Improved padlock probe for nucleic acid detection using superbranched rolling circle amplification

Abstract

The present invention relates to an HRCA method comprising the following steps: a) obtaining a nucleic acid structure formed by hybridization of at least one padlock with a target nucleic acid; b) circulating the hybridized padlock; c) subjecting the ligated circulated padlock probe to rolling circle amplification (RCA) and, if necessary, performing an MDA reaction using short primers; and d) detecting the target nucleic acid by detecting the amplified product from step c), preferably by a sequence-specific detection method.

Description

Detailed Description of the Invention

[0001] 〔Technical Field〕 The present invention relates to the field of molecular biology. In particular, the present invention relates to the fields of amplification methods and detection methods using hyperbranched rolling circle amplification. 〔Background Art〕

[0002] Rolling circle amplification (RCA) is an enzymatic process that amplifies a short DNA primer or a short RNA primer using a circular single-stranded nucleic acid template and a DNA polymerase or an RNA polymerase having strand displacement ability to form a long single-stranded DNA or RNA. This method is used for the amplification of target molecules of DNA or RNA, and a specially designed padlock probe hybridizes with the target nucleic acid, whereby the two ends of the probe are juxtaposed and ligated by a ligase to form a circular single-stranded nucleic acid serving as a template for amplification. A polymerization reaction is initiated by the addition of a polymerase and nucleotides and can be carried out under isothermal conditions. Since the circular single-stranded nucleic acid template has no ends, the product of RCA is a long single-stranded nucleic acid molecule (i.e., a concatemer) consisting of tandem repeat units or monomers complementary to the nucleic acid template.

[0003] Therefore, the RCA reaction produces linear amplification of DNA because each circular template grows at a constant rate for a constant time. Several approaches have been explored to increase yield and achieve exponential amplification. One of these is hyperbranched rolling circle amplification (HRCA), in which primers that anneal to the RCA product and the inversely complementary sequence are added and extended in a further amplification step. This amplification step can also be carried out under isothermal conditions (Multiple Displacement Amplification; MDA). In this method, the original RCA generates more templates that can be amplified and subsequently detected. Methods for detecting nucleic acids are widely known and include the use of DNA stains, beacon probes, electrochemical assays, and CRISPR-Cas-based methods.

[0004] Since its initial introduction, HRCA using padlock probes has proven to be a reliable DNA amplification technique for pathogen detection and other applications, enabling rapid detection of nucleic acid sequences with high specificity. This method has been adopted as the basis for amplification and detection of linear and cyclic nucleic acid molecules in laboratory settings.

[0005] Despite the expansion of research into point-of-care (POC) testing, most devices remain laboratory-based. This is particularly relevant in the case of diagnostic testing, as the COVID-19 pandemic highlighted the importance of developing rapid tests and diagnoses for efficient epidemic control. Therefore, there is a need to improve and optimize methods such as RCA and HRCA to move from the laboratory to the field of low-cost point-of-care diagnostics. However, this presents several hurdles to overcome and presents a challenge. On the one hand, nonspecific amplification resulting from circularized padlock probes that do not hybridize with the target nucleic acid causes background noise that masks the positive signal. On the other hand, nonspecific hybridization at low temperatures required in POC testing also causes target-independent amplification. Therefore, detection methods need to efficiently distinguish this background amplification from specific target-dependent amplification. Importantly, methods should be adapted to be rapid, portable, simple, and low-cost, but this adaptation should not negatively impact the specificity and accuracy of the method.

[0006] This invention focuses on padlock design and improved HRCA methods, providing a novel method for rapid, low-cost, yet specific and efficient detection of target nucleic acids.

[0007] [Brief explanation of the drawing] Figure 1: (A to J) Preferred padlocks of the present invention. Figure 2: Detection of HRCA-mediated RNA using a padlock probe and HRCA. (a) A scheme of the basic method, in which a ring is specifically generated in the presence of target RNA using a padlock probe, and this product is amplified by RCA-MDA, known as hyperbranched RCA (HRCA). The RCA reaction can be primed by the target RNA. The main advantage of using phi29pol / Qualiphi is that the reaction can be carried out at room temperature. (b) Principle validation experiment. A padlock targeting the human ACTB gene was incubated with the specified synthetic RNA in the presence and absence of SplintR ligase, as described in Example 1. The amplified material was electrophoresed on a 0.8% agarose gel and visualized by Gelred staining. (c) Target preparation. The experiment was carried out as described in Example 2. A human ACTB padlock probe was incubated in the presence of a specified amount of synthetic ACTB target RNA. The amplified substances were subjected to electrophoresis on a 0.8% agarose gel and visualized using Gelred staining. Figure 3: Optimization of HRCA conditions: buffer. The experiment was performed as described in Example 3. A specified amount of pre-ligated (cyclic) or linear ACTB padlock probe was subjected to the HRCA reaction. The amplified material was electrophoresed on a 0.8% agarose gel and visualized by Gelred staining. Figure 4: Optimization of HRCA conditions: primers. The experiment was performed as described in Example 4. The HRCA reaction included specified amounts of ACTB RNA in the presence or absence of ACTB padlock and / or SplintR ligase. The amplified material was electrophoresed on a 0.8% agarose gel and visualized by Gelred staining. Figure 5: HRCA using wild-type phi29pol. The experiment was performed as described in Example 5. The HRCA reaction contained the specified amount of ACTB RNA. A negative control (NC) without an ACTB padlock was included. Amplification was performed for 2 hours (B) and 6 hours (A) as indicated. The amplified material was electrophoresed on a 0.8% agarose gel and visualized by Gelred staining. Figure 6: HRCA combined with CRISPR-Cas12a detection. (a) HRCA-CRISPR-Cas12a detection was performed as described in Example 6. The reaction contained a specified amount of target RNA. It also contained a negative control (NC, negative control) that contained the maximum amount of target but no padlock, and no padlock or target. The total amplification product detected by agarose gel electrophoresis (A) and the specific detection result by CRISPR-Cas12a in the reporter substrate (B) are shown. Figure 7: Improved padlocks for HRCA CRISPR-Cas12a:skin. The HRCA reaction was performed using the specified amount of target RNA as described in Example 7, with S gene padlocks without skin (A), or S gene padlocks with skin containing a blocked 3' biotin (B) or 3'OH (C). Negative controls (NC, negative control) without padlocks or target were also included. Figure 8: Improved padlock for HRCA CRISPR-Cas12a: FLAP and ExoI. The HRCA reaction was carried out as described in Example 8, including the ExoI treatment. The specified amount of target RNA was incubated with an S gene padlock containing a poly-A extension at the 3' end. A negative control (NC) without a padlock or target was included. Figure 9: Improved padlock for HRCA CRISPR-Cas12a: FLAP. The HRCA reaction was carried out as described in Example 9. The specified amount of target RNA was incubated with the S gene padlock. A negative control (NC) without the padlock or target was included. Figure 10: Improved padlock for HRCA CRISPR-Cas12a: GAP. The HRCA reaction was carried out as described in Example 10 and is shown on the left. A specified amount of target RNA was incubated with an S gene padlock containing a gap at the breakpoint junction, and the gap was filled with additional oligonucleotides. A negative control (NC) without a padlock or target was included. Figure 11: Modification of HRCA CRISPR-Cas12a: Two-arm padlock. Results of HRCA reactions using ssDNA(a), dsDNA(b), and dsDNA-nick(c) split padlock probes are shown as described in Example 11. A specified amount of target RNA and a negative control (NC) without a padlock or target were tested. Figure 12: Modification of HRCA CRISPR-Cas12a: FLAP and skin combination. The experiment was carried out as described in Example 12 and is shown on the left. Results for a specified amount of target RNA and for a negative control (NC) without a padlock or target are shown.

[0008] [General definition] Where used herein, the singular forms “a,” “an,” and “the” are to be interpreted as including the plural form unless the context clearly indicates otherwise. Furthermore, unless otherwise specified, the term “at least” preceding a set of elements is to be understood as referring to all elements in that set. Those skilled in the art will be able to recognize or verify, by means of ordinary experimentation, many equivalents to the particular embodiments of the inventions described herein. Such equivalents are intended to be incorporated herein.

[0009] As used herein, the conjunction "and / or" between multiple enumerated elements is understood to encompass both individual and combined options. For example, in the case of two elements connected by "and / or," the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of both the first and second elements. Any one of these options is understood to fall within the meaning of the term "and / or" as used herein, and therefore satisfies the requirements of this term. Cases where more than one of the options applies simultaneously are also understood to fall within the meaning of the term "and / or," and therefore satisfies the requirements of this term.

[0010] It should be noted that, as used herein, the term “about” refers to ±30%, preferably ±20%, preferably ±15%, and more preferably ±10% of the specified reference value.

[0011] Throughout the following detailed description and claims, unless otherwise required by context, the word “comprise,” and variations such as “comprises” and “comprising,” should be understood to mean including the integer or process, or group of integers or processes, described, but not to exclude other integers or processes, or groups of integers or processes. As used herein, the term “comprising” may be replaced with the terms “containing” or “including,” or, as sometimes used herein, with the term “having.” Whenever used herein in the context of an aspect or embodiment of the present invention, any of the above terms (comprising, containing, including, having) may, for the less favorable, be replaced with the term “consisting of.”

[0012] As used herein, "consisting of" excludes any element, process, or component not expressly described in the claimed elements. As used herein, "consisting essentially of" does not exclude any substance or process that does not significantly affect the claimed basic and novel properties.

[0013] "Percent (%) amino acid sequence identity" for proteins or polypeptides described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference sequence (i.e., the protein or polypeptide from which it originates) after the sequences have been aligned and gaps introduced as necessary to achieve maximum sequence identity, and this does not consider conservative substitutions as part of the sequence identity. Sequence alignment for determining the percentage of amino acid sequence identity can be achieved in various ways within the scope of the art. For example, commonly available computer software such as BLAST can be used. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0014] Preferably, as used herein, the “percentage of identity” is determined in the context of local alignment. That is, it is determined based on the alignment of regions of local similarity between nucleobase sequences, as opposed to global alignment which aims for alignment across the entirety of two sequences. Therefore, in the context of the present invention, the percentage of identity is preferably calculated based solely on a local alignment comparison algorithm.

[0015] "Reverse complementary," "complementary," and "complementarity" are interchangeable and refer to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units within an antiparallel polynucleotide chain or region. Complementary polynucleotide chains or regions can form base pairs in the Watson-Crick type (e.g., A and T, A and U, C and G). 100% (or completely) complementary means that each nucleotide unit of one polynucleotide chain or region can form hydrogen bonds with each nucleotide unit of a second polynucleotide chain or region. Incomplete (or partially) complementary means that some, but not all, nucleotide units of two chains or regions can form hydrogen bonds with one another, and this can be expressed as a percentage. Note that, as used in this invention, the term "complementary" also encompasses the term "substantially complementary." A region is "substantially complementary" to a target region if the percentage of complementarity between the two regions is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. A region is "substantially complementary" to another region if it hybridizes with another region under low stringency conditions, preferably under medium stringency conditions, and most preferably under high stringency conditions. Similarly, when the term "incomplementary" is used herein, it should be understood to also include the term "substantially incomplementary." A region is "substantially incomplementary" to another region if the complementarity between the two regions is less than 40%, less than 30%, less than 20%, preferably less than 10%, most preferably less than 5%, or still most preferably 0%.One region is "substantially complementary" to another region if it does not hybridize with another region under high stringency conditions, preferably medium stringency conditions, and most preferably low stringency conditions.

[0016] "Functional equivalent" in this specification means another nucleotide sequence or amino acid sequence whose nucleobase or amino acid sequence differs from that of the sequence referenced in a particular embodiment, but which performs the same function and provides the same utility or technical effect as that of the sequence referenced in a particular embodiment.

[0017] The term "hybridization" refers to a structure formed by two independent strands of nucleic acid (i.e., DNA and / or RNA) that form a double-stranded structure through base pairing from one strand to the other. These base pairs are considered to be GC, AU / T, and GU (A-adenine, C-cytosine, G-guanine, U-uracil, T-thymine). As in the case of complementarity, hybridization can be complete or partial. In the context of this invention, each uracil and thymine base can be optionally replaced with either a thymine or uracil base, respectively. Generally, whether such hybridization occurs is influenced by factors such as the length of the polynucleotide, complementarity, pH, temperature, the presence of monovalent and divalent cations, the ratio of G and C nucleotides in the hybridization region, the viscosity of the medium, and the presence of denaturants. These variables affect the time required for hybridization. Therefore, preferred annealing conditions vary depending on the specific application. However, these conditions can be determined by a person skilled in the art in a normal manner, without excessive trial and error, at the usual level of technical skill.

[0018] The "percentage of sequence identity" for polynucleotides and polypeptides is determined by comparing two optimally aligned sequences within a comparison window. In this case, when comparing the two sequences to a reference sequence (without additions or deletions) to optimally align them, some of the polynucleotide or polypeptide sequences within the comparison window may contain additions or deletions (i.e., gaps). The percentage is calculated by determining the number of positions in which the same nucleobase or amino acid residue appears in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for comparison may be performed by computer implementations of known algorithms (BLAST in the National Centre for Biotechnology Information resources, CLUSTAL in the European Bioinformatics Institute resources, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.) or by visual inspection. It should be noted that, as used herein, “percentage of sequence identity” is preferably determined in the context of local alignment. That is, this is based on the alignment of local similar regions between nucleobase sequences, as opposed to global alignment, which aims to align two entire sequences. Therefore, in the context of the present invention, the percentage of identity is preferably calculated based solely on a local alignment comparison algorithm.

[0019] The term "rolling circle amplification" or "RCA" refers to an enzymatic process under isothermal conditions that amplifies short DNA or RNA primers using a circular DNA template and phi29 polymerase to form long single-stranded DNA or RNA. For diagnostic purposes, this circular template can be a so-called padlock probe that is circularized in the presence of a target nucleic acid molecule. The RCA product is a concatemer containing dozens to hundreds of tandem repeats that are complementary to the padlock probe. In the method of the present invention, it is preferable that the DNA or RNA primer corresponds to the target nucleic acid.

[0020] The term "hyper-branched rolling circle amplification" or "HRCA" refers to an RCA reaction combined with an MDA reaction.

[0021] The term "strand-displacing DNA polymerase" refers to a DNA polymerase that performs a 3'-end extension reaction while melting the corresponding portion of a nucleic acid hybridized to a template DNA strand.

[0022] As used herein, the expression "3'-region" refers to a region of a nucleotide strand that includes the 3'-end of the strand. As used herein, the term "3'-end" refers to the end of a nucleotide strand having a hydroxy group at the 3rd carbon of the sugar ring of deoxyribose at its end. As used herein, the expression "5'-region" refers to a region of a nucleotide strand that includes the 5'-end of the strand. As used herein, the term "5'-end" refers to the end of a nucleotide strand having the 5th carbon of the sugar ring of deoxyribose at its end.

[0023] As used herein, the term "portion" when referring to a sequence means a nucleotide region or fragment of the sequence. Thus, the "first portion" refers to the first nucleotide region or fragment within the sequence. Similarly, the "second portion" refers to the second nucleotide region or fragment within the sequence. As used herein, "portion" and "region" are treated as synonyms.

[0024] As used herein, the term "detecting" refers to any of a variety of methods for determining the presence and / or amount and / or identity of a target nucleic acid.

[0025] 〔Description of Embodiments〕 In the present invention, the HRCA assay is improved by applying different optimization strategies. The general concept of optimization applied herein facilitates conditions that provide a robust amplification step that ensures amplification of the target nucleic acid even when present at low concentrations, and then applies stringent conditions in the detection step, such that only the target nucleic acid is detected, rather than the background that may have been generated as a result of the amplification step. Thus, in the first step, non-stringent amplification conditions are applied by providing short primers having a low Tm and that hybridize readily with the padlock probe. As a result of this amplification step, the nucleic acid is amplified exponentially in large amounts. The amplified nucleic acid can result from target-specific amplification or non-specific (also referred to as background) amplification, where target-specific amplification is derived from the padlock hybridized to the target nucleic acid, and non-specific amplification is derived from the indiscriminate and non-specific annealing of the short primers and / or from the padlock circularized in the absence of the target nucleic acid.

[0026] Next, this bulk amplification is subjected to a rigorous detection step based on a specific nucleotide sequence, which filters out the large amount of amplification product generated during bulk amplification, allowing only the nucleic acid obtained from the specific amplification to be detected. This strategy makes it possible for the assay of the present invention to detect small amounts of target nucleic acid or analyte in a sample. Example 6 shows an example of this HRCA method, in which short primers are used, but the detection step is performed using a CRISPR-Cas12a system that specifically detects the target sequence present in the S gene region of the target SARS-CoV-2 virus genome. As shown in Figure 6, even though nonspecific amplification yielded a high yield of amplicons that were indistinguishable on the gel, the detection step enabled specific detection of the target nucleic acid. 5 We were able to detect a number of molecules of the order of magnitude. This demonstrates that the combination of short primers and a sequence-specific method results in a synergistic effect derived from the strong amplification provided by the short primers and the high specificity of the sequence-based detection method, and that this synergy allows for the efficient differentiation of amplicons with the correct sequence.

[0027] Based on the above, a first aspect of the present invention provides a method for detecting a target nucleic acid in a sample by superbranched rolling circle amplification (HRCA), the method comprising the following steps: a) A step of adding at least one padlock probe to the sample to provide a nucleic acid structure formed by hybridization of the at least one padlock with the target nucleic acid, wherein in the nucleic acid structure, the at least one padlock is in a cyclic form, b) A step of subjecting the cyclizable padlock to a ligation reaction using at least one ligase to obtain a padlock having sequence ABC as a single cyclic molecule. c) The ligated cyclic padlock from step b) is subjected to a rolling circle amplification (RCA) reaction, and optionally the RCA product is amplified in an MDA reaction using an MDA primer up to 10 nucleotides in length, and d) A step of detecting the amplification product obtained from step c) and thereby detecting the target nucleic acid, wherein the detection is performed using a sequence-specific detection method.

[0028] Each step of the method of the present invention is further described below: (Step a) A step of adding at least one padlock probe to a sample to provide a nucleic acid structure formed by hybridization of at least one padlock with a target nucleic acid, wherein the nucleic acid structure is in a cyclic form of at least one padlock.

[0029] Padlocks are known molecules that play a major role in RCA-based methods. These molecules are cyclizable oligonucleotides, and these molecules contain target-complementary regions in their 3' and 5' regions that can hybridize with a target DNA sequence or target RNA sequence. Upon hybridization with the target nucleic acid, the 3' and 5' ends of the padlock probe become close to each other, and a ligase binds them together, thereby forming a cyclic padlock probe having a sequence region hybridized with the target molecule. In the context of this invention, “padlock” and “padlock probe” are considered synonymous and are therefore used interchangeably. Specific structures of padlocks used herein are also referred to as “the padlock of this invention,” as defined below. Preferably, the padlock of this invention is a DNA molecule.

[0030] In one embodiment, at least one padlock is a group of padlocks with the same sequence, or a group of padlocks with different sequences.

[0031] Step a) includes adding at least one padlock to the sample. If the sample contains a target nucleic acid to be detected, at least one padlock and the target nucleic acid come into contact, a hybridization reaction occurs, and the formation of a nucleic acid structure is carried out. "Nucleic acid structure" means, as herein it means, the nucleotide configuration formed by the hybridization of at least one padlock and the target nucleic acid, where the 3' and 5' ends of the padlock are brought closer to each other by the hybridization, and the padlock then takes on a cyclizable form. "Cyclizable form" means, as herein it means, the padlock conformation, where the padlock includes ligable 5' and 3' ends that can hybridize with the target nucleic acid and be ligated to close into a ring. The padlock in the cyclizable form is also referred to herein as a "cyclizable padlock" and is a target-specific, ligation-dependent cyclizable probe. Therefore, the nucleic acid structure in step a) is a structure formed by a padlock of a cyclizable conformation hybridized with the target nucleic acid.

[0032] In one embodiment, the padlock is added in the form of a single oligonucleotide molecule that becomes a cyclizable probe when hybridized with the target nucleic acid, thereby obtaining the nucleic acid structure of step a). In this case, the cyclizable form of the padlock is a single target-specific ligation-dependent cyclizable probe.

[0033] In another embodiment, the padlock is added as a system of two or more different oligonucleotide molecules that are arranged cyclically when hybridized with the target nucleic acid, thereby obtaining the nucleic acid structure of step a). In this specification, “a system of two or more different oligonucleotide molecules” means independent oligonucleotide molecules containing different sequences and thus representing different parts or fragments of the padlock. In this case, the padlock in a cyclizable form is a cluster of several molecules that form a target-specific ligation-dependent cyclizable probe.

[0034] The nucleotide sequences of a system of two or more different oligonucleotide molecules are designed so that when one or more of them hybridize with a target nucleic acid, they combine to form a padlock that can be cyclically assembled. This results in the nucleic acid structure of step a). This assembly can be achieved by including complementary sequences in each of the above oligonucleotide sequences, so that they are arranged in a specific order by hybridization. This assembly can also be achieved by including other splint molecules that hybridize with two of the above molecules and unite them. Thus, a “splint nucleic acid molecule” is a nucleotide sequence that bridges both ends of the padlock molecule or system of molecules, and helps facilitate the ligation of the ends of the above molecules in step b).

[0035] When a cyclizable padlock is added as a single oligonucleotide, it has one nick located in the region that hybridizes with the target nucleic acid, i.e., between its 5' and 3' ends. When a cyclizable padlock is added as a system of two or more different oligonucleotide molecules, its sequence has one nick at each junction between the two molecules and one nick at the position that hybridizes with the target nucleic acid. Thus, a cyclizable padlock added as a system of two or more molecules may have two or more nicks in its sequence. Therefore, the padlock of the cyclizable padlock or cyclizable conformation provided in the nucleic acid structure of step a) should not be interpreted only as a single continuous molecule, but rather as a single-stranded nucleic acid circular probe in which one or more molecules are arranged in a ring, have one or more nicks in their sequence, and are ligated to each other in step b) and covalently closed when at least one ligase is added. Preferably, the covalently closed single-stranded nucleic acid circular probe described above is a single-stranded nucleic acid circular probe closed by the covalent bonds of DNA. Therefore, the padlock of the cyclizable conformation of the nucleic acid structure in step a) does not have to be a single molecule, but can be several molecules arranged in a specific order that results in a cyclizable structure. Figure 1 shows a preferred nucleic acid structure of the present invention, which is further described below.

[0036] Each of the different molecules forming the padlock system may be added to the sample simultaneously or sequentially. They may also be added in a state of preliminary hybridization with one another, as in the case of Skinlock or dsSplitlock, which are described in detail below. Suitable hybridization conditions are known in the art. Thus, step a) of the method of the present invention comprises contacting a sample containing a target nucleic acid with at least one padlock to provide a nucleic acid structure formed by hybridization of at least one padlock with the target nucleic acid, wherein the padlock takes a cyclizable form.

[0037] In one embodiment, at least one cyclizable padlock of the present invention is characterized by containing sequences A, B, C, and C in the 5' to 3' direction, as shown in Figure 1A. "Sequence A" as used herein refers to a nucleotide sequence in the 5' region of the cyclizable padlock, preferably located at the 5' end, and complementary (including substantially complementary, see definition above) to the first portion of the target nucleic acid. "Sequence C" as used herein refers to a nucleotide sequence in the 3' region of the cyclizable padlock, preferably located at the 3' end, and complementary (including substantially complementary, see definition above) to the second portion of the target nucleic acid. "Sequence B" as used herein refers to a nucleotide sequence located between sequences A and C, and not complementary (including substantially complementary, see definition above) to the target nucleic acid. Preferably, the different sequences of the padlock are defined in the context of the nucleic acid structure of step a), as shown in Figure 1, i.e., when the target nucleic acid is shown in the 5' to 3' direction.

[0038] In this specification, “in the 5' region of the padlock” and “in the 3' region of the padlock” mean that, when the padlock (or one of the molecules constituting a system of two or more different oligonucleotide molecules) is arranged in a cyclizable conformation, sequences A and C are located toward or near the 5' end or 3' end of the padlock, respectively. While it is preferable that sequences A and / or C are located at the 5' and 3' ends of the padlock, respectively, in some other embodiments, sequences A and / or C may be located near or adjacent to the 5' and 3' ends of the padlock, thereby allowing for the presence of further nucleotide sequences located at the 5' or 3' end that are not complementary to the target nucleic acid. This is possible as long as sequences A and C of the padlock hybridize to the first and second parts of the target nucleic acid, respectively, under appropriate hybridization conditions, resulting in a padlock having a cyclizable conformation. Appropriate hybridization conditions are well known in the art.

[0039] In one embodiment, sequences A and / or C of the cyclizable padlock are contained within 30 nucleotides, 25 nucleotides, 20 nucleotides, preferably 15 nucleotides, located at the 5' and / or 3' ends of the cyclizable padlock. In one embodiment, sequence A of the cyclizable padlock is located less than 50 nucleotides, less than 40 nucleotides, less than 30 nucleotides, less than 25 nucleotides, less than 20 nucleotides, less than 15 nucleotides, less than 10 nucleotides, less than 9 nucleotides, less than 8 nucleotides, less than 7 nucleotides, less than 6 nucleotides, less than 5 nucleotides, less than 4 nucleotides, less than 3 nucleotides, less than 2 nucleotides, or 1 nucleotide from the 5' end of the padlock. More preferably, sequence A is located precisely at the 5' end of the padlock of the cyclizable conformation. In one embodiment, sequence C of the cyclizable padlock is located less than 50 nucleotides, less than 40 nucleotides, less than 30 nucleotides, less than 25 nucleotides, less than 20 nucleotides, less than 15 nucleotides, less than 10 nucleotides, less than 9 nucleotides, less than 8 nucleotides, less than 7 nucleotides, less than 6 nucleotides, less than 5 nucleotides, less than 4 nucleotides, less than 3 nucleotides, less than 2 nucleotides, or 1 nucleotide from the 3' end of the padlock probe. More preferably, sequence C of the padlock probe is located precisely at the 3' end of the padlock of the cyclizable conformation.

[0040] In one embodiment, the circularly cyclizable padlock sequence A and / or sequence C comprises nucleotide sequences having at least 70%, 75%, 80%, 85%, preferably 90%, 92%, 94%, 96%, 98%, or 100% complementarity to the first and second portions of the target nucleic acid, respectively.

[0041] In a preferred embodiment, the 5' region, preferably the 5' end, of the cyclizable padlock of the present invention is characterized by containing sequence A. In a preferred embodiment, the 3' region, preferably the 3' end, of the cyclizable padlock of the present invention is characterized by containing sequence C. As will be described in detail below, sequences A and / or C of the padlock may have any suitable length, as long as the padlock stably hybridizes to the target nucleic acid sequence and results in the cyclization of the probe. For example, sequences A and / or C may be 6 to 20 nucleotides in length. To ensure binding specificity, a minimum size of 6 nucleotides is selected. Therefore, the respective size ranges of sequences A and / or C are preferably 6 to 20 nucleotides, 6 to 19 nucleotides, 6 to 18 nucleotides, 6 to 17 nucleotides, 6 to 16 nucleotides, 6 to 15 nucleotides, 6 to 14 nucleotides, 6 to 13 nucleotides, 6 to 12 nucleotides, 6 to 11 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 6 to 10 nucleotides in length. Preferably, sequence A and / or sequence C are 15 nucleotides long. Sequences A and C may be the same or different in length, and therefore, for example, the probe may have target-complementary regions of any combination of integers within the above range, such as 6+6, 6+7, 7+7, 6+8, 7+8, 8+8, 8+9, 7+9, 9+9, 10+9, 10+10, 10+11, 11+11, 11+12, 12+12, etc. Sequences A and / or C may be contained in different molecules if the padlock is added in the form of two or more different molecules.

[0042] The total length of the hybridized region (i.e., the sum of the nucleotides forming sequence A and sequence C) is 12–50 nucleotides, more specifically 12–40 nucleotides, even more specifically 12–36 nucleotides, 12–32 nucleotides, or 12–30 nucleotides, 12–28 nucleotides, 12–26 nucleotides, 12–24 nucleotides, 12–23 nucleotides, 12–22 nucleotides, 12–21 nucleotides, 12–20 nucleotides, 12–19 nucleotides, 12–18 nucleotides, 12–17 nucleotides, 12–16 nucleotides, or 10 nucleotides. 13-30 nucleotides, 13-28 nucleotides, 13-26 nucleotides, 13-24 nucleotides, 13-23 nucleotides, 13-22 nucleotides, 13-21 nucleotides, 13-20 nucleotides, 13-19 nucleotides, 13-18 nucleotides, 13-17 nucleotides, 13-16 nucleotides, 14-30 nucleotides, 14-28 nucleotides, 14-26 nucleotides, 14-24 nucleotides, 12-23 nucleotides, 14-22 nucleotides, 14-2 The number of nucleotides may be 1, 14-20, 12-19, 14-18, 14-17, 14-16, 15-30, 15-28, 15-26, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, or 15-16. Preferably, the total length of the hybridized region is 20-40 nucleotides, preferably 30 nucleotides.

[0043] Sequence A is complementary to the first portion of the target nucleotide and is hybridizable with the first portion. Sequence C is complementary to the second portion of the target nucleotide and is hybridizable with the second portion. Therefore, the first and second portions of the target nucleotide are complementary to sequences A and C of the padlock, respectively. This is essential because, as described above, when the padlock hybridizes with the target, it takes on a cyclic form, and its 5' and 3' ends unite to form the nucleic acid structure of step a). Therefore, sequences A and C located in the 5' and 3' regions of the cyclizable padlock, preferably at the 5' and 3' ends, are complementary to adjacent or substantially adjacent sequences in the target nucleic acid, are hybridizable, and hybridization with the target nucleic acid in step a) of the padlock (as a single oligonucleotide molecule or as a system of two or more different oligonucleotide molecules) results in juxtaposition of the 5' and 3' ends of the padlock, resulting in a cyclizable conformation of the padlock, but with a nucleic acid structure formed that contains at least one nick between sequences A and C. Where the term “adjacent” is used herein, it is intended to mean that there are no nucleotides in the target sequence that remain unpaired (i.e., 0 nucleotides) between the first and second parts of the target nucleic acid sequence that form base pairs with sequences A and C of the padlock, respectively. This proximity between the first and second parts of the target nucleotide allows the 5' and 3' ends of the padlock to be close to each other. Therefore, the first and second portions of the target nucleic acid are close to each other but do not overlap. Preferably, the first and second portions are in a continuous sequence, i.e., they are directly adjacent to each other, in which case the first portion of the target nucleic acid is located 5' to the second portion of the target nucleic acid, or in other words, the second portion of the target nucleic acid is located 3' to the first portion of the target nucleic acid. As a result, the first portion of the target nucleic acid is located closer to the 5' end of the target nucleic acid than the second portion, and the second portion of the target nucleic acid is located closer to the 3' end of the target nucleic acid than the first portion.

[0044] As will be readily apparent to those skilled in the art, when selecting a first and second portion of a target nucleotide to design the padlock sequences A and / or C, a gap between the first and second portions of the target nucleic acid may be introduced between the nucleotides that the probe sequences A and / or C hybridize. In this situation, the first and second portions of the target nucleic acid are described as “substantially adjacent.” This is because there may be some nucleotides that remain unpaired with the padlock sequences A and C. Preferably, “substantially adjacent” as used herein means that there may be up to four nucleotides, preferably up to three, two, or one nucleotide, in the target nucleic acid that remains unpaired with the padlock between the first and second portions. Therefore, it is preferable that the padlock sequences A and C hybridize adjacently within the target nucleic acid, i.e., that no nucleotides remain between the first and second portions of the target nucleic acid (0 nucleotides). However, the padlock sequences A and C may be separated by up to 4 nucleotides, up to 3 nucleotides, up to 2 nucleotides, or 1 nucleotide, and the term “substantially adjacent” in this invention is intended to refer to such the latter situation. In one embodiment, the first portion of the target nucleotide is located at the 5' end of the target nucleic acid, and the second portion is located at the 3' end of the target nucleic acid. See Figure 1.

[0045] The padlock of the present invention further comprises a backbone sequence that is not complementary or substantially complementary to the target nucleic acid, and is referred to herein as sequence B. The length of this sequence is not particularly limited, provided that it is long enough to hybridize with the target nucleic acid and form a ligable circular structure. Therefore, it is preferable that sequence B is longer than the combined length of sequences A and C. This gives the padlock flexibility and allows it to hybridize more easily with the target nucleotide in a cyclizable conformation. Preferably, sequence B is at least 30 nucleotides long, preferably at least 40, 50, 60, 70, or 80 nucleotides long, and more preferably 30-50, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, or 30-100 nucleotides long. In one embodiment, sequence B is characterized by being non-complementary to the target nucleic acid and complementary to the sequences of one or more primers used in the method of the present invention. That is, one or more primers used in the present invention, as further defined below, preferably hybridize or anneal at sequence B and / or its reverse complementary sequence. Similar to sequence A and / or sequence C, sequence B may be contained in different molecules if the padlock is added in the form of two or more different molecules.

[0046] Furthermore, as described above, the padlock may be added together with other molecules referred to herein as “splint nucleic acid molecules.” In one embodiment, the splint nucleic acid molecule comprises a first domain that hybridizes with a region of sequence A, B, or C, and a second domain that hybridizes with another region of sequence A, B, or C, thereby facilitating the formation of a cyclic arrangement of these regions.

[0047] To further improve the detection limits of the HRCA method, the inventors focused on implementing different modifications to the padlock that, individually or in combination, could reduce background noise and / or increase target-specific amplification. The modifications include various forms of “adding at least one padlock” in step a) and various forms of “resulting in nucleic acid structures,” as further described below.

[0048] As described above, all cyclic padlocks contain the sequence ABC in the 5' to 3' direction. However, one improvement is to add at least one padlock containing an additional sequence other than ABC, either as a single molecule or as a system of two or more different molecules. The additional sequence may be sequence E (skin) and / or sequence D (flap). The following embodiments define such an improved padlock.

[0049] (Skinlock) As described above, at least one padlock can be added in step a) as a system of two or more different oligonucleotide molecules designed to bind to each other using sequences A and C and hybridize to the target nucleic acid. In the case of a padlock having a skin or skinlock, the padlock is added as a system of at least two different oligonucleotide molecules, where one of the molecules is a skin sequence, also referred to herein as sequence E. Sequence E is complementary to sequence B or a region of sequence B. A skinlock was used in Example 7 and is shown in Figure 1B. The advantages of using a padlock having sequence E in the HRCA method of the present invention can be summarized as follows: i) Avoid premature primer annealing, which can cause nonspecific RCA or MDA amplification before the target-dependent RCA response is triggered, by completely or partially covering sequence B of the padlock. This reduces nonspecific amplification. ii) Furthermore, the skin limits hybridization between the remaining padlock and the initial RCA product, preventing it from competing with the MDA primer and inhibiting the HCRA reaction. This improves the efficiency of the amplification step. iii) Finally, when a 3'OH group is added to the skin or sequence E, it can be used as a primer to initiate RCA, thereby improving the efficiency of the method without compromising specificity. The efficiency of the amplification step is also improved. Therefore, the presence of the padlock and hybridized skin reduces the background signal during the amplification process, as shown in Example 7 and Figure 7.

[0050] In one embodiment, sequence E is complementary to the entire length of sequence B. In another preferred embodiment, sequence E is not complementary to the entire length of sequence B, but is complementary to a region contained in sequence B, and therefore not all nucleotides of sequence B are hybridized with sequence E. Preferably, sequence E is complementary to a region of sequence B, in which case the region is the primer hybridization region of the primer used in the MDA reaction of step c), and therefore, if sequence E is hybridized with sequence B, the primer cannot hybridize with sequence B. Preferably, sequence E is complementary to the central region of sequence B, but not to the 3' and 5' regions of sequence B. The fact that there are nucleotides in the 3' and 5' regions of sequence B, preferably at the 5' and 3' ends, that are not complementary to sequence E provides flexibility to the padlock during cyclization and hybridization with the target nucleic acid. Therefore, in one embodiment, sequence E is shorter than sequence B. In one embodiment, sequence E is complementary to a region of sequence B, in which case the region of sequence B is neither the 3' end nor the 5' end of sequence B. Preferably, sequence E is complementary to a region of sequence B, in which case the region of sequence B is at a distance of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 3' or 5' end of sequence B. In one embodiment, sequence E is complementary to a region of sequence B, in which case the region represents at least 50%, 60%, 70%, 80%, or 90% of the length of sequence B. In one embodiment, sequence E is DNA or RNA.

[0051] In one embodiment, sequence E is designed to hybridize with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of sequence B. In one embodiment, the length of sequence E is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of sequence B. For example, if sequence B is 50 nucleotides, then sequence E having at least 80% of the length is sequence E containing at least 40 nucleotides, thereby covering 80% of sequence B when hybridized with sequence B. In this way, sequence E protects sequence B from hybridization with undesirable primers. When referring to sequence E, note that this includes sequences E1 and E2 in the case of padlocks having splits in the sequence (see dsSplitlocks below). In this case, if sequence B is 50 nucleotides, then sequence E having a split and being at least 80% of the length is sequence E containing at least 40 nucleotides, for example, 30 nucleotides from sequence E1 and 10 nucleotides from sequence E2, thereby covering 80% of sequence B when hybridized with sequence B.

[0052] In one embodiment, sequence E includes a modified nucleotide at its 3' end, thereby preventing sequence E from priming the RCA reaction. In one embodiment, the modified nucleotide includes a molecule that prevents the nucleotide from being used as a polymerization starting point (i.e., primer). In one embodiment, the modification of the nucleotide at the 3' end of sequence E is to bind to a molecule that prevents the 3' end from being used as a primer by the polymerase of the reaction. Preferably, the 3' end of sequence E is covalently bonded to biotin. In an alternative embodiment, sequence E is used as a primer in the RCA reaction of step c). To facilitate priming using the 3' end of sequence E, the nucleotide located at the 3' end of sequence E includes a 3'OH group.

[0053] Preferably, array E is: - At least 60%, 70%, preferably 80%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of array B, and / or - It contains a modified nucleotide at the 3' end, which prevents sequence E from priming the RCA reaction.

[0054] In its most preferred embodiment, the padlock of the present invention is a Skinlock, which is added in step a) in the form of a system of two different oligonucleotide molecules. In the system, the first molecule comprises the nucleotide sequence A, B, C in the 5' to 3' direction, where: - A is a nucleotide sequence located in the 5' region of the padlock, complementary to the first portion of the target nucleic acid, and capable of hybridizing with the first portion. - B is a nucleotide sequence that is not complementary to the target nucleic acid. - C is a nucleotide sequence located in the 3' region of the padlock, complementary to the second portion of the target nucleic acid, and capable of hybridizing with the second portion. Furthermore, the second oligonucleotide molecule comprises nucleotide sequence E and is complementary to nucleotide sequence B or a portion thereof, and further, the nucleic acid structure of step a) is obtained by hybridizing the first molecule with the target nucleic acid and the second molecule with the first molecule, thereby obtaining the padlock in a cyclizable form. The cyclizable padlock has at least one nick in its sequence and is located in the junction region of sequences A and C. The first and second molecules can be added to the sample simultaneously or sequentially. When the padlock comprises sequence E, it is preferable to add the padlock to the sample with sequence E already hybridized with sequence B, i.e., the padlock is preferably added in a pre-hybridized form.

[0055] Array E is compatible with and can be added to any padlock as defined in this invention.

[0056] (Flaplock) These padlocks were examined in Example 9 and are shown in Figures 1C and D. They are added in step a) which includes an additional sequence (named herein sequence D or flap) located at the 3' and / or 5' ends of the padlock and not complementary to the target nucleic acid. Sequence D protrudes from the ends of the padlock and prevents ligation of the 3' end of sequence C and the 5' end of sequence A until sequence D is removed by cleavage after the padlock has hybridized with the target. Thus, if sequence D is located at the 3' end of sequence C and the padlock has hybridized with the target nucleic acid, the 3' end of the padlock cannot ligate with the 5' end until sequence D is cleaved. Similarly, if sequence D is located at the 5' end of sequence A and the padlock has hybridized with the target nucleic acid, the 5' end of the padlock cannot ligate with the 3' end until sequence D is cleaved. However, note that the presence of sequence D at either the 3' or 5' end of the padlock does not prevent the hybridization of sequences A and C to the target nucleic acid. Sequence D prevents the formation of the padlock in a cyclizable form and thus prevents the formation of the nucleic acid structure in step a). Therefore, when a padlock having sequence D hybridizes with the target nucleic acid, it cannot proceed with the ligation of the 5' end of sequence A and the 3' end of sequence C in step b).

[0057] In this case, the junction of the 5' end of A and the 3' end of C is "created" by nucleolytic cleavage of the 3' or 5' end of sequence D. Thus, the padlock may include an additional sequence D at its 5' or 3' end, which, when hybridized with the target nucleic acid, results in a 5' flap that makes the 5'-3' end non-ligatable. The end having the additional sequence D or flap becomes ligatable when sequence D or flap is removed by cleavage. When sequence D is located at the 5' end of the padlock, the padlock includes sequence A located inside its 5' end, and when the padlock hybridizes with the target nucleic acid, the presence of sequence D makes the 5' end of the padlock non-ligable to its 3' end. When the internal sequence A hybridizes with the target nucleic acid, sequence D is removed by cleavage due to nucleolysis, thereby creating a 5' ligable end of sequence A of the padlock.

[0058] When sequence D is located at the 3' end of the padlock, the padlock includes sequence C located inside its 3' end, and when the padlock hybridizes with the target nucleic acid, sequence D cannot hybridize with the target nucleic acid, so the 3' end of the padlock forms a 3' flap that cannot ligate with its 5' end. When the internal sequence C hybridizes with the target nucleic acid, sequence D is removed by cleavage due to nucleolysis, thereby creating a 3' ligable end of sequence C in the padlock.

[0059] A padlock having sequence D (i.e., a padlock in which sequence A or C is located inside the 5' or 3' end of the padlock, respectively) and hybridized with the target nucleotide is considered a non-cyclizable padlock because it needs to be activated (i.e., cleaved by nucleic degradation) before forming the nucleic acid structure of step a). Therefore, only if sequence D is cleaved after the padlock has hybridized with the target nucleic acid can the padlock adopt a cyclizable form, obtain the structure of step a), and allow step b) of the method to proceed. In this specification, “located inside” means that sequence A or C is located in a region adjacent to sequence D, rather than being located at the 5' or 3' end of the padlock, respectively.

[0060] The use of the flap sequence or sequence D in the padlock is advantageous when using a sequence-based detection method. This is because it allows for the distinction between target-specific amplicons and non-specific amplicons: if the target nucleic acid is present in the sample and the padlock hybridizes to the target nucleic acid, sequence D is removed by cleavage, and the 5' end of sequence A and the 3' end of sequence C in the padlock are ligated. Subsequently, the padlock is amplified in step c) without sequence D. Therefore, the target-specific amplicon will lack sequence D. However, if the padlock sequence does not hybridize with the target nucleic acid (either because the target nucleic acid is absent or because there is an excess of padlock compared to the target nucleic acid), the ligase ligates the end of sequence D to the end of sequence A or C, thereby circularizing the padlock with sequence D. This padlock containing sequence D is amplified, generating a nonspecific amplicon containing sequence D. As a result, two amplification products with different nucleotide sequences are obtained: one derived from the padlock that hybridized with the target nucleic acid and lacking sequence D, and the other derived from the nonspecific padlock that did not hybridize with the target nucleic acid and therefore containing sequence D. The sequence-specific detection method allows for efficient identification of the amplicon hybridized with the target nucleic acid, while nonspecific amplicons with different sequences are ignored.

[0061] The cleavage of sequence D after the padlock has hybridized with the target nucleic acid can be carried out by the action of a single-stranded DNA-specific exonuclease that degrades sequence D down to the hybridized sequence A or C. If sequence D is located at the 5' end of sequence A, the 5' sequence D can be cleaved using an enzyme having 5' exonuclease activity. If sequence D is located at the 3' end of sequence C, the 3' sequence D can be cleaved using an enzyme having 3' exonuclease activity. These 5' or 3' exonucleases degrade single-stranded oligonucleotides with a free 5' or 3' end from that 5' or 3' end to the constituent mononucleotides. Alternatively, structure-specific nucleases such as flap endonucleases (FENS), which are a class of enzymes that have endonucleolytic activity and can catalyze hydrolytic cleavage of phosphodiester bonds at the junctions of single-stranded and double-stranded DNA, can also be used. Similar to the exonuclease described above, the FEN used must have 3' or 5' polarity depending on the position of D. It is preferable to use an exonuclease not only to remove sequence D of the hybridized padlock but also to decompose any excess of the non-hybridized padlock.

[0062] The length of sequence D is not limited, as long as the padlock can hybridize with the target nucleic acid through sequences A and C, and as long as, in the presence of sequence D, sequence D prevents ligation of the 5' and 3' ends of the padlock when the padlock hybridizes with the target nucleic acid. In one embodiment, sequence D is 5-50, 5-40, 5-30, 10-40, 10-30, 5-10, 5-15, 10-15, or 10-20 nucleotides long. Furthermore, the specific nucleotide sequence of sequence D is not limited and may be any sequence, provided that it does not hybridize with the target nucleic acid and, preferably, does not hybridize with or exhibit complementarity with any molecules present during the reaction.

[0063] In the most preferred embodiment, the padlock of the present invention added in step a) is a flaplock comprising the nucleotide sequence ABC in the 5' to 3' direction, where: - A is a nucleotide sequence located in the 5' region of the padlock, complementary to the first portion of the target nucleic acid, and capable of hybridizing with the first portion. - B is a nucleotide sequence that is not complementary to the target nucleic acid. - C is a nucleotide sequence located in the 3' region of the padlock, complementary to the second portion of the target nucleic acid, and capable of hybridizing with the second portion. Here, the Flaplock includes a nucleotide sequence D located at the 3' end of sequence C and / or the 5' end of sequence A, characterized in that sequence D is not complementary to the target nucleic acid. Furthermore, the nucleic acid structure of step a) having the padlock in a cyclizable form is preferably obtained by hybridizing the Flaplock, which is in the form of a single oligonucleotide molecule, with the target nucleic acid, and then preferably by cleaving sequence D by nucleolysis using an ssDNA-specific exonuclease or FLAP endonuclease. The cyclizable padlock has at least one nick in its sequence and is located in the junction region of sequences A and C.

[0064] In one embodiment, the Flaplock is added to the reaction as a single oligonucleotide molecule containing sequences A, B, C, and C in the 5' to 3' direction, wherein the padlock further contains sequence D, where sequence D is located at the 5' end of sequence A or the 3' end of sequence C, wherein sequence D is characterized by being non-complementary to the target nucleic acid, and wherein the nucleic acid structure of step a) having the padlock in a cyclizable form is obtained by hybridizing the first molecule with the target nucleic acid, and then by cleaving sequence D by nucleolysis with the action of an ssDNA-specific exonuclease or FLAP endonuclease. The cyclizable padlock has at least one nick in its sequence and is located in the junction region of sequences A and C.

[0065] In other embodiments, Flaplock is added to the reaction in the form of a system of two different oligonucleotide molecules. In this system, the first molecule contains the sequence ABCD as shown in Figure 1D, or DABC as shown in Figure 1C, in the 5' to 3' direction. The second oligonucleotide molecule contains the sequence E. Where: - The first molecule and the second molecule are added separately to the sample (in any order), and the nucleic acid structure of step a) is formed by hybridizing the first molecule with the target nucleic acid and hybridizing the second molecule with the first molecule, and then by exonucleolytic cleaving of sequence D by the action of an ssDNA-specific exonuclease or FLAP endonuclease. - The first molecule and the second molecule are added to the sample in a hybridized state, and the nucleic acid structure of step a) is formed by hybridizing the first molecule with the target nucleic acid, and then by cleaving sequence D by exonucleic acid degradation using an ssDNA-specific exonuclease or FLAP endonuclease.

[0066] The cyclic padlock has at least one nick in its sequence and is located in the junction region of sequences A and C. The first and second molecules can be added to the sample simultaneously or sequentially, in a pre-hybridized state (i.e., hybridized with each other). Sequence E has the characteristics defined in the case of Skinlock, and therefore the above embodiments also apply herein and to all places throughout the document where sequence E is mentioned.

[0067] Preferably, the exonuclease is a 5' exonuclease when sequence D is located at the 5' end of sequence A. Preferably, the exonuclease is a 3' exonuclease when sequence D is located at the 3' end of sequence C. Preferably, the 3' exonuclease is selected from the 3' exonuclease activity of Exo I or phi29 polymerase.

[0068] Preferably, at least one padlock is at least one flaplock, and the method is a.1) Adding the Flaplock to the sample and hybridizing at least one padlock with the target nucleic acid, Step a.2) includes obtaining the nucleic acid structure of step a) by cleaving the 5' or 3' of sequence D by nucleic acid degradation, This includes step a). Array D is compatible with and can be added to any padlock as defined in this invention.

[0069] Preferably, the Flaplock comprises a nucleotide sequence E, where sequence E is complementary to a region of sequence B or a portion thereof, and where: - The length of array E is at least 60%, 70%, preferably 80%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of array B, and / or - Sequence E contains a modified nucleotide at its 3' end, which prevents sequence E from priming the RCA reaction. Here, the cyclizable padlock is obtained by hybridizing the first molecule with the target nucleic acid and the second molecule with the first molecule.

[0070] (A padlock applied in the form of a system of two or more different molecules, where sequence ABC is divided into two or more oligonucleotide molecules.)

[0071] In one embodiment, at least one padlock sequence ABC of the present invention is added in step a) and is divided into or contained in different oligonucleotide molecules. When sequence ABC is added in one or more parts as a system of different oligonucleotide molecules, the different oligonucleotide molecules containing sequences A and / or B and / or C each need to contain or generate ligateable 5' and 3' ends (i.e., by cleavage or extension). This allows sequence ABC to be ligated and bound in this order (5' to 3') upon addition of ligase in step b), obtaining a cyclic padlock in step b) which can then be amplified in step c). Providing the padlock sequence ABC in two or more different molecules has a general advantage in that it limits auto-cyclization by reducing the size of the molecules. Most importantly, dividing the sequences required for different steps of the reaction (primer binding sites, complementary target sequences, etc.) into different molecules facilitates amplification and detection of only correctly assembled molecules, as opposed to random ligation and spurious priming events.

[0072] The padlock system containing sequences A, B, C, and D in two or more different molecular forms preferably comprises at least two molecules, each containing sequences A and / or C, thereby enabling hybridization with the target nucleic acid. Therefore, although not essential, it is preferable that each of the different molecules has at least one target-specific binding site, sequence A and / or C, or a portion of sequence A and / or C. In these systems, if sequences A, B, C, and D are contained in two or more different molecular forms, a sprint nucleic acid molecule may be present to bridge the gap between the different molecules, functioning as a bridge and facilitating the cyclizable conformation of the padlock. The sprint nucleic acid molecule may be the target nucleic acid, sequence E as described above, and / or other molecules. The sprint molecule guides the assembly of sequences A, B, and C of the padlock in the correct direction (sequences A, B, C, and D in the 5' to 3' direction), so that the cyclizable padlock of the nucleic acid structure in step a) contains two or more nicks between sequences A, B, C, and D. However, the molecule containing sequence ABC and forming the cyclizable padlock contains a ligateable end and is therefore ligateable in step b), forming a single cyclized molecule.

[0073] In one embodiment, sequences A, B, and C of the padlock are contained in two or more different molecules, and all molecules come into contact with the sample simultaneously. In one embodiment, sequences A, B, and C of the padlock are contained in two or more different molecules, and at least two different molecules come into contact with the sample sequentially. In a preferred embodiment, the A-containing and C-containing molecules first come into contact with the sample, enabling hybridization with the target nucleic acid (if present). Subsequently, an additional sprint nucleic acid molecule or molecule (e.g., sequence E) is added to the sample to further bridge sequences A and C to obtain the nucleic acid structure of step a). In another preferred embodiment, the A-containing and C-containing molecules are pre-hybridized with sequence E before being added to the sample, and the system of the two molecules is added to the sample in a pre-hybridized form.

[0074] The following padlock is a preferred embodiment of the padlock of the present invention, wherein sequence ABC is contained in at least two, preferably two different molecules: Gaplock: These padlocks were examined in Example 10 and are shown in Figures 1E and 1D. They are added to the reaction as a system of two or more different molecules, where the first molecule of the Gaplock system contains sequence B and portions of sequences A and C, and the second and subsequent molecules contain the remainder of sequences A and / or C. Hybridization of the first molecule's portions of sequences A and C results in an unhybridized gap in the target nucleic acid, which is filled by hybridization of the second molecule or further molecules. When the first molecule and the second and subsequent molecules hybridize with the target nucleic acid, the Gaplock probe adopts a cyclizable form to obtain the nucleic acid structure of step a). In these Gaplocks, the target nucleic acid functions as a sprint nucleic acid molecule for ligation, which requires the binding of the first molecule and the second and subsequent molecules, to obtain sequences ABC in a single molecule in step b). Therefore, in this system of two or more molecules, additional molecules other than those containing A, B, and C may not be necessary.

[0075] The effectiveness of these padlock and sequence-specific detection method combinations lies in the fact that only hybridized Gaplocks contain the sequence obtained in the second or further molecule, and therefore they are readily identifiable by the sequence-specific method of step d). Gaplock probes that do not hybridize with the target nucleic acid or are ligated without the target nucleic acid do not exhibit the sequence obtained in a different second or further molecule. Thus, while target-specific amplicons derived from Flaplock are detected by the absence of sequence D or flap, target-specific amplicons derived from Gaplock are detected by the presence of the sequence obtained in the second or further molecule that fills the gap between the 3' and 5' ends of the first molecule of the padlock system.

[0076] In the Gaplock example shown in Figure 1E, the split is performed on array C: In this preferred embodiment, the sequence ABC of Gaplock is added in the form of a system of at least two different oligonucleotide molecules, where the first molecule contains sequences A, B, and the first part (C1) of sequence C in the 5'-3' direction, and the second molecule contains the second part (C2) of sequence C. Thus, in this preferred embodiment of Gaplock, the first molecule contains the sequence AB-C1 in the 5'-3' direction, and the second molecule contains the sequence C2. Adding a third molecule containing sequence E results in a system of three or more different oligonucleotide molecules, as shown in Figure 1G. In embodiments of Gaplock having a split in sequence C, it is preferable to add the first and second different molecules, each containing the sequences 5'-AB-C1-3' and 5'-C2-3' respectively, to the sample simultaneously in step a). The lengths of C1 and C2 are not limited, as long as they are long enough to hybridize with the target nucleotide. Preferably, C1 and / or C2 are at least 6, 8, 10, 12, or 15 nucleotides in length. The lengths of sequences B and E, if present, are as defined in the embodiments described above. In embodiments of Gaplock having a split in sequence C, the cyclizable padlock in step a) is formed by hybridizing the first and second molecules to the target nucleic acid. The cyclizable padlock has at least two nicks in its sequence, one located in the junction region of sequences C1 and C2 and the other located between sequences A and C2. The first and second molecules can be added to the sample simultaneously or sequentially.

[0077] In another example of Gaplock shown in Figure 1E, the splitting occurs at array A: In this preferred embodiment, the sequence ABC of Gaplock is added in the form of a system of at least two different oligonucleotide molecules, where the first molecule contains the first part of sequence A (A1) followed by sequences B and C in the 5'-3' direction, and where the second molecule contains the second part of sequence A (A2). Thus, in this preferred embodiment of Gaplock, the second molecule contains sequence A2, and the second molecule contains sequence A1-BC in the 5'-3' direction. Adding a third molecule containing sequence E results in a system of three or more different oligonucleotide molecules, as shown in Figure 1G. In embodiments of Gaplock having a split in sequence A, it is preferable to add the first and second different molecules, each containing the sequences 5'-A1-BC-3' and 5'-A2-3' respectively, to the sample simultaneously in step a). The lengths of A1 and A2 are not limited, as long as they are long enough to hybridize with the target nucleotide. Preferably, A1 and / or A2 are at least 6, 8, 10, 12, or 15 nucleotides in length. The lengths of sequences B and E, if present, are as defined in the embodiments described above. In embodiments of Gaplock having a split in sequence A, the cyclizable padlock in step a) is formed by hybridizing the first and second molecules to the target nucleic acid. The cyclizable padlock has at least two nicks in its sequence, one located in the junction region of sequences A1 and A2 and the other located between sequences A2 and C. The first and second molecules can be added to the sample simultaneously or sequentially.

[0078] In the Gaplock example shown in Figure 1E, the split is performed on sequences A and C: In this preferred embodiment, the sequence ABC of Gaplock is added in the form of a system of at least two different oligonucleotide molecules, where the first molecule comprises, in the 5' to 3' direction, a first portion of sequence A (A1), followed by sequence B and a first portion of sequence C (C1); and the second molecule comprises, in the 5' to 3' direction, a second portion of sequence C (C2) and a second portion of sequence A (A2). Thus, in this preferred embodiment of Gaplock, the first molecule comprises the sequence A1-B-C1 in the 5' to 3' direction, and the second molecule comprises the sequence C2-A2 in the 5' to 3' direction. Adding a third molecule containing sequence E results in a system of three or more different oligonucleotide molecules. In embodiments of Gaplock having splits in sequences A and C, it is preferable to add the first and second distinct molecules, each containing the sequences 5'-A1-B-C1-3' and 5'-C2-A2-3', to the sample simultaneously in step a). The lengths of A1, A2, C1, and C2 are not limited, as long as they are long enough to hybridize with the target nucleotide. Preferably, A1, A2, C1, and / or C2 are at least 6, 8, 10, 12, and 15 nucleotides in length. The lengths of sequences B and E, if present, are as defined in the embodiments described above. In embodiments of Gaplock having splits in sequences A and C, the cyclizable padlock in step a) is formed by hybridizing the first and second molecules to the target nucleic acid. The cyclizable padlock has at least two nicks in its sequence, one located in the junction region of sequences A1 and A2 and the other located between sequences C1 and C2. The first and second molecules can be added to the sample simultaneously or sequentially.

[0079] Therefore, in all Gaplocks added in the form of a system of at least two different oligonucleotide molecules, the nucleic acid structure of step a) is obtained when the first and second molecules are hybridized to the target nucleotide, and a cyclizable padlock is obtained. If the Gaplock contains sequence E and is therefore added in step a) as a system of at least three different oligonucleotide molecules, the first, second and third molecules can be added to the sample separately (in any order) or added to the sample already pre-hybridized (hybridized to each other). The nucleic acid structure of step a) having the cyclizable padlock is obtained by hybridizing the first and second molecules with the target nucleic acid and the third molecule with the first molecule. Therefore, the first, second and third molecules can be added simultaneously or sequentially in a pre-hybridized state.

[0080] It is obvious to those skilled in the art that sequences A and C can be split into two or more molecules, and that the ends of these molecules can be ligated to one another, and that the cyclizable padlock in step b) can be a single cyclizable padlock molecule, insofar as different molecules hybridize to the target nucleic acid to obtain a cyclizable padlock of the nucleic acid structure in step a). The lengths of the first, second, third, and further molecules are not limited insofar as they contain the minimum number of nucleotides that enable stable hybridization between each molecule and the target nucleic acid. Under standard conditions, the length is at least 6 nucleotides. If the conditions are modified to facilitate hybridization, this minimum length can be shortened to 5 or 4 nucleotides.

[0081] Preferably, the Gaplock comprises a nucleotide sequence E, where sequence E is complementary to a region of sequence B or a portion thereof, and where: - The length of array E is at least 60%, 70%, preferably 80%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of array B, and / or - Sequence E contains a modified nucleotide at its 3' end, which prevents sequence E from priming the RCA reaction. Here, the cyclizable padlock is obtained by hybridizing the first molecule with the target nucleic acid and the second molecule with the first molecule.

[0082] Splitlock: These padlocks are shown in Example 11 and are represented in Figures 1H, 1I, and 1J. These are padlocks in which at least one splitting occurs in sequence B. Thus, in these padlocks, sequence B is obtained in at least two different oligonucleotide molecules. Since the target nucleic acid does not hybridize with B, the addition of a skin (sequence E) that functions as a sprint nucleic acid molecule bridging the two molecules containing sequence B in the padlock is necessary to make it ligateable in step b).

[0083] One advantage of Splitlock is that two or more different oligonucleotide molecules of the padlock containing sequences ABC ligate to each other to form a circular padlock only if the first and second molecules hybridize with the target nucleotide and at least one third molecule (i.e., sequence E) hybridizes with both the first and second molecules. This brings their ends close together, yielding the cyclizable padlock of step a). This reduces the formation of a padlock dimer (5'-ABCABC-3'), where the second portion of the dimer is already occupied by sequence C of a different padlock molecule, so sequence A of one padlock hybridizes with the first portion of the target nucleic acid, but sequence C of the same padlock cannot hybridize with the second portion of the target nucleic acid. When the ABC padlock sequence is added in the form of a system of at least two molecules, where sequences A and C are obtained in different molecules, the formation of a padlock dimer is limited. This is because the ligase binds two molecules that have independently hybridized to the target molecule by sequences A and C, and to sequence E by segment B, thereby obtaining a single padlock according to the present invention. The Splitlock has the further advantage that, if the MDA primers are designed to hybridize to different molecules containing sequence B, the complete padlock has successfully hybridized and only initiates MDA amplification in the process of step c) when it has adopted a cyclizable form that obtains the nucleic acid structure of step a).

[0084] Based on the above, in a preferred embodiment, sequences A, B, C, and E of the Splitlock are added in the form of a system of at least three different molecules, where the split is located in sequence B, and sequence E (included in the third molecule) is preferably added after hybridization of sequences A and C with the target nucleic acid. This Splitlock is also referred to herein as a single-stranded Splitlock (ssSplitlock) and is shown in Figure 1H. This Splitlock is, A first molecule containing sequence A in the direction from 5' to 3', and the first part (B1) of sequence B, The second part of sequence B (B2) is located in the direction from 5' to 3', and a second molecule containing sequence C is located, and A third molecule containing sequences B1 and B2, or sequence E that hybridizes with a portion of sequences B1 and B2, Includes. Therefore, in a preferred embodiment of this ssSplitlock, the first molecule contains sequences A-B1 in the 5' to 3' direction, the second molecule contains sequences B2-C in the 5' to 3' direction, and the third molecule contains E. In the case of the ssSplitlock, the nucleic acid structure of step a) having the padlock in a cyclizable form is obtained by hybridizing the first and second molecules with the target nucleic acid and hybridizing the third molecule with the first and second molecules. When the cyclizable padlock is added as an ssSplitlock, it has at least two nicks in its sequence, one in the junction region of sequences B1 and B2 and the other located between sequences A and C. Figure 11a shows that the ssSplitlock approach is effective in reducing background signal.

[0085] In a preferred embodiment of this ssSplitlock, in an ssSplitlock having a split in sequence B, it is preferable to add the first and second molecules, each containing the sequences 5'-A-B1-3' and 5'-B2-C-3' respectively, to the sample simultaneously in step a), and then sequentially add the third molecule having sequence E. The third molecule is preferably added after the addition of the first and second molecules so as to be able to bridge (function as a sprint nucleic acid molecule) to obtain a cyclizable padlock included in the nucleic acid structure of step a), which is then joined by ligation in step b). This specific order of adding different molecules using this ssSplitlock is preferred because otherwise the formed structure would not differ significantly from a normal Skinlock, and the advantage of reducing the formation of padlock dimers associated with the cyclization event would be lost. The lengths of B1 and / or B2 are not limited as long as hybridization with E is possible, and are preferably at least 6, 7, 8, 9, 10, 12, 14, 18, 20, 22, 26, 28, or 30 nucleotides in length. The lengths of sequences A, C, and E are as defined in the embodiments described above. Preferably, sequences B1 and B2 include the hybridization and reverse complementary sequences of each MDA primer.

[0086] In a preferred embodiment, the third molecule containing sequence E is also split into at least two distinct molecules. This Splitlock is referred to herein as a double-stranded Splitlock (dsSplitlock) and is shown in Figure 1I. In the dsSplitlock, the padlock is added in the form of a system of at least four or more distinct molecules, where: - The first molecule includes sequence A in the 5' to 3' direction, and the first portion of sequence B (B1), - The second molecule contains the second part of sequence B (B2) and sequence C in the 5' to 3' direction, - The third molecule comprises a first portion (E1) of sequence E, the first portion being preferably complementary to sequence B1 of the first molecule, and - The fourth molecule includes a second portion (E2) of sequence E, the second portion being preferably complementary to sequence B2 of the second molecule.

[0087] Therefore, in this preferred embodiment of dsSplitlock, the first molecule comprises sequence A-B1 in the 5' to 3' direction, the second molecule comprises sequence B2-C in the 5' to 3' direction, the third molecule comprises sequence E1, and the fourth molecule comprises sequence E2.

[0088] In the case of dsSplitlock, the nucleic acid structure of step a) having the padlock in a cyclizable form is obtained by hybridizing the first and second molecules with the target nucleic acid, and by hybridizing the third molecule with the first molecule and the fourth molecule with the second molecule. In the case of dsSplitlock, the four molecules can be added in any order and manner, but it is preferable to add them simultaneously, most preferably by adding the first molecule in a state where it has been pre-hybridized with the third molecule (i.e., pre-hybridizing sequence B1 with sequence E1), and by adding the second molecule in a state where it has been pre-hybridized with the fourth molecule (i.e., hybridizing sequence B2 with sequence E2). Thus, when all molecules are added simultaneously, the nucleic acid structure of step a) having the padlock in a cyclizable form is obtained simply by hybridizing the first and second molecules with the target nucleic acid.

[0089] The circularizable padlock in the nucleic acid structure of step a) has at least three nicks when added as dsSplitlock: one in the junction region of sequences E1 and E2, one in the junction region of sequences B1 and B2, and another in the junction region of sequences A and C.

[0090] The lengths of E1 and E2 are not limited as long as they are long enough to hybridize with sequences B1 and B2. This is a minimum length of 6 nucleotides. Preferably, E1 and E2 are at least 5, 8, 10, 12, and 15 nucleotides long. This system of at least four molecules can be designed so that the splits in B and E coincide. In this case, the junctions between B1 / E1 and B2 / E2 “arms” contain blunt dsDNA ends. Preferably, by shifting the splits in B and E, reverse complementary or “sticky” protrusions are formed at the dsDNA junctions between B1 / E1 and B2 / E2, facilitating ligation and circularization. A padlock with a 5' protrusion at the B1-E1 / B2-E2 junction is shown in Figure 1I, and a padlock with a 3' protrusion at the B1-E1 / B2-E2 junction is shown in Figure 1J. Therefore, in a preferred embodiment, the nucleic acid structure includes a reverse complementary protrusion at the junction between B1 / E1 and B2 / E2 to facilitate ligation and cyclization. Here, the reverse complementary protrusion contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Furthermore, it is preferable that the molecule containing sequences B1 and B2 contains hybridization and reverse complementary sequences of each MDA primer.

[0091] Therefore, in a preferred embodiment, the dsSplitlock comprises sequences A, B, C and E, which are added in step a) in the form of a system of at least four different molecules, where - The first molecule contains the first portion (B1) of sequence A and sequence B in the 5' to 3' direction; - The second molecule contains the second part of sequence B (B2) and sequence C in the 5' to 3' direction, - The third molecule comprises a first portion (E1) of sequence E, where the first portion is complementary to sequence B1 of the first molecule, and - The fourth molecule includes a second portion (E2) of sequence E, where the second portion is complementary to sequence B2 of the second molecule. Here, the first and third molecules are preferably added in a pre-hybridized state, and the second and fourth molecules are also preferably added in a pre-hybridized state, thereby obtaining the nucleic acid structure of step a) having the padlock in a cyclizable form by the hybridization of the first and second molecules with the target nucleic acid and the alignment of the B1-E1 / B2-E2 junctions.

[0092] It is obvious to those skilled in the art that sequence B is divisible at any point in its sequence, as long as each resulting different molecule can hybridize with E or a fragment thereof. The lengths of the first, second, third, fourth, and further molecules are not limited, as long as they contain the minimum number of nucleotides necessary to enable stable hybridization between each molecule and the target nucleic acid.

[0093] Preferably, the sequence E of the Splitlock (including ssSplitlock and dsSplitlock) is complementary to a region of sequence B or a portion thereof, and: - The length of array E is at least 60%, 70%, preferably 80%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of array B, and / or - Sequence E contains a modified nucleotide at its 3' end, which prevents sequence E from priming the RCA reaction. Here, the cyclizable padlock is obtained by hybridizing the first molecule with the target nucleic acid and the second molecule with the first molecule.

[0094] Preferably, when sequence E is obtained in the form of sequences E1 and E2, the modified nucleotide at the 3' end is located at the 3' end of sequence E when sequences E1 and E2 are ligated.

[0095] It is understood that the aforementioned systems of different molecules are not incompatible with the presence of sequence D, as defined above. Therefore, padlocks with additional sequences (Flaplocks) can be combined with padlocks added as a form of a system of different molecules (Skinlocks, Gaplocks, and Splitlocks including ssSplitlocks and dsSplitlocks). In these systems of two or more molecules, discontinuities may be present simultaneously in regions A / C and B, resulting in a composite Gaplock-Splitlock.

[0096] (Nucleotide sequence present in padlock) Padlock sequences A and C are selected based on the sequence of the target nucleic acid molecule to which they are bound. The nucleotide sequences of the other sequences (sequences E, B, or D) are not definitive, but it is desirable that these sequences not only do not hybridize with the target nucleic acid, but also with other nucleotides present during the reaction. Therefore, the sequences should be selected to avoid intermolecular and intramolecular hybridization. Once the sequences have been selected or identified, the padlock can be synthesized using any convenient method.

[0097] The padlocks described herein may take the form of a single molecule or a system of two or more different molecules and may include one or more sequences that function to distinguish target-specific amplification from non-specific or background amplification during amplification after step c). The sequences are referred herein as “detectable templates” and are useful in sequence-based detection methods, as will be described in more detail below. The detectable templates may be present in any sequence of the padlock (including sequences A, B, C, D, and E).

[0098] Particularly preferred is that at least one detectable template is included within sequence A or C, and most preferably, at the junction between sequences A and C, it is reconstructed only in a correctly assembled and ligated padlock. This is particularly preferred in the case of Flaplock and Gaplock, where target-independent reconstruction of the AC sequence is prevented by the inclusion of an additional nucleotide (sequence D) adjacent to A and / or C, or by obtaining sequences A and / or C in different molecules, respectively. A Flaplock or Gaplock that is not properly assembled, processed, and ligated will therefore not contain the correct detectable template, thus limiting the target-independent background signal. In the case of Flaplock, non-specific ligation includes sequence D, which disrupts the AC junction and interferes with the sequence-specific recognition process. In the case of Gaplock, the target-independent reconstruction event results in the absence of sequences A1 and / or C2 from the AC junction, preventing subsequent sequence-specific recognition. Therefore, in these approaches, only correctly assembled target-dependent events result in a complete reconstruction of the detectable template at the AC junction, thereby obtaining further specificity for the system.

[0099] The padlock of the present invention may include one or more, preferably two or more primer or reverse complementary sequence binding sites, and the primer used in the RCA and / or MDA reaction of step c) hybridizes to the binding site of the padlock sequence and / or to the binding site of the chain that is the RCA and / or MDA reaction product. In one embodiment, the primer binding site is located on sequence B of the padlock. If the padlock is a Splitlock and sequence B is contained in two or more different molecules, the primer binding sites are preferably and advantageously located on different molecules containing sequence B and its reverse complementary sequence, respectively, so that only target-specific, cyclized, and ligated padlocks are amplified by MDA.

[0100] (Target nucleic acids and samples) As described above, the method of the present invention aims to detect a target nucleic acid in a sample. In a preferred embodiment, the target nucleic acid indicates the presence of a target analyte in the sample. In this specification, "analyte" means any target molecule to be detected. This includes proteins, nucleic acids, peptides, and the like. Therefore, the detection of the target nucleic acid in this method is an indicator of the presence of an analyte in the sample. In some embodiments, the target nucleic acid is used to directly or indirectly tag or label a target analyte in a sample, and the detection of the nucleic acid molecule serves to indicate the presence of the analyte in the sample. In some methods, when one or more molecules interact with (e.g., bind to) the target analyte, new nucleic acid molecules (i.e., nucleic acid molecules that were not present in the original sample and are not any of the components added to the sample) may be generated in the sample.

[0101] In some other embodiments, the target nucleic acid is itself the analyte of interest. In this case, the target nucleic acid or analyte may be any nucleotide sequence to be detected, analyzed, or amplified. The target nucleic acid or analyte may be a nucleotide sequence derived from the analyte (such as mRNA derived from a viral genome). Target nucleic acids include DNA (e.g., genomic DNA, mitochondrial DNA, plasmid DNA, viral DNA, etc.), RNA (e.g., mRNA, microRNA, rRNA, snRNA, viral RNA, etc.), and synthetic and / or modified nucleic acid molecules (e.g., those comprising or containing synthetic or modified nucleotides such as LNA, PNA, morpholino, etc., or containing nucleic acid domains), or fragments thereof. Thus, the target nucleic acid or analyte may consist of ribonucleotides and / or deoxyribonucleotides, as well as synthetic nucleotides capable of participating in Watson-Crick type or similar base-pair interactions. Therefore, the target nucleic acid or analyte may be, for example, bi-sulphite DNA, LNA, PNA, or other derivatives containing a non-nucleotide backbone.

[0102] Therefore, the target nucleic acid or analyte may be coding region DNA or non-coding region DNA (e.g., genomic DNA or a partial fraction thereof), or derived from genomic DNA (e.g., a copy or amplicon thereof), or cDNA or a partial fraction thereof, or an amplicon or copy thereof, etc.

[0103] As described above, the target nucleic acid or analyte may be a target RNA molecule. The target nucleic acid or analyte may be, for example, RNA or other nucleic acid molecules or RNA molecules in a pool of nucleotide sequences. For example, it may be a genomic nucleic acid from human or any source, a transcriptome-derived nucleic acid or any other nucleic acid (e.g., organelle nucleic acid, i.e., mitochondrial or plasmid nucleic acid). Therefore, the target RNA or analyte may be a coding region RNA sequence (i.e., precursor mRNA or mRNA) or a non-coding region RNA sequence (e.g., tRNA, rRNA, snoRNA, miRNA, siRNA, snRNA, exRNA, piRNA, and long ncRNA), or derived therefrom. The target RNA or analyte is usually an RNA molecule that we want to detect in the sample and has meaning as the target (i.e., analyte) of the assay. In one preferred embodiment, the target nucleic acid or analyte is a microRNA (miRNA). In another preferred embodiment, the target RNA or analyte is 16S RNA, preferably the 16S RNA is derived from a microorganism (e.g., a pathogenic microorganism) in the sample and identifies that microorganism.

[0104] Alternatively, the target RNA or analyte may be genomic RNA (e.g., ssRNA or dsRNA of a virus having RNA as its genetic material). Such viruses include Ebola, HIV, SARS, influenza, hepatitis C, West Nile fever, polio, and measles. Therefore, the target RNA or analyte may be positive-chain RNA, negative-chain RNA, or double-chain RNA derived from a viral genome, or positive-chain RNA derived from a retroviral RNA genome. Preferably, the target nucleic acid or analyte is a viral genome, and preferably the genome of the SARS-CoV-2 virus.

[0105] The target nucleic acid or analyte may be present in the sample. The sample may be any sample containing the target nucleic acid or analyte, including both natural and synthetic samples (i.e., naturally occurring substances or manufactured preparations). All biological and clinical samples are included, for example, cell or tissue samples of organisms, or bodily fluids or preparations derived therefrom, as well as samples such as cell cultures, cell preparations, and cell lysates. The sample may be freshly prepared or pre-treated by any convenient method, such as for preservation.

[0106] The aforementioned sample is a biological sample and may contain viruses or cellular material (including cells of all prokaryotes or eukaryotes, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles). Thus, such biological material may include all types of mammalian and non-mammalian animal cells. Examples of biological samples include tissue samples (such as tissue sections and needle biopsies); cell samples (e.g., cytological smears (e.g., cervical smears (Pap smears) or blood smears (blood smears)) or cell samples obtained by microdissection); whole organism samples (e.g., yeast or bacterial samples); or cell fractions, fragments, or organelles (e.g., obtained by lysing cells and separating their components by centrifugation or other methods). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical or needle biopsy), nipple aspirate, milk, vaginal fluid, saliva, swabs (e.g., oral swabs), or substances containing biomolecules (derived from the biological sample). In some embodiments, the biological sample may be a body fluid, which is a liquid separated from an individual's body. For example, “body fluid” may include blood, plasma, serum, mucus, bile, saliva, nasopharyngeal swabs, urine, tears, sweat (perspiration), or body cavity lavage fluid. Preferably, the sample is a biological sample, and preferably saliva.

[0107] The biological sample can be obtained from a subject requiring analysis. “Subject” is a human being (i.e., male or female of any age, e.g., a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young adult, middle-aged adult, or elderly). Alternatively, the subject may be a non-human animal. In certain embodiments, the non-human animal may be a mammal (e.g., a primate (e.g., a crab-eating macaque or rhesus macaque)), a commercially valuable mammal (e.g., a cattle, pig, horse, sheep, goat, cat, or dog)), or a bird (e.g., a commercially valuable bird (e.g., a chicken, duck, goose, or turkey)). In other examples, the non-human animal may be a fish, reptile, or amphibian. The non-human animal may also be a transgenic or genetically engineered animal, and in some examples, the subject may be a plant.

[0108] In one particular embodiment, the sample comprises microbial cells or viruses isolated from a clinical sample or a culture of a clinical sample (e.g., saliva). In one particular embodiment, the sample comprises microbial cells or viruses isolated from a food or water sample, or a culture of a food or water sample. In such a sample, Padlock's target nucleic acid sequence may be a nucleotide sequence present in the microbial cell or virus (e.g., a nucleotide sequence that characterizes, distinguishes, or identifies the microbial cell or virus at any level (e.g., type, group, rank, genus, species, or strain level)). In one embodiment, the target nucleic acid or analyte is the genome of an infectious agent. In this specification, “infectious agent” means a pathogen that can cause disease in a subject (preferably human) requiring analysis. Preferably, the target nucleic acid or analyte is a viral genome, preferably a single-stranded viral genome, and preferably an RNA single-stranded viral genome. In one embodiment according to the first aspect, or in any embodiment thereof, the viral genome is the SARS-CoV-2 viral genome.

[0109] In one embodiment, if the target is RNA and the padlock is DNA, the nucleic acid structure in a) may be a DNA-RNA structure. Also, if sequences A, B, C are obtained in DNA form but sequence E is an RNA molecule, the nucleic acid structure in a) may be a DNA-RNA hybrid.

[0110] In one embodiment, the padlock of the present invention includes, consists of, or essentially consists of sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence numbers 8, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, or 32.

[0111] (Step b of the method of the present invention includes, or consists of, circulating the hybridized padlock.) Step b) comprises subjecting the cyclizable padlock contained in the nucleic acid structure of step a) to ligation using at least one ligase to obtain a single circular molecule containing sequence ABC, which is a cyclic padlock free of nicks in its sequence. The cyclic padlock is a covalently closed single-stranded DNA circular probe. The ligation step preferably occurs after step a) (i.e., after the nucleic acid structure is obtained). However, it should be noted that reactions a) and b) may overlap (i.e., the ligation step may be initiated before step a) is completely completed).

[0112] In all padlocks, the target nucleic acid functions as a sprint nucleic acid molecule that facilitates ligation between sequences A and / or C. When the padlock comes into contact with the target nucleic acid containing sequences A, B, C, and D in a single molecule (i.e., undivided), the padlock hybridizes with the target nucleic acid, and its 3' and 5' ends are directly paralleled to ligate each other, using the target nucleic acid as a ligation template (i.e., sprint nucleic acid molecule). No further modification or processing of the padlock (e.g., cleavage or additional hybridization) is required. Preferably, if sequences A, B, C, and D are obtained as a single molecule, step b) of the present invention includes, or consists of, a ligation step, which includes ligating the 5' end of sequence A with the 3' end of sequence C. No further steps are required to obtain a ligated cyclic probe, which is a single cyclic molecule containing sequences A, B, C, and D.

[0113] However, the formation of the nucleic acid structure in step a) does not require that the sequence ABC be obtained in a single molecule, because, as described above, one or both of the ligateable ends of sequence A and / or C may be generated in one or more processing or hybridization steps. In some embodiments, the sequence ABC is obtained in two or more different molecules or parts (e.g., two, three, four, five, or more different molecules) and forms two or more ligation junctions. This is the case, for example, with Gaplock, ssSplitlock, and dsSplitlock. Each molecule in the system obtains (i.e., forms or includes) 3' and / or 5' ligateable ends. These ends can be aligned in parallel and ligated to ligate in step b) of the method of the present invention, as outlined above. In certain embodiments, step b) therefore includes two or more different ligation reactions (i.e., reactions between adjacent ligateable ends obtained by two or more different molecules). In this case, the ligation step includes one or more ligation reactions for binding two or more different molecules (containing sequences A, B, and / or C in a single cyclic molecule). As described above, the different molecules containing sequences A, B, C, and P contain ligable ends and are therefore ligable in step b). One ligation reaction is performed for each nick between molecules containing sequences A, B, and / or C.

[0114] Preferably, when the padlock is added in the form of a system of two different molecules in which sequence ABC is split into two molecules (for example, in the case of Gaplock or Splitlock), and when the nucleic acid structure of step a) is obtained, the ligation step b) includes, or consists of, obtaining a padlock having sequence ABC in a single cyclic molecule by ligating the 5' end of the first molecule with the 3' end of the second molecule and the 5' end of the second molecule with the 3' end of the first molecule using at least one ligase. In the case of a padlock in which sequence ABC is added in different oligonucleotide molecules, the ligation to obtain sequence ABC in a single cyclic molecule may be carried out simultaneously or sequentially, preferably after the molecules have hybridized with the target nucleic acid and formed the nucleic acid structure of step a).

[0115] As mentioned above, the only requirement for the system of different molecules to function is that, in step b), in the presence of the target nucleic acid, the ABC sequence is reconstructed as a single circular molecule by the action of the ligase. Therefore, sequence E does not need to be circularized, reconstructed, or ligated with any other sequence, whether present (i.e., Skinlock and Splitlock) or if E is split and added in a different molecule (i.e., dsSplitlock).

[0116] In the specific case of Flaplock (with or without skin), the target nucleic acid functions as a sprint nucleic acid molecule for ligation between sequences A and C. However, ligation occurs only after sequence D is removed from the padlock and the padlock is thus formed in a cyclizable form.

[0117] In the case of Gaplock, with or without skin or sequence E, the target nucleic acid functions as a sprint nucleic acid molecule for sequence A1 and A2, and / or C1 and C2 ligation. Alternatively, in some embodiments of Gaplock, the space between the free ends of the first molecule can be "filled" by using the target nucleic acid molecule as an extension template and extending the free 3' end (e.g., by a polymerase reaction). When the free 3' end is extended to be adjacent to the free 5' end, the two ends are joined by a ligation reaction.

[0118] In the case of ssSplitlock, sequence E will function as a sprint nucleic acid molecule to lygate the nick between B1 and B2, and the target nucleic acid will function as a sprint nucleic acid molecule to lygate the nick between A and C.

[0119] In dsSplitlock, the target nucleic acid functions as a sprint nucleic acid molecule for ligation of sequences A and C, and sequence E functions to align sequences B1 and B2 at the B1-E1 / B2-E2 junction. However, the third (E1) and fourth (E2) molecules may or may not be ligateable to each other. If they are ligateable, the third and fourth molecules are joined by ligation of the 5' end of the third molecule (E1) and the 3' end of the fourth molecule (E2), thereby obtaining sequence E as a single molecule. This is then hybridized to sequence B or its region in the padlock. However, as depicted in Figure 1J, it may be advantageous to prevent ligation of the third (E1) and fourth (E2) molecules of sequence E, obtaining a third (E1) molecule with a modified 3' end, and thus preventing priming of the RCA reaction in step c). In this method, even higher specificity is obtained because only dsSplitlock, which hybridizes with the target nucleic acid and ligated with B1 and B2, contains the 3' end of E2 and triggers the RCA reaction. Therefore, ligation of sequences E1 and E2 is not essential for this method to work. Ligation of the 5' end of the third molecule (E1) and the 3' end of the fourth molecule (E2) can be prevented by removing a phosphate group from the 5' end of E1, by including modifications that are not ligable, or by not aligning the molecules E1 and E2 when they are arranged in a cyclizable form (i.e., leaving a gap of at least one nucleotide).

[0120] Therefore, in the case of dsSplitlock, when a nucleic acid structure having a cyclizable padlock is obtained in step a), step b) includes ligation of the 5' end of the first molecule and the 3' end of the second molecule, and ligation of the 3' end of the first molecule and the 5' end of the second molecule, where: The third and fourth molecules are joined by ligation of the 5' end of the third molecule and the 3' end of the fourth molecule, thereby forming a padlock containing sequences A, B, C, and D within a single cyclic molecule, further containing sequence E as a single molecule hybridized to sequence B or its region. Or, here Because the 5' end of the third molecule has a non-ligateable 5' end, the third and fourth molecules are not joined by ligation of the 5' end of the third molecule and the 3' end of the fourth molecule, thereby forming a padlock containing sequence ABC within a single cyclic molecule, further containing sequence E as two molecules hybridized to sequence B or its region. In the context of the present invention, "non-ligateable" means a 5' or 3' end that cannot form a phosphodiester bond by ligase action between adjacent nucleotides, and / or the 5' and 3' ends are not adjacent or not adjacent enough for ligase action to occur. In a preferred embodiment, the non-ligateable 5' end of the third molecule lacks a phosphate group.

[0121] In one embodiment, ligation of the 5' and 3' ends of a molecule or group of molecules containing the padlock sequences A, B, and C of the present invention is carried out by the action of at least one ligase. As is known in the art, in template-directed ligation, the ligase catalyzes the formation of phosphodiester bonds between parallel 3'-hydroxyl and 5'-phosphate ends in two closely adjacent nucleic acids. This occurs when the nucleic acids are annealed or hybridized to a third nucleic acid sequence complementary to them (i.e., a ligation template or sprint nucleic acid molecule). Also, as is known in the art, there are non-template-directed ligations. In the ligation that occurs in the case of dsSplitlock, the splits to E1 and E2 are located within the padlock at the same positions as the splits to B1 and B2, thereby forming blunt ends that are ligated by a ligase (e.g., a T4 ligase capable of template-independent ligation).

[0122] Any convenient ligase can be used, and representative ligases for this purpose include, but are not limited to, temperature-sensitive ligases and heat-stable ligases. Temperature-sensitive ligases include, but are not limited to, bacteriophage T4 DNA ligase, bacteriophage T7 ligase, and E. coli ligase. Heat-stable ligases include, but are not limited to, Taq ligase, Tth ligase, Ampligase®, and Pfu ligase. Heat-stable ligases can be obtained from thermophilic or hyperthermophilic organisms (including, but not limited to, prokaryotes, eukaryotes, or archaea). Specific RNA ligases can also be used in the methods of the present invention.

[0123] A suitable ligase and any necessary and / or desirable reagents are mixed with the reaction mixture and maintained under conditions sufficient for ligation of the hybridized oligonucleotides to occur. Ligation reaction conditions are well known to those skilled in the art.

[0124] In a typical embodiment, the ligation reaction mixture contains 50 mM Tris pH 7.5, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 mg / ml BSA, 0.25 units / ml of an RNase inhibitor, and 0.2 units / ml of T4 DNA ligase.

[0125] It will become clear that the ligation conditions are determined by the ligase enzyme used in the method of the invention. Therefore, the ligation conditions described above are merely representative examples, and the parameters can be modified according to known protocols. The operation of such assay methods is common practice in the art.

[0126] In a preferred embodiment, when the target is single-stranded RNA, the ligase used is preferably a DNA ligase derived from Paramecium bursaria Chlorella Virus-1 (also known as SplintR), represented by Sequence ID No. 1. In one embodiment, the ligase is a DNA ligase derived from Paramecium bursaria Chlorella Virus-1 containing or consisting of Sequence ID No. 1, or a sequence having at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence defined by Sequence ID No. 1. The ligase has the ability to ligate the 5' end of A and the 3' end of C when a padlock molecule or a system of two or more different molecules hybridizes to the target ribonucleic acid. Therefore, it is preferable to use a SplintR ligase in step b) of the method of the present invention. This ligase also has the ability to ligate additional nicks within the sequence of a cyclizable padlock probe, provided that it is templated by the RNA sprint nucleic acid molecule when added as part of a system of different molecules. The SplitR ligase is preferably used to ligate sequences B1 and B2 when sequence E is obtained as an RNA molecule.

[0127] Therefore, when the sprint nucleic acid molecule (e.g., the target nucleic acid or sequence E) is an RNA molecule, SplintR ligase is preferably used in the ligation reaction of step b). However, when the sprint nucleic acid molecule is a DNA molecule (e.g., when sequence E is obtained as a DNA molecule), T4 DNA ligase is preferably used in the ligation reaction of step b). If the reaction involves both DNA and RNA sprint nucleic acid molecules (e.g., when the target nucleic acid is an RNA molecule and sequence E is a DNA molecule), two ligases (preferably SplinR and T4 DNA ligase) can be added.

[0128] (Step c of the method of the present invention includes, or comprises, providing a lygated annular padlock for rolling circle amplification (RCA).) The ligated annular padlock (or a portion thereof) obtained in step b) is then amplified by rolling circle amplification (RCA). Step c) therefore includes at least an RCA reaction. The amplification is to increase the copy number of the ligation product in the sample, thereby improving the sensitivity of the detection method of the present invention. In certain embodiments, only a portion or part of the ligated annular padlock is amplified.

[0129] Rolling circle amplification (RCA) using a circular template yields a linked RCA product containing multiple tandem repeats having sequences complementary to the circular template oligonucleotide (i.e., a circular padlock). RCA can be induced directly from the target nucleic acid of interest, provided that a hybridized 3' end is obtained. This can occur directly by designing the padlock to hybridize with the 3' end of the target nucleic acid of interest. Alternatively, it can occur directly by nucleolysis, preferably by 3' exonucleolysis activity (linkable to the polymerase used in the RCA reaction), or by nick formation by endonucleolysis of the target molecule hybridized to the padlock (e.g., mild treatment with RNAse H in the case of a target RNA molecule). Alternatively, RCA can occur by contacting the sample with at least one RCA primer that is complementary to and hybridizable with the circular padlock. Therefore, the 3' end of at least one of the primers is extended to obtain a ligated RCA product. The RCA primer is characterized by having a length of 6 to 80 nucleotides, preferably 10 to 50, 10 to 40, 10 to 30, or 10 to 20 nucleotides. Preferably, the primer or primer hybridizes to the region of sequence B.

[0130] In one embodiment, the padlock contains sequence E, and the RCA reaction uses the free 3' end of sequence E or a fragment thereof (e.g., E2) as a primer. In one embodiment, the padlock contains sequence E, but the RCA reaction is triggered by the target nucleic acid or an additional primer; preferably, this primer anneals with a portion of sequence B that is not hybridized with E. Amplification of the ligated circular padlock is carried out using a DNA polymerase enzyme capable of synthesizing DNA oligonucleotides from dNTPs. As used herein, the term "DNA polymerase" includes any enzyme capable of incorporating dNTPs, and therefore includes enzymes having both DNA and RNA polymerase activity. In particular, such DNA polymerases may have the ability to incorporate RNA nucleotides, although their primary activity or function is DNA synthesis.

[0131] The RCA reaction in step c) is preferably carried out by bacteriophage phi29 DNA polymerase. Bacteriophage phi29 DNA polymerase exhibits unique properties that enable numerous DNA amplification, DNA sequencing techniques, and platform applications: Highly processive DNA synthesis; outstanding strand-displacement enabling polymerization linked to double-stranded DNA unwinding in the absence of helicase-type enzymes; high fidelity of synthesis with a very low insertion error rate; and efficient proofreading function for insertion errors that improves overall fidelity.

[0132] In this specification, “bacteriophage phi29 DNA polymerase” includes the wild-type Phi29 DNA polymerase represented by Sequence ID No. 2, but also includes variants, chimeras, and truncated versions of said polymerase. Therefore, the term “bacteriophage phi29 DNA polymerase” includes genetically modified polymerases, including naturally occurring phi29-type DNA polymerases or polymerases modified therefrom, or polymerases substantially identical to equivalent enzymes. “Substantially identical” means that the enzyme may include amino acid substitutions that do not affect the properties of the enzyme as described above.

[0133] In a preferred embodiment, the bacteriophage phi29 polymerase is an improved chimeric phi29 polymerase containing the entire amino acid sequence of the wild-type phi29, represented by SEQ ID NO: 3, further comprising the M. kandleri Topo V (HhH)2 domain H (residues 696-751), or H and I (residues 696-802), fused to the C-terminus of the phi29 DNA polymerase. This chimeric phi29 DNA polymerase, also known as Qualiphi polymerase, has improved DNA binding ability and is therefore preferred as the phi29 DNA polymerase used in the method of the present invention. The Qualiphi polymerase is represented by SEQ ID NO: 3.

[0134] In another preferred embodiment, the bacteriophage phi29 DNA polymerase is modified by several amino acid substitutions, as described in Figure 3 of (Povilaitis, T., et al., In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self-replication technique, Protein Eng. Des. Select. 29, 617-628, 2016), and is referred to herein as Equiphi.

[0135] Therefore, in one embodiment, the bacteriophage phi29 DNA polymerase used in step c) of the method of the present invention is a bacteriophage phi29 enzyme that contains or consists of sequences having at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identity with the nucleotide sequences defined in SEQ ID NO: 2 or 3, respectively. Preferably, the bacteriophage phi29 DNA polymerase used in step c) of the method of the present invention is a bacteriophage phi29 DNA polymerase enzyme comprising or consisting of sequence number 3. Notably, a certain percentage of mutations in sequence number 2 or 3 are included in and acceptable in the method of the present invention. This is limited to the condition that the variant of the bacteriophage phi29 DNA polymerase maintains or improves the 3'-5' exonuclease activity, processivity, and strand-substitution DNA polymerase activity of the corresponding phi29 (sequence number 2 or 3).

[0136] In one embodiment, the bacteriophage phi29 DNA polymerase used in step c) of the method of the present invention is a bacteriophage phi29 DNA polymerase comprising, or consisting of, a sequence having at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 2, or the nucleotide sequence defined in SEQ ID NO: 2, wherein the sequence comprises at least the amino acid substitutions F526L, M97T, G197D, E221K, Q497P, V51A, K512E, M8R, L123S, K209E, E515A, and E239G. When referring to amino acid substitutions having a certain percentage of identity with SEQ ID NO: 2, it should be noted that such percentage of identity is understood to allow modifications to SEQ ID NO: 2, as long as the amino acid substitutions described herein are maintained. Furthermore, throughout this specification, the positions or locations of amino acid residues in the protein or polypeptide sequence are numbered sequentially, starting from the first amino acid residue at the N-terminus. For example, in a 137-amino acid protein, the residues are numbered from 1 (the first amino acid residue at the N-terminus of the protein) to 137 (the last amino acid residue at the C-terminus of the protein). Therefore, it is preferable that the numbering begins at the first amino acid residue at the N-terminus of the protein or polypeptide and ends at the C-terminus of the protein or polypeptide. Preferably, the positions of the amino acid residues are numbered using the amino acid sequence of the translated mature protein.

[0137] Step c) comprises, or consists of, carrying out at least one RCA reaction using the circularized padlock as a template and preferably using bacteriophage phi29 DNA polymerase, as defined above.

[0138] The RCA amplification comprises at least one linear RCA reaction that uses the RCA template formed in step (b) to form a first rolling circle amplification product (RCA product), which is a concatemer comprising a monomer complementary to the cyclic padlock and using its formation as a template. Suitable conditions for carrying out the RCA reaction are known in the art. The RCA product may be the product of a primary (i.e., initial) RCA reaction or a product of a further subsequent or subsequent RCA reaction. The second RCA reaction may be secondary or a further subsequent or subsequent RCA reaction. Thus, it is understood that the method of the present invention comprises multiple (e.g., two, three, four or more) consecutive rounds of RCA, where in each round a probe is used that is directly or indirectly hybridized to the reaction product of the previous round of RCA. In other words, the method of the present invention may include repeating steps (a), (b), and (c) one or more times, and / or performing steps (a), (b), and / or (c) simultaneously.

[0139] In some embodiments, for example, in an in situ assay or other assay in which the target nucleic acid is immobilized, a washing step may be included between the addition of the probe and ligation and / or amplification of the ligation product. That is, the target nucleic acid molecule may be captured or immobilized on a solid support or substrate and washed to remove unbound or nonspecifically bound probes. In some embodiments, a washing step is included between the ligation of the probe and amplification of the ligation product to remove unligated probes. In other representative embodiments, a washing step may be included before ligation is performed. If the probe is a multi-part padlock, the backbone oligonucleotide a can be brought into contact with the sample and hybridized. Subsequently, gap oligonucleotides can be added and hybridized as needed.

[0140] The product obtained by the RCA reaction described above is a linear RCA product composed of concatemers of the complementary sequence of the padlock of the present invention. This linear RCA product can optionally be used as a template for further amplification reactions (MDA reactions).

[0141] Therefore, in one embodiment, step c) comprises an RCA reaction followed by an MDA reaction, where the product of the RCA reaction is amplified exponentially. At least two primers are used in the MDA reaction. These primers are referred to herein as MDA primers and are characterized by having a maximum length of 15 nucleotides, but are preferably less than 14, 13, 12, 11, 10, 9, 8, 7, or 6 nucleotides. The advantage of using short MDA primers is that they facilitate the strong amplification characteristics of this method, allowing the target nucleic acid to be amplified even in very small amounts. At least one MDA primer (MDA1) is inversely complementary and hybridizes to the ABC sequence of the cyclized padlock, preferably region B. This primer may be the same as the primer used to induce the RCA reaction as described above. At least one MDA primer (MDA2) contains the sequence contained in the ABC cyclized padlock, preferably region B, and is therefore inversely complementary and hybridizes with the RCA product. If the B sequence is split into two or more different molecules, it is preferable that each MDA primer anneals or hybridizes to a different molecule of the B sequence or its reverse complementary sequence. For example, one primer anneals to the B1 sequence (MDA1) and the other primer anneals to the reverse complementary sequence of the B2 sequence (MDA2). Therefore, in a preferred embodiment, step c) includes amplifying the RCA product by using two primers having a maximum length of 15, preferably 10 or less nucleotides, where the padlock is added in the form of a system of two different molecules, and splitting occurs at sequence B, where one primer hybridizes to the B1 portion and the other primer hybridizes to the reverse complementary sequence of B2.

[0142] This MDA reaction can be carried out after the RCA reaction, but more preferably simultaneously with the RCA reaction. Therefore, in a preferred embodiment, step c) includes an HRCA reaction, which is an amplification reaction combining the linear RCA reaction and the MDA reaction (HRCA is also known as a strand-substitution cascade reaction). Typically, HRCA is carried out by generating a first-generation RCA product initiated from either a hybridized target molecule or a primer that anneals to the padlock (e.g., an MDA1 primer or skin as described above). The RCA product is a concatemerized ssDNA molecule containing multiple serial priming sites for the MDA2 primer, thus triggering an MDA reaction. In this MDA reaction, polymerases extending from each primer displace and release all products being generated downstream. This converts the RCA product to dsDNA, generating a second-generation ssDNA molecule with the same polarity as the padlock. Each molecule contains an ABC sequence variable ranging from 1 to the number of repeats contained in the RCA product (i.e., 1, 2, 3, ..., n). Furthermore, each of these ssDNA molecules contains a priming site for MDA1, thus triggering further MDA reactions as described above. The second generation of ssDNA molecules is converted to dsDNA, and a set of third-generation ssDNA molecules with the opposite polarity to the padlock is released, similar to the first generation of RCA products. This process can proceed over multiple cycles, with alternating priming by MDA2 and MDA1 while the RCA reaction is active and / or while ssDNA is being released by MDA. The final result is an exponentially amplified mixed dsDNA and ssDNA product.

[0143] In one embodiment, the polymerase used in the MDA reaction is the same as the one used in the RCA reaction defined above.

[0144] Therefore, in the method of the present invention, the RCA reaction in step c) may be an HRCA assay, in which the linear RCA reaction is performed as part of the HRCA reaction, and the HRCA reaction is performed before the detection step d).

[0145] (Step d of the method of the present invention includes, or comprises, detecting the target nucleic acid by detecting the amplified product.) Step d) includes the detection of the amplification product. Note that the molecule detected is the product amplified in step c) and not the target nucleic acid. However, the detection of the amplification product indicates the presence of the target nucleic acid or analyte in the sample. This step can be performed after step c) or simultaneously with the generation of the amplification product. Steps a) through c) are preferably performed for an appropriate amount of time until a detectable signal is obtained.

[0146] In this specification, the term “detection” is used in a broad sense to include any means or form of measurement that determines the presence or absence (i.e., whether or not the target nucleic acid is present). Because the amplification in step c) is strong and may have produced nonspecific amplicons, the detection step is preferably a sequence-based, i.e., sequence-specific detection step. Sequence-specific methods can specifically detect target-specific amplicons and thus enhance the specificity of the method and ignore any background that may have been generated during the strong amplification using the short primers in step c). A region specifically identified by a sequence-specific detection method is referred to herein as a “detectable sequence” and is a complementary sequence to the sequence present in the padlock probe, also referred to herein as a “detectable template.” Because the HRCA method involves branched amplification, the “detectable template or sequence” may be a sequence similar to the sequence in the padlock probe. Thus, the “detectable template” may be the sequence present in the padlock probe, its inverse complementary sequence, or its complementary sequence. The detectable template is included in the padlock sequence as a whole or in fragments, and the correctly assembled padlock product amplified in the process of step c) includes its inverse complementary sequence, i.e., includes the detectable sequence. One or more detectable templates may be included in the padlock sequence. Preferably, the detectable sequence is a sequence that includes at least a portion of the sequence formed when the A-terminus (including A1 and A2) and / or C-terminus (including C1 and C2) of the sequence are ligated in step b), or a complementary or inverse complementary sequence of the ligated sequence.

[0147] In these embodiments, the signal producing system may include nucleic acids or oligonucleotides that specifically bind to sequences present in the amplification product (i.e., detectable sequences), and such nucleic acids / oligonucleotides may be labeled directly or indirectly with detectable labels.

[0148] A directly detectable label is one that can be detected directly without the use of additional reagents, while an indirectly detectable label is one that can be detected by using one or more additional reagents. For example, the label may be a component of a signal-generating system consisting of two or more components.

[0149] In many embodiments, the label is a directly detectable label, and directly detectable labels of the target include, but are not limited to, fluorescent labels, radioisotope labels, and chemiluminescent labels. In many embodiments, the label is a fluorescent label, and the labeling reagent used in such embodiments is a fluorescently labeled nucleotide (e.g., fluorescently labeled CTP (e.g., Cy3-CTP, Cy5-CTP)). Fluorescent moieties that can be used to tag nucleotides in the preparation of labeled probe nucleic acids (i.e., detection probes) include, but are not limited to, fluorescein and cyanine dyes (e.g., Cy3, Cy5, Alexa555, Bodipy 630 / 650). Other labels as described above can also be used, as are well known in the art.

[0150] In one embodiment, step d) sequence-specific detection uses a labeled nucleic acid that binds to a detectable sequence and emits a detectable signal. In certain embodiments, the specifically labeled nucleic acid (detection probe) is labeled with an “energy transfer” label. In this specification, “energy transfer” refers to a process in which the fluorescence emission of a fluorescent group is altered by a fluorescent modifier. Energy transfer labels are well known in the art, and such labeled oligonucleotide probes include TaqMan® probes, Scorpion probes, Sunrise probes, and conformationally assisted probes.

[0151] In another embodiment, the sequence-specific detection step uses a CRISPR / Cas system and a guide RNA that specifically binds to the detectable sequence. This specific binding releases a detectable signal. Preferably, the Cas protein is the Cas12 protein, more preferably Cas12a, and most preferably LbaCas12a (Cas12a(Cpf1) derived from Lachnospiraceae bacterium ND20006). In a preferred embodiment, the detectable template contains a protospacer-adjacent motif (PAM) sequence that can recognize the detectable sequence obtained in step c) in both ssDNA and dsDNA forms. Preferably, the PAM sequence in the detectable template is included in sequences A, B and / or C of the padlock probe, and the sequence-specific detection method is CRISPR / Cas, preferably CRISPR / Cas12a, and most preferably CRISPR / LbaCas12a. Preferably, at least one detectable template containing a PAM sequence is located in regions A and / or C.

[0152] As a general rule, the detectable template is reconstructed only when sequences A and C are ligated, which characterizes the presence of the target nucleic acid, preferably by being located in the junction region of sequences A and C, or in their complementary sequences. In this specification, “junction region” refers to the region in the padlock sequence where a nick (break) exists; for example, “junction region of sequences A and C” refers to the nucleotide sequence containing the 5' end of region A and the 3' end of region C, which is formed only when regions A and C are joined by the action of a ligase. In other words, the junction region of sequences A and C is the region to which sequences A and C ligate. Since the padlock sequence may have one or more nicks, there may be one or more junction regions.

[0153] If the padlock is a Flaplock, the detectable template is preferably positioned in the junction region of sequences A and C, and only the padlock from which sequence D has been removed allows ligation between A and C, resulting in the generation of a detectable sequence in step c). Therefore, in the case of a Flaplock, the sequence-specific detection method preferably detects the following sequences: i) The array located in the junction region of array A and array C, and formed when arrays A and C are ligated in step b), and / or ii) A complementary or inverse complementary sequence of sequences located in the junction region of sequences A and C, formed when sequences A and C are ligated in step b).

[0154] If the padlock is a Gaplock, the detectable template is preferably located in at least one junction region of sequences A and / or C, and more preferably in at least one junction region of sequences obtained in different molecules. For example, in the case of a Gaplock in which splitting occurs at sequence A, the detectable template is preferably located in the junction region of sequences A1 and A2, or the junction region of sequences A2 and C, or both. In the case of a Gaplock in which splitting occurs at sequence C, the detectable template is preferably located in the junction region of sequences C1 and C2, or the junction region of sequences C2 and A, or both. In the case of a Gaplock in which splitting occurs at sequences A and C, the detectable template is preferably located in the junction region of sequences C1 and C2, or the junction region of sequences A1 and A2, or both. Since the sequences (A1 and A2, C1 and C2, or A1 and C2) are arranged in different molecules (i.e., the first molecule and the second molecule, respectively), in the case of Gaplock, the detectable template is preferably located in the junction region of the first molecule and the second molecule (which is formed by the ligation of the first molecule and the second molecule in step b). In this method, only the padlock completed and circularized by the hybridization of the target nucleic acid is amplified, and a detectable sequence is generated in the process of step c). Since Gaplock has at least two nicks, it has at least two junction regions on which the detectable sequence can be placed. Therefore, in the case of Gaplock, it is preferable that the following sequence is detected: i) an arrangement located in one of the junction regions of the first molecule and the second molecule, formed when the first molecule and the second molecule are ligated in step b), and / or ii) A complementary or inverse complementary sequence of the sequence located in one of the junction regions of the first molecule and the second molecule, which is formed when the first molecule and the second molecule are ligated in step b).

[0155] If the padlock is a Splitlock (including both ssSplitlock and dsSplitlock) and has a split in sequence B, the detectable template is still preferably located in the junction region of sequences A and C. This is because the ligation of sequences A and C is the event most directly related to the presence of the target nucleic acid. Since the sequences (A and B) are located in different molecules (i.e., the first molecule and the second molecule, respectively), in the case of a Gaplock, the detectable template is preferably located in the junction region of the first molecule and the second molecule (formed by the ligation of the first molecule and the second molecule in step b). Therefore, in the case of a Splitlock, the method in step d) is preferably used to detect the following sequences: i) A sequence located in the junction region of sequences A and C, formed when sequences A and C are ligated in step b), or ii) A complementary or inverse complementary sequence of sequences located in the junction region of sequences A and C, formed when sequences A and C are ligated in step b).

[0156] Alternatively, other non-sequence-specific detection methods can be used. This is particularly useful when using the optimized padlock as defined herein. Non-sequence-specific methods for detecting nucleic acids (such as the use of intercalation agents that label amplification products and emit detectable signals) are known in the art.

[0157] The detection step may include measuring the emitted signal. This measurement is performed by detecting and determining the presence of the target nucleic acid in the sample based on the intensity of the signal. The detectable signal may be a fluorogenic signal or a colorimetric signal, etc. The detection step may include a quantification step to obtain the level (concentration) of the target nucleic acid. To measure the concentration of the target nucleic acid in a sample, a calibration curve can be drawn using a sample containing a known concentration of the target nucleic acid molecule. The concentration of the target nucleic acid in the sample can be determined by comparing the measured parameter with a calibration standard. In some cases, a calibration curve can be prepared, in which the measured total signal is determined using substantially the same assay format for multiple samples containing the target nucleic acid at known concentrations. For example, the concentration of the target nucleic acid in a sample can be measured by comparing the total intensity of the array with the calibration curve. A calibration curve can be prepared by performing the method using multiple standardized samples of known concentrations under conditions similar to those used when analyzing test samples of unknown concentrations. A calibration curve may be used to correlate the detected signal of the target nucleic acid complex with a known concentration of the target nucleic acid. Subsequently, the assay can be performed on a sample containing an unknown concentration of the target nucleic acid or fragment, and the concentration of the target nucleic acid in the sample can be measured by comparing the detected signal with the calibration curve (or a mathematical equation that fits the same curve).

[0158] The method described herein can also be used to detect multiple targets present in a sample or to measure the concentrations of two or more targets in a sample (multiplex assay). In this case, the two or more target nucleic acids are detected using different padlocks. In one embodiment, two or more padlocks are used, where each padlock used has the ability to hybridize with a different target nucleic acid of interest. In other words, each padlock has different sequences A and C in its 5' and 3' regions. This induces the padlock to hybridize (bind) with (and thus indicate the presence of) different target nucleic acid sequences present in the sample. By using two or more padlocks in the multiplex detection method of the present invention, it is possible to detect two or more different target nucleic acids in a sample. Since each target nucleic acid is different, each padlock can be considered a "detection-only" padlock for detecting that target nucleic acid or an analyte representative of that target nucleic acid.

[0159] Multiplex assays may detect tens, hundreds, thousands, or even tens of thousands of target nucleic acids or analytes in a sample. In this case, it is preferable that each padlock used to detect each target nucleic acid or analyte contains a tag sequence (e.g., a barcode or identification motif, or a binding site for a detection probe or primer) within its sequence. Such tag sequences may be found, for example, at the 3' or 5' end of the padlock, or in one of the different molecules, or at the end of the padlock or in the middle of a different molecule (e.g., part of a cyclizable backbone oligonucleotide that is not hybridized with the target nucleic acid molecule). The tags (e.g., barcodes) can be designed to suit different needs / purposes (e.g., to introduce a general-purpose or common sequence to allow different ligated padlocks to be processed together in a multiplex setting). This allows different ligated padlocks to be amplified together (e.g., in library amplification by RCA). Alternatively, or additionally, tag / barcode sequences can be used to "label" amplified, different ligated padlocks so that they can be easily distinguished from one another (i.e., "target" tags or markers), or to tag different samples, etc., so that they can be pooled before a common / general amplification step (i.e., "sample" tags or markers). Thus, in a multiplex environment, different padlocks (e.g., padlocks for different target nucleic acids) can be assigned different tag sequences (e.g., different marker or detection sequences) and / or the same tag sequence (e.g., for the introduction of a common / general sequence).

[0160] In a multiplex reaction (for example, in a sample suspected of containing three different target nucleic acids or analytes), the detection step is: (i) measuring a first signal emitted from a first amplification product obtained from amplification of a first padlock, and determining the presence or concentration of a first target nucleic acid in the sample based on the intensity of the first signal; (ii) measuring a second signal emitted from a second amplification product obtained from amplification of a second padlock, and determining the presence or concentration of a second target nucleic acid in the sample based on the intensity of the second signal; and (iii) measuring a third signal emitted from a third amplification product obtained from amplification of a third padlock, and determining the presence or concentration of a third target nucleic acid in the sample based on the intensity of the second signal.

[0161] In other embodiments, a single target nucleic acid or analyte may be represented by two or more different target nucleic acid sequences, and the presence of the target nucleic acid in the sample can be indicated by detecting one or more target nucleic acid sequences that represent that target nucleic acid or analyte.

[0162] The amplified, ligated probe can be detected using any established method for analyzing nucleic acid molecules known in the literature (such as liquid chromatography, electrophoresis, mass spectrometry, microscopy, real-time PCR, fluorescent probes, microarrays, colorimetric analysis such as ELISA, flow cytometry, lateral flow, and mass spectrometry (CyTOF)). This may require immobilization of the nucleic acid molecule (e.g., immobilization in an in situ detection procedure, or immobilization of the padlock probe or amplification product). The use of solid-phase assays offers advantages, particularly in detecting difficult-to-detect samples: the washing step can help remove inhibitory components, such as unbound probes and / or unligated probes. It also allows for the concentration of target molecules from undesirably large sample volumes. Because washing removes unbound probes and RNA molecules, higher concentrations and larger volumes of probe can be used.

[0163] (Other improvements) The authors of the present invention have also discovered improvements to the conditions under which the method is carried out. In one embodiment, the method of the present invention is an isothermal method, thereby making all the reactions described herein potentially achievable at a single temperature. Preferably, the temperature is 20 to 37°C, preferably 25 to 30°C.

[0164] As explained in Example 3, the presence of ammonium sulfate is clearly detrimental to the amplification reaction using the Qualiphi enzyme, and when this compound is removed from the reaction, 10 7 Numerous molecules of a magnitude were clearly detected. Therefore, in one embodiment, the method of the present invention is carried out in the absence of an ammonium salt, preferably ammonium sulfate, and in step c) of the method, using the Qualiphi polymerase shown in SEQ ID NO: 3.

[0165] Since RCA and MDA, and therefore HRCA methods, are known in the art, the assay conditions and steps are known to those skilled in the art and can be used herein.

[0166] In a second embodiment, the present invention provides a padlock suitable for the method of the present invention. Preferably, the padlock is one of the padlocks defined above, preferably Gaplock, Flaplock, ssSplitlock or dsSplitlock (with or without skin), or any padlock obtained therefrom. Thus, all embodiments and definitions relating to the padlock of the present invention as defined in the first embodiment or any embodiment thereof are also included in this second embodiment. In one embodiment, the padlock is obtained as a composition (including other reagents or additives, such as stabilizers). The padlock sequence can be modified to improve the stability of the padlock. Modifications include chemical modifications (such as 2'-O-methyl (2'OMe), 2'-O-methoxyethyl (2'MOE), an additional bridge (LNA) connecting 2'-oxygen and 4'-carbon, or a phosphorothioate bond, or a combination thereof).

[0167] Preferably, the padlock probe includes, consists of, or essentially consists of sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence numbers 8, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, or 32. More preferably, the padlock of the present invention includes, consists of, or essentially consists of sequences having sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with sequence numbers 24-27 or 32. The backbone region (i.e., sequence B) is denoted by "N" and preferably contains 20 to 160, preferably 40 to 150 nucleotides that are not complementary to the target nucleic acid. The use of the padlock probe in the method of the present invention or any RCA or HRCA method is also included herein.

[0168] Preferably, the padlock is a cyclizable padlock. As defined above, “cyclizable” means a padlock conformation in which the padlock is hybridized with the target nucleic acid and includes ligateable 5' and 3' ends, which are capable of being ligated to form a ring or a “cyclical padlock”.

[0169] Preferably, the cyclizable padlock probe is characterized by comprising at least two different oligonucleotide molecules, where: i) The first molecule contains the nucleotide sequence ABC in the 5' to 3' direction, where: - A is a nucleotide sequence located at the 5' end of the padlock, complementary to the first portion of the target nucleic acid, and capable of hybridizing with the first portion. - B is a nucleotide sequence that is not complementary to the target nucleic acid, and - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and capable of hybridizing with the second portion. The first and second portions of the target nucleic acid are located adjacent to each other; and ii) The second molecule contains a nucleotide sequence E, where sequence E is complementary to sequence B or a region thereof, where: - The length of array E is at least 60%, 70%, preferably 80%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of array B, and / or - Sequence E contains a modified nucleotide at its 3' end, which prevents sequence E from priming the RCA reaction. A cyclizable padlock probe is obtained by hybridizing the first molecule to the target nucleic acid and the second molecule to the first molecule.

[0170] Preferably, the cyclizable padlock probe is a skinned flaplock, characterized in that the first molecule further comprises sequence D, where sequence D is located at the 3' end of sequence C or the 5' end of sequence A and is not complementary to the target nucleic acid, and the cyclizable padlock is obtained by first hybridizing the first molecule with the target nucleic acid and the second molecule, and then cleaving sequence D by exolysis using an ssDNA-specific exonuclease or FLAP endonuclease.

[0171] Preferably, the cyclizable padlock probe is a Gaplock having a skin and is characterized by comprising at least three different oligonucleotide molecules, where: - The first molecule contains sequence A, sequence B, and the first part of sequence C (C1) in the 5' to 3' direction; the second molecule contains the second part of sequence C (C2) in the 5' to 3' direction; the third molecule contains sequence E, or - The first molecule contains the first part of sequence A (A1), sequence B, and sequence C in the 5' to 3' direction; the second molecule contains the second part of sequence A (A2) in the 5' to 3' direction; the third molecule contains sequence E, or - The first molecule comprises the first part of sequence A (A1), sequence B, and the first part of sequence C (C1) in the 5' to 3' direction; the second molecule comprises the second part of sequence C (C2) and the second part of sequence A (A2) in the 5' to 3' direction; and the third molecule comprises sequence E, where: - Sequence A is a nucleotide sequence located at the 5' end of the padlock, complementary to the first portion of the target nucleic acid, and capable of hybridizing with the first portion. - Sequence B is a nucleotide sequence that is not complementary to the target nucleic acid. - Sequence C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and capable of hybridizing with the second portion, and - Sequence E is complementary to sequence B or its region and can hybridize with sequence B or its region, where: - The length of array E is at least 60%, 70%, preferably 80%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of array B, and / or - Sequence E contains a modified nucleotide at its 3' end, which prevents sequence E from priming the RCA reaction. A cyclizable padlock probe, wherein the first and second portions of the target nucleic acid are located adjacent to each other, and the cyclizable padlock is obtained by hybridizing the first and second molecules with the target nucleic acid and the third molecule.

[0172] Preferably, the cyclizable padlock probe is a splitlock having a skin and is characterized by comprising at least three different oligonucleotide molecules, where: A cyclizable padlock probe characterized by comprising at least three different oligonucleotide molecules, - The first molecule contains sequence A and a portion of sequence B (B1) in the 5' to 3' direction; - The second molecule comprises the second portion of sequence B (B2) and sequence C in the 5' to 3' direction; and - The third molecule contains sequence E, where sequence E is complementary to the first (B1) and / or second (B2) portion of sequence B, where: - Sequence A is a nucleotide sequence located at the 5' end of the padlock, complementary to the first portion of the target nucleic acid, and capable of hybridizing with the first portion of the target nucleic acid. - Sequence B is a nucleotide sequence that is not complementary to the target nucleic acid. - Sequence C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and capable of hybridizing with the second portion, and - Sequence E is complementary to sequence B or its region and can hybridize with sequence B or its region, where: - The length of array E is at least 60%, 70%, preferably 80%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of array B, and / or - Sequence E contains a modified nucleotide at its 3' end, and sequence E cannot prime the RCA reaction. A cyclizable padlock probe, wherein the first and second portions of the target nucleic acid are located adjacent to each other, and the cyclizable padlock is obtained by hybridizing the first and second molecules with the target nucleic acid and the third molecule.

[0173] Preferably, the cyclizable padlock probe is a dsSplitlock having a skin and is characterized by comprising at least four different oligonucleotide molecules, where: - The first molecule contains the first portion (B1) of sequence A and sequence B in the 5' to 3' direction, - The second molecule contains the second part of sequence B (B2) and sequence C in the 5' to 3' direction, - The third molecule comprises a first portion (E1) of sequence E, wherein the first portion (E1) of sequence E is complementary to the first portion (B1) of sequence B and can hybridize with the first portion (B1) of sequence B, and - A fourth molecule comprises a second portion (E2) of sequence E, wherein the second portion (E2) of sequence E is complementary to the second portion (B2) of sequence B and can hybridize with the second portion (B2) of sequence B, where: - Sequence A is a nucleotide sequence located at the 5' end of the padlock, complementary to the first portion of the target nucleic acid, and capable of hybridizing with the first portion. - Sequence B is a nucleotide sequence that is not complementary to the target nucleic acid. - Sequence C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and capable of hybridizing with the second portion, and - Array E includes E1 and E2, - At least 60%, 70%, preferably 80%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of array B, and / or - Sequence E contains a modified nucleotide at its 3' end, which prevents sequence E from priming the RCA reaction. A cyclizable padlock probe is obtained by hybridizing the first and second portions of the target nucleic acid adjacent to each other, and the cyclizable padlock is obtained by hybridizing the first and second molecules with the target nucleic acid, and the third and fourth molecules, respectively.

[0174] In a preferred embodiment of the second aspect, a composition is obtained comprising at least one padlock as defined in the second aspect, the first aspect, or any of their embodiments.

[0175] The second embodiment also includes the use of the padlock as defined herein. Preferably, the padlock of the second embodiment, or the padlock as defined in the context of the first embodiment, is used in the step of detecting the presence of a target nucleic acid in a sample by amplification.

[0176] Preferably, the amplification method includes a detection step of detecting the amplified nucleic acid by a sequence-specific detection method, thereby detecting the target nucleic acid, the detection step including detecting a complementary sequence of the sequence located in the junction region of sequences A and C of the padlock when the padlock is circularized. Preferably, the sequence-specific detection method detects at least a portion of the sequence formed when the ends of sequence A and / or sequence C are ligated in step b), or a sequence containing a complementary or inversely complementary sequence of such ligated sequence.

[0177] Preferably, the Gaplock is used in a method for detecting the presence of a target nucleic acid in a sample by amplification. The amplification method includes a detection step of detecting the amplified nucleic acid by a sequence-specific detection method, thereby detecting the target nucleic acid. The detection step includes detecting complementary sequences of sequences located in the junction region of the first molecule and the second molecule, and sequences formed when the padlock is circularized.

[0178] Preferably, the method is a rolling circle amplification (RCA) or a super-branched rolling circle amplification (HRCA) method.

[0179] More preferably, the method is defined in the first embodiment or any embodiment thereof.

[0180] In a third embodiment, the present invention relates to the use of the method of the first embodiment or the padlock of the second embodiment for diagnostic and / or prognostic purposes. Applications of the method and padlock of the present invention include the steps of detecting the presence of a target nucleic acid of interest in a suitable sample and / or measuring its concentration. In one particular embodiment, the method is used to detect a target nucleic acid in a food or water sample (e.g., to detect the presence of a human pathogen in the sample). In other embodiments, the assay method and the padlock described herein can be used in a research setting to detect the presence of a target nucleic acid in a sample or to measure its concentration. The method and the padlock can be applied in a diagnostic / prognostic setting to detect the presence of a nucleic acid biomarker associated with a target disease or to measure its level. For example, the method and the padlock can be used to detect / measure the presence of an infectious agent associated with a particular disease (e.g., COVID or flu). The method and the padlock can be used for early diagnosis in subjects that do not exhibit symptoms of the disease to detect such nucleic acid biomarkers. The assay method and the padlock can also be used to detect microbial nucleic acids. This allows it to determine whether a subject is infected with a microorganism (e.g., a virus (HBV, HCV, HPV, HIV, influenza, coronavirus, etc.)). Preferably, the method and padlock of the present invention are used to detect the presence of a virus in a sample, preferably a coronavirus, and most preferably the SARS-CoV-2 virus.

[0181] In a fourth aspect, the present invention relates to a system or apparatus for carrying out the method of the present invention. For the purposes described above, a number of known systems or apparatuses can be used. These include, but are not limited to, HRCA-based point-of-care (POC) tests for the rapid detection of at least one target polynucleotide. Detection methods in POC tests are known in the art and may include immunochromatographic assays, colorimetric assays, electrochemical assays, and the like. The POC tests are usually designed as easy-to-use membrane-based test strips and are often sealed in plastic test cassettes. Examples of POC tests include tests based on immunochromatographic assays, particularly lateral flow tests (LFTs). Essentially, these LFTs involve flowing a test sample along the surface of a pad having a reactive molecule, and showing a positive or negative result. The result may be visually displayed or displayed in response to a stimulus (e.g., by a laser). Lateral flow-based biosensors typically comprise a backing card, a sample pad, a nitrocellulose membrane, and an absorbent pad.

[0182] In the context of this invention, "lateral flow system" or "lateral flow assay" is understood to be a system comprising a lateral flow apparatus that performs a methodology for detecting the presence or absence of a target nucleic acid in a given matrix sample in a specific manner. This type of assay eliminates the need for specialized and expensive equipment traditionally used in laboratories. Lateral flow technology is widely used as a point-of-care tool (rapid and in situ) in a wide range of applications (from home pregnancy tests to more specialized tests (clinical, food safety, environmental, and industrial fields)).

[0183] In the context of this invention, "backing card" is understood as the material to which all membranes (sample pad, conjugate pad, membrane, and wicking pad) are attached and connected to one another. At the same time, the backing card provides excellent stability and ease of handling to the lateral flow apparatus.

[0184] In the context of this invention, "sample pad" is understood to be an element of a device into which a test sample is introduced or deposited, and which functions as a sponge that holds the sample liquid. The sample liquid is then transported to the membrane by capillary action.

[0185] In the context of this invention, “membrane” is understood as a material (e.g., nitrocellulose) that transports the test sample, provides support to capture molecules (antibodies, aptamers, etc.), and enables or enhances the binding ability of those molecules. Two distinct capture molecule regions are defined on the membrane: a test line region that indicates the result of the lateral flow assay (positive or negative), and a control line region that examines the correct operation of the lateral flow assay.

[0186] In the context of the present invention, "wicking pad" is understood as an element within the apparatus that holds all of the assay materials and functions as a waste container.

[0187] Furthermore, the methods disclosed herein are also applicable to other devices or systems (such as microfluidic devices, microarrays, or electrochemical biosensors). The term “microfluidic chip or device” refers to a series of microchannels etched or molded into a material (such as glass, silicon, thermoplastic material, or polymer (such as polydimethylsiloxane (PDMS))). The microchannels forming the microfluidic chip are connected to one another to achieve a desired function (mixing, pumping, sorting, control of a biochemical environment). The network of microchannels enclosed within the microfluidic chip is connected to the outside by inlets and outlets that penetrate the chip, and functions as an interface between the macro and micro worlds. Liquids are injected through these holes and removed from the microfluidic chip (through tubes, syringe adapters, or simple holes in the chip) by an external active system (pressure controller, push syringe, or peristatic pump) or by a passive method (e.g., water pressure).

[0188] A microfluidic chip or device comprises a support or substrate, wherein the support or substrate comprises at least one channel within the substrate. The channel includes an inlet, an outlet, and a flow path connecting the inlet and outlet, wherein the inlet and outlet together form a midplane; and a portion of the flow path extends across the midplane, wherein the portion of the flow path extending across the midplane includes a recognition site or sensing region for detection (e.g., target nucleic acid detection).

[0189] In the context of this invention, a "DNA microarray chip system" is understood as a system comprising a DNA microarray chip or device, wherein the chip or device is mounted on a solid surface to which DNA molecules (e.g., padlocks) are chemically bound. Light to be measured is generated by complementary base pairs between the target nucleic acid present in the test sample (preferably an isolated biological sample) and the DNA molecules bound to the chip. In this particular system, the detection is obtained when a hybridization complex comprising at least one padlock and the target nucleic acid is formed and further amplified. The region on the chip that generates light identifies the target nucleotide present in the test sample. The microarray technology is a platform for simultaneously analyzing different target sequences in complex liquids in a high-throughput manner. The microarray technology also enables multiplexing (simultaneous detection of different target polynucleotides).

[0190] In the context of this invention, “electrochemical system” means a system including an electrochemical apparatus that obtains an analytical signal as an electric current. This signal is directly proportional to the concentration of the analyte, or in this case, the concentration of the target nucleic acid. Therefore, the electrochemical system or apparatus can function as a biosensor. Detection of at least one target polynucleotide is achieved by a Faraday current generated when the target nucleotide binds to the padlock. This current triggers the method of this invention. The generation of the RCA product or the RCA reaction itself (for example, by changing the pH) produces an electroactivatable compound. The redox process of the electroactivatable compound generated on the electrochemical apparatus generates a current that is directly proportional to the concentration of the target nucleic acid when a voltage is applied.

[0191] In a fifth embodiment, the present invention provides a kit comprising reagents necessary for carrying out the method of the present invention. Preferably, the kit also includes instructions for the method. In one embodiment, the kit includes: a) At least one bacteriophage phi29 polymerase, preferably a polymerase defined in the first aspect of the present invention, or any embodiment thereof. b) At least one ligase, preferably the target nucleic acid is a ribonucleotide, SplintR, c) If the target nucleic acid is a ribonucleotide, optionally, at least one RNAase, c) Suitable MDA and / or RCA primers, preferably MDA and RCA primers as defined in the present invention. d) dNTPs, e) A buffer suitable for amplification of the target nucleic acid, preferably under isothermal conditions.

[0192] Preferably, the kit includes a padlock probe as defined in the first embodiment or any of its embodiments.

[0193] Preferred embodiments of the method of the present invention are as follows: As shown in Figure 1A, a method using a padlock in which sequence ABC is obtained in a single molecule: In one embodiment, the method includes the following steps: a) A step of adding at least one padlock probe to the sample to obtain a nucleic acid structure formed by hybridization of at least one padlock with the target nucleic acid. In the nucleic acid structure, at least one padlock is in a cyclic form and contains the nucleotide sequence A, B, C in the 5' to 3' direction. Where: - A is a nucleotide sequence located at the 5' end of the padlock, which is complementary to and hybridizes with the first portion of the target nucleic acid. - B is a nucleotide sequence that is not complementary to the target nucleic acid. - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and hybridizing with the second portion. Here, the first and second portions of the target nucleic acid are located adjacent to each other, and at least one padlock is added in the form of a single molecule containing the sequence ABC. The nucleic acid structure of step a) including the padlock in a cyclizable form is obtained by hybridizing at least one padlock in the form of a single molecule with the target nucleic acid. b) A step of ligating the 5' end of sequence A to the 3' end of sequence C using at least one ligase to obtain a padlock as a ring-closed molecule with covalent bonds containing sequences A, B, C. c) a step of subjecting the ligated padlock probe from step b) to rolling circle amplification (RCA), wherein the RCA is carried out using bacteriophage phi29 polymerase; and a step of amplifying the RCA product by an MDA reaction using at least two MDA primers, wherein the primers are up to 15 nucleotides in length, preferably up to 10 nucleotides. d) A step of detecting the amplification product of step c), thereby detecting the target nucleic acid. Here, the detection method is preferably a sequence-specific detection method, which detects complementary sequences of sequences A, B, and / or C of the padlock probe. Preferably, a sequence located in the junction region of sequences A and C, which is formed when sequences A and C are ligated in step b), is detected.

[0194] As shown in Figure 1B, the sequence ABC is obtained in a single molecule, and a method using a padlock containing a skin (SkinLock) is also used: In one embodiment, the method includes the following steps: a) A step of adding at least one padlock probe to the sample to obtain a nucleic acid structure formed by hybridization of at least one padlock with the target nucleic acid. In the nucleic acid structure, at least one padlock is in a cyclic form and contains the nucleotide sequence A, B, C in the 5' to 3' direction. Where: - A is a nucleotide sequence located at the 5' end of the padlock, which is complementary to and hybridizes with the first portion of the target nucleic acid. - B is a nucleotide sequence that is not complementary to the target nucleic acid. - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and hybridizing with the second portion. Here, the first and second portions of the target nucleic acid are located adjacent to each other, and at least one padlock is added in the form of a system of two different oligonucleotide molecules. The first molecule contains the nucleotide sequence ABC in the 5' to 3' direction. The second molecule contains sequence E, where sequence E is complementary to sequence B or a portion thereof and can be hybridized with them. The nucleic acid structure of step a) containing the padlock is obtained by hybridizing the first molecule to the target nucleic acid and the second molecule to the first molecule. The nucleic acid structure of step a) having the padlock in a cyclizable form is obtained by hybridizing the first molecule to the target nucleic acid and the second molecule to the first molecule. b) A step of ligating the 5' end of sequence A to the 3' end of sequence C using at least one ligase to obtain a padlock as a ring-closed molecule containing sequence A, B, C by covalent bonds. c) a step of subjecting the ligated padlock probe from step b) to rolling circle amplification (RCA), wherein the RCA is carried out using bacteriophage phi29 polymerase; and a step of amplifying the RCA product by an MDA reaction using at least two MDA primers, wherein the primers are up to 15 nucleotides in length, preferably up to 10 nucleotides. d) A step of detecting the amplification product of step c), thereby detecting the target nucleic acid. Here, the detection method is preferably a sequence-specific detection method, which detects complementary sequences of sequences A, B, and / or C of the padlock probe. Preferably, a sequence located in the junction region of sequences A and C, which is formed when sequences A and C are ligated in step b), is detected.

[0195] As shown in Figure 1C, a method using a Flaplock probe: In one embodiment, the method includes the following steps: a) A step of adding at least one padlock probe to the sample to obtain a nucleic acid structure formed by hybridization of at least one padlock with the target nucleic acid. In the nucleic acid structure, at least one padlock is in a cyclic form and contains the nucleotide sequence A, B, C in the 5' to 3' direction. Where: - A is a nucleotide sequence located at the 5' end of the padlock, which is complementary to and hybridizes with the first portion of the target nucleic acid. - B is a nucleotide sequence that is not complementary to the target nucleic acid. - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and hybridizing with the second portion. Here, the first and second portions of the target nucleic acid are located adjacent to each other, and at least one padlock is added in the form of a single molecule containing the nucleotide sequence DABC or ABCD in the 5' to 3' direction. Here, sequence D is not complementary to the target nucleic acid and prevents sequences A and C from being ligated to each other. The nucleic acid structure of step a) having the padlock in a cyclizable form is obtained by first hybridizing the padlock with the target nucleic acid in the form of a single oligonucleotide molecule, and then cleaving sequence D by exonucleic acid degradation using an ssDNA-specific exonuclease or FLAP endonuclease. b) A step of ligating the 5' end of sequence A to the 3' end of sequence C using at least one ligase to obtain a padlock as a ring-closed molecule containing sequence A, B, C by covalent bonds. c) a step of subjecting the ligated padlock probe from step b) to rolling circle amplification (RCA), wherein the RCA is carried out using bacteriophage phi29 polymerase; and a step of amplifying the RCA product by an MDA reaction using at least two MDA primers, wherein the primers are up to 15 nucleotides in length, preferably up to 10 nucleotides. d) A step of detecting the amplification product of step c) and thereby detecting the target nucleic acid. Here, the detection method is preferably a sequence-specific detection method, which detects the complementary sequence of the sequence located in the junction region of sequences A and C that is formed when sequences A and C are ligated in step b).

[0196] As shown in Figure 1D, a method using a Flaplock with a skin: In one embodiment, the method includes the following steps: a) A step of adding at least one padlock probe to the sample to obtain a nucleic acid structure formed by hybridization of at least one padlock with the target nucleic acid. In the nucleic acid structure, at least one padlock is in a cyclic form and contains the nucleotide sequence A, B, C in the 5' to 3' direction. Where: - A is a nucleotide sequence located at the 5' end of the padlock, which is complementary to and hybridizes with the first portion of the target nucleic acid. - B is a nucleotide sequence that is not complementary to the target nucleic acid. - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and hybridizing with the second portion. Here, the first and second portions of the target nucleic acid are located adjacent to each other, and at least one padlock is added in the form of a system of two different oligonucleotide molecules. Here: - The first molecule contains the sequence DABC or ABCD in the 5' to 3' direction, where sequence D is characterized by being non-complementary to the target nucleic acid, preventing sequences A and C from being ligated to each other; and - The second molecule contains sequence E, where sequence E is complementary to sequence B or a portion thereof. The nucleic acid structure of step a) having the padlock in a circular form is obtained by hybridizing the first molecule to the target nucleic acid and the second molecule to the first molecule, and then cleaving sequence D by exonucleic acid degradation using an ssDNA-specific exonuclease or FLAP endonuclease. b) A step of ligating the 5' end of sequence A to the 3' end of sequence C using at least one ligase to obtain a padlock as a ring-closed molecule containing sequence A, B, C by covalent bonds. c) a step of subjecting the ligated cyclic padlock probe from step b) to rolling circle amplification (RCA), wherein the RCA is carried out using bacteriophage phi29 polymerase; and a step of amplifying the RCA product by an MDA reaction using at least two MDA primers, wherein the primers are up to 15 nucleotides in length, preferably up to 10 nucleotides. d) A step of detecting the amplification product of step c) and thereby detecting the target nucleic acid. Here, the detection method is preferably a sequence-specific detection method, which detects the complementary sequence of the sequence located in the junction region of sequences A and C that is formed when sequences A and C are ligated in step b).

[0197] A method using Gaplock obtained in two different oligonucleotide molecules having a split in sequence ABC (shown in Figure 1E), a split in sequence A (shown in Figure 1F), or a split in both sequences A and C: In one embodiment, the method includes the following steps: a) A step of adding at least one padlock probe to the sample to obtain a nucleic acid structure formed by hybridization of at least one padlock with the target nucleic acid. In the nucleic acid structure, at least one padlock is in a cyclic form and contains the nucleotide sequence A, B, C in the 5' to 3' direction. Where: - A is a nucleotide sequence located at the 5' end of the padlock, which is complementary to and hybridizes with the first portion of the target nucleic acid. - B is a nucleotide sequence that is not complementary to the target nucleic acid. - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and hybridizing with the second portion. Here, the first and second portions of the target nucleic acid are located adjacent to each other, and at least one padlock is added in the form of a system of two different oligonucleotide molecules. Here: - The first molecule contains sequence A, followed by sequence B, and the first part of sequence C (C1) in the 5' to 3' direction, and the second molecule contains the second part of sequence C (C2), or - The first molecule contains the first part of sequence A (A1) in the 5' to 3' direction, followed by sequences B and C, and the second molecule contains the second part of sequence A (A2), or - The first molecule comprises the first part of sequence A (A1) in the 5'-3' direction, followed by sequence B, and the first part of sequence C (C1), and the second molecule comprises the second part of sequence C (C2) in the 5'-3' direction, followed by the second part of sequence A (A2). Here, the nucleic acid structure of step a) having the padlock in a cyclic form is obtained by hybridizing the first molecule and the second molecule with the target nucleic acid. b) A step of obtaining a padlock as a covalently ring-closed molecule containing sequence ABC by ligating the 5' end of the first molecule with the 3' end of the second molecule and the 5' end of the second molecule with the 3' end of the first molecule using at least one ligase. c) a step of subjecting the ligated cyclic padlock probe from step b) to rolling circle amplification (RCA), wherein the RCA is carried out using bacteriophage phi29 polymerase; and a step of amplifying the RCA product by an MDA reaction using at least two MDA primers, wherein the primers are up to 15 nucleotides in length, preferably up to 10 nucleotides. d) A step of detecting the amplification product of step c) thereby detecting the target nucleic acid. Here, the detection method is preferably a sequence-specific detection method, which detects a complementary sequence of a sequence located at one of the junction regions of the first molecule and the second molecule, which is formed by the ligation of the first molecule and the second molecule in step b).

[0198] As shown in Figure 1G, sequences ABC are obtained in two different oligonucleotide molecules, and a method using Gaplock such that the padlock contains sequence E: In one embodiment, the method includes the following steps: a) A step of adding at least one padlock probe to the sample to obtain a nucleic acid structure formed by hybridization of at least one padlock with the target nucleic acid. In the nucleic acid structure, at least one padlock is in a cyclic form and contains the nucleotide sequence A, B, C in the 5' to 3' direction. Where: - A is a nucleotide sequence located at the 5' end of the padlock, which is complementary to and hybridizes with the first portion of the target nucleic acid. - B is a nucleotide sequence that is not complementary to the target nucleic acid. - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and hybridizing with the second portion. Here, the first and second portions of the target nucleic acid are located adjacent to each other, and at least one padlock is added in the form of a system of three different oligonucleotide molecules. Here: - The first molecule contains sequence A in the 5' to 3' direction, followed by sequence B, and the first part of sequence C (C1); the second molecule contains the second part of C (C2); the third molecule contains sequence E, or - The first molecule contains the first part of sequence A (A1) in the 5' to 3' direction, followed by sequences B and C; the second molecule contains the second part of sequence A (A2); the third molecule contains sequence E, or - The first molecule contains the first part of sequence A (A1) in the 5'-3' direction, followed by sequence B, and the first part of sequence C (C1); the second molecule contains the second part of sequence C (C2) and the second part of sequence A (A2) in the 5'-3' direction; the third molecule contains sequence E. Here, sequence E is complementary to sequence B or a portion thereof, and the nucleic acid structure of step a) having the padlock in a cyclic form is obtained by hybridizing the first molecule and the second molecule to the target nucleic acid. b) A step of obtaining a padlock as a covalently ring-closed molecule containing sequence ABC by ligating the 5' end of the first molecule with the 3' end of the second molecule and the 5' end of the second molecule with the 3' end of the first molecule using at least one ligase. c) a step of subjecting the ligated cyclic padlock probe from step b) to rolling circle amplification (RCA), wherein the RCA is carried out using bacteriophage phi29 polymerase; and a step of amplifying the RCA product by an MDA reaction using at least two MDA primers, wherein the primers are up to 15 nucleotides in length, preferably up to 10 nucleotides. d) A step of detecting the amplification product of step c) thereby detecting the target nucleic acid. Here, the detection method is preferably a sequence-specific detection method, which detects a complementary sequence of a sequence located at one of the junction regions of the first molecule and the second molecule, which is formed by the ligation of the first molecule and the second molecule in step b).

[0199] As shown in Figure 1H, a method using ssSplitclock such that sequence ABC is obtained in two different oligonucleotide molecules having a split in sequence B, and further containing sequence E: In one embodiment, the method includes the following steps: a) A step of adding at least one padlock probe to the sample to obtain a nucleic acid structure formed by hybridization of at least one padlock with the target nucleic acid. In the nucleic acid structure, at least one padlock is in a cyclic form and contains the nucleotide sequence A, B, C in the 5' to 3' direction. Where: - A is a nucleotide sequence located at the 5' end of the padlock, which is complementary to and hybridizes with the first portion of the target nucleic acid. - B is a nucleotide sequence that is not complementary to the target nucleic acid. - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and hybridizing with the second portion. Here, the first and second portions of the target nucleic acid are located adjacent to each other, and the padlock probe is added as a system of three different oligonucleotide molecules. Here: - The first molecule contains sequence A in the 5' to 3' direction, followed by the first part of sequence B (B1), - The second molecule contains the second part of B (B2) in the 5' to 3' direction, followed by sequence C, and - The third molecule includes sequence E in the 5' to 3' direction, where sequence E is complementary to sequence B or a portion thereof (preferably a portion of both B1 and B2) and can hybridize with them. The nucleic acid structure of step a) having the padlock in a cyclizable form is obtained by hybridizing the first and second molecules to the target nucleic acid and the third molecule to the first and second molecules. b) A step of obtaining a padlock as a covalently ring-closed molecule containing sequence ABC by ligating the 5' end of the first molecule with the 3' end of the second molecule and the 5' end of the second molecule with the 3' end of the first molecule using at least one ligase. c) a step of subjecting the ligated cyclic padlock probe from step b) to rolling circle amplification (RCA), wherein the RCA is carried out using bacteriophage phi29 polymerase; and a step of amplifying the RCA product by an MDA reaction using at least two MDA primers, wherein the primers are up to 15 nucleotides in length, preferably up to 10 nucleotides. d) A step of detecting the amplification product of step c) thereby detecting the target nucleic acid. Here, the detection method is preferably a sequence-specific detection method, which detects a complementary sequence of a sequence located in one of the junction regions of the first molecule and the second molecule, which are formed by the ligation of the first molecule and the second molecule in step b). Preferably, the sequence is located in the junction region of sequences A and C.

[0200] A method using dsSplitclock, as shown in Figures 1I and 1J, in which sequence ABC is obtained in two different oligonucleotide molecules having a split in sequence B, and further comprises sequence E obtained in two different oligonucleotide molecules: In one embodiment, the method includes the following steps: a) A step of adding at least one padlock probe to the sample to obtain a nucleic acid structure formed by hybridization of at least one padlock with the target nucleic acid. In the nucleic acid structure, at least one padlock is in a cyclic form and contains the nucleotide sequence A, B, C in the 5' to 3' direction. Where: - A is a nucleotide sequence located at the 5' end of the padlock, which is complementary to and hybridizes with the first portion of the target nucleic acid. - B is a nucleotide sequence that is not complementary to the target nucleic acid. - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and hybridizing with the second portion. Here, the first and second portions of the target nucleic acid are located adjacent to each other, and the padlock probe is added as a system of four different oligonucleotide molecules. Here: - The first molecule contains sequence A (A1) and the first part of sequence B (B1) in the 5' to 3' direction, - The second molecule contains the second part of B (B2) in the 5' to 3' direction, followed by sequence C. - The third molecule comprises the first portion (E1) of sequence E, where sequence E1 is complementary to and capable of hybridizing with a portion of sequence B that contains at least a portion of the first portion (B1) of sequence B. - The fourth molecule comprises the second part (E2) of sequence E, where sequence E is complementary to and can hybridize with a portion of sequence B that contains at least a portion of the second part (B2) of sequence B. The nucleic acid structure of step a) having the padlock in a cyclic form is obtained by hybridizing the first molecule and the second molecule to the target nucleic acid, and the third molecule and the fourth molecule to the first molecule and the second molecule, respectively. Preferably, the nucleic acid structure includes an inverse complementary projection at the junction between sequences B1 / E1 and B2 / E2. b) A step of obtaining a padlock as a covalently ring-closed molecule containing sequence ABC by ligating the 5' end of the first molecule with the 3' end of the second molecule and the 5' end of the second molecule with the 3' end of the first molecule using at least one ligase. c) a step of subjecting the ligated cyclic padlock probe from step b) to rolling circle amplification (RCA), wherein the RCA is carried out using bacteriophage phi29 polymerase; and a step of amplifying the RCA product by an MDA reaction using at least two MDA primers, wherein the primers are up to 15 nucleotides in length, preferably up to 10 nucleotides. d) A step of detecting the amplification product of step c) thereby detecting the target nucleic acid. Here, the detection method is preferably a sequence-specific detection method, which detects a complementary sequence of a sequence located in one of the junction regions of the first molecule and the second molecule, which are formed by the ligation of the first molecule and the second molecule in step b). Preferably, the sequence is located in the junction region of sequences A and C.

[0201] Each embodiment disclosed herein is assumed to be applicable to each other disclosed embodiment. Therefore, all combinations of the various elements described herein fall within the scope of the invention. Furthermore, it should be understood that in any method claimed herein, unless the reverse is explicitly stated, the order of steps or actions in a method involving multiple steps or actions is not necessarily limited to the order described.

[0202] The following provisions are also included in the present invention: 1. A method for detecting the presence of a target nucleic acid in a sample by superbranched rolling circle amplification (HRCA), comprising the following steps: a) A step of adding at least one padlock probe to the sample to provide a nucleic acid structure formed by hybridization of the at least one padlock with the target nucleic acid, wherein the at least one padlock is in a cyclic form and comprises a nucleotide sequence A, B, C in the 5' to 3' direction, where: - A is a nucleotide sequence located at the 5' end of the padlock, complementary to and hybridized with the first portion of the target nucleic acid. - B is a nucleotide sequence that is not complementary to the target nucleic acid, and - C is a nucleotide sequence located at the 3' end of the padlock, complementary to and hybridized with the second portion of the target nucleic acid. The first portion and the second portion of the target nucleic acid are located adjacent to each other in the process, b) A step of subjecting the cyclizable padlock to a ligation reaction using at least one ligase to obtain a padlock having sequence ABC as a single molecule that is ring-closed by covalent bonds, c) The ligated cyclic padlock from step b) is subjected to a rolling circle amplification (RCA) reaction, and the RCA product is amplified in an MDA reaction using an MDA primer up to 10 nucleotides in length, and d) A step of detecting the target nucleic acid by detecting the amplified product obtained from step c) using a sequence-specific detection method.

[0203] 2. The at least one padlock is added in step a) in the form of a system of at least two different oligonucleotide molecules, where the first molecule comprises nucleotide sequence ABC in the 5' to 3' direction, and the second molecule comprises nucleotide sequence E, where sequence E is complementary to sequence B or a portion thereof. The nucleic acid structure having the padlock in a cyclic form of step a) is obtained by hybridizing the first molecule with the target nucleic acid and the second molecule with the first molecule. The method according to Clause 1, wherein the single ring-closed molecule by covalent bonding in step b) is obtained by ligating the 5' end of sequence A with the 3' end of sequence C using at least one ligase, and the sequence-specific detection method in step d) detects a complementary sequence of the sequence formed when sequences A and C are ligated in step b) and are located in the junction region of sequences A and C.

[0204] 3. The at least one padlock is added in step a) in the form of a single oligonucleotide molecule containing sequence DABC or sequence ABCD in the 5' to 3' direction, where sequence D is located at the 3' end of sequence C or the 5' end of sequence A and is not complementary to the target nucleic acid. The nucleic acid structure having the padlock in a cyclic form of step a) is obtained by first hybridizing the padlock in the form of a single oligonucleotide molecule with the target nucleic acid, and then cleaving sequence D by exonucleic acid degradation using an ssDNA-specific exonuclease or FLAP endonuclease. The method according to Clause 1, wherein the single molecule ring-closed by covalent bonding in step b) is obtained by ligating the 5' end of sequence A with the 3' end of sequence C using at least one ligase, and the sequence-specific detection method detects a complementary sequence of the sequence formed when sequences A and C are ligated in step b), which is located in the junction region of sequences A and C.

[0205] 4. The at least one padlock is added in step a) in the form of a system of two different oligonucleotide molecules, where: - The first molecule contains the sequence DABC or sequence ABCD in the 5' to 3' direction, where sequence D is located at the 3' end of sequence C or the 5' end of sequence A, and has features that are not complementary to the target nucleic acid. - The second molecule contains sequence E, and sequence E is complementary to sequence B or its region. The nucleic acid structure having a padlock in a cyclic form of step a) is obtained by first hybridizing the first molecule with the target nucleic acid and the second molecule, and then cleaving sequence D by exonucleic acid degradation using an ssDNA-specific exonuclease or FLAP endonuclease; The method according to Clause 1, wherein the single molecule ring-closed by covalent bonding in step b) is obtained by ligating the 5' end of sequence A with the 3' end of sequence C using at least one ligase, and the sequence-specific detection method detects a complementary sequence of the sequence formed when sequences A and C are ligated in step b) and is located in the junction region of sequences A and C.

[0206] 5. The at least one padlock is added in step a) in the form of a system of two different oligonucleotide molecules, where: - The first molecule contains sequence A, sequence B, and the first part of sequence C (C1) in the 5' to 3' direction, and the second molecule contains the second part of sequence C (C2), or - The first molecule contains the first part of sequence A (A1), sequence B, and sequence C in the 5' to 3' direction, and the second molecule contains the second part of sequence A (A2), or - The first molecule comprises the first part of sequence A (A1), sequence B, and the first part of sequence C (C1) in the 5' to 3' direction, and the second molecule comprises the second part of sequence C (C2) and the second part of sequence A (A2) in the 5' to 3' direction. The nucleic acid structure having the padlock in a cyclic form of step a) is obtained by hybridizing the first molecule and the second molecule with the target nucleic acid; The method according to Clause 1, wherein the single ring-closed molecule by covalent bonding in step b) is obtained by ligating the 3' end of the first molecule with the 5' end of the second molecule and ligating the 5' end of the first molecule with the 3' end of the second molecule, and the sequence-specific detection method in step d) detects a complementary sequence of the sequence formed when the first molecule and the second molecule are ligated in step b), located in one of the junction regions of the first molecule and the second molecule.

[0207] 6. The at least one padlock is added in step a) in the form of a system of three different oligonucleotide molecules, where: - The first molecule contains sequence A, sequence B, and the first part of sequence C (C1) in the 5' to 3' direction, the second molecule contains the second part of sequence C (C2) in the 5' to 3' direction, and the third molecule contains sequence E, or - The first molecule contains the first part of sequence A (A1), sequence B, and sequence C in the 5' to 3' direction, the second molecule contains the second part of sequence A (A2) in the 5' to 3' direction, and the third molecule contains sequence E, or - The first molecule comprises the first part of sequence A (A1), sequence B, and the first part of sequence C (C1) in the 5' to 3' direction; the second molecule comprises the second part of sequence C (C2) and the second part of sequence A (A2) in the 5' to 3' direction; and the third molecule comprises sequence E. Sequence E is complementary to sequence B or its region, and the nucleic acid structure of step a) having the padlock in a cyclic form is obtained by hybridizing the first molecule and the second molecule with the target nucleic acid and the third molecule; The method according to Clause 1, wherein the single ring-closed molecule by covalent bonding in step b) is obtained by ligating the 3' end of the first molecule with the 5' end of the second molecule and the 5' end of the first molecule with the 3' end of the second molecule, and the sequence-specific detection method in step d) detects a complementary sequence of the sequence formed when the first molecule and the second molecule were ligated in step b) and is located in one of the junction regions of the first molecule and the second molecule.

[0208] 7. The at least one padlock is added in step a) in the form of a system of three different oligonucleotide molecules, where: - The first molecule contains the first portion (B1) of sequence A and sequence B in the 5' to 3' direction; - The second molecule contains the second part of sequence B (B2) and sequence C in the 5' to 3' direction; and - The third molecule comprises sequence E, wherein sequence E is complementary to the first portion (B1) and / or the second portion (B2) of sequence B. The nucleic acid structure having the padlock in a cyclic form of step a) is obtained by hybridizing the first molecule and the second molecule with the target nucleic acid and the third molecule, The method according to Clause 1, wherein the single ring-closed molecule by covalent bonding in step b) is obtained by ligating the 3' end of the first molecule with the 5' end of the second molecule and the 5' end of the first molecule with the 3' end of the second molecule, and the sequence-specific detection method in step d) detects a complementary sequence of sequences that are located in the junction region of sequences A and C and that were formed when sequences A and C were ligated in step b).

[0209] 8. The at least one padlock is added in step a) in the form of a system of four different oligonucleotide molecules, where: - The first molecule includes sequence A in the 5' to 3' direction, and the first portion of sequence B (B1), - The second molecule contains the second part (B2) of sequence B and sequence C in the 5' to 3' direction, - The third molecule contains the first part (E1) of sequence E, and the first part (E1) of sequence E is complementary to the first part (B1) of sequence B, hybridizes with the first part (B1) of sequence B, and - The fourth molecule contains the second part (E2) of sequence E, and the second part (E2) of sequence E is complementary to the second part (B2) of sequence B, hybridizes with the second part (B2) of sequence B, The nucleic acid structure having the padlock in the cyclizable form of step a) is obtained by hybridizing the first molecule and the second molecule with the target nucleic acid as well as the third molecule and the fourth molecule, The single molecule closed in a ring by the covalent bond in step b) is obtained by ligating the 3' end of the first molecule to the 5' end of the second molecule and ligating the 5' end of the first molecule to the 3' end of the second molecule, and the sequence-specific detection method of step d) is located in the junction region of sequence A and sequence C, and detects the complementary sequence of the sequence formed when sequence A and sequence C are ligated in step b). The method according to claim 1.

[0210] 9. The method according to any one of claims 1 to 9, wherein the ligation reaction in step b) is carried out by at least one DNA ligase.

[0211] 10. The sequence-specific detection method is CRISPR / Cas, preferably CRISPR / Cas12a, the at least one padlock contains at least one protospacer adjacent motif (PAM), and the guide RNA (gRNA) hybridizes with a sequence that is reverse complementary to the region of the padlock adjacent to the PAM for targeting. The method according to any one of the preceding claims.

[0212] [[ID=二十]]11. The method according to any one of the preceding claims, wherein the target nucleic acid is a single-stranded nucleic acid.

[0213] 12. The method according to any one of the preceding clauses, wherein the target nucleic acid is the genome of a virus.

[0214] 13. The method according to any one of the preceding clauses, carried out in the absence of an ammonium salt, preferably ammonium sulfate.

[0215] 14. The method according to any one of the preceding clauses, carried out under isothermal conditions, preferably in a temperature range of 20 to 30°C.

[0216] 15. A composition comprising at least one padlock as defined in Clauses 1 to 8.

[0217] [Array List] [Sequence ID 1:SplintR:DNA ligase derived from Paramecium bursaria Chlorella Virus-1] MAITKPLLAATLENIEDVQFPCLATPKIDGIRSVKQTQMLSRTFKPIRNSVMNRLLTELL PEGSDGEISIEGATFQDTTSAVMTGHKMYNAKFSYYWFDYVTDDPLKKYIDRVEDMKNYI TVHPHILEHAQVKIIPLIPVEINNITELLQYERDVLSKGFEGVMIRKPDGKYKFGRSTLK EGILLKMKQFKDAEATIISMTALFKNTNTKTKDNFGYSKRSTHKSGKVEEDVMGSIEVDY DGVVFSIGTGFDADQRRDFWQNKESYIGKMVKFKYFEMGSKDCPRFPVFIGIRHEEDR [Sequence ID 2: Phi29 wild-type polymerase] MPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNL KFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSL KKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQ GLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIG EGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQ IKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDF IDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKD PVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYW AHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTF ENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK [Sequence ID 3:Qualiphi polymerase] MPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNL KFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSL KKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQ GLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIG EGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQ IKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDF IDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKD PVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYW AHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTF ENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGTGSGAWKEWLERKVGEGRARRLIEYFG SAGEVGKLVENAEVSKLLEVPGIGDEAVARLVPGYKTLRDAGLTPAEAERVLKRYGSVSK VQEGATPDELRELGLGDAKIARILG [SEQ ID NO: 24: Padlock (similar to SEQ ID NO: 16)] [Chem.] Here, the 5'-end of the padlock is phosphorylated (indicated by [Phos]) to facilitate ligation of the 5'-end and 3'-end of the hybridized padlock. Here, the underlined sequence represents sequence "A", the bold sequence "N" represents sequence "B", and the italicized sequence represents sequence "C".

[0218] [SEQ ID NO: 25: Padlock (similar to SEQ ID NO: 8)] [Chem.] Here, the 5'-end of the padlock is phosphorylated (indicated by [Phos]) to facilitate ligation of the 5'-end and 3'-end of the hybridized padlock. Here, the underlined sequence represents sequence "A", the bold sequence "N" represents sequence "B", and the italicized sequence represents sequence "C".

[0219] [Sequence ID 26: Padlock (similar to Sequence ID 23)] [ka] Here, the 5' end of the padlock is phosphorylated (indicated by [Phos]), facilitating ligation of the 5' and 3' ends of the hybridized padlock. Here, the underlined sequence represents sequence "A", the bold sequence "N" represents sequence "B", the italicized sequence represents sequence "C", and the highlighted font represents sequence "D".

[0220] [Sequence ID 27: Padlock (similar to Sequence IDs 17 and 18)] [ka] Here, the 5' end of the padlock is phosphorylated (indicated by [Phos]), facilitating ligation of the 5' and 3' ends of the hybridized padlock. Here, the underlined sequence represents sequence "A", the bold sequence "N" represents sequence "B", the italicized sequence represents sequence "C1", and the highlighted font represents sequence "C2".

[0221] [Sequence ID 32: Padlock (similar to Sequence IDs 19+20 and 28+29)] [ka] Here, the 5' end of the padlock is phosphorylated (indicated by [Phos]), facilitating ligation of the 5' and 3' ends of the hybridized padlock. Here, the underlined sequence represents sequence "A", the bolded sequence "N" represents sequence "B", and the italicized sequence represents sequence "C".

[0222] The present invention will be described by the following embodiments, but these embodiments are merely illustrative and do not limit the scope of the present invention in any way.

[0223] [Examples] Example 1. Demonstration of the principle As a demonstration of the principle of detecting RNA sequences at low temperatures (30°C) by hyperbranched rolling circle amplification (HRCA) mediated by phi29pol (Figure 2a), a padlock probe for human ACTB mRNA was designed. Subsequently, the HRCA reaction was performed using Qualiphi and SplintR-ligase enzymes as follows. Amplification was observed only when both SplintR-ligase and the corresponding ACTB synthetic RNA were included in the reaction (Figure 2b). This result demonstrates that Qualiphi can be used for HRCA-mediated RNA detection at low temperatures.

[0224] [Table 1]

[0225] reaction: [Table 2]

[0226] [Table 3]

[0227] [Table 4]

[0228] Example 2. Target preparation Next, to verify the sensitivity level of this method, the HRCA reaction was performed by gradually decreasing the concentration of ACTB synthetic RNA, as described in Example 1. As can be seen in Figure 2c, the sensitivity of the reaction was 10 7 It was limited to a number of target molecules exceeding a certain threshold.

[0229] Example 3. Ammonium sulfate To determine if problems in the ligation or amplification steps of the reaction were causing the decrease in sensitivity, the target and padlock probe were annealed in a 1:1 ratio, ligation was performed, and then the amplification of the resulting ring was tested by sequentially diluting the ligation product. Furthermore, the possibility that ammonium sulfate, contained in Qualiphi's commercially available reaction buffer, might limit the amplification to some extent under these conditions was tested. Firstly, the detection sensitivity of the diluted ring (Figure 3) was similar to that of the target preparation experiment (Figure 2c), confirming the existence of a clear amplification problem. Furthermore, when ammonium sulfate was removed from the reaction, 10 7 Since molecules of the order could be clearly detected, the presence of ammonium sulfate was clearly detrimental in the amplification reaction using the Qualiphi enzyme. The experiment was carried out as described in Example 1, both in the presence and absence of ammonium sulfate in Qualiphi buffer, as shown.

[0230] Example 4. Primer Since ammonium sulfate is known to affect primer annealing and is used to increase the specificity and sensitivity of PCR reactions, we decided to test the effect of primer length on the reaction and used short (10nt) primers with low Tm (Figure 4). Strong amplification was observed in all dilutions of the tested ring, but this came at the cost of significant levels of nonspecific amplification being observed in all samples, including linear padlocks, both in the absence of the target and / or ligase. The problem with Qualiphi in HRCA implementation is thought to be due to the behavior of nucleic acids at the low temperature (30°C) used in the assay, rather than to the inherent properties of the enzyme, and therefore, it is feasible to establish conditions for the required sensitivity. The experiment was performed as described in Example 3, without ammonium sulfate in the Qualiphi buffer and with the following MDA1 / 2 primers.

[0231] [Table 5]

[0232] Example 5. Wild-type phi29pol Qualiphi is a modified version of phi29pol with a fused DNA-binding domain, resulting in enhanced substrate affinity and processability. We tested whether this optimized method could also be operated with the wild-type enzyme. As can be seen in Figure 5, using wild-type phi29pol resulted in significantly delayed amplification and reduced sensitivity at a 2-hour incubation time; however, this problem was circumvented by extending the incubation time to 6 hours. Therefore, while this method works with wild-type phi29pol, an enhanced version such as Qualiphi may be necessary to shorten the diagnostic test time. The experiment was basically as described in Example 4, but performed under the amplification conditions described below. Ammonium sulfate was beneficial to the performance of the wild-type enzyme.

[0233] reaction: [Table 6]

[0234] For all of these approaches, the amplification conditions were optimized, but at the cost of losing specificity. The following examples aim to reduce nonspecific background signals while maintaining high sensitivity.

[0235] Example 6. Detection of CRISPR-Cas12a Given that target-specific (i.e., circularized and amplified, hybridized padlock) and non-specific (i.e., non-hybridized padlock) amplification events are unlikely to produce the same sequence, particularly at the padlock breakpoint regions, one possibility to avoid non-specificity in the assay while maintaining high amplification yield was considered to be combining the assay with a sequence-specific detection step, such as CRISPR-Cas12a-mediated detection (Figure 6a). Therefore, a novel padlock probe was designed to anneal to the S gene region of the SARS-CoV-2 viral genome, and it included a Cas12a PAM motif to enable detection of amplified dsDNA. A corresponding gRNA specifically targeting this sequence was also designed on the (+) viral strand, thereby enabling detection of only the amplified material and not the padlock itself. Next, HRCA-Cas12a detection was performed with progressively reduced concentrations of in vitro transcribed S gene RNA under the conditions described below (Figure 6b). In these reactions, the nucleolytic activity of Cas12a ribonucleoparticles specifically activated by the amplified target is detected by the fluorescence emission of the reporter substrate. Interestingly, specific and nonspecific amplified substances that are indistinguishable in the gel can be distinguished when combined with CRISPR-Cas12a sequence-specific detection. 5 We achieved the detection of a number of molecules on the order of magnitude.

[0236] [Table 7]

[0237] reaction: [Table 8]

[0238] [Table 9]

[0239] [Table 10]

[0240] [Table 11]

[0241] With this in mind, the inventors focused on implementing various improvements in the padlock that could reduce background noise and / or improve target-specific amplification, either individually or in combination. Both wild-type and Qualiphi versions of the enzyme were used without distinction, and the merits of the various approaches tested were evaluated.

[0242] Example 7. Skinlock Since amplification and background issues are likely due to nonspecific annealing events, we decided to cover the padlock backbone with a complementary DNA strand (referred to herein as skin or sequence E). This should protect against nonspecific priming events (reducing background), limit hybridization with the remaining padlock's initial RCA product, suppress competition with MDA primers, and limit the HCRA reaction. This skin may contain a 3'-OH end that functions as an initial point for DNA synthesis, or it may have a blocked (e.g., biotin-bound) 3' end, in which case synthesis needs to be triggered by the annealed RNA. Testing these possibilities using phi29pol (Figure 7) confirmed that the blocked skin significantly reduced background signal. Amplification was further improved by including the 3'-OH skin, while maintaining the specificity advantage. Therefore, we conclude that a skin inversely complementary to the padlock backbone can limit background amplification. Depending on the sensitivity requirements, this skin may or may not retain an extendable 3'-OH end. The experiment was conducted as described in Example 6, but under the amplification conditions described in Example 5. Furthermore, the padlock was pre-annealed with the skin described below.

[0243] [Table 12]

[0244] Example 8. Flaplock and exonuclease treatment To improve the specific detection of amplification products obtained from padlock hybridization in the presence of target nucleic acids, we explored approaches to ensure that the hybridized and amplified padlock differs as much as possible from the padlock ligated in the absence of the target. One possibility is to include a sequence at the junction of the padlock's fracture site, which is then specifically removed only upon annealing with the target. We decided to include a poly-A extension at the 3' end of the S gene padlock to form a flap upon annealing with the target RNA (Figure 8a). Some approaches have involved removing the 5'-flap padlock using a structure-specific endonuclease, but the inventors have adopted the use of a 3'-flap and treatment with E. coli exonuclease I (ExoI), a 3'ssDNA-specific exonuclease, because this approach can achieve two objectives: removal of the annealed padlock flap and removal of excess, unannealed padlock. The latter is relevant because excess padlock may interfere with the reaction by annealing with the initial RCA product, potentially resulting in some background amplification. It is also worth noting that if not removed from the reaction, the remaining padlock can function as an MDA primer. As shown in Figure 8b, this flap-ExoI approach using the Qualiphi enzyme clearly reduces the background signal, and 10 3 This enables detection of the target molecule, and this detection can be easily identified from samples that do not contain the target RNA. The experiment was carried out as described in Example 6, but using the padlock-flap described below, and included an ExoI degradation step after annealing and before ligation, as shown below.

[0245] [Table 13]

[0246] reaction: [Table 14]

[0247] Example 9. Flaplock without the use of exonuclease. Phi29pol and its derivatives (such as Qualiphi) also possess strong 3'-exonuclease activity. Therefore, we investigated whether this approach using a padlock flap would function even without ExoI treatment. In fact, it was confirmed that the ExoI treatment step was not necessary (Figure 9), and in the reaction using the flap padlock, the detection level and background signal were similar whether or not the ExoI treatment step was included. The experiment was carried out as described in Example 6, using the padlock described in Example 8.

[0248] Example 10. Gaplock Another possibility for differentiating hybridized and amplified padlocks from those that did not hybridize to the target sequence is to remove a certain number of nucleotides from the rupture junction (sequence A or C) of the padlock to form a gap, which is then filled by providing a short oligonucleotide to cover the gap, thereby circularizing the padlock through a double ligation event, and the correct sequence is reconstructed only when the molecule is fully aligned with the target RNA (Figure 10a). As shown in Figure 10b, the use of this Gaplock approach completely eliminated the background signal. The experiment was carried out as described in Example 6, but the amplification conditions were those of Example 5 (wild-type phi29pol), and the padlock (AB-C1 sequence) and gap fragment (C2 sequence) were as follows.

[0249] [Table 15]

[0250] Example 11. Splitlock with separate arms Finally, the inventors hypothesized that separating the padlock sequence B into two different molecules or arms (B1 and B2, i.e., splitting the padlock into two fragments) would also likely significantly reduce the background signal, as long as the padlock is not completely cyclized (Figure 1H-J), because the annealing regions of the MDA primers are located on different molecules.

[0251] In one embodiment (ssSplitlock; Figure 1H), two arms independently locate target regions, after which a skin (sequence E) is added to function as a sprint between the two arms. The breaks in the target RNA and skin are ligated by SplintR and T4 ligases, respectively. This approach not only reduces background signaling but is also likely to promote productive circularization events rather than padlock dimerization, which can be a significant limitation, especially under conditions of low target availability, a limitation that applies to many diagnostic approaches.

[0252] Another embodiment (dsSplitlock; Figure 1I) provides a padlock arm that is already double-stranded (pre-hybridized by the split skin) at the break point region of the backbone, which may be designed to produce an inversely complementary overhang to facilitate ligation. In this case, a padlock arm with a 5 bp 5' overhang is designed herein, but other configurations may be used. Figures 11a and 11b show that both the ssDNA and dsDNA Splitlock approaches are effective in reducing background signal, respectively.

[0253] Finally, this dsSplitlock approach can be designed so that the sequences of E1 and E2 cannot be ligated. This makes the 3'OH end of E2 available for priming in the RCA reaction only in a ligated padlock (dsSplitlock-nick; Figure 1J). In this case, it is desirable to use a design that leaves a 3' overhang to prevent it from being filled by the polymerase, and also to leave at least a 1-nucleotide gap when E1 and E2 are annealed to facilitate the action of the polymerase on the ligated template. In this case, we used a design with a 5bp 3' overhang with a 1-nucleotide gap and an E1 fragment lacking a 5' phosphate group. A key advantage of this arrangement is that a one-step reaction condition is possible because the padlock is not modified by the DNA polymerase until it is properly assembled and circularized. Furthermore, the cyclization of the padlock using the RCA product is facilitated, and this padlock can self-containedly induce subsequent RCA reactions, even in the absence of MDA, potentially leading to exponential amplification. Figure 11c shows that this approach further enhances sensitivity without increasing background signal.

[0254] The ssSplitlock experiment was carried out as described in Example 6, but the following additional components were included in the ligation process.

[0255] [Table 16]

[0256] reaction: [Table 17]

[0257] The dsSplitlock experiment was conducted as described above, but each split arm of the padlock was pre-hybridized with the corresponding inversely complementary (skin) oligonucleotide before the annealing step of the reaction.

[0258] [Table 18]

[0259] The dsSplitlock-nick experiment was performed using the following oligonucleotides. In this case, the RCA reaction was carried out for 3 hours using Equiphi29 DNA polymerase.

[0260] [Table 19]

[0261] Example 12. Combination of Approaches The implementation of each of these improvements, and combinations thereof, may depend on the specific needs of the diagnostic test to be developed. As an example, the inventors combined flap pad lock and ExoI treatment with a 3'-OH skin. The results shown in Figure 12 were obtained. Under these conditions, background signal was minimal, at just 10 3 The molecule was detected down to the level described in Example 7, including the ExoI treatment described in Example 8.

[0262] In all the examples described above, real-time fluorescence measurements were used, but one of the advantages of CRISPR-Cas12a detection is that, as mentioned above, it is easily adaptable to qualitative diagnosis in side-flow apparatuses. The high signal levels obtained in the approach described herein are fully compatible with this type of detection. [Brief explanation of the drawing]

[0263] [Figure 1-1] Figure 1: (A to J) Preferred padlocks of the present invention. [Figure 1-2] Figure 1: (A to J) Preferred padlocks of the present invention. [Figure 1-3] Figure 1: (A to J) Preferred padlocks of the present invention. [Figure 1-4] Figure 1: (A to J) Preferred padlocks of the present invention. [Figure 1-5] Figure 1: (A to J) Preferred padlocks of the present invention. [Figure 1-6] Figure 1: (A to J) Preferred padlocks of the present invention. [Figure 1-7] Figure 1: (A to J) Preferred padlocks of the present invention. [Figure 2a] Figure 2: Detection of HRCA-mediated RNA using a padlock probe and HRCA. (a) A scheme for the basic method, in which a ring is specifically generated in the presence of target RNA using a padlock probe, and this product is amplified by RCA-MDA, known as superbranched RCA (HRCA). The RCA reaction can be primed by the target RNA. The main advantage of using phi29pol / Qualiphi is that the reaction can be carried out at room temperature. [Figure 2b] Figure 2: Detection of HRCA-mediated RNA using a padlock probe and HRCA. (b) Principle verification experiment. Padlocks targeting the human ACTB gene were incubated with the specified synthetic RNA in the presence and absence of SplintR ligase, as described in Example 1. The amplified material was electrophoresed on a 0.8% agarose gel and visualized by Gelred staining. [Figure 2c] Figure 2: Detection of HRCA-mediated RNA using a padlock probe and HRCA. (c) Preparation of the target. The experiment was carried out as described in Example 2. The human ACTB padlock probe was incubated in the presence of a specified amount of synthetic ACTB target RNA. The amplified material was electrophoresed on a 0.8% agarose gel and visualized by Gelred staining. [Figure 3]Figure 3: Optimization of HRCA conditions: buffer. The experiment was performed as described in Example 3. A specified amount of pre-ligation (cyclic) or linear ACTB padlock probe was subjected to the HRCA reaction. The amplified substance was electrophoresed on a 0.8% agarose gel and visualized by Gelred staining. [Figure 4] Figure 4: Optimization of HRCA conditions: primers. The experiment was performed as described in Example 4. The HRCA reaction included specified amounts of ACTB RNA in the presence or absence of ACTB padlock and / or SplintR ligase. The amplified material was electrophoresed on a 0.8% agarose gel and visualized by Gelred staining. [Figure 5] Figure 5: HRCA using wild-type phi29pol. The experiment was performed as described in Example 5. The HRCA reaction contained the specified amount of ACTB RNA. A negative control (NC) without an ACTB padlock was included. Amplification was performed for 2 hours (B) and 6 hours (A) as shown. The amplified material was electrophoresed on a 0.8% agarose gel and visualized by Gelred staining. [Figure 6a] Figure 6: HRCA combined with CRISPR-Cas12a detection. (a) HRCA-CRISPR-Cas12a detection was performed as described in Example 6. The reaction contained a specified amount of target RNA. It also contained a target (without padlock) but with the maximum amount of target, and a negative control (NC, negative control) without padlock or target. The total amplification product detected by agarose gel electrophoresis (A) and the specific detection result by CRISPR-Cas12a in the reporter substrate (B) are shown. [Figure 6b]Figure 6: HRCA combined with CRISPR-Cas12a detection. (a) HRCA-CRISPR-Cas12a detection was performed as described in Example 6. The reaction contained a specified amount of target RNA. It also contained a target (without padlock) but with the maximum amount of target, and a negative control (NC, negative control) without padlock or target. The total amplification product detected by agarose gel electrophoresis (A) and the specific detection result by CRISPR-Cas12a in the reporter substrate (B) are shown. [Figure 7A] Figure 7: Improved padlock:skin for HRCA CRISPR-Cas12a. The HRCA reaction was carried out using the specified amount of target RNA as described in Example 7, with the S gene padlock not present (A), or with skin containing block 3' biotin (B) or skin containing 3'OH (C). A negative control (NC, negative control) without a padlock or target was also included. [Figure 7B] Figure 7: Improved padlock:skin for HRCA CRISPR-Cas12a. The HRCA reaction was carried out using the specified amount of target RNA as described in Example 7, with the S gene padlock not present (A), or with skin containing block 3' biotin (B) or skin containing 3'OH (C). A negative control (NC, negative control) without a padlock or target was also included. [Figure 7C] Figure 7: Improved padlock:skin for HRCA CRISPR-Cas12a. The HRCA reaction was carried out using the specified amount of target RNA as described in Example 7, with the S gene padlock not present (A), or with skin containing block 3' biotin (B) or skin containing 3'OH (C). A negative control (NC, negative control) without a padlock or target was also included. [Figure 8] Figure 8: Improved padlock for HRCA CRISPR-Cas12a: FLAP and ExoI. The HRCA reaction was carried out as described in Example 8, including ExoI treatment. A specified amount of target RNA was incubated with an S gene padlock containing a poly-A extension at the 3' end. A negative control (NC) without a padlock or target was included. [Figure 9] Figure 9: Improved padlock for HRCA CRISPR-Cas12a: FLAP. The HRCA reaction was carried out as described in Example 9. The specified amount of target RNA was incubated with the S gene padlock. A negative control (NC) without the padlock or target was included. [Figure 10] Figure 10: Improved padlock for HRCA CRISPR-Cas12a: GAP. The HRCA reaction was carried out as described in Example 10 and as shown on the left. A specified amount of target RNA was incubated with an S gene padlock containing a gap at the breakpoint junction, and the gap was filled with additional oligonucleotides. A negative control (NC) without a padlock or target was included. [Figure 11A] Figure 11: Modification of HRCA CRISPR-Cas12a: Two-arm padlock. Results of HRCA reactions using ssDNA(a), dsDNA(b), and dsDNA-nick(c) split padlock probes are shown as described in Example 11. A specified amount of target RNA and a negative control (NC) without a padlock or target were tested. [Figure 11B] Figure 11: Modification of HRCA CRISPR-Cas12a: Two-arm padlock. Results of HRCA reactions using ssDNA(a), dsDNA(b), and dsDNA-nick(c) split padlock probes are shown as described in Example 11. A specified amount of target RNA and a negative control (NC) without a padlock or target were tested. [Figure 11C] Figure 11: Modification of HRCA CRISPR-Cas12a: Two-arm padlock. Results of HRCA reactions using ssDNA(a), dsDNA(b), and dsDNA-nick(c) split padlock probes are shown as described in Example 11. A specified amount of target RNA and a negative control (NC) without a padlock or target were tested. [Figure 12]Figure 12: Modification of HRCA CRISPR-Cas12a: FLAP and skin combination. The experiment was carried out as described in Example 12 and is shown on the left. Results for a specified amount of target RNA and for a negative control (NC) without a padlock or target are shown.

Claims

1. A method for detecting the presence of a target nucleic acid in a sample by hyperbranched rolling circle amplification (HRCA), comprising the following steps: a) A step of adding at least one padlock probe to the sample and providing a nucleic acid structure formed by hybridization of the at least one padlock with the target nucleic acid, wherein the at least one padlock is in a cyclic form and comprises a nucleotide sequence A-B-C in the 5' to 3' direction, where: - A is a nucleotide sequence located at the 5' end of the padlock, complementary to and hybridized with the first portion of the target nucleic acid. - B is a nucleotide sequence that is not complementary to the target nucleic acid, and - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and hybridized with the second portion. The first portion and the second portion of the target nucleic acid are located adjacent to each other in the process, b) A step of subjecting the cyclizable padlock to a ligation reaction using at least one ligase to obtain a padlock having sequence A-B-C as a single molecule closed by covalent bonds, c) The ligated cyclic padlock from step b) is subjected to a rolling circle amplification (RCA) reaction, and the RCA product is amplified in an MDA reaction using an MDA primer up to 10 nucleotides in length, and d) A step of detecting the target nucleic acid by detecting the amplification product obtained from step c) using a sequence-specific detection method, wherein the sequence-specific detection method detects at least a portion of the sequence formed when the ends of sequence A and / or sequence C were ligated in step b), or a sequence including the inverse complementary sequence of such ligated sequence.

2. The at least one padlock is added in step a) in the form of a system of at least two different oligonucleotide molecules, The first molecule contains nucleotide sequences A-B-C in the 5' to 3' direction, and the second molecule contains nucleotide sequence E, where sequence E is complementary to sequence B or a portion thereof. The nucleic acid structure having a cyclically formable padlock in step a) is obtained by hybridizing the first molecule with the target nucleic acid and the second molecule with the first molecule. The method according to claim 1, wherein the single molecule ring-closed by covalent bonding in step b) is obtained by ligating the 5' end of sequence A with the 3' end of sequence C using at least one ligase, and the sequence-specific detection method in step d) detects the following sequence: i) Arrangements located in the junction region of sequence A and sequence C, formed when sequence A and sequence C are ligated in step b), and / or ii) The inverse complementary sequence of sequences located in the junction region of sequences A and C, formed when sequences A and C were ligated in step b).

3. The at least one padlock is added in step a) in the form of a single oligonucleotide molecule containing sequence D-A-B-C or sequence A-B-C-D in the 5' to 3' direction, wherein sequence D is located at the 3' end of sequence C or the 5' end of sequence A and has characteristics that are not complementary to the target nucleic acid. The nucleic acid structure having a cyclically formed padlock in step a) is obtained by first hybridizing the padlock in the form of a single oligonucleotide molecule with the target nucleic acid, and then cleaving sequence D by exonucleic acid degradation using an ssDNA-specific exonuclease or FLAP endonuclease. The method according to claim 1, wherein the single molecule ring-closed by the covalent bond in step b) is obtained by ligating the 5' end of sequence A with the 3' end of sequence C using at least one ligase, and the sequence-specific detection method detects the following sequence: i) Arrangements located in the junction region of sequence A and sequence C, formed when sequence A and sequence C are ligated in step b), and / or ii) The inverse complementary sequence of sequences located in the junction region of sequences A and C, formed when sequences A and C were ligated in step b).

4. The at least one padlock is added in step a) in the form of a system of two different oligonucleotide molecules: - The first molecule contains the sequence D-A-B-C or A-B-C-D in the 5' to 3' direction, wherein sequence D is located at the 3' end of sequence C or the 5' end of sequence A, and has characteristics that are not complementary to the target nucleic acid. - The second molecule contains sequence E, and sequence E is complementary to sequence B or its region. The nucleic acid structure having a padlock in a cyclic form of step a) is obtained by first hybridizing the first molecule with the target nucleic acid and the second molecule, and then cleaving sequence D by exonucleic acid degradation using an ssDNA-specific exonuclease or FLAP endonuclease; The method according to claim 1, wherein the single molecule ring-closed by the covalent bond in step b) is obtained by ligating the 5' end of sequence A with the 3' end of sequence C using at least one ligase, and the sequence-specific detection method detects the following sequence: i) Arrangements located in the junction region of sequence A and sequence C, formed when sequence A and sequence C are ligated in step b), and / or ii) The inverse complementary sequence of sequences located in the junction region of sequences A and C, formed when sequences A and C were ligated in step b).

5. The at least one padlock is added in step a) in the form of a system of two different oligonucleotide molecules: - The first molecule contains sequence A, sequence B, and the first part of sequence C (C1) in the 5' to 3' direction, and the second molecule contains the second part of sequence C (C2), or - The first molecule contains the first part of sequence A (A1), sequence B, and sequence C in the 5' to 3' direction, and the second molecule contains the second part of sequence A (A2), or - The first molecule comprises the first part of sequence A (A1), sequence B, and the first part of sequence C (C1) in the 5' to 3' direction, and the second molecule comprises the second part of sequence C (C2) and the second part of sequence A (A2) in the 5' to 3' direction. The nucleic acid structure having the padlock in a cyclic form of step a) is obtained by hybridizing the first molecule and the second molecule with the target nucleic acid; The method according to claim 1, wherein the single ring-closed molecule in step b) is obtained by ligating the 3' end of the first molecule with the 5' end of the second molecule and the 5' end of the first molecule with the 3' end of the second molecule, and the sequence-specific detection method in step d) detects the following sequence: i) an arrangement located in one of the junction regions of the first molecule and the second molecule, formed when the first molecule and the second molecule are ligated in step b), and / or ii) An inverse complementary sequence of the sequence formed when the first molecule and the second molecule are ligated in step b), located in one of the junction regions of the first molecule and the second molecule.

6. The at least one padlock is added in step a) in the form of a system of three different oligonucleotide molecules: - The first molecule contains sequence A, sequence B, and the first part of sequence C (C1) in the 5' to 3' direction, the second molecule contains the second part of sequence C (C2) in the 5' to 3' direction, and the third molecule contains sequence E, or - The first molecule contains the first part of sequence A (A1), sequence B, and sequence C in the 5' to 3' direction, the second molecule contains the second part of sequence A (A2) in the 5' to 3' direction, and the third molecule contains sequence E, or - The first molecule comprises the first part of sequence A (A1), sequence B, and the first part of sequence C (C1) in the 5' to 3' direction; the second molecule comprises the second part of sequence C (C2) and the second part of sequence A (A2) in the 5' to 3' direction; and the third molecule comprises sequence E. Sequence E is complementary to sequence B or a region thereof, and the nucleic acid structure having the padlock in a cyclic form of step a) is obtained by hybridizing the first molecule and the second molecule with the target nucleic acid and the third molecule; The method according to claim 1, wherein the single ring-closed molecule in step b) is obtained by ligating the 3' end of the first molecule with the 5' end of the second molecule and the 5' end of the first molecule with the 3' end of the second molecule, and the sequence-specific detection method in step d) detects the following sequence: i) an arrangement located in one of the junction regions of the first molecule and the second molecule, the first molecule and the second molecule formed when they were ligated in step b), and / or ii) A reverse complementary sequence of the sequence formed when the first molecule and the second molecule were ligated in step b), wherein the first molecule and the second molecule are located in one of the junction regions.

7. The at least one padlock is added in step a) in the form of a system of three different oligonucleotide molecules: - The first molecule contains a portion of sequence A and sequence B (B1) in the 5' to 3' direction; - The second molecule includes the second portion of sequence B (B2) and sequence C in the 5' to 3' direction; and - The third molecule contains sequence E, wherein sequence E is complementary to the first portion (B1) and / or the second portion (B2) of sequence B. The nucleic acid structure having the padlock in a cyclic form of step a) is obtained by hybridizing the first molecule and the second molecule with the target nucleic acid and the third molecule, The method according to claim 1, wherein the single ring-closed molecule in step b) is obtained by ligating the 3' end of the first molecule with the 5' end of the second molecule and the 5' end of the first molecule with the 3' end of the second molecule, and the sequence-specific detection method in step d) detects the following sequence: i) Arrangements located in the junction region of sequence A and sequence C, formed when sequence A and sequence C are ligated in step b), and / or ii) The inverse complementary sequence of sequences located in the junction region of sequences A and C, formed when sequences A and C were ligated in step b).

8. The at least one padlock is added in step a) in the form of a system of four different oligonucleotide molecules: - The first molecule includes sequence A and the first portion of sequence B (B1) in the 5' to 3' direction, - The second molecule includes the second part of sequence B (B2) and sequence C in the 5' to 3' direction. - The third molecule comprises a first portion (E1) of sequence E, the first portion (E1) of sequence E is complementary to the first portion (B1) of sequence B, and hybridizes with the first portion (B1) of sequence B, and - The fourth molecule includes a second portion (E2) of sequence E, the second portion (E2) of sequence E is complementary to the second portion (B2) of sequence B, and hybridizes with the second portion (B2) of sequence B. The nucleic acid structure having the padlock in a cyclic form of step a) is obtained by hybridizing the first molecule and the second molecule with the target nucleic acid, and the third molecule and the fourth molecule, respectively. The method according to claim 1, wherein the single ring-closed molecule in step b) is obtained by ligating the 3' end of the first molecule with the 5' end of the second molecule and the 5' end of the first molecule with the 3' end of the second molecule, and the sequence-specific detection method in step d) detects the following sequence: i) Arrangements located in the junction region of sequence A and sequence C, formed when sequence A and sequence C are ligated in step b), and / or ii) The inverse complementary sequence of sequences located in the junction region of sequences A and C, formed when sequences A and C were ligated in step b).

9. The method according to any one of claims 1 to 9, wherein the ligation reaction in step b) is carried out by at least one DNA ligase.

10. The method according to any one of the preceding claims, wherein the sequence-specific detection method is CRISPR / Cas, preferably CRISPR / Cas12a, and the at least one padlock comprises at least one protospacer adjacent motif (PAM), and the guide RNA (gRNA) hybridizes with and targets a sequence that is inversely complementary to a region of the padlock adjacent to the PAM.

11. The method according to any one of the preceding claims, wherein the target nucleic acid is a single-stranded nucleic acid.

12. The method according to any one of the preceding claims, wherein the target nucleic acid is the genome of a virus.

13. The method according to any one of the preceding claims, carried out in the absence of an ammonium salt, preferably ammonium sulfate.

14. The method according to any one of the preceding claims, which is carried out under isothermal conditions, preferably in a temperature range of 20 to 30°C.

15. A cyclizable padlock probe characterized by comprising at least two different oligonucleotide molecules, i) The first molecule contains the nucleotide sequence A-B-C in the 5' to 3' direction, where: - A is a nucleotide sequence located at the 5' end of the padlock, complementary to the first portion of the target nucleic acid, and capable of hybridizing with the first portion. - B is a nucleotide sequence that is not complementary to the target nucleic acid, and - C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and capable of hybridizing with the second portion. The first and second portions of the target nucleic acid are located adjacent to each other; and ii) The second molecule contains a nucleotide sequence E, where sequence E is complementary to sequence B or a region thereof, where: - The length of array E is at least 80% of the length of array B, and / or - Sequence E contains a modified nucleotide at its 3' end, which prevents sequence E from priming the RCA reaction. A cyclizable padlock probe is obtained by hybridizing the first molecule to the target nucleic acid and the second molecule to the first molecule.

16. The cyclizable padlock probe according to claim 15, wherein the first molecule further comprises sequence D, wherein sequence D is located at the 3' end of sequence C or at the 5' end of sequence A and is not complementary to the target nucleic acid. A cyclizable padlock probe obtained by first hybridizing the first molecule with the target nucleic acid and the second molecule, and then cleaving sequence D by exonucleic acid degradation using an ssDNA-specific exonuclease or FLAP endonuclease.

17. A cyclizable padlock probe characterized by comprising at least three different oligonucleotide molecules, - The first molecule contains sequence A, sequence B, and the first part of sequence C (C1) in the 5' to 3' direction; the second molecule contains the second part of sequence C (C2) in the 5' to 3' direction; the third molecule contains sequence E, or - The first molecule includes the first part of sequence A (A1), sequence B, and sequence C in the 5' to 3' direction; the second molecule includes the second part of sequence A (A2) in the 5' to 3' direction; the third molecule includes sequence E, or - The first molecule comprises the first part of sequence A (A1), sequence B, and the first part of sequence C (C1) in the 5'-3' direction; the second molecule comprises the second part of sequence C (C2) and the second part of sequence A (A2) in the 5'-3' direction; the third molecule comprises sequence E, where: - Sequence A is a nucleotide sequence located at the 5' end of the padlock, complementary to the first portion of the target nucleic acid, and capable of hybridizing with the first portion. - Sequence B is a nucleotide sequence that is not complementary to the target nucleic acid. - Sequence C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and capable of hybridizing with the second portion, and - Sequence E is complementary to sequence B or its region and can hybridize with sequence B or its region, where: - The length of array E is at least 80% of the length of array B, and / or - Sequence E contains a modified nucleotide at its 3' end, and sequence E cannot prime the RCA reaction. A cyclizable padlock probe, wherein the first and second portions of the target nucleic acid are located adjacent to each other, and the cyclizable padlock is obtained by hybridizing the first and second molecules with the target nucleic acid and the third molecule.

18. A cyclizable padlock probe characterized by comprising at least three different oligonucleotide molecules, - The first molecule contains a portion of sequence A and sequence B (B1) in the 5' to 3' direction; - The second molecule includes the second portion of sequence B (B2) and sequence C in the 5' to 3' direction; and - The third molecule comprises sequence E, which is complementary to the first (B1) and / or second (B2) portion of sequence B, where: - Sequence A is a nucleotide sequence located at the 5' end of the padlock, complementary to the first portion of the target nucleic acid, and capable of hybridizing with the first portion of the target nucleic acid. - Sequence B is a nucleotide sequence that is not complementary to the target nucleic acid. - Sequence C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and capable of hybridizing with the second portion, and - Sequence E is complementary to sequence B or its region and can hybridize with sequence B or its region, where: - The length of array E is at least 80% of the length of array B, and / or - Sequence E contains a modified nucleotide at its 3' end, and sequence E cannot prime the RCA reaction. A cyclizable padlock probe, wherein the first and second portions of the target nucleic acid are located adjacent to each other, and the cyclizable padlock is obtained by hybridizing the first and second molecules with the target nucleic acid and the third molecule.

19. A cyclizable padlock probe characterized by comprising at least four different oligonucleotide molecules, - The first molecule includes the first portion (B1) of sequence A and sequence B in the 5' to 3' direction, - The second molecule includes the second part of sequence B (B2) and sequence C in the 5' to 3' direction, - The third molecule comprises a first portion (E1) of sequence E, the first portion (E1) of sequence E is complementary to the first portion (B1) of sequence B, and can hybridize with the first portion (B1) of sequence B, and - The fourth molecule comprises a second portion (E2) of sequence E, the second portion (E2) of sequence E being complementary to the aforementioned second portion (B2) of sequence B and capable of hybridizing with the aforementioned second portion (B2) of sequence B, where: - Sequence A is a nucleotide sequence located at the 5' end of the padlock, complementary to the first portion of the target nucleic acid, and capable of hybridizing with the first portion. - Sequence B is a nucleotide sequence that is not complementary to the target nucleic acid. - Sequence C is a nucleotide sequence located at the 3' end of the padlock, complementary to the second portion of the target nucleic acid, and capable of hybridizing with the second portion, and - Sequence E includes E1 and E2, - At least 80% of the length of array B, and / or - The sequence E contains a modified nucleotide at its 3' end, which prevents it from priming the RCA reaction. A cyclizable padlock probe is obtained by hybridizing the first and second portions of the target nucleic acid adjacent to each other, and the cyclizable padlock is obtained by hybridizing the first and second molecules with the target nucleic acid, and the third and fourth molecules, respectively.

20. A padlock according to any one of claims 15 to 19, wherein the nucleotide modification at the 3' end of sequence E includes adding a molecule that prevents the 3' end from being used as a primer by the polymerase of the reaction, preferably including the addition of biotin.

21. Use of a padlock as defined in any one of claims 15 to 20 in a method for detecting the presence of a target nucleic acid in a sample by amplification.

22. A method for detecting the presence of a target nucleic acid in a sample by amplification, using a padlock as defined in any one of claims 15 to 20, The amplification method includes a detection step of detecting the amplified nucleic acid by a sequence-specific detection method, thereby detecting the target nucleic acid, wherein the detection step includes detecting at least a portion of the sequence formed when the ends of sequence A and / or sequence C were ligated in step b), or a sequence containing a sequence that is inversely complementary to such ligated sequence.

23. The use according to any one of claims 21 to 22, wherein the method is a rolling circle amplification (RCA) method or a hyperbranched rolling circle amplification (HRCA) method.

24. The use according to any one of claims 21 to 22, wherein the method is one of the methods defined in any one of claims 1 to 14.

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