WO2022060939A1 - Compositions and methods for nucleic acid preparation - Google Patents

Compositions and methods for nucleic acid preparation Download PDF

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Publication number
WO2022060939A1
WO2022060939A1 PCT/US2021/050604 US2021050604W WO2022060939A1 WO 2022060939 A1 WO2022060939 A1 WO 2022060939A1 US 2021050604 W US2021050604 W US 2021050604W WO 2022060939 A1 WO2022060939 A1 WO 2022060939A1
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nucleic acid
composition
nucleic acids
amplification
target nucleic
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PCT/US2021/050604
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French (fr)
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Rose Anne LEE
Helena De Puig Guixe
Nira POLLOCK
Catherine M. Klapperich
James J. Collins
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President And Fellows Of Harvard College
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Publication of WO2022060939A1 publication Critical patent/WO2022060939A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the field of the invention relates to preparation of nucleic acids for use in amplification reactions and for point-of-care diagnostics.
  • Non-falciparum clinical diagnostics are a second gap, as these infections have a low parasite density and are commonly undetected.
  • compositions and methods described herein are based, in part, on the discovery of a nucleic acid preparation protocol that does not require the use of a nucleic acid extraction kit and for which the resulting nucleic acids can be added directly to isothermal nucleic acid amplification methods (e.g., recombinase polymerase amplification (RPA), among others) and/or probe cleavage-based detection reactions, e.g. SHERLOCK, among others) without a step of purifying or isolating the nucleic acids.
  • RPA recombinase polymerase amplification
  • probe cleavage-based detection reactions e.g. SHERLOCK
  • compositions for nucleic acid preparation comprising a reducing agent and a metal ion chelating resin in aqueous suspension.
  • the metal ion chelating resin comprises paired iminodiacetate ions.
  • the resin is present at a concentration of about 10% to 30% w/v.
  • the reducing agent is dithiothreitol (DTT).
  • the reducing agent is present at a concentration of 20-150 mM.
  • the resin comprises styrene divinylbenzidine copolymer.
  • the reducing agent is DTT in the range of 20-150 mM
  • the resin is a styrene divinylbenzidine copolymer resin with paired iminidiacetate ions at a concentration of 10-30% w/v.
  • the reducing agent is 50 mM DTT, and the resin is present at a concentration of 20% w/v.
  • the composition releases nucleic acid from a biological sample upon heating, without need for a proteolytic enzyme.
  • the composition does not comprise proteinase K.
  • the biological sample comprises blood or a blood fraction, a nasopharyngeal swab, an oropharyngeal swab, sputum or saliva.
  • the blood fraction comprises erythrocytes.
  • composition consisting essentially of an aqueous buffer, DTT and a metal ion chelating resin.
  • the composition consisting essentially of an aqueous buffer, DTT and a metal ion chelating resin is in an admixture with a biological sample.
  • composition for nucleic acid preparation is present at about a 3: 1 ratio relative to biological sample by volume.
  • the biological sample comprises blood or a blood fraction, a nasopharyngeal swab, an oropharyngeal swab, sputum or saliva.
  • the blood fraction comprises erythrocytes.
  • the blood or blood fraction has previously been dried on a solid support.
  • the solid support is present in the admixture.
  • composition or admixture thereof further comprises reagents sufficient to perform an isothermal nucleic acid amplification.
  • composition or admixture thereof further comprises reagents sufficient to perform Specific High sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) detection of a target nucleic acid.
  • SHERLOCK Specific High sensitivity Enzymatic Reporter unLOCKing
  • the composition substantially prevents target-independent cleavage of a SHERLOCK reporter nucleic acid in the presence of a biological sample or when in admixture with a biological sample.
  • a method of preparing a biological sample for nucleic acid analysis comprising contacting a composition comprising a reducing agent and a metal ion chelating resin in aqueous suspension with the biological sample, and heating the resulting mixture to at least 80°C.
  • the ratio of the composition comprising a reducing agent and a metal ion chelating resin in aqueous suspension to biological sample is about 3: 1.
  • heating is performed for 2-20 minutes.
  • heating is performed at about 95°C for about 10 minutes.
  • the biological sample comprises: (i) blood or a fraction thereof comprising erythrocytes, (ii) a nasopharyngeal swab, (iii) an oropharyngeal swab, (iv) sputum, or (v) saliva.
  • the biological sample had previously been dried on a solid support.
  • the solid support is included in the mixture.
  • the biological sample comprises or is suspected of comprising an intracellular parasite, bacterium, or a virus.
  • the intracellular parasite comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Babesia sp. Leishmaniasis spp. Toxoplasmosis spp. or filarial nematodes.
  • the virus comprises Dengue virus, Zika virus, Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoV 1), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Heptatis E,
  • the bacterium comprises a gram negative bacterium, a gram positive bacterium or an intracellular bacterium.
  • heating the mixture promotes red blood cell lysis and lysis of intracellular parasite or virus present and releases nucleic acids from the parasite or virus if present, and wherein the resin chelates multivalent metal ions, thereby inhibiting nuclease degradation of parasite and/or viral nucleic acid.
  • the method further comprises, after the heating step, contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids.
  • the method further comprises incubating the mixture under conditions and for a time sufficient to amplify the one or more target nucleic acids if present.
  • the isothermal amplification is a recombinase polymerase amplification (RPA) reaction.
  • RPA recombinase polymerase amplification
  • the reagents sufficient for isothermal amplification are lyophilized prior to the step of contacting the mixture with the reagents.
  • the reagents sufficient for SHERLOCK detection are lyophilized prior to the step of contacting the mixture with the reagents.
  • the method further comprises, after the heating step, contacting the mixture with reagents sufficient for isothermal
  • SUBSTITUTE SHEET (RULE 26) amplification of one or more target nucleic acids and reagents sufficient for SHERLOCK detection of one or more target nucleic acids.
  • the reagents sufficient for isothermal amplification and the reagents sufficient for SHERLOCK detection are lyophilized prior to the contacting step, wherein the contacting step reconstitutes the lyophilized reagents and permits amplification and SHERLOCK detection of one or more target nucleic acid molecules if present.
  • the method further comprises incubating the mixture under conditions and for a time sufficient to amplify one or more target nucleic acids and to generate a SHERLOCK detection signal for one or more target nucleic acids if present.
  • Another aspect provided herein relates to a method of amplifying one or more target nucleic acids in a biological sample comprising nucleic acids, the method comprising: (i) contacting a composition comprising a reducing agent and a metal ion chelating resin in aqueous suspension with the biological sample; (ii) heating the mixture resulting from step (i) to at least 80°C for a time sufficient to release nucleic acids in the biological sample; (iii) after step (ii), contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids; (iv) incubating the mixture of step (iii) under conditions and for a time sufficient to generate an isothermal amplification product for one or more target nucleic acids.
  • Another aspect provided herein relates to a method of detecting one or more target nucleic acids in a biological sample, the method comprising: (i) contacting a composition of any one of claims 1-13 with the biological sample; (ii) heating the mixture resulting from step (i) to at least 80°C for a time sufficient to release nucleic acids in the biological sample; (iii) after step (ii), contacting the mixture with reagents sufficient for isothermal amplification and SHERLOCK detection of one or more target nucleic acids; and (iv) incubating the mixture of step (iii) under conditions and for a time sufficient to permit isothermal amplification and production of a SHERLOCK detection signal for one or more target nucleic acids present in the sample.
  • kits for nucleic acid preparation and/or detection comprising a reducing agent and a metal ion chelating resin in aqueous suspension, and packaging materials therefor.
  • the metal ion chelating resin comprises paired iminodiacetate ions.
  • the reducing agent is dithiothreitol
  • the kit does not contain proteinase
  • the reducing agent is present at a concentration of 20-150 mM.
  • the resin comprises styrene divinylbenzidine copolymer.
  • the kit further comprises reagents sufficient for an isothermal nucleic acid amplification reaction.
  • the reagents sufficient for an isothermal nucleic acid amplification reaction are lyophilized.
  • the kit further comprises reagents sufficient for a SHERLOCK detection reaction.
  • the reagents sufficient for a SHERLOCK detection reaction are lyophilized.
  • the reagents sufficient for an isothermal nucleic acid amplification reaction and reagents sufficient for a SHERLOCK detection reaction are lyophilized in one composition.
  • FIG. 1 SHERLOCK diagnostic workflow: (1) human serum, whole blood, or dried blood spot samples undergo a 10 minute S-PREP protocol where the sample is suspended in 20% w/v Chelex®-100 in TE buffer with 50mM DTT and incubated at 95°C for 10 minutes; and (2) transfer of suspended sample to lyophilized SHERLOCK pellet followed by incubation at 40°C for 60 minutes prior to endpoint analysis via fluorescence or lateral flow strip.
  • FIG. 2 Schematic of one-pot SHERLOCK assay.
  • Reverse-transcriptase recombinase polymerase amplification (RT-RPA) amplifies Plasmodium species target sequences and occurs in parallel with programmed Casl2a detection, resulting in cleavage of target sequences and collateral cleavage of spiked fluorophore -labeled ssDNA reporter detectable by fluorescent measurement or lateral flow readout.
  • FIGs. 3A-3B Sample preparation methods tested with SHERLOCK P. falciparum assay using simulated malaria samples of live intraerythrocytic P. falciparum spiked into whole blood at IfM (602 parasites/ LIL) concentration.
  • FIG. 3A Detergents and heating methods assessed for SHERLOCK compatibility.
  • FIG. 3B Combinations of chelating and reducing agents tested for optimization of chemical deactivation of nucleases and inhibitors.
  • Asterisks indicate significant differences from untreated simulated whole blood sample assessed by Student’s two-tailed t-test. Bars: mean +/- S.D. of three technical replicates. *p ⁇ 0.05, p ⁇ 0.01, *** ⁇ 0.001, ****p ⁇ 0.0001.
  • FIGs. 4A-4E Specificity of SHERLOCK assays.
  • FIG. 4A Using P. falciparum assay and DTT/EGTA/95°C sample preparation, P. falciparum and P. vivax patient serum in SHERLOCK diagnostic display similar fluorescent kinetics that are eliminated when an aliquot of the same P. vivax serum undergoes nucleic acid extraction via commercial kit.
  • FIG. 4B Using P. vivax assay and DTT/EGTA/95°C sample preparation, P. falciparum serum demonstrates false positive signal that is eliminated when an aliquot of the same P. falciparum serum undergoes nucleic acid extraction via commercial kit.
  • FIG. 4C False positive P.
  • FIG. 4D False positive P. falciparum signal is eliminated with S-PREP.
  • FIG. 4E Performance of SHERLOCK diagnostic on clinical patient serum and whole blood samples prepared with S-PREP: five P. falciparum samples (four serum, one whole blood), 10 P. vivax serum samples, and five serum samples from healthy controls.
  • FIGs. 5A-5D SHERLOCK Performance.
  • FIG. 5A Sensitivity of SHERLOCK diagnostic for detection of Plasmodium species by comparison of probit regression curves obtained from 21 replicates of five dilutions.
  • FIG. 5B Fluorescence kinetics of P. falciparum SHERLOCK assay at lOOaM (60 parasites/pL) and 2aM (1 parasite/pL) concentrations.
  • FIG. 5C Specificity of SHERLOCK diagnostic using at 10 fM (6020 parasites/pL) concentrations of parasite.
  • FIG. 5D Comparison of performance between simulated dried blood spot and whole blood samples. All experiments used simulated whole blood samples. Asterisks indicate p-value ⁇ 0.0001 for Student’s t- test between fluorescent output of sample type versus no-template control.
  • FIGs. 6A-6E SHERLOCK Lateral Flow Assay Performance.
  • FIGs. 6A-6D Detection of 1 fM (-602 parasites/pL), 100 aM (60 parasites/pL), 50 aM (30 parasites/pL), and 2 aM (1 parasite/pL) concentrations of P. falciparum, P. vivax, P. ovale, and P. malariae respectively, and comparison to 1 femtomolar concentrations of off-target Plasmodium species for each assay.
  • FIG. 6E Background-subtracted grayscale intensity averages of test line for 3 separate flow tests +/- standard deviation. All experiments used simulated whole blood samples.
  • compositions and methods useful for preparing nucleic acids obtained from a biological sample, such as blood, serum or saliva, for assays utilizing nucleic acids as a starting material permit conventional multi-step nucleic acid protocols to be performed in a single reaction or ‘pot’ and do not require a prior step of purifying or isolating the nucleic acids.
  • this nucleic acid preparation protocol is used in conjunction with an isothermal amplification method (e g., RPA, LAMP, among others).
  • detection based upon probe cleavage permits very sensitive detection of target nucleic acids.
  • isothermal amplification such as RPA, among others, is combined with detection based, for example, on SHERLOCK to produce an ultra-sensitive diagnostic for, e.g., malaria that can be used as a point-of-care diagnostic.
  • biological sample refers to a cell or population of cells or a quantity of tissue or fluid from a subject. Often, a “biological sample” will contain cells from an animal or subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine, that can be used to prepare nucleic acids using the methods and compositions described herein. Biological samples include, but are not limited to, whole blood, plasma, serum, saliva, sputum, nasopharyngeal swab, oropharyngeal swab, urine, cell culture, tissue biopsies, scrapes (e.g., buccal scrapes), or cerebrospinal fluid.
  • a biological sample When a biological sample is intended for point-of-care diagnostics, it is preferred that the sample be easily and non-invasibly obtained, for example, blood, serum, saliva, sputum, nasopharyngeal swab, oropharyngeal swab, or urine.
  • a biological sample or tissue sample can refer to a sample of tissue or fluid isolated from an individual including, but not limited to, blood, plasma, serum, tumor biopsy, urine, stool, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, cells (including, but not limited to, blood cells), tumors, organs, and also samples of in vitro cell culture constituent.
  • nucleic acid sample preparation refers to a method of preparing nucleic acids such that they can be used in a downstream reaction, such as an amplification and/or detection reaction, without the need for additional isolation steps.
  • Nucleic acid sample preparation can include inactivation of endogenous enzymes from the biological sample, nicking or destabilizing DNA, or releasing nucleic acids from the biological sample such that they are available for binding with a primer, etc.
  • the nucleic acid sample preparation method described herein does not include a proteinase, such as proteinase K, or a collagenase enzyme to disrupt tissue or cells.
  • the term “isothermal amplification reaction” refers to a nucleic acid amplification method that does not require thermal cycling to permit amplification.
  • the term distinguishes amplification methods, such as the polymerase chain reaction (PCR), that use cycles of thermal denaturation of nucleic acid by incubation at a temperature above the melting temperature (T m ), annealing of nucleic acid primers by incubation at another temperature generally below the T m , and most often, incubation at another temperature optimal for a polymerase enzyme to extend the annealed primers.
  • PCR polymerase chain reaction
  • An isothermal amplification reaction can be performed at a single temperature, and generally relies upon, for example, DNA binding and strand-displacing enzyme factors to permit target nucleic acid amplification.
  • the temperature of the isothermal amplification reaction does not deviate by more than 10° C in either direction (e.g., deviates by less than 5° C, by less than 2° C, by less than 1°C) or is performed at a single temperature with no temperature deviation required for amplification
  • solid support refers to beads, membranes, filters, matrices, columns, chips, arrays etc. that can bind to e.g., nucleic acids, antibodies etc.
  • Exemplary solid supports include, but are not limited to, magnetic beads (e g., micron-sized magnetic beads), Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix.
  • a solid support is one on which a biological sample as described herein is spotted on and dried to preserve the sample for analysis at a later time.
  • the solid support can include, for example, paper, nitrocellulose or nylon sheets and the like.
  • the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • compositions, methods, and respective components thereof refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • Nucleic acids that can be prepared using the methods and compositions described herein can include RNA, DNA, mRNA (messenger RNA), mtRNA (mitochondrial RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), nRNA (nuclear RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNA (Small Cajal Body specific RNA), microRNA, chromosomal DNA, nDNA (nuclear DNA), snDNA (small nuclear DNA), viral RNA, dsDNA (double-stranded DNA), ssDNA (single-stranded DNA), as well as cDNA (complementary DNA), ribozyme and riboswitch RNAs, LNA (locked nucleic acid), siRNA (short interfering RNA), plasmid DNA, cosmid DNA, or the like.
  • mRNA messenger RNA
  • mtRNA mito
  • a nucleic acid can be a nucleic acid of a pathogen, such as from a virus (e.g., RNA, DNA), a parasite (including intracellular parasites), or a bacterium.
  • a pathogen i.e., a parasite, bacterium or virus
  • the nucleic acids to be prepared are typically pathogen DNA, RNA, cDNA or mRNA.
  • the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • the nucleic acids prepared using the methods and compositions described herein are used as templates or targets for nucleic acid amplification, particularly those using isothermal nucleic acid amplification methods.
  • Target nucleic acids can also be referred to as target sequences, trigger nucleic acids, target polynucleotides, and/or target polynucleotide sequences, and can include double -stranded and single-stranded nucleic acid molecules.
  • the target nucleic acid is double stranded
  • the target nucleic acid generally includes a first strand and a second strand.
  • a first strand and a second strand may be referred to as a forward strand and a reverse strand and generally are complementary to each other.
  • a complementary strand may be generated, for example by polymerization and/or reverse transcription, rendering the target nucleic acid double stranded and having a first/forward strand and a second/reverse strand.
  • a target nucleic acid sequence can refer to either the sense or antisense strand of a nucleic acid sequence, and can also refer to sequences as they exist on target nucleic acids, amplified copies, or amplification products, of the original target sequence.
  • a target sequence can be a subsequence within a larger polynucleotide.
  • a target sequence can be a short sequence (e.g., 20 to 50 bases) within a nucleic acid fragment, a viral genome, a bacterial genome or a genome of a parasite, that is targeted for amplification.
  • a target sequence can refer to a sequence in a target nucleic acid that is complementary to an oligonucleotide (e.g., primer) used for amplifying a nucleic acid.
  • a target sequence can refer to the entire sequence targeted for amplification or can refer to a subsequence in the target nucleic acid where an oligonucleotide binds.
  • Samples from which the nucleic acids are prepared using the methods and compositions described herein can be obtained from any suitable biological specimen or sample, and often are isolated from a sample obtained from a subject.
  • a subject can be any living or non-living organism, including but not limited to a human, a non-human animal, a plant, a bacterium, a fungus, a virus, a parasite, or a
  • SUBSTITUTE SHEET protist.
  • Any human or non-human animal can be selected, including but not limited to a mammal, reptile, avian, amphibian, fish, ungulate, ruminant, bovine (e.g., cattle), equine (e.g., horse), caprine and ovine (e g., sheep, goat), swine (e.g., pig), camelid (e g., camel, llama, alpaca), monkey, ape (e g., gorilla, chimpanzee), ursid (e.g, bear), poultry, dog, cat, mouse, rat, fish, dolphin, whale and shark.
  • a subject can be a male or female, and a subject can be any age (e.g., an embryo, a fetus, infant, child, adult).
  • a biological sample can be any specimen that is isolated or obtained from a subject or part thereof.
  • specimens include fluid or tissue from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, or the like), a nasopharyngeal swab sample, an oropharyngeal swab sample, nasal mucus, sputum, saliva, urine, umbilical cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), biopsy sample, cells (e.g., blood cells) or parts thereof (e.g., mitochondrial, nucleus, extracts, or the like), washings of female reproductive tract, feces, prostate fluid, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, hard
  • blood encompasses whole blood, blood product or any fraction of blood, such as serum, plasma, buffy coat, or the like as conventionally defined.
  • Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants.
  • Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. Fluid or tissue samples often are collected in accordance with standard protocols hospitals or clinics generally follow. For blood, an appropriate amount of peripheral blood (e.g., between 3-40 milliliters) often is collected and can be stored according to standard procedures prior to or after preparation.
  • a biological sample can include samples containing parasites, viruses, bacteria, spores, cells, nucleic acid from prokaryotes or eukaryotes, or any free nucleic acid.
  • a sample can be isolated from any material suspected of containing a target sequence, such as from a subject described above. In certain instances, a target sequence can be present in air, plant, soil, or other materials suspected of containing biological organisms of interest.
  • nucleic acids prepared using the methods and compositions described herein do not require isolation prior to amplification; that is, the prepped nucleic acid sample can be subjected to an isothermal or other
  • the methods and compositions for nucleic acid preparation can include a step of inactivation of nucleases and other inhibitors present in the biological sample
  • a ‘dry’ sample such as a swab or dried blood, is specifically contemplated for processing and use with the methods and compositions described herein.
  • compositions and methods for use in preparing nucleic acids as described herein comprise one or more reducing agents.
  • the reducing agent is dithiothreitol (DTT).
  • DTT dithiothreitol
  • Other reducing agents contemplated for use as described herein include, but are not limited to, dimethyl sulfoxide (DMSO), tris(2-carboxyethyl)phosphine, tertiary butyl alcohol, beta-mercaptoethanol, among others.
  • the reducing agent can introduce nicks into the nucleic acids to aid in downstream methods, such as isothermal amplification reactions.
  • the concentration of DTT or other reducing agent used in nucleic acid sample preparation is in the range of l-100mM, 1-75 mM, l-50mM, 25-100mM, 50-100mM, 25-75mM, 25-70mM, 25-65mM, 25-60mM, 25-55mM, 25- 50mM, 40-60nM, 40-50nM, 50-60nM, 40-65mM, 40-70mM, 40-75mM, or 40-100mM, inclusive. In certain embodiments, the concentration of DTT is 50mM.
  • compositions and methods for use in preparing nucleic acids as described herein comprise a metal ion chelating resin.
  • resins typically comprise a polymer matrix and a chelating group.
  • a polymer matrix can include synthetic and/or natural organic polymers. Synthetic organic polymers, which are usually cross-linked polymers such as styrene -di vinylbenzene copolymer, are widely used as matrices. Chitin/chitosan, cellulose, and agarose as natural organic polymers are also sometimes utilized.
  • As chelating groups various ligands, which have nitrogen (N), oxygen (0), and/or sulfur (S) donor atoms, are immobilized on the matrix.
  • the metal ion chelating resin used herein comprises iminodiacetate ions and/or comprises styrene divinylbenzidine copolymer. In some embodiments, the metal ion chelating resin is a cation binding ion-exchange resin.
  • Exemplary ion-exchange resins for use in the methods and compositions described herein include ChelexTM resin (BioRad, Hercules, CA), DionexTM (ThermoFisher Scientific, Waltham, MA), DowexTM (Millipore Sigma, Burlington, MA), sodium polystyrene sulfonate, colestipol, cholestyramine, sodium polystyrene sulfonate, iminodiacetic acid-based resins, and thiourea-based resins, among others.
  • the metal ion chelating resin comprises cross-linked polystyrene or divinylbenzidine. Chelating resins can be obtained commercially from e.g., BioRad, Sigma Aldrich, Mitsubishi Chemical, Purolite, and the like.
  • the concentration/amount of metal ion chelating resin for use with the methods described herein can vary depending on the resin but should not be in such large concentrations that would interfere with downstream enzymatic reactions.
  • the concentration of the metal ion chelating resin e.g., a resin comprising paired iminodiacetate ions; ChelexTM
  • the concentration of the metal ion chelating resin is in the range of 5-50% w/v, for example, 5-40%w/v, 5-30%w/v, 5-25%w/v, 5-20%w/v, 5-10%w/v, 10-20%w/v, 10-
  • SUBSTITUTE SHEET (RULE 26) 30%w/v, 10-40%w/v, 20-30%w/v, 25-30%w/v, 25-35%w/v, or any integer therebetween (fore.g., 10%, 20%, or 30%).
  • the nucleic acid sample preparation methods can be performed at a temperature of at least 80°C, for example, at least 85°C, at least 90°C, at least 92°C, at least 95°C, at least 100°C, at least 105°C, or higher.
  • the method of nucleic acid preparation is performed at or includes incubation, e.g, at or about 80°C, at or about 81°C, at or about 82°C, at or about 83°C, at or about 84°C, at or about 85 °C, at or about 86°C, at or about 87°C, at or about 88°C, at or about 89°C, at or about 90°C, at or about 91°C, at or about 92°C, at or about 93°C, at or about 94°C, at or about 95°C, at or about 96°C, at or about 97°C, at or about 98°C, at or about 99°C, at or about 100°C, at or about 101°C, at or about 102°C, at or about 103°C, at or about 104°C, or at or about 105°C.
  • incubation e.g, at or about 80°C, at or about 81°C, at or about
  • the method of nucleic acid preparation is performed at or includes incubation at about 80°C to about 100°C, e.g., about 80°C to about 95°C, about 80°C to about 90°C, about 80°C to about 85°C, about 85°C to about 95°C, about 85°C to about 100°C, about 90°C to about 100°C or about 95°C to about 100°C.
  • the nucleic acid sample preparation can be performed for any desired time or until sufficient or complete inactivation of endogenous enzymes and cell lysis or liberation of nucleic acid is achieved.
  • the method of nucleic acid sample preparation is performed for at least 1 min, at least 2 min, at least 3 min, at least 4 min, at least 5 min, at least 6 min, at least 7 min, at least 8 min, at least 9 min, at least 10 min, at least 11 min, at least 12 min, at least 13 min, at least 14 min, at least 15 min, at least 16 min, at least 17 min, at least 18 min, at least 19 min, at least 20 min or more.
  • the method is performed for a time within the range of 1-20 min, 1-15 min, 1- 10 min, 1-5 min, 1-2 min, 15-20 min, 10-20 min, 5-20 min, 5-15 min, 5-10 min, 10-15 min, etc.
  • the nucleic acid sample preparation will be performed in an enclosed reaction vessel (e.g., test tube, Eppendorf tube etc) to prevent evaporation, concentration of reagents, contamination by nucleic acid degrading enzymes (e.g., RNAses), and for maintenance of reaction temperature etc. It is specifically contemplated that the nucleic acid sample preparation step is performed in a vessel that is compatible with an isothermal amplification method, such that the reaction mixture necessary for amplification can be added directly to the vessel containing the prepared nucleic acids (i.e, a ‘one-pot’ reaction system).
  • an enclosed reaction vessel e.g., test tube, Eppendorf tube etc
  • nucleic acids prepared using the methods and compositions described herein can be used with essentially any isothermal nucleic acid amplification method.
  • Exemplary isothermal nucleic acid amplification methods include, but are not limited to, Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), recombinase polymerase amplification (RPA), Loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), reverse transcription recombinase polymerase amplification (RT-RPA), reverse transcription Loop-mediated isothermal amplification (RT-LAMP), reverse transcription nucleic acid sequence-based amplification (RT-NASBA), transcription mediated amplification (TMA), helicase dependent amplification (HD A), multiple displacement amplification (MDA), strand displacement amplification (SDA), rolling circle amplification (RCA), single primer isothermal amplification (SPIA), restriction
  • SHERLOCK Specific High Sensitivity Enzymatic
  • Nucleic acids prepared using the methods and compositions described herein can be used in isothermal amplification reactions with or without modifying the nucleic acid.
  • Optional modifications can include, for example, denaturation, digestion, nicking, unwinding, incorporation and/or ligation of heterogeneous sequences, addition of epigenetic modifications, addition of labels (e.g., radiolabels such as 32 P, 33 P, "'I. or 35 S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, fluorochromes), and the like.
  • labels e.g., radiolabels such as 32 P, 33 P, "'I. or 35 S
  • enzyme labels such as alkaline phosphatase
  • fluorescent labels such as fluorescein isothiocyanate (FITC)
  • FITC fluorescein isothi
  • amplifying refer to any in vitro process for multiplying the copies of a target nucleic acid using an isothermal amplification method. Amplification can refer to an “exponential” increase in target nucleic acid. However, “amplifying” can also refer to linear increases in the numbers of a target nucleic acid, but is different than a one-time, single primer extension step.
  • Components of an isothermal amplification reaction can include, for example, one or more primers (e.g., individual primers, primer pairs, primer sets, oligonucleotides, multiple primer sets for multiplex amplification, and the like), nucleic acid target(s) or templates (e.g., target nucleic acid from a sample), one or more polymerases, nucleotides (e.g., dNTPs and the like), and a suitable buffer (e.g., a buffer comprising a detergent, a reducing agent, monovalent ions, and divalent ions as appropriate).
  • An amplification reaction can further include a reverse transcriptase, in some embodiments.
  • An amplification reaction can further include one or more detection agents, including but not limited to a probe that generates a signal when cleaved by an enzyme activated in a targetdependent manner.
  • components of an amplification reaction can include, but not limited to components and/or common additives such as salts, buffers, detergents, ions, oils, proteins, polymers and the like.
  • components of an amplification reaction can include non-enzymatic components and enzymatic components.
  • Non-enzymatic components can include, for example, primers, nucleotides, buffers, salts, reducing agents, detergents,
  • SUBSTITUTE SHEET (RULE 26) and ions; and generally do not include proteins (e.g., nucleic acid binding proteins), enzymes, or proteins having enzymatic activity such as, for example, polymerases, reverse transcriptases, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases and the like.
  • an enzymatic component can comprise a polymerase, either in the presence or absence of a reverse transcriptase.
  • polymerase enzymatic components are distinguished from other proteins (e.g., nucleic acid binding proteins and/or proteins having other enzymatic activities) such as, for example, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases, and the like.
  • proteins e.g., nucleic acid binding proteins and/or proteins having other enzymatic activities
  • proteins e.g., nucleic acid binding proteins and/or proteins having other enzymatic activities
  • proteins e.g., nucleic acid binding proteins and/or proteins having other enzymatic activities
  • proteins e.g., nucleic acid binding proteins and/or proteins having other enzymatic activities
  • proteins e.g., nucleic acid binding proteins and/or proteins having other enzymatic activities
  • helicases e.g., topoisomerases, ligases, exonucleases,
  • Essential co-factors of isothermal amplification reactions are known to those of skill in the art and are dependent on the enzyme(s) used. They can be organic or inorganic chemical compounds. Inorganic chemical compounds, for example, can be selected from the group comprising metal ions, e.g., Mg, Mn, Ca, Fe, Cu and Ni. Organic co-factors can include vitamins, proteins, biotin, nicotinamide adenine dinucleotide, and nucleotides, e.g. ATP.
  • Isothermal amplification reactions can be conducted at a range of temperatures, depending upon the exact reaction chosen and the enzyme (s) or factors used in the amplification. Some isothermal amplification reactions can proceed at room temperature. More often, an elevated temperature, e.g., around 37 degrees Celsius or higher, e.g., 37 degrees Celsius to about 75 degrees Celsius, e.g., about 38 degrees Celsius, about 39 degrees Celsius, about 40 degrees Celsius, about 41 degrees Celsius, about 42 degrees Celsius, about 43 degrees Celsius, about 44 degrees Celsius, about 45 degrees Celsius, about 46 degrees Celsius, about 47 degrees Celsius, about 48 degrees Celsius, about 49 degrees Celsius, about 50 degrees Celsius, about 51 degrees Celsius, about 52 degrees Celsius, about 53 degrees Celsius, about 54 degrees Celsius, about 55 degrees Celsius, about 56 degrees Celsius, about 57 degrees Celsius, about 58 degrees Celsius, about 59 degrees Celsius, about 60 degrees Celsius, about 61 degrees Celsius, about 62 degrees Celsius, about 63 degrees Celsius, about 64 degrees Celsius, about 65 degrees Celsius, about 66 degrees Celsius, about 67 degrees Celsius.
  • the isothermal amplification methods used herein can be conducted over a certain length of time and will typically be conducted until a detectable nucleic acid amplification product is generated.
  • a nucleic acid amplification product can be detected by any suitable detection process and/or a detection process compatible with isothermal amplification methods.
  • an amplification process is conducted over a length of time within about 2 hours or less, 2.5 hours or less, 60 minutes or less, for example 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less or 5 minutes or less.
  • the amplification process is conducted for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes,
  • SUBSTITUTE SHEET (RULE 26) about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes.
  • an amplification process is conducted over a length of time within about 10 minutes or less.
  • the nucleic acid targets are amplified without exposure to agents or conditions that denature or destabilize nucleic acids in the preparation, including, but not limited to thermal conditions (e.g., high temperatures), pH conditions (e.g., high or low pH), chemical agents (e.g., formamide, urea, dimethyl sulfoxide (DMSO), betaine sodium hydroxide, hydrochloric acid), proteins (e.g., enzymatic agents, such as helicases), and the like.
  • thermal conditions e.g., high temperatures
  • pH conditions e.g., high or low pH
  • chemical agents e.g., formamide, urea, dimethyl sulfoxide (DMSO), betaine sodium hydroxide, hydrochloric acid
  • proteins e.g., enzymatic agents, such as helicases
  • the nucleic acid amplification product can be up to 50 bases in length (e.g., about 15 to about 40 bases long or 15 bases long, 16 bases long, 17 bases long, 18 bases long, 19 bases long, 20 bases long, 21 bases long, 22 bases long, 23 bases long, 24 bases long, 25 bases long, 26 bases long, 27 bases long, 28 bases long, 29 bases long, 30 bases long, 31 bases long, 32 bases long, 33 bases long, 34 bases long, 35 bases long, 36 bases long, 37 bases long, 38 bases long, 39 bases long, or 40 bases long.
  • an amplification product is about 20 to about 40 bases long.
  • an amplification product is about 20 to about 30 bases long.
  • nucleic acids generated using the methods and compositions described herein are contemplated for use in a multiplex amplification format (i.e., amplification or more than one target sequence).
  • nucleic acids prepared as described herein are not considered ideal for conventional nucleic acid amplification methods using cycling temperatures (e.g., PCR), it is thought that this is due to a sensitivity of the PCR thermocycling enzymes to divalent metal ion concentration.
  • compositions and methods described herein chelate divalent metal ions, e.g., to limit targetindependent nuclease cleavage of detection probes, it can be exacting to achieve, for example, a magnesium ion concentration optimal for PCR while also limiting target-independent nuclease cleavage.
  • nucleic acid preparation methods and compositions described herein can be used in thermocycling nucleic acid amplification methods when a thermostable enzyme that maintains activity or specificity at reduced divalent metal cation concentrations is used.
  • a primer is generally characterized as an oligonucleotide that includes a nucleotide sequence capable of hybridizing or annealing to a target nucleic acid, at or near (e g., adjacent to) a specific region of interest (i.e., target sequence). Primers can allow for specific determination of a target nucleic acid nucleotide sequence or detection of the target nucleic acid (e.g., presence or absence of a sequence), or feature thereof, for example. A primer can be naturally occurring or synthetic.
  • the term “specific,” or “specificity”, generally refers to the binding or
  • SUBSTITUTE SHEET (RULE 26) hybridization of one molecule to another molecule, such as a primer for a target polynucleotide. That is, specific or specificity refers to the recognition, contact, and formation of a stable complex between two molecules, as compared to substantially less recognition, contact, or complex formation of either of those two molecules with other molecules.
  • anneal or “hybridize” generally refers to the formation of a stable base-paired nucleic acid complex, e.g., via hydrogen bonding, between two nucleic acid molecules or, where relevant, between complementary portions of a single nucleic acid molecule.
  • primer, oligo, or oligonucleotide can be used interchangeably herein, when referring to primers.
  • a primer can be designed and synthesized using suitable processes, and can be of any length suitable for hybridizing to a target sequence and permitting extension for an amplification process described herein.
  • Primers are generally designed according to a sequence in a target nucleic acid.
  • a primer in some embodiments can be about 5 bases in length to about 30 bases in length.
  • a primer can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases in length.
  • a primer is less than 28 bases in length.
  • a primer is about 8 to about 16 bases in length.
  • a primer is about 10 to about 12 bases in length.
  • a primer can be composed of naturally occurring and/or non-naturally occurring nucleotides (e g., labeled nucleotides), or a mixture thereof.
  • Primers suitable for use with methods described herein can be synthesized and labeled using any suitable technique.
  • primers can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts., 22: 1859-1862, 1981, using an automated synthesizer, as described in Needham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168, 1984.
  • primers can be effected, for example, by native acrylamide gel electrophoresis or by anion-exchange high-performance liquid chromatography (HPLC), for example, as described in Pearson and Regnier, J. Chrom., 255: 137-149, 1983.
  • HPLC high-performance liquid chromatography
  • All or a portion of a primer sequence can be complementary or substantially complementary to a target nucleic acid, in some embodiments.
  • Substantially complementary with respect to sequences generally refers to nucleotide sequences that will hybridize with each other. The stringency of the hybridization conditions can be altered to tolerate varying amounts of sequence mismatch.
  • target and primer sequences are at least 75% complementary to each other.
  • target and primer sequences can be 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more complementary to each other.
  • primers comprise a pair of primers.
  • a pair of primers can include a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of a target nucleic acid).
  • primers consist of a pair of primers, however, in certain
  • an amplification reaction can include additional primer pairs for amplifying different target sequences, such as in a multiplex amplification.
  • primers consist of a pair of primers, however, in certain instances, an amplification reaction can include additional primers, oligonucleotides or probes for a detection process that are not considered part of amplification.
  • a primer in certain embodiments, can contain a modification such as one or more inosines, abasic sites, locked nucleic acids, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), T m modifiers or any modifier that changes the binding properties of the primer.
  • a primer in certain embodiments, can contain a detectable molecule or entity (e.g., a fluorophore, radioisotope, colorimetric agent, particle, enzyme and the like).
  • Polymerases are proteins capable of catalyzing the specific incorporation of nucleotides to extend a 3' hydroxyl terminus of a primer molecule, such as, for example, an amplification primer described herein, against a nucleic acid target sequence (e.g., to which a primer is annealed). While not necessarily a required property, polymerases useful in the compositions and methods described herein can include, for example, thermophilic or hyperthermophilic polymerases that can have activity at an elevated reaction temperature (e.g., above 55 degrees Celsius, above 60 degrees Celsius, above 65 degrees Celsius, above 70 degrees Celsius, above 75 degrees Celsius, above 80 degrees Celsius, above 85 degrees Celsius, or higher).
  • an elevated reaction temperature e.g., above 55 degrees Celsius, above 60 degrees Celsius, above 65 degrees Celsius, above 70 degrees Celsius, above 75 degrees Celsius, above 80 degrees Celsius, above 85 degrees Celsius, or higher.
  • a polymerase can incorporate about 1 to about 50 nucleotides in a single synthesis.
  • a polymerase can incorporate about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in a single synthesis.
  • a polymerase is more processive, and can incorporate 50 or more nucleotides in a single synthesis.
  • amplification reaction components comprise one or more DNA polymerases.
  • Exemplary DNA polymerases can be obtained commercially and include, but are not limited to, a 9° N DNA polymerase; 9°NmTM DNA polymerase; TherminatorTM DNA Polymerase; TherminatorTM II DNA Polymerase; TherminatorTM III DNA Polymerase; TherminatorTM y DNA Polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (A.
  • DNA polymerase I large (Klenow) fragment; Klenow fragment (3 '-5' exo- ); T4 DNA polymerase; T7 DNA polymerase; Deep VentRTM (exo-) DNA Polymerase; Deep VentRTM DNA Polymerase; DyNAzymeTM EXT DNA; DyNAzymeTM II Hot Start DNA Polymerase; PhusionTM High-Fidelity DNA Polymerase; VentR® DNA Polymerase; VentR® (exo-) DNA Polymerase; RepliPHITM Phi29 DNA Polymerase; rBst DNA Polymerase, large fragment (IsoThermTM DNA Polymerase); MasterAmpTM AmpliThermTM DNA Polymerase; Tag DNA polymerase; Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNA polymerase; Tbr DNA polymerase; DNA polymerase Beta; and ThermoPhi DNA polymerase..
  • a polymerase can possess reverse transcription capabilities.
  • an amplification reaction can amplify RNA targets, for example, in a single step without the use of a separate reverse transcriptase.
  • transcriptase capabilities include Bst (large fragment), 9° N DNA polymerase, 9°NmTM DNA polymerase, TherminatorTM, TherminatorTM II, and the like.
  • amplification reaction components comprise one or more separate reverse transcriptases.
  • more than one polymerase can be included in an amplification reaction.
  • an amplification reaction can comprise a polymerase having reverse transcriptase activity and a second polymerase having no reverse transcriptase activity.
  • Nucleic acids prepared using the methods and compositions described herein can be subjected to reactions that detect one or more target nucleic acids.
  • the detection can be performed without a prior amplification.
  • the detection is performed on amplified nucleic acid products.
  • an amplification method as described herein, including but not limited to an isothermal amplification method can further comprise detecting and/or quantifying a nucleic acid amplification product using any detection method or quantification method known to those of skill in the art.
  • Non-limiting examples of detection and/or quantification methods include RNA-guided nuclease (e.g., Cas) mediated cleavage and activation of a fluorophore, molecular beacon (e.g., real-time, endpoint), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface capture, 5' to 3' exonuclease hydrolysis probes (e.g., TAQMAN), intercalating/binding dyes, absorbance methods (e.g., colorimetric, turbidity), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, a primer extension method (e.g., iPLEXTM), Molecular Inversion Probe (MIP) technology from Affymetrix, restriction fragment length polymorphism (RFLP) analysis, allele specific oligonucleotide (ASO) analysis,
  • detecting a nucleic acid amplification product comprises use of a real-time detection method (i.e., product is detected and/or continuously monitored during an amplification process). In some embodiments, detecting a nucleic acid amplification product comprises use of an endpoint detection method (i.e., product is detected after completing or stopping an amplification process). Nucleic acid detection methods can also employ the use of labeled nucleotides incorporated directly into a target sequence or into probes containing complementary sequences to a target. Such labels can be radioactive and/or fluorescent in nature and can be resolved in any of the maimers discussed herein.
  • quantification of a nucleic acid amplification product can be achieved using certain detection methods described below.
  • a detection method can be used in conjunction with a measurement of signal intensity, and/or generation of (or reference to) a standard curve and/or look-up table for quantification of a nucleic acid amplification product.
  • detecting a nucleic acid amplification product comprises use of lateral flow or a lateral flow device.
  • Such devices generally include a solid phase fluid permeable flow path through which fluid flows by capillary force.
  • Example devices include, but are not limited to, dipstick assays and thin layer chromatographic plates with various appropriate coatings. Immobilized on the flow path are various binding reagents for the sample, binding partners or conjugates involving binding partners for the sample and signal producing systems. Detection can be achieved in several manners including, for example, enzymatic detection, nanoparticle detection, colorimetric detection, and fluorescence detection.
  • Enzymatic detection can involve enzyme-labeled probes that are hybridized to complementary nucleic acid targets on the surface of the lateral flow device. The resulting complex can be treated with appropriate markers to develop a readable signal.
  • Nanoparticle detection involves bead technology that can use colloidal gold, latex and/or paramagnetic nanoparticles. In one example, beads can be conjugated to an anti-biotin antibody. Target sequences can be directly biotinylated, or target sequences can be hybridized to sequence-specific biotinylated probes. Gold and latex give rise to colonmetric signals visible to the naked eye, and paramagnetic particles give rise to a non-visual signal when excited in a magnetic field and can be interpreted by a specialized reader.
  • Fluorescence-based lateral flow detection methods also can be used and include, for example, dual fluorescein and biotin- labeled oligo probe methods, UPT-N ALP utilizing up-convertmg phosphor reporters composed of lanthanide elements embedded in a crystal (Corstjens et al., Clinical Chemistry, 47: 10, 1885-1893, 2001), and the use of quantum dots.
  • Nucleic acids can be captured on lateral flow devices.
  • Means of capture can include antibody-dependent and antibody-independent methods.
  • Antibody-dependent capture generally comprises an antibody capture line and a labeled probe of complementary sequence to the target.
  • Antibody -independent capture generally uses non-covalent interactions between two binding partners, for example, the high affinity and irreversible linkage between a biotinylated probe and a streptavidin line.
  • Capture probes can be immobilized directly on lateral flow membranes. Both antibody-dependent and antibody-independent methods can be used, for example, for detecting amplification products generated in a multiplex reaction.
  • detecting a nucleic acid amplification product comprises use of molecular beacon technology.
  • molecular beacon generally refers to a detectable molecule, where the detectable property of the molecule is detectable under certain conditions, thereby enabling the molecule to function as a specific and informative signal.
  • detectable properties include, optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties and time or speed through an opening of known size.
  • SUBSTITUTE SHEET (RULE 26) detecting nucleic acid molecules can be, for example, hair-pin shaped oligonucleotides containing a fluorophore on one end and a quenching dye on the opposite end.
  • the loop of the hair-pin can contain a probe sequence that is complementary to a target sequence and the stem is formed by annealing of complementary arm sequences located on either side of the probe sequence.
  • a fluorophore and a quenching molecule can be covalently linked at opposite ends of each arm.
  • the fluorescent and quenching molecules are proximal to one another preventing fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product)
  • hybridization can occur, and the loop structure is converted to a stable more rigid conformation causing separation of the fluorophore and quencher molecules leading to fluorescence (Tyagi et al. Nature Biotechnology 14: March 1996, 303- 308). Due to the specificity of the probe, the generation of fluorescence generally is exclusively due to the synthesis of the intended amplified product.
  • a molecular beacon probe sequence hybridizes to a sequence in an amplification product that is identical to or complementary to a sequence in a target nucleic acid. In some instances, a molecular beacon probe sequence hybridizes to a sequence in an amplification product that is not identical to or complementary to a sequence in a target nucleic acid (e.g., hybridizes to a sequence added to an amplification product by way of a tailed amplification primer or ligation).
  • Molecular beacons are highly specific and can discern a single nucleotide polymorphism. Molecular beacons also can be synthesized with different colored fluorophores and different target sequences, enabling simultaneous detection of several products in the same reaction (e.g., in a multiplex reaction). For quantitative amplification processes, molecular beacons can specifically bind to the amplified target following each cycle of amplification, and because nonhybridized molecular beacons are dark, it is not necessary to isolate the probe-target hybrids to quantitatively determine the amount of amplified product. The resulting signal is proportional to the amount of amplified product. Detection using molecular beacons can be done in real time or as an endpoint detection method. In some instances, certain reaction conditions can be optimized for each primer/probe set to ensure accuracy and precision.
  • detecting a nucleic acid amplification product comprises use of fluorescence resonance energy transfer (FRET).
  • FRET is an energy transfer mechanism between two chromophores: a donor and an acceptor molecule. Briefly, a donor fluorophore molecule is excited at a specific excitation wavelength. The subsequent emission from the donor molecule as it returns to its ground state can transfer excitation energy to the acceptor molecule through a long range dipole-dipole interaction. The emission intensity of the acceptor molecule can be monitored and is a function of the distance between the donor and the acceptor, the overlap of the donor emission spectrum and the acceptor absorption spectrum and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment.
  • FRET can be useful for quantifying molecular dynamics, for example, in
  • SUBSTITUTE SHEET (RULE 26) DNA-DNA interactions as described for molecular beacons.
  • a probe can be labeled with a donor molecule on one end and an acceptor molecule on the other Probe-target hybridization brings a change in the distance or orientation of the donor and acceptor and FRET change is observed.
  • detecting a nucleic acid amplification product comprises use of fluorescence polarization (FP).
  • Fluorescence polarization techniques generally are based on the principle that a fluorescently labeled compound when excited by linearly polarized light will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Therefore, when a molecule such as a tracer-nucleic acid conjugate, for example, having a fluorescent label is excited with linearly polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time light is absorbed and emitted.
  • fluorescence polarization provides a quantitative means for measuring the amount of tracer-nucleic acid conjugate produced in an amplification reaction.
  • detecting a nucleic acid amplification product comprises use of surface capture. This can be accomplished by the immobilization of specific oligonucleotides to a surface producing a biosensor that is both highly sensitive and selective.
  • Example surfaces that can be used include gold and carbon, and a surface capture method can use a number of covalent or noncovalent coupling methods to attach a probe to the surface. The subsequent detection of a target nucleic acid can be monitored by a variety of methods.
  • detecting a nucleic acid amplification product comprises use of
  • TAQMAN probes are hydrolysis probes that can increase the specificity of a quantitative amplification method (e.g., quantitative PCR).
  • the TAQMAN probe principle relies on 1) the 5' to 3' exonuclease activity of Taq polymerase to cleave a dual -labeled probe during hybridization to a complementary target sequence and 2) fluorophore-based detection.
  • a resulting fluorescence signal permits quantitative measurements of the accumulation of amplification product during the exponential stages of amplification, and the TAQMAN probe can significantly increase the specificity of the detection.
  • detecting a nucleic acid amplification product comprises use of intercalating and/or binding dyes. In some embodiments, detecting a nucleic acid amplification product comprises use of dyes that specifically stain nucleic acid. For example, intercalating dyes exhibit enhanced fluorescence upon binding to DNA or RNA.
  • Dyes can include DNA or RNA intercalating fluorophores and can include for example, SYTO® 82, acridine orange, ethidium bromide, Hoechst dyes, PicoGreen®, propidium iodide, SYBR® I (an asymmetrical cyanine dye), SYBR® II, TOTO (a thiaxole orange dimer) and YOYO (an oxazole yellow dimer). Dyes provide an opportunity for increasing the sensitivity of nucleic acid detection when used in conjunction with various detection
  • SUBSTITUTE SHEET (RULE 26) methods.
  • ethidium bromide can be used for staining DNA in agarose gels after gel electrophoresis; propidium iodide and Hoechst 33258 can be used in flow cytometry to determine DNA ploidy of cells; SYBR® Green 1 can be used in the analysis of double-stranded DNA by capillary electrophoresis with laser induced fluorescence detection; and PicoGreen® can be used to enhance the detection of double-stranded DNA after matched ion pair polynucleotide chromatography.
  • detecting a nucleic acid amplification product comprises use of absorbance methods (e.g., colorimetric, turbidity).
  • detection and/or quantitation of nucleic acid can be achieved by directly converting absorbance (e.g., UV absorbance measurements at 260 nm) to concentration, for example.
  • Direct measurement of nucleic acid can be converted to concentration using the Beer Lambert law which relates absorbance to concentration using the path length of the measurement and an extinction coefficient.
  • detecting a nucleic acid amplification product comprises use of a colorimetric detection method.
  • any suitable colorimetric detection can be used, and non-limiting examples include assays that use nanoparticles (e.g., metallic nanoparticles, modified nanoparticles, unmodified nanoparticles) and/or peptide nucleic acid (PNA) probes.
  • nanoparticles e.g., metallic nanoparticles, modified nanoparticles, unmodified nanoparticles
  • PNA peptide nucleic acid
  • certain gold nanoparticle-based methods typically rely on a quantitative coupling between target recognition and the aggregation of the nanoparticles, which, in turn, can lead to a change in the photonic properties (e.g., color) of a nanoparticle solution.
  • detecting a nucleic acid amplification product comprises use of electrophoresis (e.g., gel electrophoresis, capillary electrophoresis).
  • Gel electrophoresis involves the separation of nucleic acids through a matrix, generally a cross-linked polymer, using an electromotive force that pulls the molecules through the matrix. Molecules of different sizes or configurations move through the matrix at different rates causing a separation between products that can be visualized and interpreted via a number of methods including but not limited to; autoradiography, phosphorimaging, and staining with nucleic acid chelating dyes.
  • Capillary-gel electrophoresis is a combination of traditional gel electrophoresis and liquid chromatography that employs a medium such as polyacrylamide in a narrow bore capillary to generate fast, high-efficient separations of nucleic acid molecules with up to single base resolution.
  • CGE can be combined with laser induced fluorescence (LIF) detection where as few as six molecules of stained DNA can be detected.
  • LIF detection generally involves the use of fluorescent DNA intercalating dyes including ethidium bromide, YOYO and SYBR® Green 1, and also can involve the use of fluorescent DNA derivatives where fluorescent dye is covalently bound to DNA. Simultaneous identification of several different target sequences (e.g., products from a multiplex reaction) can be made using this method.
  • detecting a nucleic acid amplification product comprises use of mass spectrometry.
  • Mass Spectrometry is an analytical technique that can be used to determine the structure and quantity of a nucleic acid and can be used to provide rapid analysis of complex mixtures.
  • samples can be ionized, the resulting ions separated in electric and/or magnetic fields according to their mass-to-charge ratio, and a detector measures the mass-to -charge ratio of ions
  • Mass spectrometry methods include, for example, MALDI, MALDI-TOF, or Electrospray. These methods can be combined with gas chromatography (GC/MS) and liquid chromatography (LC/MS) Mass spectrometry (e.g., matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS)) can be high throughput due to high-speed signal acquisition and automated analysis off solid surfaces.
  • MALDI MS matrix-assisted laser desorption/ionization mass spectrometry
  • detecting a nucleic acid amplification product comprises use of nucleic acid sequencing.
  • the entire sequence or a partial sequence of an amplification product can be determined, and the determined nucleotide sequence can be referred to as a read.
  • linear amplification products can be analyzed directly without further amplification in some embodiments (e.g., by using single-molecule sequencing methodology).
  • linear amplification products can be subject to further amplification and then analyzed (e.g., using sequencing by ligation or pyrosequencing methodology).
  • Reads can be subject to different types of sequence analysis. Any suitable sequencing method can be utilized to detect, and in some instances determine the amount of, detectable products generated by the amplification methods described herein.
  • Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, reversible terminatorbased sequencing, sequencing by ligation, pyrosequencing, sequencing by synthesis, single-molecule sequencing, multiplex sequencing, solid phase single nucleotide sequencing, and nanopore sequencing.
  • detecting a nucleic acid amplification product comprises use of digital amplification (e.g., digital PCR).
  • Digital PCR takes advantage of nucleic acid (DNA, cDNA or RNA) amplification on a single molecule level, and offers a highly sensitive method for quantifying low copy number nucleic acid.
  • Systems for digital amplification and analysis of nucleic acids are available (e.g., Fluidigm® Corporation).
  • Isothermal nucleic acid amplification methods can be conducted in the presence of native nucleotides, such as, for example, dideoxyribonucleoside triphosphates (dNTPs), and/or derivatized nucleotides.
  • a native nucleotide generally refers to adenylic acid, guanylic acid, cytidylic acid, thymidylic acid, or uridylic acid.
  • a derivatized nucleotide generally is a nucleotide other than a native nucleotide. Nucleotides typically are designated as follows.
  • a ribonucleoside triphosphate is referred to as NTP or rNTP, where N can be A, G, C, U.
  • a deoxynucleoside triphosphate substrate is referred to as dNTP, where N can be A, G, C, T, or U.
  • Monomeric nucleotide subunits can be denoted as A, G, C, T, or U herein with no particular reference to DNA or RNA.
  • non- naturally occurring nucleotides or nucleotide analogs such as analogs containing a detectable label (e.g., fluorescent or colorimetric label), can be used.
  • nucleic acid amplification can be carried out in the presence of labeled dNTPs, such as, for example, radiolabels such as 32 P, 33 P, 125 I, or 35 S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, or fluorochromes.
  • labeled dNTPs such as, for example, radiolabels such as 32 P, 33 P, 125 I, or 35 S
  • enzyme labels such as alkaline phosphatase
  • fluorescent labels such as fluorescein isothiocyanate (FITC)
  • FITC fluorescein isothiocyanate
  • nucleic acid amplification can be carried out in the presence of modified dNTPs, such as, for example, heat activated dNTPs (e.g., CleanAmpTM dNTPs from TriLink
  • CRISPR Cas- mediated detection of a target sequence In some embodiments, the isothermal amplification methods use a CRISPR-Cas method for detecting the presence of a target sequence in the pool of amplified nucleic acids.
  • the CRISPR-Cas enzyme can be from an organism from a genus comprising, for example, Streptococcus, Campylobacter, Ni trati fr actor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methyl obacterium or
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • a target sequence can be DNA or RNA.
  • target nucleic acid refers to a polynucleotide being or comprising the target sequence.
  • the target nucleic acid can be a polynucleotide or a part of a polynucleotide to which a part of the gRNA, i.e. the guide sequence, is designed to have complementarity and to which the effector function mediated by the complex comprising CRISPR effector protein and a gRNA is to be directed.
  • the enzyme system can be a DNA targeting CRISPR-Cas protein or an
  • CRISPR-Cas proteins include, but are not limited to, Cas 13, Cas 12a, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof.
  • CsxlO Csxl6, Csa
  • guide nucleic acid As used herein, the terms “guide nucleic acid,” “guide sequence,” “crRNA,” “guide
  • RNA refers to a polynucleotide comprising any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and to direct sequence-specific binding of a CRISPR complex comprising the guide sequence and a CRISPR effector protein to the target nucleic acid sequence.
  • the degree of complementarity when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%), or more.
  • Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences.
  • Exemplary algorithms for determining optimal alignment include, but are not limited to, the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft
  • the guide nucleic acid strand can be any length.
  • the guide nucleic acid strand can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
  • a nucleic acid strand is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.
  • the guide nucleic acid sequence is 10-30 nucleotides long.
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • the section of the guide sequence through which complementarity to the target sequence is important for cleavage activity is referred to herein as the seed sequence.
  • a target sequence can comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in a parasite, bacterial or viral genome.
  • the presence of a target nucleic acid sequence will activate the Cas enzyme to non-specifically cleave a reporter molecule, for example, releasing or activating a fluorophore that indicates the presence of the target nucleic acid.
  • .ST/ ERL I ('K amplification: SHERLOCK, also known as “Specific high-sensitivity enzymatic reporter unlocking,” is a nucleic acid detection method that is used to detect RNA or DNA target sequences depending upon the particular enzymes used. In each instance, SHERLOCK detection is based on the target sequence -dependent activation of an RNA- guided nuclease, which, once activated, cleaves not only the target sequence, but other nucleic acids.
  • a sensitive target-sequence detection assay is provided.
  • a SHERLOCK detection assay includes the use of an RNA-guided nuclease, a guide RNA including complementarity to a desired target nucleic acid, and a labeled probe that generates a signal upon cleavage by the promiscuous activity of the target-sequence-activated RNA-guided nuclease.
  • targetsequence detection assay When that targetsequence detection assay is coupled with a target-specific isothermal nucleic acid amplification reaction, a single-pot amplification and detection assay is provided that has extremely high sensitivity.
  • This combined isothermal amplification/RNA-guided nuclease detection approach is the assay initially published as SHERLOCK - see Gootenberg et al., Science 356: 438-442 (2017), incorporated herein by reference.
  • the isothermal amplification approach can be coupled with reverse transcription to detect RNA targets in a highly sensitive manner by first generating amplified cDNA, and then detecting with RNA-guided nuclease that targets DNA.
  • RNA-guided nuclease is one that cleaves RNA, e.g., a Casl3a enzyme
  • a single -stranded RNA probe can be used (Gootenberg et al., Science 356: 438- 442 (2017)).
  • a DNA probe can be used; see, e.g., Gootenberg et al., Science 360: 439-444 (2016), and Li et al., Cell
  • nucleic acid sample preparation compositions and methods described herein are well-suited for preparing biological samples for use in isothermal nucleic acid amplification reactions, as well as for SHERLOCK detection of target nucleic acid sequences.
  • a pathogen can be, for example, an intracellular parasite, a virus, a bacterium, or a fungus.
  • Exemplary intracellular parasites that can be detected using the methods and compositions described herein include, but are not limited to Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Babesia sp. Leishmaniasis spp. Toxoplasmosis spp. and filarial nematodes.
  • a pathogenic virus that can be detected includes the Dengue virus, Zika virus, Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoVl), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Heptatis E, Herpes virus, Varicella virus, Cytomegalovirus, Epstein-Barr virus, Human herpesvirus 6, Human herpesvirus8, adenovirus, influenza, parainfluenza, respiratory syncytial virus, or Chikungunya virus.
  • SARS-CoVl Severe Acute Respiratory Syndrome Coronavirus 1
  • MERS-CoV Middle Eastern Respiratory Syndrome Coronavirus
  • SARS-CoV2 Severe Acute Respiratory Syndrome Coronavirus 2
  • Hepatitis A Hepatitis B
  • Hepatitis C Hepatitis D
  • viruses that can be detected as described herein include genera of viruses: Adenoviridae, Alfamovirus, Allexivirus, Allolevivirus, Alphacryptovirus, Alphaherpesvirinae, Alphanodavirus, Alpharetrovirus, Alphavirus, Aphthovirus, Apscaviroid, Aquabirnavirus, Aquareovirus, Arenaviridae, Arenavirus, Arteriviridae, Arterivirus, Ascoviridae, Ascovirus, Asfarviridae, Asfivirus, Astroviridae, Astrovirus, Aureusvirus, Avenavirus, Aviadenovirus, Avibirnavirus, Avihepadnavirus, Avipoxvirus, Avsunviroid, Avsunviroidae, Baculoviridae, Badnavirus, Barnaviridae, Barnavirus, Bdellomicrovirus, Begomovirus, Benyvirus, Betacryptovirus, Betaherpesvirinae, Betanodavirus,
  • Entomopoxvirus B Entomopoxvirus C
  • Ephemerovirus Epsilonretrovirus
  • Errantivirus Erythrovirus
  • Fabavirus Fijivirus
  • Filoviridae Flaviviridae
  • Flavivirus Foveavirus
  • Furovirus Fuselloviridae
  • Fusellovirus Gammaherpesvirinae
  • Gammaretrovirus Geminiviridae
  • Giardiavirus Granulovirus
  • Hantavirus Hemivirus, Hepacivirus, Hepadnaviridae
  • Hepatitis E-like viruses Hepatovirus
  • Herpesviridae Hordeivirus, Hostuviroid
  • Hypoviridae Hypovirus, Ichnovirus, "Ictalurid herpes-like viruses
  • Idaeovirus liarvirus
  • Infectious laryngotracheitis-like viruses
  • a bacterium that can be detected using the methods and compositions described herein can be a gram negative bacterium, a gram positive bacterium, an anaerobic bacterium, an aerobic bacterium, a facultative anaerobic bacterium, or an intracellular bacterium.
  • Examples of gram-negative bacteria include cocci, nonenteric rods, and enteric rods.
  • the genera of Gram-negative bacteria include,
  • SUBSTITUTE SHEET for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.
  • Exemplary gram -positive bacteria include, but are not limited to, cocci, nonsporulating rods, and sporulating rods.
  • the genera of Gram -positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Coryneb cterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces .
  • Additional bacteria that can be detected as described herein include bacteria from one or more of the following genera: genera of the domain of Bacteria (or Eubacteria): Abiotrophia, Acetitomaculum, Acetivibrio, Acetoanaerobium, Acetobacter, Acetobacterium, Acetofilamentum, Acetogenium, Acetohalobium, Acetomicrobium, Acetonema, Acetothermus, Acholeplasma, Achromatium, Achromobacter, Acidaminobacter, Acidaminococcus, Acidimicrobium, Acidiphilium, Acidisphaera, Acidithiobacillus, Acidobacterium, Acidocella, Acidomonas, Acidothermus, Acidovorax, Acinetobacter, Acrocarpospora, Actinoalloteichus, Actinobacillus, Actinobaculum, Actinobispora, Actinocorallia, Actinokineospora, Actinomadura, Actinom
  • SUBSTITUTE SHEET (RULE 26) Brevibacillus, Brevibacterium, Brevinema, Brevundimonas, Brochothrix, Brucella, Brumimicrobium, Buchnera, Budvicia, Bulleidia, Burkholderia, Buttiauxella, Butyrivibrio, Caedibacter, Caenibacterium, Calderobacterium, Caldicellulosiruptor, Caldilinea, Caldimonas, Caldithrix, Caloramator, Caloranaerobacter, Calymmatobacterium, Caminibacter, Caminicella, Campylobacter, Capnocytophaga, Capsularis, Carbophilus, Carboxydibrachium, Carboxydobrachium, Carboxydocella, Carboxydothermus, Cardiobacterium, Carnimonas, Carnob acterium, Caryophanon, Caseobacter, Catellatospora, Catenibacterium
  • Neorickettsia Neorickettsia, Neptunomonas, Nesterenkonia, Nevskia, Nitrobacter, Nitrococcus, Nitrosococcus, Nitrosolobus, Nitrosomonas, Nitrosospira, Nitrospina, Nitrospira, Nocardia, Nocardioides, Nocardiopsis, Nonomuraea, Nonomuria, Novosphingobium, Obesumbacterium, Oceanicaulis, Oceanimonas, Oceanisphaera, Oceanithermus, Oceanobacillus, Oceanobacter, Oceanomonas, Oceanospirillum, Ochrobactrum, Octadecabacter, Oenococcus, Oerskovia, Okibacterium, Oleiphilus, Oleispira, Oligella, Oligotropha, Olsenella, Opitutus, Orenia, Oribaculum, Orientia, Omithinicoccus, Ornithinimicrobium, Orni
  • SUBSTITUTE SHEET (RULE 26) Selenomonas, Seliberia, Serpens, Serpula, Serpulina, Serratia, Shewanella, Shigella, Shuttleworthia, Silicibacter, Simkania, Simonsiella, Sinorhizobium, Skermanella, Skermania, Slackia, Smithella, Sneathia, Sodalis, Soehngenia, Solirubrobacter, Solobacterium, Sphaerobacter, Sphaerotilus, Sphingobact rium, Sphingobium, Sphingomonas, Sphingopyxis, Spirilliplanes, Spirillospora, Spirillum, Spirochaeta, Spiroplasma, Spirosoma, Sporanaerobacter, Sporichthya, Sporobacter, Sporobacterium, Sporocytophaga, Sporohalobacter, Sporolactobacillus, Sporomusa, Sporos
  • RDT strengths include point- of-care (POC) utility and an intuitive format, but most tests target Plasmodium falciparum and are incapable of species-specific identification - a critical clinical limitation as P.vivax and P. ovale uniquely require an 8 -aminoquinolone (i.e., primaquine or tafenoquine) therapy to prevent relapse. Although there are sustained calls for more sensitive, non-falciparum malaria diagnostics, this remains an ongoing diagnostic gap (5). Additionally, the most common RDT antigen target for P.
  • POC point- of-care
  • HRP2 histidine-rich protein 2
  • PCR polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • SUBSTITUTE SHEET (RULE 26) [0152] Most NAATs for pathogen detection require nucleic acid extraction via multi-step commercial kits involving numerous specimen transfers, laboratory infrastructure (flow-columns, management of biohazardous wastes such as chaotropic agents, etc ), and 30 minutes or more of preassay preparation time This is not practically implementable for POC testing, and sample preparation remains a general bottleneck for adoption of nucleic acid technologies, particularly for RLS (13, 14).
  • SHERLOCK Specific High-Sensitivity Enzymatic Reporter UnLOCKing
  • a streamlined, field-applicable diagnostic comprised of 10-minute S-PREP (SHERLOCK Parasite Rapid Extraction Protocol) followed by SHERLOCK for 60 minutes for Plasmodium species-specific detection via fluorescent or lateral flow strip readout.
  • the inventors optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization.
  • These P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (five P. falciparum and ten P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid POC clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections.
  • kits for nucleic acid preparation which at a minimum include a reducing agent and a metal ion chelating resin in aqueous suspension or for preparation of an aqueous suspension.
  • the reducing agent is dithiothreitol (DTT) and the metal ion chelating resin comprises paired iminodiacetate ions.
  • DTT dithiothreitol
  • the reducing agent and chelating resin can be provided in an admixture or can be provided separately with instructions to generate an admixture, for example, comprising 10-30% w/v of the chelating resin.
  • the reducing agent and metal ion chelating agent can be provided in concentrated stock solutions or in pre-measured aliquots.
  • a kit can comprise one or more reagents for performing an isothermal amplification method, for example, one or more polymerases and one or more primers, and optionally one or more reverse transcriptases.
  • a pair of primers forward and reverse
  • a pair of primers can be included in the kit for a desired target sequence, such as for detection of a parasite that causes malaria.
  • a plurality of primer pairs can be included in the kit.
  • a kit can include a control polynucleotide, and where multiple target sequences are amplified, a plurality of control polynucleotides can be included in the kit.
  • Kits can also comprise one or more of the components in any number of separate vessels, chambers, containers, packets, tubes, vials, microtiter plates and the like, or the components can be combined in various combinations in such containers
  • Components of the kit can, for example, be present in one or more containers. In some embodiments, all of the components are provided in one container.
  • the enzymes e.g., polymerase(s) and/or reverse transcriptase(s)
  • the components can, for example, be lyophilized, freeze dried, or in a stable buffer.
  • polymerase(s) and/or reverse transcriptase(s) are in lyophilized form in a single container, and the primers are either lyophilized, freeze dried, or in buffer, in a different container. In some embodiments, polymerase(s) and/or reverse transcriptase(s), and the primers are, in lyophilized form, in a single container.
  • Kits can further comprise, for example, dNTPs used in the reaction, or modified nucleotides, vessels, cuvettes or other containers used for the reaction, or a vial of water or buffer for re-hydrating lyophilized components.
  • the buffer used can, for example, be appropriate for both polymerase and primer annealing activity.
  • Kits can also comprise instructions for performing one or more methods described herein and/or a description of one or more components described herein. Instructions and/or descriptions can be in printed form and can be included in a kit insert. A kit also can include a written description of an internet location that provides such instructions or descriptions.
  • Kits can further comprise reagents used for detection methods, such as, for example, reagents used for FRET, lateral flow devices, dipsticks, fluorescent dye, colloidal gold particles, latex particles, a molecular beacon, or polystyrene beads.
  • reagents used for detection methods such as, for example, reagents used for FRET, lateral flow devices, dipsticks, fluorescent dye, colloidal gold particles, latex particles, a molecular beacon, or polystyrene beads.
  • kits for detecting malaria comprising the components described in the Examples section is specifically contemplated.
  • composition for nucleic acid preparation comprising a reducing agent and a metal ion chelating resin in aqueous suspension.
  • composition of paragraph 9 or paragraph 10, wherein the biological sample comprises blood or a blood fraction, a nasopharyngeal swab, an oropharyngeal swab, sputum or saliva.
  • a composition consisting essentially of an aqueous buffer, DTT and a metal ion chelating resin.
  • composition of paragraph 14 wherein the composition of any one of paragraphs 1-13 is present at about a 3: 1 ratio relative to biological sample by volume.
  • composition of paragraph 14 or paragraph 15, wherein the biological sample comprises blood or a blood fraction, a nasopharyngeal swab, an oropharyngeal swab, sputum or saliva.
  • composition of paragraph 21, wherein the detection of a target nucleic acid comprises SHERLOCK detection.
  • [0185] 24 A method of preparing a biological sample for nucleic acid analysis, the method comprising contacting a composition of any one of paragraphs 1-13 with the biological sample, and heating the resulting mixture to at least 80°C.
  • the intracellular parasite comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Babesia sp. Leishmaniasis spp. Toxoplasmosis spp. or filarial nematodes.
  • the virus comprises Dengue virus, Zika virus, Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoVl), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Heptatis E, Herpes virus, Varicella virus, Cytomegalovirus, Epstein-Barr virus, Human herpesvirus 6, Human herpesvirus8, adenovirus, influenza, parainfluenza, respiratory syncytial virus, or Chikungunya virus,
  • SARS-CoVl Severe Acute Respiratory Syndrome Coronavirus 1
  • MERS-CoV Middle Eastern Respiratory Syndrome Coronavirus
  • SARS-CoV2 Severe Acute Respiratory Syndrome Coronavirus 2
  • Hepatitis A Hepatitis B
  • Hepatitis C Hepatitis D
  • Heptatis E Herpes
  • a method of amplifying one or more target nucleic acids in a biological sample comprising nucleic acids comprising:
  • step (ii) heating the mixture resulting from step (i) to at least 80°C for a time sufficient to release nucleic acids in the biological sample;
  • step (iii) after step (ii), contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids;
  • step (iv) incubating the mixture of step (iii) under conditions and for a time sufficient to generate an isothermal amplification product for one or more target nucleic acids.
  • a method of detecting one or more target nucleic acids in a biological sample comprising:
  • step (ii) heating the mixture resulting from step (i) to at least 80°C for a time sufficient to release nucleic acids in the biological sample;
  • step (iii) after step (ii), contacting the mixture with reagents sufficient for isothermal amplification and SHERLOCK detection of one or more target nucleic acids;
  • step (iv) incubating the mixture of step (iii) under conditions and for a time sufficient to permit isothermal amplification and production of a SHERLOCK detection signal for one or more target nucleic acids present in the sample.
  • kits for nucleic acid preparation and/or detection comprising a reducing agent and a metal ion chelating resin in aqueous suspension, and packaging materials therefor.
  • SUBSTITUTE SHEET (RULE 26) [0230] 64.
  • Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections ( ⁇ 100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care (POC) diagnostics are also lacking for non-falciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods such as polymerase chain reaction (PCR) have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS.
  • PCR polymerase chain reaction
  • SHERLOCK Specific High-Sensitivity Enzymatic Reporter UnLOCKing
  • falciparum SHERLOCK assay a simplified field-ready SHERLOCK diagnostic was demonstrated, and the accuracy of the diagnostic was confirmed on simulated whole blood, serum, and dried blood spot samples, as well as clinical samples from patients with P. falciparum and P. vivax infection.
  • FIG 1 illustrates the workflow of a simplified SHERLOCK diagnostic. This test combines a 10-minute sample preparation step and a 60-minute SHERLOCK assay prior to endpoint analysis via lateral flow strip or fluorescence measurement.
  • CRISPR-based diagnostics utilize the programmable endonucleases (Cas enzymes) of CRISPR-associated microbial adaptive immune systems. Casl2a (also known as Cpfl) is one such RNA -guided, DNA-cleaving enzyme, which can be programmed with CRISPR guide RNAs (gRNA) to construct highly sensitive and specific nucleic acid detection platforms (15-19).
  • Cas enzymes programmable endonucleases
  • Casl2a also known as Cpfl
  • gRNA CRISPR guide RNAs
  • Programmed Casl2a is activated through recognition of its dsDNA target and exhibits indiscriminate, non-specific DNase activity that cleaves non-target DNAs. This non-specific degradation of fluorophore -quencher labeled reporter ssDNA is exploited to detect the presence ofthe dsDNA target that activated Cas 12a. To further decrease the limit of detection (LOD), a reverse-transcriptase recombinase polymerase amplification
  • RPA SUBSTITUTE SHEET (RULE 26) (RT-RPA) step is added before Casl2a detection to increase target DNA concentrations (FIG. 2).
  • RPA is a powerful isothermal nucleic acid amplification tool comprised of three core enzymes: a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing polymerase that coordinates DNA synthesis from primer-paired target DNA (20).
  • released fluorophore from cleaved reporter ssDNA was measured by a plate reader or a handheld fluorimeter. Particularly in RLS, use of a handheld fluorimeter enables a field-applicable readout method. The inventors did not find a significant difference in the sensitivity performance between machines and observed a similar 7-10 fold-change in fluorescence between platforms although they had different baselines (data not shown).
  • SHERLOCK reactions 50 pL
  • triplicate 150 pL
  • the assays are also adapted for endpoint detection via lateral flow strip based upon degradation of ssDNA reporter that is labeled on opposing ends with FAM and biotin.
  • Casl2a was selected (as opposed to the Casl3 family (21, 22), which also has non-specific nuclease activity) so DNA targets could be directly detected instead of RNA, particularly in dried blood spots where RNA may be degraded.
  • the rapid enzymatic kinetics of Cas 12a also make this nucleic-acid based technology comparable to the POC format of antigen -based lateral flow immunoassays.
  • Cas 12a bound to its dsDNA activator is capable of -1250 turnovers per second with a catalytic efficiency (C ca t/K M - I.7x 10 9 s' 1 M 1 ) approaching the rate of diffusion (17).
  • the addition of a reverse transcriptase enzyme further enhances the sensitivity by transcribing multiple-copy RNAs from a target sequence into DNA for detection.
  • SHERLOCK parameters including reaction temperature, RPA primer concentration, RT commercial brand, and ssDNA reporter concentration were optimized (data not shown). The reactions was also lyophilized into a pellet to be resuspended with S-PREP treated sample for cold-chain independence in the field, and importantly, also improved the LOD by increasing sample input volume.
  • RPA primer and gRNA selection were designed to detect four of the most common pathogenic species of malaria. The inventors iterated a two-step design process of RPA primer screen followed by gRNA screen.
  • RPA primer targets were identified by reviewing the literature for the best-performing NAATs and searching for conserved and specific sequences from alignment of species-specific strains available from the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • Pfr364 target For P . falciparum 18S rRNA, mitochondrial (cytochrome oxidase III, cytochrome B), and subtelomeric (Pfr364) targets were screened (23-29).
  • the Pfr364 target which
  • SUBSTITUTE SHEET (RULE 26) is a species-specific, non-coding subtelomeric repeat sequence present in 41 copies on the P. falciparum genome, had the best signal in comparison to the othertargets (data not shown). Moreover, the selected gRNA had >90% sequence homology among all assembled P. falciparum genomes available in NCBI as well as 86% of sequences from the Pf3k dataset (an open-access collaboration and deep-genomic sequencing database) accessed via IGV (Integrative Genomics Viewer) (30). For P. vivax, an 18S rRNA and mitochondrial target were tested, and it was found that the mitochondrial target worked best (copy number per parasite can be as high as 20) (27, 31). For P.
  • F1-F5 and R1-R5 Five forward (F1-F5) and five reverse primers (R1-R5) per sequence target were constructed using guidance provided by the TwistDx manufacturer; primers were 30-40 nucleotides long, with goal amplicons of 100-200 base-pairs in length. Forward and reverse primers were paired for a total of 25 combinations (FLR1-5, F2:Rl-5, F3:Rl-5, F4:Rl-5, F5:Rl-5) for each sequence target and 2-3 of the best-performing pairs were selected for the optimization of gRNA design (data not shown). RPA was performed according to the manufacturer’s instructions as described in the Methods.
  • Casl2a recognizes a short nucleotide sequence (TTTN) called the protospacer adjacent motif (PAM) for generation of distal dsDNA cleavage, and 2-4 gRNAs based upon the TTTN PAM were designed within the RPA amplicon.
  • the RPA reaction for each primer set was then transferred to a Cas reaction as described in the Methods, and fluorescent kinetics were monitored for selection of best performing gRNAs (Table 1).
  • Sample preparation Accessing sample nucleic acids in a field-applicable manner involves overcoming several challenges. Preparation requires lysing the red blood cell and parasite membrane (with the exception of the invasive merozoite form, all blood-stage parasites are intraerythrocytic), deactivating multiple inhibitory blood components, and importantly, appropriately deactivating nucleases that could shear the ssDNA reporter and lead to a false positive signal. The requirement for simplicity and low cost ruled out commercial nucleic acid extraction kits. To test sample preparation methods, simulated whole blood samples of live intraerythrocytic P.
  • falciparum spiked into purchased EDTA-treated human blood were used to a final 1 fM (602 parasites/pL) concentration for rehydration of the one-pot lyophilized P. falciparum SHERLOCK assay described herein.
  • the inventors therefore assessed alternative simplified sample preparation protocols described in the Methods including various detergents, thermal lysis, and chemical deactivation protocols (FIGs. 3A-B). It was discovered that treating samples with 50mM DTT and lOmM EGTA followed by 95°C incubation for 10 minutes resulted in a robust SHERLOCK signal, although some variability was observed in the no-template control signal that was attributed to background nucleases in different blood aliquots . However, when the DTT/EGTA/95 °C sample preparation method was tested on patient P. falciparum and P. vivax serum samples from the Dominican Republic, bidirectional crossreactivity of the species-specific SHERLOCK assays was observed. Using the P.
  • P. vivax patient serum samples produced a false positive signal (FIG. 4A).
  • P. falciparum patient samples also produced a false positive signal using the P. vivax-specific assay (FIG. 4B).
  • Extracted nucleic acid reflected combined human and parasite DNA, with numbers of human sequences dwarfing numbers of parasite sequences, and the highly sensitive and specific performance of the appropriate SHERLOCK assay on the extracted nucleic acid made cross-reactivity due to human DNA unlikely. These results were also observed on all five P. falciparum and all ten P. vivax specimens making co-infection unlikely and the specimens had all undergone species-specific qualitative PCR testing (ARUP, Salt Lake City, UT).
  • Chelex®-100 is a resin containing styrene divinylbenzene copolymers with paired iminodiacetate ions that act as chelating groups in binding polyvalent metal ions (34). Nucleases require metal ions as cofactors and therefore chelating agents inhibit their activity.
  • S-PREP is a simplified sample preparation method where sample is diluted
  • SUBSTITUTE SHEET (RULE 26) 1:3 (5 pL into 15 pL of S-PREP buffer) followed by heating to 95°C for 10 minutes. False positive signals of serum samples were eliminated using S-PREP (FIGs. 4C-4D). It was concluded that higher concentrations of nucleases present in “sick” serum (patients sick with another disease but not the target disease) necessitate stronger nuclease deactivation procedures. The inventors are the first to report on this cross-reactivity in non-nucleic-acid extracted clinical samples for SHERLOCK as the inventors are not aware of other studies comparing performance using unextracted samples against controls from patients sick with a different disease (instead of only comparing to healthy control specimens).
  • the CRISPR diagnostic described herein can also detect clinically relevant levels of parasitemia in 40 minutes or less from unextracted blood samples (10 minute S-PREP followed by 30 minute SHERLOCK) with better sensitivity than existing POC antigen-based RDTs - filling an important clinical diagnostic gap for HRP-2 deletion P. falciparum, and non-falciparum malaria.
  • a 0.001% parasitemia (assuming a red blood cell mean corpuscular volume of 80 femtoliters and hematocrit of 45%) corresponds to ⁇ 60 parasites/pL (lOOaM concentration), for which a 30-minute detectable signal difference between the no-template control and infected blood is readily apparent (FIG. 5B).
  • simulated dried blood spots were prepared to a 2aM (one parasite per microliter blood) concentration for each of the four Plasmodium species and tested with the S- PREP/SHERLOCK protocol with modifications as described in the methods.
  • a robust fluorescence signal was demonstrated at the one-hour time point that was significantly different from the no-template control.
  • the only notable difference in assay performance compared with whole blood samples was a greater no-template control signal in simulated DBS samples - likely from autofluorescence from the paper substrate (FIG. 5D).
  • SHERLOCK is a cost-effective technology estimated at $0.61 (USD) per test (15) given its lyophilizable format and lateral flow readout capability This work brings the platform closer to clinical care in demonstrating a field-ready SHERLOCK diagnostic.
  • Key features include simplified sample preparation without nucleic acid extraction, isothermal assay conditions (40°C) independent of a thermocycler, a lyophilized integrated assay, and field-applicable readouts including use of a handheld fluorometer or lateral flow strip. The ultrasensitive LOD of these assays was validated using industry standard protocols of replicate testing.
  • SHERLOCK assays in general, are highly susceptible to false positives in the presence of contaminating nucleases While all NAATs are at risk of target degradation in the presence of nucleases, appropriate deactivation is crucially important for SHERLOCK assays, and it was observed that specimens may very well have differing levels of nucleases depending on disease state, sample type, and even blood aliquot.
  • Plasmodium species identification is a promising new tool that moves this technology closer to clinical POC application in resource-limited settings. Future work will be needed to optimize performance in field settings and define the utilization of ultrasensitive detection for clinical and policy decisionmaking.
  • Table 1 Best performing RPA primers and gRNA sequences for development of Plasmodium SHERLOCK assays.
  • Table 2 Analytical sensitivity of Plasmodium species SHERLOCK. Results of replicate testing at five different calibration standard concentrations near the expected LOD (replicates testing positive/replicates tested for determination of 95% LOD by probit analysis).
  • P. falciparum simulated samples were prepared by either serially diluting live parasites into whole blood or serially diluting purified whole genomic DNA into whole blood.
  • the 3D7 strain obtained from the Walter & Eliza Hall Institute
  • Plasmodium falciparum was cultured in human red blood cells (RBCs) at 4% hematocrit to ⁇ 2% parasitemia in RPMI 1640 supplemented with 0.5% Albumax II, 50 mg/liter hypoxanthine, 0.21% sodium bicarbonate, and 25 mM HEPES, as previously described (39).
  • SUBSTITUTE SHEET (RULE 26) the number of base pairs of dsDNA by the average molecular weight of a base-pair (650 g/mol) (41). The molar concentration calculated by dividing the mass of a sample by its molecular weight can be translated to copies of target (parasites) per unit volume by multiplying by Avogadro’s number (6.022 x 10 23 molecules/mole).
  • nucleic acids were obtained from patient clinical samples via QIAamp
  • plasmids containing the small subunit ribosomal RNA genes ( 18S) MRA-179 and MRA-180 were obtained. After quantification of plasmid on Nanodrop and using the estimated molecular weight based on known plasmid size (5100 base-pairs and 5000 basepairs, respectively) for calculation of molar concentration, diluted plasmids were serially diluted into whole blood to determine the LOD (FIG. 5).
  • Dried blood spots were simulated by deposition of 50pL of simulated blood samples (live intracellular P. falciparum spiked into whole blood, P. vivax purified whole genomic DNA spiked into whole blood, P. malariae MRA-179 plasmid spiked into whole blood, P. ovale MRA- 180 plasmid spiked into whole blood) x2 onto Whatman 903 Protein saver cards (Thermo Fisher Scientific, Waltham, MA).
  • the DBS were dried in ambient conditions for 3 hours and then tested as described below in the sample preparation and SHERLOCK reaction procedure.
  • P. falciparum and ten serum collected in serum separator tubes
  • P. vivax samples from deidentified symptomatic patients in the Dominican Republic were purchased from BocaBiolistics (Pompano Beach, FL). Samples had been previously characterized by Alere BinaxNOW Malaria RDT (Waltham, MA) and qualitative species-specific PCR (ARUP, Salt Lake City, USA). All clinical samples and human red blood cell aliquots used had been previously deidentified prior to purchase.
  • RPA primer, gRNA screen and construction conserved Plasmodium regions identified from the literature and publicly accessible databases (NCBI, Pf3k, and PlasmoDB) were used to generate target RPA primers and gRNA sequences. Alignments to ensure conservation of targets across available individual species’ genome assemblies as well as exclusivity between Plasmodium species were performed using MAFFT (43) and visualized with Jalview 2.11.1.0 (44). RPA primers were purchased from Integrated DNA Technologies (IDT, Coralville, Iowa).
  • the CRISPR gRNA was produced by in vitro transcription from synthetic DNA sequences purchased from IDT using the HiScribeTM T7 Quick High Yield RNA Synthesis kit (New England Biolabs, Ipswich, MA) and purified using the RNA Clean and Concentrator kit (Zymo Research, Irvine, MA).
  • a quenched fluorescent single-stranded DNA (ssDNA) reporter with a 5’ end labeled FAM group and a 3’ end attached to an Iowa Black® quencher (56-FAM/TTATT/3IABkFQ) was purchased from IDT (Coralville, IA).
  • RPA primer screens were conducted using 7.5 pl reaction volumes of RPA basic kit (TwistDx, Cambridge,
  • SUBSTITUTE SHEET (RULE 26) UK) spiked with unique primer sets to final concentrations as recommended per the manufacturer’s instructions: 14 mM magnesium acetate, 490 pM RPA primers each, and 0.6x rehydration buffer incubated at 40°C for 30 minutes.
  • Initial screen gRNAs were constructed for expected RPA amplicons of different sequence targets Collateral degradation of ssDNA reporter upon Casl2a activation was measured by mixing 2 pl of a RPA primer screen reaction into a 10 pl reaction volume with final concentrations of lOOnM Casl2a (New England Biolabs, Ipswich, MA), 200nM gRNA, IxNEB 2.1 buffer (New England Biolabs, Ipswich, MA), and 1 pM ssDNA reporter.
  • Sample preparation testing Using live intraerythrocytic P. falciparum spiked into whole blood as a simulated malaria sample, multiple sample preparation methods were tested. All sample preparation methods tested had a final volume of 20 pL with a final P. falciparum concentration of I fM or 602 copies/pL (various methods had different dilution steps and so initial spiked concentration varied) and were tested via rehydration of the one-pot lyophilized SHERLOCK P. falciparum pellet described below. Fluorescence was measured over 1 hour at 40°C using a BioTek NEO HTS plate reader with readings every 3 minutes (Ex: 485 nm; Em 535 nm).
  • Detergents at varying w/v% (SDS 0.5%, saponin 1%, Tween-20 1%, Triton-X 100 1%) were added to a 20 pL simulated whole blood sample along with lOOmM TCEP.
  • Two heating sample preparation protocols were tested: (1) dilution of simulated sample 1:4 in nuclease-free water followed by 10-minute 95°C incubation (1:4 dilution required to prevent solidification when diluting with water), and (2) addition of lOOmM TCEP into the diluted simulated sample prior to 10-minute 95°C incubation.
  • combinations of chelators and reducing agents added to 20 pL simulated samples at concentrations demonstrated in FIG. 3B were tested.
  • S-PREP sample preparation Inactivation (nucleases and inhibitors) of whole blood and serum samples was performed by dilution of sample in 1:3 ratio (12.5 pL sample: 37.5 pL S-PREP buffer); S-PREP buffer comprised Tris-EDTA buffer (Invitrogen, Carlsbad, CA) with 50mM DTT (Sigma Aldrich, St. Louis, MO) and 20% w/v Chelex®-100 (Bio-Rad, Hercules, CA). Samples were then heated to 95°C for 10 minutes.
  • Tris-EDTA buffer Invitrogen, Carlsbad, CA
  • DTT Sigma Aldrich, St. Louis, MO
  • Chelex®-100 Bio-Rad, Hercules, CA
  • a disposable biopsy punch VWR International, Radnor, PA
  • 50 pL of S-PREP buffer was added to the tube followed by 95°C heat inactivation for 10 minutes.
  • live intraerythrocytic P live intraerythrocytic P.
  • SHERLOCK reactions were prepared to 50 pL using 100 nM Casl2a, 200 nM gRNA, 0.8x NEB buffer 2.1, 430 nM of each RPA primer, 2 U/pL ProtoScript II reverse-transcriptase (NEB, Ipswich, USA), 0.6x RPA re hydration buffer, 14 mM MgOAc, 10 mM EGTA, and 1 pM FAM-Iowa Black® quenched ssDNA fluorescent reporter.
  • IpM fluorophore-biotin labeled ssDNA reporter 56- FAM/TTATT/3Bio, IDT, Coralville, IA was used instead of fluorophore-quencher reporter.
  • Reactions were prepared in 200 pL PCR-compatible tubes and a small opening was pierced in the cap with a 25-gauge x 5/8 (0.5 mm x 16 mm) BD PrecisionGlide Needle (Becton, Dickinson and Company, Franklin Lakes, NJ) to allow for sublimation during lyophilization.
  • Reaction tubes were placed in a chilled metallic tube rack and submerged for 1 minute in liquid nitrogen.
  • the snap frozen tubes and rack were wrapped in Kimwipes (Kimberly-Clark, Irving, TX) and three layers of aluminum foil. The entire bundle was then placed inside a sealed glass lyophilization chamber and connected to a freeze-drying machine (Labconco, Kansas City, MO).
  • Lyophilization was performed for 6 hours. Activation of reaction was performed by rehydration in 50 pL of sample prepared by S-PREP (12.5 pL of sample into 37.5 pL of buffer followed by 95°C incubation). Notably, for testing of simplified sample preparation methods, lyophilization reactions were scaled to a 20 pL sample input volume, so 20 pL SHERLOCK reactions were lyophilized and 20 pL of simulated sample prepared by tested preparation method were used for rehydration of reaction. Fluorescence was measured over 1-3 hours at 40°C using a BioTek NEO HTS plate reader with readings every 3 minutes (Ex: 485 nm; Em 535 nm).
  • SUBSTITUTE SHEET (RULE 26) SHERLOCK lyophilized pellet as described above for resuspension of reaction. Fluorescence was measured over 1 hour at 40°C using a BioTek NEO HTS plate reader with readings every 3 minutes (Ex: 485 nm; Em 535 nm).
  • the analytical limit of detection was defined as the lowest Plasmodium species concentration that was successfully detected with a probability of 95% or greater.
  • Calibration standards near the estimated LOD were prepared by serial dilutions of simulated samples described above to the following concentrations: 50 zM (0.03 copies/pL sample), 200 zM (0.12 copies/pL), 500 zM (0.3 copies/pL), 5 aM (3 copies/pL), 50 aM (30 copies/pL).
  • the LOD was evaluated by testing the calibration standard over three separate runs performed on different days with seven replicates for each concentration, for a total of 21 replicate results at each concentration level.
  • Plasmodium log concentration was examined using Probit regression analysis to estimate 95% LOD and 95% confidence intervals of each target (GraphPad 8.4.1, San Diego, USA). Lateral flow test line signal intensities were quantified to grayscale pixel values using Image J software (National Institutes of Health). Background-subtracted intensity was calculated from line scans that spanned the 1mm test line subtracted from background blank (white) area to normalize to ambient background grayscale value of the lateral flow strip.

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Abstract

Provided herein are compositions and methods for the preparation of biological samples for nucleic acid detection. The methods and compositions described permit the detection of target nucleic acid sequences without the need to isolate nucleic acids from the biological samples, and are well suited for use with isothermal nucleic acid amplification, and detection methods based on RNA-guided nuclease cleavage, among others.

Description

COMPOSITIONS AND METHODS FOR NUCLEIC ACID PREPARATION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. § 119(e) to U.S Provisional
Application No. 63/079,085, filed September 16, 2020, the contents of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The field of the invention relates to preparation of nucleic acids for use in amplification reactions and for point-of-care diagnostics.
BACKGROUND
[0003] Detection of submicroscopic malaria in asymptomatic individuals is needed for eradication of malaria and remains a diagnostic gap in resource-limited settings. Non-falciparum clinical diagnostics are a second gap, as these infections have a low parasite density and are commonly undetected.
[0004] Accessing sample nucleic acids in a field-applicable manner involves overcoming several challenges. Preparation requires lysing the red blood cell and parasite membrane (with the exception of the invasive merozoite form, all blood-stage parasites are intraerythrocytic), deactivating multiple inhibitory blood components, and importantly, appropriately deactivating nucleases.
SUMMARY
[0005] The compositions and methods described herein are based, in part, on the discovery of a nucleic acid preparation protocol that does not require the use of a nucleic acid extraction kit and for which the resulting nucleic acids can be added directly to isothermal nucleic acid amplification methods (e.g., recombinase polymerase amplification (RPA), among others) and/or probe cleavage-based detection reactions, e.g. SHERLOCK, among others) without a step of purifying or isolating the nucleic acids. This method was shown to eliminate false positives in a proof-of-concept ultrasensitive assay for malaria utilizing, in part, SHERLOCK detection methods.
[0006] One aspect provided herein relates to a composition for nucleic acid preparation, the composition comprising a reducing agent and a metal ion chelating resin in aqueous suspension.
[0007] In one embodiment of this aspect and all other aspects provided herein, the metal ion chelating resin comprises paired iminodiacetate ions.
[0008] In another embodiment of this aspect and all other aspects provided herein, the resin is present at a concentration of about 10% to 30% w/v.
[0009] In another embodiment of this aspect and all other aspects provided herein, the reducing agent is dithiothreitol (DTT).
SUBSTITUTE SHEET (RULE 26) [0010] In another embodiment of this aspect and all other aspects provided herein, the reducing agent is present at a concentration of 20-150 mM.
[0011] In another embodiment of this aspect and all other aspects provided herein, the resin comprises styrene divinylbenzidine copolymer.
[0012] In another embodiment of this aspect and all other aspects provided herein, the reducing agent is DTT in the range of 20-150 mM, and the resin is a styrene divinylbenzidine copolymer resin with paired iminidiacetate ions at a concentration of 10-30% w/v.
[0013] In another embodiment of this aspect and all other aspects provided herein, the reducing agent is 50 mM DTT, and the resin is present at a concentration of 20% w/v.
[0014] In another embodiment of this aspect and all other aspects provided herein, the composition releases nucleic acid from a biological sample upon heating, without need for a proteolytic enzyme.
[0015] In another embodiment of this aspect and all other aspects provided herein, the composition does not comprise proteinase K.
[0016] In another embodiment of this aspect and all other aspects provided herein, the biological sample comprises blood or a blood fraction, a nasopharyngeal swab, an oropharyngeal swab, sputum or saliva.
[0017] In another embodiment of this aspect and all other aspects provided herein, the blood fraction comprises erythrocytes.
[0018] Another aspect provided herein relates to a composition consisting essentially of an aqueous buffer, DTT and a metal ion chelating resin.
[0019] In one embodiment of this aspect and all other aspects provided herein, the composition consisting essentially of an aqueous buffer, DTT and a metal ion chelating resin is in an admixture with a biological sample.
[0020] In another embodiment of this aspect and all other aspects provided herein, the composition for nucleic acid preparation is present at about a 3: 1 ratio relative to biological sample by volume.
[0021] In another embodiment of this aspect and all other aspects provided herein, the biological sample comprises blood or a blood fraction, a nasopharyngeal swab, an oropharyngeal swab, sputum or saliva.
[0022] In another embodiment of this aspect and all other aspects provided herein, the blood fraction comprises erythrocytes.
[0023] In another embodiment of this aspect and all other aspects provided herein, the blood or blood fraction has previously been dried on a solid support.
[0024] In another embodiment of this aspect and all other aspects provided herein, the solid support is present in the admixture.
SUBSTITUTE SHEET (RULE 26) [0025] In another embodiment of this aspect and all other aspects provided herein, the composition or admixture thereof further comprises reagents sufficient to perform an isothermal nucleic acid amplification.
[0026] In another embodiment of this aspect and all other aspects provided herein, the composition or admixture thereof further comprises reagents sufficient to perform Specific High sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) detection of a target nucleic acid.
[0027] In another embodiment of this aspect and all other aspects provided herein, the composition substantially prevents target-independent cleavage of a SHERLOCK reporter nucleic acid in the presence of a biological sample or when in admixture with a biological sample.
[0028] Also provided herein, in another aspect, is a method of preparing a biological sample for nucleic acid analysis, the method comprising contacting a composition comprising a reducing agent and a metal ion chelating resin in aqueous suspension with the biological sample, and heating the resulting mixture to at least 80°C.
[0029] In one embodiment of this aspect and all other aspects provided herein, the ratio of the composition comprising a reducing agent and a metal ion chelating resin in aqueous suspension to biological sample is about 3: 1.
[0030] In another embodiment of this aspect and all other aspects provided herein, heating is performed for 2-20 minutes.
[0031] In another embodiment of this aspect and all other aspects provided herein, heating is performed at about 95°C for about 10 minutes.
[0032] In another embodiment of this aspect and all other aspects provided herein, the biological sample comprises: (i) blood or a fraction thereof comprising erythrocytes, (ii) a nasopharyngeal swab, (iii) an oropharyngeal swab, (iv) sputum, or (v) saliva.
[0033] In another embodiment of this aspect and all other aspects provided herein, the biological sample had previously been dried on a solid support.
[0034] In another embodiment of this aspect and all other aspects provided herein, the solid support is included in the mixture.
[0035] In another embodiment of this aspect and all other aspects provided herein, the biological sample comprises or is suspected of comprising an intracellular parasite, bacterium, or a virus.
[0036] In another embodiment of this aspect and all other aspects provided herein, the intracellular parasite comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Babesia sp. Leishmaniasis spp. Toxoplasmosis spp. or filarial nematodes.
[0037] In another embodiment of this aspect and all other aspects provided herein, the virus comprises Dengue virus, Zika virus, Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoV 1), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Heptatis E,
SUBSTITUTE SHEET (RULE 26) Herpes virus, Varicella virus, Cytomegalovirus, Epstein-Barr virus, Human herpesvirus 6, Human herpesvirusS, adenovirus, influenza, parainfluenza, respiratory syncytial virus, or Chikungunya virus. [0038] In another embodiment of this aspect and all other aspects provided herein, the bacterium comprises a gram negative bacterium, a gram positive bacterium or an intracellular bacterium.
[0039] In another embodiment of this aspect and all other aspects provided herein, heating the mixture promotes red blood cell lysis and lysis of intracellular parasite or virus present and releases nucleic acids from the parasite or virus if present, and wherein the resin chelates multivalent metal ions, thereby inhibiting nuclease degradation of parasite and/or viral nucleic acid.
[0040] In another embodiment of this aspect and all other aspects provided herein, the method further comprises, after the heating step, contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids.
[0041] In another embodiment of this aspect and all other aspects provided herein, the method further comprises incubating the mixture under conditions and for a time sufficient to amplify the one or more target nucleic acids if present.
[0042] In another embodiment of this aspect and all other aspects provided herein, the isothermal amplification is a recombinase polymerase amplification (RPA) reaction.
[0043] In another embodiment of this aspect and all other aspects provided herein, no additional sample processing or liquid transfer steps are required to permit isothermal amplification of one or more target nucleic acids.
[0044] In another embodiment of this aspect and all other aspects provided herein, the reagents sufficient for isothermal amplification are lyophilized prior to the step of contacting the mixture with the reagents.
[0045] In another embodiment of this aspect and all other aspects provided herein, further comprising, after the heating step, contacting the mixture with reagents sufficient for SHERLOCK detection of one or more target nucleic acids.
[0046] In another embodiment of this aspect and all other aspects provided herein, further comprising incubating the mixture under conditions and for a time sufficient to generate a SHERLOCK detection signal for one or more target nucleic acids if present.
[0047] In another embodiment of this aspect and all other aspects provided herein, the reagents sufficient for SHERLOCK detection are lyophilized prior to the step of contacting the mixture with the reagents.
[0048] In another embodiment of this aspect and all other aspects provided herein, no additional sample processing or liquid transfer steps are required to permit SHERLOCK detection of one or more target nucleic acids.
[0049] In another embodiment of this aspect and all other aspects provided herein, the method further comprises, after the heating step, contacting the mixture with reagents sufficient for isothermal
SUBSTITUTE SHEET (RULE 26) amplification of one or more target nucleic acids and reagents sufficient for SHERLOCK detection of one or more target nucleic acids.
[0050] In another embodiment of this aspect and all other aspects provided herein, the reagents sufficient for isothermal amplification and the reagents sufficient for SHERLOCK detection are lyophilized prior to the contacting step, wherein the contacting step reconstitutes the lyophilized reagents and permits amplification and SHERLOCK detection of one or more target nucleic acid molecules if present.
[0051] In another embodiment of this aspect and all other aspects provided herein, the method further comprises incubating the mixture under conditions and for a time sufficient to amplify one or more target nucleic acids and to generate a SHERLOCK detection signal for one or more target nucleic acids if present.
[0052] Another aspect provided herein relates to a method of amplifying one or more target nucleic acids in a biological sample comprising nucleic acids, the method comprising: (i) contacting a composition comprising a reducing agent and a metal ion chelating resin in aqueous suspension with the biological sample; (ii) heating the mixture resulting from step (i) to at least 80°C for a time sufficient to release nucleic acids in the biological sample; (iii) after step (ii), contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids; (iv) incubating the mixture of step (iii) under conditions and for a time sufficient to generate an isothermal amplification product for one or more target nucleic acids.
[0053] In another embodiment of this aspect and all other aspects provided herein, no additional sample processing or liquid transfer steps are required to generate an amplification product for one or more target nucleic acid.
[0054] In another embodiment of this aspect and all other aspects provided herein, steps (i)-
(iv) of the method of amplifying one or more target nucleic acids are performed in the same reaction or sample container.
[0055] Another aspect provided herein relates to a method of detecting one or more target nucleic acids in a biological sample, the method comprising: (i) contacting a composition of any one of claims 1-13 with the biological sample; (ii) heating the mixture resulting from step (i) to at least 80°C for a time sufficient to release nucleic acids in the biological sample; (iii) after step (ii), contacting the mixture with reagents sufficient for isothermal amplification and SHERLOCK detection of one or more target nucleic acids; and (iv) incubating the mixture of step (iii) under conditions and for a time sufficient to permit isothermal amplification and production of a SHERLOCK detection signal for one or more target nucleic acids present in the sample.
[0056] In another embodiment of this aspect and all other aspects provided herein, no additional sample processing or liquid transfer steps are required to generate SHERLOCK detection signal for one or more target nucleic acids present in the sample.
SUBSTITUTE SHEET (RULE 26) [0057] In another embodiment of this aspect and all other aspects provided herein, steps (i)-
(iv) are performed in the same reaction or sample container.
[0058] In another aspect, described herein is a kit for nucleic acid preparation and/or detection, the kit comprising a reducing agent and a metal ion chelating resin in aqueous suspension, and packaging materials therefor.
[0059] In one embodiment, the metal ion chelating resin comprises paired iminodiacetate ions.
[0060] In another embodiment of this or any other aspect, the reducing agent is dithiothreitol
(DTT).
[0061] In another embodiment of this or any other aspect, the kit does not contain proteinase
K
[0062] In another embodiment of this or any other aspect, the reducing agent is present at a concentration of 20-150 mM.
[0063] In another embodiment of this or any other aspect, the resin comprises styrene divinylbenzidine copolymer.
[0064] In another embodiment of this or any other aspect, the kit further comprises reagents sufficient for an isothermal nucleic acid amplification reaction.
[0065] In another embodiment of this or any other aspect, the reagents sufficient for an isothermal nucleic acid amplification reaction are lyophilized.
[0066] In another embodiment of this or any other aspect, the kit further comprises reagents sufficient for a SHERLOCK detection reaction.
[0067] In another embodiment of this or any other aspect, the reagents sufficient for a SHERLOCK detection reaction are lyophilized.
[0068] In another embodiment of this or any other aspect, the reagents sufficient for an isothermal nucleic acid amplification reaction and reagents sufficient for a SHERLOCK detection reaction are lyophilized in one composition.
BRIEF DESCRIPTION OF THE FIGURES
[0069] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0070] FIG. 1 SHERLOCK diagnostic workflow: (1) human serum, whole blood, or dried blood spot samples undergo a 10 minute S-PREP protocol where the sample is suspended in 20% w/v Chelex®-100 in TE buffer with 50mM DTT and incubated at 95°C for 10 minutes; and (2) transfer of suspended sample to lyophilized SHERLOCK pellet followed by incubation at 40°C for 60 minutes prior to endpoint analysis via fluorescence or lateral flow strip.
SUBSTITUTE SHEET (RULE 26) [0071] FIG. 2 Schematic of one-pot SHERLOCK assay. Reverse-transcriptase recombinase polymerase amplification (RT-RPA) amplifies Plasmodium species target sequences and occurs in parallel with programmed Casl2a detection, resulting in cleavage of target sequences and collateral cleavage of spiked fluorophore -labeled ssDNA reporter detectable by fluorescent measurement or lateral flow readout.
[0072] FIGs. 3A-3B Sample preparation methods tested with SHERLOCK P. falciparum assay using simulated malaria samples of live intraerythrocytic P. falciparum spiked into whole blood at IfM (602 parasites/ LIL) concentration. FIG. 3A, Detergents and heating methods assessed for SHERLOCK compatibility. FIG. 3B, Combinations of chelating and reducing agents tested for optimization of chemical deactivation of nucleases and inhibitors. Asterisks indicate significant differences from untreated simulated whole blood sample assessed by Student’s two-tailed t-test. Bars: mean +/- S.D. of three technical replicates. *p<0.05, p<0.01, ***<0.001, ****p<0.0001.
[0073] FIGs. 4A-4E Specificity of SHERLOCK assays. FIG. 4A, Using P. falciparum assay and DTT/EGTA/95°C sample preparation, P. falciparum and P. vivax patient serum in SHERLOCK diagnostic display similar fluorescent kinetics that are eliminated when an aliquot of the same P. vivax serum undergoes nucleic acid extraction via commercial kit. FIG. 4B, Using P. vivax assay and DTT/EGTA/95°C sample preparation, P. falciparum serum demonstrates false positive signal that is eliminated when an aliquot of the same P. falciparum serum undergoes nucleic acid extraction via commercial kit. FIG. 4C, False positive P. vivax signal is eliminated with S-PREP. FIG. 4D, False positive P. falciparum signal is eliminated with S-PREP. FIG. 4E, Performance of SHERLOCK diagnostic on clinical patient serum and whole blood samples prepared with S-PREP: five P. falciparum samples (four serum, one whole blood), 10 P. vivax serum samples, and five serum samples from healthy controls.
[0074] FIGs. 5A-5D SHERLOCK Performance. FIG. 5A, Sensitivity of SHERLOCK diagnostic for detection of Plasmodium species by comparison of probit regression curves obtained from 21 replicates of five dilutions. FIG. 5B, Fluorescence kinetics of P. falciparum SHERLOCK assay at lOOaM (60 parasites/pL) and 2aM (1 parasite/pL) concentrations. FIG. 5C, Specificity of SHERLOCK diagnostic using at 10 fM (6020 parasites/pL) concentrations of parasite. FIG. 5D, Comparison of performance between simulated dried blood spot and whole blood samples. All experiments used simulated whole blood samples. Asterisks indicate p-value <0.0001 for Student’s t- test between fluorescent output of sample type versus no-template control.
[0075] FIGs. 6A-6E SHERLOCK Lateral Flow Assay Performance. FIGs. 6A-6D, Detection of 1 fM (-602 parasites/pL), 100 aM (60 parasites/pL), 50 aM (30 parasites/pL), and 2 aM (1 parasite/pL) concentrations of P. falciparum, P. vivax, P. ovale, and P. malariae respectively, and comparison to 1 femtomolar concentrations of off-target Plasmodium species for each assay. FIG. 6E, Background-subtracted grayscale intensity averages of test line for 3 separate flow tests +/- standard deviation. All experiments used simulated whole blood samples.
SUBSTITUTE SHEET (RULE 26) DETAILED DESCRIPTION
[0076] Provided herein are compositions and methods useful for preparing nucleic acids obtained from a biological sample, such as blood, serum or saliva, for assays utilizing nucleic acids as a starting material. The compositions and methods provided herein permit conventional multi-step nucleic acid protocols to be performed in a single reaction or ‘pot’ and do not require a prior step of purifying or isolating the nucleic acids. In some embodiments, this nucleic acid preparation protocol is used in conjunction with an isothermal amplification method (e g., RPA, LAMP, among others). In other embodiments, detection based upon probe cleavage permits very sensitive detection of target nucleic acids. In yet further embodiments, isothermal amplification, such as RPA, among others, is combined with detection based, for example, on SHERLOCK to produce an ultra-sensitive diagnostic for, e.g., malaria that can be used as a point-of-care diagnostic.
Definitions
[0077] The term "biological sample" as used herein refers to a cell or population of cells or a quantity of tissue or fluid from a subject. Often, a "biological sample" will contain cells from an animal or subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine, that can be used to prepare nucleic acids using the methods and compositions described herein. Biological samples include, but are not limited to, whole blood, plasma, serum, saliva, sputum, nasopharyngeal swab, oropharyngeal swab, urine, cell culture, tissue biopsies, scrapes (e.g., buccal scrapes), or cerebrospinal fluid. When a biological sample is intended for point-of-care diagnostics, it is preferred that the sample be easily and non-invasibly obtained, for example, blood, serum, saliva, sputum, nasopharyngeal swab, oropharyngeal swab, or urine. A biological sample or tissue sample can refer to a sample of tissue or fluid isolated from an individual including, but not limited to, blood, plasma, serum, tumor biopsy, urine, stool, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, cells (including, but not limited to, blood cells), tumors, organs, and also samples of in vitro cell culture constituent.
[0078] As used herein, the term “nucleic acid sample preparation” refers to a method of preparing nucleic acids such that they can be used in a downstream reaction, such as an amplification and/or detection reaction, without the need for additional isolation steps. Nucleic acid sample preparation can include inactivation of endogenous enzymes from the biological sample, nicking or destabilizing DNA, or releasing nucleic acids from the biological sample such that they are available for binding with a primer, etc. In certain embodiments, the nucleic acid sample preparation method described herein does not include a proteinase, such as proteinase K, or a collagenase enzyme to disrupt tissue or cells.
SUBSTITUTE SHEET (RULE 26) [0079] The term “isothermal amplification reaction” refers to a nucleic acid amplification method that does not require thermal cycling to permit amplification. The term distinguishes amplification methods, such as the polymerase chain reaction (PCR), that use cycles of thermal denaturation of nucleic acid by incubation at a temperature above the melting temperature (Tm), annealing of nucleic acid primers by incubation at another temperature generally below the Tm, and most often, incubation at another temperature optimal for a polymerase enzyme to extend the annealed primers. An isothermal amplification reaction, by contrast, can be performed at a single temperature, and generally relies upon, for example, DNA binding and strand-displacing enzyme factors to permit target nucleic acid amplification. In one embodiment, the temperature of the isothermal amplification reaction does not deviate by more than 10° C in either direction (e.g., deviates by less than 5° C, by less than 2° C, by less than 1°C) or is performed at a single temperature with no temperature deviation required for amplification
[0080] As used herein, the term “solid support” refers to beads, membranes, filters, matrices, columns, chips, arrays etc. that can bind to e.g., nucleic acids, antibodies etc. Exemplary solid supports include, but are not limited to, magnetic beads (e g., micron-sized magnetic beads), Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix. In some embodiments, a solid support is one on which a biological sample as described herein is spotted on and dried to preserve the sample for analysis at a later time. In such embodiments, the solid support can include, for example, paper, nitrocellulose or nylon sheets and the like.
[0081] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.
[0082] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
[0083] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[0084] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[0085] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.
Nucleic Acids
SUBSTITUTE SHEET (RULE 26) [0086] Nucleic acids that can be prepared using the methods and compositions described herein can include RNA, DNA, mRNA (messenger RNA), mtRNA (mitochondrial RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), nRNA (nuclear RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNA (Small Cajal Body specific RNA), microRNA, chromosomal DNA, nDNA (nuclear DNA), snDNA (small nuclear DNA), viral RNA, dsDNA (double-stranded DNA), ssDNA (single-stranded DNA), as well as cDNA (complementary DNA), ribozyme and riboswitch RNAs, LNA (locked nucleic acid), siRNA (short interfering RNA), plasmid DNA, cosmid DNA, or the like. A nucleic acid can be a nucleic acid of a pathogen, such as from a virus (e.g., RNA, DNA), a parasite (including intracellular parasites), or a bacterium. When nucleic acids are to be used in downstream diagnostics for a pathogen (i.e., a parasite, bacterium or virus), the nucleic acids to be prepared are typically pathogen DNA, RNA, cDNA or mRNA. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
[0087] In some embodiments, the nucleic acids prepared using the methods and compositions described herein are used as templates or targets for nucleic acid amplification, particularly those using isothermal nucleic acid amplification methods. Target nucleic acids can also be referred to as target sequences, trigger nucleic acids, target polynucleotides, and/or target polynucleotide sequences, and can include double -stranded and single-stranded nucleic acid molecules. Where the target nucleic acid is double stranded, the target nucleic acid generally includes a first strand and a second strand. A first strand and a second strand may be referred to as a forward strand and a reverse strand and generally are complementary to each other. Where the target nucleic acid is single stranded, a complementary strand may be generated, for example by polymerization and/or reverse transcription, rendering the target nucleic acid double stranded and having a first/forward strand and a second/reverse strand.
[0088] A target nucleic acid sequence can refer to either the sense or antisense strand of a nucleic acid sequence, and can also refer to sequences as they exist on target nucleic acids, amplified copies, or amplification products, of the original target sequence. A target sequence can be a subsequence within a larger polynucleotide. For example, a target sequence can be a short sequence (e.g., 20 to 50 bases) within a nucleic acid fragment, a viral genome, a bacterial genome or a genome of a parasite, that is targeted for amplification. In some embodiments, a target sequence can refer to a sequence in a target nucleic acid that is complementary to an oligonucleotide (e.g., primer) used for amplifying a nucleic acid. Thus, a target sequence can refer to the entire sequence targeted for amplification or can refer to a subsequence in the target nucleic acid where an oligonucleotide binds. [0089] Samples from which the nucleic acids are prepared using the methods and compositions described herein can be obtained from any suitable biological specimen or sample, and often are isolated from a sample obtained from a subject. A subject can be any living or non-living organism, including but not limited to a human, a non-human animal, a plant, a bacterium, a fungus, a virus, a parasite, or a
SUBSTITUTE SHEET (RULE 26) protist. Any human or non-human animal can be selected, including but not limited to a mammal, reptile, avian, amphibian, fish, ungulate, ruminant, bovine (e.g., cattle), equine (e.g., horse), caprine and ovine (e g., sheep, goat), swine (e.g., pig), camelid (e g., camel, llama, alpaca), monkey, ape (e g., gorilla, chimpanzee), ursid (e.g, bear), poultry, dog, cat, mouse, rat, fish, dolphin, whale and shark. A subject can be a male or female, and a subject can be any age (e.g., an embryo, a fetus, infant, child, adult).
Biological Samples
[0090] A biological sample can be any specimen that is isolated or obtained from a subject or part thereof. Non-limiting examples of specimens include fluid or tissue from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, or the like), a nasopharyngeal swab sample, an oropharyngeal swab sample, nasal mucus, sputum, saliva, urine, umbilical cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), biopsy sample, cells (e.g., blood cells) or parts thereof (e.g., mitochondrial, nucleus, extracts, or the like), washings of female reproductive tract, feces, prostate fluid, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, hard tissues (e.g., liver, spleen, kidney, lung, or ovary), the like or combinations thereof. The term “blood” encompasses whole blood, blood product or any fraction of blood, such as serum, plasma, buffy coat, or the like as conventionally defined. Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants. Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. Fluid or tissue samples often are collected in accordance with standard protocols hospitals or clinics generally follow. For blood, an appropriate amount of peripheral blood (e.g., between 3-40 milliliters) often is collected and can be stored according to standard procedures prior to or after preparation.
[0091] A biological sample can include samples containing parasites, viruses, bacteria, spores, cells, nucleic acid from prokaryotes or eukaryotes, or any free nucleic acid. A sample can be isolated from any material suspected of containing a target sequence, such as from a subject described above. In certain instances, a target sequence can be present in air, plant, soil, or other materials suspected of containing biological organisms of interest.
Nucleic Acid Sample Prep (also referred to as S-PREP herein)
[0092] Provided herein are methods and compositions for the preparation of nucleic acids using reagents that are compatible with downstream nucleic acid amplification reactions. Nucleic acids prepared using the methods and compositions described herein do not require isolation prior to amplification; that is, the prepped nucleic acid sample can be subjected to an isothermal or other
SUBSTITUTE SHEET (RULE 26) amplification reaction without need for additional processing or isolation steps, or can be added directly to e g., an isothermal amplification reaction. The methods and compositions for nucleic acid preparation can include a step of inactivation of nucleases and other inhibitors present in the biological sample A ‘dry’ sample, such as a swab or dried blood, is specifically contemplated for processing and use with the methods and compositions described herein.
[0093] The compositions and methods for use in preparing nucleic acids as described herein comprise one or more reducing agents. In one embodiment, the reducing agent is dithiothreitol (DTT). Other reducing agents contemplated for use as described herein include, but are not limited to, dimethyl sulfoxide (DMSO), tris(2-carboxyethyl)phosphine, tertiary butyl alcohol, beta-mercaptoethanol, among others. In some embodiments, the reducing agent can introduce nicks into the nucleic acids to aid in downstream methods, such as isothermal amplification reactions. In some embodiments, the concentration of DTT or other reducing agent used in nucleic acid sample preparation is in the range of l-100mM, 1-75 mM, l-50mM, 25-100mM, 50-100mM, 25-75mM, 25-70mM, 25-65mM, 25-60mM, 25-55mM, 25- 50mM, 40-60nM, 40-50nM, 50-60nM, 40-65mM, 40-70mM, 40-75mM, or 40-100mM, inclusive. In certain embodiments, the concentration of DTT is 50mM.
[0094] The compositions and methods for use in preparing nucleic acids as described herein comprise a metal ion chelating resin. Such resins typically comprise a polymer matrix and a chelating group. A polymer matrix can include synthetic and/or natural organic polymers. Synthetic organic polymers, which are usually cross-linked polymers such as styrene -di vinylbenzene copolymer, are widely used as matrices. Chitin/chitosan, cellulose, and agarose as natural organic polymers are also sometimes utilized. As chelating groups, various ligands, which have nitrogen (N), oxygen (0), and/or sulfur (S) donor atoms, are immobilized on the matrix. Other types of chelating resins are prepared by copolymerizing two types of ligands or ligand and other compounds such as formaldehyde. In one embodiment, the metal ion chelating resin used herein comprises iminodiacetate ions and/or comprises styrene divinylbenzidine copolymer. In some embodiments, the metal ion chelating resin is a cation binding ion-exchange resin. Exemplary ion-exchange resins for use in the methods and compositions described herein include Chelex™ resin (BioRad, Hercules, CA), Dionex™ (ThermoFisher Scientific, Waltham, MA), Dowex™ (Millipore Sigma, Burlington, MA), sodium polystyrene sulfonate, colestipol, cholestyramine, sodium polystyrene sulfonate, iminodiacetic acid-based resins, and thiourea-based resins, among others. In some embodiments, the metal ion chelating resin comprises cross-linked polystyrene or divinylbenzidine. Chelating resins can be obtained commercially from e.g., BioRad, Sigma Aldrich, Mitsubishi Chemical, Purolite, and the like.
[0095] The concentration/amount of metal ion chelating resin for use with the methods described herein can vary depending on the resin but should not be in such large concentrations that would interfere with downstream enzymatic reactions. In some embodiments, the concentration of the metal ion chelating resin (e.g., a resin comprising paired iminodiacetate ions; Chelex™) is in the range of 5-50% w/v, for example, 5-40%w/v, 5-30%w/v, 5-25%w/v, 5-20%w/v, 5-10%w/v, 10-20%w/v, 10-
SUBSTITUTE SHEET (RULE 26) 30%w/v, 10-40%w/v, 20-30%w/v, 25-30%w/v, 25-35%w/v, or any integer therebetween (fore.g., 10%, 20%, or 30%).
[0096] The nucleic acid sample preparation methods can be performed at a temperature of at least 80°C, for example, at least 85°C, at least 90°C, at least 92°C, at least 95°C, at least 100°C, at least 105°C, or higher. In some embodiments, the method of nucleic acid preparation is performed at or includes incubation, e.g, at or about 80°C, at or about 81°C, at or about 82°C, at or about 83°C, at or about 84°C, at or about 85 °C, at or about 86°C, at or about 87°C, at or about 88°C, at or about 89°C, at or about 90°C, at or about 91°C, at or about 92°C, at or about 93°C, at or about 94°C, at or about 95°C, at or about 96°C, at or about 97°C, at or about 98°C, at or about 99°C, at or about 100°C, at or about 101°C, at or about 102°C, at or about 103°C, at or about 104°C, or at or about 105°C. In some embodiments, the method of nucleic acid preparation is performed at or includes incubation at about 80°C to about 100°C, e.g., about 80°C to about 95°C, about 80°C to about 90°C, about 80°C to about 85°C, about 85°C to about 95°C, about 85°C to about 100°C, about 90°C to about 100°C or about 95°C to about 100°C.
[0097] The nucleic acid sample preparation can be performed for any desired time or until sufficient or complete inactivation of endogenous enzymes and cell lysis or liberation of nucleic acid is achieved. In some embodiments, the method of nucleic acid sample preparation is performed for at least 1 min, at least 2 min, at least 3 min, at least 4 min, at least 5 min, at least 6 min, at least 7 min, at least 8 min, at least 9 min, at least 10 min, at least 11 min, at least 12 min, at least 13 min, at least 14 min, at least 15 min, at least 16 min, at least 17 min, at least 18 min, at least 19 min, at least 20 min or more. In some embodiments, the method is performed for a time within the range of 1-20 min, 1-15 min, 1- 10 min, 1-5 min, 1-2 min, 15-20 min, 10-20 min, 5-20 min, 5-15 min, 5-10 min, 10-15 min, etc.
[0098] It will be appreciated by those of skill in the art that a lower temperature can require a longer incubation time to achieve the same degree of endogenous enzyme inactivation and nucleic acid liberation. For example, incubation at 80°C performed for 20 min can be as effective as or used in place of incubation at 95° for 10 min. It is within the skill set of one of ordinary skill in the art to optimize the temperature and reaction time to achieve suitable nucleic acid preparation as described herein.
[0099] Typically, the nucleic acid sample preparation will be performed in an enclosed reaction vessel (e.g., test tube, Eppendorf tube etc) to prevent evaporation, concentration of reagents, contamination by nucleic acid degrading enzymes (e.g., RNAses), and for maintenance of reaction temperature etc. It is specifically contemplated that the nucleic acid sample preparation step is performed in a vessel that is compatible with an isothermal amplification method, such that the reaction mixture necessary for amplification can be added directly to the vessel containing the prepared nucleic acids (i.e, a ‘one-pot’ reaction system).
Isothermal Nucleic Acid Amplification Methods
SUBSTITUTE SHEET (RULE 26) [0100] The nucleic acids prepared using the methods and compositions described herein can be used with essentially any isothermal nucleic acid amplification method. Exemplary isothermal nucleic acid amplification methods include, but are not limited to, Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), recombinase polymerase amplification (RPA), Loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), reverse transcription recombinase polymerase amplification (RT-RPA), reverse transcription Loop-mediated isothermal amplification (RT-LAMP), reverse transcription nucleic acid sequence-based amplification (RT-NASBA), transcription mediated amplification (TMA), helicase dependent amplification (HD A), multiple displacement amplification (MDA), strand displacement amplification (SDA), rolling circle amplification (RCA), single primer isothermal amplification (SPIA), restriction aided rolling circle amplification, and nicking enzyme amplification reaction (NEAR).
[0101] Nucleic acids prepared using the methods and compositions described herein can be used in isothermal amplification reactions with or without modifying the nucleic acid. Optional modifications can include, for example, denaturation, digestion, nicking, unwinding, incorporation and/or ligation of heterogeneous sequences, addition of epigenetic modifications, addition of labels (e.g., radiolabels such as 32P, 33P, "'I. or 35S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, fluorochromes), and the like.
[0102] As used herein, the terms “amplify”, “amplification”, “amplification reaction”, or
“amplifying” refer to any in vitro process for multiplying the copies of a target nucleic acid using an isothermal amplification method. Amplification can refer to an “exponential” increase in target nucleic acid. However, “amplifying” can also refer to linear increases in the numbers of a target nucleic acid, but is different than a one-time, single primer extension step.
[0103] Components of an isothermal amplification reaction can include, for example, one or more primers (e.g., individual primers, primer pairs, primer sets, oligonucleotides, multiple primer sets for multiplex amplification, and the like), nucleic acid target(s) or templates (e.g., target nucleic acid from a sample), one or more polymerases, nucleotides (e.g., dNTPs and the like), and a suitable buffer (e.g., a buffer comprising a detergent, a reducing agent, monovalent ions, and divalent ions as appropriate). An amplification reaction can further include a reverse transcriptase, in some embodiments. An amplification reaction can further include one or more detection agents, including but not limited to a probe that generates a signal when cleaved by an enzyme activated in a targetdependent manner.
[0104] Additional components can be used in a typical isothermal amplification reaction including, but not limited to components and/or common additives such as salts, buffers, detergents, ions, oils, proteins, polymers and the like. In some embodiments, components of an amplification reaction can include non-enzymatic components and enzymatic components. Non-enzymatic components can include, for example, primers, nucleotides, buffers, salts, reducing agents, detergents,
SUBSTITUTE SHEET (RULE 26) and ions; and generally do not include proteins (e.g., nucleic acid binding proteins), enzymes, or proteins having enzymatic activity such as, for example, polymerases, reverse transcriptases, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases and the like. In some embodiments, an enzymatic component can comprise a polymerase, either in the presence or absence of a reverse transcriptase. Accordingly, polymerase enzymatic components are distinguished from other proteins (e.g., nucleic acid binding proteins and/or proteins having other enzymatic activities) such as, for example, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases, and the like. Typically, the methods and compositions described herein for the preparation of nucleic acids will inactivate endogenous enzymes prior to initiation of an isothermal amplification reaction.
[0105] Essential co-factors of isothermal amplification reactions are known to those of skill in the art and are dependent on the enzyme(s) used. They can be organic or inorganic chemical compounds. Inorganic chemical compounds, for example, can be selected from the group comprising metal ions, e.g., Mg, Mn, Ca, Fe, Cu and Ni. Organic co-factors can include vitamins, proteins, biotin, nicotinamide adenine dinucleotide, and nucleotides, e.g. ATP.
[0106] Isothermal amplification reactions can be conducted at a range of temperatures, depending upon the exact reaction chosen and the enzyme (s) or factors used in the amplification. Some isothermal amplification reactions can proceed at room temperature. More often, an elevated temperature, e.g., around 37 degrees Celsius or higher, e.g., 37 degrees Celsius to about 75 degrees Celsius, e.g., about 38 degrees Celsius, about 39 degrees Celsius, about 40 degrees Celsius, about 41 degrees Celsius, about 42 degrees Celsius, about 43 degrees Celsius, about 44 degrees Celsius, about 45 degrees Celsius, about 46 degrees Celsius, about 47 degrees Celsius, about 48 degrees Celsius, about 49 degrees Celsius, about 50 degrees Celsius, about 51 degrees Celsius, about 52 degrees Celsius, about 53 degrees Celsius, about 54 degrees Celsius, about 55 degrees Celsius, about 56 degrees Celsius, about 57 degrees Celsius, about 58 degrees Celsius, about 59 degrees Celsius, about 60 degrees Celsius, about 61 degrees Celsius, about 62 degrees Celsius, about 63 degrees Celsius, about 64 degrees Celsius, about 65 degrees Celsius, about 66 degrees Celsius, about 67 degrees Celsius, about 68 degrees Celsius, about 69 degrees Celsius, about 70 degrees Celsius, about 71 degrees Celsius, about 72 degrees Celsius, about 73 degrees Celsius, about 74 degrees Celsius, or about 75 degrees Celsius.
[0107] The isothermal amplification methods used herein can be conducted over a certain length of time and will typically be conducted until a detectable nucleic acid amplification product is generated. A nucleic acid amplification product can be detected by any suitable detection process and/or a detection process compatible with isothermal amplification methods. In some embodiments, an amplification process is conducted over a length of time within about 2 hours or less, 2.5 hours or less, 60 minutes or less, for example 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less or 5 minutes or less. In some embodiments, the amplification process is conducted for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes,
SUBSTITUTE SHEET (RULE 26) about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes. In some embodiments, an amplification process is conducted over a length of time within about 10 minutes or less.
[0108] In some embodiments, the nucleic acid targets are amplified without exposure to agents or conditions that denature or destabilize nucleic acids in the preparation, including, but not limited to thermal conditions (e.g., high temperatures), pH conditions (e.g., high or low pH), chemical agents (e.g., formamide, urea, dimethyl sulfoxide (DMSO), betaine sodium hydroxide, hydrochloric acid), proteins (e.g., enzymatic agents, such as helicases), and the like.
[0109] In some embodiments, it is desirable to design an isothermal amplification reaction to generate a short amplification product, for example, for rapid detection of a target sequence. In such embodiments, the nucleic acid amplification product can be up to 50 bases in length (e.g., about 15 to about 40 bases long or 15 bases long, 16 bases long, 17 bases long, 18 bases long, 19 bases long, 20 bases long, 21 bases long, 22 bases long, 23 bases long, 24 bases long, 25 bases long, 26 bases long, 27 bases long, 28 bases long, 29 bases long, 30 bases long, 31 bases long, 32 bases long, 33 bases long, 34 bases long, 35 bases long, 36 bases long, 37 bases long, 38 bases long, 39 bases long, or 40 bases long. In some embodiments, an amplification product is about 20 to about 40 bases long. In some embodiments, an amplification product is about 20 to about 30 bases long.
[0110] The nucleic acids generated using the methods and compositions described herein are contemplated for use in a multiplex amplification format (i.e., amplification or more than one target sequence).
[0111] Although the nucleic acids prepared as described herein are not considered ideal for conventional nucleic acid amplification methods using cycling temperatures (e.g., PCR), it is thought that this is due to a sensitivity of the PCR thermocycling enzymes to divalent metal ion concentration. Where the compositions and methods described herein chelate divalent metal ions, e.g., to limit targetindependent nuclease cleavage of detection probes, it can be exacting to achieve, for example, a magnesium ion concentration optimal for PCR while also limiting target-independent nuclease cleavage. Nonetheless, it is also specifically contemplated that the nucleic acid preparation methods and compositions described herein can be used in thermocycling nucleic acid amplification methods when a thermostable enzyme that maintains activity or specificity at reduced divalent metal cation concentrations is used.
[0112] Primers for isothermal amplification: A primer is generally characterized as an oligonucleotide that includes a nucleotide sequence capable of hybridizing or annealing to a target nucleic acid, at or near (e g., adjacent to) a specific region of interest (i.e., target sequence). Primers can allow for specific determination of a target nucleic acid nucleotide sequence or detection of the target nucleic acid (e.g., presence or absence of a sequence), or feature thereof, for example. A primer can be naturally occurring or synthetic. The term “specific,” or “specificity”, generally refers to the binding or
SUBSTITUTE SHEET (RULE 26) hybridization of one molecule to another molecule, such as a primer for a target polynucleotide. That is, specific or specificity refers to the recognition, contact, and formation of a stable complex between two molecules, as compared to substantially less recognition, contact, or complex formation of either of those two molecules with other molecules. The term “anneal” or “hybridize” generally refers to the formation of a stable base-paired nucleic acid complex, e.g., via hydrogen bonding, between two nucleic acid molecules or, where relevant, between complementary portions of a single nucleic acid molecule. The terms primer, oligo, or oligonucleotide can be used interchangeably herein, when referring to primers.
[0113] A primer can be designed and synthesized using suitable processes, and can be of any length suitable for hybridizing to a target sequence and permitting extension for an amplification process described herein. Primers are generally designed according to a sequence in a target nucleic acid. A primer in some embodiments can be about 5 bases in length to about 30 bases in length. For example, a primer can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases in length. In some embodiments, a primer is less than 28 bases in length. In some embodiments, a primer is about 8 to about 16 bases in length. In some embodiments, a primer is about 10 to about 12 bases in length. A primer can be composed of naturally occurring and/or non-naturally occurring nucleotides (e g., labeled nucleotides), or a mixture thereof. Primers suitable for use with methods described herein can be synthesized and labeled using any suitable technique. For example, primers can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts., 22: 1859-1862, 1981, using an automated synthesizer, as described in Needham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168, 1984. Purification of primers can be effected, for example, by native acrylamide gel electrophoresis or by anion-exchange high-performance liquid chromatography (HPLC), for example, as described in Pearson and Regnier, J. Chrom., 255: 137-149, 1983.
[0114] All or a portion of a primer sequence can be complementary or substantially complementary to a target nucleic acid, in some embodiments. Substantially complementary with respect to sequences generally refers to nucleotide sequences that will hybridize with each other. The stringency of the hybridization conditions can be altered to tolerate varying amounts of sequence mismatch. In some embodiments, target and primer sequences are at least 75% complementary to each other. For example, target and primer sequences can be 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more complementary to each other.
[0115] In some embodiments, primers comprise a pair of primers. A pair of primers can include a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of a target nucleic acid). In some embodiments, primers consist of a pair of primers, however, in certain
SUBSTITUTE SHEET (RULE 26) instances, an amplification reaction can include additional primer pairs for amplifying different target sequences, such as in a multiplex amplification. In some embodiments, primers consist of a pair of primers, however, in certain instances, an amplification reaction can include additional primers, oligonucleotides or probes for a detection process that are not considered part of amplification.
[0116] A primer, in certain embodiments, can contain a modification such as one or more inosines, abasic sites, locked nucleic acids, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiers or any modifier that changes the binding properties of the primer. A primer, in certain embodiments, can contain a detectable molecule or entity (e.g., a fluorophore, radioisotope, colorimetric agent, particle, enzyme and the like).
[0117] Polymerases: Polymerases are proteins capable of catalyzing the specific incorporation of nucleotides to extend a 3' hydroxyl terminus of a primer molecule, such as, for example, an amplification primer described herein, against a nucleic acid target sequence (e.g., to which a primer is annealed). While not necessarily a required property, polymerases useful in the compositions and methods described herein can include, for example, thermophilic or hyperthermophilic polymerases that can have activity at an elevated reaction temperature (e.g., above 55 degrees Celsius, above 60 degrees Celsius, above 65 degrees Celsius, above 70 degrees Celsius, above 75 degrees Celsius, above 80 degrees Celsius, above 85 degrees Celsius, or higher). In some embodiments, a polymerase can incorporate about 1 to about 50 nucleotides in a single synthesis. For example, a polymerase can incorporate about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in a single synthesis. In other embodiments, a polymerase is more processive, and can incorporate 50 or more nucleotides in a single synthesis. In some embodiments, amplification reaction components comprise one or more DNA polymerases.
[0118] Exemplary DNA polymerases can be obtained commercially and include, but are not limited to, a 9° N DNA polymerase; 9°Nm™ DNA polymerase; Therminator™ DNA Polymerase; Therminator™ II DNA Polymerase; Therminator™ III DNA Polymerase; Therminator™ y DNA Polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (A. colt), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3 '-5' exo- ); T4 DNA polymerase; T7 DNA polymerase; Deep VentR™ (exo-) DNA Polymerase; Deep VentR™ DNA Polymerase; DyNAzyme™ EXT DNA; DyNAzyme™ II Hot Start DNA Polymerase; Phusion™ High-Fidelity DNA Polymerase; VentR® DNA Polymerase; VentR® (exo-) DNA Polymerase; RepliPHI™ Phi29 DNA Polymerase; rBst DNA Polymerase, large fragment (IsoTherm™ DNA Polymerase); MasterAmp™ AmpliTherm™ DNA Polymerase; Tag DNA polymerase; Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNA polymerase; Tbr DNA polymerase; DNA polymerase Beta; and ThermoPhi DNA polymerase..
[0119] In some embodiments, a polymerase can possess reverse transcription capabilities. In such instances, an amplification reaction can amplify RNA targets, for example, in a single step without the use of a separate reverse transcriptase. Non-limiting examples of polymerases that possess reverse
SUBSTITUTE SHEET (RULE 26) transcriptase capabilities include Bst (large fragment), 9° N DNA polymerase, 9°Nm™ DNA polymerase, Therminator™, Therminator™ II, and the like. In some embodiments, amplification reaction components comprise one or more separate reverse transcriptases. In some embodiments, more than one polymerase can be included in an amplification reaction. For example, an amplification reaction can comprise a polymerase having reverse transcriptase activity and a second polymerase having no reverse transcriptase activity.
[0120] Detection and Quantification: Nucleic acids prepared using the methods and compositions described herein can be subjected to reactions that detect one or more target nucleic acids. In one embodiment, the detection can be performed without a prior amplification. In other embodiments, the detection is performed on amplified nucleic acid products. Thus, an amplification method as described herein, including but not limited to an isothermal amplification method, can further comprise detecting and/or quantifying a nucleic acid amplification product using any detection method or quantification method known to those of skill in the art. Non-limiting examples of detection and/or quantification methods include RNA-guided nuclease (e.g., Cas) mediated cleavage and activation of a fluorophore, molecular beacon (e.g., real-time, endpoint), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface capture, 5' to 3' exonuclease hydrolysis probes (e.g., TAQMAN), intercalating/binding dyes, absorbance methods (e.g., colorimetric, turbidity), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, a primer extension method (e.g., iPLEX™), Molecular Inversion Probe (MIP) technology from Affymetrix, restriction fragment length polymorphism (RFLP) analysis, allele specific oligonucleotide (ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, Reverse dot blot, GeneChip microarrays, Dynamic allele-specific hybridization (DASH), Peptide nucleic acid (PNA) and locked nucleic acids (LNA) probes, AlphaScreen, SNPstream, genetic bit analysis (GBA), Multiplex minisequencing, SNaPshot, GOOD assay, Microarray miniseq, arrayed primer extension (APEX), Microarray primer extension, Tag arrays, Coded microspheres, Template-directed incorporation (TDI), colorimetric oligonucleotide ligation assay (OLA), sequence-coded OLA, microarray ligation, ligase chain reaction, padlock probes, invader assay, hybridization using at least one probe, hybridization using at least one fluorescently labeled probe, cloning and sequencing, the use of hybridization probes and quantitative real time polymerase chain reaction (QRT-PCR), nanopore sequencing, chips and combinations thereof. In some embodiments, detecting a nucleic acid amplification product comprises use of a real-time detection method (i.e., product is detected and/or continuously monitored during an amplification process). In some embodiments, detecting a nucleic acid amplification product comprises use of an endpoint detection method (i.e., product is detected after completing or stopping an amplification process). Nucleic acid detection methods can also employ the use of labeled nucleotides incorporated directly into a target sequence or into probes containing complementary sequences to a target. Such labels can be radioactive and/or fluorescent in nature and can be resolved in any of the maimers discussed herein.
SUBSTITUTE SHEET (RULE 26) In some embodiments, quantification of a nucleic acid amplification product can be achieved using certain detection methods described below. In certain instances, a detection method can be used in conjunction with a measurement of signal intensity, and/or generation of (or reference to) a standard curve and/or look-up table for quantification of a nucleic acid amplification product.
[0121] In some embodiments, detecting a nucleic acid amplification product comprises use of lateral flow or a lateral flow device. Such devices generally include a solid phase fluid permeable flow path through which fluid flows by capillary force. Example devices include, but are not limited to, dipstick assays and thin layer chromatographic plates with various appropriate coatings. Immobilized on the flow path are various binding reagents for the sample, binding partners or conjugates involving binding partners for the sample and signal producing systems. Detection can be achieved in several manners including, for example, enzymatic detection, nanoparticle detection, colorimetric detection, and fluorescence detection. Enzymatic detection can involve enzyme-labeled probes that are hybridized to complementary nucleic acid targets on the surface of the lateral flow device. The resulting complex can be treated with appropriate markers to develop a readable signal. Nanoparticle detection involves bead technology that can use colloidal gold, latex and/or paramagnetic nanoparticles. In one example, beads can be conjugated to an anti-biotin antibody. Target sequences can be directly biotinylated, or target sequences can be hybridized to sequence-specific biotinylated probes. Gold and latex give rise to colonmetric signals visible to the naked eye, and paramagnetic particles give rise to a non-visual signal when excited in a magnetic field and can be interpreted by a specialized reader. Fluorescence-based lateral flow detection methods also can be used and include, for example, dual fluorescein and biotin- labeled oligo probe methods, UPT-N ALP utilizing up-convertmg phosphor reporters composed of lanthanide elements embedded in a crystal (Corstjens et al., Clinical Chemistry, 47: 10, 1885-1893, 2001), and the use of quantum dots.
[0122] Nucleic acids can be captured on lateral flow devices. Means of capture can include antibody-dependent and antibody-independent methods. Antibody-dependent capture generally comprises an antibody capture line and a labeled probe of complementary sequence to the target. Antibody -independent capture generally uses non-covalent interactions between two binding partners, for example, the high affinity and irreversible linkage between a biotinylated probe and a streptavidin line. Capture probes can be immobilized directly on lateral flow membranes. Both antibody-dependent and antibody-independent methods can be used, for example, for detecting amplification products generated in a multiplex reaction.
[0123] In some embodiments, detecting a nucleic acid amplification product comprises use of molecular beacon technology. The term molecular beacon generally refers to a detectable molecule, where the detectable property of the molecule is detectable under certain conditions, thereby enabling the molecule to function as a specific and informative signal. Non-limiting examples of detectable properties include, optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties and time or speed through an opening of known size. Molecular beacons for
SUBSTITUTE SHEET (RULE 26) detecting nucleic acid molecules can be, for example, hair-pin shaped oligonucleotides containing a fluorophore on one end and a quenching dye on the opposite end. The loop of the hair-pin can contain a probe sequence that is complementary to a target sequence and the stem is formed by annealing of complementary arm sequences located on either side of the probe sequence. A fluorophore and a quenching molecule can be covalently linked at opposite ends of each arm. Under conditions that prevent the oligonucleotides from hybridizing to its complementary target or when the molecular beacon is free in solution, the fluorescent and quenching molecules are proximal to one another preventing fluorescence resonance energy transfer (FRET). When the molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product), hybridization can occur, and the loop structure is converted to a stable more rigid conformation causing separation of the fluorophore and quencher molecules leading to fluorescence (Tyagi et al. Nature Biotechnology 14: March 1996, 303- 308). Due to the specificity of the probe, the generation of fluorescence generally is exclusively due to the synthesis of the intended amplified product. In some instances, a molecular beacon probe sequence hybridizes to a sequence in an amplification product that is identical to or complementary to a sequence in a target nucleic acid. In some instances, a molecular beacon probe sequence hybridizes to a sequence in an amplification product that is not identical to or complementary to a sequence in a target nucleic acid (e.g., hybridizes to a sequence added to an amplification product by way of a tailed amplification primer or ligation).
[0124] Molecular beacons are highly specific and can discern a single nucleotide polymorphism. Molecular beacons also can be synthesized with different colored fluorophores and different target sequences, enabling simultaneous detection of several products in the same reaction (e.g., in a multiplex reaction). For quantitative amplification processes, molecular beacons can specifically bind to the amplified target following each cycle of amplification, and because nonhybridized molecular beacons are dark, it is not necessary to isolate the probe-target hybrids to quantitatively determine the amount of amplified product. The resulting signal is proportional to the amount of amplified product. Detection using molecular beacons can be done in real time or as an endpoint detection method. In some instances, certain reaction conditions can be optimized for each primer/probe set to ensure accuracy and precision.
[0125] In some embodiments, detecting a nucleic acid amplification product comprises use of fluorescence resonance energy transfer (FRET). FRET is an energy transfer mechanism between two chromophores: a donor and an acceptor molecule. Briefly, a donor fluorophore molecule is excited at a specific excitation wavelength. The subsequent emission from the donor molecule as it returns to its ground state can transfer excitation energy to the acceptor molecule through a long range dipole-dipole interaction. The emission intensity of the acceptor molecule can be monitored and is a function of the distance between the donor and the acceptor, the overlap of the donor emission spectrum and the acceptor absorption spectrum and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET can be useful for quantifying molecular dynamics, for example, in
SUBSTITUTE SHEET (RULE 26) DNA-DNA interactions as described for molecular beacons. For monitoring the production of a specific product, a probe can be labeled with a donor molecule on one end and an acceptor molecule on the other Probe-target hybridization brings a change in the distance or orientation of the donor and acceptor and FRET change is observed.
[0126] In some embodiments, detecting a nucleic acid amplification product comprises use of fluorescence polarization (FP). Fluorescence polarization techniques generally are based on the principle that a fluorescently labeled compound when excited by linearly polarized light will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Therefore, when a molecule such as a tracer-nucleic acid conjugate, for example, having a fluorescent label is excited with linearly polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time light is absorbed and emitted. When a free tracer compound (i.e., unbound to a nucleic acid) is excited by linearly polarized light, its rotation is much faster than the corresponding tracer-nucleic acid conjugate and the molecules are more randomly oriented, therefore, the emitted light is depolarized. Thus, fluorescence polarization provides a quantitative means for measuring the amount of tracer-nucleic acid conjugate produced in an amplification reaction.
[0127] In some embodiments, detecting a nucleic acid amplification product comprises use of surface capture. This can be accomplished by the immobilization of specific oligonucleotides to a surface producing a biosensor that is both highly sensitive and selective. Example surfaces that can be used include gold and carbon, and a surface capture method can use a number of covalent or noncovalent coupling methods to attach a probe to the surface. The subsequent detection of a target nucleic acid can be monitored by a variety of methods.
[0128] In some embodiments, detecting a nucleic acid amplification product comprises use of
5' to 3' exonuclease hydrolysis probes (e.g., TAQMAN). TAQMAN probes, for example, are hydrolysis probes that can increase the specificity of a quantitative amplification method (e.g., quantitative PCR). The TAQMAN probe principle relies on 1) the 5' to 3' exonuclease activity of Taq polymerase to cleave a dual -labeled probe during hybridization to a complementary target sequence and 2) fluorophore-based detection. A resulting fluorescence signal permits quantitative measurements of the accumulation of amplification product during the exponential stages of amplification, and the TAQMAN probe can significantly increase the specificity of the detection.
[0129] In some embodiments, detecting a nucleic acid amplification product comprises use of intercalating and/or binding dyes. In some embodiments, detecting a nucleic acid amplification product comprises use of dyes that specifically stain nucleic acid. For example, intercalating dyes exhibit enhanced fluorescence upon binding to DNA or RNA. Dyes can include DNA or RNA intercalating fluorophores and can include for example, SYTO® 82, acridine orange, ethidium bromide, Hoechst dyes, PicoGreen®, propidium iodide, SYBR® I (an asymmetrical cyanine dye), SYBR® II, TOTO (a thiaxole orange dimer) and YOYO (an oxazole yellow dimer). Dyes provide an opportunity for increasing the sensitivity of nucleic acid detection when used in conjunction with various detection
SUBSTITUTE SHEET (RULE 26) methods. For example, ethidium bromide can be used for staining DNA in agarose gels after gel electrophoresis; propidium iodide and Hoechst 33258 can be used in flow cytometry to determine DNA ploidy of cells; SYBR® Green 1 can be used in the analysis of double-stranded DNA by capillary electrophoresis with laser induced fluorescence detection; and PicoGreen® can be used to enhance the detection of double-stranded DNA after matched ion pair polynucleotide chromatography.
[0130] In some embodiments, detecting a nucleic acid amplification product comprises use of absorbance methods (e.g., colorimetric, turbidity). In some instances, detection and/or quantitation of nucleic acid can be achieved by directly converting absorbance (e.g., UV absorbance measurements at 260 nm) to concentration, for example. Direct measurement of nucleic acid can be converted to concentration using the Beer Lambert law which relates absorbance to concentration using the path length of the measurement and an extinction coefficient. In some embodiments, detecting a nucleic acid amplification product comprises use of a colorimetric detection method. Any suitable colorimetric detection can be used, and non-limiting examples include assays that use nanoparticles (e.g., metallic nanoparticles, modified nanoparticles, unmodified nanoparticles) and/or peptide nucleic acid (PNA) probes. For example, certain gold nanoparticle-based methods typically rely on a quantitative coupling between target recognition and the aggregation of the nanoparticles, which, in turn, can lead to a change in the photonic properties (e.g., color) of a nanoparticle solution.
[0131] In some embodiments, detecting a nucleic acid amplification product comprises use of electrophoresis (e.g., gel electrophoresis, capillary electrophoresis). Gel electrophoresis involves the separation of nucleic acids through a matrix, generally a cross-linked polymer, using an electromotive force that pulls the molecules through the matrix. Molecules of different sizes or configurations move through the matrix at different rates causing a separation between products that can be visualized and interpreted via a number of methods including but not limited to; autoradiography, phosphorimaging, and staining with nucleic acid chelating dyes. Capillary-gel electrophoresis (CGE) is a combination of traditional gel electrophoresis and liquid chromatography that employs a medium such as polyacrylamide in a narrow bore capillary to generate fast, high-efficient separations of nucleic acid molecules with up to single base resolution. CGE can be combined with laser induced fluorescence (LIF) detection where as few as six molecules of stained DNA can be detected. CGE/LIF detection generally involves the use of fluorescent DNA intercalating dyes including ethidium bromide, YOYO and SYBR® Green 1, and also can involve the use of fluorescent DNA derivatives where fluorescent dye is covalently bound to DNA. Simultaneous identification of several different target sequences (e.g., products from a multiplex reaction) can be made using this method.
[0132] In some embodiments, detecting a nucleic acid amplification product comprises use of mass spectrometry. Mass Spectrometry is an analytical technique that can be used to determine the structure and quantity of a nucleic acid and can be used to provide rapid analysis of complex mixtures. Following amplification, samples can be ionized, the resulting ions separated in electric and/or magnetic fields according to their mass-to-charge ratio, and a detector measures the mass-to -charge ratio of ions
SUBSTITUTE SHEET (RULE 26) (Crain, P. F. and McCloskey, J. A., Current Opinion in Biotechnology 9: 25-34 (1998)). Mass spectrometry methods include, for example, MALDI, MALDI-TOF, or Electrospray. These methods can be combined with gas chromatography (GC/MS) and liquid chromatography (LC/MS) Mass spectrometry (e.g., matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS)) can be high throughput due to high-speed signal acquisition and automated analysis off solid surfaces.
[0133] In some embodiments, detecting a nucleic acid amplification product comprises use of nucleic acid sequencing. The entire sequence or a partial sequence of an amplification product can be determined, and the determined nucleotide sequence can be referred to as a read. For example, linear amplification products can be analyzed directly without further amplification in some embodiments (e.g., by using single-molecule sequencing methodology). In certain embodiments, linear amplification products can be subject to further amplification and then analyzed (e.g., using sequencing by ligation or pyrosequencing methodology). Reads can be subject to different types of sequence analysis. Any suitable sequencing method can be utilized to detect, and in some instances determine the amount of, detectable products generated by the amplification methods described herein. Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, reversible terminatorbased sequencing, sequencing by ligation, pyrosequencing, sequencing by synthesis, single-molecule sequencing, multiplex sequencing, solid phase single nucleotide sequencing, and nanopore sequencing. [0134] In some embodiments, detecting a nucleic acid amplification product comprises use of digital amplification (e.g., digital PCR). Digital PCR, for example, takes advantage of nucleic acid (DNA, cDNA or RNA) amplification on a single molecule level, and offers a highly sensitive method for quantifying low copy number nucleic acid. Systems for digital amplification and analysis of nucleic acids are available (e.g., Fluidigm® Corporation).
[0135] Isothermal nucleic acid amplification methods can be conducted in the presence of native nucleotides, such as, for example, dideoxyribonucleoside triphosphates (dNTPs), and/or derivatized nucleotides. A native nucleotide generally refers to adenylic acid, guanylic acid, cytidylic acid, thymidylic acid, or uridylic acid. A derivatized nucleotide generally is a nucleotide other than a native nucleotide. Nucleotides typically are designated as follows. A ribonucleoside triphosphate is referred to as NTP or rNTP, where N can be A, G, C, U. A deoxynucleoside triphosphate substrate is referred to as dNTP, where N can be A, G, C, T, or U. Monomeric nucleotide subunits can be denoted as A, G, C, T, or U herein with no particular reference to DNA or RNA. In some embodiments, non- naturally occurring nucleotides or nucleotide analogs, such as analogs containing a detectable label (e.g., fluorescent or colorimetric label), can be used. For example, nucleic acid amplification can be carried out in the presence of labeled dNTPs, such as, for example, radiolabels such as 32P, 33P, 125I, or 35S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, or fluorochromes. In some embodiments, nucleic acid amplification can be carried out in the presence of modified dNTPs, such as, for example, heat activated dNTPs (e.g., CleanAmp™ dNTPs from TriLink).
SUBSTITUTE SHEET (RULE 26) [0136] CRISPR Cas- mediated detection of a target sequence: In some embodiments, the isothermal amplification methods use a CRISPR-Cas method for detecting the presence of a target sequence in the pool of amplified nucleic acids. The CRISPR-Cas enzyme can be from an organism from a genus comprising, for example, Streptococcus, Campylobacter, Ni trati fr actor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methyl obacterium or Acidaminococcus.
[0137] In the context of formation of a CRISPR complex, the term “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. A target sequence can be DNA or RNA. Generally, the term ‘target nucleic acid’ refers to a polynucleotide being or comprising the target sequence. In other words, the target nucleic acid can be a polynucleotide or a part of a polynucleotide to which a part of the gRNA, i.e. the guide sequence, is designed to have complementarity and to which the effector function mediated by the complex comprising CRISPR effector protein and a gRNA is to be directed.
[0138] It is noted that the enzyme system can be a DNA targeting CRISPR-Cas protein or an
RNA targeting CRISPR-Cas protein. Exemplary CRISPR-Cas proteins include, but are not limited to, Cas 13, Cas 12a, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof. In some embodiments, the effector protein Cas 9, Casl2a, Casl3a, or Casl4. In some embodiments, the effector protein is Cas 13.
[0139] As used herein, the terms “guide nucleic acid,” “guide sequence,” “crRNA,” “guide
RNA,” or “single guide RNA,” or “gRNA” refers to a polynucleotide comprising any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and to direct sequence-specific binding of a CRISPR complex comprising the guide sequence and a CRISPR effector protein to the target nucleic acid sequence. In some example embodiments, the degree of complementarity, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%), or more. Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences. Exemplary algorithms for determining optimal alignment include, but are not limited to, the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft
SUBSTITUTE SHEET (RULE 26) Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
[0140] The guide nucleic acid strand can be any length. For example, the guide nucleic acid strand can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a nucleic acid strand is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Preferably the guide nucleic acid sequence is 10-30 nucleotides long.
[0141] In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. The section of the guide sequence through which complementarity to the target sequence is important for cleavage activity is referred to herein as the seed sequence. A target sequence can comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in a parasite, bacterial or viral genome.
[0142] Typically, the presence of a target nucleic acid sequence will activate the Cas enzyme to non-specifically cleave a reporter molecule, for example, releasing or activating a fluorophore that indicates the presence of the target nucleic acid..ST/ ERL I )('K amplification: SHERLOCK, also known as “Specific high-sensitivity enzymatic reporter unlocking,” is a nucleic acid detection method that is used to detect RNA or DNA target sequences depending upon the particular enzymes used. In each instance, SHERLOCK detection is based on the target sequence -dependent activation of an RNA- guided nuclease, which, once activated, cleaves not only the target sequence, but other nucleic acids. When the other nucleic acid is a labeled probe that generates a signal upon cleavage, e.g., via separation of a fluorophore and a quencher, a sensitive target-sequence detection assay is provided. These are the hallmarks of a SHERLOCK detection assay as described herein - that is, as used herein, a SHERLOCK detection assay includes the use of an RNA-guided nuclease, a guide RNA including complementarity to a desired target nucleic acid, and a labeled probe that generates a signal upon cleavage by the promiscuous activity of the target-sequence-activated RNA-guided nuclease. When that targetsequence detection assay is coupled with a target-specific isothermal nucleic acid amplification reaction, a single-pot amplification and detection assay is provided that has extremely high sensitivity. This combined isothermal amplification/RNA-guided nuclease detection approach is the assay initially published as SHERLOCK - see Gootenberg et al., Science 356: 438-442 (2017), incorporated herein by reference. The isothermal amplification approach can be coupled with reverse transcription to detect RNA targets in a highly sensitive manner by first generating amplified cDNA, and then detecting with RNA-guided nuclease that targets DNA. When the RNA-guided nuclease is one that cleaves RNA, e.g., a Casl3a enzyme, a single -stranded RNA probe can be used (Gootenberg et al., Science 356: 438- 442 (2017)). When the RNA-guided nuclease is one that cleaves DNA, e.g., a Casl2a enzyme, a DNA probe can be used; see, e.g., Gootenberg et al., Science 360: 439-444 (2018), and Li et al., Cell
SUBSTITUTE SHEET (RULE 26) Discovery 4, 20 (2018) see https:// at doi.org/10.1038/s41421-018-0028-z, each of which is incorporated herein by reference. The nucleic acid sample preparation compositions and methods described herein are well-suited for preparing biological samples for use in isothermal nucleic acid amplification reactions, as well as for SHERLOCK detection of target nucleic acid sequences.
Ultrasensitive Detection of a Pathogen
[0143] The methods and compositions used for nucleic acid preparation described herein can be used in conjunction with an isothermal amplification method to permit detection of a pathogen in a biological sample. A pathogen can be, for example, an intracellular parasite, a virus, a bacterium, or a fungus.
[0144] Exemplary intracellular parasites that can be detected using the methods and compositions described herein include, but are not limited to Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Babesia sp. Leishmaniasis spp. Toxoplasmosis spp. and filarial nematodes.
[0145] In some embodiments, a pathogenic virus that can be detected includes the Dengue virus, Zika virus, Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoVl), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Heptatis E, Herpes virus, Varicella virus, Cytomegalovirus, Epstein-Barr virus, Human herpesvirus 6, Human herpesvirus8, adenovirus, influenza, parainfluenza, respiratory syncytial virus, or Chikungunya virus. Other exemplary viruses that can be detected as described herein include genera of viruses: Adenoviridae, Alfamovirus, Allexivirus, Allolevivirus, Alphacryptovirus, Alphaherpesvirinae, Alphanodavirus, Alpharetrovirus, Alphavirus, Aphthovirus, Apscaviroid, Aquabirnavirus, Aquareovirus, Arenaviridae, Arenavirus, Arteriviridae, Arterivirus, Ascoviridae, Ascovirus, Asfarviridae, Asfivirus, Astroviridae, Astrovirus, Aureusvirus, Avenavirus, Aviadenovirus, Avibirnavirus, Avihepadnavirus, Avipoxvirus, Avsunviroid, Avsunviroidae, Baculoviridae, Badnavirus, Barnaviridae, Barnavirus, Bdellomicrovirus, Begomovirus, Benyvirus, Betacryptovirus, Betaherpesvirinae, Betanodavirus, Betaretrovirus, Betatetravirus, Birnaviridae, Bornaviridae, Bornavirus, Bracovirus, Brevidensovirus, Bromoviridae, Bromovirus, Bunyaviridae, Bunyavirus, Bymovirus, ”c2-like viruses, ” Caliciviridae, Capillovirus, Capripoxvirus, Cardiovirus, Carlavirus, Carmovirus, "Cassava vein mosaic-like viruses, " Caulimoviridae, Caulimovirus, Chlamydiamicrovirus, Chloriridovirus, Chlorovirus, Chordopoxyirinae, Chrysovirus, Circoviridae, Circovirus, Closteroviridae, Closterovirus, Cocadviroid, Coleviroid, Coltivirus, Comoviridae, Comovirus, Coronaviridae, Coronavirus, Corticoviridae, Corticovirus, "Cricket paralysis-like viruses, " Crinivirus, Cucumovirus, Curtovirus, Cypovirus, Cystoviridae, Cystovirus, Cytomegalovirus, Cytorhabdovirus, Deltarelrovirus, Deltavirus, Densovirinae, Densovirus, Dependovirus, Dianthovirus, "Ebola-like viruses, " Enamovirus, Enterovirus, Entomobirnavirus, Entomopoxyirinae, Entomopoxvirus
SUBSTITUTE SHEET (RULE 26) A, Entomopoxvirus B, Entomopoxvirus C, Ephemerovirus, Epsilonretrovirus, Errantivirus, Erythrovirus, Fabavirus, Fijivirus, Filoviridae, Flaviviridae, Flavivirus, Foveavirus, Furovirus, Fuselloviridae, Fusellovirus, Gammaherpesvirinae, Gammaretrovirus, Geminiviridae, Giardiavirus, Granulovirus , Hantavirus, Hemivirus, Hepacivirus, Hepadnaviridae, "Hepatitis E-like viruses, " Hepatovirus, Herpesviridae, Hordeivirus, Hostuviroid, Hypoviridae, Hypovirus, Ichnovirus, "Ictalurid herpes-like viruses, " Idaeovirus, liarvirus, "Infectious laryngotracheitis-like viruses, " Influenzavirus A, Influenzavirus B, Influenzavirus C, Inoviridae, Inovirus, Ipomovirus, Iridoviridae, Iridovirus, Iteravirus, "L5-like viruses, " Lagovirus, "-like viruses, " Leishmaniavirus, Lentivirus, Leporipoxvirus, Leviviridae, Levivirus, Lipothrixviridae, Lipothrixvirus, Luteoviridae, Luteovirus, Lymphocryptovirus, Lymphocystivirus, Lyssavirus, Machlomovirus, Macluravirus, Marafivirus, "Marburg-like viruses," "Marek's disease-like viruses," Mastadenovirus, Mastrevirus, Metapneumovirus, Metaviridae, Metavirus, Microviridae, Microvirus, Mitovirus, Molluscipoxvirus, Morbillivirus, "Mu-like viruses," Muromegalovirus, Myoviridae, Nairovirus, Nanovirus, Narnaviridae, Narnavirus, Necrovirus, Nepovirus, Nodaviridae, "Norwalk-like viruses, " Novirhabdovirus, Nucleopolyhedrovirus, Nucleorhabdovirus, Oleavirus, Omegatetravirus, Ophiovirus, Orbivirus, Orthohepadnavirus, Orthomyxoviridae, Orthopoxvirus, Orthoreovirus, Oryzavirus, Ourmiavirus, "Pl -like viruses, " "P2- like viruses, " "P22-like viruses, " Panicovirus, Papillomaviridae, Papillomavirus, Paramyxoviridae, Paramyxovirinae, Parapoxvirus, Parechovirus, Partitiviridae, Partitivirus, Parvoviridae, Parvovirinae, Parvovirus, Pecluvirus, Pelamoviroid, Pestivirus, "Petunia vein clearing-like viruses, " Phaeovirus, "-29-like viruses, " "-H-like viruses, " Phlebovirus, Phycodnaviridae, Phytoreovirus, Picornaviridae, Plasmaviridae, Plasmavirus, Plectrovirus, Pneumovirinae, Pneumovirus, Podoviridae, Polerovirus, Polydnaviridae, Polyomaviridae, Polyomavirus, Pomovirus, Pospiviroid, Pospiviroidae, Potexvirus, Potyviridae, Potyvirus, Poxyiridae, Prasinovirus, Prions, Prymnesiovirus, Pseudoviridae, Pseudovirus, "Ml -like viruses", Ranavirus, Reoviridae, Respirovirus, Retroviridae, Rhabdoviridae, Rhadinovirus, Rhinovirus, Rhizidiovirus, "Rice tungro bacilliform-like viruses," Roseolovirus, Rotavirus, Rubivirus, Rubulavirus, Rudiviridae, Rudivirus, Rymovirus, "Sapporo-like viruses," Satellites, Sequiviridae, Sequivirus, Simplexvirus, Siphoviridae, Sobermovirus, "Soybean chlorotic mottle-like viruses, " Spiromicrovirus, "SPOl-like viruses, " Spumavirus, Suipoxvirus, "Sulfolob us SNDV-like viruses," "Tl-like viruses," "T4-like viruses," "T 5 -like viruses," "T7-like viruses, " Tectiviridae, Tectivirus, Tenuivirus, Tetraviridae, Thogotovirus, Tobamovirus, Tobravirus, Togaviridae, Tombusviridae, Tombusvirus, Torovirus, Tospovirus, Totiviridae, Totivirus, Trichovirus, Tritimovirus, Tymovirus, Umbravirus, Varicellovirus, Varicosavirus, Vesiculovirus, Vesivirus, Viroids, Vitivirus, Wakavirus, and Yatapoxvirus.
[0146] A bacterium that can be detected using the methods and compositions described herein can be a gram negative bacterium, a gram positive bacterium, an anaerobic bacterium, an aerobic bacterium, a facultative anaerobic bacterium, or an intracellular bacterium. Examples of gram-negative bacteria include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include,
SUBSTITUTE SHEET (RULE 26) for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium. Exemplary gram -positive bacteria include, but are not limited to, cocci, nonsporulating rods, and sporulating rods. The genera of Gram -positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Coryneb cterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces .
[0147] Additional bacteria that can be detected as described herein include bacteria from one or more of the following genera: genera of the domain of Bacteria (or Eubacteria): Abiotrophia, Acetitomaculum, Acetivibrio, Acetoanaerobium, Acetobacter, Acetobacterium, Acetofilamentum, Acetogenium, Acetohalobium, Acetomicrobium, Acetonema, Acetothermus, Acholeplasma, Achromatium, Achromobacter, Acidaminobacter, Acidaminococcus, Acidimicrobium, Acidiphilium, Acidisphaera, Acidithiobacillus, Acidobacterium, Acidocella, Acidomonas, Acidothermus, Acidovorax, Acinetobacter, Acrocarpospora, Actinoalloteichus, Actinobacillus, Actinobaculum, Actinobispora, Actinocorallia, Actinokineospora, Actinomadura, Actinomyces, Actinoplanes, Actinopolymorpha, Actinopolyspora, Actinopycnidium, Actinosporangium, Actinosynnema, Aegyptianella, Aequorivita, Aerococcus, Aeromicrobium, Aeromonas, Aflpia, Agitococcus, Agreia, Agrobacterium, Agrococcus, Agromonas, Agromyces, Ahrensia, Albibacter, Albidovulum, Alcaligenes, Alcalilimnicola, Alcanivorax, Algoriphagus, Alicycliphilus, Alicyclobacillus, Alishew nella, Alistipes, Alkalibacterium, Alkalilimnicola, Alkaliphilus, Alkalispirillum, Alkanindiges, Allisonella, Allochromatium, Allofustis, Alloiococcus, Allomonas, Allorhizobium, Alterococcus, Alteromonas, Alysiella, Amaricoccus, Aminobacter, Aminobacterium, Aminomonas, Ammonifex, Ammoniphilus, Amoebobacter, Amorphosphorangium, Amphibacillus, Ampullariella, Amycolata, Amycolatopsis, Anaeroarcus, Anaerobacter, Anaerobaculum, Anaerobiospirillum, Anaerobranca, Anaerococcus, Anaerofilum, Anaeroglobus, Anaerolinea, Anaeromusa, Anaeromyxobacter, Anaerophaga, Anaeroplasma, Anaerorhabdus, Anaerosinus, Anaerostipes, Anaerovibrio, Anaerovorax, Anaplasma, Ancalochloris, Ancalomicrobium, Ancylobacter, Aneurinibacillus, Angiococcus, Angulomicrobium, Anoxybacillus, Anoxynatronum, Antarctobacter, Aquabacter, Aquabacterium, Aquamicrobium, Aquaspirillum, Aquifex, Arachnia, Arcanobacterium, Archangium, Arcobacter, Arenibacter, Arhodomonas, Arsenophonus, Arthrobacter, Asaia, Asanoa, Asteroleplasma, Asticcacaulis, Atopobacter, Atopobium, Aurantimonas, Aureobacterium, Azoarcus, Azomonas, Azomonotrichon, Azonexus, Azorhizobium, Azorhizophilus , Azospira, Azospirillum, Azotobacter, Azovibrio, Bacillus, Bacterionema, Bacteriovorax, Bacteroides, Bactoderma, Balnearium, Balneatrix, Bartonella, Bdellovibrio, Beggiatoa, Beijerinckia, Beneckea, Bergeyella, Beutenbergia, Bifidobacterium, Bilophila, Blastobacter, Blastochloris, Blastococcus, Blastomonas, Blattabacterium, Bogoriella, Bordetella, Borrelia, Bosea, Brachybacterium, Brachymonas, Brachyspira, Brackiella, Bradyrhizobium, Branhamella, Brenneria,
SUBSTITUTE SHEET (RULE 26) Brevibacillus, Brevibacterium, Brevinema, Brevundimonas, Brochothrix, Brucella, Brumimicrobium, Buchnera, Budvicia, Bulleidia, Burkholderia, Buttiauxella, Butyrivibrio, Caedibacter, Caenibacterium, Calderobacterium, Caldicellulosiruptor, Caldilinea, Caldimonas, Caldithrix, Caloramator, Caloranaerobacter, Calymmatobacterium, Caminibacter, Caminicella, Campylobacter, Capnocytophaga, Capsularis, Carbophilus, Carboxydibrachium, Carboxydobrachium, Carboxydocella, Carboxydothermus, Cardiobacterium, Carnimonas, Carnob acterium, Caryophanon, Caseobacter, Catellatospora, Catenibacterium, Catenococcus, Catenuloplanes, Catonella, Caulobacter, Cedecea, Cellulomonas, Cellulophaga, Cellulosimicrobium, Cellvibrio, Centipeda, Cetobacterium, Chainia, Chelatobacter, Chelatococcus, Chitinophaga, Chlamydia, Chlamydophila, Chlorobaculum, Chlorobium, Chloroflexus , Chloroherpeton, Chloronema, Chondromyces, Chromatium, Chromobacterium, Chromohalobacter, Chryseobacterium, Chryseomonas, Chrysiogenes, Citricoccus, Citrobacter, Clavibacter, Clevelandina, Clostridium, Cobetia, Coenonia, Collinsella, Colwellia, Comamonas, Conexibacter, Conglomeromonas, Coprobacillus, Coprococcus, Coprothermobacter, Coriobacterium, Corynebacterium, Couchioplanes, Cowdria, Coxiella, Craurococcus, Crenothrix, Crinalium (not validly published), Cristispira, Croceibacter, Crocinitomix, Crossiella, Cryobacterium, Cryomorpha, Cryptobacterium, Cryptosporangium, Cupriavidus, Curtobacterium, Cyclobacterium, Cycloclasticus, Cystobacter, Cytophaga, Dactylosporangium, Dechloromonas, Dechlorosoma, Deferribacter, Defluvibacter, Dehalobacter, Dehalospirillum, Deinobacter, Deinococcus, Deleya, Delftia, Demetria, Dendrosporobacter, Denitrobacterium, Denitrovibrio, Dermabacter, Dermacoccus, Dermatophilus, Derxia, Desemzia, Desulfacinum, Desulfitobacterium, Desulfobacca, Desulfobacter, Desulfobacterium, Desulfobacula, Desulfobulbus, Desulfocapsa, Desulfocella, Desulfococcus, Desulfofaba, Desulfofrigus, Desulfofustis, Desulfohalobium, Desulfomicrobium, Desulfomonas, Desulfomonile, Desulfomusa, Desulfonatronovibrio, Desulfonatronum, Desulfonauticus, Desulfonema, Desulfonispora, Desulforegula, Desulforhabdus, Desulforhopalus, Desulfosarcina, Desulfospira, Desulfosporosinus, Desulfotalea, Desulfotignum, Desulfotomaculum, Desulfovibrio, Desulfovirga, Desulfurella, Desulfurobacterium, Desulfuromonas, Desulfuromusa, Dethiosulfovibrio, Devosia, Dialister, Diaphorobacter, Dichelobacter, Dichotomicrobium, Dictyoglomus, Dietzia, Diplocalyx, Dolosicoccus, Dolosigranulum, Dorea, Duganella, Dyadobacter, Dysgonomonas, Ectothiorhodospira, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Elytrosporangium, Empedobacter, Enhydrobacter, Enhygromyxa, Ensifer, Enterobacter, Enterococcus, Enterovibrio, Entomoplasma, Eperythrozoon, Eremococcus, Erwinia, Erysipelothrix, Erythrobacter, Erythromicrobium, Erythromonas, Escherichia, Eubacterium, Ewingella, Excellospora, Exiguobacterium, Facklamia, Faecalibacterium, Faenia, Falcivibrio, Ferribacterium, Ferrimonas, Fervidobacterium, Fibrobacter, Filibacter, Filifactor, Filobacillus, Filomicrobium, Finegoldia, Flammeovirga, Flavimonas, Flavobacterium, Flectobacillus, Flexibacter, Flexistipes, Flexithrix, Fluoribacter, Formivibrio, Francisella, Frankia, Frateuria, Friedmanniella, Frigoribacterium, Fulvimarina, Fulvimonas, Fundibacter, Fusibacter, Fusobacterium,
SUBSTITUTE SHEET (RULE 26) Gallibacterium, Gallicola, Gallionella, Garciella, Gardnerella, Gelidibacter, Gelria, Gemella, Gemmata, Gemmatimonas, Gemmiger, Gemmobacter, Geobacillus, Geobacter, Geodermatophilus, Georgenia, Geothrix, Geotoga, Geovibrio, Glaciecola, Globicatella, Gluconacetobacter, Gluconoacetobact r, Gluconobacter, Glycomyces, Gordonia, Gordonia, Gracilibacillus, Grahamella, Granulicatella, Grimontia, Haemobartonella, Haemophilus, Hafnia, Hahella, Halanaerobacter, Halanaerobium, Haliangium, Haliscomenobacter, Hallella, Haloanaerobacter, Haloanaerobium, Halobacillus, Halobacteroides, Halocella, Halochromatium, Haloincola, Halomicrobium, Halomonas, Halonatronum, Halorhodospira, Halospirulina, Halothermothrix, Halothiobacillus, Halovibrio, Helcococcus, Heliobacillus, Helicobacter, Heliobacterium, Heliophilum, Heliorestis, Heliothrix, Herbaspirillum, Herbidospora, Herpetosiphon, Hippea, Hirschia, Histophilus, Holdemania, Hollandina, Holophaga, Holospora, Hongia, Hydrogenobacter, Hydrogenobaculum, Hydrogenophaga, Hydrogenophilus, Hydrogenothermus, Hydrogenovibrio, Hymenobacter, Hyphomicrobium, Hyphomonas, Ideonella, Idiomarina, Ignavigranum, llyobacter, Inquilinus, Intrasporangium, lodobacter, Isobaculum, Isochromatium, Isosphaera, Janibacter, Jannaschia, Janthinobacterium, Jeotgalibacillus, Jeotgalicoccus, Johnsonella, Jonesia, Kerstersia, Ketogulonicigenium, Ketogulonigenium, Kibdelosporangium, Kineococcus, Kineosphaera, Kineosporia, Kingella, Kitasatoa, Kitasatospora, Kitasatosporia, Klebsiella, Kluyvera, Knoellia, Kocuria, Koserella, Kozakia, Kribbella, Kurthia, Kutzneria, Kytococcus, Labrys, Lachnobacterium, Lachnospira, Lactobacillus, Lactococcus, Lactosphaera, Lamprobacter, Lamprocystis, Lampropedia, Laribacter, Lautropia, Lawsonia, Lechevalieria, Leclercia, Legionella, Leifsonia, Leisingera, Leminorella, Lentibacillus, Lentzea, Leptonema, Leptospira, Leptospirillum, Leptothrix, Leptotrichia, Leucobacter, Leuconostoc, Leucothrix, Levinea, Lewinella, Limnobacter, Limnothrix, Listeria, Listonella, Lonepinella, Longispora, Lucibacterium, Luteimonas, Luteococcus, Lysobacter, Lyticum, Macrococcus, Macromonas, Magnetospirillum, Malonomonas, Mannheimia, Maricaulis, Marichromatium, Marinibacillus, Marinilabilia, Marinilactibacillus, Marinithermus, Marinitoga, Marinobacter, Marinobacterium, Marinococcus, Marinomonas, Marinospirillum, Marmoricola, Massilia, Megamonas, Megasphaera, Meiothermus, Melissococcus, Melittangium, Meniscus, Mesonia, Mesophilobacter, Mesoplasma, Mesorhizobium, Methylarcula, Methylobacillus, Methylobacter, Methylobacterium, Methylocaldum, Methylocapsa, Methylocella, Methylococcus, Methylocystis, Methylomicrobium, Methylomonas, Methylophaga, Me thy lophilus, Methylopila, Methylorhabdus, Methylosarcina, Methylosinus, Methylosphaera, Methylovorus, Micavibrio, Microbacterium, Microbispora, Microbulbifer, Micrococcus, Microcyclus, Microcystis, Microellobosporia, Microlunatus, Micromonas, Micromonospora, Micropolyspora, Micropruina, Microscilla, Microsphaera, Microtetraspora, Microvirga, Microvirgula, Mitsuokella, Mobiluncus, Modestobacter, Moellerella, Mogibacterium, Moorella, Moraxella, Morganella, Moritella, Morococcus, Muricauda, Muricoccus, Mycetocola, Mycobacterium, Mycoplana, Mycoplasma, Myroides, Myxococcus, Nannocystis, Natroniella, Natronincola, Natronoincola, Nautilia, Neisseria, Neochlamydia,
SUBSTITUTE SHEET (RULE 26) Neorickettsia, Neptunomonas, Nesterenkonia, Nevskia, Nitrobacter, Nitrococcus, Nitrosococcus, Nitrosolobus, Nitrosomonas, Nitrosospira, Nitrospina, Nitrospira, Nocardia, Nocardioides, Nocardiopsis, Nonomuraea, Nonomuria, Novosphingobium, Obesumbacterium, Oceanicaulis, Oceanimonas, Oceanisphaera, Oceanithermus, Oceanobacillus, Oceanobacter, Oceanomonas, Oceanospirillum, Ochrobactrum, Octadecabacter, Oenococcus, Oerskovia, Okibacterium, Oleiphilus, Oleispira, Oligella, Oligotropha, Olsenella, Opitutus, Orenia, Oribaculum, Orientia, Omithinicoccus, Ornithinimicrobium, Ornithobacterium, Oscillochloris, Oscillospira, Oxalicibacterium, Oxalobacter, Oxalophagus, Oxobacter, Paenibacillus, Pandoraea, Pannonibacter, Pantoea, Papillibacter, Parachlamydia, Paracoccus, Paracraurococcus, Paralactobacillus, Paraliobacillus, Parascardovia, Parvularcula, Pasteurella, Pasteuria, Paucimonas, Pectinatus, Pectobacterium, Pediococcus, Pedobacter, Pedomicrobium, Pelczaria, Pelistega, Pelobacter, Pelodictyon, Pelospora, Pelotomaculum, Peptococcus, Peptoniphilus, Peptostreptococcus, Persephonella, Persicobacter, Petrotoga, Pfennigia, Phaeospirillum, Phascolarctobacterium, Phenylobacterium, Phocoenobacter, Photobacterium, Photorhabdus, Phyllobacterium, Pigmentiphaga, Pilimelia, Pillotina, Pimelobacter, Pirella, Pirellula, Piscirickettsia, Planctomyces, Planktothricoides, Planktothrix, Planobispora, Pianococcus, Planomicrobium, Planomonospora, Planopolyspora, Planotetraspora, Plantibacter, Pleisomonas, Plesiocystis, Plesiomonas, Polaribacter, Polaromonas, Polyangium, Polynucleobacter, Porphyrobacter, Porphyromonas, Pragia, Prauserella, Prevotella, Prochlorococcus, Prochloron, Prochlorothrix, Prolinoborus, Promicromonospora, Propionibacter, Propionibacterium, Propionicimonas, Propioniferax, Propionigenium, Propionimicrobium, Propionispira, Propionispora, Propionivibrio, Prosthecobacter, Prosthecochloris, Prosthecomicrobium, Proteus, Protomonas, Providencia, Pseudaminobacter, Pseudoalteromonas, Pseudoamycolata, Pseudobutyrivibrio, Pseudocaedibacter, Pseudomonas, Pseudonocardia, Pseudoramibacter, Pseudorhodobacter, Pseudospirillum, Pseudoxanthomonas, Psychrobacter, Psychroflexus, Psychromonas, Psychroserpens, Quadricoccus, Quinella, Rahnella, Ralsto ia, Ramlibacter, Raoultella, Rarobacter, Rathayibacter, Reichenbachia, Renibacterium, Rhabdochromatium, Rheinheimera, Rhizobacter, Rhizobium, Rhizomonas, Rhodanobacter, Rhodobaca, Rhodobacter, Rhodobium, Rhodoblastus, Rhodocista, Rhodococcus, Rhodocyclus, Rhodoferax, Rhodoglobus, Rhodomicrobium, Rhodopila, Rhodoplanes, Rhodopseudomonas, Rhodospira, Rhodospirillum, Rhodothalassium, Rhodothermus, Rhodovibrio, Rhodovulum, Rickettsia, Rickettsiella, Riemerella, Rikenella, Rochalimaea, Roseateles, Roseburia, Roseibium, Roseiflexus, Roseinatronobacter, Roseivivax, Roseobacter, Roseococcus, Roseomonas, Roseospira, Roseospirillum, Roseovarius, Rothia, Rubrimonas, Rubritepida, Rubrivivax, Rubrobacter, Ruegeria, Rugamonas, Ruminobacter, Ruminococcus, Runella, Saccharobacter, Saccharococcus, Saccharomonospora, Saccharopolyspora, Saccharospirillum, Saccharothrix, Sagittula, Salana, Salegentibacter, Salibacillus, Salinibacter, Salinibacterium, Salinicoccus, Salinisphaera, Salinivibrio, Salmonella, Samsonia, Sandaracinobacter, Sanguibacter, Saprospira, Sarcina, Sarcobium, Scardovia, Schineria, Schlegelella, Schwartzia, Sebaldella, Sedimentibacter, Selenihalanaerobacter,
SUBSTITUTE SHEET (RULE 26) Selenomonas, Seliberia, Serpens, Serpula, Serpulina, Serratia, Shewanella, Shigella, Shuttleworthia, Silicibacter, Simkania, Simonsiella, Sinorhizobium, Skermanella, Skermania, Slackia, Smithella, Sneathia, Sodalis, Soehngenia, Solirubrobacter, Solobacterium, Sphaerobacter, Sphaerotilus, Sphingobact rium, Sphingobium, Sphingomonas, Sphingopyxis, Spirilliplanes, Spirillospora, Spirillum, Spirochaeta, Spiroplasma, Spirosoma, Sporanaerobacter, Sporichthya, Sporobacter, Sporobacterium, Sporocytophaga, Sporohalobacter, Sporolactobacillus, Sporomusa, Sporosarcina, Sporotomaculum, Staleya, Staphylococcus, Stappia, Starkeya, Stella, Stenotrophomonas, Sterolibacterium, Stibiobacter, Stigmatella, Stomatococcus, Str eptaci diphilus, Streptimonospora, Str ptoalloteichus, Streptobacillus, Streptococcus, Streptomonospora, Streptomyces : S. abikoensis, S. erumpens, S. erythraeus, S. michiganensis, S. microflavus, S. zaomyceticus, Streptosporangium, Streptoverticillium, Sub tercola, Succi iclasticum, Succinimonas, Succinispira, Succinivibrio, Sulfitobacter, Sulfobacillus, Sulfurihydrogenibium, Sulfurimonas, Sulfurospirillum, Sutterella, Suttonella, Symbiobacterium, Symbiotes, Synergistes, Syntrophobacter, Syntrophobotulus, Syntrophococcus, Syntrophomonas, Syntrophosphora, Syntrophothermus, Syntrophus, Tannerella, Tatlockia, Tatumella, Taylorella, Tectibacter, Teichococcus, Telluria, Tenacibaculum, Tepidibacter, Tepidimonas, Tepidiphilus, Terasakiella, Teredinibacter, Terrabacter, Terracoccus, Tessaracoccus, Tetragenococcus, Tetrasphaera, Thalassomonas, Thalassospira, Thauera, Thermacetogenium, Thermaerobacter, Thermanaeromonas, Thermanaerovibrio, Thermicanus, Thermithiobacillus, Thermoactinomyces, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobium, Thermobacillus, Thermobacteroides, Thermobifida, Thermobispora, Thermobrachium, Thermochromatium, Thermocrinis, Thermocri spurn, Thermodesulfobacterium, Thermodesulforhabdus, Thermodesulfovibrio, Thermohalobacter, Thermohydrogenium, Thermoleophilum, Thermomicrobium, Thermomonas, Thermomonospora, Thermonema, Thermosipho, Thermosyntropha, Thermoterrabacterium, Thermothrix, Thermotoga, Thermovenabulum, Thermovibrio, Thermus, Thialkalicoccus, Thialkalimicrobium, Thialkalivibrio, Thioalkalicoccus, Thioalkalimicrobium, Thioalkalispira, Thioalkalivibrio, Thiobaca, Thiobacillus, Thiobacterium, Thiocapsa, Thiococcus, Thiocystis, Thiodictyon, Thioflavicoccus, Thiohalocapsa, Thiolamprovum, Thiomargarita, Thiomicrospira, Thiomonas, Thiopedia, Thioploca, Thiorhodococcus, Thiorhodospira, Thiorhodovibrio, Thiosphaera, Thiospira, Thiospirillum, Thiothrix, Thiovulum, Tindallia, Tissierella, Tistrella, Tolumonas, Toxothrix, Trabulsiella, Treponema, Trichlorobacter, Trichococcus, Tropheryma, Tsukamurella, Turicella, Turicibacter, Tychonema, Ureaplasma, Ureibacillus, Vagococcus, Vampirovibrio, Varibaculum, Variovorax, Veillonella, Verrucomicrobium, Verrucosispora, Vibrio, Victivallis, Virgibacillus, Virgisporangium, Virgosporangium, Vitellibacter, Vitreoscilla, Vogesella, Volcaniella, Vulcanithermus, Waddlia, Weeksella, Weissella, Wigglesworthia, Williamsia, Wolbachia, Wolinella, Xanthobacter, Xanthomonas, Xenophilus, Xenorhabdus, Xylanimonas, Xylella, Xylophilus, Yersinia, Yokenella, Zavarzinia, Zobellia, Zoogloea, Zooshikella, Zymobacter, Zymomonas, and Zymophilus.
SUBSTITUTE SHEET (RULE 26) Malaria
[0148] Malaria has an enormous global health impact, with an estimated 228 million cases and
405,000 deaths in 2018 (1). In 2007, the World Health Organization (WHO) endorsed the ambitious goal of eradicating malaria, but the decline has stalled and even reversed in some regions since 2014 (1). Malaria control strategies are thwarted in part by asymptomatic carriers, who serve as parasite reservoirs for ongoing spread. Low density infections (< 100 parasites per microliter blood) are particularly common in low-endemnicity settings, and fall below the limit of detection (LOD) of both light microscopy and antigen-based malaria rapid diagnostic tests (RDT), which are the primary diagnostics used worldwide. Submicroscopic carriers may be responsible for 20-50% of all human-to- mosquito transmission (2).
[0149] Highly sensitive, field-applicable diagnostic devices compatible for use in RLS are required for detection of residual infections in pre-elimination areas. The Malaria Eradication Research Agenda (malERA) Consultative Group on Diagnoses and Diagnostics and the WHO determined that a diagnostic capable of detecting two parasites per microliter would be a “significant improvement on expert microscopy” (3, 4).
[0150] While light microscopy remains the gold standard for distinguishing Plasmodium species, it requires skilled technician interpretation and is time -intensive. RDT strengths include point- of-care (POC) utility and an intuitive format, but most tests target Plasmodium falciparum and are incapable of species-specific identification - a critical clinical limitation as P.vivax and P. ovale uniquely require an 8 -aminoquinolone (i.e., primaquine or tafenoquine) therapy to prevent relapse. Although there are sustained calls for more sensitive, non-falciparum malaria diagnostics, this remains an ongoing diagnostic gap (5). Additionally, the most common RDT antigen target for P. falciparum, histidine-rich protein 2 (HRP2), persists for several weeks after resolution of infection contributing to false positives and limited surveillance utility (6). A worrisome rise in HRP2 gene deletions over the past two decades also renders many RDTs obsolete (40% of parasites in some areas of South America) (7, 8).
[0151] Molecular methods for DNA detection, such as polymerase chain reaction (PCR), are capable of much higher sensitivity and specificity, confirmed by surveillance surveys where the prevalence of infection estimated by light microscopy was half of that measured by PCR (9). Yet PCR remains a high-complexity technology requiring expensive laboratory equipment, personnel training, and nucleic acid extraction sample preparation, making it impractical for resource-limited settings (RLS). The furthest developed commercial nucleic acid amplification tests (NAAT) for malaria are loop-mediated isothermal amplification (LAMP)-based assays, but they have exhibited disappointing sensitivity in field studies in comparison to PCR and require separate nucleic acid extraction steps (10- 12).
SUBSTITUTE SHEET (RULE 26) [0152] Most NAATs for pathogen detection require nucleic acid extraction via multi-step commercial kits involving numerous specimen transfers, laboratory infrastructure (flow-columns, management of biohazardous wastes such as chaotropic agents, etc ), and 30 minutes or more of preassay preparation time This is not practically implementable for POC testing, and sample preparation remains a general bottleneck for adoption of nucleic acid technologies, particularly for RLS (13, 14).
[0153] Described herein, in one embodiment, is a CRISPR (clustered regularly interspaced short palindromic repeats)-based diagnostic for ultrasensitive detection and differentiation of Plasmodium falciparum, vivax, ovale, and malariae. using the nucleic acid detection platform SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing). Provided herein, in one embodiment, is a streamlined, field-applicable diagnostic comprised of 10-minute S-PREP (SHERLOCK Parasite Rapid Extraction Protocol) followed by SHERLOCK for 60 minutes for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. The inventors optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. These P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (five P. falciparum and ten P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid POC clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections.
Kits
[0154] Also provided herein are kits for nucleic acid preparation (i.e., S-PREP), which at a minimum include a reducing agent and a metal ion chelating resin in aqueous suspension or for preparation of an aqueous suspension. In some embodiments, the reducing agent is dithiothreitol (DTT) and the metal ion chelating resin comprises paired iminodiacetate ions. The reducing agent and chelating resin can be provided in an admixture or can be provided separately with instructions to generate an admixture, for example, comprising 10-30% w/v of the chelating resin. The reducing agent and metal ion chelating agent can be provided in concentrated stock solutions or in pre-measured aliquots.
[0155] In addition, a kit can comprise one or more reagents for performing an isothermal amplification method, for example, one or more polymerases and one or more primers, and optionally one or more reverse transcriptases. In some embodiments, a pair of primers (forward and reverse) can be included in the kit for a desired target sequence, such as for detection of a parasite that causes malaria. Where multiple target sequences are amplified, a plurality of primer pairs can be included in the kit. A kit can include a control polynucleotide, and where multiple target sequences are amplified, a plurality of control polynucleotides can be included in the kit.
SUBSTITUTE SHEET (RULE 26) [0156] Kits can also comprise one or more of the components in any number of separate vessels, chambers, containers, packets, tubes, vials, microtiter plates and the like, or the components can be combined in various combinations in such containers Components of the kit can, for example, be present in one or more containers. In some embodiments, all of the components are provided in one container. In some embodiments, the enzymes (e.g., polymerase(s) and/or reverse transcriptase(s)) can be provided in a separate container from the primers. The components can, for example, be lyophilized, freeze dried, or in a stable buffer. In one example, polymerase(s) and/or reverse transcriptase(s) are in lyophilized form in a single container, and the primers are either lyophilized, freeze dried, or in buffer, in a different container. In some embodiments, polymerase(s) and/or reverse transcriptase(s), and the primers are, in lyophilized form, in a single container.
[0157] Kits can further comprise, for example, dNTPs used in the reaction, or modified nucleotides, vessels, cuvettes or other containers used for the reaction, or a vial of water or buffer for re-hydrating lyophilized components. The buffer used can, for example, be appropriate for both polymerase and primer annealing activity.
[0158] Kits can also comprise instructions for performing one or more methods described herein and/or a description of one or more components described herein. Instructions and/or descriptions can be in printed form and can be included in a kit insert. A kit also can include a written description of an internet location that provides such instructions or descriptions.
[0159] Kits can further comprise reagents used for detection methods, such as, for example, reagents used for FRET, lateral flow devices, dipsticks, fluorescent dye, colloidal gold particles, latex particles, a molecular beacon, or polystyrene beads.
[0160] In one embodiment, a kit for detecting malaria comprising the components described in the Examples section is specifically contemplated.
[0161] The invention may be as described in any one of the following numbered paragraphs:
[0162] 1 A composition for nucleic acid preparation, the composition comprising a reducing agent and a metal ion chelating resin in aqueous suspension.
[0163] 2 The composition of paragraph 1, wherein the metal ion chelating resin comprises paired iminodiacetate ions.
[0164] 3 The composition of paragraph 1 or paragraph 2, wherein the resin is present at a concentration of about 10% to 30% w/v.
[0165] 4 The composition of any one of paragraphs 1-3, wherein the reducing agent is dithiothreitol (DTT).
[0166] 5. The composition of any one of paragraphs 1-4, wherein the reducing agent is present at a concentration of 20-150 mM.
[0167] 6 The composition of any one of paragraphs 1-5, wherein the resin comprises styrene divinylbenzidine copolymer.
SUBSTITUTE SHEET (RULE 26) [0168] 7 The composition of any one of paragraphs 1-6, wherein the reducing agent is DTT in the range of 20-150 mM, and the resin is a styrene divinylbenzidine copolymer resin with paired iminidiacetate ions at a concentration of 10-30% w/v.
[0169] 8 The composition of any one of paragraphs 1-7, wherein the reducing agent is 50 mM DTT, and the resin is present at a concentration of 20% w/v.
[0170] 9 The composition of any one of paragraphs 1-8, wherein the composition releases nucleic acid from a biological sample upon heating, without need for a proteolytic enzyme.
[0171] 10. The composition of any one of paragraphs 1-9, which does not comprise proteinase K.
[0172] 11. The composition of paragraph 9 or paragraph 10, wherein the biological sample comprises blood or a blood fraction, a nasopharyngeal swab, an oropharyngeal swab, sputum or saliva.
[0173] 12. The composition of any one of paragraphs 9-11, wherein the blood fraction comprises erythrocytes.
[0174] 13. A composition consisting essentially of an aqueous buffer, DTT and a metal ion chelating resin.
[0175] 14. The composition of any one of paragraphs 1-13, in admixture with a biological sample.
[0176] 15. The composition of paragraph 14, wherein the composition of any one of paragraphs 1-13 is present at about a 3: 1 ratio relative to biological sample by volume.
[0177] 16. The composition of paragraph 14 or paragraph 15, wherein the biological sample comprises blood or a blood fraction, a nasopharyngeal swab, an oropharyngeal swab, sputum or saliva.
[0178] 17. The composition of paragraph 16, wherein the blood fraction comprises erythrocytes.
[0179] 18. The composition of paragraph 16 or paragraph 17, wherein the blood or blood fraction has previously been dried on a solid support.
[0180] 19. The composition of paragraph 18, wherein the solid support is present in the admixture.
[0181] 20. The composition of any one of paragraphs 1-19, further comprising reagents sufficient to perform an isothermal nucleic acid amplification.
[0182] 21. The composition of any one of paragraphs 1-20, further comprising reagents sufficient to perform detection of a target nucleic acid.
[0183] 22. The composition of paragraph 21, wherein the detection of a target nucleic acid comprises SHERLOCK detection.
[0184] 23. The composition of any one of paragraphs 1-22, which substantially prevents targetindependent cleavage of a SHERLOCK reporter nucleic acid in the presence of a biological sample.
[0185] 24. A method of preparing a biological sample for nucleic acid analysis, the method comprising contacting a composition of any one of paragraphs 1-13 with the biological sample, and heating the resulting mixture to at least 80°C.
[0186] 25. The method of paragraph 24, wherein the ratio of the composition of any one of paragraphs 1-13 to biological sample is about 3: 1.
SUBSTITUTE SHEET (RULE 26) [0187] 26. The method of paragraph 24 or 25, wherein heating is performed for 2-20 minutes.
[0188] 27. The method of any one of paragraphs 24-26, wherein heating is performed at about 95°C for about 10 minutes.
[0189] 28. The method of any one of paragraphs 24-27, wherein the biological sample comprises:
(i) blood or a fraction thereof comprising erythrocytes,
(ii) a nasopharyngeal swab,
(iii) an oropharyngeal swab,
(iv) sputum, or
(v) saliva.
[0190] 29. The method of any one of paragraphs 24-28, wherein the biological sample had previously been dried on a solid support.
[0191] 30. The method of paragraph 29, wherein the solid support is included in the mixture.
[0192] 31. The method of any one of paragraphs 24-30, wherein the biological sample comprises or is suspected of comprising an intracellular parasite, bacterium, or a virus.
[0193] 32. The method of paragraph 31, wherein the intracellular parasite comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Babesia sp. Leishmaniasis spp. Toxoplasmosis spp. or filarial nematodes.
[0194] 33. The method of paragraph 31 , wherein the virus comprises Dengue virus, Zika virus, Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoVl), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Heptatis E, Herpes virus, Varicella virus, Cytomegalovirus, Epstein-Barr virus, Human herpesvirus 6, Human herpesvirus8, adenovirus, influenza, parainfluenza, respiratory syncytial virus, or Chikungunya virus,
[0195] 34. The method of paragraph 31, wherein the bacterium comprises a gram negative bacterium, a gram positive bacterium or an intracellular bacterium.
[0196] 35. The method of any one of paragraphs 28-34, wherein heating the mixture promotes red blood cell lysis and lysis of intracellular parasite or virus present and releases nucleic acids from the parasite or virus if present, and wherein the resin chelates multivalent metal ions, thereby inhibiting nuclease degradation of parasite and/or viral nucleic acid.
[0197] 36. The method of any one of paragraphs 24-35, further comprising, after the heating step, contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids.
[0198] 37. The method of paragraph 36, further comprising incubating the mixture under conditions and for a time sufficient to amplify the one or more target nucleic acids if present.
[0199] 38. The method of any one of paragraphs 36-37, wherein the isothermal amplification is a recombinase polymerase amplification (RPA) reaction.
SUBSTITUTE SHEET (RULE 26) [0200] 39. The method of any one of clams 36-38, wherein no additional sample processing or liquid transfer steps are required to permit isothermal amplification of one or more target nucleic acids.
[0201] 40. The method of any one of paragraphs 36-39, wherein the reagents sufficient for isothermal amplification are lyophilized prior to the step of contacting the mixture with the reagents.
[0202] 41. The method of any one of paragraphs 24-40, further comprising, after the heating step, contacting the mixture with reagents sufficient for SHERLOCK detection of one or more target nucleic acids.
[0203] 42. The method of paragraph 41, further comprising incubating the mixture under conditions and for a time sufficient to generate a SHERLOCK detection signal for one or more target nucleic acids if present.
[0204] 43. The method of paragraph 41 or 42, wherein the reagents sufficient for SHERLOCK detection are lyophilized prior to the step of contacting the mixture with the reagents.
[0205] 44. The method of any one of clams 41-43, wherein no additional sample processing or liquid transfer steps are required to permit SHERLOCK detection of one or more target nucleic acids.
[0206] 45. The method of any one of paragraphs 24-35, further comprising, after the heating step, contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids and reagents sufficient for SHERLOCK detection of one or more target nucleic acids.
[0207] 46. The method of paragraph 45, wherein the reagents sufficient for isothermal amplification and the reagents sufficient for SHERLOCK detection are lyophilized prior to the contacting step, wherein the contacting step reconstitutes the lyophilized reagents and permits amplification and SHERLOCK detection of one or more target nucleic acid molecules if present.
[0208] 47. The method of paragraph 45 or paragraph 46, further comprising incubating the mixture under conditions and for a time sufficient to amplify one or more target nucleic acids and to generate a SHERLOCK detection signal for one or more target nucleic acids if present.
[0209] 48. A method of amplifying one or more target nucleic acids in a biological sample comprising nucleic acids, the method comprising:
[0210] (i) contacting a composition of any one of paragraphs 1-13 with the biological sample;
[0211] (ii) heating the mixture resulting from step (i) to at least 80°C for a time sufficient to release nucleic acids in the biological sample;
[0212] (iii) after step (ii), contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids;
[0213] (iv) incubating the mixture of step (iii) under conditions and for a time sufficient to generate an isothermal amplification product for one or more target nucleic acids.
[0214]
SUBSTITUTE SHEET (RULE 26) [0215] 49. The method of paragraph 48, wherein no additional sample processing or liquid transfer steps are required to generate an amplification product for one or more target nucleic acids.
[0216] 50. The method of paragraph 48 or paragraph 49, wherein steps (i)-(iv) are performed in the same reaction or sample container.
[0217] 51. A method of detecting one or more target nucleic acids in a biological sample, the method comprising:
(i) contacting a composition of any one of paragraphs 1-13 with the biological sample;
(ii) heating the mixture resulting from step (i) to at least 80°C for a time sufficient to release nucleic acids in the biological sample;
(iii) after step (ii), contacting the mixture with reagents sufficient for isothermal amplification and SHERLOCK detection of one or more target nucleic acids; and
(iv) incubating the mixture of step (iii) under conditions and for a time sufficient to permit isothermal amplification and production of a SHERLOCK detection signal for one or more target nucleic acids present in the sample.
[0218] 52. The method of paragraph 51, wherein no additional sample processing or liquid transfer steps are required to generate SHERLOCK detection signal for one or more target nucleic acids present in the sample.
[0219] 53. The method of paragraph 51 or paragraph 52, wherein steps (i)-(iv) are performed in the same reaction or sample container.
[0220] 54. A kit for nucleic acid preparation and/or detection, the kit comprising a reducing agent and a metal ion chelating resin in aqueous suspension, and packaging materials therefor.
[0221] 55. The kit of paragraph 54, wherein the metal ion chelating resin comprises paired iminodiacetate ions.
[0222] 56. The kit of paragraph 54 or 55, wherein the reducing agent is dithiothreitol (DTT).
[0223] 57. The kit of any one of paragraphs 54-56, which does not contain proteinase K.
[0224] 58. The kit of any one of paragraphs 54-57, wherein the reducing agent is present at a concentration of 20-150 mM.
[0225] 59. The kit of any one of paragraphs 54-58, wherein the resin comprises styrene divinylbenzidine copolymer.
[0226] 60. The kit of any one of paragraphs 54-59, further comprising reagents sufficient for an isothermal nucleic acid amplification reaction.
[0227] 61. The kit of paragraph 60, wherein the reagents sufficient for an isothermal nucleic acid amplification reaction are lyophilized.
[0228] 62. The kit of any one of paragraphs 54-60, further comprising reagents sufficient for a SHERLOCK detection reaction.
[0229] 63. The kit of any one of paragraphs 54-61, wherein the reagents sufficient for a SHERLOCK detection reaction are lyophilized.
SUBSTITUTE SHEET (RULE 26) [0230] 64. The kit of any one of paragraphs 60 or 61, wherein reagents sufficient for an isothermal nucleic acid amplification reaction and reagents sufficient for a SHERLOCK detection reaction are lyophilized in one composition.
EXAMPLES
EXAMPLE 1
[0231] Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care (POC) diagnostics are also lacking for non-falciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods such as polymerase chain reaction (PCR) have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS.
[0232] Here, the inventors describe the development of field-applicable, 60-minute, ultrasensitive malaria diagnostic tools using the CRISPR (clustered regularly interspaced short palindromic repeats)-based nucleic acid detection platform SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing) (15-19) for detection of P. falciparum, P. vivax, P. ovale, and P. malariae. These isothermal, lyophilized, one-pot SHERLOCK assays for ultrasensitive detection are coupled with a novel simplified sample preparation method: S-PREP (SHERLOCK Parasite Rapid Extraction Protocol) that eliminates the need for commercial kit nucleic acid extraction. Building from prior work on aP. falciparum SHERLOCK assay, a simplified field-ready SHERLOCK diagnostic was demonstrated, and the accuracy of the diagnostic was confirmed on simulated whole blood, serum, and dried blood spot samples, as well as clinical samples from patients with P. falciparum and P. vivax infection.
[0233] Design and optimization of malaria SHERLOCK diagnostic. FIG 1 illustrates the workflow of a simplified SHERLOCK diagnostic. This test combines a 10-minute sample preparation step and a 60-minute SHERLOCK assay prior to endpoint analysis via lateral flow strip or fluorescence measurement. CRISPR-based diagnostics utilize the programmable endonucleases (Cas enzymes) of CRISPR-associated microbial adaptive immune systems. Casl2a (also known as Cpfl) is one such RNA -guided, DNA-cleaving enzyme, which can be programmed with CRISPR guide RNAs (gRNA) to construct highly sensitive and specific nucleic acid detection platforms (15-19). Programmed Casl2a is activated through recognition of its dsDNA target and exhibits indiscriminate, non-specific DNase activity that cleaves non-target DNAs. This non-specific degradation of fluorophore -quencher labeled reporter ssDNA is exploited to detect the presence ofthe dsDNA target that activated Cas 12a. To further decrease the limit of detection (LOD), a reverse-transcriptase recombinase polymerase amplification
SUBSTITUTE SHEET (RULE 26) (RT-RPA) step is added before Casl2a detection to increase target DNA concentrations (FIG. 2). RPA is a powerful isothermal nucleic acid amplification tool comprised of three core enzymes: a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing polymerase that coordinates DNA synthesis from primer-paired target DNA (20).
[0234] For endpoint analysis, released fluorophore from cleaved reporter ssDNA was measured by a plate reader or a handheld fluorimeter. Particularly in RLS, use of a handheld fluorimeter enables a field-applicable readout method. The inventors did not find a significant difference in the sensitivity performance between machines and observed a similar 7-10 fold-change in fluorescence between platforms although they had different baselines (data not shown). For use of the handheld fluorimeter, SHERLOCK reactions (50 pL) were performed combined in triplicate (150 pL) to increase the volume size for appropriate instrument reading.
[0235] The assays are also adapted for endpoint detection via lateral flow strip based upon degradation of ssDNA reporter that is labeled on opposing ends with FAM and biotin. The FAM- biotinylated reporter conjugates to anti-FAM gold nanoparticles contained within commercial lateral flow strips. If the reporter remains intact, FAM-labeled-reporter/anti-FAM conjugates accumulate at the first line of the strip immobilized by streptavidin (control line). In presence of activated Casl2a, the reporter is cleaved and freed FAM/anti-FAM conjugates are released to collect at the second line of the lateral flow strip containing anti-rabbit antibody (test line), which binds anti-FAM antibodies (FIG. 2). [0236] There are many Cas enzymes that could have been used. For proof of concept, Casl2a was selected (as opposed to the Casl3 family (21, 22), which also has non-specific nuclease activity) so DNA targets could be directly detected instead of RNA, particularly in dried blood spots where RNA may be degraded. The rapid enzymatic kinetics of Cas 12a also make this nucleic-acid based technology comparable to the POC format of antigen -based lateral flow immunoassays. Cas 12a bound to its dsDNA activator is capable of -1250 turnovers per second with a catalytic efficiency (Ccat/KM - I.7x 109 s'1 M 1 ) approaching the rate of diffusion (17). The addition of a reverse transcriptase enzyme further enhances the sensitivity by transcribing multiple-copy RNAs from a target sequence into DNA for detection. SHERLOCK parameters, including reaction temperature, RPA primer concentration, RT commercial brand, and ssDNA reporter concentration were optimized (data not shown). The reactions was also lyophilized into a pellet to be resuspended with S-PREP treated sample for cold-chain independence in the field, and importantly, also improved the LOD by increasing sample input volume.
[0237] RPA primer and gRNA selection. The SHERLOCK assays used herein were designed to detect four of the most common pathogenic species of malaria. The inventors iterated a two-step design process of RPA primer screen followed by gRNA screen. RPA primer targets were identified by reviewing the literature for the best-performing NAATs and searching for conserved and specific sequences from alignment of species-specific strains available from the National Center for Biotechnology Information (NCBI). For P . falciparum 18S rRNA, mitochondrial (cytochrome oxidase III, cytochrome B), and subtelomeric (Pfr364) targets were screened (23-29). The Pfr364 target, which
SUBSTITUTE SHEET (RULE 26) is a species-specific, non-coding subtelomeric repeat sequence present in 41 copies on the P. falciparum genome, had the best signal in comparison to the othertargets (data not shown). Moreover, the selected gRNA had >90% sequence homology among all assembled P. falciparum genomes available in NCBI as well as 86% of sequences from the Pf3k dataset (an open-access collaboration and deep-genomic sequencing database) accessed via IGV (Integrative Genomics Viewer) (30). For P. vivax, an 18S rRNA and mitochondrial target were tested, and it was found that the mitochondrial target worked best (copy number per parasite can be as high as 20) (27, 31). For P. ovale and P. malariae, different regions of the 18S rRNA gene known to be conserved species-specific targets (27, 32, 33) typically present in 4- 8 copies per genome (notably, copy number is variable and depends on the parasite life cycle stage) were tested. The sequence targets’ primers and gRNA were mapped and aligned to the corresponding regions in off-target Plasmodium species (either homologous genes, or analogous sequences identified using NCBI’s Basic Local Alignment Search Tool (BLAST) with the lowest E-values). Despite overlap in RPA primers, which can tolerate significant sequence mismatch, it was found that few-nucleotide differences in gRNA sequence were sufficient to obtain discriminating species-specific detection.
[0238] Five forward (F1-F5) and five reverse primers (R1-R5) per sequence target were constructed using guidance provided by the TwistDx manufacturer; primers were 30-40 nucleotides long, with goal amplicons of 100-200 base-pairs in length. Forward and reverse primers were paired for a total of 25 combinations (FLR1-5, F2:Rl-5, F3:Rl-5, F4:Rl-5, F5:Rl-5) for each sequence target and 2-3 of the best-performing pairs were selected for the optimization of gRNA design (data not shown). RPA was performed according to the manufacturer’s instructions as described in the Methods. Casl2a recognizes a short nucleotide sequence (TTTN) called the protospacer adjacent motif (PAM) for generation of distal dsDNA cleavage, and 2-4 gRNAs based upon the TTTN PAM were designed within the RPA amplicon. The RPA reaction for each primer set was then transferred to a Cas reaction as described in the Methods, and fluorescent kinetics were monitored for selection of best performing gRNAs (Table 1).
[0239] Sample preparation. Accessing sample nucleic acids in a field-applicable manner involves overcoming several challenges. Preparation requires lysing the red blood cell and parasite membrane (with the exception of the invasive merozoite form, all blood-stage parasites are intraerythrocytic), deactivating multiple inhibitory blood components, and importantly, appropriately deactivating nucleases that could shear the ssDNA reporter and lead to a false positive signal. The requirement for simplicity and low cost ruled out commercial nucleic acid extraction kits. To test sample preparation methods, simulated whole blood samples of live intraerythrocytic P. falciparum spiked into purchased EDTA-treated human blood (VWR International, Radnor, PA) were used to a final 1 fM (602 parasites/pL) concentration for rehydration of the one-pot lyophilized P. falciparum SHERLOCK assay described herein.
[0240] One approach that did not work was HUDSON (Heating Unextracted Diagnostic
Samples to Obliterate Nucleases), a simplified sample preparation method for viral nucleic acid
SUBSTITUTE SHEET (RULE 26) extraction (16) compatible with Cast 3 SHERLOCK. In HUDSON, whole blood samples are pre-treated with lOOmM TCEP (tris(carboxyethyl)phosphine) and ImM EDTA (ethylenediaminetetraacetic acid) to augment protein deactivation, followed by a two-step process of nuclease deactivation (heating for 5 min at 50°C) followed by viral inactivation (heating for 5min at 64°C). HUDSON -treated simulated whole blood samples produced minimal signal, likely from not accessing the intracellular parasitic nucleic acid.
[0241] The inventors therefore assessed alternative simplified sample preparation protocols described in the Methods including various detergents, thermal lysis, and chemical deactivation protocols (FIGs. 3A-B). It was discovered that treating samples with 50mM DTT and lOmM EGTA followed by 95°C incubation for 10 minutes resulted in a robust SHERLOCK signal, although some variability was observed in the no-template control signal that was attributed to background nucleases in different blood aliquots . However, when the DTT/EGTA/95 °C sample preparation method was tested on patient P. falciparum and P. vivax serum samples from the Dominican Republic, bidirectional crossreactivity of the species-specific SHERLOCK assays was observed. Using the P. falciparum-specific assay, P. vivax patient serum samples produced a false positive signal (FIG. 4A). P. falciparum patient samples also produced a false positive signal using the P. vivax-specific assay (FIG. 4B).
[0242] The false positive signals were eliminated, however, when DNA from the same crossreacting P. vivax and P. falciparum patient serum samples was extracted via QIAamp™ DNA mini kit (Qiagen, Hilden, Germany), spiked into a healthy commercial serum no-template control [lOng extracted DNA into 20pL serum (Sigma Aldrich, St. Louis, MO)], and retested (FIGs. 4A, 4B). Furthermore, the extracted patient serum DNA maintained a robust species-specific signal with the appropriate Plasmodium species-specific assay. Extracted nucleic acid reflected combined human and parasite DNA, with numbers of human sequences dwarfing numbers of parasite sequences, and the highly sensitive and specific performance of the appropriate SHERLOCK assay on the extracted nucleic acid made cross-reactivity due to human DNA unlikely. These results were also observed on all five P. falciparum and all ten P. vivax specimens making co-infection unlikely and the specimens had all undergone species-specific qualitative PCR testing (ARUP, Salt Lake City, UT). Without wishing to be bound by theory, it was hypothesized that the cross-reactivity could be secondary to non-specific ssDNA reporter cleavage from higher concentrations of nucleases in “sick” versus “healthy” serum that resulted in incomplete deactivation of nucleases in “sick serum” by the DTT/EGTA/95°C simplified preparation method.
[0243] This hypothesis was confirmed when S-PREP (SHERLOCK Parasite Rapid Extraction
Protocol) was developed using a buffer comprised of a stronger chelating agent: 20% w/v Chelex®-100 (Bio-Rad, Hercules, USA) suspended in TE buffer with 50mM DTT. Chelex®-100 is a resin containing styrene divinylbenzene copolymers with paired iminodiacetate ions that act as chelating groups in binding polyvalent metal ions (34). Nucleases require metal ions as cofactors and therefore chelating agents inhibit their activity. S-PREP is a simplified sample preparation method where sample is diluted
SUBSTITUTE SHEET (RULE 26) 1:3 (5 pL into 15 pL of S-PREP buffer) followed by heating to 95°C for 10 minutes. False positive signals of serum samples were eliminated using S-PREP (FIGs. 4C-4D). It was concluded that higher concentrations of nucleases present in “sick” serum (patients sick with another disease but not the target disease) necessitate stronger nuclease deactivation procedures. The inventors are the first to report on this cross-reactivity in non-nucleic-acid extracted clinical samples for SHERLOCK as the inventors are not aware of other studies comparing performance using unextracted samples against controls from patients sick with a different disease (instead of only comparing to healthy control specimens). This highlights the importance of considering baseline nuclease activity in specimen types with CRISPR- based assays as the readout is dependent on reporter nucleic acid cleavage and contaminating nucleases are a major concern for false positives. Importantly, it was demonstrated that S-PREP can deactivate high levels of nucleases and the absence of false positives was confirmed in the clinical sample set with 100% specificity (P. falciparum n = 4 serum, n = 1 whole blood; P. vivax n = 10 serum; healthy serum patient controls n =5, FIG. 4E). It was additionally found that S-PREP was compatible with RNA-only simulated samples, despite its increased susceptibility to hydrolysis in comparison to DNA, demonstrated by detection of an RNA-only synthetic target prepared using S-PREP (SARS-CoV-2 RNA target detected in novel SHERLOCK assay, data not shown).
[0244] To further assess the field versatility of this work, the S-PREP/SHERLOCK diagnostic was tested on simulated samples from multiple specimen collection types. Live intraerythrocytic P. falciparum was spiked into whole blood and plasma stored in multiple different specimen collection tubes (acid-citrate dextrose, EDTA K-2, EDTA K-3, Na heparin, Na citrate, plasma heparin, and plasma EDTA) to a final 1 fM (602 parasites/pL) concentration and prepared these samples with S-PREP for rehydration of our SHERLOCK assay as described herein. Although many of these additives are known PCR inhibitors, all simulated samples were able to produce a distinguishable signal from the notemplate control (data not shown). The compatibility of SHERLOCK with unextracted samples from multiple specimen tube types emphasizes its unique robustness, versatility, and ultimately suitability for RLS.
[0245] Performance and readout of malaria SHERLOCK diagnostic. The analytical sensitivity of these assays was determined using industry standard definitions of the diagnostic LOD to guarantee a 95% probability of successful detection. Septet replicate testing was performed on three different runs on simulated whole blood samples (described in Methods) for each Plasmodium species and probit analysis was used to establish: P. falciparum 0.36 parasite/pL blood [95% confidence interval (CI) 0.23 - 1.0], P. vivax 1.2 parasites/pL (95% CI 0.52 - 6.2), P. ovale 2.4 parasites/pL (95% CI 0.81 - 19), andP malariae 1.9 parasite/pL (95% CI 1.1 - 12) (Table 2, FIG. 5A). This reaches the WHO LOD goal for low endemnicity (asymptomatic carriage) settings and is notable in that SHERLOCK was capable of attomolar to subattomolar detection in the absence of commercial kit nucleic acid extraction and sample nucleic acid concentration. This also emphasizes the ultrasensitive capacity of SHERLOCK in that the best detection level (0.36 parasite/pL for P. falciparum) closes in
SUBSTITUTE SHEET (RULE 26) on the theoretical LOD of the engineering design. Using 12.5 nL of sample input, a 0.3 parasite/pL concentration sample has a 95% probability of containing at least one parasite (and therefore being detectable) following the Poisson distribution (data not shown).
[0246] The CRISPR diagnostic described herein can also detect clinically relevant levels of parasitemia in 40 minutes or less from unextracted blood samples (10 minute S-PREP followed by 30 minute SHERLOCK) with better sensitivity than existing POC antigen-based RDTs - filling an important clinical diagnostic gap for HRP-2 deletion P. falciparum, and non-falciparum malaria. A 0.001% parasitemia (assuming a red blood cell mean corpuscular volume of 80 femtoliters and hematocrit of 45%) corresponds to ~60 parasites/pL (lOOaM concentration), for which a 30-minute detectable signal difference between the no-template control and infected blood is readily apparent (FIG. 5B). This level of parasitemia would likely be missed on RDT or light microscopy (a technician would have to view 100,000 RBCs to view an infected RBC, which is theoretically possible though would require considerable effort). Lastly, while there is no consensus definition of asymptomatic malaria, some have used parasite density cutoffs of 5,000 parasites/pL blood (~8.5 fM) as a threshold (vaccine trials and epidemiological studies), which is a rapidly detectable concentration with SHERLOCK (35-37).
[0247] The analytical specificity of the assays in this study was determined using simulated clinical samples at a 10 fM concentration (6,020 parasites/pL) and no detection of non-target Plasmodium species was determined, confirming high specificity (FIG. 5C). Without wishing to be bound by theory, it is surmised that the highly specific performance of these assays is likely attributable to a two-step target selection via RPA primer match and amplification, followed by gRNA match and Cas activation. For clinical sensitivity and specificity, the inventors were able to detect and differentiate five P. falciparum (four serum, one whole blood) and 10 P. vivax samples with 100% accuracy (FIG. 4E). Deidentified clinical samples were purchased from BocaBiolistics (Pompano Beach, USA) and came from symptomatic patients from the Dominican Republic. They had been previously characterized by both the BinaxNOW Malaria RDT (Alere, Waltham, MA) and species-specific qualitative PCR (ARUP, Salt Lake City, USA), demonstrating that this diagnostic had 100% concordance with these methods in the limited clinical set.
[0248] In addition, simulated dried blood spots were prepared to a 2aM (one parasite per microliter blood) concentration for each of the four Plasmodium species and tested with the S- PREP/SHERLOCK protocol with modifications as described in the methods. A robust fluorescence signal was demonstrated at the one-hour time point that was significantly different from the no-template control. The only notable difference in assay performance compared with whole blood samples was a greater no-template control signal in simulated DBS samples - likely from autofluorescence from the paper substrate (FIG. 5D).
SUBSTITUTE SHEET (RULE 26) [0249] In addition to establishing analytical LOD via fluorescent measurement, the inventors also demonstrated a lateral flow readout given its ease of use in RLS. It was found that a clearly visible band was distinguishable at 50 aM (30 parasites/pL) for all of the Plasmodium species assays (FIGs. 6A-6D); this LOD is higher than that of the fluorescent readout, but it is still lower than best-in-class contemporary RDTs (38).
[0250] Demonstrated herein is a simplified SHERLOCK diagnostic comprised of a 10-minute
S-PREP followed by SHERLOCK for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. These advancements could fill significant gaps in malaria diagnostics by establishing a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers and malaria eradication, and a POC clinical diagnostic for HRP-2 deletion P. falciparum infections and nonfalciparum malaria species. This is a particularly important goal for P. vivax - the most widely distributed malaria pathogen worldwide, missed by many contemporary RDTs, and requiring different therapy than P. falciparum.
[0251] The assays described herein were rigorously optimized for field implementation. The inventors demonstrated a fully lyophilized one-pot SHERLOCK protocol on clinical samples only requiring rehydration of the reaction with sample, eliminating the labor and contamination risk of multiple specimen transfer steps. Lyophilization also enables cold-chain independence, and improves the LOD by increasing sample input volume (12.5 pL versus 4.25 pL blood input in a non-lyophilized reaction).
[0252] These results highlight the applicability of SHERLOCK platforms to the arena of global health and RLS. SHERLOCK is a cost-effective technology estimated at $0.61 (USD) per test (15) given its lyophilizable format and lateral flow readout capability This work brings the platform closer to clinical care in demonstrating a field-ready SHERLOCK diagnostic. Key features include simplified sample preparation without nucleic acid extraction, isothermal assay conditions (40°C) independent of a thermocycler, a lyophilized integrated assay, and field-applicable readouts including use of a handheld fluorometer or lateral flow strip. The ultrasensitive LOD of these assays was validated using industry standard protocols of replicate testing.
[0253] In addition, these studies provided critical insight into engineering design considerations for ultrasensitive microvolume and SHERLOCK-based diagnostics. Firstly, the inventors highlighted an underappreciated concept that when reaching attomolar and subattomolar concentrations where assays are capable of one copy per assay detection levels, the rate-limiting consideration is the probability of pathogen presence in the sample input volume - no longer approximated by a Gaussian but instead Poisson distribution as the probability of blank inputs is significant. For the cumulative distribution function to reach 100% for a 2aM pathogen concentration (guaranteeing at least 1 target copy in the sample volume), the sample input volume must be at least 12
SUBSTITUTE SHEET (RULE 26) microliters (data not shown). Secondly, it was demonstrated that a key limitation of SHERLOCK assays, in general, is that their readout dependence on ssDNA cleavage makes the assays highly susceptible to false positives in the presence of contaminating nucleases While all NAATs are at risk of target degradation in the presence of nucleases, appropriate deactivation is crucially important for SHERLOCK assays, and it was observed that specimens may very well have differing levels of nucleases depending on disease state, sample type, and even blood aliquot.
[0254] Notably, as diagnostics become increasingly capable of ultrasensitive limits of detection, it is important to consider whether technologies may detect pathogens below the level of clinical and epidemiological relevance. Future studies will also be needed to better characterize the bloodstream clearance kinetics of ultra-low parasitemia. Currently, it is unknown whether trace amounts of DNA may persist for several days after treatment or prophylactic therapy, and falsely raise concerns of drug failure. Furthermore, while evidence suggests that asymptomatic carriers are likely contributing to ongoing spread of malaria (2), it is unclear if there is a pathogen burden cutoff below which transmission is unlikely.
[0255] In summary, this malaria SHERLOCK diagnostic for ultrasensitive and specific
Plasmodium species identification is a promising new tool that moves this technology closer to clinical POC application in resource-limited settings. Future work will be needed to optimize performance in field settings and define the utilization of ultrasensitive detection for clinical and policy decisionmaking.
Table 1 : Best performing RPA primers and gRNA sequences for development of Plasmodium SHERLOCK assays.
Figure imgf000050_0001
SUBSTITUTE SHEET (RULE 26)
Figure imgf000051_0001
Table 2: Analytical sensitivity of Plasmodium species SHERLOCK. Results of replicate testing at five different calibration standard concentrations near the expected LOD (replicates testing positive/replicates tested for determination of 95% LOD by probit analysis).
Figure imgf000051_0002
* p/pL: parasites per microliter in contrived calibration sample (prior to S-PREP dilution)
Materials and Methods
[0256] Simulated samples and clinical samples. P. falciparum simulated samples were prepared by either serially diluting live parasites into whole blood or serially diluting purified whole genomic DNA into whole blood. To prepare simulated infected whole blood with live intraerythrocytic P. falciparum, the 3D7 strain (obtained from the Walter & Eliza Hall Institute) of Plasmodium falciparum was cultured in human red blood cells (RBCs) at 4% hematocrit to ~2% parasitemia in RPMI 1640 supplemented with 0.5% Albumax II, 50 mg/liter hypoxanthine, 0.21% sodium bicarbonate, and 25 mM HEPES, as previously described (39). Aliquots of cultures with known parasitemia (parasites per microliter RPMI 1640) determined by microscopy via triplicate field stained blood smears with average parasitemia calculated were spiked into uninfected whole blood (VWR International, Radnor PA) stored with EDTA anticoagulant to make serial dilutions. For LOD calculations, extracted whole genomic DNA harvested from cultured Plasmodium falciparum via QIAamp Blood Mini Kit (Qiagen, Hilden, Germany) was quantified (ng/uL) on the NanoDrop 2000 (Thermo Fisher Scientific Inc, Waltham, MA), and spiked into uninfected whole blood or serum and serially diluted. Molar concentration was calculated by the estimated molecular weight of a 22.8 megabase genome (40). dsDNA molecular weight can be estimated from genome size by multiplying
SUBSTITUTE SHEET (RULE 26) the number of base pairs of dsDNA by the average molecular weight of a base-pair (650 g/mol) (41). The molar concentration calculated by dividing the mass of a sample by its molecular weight can be translated to copies of target (parasites) per unit volume by multiplying by Avogadro’s number (6.022 x 1023 molecules/mole).
[0257] For P. vivax, nucleic acids were obtained from patient clinical samples via QIAamp
Blood Mini Kit, measured DNA concentration via Nanodrop, and the estimated molecular weight presuming a 26.8 megabase genome (42) was used to calculate a molar concentration. Serial dilutions of concentrated DNA into whole blood were used for LOD measurements (FIG. 5).
[0258] For P. malariae and P. ovale, plasmids containing the small subunit ribosomal RNA genes ( 18S) MRA-179 and MRA-180 were obtained. After quantification of plasmid on Nanodrop and using the estimated molecular weight based on known plasmid size (5100 base-pairs and 5000 basepairs, respectively) for calculation of molar concentration, diluted plasmids were serially diluted into whole blood to determine the LOD (FIG. 5).
[0259] Dried blood spots (DBS) were simulated by deposition of 50pL of simulated blood samples (live intracellular P. falciparum spiked into whole blood, P. vivax purified whole genomic DNA spiked into whole blood, P. malariae MRA-179 plasmid spiked into whole blood, P. ovale MRA- 180 plasmid spiked into whole blood) x2 onto Whatman 903 Protein saver cards (Thermo Fisher Scientific, Waltham, MA). The DBS were dried in ambient conditions for 3 hours and then tested as described below in the sample preparation and SHERLOCK reaction procedure.
[0260] Four serum (collected in serum separator tubes), and one whole blood (collected in K2-
EDTA tube) P. falciparum and ten serum (collected in serum separator tubes) P. vivax samples from deidentified symptomatic patients in the Dominican Republic were purchased from BocaBiolistics (Pompano Beach, FL). Samples had been previously characterized by Alere BinaxNOW Malaria RDT (Waltham, MA) and qualitative species-specific PCR (ARUP, Salt Lake City, USA). All clinical samples and human red blood cell aliquots used had been previously deidentified prior to purchase.
[0261] RPA primer, gRNA screen and construction. Conserved Plasmodium regions identified from the literature and publicly accessible databases (NCBI, Pf3k, and PlasmoDB) were used to generate target RPA primers and gRNA sequences. Alignments to ensure conservation of targets across available individual species’ genome assemblies as well as exclusivity between Plasmodium species were performed using MAFFT (43) and visualized with Jalview 2.11.1.0 (44). RPA primers were purchased from Integrated DNA Technologies (IDT, Coralville, Iowa). The CRISPR gRNA was produced by in vitro transcription from synthetic DNA sequences purchased from IDT using the HiScribe™ T7 Quick High Yield RNA Synthesis kit (New England Biolabs, Ipswich, MA) and purified using the RNA Clean and Concentrator kit (Zymo Research, Irvine, MA). A quenched fluorescent single-stranded DNA (ssDNA) reporter with a 5’ end labeled FAM group and a 3’ end attached to an Iowa Black® quencher (56-FAM/TTATT/3IABkFQ) was purchased from IDT (Coralville, IA). RPA primer screens were conducted using 7.5 pl reaction volumes of RPA basic kit (TwistDx, Cambridge,
SUBSTITUTE SHEET (RULE 26) UK) spiked with unique primer sets to final concentrations as recommended per the manufacturer’s instructions: 14 mM magnesium acetate, 490 pM RPA primers each, and 0.6x rehydration buffer incubated at 40°C for 30 minutes. Initial screen gRNAs were constructed for expected RPA amplicons of different sequence targets Collateral degradation of ssDNA reporter upon Casl2a activation was measured by mixing 2 pl of a RPA primer screen reaction into a 10 pl reaction volume with final concentrations of lOOnM Casl2a (New England Biolabs, Ipswich, MA), 200nM gRNA, IxNEB 2.1 buffer (New England Biolabs, Ipswich, MA), and 1 pM ssDNA reporter. We incubated the mixture at 40°C for 120 minutes and measured fluorescence kinetics in a BioTek NEO HTS plate reader (BioTek Instruments, Inc., Winooski, VT) with readings every 3 minutes (Ex: 485 nm; Em 535 nm). Bestperforming RPA primer sets from sequence targets were selected for testing of 2-3 gRNAs constructed from RPA amplicon region, using the same protocol.
[0262] Sample preparation testing. Using live intraerythrocytic P. falciparum spiked into whole blood as a simulated malaria sample, multiple sample preparation methods were tested. All sample preparation methods tested had a final volume of 20 pL with a final P. falciparum concentration of I fM or 602 copies/pL (various methods had different dilution steps and so initial spiked concentration varied) and were tested via rehydration of the one-pot lyophilized SHERLOCK P. falciparum pellet described below. Fluorescence was measured over 1 hour at 40°C using a BioTek NEO HTS plate reader with readings every 3 minutes (Ex: 485 nm; Em 535 nm). Detergents at varying w/v% (SDS 0.5%, saponin 1%, Tween-20 1%, Triton-X 100 1%) were added to a 20 pL simulated whole blood sample along with lOOmM TCEP. Two heating sample preparation protocols were tested: (1) dilution of simulated sample 1:4 in nuclease-free water followed by 10-minute 95°C incubation (1:4 dilution required to prevent solidification when diluting with water), and (2) addition of lOOmM TCEP into the diluted simulated sample prior to 10-minute 95°C incubation. For optimization of chemical deactivation methods of nucleases and SHERLOCK inhibitors, combinations of chelators and reducing agents added to 20 pL simulated samples at concentrations demonstrated in FIG. 3B were tested.
[0263] S-PREP sample preparation. Inactivation (nucleases and inhibitors) of whole blood and serum samples was performed by dilution of sample in 1:3 ratio (12.5 pL sample: 37.5 pL S-PREP buffer); S-PREP buffer comprised Tris-EDTA buffer (Invitrogen, Carlsbad, CA) with 50mM DTT (Sigma Aldrich, St. Louis, MO) and 20% w/v Chelex®-100 (Bio-Rad, Hercules, CA). Samples were then heated to 95°C for 10 minutes. For simulated dried blood specimens, a disposable biopsy punch (VWR International, Radnor, PA) was used to make 2 mm diameter disks from DBS simulated samples that were dropped into 200 pL PCR-compatible tubes. 50 pL of S-PREP buffer was added to the tube followed by 95°C heat inactivation for 10 minutes. For testing of compatibility of S-PREP and SHERLOCK with different collection tube types, live intraerythrocytic P. falciparum spiked into whole blood collected from different collection tubes to a final 1 fM (602 parasites/ pL) concentration was prepared via S-PREP (5 pL simulated sample into 15 pL S-PREP buffer followed by 10 minute 95°C heating) and used to rehydrate the SHERLOCK lyophilized reaction described below. To demonstrate
SUBSTITUTE SHEET (RULE 26) compatibility of S-PREP with RNA, synthetic SARS-CoV-2 RNA SKU 103086 (Twist Bioscience, San Francisco, CA) was prepared using S-PREP and tested in SARS-CoV-2 SHERLOCK assay in development.
[0264] Preparation of lyophilized SHERLOCK reactions and procedure. SHERLOCK reactions were prepared to 50 pL using 100 nM Casl2a, 200 nM gRNA, 0.8x NEB buffer 2.1, 430 nM of each RPA primer, 2 U/pL ProtoScript II reverse-transcriptase (NEB, Ipswich, USA), 0.6x RPA re hydration buffer, 14 mM MgOAc, 10 mM EGTA, and 1 pM FAM-Iowa Black® quenched ssDNA fluorescent reporter. For lateral flow readout, IpM fluorophore-biotin labeled ssDNA reporter (56- FAM/TTATT/3Bio, IDT, Coralville, IA) was used instead of fluorophore-quencher reporter.
[0265] Reactions were prepared in 200 pL PCR-compatible tubes and a small opening was pierced in the cap with a 25-gauge x 5/8 (0.5 mm x 16 mm) BD PrecisionGlide Needle (Becton, Dickinson and Company, Franklin Lakes, NJ) to allow for sublimation during lyophilization. Reaction tubes were placed in a chilled metallic tube rack and submerged for 1 minute in liquid nitrogen. The snap frozen tubes and rack were wrapped in Kimwipes (Kimberly-Clark, Irving, TX) and three layers of aluminum foil. The entire bundle was then placed inside a sealed glass lyophilization chamber and connected to a freeze-drying machine (Labconco, Kansas City, MO). Lyophilization was performed for 6 hours. Activation of reaction was performed by rehydration in 50 pL of sample prepared by S-PREP (12.5 pL of sample into 37.5 pL of buffer followed by 95°C incubation). Notably, for testing of simplified sample preparation methods, lyophilization reactions were scaled to a 20 pL sample input volume, so 20 pL SHERLOCK reactions were lyophilized and 20 pL of simulated sample prepared by tested preparation method were used for rehydration of reaction. Fluorescence was measured over 1-3 hours at 40°C using a BioTek NEO HTS plate reader with readings every 3 minutes (Ex: 485 nm; Em 535 nm). For field simulation a start and 1-hour fluorescence measurement was made with a Quantus™ fluorometer (due to a minimum volume instrument input the reaction was performed in triplicate, although could have been diluted albeit with lower signal output). For DBS assays, the supernatant from the DBS-S-PREP reaction was transferred to lyophilized SHERLOCK pellets for resuspension of reaction; the 2mm DBS punch and re-suspended SHERLOCK reactions were then transferred to a 384- well plate for fluorescence measurement by same protocol as non-DBS reactions. For lateral flow readout, 20 pL of the SHERLOCK endpoint reaction was added to 100 pL of HybriDetect™ 1 assay buffer and run on HybriDetect™ 1 lateral flow strips (Milenia, Gieben, Germany).
[0266] Clinical and analytical specificity of patient serum samples. For demonstration of specificity on clinical samples (P . falciparum n = 4 serum, n = 1 whole blood; P. vivax n = 10), 12.5pL of serum (or whole blood) was diluted into 37.5pL S-PREP buffer (20% w/v Chelex®-100 in TE buffer with 50mM DTT). For determination of analytical specificity, three replicates of P. falciparum, P. vivax, P. ovale, and P. malariae simulated whole blood samples were prepared to a final concentration of 10 f (6020 parasites/ pL) as described above and similarly diluted in S-PREP buffer. Prepared simulated or real patient samples were then incubated at 95°C for 10 minutes and transferred to a
SUBSTITUTE SHEET (RULE 26) SHERLOCK lyophilized pellet as described above for resuspension of reaction. Fluorescence was measured over 1 hour at 40°C using a BioTek NEO HTS plate reader with readings every 3 minutes (Ex: 485 nm; Em 535 nm).
[0267] Determination of analytical sensitivity, limit of detection. The analytical limit of detection (LOD) was defined as the lowest Plasmodium species concentration that was successfully detected with a probability of 95% or greater. Calibration standards near the estimated LOD were prepared by serial dilutions of simulated samples described above to the following concentrations: 50 zM (0.03 copies/pL sample), 200 zM (0.12 copies/pL), 500 zM (0.3 copies/pL), 5 aM (3 copies/pL), 50 aM (30 copies/pL). The LOD was evaluated by testing the calibration standard over three separate runs performed on different days with seven replicates for each concentration, for a total of 21 replicate results at each concentration level.
[0268] Data analysis. Background-subtracted fluorescence was calculated by subtraction of the fluorescence of no-input (water only as “template” input into SHERLOCK reaction) control wells on the plate from target fluorescence values evaluated in the assay run at the same time points in the assay. Water-only control wells were therefore subtracted from both no-template controls (such as whole blood or serum) and samples or simulated sample wells. Student’s t-tests were used for comparison of background-subtracted fluorescence between Plasmodium targets and controls. A P value of < 0.05 was considered statistically significant. The relationship between the proportion of replicates testing positive and the corresponding sensitivity standard Plasmodium log concentration was examined using Probit regression analysis to estimate 95% LOD and 95% confidence intervals of each target (GraphPad 8.4.1, San Diego, USA). Lateral flow test line signal intensities were quantified to grayscale pixel values using Image J software (National Institutes of Health). Background-subtracted intensity was calculated from line scans that spanned the 1mm test line subtracted from background blank (white) area to normalize to ambient background grayscale value of the lateral flow strip.
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[0309] 40. M. J. Gardner, et al. , Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 419, 498-511 (2002).
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[0311] 42. C. Bourgard, L. Albrecht, A. C. A. V. Kayano, P. Sunnerhagen, F. T. M. Costa, Plasmodium vivax Biology: Insights Provided by Genomics, Transcriptomics and Proteomics. Front. Cell. Infect. Microbiol. 8, 34 (2018).
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[0313] 44. A. M. Waterhouse, J. B. Procter, D. M. A. Martin, M. Clamp, G. J. Barton, Jalview Version 2 — a multiple sequence alignment editor and analysis workbench. Bioinformatics. 25, 1189— 1191 (2009).
SUBSTITUTE SHEET (RULE 26)

Claims

57 CLAIMS
1. A composition for nucleic acid preparation, the composition comprising a reducing agent and a metal ion chelating resin in aqueous suspension.
2. The composition of claim 1, wherein the metal ion chelating resin comprises paired iminodiacetate ions.
3. The composition of claim 1 or claim 2, wherein the resin is present at a concentration of about 10% to 30% w/v.
4. The composition of any one of claims 1-3, wherein the reducing agent is dithiothreitol (DTT).
5. The composition of any one of claims 1-4, wherein the reducing agent is present at a concentration of 20-150 mM.
6. The composition of any one of claims 1-5, wherein the resin comprises styrene divinylbenzidine copolymer.
7. The composition of any one of claims 1-6, wherein the reducing agent is DTT in the range of 20- 150 mM, and the resin is a styrene divinylbenzidine copolymer resin with paired iminidiacetate ions at a concentration of 10-30% w/v.
8. The composition of any one of claims 1-7, wherein the reducing agent is 50 mM DTT, and the resin is present at a concentration of 20% w/v.
9. The composition of any one of claims 1-8, wherein the composition releases nucleic acid from a biological sample upon heating, without need for a proteolytic enzyme.
10. The composition of any one of claims 1-9, which does not comprise proteinase K.
11. The composition of claim 9 or claim 10, wherein the biological sample comprises blood or a blood fraction, a nasopharyngeal swab, an oropharyngeal swab, sputum or saliva.
12. The composition of any one of claims 9-11, wherein the blood fraction comprises erythrocytes.
13. A composition consisting essentially of an aqueous buffer, DTT and a metal ion chelating resin.
14. The composition of any one of claims 1-13, in admixture with a biological sample.
15. The composition of claim 14, wherein the composition of any one of claims 1-13 is present at about a 3: 1 ratio relative to biological sample by volume.
SUBSTITUTE SHEET (RULE 26) 58
16. The composition of claim 14 or claim 15, wherein the biological sample comprises blood or a blood fraction, a nasopharyngeal swab, an oropharyngeal swab, sputum or saliva.
17. The composition of claim 16, wherein the blood fraction comprises erythrocytes.
18. The composition of claim 16 or claim 17, wherein the blood or blood fraction has previously been dried on a solid support.
19. The composition of claim 18, wherein the solid support is present in the admixture.
20. The composition of any one of claims 1-19, further comprising reagents sufficient to perform an isothermal nucleic acid amplification.
21. The composition of any one of claims 1-20, further compnsing reagents sufficient to perform detection of a target nucleic acid.
22. The composition of claim 21, wherein the detection of atarget nucleic acid comprises SHERLOCK detection.
23. The composition of any one of claims 1-22, which substantially prevents target-independent cleavage of a SHERLOCK reporter nucleic acid in the presence of a biological sample.
24. A method of preparing a biological sample for nucleic acid analysis, the method comprising contacting a composition of any one of claims 1-13 with the biological sample, and heating the resulting mixture to at least 80°C.
25. The method of claim 24, wherein the ratio of the composition of any one of claims 1-13 to biological sample is about 3: 1.
26. The method of claim 24 or 25, wherein heating is performed for 2-20 minutes.
27. The method of any one of claims 24-26, wherein heating is performed at about 95°C for about 10 minutes.
28. The method of any one of claims 24-27, wherein the biological sample comprises:
(i) blood or a fraction thereof comprising erythrocytes,
(ii) a nasopharyngeal swab,
(iii) an oropharyngeal swab,
SUBSTITUTE SHEET (RULE 26) 59
(iv) sputum, or
(v) saliva.
29. The method of any one of claims 24-28, wherein the biological sample had previously been dried on a solid support.
30. The method of claim 29, wherein the solid support is included in the mixture.
31. The method of any one of claims 24-30, wherein the biological sample comprises or is suspected of comprising an intracellular parasite, bacterium, or a virus.
32. The method of claim 31, wherein the intracellular parasite comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Babesia sp. Leishmaniasis spp. Toxoplasmosis spp. or filarial nematodes.
33. The method of claim 31, wherein the virus comprises Dengue virus, Zika virus, Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoVl), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Heptatis E, Herpes virus, Varicella virus, Cytomegalovirus, Epstein-Barr virus, Human herpesvirus 6, Human herpesvirus8, adenovirus, influenza, parainfluenza, respiratory syncytial virus, or Chikungunya virus,
34. The method of claim 31, wherein the bacterium comprises a gram negative bacterium, a gram positive bacterium or an intracellular bacterium.
35. The method of any one of claims 28-34, wherein heating the mixture promotes red blood cell lysis and lysis of intracellular parasite or virus present and releases nucleic acids from the parasite or virus if present, and wherein the resin chelates multivalent metal ions, thereby inhibiting nuclease degradation of parasite and/or viral nucleic acid.
36. The method of any one of claims 24-35, further comprising, after the heating step, contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids.
37. The method of claim 36, further comprising incubating the mixture under conditions and for a time sufficient to amplify the one or more target nucleic acids if present.
38. The method of any one of claims 36-37, wherein the isothermal amplification is a recombinase polymerase amplification (RPA) reaction.
SUBSTITUTE SHEET (RULE 26) 60
39. The method of any one of clams 36-38, wherein no additional sample processing or liquid transfer steps are required to permit isothermal amplification of one or more target nucleic acids.
40. The method of any one of claims 36-39, wherein the reagents sufficient for isothermal amplification are lyophilized prior to the step of contacting the mixture with the reagents.
41. The method of any one of claims 24-40, further comprising, after the heating step, contacting the mixture with reagents sufficient for SHERLOCK detection of one or more target nucleic acids.
42. The method of claim 41 , further comprising incubating the mixture under conditions and for a time sufficient to generate a SHERLOCK detection signal for one or more target nucleic acids if present.
43. The method of claim 41 or 42, wherein the reagents sufficient for SHERLOCK detection are lyophilized prior to the step of contacting the mixture with the reagents.
44. The method of any one of clams 41-43, wherein no additional sample processing or liquid transfer steps are required to permit SHERLOCK detection of one or more target nucleic acids.
45. The method of any one of claims 24-35, further comprising, after the heating step, contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids and reagents sufficient for SHERLOCK detection of one or more target nucleic acids.
46. The method of claim 45, wherein the reagents sufficient for isothermal amplification and the reagents sufficient for SHERLOCK detection are lyophilized prior to the contacting step, wherein the contacting step reconstitutes the lyophilized reagents and permits amplification and SHERLOCK detection of one or more target nucleic acid molecules if present.
47. The method of claim 45 or claim 46, further comprising incubating the mixture under conditions and for a time sufficient to amplify one or more target nucleic acids and to generate a SHERLOCK detection signal for one or more target nucleic acids if present.
48. A method of amplifying one or more target nucleic acids in a biological sample comprising nucleic acids, the method comprising:
(i) contacting a composition of any one of claims 1-13 with the biological sample;
(ii) heating the mixture resulting from step (i) to at least 80°C for a time sufficient to release nucleic acids in the biological sample;
(iii) after step (ii), contacting the mixture with reagents sufficient for isothermal amplification of one or more target nucleic acids;
SUBSTITUTE SHEET (RULE 26) 61
(iv) incubating the mixture of step (iii) under conditions and for a time sufficient to generate an isothermal amplification product for one or more target nucleic acids.
49. The method of claim 48, wherein no additional sample processing or liquid transfer steps are required to generate an amplification product for one or more target nucleic acids.
50. The method of claim 48 or claim 49, wherein steps (i)-(iv) are performed in the same reaction or sample container.
51. A method of detecting one or more target nucleic acids in a biological sample, the method comprising:
(i) contacting a composition of any one of claims 1-13 with the biological sample;
(ii) heating the mixture resulting from step (i) to at least 80°C for a time sufficient to release nucleic acids in the biological sample;
(iii) after step (ii), contacting the mixture with reagents sufficient for isothermal amplification and SHERLOCK detection of one or more target nucleic acids; and
(iv) incubating the mixture of step (iii) under conditions and for a time sufficient to permit isothermal amplification and production of a SHERLOCK detection signal for one or more target nucleic acids present in the sample.
52. The method of claim 51, wherein no additional sample processing or liquid transfer steps are required to generate SHERLOCK detection signal for one or more target nucleic acids present in the sample.
53. The method of claim 51 or claim 52, wherein steps (i)-(iv) are performed in the same reaction or sample container.
54. A kit for nucleic acid preparation and/or detection, the kit comprising a reducing agent and a metal ion chelating resin in aqueous suspension, and packaging materials therefor.
55. The kit of claim 54, wherein the metal ion chelating resin comprises paired iminodiacetate ions.
SUBSTITUTE SHEET (RULE 26)
56. The kit of claim 54 or 55, wherein the reducing agent is dithiothreitol (DTT).
57. The kit of any one of claims 54-56, which does not contain proteinase K.
58. The kit of any one of claims 54-57, wherein the reducing agent is present at a concentration of 20- 150 mM.
59. The kit of any one of claims 54-58, wherein the resin comprises styrene divinylbenzidine copolymer.
60. The kit of any one of claims 54-59, further comprising reagents sufficient for an isothermal nucleic acid amplification reaction.
61. The kit of claim 60, wherein the reagents sufficient for an isothermal nucleic acid amplification reaction are lyophilized.
62. The kit of any one of claims 54-60, further comprising reagents sufficient for a SHERLOCK detection reaction.
63. The kit of any one of claims 54-61, wherein the reagents sufficient for a SHERLOCK detection reaction are lyophilized.
64. The kit of any one of claims 60 or 61, wherein reagents sufficient for an isothermal nucleic acid amplification reaction and reagents sufficient for a SHERLOCK detection reaction are lyophilized in one composition.
SUBSTITUTE SHEET (RULE 26)
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