Improved detection of LAMP amplification products

Abstract

The present invention relates to a method for improving detection of amplification products from loop-mediated amplification (LAMP) reactions through the use of nucleotide analogues that form duplex structures that are not efficiently recognized by nucleic acid polymerase enzymes.

Description

[Technical field]

[0001] The present invention relates to the field of nucleic acid amplification and detection, in particular to the field of isothermal nucleic acid amplification and detection of amplification products. [Background technology]

[0002] Loop-mediated isothermal amplification (LAMP) is a unique isothermal amplification method with many advantages (see, for example, U.S. Pat. No. 6,410,278 and U.S. Pat. No. 7,374,913). First, LAMP is an isothermal nucleic acid amplification method, which eliminates the need for specific equipment to control temperature cycles, such as those required for PCR, and is therefore ideally suited for point-of-care testing. Second, LAMP can produce large amounts of amplification products in a short period of time with high specificity. These two advantages allow for relatively easy detection and potential wide applications in point-of-care testing. Another advantage is that, compared to other isothermal amplification methods, LAMP requires only one enzyme, a strand-displacing DNA polymerase, making the system easier to handle.

[0003] In addition to DNA polymerase, LAMP uses four core primers (FIP, BIP, F3, B3) (Figure 1) that recognize six different sequence regions on the target, and two primers (F1C, B1C) that contain sequences that result in a loop structure that promotes exponential amplification (Notomi et al., Nucleic Acids Res., 28:E63 (2000)). The use of multiple target sequence regions confers a high degree of specificity to the reaction. To increase the reaction rate, two additional primers, called loop primers, can be added, resulting in a total of six primers being used per target sequence (Nagamine et al., Mol. Cel. Probes, 16:223-9 (2002)). The LAMP reaction rapidly generates amplification products as multimers of target regions of various sizes and is important for total DNA synthesis (>10 μg, >50× PCR yield) (Notomi et al. (2000); Nagamine et al., Clin. Chem., 47:1742-3 (2001)).

[0004] Measurement of LAMP amplification products may be performed using fluorescent detection of double-stranded DNA, but these methods are limited to the detection of a single target because they detect the total DNA amplification in the reaction. To utilize LAMP to detect multiple targets in a single sample, Tanner et al. (BioTechniques 53:81-89, 2012, and U.S. Patent No. 9,074,249, each of which is incorporated herein by reference in its entirety) have developed a DARQ (DARQ) probe that allows for the incorporation of fluorescent probes. D etection of A Mplification by R elease of Q They developed a simple and ingenious modification called LAMP (LAMP) (Quenching). In addition to improving sensitivity, fluorescent detection also allowed multiplexing of individually resolvable targets. In DARQ, standard FIP primers are modified with a 5' quencher and annealed to the F1c complementary detection probe Fd (see top of Figure 2). To test DARQ LAMP detection, four sets of LAMP primers were designed with Q-FIP and accompanying Fd probes, each with a different fluorophore and quencher pair. The fluorescent probe used by Tanner forms a quenched duplex with one of the LAMP primers. Subsequent displacement of this probe by strand-displacing Bst 2.0 DNA polymerase (New England Biolabs) during amplification results in accumulation of fluorescent signal. Figure 2 shows a graphical representation of the DARQ LAMP method.

[0005] One limitation of the DARQ LAMP method is the inhibition of amplification observed at high concentrations of quenched FIP primer (Q-FIP):Fd probe duplex. This inhibition could be significantly reduced by using equimolar standard FIP primer and Q-FIP:Fd duplex, but the fluorescence signal was also reduced by this process and could be reduced to undetectable levels. A method to perform DARQ LAMP without reducing either amplification efficiency or signal generation is highly desirable, which would increase the usefulness of the DARQ LAMP method for molecular diagnostics. Summary of the Invention

[0006] The present invention relates to an improved method of performing a DARQ LAMP assay by using an Fd detection probe that contains nucleotides at the 2' position of the ribose ring substituted with an O-methylated (OMe) moiety to form a duplex structure that is not efficiently recognized by DNA polymerase enzymes. Accordingly, in one aspect, the present invention provides a method for determining the presence or amount of a target nucleic acid molecule in a sample using loop-mediated isothermal amplification (LAMP), the method comprising: (a) combining a LAMP reaction mixture, a DNA polymerase having strand displacement properties, a sample comprising the target nucleic acid molecule, the LAMP reaction mixture comprising: (i) a LAMP primer set of 4-6 primers comprising two internal primers (FIP and BIP), two external primers (F3 and B3), and optionally one or two loop primers (loop F and / or loop B), wherein the FIP primer comprises a primer sequence (F2) that is complementary to a sequence in the target nucleic acid molecule, and a tail sequence (F1c) that is not complementary to a sequence in the target nucleic acid molecule and is 5' of the primer sequence, wherein the tail sequence is labeled with a quencher molecule; and The method includes: (ii) combining with a detection probe (Fd) that can hybridize to the tail sequence of FIP primer to form a duplex, the detection probe is labeled with a fluorescent molecule that is quenched by a quencher molecule when the detection probe hybridizes to the tail sequence, and more than 80% of the nucleotides in the detection probe are substituted with a methoxy group at the 2' position of their ribose ring (OMe-substituted); amplifying the target nucleic acid molecule by LAMP under suitable assay conditions that allow the LAMP reaction to generate target amplicons and multiple cycles that displace the detection probe from the tail sequence, thereby releasing the quenching of the fluorescent molecule to generate a detectable signal; and detecting and optionally quantifying the signal from the detection probe, and determining the presence or amount of the target nucleic acid molecule in the sample therefrom. In one embodiment, more than 90% of the nucleotides in the detection probe are OMe-substituted. In another embodiment, all of the nucleotides in the detection probe are OMe-substituted.In a particular embodiment, the DNA polymerase with strand displacement properties is Bst polymerase. In another embodiment, the concentration of the detection probe is 200 nM or less. In a further embodiment, the sample comprises a plurality of target nucleic acid molecules, and the LAMP reaction mixture is a multiplex reaction mixture that can amplify and detect a plurality of target nucleic acid molecules. In yet another embodiment, the multiplex reaction mixture comprises a plurality of detection probes, each probe being labeled with a different fluorescent molecule.

[0007] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic illustration of the basic LAMP reaction. [Diagram 2] FIG. 2 is a diagrammatic representation of the DARQ LAMP reaction. [Diagram 3] FIG. 3 shows the growth curves of the DARQ LAMP singleplex assay described in Example 1. [Figure 4] FIG. 4 shows the growth curves of the DARQ LAMP singleplex assay described in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] definition The term "sample" as used herein includes specimens or cultures (e.g., microbial cultures) that contain nucleic acids. The term "sample" is also meant to include both biological samples and environmental samples. Samples may include specimens of synthetic origin. Biological samples include whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar epithelium, stomach, peritoneum, duct, ear, arthroscopic), biopsy samples, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, semen, lymph, bile, tears, sweat, breast milk, breast fluid, embryonic cells, and fetal cells. In a preferred embodiment, the biological sample is blood, more preferably plasma. As used herein, the term "blood" encompasses whole blood or any fraction of blood (e.g., serum and plasma) as generally defined. Plasma refers to the fraction of whole blood resulting from centrifugation of blood that has been treated with an anticoagulant. Serum refers to the aqueous portion of bodily fluids remaining after a blood sample has clotted. Environmental samples include environmental materials such as surface matter, soil, water, and industrial samples, as well as samples obtained from food and dairy processing equipment, devices, instruments, facilities, utensils, disposable and non-disposable items. These examples should not be construed as limiting the types of samples applicable to the present invention.

[0010] The term "target" or "target nucleic acid" as used herein is intended to mean any molecule whose presence is to be detected or measured, or whose function, interaction or properties are to be studied. Thus, a target includes essentially any molecule for which a detectable probe (e.g., an oligonucleotide probe) or assay exists or can be produced by one of skill in the art. For example, a target can be a biomolecule, such as a nucleic acid molecule, a polypeptide, a lipid, or a carbohydrate, that can bind to or otherwise contact a detectable probe (e.g., an antibody), and a detectable probe also includes a nucleic acid that can be detected by the methods of the present invention. As used herein, a "detectable probe" refers to any molecule or agent that can hybridize or anneal to a target biomolecule of interest and allow for specific detection of the target biomolecule described herein. In one aspect of the present invention, the target is a nucleic acid and the detectable probe is an oligonucleotide. The terms "nucleic acid" and "nucleic acid molecule" can be used interchangeably throughout this disclosure. The term refers to oligonucleotides, oligos, polynucleotides, deoxyribonucleotides (DNA), genomic DNA, mitochondrial DNA (mtDNA), complementary DNA (cDNA), bacterial DNA, viral DNA, viral RNA, RNA, message RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), siRNA, catalytic RNA, clones, plasmids, M13, P1, cosmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), amplified nucleic acids, amplicons, PCR products and other types of amplified nucleic acids, RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which may be in either single-stranded or double-stranded form, and unless otherwise limited, may include known analogs of natural nucleotides that may perform similar functions as naturally occurring nucleotides, as well as combinations and / or mixtures thereof. Thus, the term "nucleotide" refers to both naturally occurring and modified / non-naturally occurring nucleotides, including nucleoside triphosphates, diphosphates and monophosphates, as well as monophosphate monomers present within polynucleic acids or oligonucleotides.Nucleotides can also be ribo; 2'-deoxy; 2',3'-deoxy, and many other nucleotide mimics well known in the art. Mimics include chain-terminating nucleotides, such as 3'-O-methyl, halogenated bases or sugar substitutes; non-sugar, alternative sugar structures, including alkyl ring structures; alternative bases, including inosine; deaza modifications; chi, and psi, linker modifications; mass label modifications; phosphodiester modifications or replacements, including phosphorothioates, methylphosphonates, boranophosphates, amides, esters, ethers; and basic or complete internucleotide replacements, including cleavable linkages, such as photocleavable nitrophenyl moieties.

[0011] The presence or absence of a target can be measured quantitatively or qualitatively. Targets can be in a variety of different forms, including, for example, simple or complex mixtures, or in substantially purified form. For example, targets can be part of a sample that contains other components, or can be the only or major component of a sample. Thus, targets can be components of whole cells or tissues, cell or tissue extracts, fractionated lysates thereof, or substantially purified molecules. Targets can also have either known or unknown sequences or structures.

[0012] The term "amplification reaction" refers to any in vitro means for increasing the copies of a target sequence of nucleic acid.

[0013] "Amplifying" refers to subjecting a solution to sufficient conditions to allow amplification. Components of an amplification reaction may include, but are not limited to, for example, primers, polynucleotide templates, polymerase, nucleotides, dNTPs, etc. The term "amplifying" typically refers to the "exponential" increase of target nucleic acid. However, as used herein, "amplifying" may refer to a linear increase in the number of selected target sequences of nucleic acid, but is different from a one-off single primer extension step.

[0014] "Polymerase chain reaction" or "PCR" refers to a method in which a specific segment or subsequence of a target double-stranded DNA is amplified in a geometric progression. PCR is well known to those skilled in the art. See, for example, U.S. Patent Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, edited by Innis et al., 1990.

[0015] As used herein, "oligonucleotide" refers to a linear oligomer of natural or modified nucleoside monomers linked by phosphodiester bonds or analogs thereof. Oligonucleotides include deoxyribonucleosides, ribonucleosides, their anomeric forms, peptide nucleic acids (PNAs), and the like, that can specifically bind to a target nucleic acid. Typically, the monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides that range in size from a few monomer units (e.g., 3-4) to dozens of monomer units (e.g., 40-60). When an oligonucleotide is represented by a sequence of letters such as "ATGCCTG," it will be understood that the nucleotides are in 5'-3' order from left to right, with "A" indicating deoxyadenosine, "C" indicating deoxycytidine, "G" indicating deoxyguanosine, "T" indicating deoxythymidine, and "U" indicating uridine, a ribonucleoside, unless otherwise indicated. Typically, oligonucleotides contain the four natural deoxynucleotides, but they may also contain ribonucleosides or non-natural nucleotide analogs. If an enzyme has a specific oligonucleotide or polynucleotide substrate requirement for activity (e.g., single-stranded DNA, RNA / DNA duplex, etc.), the selection of the appropriate composition of the oligonucleotide or polynucleotide substrate is well within the knowledge of one of ordinary skill in the art.

[0016] As used herein, "oligonucleotide primer" or simply "primer" refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid template and facilitates detection of an oligonucleotide probe. In an amplification embodiment of the invention, an oligonucleotide primer serves as a starting point for nucleic acid synthesis. In a non-amplification embodiment, an oligonucleotide primer may be used to create a structure that can be cleaved by a cleavage agent. Primers may be of various lengths, often less than 50 nucleotides long, for example 12-25 nucleotides long. The length and sequence of primers for use in PCR may be designed based on principles known to those of skill in the art.

[0017] As used herein, the term "oligonucleotide probe" refers to a polynucleotide sequence that is capable of hybridizing or annealing to a target nucleic acid of interest and allowing for the specific detection of the target nucleic acid.

[0018] A "mismatched nucleotide" or "mismatch" refers to a nucleotide that is not complementary to the target sequence at that position or positions. An oligonucleotide probe can have at least one mismatch, and can also have 2, 3, 4, 5, 6, or 7 or more mismatched nucleotides.

[0019] The term "polymorphism" as used herein refers to allelic variants.Polymorphisms can include single nucleotide polymorphisms (SNPs) and simple sequence length polymorphisms.Polymorphisms can be due to one or more nucleotide substitutions at one allele compared to another, or can be due to insertions or deletions, duplications, inversions, and other modifications known in the art.

[0020] As used herein, the term "modification" refers to alterations of the oligonucleotide probe at the molecular level (e.g., base moiety, sugar moiety, or phosphate backbone). Nucleoside modifications include, but are not limited to, the introduction of cleavage blockers or cleavage inducers, the introduction of minor groove binders, isotopic enrichment, isotopic depletion, the introduction of deuterium, and halogen modifications. Nucleoside modifications may also include moieties that increase the stringency of hybridization or increase the melting temperature of the oligonucleotide probe. For example, nucleotide molecules may be modified with an extra bridge connecting the 2' and 4' carbons to result in a locked nucleic acid (LNA) nucleotide that is resistant to cleavage by nucleases. The composition of the tag portion of the oligonucleotide probe and capture oligonucleotide molecule is limited only by the ability to form a stable duplex. Thus, these oligonucleotides may contain DNA, L-DNA, RNA, L-RNA, LNA, L-LNA, PNA, BNA, L-BNA, etc. (where "L-XXX" refers to the L-enantiomer of the sugar unit of the nucleic acid) or any other known variations and modifications on the nucleotide base, sugar or phosphodiester backbone.

[0021] Other examples of nucleoside modifications include various 2'-substitutions such as halo, alkoxy and allyloxy groups introduced into the sugar portion of oligonucleotides. Evidence has been presented that 2'-substituted-2'-deoxyadenosine polynucleotides resemble double-stranded RNA more than DNA. Ikehara et al. (Nucleic Acids Res., 1978, 5, 3315) showed that 2'-fluoro substituents in polyA, polyI, or polyC duplexed to their complements were significantly more stable than ribonucleotide or deoxyribonucleotide polyduplexes as determined by standard melting assays. Inoue et al. (Nucleic Acids Res., 1987, 15, 6131) described the synthesis of mixed oligonucleotide sequences containing 2'-OMe (O-methyl) substituents on all nucleic acid nucleotides. The mixed 2'-OMe-substituted oligonucleotides hybridized to their RNA complements as strongly as RNA-RNA duplexes, which were significantly stronger than the same sequence RNA-DNA heteroduplexes. Thus, examples of substitutions at the 2' position of the sugar include F, CN, CF3, OCF3, OMe, OCN, O-alkyl, S-alkyl, SMe, SO2Me, ONO2, NO2, NH3, NH2, NH-alkyl, OCH3=CH2 and OCCH.

[0022] The terms "specific" or "specificity" in reference to the binding of one molecule to another, such as a probe for a target polynucleotide, refer to the recognition, contact, and formation of a stable complex between the two molecules, as well as the significantly weaker recognition, contact, or complex formation of that molecule with another molecule. As used herein, the term "anneal" refers to the formation of a stable complex between two molecules.

[0023] A probe can "anneal" to a nucleic acid sequence if at least one region of the probe shares substantial sequence identity with at least one region of the complement of the nucleic acid sequence. "Substantial sequence identity" is at least about 80%, preferably at least about 85%, more preferably at least about 90%, 95% or 99%, and most preferably 100% sequence identity. For the purpose of determining sequence identity of DNA and RNA sequences, U and T are often considered to be the same nucleotide. For example, a probe containing the sequence ATCAGC can hybridize to a target RNA sequence containing the sequence GCUGAU.

[0024] "Nucleic acid polymerase" refers to an enzyme that catalyzes the incorporation of nucleotides into a nucleic acid. Exemplary nucleic acid polymerases include DNA polymerase, RNA polymerase, terminal transferase, reverse transcriptase, telomerase, and the like.

[0025] Fluorescence Resonance Energy Transfer (FRET) FRET technology (e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are placed within a certain distance from each other, energy transfer occurs between two fluorescent moieties that can be visualized or otherwise detected and / or quantified. Typically, the donor transfers energy to the acceptor when excited by light radiation of a suitable wavelength. Typically, the acceptor re-emits the transferred energy in the form of light radiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule that includes a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0026] In one example, an oligonucleotide probe may contain a donor fluorescent moiety and a corresponding quencher that may or may not be fluorescent and dissipates the transferred energy in a form other than light. When the probe is intact, energy transfer typically occurs between the two fluorescent moieties, resulting in quenching of the fluorescence emission from the donor fluorescent moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase, so that the fluorescence emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Calif.), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry® Quencher 650 (BBQ-650) (Berry & Assoc, Dexta, Michigan).

[0027] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at a specific position determined by the complementarity of the oligonucleotide probe to the target nucleic acid sequence. When the oligonucleotide probe hybridizes to the nucleic acid of the amplification product at the appropriate position, a FRET signal is generated. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0028] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor fluorescence emission in a specific range), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore can be performed with an argon ion laser, a high-intensity mercury (Hg) arc lamp, a fiber optic light source, or other high-intensity light source appropriately filtered for excitation of the desired range.

[0029] As used herein with respect to a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, "corresponding" refers to an acceptor fluorescent moiety that has an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Thus, efficient non-radiative energy transfer can be generated between them.

[0030] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) high efficiency Forster energy transfer, (b) large final Stokes shift (>100 nm), (c) emission shift as far as possible to the red portion of the visible spectrum (>600 nm); and (d) emission shift to a wavelength higher than the Raman water fluorescence emission caused by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety may be selected that has its maximum excitation wavelength near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety may be selected that has a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0031] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, lucifer yellow, B-phycoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red640, LC Red705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriaminepentaacetate, or other chelates of lanthanide ions (e.g., europium, or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0032] The donor and acceptor fluorescent moieties may be attached to the appropriate probe oligonucleotide via a linker arm. The length of each linker arm is important since it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm may be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Generally, the linker arm is from about 10 Å to about 25 Å. The linker arm may be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods of attaching the linker arm to a particular nucleotide base and attaching the fluorescent moiety to the linker arm.

[0033] Acceptor fluorescent moieties such as LC Red640 can be combined with oligonucleotides containing amino linkers (e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.)) to produce, for example, LC Red640-labeled oligonucleotides. Linkers frequently used to couple donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (derived from fluorescein-NHS-ester, e.g., CX-Fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3'-amino-CPG, which requires coupling of the fluorescein-NHS-ester after oligonucleotide synthesis.

[0034] LAMP and DARQ LAMP methods "LAMP" or " R -Mediated isothermal Increase"LAMP" refers to isothermal amplification methods, i.e., methods performed at essentially constant temperature without the need for a thermocycler. In LAMP, target sequences are typically amplified at 60°C-65°C using two or three sets of primers (i.e., 4-6 primers) and a polymerase that has high strand displacement activity in addition to replicative activity. A DNA polymerase with strand displacement activity / property is known to those skilled in the art as the ability of a polymerase to displace downstream DNA strands encountered during synthesis along the target strand. Typically, four different primers are used to identify six different regions on the target gene, greatly increasing specificity (Figure 1). Additional "loop primer" or "multiple loop primer" pairs can further accelerate the reaction. Due to the specific nature of the action of these primers, the amount of DNA generated in LAMP is considerably higher than PCR-based amplification. The LAMP method is described in U.S. Patent No. 6,410,278 and U.S. Patent No. 7,374,913. In general, this method uses two internal primers (forward internal primer=FIP and backward internal primer=BIP), two external primers (F3 and B3), and optionally one or two, preferably two, loop primers (loop forward=LF and / or loop backward=LB). When two loop primers are used, one is preferably a loop forward primer and the other is a loop backward primer. The internal primer contains a target complementary region (typically called F2 and B2) that facilitates hybridization, and at its 5', a sequence that is identical to a sequence in the target nucleic acid located upstream (5') relative to the sequence of the target bound by the target complementary region of the internal primer (typically called F1c and B1c). Thus, extension of the internal primer by polymerase forms a sequence that contains a region of self-complementarity, in that the target identical sequence (B1c) on the 5' end of the internal primer binds to a sequence synthesized downstream of the target complementary region of the internal primer (called B1) after extension and can act as a primer for further extension.The outer primer binds to a target region in the target nucleic acid downstream (i.e., 3') of the target region bound by the inner primer (called F3c and B3c), and is thus responsible for the displacement of the extended inner primer sequence from the template strand. The extended inner primer is recognized and hybridized by the other primer of the inner primer pair, resulting in the generation of a dumbbell-shaped starting amplicon. The dumbbell structure is then used for the next amplification, and the amplicon takes the form of a concatemer. The principle of LAMP has been disclosed, for example, by Eiken Chemical Co., Ltd. in the publications of Nagamine et al. (Mol. Cell. Probes (2002) 16:223-229) and Notomi et al. (Nucleic Acids Res. (2000), 28(12):e63), and the structure of the LAMP target and primer is also shown diagrammatically in FIG. 1.

[0035] DARQ (Detection of Amplification by Release of Quenching) LAMP is an improvement of the LAMP method that allows for fluorescent detection of LAMP amplification products in multiplex reactions. LAMP forward and back internal primers (FIP and BIP) contain a 5' flap (Figure 2, F1c sequence) that anneals to a complementary downstream region (F1) upon synthesis and displacement. This region was chosen for the development of detection probes because it is unique to LAMP and contains sequences specific to each target, eliminating the need for probe sequence optimization. LAMP also requires a strand-displacing DNA polymerase (typically Bst DNA polymerase, large fragment), a component utilized for detection by strand displacement. Using previously designed LAMP primers as a basis, FIPs modified at the 5' end were synthesized with a dark quencher. For probe creation (Fd), an oligonucleotide complementary to the flap region (F1c) was annealed with a fluorophore that spectrally overlaps with the dark quencher of FIP (Figure 2, Q-FIP:Fd duplex). This duplex primer retains its function as a LAMP primer, but when synthesized from the reverse orientation, the flap duplex is separated, resulting in detection of amplification by release of the quench (Figure 2, step 3).

[0036] However, it has been shown that in DARQ LAMP, the concentration of the Fd probe must be reduced because a high concentration (1.6 μM) of Q-FIP:Fd duplex inhibits the LAMP amplification process, which reduces the detectable fluorescent signal. One possible reason that amplification is inhibited is that DNA polymerase binds non-productively to the FIP:Fd duplex. A way to eliminate this binding of polymerase to the duplex is to synthesize the Fd probe with nucleotide analogs such as L-DNA or 2'-O-methylated nucleotides that form a double-stranded structure that the polymerase enzyme does not recognize efficiently. Although the 2'-O-methyl-substituted (OMe) Fd probe hybridizes with the normal bases in the F1c portion of the primer, the polymerase should not bind to the OMe:DNA duplex.

[0037] Embodiments of the present invention are further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0038] Example 1: Performance of OMe DARQ LAMP probes on native DNA For the amplification and detection of three of the target genes: E. coli dnaE, Caenorhabditis elegans lec10, and human BRCA-1, DARQ LAMP experiments were performed according to the method described in Tanner et al., BioTechniques 53:81-89, 2012. The experimental conditions were as follows: the Q-FIP:Fd duplex was annealed by heating 50 μM Q-FIP and 50 μM Fd to 98 °C and slowly cooling the mixture to room temperature. LAMP reactions with Bst2.0 DNA polymerase or Bst2.0 WarmStart DNA polymerase (New England Biolabs, Ipswich, Massachusetts, USA) were carried out in 1× isothermal amplification buffer (New England Biolabs): 20 mM Tris-HCl (pH 8.8, 25° C.), 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween-20®, supplemented with 8 mM MgSO4, and 1.4 mM each of dATP, dCTP, dGTP, and dTTP. LAMP reactions contained 1.6 μM FIP (or 0.8 μM FIP and 0.8 μM Q-FIP:Fd), 1.6 μM BIP, 0.2 μM F3 and B3, 0.4 μM Loop F and Loop B, and 0.64 U / μL Bst2.0 DNA polymerase or Bst2.0 WarmStart DNA polymerase. Both native and OMe-substituted Fd probes were labeled with BHQ-2 dark quencher, while FIP primers annealing to dnaE, lec10, and BRCA-1 were labeled with the fluorescent dyes coumarin (COU), FAM, and HEX, respectively. Synthetic templates were added at 2,500 copies per reaction.

[0039] [Table 1]

[0040] The results of the experiment are shown as detection times in Table 1 and growth curves in Figure 3. Compared to the "native" D-DNA Fd probe, the Fd probe with the OMe substitution showed approximately two-fold faster detection times and also generated a significantly stronger fluorescent signal.

[0041] Example 2: Multiplexing of 5 targets by OMe DARQ LAMP To demonstrate the performance of the DARQ LAMP assay with 2'-OMe substituted Fd probes, a 5-target multiplex assay was designed with four gene targets: SARS-CoV-2 N gene (SC2), FluA hemagglutinin H1 gene (AH1), FluA hemagglutinin H3 gene (AH3), FluB neuraminidase gene (BNA), and one internal control (GIC). DARQ LAMP 5-plex reactions were carried out in a volume of 50 μL with 400 nM each FIP / BIP, 100 nM each F3 / B3, 400 nM each Loop F / Loop B, and 200 nM each Fd probe in 1×IsoAmp buffer (New England Biolabs), 1.5 mM dATP, 1.5 mM dCTP, 1.5 mM dGTP, 1.5 mM dTTP, 6 mM MgSO4, 40 mM guanine hydrochloride, and 64 U Bst 2.0 Warm Start polymerase (New England Biolabs). Reactions were incubated at 65° C. for 60 min in a LightCycler 480 (Roche) set to collect fluorescence data every 0.5 min. For each Fd probe, the following fluorescent dyes were used: Coumarin for SC2, FAM for AH1, HEX for AH3, JA270 for BNA, and Cy5.5 for GIC. When present, single-stranded K562 human genomic DNA (Promega) was added at 100 ng / reaction.

[0042] The performance of the OMe DARQ LAMP multiplex reaction was tested using single-stranded synthetic templates at concentrations of 10, 100, 1000, 10,000 and 100,000 copies per reaction, and the results are shown as detection times in Table 2 and growth curves in Figure 4. Three of the templates (AH3, BNA, GIC) showed reliable detection up to 10 copies per reaction, while two of the templates (SC2, AH1) showed detection up to 100 copies per reaction.

[0043] [Table 2]

[0044] Although the foregoing invention has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the invention. For example, the methods described above may be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually indicated to be incorporated by reference for all purposes.

[0045] [Table 3-1] [Table 3-2]

Claims

1. 1. A method for determining the presence or amount of a target nucleic acid molecule in a sample using loop-mediated isothermal amplification (LAMP), said method comprising: (a) combining a LAMP reaction mixture, a DNA polymerase having strand displacement properties, and a sample containing the target nucleic acid molecule, wherein the LAMP reaction mixture comprises: (i) a LAMP primer set comprising two internal primers (FIP and BIP) and two external primers (F3 and B3), wherein the FIP primers comprise a primer sequence (F2) that is complementary to a sequence within the target nucleic acid molecule and a tail sequence (F1c) that is not complementary to a sequence within the target nucleic acid molecule and is 5' of the primer sequence, wherein the tail sequence is labeled with a quencher molecule; and (ii) combining a detection probe (Fd) capable of hybridizing to the tail sequence of the FIP primer to form a duplex, wherein the detection probe is labeled with a fluorescent molecule that is quenched by the quencher molecule when the detection probe hybridizes to the tail sequence, and more than 80% of the nucleotides in the detection probe are substituted with a methoxy group at the 2' position of the ribose ring (OMe substituted); (b) amplifying the target nucleic acid molecule by LAMP under suitable assay conditions that allow the LAMP reaction to generate a target amplicon and multiple cycles that displace the detection probe from the tail sequence, thereby unquenching the fluorescent molecule and generating a detectable signal; and (c) detecting and optionally quantifying the signal from the detection probe and determining therefrom the presence or amount of the target nucleic acid molecule in the sample. A method comprising:

2. 2. The method of claim 1, wherein more than 90% of the nucleotides in said detection probe (Fd) are OMe substituted.

3. 3. The method of claim 2, wherein all of the nucleotides in said detection probe (Fd) are OMe substituted.

4. The method described in claim 1, wherein the LAMP primer set further includes at least one of a first loop primer (loop F) and a second loop primer (loop B).

5. The method of claim 1, wherein the DNA polymerase having strand displacement properties is Bst polymerase.

6. 2. The method of claim 1, wherein the concentration of the detection probe is 200 nM or less.

7. The method of claim 1 , wherein the sample contains a plurality of target nucleic acid molecules.

8. 8. The method of claim 7, wherein the LAMP reaction mixture is a multiplex reaction mixture capable of amplifying and detecting multiple target nucleic acid molecules.

9. 9. The method of claim 8, wherein the multiplex reaction mixture comprises multiple detection probes, each probe labeled with a different fluorescent molecule.

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