MnCl 2 .
  • the LAMP primer set was BRCA2b including FIP/BIP/F3/B3/LF/LB (see below). 1 ⁇ l Hela genomic DNA (100 ng/ ⁇ l) was used in the positive samples and no DNA in the controls. The reaction was performed at 65°C for 1 hour. A positive endpoint was yellow corresponding to the reaction of manganese with pyrophosphate. The negative control was orange corresponding to the reaction of manganese with PAR.
  • Example 7 Non-ionic detergent increases the positive signal in a LAMP reaction using PAR to detect sample
  • Triton X-100 An example of a non-ionic detergent (Triton X-100) was added to the LAMP 2X master mix containing PAR.
  • the reaction mix contained Bst 2.0, PAR (200 ⁇ M), Manganese (0.8 mM), Isothermal amplification buffer with 2% Triton X-100, the BRCA 2b primer set as used in Example 5 and 1 ⁇ l Hela gDNA.
  • the beneficial effect of adding Triton X-100 to the colorimetric PAR LAMP reaction is shown here, any non-ionic detergent from the Triton series or from the Brij series is expected to show similar benefits.
  • the results are shown in FIG. 11A-11B.
  • 2% Triton X-100 was used in FIG. 14 to provide enhanced signal without adversely affecting polymerase activity although l%-3% Triton X-100 also showed enhanced signal in visible wave lengths in FIG. 13.
  • Guanidine hydrochloride (10 mM-60 mM) not only increased the rate of the LAMP reaction performed according to Example 1, but also improved the limit of detection sensitivity (see FIG. 16, FIG. 17A-17C and FIG. 18A-18D).
  • BRCA and CFTR detection Standard LAMP amplification was performed in lx ThermoPol buffer and Bst 2.0 DNA polymerase at 65°C using 10 ng of genomic DNA isolated from Hela cells as template. Two amplification targets were tested: BRCA gene fragment and CFTR gene fragment. Guanidine hydrochloride at a final concentration of 0 mM -60 mM were added to the reactions. The amplification reactions contained 1 ⁇ M dsDNA binding dye-Syto9 and the reaction was performed and the reaction speed was monitored on a Bio-Rad IQ5 Real Time PCR machine.
  • SARS-CoV-2 detection single primer set in a single LAMP assay: As shown in FIG. 16, guanidine significantly increased the LAMP reaction speed for both primer sets with optimal concentration range between 20 mM-40 mM for primer sets 3 and 4 for coronavirus detection.
  • the primer sets were tested against an AccuPlexTM SARS-CoV-2 Verification Panel (SeraCare Milford, MA) where the viral RNA is contained in a noninfectious viral protein coat.
  • Primer set 1 or 5 Asle/Orfla (5'-3'):
  • LAMP reactions were set up in ThermoPol buffer (lOmM KCI) for Bst2.0 (see FIG. 17B or in isothermal amplification buffer (50mM KCI) for Bst 3.0 (see FIG. 17C) using a Iambda2 primer set with 0.5 ng lambda DNA with or without 30 mM guanidine hydrochloride. Addition of guanidine stimulated the LAMP amplification rate significantly at the lower end of the KCI concentration both with Bst 2.0 and Bst 3.0.
  • Standard HDA in IsoAmp II kit H0110 was performed using 0.1 ng plasmid provided in the kit but added guanidine hydrochloride at a final concentration of 0 mM -60 mM. The reactions were performed at 65°C and EvaGreen dye was included to monitor the progression of amplification. The Tt (time to threshold) was used to estimate the rate of amplification. Tt was shown to be 5 minutes shorter than standard HDA reactions. It was concluded that guanidine HCI increase melt temp for 0.5°C per 10 mM up to 60 mM guanidine hydrochloride. Isothermal reactions were monitored in the presence of reduced NaCI in the buffer. Increased rates were observed when NaCI was reduced from the standard amount of 40 mM to 10 mM NaCI. Less NaCI gave a higher RFU signal (see FIG. 17A).
  • Example 9 Detection of polynucleotides in saliva samples using pH-dependent colorimetric LAMP
  • the lysis buffer was tested to determine an optimal formula for enhanced sensitivity of a LAMP assay for SARS-CoV-2.
  • a lysis buffer containing guanidine hydrochloride (GnHCL) (Millipore Sigma, Burlington, MA) was tested at various concentrations in the range of 10 mM-400 mM (IX) in combination with 1 mM, 4 mM and 8 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)(Millipore Sigma, Burlington, MA) (IX) with or without 75 mM LiCI (NEB product B20151) (IX) against a SARS-CoV-2 virus titer that was varied for different samples containing 5,000 cps/ml, 10,000 cps/ml, 20,000 cps/ml or 40,000 cps/ml (from a stock solution of 100,000 cps/ml from SeraCare, Milford, MA).
  • GnHCL guanidine hydrochloride
  • the virus was spiked into saliva of 20 ⁇ l, 30 ⁇ l, 35 ⁇ l and 37.5 ⁇ l volumes with TCEP and GnHCL. Copies of actin RNA at 100 copies/ ⁇ l saliva was used as a control. Lysis of the virus in saliva added to the saliva lysis mix occurred at 95°C for 5 minutes (the 10X lysis buffer contained 100 mM-4 M GnHCL, 10 mM-80 mM TCEP and 750 mM LiCI)
  • N2+E1 primer sets were added to a LAMP master mix (New England Biolabs, Ipswich, MA) containing reverse transcriptase to amplify SARS-CoV-2 virus derived RNA.
  • FIG. 21A and FIG. 21B 400 mM GnHCL and varying TCEP concentration, pH and LiCI concentration for 20,000 cps/ml where final concentrations are given
  • FIG. 22 showing the effect of increasing the concentration of TCEP
  • FIG. 23A-FIG. 23D where the LAMP reaction time was varied from 35 minutes to 60 minutes with and without LiCI.
  • a LAM P master mix (10 ⁇ l of 2X stock from NEB product M1800, 0.8 ⁇ l 25X primer set N2 and 0.8 ⁇ l 25X primer set El, 0.4ul 50X dye and 6 ⁇ l water to a total of 18 ⁇ l) and incubated at 65°C for 35 minutes.
  • the lysis buffer was found to be compatible with colorimetric LAMP (FIG. 21A-21D to FIG. 23A- 23D), fluorescent LAMP and RT-qPCR (FIG. 24). It should be noted that where a lysis buffer is used that contained guanidine salt, it was not necessary to add the guanidine salt to the master mix because of the carryover of this salt from the lysis buffer.
  • ACTB-LoopF TGTGGTGCCAGATTTTCTCCA (SEQ ID NQ:100)
  • ACTB-LoopB CGAGAAGATGACCCAGATCATGT SEQ ID NO:102
  • Example 10 Detection of polynucleotides in saliva samples using RT-qPCR
  • the RT-QPCR reaction was set up as follows:
  • the thermal cycler was set up as follows:
  • SARS-CoV-2 RNA (Twist Biosciences, San Francisco, CA) and virus (SeraCare, Milford, MA) were spiked separately into different tubes of 2X saliva lysis buffer. The virus was previously spiked into saliva. A positive control contained purified RNA and a negative control contained water only. The results are consisted with those reported for RT Q.PCR from saliva or nasopharyngeal swabs where detection limits were ascertained from 10 copies/sample (5000 cps/ml) up to 100,000 cps/ml.
  • Lysis buffer has minimal or no adverse effects on RT-qPCR or on LAMP
  • the lysis buffer as a whole had no adverse effects on the sensitivity of RT-QPCR or pH-dependent colorimetric LAMP. The results are shown in FIG. 24.
  • Example 12 Automation of a workflow to achieve a throughput of 100,000 reactions in about 20 hours
  • a workflow that is capable of delivering high volume throughput is illustrated in FIG. 28A-FIG. 28F and may include the following instruments: RNA collection tubes from Ora (Ottawa, Canada) (ORE- 100), 96-384 tube to plate sample transfer (Bravo with 96 or 384 ST Head from Agilent, Santa Clara, CA), 384 well plate consumables (Corning, NY), 384 well filling LAMP master mix into detection plates (BioTek, Winooski, VT), heat sealing of plates (Thermo Fisher, Waltham, MA), Automated 65°C timed incubation (StoreX -Liconic Instruments) or Intek conveyer (Intek WA), Endpoint fluorescence (BioTek, Winooski, VT) or SpectraMax. Although any of these instruments can be switched out for other comparable devices, the workflow illustrates the suitability of colorimetric LAMP for high throughput workflows that are relatively simple, cost effective,
  • Example 13 A surfactant containing buffer for use in a sensitive nucleic acid assay for detecting SARS- CoV-2 in saliva
  • a buffer was formulated at a concentration that was a suitable multiple of the final concentration for adding to a volume of sample.
  • the buffer may be formulated in high concentrations such as 10X or 5X, a concentration of 2X sample buffer was used here as this offered ease of mixing with the relatively viscous saliva sample.
  • the NEB sample 2X buffer (2X contains 8 mM TCEP/NaOH (pH 8.2) 800 mM GuHCI, 150 mM LiCI, 0.2 mM EDTA, 0.4% PF68 ) was found to be stable over months at 2X concentration.
  • the final concentration of the sample buffer (NEB buffer) combined with saliva was: 4mM TCEP (pH 8.2) 400 mM GuHCI, 75 mM LiCI, 0.1 mM EDTA, 0.2% PF68.
  • the CEPKO buffer (IX contains 2.5 mM TCEP, 11 mM NaOH, 1 mM EDTA) was found to be stable at a 100X concentration but was not stable at 2X or 5X concentration and 100X Cepko buffer was made freshly for each experiment.
  • a reducing agent in the buffer could be used within the range of 1 mM-8 mM and pH adjusted to pH 8 with NaOH.
  • Guanidinium hydrochloride was preferably included in the sample buffer for testing using pH colorimetric LAMP.
  • the surfactant used in this example was the poloxamer PF68. Although used here at 0.2%, the concentration can be anywhere in the range of 0.1%-l%.
  • EDTA was used at 0.1 mM but the concentration of EDTA can be in the range of 0.1%-l%.
  • heat inactivated SARS-CoV-2 (3.75 x 10 8 / cps/ml) or gamma irradiated SARS- CoV-2 (1.75 x10 9 cps/ml) supplied by BEI Resources or from ATCC was serially diluted to determine whether the presence of saliva could adversely affect the sensitivity of the assay.
  • the assay was carried out as follows:
  • 16 ⁇ L virus either in saliva or combined with 4 ⁇ L 5X NEB sample buffer was serially diluted 1:10 in IX buffer. All samples were heated at 95°C for 5 minutes and 2 ⁇ L was added to each LAMP reaction using El and N2 sets of primers as provided in Example 3 and WarmStart® Colorimetric LAMP 2X Master Mix with UDG (M1804 from New England Biolabs) or WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA) (M1800 from New England Biolabs).
  • Virus was heat inactivated SARS-CoV-2 (not in saliva). 60 copies of virus could be detected in 100% of the samples and 6 copies of virus could be detected in about 85% of the samples tested using colorimetric LAMP (observing pH dependent color change) and also using fluorescence based LAMP (measuring Cq).
  • Virus was Gamma irradiated SARS-CoV-2 (not in saliva).
  • Example 14 Improvements in isothermal amplification of nucleic acids from RNA viruses in saliva
  • NEB buffer containing an alkoxylated alcohol e.g. PF68 was added to the sample at the start of analysis.
  • the sample was found to be stable at room temperature for up to 6 hours. This is helpful when large numbers of samples are being processed so that the samples may be maintained on the bench until ready to perform the LAMP reaction.
  • NEB IX buffer formulation in the saliva sample in this example was 400 mM GnHCL, 4.5 mM TCEP pH 8.2, 75 mM LiCI and 0.2% PF68.
  • the reagent buffer was prepared as a 2X formulation containing 800 mM GnHCL, 9 mM TCEP pH 8.2, 150 mM LiCI, and 0.4% PF68.
  • RNA sample Heat saliva sample to 95°C for 5 minutes to release RNA from the capsids in the saliva. After heat treatment, the sample should be used in LAMP detected as soon as possible. If samples have to be stored, they should be stored at -20°C or preferably -80C. Store 1A or IB on ice for up to a couple of days as needed, longer at -20°C.
  • Results 20 cps of virus mixed with saliva could be detected 100% sensitivity for 12/12 samples tested using saliva from a single source.
  • Saliva samples were collected from multiple donors and spiked with attenuated RNA virus particles. 5 ⁇ L of SeraCare (mock virus with 100 cps/ ⁇ L) was added to 30 ⁇ L of Saliva, 5 ⁇ L of water and 10 ⁇ L of 5X NEB buffer. Therefore, the original titer in mixed SeraCare-Saliva sample was 14.3 copies/ ⁇ L and after adding buffer and water, the titer was 10 copies/ ⁇ L.
  • NEBuffer 2X NEB inactivation buffer formulation as a reagent: 800mM GnHCL, 9 mM TCEP pH 8.2.150mM LiCI and 0.4% PF68 (no Proteinase K included here) was added to the saliva. Samples were stable with no observable decrease in detectable copies of viral RNA even when stored at room temperature for at least 6 hours. Samples were then heat at 95°C for 5 minutes and stored on ice. Aliquots were then used for LAMP detection or RT-qPCR as described in previous examples.
  • PF68 improved the sensitivity of the LAMP reaction where 20 copies of virus mixed with saliva from different donors could be detected with 100% sensitivity.
  • buffer should contain preferred non-ionic detergent e.g. PF68
  • sample is good at RT for up to 6 hours
  • heat to 95°C for 5 minutes This step releases RNA from inactivated virus and detergent in buffer with detergent substantially reduces RNase activity.
  • RNase activity remains very high after the samples were heated at 95°C for 5 minutes in the absence of buffer, therefore no SARS-CoV-2 signal can be detected.
  • other detergents such as Triton X-100 or Tween-20 were used in buffer, even though after the heating step RNase activity was significantly reduced, the remaining RNase activity requires the samples to be stored on ice to prevent the virus RNA degradation.
  • sample should be used in LAMP detected as soon as possible. If samples have to be stored, they should be stored at -20°C or preferably -80°C. Store 1A or IB on ice for up to a couple of days as needed, longer at -20°C.
  • LAMP reaction kit e.g. 2 ul of saliva sample into 18 ⁇ l of LAMP reaction mix.
  • Results 20 cps of virus mixed with saliva can be detected 100% sensitivity for 12/12 samples tested using saliva from 1 person.
  • Example 15 An alternate protocol for SARS-CoV-2 screening
  • Saliva was collected in 1.5 ml barcoded tubes and heated to 65°C for 30 minutes at the collection site in a buffer that contained 0.2% PF68 or Triton X-100 or no detergent.
  • the sample was spiked with non-infectious heat inactivated SARS-CoV-2 virus grown in cell culture (ATCC).
  • a space change multichannel pipette was used to transfer saliva into 96-well plate with buffer and mixed samples were heated at 95°C for 5 minutes. 2 ⁇ L of sample was transferred into a LAMP reaction followed by a 65°C 30 minutes amplification and data collection by observing a color change in a tube. The protocol is described in FIG. 30. Examples of the results obtained are shown in FIG. 31A-31C.
  • Example 16 Immobilization of oligonucleotide reagents to enhance sensitivity and/or improve efficiency of workflow for diagnosis of SARS-CoV-2
  • Immobilized reagents provide an opportunity to achieve enrichment of target nucleic acid from a biological sample and/or reduce the number of steps in a workflow.
  • This example describes the application of bead immobilized reagents to improve a coronavirus testing workflow that utilizes saliva as a starting material to test for the presence of SARS-CoV-2 virus, and uses LAMP to amplify cDNA derived from any SARS-CoV-2 RNA from virus present in the saliva to determine whether an individual is infected. Human saliva is used and an attenuated SARS-CoV-2 is spiked into the saliva at known concentrations.
  • the saliva is collected in a first receiving vessel containing a buffer that includes a surfactant/detergent.
  • Enrichment of the SARS-CoV-2 RNA is initiated by the release of viral nucleic acid from virus followed by hybridization of the released RNA to biotinylated oligonucleotides immobilized on streptavidin coated magnetic beads or oligonucleotide coated glass beads.
  • a non- ionic detergent -Pluronic F-68 as the surfactant in the receiving vessel.
  • the contents of the receiving tube is varied as described below and may additionally contain one or more RNase inhibitors and a proteolytic agent.
  • Buffer contains PF-68, magnetic beads coated with a DNA oligonucleotide primer for initiating reverse transcription plus heat labile Proteinase K.
  • Buffer contains the Pf-68, magnetic beads coated with a DNA oligonucleotide primer for initiating reverse transcription absent heat labile Proteinase K, plus guanidinium chloride.
  • Buffer contains PF-68, glass (or any silica-derived) beads of at least 1 mm diameter optionally coated with a DNA oligonucleotide primer for initiating reverse transcription plus heat labile Proteinase K.
  • Buffer contains PF-68, glass (or any silica-derived) beads of at least 1 mm diameter optionally coated with a DNA oligonucleotide primer for initiating reverse transcription absent heat labile Proteinase K, plus guanidinium chloride.
  • Buffer contains PF-68, magnetic beads coated with an RNA or DNA oligonucleotide primer for binding the viral RNA at a predetermined location (e.g. EIA) plus heat labile Proteinase K.
  • Buffer contains PF-68, magnetic beads coated with coated with an RNA or DNA oligonucleotide primer for binding the viral RNA at a predetermined location (e.g. EIA) absent heat labile Proteinase K, plus guanidinium chloride.
  • EIA heat labile Proteinase K
  • Buffer contains PF-68, glass (or any silica-derived) beads of at least 1 mm diameter optionally coated with an RNA or DNA oligonucleotide primer for binding the viral RNA at a predetermined location (e.g. EIA) plus heat labile Proteinase K.
  • Buffer contains PF-68, glass beads (or any silica-derived) of at least 1 mm diameter optionally coated with an RNA or DNA oligonucleotide primer for binding the viral RNA at a predetermined location (e.g. EIA) absent heat labile Proteinase K, plus guanidinium chloride.
  • the magnetic beads containing the hybridized viral genome are separated from the buffer by means of a magnet, where the buffer containing unwanted biological material is discarded and the beads suspended in a buffer containing reagents for reverse transcription and amplification by LAMP (see RT-LAMP from New England Biolabs, Ipswich, MA).
  • a filter with a pore size less than the diameter of the beads is placed in a sleeve within a second receiving vessel (see US patent application serial no. 16/547,844 and New England Biolabs, Ipswich, MA)
  • the receiving vessel has an exit that has an open and shut position.
  • the buffer containing the unwanted biological material is drained away and the sleeve placed into a second tube containing a buffer suitable for amplification (e.g., RT-LAMP, RT-qPCR, etc.).
  • the saliva is expected to contain between 10,000-20,000 viral particles.
  • LAMP low-density lipoprotein
  • the immobilization of oligonucleotide probes and enrichment of target nucleic acids from saliva is expected to deliver this degree of sensitivity even if hybridization of target viral RNA to immobilized probes is not 100% efficient. In fact, even if this were less than 5% efficient, the sensitivity and reproducibility of the LAMP based test would be expected to increase besides any loss of efficiency in handling large numbers of samples or significant increase in workflow complexity.
  • Example 17 Detection of Influenza and SARS-CoV-2 in a multiplex reaction
  • DARQ LAMP primer sets F3, B3, FIP, BIP, LF and LB
  • a duplex oligo consisting of the FIP modified at its 5'-end with a dark quencher (Q-FIP) annealed to a complementary Flc oligo with 3' fluorophore (Fd) (Table 1) were selected for target RNAs.
  • the El LAMP primers targeting SARS-CoV-2 sequence GenBank accession number MN908947
  • IAV and IBV primer sets Takayama, et al
  • Synthetic SARS-CoV-2 RNA containing equal ratio of the viral genome regions was purchased from Twist Bioscience, California (Twist Synthetic SARS- CoV-2 RNA Control 2 #102024, MN908947.3).
  • RNAs for Influenza A H1N1 (VR-1737D, Strain A/Virginia/ATCC/2009), A (H3N2) (VR-1811D, Strain A/Virginia/ATCC6/2012) and B (VR-1885DQ, Strain B/Wisconsin/1/2010 BX-41A) were purchased from ATCC.
  • Viral RNA was diluted to lower concentrations in 10 ng/ ⁇ l Jurkat total RNA (BioChain) based on quantification provided by the manufacturers.
  • RNA used was 50 copies of SARS-CoV-2 RNA, 1 ul of 1:1000 diluted Influenza A RNA and approximately 21 copies for Influenza B RNA. This amount of viral RNAs was sufficient for more than half but not all the 24 repeats to show positive amplification thus allowing detection of sensitivity change under different conditions.
  • DARQ LAMP reactions contained lx E1700 (New England Biolabs), with an additional 0.32 U/ ⁇ L Bst 2.0 WarmStart DNA Polymerase (New England Biolabs, M0538), 40mM guanidine chloride (Sigma, RDD001) and standard concentrations of LAMP primers (0.2 ⁇ M F3, 0.2 ⁇ M B3, 1.6 ⁇ M FIP, 1.6 ⁇ M BIP, 0.4 ⁇ M LoopF, 0.4 ⁇ M Loop B) supplemented with DARQ.
  • FIP duplex (0.22 ⁇ M QFIP: 0.18 ⁇ M Fd, pre- annealed as 55 ⁇ M QFIP: 45 ⁇ M Fd by heating to 95°C and slowly cooling to room temperature).
  • the concentration for ACTB primers was reduced to 0.25X the standard concentrations for LAMP primers (0.05 ⁇ M F3, 0.05 ⁇ M B3, 0.4 ⁇ M FIP, 0.4 ⁇ M BIP, 0.1 ⁇ M LoopF, 0.1 ⁇ M Loop B) with DARQ FIP duplex added as 0.066 ⁇ M QFIP and 0.054 ⁇ M Fd.
  • the reactions were incubated at 60°C in half-skirted plates (BioRad, HSP9601) on a real-time qPCR machine (Bio-Rad CFX96). Real time LAMP signal was acquired every 15 seconds for 108 cycles (total incubation time ⁇ 40 minutes for single channel or 49 minutes for 4-channel acquisition).
  • Example 18 SARS -Cov-2 RT LAMP assay validation for saliva samples
  • RNA virus namely Eastern equine encephalitis using crushed mosquitos that contain virus infected blood from mammalian hosts using a single set of LAMP primers.
  • RT-LAMP detects the genetic material present in the SARS-CoV- 2 virus.
  • the RNA genome is first reverse transcribed to create a complementary DNA (cDNA) strand.
  • Specific cDNA primers then amplify conserved SARS-CoV-2 regions within two targets, the N and E genes.
  • Amplification is detected by increased fluorescence as the included dye intercalates in the newly created cDNA.
  • a pH indicator dye signals a change in pH resulting from the release of H+ as nucleotide triphosphates are incorporated into the cDNA amplification product.
  • An analogous reaction targeting human actin RNA is run separately on each sample to ensure the sample is devoid of reaction inhibitors and contains sufficient material for baseline detection.
  • Cq refers to the cycle number in which the fluorescence signal passes a threshold. If no signal above threshold can be detected, Cq is reported as N/A (not available). The more target RNA present, the smaller the Cq value obtained. In the current protocol, the fluorescence signal is read at 15 second intervals throughout the reaction period of 36 minutes.
  • Color change indicates the presence of a target nucleic acid.
  • Color tone of LAMP without target is pink and with detectable target is yellow. Amplification is determined visually and recorded using a scanner where yellow indicates virus is present and pink indicates that the virus is absent. Sometimes the color is orange if the reaction contains an inhibitory substance or a very low copy number of virus.
  • SARS-CoV-2 Positive Control was obtained from BEI resources (Manassas, VA) Pooled human saliva samples negative for SARS-CoV-2 (from NEB research study) were also tested. Human total RNA was monitored with a sample extraction and inhibition control using detection of actin mRNA where actin mRNA was obtained from ThermoFisher (MA). SARS-CoV-2 positive and negative clinical saliva samples provide by Mirimus Clinical Labs, a CLIA-certified facility where SARS- CoV-2 sample status was classified by Mirimus following RNA extraction using ThermoFisher TaqPath. 30 positive and 30 negative samples were analyzed in this example. The primers used in LAMP targeting gene N, Orf lab, and S genes.
  • Saliva samples were heated in 1.5mL labelled tubes at 65°C for 30min to inactivate any SARS- CoV2, and subsequently stored on ice or in the refrigerator for up to 72 hours.
  • 15 ⁇ L of individual heat- inactivated test samples were transferred into 94 of the individual wells of a 96 well SLB plate. Up to 94 test samples were added into each 96 well SLB plate.
  • One well was reserved for the negative control (NC) and one for the positive control (PC). See an example plate in FIG. 33.
  • a 2 mL Eppendorf tube was labelled for SARS-CoV-2 reaction mix and another for human actin reaction mix and both were placed on ice.
  • E2019 RT-LAMP kit contains two 10X Primer mixes.
  • the SARS-CoV-2 LAMP reaction uses primer mix S1883, and the actin LAMP reaction uses primer mix S0164.
  • 18 ⁇ L SARS-CoV-2 LAMP reaction mix was distributed to each well of the SARS-CoV-2 reaction plate.
  • 18 ⁇ L actin LAMP reaction mix was distributed to each well of the actin reaction plate.
  • SARS-CoV-2 Positive Control was obtained from BEI resources (Manassas, VA) Pooled human saliva samples negative for SARS-CoV-2 (from NEB research study) were also tested. Human total RNA was monitored with a sample extraction and inhibition control using detection of actin mRNA where actin mRNA was obtained from ThermoFisher (MA). SARS-CoV-2 positive and negative clinical saliva samples provide by Mirimus Clinical Labs, a CLIA-certified facility where SARS- CoV-2 sample status was classified by Mirimus following RNA extraction using ThermoFisher TaqPath. 30 positive and 30 negative samples were analyzed in this example. The primers used in LAMP targeting gene N, Orf lab, and S genes.
  • the saliva assay was tested using negative saliva samples spiked with inactivated SARS-CoV-2 (contingent samples) and clinical samples. No RNA purification was performed.
  • This spiked saliva was then diluted by a 1:2 serial dilution: 7 fold) in negative saliva.
  • Triplicate saliva samples were prepared for each dilution. 15uL of saliva was combined with 15uL of 2X sample buffer and incubated at 95°C for 5 minutes. 2uL of sample was added to 20uL of a LAMP reaction mix for triplicate LAMP reactions at each dilution. Cq values were obtained.
  • the LOD of SARS-Cov-2 detection was 40 copies/uL
  • hRNA indicates 2 ng human total RNA positive control for actin mRNA.
  • the anticipated viral load in saliva ranged between 104-1010 copies/mL in patients, with decreasing viral loads as infection progresses.
  • the LOD for this method was 40 copies/ ⁇ L, or 4x104 copies/mL, covering the vast majority of expected infection viral titers, particularly in the early stages of infection.
  • Example 19 Universal assay for a pathogen and its known and unknown variants
  • a variant-specific LAMP detection method based on molecular beacons targeting a 9- base deletion in Orfla that is more widely observed (SGF deletion), enabling the detection of multiple variants of concern including B.1.1.7, B.1.351, and P.l.
  • Target nucleic acids with (i) a known sequence or (ii) a known sequence with a plurality of undefined mutations therein refers to variants of a target nucleic acid in a cell or virus that have "undefined mutations".
  • Undefined mutations may arise in a cell or virus pathogen to evade host immune mechanisms or medications or for other reasons.
  • the mutations that arise may result in changed properties relating to virulence and pathogenicity usually by increasing one or both. Often these variants coexist with other mutants or variants. It is beneficial to an animal population such as humans if the appearance of a new variant is rapidly detected before extensive spreading.
  • PCR is an established, sensitive method of detecting specific target nucleic acids, that require two primers only and a thermocycling protocol. If novel mutations arise in a target nucleic particularly at the 3' end of the primer binding site, PCR will take longer to amplify the nucleic acid to the point that a positive sample will register as negative.
  • a negative PCR result is one that does not yield a positive signal after a predetermined number of thermocycles. This can have negative repercussions for detecting infected individuals in a population in which multiple variants coexist where an infected person may receive a negative result on the basis of less than ideal PCR primers. Efficiency of primer annealing is therefore a factor in considering sensitivity of a PCR reaction.
  • PCR differs from LAMP amplification in ways that include the nature of the amplification itself. Without being limited by theory it is proposed here that the inefficiencies in primer hybridization for PCR are amplified by the protocol compared with LAMP.
  • amplification is a series of discrete events in which primers are required to bind to each molecule in the population and then to bind to each of the amplicons in every cycle. Therefore any inefficiency in binding due to mutations is itself amplified.
  • LAMP uses 4, 5 or 6 primers which create intermediate species that are then amplified from multiple primer binding events and locations, These multiple binding and amplification sites reduce the impact of primer binding efficiencies.
  • universal primer set is intended to refer to a primer set used in LAMP to detect a specific gene or specific sequence in a target nucleic acid or the entire nucleic acid.
  • the specific gene, sequence or the entire target nucleic acid may contain mutations including one or more, or a plurality of previously unknown mutations.
  • the presence of mutations in the target nucleic acid is referred to here as target nucleic acid variants. These mutations may be additions, deletions or point mutations that occur at primer binding sites for primers used in nucleic acid amplification assays to determine the presence of a pathogenic cell or virus.
  • the universal primer set is so named because the primers can initiate amplification reactions to detect a specific target with a similar sensitivity and specificity regardless of known or unknown mutations in the primer binding sites of that target.
  • the deletions or additions will not exceed 10 nucleotides or more particularly will not exceed 9 nucleotides for example, will not exceed 8 nucleotides for example, will not exceed 6 nucleotides within primer binding regions.
  • deletions of 9 nucleotides and 6 nucleotides in the FIP and BIP primer binding sites could be detected by LAMP using the universal LAMP primer set although it was noted that a deletion of 9 nucleotides could reduce sensitivity of a diagnostic test.
  • the universal primer set is also able to detect a mixture of nucleic acids where some nucleic acids may have a particular target sequence and others are variants thereof.
  • the universal primer set may include 4, 5 or 6 primers. In some circumstances, a plurality of universal primer sets will be used to detect a target nucleic acid for amplifying different regions of the target nucleic acid.
  • LAMP is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions of the target gene.
  • the four primers include 2 inner primers (Forward inner primer (FIP) and Backward inner primer) and 2 outer primers (Forward outer primer (F3) and Backward outer primer (B3).
  • a universal primer set may also include 2 additional primers- Loop primers (Loop forward (LF) and Loop backward (LB) primers. Although the loop primers are optional, they can increase sensitivity and specificity of the amplification reaction.
  • reaction positive period of time refers to the reaction time for amplification prior to an established cut-off time in which a sample is determined to be positive.
  • the cut-off time for amplification to occur has been established by diagnostic laboratories to determine if a sample is positive or negative.
  • LAMP primers and molecular beacons LAMP primers were designed with the SGF deletion located between Bic and LB (FIG. 37A and 37B) using LAMP Primer Design Tool at NEB https://lamp.neb.eom/#l/) and with enough length between Bic and LB to accommodate the location of molecular beacons in this region.
  • Molecular beacons targeting either wild-type or the SGF deletion sequence were designed using principles according to Sherrill-Mix et al., Genome Biol, 22(1), 169 (2021).
  • the annealing temperatures for these beacons are lower: the calculated Tm of the annealed beacon-target for wt MB and SGFdel M B is 63.9°C and 62.5°C, and the stem is 55.1°C and 54.7°C, respectively.
  • These beacons were synthesized as Affinity Plus qPCR Probes by IDT with sequences shown in Table 9.
  • RT-LAMP reactions were performed using WarmStart® LAM P Kit (DNA & RNA) (E1700) with standard primer concentrations (0.2 ⁇ M F3, 0.2 ⁇ M B3, 1.6 ⁇ M FIP, 1.6 ⁇ M BIP, 0.4 ⁇ M Loop F, 0.4 ⁇ M Loop B) in the presence of 40 mM guanidine hydrochloride in 25 ⁇ l on 96-well plates at 65 °C in a Bio-Rad CFX96 instrument.
  • LAMP amplification was measured by including 1 uM SYTOTM-9 fluorescent dye (ThermoFisher S34854), 0.5 ⁇ M SGFdel or 1.0 ⁇ M wt beacon and signal was acquired at 15 second intervals.
  • Synthetic SARS-CoV-2 RNAs were obtained from Twist Bioscience (Control 2 for WT M N908947.3, Control 14 for B.1.1.7, Control 16 for B.1.351 and Control 17 for P.l) and diluted in 10 ng/ ⁇ l Jurkat total RNA based on the copy number provided by the manufacturer.
  • each of the 527 variant primers from the 3 assays was tested in RT-LAMP reactions with three different SARS-CoV-2 RNA copy number concentrations: 100, 200, and 10,000 copies in order to gain a sense of the mutation effect on reaction speed and sensitivity. Both lower concentrations allowed for amplification effects to be confidently determined outside of stochastic performance when close to the limit of detection in the 100 copy reactions, particularly for the Asle primer set which displays slightly lower sensitivity in our testing.
  • reaction output speed was measured relative to the fully-complementary wild-type primer, and relative effect plotted against the position in the primer sequence (FIG. 36A-36F).
  • the most common result across the primers was a 5%-10% reduction in amplification speed in the presence of a mismatch.
  • the B3 primers showed remarkably little impact in any of the 69 variant primer sets for the 3 amplicons, though the F3 primer did see some slowing when mismatches were present away from the 5' end in the El and Asle sets (FIG. 36A, 36B).
  • These primers are the least critical to the reaction, but the difference between the two may indicate differential tolerances to mismatches by the reaction initiation (B3 annealing to single-stranded RNA and extension by RTx reverse transcriptase) vs. the strand invasion and displacement via Bst 2.0 polymerase that occurs at the F3 primer.
  • the 3' half of the primer serves to invade and primer double-stranded DNA, with the 5' half annealing to displaced product strands to form the 'loop' dumbbell shapes for amplification.
  • the results in Figure 36C-D clearly indicate more impact of mutations on the F2/B2 regions, with more variant oligos causing amplification delays relative to fully base-paired control toward the 3' end of the FIP and BIP in all 3 gene assays.
  • the extreme 5' end was more likely to display sensitivity to mismatch, likely indicating an impact on extension from a mismatch in the looped- back LAMP hairpin structure, but sensitivity was not impacted in these tests.
  • Table 10 lists the number of positions from each primer that resulted in a detection cycle change of more than 10% from the WT primer. Though overall effects on amplification were minimal, the greater impact of 3' mutations is clear from the trends in Figure 36A-F. And while a significant number of variant primers resulted in slowing of the reaction, in all 527 variant primers tested no mutation position prevented amplification in the RT-LAMP reaction with SARS-CoV-2 RNA even with lower RNA copy numbers. Table 10 - Effect of Single Point Mutations
  • RT-LAMP assays We investigated a single base mutation at every position of every primer in three prominent SARS-CoV-2 RT-LAMP assays. Remarkably, we find very little impact of the single base changes, with only marginal effect on speed in most positions. The robustness of RT-LAMP to sequence variation is a significant benefit to its adoption, with reduced worry about deleterious effects from the commonly emerging single-base changes that could occur with some frequency in the regions targeted by the LAMP primers. Additionally, many RT-LAMP assays combine primer sets for added speed and sensitivity [4], adding an additional layer of protection against possible sequence variation.

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