Kit and Device

Abstract

Provided herein are kits and devices that can be used for improved polynucleotide detection.

Description

Technical Field

[0001] The present invention relates to kits and devices suitable for use in testing for the presence of multiple diagnostic markers, including those used for the identification of cancer, infectious diseases, and transplant organ rejection. These are also useful for companion diagnostic tests that must reliably and at low cost identify marker panels.

Summary of the Invention

[0002] Polymerase chain reaction (PCR) is a well-known and powerful technique for amplifying DNA or RNA present in laboratory and diagnostic samples to levels that can be reliably detected and / or quantified. However, when applied for the purpose of examining analyte samples containing low levels of such molecules, it suffers from several limitations. First, this technique can detect as few molecules as a single target molecule, but tends to produce false positive results due to unwanted amplification of other nucleic acid sequences present in the sample. For this reason, the selection of oligonucleotide primers used to initiate the reaction is important; this in turn makes it relatively complex to design primers with the required level of specificity. As a result, many currently available PCR-based tests on the market have limited specificity.

[0003] A second drawback is that multiplexing of PCR-based methods is effectively limited to a maximum of several dozen target sequences (often 10 or less) due to the need to avoid primer-primer interactions, resulting in a relatively narrow operating window.

[0004] Another problem is that PCR reactions cycle in an exponential fashion, making it difficult to quantify the target; small variations in reaction efficiency have a large impact on the amount of detectable material produced. Thus, even with appropriate controls and calibrations in place, quantification is typically limited to an accuracy of approximately 1 / 3 to approximately 3-fold.

[0005] Finally, mutations in the regions targeted for research by the PCR amplification method can have undesirable side effects. For example, there was an example where a test approved by the FDA had to be withdrawn because multiple false negatives occurred in the target organism through mutations in the gene region targeted by the test primers. Conversely, when targeting a specific single nucleotide polymorphism (SNP) for amplification, the PCR method will often produce false positives if wild-type mutants are present. To avoid this, very careful primer design is required, and furthermore, the effectiveness of multiplexing is limited. This is particularly relevant when searching for SNP panels, which are common requirements in cancer testing / screening or companion diagnostics.

[0006] US2006 / 110765A1 (Wang et al.) describes enzymatic cleavage at mismatch sites, which is typically inefficient and not a very specific reaction. Furthermore, off-target hybridization of probes as disclosed by Wang et al. to similar sequences in the sample produces false positive results due to the use of cleavage at mismatch sites. Since two different gene mutants at the same or nearby positions all result in cleavage and amplification of the same probe, it is also impossible to distinguish between such mutants. The description by Wang et al. will thus result in a reaction scheme with low sensitivity and low specificity. In contrast, the technical effect of the method as disclosed by the present invention is to provide a rapid and efficient method with high specificity for dsDNA that can be effectively blocked by mismatches. Furthermore, the method of the present invention is very specific for the targeted gene mutants and enables discrimination between different mutants at the same or nearby positions.

[0007] US2009 / 239283 A1 (Liu et al.) describes the use of non-extendable 3' ends that are removed by pyrophosphorolysis, which requires genetic engineering of custom polymerases capable of removing 3'-blocking modifications. In contrast, the present invention utilizes the native pyrophosphorolytic activity inherent in existing polymerases and does not use 3'-blocking modifications. The methods as disclosed by Liu et al. also rely on the removal of only the terminal bases from a portion of the probe to enable subsequent amplification and are limited in this regard by the use of 3'-blocking modifications. In contrast, the methods disclosed in the present invention enable embodiments where progressive removal of multiple bases from the probe is required to initiate the reaction, rendering the reaction substantially more robust against transient off-target annealing to either background DNA or other probes that can result in unwanted removal of terminal bases.

[0008] Summary of the Invention The inventors are currently developing kits and devices for use in a novel method that builds on the inventors' experience using the pyrophosphorolytic method used in the inventors' previous patent (see PCT / GB2019 / 052017) to overcome many of these limitations. In doing so, the inventors utilize the double-stranded specificity of pyrophosphorolysis, a reaction that does not proceed efficiently with single-stranded oligonucleotide substrates or double-stranded substrates containing blocking groups or nucleotide mismatches. Thus, in accordance with the present invention: (a) A single-stranded probe oligonucleotide A capable of forming a first intermediate with a target polynucleotide sequence 0 wherein the intermediate is at least partially double-stranded, said oligonucleotide A 0 ; (b) A ligase; (c) A pyrophosphorolytic enzyme capable of digesting the first intermediate in the 3'-5' direction from the end of A 0 to produce a partially digested strand A 1 ; (d) A 0At least one single-stranded primer oligonucleotide that is substantially complementary to a part of; (e) An amplification enzyme; (f) A suitable buffer To provide a kit containing.

[0009] And: A fluid path between the first region, the second region and the third region A device comprising at least, The first region contains one or more wells, and each well: dNTP; At least one single-stranded primer oligonucleotide; An amplification enzyme for the initial amplification of DNA present in the sample And The second region contains one or more wells, and each well: A single-stranded probe oligonucleotide A capable of forming a target polynucleotide sequence and a first intermediate product 0 Wherein the intermediate product is at least partially double-stranded, said oligonucleotide A 0 ; A 0 A pyrophosphate-decomposing enzyme capable of digesting the first intermediate product in the 3'-5' direction from the end of A to produce a partially digested strand A 1 ; At least one source of pyrophosphate ions; Ligase And The third region contains one or more wells, and each well: dNTP; Buffer; Amplification enzyme; A 1 Or a part thereof, or means for detecting a signal derived from multiple copies of A 1 Or multiple copies of a part thereof Including; and The wells in the second region or the wells in the third region are A 0further comprising at least one single-stranded primer oligonucleotide that is substantially complementary to a portion of said device.

[0010] Analytes to which the methods of the invention may be applied are nucleic acids containing the target polynucleotide sequence(s) being sought, such as naturally occurring or synthetic DNA or RNA molecules. In one embodiment, the analyte is typically present in an aqueous solution containing the analyte and other biological materials, and in one embodiment, the analyte will be present with other background nucleic acid molecules that are not of interest for the purposes of the assay. In some embodiments, the analyte will be present in small amounts compared to these other nucleic acid components. Preferably, for example, if the analyte is derived from a biological specimen containing cellular material, to some extent or all of these other nucleic acids and exogenous biological materials will have been removed using sample preparation techniques such as filtration, centrifugation, chromatography or electrophoresis prior to performing step (b) of the method. Suitably, the analyte is derived from a biological sample taken from a mammalian subject (particularly a human patient), such as blood, plasma, sputum, urine, skin or biopsy. In one embodiment, the biological sample will be subjected to lysis to release the analyte by disrupting any cells present. In other embodiments, the analyte may already be in a free form within the sample; for example, as cell-free DNA circulating in blood or plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0011]

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Mode for Carrying Out the Invention

[0012] In one aspect of the present invention, a method for detecting a target polynucleotide sequence in a predetermined nucleic acid analyte present in a sample, comprising: (a) i. a single-stranded probe oligonucleotide A 0 ; ii. pyrophosphorolysis enzyme; and iii. ligase introduce one or more nucleic acid analytes into a first reaction mixture containing, where A 0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to generate a chain A 1 that is at least partially digested, and A 1forms A through ligation 2 ; (b) detecting a signal from the product of the previous step, where the product is A 2 or a part thereof, or multiple copies of A 2 or multiple copies of a part thereof, and inferring the presence or absence of a polynucleotide target sequence in the analyte therefrom to provide said method.

[0013] In some embodiments, the first reaction mixture further comprises a source of pyrophosphate ions. In some embodiments, the first reaction mixture further comprises at least one single-stranded primer oligonucleotide that is substantially complementary to a part of A 0 and deoxyribonucleotide triphosphates (dNTPs).

[0014] In some embodiments, the dNTPs are optional. In some embodiments, the first reaction mixture further comprises an amplification enzyme. In some embodiments, the product of step (a) is introduced into a second reaction mixture before step (b), and said second reaction mixture comprises at least one single-stranded primer oligonucleotide and dNTPs.

[0015] In some embodiments, the second reaction mixture further comprises an amplification enzyme. In some embodiments, one or more nucleic acid analytes may be introduced into the first and second reaction mixtures simultaneously.

[0016] In some embodiments, one or more nucleic acid analytes may be introduced into the first and second reaction mixtures sequentially. In some embodiments, the dNTPs are hot start dNTPs.

[0017] Hot start dNTPs are dNTPs that are modified with a thermolabile protecting group at the 3'-end. The presence of this modification blocks DNA polymerase nucleotide incorporation until the nucleotide protecting group is removed using a thermal activation step.

[0018] In some embodiments, during step (a), the analyte anneals to single-stranded probe oligonucleotide A 0 to form a first intermediate that is at least partially double-stranded and in which the 3'-end of A 0 forms a double-stranded complex with the analyte target sequence.

[0019] In some embodiments, during step (a), the first intermediate is pyrophosphorolytically digested in the 3'-5' direction from the 3'-end of A 0 to produce a partially digested strand A 1 and the analyte.

[0020] In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C, and the partially digested strand A 1 is ligated to the 5'-end of C at its 3'-end to produce oligonucleotide A 2

[0021] In some embodiments, the partially digested strand A 1 is circularized through ligation of its 3' and 5' ends. In some embodiments, the ligation of A 1 occurs: during step (a); or during step (b); or between steps (a) and (b)

[0022] In one embodiment, A 1 is circularized relative to the analyte target sequence. In this embodiment, the 3'-5' digestion of A 1 to form A 0 from the 3'-end of A 0 ​​The target region revealed by the progressive digestion of is A 0 / A 1 is complementary to the 5' end of. In this embodiment, using a ligase, the 3' and 5' ends of A 1 are ligated to form a circularized oligonucleotide A 2 . This is shown, for example, in FIG. 15. In one embodiment, the 5' end of A 0 / A 1 is complementary to the target over a region 5 to 50 nucleotides in length. In one embodiment, this is 5 to 25 nucleotides in length. In one embodiment, this is 5 to 20 nucleotides in length. In one embodiment, this is 5 to 15 nucleotides in length. In one embodiment, this is 5 to 12 nucleotides in length. In one embodiment, this is 5 to 10 nucleotides in length.

[0023] In some embodiments, the first reaction mixture further comprises a 5'-3' exonuclease, and the 5' end of A 0 is rendered resistant to 5'-3' exonuclease digestion.

[0024] In some embodiments, the sample is further treated with proteinase after amplification of the predetermined nucleic acid analyte and prior to addition of the first reaction mixture (step (a)). In some embodiments, the first reaction mixture further comprises a phosphatase or phosphohydrolase.

[0025] In some embodiments, prior to or during step (b), the product of the previous step is treated with pyrophosphatase. In some embodiments, prior to or during step (b), the product of the previous step is treated with exonuclease.

[0026] In some embodiments, oligonucleotide C further comprises a 3' or internal modification that protects from 3'-5' exonuclease digestion. In some embodiments, oligonucleotide C further comprises a 5' modification that protects against 5'-3' exonuclease digestion.

[0027] In some embodiments, the first or second reaction mixture further comprises a sprint oligonucleotide D. In some embodiments, D is 1 an oligonucleotide region complementary to the 3' end of A, and a region complementary to either the 5' end of oligonucleotide C or the 5' end of A 1 is included.

[0028] In some embodiments, D is unable to undergo extension relative to A, either by a 3' modification or through a mismatch between the 3' end of D and the corresponding region of A 1 1 .

[0029] In some embodiments, the enzyme that performs pyrophosphate cleavage of A to form partially digested strand A 0 1 also amplifies A. 2 In some embodiments, detection is achieved using one or more oligonucleotide fluorescent binding dyes or molecular probes.

[0030] In some embodiments, the concentration of the target sequence in the analyte is inferred using the increase in signal over time resulting from the generation of an amplicon of A 2 . In some embodiments, a plurality of probes A 0 each selective for a different target sequence and each containing an identification region, and the amplicon derived from A 2 contains this identification region, and thus the target sequence present in the analyte is further characterized by being inferred through detection of the identification region(s), said A 0 is used.

[0031] ​​​In some embodiments, detection of the identified region(s) is performed using molecular probes or via sequencing. In some embodiments, the final step of the method is: i. Labeling the product of step (b) with one or more oligonucleotide fluorescent binding dyes or molecular probes; ii. Measuring the fluorescence signal of the product; iii. Exposing the product to a set of denaturing conditions; and Identifying the polynucleotide target sequence in the analyte by monitoring changes in the fluorescence signal of the product during exposure to the denaturing conditions further comprising the step.

[0032] In some embodiments, one or more nucleic acid analytes are divided into a plurality of reaction volumes, each volume containing one or more probe oligonucleotides A 0 introduced to detect different target sequences.

[0033] In some embodiments, different probe As 0 contain a common priming site and allow for amplification of the region of A 2 using a single primer or a single set of primers.

[0034] In some embodiments, a method for detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample, comprising: (a) Amplifying the given nucleic acid analyte present in the sample; (b) i. Single-stranded probe oligonucleotide A 0 ; ii. Pyrophosphatase; and iii. Ligase Introducing the product of step (a) into a first reaction mixture containing, where A 0 is pyrophosphorylyzed from the 3'-end in the 3'-5' direction to produce at least partially digested strand A 1 and A1 forms A through ligation 2 ; (c) detecting a signal from the product of the previous step, where the product is A 2 or a part thereof, or multiple copies of A 2 or multiple copies of a part thereof, and inferring the presence or absence of the polynucleotide target sequence in the analyte therefrom including the step, provides the method.

[0035] According to the present invention, provided is a method for detecting a target polynucleotide sequence in a given nucleic acid analyte. Analytes to which various methods of the present invention may be applied may be prepared from the biological sample described above by a series of preliminary steps designed to amplify the analyte, and the analyte may typically be separated from the background genomic DNA present in significant excess. This method is generally applicable to the production of single-stranded target analytes and is thus useful in situations other than when incorporated with or further including parts of the method of the first aspect of the present invention. Thus, (1) a method for preparing at least one single-stranded analyte of a nucleic acid containing a target polynucleotide region, characterized by a step of producing an amplicon of the analyte(s) by subjecting a biological sample containing the analyte(s) and optionally background genomic DNA to an amplification cycle, is provided.

[0036] In some preferred embodiments, amplification is performed using polymerase chain reaction (PCR) in the presence of a polymerase, nucleoside triphosphates, and at least one corresponding primer pair, wherein one of the primers contains a 5'-3' exonuclease blocking group, and (2) optionally, the product of step (1) is digested with an exonuclease having 5'-3' exonuclease activity. In one embodiment, the method further comprises (3) reacting the product of step (2) with a protease to destroy the polymerase, and then (4) inactivating the protease by heating the product of step (3) to a temperature above 50°C.

[0037] In some preferred embodiments, steps (1)-(4) are performed prior to step (a) of the method of the first aspect of the present invention, resulting in an integrated method for detecting a target sequence derived from a biological sample. In another embodiment, the biological sample has undergone cell lysis prior to performing step (1).

[0038] In some embodiments of step (1), the nucleoside triphosphates are a mixture of the four deoxynucleoside triphosphates characteristic of naturally occurring DNA. In a preferred embodiment, the mixture of deoxynucleoside triphosphates contains deoxyuridine triphosphate (dUTP) instead of deoxythymidine triphosphate (dTTP), and step (1) is further performed in the presence of the enzyme dUTP-DNA glycosylase (UDG) to remove any contaminating amplicons from a previous assay. In yet another embodiment, a high-fidelity polymerase, such as one sold under the trade name Phusion® or Q5, is used in step (1). In yet another embodiment, the polymerase may be KAPA HiFi uracil+ DNA polymerase.

[0039] High-fidelity DNA polymerases have several safeguards to protect against both the creation and amplification of errors during DNA copying. Such enzymes have a significant binding preference for the correct nucleoside triphosphates over incorrect ones during polymerization. When an incorrect nucleotide binds to the polymerase active site, incorporation is slowed due to a sub-optimal structure of the active site complex. This delay increases the chance that the incorrect nucleotide will dissociate before polymerase progression, thereby allowing the process to start again with the correct nucleoside triphosphate. If an incorrect nucleotide is inserted, proofreading DNA polymerase provides an additional line of defense. A perturbation caused by the mispaired base is detected, and the polymerase moves the 3' end of the growing DNA strand into the proofreading 3'→5' exonuclease domain. Here, the incorrect nucleotide is removed by 3'→5' exonuclease activity, at which point the strand is returned to the polymerase domain where polymerization can continue.

[0040] In some embodiments, the nucleoside triphosphates are a mixture of synthetic or modified deoxynucleoside triphosphates. In some embodiments, the nucleoside triphosphates are a mixture of the four deoxynucleoside triphosphates and synthetic or modified deoxynucleotide triphosphates.

[0041] In some embodiments, step (1) is performed using a limited amount of primer and an excess number of amplification cycles. This means that a fixed amount of amplicon is produced regardless of the initial amount of analyte. Thus, the need for analyte quantification prior to subsequent steps is avoided. In another embodiment of step (1), which has the advantage of removing the need for step (2), amplification is performed in the presence of a primer pair in which one of the two primers is present in excess over the other and generates single-stranded amplicons when one primer has been completely utilized.

[0042] In some preferred embodiments of step (2), the 5' primer is blocked with an exonuclease blocking group selected from phosphorothioate linkages, inverted bases, DNA spacers and other oligonucleotide modifications commonly known in the art. In another embodiment, the other primer of the pair has a phosphate group at the 5' end.

[0043] In some embodiments, in step (3), the proteinase used is proteinase K, and step (4) is carried out by heating at a temperature of 80 - 100 °C for a maximum of 30 minutes. In one embodiment, in step (3), the proteinase used is proteinase K, step (3) is carried out by heating to a temperature of 55 °C for 5 minutes, and step (4) is carried out by heating to a temperature of 95 °C for 10 minutes. In another embodiment, at some point after step (2), the reaction medium is treated with phosphatase or phosphodiesterase to remove any residual nucleoside triphosphates that may be present.

[0044] In some embodiments, the target polynucleotide sequence in the analyte is a gene or chromosomal region within the DNA or RNA of cancerous tumor cells and is characterized by the presence of one or more mutations; for example, mutations in the form of one or more single nucleotide polymorphisms (SNPs). Thus, the present invention would be useful in monitoring disease recurrence and / or during treatment. Patients declared to be disease-free after treatment may be monitored over a long period to detect disease recurrence. This needs to be done non-invasively and requires highly sensitive detection of the target sequence from a blood sample. Similarly, for some cancers, there are residual cancer cells remaining in the patient after treatment. Monitoring the levels of these cells (or cell-free DNA) present in the patient's blood using the present invention enables detection of disease recurrence or failure of current therapy and the need to switch to an alternative therapy.

[0045] In some embodiments, detection of the target polynucleotide sequence will enable repeated testing of patient samples during disease treatment and will enable early detection of the development of resistance to therapy. For example, epidermal growth factor receptor (EGFR) inhibitors, such as gefitinib and erlotinib, are commonly used as first-line treatments for non-small cell lung cancer (NSCLC). During treatment, tumors will often develop mutations (e.g., T790M, C797S) in the EGFR gene that confer resistance to the therapy. Early detection of these mutations will enable conversion of the patient to an alternative therapy.

[0046] In some embodiments, the target polynucleotide sequence in the analyte is in a gene or chromosomal region within DNA or RNA of fetal origin and will be characterized by the presence of one or more mutations; e.g., mutations in the form of one or more single nucleotide polymorphisms (SNPs). Thus, the present invention can be used to detect mutations at very low allele frequencies at earlier stages of pregnancy than other available test technologies.

[0047] In another embodiment, the target polynucleotide sequence may be a gene or genomic region from an individual who is healthy except for the resulting genetic information, and may assist in generating valuable companion diagnostic information and may enable drawing medical or therapeutic conclusions across one or more defined groups within the human population.

[0048] In yet another embodiment, the target polynucleotide sequence may be characteristic of an infectious disease, or of resistance of an infectious disease to treatment with a particular therapy; e.g., a polynucleotide sequence characteristic of a gene or chromosomal region of a bacterium or virus, or a mutation therein that confers resistance to the therapy.

[0049] In some embodiments, the target polynucleotide sequence may be characteristic of the donor DNA. When the transplanted organ is rejected by the patient, the DNA from this organ will shed into the patient's bloodstream. Early detection of this DNA would enable early detection of rejection. This can be achieved by using a custom panel of donor-specific markers or, in some cases, a panel of variants that are known to be common in the population, some of which are present in the donor and some in the recipient. Thus, routine monitoring of long-term organ recipients is possible by the claimed method.

[0050] In yet another embodiment, multiple versions of the method using different combinations of probes (see below) are used in parallel to simultaneously screen an analyte for multiple target sequences; for example, sources of cancer, cancer markers, or multiple sources of infection. In this approach, the amplified products obtained by the parallel application of the method are contacted with a detection panel composed of one or more oligonucleotide-binding dyes or sequence-specific molecular probes, such as molecular beacons, hairpin probes, etc. Thus, in another aspect of the invention, provided is the use of at least one probe and optionally one ligation oligonucleotide, in combination with one or more chemical and biological probes that are selective for the target polynucleotide sequence or the use of sequencing to identify the amplified probe region.

[0051] In some embodiments, single-stranded probe oligonucleotide A 0 comprises a priming region and a 3' end that is complementary to the target polynucleotide sequence to be detected. By this means, a first intermediate product that is at least partially double-stranded is generated. In one embodiment, this step is performed in the presence of excess A 0 and in an aqueous medium containing the analyte and any other nucleic acid molecules.

[0052] In step (a), the double-stranded region of the first intermediate product is pyrophosphorolytically digested in the 3'-5' direction from the 3' end of its A 0 strand. As a result, the A 0 strand is gradually digested to produce a partially digested strand; hereinafter this is referred to as A 1 . If the probe oligonucleotide accidentally hybridizes to a non-target sequence, the pyrophosphorolytic digestion reaction stops at any mismatch, preventing the subsequent steps of the method from proceeding. In another embodiment, the digestion is continued until A 1 lacks sufficient complementarity to form a stable duplex with the analyte or a target region therein. At this point, the various strands then separate by melting, thereby producing single-stranded A 1 . Under typical pyrophosphorolytic digestion conditions, this separation occurs when there are 6 to 20 complementary nucleotides between the analyte and A 0 .

[0053] In another embodiment, the digestion is continued until A 1 lacks sufficient complementarity to bind the pyrophosphorolytic enzyme or for the pyrophosphorolytic reaction to continue. This typically occurs when there are 6 to 20 complementary nucleotides between the analyte and the probe. In some embodiments, this occurs when there are 6 to 40 complementary nucleotides remaining.

[0054] A 1 Using a sprint oligonucleotide D having complementarity at the 5' and 3' ends of A 1 (see below), in another embodiment, the digestion continues until the length of complementarity between A 1 and the target decreases to the point where it is energetically favorable for oligo D to displace the analyte molecule from A 1 . This typically occurs when the complementary region between A 1 and the analyte molecule is similar to or shorter than the complementary region between oligo D and the 3' end of A 1For the favorability of intramolecular hybridization of oligo D, which may already be hybridized to the 5' end of [[ID=]], A 1 Complementarity between and the analyte molecule may also occur if it is longer than this.

[0055] A 1 In another embodiment, the ligation of [[ID=]] is carried out using the analyte molecule as a splint (see Figure 16), where the 3' and 5' ends of [[ID=]] are adjacent and separated only by a nick, and digestion continues until the 5' end of [[ID=]] is capable of hybridizing to the analyte molecule, at which point they are ligated together by ligase and digestion can no longer proceed. 1 A 1 A

[0056] Suitably, pyrophosphorolysis is carried out in the reaction medium at a temperature in the range of 20 - 90 °C in the presence of at least one polymerase showing pyrophosphorolytic activity and a source of pyrophosphate ions. Further information regarding the pyrophosphorolysis reaction as applied to polynucleotide digestion can be found, for example, in J. Biol. Chem. 244 (1969) pp.3019 - 3028 or in the inventors' previous patent applications.

[0057] In some embodiments, the pyrophosphorolysis step is driven by the presence of an excess source of polyphosphate, and suitable sources include compounds containing 3 or more phosphorus atoms.

[0058] In some embodiments, the first reaction mixture contains an excess source of polyphosphate. In some embodiments, the pyrophosphate cleavage step is driven by the presence of a source of excess modified pyrophosphate. Suitable modified pyrophosphates include those having other atoms or groups substituted in place of the bridging oxygen, or pyrophosphates (or polyphosphates) containing substituents or modifying groups on other oxygens. One of ordinary skill in the art will understand that there are many such examples of modified pyrophosphates suitable for use in the present invention, and a non-limiting selection thereof is:

[0059]

Chemical formula

[0060] is as follows. In some embodiments, the first reaction mixture contains a source of excess modified polyphosphate.

[0061] In some preferred embodiments, the source of pyrophosphate ions is PNP, PCP or tripolyphosphate (PPPi). Furthermore, non-limiting examples of sources of pyrophosphate ions for use in the pyrophosphate cleavage step (b) can be found in WO2014 / 165210 and WO00 / 49180.

[0062] In some embodiments, the source of excess modified pyrophosphate can also be represented as Y-H, wherein Y corresponds to the general formula (X-O) 2 P(=B)-(Z-P(=B)(O-X)) n -, where n is an integer from 1 to 4; each Z- is independently selected from -O-, -NH- or -CH 2 -, independently; each B is independently either O or S; the X groups are independently selected from -H, -Na, -K, alkyl, alkenyl, or heterocyclic groups, provided that when both Z and B correspond to -O- and n is 1, at least one X group is not H.

[0063] In some embodiments, Y has the general formula (X-O) 2 P(=B)-(Z-P(=B)(O-X))n - corresponding, where n is 1, 2, 3 or 4. In another embodiment, the Y group has the general formula (X-O) 2 corresponding to P(=O)-Z-P(=O)(O-H)-, where one of the X groups is -H. In yet another preferred embodiment, Y has the general formula (X-O) 2 corresponding to P(=O)-Z-P(=O)(O-X)--, where at least one of the X groups is selected from methyl, ethyl, allyl or dimethylallyl.

[0064] In another embodiment, Y has the general formula (H-O) 2 P(=O)-Z-P(=O)(O-H)-, where Z is -NH- or -CH 2 -, or (X-O) 2 P(=O)-Z-P(=O)(O-X)--, where all of the X groups are either -Na or -K, and Z is either -NH- or -CH 2 - of either.

[0065] In other embodiments, Y has the general formula (H-O) 2 corresponding to P(=B)-O-P(=B)(O-H)-, where each B group is independently either O or S, and at least one is S.

[0066] Specific examples of preferred embodiments of Y include those of the formula (X1-O)(HO)P(=O)-Z-P(=O)(O-X2), where Z is O, NH or CH 2 and (a) X1 is γ,γ-dimethylallyl and X2 is -H; or (b) both X1 and X2 are methyl; or (c) both X1 and X2 are ethyl; or (d) X1 is methyl and X2 is ethyl or vice versa.

[0067] In some embodiments, when attempting to use a molecular probe for detection, probe oligonucleotide A 0is configured to include an oligonucleotide identification region on the 5'-side of the region complementary to the target sequence, and the molecular probe used is designed to anneal to this identification region. In one embodiment, A 0 only the 3'-region of can anneal to the target; that is, none of the other regions have sufficient complementarity with the analyte to form a stable duplex at the temperature at which the pyrophosphate cleavage step is performed. Here, and throughout, the term "sufficient complementarity" means that the complementary region is longer than 10 nucleotides in length, as long as the given region has complementarity with the given region on the analyte.

[0068] In a further aspect of the method of the invention, an alternative embodiment is provided in which the pyrophosphate cleavage step of any previous embodiment is replaced with an exonuclease digestion step using a double-strand specific exonuclease. Those skilled in the art will understand that double-strand specific exonucleases include, inter alia, those that read in the 3'-5' direction, such as ExoIII, and those that read in the 5'-3' direction, such as lambda exon.

[0069] In some embodiments of the invention where the exonuclease digestion step utilizes a double-strand specific 5'-3' exonuclease, it is the 5'-end of A 0 that is complementary to the target analyte, and the common priming sequence and the blocking group are located on the 3'-side of the region complementary to the target. In a further embodiment where a molecular probe is to be used for detection, the probe oligonucleotide A 0 is configured to include an oligonucleotide identification region on the 3'-side of the region complementary to the target sequence, and the molecular probe used is designed to anneal to this identification region.

[0070] In embodiments of the invention where the exonuclease digestion step utilizes a double-strand specific 5'-3' exonuclease, any other nucleic acid molecules present are digested, while A 0 and the partially digested strand A 1For the purpose of leaving any substances containing it intact, an exonuclease having 3'-to-5' exonuclease activity may optionally be added to the reaction mixture. Appropriately, this resistance to exonuclease digestion is achieved as described elsewhere in this application.

[0071] In one preferred embodiment of the invention, 0 the 5'-end of A or an internal site on the 5'-side of the priming region is rendered resistant to exonuclease digestion. By this means, and after or simultaneously with the pyrophosphate addition step, while digesting any other nucleic acid molecules present, 0 A and the partially digested strand A 1 For the purpose of leaving any substances containing it intact, an exonuclease having 5'-3' exonuclease activity may optionally be added to the reaction medium. Appropriately, this resistance to exonuclease digestion is achieved at the required points by introducing one or more blocking groups within the oligonucleotide A 0 In one embodiment, these blocking groups may be selected from phosphorothioate linkages and other backbone modifications commonly used in the art, C3 spacers, phosphate groups, modified bases, etc.

[0072] In some embodiments, the identification region has a unique sequence and is adapted to be indirectly identified using sequence-specific molecular probes applied to the amplified component A 2 or directly identified by sequencing of these components, and may include a barcode region or be embedded within such a barcode region. Examples of molecular probes that may be used include, but are not limited to, molecular beacons, TaqMan® probes, Scorpion® probes, etc.

[0073] In all embodiments, multiple, typically millions of copies are made of 2The lock or its desired region is made to undergo amplification. This is achieved by 2 priming the region of A and any subsequent amplicons derived therefrom, for example, in the form of forward / reverse or sense / antisense pairs, with single-stranded primer oligonucleotides that are capable of annealing to complementary regions thereon. The primed strand then serves as a starting point for amplification. Amplification methods include, but are not limited to, thermal cycling and isothermal methods such as polymerase chain reaction, recombinase polymerase amplification, and rolling circle amplification; the last of these is applicable when A 2 is circularized. By any of these means, multiple amplicon copies of the region of A 2 and, in some cases, its sequence complement can be rapidly generated. The exact methodology for performing any of these amplification methods is well known to those of ordinary skill in the art, and the exact conditions and temperature control regimes to be used are readily available in the general literature; see there for reference. In particular, in the case of polymerase chain reaction (PCR), the methodology generally involves using a source of polymerase and various single nucleoside triphosphates to extend the primer oligonucleotide in the 5'-3' direction along the A 2 strand until a complementary strand is produced; denaturing the resulting double-stranded product, 2 regenerating the A 2 strand and the complementary strand; re-priming both the A 2 strand and its amplicons, and then repeating these extension / denaturation / re-priming steps multiple times to increase the concentration of the amplicons to a level that can be reliably detected.

[0074] In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C, and the partially digested strand A 1 is ligated to the 5' end of C at its 3' end, while in another embodiment, A 1 is circularized through ligation of its 3' and 5' ends; in each case, the oligonucleotide A2 is formed.

[0075] In one embodiment, the ligation of A 1 occurs: during step (a); or during step (b); or between steps (a) and (b). takes place.

[0076] In one embodiment, A 1 is optionally extended in the 5'-3' direction prior to ligation. In some embodiments, this optional extension and ligation are performed on the target oligonucleotide, while in another embodiment, they are performed through the addition of an additional sprint oligonucleotide D to which A 1 anneals. In one embodiment, D comprises an oligonucleotide region complementary to the 3' end of A 1 and a region complementary to either the 5' end of oligonucleotide C or the 5' end of A 1 . In another embodiment, D is non-extendable with respect to A 1 either due to a 3' end modification or through a nucleotide mismatch between the 3' end of D and the corresponding region of A 1 .

[0077] In some embodiments, the ligation probe C has at least a portion of the 5' end region of the sprint oligonucleotide D or a 5' region complementary to the target oligonucleotide. By such means, a second intermediate product is formed, where the A 2 strand is composed of A 1 , C and optionally an intermediate region formed by the extension of A 1 in the 5'-3' direction such that it meets the 5' end of C. In such embodiments, the primer used in step (c) (see below) comprises at least the site where the ligation of A 1 to C occurs, of A 2is selected to amplify the region. In this embodiment, the inventors have found it preferable to include a 3'-blocking group on the C so that any unligated A can be digested by a 3'-5' exonuclease prior to amplification. Suitable polymerases that can be used for the elongation of A prior to ligation include, but are not limited to, Hemo KlenTaq, Mako, and Stoffel fragment. 1 In some embodiments, the first reaction mixture further comprises a phosphatase or phosphohydrolase to remove the nucleoside triphosphates produced by the pyrophosphorolysis reaction by hydrolysis, thereby allowing the pyrophosphorolysis reaction to continue and ensuring that it is not overcome by the forward polymerization reaction. 1 In some embodiments, prior to or during step (b), the product of the previous step is treated with pyrophosphatase to hydrolyze pyrophosphate ions, prevent further pyrophosphate reaction from occurring, and support the forward polymerization reaction.

[0078] In some embodiments, prior to or during step (b), the product of the previous step is treated with exonuclease.

[0079] In some embodiments, the enzyme that performs pyrophosphorolysis of A to form a partially digested strand A also amplifies A. One of ordinary skill in the art will recognize that many such enzymes exist.

[0080] In some embodiments, prior to or during step (b), the product of the previous step is treated with exonuclease. In some embodiments, A 0 is pyrophosphorolyzed to form a partially digested strand A 1 The enzyme that forms also amplifies A 2 One of ordinary skill in the art will recognize that many such enzymes exist.

[0081] Detect the amplicon and use the information obtained to infer whether the polynucleotide target sequence is present or absent in the original analyte and / or related characteristics thereof. For example, by this means, a target sequence characteristic of cancerous tumor cells may be detected with reference to a specific SNP being sought. In another embodiment, a target sequence characteristic of a viral or bacterial genome (including its novel mutations) may be detected. For example, many methods for detecting amplicons or regions of interest may be used, including oligonucleotide-binding dyes, sequence-specific molecular probes, such as fluorescently labeled molecular beacons or hairpin probes. Alternatively, using one of the direct sequencing methods used or reported in the art, A 2 Direct sequencing of the amplicon may be performed. When using oligonucleotide-binding dyes, fluorescently labeled beacons or probes, a source of stimulating electromagnetic radiation (laser, LED, lamp, etc.), and detecting the emitted fluorescence, and therefrom, using a specially designed algorithm, a signal including a data stream analyzable by a microprocessor or computer is generated. It is preferred to detect the amplicon using an arrangement including a light detection device arranged to generate the signal.

[0082] In some embodiments, detection is achieved using one or more oligonucleotide fluorescent binding dyes or molecular probes. In such embodiments, A 2 the increase in the signal over time resulting from the generation of the amplicon is used to infer the concentration of the target sequence in the analyte. In one embodiment, the final step of the method is: i. Label the product of step (b) using one or more oligonucleotide fluorescent binding dyes or molecular probes; ii. Measure the fluorescence signal of the product; iii. Expose the product to a set of denaturing conditions; and identifying the polynucleotide target sequence in the analyte by monitoring the change in the fluorescence signal of the product during exposure to the denaturing conditions further comprising the step of.

[0083] In some embodiments, a plurality of probe A 0 each of which is selective for a different target sequence, and each of which contains an identification region, and the identification region is further characterized by being included in an amplicon of A 2 and thus the target sequence present in the analyte is inferred through detection of the identification region(s), said probe A 0 is used. In such embodiments, detection of the identification region(s) is performed using molecular probes or through sequencing.

[0084] In some embodiments, one or more nucleic acid analytes are divided into a plurality of reaction volumes, each volume being introduced with one or more probe oligonucleotides A for detecting different target sequences 0 having.

[0085] Different probe A 0 In some embodiments using different probe A 0 contains one or more common priming sites, allowing a single primer or a single primer set to be used for amplification.

[0086] In another aspect of the invention, a method for identifying a target polynucleotide sequence in a given nucleic acid analyte characterized by the steps of any of the previous embodiments of the invention, comprising A 2 or A 2 a large number of copies of the region of are labeled with one or more oligonucleotide fluorescent binding dyes or molecular probes, said method is provided. Measure the fluorescence signals of these multiple copies and expose the multiple copies to a set of denaturing conditions. The target polynucleotide sequence is identified by monitoring the change in the fluorescence signal of the multiple copies during exposure to the denaturing conditions.

[0087] In some embodiments, denaturing conditions may be provided by changing the temperature, for example, increasing the temperature up to the point at which the double strands begin to dissociate. Additionally or alternatively, denaturing conditions may also be provided by changing the pH such that the conditions are acidic or alkaline, or by adding an additive or agent, such as a strong acid or strong base, a concentrated inorganic salt, or an organic solvent, such as alcohol.

[0088] In another aspect of the invention, there is provided the use of the above-described method for screening a mammalian subject, particularly a human patient, for the presence of an infectious disease, cancer, or for the purpose of generating companion diagnostic information.

[0089] In a further aspect of the invention, there is provided a control probe for use in a method as described above. Embodiments of the invention include those in which the presence of one or more specific target sequences is elucidated by the generation of a fluorescent signal. In such embodiments, there may necessarily be a level of signal generated from non-target DNA present in the sample. For a given sample, this background signal starts later than the "true" signal, but this start can vary between samples. The accurate detection of the presence of low concentrations of one or more target sequences therefore relies on knowledge of what signal is expected in the absence thereof. A reference is available for the intended samples, but not for true "blind" samples from patients. Control probe (E 0 ) is utilized to determine the expected background signal profile for each assay probe. The control probe targets a sequence expected not to be present in the sample and may then use the signal generated from this probe to infer the expected rate of signal generation from the sample in the absence of the target sequence.

[0090] Accordingly: a. A second single-stranded probe oligonucleotide having a 3' end region that is at least partially mismatched to the target sequence E 0Using either separate aliquots of the sample or within the same aliquot and using a second detection channel, repeat the steps of the method either subsequently or simultaneously; b. Infer the background signal expected to be generated from A 0 in the absence of any target analyte in the sample; and c. Infer the presence or absence of the polynucleotide target sequence in the analyte through comparison of the expected background signal inferred in (a) with the actual signal observed in the presence of the target analyte A method for detecting a target polynucleotide sequence in a given nucleic acid analyte is provided according to any of the methods described above characterized by the steps.

[0091] In some embodiments, a control probe (E 0 ) and A 0 are added to separate portions of the sample, while in another embodiment, E 0 and A 0 are added to the same portion of the sample and the respective signals are measured using different detection channels (e.g., different colored dyes). Then, the signal generated by E 0 is utilized to infer the background signal expected to be generated by A 0 in the absence of the polynucleotide target sequence in the sample, and it may be corrected in relation thereto. For example, correction of the background signal may involve subtracting the signal observed from E 0 from that observed from A 0 , or through calibration of the signal observed from A 0 using a calibration curve of the relative signals generated by A 0 and E 0 under various conditions.

[0092] In some embodiments, a single E 0 may be used to calibrate all of the assay probes that can be produced. In some embodiments, separate E 0 may be used to calibrate each amplicon of the sample DNA generated in the first amplification step. Each amplicon may contain multiple mutations / target sequences of interest, but a single E 0 will be sufficient to calibrate all of the assay probes for a single amplicon.

[0093] In a further embodiment, for each target sequence, separate E 0 may be used. For example, when targeting a C>T mutation, an E 0 targeting a C>G mutation at the same site that is not known to occur in the patient may be designed. Under various conditions, the signal profiles generated by E 0 are evaluated in a calibration reaction, and these data are used to infer the signal expected from an assay probe targeting the C>T mutant in the absence of the variant.

[0094] Some embodiments of the methods of the invention can be seen in FIGS. 17-20. In FIG. 17, single-stranded probe oligonucleotide A 0 anneals to a target polynucleotide sequence to form at least partially double-stranded, and a first intermediate product is generated in which the 3' end of A 0 forms a double-stranded complex with the target polynucleotide sequence. In this simplified embodiment of the invention, to illustrate how A 0 that does not anneal to the target is not involved in further steps of the method, there are two molecules of A 0 present and one target polynucleotide sequence. In this exemplary example, the 3' end of A 0 anneals to the target polynucleotide sequence while the 5' end of A 0 does not. The 5' end of A 0 contains a 5' chemical blocking group, a common priming sequence, and a barcode region.

[0095] The first intermediate product that is partially double-stranded, in the presence of pyrophosphorolytic exonucleases, undergoes pyrophosphorolytic digestion in the 3'-5' direction from the 3' end of A 0 to generate a partially digested strand A 1 that does not anneal to the analyte and the target, and undigested A 0 molecules.

[0096] In Figure 18, A 1 is annealed to the single-stranded trigger oligonucleotide B, and the A 1 strand is extended in the 5'-3' direction relative to B to generate oligonucleotide A 2 . In this exemplary example, the trigger oligonucleotide B has a 5' chemical block. Neither of the undigested A 0 can anneal to the trigger oligonucleotide B and extend in the 5'-3' direction relative to B to generate the sequence that is the target for a later part of the method. In this example, A 2 is primed with at least one single-stranded primer oligonucleotide, and multiple copies of A 2 or A 2 regions are generated.

[0097] In Figure 19, A 1 is annealed to the sprint oligonucleotide D and then circularized by ligation of its 3' and 5' ends. Here, the circularized A 2 is primed with at least one single-stranded primer oligonucleotide, and multiple copies of A 2 or A 2 regions are generated. In this exemplary example, the sprint oligonucleotide D cannot extend relative to A 2 due to a 3' modification (in this example a chemical) or a nucleotide mismatch between the 3' end of D and the corresponding region of A 1 .

[0098] In Figure 20, the 3' region of the sprint oligonucleotide D is A 1anneals to the 3' region of, while the 5' region of the sprint oligonucleotide D anneals to the 5' region of the ligation probe C. Thus, A 1 , C, and optionally A in the 5'-3' direction to meet the 5' end of C 1 A second intermediate product A composed of an intermediate region formed by the extension of 2 is formed. In this exemplary example, the ligation probe C has a 3' chemical blocking group so that any unligated A 1 can be digested using a 3'-5' exonuclease.

[0099] A 2 is primed with at least one single-stranded primer oligonucleotide, and multiple copies of A 2 or the region of A 2 are generated. The specificity of the method of the present invention may be improved by the introduction of a blocking oligonucleotide. For example, a blocking oligonucleotide is introduced so as to hybridize to at least a part of the wild-type DNA, promoting the annealing of A 0 only to the target polynucleotide sequence and not promoting annealing to the wild-type. Alternatively or additionally, a blocking oligonucleotide may be used to improve the specificity of the polymerase chain reaction (PCR) and prevent the amplification of any wild-type sequences present. The general technique used is to design an oligonucleotide that anneals between the PCR primers and cannot be displaced or digested by the PCR polymerase. The oligonucleotide is designed to anneal to non-target (usually healthy) sequences, while having a mismatch (often a single base) to the target (mutant) sequence. This mismatch results in different melting temperatures for the two sequences, and the oligonucleotide is designed to remain annealed to the non-target sequence at the PCR extension temperature while dissociating from the target sequence.

[0100] Blocking oligonucleotides may often have modifications such as preventing digestion by the exonuclease activity of PCR polymerase or enhancing the melting temperature difference between target and non-target sequences.

[0101] Incorporation of locked nucleic acid (LNA) or other melting temperature-modifying modifications into blocking oligonucleotides can significantly increase the difference in melting temperature of the oligonucleotide for target and non-target sequences.

[0102] Accordingly, aspects of the present invention using blocking oligonucleotides are provided. The blocking oligonucleotide must be resistant to the pyrophosphorolysis (PPL) reaction in order to ensure that it is not digested and not replaced. This can be achieved in several different ways, for example, either through a 3'-end mismatch or through modifications such as phosphorothioate linkages or spacers.

[0103] In such aspects or facets of the present invention using blocking oligonucleotides, a method for detecting a target polynucleotide sequence in a given nucleic acid analyte is characterized by annealing the analyte target sequence to a single-stranded probe oligonucleotide A 0 to produce a first intermediate product that is at least partially double-stranded and where the 3'-end of A 0 forms a double-stranded complex with the analyte target sequence, and annealing a single-stranded blocking oligonucleotide to at least a subset of the non-target polynucleotide sequences either before or during the same step of generating the first intermediate product.

[0104] In some embodiments, the blocking oligonucleotide is made to be resistant to the pyrophosphorolysis reaction through a mismatch at the 3' end. In another embodiment, the blocking oligonucleotide is made to be resistant through the presence of a 3' blocking group. In another embodiment, the blocking oligonucleotide is made to be resistant through the presence of a spacer or other internal modification. In a further embodiment, the blocking oligonucleotide includes both a modification or modified nucleotide base that increases the melting temperature and is rendered resistant to pyrophosphorolysis.

[0105] As used herein, reference to a "phosphatase enzyme" refers to any enzyme or functional fragment thereof having the ability to remove nucleoside triphosphates produced by the methods of the invention by hydrolysis. This includes any enzyme or functional fragment thereof having the ability to cleave a phosphate monoester into a phosphate ion and an alcohol.

[0106] As used herein, reference to a "pyrophosphatase enzyme" refers to any enzyme or functional fragment thereof having the ability to catalyze the conversion of one ion of pyrophosphate into two phosphate ions.

[0107] This also includes inorganic pyrophosphatase and inorganic diphosphatase. Non-limiting examples are thermostable inorganic pyrophosphate (TIPP).

[0108] In some embodiments, a modified form of any of the previously described embodiments is provided where the use of pyrophosphatase is optional. In some embodiments of the invention, a kit for use in a method of detecting a target polynucleotide sequence in a predetermined nucleic acid analyte present in a sample, comprising: (a) a single-stranded probe oligonucleotide A capable of forming a first intermediate product with the target polynucleotide sequence 0 wherein the intermediate product is at least partially double-stranded, the probe oligonucleotide A0 ; (b) ligase; (c) The first intermediate product is A 0 The partially digested strand A is digested in the 3'-5' direction from the end of 1 pyrophosphorolytic enzymes capable of producing (d)A 0 at least one single-stranded primer oligonucleotide substantially complementary to a portion of (e) an amplifying enzyme; and (f) Suitable buffers The kit further comprises:

[0109] In one embodiment, A 0 The 3' end of the is perfectly complementary to the target polynucleotide sequence. In one embodiment, the ligase substantially lacks single-stranded ligation activity.

[0110] In some embodiments, the kit comprises a single-stranded probe oligonucleotide A capable of forming a first intermediate product with a target polynucleotide sequence. 0 wherein the intermediate product is at least partially double-stranded, 0 ; (a) ligase; (b)A 0 3'-5' from the end of the first intermediate product to produce a partially digested strand A 1 pyrophosphorylase, capable of producing (c) Suitable buffers Includes.

[0111] In some embodiments, the kit further comprises: -A 1 two or more ligation chain reaction (LCR) probe oligonucleotides that are complementary to the above flanking sequences, such that when a probe successfully anneals to the 5' phosphate of one LCR probe, it is immediately adjacent to the 3' OH of the other LCR probe; and - One or more ligases is further included.

[0112] In some embodiments, in the presence of A 2 , two LCR probes successfully anneal to A 2 , and are ligated together to form one oligonucleotide molecule, which then serves as a new target for a second round of covalent ligation, leading to geometric amplification of the target of interest, in this case A 2 . The ligated product or amplicon is complementary to A 2 and functions as a target in the next cycle of amplification. Thus, exponential amplification of a specific target DNA sequence is achieved through repeated cycles of denaturation, hybridization, and ligation in the presence of an excess of LCR probes. From this, the presence of A 2 , and thus the presence of the target polynucleotide sequence, is inferred.

[0113] In some embodiments, in the presence of A 2 , two PCR probes successfully anneal to A 2 , and are ligated together to form one oligonucleotide molecule, which then serves as a new target for a second round of covalent ligation, leading to geometric amplification of the target of interest, in this case A 2 , which is then detected.

[0114] In some embodiments, the kit optionally further comprises: - A ligation probe oligonucleotide C; - A sprint oligonucleotide D ; where C has a 5' phosphate, and the 3' end of the sprint oligonucleotide D is complementary to the 5' of C and the 5' end of D is complementary to the 3' end of A such that A 1 and C can be ligated together to form A 2 . 1

[0115] In some embodiments, the kit comprises: - Hairpin oligonucleotide 1 (HO1) comprising a fluorophore-quencher pair, wherein HO1 is 2 complementary to A 2 and when annealed to A, the hairpin structure of HO1 opens and the fluorophore-quencher pair separates; said HO1; and - Hairpin oligonucleotide 2 (HO2) comprising a fluorophore-quencher pair, wherein HO2 is complementary to the opened HO1 and when annealed to HO1, the hairpin structure of HO2 opens and the fluorophore-quencher pair separates; said HO2 may further be included.

[0116] In some embodiments, the kit may further comprise a plurality of HO1 and HO2. In some embodiments, the kit separately comprises: oligonucleotide A comprising a substrate arm, a partial catalytic core and a sensor arm; - oligonucleotide B comprising a substrate arm, a partial catalytic core and a sensor arm; and - a substrate comprising a fluorophore-quencher pair may further be included; wherein the sensor arms of oligonucleotides A and B are complementary to the adjacent region of A 2 such that in the presence of A 2 oligonucleotides A and B combine to form a catalytic multi-component nucleic acid enzyme (MNA enzyme).

[0117] In some embodiments, the kit may separately further comprise a nucleic acid construct that is partially double-stranded, wherein: - one strand comprises at least one RNA base, at least one fluorophore, and one region of this strand is complementary to the region of A 2 and this strand may be referred to as the "substrate" strand; - the other strand comprises at least one quencher, and one region of this strand is complementary to A 2In the presence of A, a nucleic acid construct that is partially double-stranded forms a substantially larger double-stranded portion adjacent to a region where the substrate strand is complementary so that it forms a more double-stranded structure. 2 is complementary to the region of A.

[0118] In other words, a nucleic acid construct that is partially double-stranded has a larger double-stranded portion in the presence of A. 2 In some embodiments, the kit may further comprise an enzyme for removing at least one RNA base. In some embodiments, the enzyme is uracil-DNA glycosylase (UDG) and the RNA base is uracil.

[0119] In some embodiments, the kit may further comprise, separately: - an oligonucleotide complementary to the region of A containing the ligation site and comprising one or more fluorophores arranged such that their fluorescence is quenched either by proximity to each other or to one or more fluorescence quenchers; - a double-strand specific DNA digesting enzyme 2 wherein, in the presence of A, the fluorophore is separated from each other or from the corresponding quencher and the labeled oligonucleotide is digested such that the fluorescence signal and thus the presence of A is detectable. - a double-strand specific DNA digesting enzyme may further be included; wherein, in the presence of A, the fluorophore is separated from each other or from the corresponding quencher and the labeled oligonucleotide is digested such that the fluorescence signal and thus the presence of A is detectable. 2 In some embodiments, the double-strand specific DNA digesting enzyme is an exonuclease. 2 In some embodiments, the double-strand specific DNA digesting enzyme is a polymerase with proofreading activity.

[0120]

[0121] ​​In some embodiments, the fluorophores of the kit may be selected from fluorescein family, carboxyrhodamine family, cyanine family, rhodamine family dyes, polyhalofluorescein family dyes, hexachlorofluorescein family dyes, coumarin family dyes, oxazine family dyes, thiazine family dyes, squaraine family dyes, and chelated lanthanide family dyes.

[0122] In some embodiments, the fluorophores of the kit may be selected from any commercially available dyes. In some embodiments, the quencher of the kit has the trade name Black Hole TM , Eclipse TM . Dark, Qx1J, and Iowa Black TM and may be selected from those available under these names.

[0123] In some embodiments, the quencher of the kit may be selected from any commercially available quencher. In some embodiments, the kit may further comprise one or more partially double-stranded DNA constructs, each construct containing one or more fluorophores and one or more quenchers. In some embodiments, when the construct is partially double-stranded, the one or more fluorophores and the one or more quenchers are positioned in close proximity such that sufficient quenching of the one or more fluorophores occurs.

[0124] In some embodiments, the construct is a single-stranded DNA having self-complementary regions that loop back on itself. In some embodiments, the construct comprises one of the primers of a primer pair.

[0125] In some embodiments, the kit may further comprise the other primer of the primer pair. In some embodiments, a portion of a single-stranded section of a construct hybridizes to A 2 and is extended by DNA polymerase against A 2 . In some embodiments, the other primer of a primer pair then hybridizes to the extended construct. This primer is then extended against the construct to replace the self-complementary region. Thus, one or more fluorophores and one or more dyes are sufficiently separated for a fluorescence signal to be detected, indicating the presence of A 2 .

[0126] In such embodiments, the construct may be known as a Sunrise primer. In some embodiments, the construct comprises two separate DNA strands. In some embodiments, a portion of a single-stranded section of a construct hybridizes to A 2 and is extended by DNA polymerase against A 2 . In some embodiments, the other primer of a primer pair then hybridizes to the extended construct. This primer is then extended against the construct in the direction of the double-stranded section, replacing the shorter DNA strand, and thus one or more fluorophores and one or more dyes are sufficiently separated for a fluorescence signal to be detected, indicating the presence of A 2 .

[0127] In such embodiments, the construct may be known as a Molecular Zipper. One of ordinary skill in the art will recognize that, with respect to both Sunrise primers and Molecular Zippers, one or more fluorophores and one or more quencher pairs can be positioned at various positions within each respective construct. The important feature is that each pair is positioned sufficiently close to each other such that, in the absence of A 2 , i.e., when extension and strand displacement do not occur, no fluorescence signal is emitted.

[0128] In one embodiment, the kit further includes a source of pyrophosphate ions. Suitable source(s) of pyrophosphate ions are as described above. In some embodiments, the kit further includes suitable positive and negative controls.

[0129] In some embodiments, the kit may further include one or more control probes (E 0 ) as described above. In some embodiments, the kit may further include one or more blocking oligonucleotides as described above.

[0130] In some embodiments, the kit may further include one or more control probes (E 0 ) and one or more blocking oligonucleotides. In some embodiments, the 5’ end of A 0 may be rendered resistant to 5’-3’ exonuclease digestion, and the kit may further include 5’-3’ exonuclease.

[0131] In some embodiments, the kit may further include a ligation probe oligonucleotide C. In some embodiments, the kit may further include a sprint oligonucleotide D.

[0132] In some embodiments, the kit may include both C and D. The ligation probe C may include a 3’ or internal modification that protects the probe from 3’-5’ exonuclease digestion.

[0133] D may include an oligonucleotide region complementary to the 3’ end of A 1 , and a region complementary to either the 5’ end of oligonucleotide C or the 5’ end of A 1 . In some embodiments, D may be unable to undergo extension relative to A due to 3’ modification or through a mismatch between the 3’ end of D and the corresponding region of A 1 or C. 1 even if it is impossible to undergo extension relative to A.

[0134] In some embodiments, the kit may further comprise dNTPs, polymerase, and a buffer suitable for the initial amplification of the target polynucleotide sequence present in the sample. In some embodiments, the kit may further comprise a high-fidelity polymerase that incorporates dUTP, dUTP, and uracil-DNA N-glycosylase (UDG).

[0135] In some embodiments, the kit may further comprise a phosphatase or phosphohydrolase. In some embodiments, the kit may further comprise pyrophosphatase. The pyrophosphatase may be hot start.

[0136] In some embodiments, the kit may further comprise a protease. In some embodiments, the kit may further comprise one or more oligonucleotide-binding dyes or molecular probes.

[0137] In some embodiments, the kit may further comprise a plurality of As that are each selective for a different target sequence and each contain an identification region. 0 In some embodiments, the kit may further comprise an enzyme for forming DNA from an RNA template.

[0138] In some embodiments, the enzyme is reverse transcriptase. In some embodiments, one or more of the enzymes of the kit may be hot start.

[0139] In some embodiments, one or more of the enzymes in the kit may be heat stable. In some embodiments, the kit may further include suitable washing and buffering reagents.

[0140] In some embodiments, the amplification enzyme and pyrophosphate-adding enzyme in (e) are the same. In some embodiments, the amplification enzyme and pyrophosphate-adding enzyme are the same.

[0141] The kit may further include purification devices and reagents for isolating and / or purifying a portion of the polynucleotide according to the processes described herein. Suitable reagents are well known in the art and include, for example, gel filtration columns and wash buffers.

[0142] In some embodiments: (a) A single-stranded probe oligonucleotide A capable of forming a first intermediate with the target polynucleotide sequence, 0 wherein the intermediate is at least partially double-stranded, said probe oligonucleotide A 0 ; (b) A ligase; (c) A pyrophosphate-adding enzyme capable of digesting the first intermediate in the 3'-5' direction from the end of A to produce a partially digested strand A 0 ; 1 ; (d) A suitable buffer is provided in the kit.

[0143] In some embodiments, the kit may further include a source of pyrophosphate ions. In some embodiments, the kit may include suitable positive and negative controls.

[0144] In some embodiments of the kit, A 0The 5' end is resistant to 5'-3' exonuclease digestion, and the kit may further contain 5'-3' exonuclease.

[0145] In some embodiments, the kit may further contain dNTPs, a polymerase, and a buffer suitable for the initial amplification of the target polynucleotide sequence present in the sample. In some embodiments, the kit may further contain a high-fidelity polymerase that incorporates dUTP, dUTP, and uracil-DNA N-glycosylase (UDG).

[0146] In some embodiments, the kit may further contain a protease. In some embodiments, the kit may further contain a ligation probe oligonucleotide C.

[0147] In some embodiments, the kit may further contain a sprint oligonucleotide D. In some embodiments, the kit may further contain a ligation probe oligonucleotide C and a sprint oligonucleotide D.

[0148] In some embodiments of the kit, oligonucleotide C contains a 3' or internal modification that protects the oligonucleotide from 3'-5' exonuclease digestion. In some embodiments of the kit, D is A 1 an oligonucleotide region complementary to the 3' end of, and a region complementary to either the 5' end of oligonucleotide C or the 5' end of A 1 is included.

[0149] In some embodiments of the kit, D is unable to undergo extension relative to A, either by a 3' modification or through a mismatch between the 3' end of D and the corresponding region of A 1 or C. 1 is impossible.

[0150] In some embodiments, the kit comprises A 0 and further comprises at least one single-stranded primer oligonucleotide that is substantially complementary to a portion of A, an amplification enzyme, and dNTPs. In some embodiments, the kit further comprises one or more oligonucleotide-binding dyes or molecular probes.

[0151] In some embodiments, the kit further comprises a plurality of A, each of which is selective for a different target sequence and each of which comprises an identification region. 0 In some embodiments, the kit comprises: - A 1 two or more ligation chain reaction (LCR) probe oligonucleotides that are complementary to adjacent sequences on A, wherein when the probe successfully anneals to the 5' phosphate of one LCR probe, it is immediately adjacent to the 3' OH of the other LCR probe; and - one or more ligases

[0152] In some embodiments, the kit further comprises one or more polymerases. In some embodiments of the kit, one or more polymerases are the same as pyrophosphate-degrading enzymes.

[0153] In some embodiments, the kit comprises: - ligation probe oligonucleotide C; - splint oligonucleotide D wherein E has a 5' phosphate, and the 3' end of splint oligonucleotide D is complementary to the 5' of E and the 5' end of D is complementary to the 3' end of A 1 such that A and E can be ligated together to form A 2 1

[0154] In some embodiments, the kit comprises: - A hairpin oligonucleotide 1 (HO1) containing a fluorophore-quencher pair, wherein HO1 is complementary to A 2 and, when annealed to A 2 , the hairpin structure of HO1 opens and the fluorophore-quencher pair separates; said HO1; and - A hairpin oligonucleotide 2 (HO2) containing a fluorophore-quencher pair, wherein HO2 is complementary to the opened HO1 and, when annealed to HO1, the hairpin structure of HO2 opens and the fluorophore-quencher pair separates; said HO2 further comprises.

[0155] In some embodiments, the kit further comprises a plurality of HO1 and HO2. In some embodiments, the kit comprises: - An oligonucleotide A comprising a substrate arm, a partial catalytic core and a sensor arm; - An oligonucleotide B comprising a substrate arm, a partial catalytic core and a sensor arm; and - A substrate containing a fluorophore-quencher pair further comprises; wherein the sensor arms of oligonucleotides A and B are complementary to the adjacent region of A 2 such that in the presence of A, oligonucleotides A and B combine to form a catalytic multi-component nucleic acid enzyme (MNA enzyme). 2

[0156] In some embodiments, the kit further comprises a nucleic acid construct that is partially double-stranded, wherein: - One strand contains at least one RNA base, at least one fluorophore, and one region of this strand is complementary to the region of A 2 and this strand may be referred to as the "substrate" strand; and - The other strand contains at least one quencher, and one region of this strand is complementary to A 2 ​In the presence of, a substrate strand is complementary to a region adjacent to a region where a partially strand-formed nucleic acid construct forms substantially more double strands such that A 2 is complementary to the region of.

[0157] In some embodiments, the kit further comprises an enzyme for removing at least one RNA base. In some embodiments of the kit, the enzyme is uracil-DNA glycosylase (UDG), and the RNA base is uracil.

[0158] In some embodiments, the kit comprises: - an oligonucleotide complementary to the region of A containing the ligation site, the oligonucleotide comprising one or more fluorophores arranged such that the fluorescence thereof is quenched by proximity to each other or to one or more fluorescence quenchers relative to each other; 2 - a double-strand specific DNA digesting enzyme and further comprises, wherein, in the presence of A the fluorophore is separated from each other or from the corresponding quencher, and the labeled oligonucleotide is digested such that the fluorescence signal, and thus the presence of A 2 is detectable. 2

[0159] In some embodiments of the kit, the double-strand specific DNA digesting enzyme is an exonuclease. In some embodiments of the kit, the double-strand specific DNA digesting enzyme is a polymerase having proofreading activity.

[0160] ​In some aspects of the kit, the fluorophore is selected from dyes of the fluorescein family, carboxyrhodamine family, cyanine family, rhodamine family, polyhalofluorescein family dyes, hexachlorofluorescein family dyes, coumarin family dyes, oxazine family dyes, thiazine family dyes, squaraine family dyes, and chelated lanthanide family dyes.

[0161] In some aspects of the kit, the quencher is the trade name Black Hole TM , Eclipse TM . Dark, Qx1J, and Iowa Black TM selected from those available under.

[0162] In some aspects, the kit further comprises a phosphatase or a phosphohydrolase. In some aspects, the kit further comprises a pyrophosphatase.

[0163] In some aspects, the kit further comprises an enzyme for forming DNA from an RNA template. In some aspects of the kit, the enzyme is a reverse transcriptase.

[0164] In some aspects of the kit, one or more enzymes are hot start. In some aspects of the kit, one or more enzymes are thermostable.

[0165] In some aspects, the kit may further comprise appropriate washing and buffering reagents. In one aspect of the present invention: A device comprising at least a fluid path between a first region, a second region and a third region comprising: The first region comprises one or more wells, each well comprising: dNTP; At least one single-stranded primer oligonucleotide; An amplification enzyme for the first amplification of DNA present in a sample and; The second region contains one or more wells, each well being: A single-stranded probe oligonucleotide A capable of forming a first intermediate product with a target polynucleotide sequence 0 wherein the intermediate product is at least partially double-stranded, said probe oligonucleotide A 0 ; A 0 A pyrophosphate-adding enzyme capable of digesting the first intermediate product in the 3'-5' direction from the end of A to produce a partially digested strand A 1 is included; and and; The third region contains one or more wells, each well being: dNTP; A buffer; An amplification enzyme; A 2 or a part thereof, or means for detecting a signal derived from multiple copies of A 2 or multiple copies of a part thereof are included; and and; The wells in the second region or the wells in the third region further contain at least one single-stranded primer oligonucleotide substantially complementary to a part of A 0 to provide said device. In some embodiments, the means for detecting a signal is located within one or more wells of the third region.

[0166] In some embodiments, the means for detecting a signal is located within the third region of the device. In some embodiments, the means for detecting a signal is located within an adjacent region of the device.

[0167] In some embodiments, the means for detecting a signal is located within an adjacent region of the device. In some embodiments, the dNTPs of each well in the first region may be dUTP, dGTP, dATP, and dCTP, and each well may further include a high-fidelity polymerase that incorporates dUTP and uracil-DNA N-glycosylase (UDG).

[0168] In some embodiments, the dNTPs of each well in the third region may be dUTP, dGTP, dATP, and dCTP, and each well may further include a high-fidelity polymerase that incorporates dUTP and uracil-DNA N-glycosylase (UDG).

[0169] In some embodiments, each well in the second region may further include a source of pyrophosphate ions. In some embodiments, A 0 The 5' end of may be rendered resistant to 5'-3' exonuclease digestion, and the wells in the second region may further include 5'-3' exonuclease.

[0170] In some embodiments, each well in the second or third region may further include ligase and ligation probe oligonucleotide C or splint oligonucleotide D.

[0171] Ligation probe C may include a 3' or internal modification that protects the probe from 3'-5' exonuclease digestion. Splint oligonucleotide D is A 1 An oligonucleotide region complementary to the 3' end of, and a region complementary to either the 5' end of oligonucleotide C or the 5' end of A 1 May include a region complementary to either.

[0172] D may be unable to undergo extension relative to A either by 3' modification or through a mismatch between the 3' end of D and the corresponding region of A 1 Or C. 1 May be impossible. In some embodiments, the dNTPs may be hot start, and each well of the second region may further contain a phosphatase or phosphohydrolase.

[0173] In some embodiments, each well of the second region may further contain pyrophosphatase. In some embodiments, the pyrophosphatase may be hot start.

[0174] In some embodiments, each well of the third region may further contain one or more oligonucleotide-binding dyes or molecular probes. In some embodiments, each well of the second region contains at least one or more different As that are selective for a target sequence containing an identification region. 0 may include.

[0175] In some embodiments, the amplification enzyme and pyrophosphate-degrading enzyme in the second region may be the same. In some embodiments, there may be a fourth region containing one or more wells, where each well may contain a protease, and the fourth region may be located between the first and second regions.

[0176] In some embodiments, the second and third regions of the device are such that the wells of the second region are: dNTP; buffer; amplification enzyme; and A 1 or a part thereof, or means for detecting a signal derived from multiple copies of A 1 or multiple copies of a part thereof may be further combined to include.

[0177] In some embodiments, the means for detecting the signal is located within one or more wells of the second region. In some embodiments, the means for detecting the signal is located within a second region of the device.

[0178] In some embodiments, the means for detecting the signal is located within an adjacent region of the device. In some embodiments: A device comprising a fluid path between a first region and a second region, the first region comprising one or more wells, the one or more wells being: A single-stranded probe oligonucleotide A capable of forming a target polynucleotide sequence and a first intermediate product 0 wherein the intermediate product is at least partially double-stranded, the oligonucleotide A 0 ; A 0 Pyrophosphatase capable of digesting the first intermediate product in the 3'-5' direction from the end of A to produce a partially digested strand A 1 ; and A 1 One or more ligases capable of ligating to produce oligonucleotide A 2 Including, The second region includes one or more wells, Providing the device.

[0179] In some embodiments, one or more wells of the first region may further include a source of ions to drive the pyrophosphorolysis reaction in the forward direction. In some embodiments, the ion is a pyrophosphate ion.

[0180] In some embodiments, the 5' end of A 0 is resistant to 5'-3' exonuclease digestion, and the wells of the first region further include 5'-3' exonuclease. In some embodiments, the device may further include a third region including one or more wells that are connected to the first region by a fluid path, and one or more wells of the third region may be: dNTP; single-stranded primer oligonucleotide; and amplification enzyme comprising.

[0181] In some embodiments, the dNTP of the third region may be dUTP, dGTP, dCTP, and dATP; the amplification enzyme may be a high-fidelity polymerase that incorporates dUTP; and one or more wells of the third region may further include uracil-DNA N-glycosylase.

[0182] In some embodiments, the device may further include a fourth region located between the first and third regions and including one or more wells, and one or more wells may include protease.

[0183] In some embodiments, one or more wells of the first or second region may further include ligase, and a ligation probe oligonucleotide C that is complementary to the region of A 0 may be further included.

[0184] In some embodiments, one or more wells of the first or second region may further include ligase, and a splint oligonucleotide D that is complementary to the region of A 0 may be further included.

[0185] In some embodiments, one or more wells of the first or second region may further include ligase, splint oligonucleotide D, and ligation probe oligonucleotide C.

[0186] In some embodiments, the linking probe oligonucleotide C may include a 3' or internal modification that protects the oligonucleotide from 3'-5' exonuclease digestion.

[0187] In some embodiments, D is an oligonucleotide region complementary to the 3' end of A 1 and may include a region complementary to either the 5' end of oligonucleotide C or the 5' end of A 1 or the 5' end of A.

[0188] In some embodiments, D may be unable to undergo extension relative to A either by a 3' modification or through a mismatch between the 3' end of D and the corresponding region of A 1 or C. 1

[0189] In some embodiments, one or more wells of the first region are each selective for a different target sequence and each contain at least one or more different A 0 that each contain an identification region.

[0190] In some embodiments, the wells of the second region contain: dNTP; a buffer; an amplification enzyme; A 1 or a portion thereof, or means for detecting a signal derived from multiple copies of A 1 or a portion of multiple copies thereof.

[0191] In some embodiments, the means for detecting a signal is located within one or more wells of the second region. In some embodiments, the means for detecting a signal is located within the second region of the device.

[0192] In some embodiments, the means for detecting a signal is located within an adjacent region of the device. In some embodiments, one or more wells of the second region may further comprise one or more oligonucleotide-binding dyes or molecular probes.

[0193] In some embodiments, the amplification enzyme and pyrophosphate-degrading enzyme of the device are the same. In some embodiments, the wells of the second region are: -A 1 Two or more ligation chain reaction (LCR) probe oligonucleotides complementary to the adjacent sequence above, wherein when the probe successfully anneals to the 5'-phosphate of one LCR probe, it is immediately adjacent to the 3'-OH of the other LCR probe, said probe oligonucleotide; and -One or more ligases and further comprise.

[0194] In some embodiments, the wells of the second region are: -Ligation probe oligonucleotide C; -Sprint oligonucleotide D may further comprise; wherein C has a 5'-phosphate and A 1 and C are ligated together to form oligonucleotide A 2 such that the 3' end of the sprint oligonucleotide D is complementary to the 5' of C, and the 5' end of D is complementary to the 3' end of A 1 is complementary.

[0195] In some embodiments, the wells of the second region are: -Hairpin oligonucleotide 1 (HO1) containing a fluorophore-quencher pair, wherein HO1 is complementary to A 2 and when annealed to A 2 the hairpin structure of HO1 opens and the fluorophore-quencher pair separates, said HO1; and -A hairpin oligonucleotide 2 (HO2) containing a fluorophore-quencher pair, wherein HO2 is complementary to the opened HO1, and when annealed to HO1, the hairpin structure of HO2 opens and the fluorophore-quencher pair separates, said HO2 may further be included.

[0196] In some embodiments, the wells of the second region may further include a plurality of HO1 and HO2. In some embodiments, the wells of the second region are: - An oligonucleotide A comprising a substrate arm, a partial catalytic core, and a sensor arm; - An oligonucleotide B comprising a substrate arm, a partial catalytic core, and a sensor arm; and - A substrate containing a fluorophore-quencher pair may further be included; wherein the sensor arms of oligonucleotides A and B are adjacent to A 2 such that in the presence of A, oligonucleotides A and B combine to form a catalytic multi-component nucleic acid enzyme (MNA enzyme). 2 are complementary to the adjacent region of A.

[0197] In some embodiments, the wells of the second region may further include a nucleic acid construct that is partially double-stranded, where: - One strand contains at least one RNA base, at least one fluorophore, and one region of this strand is complementary to the region of A 2 and this strand may be referred to as the "substrate" strand; - The other strand contains at least one quencher, and one region of this strand is adjacent to the region of A 2 such that in the presence of A, the nucleic acid construct that forms a partial strand forms a substantially more double-stranded structure, 2 is complementary to the region of A that is complementary to the substrate strand.

[0198] In some embodiments, the wells of the second region may further contain an enzyme for removing at least one RNA base. In some embodiments, the enzyme is uracil-DNA glycosylase (UDG), and the RNA base is uracil.

[0199] In some embodiments, one or more wells of the second region are: A oligonucleotide complementary to the region of A 2 containing one or more fluorophores arranged such that their fluorescence is quenched either by proximity to each other or to one or more fluorescence quenchers, said oligonucleotide; double-strand specific DNA digesting enzyme may further contain; wherein, in the presence of A 2 the fluorophores are separated from each other or from the corresponding quenchers, and the labeled oligonucleotide is digested such that the fluorescence signal, and thus the presence of A 2 is detectable.

[0200] In some embodiments, the double-strand specific DNA digesting enzyme is an exonuclease. In some embodiments, the double-strand specific DNA digesting enzyme is a polymerase with proofreading activity.

[0201] In some embodiments, the fluorophore is selected from dyes of the fluorescein family, carboxyrhodamine family, cyanine family, rhodamine family, polyhalofluorescein family dyes, hexachlorofluorescein family dyes, coumarin family dyes, oxazine family dyes, thiazine family dyes, squaraine family dyes, and chelated lanthanide family dyes.

[0202] In some embodiments, the fluorophore of the device may be selected from any commercially available dye. In some embodiments, the quencher of the device is selected from those available under the trade names Black Hole TM , Eclipse TM Dark, Qx1J, Iowa Black TM , ZEN and / or TAO.

[0203] In some embodiments, the quencher of the device may be selected from any commercially available quencher. In some embodiments, one or more wells of the second region may further contain one or more partially double-stranded DNA constructs, each construct containing one or more fluorophores and one or more quenchers. In some embodiments, when the construct is partially double-stranded, the one or more fluorophores and the one or more quenchers are positioned sufficiently close so that sufficient quenching of the one or more fluorophores occurs.

[0204] In some embodiments, the construct is a single-stranded DNA having self-complementary regions that fold upon themselves. In some embodiments, the construct contains one primer of a primer pair.

[0205] In some embodiments, one or more wells of the second region may further contain the other primer of the primer pair. In some embodiments, a portion of the single-stranded section of the construct hybridizes to A 2 and is extended by DNA polymerase against A 2 . In some embodiments, the other primer of the primer pair then hybridizes to the extended construct to display A 2 . This primer is then extended against the construct to replace the self-complementary region. Thus, the one or more fluorophores and the one or more dyes are sufficiently separated for a fluorescent signal to be detected, A2 indicates the presence of.

[0206] In such embodiments, the construct may be known as a Sunrise primer. In some embodiments, the construct comprises two separate DNA strands. In some embodiments, a portion of a single-stranded section of the construct hybridizes to A 2 and is extended by DNA polymerase to A 2 In some embodiments, the other primer of the primer pair then hybridizes to the extended construct to display A 2 This primer is then extended in the direction of the double-stranded section with respect to the construct, displacing the shorter DNA strand, and thus one or more fluorophores and one or more dyes are sufficiently separated for a fluorescence signal to be detected, indicating the presence of A 2 indicates the presence of.

[0207] In such embodiments, the construct may be known as a Molecular Zipper. One of ordinary skill in the art will recognize that with respect to both the Sunrise primer and the Molecular Zipper, one or more fluorophores and one or more quencher pairs can be located at various positions within each respective construct. The important feature is that each pair is located sufficiently close to each other such that in the absence of A 2 i.e., when extension and strand displacement do not occur, no fluorescence signal is emitted.

[0208] In some embodiments, one or more wells of one or more regions may further comprise pyrophosphatase. In some embodiments, one or more wells of one or more regions of the device may further comprise phosphatase or phosphohydrolase.

[0209] In some embodiments, one or more wells in the first region of the device may further contain an enzyme for forming DNA from an RNA template. In some embodiments, the enzyme is a reverse transcriptase.

[0210] In some embodiments, one or more enzymes present in the device are hot start. In some embodiments, one or more enzymes present in the device are thermostable.

[0211] In some embodiments, the first and second regions of the device are combined. In some embodiments, one or more fluid pathways are located between one or more wells of the region and / or between one or more regions of the device.

[0212] In some embodiments, the first region may be fluidly connected to the sample container through a fluid interface. In some embodiments, one or more heating and / or cooling elements may be present in one or more regions of the device.

[0213] In some embodiments, heating and / or cooling may be applied to one or more regions of the device. In some embodiments, each region of the device may independently contain at least 100 or 200 wells.

[0214] In some embodiments, each region of the device may independently contain between about 100 and 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or more wells. The wells may be of any shape and their positions may be arranged in any format or pattern on the support.

[0215] In some embodiments, the well support may be constructed from metal (e.g., by way of non-limiting example, gold, platinum, or nickel alloy), ceramic, glass, or other PCR-compatible polymeric material, or a composite material. The well support includes a plurality of wells.

[0216] In some embodiments, the wells may be formed as blind holes or through holes in the well support. The wells may be generated within the well support by, for example, laser drilling (e.g., excimer or solid-state lasers), ultrasonic embossing, hot embossing lithography, electroforming of nickel molds, injection molding, and injection compression molding.

[0217] In some embodiments, the individual well volumes may range from 0.1 to 1500 nl. In one embodiment, it is from 0.5 to 50 nL. Each well may have a volume of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nL.

[0218] In some embodiments, the well dimensions may have any shape, such as circular, elliptical, square, rectangular, oval, hexagonal, octagonal, conical, and other shapes well-known to those skilled in the art. In some embodiments, the well shape may have a variety of cross-sectional areas along an axis. For example, a square hole may gradually decrease in size from a first size to a second size that is a portion of the first size.

[0219] In some embodiments, the well dimensions may be a square with approximately equal diameter and depth. In some embodiments, the walls defining the well may be non-parallel.

[0220] In some embodiments, the walls defining the well may converge to a point. The well dimensions may be obtained from the total volume capacity of the well support. In some embodiments, the depth of the well may range from 25 μm to 1000 μm.

[0221] In some embodiments, the well may have a depth of 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μm.

[0222] In some embodiments, the well diameter may range from about 25 μm to about 500 μm. In some embodiments, the well may have a width of 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 μm.

[0223] In some embodiments, a portion of one or more regions of the device may be modified to promote or prevent fluid adhesion. The surface defining the well may be coated with a hydrophilic substance (or modified to be hydrophilic) and thus promote fluid retention.

[0224] In some embodiments, a portion of one or more regions of the device may be coated with a hydrophobic substance (or modified to be hydrophobic) and thus prevent fluid retention. One of ordinary skill in the art will understand that other surface treatments may be performed to promote the drainage of excess fluid such that the fluid is preferably retained within the well but not on the upper surface.

[0225] In some embodiments, the wells of the well support may be patterned such that they have a simple geometric pattern of aligned rows and columns, or a pattern arranged diagonally or hexagonally. In one embodiment, the wells of the well support may be patterned such that they have a complex geometric pattern, such as a chaotic pattern or an isodimetric design pattern.

[0226] In some embodiments, the wells may be geometrically separated from each other and / or may feature a large depth-to-width ratio to assist in preventing cross-contamination of reagents. In some embodiments, the device may include one or more auxiliary regions that can be used to provide a process fluid, such as oil or another chemical solution, to one or more of the regions of the device. Such auxiliary regions may be fluidly connected to one or more of the regions of the device through one or more membranes, valves, and / or pressure-cuttable substrates (i.e., materials that are broken when subjected to a predetermined amount of pressure from the fluid within the auxiliary region or within an adjacent portion of the fluid path), such as metal foils or thin films.

[0227] In some embodiments, the fluid path of the device may include torturous portions. The torturous path between the inlet passage of the fluid path and one or more of the regions of the device may be useful for controlling and handling the fluid process. The torturous path may also assist in reducing the formation of gas bubbles that can interfere with the flow of oil through the fluid path.

[0228] In some embodiments, the device may further include a gas-permeable membrane that allows gas to be discharged from wells in one or more regions of the device while not allowing fluid to pass through. The gas-permeable membrane may be adhered to the well support of the device by a gas-permeable adhesive. In one embodiment, the membrane may be constructed from polydimethylsiloxane (PDMS) and have a thickness in the range of 20 - 1000 μm. In some embodiments, the membrane may have a thickness in the range of 100 - 200 μm.

[0229] In some embodiments, all or part of the well support may contain a conductive metal portion (such as gold) to allow heat transfer from the metal to the wells. In one embodiment, the inner surface of the well may be coated with metal to allow heat transfer.

[0230] In some embodiments, after a suitable reagent fills the wells in one or more regions of the device, an insulating oil or a thermally conductive liquid may be applied to the device to prevent crosstalk.

[0231] In some embodiments, the wells in one or more regions of the device may be shaped to taper from a larger diameter to a smaller diameter, like a cone. Conical wells with sloped walls allow for the use of non-contact deposition methods (such as inkjet) for reagents. The conical shape has also been found to assist in drying and, when a gas-permeable membrane is present, prevent bubbles and leakage.

[0232] In some embodiments, wells in one or more regions of the device may be filled by advancing a sample fluid (e.g., by pressure) along a fluid path of the device. As the fluid passes over the wells in one or more regions of the device, each well is filled with fluid, which is then retained within the well through surface tension. As previously described, portions of the well support of the device may be coated with hydrophilic / hydrophobic materials, as desired, to facilitate complete and uniform filling of the wells as the sample fluid passes through.

[0233] In some embodiments, the wells in one or more regions of the device may be "capped" with oil after filling. This may then assist in reducing evaporation when the well support is subjected to thermal cycling. In one embodiment, after oil capping, an aqueous solution may be filled into one or more regions of the device to improve thermal conductivity.

[0234] In some embodiments, a steady aqueous solution may be pressurized within one or more regions of the device to halt the movement of fluid and any bubbles. In some embodiments, an oil, such as mineral oil, may be used for isolation of the wells in one or more regions of the device and to provide thermal conductivity. However, any heat transfer fluid, such as a fluorinated liquid (e.g., 3M FC-40), may be used. References to oil in the present disclosure are to be understood to include such alternatives as would be recognized by one of ordinary skill in the art as applicable.

[0235] In some embodiments, the device may further include one or more sensor assemblies. In some embodiments, one or more sensor assemblies may include a charge-coupled device (CCD) / complementary metal-oxide semiconductor (CMOS) detection device coupled to an optical fiber faceplate (FOFP). Filters may be disposed in layers on top of the FOPF and may be disposed on or adjacent to the well support. In one embodiment, the filter may be disposed (bonded) directly in layers on top of the top of the CCD with the FOPF on top.

[0236] In some embodiments, a hydrating liquid, such as distilled water, may be heated within one of the first regions or auxiliary regions so that one or more regions of the device have up to 100% humidity or at least enough humidity to prevent evaporation during at least thermal cycling.

[0237] In some embodiments, after the device is filled, the well support may be heated by an external device in thermal contact with the device to perform thermal cycling for PCR.

[0238] In some embodiments, non-contact methods of heating, such as radio frequency identification (RFID), Curie point, inductive, or microwave heating, may be used. These and other non-contact methods of heating will be well known to those skilled in the art. During thermal cycling, the device may be monitored for chemical reactions through the sensor arrangements described above.

[0239] In some embodiments, reagents deposited in one or more of one or more wells of a region of the device are deposited in a predetermined arrangement. In some embodiments: providing a sample fluid to a fluid path of the device, where the device includes at least a fluid path between a first region, a second region, and a third region, the first, second, and third regions each independently including one or more wells; Fill the second region with the amplified fluid from the first region such that one or more wells in the second region are coated with the amplified fluid; Drain the amplified fluid from the second region such that one or more wells remain at least somewhat wet with the amplified fluid; Fill the third region with the fluid drained from the second region such that one or more wells in the third region are coated with this fluid; and Drain fluid from the third chamber such that one or more wells remain at least somewhat wet with this fluid Provide a method comprising the steps.

[0240] In some embodiments of the method, the fluid path may be valve - less. In some embodiments of the method, the drained second region may be filled with a hydrophobic substance.

[0241] In some embodiments of the method, the drained third region may be filled with a hydrophobic substance. In some embodiments of the method, the hydrophobic substance may be supplied from an oil chamber in fluid communication with the second and third regions.

[0242] In some embodiments of the method, the sample fluid may be sent along the fluid path in a meandering fashion. In some embodiments, the method may further comprise applying heating and cooling cycles to one or more of the first, second, or third regions.

[0243] Various further aspects and embodiments of the invention will be apparent to those skilled in the art from the perspective of this disclosure. As used herein, "and / or" shall be construed as a specific disclosure of each of two recited features or components, with or without the other. For example, "A and / or B" shall be construed as (i) A, (ii) B, and (iii) each specific disclosure of A and B, in the same way as each is individually shown herein.

[0244] Unless otherwise indicated, the descriptions and definitions of the features set forth above are not limited to any particular aspect or embodiment of the present invention and apply equally to all aspects and embodiments described.

[0245] Those skilled in the art will further recognize that although the present invention has been described by way of example with reference to several aspects, the present invention is not limited to the disclosed aspects and other aspects can be constructed without departing from the scope of the present invention as defined in the appended claims.

Examples

[0246] Example 1 - Simplified Protocol For this purpose and the following sections, aspects of the present invention are illustrated and referred to as Protocols 1 - 5, respectively.

[0247] Figure 1 provides an overview of different protocols. The following table shows an overview of the time taken to execute each protocol:

[0248]

Table 1

[0249] In one aspect, TIPP is not present in any one of the protocols, methods, kits, and / or devices of the present invention. In one aspect, 5'-3' exonuclease is not present in any one of the protocols, methods, kits, and / or devices of the present invention.

[0250] The following table shows an overview of the enzymes that may be used in each protocol:

[0251]

Table 2

[0252] In one embodiment, the presence of pyrophosphatase is optional. In one embodiment, the presence of 5'-3' exonuclease is optional. In one embodiment, the presence of UDG is optional.

[0253]

Table 3

[0254] As can be seen, the inventors have reduced the total number of enzymes required and thus the cost and complexity of the method. Surprisingly, the inventors have found that when moving the 5'-3' exonuclease addition from the pre-amplification step of the protocol to the pyrophosphate decomposition / ligation step (as in Protocols 3-5), a higher fluorescence signal (corresponding to the detection of a specific target analyte sequence) is generated, as shown in Figure 2.

[0255] Example 2: Pyrophosphate decomposition (PPL) enzyme The inventors tested the method of Protocol 3 of the present invention using a range of different PPL enzymes, and the results can be seen in Figure 3. Figure 3(A) shows the detection of 1% MAF T790M using Mako, Klenow, and Bsu. Figure 3(B) shows the detection of 0.5% MAF T790M using Bst LF at a range of different PPi concentrations. The inventors tested the method of Protocol 4 of the present invention using a range of different PPL enzymes, and the results can be seen in Figure 4.

[0256] Example 3: Protocol 1 vs Protocol 4 The inventors detected exon 19 del_6223 at 0.5%, 0.10% and 0.05% MAF using both Protocol 1 and Protocol 4, and the results can be seen in Figure 5. As can be seen, the fluorescence peak is larger when Protocol 4 is used.

[0257] Example 4: Protocol 4 - Sensitivity The inventors detected the EGFR exon 20 T790M mutation at 0.10%, 0.50% and 1% MAF according to Protocol 4, as shown in Figure 6.

[0258] Example 5: Protocol 4 - Is an exonuclease digestion step necessary during RCA? The inventors showed that the exonuclease digestion step during RCA is not essential. However, the detectable signal is detected later during RCA when the exonuclease digestion step is omitted. Figure 7 shows the detection of EGFR exon 20 T790M at 1% MAF with and without the presence of exonuclease during the RCA process.

[0259] Example 6: Protocol 4 - PPL:RCA mixing ratio The inventors investigated the effect of the PPL:RCA mixing ratio on the signal intensity detected for 0.5% MAF EGFR exon 20 T790M, and the results are shown in Figure 8. As can be seen, the 1:2 PPL:RCA mixing ratio results in the lowest signal intensity, which is at the earliest time point. The maximum signal intensity is seen for the 1:8 PPL:RCA mixture at the latest time point of the reaction.

[0260] Example 7: Protocol 4 - Dye selection The inventors investigated whether the dyes used in RCA could be optimized. Figure 9 shows the results of comparative experiments performed according to Protocol 4 using SybrGreenI (50 °C and 60 °C) and Syto82 (50 °C and 60 °C). The Syto82 dye allows RCA to be performed at a lower temperature of 50 °C, while SybrGreenI requires a higher temperature of 60 °C. For Protocol 5, which omits the addition of proteinase K to the reaction mixture, a lower RCA temperature is required. A temperature higher than 50 °C is required for detection using the method of the present invention in order for the amplification enzyme used to prepare at least one single-stranded analyte of nucleic acid containing the target polynucleotide region to function. The use of SybrGreenI requires a reaction temperature of 60 °C and thus proteinase K must be added at some point in the method to inactivate the amplification enzyme prior to RCA.

[0261] A lower RCA temperature may enable the method of the present invention to be performed in a plate reader instead of qPCR. The reaction using Syto82 is faster as can be seen in Figure 9, and the total fluorescence is lower for Syto82, which can be alleviated by using a higher concentration of Syto82 dye.

[0262] Example 8: Protocol 4 - BST L.F. vs BST 2.0WS To detect the 0.5% MAF EGFR exon 20 T790M mutation, the inventors examined the use of two different enzymes, BST L.F and BST 2.0WS, for RCA according to protocol 4. The results are shown in Figure 10, from which it can be seen that the reaction is fastest with BST 2.0WS. BST 2.0 WS is designed to incorporate dUTP, which aids in the reaction rate. There is only a negligible difference in the total signal intensity achieved between BST L.F. and BST 2.0WS. According to the description provided by New England Biolabs (NEB), BST 2.0WS should be more stable and should be active only at temperatures higher than 45°C.

[0263] Example 9: Influence of PPL enzymes on signal detection The inventors examined the influence of different PPL enzymes on the RCA reaction at different PPL:RCA reaction mixing ratios. The results can be seen in Figure 11(A) 1:4 PPL:RCA and Figure 11(B) 1:8 PPL:RCA. Except for BST, all PPL enzymes affect the RCA reaction at a 1:4 PPL:RCA ratio. At a 1:8 PPL:RCA ratio, BST and Klenow do not affect the RCA reaction.

[0264] Example 10: Pyrophosphate decomposition and ligation specificity for single-base mismatches A single-stranded first oligonucleotide (SEQ ID NO: 1) having the following nucleotide sequence was prepared:

[0265]

Chemical formula

[0266] A single-stranded ligated oligonucleotide 2 (SEQ ID NO: 2) having the following nucleotide sequence was prepared:

[0267]

Chemical formula

[0268] In the formula, A, C, G, and T represent nucleotides having the relevant characteristic nucleobases of DNA, / 5Phos / represents a 5' end phosphate, * represents a phosphorothioate bond.

[0269] A set of single-stranded oligonucleotides 3 to 4 (SEQ ID NOs: 3 to 4) having the following nucleotide sequences were also prepared in the 5' to 3' direction:

[0270]

Chemical formula

[0271] In the formula, oligonucleotide 3 contains a 17-base region complementary to the 17 bases at the 3' end of oligonucleotide 1, and oligonucleotide 4 contains the same region with a single-base mismatch at the 3rd position. SEQ ID NOs: 3 and 4 are each a part of the human EGFR gene with / without the C797S mutation.

[0272] Next, a first reaction mixture was prepared having a composition corresponding to that obtained from the following formulation: 0.5 μL of 20x buffer pH 7.0 0.25 μL of 5x buffer pH 8.0 0.25 μL of 5x HF buffer 0.2 μL of oligonucleotide 1, 1000 nM 0.3 μL of oligonucleotide 2, 1000 nM 1 μL of oligonucleotide 2 (500 nM) or a mixture of oligonucleotides 2 and 3 (500 and 0.5 nM respectively) 0.3 U of Klenow fragment exo- (NEB) 0.01 μL of inorganic pyrophosphate, 10 mM 0.0132 U of apyrase (e.g., NEB) 1 U of Escherichia coli (E. coli) DNA ligase (e.g., NEB) Water up to 10 μL Here, the 20x buffer contained the following mixture: 200 μL Tris acetate, 1 M, pH 7.0 342.5 μL magnesium acetate aqueous solution, 1 M 120 μL potassium acetate aqueous solution, 5 M 50 μL Triton X-100 surfactant (10%) Water up to 1 mL Here, the 5x buffer contained the following mixture: 50 μL Trizma acetate, 1 M, pH 8.0 25 μL magnesium acetate aqueous solution, 1 M 25 μL potassium acetate aqueous solution, 5 M 50 μL Triton X-100 surfactant (10%) Water up to 1 mL Then, by incubating the mixture at 45 °C for 15 minutes, depyrophosphorylation of oligonucleotide 1 and subsequent cyclization through ligation were performed, and the resulting product mixture was used in the amplification reaction (Example 11).

[0273] Example 11: Amplification of Cyclized Probe A pair of single-stranded oligonucleotide primers 1 (SEQ ID NO: 5) and 2 (SEQ ID NO: 6) having the following nucleotide sequences was prepared:

[0274]

Chemical formula

[0275] Wherein A, C, G, and T represent nucleotides having the relevant characteristic nucleobases of DNA. Then, a second reaction mixture having a composition corresponding to that obtained from the following formulation was prepared: 3 μL 10x Thermopol buffer 3.2 U BST 2.0 WS 0.32 μL oligonucleotide 1, 10 μM 0.32 μL oligonucleotide 2, 10 μM 1.125 μL Syto82, 30 μM 0.165 U inorganic pyrophosphatase 1.2 μL dNTP mixture, 10 mM 1.25 μL of the reaction mixture of Example 10 Water up to 11.25 μL Here, the 10x Thermopol buffer contained the following mixture: 200 μL Tris-HCl, pH = 8.8, 1 M 100 μL (NH4)2SO4, 1 M 100 μL mM KCl, 1 M 20 mM MgSO4, 1 M 10 μL Triton® X-100, 10% Water up to 1 mL The reaction mixture was then incubated at 50 °C for 40 minutes, and the resulting reaction products were analyzed by real-time fluorescence. The results are shown in Figure 12.

[0276] From this analysis, when both oligonucleotides 3 and 4 were present, the fluorescence signal appeared more rapidly during the reaction, indicating that the pyrophosphate decomposition and ligation of oligonucleotide 3 occurred in the first reaction mixture.

[0277] Example 12: Multicolor Detection Using Sunrise Primers 1. Target Oligo Dilution The WT oligo dilution was composed of the following components: 0.5x A7 buffer 0.5x Phusion U buffer 200 nM WT oligonucleotide (SEQ ID NO: 7) Total volume: 5 μL T790M and C797S 1% AF Mutant Oligo Mixture: 0.5x A7 buffer 0.5x Phusion U buffer 100 nM WT oligonucleotide (SEQ ID NO: 7) 2 nM T790M oligonucleotide (SEQ ID NO: 8) 2 nM C797S_2389 oligo (SEQ ID NO: 9) Total volume: 5 μL

[0278]

Chemical formula

[0279] 1xA7 composition Tris acetate pH = 8.0, 10 mM Potassium acetate 25 mM Magnesium acetate 5 mM Triton-X 0.01% PhusionU buffer The composition of PhusionU buffer is not publicly available.

[0280] 2. PPL The following corresponding mixtures were prepared: 1X BFF1 37.5 U / mL Mako DNA polymerase (3’→5’ exo-) 100 U / mL E. coli ligase 1.2 U / mL Apyrase 0.6 mM PPi 20 nM T790M probe 20 nM C797S_2389 probe 30 nM T790M sprint oligonucleotide 30 nM C797S_2389 sprint oligonucleotide 5 μL of the WT or 1% AF mutant dilution of point 1 Total volume 10 μL Then, this mixture was incubated at 41 °C for 30 minutes.

[0281] 1xBFF1 composition Tris acetate pH = 7.0, 10 mM Potassium acetate 30 mM Magnesium acetate 17.125 mM Triton-X 0.01%

[0282] [Chemical]

[0283] In the formula, * represents a phosphorothioate bond.

[0284] 3. TIPP The following corresponding mixture was prepared: 1 x A7 66.6 U / mL TIPP 10 μL of the mixture from Point 2 Total volume 20 μL Then, this mixture was incubated at 25°C for 5 minutes and at 95°C for 5 minutes.

[0285] 4. Ligation The following corresponding mixture was prepared: 1 x A7 100 U / mL E. coli ligase 20 μL of the mixture from Point 3 10 nM T790M sprint oligonucleotide 10 nM C797S_2389 sprint oligonucleotide Total volume 30 μL Then, this mixture was incubated at 37°C for 10 minutes and at 95°C for 10 minutes.

[0286] [Chemical]

[0287] 5. Exonuclease treatment The following corresponding mixture was prepared: 1 x A7 100 U / mL E. coli ligase 30 μL of the mixture from Point 4 625 U / mL exonuclease III 62.5 U / mL T5 exonuclease Total volume 40 μL Next, this mixture was incubated at 30 °C for 5 minutes and then at 95 °C for 5 minutes.

[0288] 6.RCA The following corresponding mixtures were prepared: 1x Thermopol buffer (53.2 mM Tris-HCl, 26.6 mM (NH 4 ) 2 SO 4 , 26.6 mM KCl, 5.32 mM MgSO 4 , 0.266% Triton® X-100, pH 8.8) 0.2 μM primer mixture 1 0.4 μM reverse primer 533.3 U / mL BST L.F. 0.4 mM dNTP 10 μL of the reaction mixture from point 5 Total volume 15 μL

[0289]

Chemical formula

[0290] Next, the mixture was incubated at 60 °C for 90 minutes. Fluorescence measurements were taken every minute. Cq was obtained based on the automatic threshold provided by the Bio-rad device. This result can be seen in Figure 13.

[0291] Example 13: Multicolor Detection Using Molecular Zipper 1. Target Oligo Dilution The WT oligo dilution consists of the following components: 0.5x A7 buffer 0.5x Q5U buffer 100 nM WT oligonucleotide (SEQ ID NO: 17) Total volume: 1.25 μL G719X_6239, G719X_6252, G719X_6253 0.5% AF mutant oligonucleotide mixture: 0.5x A7 buffer 0.5x Q5U Buffer 100 nM WT oligonucleotide (SEQ ID NO: 17) 0.5 nM G719X_6239 oligonucleotide (SEQ ID NO: 18) 0.5 nM G719X_6252 oligonucleotide (SEQ ID NO: 19) 0.5 nM G719X_6253 oligonucleotide (SEQ ID NO: 20) Total volume: 1.25 μL

[0292]

Chem.

[0293] 1xA7 composition 10 mM Tris acetate, pH = 8.0 25 mM potassium acetate 5 mM magnesium acetate 0.01% Triton-X Q5U buffer The Q5U buffer composition is not publicly available.

[0294] 2. Pyrophosphate decomposition (PPL) and ligation The following corresponding mixtures were prepared: 1X BFF1 10 U / mL Klenow (exo-) 100 U / mL E. coli ligase 1.2 U / mL apyrase 100 U / mL lambda exonuclease 0.25 mM PPi 6.6 nM G719X_6239 probe oligonucleotide (SEQ ID NO: 21) 6.6 nM G719X_6252 probe oligonucleotide (SEQ ID NO: 22) 6.6 nM G719X_6253 probe oligonucleotide (SEQ ID NO: 23) 30 nM sprint oligonucleotide (SEQ ID NO: 24)

[0295] 1.25 μL of the mixture from Point 2 Total volume 10 μL This mixture was then incubated at 45 °C for 15 minutes.

[0296] 1xBFF1 composition 10 mM Tris acetate pH = 7.0 30 mM potassium acetate 17.125 mM magnesium acetate 0.01% Triton-X

[0297]

Chem.

[0298] wherein * represents a phosphorothioate bond.

[0299] 3. Detection - RCA The following corresponding mixtures were prepared: 2.66x Thermopol buffer (53.2 mM Tris-HCl, 26.6 mM (NH 4 ) 2 SO 4 4, 26.6 mM KCl, 5.32 mM MgSO 4 4, 0.266% Triton® X-100, pH 8.8) 0.28 μM dye primer mixture 1 0.56 μM quencher primer 1 0.28 μM quencher primer 2 0.84 μM reverse primer 568.8 U / mL BST 2.0 WarmStart 14.67 U / mL TIPP 1.06 mM dNTP 1.25 μL of the reaction mixture from Point 2 Total volume 11.25 μL Dye primer mixture 1 consisted of the following

[0300]

Chem.

[0301] Subsequently, the mixture was incubated at 58 °C for 150 minutes. Fluorescence measurements were taken every minute. The results can be seen in Figure 14.

[0302] Example 14: Pyrophosphate cleavage and ligation to a target 1. Preparation of oligonucleotide dilution Dilutions of oligonucleotides were prepared in 0.5 x A7 and 0.5 x Q5 buffers: WT oligonucleotide 200 nM + / - mutant oligonucleotide 500 pM Total volume 1.25 μL

[0303]

Chemical formula

[0304] 2. Pyrophosphate cleavage and ligation A PPL mixture consisting of the following was prepared: 1 x BFF1 10 U / mL Klenow (exo-) 100 U / mL E. coli ligase 1.2 U / mL apyrase 100 U / mL lambda exonuclease 0.25 mM PPi 20 nM probe A 0 1.25 μL of oligo from point 1 Total volume 10 μL

[0305]

Chemical formula

[0306] wherein * is a phosphorothioate bond.

[0307] 1xBFF1 composition 10 mM Tris acetate, pH = 7.0 30 mM potassium acetate 17.125 mM magnesium acetate 0.01% Triton-X 1xA7 composition 10 mM Tris acetate, pH = 8.0 25 mM potassium acetate 5 mM magnesium acetate 0.01% Triton-X Q5 buffer The Q5 buffer composition is not publicly available. The resulting mixture was incubated at 45 °C for 15 minutes.

[0308] 3. Detection - RCA An RCA mixture consisting of the following was prepared: 2.66x Thermopol buffer (53.2 mM Tris-HCl, 26.6 mM (NH 4 ) 2 SO 4 4, 26.6 mM KCl, 5.32 mM MgSO 4 4, 0.266% Triton® X-100, pH 8.8) 0.28 μM primer mixture 284.4 U / mL BST 2.0 WarmStart 14.67 U / mL TIPP 1.06 mM dNTP 3 μM Syto82 dye 1.25 μL of the reaction from Point 2 Total volume 11.25 μL Primer mixture:

[0309]

Chemical formula

[0310] The resulting mixture was incubated at 50 °C for 70 minutes. Fluorescence readings were taken every minute. The results can be seen in Figure 16.

[0311] Example 15: Further Selected Applications and Aspects of the Invention KRAS Detection The KRAS gene controls cell proliferation, and when mutated, this negative signaling is disrupted and the cells can continue to proliferate, often developing into cancer. Single amino acid substitutions, and particularly single nucleotide substitutions, are the cause of activating mutations associated with various cancers: lung adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, and colorectal cancer. KRAS mutations have been used, for example, as prognostic biomarkers in lung cancer.

[0312] Driver mutations in KRAS are associated with up to 20% of human cancers, and there are developing targeting therapies for this mutation and its associated disease(s). A non - exhaustive list of some such therapies can be found in the following table:

[0313] [Table 4]

[0314] The presence of KRAS mutations has been found to reflect a very poor response to EGFR inhibitors, panitumumab (Vectibix) and cetuximab (Erbitux). Activating mutations in the gene encoding KRAS occur in 30% - 50% of colorectal cancers, and studies have shown that patients whose tumors express this mutant form of the KRAS gene do not respond to panitumumab and cetuximab. The presence of the wild - type KRAS gene does not guarantee that a patient will respond to these drugs, but studies have shown that cetuximab has significant efficacy in metastatic colorectal cancer patients with wild - type KRAS tumors. Lung cancer patients who are positive for KRAS mutations (wild - type EGFR) have an estimated response rate of 5% or less compared to a 60% response rate in patients without KRAS mutations with respect to the EGFR antagonists, erlotinib or gefitinib.

[0315] Early detection of the emergence of KRAS mutations (activation or overexpression), which are frequent drivers of acquired resistance to cetuximab therapy (anti-EGFR therapy) in colorectal cancer, allows for modification of treatment (e.g., early initiation of a mitogen-activated protein kinase kinase (MEK) inhibitor) to delay or reverse resistance, and thus it is preferred that the method of the present invention allows for rapid and inexpensive detection of the patient's KRAS status.

[0316] A non-limiting list of mutations is: G12D, G12A, G12C, G13D, G12V, G12S, G12R, A59T / E / G, Q61H, Q61K, Q61R / L, K117N and A146P / T / V.

[0317] A further non-limiting list of mutations is shown in the following table:

[0318]

Table 5

[0319] BRAF detection BRAF is a human gene that encodes a protein called B-Raf, which is involved in sending signals inside cells that are involved in instructing cell growth. BRAF has been shown to be mutated in several human cancers. B-Raf is a member of the Raf kinase family of growth signal-transducing protein kinases and, among other things, plays a role in the control of the MAP kinase / ERK signal transduction pathway, which affects cell division.

[0320] Certain other hereditary BRAF mutations cause birth defects. More than 30 mutations of the BRAF gene have been identified that are associated with human cancer. In 90% of cases, thymine is substituted by adenine at nucleotide 1799. This leads to a substitution of valine (V) with glutamic acid (E) at codon 600 in the activation segment found in human cancer (herein referred to as V600E). This mutation is widespread: - Colorectal cancer - Melanoma - Papillary thyroid carcinoma - Non-small cell lung cancer - Ameloblastoma is observed in.

[0321] A non-exhaustive list of other mutations that have been found are: R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600K and A727V.

[0322] Drugs have been developed to treat cancers driven by BRAF mutations; vemurafenib and dabrafenib have been approved by the FDA for the treatment of advanced melanoma. The response rate to treatment with vemurafenib for metastatic melanoma was 53%, compared to 7 - 12% for the previous optimal chemotherapeutic agent dacarbazine.

[0323] ERBB2 / HER2 detection Human epidermal growth factor receptor 2 (HER2), also known as CD340 (surface antigen classification 340), the proto-oncogene Neu, Erbb2 (rodent) or ERBB2 (human), is a protein encoded by the ERBB2 gene. Amplification or overexpression of this oncogene plays an important role in the invasive progression of breast cancer. Overexpression of the ERBB2 gene is also known to occur in 30% of invasive ovarian, gastric, lung adenocarcinomas, and uterine cancers and salivary duct carcinomas. Structural alterations that cause ligand-independent firing of the receptor in the absence of overexpression have also been identified.

[0324] There are many targeting therapies that are approved for this mutation and its associated disease(s) and are in development, and some non-limiting lists of such therapies can be found in the following table:

[0325]

Table 6

[0326] HER2 testing is routinely performed in breast cancer patients to assess prognosis, monitor response to treatment, and determine the appropriateness of targeting therapies (such as trastuzumab). Since trastuzumab is expensive and associated with significant side effects (cardiotoxicity), it is important that only HER2+ patients are selected to receive this drug, and thus it is advantageous that the method of the present invention enables rapid and inexpensive detection of a patient's HER2 status.

[0327] In one aspect, the method of the present invention is used to detect the presence or absence of an ERBB2 exon 20 insertion mutation. A further non-limiting list of ERBB2 mutations is shown in the following table:

[0328]

Table 7

[0329] EML4-ALK detection EML4-ALK is an abnormal gene fusion of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene. This gene fusion leads to the production of the protein EML4-ALK, which appears to promote and maintain the malignant behavior of cancer cells. EML4-ALK-positive lung cancer is a primary malignant lung tumor whose cells contain this mutation.

[0330] There are many targeting therapies approved and in development for this mutation and its associated disease(s), and some non-limiting lists of such therapies can be found in the table below:

[0331]

Table 8

[0332] The EML4-ALK gene fusion is involved in approximately 5% of non-small cell lung cancers (NSCLCs), which have approximately 9,000 new cases per year in the United States and approximately 45,000 cases worldwide.

[0333] There are many variants of EML4-ALK, and all variants have an essential coiled-coil domain in the EML4 N-terminal portion and the kinase domain of ALK exon 20 required for transforming activity. The fusions of exon 13 of EML4 and exon 20 of ALK (variant 1: V1) (this detection can be seen in Figure 20), exon 20 of EML4 and exon 20 of ALK (V2), and exon 6 of EML4 and exon 20 of ALK (V3) are some of the more common variants. The clinical importance of these different variants is only recently beginning to become clear.

[0334] V3 has emerged as a marker suitable for the selection of patients who are likely to have a shorter progression-free survival (PFS) after non-tyrosine kinase inhibitor (TKI) treatments, such as chemotherapy and radiotherapy. There is further evidence that V3 is associated with a shorter PFS and a worsening of overall survival (OS) in patients administered first- and second-generation treatment lines compared to V1 and V2 of EML4-ALK.

[0335] V3-positive patients have been found to develop resistance to first- and second-line therapies through the development of resistance mutations, and this is also likely to be promoted by incomplete tumor cell suppression due to the higher IC50 of wild-type V3. Detection of unfavorable V3 may be used to select patients who require more aggressive monitoring and treatment strategies. Administration of third-generation lorlatinib to patients with V3 appears to provide a longer PFS than those with V1, and thus it is preferred that the method of the present invention enables rapid and inexpensive detection of which variant a patient may have.

[0336] The method of the present invention further enables the detection of resistance mutations such as, but not limited to, for example: G1202R, G1269A, E1210K, D1203, S1206C, L1196M, F1174C, I1171T, I1171N / S, V1180L, T1151K, and C1156Y.

[0337] For example, G1202R is a solvent-front mutation that causes interference with drug binding and confers a high level of resistance to first- and second-generation ALK inhibitors. Thus, it is preferred that the method of the present invention enables the identification of patients who may possess this mutation and who may benefit from initiation of third-generation therapy rather than first or second.

[0338] Further non-limiting examples of EML4-ALK mutations are shown in the following table:

[0339] [Table 9]

[0340] EGFR Detection The identification of the epidermal growth factor receptor (EGFR) as an oncogene has led to the development of targeted therapies such as gefitinib, erlotinib, afatinib, brigatinib, and icotinib for lung cancer, and cetuximab for colon cancer. However, many people develop resistance to these therapies. Two major sources of resistance are the T790M mutation and the MET oncogene.

[0341] EGFR mutations occur in EGFR exons 18 - 21, and mutations in exons 18, 19, and 21 indicate the appropriateness of treatment with EGFR-TKIs (tyrosine kinase inhibitors). Mutations in exon 20 (except for some mutations) indicate that the tumor is resistant to EGFR-TKIs and is not suitable for treatment with EGFR-TKIs.

[0342] The two most common EGFR mutations are a short in-frame deletion in exon 19 and a point mutation in exon 21 at nucleotide 2573 (CTG to CGG) that results in a substitution of arginine for leucine at codon 858 (L858R). These two mutations together account for ~90% of all EGFR mutations in non-small cell lung cancer (NSCLC). Screening for these mutations in NSCLC patients may be used to predict which patients will respond to TKIs.

[0343] Therefore, it is preferred that the method of the present invention enables the identification of patients who possess these mutations and who may benefit from the initiation of treatment with TKIs. Those skilled in the art will recognize that the method of the present invention enables the identification of a range of EGFR mutations, and a non-exhaustive list of such mutations is: G719X, Ex19Del, S768I, Ex20Ins, and L861Q.

[0344] A further non-limiting list of mutations is shown in the following table:

[0345]

Table 10-1

[0346]

Table 10-2

[0347] ROS1 ROS1 is a receptor tyrosine kinase (encoded by the gene ROS1) that has structural similarity to the anaplastic lymphoma kinase (ALK) protein; it is encoded by the c-ros oncogene.

[0348] A non-exhaustive list of ROS1 mutations is shown in the following table:

[0349]

Table 11

[0350] RET proto-oncogene The RET proto-oncogene encodes a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor (GDNF) family of extracellular signaling molecules.

[0351] A non-exhaustive list of RET mutations is shown in the following table:

[0352]

Table 12

[0353] MET exon 14 MET exon 14 skipping occurs at a frequency of approximately 5% in NSCLC and is seen in both squamous and adenocarcinoma histologies.

[0354] A non-exhaustive list of MET mutations is shown in the following table:

[0355]

Table 13

[0356] NTRK proto-oncogene NTRK gene fusion leads to abnormal proteins called TRK fusion proteins, which can cause cancer cell proliferation. NTRK gene fusions can be found in several types of cancer, including brain, head and neck, thyroid, soft tissue, lung, and colon cancer. Also called neurotrophic tyrosine receptor kinase gene fusion.

[0357] A non-limiting list of NTRK mutations is provided in the table below:

[0358] [Table 14]

[0359] panel In one embodiment of the present invention, each A 0 A plurality of probe molecules (A 0 ) The mutations may be selected from any of the mutations described above or below or known. Thus, one of skill in the art will recognize that within the scope of the present invention are panels that may be useful in detecting one or more mutations to any of the proto-oncogenes or oncogenes described above or below or known.

[0360] In one embodiment, the panel comprises from 5 to 500 individual probe molecules, each of which is complementary to a specific target mutation. In one embodiment, the panel comprises from 5 to 400 individual probe molecules, each of which is complementary to a specific target mutation. In one embodiment, the panel comprises from 5 to 300 individual probe molecules, each of which is complementary to a specific target mutation. In one embodiment, the panel comprises from 5 to 200 individual probe molecules, each of which is complementary to a specific target mutation. In one embodiment, the panel comprises from 5 to 100 individual probe molecules, each of which is complementary to a specific target mutation. In one embodiment, the panel comprises from 5 to 50 individual probe molecules, each of which is complementary to a specific target mutation.

[0361] In one embodiment, there may be multiple probe molecules specific to the same mutation. In one embodiment, there may be only a single probe molecule specific to each mutation of the panel.

[0362] Provided is a panel comprising a plurality of probe molecules, wherein one or more probes are complementary to an EGFR mutation, one or more probes are complementary to a KRAS mutation, one or more probes are complementary to an ERBB2 / HER2 mutation, one or more probes are complementary to an EML4-ALK mutation, one or more probes are complementary to a ROS1 mutation, one or more probes are complementary to a RET mutation, and one or more probes are complementary to a MET mutation.

[0363] In one aspect, there is provided a panel comprising a plurality of probe molecules, wherein one or more probes may be complementary to an EGFR mutation, one or more probes may be complementary to a KRAS mutation, one or more probes may be complementary to an ERBB2 / HER2 mutation, one or more probes may be complementary to an EML4-ALK mutation, one or more probes may be complementary to a ROS1 mutation, one or more probes may be complementary to a RET mutation, and one or more probes may be complementary to a MET mutation.

[0364] In one aspect, there is provided a panel of probes that are selective for one or more of EGFR, KRAS, BRAF, ERBB2 / HER2, EML4-ALK, ROS1, RET, MET mutations.

[0365] In one aspect, there is provided a panel of probe molecules that are selective for an EGFR mutation. In one aspect, there is provided a panel of probe molecules that are selective for a KRAS mutation.

[0366] In one aspect, there is provided a panel of probe molecules that are selective for a BRAF mutation. In one aspect, there is provided a panel of probe molecules that are selective for an ERBB2 / HER2 mutation.

[0367] In one aspect, there is provided a panel of probe molecules that are selective for an EML4-ALK mutation. In one aspect, there is provided a panel of probe molecules that are selective for a ROS1 mutation.

[0368] In one aspect, there is provided a panel of probe molecules that are selective for a RET mutation. In one aspect, a panel of probe molecules that are selective for NTRK mutations is provided.

[0369] In one aspect, a panel of probe molecules that are selective for ROS1 mutations is provided. In one aspect, a panel of probe molecules that are selective for MET exon 14 mutations is provided.

[0370] In one aspect, a panel is provided that includes a plurality of probe molecules that are selective for one or more coding sequences (CDSs). In one aspect, a method is provided for detecting one or more mutations using one or more of the panels described above.

[0371] In one aspect, a method is provided for detecting the presence or absence of one or more mutations using one or more of the panels described above. In one aspect, a kit is provided that includes a panel that may be as described above or below, in combination with one or more reagents that may be as described above or below.

[0372] One of ordinary skill in the art would recognize that 0 aspects of the kits that disclose A 0 include within their scope aspects that include a panel that includes a plurality of A In one aspect, a device is provided in which one or more regions of the device include one or more panels, which may be as described above or subsequently.

[0373] Companion diagnostic Using the method of the present invention, specific gene markers in a sample may be detected, which can be used to assist in guiding the selection of an appropriate therapy. These markers may be tumor-specific mutations or may be wild-type genomic sequences, and may be detected using tissue, blood or any other patient sample type.

[0374] Resistance monitoring By repeated testing of patient samples during the treatment of a disease, early detection of resistance developing to a therapy may be possible. An example of this application is in non-small cell lung cancer (NSCLC) where epidermal growth factor receptor (EGFR) inhibitors (e.g., gefitinib, erlotinib) are commonly used as first-line treatment. During treatment, tumors often have the potential to develop mutations (e.g., T790M, C797S) in the EGFR gene, which confer resistance to the drug. Early detection of these mutations may enable conversion of the patient to an alternative therapy (e.g., Tagrisso).

[0375] Typically, patients monitored for the onset of resistance may be too ill to undergo repeated tissue biopsies. Repeated tissue biopsies are also costly, invasive and may carry associated risks. Testing from blood is preferred, but in a suitable blood sample, the mutations of interest may only be present at very low copy numbers. Therefore, monitoring requires a highly sensitive test from a blood sample using the method of the present invention that is simple and cost-effective to perform so that it can be carried out regularly.

[0376] Recurrence monitoring In this application example, patients declared disease-free after treatment may be monitored over the long term to detect disease recurrence. This needs to be done non-invasively and requires highly sensitive detection of target sequences from blood samples. By using the method of the present invention, a simple and low-cost method that can be performed regularly is provided. The targeted sequences may be gene mutations known to be common in the disease of interest or, alternatively, a custom panel of targets designed for a particular patient based on the detection of variants in pre-remission tumor tissue.

[0377] Minimal Residual Disease (MRD) Monitoring After treatment, for some cancers where residual cancer cells remain in the patient, this is a major cause of cancer and leukemia recurrence. MRD monitoring and testing have several important roles: determining whether treatment has eradicated the cancer or left a trace, comparing the effectiveness of different treatments, monitoring the patient's remission status, and detecting leukemia recurrence, and selecting a treatment that will optimally meet these needs.

[0378] Screening Population screening for early detection of disease has long been an objective, particularly in cancer diagnosis. The difficulties are twofold: the identification of a marker panel that enables reliable detection of the disease without too many false negatives, and the development of a method with sufficient sensitivity and a sufficiently low cost. Using the method of the present invention, it is possible to work with a larger mutation panel than PCR-based tests, but with a much simpler workflow and lower cost than sequencing-based diagnostic methods.

[0379] Organ Transplant Rejection If a transplanted organ is rejected by the recipient, DNA from this organ will shed into the recipient's bloodstream. Early detection of this DNA enables early detection of rejection. This can be achieved by using a custom panel of donor-specific markers or a panel of variants known to be common in the population, some of these variants being present in the donor and some in the recipient. Long-term routine monitoring of organ recipients could be enabled by the low-cost and simple workflow of the invention disclosed herein.

[0380] Non-invasive prenatal testing (NIPT) It has long been known that fetal DNA exists in maternal blood, and the NIPT market is currently fairly saturated by companies that use sequencing to identify mutations and count the copy number of specific chromosomes to enable detection of fetal abnormalities. The method of the invention as disclosed herein has the ability to detect mutations at very low allele frequencies, potentially enabling earlier detection of fetal DNA. Identification of common mutations in a given population would allow targeting of mutations that may be present in either maternal or fetal DNA, or the development of assays that enable detection of abnormalities at earlier stages of pregnancy.

[0381] Various further aspects and embodiments of the invention will be apparent to those skilled in the art in view of the present disclosure. "And / or" shall be construed herein as a specific disclosure of each of the two recited features or components, with or without the other. For example, "A and / or B" shall be construed as (i) A, (ii) B, and (iii) A and B, each being specifically disclosed herein as if individually set forth.

[0382] Unless the context otherwise indicates, the descriptions and definitions of the features given above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments described.

[0383] Those skilled in the art will further recognize that, although the present invention has been described by way of example with reference to several embodiments, it is not limited to the disclosed embodiments and alternative embodiments can be constructed without departing from the scope of the invention as defined by the appended claims.

[0384] Those skilled in the art will understand that a reference to "partially digested strand A" 1 may refer to a single-stranded oligonucleotide formed by progressive digestion of A in the 3'-5' direction until the strand dissociates due to lack of complementarity when hybridized to the target analyte sequence. 0

[0385] Those skilled in the art will understand that a reference to a nucleic acid that is "partially double-stranded" may refer to a nucleic acid in which one or more portions are double-stranded and one or more portions are single-stranded.

[0386] Those skilled in the art will understand that a reference to a nucleic acid that is "substantially double-stranded" may refer to a nucleic acid in which one or more portions are double-stranded and one or more smaller portions are single-stranded.

Claims

1. (a) A single-stranded probe oligonucleotide A capable of forming a target polynucleotide sequence and a first intermediate product ０ wherein the first intermediate product is a single-stranded probe oligonucleotide A to the target polynucleotide sequence ０ formed by hybridization of the 3'-end, and the oligonucleotide A wherein the intermediate product is at least partially double-stranded ０ ; (b) Ligase; (c) A ０ An enzyme capable of digesting the first intermediate product in the 3'-5' direction from the end of １ to generate a partially digested strand A; (d) An ion source that drives the pyrophosphate decomposition reaction, wherein the ion is a pyrophosphate ion, said ion source; and (e) A suitable buffer A kit comprising.

2. The kit according to claim 1, further comprising positive and negative controls.

3. A ０ The kit according to claim 1 or 2, wherein the 5' end of A is resistant to 5'-3' exonuclease digestion and the kit further comprises a 5'-3' exonuclease.

4. The kit according to any one of claims 1 to 3, further comprising deoxynucleotide triphosphate (dNTP), polymerase, and a buffer for the first amplification of the target polynucleotide sequence present in the sample.

5. The kit according to claim 4, further comprising a high-fidelity polymerase that incorporates dUTP, dUTP, and uracil-DNA N-glycosylase (UDG).

6. The kit according to any one of claims 1 to 5, further comprising proteinase.

7. The kit according to any one of claims 1 to 6, further comprising ligation probe oligonucleotide C.

8. The kit according to any one of claims 1 to 6, further comprising sprint oligonucleotide D.

9. The kit according to any one of claims 1 to 6, further comprising ligation probe oligonucleotide C and sprint oligonucleotide D.

10. The kit according to claim 7 or 9, wherein the ligation probe oligonucleotide C comprises a 3' or internal modification that protects the oligonucleotide from 3'-5' exonuclease digestion, and the kit further comprises 3'-5' exonuclease.

11. D is A １ An oligonucleotide region complementary to the 3'-end of, and a region complementary to either the 5'-end of oligonucleotide C or the 5'-end of A １ The kit according to claim 8, 9 or 10, comprising

12. D is unable to undergo extension relative to A, either by 3' modification or through a mismatch between the 3' end of D and the corresponding region of A １ or C. The kit according to claim 11 １ which is unable to undergo extension relative to A

13. A ０ The kit according to any one of claims 1 to 12, further comprising at least one single-stranded primer oligonucleotide complementary to a part of A , an amplification enzyme, dNTPs, and one or more oligonucleotide-binding dyes or molecular probes.

14. Plural A, which are selective for different target sequences and each contain an identification region ０ The kit according to any one of claims 1 to 13, further comprising

15. -A １ Two or more concatenated ligation chain reaction (LCR) probe oligonucleotides that are complementary to the adjacent array above, wherein when the probe successfully anneals to the 5' phosphate of one LCR probe, it is immediately adjacent to the 3' OH of the other LCR probe, said probe oligonucleotide; and - One or more ligases The kit according to any one of claims 1 to 12, further comprising.

16. The kit according to claim 13, wherein the amplification enzyme is the same as the pyrophosphate-degrading enzyme.

17. - Ligation probe oligonucleotide C; - Sprint oligonucleotide D Further comprising; Here, C has a 5'-phosphate, and A １ and E are both linked to form A ２ such that the 3'-end of the splint oligonucleotide D is complementary to the 5'-end of C, and the 5'-end of D is １ complementary to the 3'-end of A The kit according to any one of claims 1 to 12.

18. - A hairpin oligonucleotide 1 (HO1) containing a fluorophore - quencher pair, wherein HO1 is A ２ complementary to, and when annealed to A ２ the hairpin structure of HO1 opens and the fluorophore - quencher pair separates; said HO1; and - Hairpin oligonucleotide 2 (HO2) comprising a fluorophore-quencher pair, wherein HO2 is complementary to the open HO1, and when annealed to HO1, the hairpin structure of HO2 opens and the fluorophore-quencher pair separates, said HO2 The kit according to claim 17, further comprising.

19. The kit according to claim 18, further comprising a plurality of HO1 and HO2.

20. - Oligonucleotide A comprising a substrate arm, a partial catalytic core, and a sensor arm; - Oligonucleotide B comprising a substrate arm, a partial catalytic core, and a sensor arm; and - A substrate comprising a fluorophore - quencher pair further comprising; Here, the sensor arms of oligonucleotides A and B are such that, in the presence of A ２ oligonucleotides A and B combine to form a catalytic multi-component nucleic acid enzyme (MNAzyme), and are complementary to the adjacent region of A ２ ​ The kit according to any one of claims 1 to 12.

21. further comprising a nucleic acid construct that is partially double - stranded, wherein: - One strand contains at least one RNA base, at least one fluorophore, and one region of this strand is complementary to the region of A ２ and this strand may be referred to as the "substrate" strand; and - The other strand contains at least one quencher, and one region of this strand is A ２ in the presence of A, such that the nucleic acid construct that has formed a partial double-stranded structure has a larger double-stranded portion, is complementary to the region of A adjacent to the region where the substrate strand is complementary ２ to the region of A The kit according to any one of claims 1 to 12.

22. The kit according to claim 21, further comprising an enzyme for removing at least one RNA base.

23. - A including the connecting part ２ An oligonucleotide complementary to the region of, comprising one or more fluorophores arranged such that their fluorescence is quenched either by proximity to each other or to one or more fluorescence quenchers; - A double - strand - specific DNA - digesting enzyme further comprising; Here, A ２ in the presence of, the fluorophore is separated from each other or from the corresponding quencher, and the labeled oligonucleotide is digested such that the fluorescence signal, and thus the presence of A ２ is detectable The kit according to any one of claims 1 to 12.

24. The kit according to any one of claims 1 to 23, further comprising a phosphatase or a phosphohydrolase.

25. The kit according to any one of claims 1 to 24, further comprising pyrophosphatase.

26. The kit according to any one of claims 1 to 25, further comprising an enzyme for forming DNA from an RNA template.

27. A device comprising a fluid path between a first region and a second region, wherein the first region comprises one or more wells, and the one or more wells are: Single-stranded probe oligonucleotide A capable of forming a target polynucleotide sequence and a first intermediate product ０ wherein the first intermediate product is formed by hybridization of the 3'-end of the single-stranded probe oligonucleotide A0 to the target polynucleotide sequence, and the oligonucleotide A wherein the intermediate product is at least partially double-stranded ０ ; A ０ A pyrophosphate-degrading enzyme that digests the first intermediate product in the 3'-5' direction from the end of １ to produce a partially digested strand A; an ion source that drives the pyrophosphate decomposition reaction in the forward direction, wherein the ion is a pyrophosphate ion, said ion source; and A １ to ligate to form oligonucleotide A ２ and includes one or more ligases capable of generating The second region comprises one or more wells, Said device.

28. A ０ The device according to claim 27, wherein the 5' end of A is resistant to 5'-3' exonuclease digestion and the well of the first region further contains 5'-3' exonuclease.

29. further comprising a third region comprising one or more wells connected to the first region by a fluid path, and the one or more wells in the third region are: dNTP; At least one single - stranded primer oligonucleotide; and An amplification enzyme The device according to any one of claims 27 or 28.

30. The dNTP in the third region is dUTP, dGTP, dCTP, and dATP; The amplification enzyme is a high - fidelity polymerase that incorporates dUTP; and One or more wells in the third region further comprise uracil - DNA N - glycosylase, The device according to claim 29.

31. further comprising a fourth region located between the first and third regions and comprising one or more wells, and the one or more wells comprise a protease, the device according to claim 29 or claim 30.

32. The device according to any one of claims 27 to 31, wherein one or more wells of the first or second region further comprise a ligase and a ligation probe oligonucleotide C.

33. One or more wells of the first or second region further comprise a ligase and a splint oligonucleotide D that is complementary to region A ０ The device according to any one of claims 27 to 31, further comprising a ligase and a splint oligonucleotide D that is complementary to region A of ０ .

34. The device according to any one of claims 27 to 31, wherein one or more wells of the first or second region further comprise a ligase, a splint oligonucleotide D and a ligation probe oligonucleotide C.

35. The device according to claim 32 or claim 34, wherein the ligation probe oligonucleotide C comprises a 3' or internal modification that protects the oligonucleotide from 3'-5' exonuclease digestion.

36. D is A １ an oligonucleotide region complementary to the 3'-end of, and a region complementary to either the 5'-end of oligonucleotide C or the 5'-end of A １ The device according to any one of claims 33, 34, or 35, comprising

37. One or more wells of the first region are each selective for a different target sequence, at least one or more different A ０ The device according to any one of claims 27 to 36, comprising

38. The wells of the second region are: dNTP; Buffer; Amplification enzyme; One or more oligonucleotide-binding dyes or molecular probes; and A １ or a part thereof, or a signal derived from multiple copies of A １ means for detecting a signal derived from multiple copies of a part thereof The device according to claim 37, comprising.

39. The wells of the second region are: -A １ Two or more ligase chain reaction (LCR) probe oligonucleotides that are complementary to the adjacent array above, wherein when the probe successfully anneals to the 5' phosphate of one LCR probe, it is immediately adjacent to the 3' OH of the other LCR probe; and - One or more ligases The device according to any one of claims 27 to 36, further comprising.

40. The wells of the second region are: - Ligation probe oligonucleotide C; - Splint oligonucleotide D Further comprising; Here, C has a 5'-phosphate, and A １ and C are both linked to form oligonucleotide A ２ such that the 3'-end of the splint oligonucleotide D is complementary to the 5'-end of C, and the 5'-end of D is １ complementary to the 3'-end of A The device according to any one of claims 27 to 36.

41. The wells of the second region are: - A hairpin oligonucleotide 1 (HO1) containing a fluorophore - quencher pair, wherein HO1 is A ２ complementary to, and when annealed to A ２ the hairpin structure of HO1 opens and the fluorophore - quencher pair separates, said HO1; and - A hairpin oligonucleotide 2 (HO2) containing a fluorophore-quencher pair, wherein HO2 is complementary to the opened HO1 and, when annealed to HO1, the hairpin structure of HO2 opens and the fluorophore-quencher pair separates, said HO2 The device according to claim 40, further comprising.

42. The wells of the second region are: - An oligonucleotide A comprising a substrate arm, a partial catalytic core and a sensor arm; - An oligonucleotide B comprising a substrate arm, a partial catalytic core and a sensor arm; and - A substrate containing a fluorophore-quencher pair Further comprising; Here, the sensor arms of oligonucleotides A and B are such that in the presence of A ２ oligonucleotides A and B combine to form a catalytic multi-component nucleic acid enzyme (MNAzyme), and are complementary to the adjacent region of A ２ ​ The device according to any one of claims 27 to 36.

43. The wells of the second region further comprise a nucleic acid construct that is partially double-stranded, wherein: - One strand contains at least one RNA base and at least one fluorophore, and one region of this strand is complementary to the region of A ２ and this strand may be referred to as the "substrate" strand; - The other strand contains at least one quencher, and one region of this strand is A ２ in the presence of A, the substrate strand is complementary to the region of A adjacent to the region where the nucleic acid construct that has partially formed a strand forms a more double-stranded structure; and ２ is complementary to the region of A; and The wells of the second region further comprise an enzyme for removing at least one RNA base, the device according to any one of claims 27 to 36.

44. One or more wells of the second region are: A including the connecting part ２ The oligonucleotide complementary to the region of ２ , comprising one or more fluorophores arranged such that their fluorescence is quenched by proximity to each other or to one or more fluorescence quenching agents; Double-strand specific DNA digestion enzyme Further comprising; Here, A ２ in the presence of, the fluorophore is separated from each other or from the corresponding quencher, and the fluorescent signal, and thus the presence of A ２ is detected, the labeled oligonucleotide is digested The device according to any one of claims 27 to 36.

45. The device according to any one of claims 27 to 44, wherein one or more wells of one or more regions further comprise pyrophosphatase.

46. The device according to any one of claims 27 to 45, wherein one or more wells of one or more regions further comprise phosphatase or phosphohydrolase.

47. The device according to any one of claims 27 to 46, wherein one or more wells of the first region further comprise an enzyme for forming DNA from an RNA template.

48. The device according to any one of claims 27 to 47, wherein the first and second regions are combined.

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