Methods and reagents for nucleic acid amplification and / or detection

Abstract

To provide novel amplification and / or detection of nucleic acid molecules.SOLUTION: The present invention relates to amplification and / or detection of nucleic acid molecules. More specifically, the present invention relates to sensitive amplification, detection, and / or quantification of nucleic acid molecules.

Description

[Technical Field]

[0001] The present invention relates to the amplification and / or detection of nucleic acid molecules. More particularly, the present invention relates to the sensitive amplification, detection and / or quantification of nucleic acid molecules. [Background technology]

[0002] Infectious diseases caused by pathogenic microorganisms, such as bacteria, viruses, and eukaryotic parasites, are among the most serious public health concerns worldwide. Therefore, successful methods for disease diagnosis and treatment, food safety control, and environmental monitoring require rapid and specific identification of infectious agents. Simplicity and low cost are equally important. For example, methods that do not rely on sophisticated instrumentation and skilled personnel can be employed in settings where outbreaks of COVID-19, HIV, TB, malaria, certain types of influenza A, and Ebola virus pose a significant risk to patient care, and where advanced diagnostic technologies are limited or nonexistent due to economic constraints. 1,2 . Traditional methods for pathogen detection involve culturing microorganisms on agar plates, followed by standard biochemical identification, which, although inexpensive and simple, are laborious and time-consuming. 3 They often require 2-3 days of preliminary identification and more than a week for pathogen identification confirmation, which delays effective diagnosis. 4,5 This delay has a significant impact on morbidity and mortality. Misdiagnosed therapy has been shown to reduce survival for serious infections by five-fold. 6 Furthermore, these methods may be limited by their low sensitivity. 7,8 Also, culture methods can only identify organisms that can grow in culture and cannot detect viable but culture-negative pathogens. 6,4 However, they can be identified using molecular nucleic acid detection methods.

[0003] A variety of applications involving pathogen detection widely use nucleic acids as biomarkers.5,1,4 Due to their versatile functions and wide range of applications, many methods have been developed to detect extremely small amounts of nucleic acids in complex biological samples. 4 Among RNA and DNA, RNA detection is of particular interest because many live pathogens carry multiple copies of RNA (thousands in the case of ribosomal RNA), which provides a greater initial template concentration for amplification and is the only source of genetic information in some high-profile viral pathogens (measles virus, influenza, and HIV, to name a few). Nucleic acid testing (NAT) is faster than traditional methods and is inherently more specific and sensitive. In addition, it can be used to directly identify microorganisms in clinical specimens without culture, significantly shortening detection time. Rapid pathogen detection results in shorter hospital stays, improved patient care, and the prevention of epidemics of both local outbreaks and global nature. The goal of NAT is to identify and potentially quantify specific nucleic acid sequences from clinical samples. This technique traditionally involves three steps: nucleic acid isolation, amplification, and detection. However, with the advent of fluorescent DNA probes and intercalating dyes that allow real-time quantification of amplification products, amplification and detection can now be combined into a single step, significantly shortening detection time.

[0004] Polymerase chain reaction (PCR) was the first amplification technique for amplifying and detecting low-abundance nucleic acids and remains the most popular amplification technique. It was invented nearly 30 years ago. 9 , which can detect specific target DNA sequences corresponding to a single bacterial pathogen. 10,11PCR amplification products can be visualized using electrophoresis gels stained with intercalating fluorescent dyes. PCR variations include multiplex PCR (mPCR) and real-time or quantitative PCR (qPCR). Multiplex PCR uses several sets of primers to simultaneously amplify multiple targets, allowing for more rapid detection compared to simple PCR. Primer design and concentration are particularly important in avoiding primer dimerization and producing reliable PCR products. In comparison, qPCR does not require gel electrophoresis for detection, but instead continuously monitors product formation by measuring fluorescence produced by intercalating dyes (such as SYBR Green), dual-labeled probes (Taqman), or molecular beacons. 12 For pathogens with RNA genomes, RT-PCR is employed, which uses RNA as a template to produce cDNA, which is then amplified by PCR. 13 Although highly sensitive and specific, various PCR methods are affected by PCR inhibitors present in nucleic acid preps and are costly and time-consuming due to the need for thermal cycling equipment and fluorescent probes. 4 .

[0005] Isothermal amplification of nucleic acids (INA) is an alternative to PCR in which amplification is achieved at a constant temperature without the need for thermal cycling, making it both simpler and less expensive. 14,15 INA methods can be performed in a wide range of conditions, such as in a water bath or equivalent fixed temperature heating device, and can be performed inside or on the surface of cells, unlike PCR. 14 INA reactions are characterized by their kinetics as being comparable to exponential amplification (e.g., nucleic acid sequence-based amplification, NASBA). 16 , Rolling Circle Amplification (RCA) 14 , Loop-mediated isothermal amplification (LAMP) 17 , Recombinase Polymerase Amplification (RPA) 18 , helicase-dependent amplification (HDA) 19, Nicking Enzyme Amplification (NEAR) 35 , Strand Displacement Amplification (SDA) 36 INA reactions can be classified as either linear or cascade amplification methods. Like qPCR, INA reactions can be analyzed as the reaction proceeds, which makes it more complex in terms of instrumentation but may shorten reaction times.

[0006] NASBA 16 utilizes three enzymes to amplify RNA products isothermally at 41°C. First, a primer containing a T7 promoter hybridizes to the target RNA and is extended by reverse transcriptase (RT). RNase H then degrades the hybridized RNA, leaving naked cDNA. A second primer then hybridizes to the cDNA and is extended by RT to the end of the first hybridized primer, producing dsDNA containing the T7 promoter. T7 RNA polymerase then transcribes the RNA encoded between the regions used by the originally annealed primers. As multiple copies of RNA are made, free primers can hybridize and be extended, continuing to produce more template. This results in exponential amplification of the DNA template and RNA product.

[0007] Rolling Circle Amplification (RCA) 14Exponential amplification involves a DNA or RNA polymerase using a circular DNA template to generate long RNA / DNA products. The circular template typically contains a polymerase promoter, a hybridization site, and a template for the product that can act as a reporter (commonly a target site for a hybridization-based reporter such as a molecular beacon). Unlike transcription using a linear target, which produces a single copy of the product, the polymerase can complete a full cycle of the circular template to generate many copies. Methods for exponential amplification involve hybridization oligonucleotides that hybridize to the target sequence, their ligation to form a closed circular template, and the polymerase producing multiple copies; the newly generated product contains multiple copies of the target sequence, which can serve as new templates for linear template hybridization.

[0008] Loop-mediated isothermal amplification (LAMP) 17 utilizes two or three sets of primers along with a strand-displacing DNA polymerase to isothermally generate multiple mixed species of DNA products at 60-65°C. This method relies on generating DNA products containing single-stranded loop regions that allow hybridization of primers to the already extended DNA products. The addition of reverse transcriptase allows for detection of RNA samples. Recombinase polymerase amplification (RPA) 18 RPA relies on three enzymes and can amplify a DNA product isothermally at 37°C, producing many DNA copies. First, a recombinase protein guides the primer strand to hybridize to the DNA template. Single-strand binding protein (SSB) binds to the strand of the DNA duplex that is being displaced, and also to the displacement. Next, DNA polymerase extends the primer, forming a new duplex. The same reaction occurs on the opposite strand, thus leading to the complete duplication of the DNA molecule. These steps continue cyclically for exponential amplification. RPA has been multiplexed with LAMP to simultaneously detect multiple targets. 20 .

[0009] Helicase-dependent amplification (HDA) 19 HDA is an isothermal amplification method that requires the use of DNA helicase. Essentially, this system functions similarly to PCR in that it relies on strand melting, primer annealing, and polymerase extension. While PCR requires a temperature change to aid the amplification process, HDA relies on enzymatic processing. First, DNA helicase melts the double-stranded DNA complex. Second, primers hybridize to the target DNA. Third, a strand-displacing DNA polymerase extends the primers to complete the new DNA duplex. This process repeats at 37°C, resulting in exponential amplification. Nicking Enzyme Amplification (NEAR) 35 and strand displacement amplification (SDA) 36 is an isothermal method for amplifying DNA at a constant temperature (55°C-59°C) using a strand-displacing DNA polymerase (Bst DNA polymerase, large fragment or Klenow fragment (3'-5' exo)) and a nicking enzyme. Nicks are created by a strand-limiting restriction endonuclease at sites contained within the primers. Nicks are generated with each polymerase displacement step, resulting in exponential amplification.

[0010] Amplifying very low concentrations of nucleic acids is a challenging task, and it has been known for some time that it is difficult to amplify RNA templates isothermally without concomitant amplification artifacts. 15 One attempt to address this problem uses the SHERLOCK approach, in which the products of RPA-based amplification are screened for the desired amplicon using a CRISPR-mediated cleavage mechanism that specifically cleaves a fluorescently tagged reporter construct. 21,22 Although increasing sensitivity and enabling SNP-based specificity, the requirement for an additional enzymatic step and fluorescent reporter adds significant complexity to RNA detection. SHERLOCK and DETECTR 23,21utilizes an initial isothermal amplification system (RPA) to amplify the target using a primer set containing a T7 promoter and a guide RNA cassette sequence. The RPA product is transcribed using T7 RNA polymerase, leading to the production of multiple copies of the guide RNA. The guide RNA then guides the Cas13a protein to detect the RNA species, resulting in activation of Cas13a for nonspecific degradation of the RNA species, in this case degrading an RNA molecular beacon and emitting a fluorescent signal (SHERLOCK). Alternatively, the guide RNA can guide Cas12a to target an RNA molecule, activating the enzyme for nonspecific cleavage of a DNA molecular beacon, also resulting in fluorescence (DETECTR). These techniques can be adapted to detect RNA species by adding reverse transcriptase to the initial RPA reaction. In total, these systems require five enzymes, plus a reporter for detection of DNA and an additional enzyme for detection of the RNA species.

[0011] RNA tags, such as fluorogenic RNA aptamers, can be used to label RNAs of interest. RNA aptamers to fluorogenic compounds that produce fluorescence upon binding can enhance the fluorescence (F) of fluorogenic aptamer systems. E ) and K of the aptamer-fluorophore interaction D can be selected using in vitro selection that optimizes both 24,25 Maximizing both parameters gives fluorogenic aptamers higher intrinsic contrast than MS2 fluorescent protein-recruited systems. 26,27,28 Because fluorophore ligands are inexpensive and RNA fluorogenic aptamers can be generated by transcription, fluorogenic aptamers potentially have many inherent advantages as reporters. The RNA Mango aptamer series has extremely high contrast, making them useful in vitro fluorescent reporters. These aptamers have nanomolar binding affinity for a thiazole orange-based ligand (TO1-biotin), which can be up to 4,000-fold brighter when bound to the RNA Mango aptamer.29,30,31 Of particular note, the second-generation RNA Mango aptamers (Mango II, III, and IV) are highly tolerant to magnesium ion concentrations typically found in in vitro assays and function across a wide range of monovalent metal ion concentrations. 30 The Mango III has also been recently improved with structure-guided manipulation to make it even brighter. 32 . Summary of the Invention

[0012] The present invention relates to the amplification and / or detection of nucleic acid molecules. In one aspect, the present invention provides a nucleic acid molecule or analog thereof comprising: a first nucleic acid sequence capable of hybridizing to at least a portion of a target nucleic acid sequence or its reverse complement and further comprising an aptamer-encoding template sequence, wherein the aptamer-encoding template sequence is located at the 3' end of the first nucleic acid sequence; and a second nucleic acid sequence capable of hybridizing to at least a portion of the target nucleic acid sequence or its reverse complement, wherein the 5' end of the second nucleic acid sequence is covalently linked to the 3' end of the first nucleic acid sequence and the 3' end of the second nucleic acid sequence does not substantially hybridize to the first nucleic acid sequence. In some embodiments, at least the terminal 3 nucleotides at the 3' end of the second nucleic acid sequence do not hybridize to the first nucleic acid sequence. In some embodiments, the first nucleic acid sequence can be from about 20 to about 100 nucleotides in length.

[0013] In some embodiments, the aptamer-encoding template sequence may encode a fluorogenic aptamer sequence. In some embodiments, the fluorogenic aptamer sequence has a fluorophore binding dissociation constant (K) of about 0.01 nM to about 100 nM. D ). In some embodiments, the nucleic acid molecule may comprise a terminal stem structure, and at least the terminal nucleotide at the 5' end of the second nucleic acid sequence may be complementary to at least the terminal nucleotide at the 5' end of the first nucleic acid and form at least a portion of the terminal stem structure. In some embodiments, at least the terminal 2 or 3 nucleotides at the 5' end of the second nucleic acid sequence may be complementary to at least the terminal 2 or 3 nucleotides at the 5' end of the first nucleic acid and form at least a portion of the terminal stem structure. In some embodiments, the nucleic acid molecule or analog thereof may be DNA-based or RNA-based.

[0014] In some embodiments, the second nucleic acid sequence may comprise a degenerate sequence. In some embodiments, the nucleic acid molecule does not include an RNA polymerase promoter sequence. In some embodiments, the target nucleic acid sequence may be of viral, microbial, fungal, animal, or plant origin, or may be a synthetic construct. In some embodiments, the target nucleic acid sequence may be from a pathogenic virus or pathogenic bacterium. In another aspect, the present invention provides a composition comprising a first nucleic acid molecule described herein. In some embodiments, the composition may further comprise a second nucleic acid molecule capable of hybridizing to at least a portion of the target nucleic acid sequence or its reverse complement and comprising a first RNA polymerase promoter sequence, wherein the first and second nucleic acid molecules form a first primer pair capable of amplifying a first sequence of the target nucleic acid sequence. In some embodiments, the 3' end of a first nucleic acid molecule is substantially unable to hybridize to a second nucleic acid molecule or to itself.

[0015] In some embodiments, the first and second nucleic acid molecules are substantially non-hybridizable to one another. In some embodiments, the terminal 1, 2, or 3 bases at the 3' end of a first nucleic acid molecule may hybridize to the terminal 1, 2, or 3 bases at the 3' end of a second nucleic acid molecule. In some embodiments, the 3' end of a first nucleic acid molecule may be adjacent to the 3' end of a second nucleic acid molecule when aligned with the sequence of a target nucleic acid. In some embodiments, the compositions described herein may further comprise a third nucleic acid molecule and a fourth nucleic acid molecule, wherein the third and fourth nucleic acid molecules form a second primer pair capable of amplifying a second sequence of the target nucleic acid molecule, and wherein either the third nucleic acid molecule or the fourth nucleic acid molecule may comprise a second RNA polymerase promoter sequence, and wherein the second primer pair may hybridize to the target nucleic acid molecule at a position outside that of the first primer pair and amplify the first sequence and the second sequence. In some embodiments, the second RNA polymerase promoter sequence is capable of transcribing the second sequence of the target nucleic acid molecule in the opposite direction to that of the second nucleic acid molecule.

[0016] In some embodiments, when the third nucleic acid molecule comprises a second RNA polymerase promoter sequence, the fourth nucleic acid molecule comprises a second aptamer coding sequence, or when the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence, the third nucleic acid molecule comprises a second aptamer coding sequence. In some embodiments, the 3' end of the third nucleic acid molecule is substantially non-hybridizable to the fourth nucleic acid molecule. In some embodiments, the third and fourth nucleic acid molecules are substantially non-hybridizable to one another. In some embodiments, the 3' ends of the first, second, third, and fourth nucleic acid molecules are substantially non-hybridizable to one another. In some embodiments, the first, second, third, and fourth nucleic acid molecules are substantially non-hybridizable with one another. In some embodiments, the compositions described herein may further comprise a fifth nucleic acid molecule and a sixth nucleic acid molecule, wherein the fifth and sixth nucleic acid molecules may form a third primer pair capable of amplifying a third sequence of the target nucleic acid molecule, wherein either the fifth nucleic acid molecule or the sixth nucleic acid molecule may comprise a third RNA polymerase promoter sequence, and wherein the third primer pair may hybridize to the target nucleic acid molecule at a position outside those of the first and second primer pairs and amplify the first, second, and third sequences.

[0017] In some embodiments, the third RNA polymerase promoter sequence is capable of transcribing a third sequence of the target nucleic acid molecule in the same direction as the second nucleic acid molecule. In some embodiments, when the fifth nucleic acid molecule comprises a third RNA polymerase promoter sequence, the fourth nucleic acid molecule comprises a third aptamer coding sequence, or when the fourth nucleic acid molecule comprises a third RNA polymerase promoter sequence, the fifth nucleic acid molecule comprises a third aptamer coding sequence. In some embodiments, the 3' end of the fifth nucleic acid molecule is substantially non-hybridizable to the 3' end of the fourth nucleic acid molecule. In some embodiments, the fifth and fourth nucleic acid molecules are substantially non-hybridizable to each other. In some embodiments, the 3' ends of the first, second, third, fourth, fifth, and sixth nucleic acid molecules are substantially non-hybridizable to one another. In some embodiments, the first, second, third, fourth, fifth, and sixth nucleic acid molecules are substantially non-hybridizable with one another. In some embodiments, the compositions described herein may comprise one or more nucleic acid molecules comprising a sequence shown in Table 3.

[0018] In some embodiments, one or more of the nucleic acid molecules may be premixed. In some embodiments, one or more of the nucleic acid molecules may be provided in a liquid. In some embodiments, one or more of the nucleic acid molecules may be lyophilized. In another aspect, the invention provides kits comprising one or more of the nucleic acid molecules or compositions described herein together with instructions for amplification of a target nucleic acid sequence. In some embodiments, the amplification can be an isothermal amplification, such as nucleic acid sequence-based amplification, rolling circle amplification, loop-mediated isothermal amplification, helicase-dependent amplification, or strand displacement amplification. In another aspect, the present invention provides a method of amplifying a target nucleic acid sequence, the method comprising the steps of: providing a sample suspected of containing a target nucleic acid molecule; providing a first nucleic acid molecule as described herein; providing a second nucleic acid molecule capable of hybridizing to at least a portion of the target nucleic acid sequence or its complement and comprising a first RNA polymerase promoter sequence, wherein the first and second nucleic acid molecules form a first primer pair capable of amplifying a first sequence of the target nucleic acid sequence; and performing a first amplification reaction comprising the target nucleic acid molecule and the first primer pair to obtain a first amplification product, wherein the first amplification product comprises the first sequence of the target nucleic acid sequence.

[0019] In some embodiments, the 3' end of the first nucleic acid molecule is substantially non-hybridizable to the 3' end of the second nucleic acid molecule. In some embodiments, the first and second nucleic acid molecules are substantially non-hybridizable to one another. In some embodiments, the terminal 1, 2, or 3 bases at the 3' end of a first nucleic acid molecule may hybridize to the terminal 1, 2, or 3 bases at the 3' end of a second nucleic acid molecule. In some embodiments, the 3' end of a first nucleic acid molecule may be adjacent to the 3' end of a second nucleic acid molecule when aligned with the sequence of a target nucleic acid. In some embodiments, the method may further include providing a third nucleic acid molecule and a fourth nucleic acid molecule, wherein the third and fourth nucleic acid molecules form a second primer pair capable of amplifying a second sequence of the target nucleic acid molecule, wherein either the third nucleic acid molecule or the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence, and wherein the second primer pair is capable of hybridizing to the target nucleic acid molecule at a position outside that of the first primer pair and amplifying the first and second sequences of the target nucleic acid molecule; and performing a second amplification reaction comprising the first amplification product and the second primer pair to obtain a second amplification product, wherein the second amplification reaction may be performed before the first amplification reaction, and the second amplification product may comprise the first and second sequences of the target nucleic acid molecule.

[0020] In some embodiments, the second RNA polymerase promoter sequence is capable of transcribing the second sequence of the target nucleic acid molecule in the opposite direction to that of the second nucleic acid molecule. In some embodiments, when the third nucleic acid molecule comprises a second RNA polymerase promoter sequence, the fourth nucleic acid molecule comprises a second aptamer coding sequence, or when the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence, the third nucleic acid molecule comprises a second aptamer coding sequence. In some embodiments, the 3' end of the third nucleic acid molecule is substantially non-hybridizable to the 3' end of the fourth nucleic acid molecule. In some embodiments, the third and fourth nucleic acid molecules are substantially non-hybridizable to one another. In some embodiments, the 3' ends of the first, second, third, and fourth nucleic acid molecules are substantially non-hybridizable to one another. In some embodiments, the first, second, third, and fourth nucleic acid molecules are substantially non-hybridizable with one another.

[0021] In some embodiments, the methods described herein further comprise detecting a target nucleic acid sequence. In some embodiments, the methods described herein further comprise quantifying the target nucleic acid sequence. In some embodiments, the amplification can be an isothermal amplification, such as nucleic acid sequence-based amplification, rolling circle amplification, loop-mediated isothermal amplification, helicase-dependent amplification, strand displacement amplification, or a combination thereof. In some embodiments, the amplification may be RNA-based or DNA-based. In some embodiments, the amplification may be multiplexed. In some embodiments, the amplification may include color imaging in at least two colors. In some embodiments, the amplification may include color imaging in at least three colors. In some embodiments, the sample may be of viral, microbial, fungal, animal, plant, or environmental origin. In some embodiments, the sample may be from a pathogenic virus, such as a coronavirus (e.g., SARS, MERS, or SARS-CoV-2), or a pathogenic bacterium. In some embodiments, the sample is obtained from water, soil, saliva, feces, urine, blood, tracheal aspirate, or nasal aspirate. In some embodiments, the animal may be a human.

[0022] In another aspect, the present invention provides a method for detecting a target nucleic acid molecule by providing a sample containing the nucleic acid molecule; and amplifying the nucleic acid molecule by isothermal nucleic acid amplification (INA), wherein the amplifying step comprises the use of nested oligonucleotide primer pairs. In some embodiments, the nested oligonucleotide primer pair may include a fluorogenic aptamer sequence. In some embodiments, detection may be highly sensitive. In an alternative aspect, the present invention provides a kit comprising a nested oligonucleotide primer pair together with instructions for use in an isothermal nucleic acid amplification method, wherein the nested oligonucleotide primer pair may comprise a fluorogenic aptamer sequence. This summary of the invention does not necessarily describe all aspects of the invention. These and other features of the present invention will become more apparent from the following description, in which reference is made to the accompanying drawings. [Brief explanation of the drawings]

[0023] [Figure 1]Figure 1 shows the insertion of an RNA fluorogenic aptamer into an RNA-producing isothermal amplification system. A. Traditional NASBA uses two primers to generate an RNA product. Artifacts are also commonly produced (black and gray products). B. The fluorogenic aptamer-NASBA system features the addition of a fluorogenic aptamer template sequence to the top strand (PB) primer, which results in the production of an RNA product containing a fluorescent fluorogenic aptamer tag after T7 transcription. C. Nested fluorogenic aptamer-NASBA features an outer primer NASBA reaction whose product is then diluted and placed into an inner fluorogenic aptamer-NASBA reaction (shown here using, but not limited to, the Mango aptamer). [Figure 2] Nested fluorogenic aptamer NASBA is highly sensitive and specific to the target RNA sequence, even when extraneous nucleic acid background is added. A. Non-nested outer (E. coli ClpB RNA) and B. inner non-nested fluorogenic aptamer NASBA (P. fluorescens ClpB RNA) reactions. C. Nested RNA fluorogenic aptamer NASBA dramatically improves sensitivity. E. coli primers with E. coli target (Ec / Ec, gray bars in the left set). The same E. coli primers were used with P. fluorescens target added instead of E. coli target (Ec / Pf, dark gray bars in the middle set). P. fluorescens primers with P. fluorescens target (Pf / Pf, lightest gray bars in the right set). D. Nested fluorogenic aptamer NASBA using E. coli primers was performed with the inner NASBA time course shown. Black - no template added, lightest grey at the top - 150 E. coli target molecules / µL reaction, lighter grey at the bottom - 5ng / µL A549 human lung carcinoma total nucleic acid, grey - 150 E. coli target molecules / µL in the presence of 5ng / µL A549 human lung carcinoma total nucleic acid. Error bars reflect the standard deviation of triplicates for all panels. [Figure 3]Schematic showing how nesting NASBA primers increases specificity. A: Nested PCR (left) results in higher specificity due to the requirement for a second set of inner primers to hybridize within the first pair of outer primers. Nested isothermal reactions (right) can improve target specificity. B: Schematic of the nested fluorogenic aptamer NASBA process. [Figure 4] Figures and graphs showing the relative insensitivity of non-nested outside fluorogenic aptamer NASBA fluorescence appearance. A. Schematic of outside fluorogenic aptamer NASBA; B. Non-nested E. coli outside fluorogenic aptamer NASBA primer with E. coli target (Ec O / Ec); Values ​​at the dotted line (40 min time point) for Ec O / Ec were taken and plotted as in Figure 2A. [Figure 5] Figures and graphs show that non-nested inner fluorogenic aptamer NASBA fluorescence emergence with P. fluorescens is also relatively insensitive. A. Schematic of inner fluorogenic aptamer NASBA. P. fluorescens inner fluorogenic aptamer NASBA primer (Pf / Pf / inner) with P. fluorescens inner target. Ec. The value at the dotted line (40 min) for O / Ec was taken and plotted as in Figure 2B. [Figure 6] This graph shows that nested fluorogenic aptamer NASBA fluorescence is highly sensitive and specific. E. coli primers with E. coli ClpB RNA target (Ec / Ec table heading). The same E. coli primers were used, but with P. fluorescens target instead of E. coli target (Ec / Pf table heading). P. fluorescens primers with P. fluorescens target (Pf / Pf table heading). All traces show the time dependence of the associated inner fluorogenic aptamer NASBA reaction. Template concentrations are specified in RNA molecules / µL of the associated outer reaction. The value at the indicated dotted line (100 min time point) was taken and plotted as in Figure 2C. [Figure 7]Figure 1 shows the detection of E. coli RNA in MCF7 human tissue culture medium using the nested fluorogenic aptamer NASBA. Nested fluorogenic aptamer NASBA for a dilution series of E. coli cell extract from human tissue culture. Nucleic acid extract from depleted medium (0 ng) and 27 nM final ClpB short target E. coli (PC) were used as negative and positive controls, respectively. Nanogram (as determined by Nanodrop) amounts of E. coli cell extract per 20 μL reaction are shown. The estimated total number of bacterial cells in the 20 μL reaction is shown in parentheses. [Figure 8] Images and graphs show that fluorogenic aptamer non-nested NASBA generates nonspecific products at low template concentrations, regardless of primer concentration. A. Fluorogenic aptamer NASBA reactions outside Ec were performed for 2 hours, and samples were denatured and run on 8% PAGE followed by staining with TO1-biotin in buffer. E. coli ClpB target concentrations are indicated, with 250 nM primers. B. Serial dilutions of the Ec outside primer set in fluorogenic aptamer NASBA reveal that nonspecific products are not significantly dependent on primer concentration and occur over a wide range of primer concentrations. Primer concentrations: 125 nM, 25 nM, 5 nM, 1 nM, 0 M. The addition or absence of 25 pM E. coli ClpB target (Yes / No target) corresponds to a light or dark trace, respectively. [Figure 9] Images showing that only the expected RNA-sized product is fluorescent in nested fluorogenic aptamer NASBA, as opposed to non-nested NASBA. A. The product of the outer NASBA reaction (at 40 min) in nested fluorogenic aptamer NASBA was loaded onto an 8% denaturing PAGE and subsequently stained with SYBR Safe; the inner NASBA sample, shown in Figure S5, was collected after 240 min of incubation, denatured, and then run on two 8% PAGE gels and subsequently stained with either TO1-biotin (B) or SYBR Safe (C) in buffer. [Figure 10]Figure 1 shows the alignment of ClpB short targets from E. coli (SEQ ID NO: 1) and P. fluorescens (SEQ ID NO: 2). E. coli primer hybridization site, P. fluorescens hybridization site. Primer numbering corresponds to that found in Table 1. [Figure 11] Schematic for nucleic acid detection using fluorogenic aptamer-templated rolling circle amplification. A. Using a two-step ligation-rolling circle amplification (RCA) method, RNA and DNA can be conveniently and isothermally detected. The template can hybridize to the target, followed by its ligation to a circular template. B. This template can now be transcribed to mass-produce RNA fluorogenic aptamers. C. The RNA produced as described is expected to generate additional target sites that can be used for further nested ligations, effectively converting the reaction into an exponential amplification process. [Figure 12] Images showing that transcription using circular (left lane) and linear (right lane) templates results in long and short RNA products, respectively, as a function of time. Time points are 0, 30 s doubling time up to 64 min. [Figure 13] 1 is a graph showing that ligation with either DNA or RNA targets followed by transcription (t=5 min) results in the rapid appearance of fluorogenic aptamer fluorescence. [Figure 14] 1 is a graph showing that 4 / 5 primer sets targeting SARS-CoV-2 sequences were able to successfully detect 1 fM of target RNA in nested mango NASBA. 40-minute outer reactions. P1 and P2 from each set in Table 2 were used as the outer reactions, and P3 and P4 from each primer set in Table 2 were used as the inner reactions. [Figure 15]Graphs showing the sensitivity of RNA detection using the liquid NASBA kit and the detection of SARS-CoV-2 Target 4 RNA in a background of total human RNA. A. The outer reaction was performed for 40 minutes. Human RNA (HNA) was added at 5 ng / μL final (gray - positive; black - negative). B. The slope of the dataset shown in (A) delivers a robust positive signal. Primer set 4 (Table 2) P1 and P2 (outer) followed by P3 and P4 (inner reaction above). [Figure 16] 1 shows the sensitivity of SARS-CoV-2 Target 4 RNA detection using liquid (wet) and lyophilized (dry) NASBA kits. The outer reaction was performed for 40 minutes. Primer set 4 (Table 2) P1 and P2 (outer) followed by P3 and P4 (inner reaction as above). [Figure 17] Figure 1 shows that EDTA and heating are not required for low-copy SARS-CoV-2 Target 4 RNA detection. 100 aM RNA is equally detected when EDTA is omitted (T1) and when the sample is not heated and no EDTA is added (T2). The outer reaction was performed for 40 minutes. Wet data from Figure 1. Primer set 4 (Table 2) P1 and P2 (outer) followed by P3 and P4 (inner reaction as above). [Figure 18] Figure 1 shows the optimization of the outer reaction time using the lyophilized NASBA kit and SARS-CoV-2 Target 4 RNA. The template concentration was 10 aM. A 20-minute outer reaction was found to be the shortest time that produced the same robust signal as a 40-minute outer incubation. Primer set 4 (Table 2) P1 and P2 (outer reaction), followed by P3 and P4 (inner reaction as above). [Figure 19] 10 is a graph showing the sensitivity of the single-step Mango NASBA to detect SARS-CoV-2 Target 4 RNA using a lyophilized NASBA kit: P5 and P6 of Primer Set 4 (Table 2). [Figure 20]1 is a graph showing successful detection of cultured SARS-CoV-2 RNA using the Liquid LS Kit. The Liquid LS Kit was used with the dilutions shown in a nested format: Primer Set 4 (Table 2) P1 and P2 (outer) followed by P3 and P4 (inner reaction as above). [Figure 21] This graph shows the successful detection of cultured SARS-CoV-2 RNA using the LS Lyophilized Kit. 1 fM of synthetic SARS-CoV-2 Target 4 RNA was used as a positive control. Detection of cultured viral RNA was tested under various conditions: NOD - 1 / 20 dilution from the outer reaction to the inner reaction (typically, a 1 / 100 dilution is used); NOR - RNA template only heated, no primers; NH - no heating; and P1 and P2 (outer) of Primer Set 4 (Table 2), followed by P3 and P4 (inner reaction as above). [Figure 22] Graphs showing successful detection of SARS-CoV-2 RNA from patient samples using the lyophilized NASBA kit. A. Raw data showing some initial turbidity at low times. B. Plotting the slope of this data provides clear emergence times. Synthesis corresponds to target 4β RNA. Primer set 4 (Table 2) P1 and P2 (outer) followed by P3 and P4 (inner reaction above). [Figure 23] This graph shows that heating with template alone or with primers is not required for detection of SARS-CoV-2 in patients. Nested mango samples were subjected to NASBA using the lyophilized LS kit with and without prior heating with template (NH). POS represents a patient sample known to have COVID-19. Primer set 4 (Table 2) P1 and P2 (outer) followed by P3 and P4 (inner reaction as above). [Figure 24] Schematic of fluorogenic aptamer NASBA using an internal control reaction. The liquid vessel containing the reaction mixture can have an internal control RNA (such as human 18S ribosomal RNA) as well as oligomers targeting a target RNA (such as SARS-CoV-2 RNA). [Figure 25] 1 is a schematic of an exemplary aptamer fusion primer. DETAILED DESCRIPTION OF THE INVENTION

[0024] This disclosure relates, in part, to the amplification, detection, and / or quantification of nucleic acid molecules. In some embodiments, the present disclosure provides methods for nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), nicking enzyme amplification (NEAR). 35 , Strand Displacement Amplification (SDA) 36 The present invention provides methods for amplifying target nucleic acid molecules using non-nested and / or nested oligonucleotide primer pairs in isothermal nucleic acid amplification (INA) reactions, such as linear and cascade amplification methods.

[0025] In some embodiments, the present disclosure provides methods for nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), nicking enzyme amplification (NEAR). 35 , Strand Displacement Amplification (SDA) 36 Further provided are methods for detecting target nucleic acid molecules using non-nested and / or nested oligonucleotide primer pairs in isothermal nucleic acid amplification (INA) reactions, such as linear and cascade amplification methods. In some embodiments, the present disclosure provides methods for nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), nicking enzyme amplification (NEAR). 35 , Strand Displacement Amplification (SDA) 36 Further provided are methods for quantifying target nucleic acid molecules using non-nested and / or nested oligonucleotide primer pairs in isothermal nucleic acid amplification (INA) reactions, such as linear and cascade amplification methods.

[0026] In one aspect, the present disclosure provides a nucleic acid molecule or analog thereof comprising: a first nucleic acid sequence capable of hybridizing to at least a portion of a target nucleic acid sequence or its reverse complement and further comprising an aptamer-encoding template sequence, wherein the aptamer-encoding template sequence is located at the 3' end of the first nucleic acid sequence; and a second nucleic acid sequence capable of hybridizing to at least a portion of the target nucleic acid sequence or its reverse complement, wherein the 5' end of the second nucleic acid sequence is covalently linked to the 3' end of the first nucleic acid sequence. In some embodiments, the 3' end of the second nucleic acid sequence does not substantially hybridize to the first nucleic acid sequence. In some embodiments, at least the terminal 3 nucleotides of the 3' end of the second nucleic acid sequence do not hybridize to the first nucleic acid sequence. In some embodiments, the nucleic acid molecule may comprise a terminal stem structure, and at least the terminal nucleotides of the 5' end of the second nucleic acid sequence may be complementary to at least the terminal nucleotides of the 5' end of the first nucleic acid, forming at least a portion of the terminal stem structure. In some embodiments, at least the terminal 2 or 3 nucleotides of the 5' end of the second nucleic acid sequence may be complementary to at least the terminal 2 or 3 nucleotides of the 5' end of the first nucleic acid, forming at least a portion of the terminal stem structure.

[0027] In another aspect, the disclosure provides a composition comprising a first nucleic acid molecule described herein. In some embodiments, the composition may further comprise a second nucleic acid molecule capable of hybridizing to at least a portion of the target nucleic acid sequence or its reverse complement and comprising a first RNA polymerase promoter sequence, wherein the first and second nucleic acid molecules form a first primer pair capable of amplifying a first sequence of the target nucleic acid sequence. In some embodiments, the 3' end of the first nucleic acid molecule may not substantially hybridize to the second nucleic acid molecule or to itself. In some embodiments, the first and second nucleic acid molecules may not substantially hybridize to each other. In some embodiments, the terminal 1, 2, or 3 bases of the 3' end of the first nucleic acid molecule may hybridize to the terminal 1, 2, or 3 bases of the 3' end of the second nucleic acid molecule. In some embodiments, when aligned with the sequence of the target nucleic acid, the 3' end of the first nucleic acid molecule may be adjacent to the 3' end of the second nucleic acid molecule. In some embodiments, the compositions described herein may further comprise a third nucleic acid molecule and a fourth nucleic acid molecule, wherein the third and fourth nucleic acid molecules form a second primer pair capable of amplifying a second sequence of the target nucleic acid molecule, and wherein either the third nucleic acid molecule or the fourth nucleic acid molecule may comprise a second RNA polymerase promoter sequence, and wherein the second primer pair may hybridize to the target nucleic acid molecule at a position outside that of the first primer pair and amplify the first sequence and the second sequence.

[0028] In some embodiments, the second RNA polymerase promoter sequence can transcribe the second sequence of the target nucleic acid molecule in the opposite direction to that of the second nucleic acid molecule. In some embodiments, when the third nucleic acid molecule comprises the second RNA polymerase promoter sequence, the fourth nucleic acid molecule comprises a second aptamer coding sequence, or when the fourth nucleic acid molecule comprises the second RNA polymerase promoter sequence, the third nucleic acid molecule comprises a second aptamer coding sequence. In some embodiments, the 3' end of the third nucleic acid molecule cannot substantially hybridize to the fourth nucleic acid molecule. In some embodiments, the third and fourth nucleic acid molecules cannot substantially hybridize to each other. In some embodiments, the 3' ends of the first, second, third, and fourth nucleic acid molecules cannot substantially hybridize to each other. In some embodiments, the first, second, third, and fourth nucleic acid molecules cannot substantially hybridize to each other.

[0029] In some embodiments, the compositions described herein may further include a fifth nucleic acid molecule and a sixth nucleic acid molecule, where the fifth and sixth nucleic acid molecules may form a third primer pair capable of amplifying a third sequence of the target nucleic acid molecule, and either the fifth nucleic acid molecule or the sixth nucleic acid molecule may include a third RNA polymerase promoter sequence, where the third primer pair may hybridize to the target nucleic acid molecule at a position outside those of the first and second primer pairs and amplify the first, second, and third sequences. In some embodiments, the third RNA polymerase promoter sequence may transcribe the third sequence of the target nucleic acid molecule in the same direction as the second nucleic acid molecule. In some embodiments, when the fifth nucleic acid molecule includes a third RNA polymerase promoter sequence, the fourth nucleic acid molecule includes a third aptamer coding sequence, or when the fourth nucleic acid molecule includes a third RNA polymerase promoter sequence, the fifth nucleic acid molecule includes a third aptamer coding sequence. In some embodiments, the 3' end of the fifth nucleic acid molecule is substantially non-hybridizable to the 3' end of the fourth nucleic acid molecule. In some embodiments, the fifth and fourth nucleic acid molecules are substantially non-hybridizable to each other. In some embodiments, the 3' ends of the first, second, third, fourth, fifth, and sixth nucleic acid molecules are substantially non-hybridizable to each other. In some embodiments, the first, second, third, fourth, fifth, and sixth nucleic acid molecules are substantially non-hybridizable to each other.

[0030] In some embodiments, the compositions described herein may include one or more nucleic acid molecules comprising a sequence set forth in Table 3. In some embodiments, one or more of the nucleic acid molecules may be premixed. In some embodiments, one or more of the nucleic acid molecules may be provided in a liquid form. In some embodiments, one or more of the nucleic acid molecules may be lyophilized. In another aspect, the present disclosure provides a method of amplifying a target nucleic acid sequence, the method comprising the steps of: providing a sample suspected of containing a target nucleic acid molecule; providing a first nucleic acid molecule as described herein; providing a second nucleic acid molecule capable of hybridizing to at least a portion of the target nucleic acid sequence or its complement and comprising a first RNA polymerase promoter sequence, wherein the first and second nucleic acid molecules form a first primer pair capable of amplifying a first sequence of the target nucleic acid sequence; and performing a first amplification reaction comprising the target nucleic acid molecule and the first primer pair to obtain a first amplification product, wherein the first amplification product comprises the first sequence of the target nucleic acid sequence.

[0031] In some embodiments, the 3' end of the first nucleic acid molecule may not substantially hybridize to the 3' end of the second nucleic acid molecule. In some embodiments, the first and second nucleic acid molecules may not substantially hybridize to each other. In some embodiments, the terminal 1, 2, or 3 bases of the 3' end of the first nucleic acid molecule may hybridize to the terminal 1, 2, or 3 bases of the 3' end of the second nucleic acid molecule. In some embodiments, when aligned with the sequence of the target nucleic acid, the 3' end of the first nucleic acid molecule may be adjacent to the 3' end of the second nucleic acid molecule. In some embodiments, the method may further include providing a third nucleic acid molecule and a fourth nucleic acid molecule, where the third and fourth nucleic acid molecules form a second primer pair capable of amplifying a second sequence of the target nucleic acid molecule, and either the third nucleic acid molecule or the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence, where the second primer pair can hybridize to the target nucleic acid molecule at a position outside that of the first primer pair and amplify the first and second sequences of the target nucleic acid molecule; and performing a second amplification reaction comprising the first amplification product and the second primer pair to obtain a second amplification product, where the second amplification reaction can be performed before the first amplification reaction, and the second amplification product can comprise the first and second sequences of the target nucleic acid molecule. In some embodiments, the second RNA polymerase promoter sequence can transcribe the second sequence of the target nucleic acid molecule in the opposite direction to that of the second nucleic acid molecule. In some embodiments, when the third nucleic acid molecule comprises a second RNA polymerase promoter sequence, the fourth nucleic acid molecule comprises a second aptamer coding sequence, or when the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence, the third nucleic acid molecule comprises a second aptamer coding sequence. In some embodiments, the 3' end of the third nucleic acid molecule is substantially non-hybridizable with the 3' end of the fourth nucleic acid molecule. In some embodiments, the third and fourth nucleic acid molecules are substantially non-hybridizable with each other. In some embodiments, the 3' ends of the first, second, third, and fourth nucleic acid molecules are substantially non-hybridizable with each other. In some embodiments, the first, second, third, and fourth nucleic acid molecules are substantially non-hybridizable with each other. A "nucleic acid" or "nucleic acid molecule" is a chain of nucleotides, each of which consists of a nitrogen-containing aromatic base attached to a pentose sugar, which in turn is attached to a phosphate group, connecting successive sugar residues by bridging the 5'-hydroxyl group of one sugar to the 3'-hydroxyl group of the next sugar in the chain via a phosphodiester bond. Nucleic acids thus have a directionality, having a 5' end and a 3' end, and by convention, new nucleotides are added to the 3' end. By convention, nucleic acid "sequences" are written in the 5' to 3' direction.

[0032] Nucleic acids can be double-stranded or single-stranded. If single-stranded, the nucleic acid can be the sense or antisense strand. A nucleic acid molecule can be any strand of two or more covalently linked nucleotides, including naturally occurring or modified nucleotides. By "RNA" is meant a sequence of two or more covalently linked, naturally occurring or modified ribonucleotides. By "DNA" is meant a sequence of two or more covalently linked, naturally occurring or modified deoxyribonucleotides. By "cDNA" is meant complementary or copy DNA produced from an RNA template by the action of an RNA-dependent DNA polymerase (reverse transcriptase). The terms "nucleic acid" or "nucleic acid molecule" encompass both RNA (plus and minus strands) and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA.

[0033] As used herein, a nucleic acid "analog" is a nucleic acid containing at least one modified nucleotide that can be amplified by an enzyme such as a polymerase. In some embodiments, nucleic acid analogs can be amplified by RNA polymerases such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and bacterial DNA-dependent RNA polymerase. In some embodiments, nucleic acid analogs can incorporate locked nucleic acid (LNA) nucleotides (Latorra et al., Hum. Mutat. 22:79-85 2003) or peptide nucleic acids. "Modified ribonucleotides" or "modified RNA" include, but are not limited to, RNAs with modifications of the 2'-OH group of ribose (such as 2'-NH2, 2'-fluoro, or 2'-O-methyl) and modifications of the nucleobases that do not interfere with standard Watson-Crick hybridization.

[0034] "Modified deoxyribonucleotides" or "modified DNA" include, but are not limited to, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine), and N8-(7-deaza-8-aza-adenine). By "complementary" or "complementarity," it is meant that two nucleic acids, e.g., DNA and / or RNA, contain a sufficient number of nucleotides that can form Watson-Crick base pairs, creating a region of double-strandedness between the two nucleic acids. Thus, an adenine in one strand of DNA and / or RNA pairs with a thymine in the opposite complementary DNA strand or with a uracil in the opposite complementary RNA strand. It will be understood that not every single nucleotide in a nucleic acid molecule needs to form a matching Watson-Crick base pair with a nucleotide in the opposite complementary strand to form a duplex. A nucleic acid is also "complementary" to another nucleic acid if it hybridizes or is "capable of hybridizing" to that other nucleic acid.

[0035] As used herein, a "reverse complement" or "complement" sequence is the complementary sequence of a nucleic acid strand, presented 5' to 3'. As used herein, "capable of hybridizing" means that a nucleic acid can base pair with another nucleic acid having a substantially complementary sequence. In some embodiments, "capable of hybridizing" means that a nucleic acid can base pair with another nucleic acid having a substantially complementary sequence under conditions suitable for amplification, such as isothermal amplification. "Substantially" complementary means that base pairing can be partial, i.e., not all nucleotides in one nucleic acid need to be properly base paired with all nucleotides in the other nucleic acid, and there can be one or more base pairing mismatches between the two nucleic acids. "A portion of" means that hybridization need not occur over the entire length of the nucleic acid.

[0036] It should be understood that the stability of the resulting double-stranded molecule depends on the degree of base pairing that occurs and is affected by parameters such as the degree of complementarity between the two nucleic acids and the degree of stringency of the hybridization conditions. The degree of stringency of hybridization can be affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and can be determined by methods known to those skilled in the art. By "substantially non-hybridizing" it is meant that a nucleic acid does not substantially base pair with another nucleic acid under conditions suitable for amplification, such as isothermal amplification. Thus, in some embodiments, by "substantially non-hybridizing" it is meant that nucleic acids described herein, such as a first, second, third, fourth, fifth, or sixth nucleic acid or a first, second, or third primer pair, do not hybridize with each other or internally. In some embodiments, by "substantially non-hybridizing" it is meant that the 3' end of the nucleic acid, e.g., the terminal nine (9), e.g., the terminal 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides, do not base pair within the sequence of any other nucleic acid in the system. In some embodiments, by "does not substantially hybridize" it is meant that the 3' end of a nucleic acid, e.g., the terminal nine (9) nucleotides, such as the terminal 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides, do not base pair with the terminal nine (9) nucleotides, such as the terminal 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides, of another nucleic acid.

[0037] By "amplification" is meant a process by which additional copies of a nucleic acid sequence are produced. Nucleic acid amplification processes are known in the art and can include, but are not limited to, polymerase chain reactions (PCRs), such as methylation-sensitive PCR, nested PCR, cold-PCR, digital PCR, droplet digital PCR, ICE-cold-PCR, multiplex PCR (mPCR), real-time or quantitative PCR (qPCR), reverse transcriptase (RT)-PCR, or quantitative reverse transcriptase (RT)-PCR. In some embodiments, the "amplification" process can be isothermal, i.e., amplification is carried out at a constant temperature. Isothermal amplification of nucleic acids (INA) can include, but is not limited to, nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), nicking enzyme amplification (NEAR), strand displacement amplification (SDA), or linear and cascade amplification methods. In some embodiments, suitable isothermal amplification methods, such as those involving RNA intermediates, can be used, as exemplified by the NASBA or TMA methods described herein or known in the art. Thus, in some embodiments, RNA-producing isothermal amplification can produce antisense sequences using a reverse primer containing an RNA polymerase promoter sequence, such as T7, T3, or SP6. This can produce an RNA output that is the reverse complement of the input sequence. Thus, in some embodiments, when nesting reactions, the inner nested reaction requires an RNA polymerase promoter sequence in the opposite primer. In some embodiments, RNA-producing isothermal amplification can be used with a fluorogenic aptamer template, as described herein or known in the art.

[0038] In some embodiments, other isothermal amplification methods can be readily adapted and used as described herein. For example, RCA can be adapted by transcribing RNA off of DNA circles as described herein. In alternative embodiments, other DNA-based isothermal methods, such as LAMP, RPA, NEAR, HDA, or SDA, can be similarly adapted by adding an RNA polymerase promoter to the DNA oligonucleotide, depending on the isothermal amplification method being used, so that RNA transcription serves to report the DNA amplification products produced by the isothermal method. In some embodiments, HDA, RPA, or NEAR primers can be modified to have an RNA polymerase promoter and enzyme and a fluorogenic aptamer template, allowing for RNA aptamer production as a reporter of successful amplification. In some embodiments, HDA, RPA, or NEAR primers can be modified to have a DNA fluorogenic aptamer template, allowing for DNA aptamer production as a reporter of successful amplification.

[0039] In some embodiments, the conditions suitable for amplification may be conditions suitable for PCR, as known in the art. In some embodiments, the conditions suitable for isothermal amplification may be conditions suitable for a particular isothermal amplification method selected, such as NASBA, RCA, LAMP, HAD, SDA, etc., as described herein or known in the art. For example, with respect to NASBA, conditions suitable for amplification can include isothermal amplification of RNA products at about 41°C using a primer containing an RNA polymerase promoter (e.g., a T7 promoter) described herein or known in the art that hybridizes to the target nucleic acid, e.g., RNA, and is extended by reverse transcriptase (RT). RNase H then degrades the hybridized RNA, leaving behind exposed cDNA. A second primer described herein or known in the art then hybridizes to the cDNA and is extended by RT to the end of the first hybridized primer, producing dsDNA containing the T7 promoter. RNA polymerase then transcribes the RNA encoded between the regions used by the originally annealed primers. As multiple copies of RNA are made, free primers can hybridize and be extended, continuing to produce more template, resulting in exponential amplification of the DNA template and RNA product.

[0040] In some embodiments, for example, the amplification parameters for NASBA using the Mango aptamer are as follows: a. No heating of the RNA sample before performing the NASBA reaction; b. EDTA-free; c. A shorter reaction time, for example, about 20 minutes; d. In the case of nested reactions, approximately a 20-fold dilution from the outer to the inner reaction; and / or e. Freeze-drying of reagents may include one or more of: Generally, an isothermal reaction consists of a single set of isothermal amplification nucleic acids, designated as a set, required to complete the exponential amplification process. In contrast, in some embodiments, the present disclosure provides isothermal amplification reactions that can be multiplexed, as described herein. For example, "n" sets of isothermal amplification primers or "primer pairs" can be generated, where n can be 1, 2, 3, or greater. A separate primer set or pair can be used for each target nucleic acid to be amplified. This allows for the amplification, detection, and / or quantification of "n" target nucleic acids by "multiplexing," i.e., simultaneous detection of multiple target nucleic acids within the same reaction, which can enable important internal control and validation. In this example, an appropriate number of primer sets are provided, for example, in a reaction mixture. For example, two primer sets can be provided to preferentially amplify two target nucleic acid sequences, three primer sets can be provided to preferentially amplify three target nucleic acid sequences, etc. In some embodiments, detection can be based on the unique sequence of each primer set used. In some embodiments, fluorogenic detection can be used in multiplexed amplification methods, as described herein or known in the art. In some embodiments, for example, different fluorophores with distinct emission spectra can be used. In some embodiments, orthogonal two- or three-color fluorogenic aptamers and their corresponding ligands can be used, as described herein.

[0041] In some embodiments, isothermal amplification reactions can be "nested" as described herein. In such embodiments, dilution of the amplification product, for example, before performing a subsequent amplification using nested primer pairs, can substantially improve sensitivity and specificity and reduce amplification artifacts. In some embodiments, nested amplification reactions, e.g., nested isothermal amplification reactions, can be "multiplexed." In some embodiments, such nested and multiplexed amplification reactions can be used in conjunction with fluorogenic detection methods. Generally, amplification reactions are carried out in a reaction mixture. As used herein, "reaction mixture" refers to a composition containing relevant components that allow an amplification reaction to occur. An exemplary reaction mixture may include, but is not limited to, a nucleic acid sample, a primer pair, and a suitable enzyme, such as a polymerase. Those skilled in the art will understand that a reaction mixture may also include other components, such as buffers, stabilizers, templates, nucleotides, etc., and that these components may be determined by the amplification reaction being performed.

[0042] It should be understood that amplification parameters such as nucleotide concentration, nucleic acid polymerase used for amplification, buffer composition, number of amplification cycles, temperature during cycling, etc., can be optimized as described herein or as known in the art. "Target nucleic acid," "target nucleic acid molecule," or "target nucleic acid sequence" refers to any nucleic acid that can be amplified, for example, as described herein. In some embodiments, the target nucleic acid can be detected. In some embodiments, the target nucleic acid can be quantified. It should be understood that the target nucleic acid can be of any size, so long as it can be amplified using a polymerase, such as, for example, an RNA polymerase. In some embodiments, the target nucleic acid can be about 100 to about 10,000 nucleotides in length, or any value therebetween. In another example, the target nucleic acid can be about 100 to about 5,000 nucleotides in length, or any value therebetween. In another example, the target nucleic acid can be about 100 to about 3,000 nucleotides in length, or any value therebetween. In another example, the target nucleic acid can be about 100 to about 2,000 nucleotides in length, or any value therebetween. In another example, the target nucleic acid can be about 100 to about 1,000 nucleotides in length, or any value therebetween. In another example, the target nucleic acid can be about 100 to about 500 nucleotides in length, or any value therebetween.

[0043] Target nucleic acid molecules include, but are not limited to, RNA or DNA, such as chromosomal DNA, mitochondrial DNA, messenger RNA, ribosomal RNA, transfer RNA, viral RNA, and extrachromosomal DNA such as toxic plasmids. Target nucleic acid molecules can be present in a sample, such as a biological sample, a forensic sample, a synthetic sample, or an environmental sample.

[0044] As used herein, "aptamer" refers to a nucleic acid molecule that can bind with high selectivity and specificity to a ligand, such as a peptide, small molecule (e.g., an antibiotic), or carbohydrate, i.e., "specifically binds" to the ligand. In some embodiments, an aptamer can contain modified nucleotides that can be amplified by an enzyme, such as a polymerase. In some embodiments, an aptamer can contain modified nucleotides that can be amplified by an RNA polymerase, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or a bacterial or eukaryotic RNA polymerase. It should be understood that RNA polymerases can be obtained from any suitable source, such as viruses, bacteriophages, bacteria, or eukaryotes, such as plants or animals. In some embodiments, an aptamer can be a single-stranded (ss) nucleic acid (e.g., ssRNA or ssDNA). Single-stranded nucleic acid aptamers can take on a variety of shapes, including helices and single-stranded loops. Thus, aptamer-ligand binding can be determined by tertiary structure rather than primary structure. In some embodiments, the aptamer comprises a terminal stem structure, i.e., a duplex structure comprising the 3' and 5' ends of the aptamer. In some embodiments, the terminal stem structure can be as short as 2 bp or arbitrarily long. In some embodiments, the terminal stem structure can be about 6 bp to about 8 bp.

[0045] As used herein, an "aptamer-encoding template sequence" is a nucleic acid sequence that is the reverse complement of a nucleic acid aptamer sequence. In some embodiments, the ligand can be a signal-generating ligand that generates, for example, a fluorescent signal (e.g., from a fluorophore) or a colorimetric signal. The fluorogenic RNA aptamer sequence can be used to enhance the fluorescence (F) of the fluorogenic aptamer system. E ) and K of the aptamer-fluorophore interaction D Examples of fluorophore-binding aptamers include, but are not limited to, mango, pepper, broccoli, corn, spinach, and spinach2 (Strack et al., Nature Methods 2013, 10: 1219-1224), carrot and radish (Paige et al., Science 2011, 333: 642-646), RT aptamers (Sato et al., Angew. Chem. Int. Ed. 2014, 54: 1855-1858), hemin-binding G-quadruplex DNA and RNA aptamers, or malachite green-binding aptamers (Babendure et al., J. Am. Chem. Soc. 2003). Fluorophores include, but are not limited to, infrared (IR) dyes, Dyomics dyes, phycoerythrine, Cascade Blue, Oregon Green 488, Pacific Blue, rhodamine derivatives such as rhodamine green, 5(6)-carboxyfluorescein, cyanine dyes (i.e., Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7), (diethyl-amino)coumarin, fluorescein (i.e., FITC), tetramethylrhodamine, Lissamine, Texas Red, AMCA, TRITC, bodipy dyes, or Alexa dyes.

[0046] "Mango" or "Mango aptamer" refers to RNA aptamers. The RNA Mango aptamer series has extremely high contrast, making them useful in vitro fluorescent reporters. These aptamers have nanomolar binding affinity for a thiazole orange-based ligand (TO1-biotin), which can be up to 4,000-fold brighter upon binding to the RNA Mango aptamer. The RNA Mango aptamers Mango II, III, and IV are highly tolerant to magnesium ion concentrations found in in vitro assays and also function across a wide range of monovalent metal ion concentrations. Mango III has also recently been improved through structure-guided engineering to become even brighter. "Broccoli" or "Broccoli aptamer" refers to a 49-nt fluorescent RNA aptamer that confers fluorescence to a target analyte of interest (e.g., a target RNA) through activation of an attached DFHBI fluorophore or DFHBI-derived fluorophore, such as (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one) (DFHBI-IT) described by Song et al., J. Am. Chem. Soc. 2014, 136: 1198 (see, e.g., Filonov et al., J. Am. Chem. Soc. 2014, 136(46): 16299-16308).

[0047] An aptamer "specifically binds" a ligand when it recognizes and binds to the ligand, e.g., a fluorophore, but does not substantially recognize and bind to other molecules in the sample. In some embodiments, an aptamer may have an affinity for the ligand that is, for example, at least 10, 100, 1000, or 10,000 times greater than the affinity of the aptamer for another reference molecule in the sample. In some embodiments, the aptamer sequence has a ligand binding dissociation constant (K) between about 0.01 nM and about 100 nM. D), or any value therebetween, such as 0.2 nM. In some embodiments, the fluorogenic aptamer sequence has a fluorophore binding dissociation constant (K) between about 0.01 nM and about 100 nM. D ), or any value therebetween, such as 0.2 nM. In some embodiments, the fluorogenic aptamer coding sequence has a fluorophore binding dissociation constant (K) between about 0.01 nM and about 100 nM. D ), or 0.2 nM, or any value therebetween. Selection of an appropriate aptamer, such as a fluorescent RNA aptamer-fluorophore conjugate for use as described herein, can depend on a variety of parameters depending on the characteristics of the aptamer, such as binding affinity, brightness, secondary structure, amenability to sequence modification, etc.

[0048] In some embodiments, orthogonal two-color fluorogenic aptamers and ligands can be used as described herein. Two fluorogenic ligand-binding aptamers are orthogonal to each other in terms of binding when a first aptamer specifically binds to its ligand and a second aptamer specifically binds to its ligand. It should be understood that some overlap in binding may occur. In some embodiments, each fluorogenic ligand has an emission spectrum that is distinct from the other, allowing for robust two-color quantification of each aptamer concentration. In some embodiments, orthogonal three-color fluorogenic aptamers and ligands can be used based on the same concept. As will be understood by those skilled in the art, this concept of orthogonality can be easily extended to three-color imaging or higher. The term "primer" refers to a relatively short nucleic acid sequence that is complementary to at least a portion of a target nucleic acid molecule or sequence. It should be understood that a primer can additionally be complementary to the reverse complement of at least a portion of a target nucleic acid molecule or sequence.

[0049] In some embodiments, the primers have a "degenerate" sequence, i.e., the nucleic acid sequence is a composition of sequences with different nucleotides at the same locations, such that the primer is a mixture of different sequences that can hybridize to multiple different target nucleic acids. In other words, the degenerate sequence can be complementary to multiple target nucleic acid sequences. In some embodiments, a primer may include a first nucleic acid sequence capable of hybridizing to at least a portion of a target nucleic acid sequence or its complement and an aptamer-encoding template sequence, the first nucleic acid sequence being located at the 3' end of the first nucleic acid sequence; and a second nucleic acid sequence capable of hybridizing to at least a portion of a target nucleic acid sequence or its complement, the 5' end of the second nucleic acid sequence being covalently linked to the 3' end of the first nucleic acid sequence, and the 3' end of the second nucleic acid sequence not substantially hybridizing to the first nucleic acid sequence. Such a primer may be referred to herein as an "aptamer fusion primer." In some embodiments, at least the terminal nucleotide at the 5' end of the second nucleic acid sequence may be complementary to at least the terminal nucleotide at the 5' end of the first nucleic acid, forming at least a portion of a terminal stem structure. In some embodiments, at least the terminal 2 or 3 nucleotides at the 5' end of the second nucleic acid sequence can be complementary to at least the terminal 2 or 3 nucleotides at the 5' end of the first nucleic acid to form at least a portion of the terminal stem structure. A schematic diagram of an exemplary aptamer fusion primer is shown in Figure 25.

[0050] In some embodiments, the first nucleic acid sequence is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, It can be from about 20 to about 100 nucleotides in length, such as 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, or any value therebetween. In some embodiments, the first nucleic acid sequence can be greater than about 100 nucleotides in length, such as 200 nt in length.

[0051] In some embodiments, the second nucleic acid sequence is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, or any value therebetween. In some embodiments, the aptamer fusion primer can include a linker sequence between the first and second nucleic acid sequences, which can be 0 to about 65 nucleotides in length, such as 0, 1, 2, 3, 4, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65, or any value therebetween.

[0052] It should be understood that similar considerations apply to the third, fourth, fifth, or sixth nucleic acid, or additional nucleic acids, depending on whether they are designed to form an aptamer fusion primer or to include a polymerase promoter, as would be understood by one of skill in the art or as described herein. By "primer pair" is meant two optimally designed nucleic acid sequences as described herein that can serve to prime an amplification reaction, such as an isothermal amplification reaction, where the nucleic acid sequences anneal to complementary sequences on a target nucleic acid sequence.

[0053] A "sample" can be any organ, tissue, body fluid, cell, or cell extract isolated or extracted from an organism, or any material containing, potentially containing, or suspected of containing nucleic acids from an organism. For example, a sample from an animal, such as a mammal, can include, but is not limited to, bone, brain, breast, colon, muscle, nerve, ovary, prostate, retina, skin, skeletal muscle, intestine, testes, heart, liver, lung, kidney, stomach, pancreas, uterus, adrenal gland, tonsils, spleen, soft tissue, peripheral blood, whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, blood cell proteins, plasma, platelet-rich plasma, obtained from an organism (e.g., human or animal), test subject, or experimental animal. Samples may include, but are not limited to, cells or tissues derived from normal or transformed cells (e.g., by recombinant DNA or monoclonal antibody technology) in cell culture. Samples may also include, but are not limited to, any organ, tissue, cell, or cell extract isolated from non-mammalian animals, such as birds, fish, insects, or worms. In another example, the sample may be a fungal sample. In another example, the sample is obtained from a plant.

[0054] A "sample" can also be a cell or cell line created under experimental conditions that is not directly isolated from an organism. Samples can be obtained using standard techniques such as brushes, swabs, spatulas, rinses / washings, punch biopsy devices, puncture of a cavity with a needle, or surgical instrumentation. Tissue or organ samples can be obtained from any tissue or organ, for example, by biopsy or other surgical procedure. Separated cells can be obtained from body fluids or tissues or organs by separation techniques such as filtration, centrifugation, or cell sorting. In some embodiments, a "sample" may be recovered or extracted from the environment, such as, but not limited to, air, water, or soil; from materials intended for human or animal consumption, such as meat, fish, dairy products, or feed; from cosmetics, agricultural products, plastics and packaging materials, paper, clothing fibers, metal surfaces, etc. Samples may also be cell-free, artificially derived, or synthetic, e.g., synthetic constructs such as synthetic nucleic acids. Samples may be in liquid form, including, but not limited to, the conventional definition of a liquid, as well as colloids, suspensions, slurries, and dispersions.

[0055] Methods for obtaining or extracting nucleic acids, such as DNA or RNA, are well known in the art and include, but are not limited to, RNA extraction spin columns, phenyl / chloroform-based extraction methods, etc. In some embodiments, the nucleic acid may be a DNA or RNA target that can be extracted using automated techniques and equipment. A "control" includes a sample obtained for use in determining baseline expression or activity. A control also includes a previously established standard or reference. Thus, any test or assay performed in accordance with the present invention can be compared to an established standard or reference, and it may not be necessary to obtain a control sample each time for comparison. The organism may be, without limitation, a virus, a microorganism, a mycoplasma, a fungus, an animal (e.g., a mammal), a plant, a bacterium, an algae, a parasite, a fungus, or a protozoan. In some embodiments, the animal may be a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc. The organism may be a clinical patient, a clinical trial volunteer, an experimental animal, a domesticated animal, etc.

[0056] Exemplary plants include monocotyledons, dicotyledons, and conifers. For example, plants may include, but are not limited to, cereals, grapes, beets, pome fruits, stone fruits, and soft fruits; legumes, oil plants, cucumber plants, fiber plants, citrus fruits, vegetables, lauraceae, and plants such as corn, tobacco, nuts, coffee, sugarcane, tea, vines, hops, turf, bananas, rubber plants, or ornamental plants. Examples of fungi include, but are not limited to, yeast, Aspergillus species; Blastomyces dermatitidis; Candida; Coccidioides immitis; Coccidioides posadasii; Cryptococcus neoformans; Histoplasma capsulatum; and Pneumocystis species.

[0057] Maize rust, rice blast, rice brown spot disease, rye leaf blight, Sporothrix schenckii, wheat fungus, etc. Examples of protozoa and helminths include, but are not limited to, parasitic protozoa and helminths such as Acanthamoeba and other free-living amoebae; Anisakis species and other related helminths; Cryptosporidium parvum; Cyclospora cayetanensis; Diphyllobothrium species; Entamoeba histolytica; Eustrongylides species; Giardia lamblia; Nanophyetus species; Shistosoma species; Toxoplasma gondii; or Trichinella.

[0058] Examples of analytes include, but are not limited to, allergens such as plant pollen and wheat gluten. In some embodiments, the organism may be pathogenic, such as a bacterial or viral pathogen.

[0059] Examples of bacterial pathogens include, but are not limited to, Aeromonas hydrophila; Bacillus anthracis; Bacillus cereus; botulinum neurotoxin-producing species of Clostridium; Brucella abortus; Brucella melitensis; Brucella suis; Burkholderia mallei (formerly Pseudomonas mallei); Burkholderia pseudomallei (formerly Pseudomonas pseudomallei); Campylobacter jejuni; Chlamydia psittaci psittaci); Clostridium botulinum; Clostridium botulinum; Clostridium perfringens; Coccidioides immitis; Coccidioides posadaci; Cowdria ruminantium; Coxiella burnetii; Enterotoxigenic coliforms (EECs), such as Escherichia coli enterotoxigenic (ETEC), E. coli enteropathogenic (EPEC), E. coli O157:H7 enterohemorrhagic (EHEC), and E. coli enteroinvasive (EIEC); Ehrlichia species, such as Ehrlichia chaffeensis; Francisella tularensis tularensis); Legionella pneumophilia; Liberobacter africanus; Liberobacter asiaticus;Listeria monocytogenes; miscellaneous enterobacteria such as Klebsiella, Enterobacter, Proteus, Citrobacter, Aerobacter, Providencia, and Serratia; Mycobacterium bovis; Mycobacterium tuberculosis; Mycoplasma capricolum; Mycoplasma mycoides ssp mycoides; Rickettsia prowazekii; Rickettsia rickettsii rickettsii; Salmonella species; Schlerophthora rayssiae varzeae; Shigella species; Staphylococcus aureus; Streptococcus; Synchytrium endobioticum; Vibrio cholerae non-O1; Vibrio cholerae O1; Vibrio parahaemolyticus and other Vibrio; Vibrio vulnificus; Xanthomonas oryzae; Xylella fastidiosa; Yersinia enterocolitica enterocolitica and Yersinia pseudotuberculosis; or Yersinia pestis;

[0060] Examples of viral pathogens include, but are not limited to, single-stranded RNA viruses, single-stranded DNA viruses, double-stranded RNA viruses, or double-stranded DNA viruses. In some embodiments, the pathogenic virus is, but is not limited to, African horse sickness virus; African swine fever virus; Akabane virus; Banja virus; calicivirus (e.g., human enteric viruses such as norovirus and sapovirus), cercopithecine herpesvirus 1; chikungunya virus; classical swine fever virus; coronavirus (e.g., severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)); dengue viruses such as serotypes 1 (DENV1) and 3 (DENV3) and related viruses such as chikungunya virus (CHIKV); Djugbe virus; Ebola virus; Eastern equine encephalitis virus, Japanese encephalitis virus, Marek's encephalitis virus, and the like. Encephalitis viruses, such as Bay Valley encephalitis and Venezuelan equine encephalitis viruses; equine morbillivirus; flavirus, flexar virus; foot-and-mouth disease virus; Germiston virus; goat pox virus; Hantaan or other hantaviruses; Hendra virus; human immunodeficiency virus (HIV); influenza viruses (e.g., H1N1, H5N1, avian influenza viruses); Lassa fever virus; louping ill virus; lymphocytic choriomeningitis virus; poliovirus; potato virus; poxvirus; South American hemorrhagic fever viruses; variola major virus (smallpox virus); vesicular stomatitis virus; West Nile virus; yellow fever virus; and human pathogenic flaviviruses, such as Zika virus.

[0061] In some embodiments, the target nucleic acid may be detected simultaneously with or after amplification. As used herein, the term "detect" or "detection" refers to the determination of the existence, presence, or fact of a target nucleic acid or signal in a sample or reaction mixture. In some embodiments, target nucleic acids may be quantified simultaneously with or after amplification and detection. Quantification may include, but is not limited to, measuring the amount or quantity (also referred to as quantification) of a target or signal, including, but not limited to, any analysis designed to determine the amount or proportion of a target or signal. Detection is "qualitative" when it refers to, relates to, or involves identifying the quality or type of a target or signal in terms of its relative abundance relative to another target or signal, which is not quantified. "Optical detection" refers to detection performed through a visually detectable signal: fluorescence, spectrum, or image from the target of interest or a probe attached to the target.

[0062] In some embodiments, the methods of the present disclosure may be incorporated into methods of diagnosis by amplifying, detecting, and / or quantifying levels of target sequences indicative of a disease, disorder, or condition. In some embodiments, the methods of the present disclosure may be incorporated into methods of forensic or environmental analysis by amplifying, detecting, and / or quantifying levels of target sequences indicative of crime or contamination.

[0063] In various embodiments, primer design can be optimized. In some embodiments, non-nested and / or nested oligonucleotide primer pairs can have reduced chances of primer-dimer formation and reduced chances of non-specific hybridization to target nucleic acid molecules. Thus, in some embodiments, non-nested and / or nested oligonucleotide primer pairs can have one or more of the following characteristics: 1. Primer pairs can be designed to have the lowest possible hybridization with each other. For example, in some embodiments, primer pairs can be designed to have 3 or fewer nucleotides that can hybridize with each other. In alternative embodiments, primer pairs should not hybridize with each other. 2. Primers may have as few alternative target sites as possible for the nucleic acid (e.g., RNA) sequence of interest. In some embodiments, primers may be designed to eliminate hybridization at their 3' ends to undesired target sites or to other primer sequences in the design. In some embodiments, the 3' ends should not allow primer self-extension (e.g., by back-hybridization).

[0064] 3. For non-nested situations, a DNA primer capable of hybridizing to the 3' region of the RNA of interest at the isothermal temperature of interest is used, using the 3' sequence of the DNA primer. In some embodiments, the 3' end of this primer can be sufficiently hybridized to the RNA of interest by at least 1-3 nt of the terminal sequence. In some embodiments, an RNA polymerase promoter sequence can be included in the primer sequence at the 5' end of this hybridization region, which may or may not be sufficiently hybridized to the RNA of interest (e.g., T7, T3, or SP6) (PA, Figures 1A and 1B). Hybridization of the PA primer to the RNA target can be predicted by thermodynamic calculations using standard techniques to be stable under the salt and buffer conditions used in isothermal amplification systems. In some embodiments, hybridization of 15-30 bp can occur, but is not limited to such. 4. A second primer (PB, Figures 1A and 1B), which can hybridize to the reverse complement of the RNA target sequence and is otherwise designed similarly to primer PA, can hybridize to the reverse complement sequence of the RNA found 5' to the hybridization position of the PA primer. A fluorogenic aptamer reporter should be included in the design, and the reverse complement of such an aptamer sequence can be included within the 5' region of the PB primer (PB, Figure 1B). In some embodiments, the hybridization sites for primers PA and PB can be designed to be as close as possible for the most efficient isothermal amplification. In some embodiments, the 3' ends of the primers do not overlap. In an alternative embodiment, the 3' ends of the primers can be within 500 nt of each other to allow for effective nesting of inner primer pairs.

[0065] 5. With respect to nested primer design, the "outer" primer pair may be designed as for non-nested primers described herein, with the following additional criteria: The distance between the PA and PB outer primers may be sufficient to allow the inner primer to hybridize between the 3' ends of the outer PA and PB primers. Primer PB in such cases may be designed to include a fluorogenic aptamer sequence, or in some applications, no aptamer sequence (e.g., Mango, Figure 1C). Inner primers PC and PD (Figure 1C) may be designed to hybridize according to the same criteria as PA and PB, respectively. Note that these primers are amplifying RNA that is the reverse complement of the original RNA target, and PC may include a promoter sequence as described herein for PA, and primer PD may or may not include a fluorogenic aptamer sequence. In some embodiments, this is not required due to the associated leakage of RNA polymerase. In some embodiments, the hybridization regions for PC and PD may overlap with the PA and PB hybridization regions, eg, to minimize the potential for artefactual sequence amplification.

[0066] In some embodiments, if the inner primer PD contains a fluorogenic aptamer (e.g., Mango, FIG. 1C ), the outer primer PB does not contain a fluorogenic aptamer. In some embodiments, if a fluorogenic aptamer is included in PB, a distinct fluorogenic aptamer sequence may be included in the inner primer PD. In some embodiments, the distinct aptamer may have spectrally distinct properties relative to the fluorogenic aptamer found in the outer primer PB (e.g., a pepper, broccoli, or corn aptamer on PB and a Mango aptamer on PD). In some embodiments, the fluorogenic aptamer may be fully functional in the isothermal buffer of an isothermal amplification system (e.g., an RNA Mango aptamer, which is tolerant of a wide range of salt, pH, and chemical conditions). In some embodiments, the use of nested oligonucleotide primers can increase the sensitivity and / or specificity of INA. In some embodiments, the use of nested oligonucleotide primer pairs described herein can increase the sensitivity and / or specificity of INA. -19 M ~ about 10 -6 In some embodiments, the use of nested oligonucleotide primer pairs described herein provides sensitivity to attomoles of 10 -18 This results in sensitivity to M concentration.

[0067] In some embodiments, INA detection methods using nested oligonucleotide primer pairs described herein can be used in, but are not limited to, fluorogenic aptamers such as Mango, molecular beacons, nonspecific NA intercalation fluorescent staining, and / or gel-based detection methodologies. In some embodiments, INA detection methods using nested oligonucleotide primer pairs described herein are insensitive or have low sensitivity to non-specific amplification artifacts (off-target effects). In some embodiments, INA detection methods using nested oligonucleotide primer pairs described herein are rapid and convenient and can be configured to directly give a real-time fluorescent readout. In some embodiments, the nested oligonucleotide primer pairs described herein may contain fluorogenic aptamer sequences, such as, but not limited to, RNA Mango. Introduction of a fluorogenic ligand to the corresponding aptamer can result in the creation of a real-time fluorescent reporter system. Thus, an INA detection method (INAF) using oligonucleotide primers containing fluorogenic aptamer sequences can enable real-time isothermal NA detection. In some embodiments, the INAF method can be used to detect relatively high abundance nucleic acid target sequences, such as, but not limited to, template concentrations in the micromolar to picomolar concentration range.

[0068] In some embodiments, primers and / or targets according to the present disclosure may include nucleic acid sequences set forth herein, such as, but not limited to, Table 3, or sequences having at least 90% to 99.9% similarity, or any value therebetween, to a sequence in Table 3, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity. In some embodiments, primers and / or targets according to the present disclosure may include, but not limited to, nucleic acid sequences set forth in Table 3, or sequences having at least 90% to 99.9% identity, or any value therebetween, to a sequence in Table 3, such as, but not limited to, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0069] In some embodiments, the INAF method provides 10 attomoles per microliter of sample. -18This method can be used to detect low concentrations or low abundance nucleic acid target sequences, such as, but not limited to, M or 1NA molecules. Such a method, called isothermal nested fluorogenic amplification and detection (INFAD), involves an initial outer primer isothermal amplification step, followed by a subsequent nested inner primer isothermal reaction using a primer containing a fluorogenic aptamer-tagged primer. It should be understood that the method is not limited to a single nesting event, and that, for example, one or more nesting events can occur. Without being bound by any particular hypothesis, a single nesting event can eliminate many amplification artifacts. In some embodiments, a second or additional nesting event can further improve sensitivity. INFAD inner and outer primer pairs are engineered to maximize sensitivity, including but not limited to methodologies in which primers are configured so that nucleic acid fluorogenic aptamers are generated at the end of the innermost exponential isothermal amplification cycle and not at earlier steps in the amplification process.

[0070] For non-nested RCA, DNA or RNA target sequences can be detected. In some embodiments, a linear DNA oligonucleotide should contain a fluorogenic aptamer (e.g., Mango, Figure 11), a DNA promoter sequence (e.g., T7, T3, SP6) oriented to allow production of the aptamer sequence, and the ability to be ligated. This can be done, for example, by adding a 5' phosphate to the DNA oligo and using T4 DNA ligase in the process. The 5' and 3' regions of this oligonucleotide (when a "linear RCA template" is used, but not limited to, Tables 1 or 3) should contain hybridization regions to the desired DNA or RNA target (when a "target RCA splint" is used, but not limited to, Tables 1 or 3) to allow hybridization of the DNA oligonucleotide, so that the 5' and 3' ends are in close proximity to allow ligation (which can be enzymatic or chemical, for example, when imidazole activation of the 5' phosphate is performed). Addition of an upper strand promoter sequence (such as, but not limited to, a "T7 promoter complement" is used; see Tables 1 or 3) after or before ligation then allows transcription by, for example, T7 or SP6 polymerase (T7, Figures 11 and 12). The resulting amplification of DNA or RNA targets can be achieved by the creation of long, repetitive RNA sequences with the reverse complement of the DNA oligonucleotide sequence (Figures 11-13). The system maintains exponential amplification potential because each rotation of the circle by the RNA polymerase generates a new ligation target site that can promote further circle production.

[0071] Nesting RCA can be easily envisioned by hybridizing the 5' and 3' oligonucleotides just described, including a gap sequence between the oligonucleotide hybridization sites. This implies that the length of the oligonucleotides is sufficient to allow for such a gap, which can be imagined as 20-50 nt of the RNA target sequence. Addition of a non-strand-displacing RT enzyme can fill this gap, allowing ligation as just described. The resulting repeat sequence will not contain a region of RNA sequence complementary to the target RNA sequence found between the two oligonucleotide hybridization sites. Therefore, a second amplification cycle can be designed using a DNA oligonucleotide sequence designed herein to hybridize to this inner region of the sequence. In some embodiments, it is effective to include a fluorogenic aptamer only on the inner primer oligonucleotide, or to have separate fluorogenic aptamers on the "outer" and "inner" oligonucleotides of the design.

[0072] In some embodiments, the INFAD method can involve a two-color aptamer-fluorophore system, where nucleic acid aptamer 1 specifically binds to fluorophore 1 (A1:F1 fluorogenic complex) and aptamer 2 specifically binds to fluorophore 2 (A2:F2 fluorogenic complex), and the fluorescence emissions from A1:F1 and A2:F2 are distinguishable using a fluorometer, allowing for a highly sensitive, two-channel INFAD system. Such systems include, but are not limited to, mango and pepper, mango and broccoli and pepper aptamers, etc. In some embodiments, a two-color INFAD system allows for the detection of two nucleic acid templates, one of which can be an internal control for the INFAD method. Such a two-channel INFAD system can have increased reliability compared to a single channel. In some embodiments, the internal control in a two-channel INFAD system can be used to distinguish true negatives from false negatives. This can allow users to determine whether a failed reaction is the result of an internal reaction and / or an external reaction. Therefore, INFAD primers can be modified to encode two-color aptamer sequences. Furthermore, the addition of two fluorophores allows for two-channel imaging. Similar techniques can be used for three or more channel imaging using three or more color aptamer sequences.

[0073] In some embodiments, two simultaneous isothermal reactions (e.g., NASBA reactions) can be performed in the same tube, as shown in Figure 24, which outlines possible outcomes of an internally controlled two-color assay. One reaction would have an oligo targeting an RNA of interest (such as SARS-CoV-2) that produces an aptamer with fluorescence (such as green fluorescence, represented by the solid line in Figure 24). The second reaction would have an oligo targeting an internal control RNA (such as human 18S ribosomal RNA) that produces an aptamer with fluorescence (such as red fluorescence, represented by the dashed line in Figure 24, but not the same fluorescence as the first aptamer). Possible outcomes from the reaction are: A. SARS-CoV-2 RNA is detected (bar, Figure 24A) and human ribosomal RNA is detected (dashed line, Figure 24A); B. SARS-CoV-2 RNA is not detected (absence of bar, b) and human ribosomal RNA is detected (dashed line, Figure 24B); C. SARS-CoV-2 RNA is detected but the internal control human ribosomal RNA is not, implying either a false positive or simply test failure (Figure 24C); or assay failure (Figure 24D), where it is not possible to determine whether SARS-CoV-2 RNA is present because the internal control has failed. The RNAs described above are merely examples; any RNA may be substituted above for the desired RNA. It should be understood that this approach can be expanded to three or more simultaneous isothermal reactions (e.g., NASBA reactions).

[0074] In some embodiments, the Mango aptamer can be inserted into a NASBA DNA primer to monitor the exponential synthesis of an RNA reporter in an isothermal manner. NASBA uses two primers: the first serves as the initial reverse transcription primer and contains a T7 promoter. After cDNA production, the RNA in the newly formed heteroduplex can be degraded by RNase H, allowing the second DNA primer to bind and be extended again by reverse transcriptase (RT). This produces a double-stranded DNA template that can be transcribed by T7 RNA polymerase. The resulting RNA can be utilized by RT, resulting in exponential amplification (Figure 1A). By modifying the second or bottom strand NASBA primer to encode a fluorescent Mango aptamer, exponential RNA growth can be directly monitored by fluorescence (Figure 1B). Altering the DNA primer can dramatically reduce the complexity of NASBA, enabling real-time monitoring. When coupled with a nesting approach (Figure 1C), this method can detect as few as 1.5 RNA molecules per μl of reaction.

[0075] In some embodiments, the present disclosure provides a composition that can be used in the methods described herein.The composition can include fluorogenic aptamers conjugated to oligonucleotide primers for isothermal amplification described herein (e.g., one or more of the nucleic acid molecules or compositions described herein) and their corresponding ligands, such as dyes. In some embodiments, the present disclosure provides a kit that can be used in the methods described herein.In some embodiments, the kit can include one or more of the nucleic acid molecules or compositions described herein, along with instructions for amplifying target nucleic acid sequences.In some embodiments, the amplification can be isothermal amplification, such as nucleic acid sequence-based amplification, rolling circle amplification, loop-mediated isothermal amplification, helicase-dependent amplification, or strand displacement amplification.

[0076] In some embodiments, the kit may include fluorogenic aptamers conjugated to oligonucleotide primers for isothermal amplification described herein (e.g., one or more of the nucleic acid molecules or compositions described herein) and their corresponding ligands, e.g., dyes. In some embodiments, the kit may be used to amplify nucleic acid target sequences to an extent that allows for detection of the target sequence in a sample. In some embodiments, the kit may include instructions for use or for performing the methods described herein. In some embodiments, the compositions, kits, and methods described herein may be used in the detection of, for example, very low and high concentrations of RNA and / or DNA templates in the field or in the laboratory for applications including, but not limited to, specific target gene detection and quantification, pathogen detection in clinically or scientifically relevant samples such as tissue culture, serum, and plasma, disease marker detection in clinical samples, contaminant detection in in vitro samples such as environmental samples and controlled tissue culture samples, in vivo imaging, and localization.

[0077] In some embodiments, the compositions, kits, and methods described herein can be used in food safety and food biosecurity applications, such as screening foodstuffs and materials used in food processing or packaging for the presence of pathogens in biological and / or non-biological samples. In other embodiments, the methods provided herein can be used in anti-counterfeiting applications, such as verifying the authenticity of pharmaceutical products or verifying the identity of high-value items manufactured to contain or known to contain specific nucleic acid species. In some embodiments, the compositions, kits, and methods described herein may be used in conjunction with a point-of-care device. The present invention will be further illustrated in the following examples. [Example]

[0078] Example 1 Materials and Methods Target RNA generation Colony PCR reactions were performed using 5 pM plasmid template, Taq (NEB, 10 U), 0.2 mM of each dNTP, 10 mM TRIS buffer pH 8.3, 50 mM KCl, 1.5 mM MgCl, and 0.01% gelatin, with the respective PCR primers shown in Table 1, followed by cloning into the pGEM-T Easy vector (Promega). The sequence was confirmed by Eurofins tube sequencing. PCR reactions were performed using the plasmid as a template, followed by ethanol precipitation in 300 mM NaCl and 70% ethanol. The pellet was suspended in 1 / 10 the PCR reaction volume for a 10x stock. Transcription was performed using 2x template, T7 RNA polymerase (ABM) in 8 mM GTP, 5 mM CTP and ATP, 2 mM UTP, 40 mM TRIS buffer pH 7.9, 2.5 mM spermidine, 26 mM MgCl, and 0.01% Triton X-100. RNA was purified via 5% PAGE (19:1 acrylamide:bis), overnight rotation in 300 mM NaCl, and ethanol precipitation. Concentration was determined using a Shimadzu dual-beam spectrophotometer.

[0079] [Table 1] JPEG2025162555000002.jpg161162

[0080] [Table 2]

[0081] Mango-NASBA NASBA primers were selected for RNA amplification using short segments of E. coli or P. fluorescens ClpB mRNA as detection templates (represented as "ClpB short target E. coli" and "ClpB short target P. fluorescens," respectively; Table 1). Reactions were performed using NASBA buffer mix (Life Sciences, NECB-1-24), nucleotide mix (Life Sciences, NECN-1-24), 250 nM of each primer (IDT), T7-containing cDNA primer P1 and Mango template-containing reverse primer P2A (adapted from Heijnen and Medema (2009)), 480 nM TO1-biotin (ABM), and NASBA enzyme mix (Life Sciences, NEC-1-24). The NASBA reaction mixture was mixed without the enzyme mix, and the RNA target was added to a final concentration of 0, 25 aM, 25 fM, or 25 pM. In an MJ research PTC-100 thermocycler, the RNA was heated to 65°C for 2 minutes and ramped down to 41°C for 5 minutes. To initiate the reaction, enzyme mix was added to the reaction mixture, and they were incubated at 41°C in 8-tube strips with optical caps (Applied Biosystems, catalog numbers 4358293 and 4323032) in a StepOne Real-Time PCR system (Applied Biosystems). The SYBR Green reagent was set to read using the following program: 1. Ramp to 41°C and read; 2. Hold at 41°C for 30 seconds and read; 3. Repeat step 2 until 480 cycles are completed. The experiment in Supplementary Figure S6 was performed using P2A without the A10U mutation (WT Mango III was used).

[0082] Nested Mango - NASBA Outer amplification reactions were performed as described above, except that P2A was replaced with P2B lacking the Mango template. Reactions were stopped for 40 minutes by the addition of 5 μL EDTA to a final concentration of 10 mM in a final volume of 25 μL and flash-frozen in liquid nitrogen or cooled ethanol. Aliquots from these reactions were diluted 100-fold into inner nested Mango-NASBA reactions (20 μL) prepared as above, except using the T7 promoter-containing cDNA primer P3 and the Mango template-containing reverse primer P4. Reactions were again monitored in real time for TO1-biotin fluorescence using the instrumentation described above.

[0083] Detection of Escherichia coli in the presence of conditioned mammalian cell culture medium LB medium was inoculated with E. coli and the concentration was monitored by absorbance at 600 nm (cell number was calculated using Agilent online tools). 8 An aliquot of single cells was heat-shocked at 41°C for 10 minutes to induce ClpB RNA in the cells, followed by pelleting at 4000g for 4 minutes. Cells were resuspended in 50 μL of depleted cell culture medium (MCF7, medium discarded during cell passaging) and incubated at 41°C for 3 minutes. The sample was pelleted at 4000g for 4 minutes and then subjected to Nucleospin RNA Kit (Macherey-Nagel) using the recommended protocol, except for avoiding the DNase step, and elution was performed using 2 mM EDTA. Total nucleic acid samples were used in the Nested Mango-NASBA reaction described above. The negative control sample for Nested Mango-NASBA was nucleic acid extraction from depleted medium without E. coli cells, which was subjected to the same extraction procedure.

[0084] PAGE Mango Visualization Samples to be visualized by PAGE were added to 3 volumes of formamide supplemented with 20 mM EDTA and heated to 90°C for 5 minutes. Samples were loaded and run through an 8% PAGE (19:1 acrylamide:bis). Gels were post-stained in 100 mL of 1x WB (140 mM KCl, 1 mM MgCl, 10 mM NaHPO pH 7.2, 0.05% Tween-20) containing 20 nM TO1-biotin, and Mango-NASBA bands were visualized as previously described. 34 Images were taken with a GE AI600RGB imager. Alternatively, gels were stained with 1x SYBR Safe under the same conditions. Sequence alignment Sequences were aligned using Geneious software and alignment using the Clustal method.

[0085] result Sensitivity of fluorogenic aptamer-NASBA Using a commercially available NASBA enzyme mix, we were able to detect only approximately 25 pM (15,000,000 RNAs / μL reaction; Figures 2A, 4, 5, primers P1 / P2A) of E. coli ClpB RNA template using Mango NASBA, above background signals that rapidly amplified even in the complete absence of RNA template (Figures 4, 8). This inherent level of sensitivity was not primer- or template-specific, as the P. fluorescens ClpB RNA template could be detected with similar sensitivity using primers that hybridize much closer together (Figure 2B, primers P7 / P8).

[0086] Sensitivity of nested fluorogenic aptamer-NASBA The outer primers were identical in sequence to those used in the Mango NASBA, except that P2A here lacked the Mango III tag (P1 / P2B, Table 1). At a 0.25 μM concentration of the primers used in the outer NASBA reaction, we found that a 100-fold dilution was sufficient to suppress NASBA activity (Figure 8B). After 40 min of incubation of this outer NASBA reaction and a 100-fold dilution into a new inner NASBA reaction, and using the inner NASBA Mango primers (P3 / P4), we were able to easily detect 15 RNA molecules / μL of E. coli ClpB template sequence (Ec / Ec reaction, Figure 2C, 6). Using the same dilution strategy, we tested nested Mango NASBA against P. fluorescens templates and were able to detect 1.5 RNA molecules / μL using P. fluorescens-specific nested Mango NASBA primers (outer: P5 / P6, inner: P7 / P8, Figure 2C, 6). This approach improved sensitivity by six orders of magnitude when using the same E. coli target RNA.

[0087] Nested fluorogenic aptamer NASBA specificity and robustness The E. coli and P. fluorescens ClpB templates differ by 78 nt in the region to be amplified, 65 nt of which are in the primer hybridization region (alignment Figure 10). When primers designed to target E. coli were used with the P. fluorescens target, fluorescence remained within the same error range as the 0 RNA / µL reaction control (Ec / Pf, Figure 2C, 6). To verify whether the nested Mango NASBA reaction remained viable in a large background of human nucleic acids, we mixed the E. coli ClpB target with or without a very large excess of human total nucleic acid (150 RNA molecules / µL final ClpB short target E. coli, 5 ng / µL human total nucleic acid) and performed nested RNA Mango using Ec primers (outer: P1 / P2B, inner: P3 / P4, Figure 2D). Although the appearance of the time-dependent signal was slightly reduced, a robust signal was still observed under these conditions, suggesting that the Nested Mango NASBA is largely robust to nucleic acid amplification artifacts despite the addition of such large amounts of human RNA and DNA.

[0088] Example 2 RT-PCR primers were designed to amplify a 1 kb fragment from cultured SARS-CoV-2 (COVID-19; Table 2). [Table 3] JPEG2025162555000005.jpg114147

[0089] After transcription, the resulting RNA (1 fM) was subjected to nested mango NASBA using the NASBA Life Sciences (LS) liquid NASBA kit. Five sets of primers were designed to amplify 100-nt regions centered within these regions. Four of the five primer sets (see Table 3) were successful in amplifying COVID-19 RNA, with the set producing the fastest rise time and highest fluorescent signal (Figure 14).

[0090] [Table 4] JPEG2025162555000007.jpg203149

[0091] By performing a dilution series of COVID-19 RNA (1 fM to 1 aM) and subjecting it to nested NASBA, we achieved a sensitivity of 1 aM (Figure 15A). The addition of 100 ng of exogenous nucleic acid per 20 μl outer reaction did not affect either the positive (gray) or negative (black) signal (Figure 15A). The LS Lyophilized Kit was also tested in the single-step Mango NASBA and demonstrated a sensitivity of 10 pM (Figure 19). The LS lyophilized kit was compared to the liquid kit results. Serially diluted SARS-CoV-2 RNA (1 fM to 1 aM) was subjected to nested NASBA as shown in Figure 15A. Figure 16 shows that using lyophilized reagents resulted in sensitivity at concentrations of 10 aM and higher.

[0092] FIG. 17 shows that neither EDTA nor heating the RNA sample is required before performing the outer nested NASBA reaction. To shorten the overall Mango NASBA reaction time, an external reaction time was tested using 10 aM of SARS-CoV-2 RNA target 4 (Figure 18). A 20 minute external reaction time was demonstrated to maintain the sensitivity and robustness of a 40 minute external incubation time.

[0093] Following successful detection of the synthetic viral sequence, both the liquid and lyophilized LS reagents were tested in the Mango NASBA against total RNA extracted from SARS-CoV-2 virus cultured in eukaryotic cells. Figure 20 (liquid) and Figure 21 (dry) demonstrate successful detection of cultured virus after a 100-fold dilution of the culture sample (liquid). Figure 21 also shows that no preheating and a 20-fold dilution from the outside to the inside reaction were fully feasible. Tracheal aspirates from SARS-CoV-2 infected patients from the ICU unit at St. Paul's Hospital were tested using the Nested Mango NASBA (Figure 22). The patients were originally diagnosed by performing a Roche RT-PCR test. SARS-CoV-2 RNA was successfully detected in the 20-minute outer and 12-minute inner NASBA reactions from the infected patient (1A and 2A), while the curve for the uninfected patient (5A) rose at approximately the same time as the negative control water sample. As with synthetic SARS-CoV-2, heating of the viral RNA sample prior to Nested Mango NASBA was not required (Figure 23).

[0094] The commercial lyophilized reagent was slightly turbid at the beginning of the incubation, however, this turbidity did not interfere with the analysis, and the appearance time for each sample could be monitored by plotting the slope of the data as in Figures 15B and 22B. References JPEG2025162555000008.jpg132155 JPEG2025162555000009.jpg237164 JPEG2025162555000010.jpg238162 JPEG2025162555000011.jpg51151

[0095] Other embodiments The present invention has been described with respect to one or more embodiments. However, it will be apparent to those skilled in the art that numerous variations and modifications can be made without departing from the scope of the invention, as defined in the claims. Thus, while various embodiments of the present invention are disclosed herein, many adaptations and modifications can be made within the scope of the invention in accordance with the common general knowledge of those skilled in the art. Such modifications include the substitution of known equivalents for any aspect of the invention, which achieve the same result in substantially the same way. Numerical ranges are inclusive of the numbers defining the range. By "about," we mean a deviation (plus or minus) from a value or range of values ​​of 5% or less, e.g., 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, etc. In the description, the word "comprising" is used as an open-ended term substantially equivalent to the phrase "including but not limited to," and the word "comprises" has a corresponding meaning. However, it should be understood that where the words "comprising" or "comprises," or variations of the same root, are used herein, variations or modifications to "consisting" or "consists" that exclude any unspecified element, step, or ingredient, or to "consisting essentially of" or "consists essentially of," which are limited to the specified materials or recited steps, along with those that do not materially affect the basic and novel characteristics of the claimed invention, are also contemplated. The citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention. All publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference herein and as if fully set forth herein. The present invention includes all embodiments and modifications substantially as described above and with reference to the examples and drawings.

Claims

1. i) a first nucleic acid sequence capable of hybridizing to at least a portion of a target nucleic acid sequence or its reverse complement and further comprising an aptamer-encoding template sequence, wherein the aptamer-encoding template sequence is located at the 3' end of the first nucleic acid sequence; and ii) a second nucleic acid sequence capable of hybridizing to at least a portion of the target nucleic acid sequence or its reverse complement, wherein the 5' end of the second nucleic acid sequence is covalently linked to the 3' end of the first nucleic acid sequence, and the 3' end of the second nucleic acid sequence does not substantially hybridize to the first nucleic acid sequence. A nucleic acid molecule or analog thereof comprising:

2. 2. The nucleic acid molecule of claim 1, wherein at least the terminal three nucleotides at the 3' end of the second nucleic acid sequence do not hybridize to the first nucleic acid sequence.

3. 3. The nucleic acid molecule of claim 1, wherein the first nucleic acid sequence is about 20 to about 100 nucleotides in length.

4. The nucleic acid molecule of any one of claims 1 to 3, wherein the aptamer-encoding template sequence encodes a fluorogenic aptamer sequence.

5. The fluorogenic aptamer sequence has a fluorophore binding dissociation constant (K) between about 0.01 nM and about 100 nM. D 5. The nucleic acid molecule of claim 4, having:

6. 6. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a terminal stem structure, and at least the terminal nucleotide at the 5' end of the second nucleic acid sequence is complementary to at least the terminal nucleotide at the 5' end of the first nucleic acid and forms at least a part of the terminal stem structure.

7. 7. The nucleic acid molecule of claim 6, wherein at least the terminal two nucleotides at the 5' end of the second nucleic acid sequence are complementary to at least the terminal two nucleotides at the 5' end of the first nucleic acid and form at least a portion of the terminal stem structure.

8. 7. The nucleic acid molecule of claim 6, wherein at least the terminal three nucleotides at the 5' end of the second nucleic acid sequence are complementary to at least the terminal three nucleotides at the 5' end of the first nucleic acid and form at least a portion of the terminal stem structure.

9. The nucleic acid molecule according to any one of claims 1 to 8, wherein the nucleic acid molecule or analog thereof is DNA or RNA.

10. The nucleic acid molecule of any one of claims 1 to 9, wherein the second nucleic acid sequence comprises a degenerate sequence.

11. The nucleic acid molecule of any one of claims 1 to 10, wherein the nucleic acid molecule does not contain an RNA polymerase promoter sequence.

12. The nucleic acid molecule of any one of claims 1 to 11, wherein the target nucleic acid sequence is of viral, microbial, fungal, animal, or plant origin, or is a synthetic construct.

13. The nucleic acid molecule according to any one of claims 1 to 11, wherein the target nucleic acid sequence is derived from a pathogenic virus or a pathogenic bacterium.

14. A composition comprising the first nucleic acid molecule of any one of claims 1 to 13.

15. 15. The composition of claim 14, further comprising a second nucleic acid molecule capable of hybridizing to at least a portion of the target nucleic acid sequence or its reverse complement and comprising a first RNA polymerase promoter sequence, wherein the first and second nucleic acid molecules form a first primer pair capable of amplifying a first sequence of the target nucleic acid sequence.

16. 16. The composition of claim 14 or 15, wherein the 3' end of the first nucleic acid molecule does not substantially hybridize to the second nucleic acid molecule or to itself.

17. 17. The composition of claim 16, wherein the first and second nucleic acid molecules do not substantially hybridize to each other.

18. 16. The composition of claim 14 or 15, wherein the terminal 1, 2, or 3 bases at the 3' end of the first nucleic acid molecule hybridize to the terminal 1, 2, or 3 bases at the 3' end of the second nucleic acid molecule.

19. 16. The composition of claim 14 or 15, wherein the 3' end of the first nucleic acid molecule is adjacent to the 3' end of the second nucleic acid molecule when aligned with the sequence of the target nucleic acid.

20. further comprising a third nucleic acid molecule and a fourth nucleic acid molecule; the third and fourth nucleic acid molecules form a second primer pair capable of amplifying a second sequence of the target nucleic acid molecule; either the third nucleic acid molecule or the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence; 20. The composition of any one of claims 14 to 19, wherein the second primer pair hybridizes to the target nucleic acid molecule at a position outside that of the first primer pair and is capable of amplifying the first sequence and the second sequence.

21. 21. The composition of claim 20, wherein the second RNA polymerase promoter sequence transcribes the second sequence of the target nucleic acid molecule in an opposite direction to that of the second nucleic acid molecule.

22. if the third nucleic acid molecule comprises a second RNA polymerase promoter sequence, the fourth nucleic acid molecule comprises a second aptamer coding sequence; or 22. The composition of claim 20 or 21, wherein the third nucleic acid molecule comprises a second aptamer-encoding sequence, and the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence.

23. The composition of any one of claims 20 to 22, wherein the 3' end of the third nucleic acid molecule does not substantially hybridize to the fourth nucleic acid molecule.

24. 24. The composition of claim 23, wherein the third and fourth nucleic acid molecules do not substantially hybridize to each other.

25. The composition of any one of claims 20 to 24, wherein the 3' ends of the first, second, third, and fourth nucleic acid molecules do not substantially hybridize to each other.

26. 26. The composition of claim 25, wherein the first, second, third, and fourth nucleic acid molecules do not substantially hybridize to one another.

27. further comprising a fifth nucleic acid molecule and a sixth nucleic acid molecule; the fifth and sixth nucleic acid molecules form a third primer pair capable of amplifying a third sequence of the target nucleic acid molecule; either the fifth nucleic acid molecule or the sixth nucleic acid molecule comprises a third RNA polymerase promoter sequence; 27. The composition of any one of claims 14 to 26, wherein the third primer pair hybridizes to the target nucleic acid molecule at a position outside those of the first and second primer pairs and is capable of amplifying the first, second, and third sequences.

28. 28. The composition of claim 27, wherein the third RNA polymerase promoter sequence transcribes the third sequence of the target nucleic acid molecule in the same direction as the second nucleic acid molecule.

29. if the fifth nucleic acid molecule comprises a third RNA polymerase promoter sequence, the fourth nucleic acid molecule comprises a third aptamer coding sequence; or 29. The composition of claim 27 or 28, wherein the fourth nucleic acid molecule comprises a third RNA polymerase promoter sequence, and the fifth nucleic acid molecule comprises a third aptamer-encoding sequence.

30. The composition of any one of claims 27 to 29, wherein the 3' end of the fifth nucleic acid molecule does not substantially hybridize to the 3' end of the fourth nucleic acid molecule.

31. 31. The composition of claim 30, wherein the fifth and fourth nucleic acid molecules do not substantially hybridize to each other.

32. The composition of any one of claims 27 to 31, wherein the 3' ends of the first, second, third, fourth, fifth, and sixth nucleic acid molecules are substantially non-hybridizable to each other.

33. 33. The composition of claim 32, wherein the first, second, third, fourth, fifth, and sixth nucleic acid molecules do not substantially hybridize to one another.

34. 34. The composition of any one of claims 14 to 33, comprising one or more nucleic acid molecules comprising a sequence as set out in Table 3.

35. The composition of any one of claims 14 to 34, wherein the nucleic acid molecules are premixed.

36. The composition of any one of claims 14 to 35, wherein the one or more nucleic acid molecules are provided in a liquid form.

37. The composition of any one of claims 14 to 35, wherein the one or more nucleic acid molecules are lyophilized.

38. A kit comprising a nucleic acid molecule according to any one of claims 1 to 13 or a composition according to any one of claims 14 to 37 together with instructions for the amplification of a target nucleic acid sequence.

39. 39. The kit of claim 38, wherein the amplification is an isothermal amplification.

40. 40. The kit of claim 38 or 39, wherein the isothermal amplification is nucleic acid sequence-based amplification, rolling circle amplification, loop-mediated isothermal amplification, helicase-dependent amplification, or strand displacement amplification.

41. 1. A method for amplifying a target nucleic acid sequence, comprising: i) providing a sample suspected of containing a target nucleic acid molecule; ii) providing a first nucleic acid molecule according to any one of claims 1 to 13; iii) providing a second nucleic acid molecule capable of hybridizing to at least a portion of the target nucleic acid sequence or its complement and comprising a first RNA polymerase promoter sequence; the first and second nucleic acid molecules form a first primer pair capable of amplifying a first sequence of the target nucleic acid sequence; and iv) performing a first amplification reaction comprising the target nucleic acid molecule and a first primer pair to obtain a first amplification product, wherein the first amplification product comprises a first sequence of the target nucleic acid sequence; The method comprising:

42. 42. The method of claim 41, wherein the 3' end of the first nucleic acid molecule does not substantially hybridize to the 3' end of the second nucleic acid molecule.

43. 43. The method of claim 42, wherein the first and second nucleic acid molecules do not substantially hybridize to each other.

44. 42. The method of claim 41, wherein the terminal 1, 2, or 3 bases at the 3' end of the first nucleic acid molecule hybridize to the terminal 1, 2, or 3 bases at the 3' end of the second nucleic acid molecule.

45. 42. The method of claim 41, wherein the 3' end of the first nucleic acid molecule is adjacent to the 3' end of the second nucleic acid molecule when aligned with the sequence of the target nucleic acid.

46. v) providing a third nucleic acid molecule and a fourth nucleic acid molecule, the third and fourth nucleic acid molecules form a second primer pair capable of amplifying a second sequence of the target nucleic acid molecule; either the third nucleic acid molecule or the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence; a second primer pair that hybridizes to the target nucleic acid molecule at a position outside that of the first primer pair and is capable of amplifying a first sequence and a second sequence of the target nucleic acid molecule; and vi) performing a second amplification reaction comprising the first amplification product and a second primer pair to obtain a second amplification product, wherein the second amplification reaction is performed before the first amplification reaction, and the second amplification product comprises a first sequence and a second sequence of the target nucleic acid molecule; The method of any one of claims 41 to 45, further comprising:

47. 47. The method of claim 46, wherein the second RNA polymerase promoter sequence transcribes the second sequence of the target nucleic acid molecule in an opposite direction to that of the second nucleic acid molecule.

48. if the third nucleic acid molecule comprises a second RNA polymerase promoter sequence, the fourth nucleic acid molecule comprises a second aptamer coding sequence; or 48. The method of claim 46 or 47, wherein the third nucleic acid molecule comprises a second aptamer coding sequence, and the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence.

49. The method of any one of claims 46 to 48, wherein the 3' end of the third nucleic acid molecule does not substantially hybridize to the 3' end of the fourth nucleic acid molecule.

50. 50. The method of claim 49, wherein the third and fourth nucleic acid molecules do not substantially hybridize to each other.

51. 49. The method of any one of claims 46 to 48, wherein the 3' ends of the first, second, third, and fourth nucleic acid molecules do not substantially hybridize to each other.

52. 52. The method of claim 51, wherein the first, second, third, and fourth nucleic acid molecules do not substantially hybridize to one another.

53. 53. The method of any one of claims 41 to 52, further comprising the step of detecting a target nucleic acid sequence.

54. 54. The method of any one of claims 41 to 53, further comprising the step of quantifying the target nucleic acid sequence.

55. The method of any one of claims 41 to 54, wherein the amplification is an isothermal amplification.

56. The method of any one of claims 41 to 55, wherein the amplification is RNA-based or DNA-based.

57. 56. The method of claim 55, wherein the isothermal amplification is nucleic acid sequence-based amplification, rolling circle amplification, loop-mediated isothermal amplification, helicase-dependent amplification, strand displacement amplification, or a combination thereof.

58. The method of any one of claims 41 to 57, wherein the amplification is multiplexed.

59. The method of any one of claims 41 to 58, wherein the amplification comprises color imaging in at least two colors.

60. 60. The method of claim 59, wherein the amplification comprises color imaging in at least three colors.

61. 61. The method of any one of claims 41 to 60, wherein the sample is of viral, microbial, fungal, animal, plant or environmental origin.

62. 61. The method of any one of claims 41 to 60, wherein the sample is derived from a pathogenic virus or a pathogenic bacterium.

63. 63. The method of claim 62, wherein the pathogenic virus is a coronavirus.

64. 64. The method of claim 63, wherein the coronavirus is SARS, MERS, or SARS-CoV-2.

65. 65. The method of any one of claims 41 to 64, wherein the sample is obtained from water, soil, saliva, faeces, urine, blood, tracheal aspirate, or nasal aspirate.

66. 62. The method of claim 61, wherein the animal is a human.

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