Method for detecting nucleic acid amplification products using blockers

Abstract

The present invention provides a method of detecting two or more differently labeled high molecular weight nucleic acid products in a mixture wherein the products are detected by detecting one or more labels attached directly or indirectly to each product, the method comprising adding to the detection mixture an analytic agent to facilitate the analysis of the differently labeled products, wherein the analysis agent is used for inhibiting interaction between different labels. The product is preferably a rolling circle amplification (RCA) or super RCA product, and the resolving agent is preferably bovine serum albumin (BSA). BSA may be added to the RCA reaction mixture prior to detection.

Description

Technical Field

[0001] The present disclosure and invention relate to the field of nucleic acid detection. Specifically, the present disclosure and invention relate to methods for detecting products of rolling circle amplification (RCA) or other amplification or hybridization reactions that produce large nucleic acid products. According to the methods herein, the resolution of differentially labeled RCA or other nucleic acid amplification products in the same reaction mixture can be improved. This has specific applicability in the detection of target nucleic acid sequences. Background Art

[0002] Detection of target nucleic acid sequences has applications in many different areas, including, inter alia, personalized medicine and diagnosis, prognosis and / or treatment of diseases (e.g., cancer, infectious diseases and hereditary or genetic diseases) in the clinic, as well as in research and biosafety.

[0003] The target nucleic acid sequence can be easily detected using a labeled hybridization probe, but simple hybridization probes have a relatively high detection limit and cannot be easily used to distinguish similar nucleic acid sequences. In order to increase sensitivity, the target nucleic acid molecule containing the target sequence can usually be amplified to increase the amount of the target sequence that can be used for detection. Any of the various techniques known in the art can be used for amplification, and specifically, rolling circle amplification (RCA) has many advantages.

[0004] RCA is an isothermal amplification technique that utilizes a strand displacement polymerase and requires a circular amplification template. The amplification of the circular template provides a series of RCA products that contain multiple copies of a sequence that is complementary to the amplification template. This concatemer usually forms a ball or "blob" that is easy to observe and detect, and therefore, analysis based on RCA has been used to detect nucleic acids, and is even commonly used as a reporter system for detecting any target analyte. Two types of target nucleic acids that can be directly cyclized, or probes, or more general reporter nucleic acids can provide template nucleic acid rings for RCA.

[0005] The above-mentioned RCA-based analysis relies on the secondary amplification of the initial RCA product to increase the amount of the product detected, thereby providing signal amplification in the detection. These include, for example, hyperbranched RCA, which produces many unclustered subsequent RCA products by chain replacement activity. Recently, a so-called "SuperRCA" (sRCA) reaction has been developed, which includes 2 or more rounds of RCA amplification, wherein the product (second RCP) of the second round of RCA reaction is connected to the product (called the first RCP) of the first round of reaction. A version of this sRCA method is described in WO2014 / 0796209. In this method, a probe that can provide or act as a primer hybridizes with the initial RCA product and is used to initiate the amplification of the second RCA template ring hybridized with the "primer-probe". The second RCA template can be generated by the cyclization of a padlock probe hybridized with the "primer-probe".

[0006] In WO 2015 / 071445, a method for alternative sRCA, called "Padlock sRCA", is described, in which a padlock probe is used to bind directly to the initial RCA product (RCP). Thus, the padlock probe can hybridize to each monomer repeat of the first RCP (i.e. each complementary copy of the first RCA template used to generate the first RCP), or at least to a large portion of them. The hybridized padlock probe can then be circularized by intramolecular ligation and used as a second RCA template to generate a second RCP connected to the first RCP.

[0007] The sensitivity afforded by such RCA (particularly sRCA) methods means that they have applications in the detection of rare or low-abundance targets, particularly where mutations are being detected or where limited numbers of samples are available, which may be rare events. It is often convenient or desirable to perform the assay in a multiplex format, where multiple target nucleic acid sequences are assayed together in the same sample or reaction mixture. This requires that the individual RCPs generated by each individual target be detected in the mixture in a manner that allows them to be distinguished from one another.

[0008] RCPs are usually labeled for detection. This may involve incorporating the label into the product, for example, by incorporating labeled nucleotides during the RCP synthesis process, or by labeling the RCP using a chimeric dye. A common method for labeling RCPs is to use a labeled detection oligonucleotide that hybridizes with the RCP. Therefore, RCPs can be labeled directly or indirectly. RCPs are long concatemers that contain many (usually hundreds, and in some cases thousands) of complementary copies of the RCA template loop, and also provide a large number of sites where labels can be incorporated or connected. sRCA products contain a very large number of such concatemers because the padlock probe can be repeatedly hybridized (directly or indirectly) with a high proportion of the monomers of the first RCP, and each such padlock probe can produce a second RCP. Therefore, the sRCA product (sRCP) produced can be labeled by many labeling molecules.

[0009] A common problem with any multiplex detection technique that relies on the use of tags to distinguish between large nucleic acid products is the potential for interference between tags in or on different products. Specifically, it has been found that tags attached to individual RCPs in the same mixture may stick to each other, thus preventing resolution of individual RCPs. Since a particular advantage of RCA-based detection techniques is that individual RCPs can be detected and quantified individually, this presents a problem of potential interference in the analysis and reduces the level of multiplexing that can be performed.

[0010] This problem is not limited to the detection of RCP, but also applies to any large or high molecular weight nucleic acid product containing or providing a large number of labeled sites. This may include, for example, the product of an amplification method of a hybridization chain reaction (HCR), in which a large number of monomers are incorporated, each of which contains a label or detectable sequence that can hybridize with a detection oligonucleotide. For example, known signal amplification methods may involve the establishment of mutually hybridized probe sequences to provide multiple binding sites for labeled detection probes or other labeling systems, such as the RNAscopeTM technology described in WO2011 / 094669. Although RNAscopeTM was developed for in situ hybridization detection of RNA, it illustrates the principle of using sandwich or intermediate hybridization probes, each of which provides multiple binding sites for binding to labeled detection probes, thereby generating a detectable nucleic acid product containing multiple labels. This nucleic acid product can be regarded as a so-called "nucleic acid accumulation" (NAA), which includes multiple target sites for detection, or in other words, multiple detectable sites. Summary of the invention

[0011] Overview

[0012] The present inventors have discovered that resolution of differentially labeled nucleic acid products (eg, RCP and other NAAs) can be improved by using a resolving agent, which, when included with the labeled nucleic acid products, reduces interference between labels on the different products.

[0013] Such analytical agents are usually proteins, in particular inert proteins, such as proteins commonly used as blocking agents in analytical methods to prevent or reduce non-specific binding of analytical reagents. However, they may also include non-protein reagents, such as detergents or synthetic polymers. Although the use of such blocking agents is well known, their use in steps for labeling or detecting analytical reaction products has not been described before. The currently proposed method shows a new use for such blocking agents.

[0014] Therefore, in a first aspect, the present invention provides a method for detecting two or more differently labeled high molecular weight nucleic acid products in a mixture, wherein the products are detected by detecting one or more labels directly or indirectly attached to each product, and the method includes including a resolving agent in the mixture for detection to facilitate the resolution of the differently labeled products, wherein the resolving agent is used to inhibit interactions between different labels.

[0015] Specifically, interactions between tags on different nucleic acid products will be inhibited.

[0016] In particular, the tags are attached at high copy numbers.

[0017] Therefore, the nucleic acid product to be detected is a high molecular weight nucleic acid product, each product comprising multiple tags. Specifically, the nucleic acid product comprises multiple sites for incorporating or connecting tags. The labeled nucleic acid product carries or comprises multiple copies of the tag.

[0018] Rather than referring to them as "high molecular weight" nucleic acid products, the nucleic acid products to be detected are optionally referred to as nucleic acid aggregates (NAAs), accumulated nucleic acid products, or nucleic acid clusters. Specifically, the nucleic acid products to be detected can be defined as NAAs, accumulated nucleic acid products, or nucleic acid clusters, all of which contain multiple copies of tags that can distinguish them from each other.

[0019] Differentiate different nucleic acid products so that they can be distinguished or identified from each other. In one embodiment, each nucleic acid product to be detected can be labeled with a label (in the sense of a label type), and different products have different labels. However, in order to improve the multiplexing capability and enable a greater number of different products in the mixture to be distinguished from each other, two or more labels (in the sense of label types) in different combinations and / or ratios can be used for each product, so that each product has a different combination and / or different ratio of labels.

[0020] In one embodiment, the nucleic acid product is a nucleic acid amplification, nucleic acid polymerization or nucleic acid hybridization product. Specifically, they can be products containing multiple copies or repeats of nucleic acid monomer units or oligonucleotide molecules.

[0021] In one embodiment, the nucleic acid products are RCA products (RCPs) or hybridization chain reaction (HCR) products, or hybridization products produced by hybridization of multiple hybridization probes to proximal positions on the same nucleic acid molecule.

[0022] Therefore, in one embodiment, the nucleic acid product is a hybridized nucleic acid product formed by hybridizing a plurality of hybridization probes together. Therefore, the nucleic acid product may represent the accumulation of a plurality of hybridization probes. Specifically, the nucleic acid product may include a scaffold nucleic acid hybridized with a plurality of hybridization probes.

[0023] Nucleic acid products can be produced as detectable signals or markers for detection of target analytes in detection methods (i.e., target analytes for analysis). In other words, nucleic acid products can be produced in methods for detecting target analytes. They can be considered as reaction products for detection analysis. In other words, they can represent signal products or detectable products of analysis.

[0024] More specifically, nucleic acid products can be produced in methods for detecting a target nucleic acid sequence in a target nucleic acid molecule. A target nucleic acid sequence or target nucleic acid molecule can be a target analyte of a detection method, or a reporter nucleic acid of an analyte of a detection method. Thus, nucleic acid products can be produced in detection methods for detecting any analyte, nucleic acid or non-nucleic acid (e.g., protein analyte).

[0025] In one embodiment, the nucleic acid product is labeled by hybridized detection oligonucleotides, each oligonucleotide comprising a detectable label.

[0026] In one embodiment, the monomers comprising the nucleic acid product (eg, HCR monomers or hybridization probes) comprise a tag.

[0027] In one embodiment, the label is detectable spectrophotometrically. In one embodiment, the label is a fluorescent label, a colored label, a colorimetric label, a particle label or a metal label.

[0028] In one embodiment, the nucleic acid product is detected by microscopy or flow cytometry.

[0029] In one embodiment, a resolving agent is added to the mixture of nucleic acid products.

[0030] In one embodiment, when the nucleic acid product is labeled, a resolving agent is present in the reaction mixture.

[0031] In one embodiment, the resolving agent is present in the reaction mixture as the nucleic acid product is produced and remains in the labeled mixture to be detected.

[0032] In another aspect, the present invention provides a reaction mixture comprising: (i) two or more differentially labeled high molecular weight nucleic acid products, wherein the products are labeled with multiple copies of one or more tags directly or indirectly attached to each product; and (ii) a resolving agent that facilitates the resolution of the differently labeled products, wherein the function of the resolving agent is to inhibit interactions between different tags.

[0033] In yet another aspect, provided herein is a method for detecting two or more target nucleic acid sequences in one or more nucleic acid molecules in a sample, the method comprising:

[0034] (a) for each target nucleic acid sequence, generating a high molecular weight nucleic acid product as a detection analysis reaction product of the sequence, and generating a reaction mixture containing the resulting nucleic acid products for two or more target nucleic acid sequences;

[0035] (b) providing multiple copies of one or more labels for each nucleic acid product of (a), wherein each nucleic acid product is differentially labeled and can be distinguished from another nucleic acid product, and wherein the labels are provided during or after the production of the nucleic acid products in (a), thereby producing a detection mixture comprising two or more differently labeled nucleic acid products, wherein a resolving agent is included in the detection mixture to facilitate the resolution of the differentially labeled products, wherein the resolving agent is used to inhibit interactions between different labels.

[0036] (c) detecting the labeled nucleic acid product in the detection reaction mixture, thereby detecting the target nucleic acid sequence.

[0037] In one embodiment, in step (b), the label is provided after the nucleic acid product is generated. Therefore, in this embodiment, step (b) is a separate labeling step performed after step (a). In this embodiment, step (b) may include contacting the reaction mixture from (a) with a labeling mixture comprising an analytical agent. Alternatively or in addition, an analytical agent may be contained in the reaction mixture to produce a nucleic acid product (i.e., in step (a)).

[0038] In one embodiment, the labeling mixture comprises a labeled detection oligonucleotide capable of hybridizing with the nucleic acid product. It can be understood that different detection oligonucleotides are provided for each nucleic acid product, wherein different detection oligonucleotides hybridize with different nucleic acid products. In other words, each nucleic acid product has a homologous detection oligonucleotide capable of specific hybridization with it.

[0039] The nucleic acid product may be any of the nucleic acid products described above. In one embodiment, the nucleic acid reaction product is a RCP.

[0040] In one embodiment of any of the above aspects, RCP is produced by RCA of a circularized padlock probe specific for a target nucleic acid sequence. In another embodiment, RCP is produced by RCA of a circularized target nucleic acid sequence or its amplicon (such as a PCR amplicon). In any of the above embodiments, RCA may include two or more rounds of RCA reactions. In other words, the RCA reaction may be an sRCA reaction. Therefore, the nucleic acid product may be a second or more generation RCP or sRCA product. DETAILED DESCRIPTION OF THE INVENTION

[0042] The method herein solves the problem of interference between labels in or on different nucleic acid products generated from different nucleic acid targets in detection analysis.

[0043] As mentioned above, multiplexed assays are often performed to detect multiple analytes or targets in a single sample or mixture. This requires that the signals generated by different targets be distinguished from each other. This can often be achieved by using different labels or different combinations or ratios of labels for different targets, so that each target has a specific signal associated with it.

[0044] As mentioned above, many detection methods rely on signal amplification to improve the sensitivity and accuracy of the analysis, and generate large nucleic acid products that can be easily labeled and detected, such as RCP, which is used as the final reaction product in many analytical methods and is a method of detecting the target analyte in the analysis. In other words, the reaction product generated as a signal or marker for the analysis is usually a large nucleic acid product, which may contain or be labeled with a high copy number of tags.

[0045] This multiplex detection method is often used to detect variants of a target nucleic acid sequence. The target nucleic acid sequence may often appear in variant forms, such as allelic variants, or mutant and wild-type sequences, and it may be necessary to detect which variant is present. Therefore, the target nucleic acid sequence can be one of many different variants of the nucleic acid sequence that may appear in the target nucleic acid molecule. It is important to be able to distinguish different variants from each other, so that the signal generated for each variant can be clearly detected. For example, a sample may contain mutant and wild-type sequences at the same time, and it may be important to detect and quantify these sequences separately, and this is also important for being able to correctly determine the allele frequency.

[0046] During the development of the methods herein, it was found that double positives with signals for both mutant and wild-type sequences were frequently observed. These are caused by the label (e.g., fluorophore) on the "mutant" nucleic acid product adhering to or otherwise interfering with the label on the "wild-type" product present in the same reaction mixture. This interference can be reduced or prevented by adding blocking agents to the detection reaction mixture that are commonly used to prevent nonspecific binding of proteins to surfaces. Thus, the accuracy of the detection, as well as the sensitivity and specificity of the detection method, can be improved.

[0047] As described above, the nucleic acid products used for detection that are produced as signals or markers for detecting targets or detecting analytes are defined as high molecular weight nucleic acid products, or alternatively as nucleic acid aggregates (NAAs) or aggregated nucleic acids. They are actually nucleic acid clusters that can be composed of a single nucleic acid molecule or a plurality of separate nucleic acid molecules or oligonucleotides that are hybridized together.

[0048] So-called "high molecular weight" refers to large nucleic acid products. Typically, this may include products in the micron size range (e.g., at least 0.5 micron), which can be easily observed by a microscope, or detected by, for example, flow cytometry as a particle. Therefore, nucleic acid products can be alternatively defined as the size detectable by a microscope or flow cytometry itself. In general, "high molecular weight" can represent at least 500kDa, such as at least 1 megadalton, or at least 10,50,100,200 or 500 megadalton, or even at least 1 gigadalton. Alternatively, it can be defined as a nucleic acid product comprising at least 1,5,10,20,50 or 100 megabases. Alternatively, high molecular weight nucleic acid products can be defined as labels or labeling agents that can carry at least 15000, 20000 or 25000 copies, for example, even including copies in the range of 100000 to 1,000,000.

[0049] In one embodiment, if the nucleic acid product includes or consists of monomer units (monomer repeats such as monomer repeats in RCP, or connected monomers such as monomers connected in HCR products, etc.), it can be at least 100, 200, 300, 400 or 500 monomers. For sRCA products comprising at least two generations of RCP, it can be at least 100x100 monomers, and so on, up to 500x500 monomers. However, with the extension of the RCA reaction time (e.g., up to 1 hour), it is not excluded that larger products can be obtained, for example, each RCA cycle has up to 700, 800, 900 or 1,000 monomers.

[0050] In one embodiment, the term "high molecular weight nucleic acid product" or NAA generally excludes PCR products. In particular, it excludes PCR products that contain a single repeat or copy of the target sequence to be detected.

[0051] Because nucleic acid product is very large, therefore can hold multiple copies of the label for marking them.In other words, they comprise multiple sites for incorporating or connecting labels, or the part (labeling part) carrying the label.In one embodiment, this site can be a sequence, referred to as a detection sequence in this article, which provides a binding site for the detection probe (i.e., the detection sequence referred to in this article). The detection sequence can be regarded as a label sequence. The detection probe can include a label. It can be seen that when the nucleic acid product includes multiple monomers, each monomer can include a detection sequence, and therefore includes a binding site for a label (specifically a labeled detection oligonucleotide).Similarly, the nucleic acid product can include multiple hybridization probes, and each hybridization probe includes a detection sequence.

[0052] As used herein, the term "plurality" or "multiplicity" refers to two or more, for example, 3, 4, 5, 6, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 or more. In fact, for many types of nucleic acid products, it may contain one thousand or more detection sequences, so that the binding site of the tag, or the detection oligonucleotide of the label is more accurate. Therefore, in terms of the number of copies of the tag, the binding / linkage site of the tag or the detection sequence, "multiple" may include the possible number of monomers as described above, at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000, or multiples thereof, for example in a sRCA product comprising a first and a second RCP.

[0053] The characteristic of nucleic acid product (or NAA) is that it contains detection sequence, or more broadly, contains labeling site, exists in high concentration or high copy number at well-defined site or position in space respectively. As mentioned above, such product can be derived from local amplification or polymerization reaction (such as RCA or HCR), or from the hybridization of multiple hybridization probes at proximal position on the same nucleic acid molecule. Such hybridization system can allow the establishment of branched nucleic acid structure, for example, similar to RNAscopeTM product mentioned above. Therefore, a template or scaffold molecule can be provided, which can hybridize with target nucleic acid sequence and contain multiple binding sites for further hybridization probes. These can contain detection sequence, or they can contain binding sites for further hybridization probes, etc. Therefore, a "layered" or "branched" structure composed of multiple hybridization probes can be constituted. Hybridization probe or its subset (such as the last or final hybridization probe added to the structure) can contain detection sequence. In such an embodiment, nucleic acid product can be referred to as hybridization assembly.

[0054] Thus, a nucleic acid product can be a nucleic acid amplification, nucleic acid polymerization or nucleic acid hybridization product.

[0055] In certain embodiments, they may be RCA or HCR products (RCP or HCRP), but they may be produced by any amplification reaction that can be used or adapted to produce such products, including, for example, PCR, recombinase polymerase amplification (RPA), helicase-dependent amplification (HAD), LAMP, multiple strand displacement amplification (MDA), etc. Amplification methods such as MDA can be used to produce branched amplification products.

[0056] Whether produced by amplification, polymerization or hybridization reaction, the nucleic acid product can be considered to include multiple monomers, covalently linked or linked together by hybridization, wherein each monomer can contain a detection sequence that can be hybridized by, for example, a labeled detection probe (also referred to herein as a "detection oligonucleotide"). The monomer can be a repeat in an RCP or other amplification product, an HCR monomer in an HCRP, or a hybridization probe in a hybridization product.

[0057] The nucleic acid product may have a linear or branched structure. As described above, for a linear product such as RCP, it may be coiled into a ball or "blob", which is a discrete visual entity.

[0058] The nucleic acid product is typically a DNA product. However, it may also consist of or include other natural or synthetic nucleic acids. Thus, for example, it may be a chimeric construct consisting of RNA and DNA. The nucleic acid product may consist of ribonucleotides and / or deoxyribonucleotides and synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. Thus, the nucleic acid product may be or may include, for example, LNA, PNA or any other derivative containing a non-nucleotide backbone.

[0059] The analytical reagent may be any blocking agent commonly used to block non-specific binding of analytical reagents in detection methods (e.g., in immunoassays), for example, coating or blocking non-specific binding sites on the surface of a reaction vessel, etc. Such blocking agents are typically proteins, but non-proteinaceous blocking agents are also known.

[0060] Thus, the analytical agent may be proteinaceous, i.e., it may be or may contain a protein. For convenience, this will be an inert protein. By "inert" is meant that the protein does not interfere with or participate in any reaction in the detection method, or more generally the detection mechanism. However, it may inhibit (i.e., reduce or prevent) any interaction that may occur between the tag molecules or parts. Specifically, it may inhibit the tags from binding or associating with each other.

[0061] The analytical agent can be a protein with very low, negligible or no specific binding affinity for the target analyte of the detection method, i.e., it cannot specifically bind to the target analyte. It can be a single species or type of protein, or a mixture of different proteins, such as different protein types or different species of the same type (the term "species" as used herein does not have a taxonomic meaning, but is intended to represent a specific specific type of protein).

[0062] The analytical agent can generally be a stable and abundant or common protein, such as serum protein, milk protein or gelatin. Thus, for example, it can be provided as casein, or in the form of dry milk powder, such as skim dry milk powder commonly used for blocking applications. The serum protein can be a single specific type of serum protein, or can contain serum proteins of multiple different types and structures. It can be or contain serum albumin and / or globulin, and can be provided as a single protein, a protein mixture or as serum.

[0063] Serum protein includes globulin and albumin as upper components, and is derived from plasma. Serum can be defined as the remaining blood portion after removing cells and platelets. More specifically, serum can be defined as the plasma portion without cells, fibrinogen or any other coagulation factor. Therefore, serum protein can be any protein that can be obtained (or obtainable) from serum or can be present in serum. It can be a single protein present in serum or obtained from serum, or a mixture of such proteins, or a protein part or protein component in serum. Generally, the analytical agent can include serum protein, that is, it can usually represent the serum protein component of serum without the need to separate specific protein components. In other words, it can be a mixture of proteins present in serum, and can be separated therefrom. Therefore, the analytical agent can include globulin and albumin, and preferably includes gamma-globulin (immunoglobulin) and / or serum albumin. The serum protein can be from a single blood source or multiple blood sources, i.e. from different animal individuals and / or from different types of animals. Therefore, serum proteins of different species can be used, such as serum proteins of any mammalian species. Although natural sources of serum proteins are convenient, serum proteins may also be derived or obtained synthetically, such as by recombinant expression or by derivatization of naturally occurring proteins. Thus, the term "serum protein" includes not only any protein that may be naturally present in serum, but also variants and derivatives thereof, such as fragments or truncated proteins, chemically modified proteins or polypeptides, or variants obtained by genetic engineering, such as polypeptides based on the amino acid sequence of a naturally occurring serum protein, but containing one or more amino acid substitutions, additions and / or deletions, etc. In addition, equivalent or corresponding proteins are also included, which are not necessarily present in serum but are structurally or functionally equivalent.

[0064] Globulins, pseudoglobulins and euglobulins are widely present in the animal and plant kingdoms and are characterized by their physical properties, such as solubility and electrophoretic migration, for example pseudoglobulins are soluble in water and dilute salt solutions, while euglobulins are insoluble in water and soluble in dilute salt solutions. Both globulin subclasses can coagulate when heated. Any such globulin can be used as a resolving agent. Therefore, in general, the resolving agent can include any globulin. As mentioned above, albumin can also be used, and accordingly, the resolving agent can be or can include any albumin.

[0065] The major sources of globulins are plasma and serum, milk, muscle, and plant seeds. Specifically, the term "globulin" includes a heterogeneous group of proteins found in serum that are classified as having a high molecular weight and lower solubility and electrophoretic mobility than albumin.

[0066] Thus, in one embodiment, the analytical agent is serum globulin, which may contain four major types of proteins, namely: alpha-1 globulin, alpha-2 globulin, beta-globulin and gamma-globulin. However, it will be appreciated by those skilled in the art that when serum is processed to remove fibrinogen and other coagulation factors, serum proteins contain only a subset of globulins, primarily gamma-globulin. Thus, the analytical agent may comprise a serum protein preparation, wherein the globulin portion of the serum protein contains at least 70% gamma-globulin, such as at least 80% or at least 90% gamma-globulin.

[0067] The alpha-globulins are characterized by their ability to be highly mobile in alkaline or charged solutions and include alpha-1 antitrypsin and serum amyloid A (alpha-1 globulins) as well as haptoglobin and ceruloplasmin (alpha-2 globulins). The beta-globulins are characterized by lower mobility in alkaline or charged solutions than the alpha-globulins, but higher than the gamma-globulins, which include plasminogen and transferrin. Thus, the gamma-globulins are less mobile in alkaline or charged solutions than both the alpha-globulins and the beta-globulins. The most predominant class of proteins is the immunoglobulins (antibodies). Antibodies are well described in the art and can be divided into different groups, generally IgG, IgE, IgD (all monomers), IgA (dimers), and IgM (pentamers).

[0068] In a specific embodiment, the analytical agent comprises γ-globulin, specifically immunoglobulin, more specifically IgG. In a convenient embodiment, the analytical agent is bulk IgG, i.e., immunoglobulin purified from serum, which comprises a variety of immunoglobulin (IgG) proteins with a certain range of binding specificity and affinity. Therefore, Bulk IgG comprises different IgG proteins with different specificities, i.e., a mixture of IgG proteins with different specificities. Although it is contemplated to use immunoglobulins with specific structures (i.e., binding properties) as analytical agents, this property is only feasible when the binding properties of immunoglobulins do not interfere with the detection of target analytes, such as when the target analyte of the detection method is a protein, and the analyte remains in the reaction mixture for the labeling and detection steps. The important point is that any combination of blocking agents with target analytes will not interfere with the performance of the analysis. Therefore, according to the analytical format, if the immunoglobulin component is, for example, IgG or Bulk IgG, it may be advantageous to have no or low (or very low, or negligible or undetectable) binding activity to the target analyte.

[0069] In an alternative embodiment, the resolving agent is or comprises serum albumin. The serum albumin may be from a single source, such as bovine serum albumin (BSA), human serum albumin (HSA), porcine serum albumin (PSA), etc., or may be a combination of different serum albumin proteins.

[0070] In other embodiments, the resolving agent can be a DNA binding protein, such as a single-strand binding protein (SSB).

[0071] Alternatively, the analytical agent can be non-protein. In such an embodiment, it can be or can include a detergent, such as a neutral detergent such as CHAPS, TritonX-100 or Tween-20. It can also be a non-protein polymer, such as polyvinyl alcohol, such as polyvinyl pyrrolidone (PVP).

[0072] Although the main advantage of the analytical agent is to reduce interference between labels in the detection step, it may also be beneficial to add the analytical agent in the early steps, especially in the step of producing high molecular weight nucleic acid products. Therefore, the analytical agent present in the reaction solution can play a role in the entire analytical reaction, thereby producing nucleic acid products for detection. For example, the analytical agent may also be beneficial to prevent nucleic acid products (such as RCA products) from colliding with each other, so as to optimize the rate of the generation (such as RCA) reaction and reduce errors in the process. Therefore, the analytical agent can be added before the labeling and detection steps, for example, before or during the step of producing the product (such as an amplification / polymerization / hybridization reaction that produces the product, such as an RCA reaction).

[0073] However, in certain reactions, such as in the RCA reaction mixture, when the analytical agent is present in high concentrations, the analytical agent may reduce the rate of the reaction (such as RCA) or inhibit the reaction (such as RCA). Therefore, in a preferred embodiment, the concentration of the analytical agent is at a level that does not affect or interfere with the reaction (such as RCA) of the nucleic acid product. For example, the maximum concentration of the analytical agent during the step of producing the nucleic acid product (such as RCA reaction) can be at most 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1% (by weight or w / v). In one embodiment, the maximum concentration of the analytical agent is 0.1% (by weight or w / v). Alternatively, in another embodiment, the analytical agent is present at a concentration of less than 0.1% (by weight or w / v). In another embodiment, the analytical agent is present at a concentration of at most (or not more than) 0.09 or 0.08 or 0.075% (by weight or w / v). This is especially true when the analytical agent is a protein.

[0074] In contrast, during the labeling or detection step, the concentration of the analytical agent need not be limited, and thus the concentration of the analytical agent during the labeling or detection step can be higher than the concentration in the reaction step in which the nucleic acid product is generated, for example, if additional analytical agent is added to the mixture during the labeling or detection step.

[0075] During the detection step, the concentration of the resolving agent should be high enough to improve resolution of nucleic acid product detection.

[0076] In one embodiment, particularly when a protein analytical agent is used, the minimum effective concentration of the analytical agent in the detection step can be, for example, at least 0.001, 0.002, 0.003, 0.004 or 0.005% (by weight or w / v). In one embodiment, the minimum concentration of the protein analytical agent is 0.001% (by weight or w / v).

[0077] Maintaining similar analytical reagent concentrations throughout an analytical or detection method or procedure may be advantageous for ease of use.

[0078] Therefore, the concentration range of the protein analytical agent in the reaction mixture can be 0.001-0.1%, or more specifically 0.001-0.09% or 0.001-0.08%, such as 0.001-0.08%, 0.002-0.09%, 0.002-0.08%, 0.002-0.07%, 0.002-0.06%, 0.002-0.05%, 0.003-0.08%, such as 0.004-0.07% (by weight or w / v). In fact, the range can be between any of the above integers, such as between any of the above minimum and maximum values. In a specific embodiment, the concentration range of the protein analytical agent can be, for example, 0.004-0.02% (by weight or w / v). In a preferred embodiment, the concentration of the protein sanctioning agent may be 0.01-0.02%, such as 0.01 or 0.02% (by weight or w / v).

[0079] In one embodiment, serum albumin can be used as a resolving agent. For example, during the reaction period (e.g., during the RCA reaction period) in which the nucleic acid product is produced, the maximum concentration of serum albumin can be up to 0.05, 0.06, 0.07, 0.075, 0.08, 0.09, or 0.1% (by weight or w / v). In one embodiment, the maximum concentration of serum albumin is 0.1% (by weight or w / v). The minimum concentration of serum albumin during the detection step can be, for example, at least 0.001, 0.002, 0.003, 0.004, or 0.005% (by weight or w / v). In one embodiment, the minimum concentration of serum albumin is 0.001% (by weight or w / v). In another embodiment, it is 0.0015 or 0.002% (by weight or w / v). The concentration of serum albumin in the reaction mixture may range from 0.001-0.1%, such as 0.002-0.09%, 0.002-0.08%, 0.002-0.07%, 0.002-0.06%, 0.002-0.05%, such as 0.003-0.08%, such as 0.004-0.07% (by weight or w / v). In a specific embodiment, the concentration of serum albumin may be, for example, in the range of 0.004-0.02% (by weight or w / v). In a preferred embodiment, the concentration of serum albumin may be 0.01-0.02%, such as 0.01 or 0.02% (by weight or w / v).

[0080] The above ranges may apply, for example, to a protein resolving agent selected from BSA, PSA or HSA, or to another serum protein such as globulin or milk protein.

[0081] Alternatively, in another embodiment, a non-protein analytical agent can be used. For example, the maximum concentration of the non-protein analytical agent during the nucleic acid product generation (e.g., RCA) reaction can be up to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% (by weight or w / v). In one embodiment, the maximum concentration of the non-protein analytical agent is 0.5% (by weight or w / v). The minimum concentration of the non-protein analytical agent during the detection step can be, for example, at least 0.01, 0.02, 0.03, 0.04, or 0.05% (by weight or w / v). In one embodiment, the minimum concentration of the non-protein analytical agent is 0.01% (by weight or w / v). The concentration of the non-protein dissolving agent in the reaction mixture may range from 0.01-1.0%, such as 0.01-0.08%, or 0.01-0.5%, such as 0.01-0.5%, such as 0.02-0.5, or 0.02-0.4%, such as 0.03-0.3%, such as 0.04-0.2% (by weight or w / v).

[0082] In one embodiment, a detergent may be used as a resolving agent. For example, the maximum concentration of the detergent during the RCA reaction may be up to 0.1, 0.2, 0.3, 0.4 or 0.5% (by weight or w / v). In one embodiment, the maximum concentration of the detergent is 0.5% (by weight or w / v). The minimum concentration of the detergent during the detection step may be, for example, at least 0.01, 0.02, 0.03, 0.04 or 0.05% (by weight or w / v). In one embodiment, the minimum concentration of the detergent is 0.01% (by weight or w / v). The concentration range of the detergent in the reaction mixture may be 0.01-0.5%, such as 0.02-0.4%, such as 0.03-0.3%, such as 0.04-0.2% (by weight or w / v).

[0083] Label can be any detectable label, it can directly or indirectly send out signal.Therefore, it can be directly or indirectly detectable.Directly detectable label is a label that can be directly detected without using additional reagents, and indirectly detectable label is a label that can be detected by using one or more additional reagents, for example, a label is a member of a signal generation system composed of two or more components.For example, the label can be detected by spectroscopy or microscopy.Any label used in immunohistochemistry can be used.In many embodiments, the label is a directly detectable label, wherein the directly detectable label of interest includes but is not limited to: fluorescent label, radioisotope label, chemiluminescent label, colored label, particle (such as magnetic bead, including colored magnetic bead), metal label (such as gold or silver nanoparticles), metal chelate (such as fluorescent or chemiluminescent lanthanide chelate) etc.Indirectly detectable label includes enzyme label, which can produce detectable label, such as colored or chemiluminescent product.An example is horseradish peroxidase (HRP) widely used as label. In the presence of peroxide, HRP oxidizes luminol to 3-aminophthalate, an excited product that emits light at a wavelength of 425 nm, making it detectable.

[0084] In many embodiments, the label is a fluorescent label. Fluorophores used as or for such labels are widely known and used in the art. For example, these include, but are not limited to, fluorescein, cyano dyes, such as Cy3, Cy5, Alexa555, Bodipy630 / 650, etc.

[0085] The label can be introduced into the nucleic acid product or provided to the nucleic acid product in different ways. For example, the label can be incorporated into the nucleic acid product by incorporating the nucleotide of the label in the process of synthesizing the nucleic acid product. For example, the nucleotide (dNTP) can be fluorescently labeled, such as fluorescently labeled CTP (such as Cy3-CTP, Cy5-CTP) etc. In another embodiment, the nucleic acid product can be constructed using the monomer of the label (such as the HCR monomer of the label) or the hybridization oligonucleotide of the label.

[0086] Alternatively, nucleic acid products can also be labeled using non-sequence specific nucleic acid labeling methods, for example, using DNA binding stains or dyes, which methods are well known and reported in the literature.

[0087] Conveniently, as described above, a label can be introduced by hybridizing a labeled oligonucleotide (e.g., a detection oligonucleotide) with a nucleic acid product. Therefore, as described above, a nucleic acid product can include multiple copies of a detection sequence. A detection oligonucleotide complementary to or capable of hybridizing with the detection sequence is used to provide a label for the nucleic acid product. The detection oligonucleotide can be labeled. Therefore, it can carry a label, for example, by means of a nucleotide comprising one or more labels. However, the detection oligonucleotide need not be directly labeled, but can be indirectly labeled by a second labeled oligonucleotide hybridized with the detection oligonucleotide. Therefore, the labeled oligonucleotide can be indirectly hybridized to the detection sequence by a detection oligonucleotide acting as an intermediate oligonucleotide or a sandwich oligonucleotide. This labeling method is particularly suitable for the situation of RCP.

[0088] In the case of RCP, a detection sequence can be easily added by adding a detection sequence (strictly speaking, a complementary copy of the detection sequence) to the RCA template ring used to generate the RCP. For example, this can be a padlock probe, which is cyclized to form an RCA template after target binding. For sRCA reactions, padlock probes can be used to generate second or further generation RCPs, which are ultimately labeled to detect sRCA products. This will be further described below. For HCR products, the detection sequence may be included in one or more HCR monomers. For hybridization products, the detection sequence may be included in one or more groups of hybridization probes used to generate the product.

[0089] Therefore, the detection sequence is simply a sequence that can be used to detect nucleic acid products. For RCP, the detection sequence can be included in the padlock probe. The complementary sequence of the detection sequence will be incorporated into the RCP. The detection sequence can be specific to the padlock probe, thereby being specific to the target sequence or the sequence variant that needs to be detected. Therefore, each padlock probe can have a different detection sequence. The detection sequence can be detected to detect or identify the padlock probe amplified in the RCA, thereby determining the target sequence present. For example, such a scheme can be applied to the context of a method for detecting a target sequence variant, wherein a detection sequence specific to a specific variant is provided for each padlock. Therefore, the detection sequence can be regarded as a marker or recognition sequence. The term "detection sequence" used herein includes the detection sequence that appears in the padlock probe and the complementary copy that appears in the RCP.

[0090] Generally speaking, detection reagents (including labeling reagents) can be included in the reagent mixture used in the step of generating the nucleic acid product, or can be contacted with the reaction mixture after the generation step.

[0091] The analytical agent is contained in a mixture containing the labeled nucleic acid product, and the mixture is subjected to a detection step (i.e., a mixture for detection, or in other words, a detection mixture). This can be achieved in different ways, depending on the generation and labeling method of the product, etc. For example, in one embodiment, the analytical agent is contacted with the nucleic acid product mixture (such as added to the mixture). In another embodiment, when the nucleic acid product is labeled, the analytical agent is present in the reaction mixture. In another embodiment, when the nucleic acid product is produced, the analytical agent is present in the reaction mixture and is retained in the labeled mixture being detected.

[0092] The term "contacting" is used broadly herein to include contacting the relevant reagents. Thus, one may be added to the other, or vice versa, or they may be introduced into each other, etc.

[0093] For example, the analytical agent can be included in a labeling mixture, which contacts the nucleic acid product after it is produced. Or it can be added separately to a reaction mixture comprising a nucleic acid product, such as before, at the same time or after the labeling mixture. Before detecting the nucleic acid product of the label, it can be contacted with a mixture containing the nucleic acid product in any way. Conveniently, a labeling mixture comprising a labeled detection oligonucleotide, or a detection oligonucleotide and a complementary labeling oligonucleotide together can include an analytical agent. Alternatively or in addition, an analytical agent may be included in a reaction mixture when generating a nucleic acid product, such as an RCA reaction mixture, or in a reaction mixture comprising an HCR monomer when generating an HCR polymer product, or in a reaction mixture when a hybridization probe hybridizes to generate a hybrid nucleic acid product. For sRCA products, an analytical agent may be included in the reaction mixture of the first and / or second or further (such as final) RCA step, or in all RCA steps. For example, it can be included in an RCA reagent mixture for an RCA reaction.

[0094] Thus, in a convenient embodiment, the analytical agent may be included in the reaction mixture of the first RCA of the sRCA reaction. It may be added in excess so that it remains in the reaction mixture and is present in the detection mixture comprising the labeled sRCA product. It may be included in the reagent mixture of the first RCA step, or may be added separately with the other reagents of the first RCA.

[0095] As mentioned above, the detection method herein can include the steps of producing nucleic acid products and labeling them, i.e., producing a mixture of nucleic acid products comprising different labels. The generation step can be carried out in a multiple manner. In other words, different nucleic acid products can be produced in the same reaction mixture. However, it is not excluded that a single reaction is carried out in parallel to produce each different nucleic acid product, and then the product is combined or pooled to produce a reaction mixture.

[0096] In specific embodiments, nucleic acid products are produced as surrogates or markers for detecting target analytes in a detection assay. Specifically, they can be produced during a method for detecting a target nucleic acid sequence that may be present in one or more target nucleic acid molecules.

[0097] The term "detection" is widely used herein and includes any method for determining the presence of a target nucleic acid sequence in a target nucleic acid molecule. In the methods herein, the target nucleic acid is detected by detecting the presence or amount of the nucleic acid product produced, and may include simply detecting whether it exists, or any measurement form of the nucleic acid product. Therefore, the nucleic acid product can be detected as a "signal" of the target nucleic acid sequence. Therefore, detecting the nucleic acid product includes determining, measuring, evaluating or analyzing the presence or absence or amount or position of the nucleic acid product in any way. The presence of the nucleic acid product (i.e., confirmation of its presence or amount) indicates or identifies the presence of the target nucleic acid sequence, because the successful generation of the nucleic acid product ultimately depends on the presence of the target nucleic acid molecule, more specifically the target nucleic acid sequence therein.

[0098] Including quantitative and qualitative determination, measurement or evaluation, including semi-quantitative. This determination, measurement or evaluation can be relative, for example, when detecting two or more different target nucleic acid sequences or target molecules in a sample; it can also be absolute. Therefore, in one embodiment, the method can be used to quantify or determine the amount of target nucleic acid sequences present. When used in the context of a target nucleic acid sequence in a quantitative sample, the term "quantitative" can refer to absolute quantification or relative quantification. Absolute quantification can be achieved by including one or more control nucleic acid molecules of known concentration, and / or by referencing the detection level of the target nucleic acid sequence to a known control nucleic acid molecule or sequence (e.g., by generating a standard curve). Alternatively, relative quantification can be achieved by comparing the detection level or amount between two or more different target nucleic acid molecules or different target sequences to provide respective relative quantification in two or more different nucleic acid molecules or sequences, i.e., relative to each other. Therefore, as described above, the ratio of the target nucleic acid sequence present in the sample can be determined. Therefore, the copy number of the target nucleic acid molecule, such as a chromosome, can be compared.

[0099] The method herein can be specifically used for detecting variant target nucleic acid sequences in target nucleic acid molecules in samples. The target nucleic acid sequence can usually exist in variant forms, such as allelic variants, or mutant and wild-type sequences, and it may be necessary to detect which variant is present. Therefore, in one embodiment, the target nucleic acid molecule is the analyte in the sample. However, in another embodiment, the target nucleic acid molecule itself is not the target analyte, but is detected as a part of the analysis of another target analyte. Therefore, in such an embodiment, the target nucleic acid molecule can be a reporter molecule of the target analyte.

[0100] Therefore, the target analyte can be any analyte that needs to be detected. As described above, in some embodiments, the target nucleic acid molecule of the method herein is a target analyte. In other embodiments, when the target nucleic acid molecule is a reporter, the target analyte can be any analyte that needs to be detected. The analyte can be a nucleic acid, a protein (this term includes peptides and polypeptides) or any other chemical or biological molecule or part, including, for example, carbohydrates, such as may appear as sugar groups on proteins. Therefore, the target analyte can be a modified protein, for example, with a post-translational modification detected in the analyte analysis.

[0101] In one embodiment, the target analyte can be a component of a protein or protein molecule detected on the surface of a cell, or a vesicle, or other cell or subcellular compartment. For example, an extracellular vesicle or exosome can be detected and distinguished by the different proteins present on its surface. Prostatic bodies have been proposed as biomarkers for prostate cancer, and specific or selected prostatic bodies or other extracellular vesicles can be detected and distinguished by detecting one or more surface proteins thereon.

[0102] The target nucleic acid sequence can be any sequence that needs to be detected or identified. It can be DNA or RNA, or a modified variant thereof. Therefore, the nucleic acid can be composed of ribonucleotides and / or deoxyribonucleotides and synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. Therefore, the nucleic acid can be or can include, for example, bisulfite-converted DNA, LNA, PNA or any other derivative containing a non-nucleotide backbone.

[0103] Typically, the target sequence is an analyte to be detected, such as a nucleic acid present in a sample, such as a cell or tissue sample or any biological sample, etc. Therefore, the target sequence can be a naturally occurring sequence, or a derivative, copy or amplicon thereof.

[0104] Alternatively, as described above, the target sequence can be a reporter of the analyte analyzed. The reporter nucleic acid can be used or generated in the process of analyzing any analyte (e.g., protein or other biomolecule or small molecule) in the sample. Therefore, the reporter nucleic acid can be used as a marker or label for the binding probe of the analyte, and can be detected in the detection of the analyte, for example, in immunoassays, such as immuno-PCR or immuno-RCA reactions. The reporter nucleic acid can be generated during the detection process, for example, by a ligation reaction in a proximity ligation assay (PLA), or an extension reaction in a proximity extension assay (PEA), or by a cleavage reaction, etc. Therefore, such a reporter target nucleic acid can be a synthetic or artificial sequence. It can be a linear or cyclic or cyclized or cyclizable molecule.

[0105] In one embodiment, the target nucleic acid is a natural or synthetic DNA molecule. The target nucleic acid molecule can be a coding or non-coding DNA, such as a genomic DNA or a subfraction thereof, or can be derived from a genomic DNA, such as a copy thereof or an amplicon thereof, or it can be a cDNA or a subfraction thereof, or an amplicon thereof or a copy thereof, etc.

[0106] In another embodiment, the target nucleic acid molecule is a target RNA molecule. It can be an RNA molecule in an RNA pool or other nucleic acid molecule pool, such as a genomic nucleic acid, whether human or any source, from a transcriptome, or any other nucleic acid (such as an organelle nucleic acid, i.e., a mitochondrial or plastid nucleic acid), whether naturally occurring or synthetic. Therefore, the target RNA molecule can be or can be derived from a coding (i.e., a precursor mRNA or mRNA) or a non-coding RNA sequence (such as tRNA, rRNA, snoRNA, miRNA, siRNA, snRNA, exRNA, piRNA, and long ncRNA). In a preferred embodiment, the target nucleic acid molecule is a microRNA (miRNA). In one embodiment, the target RNA molecule is a 16SRNA, for example, 16SRNA is from a microorganism (such as a pathogenic microorganism) in a sample and can be identified. Alternatively, the target RNA molecule can be a genomic RNA, such as ssRNA or dsRNA of a virus with RNA as genetic material. Such viruses worth noting include Ebola virus, HIV, SARS, SARS-CoV2, influenza, hepatitis C, West Nile fever, poliomyelitis, and measles. Thus, the target RNA molecule can be positive-sense RNA, negative-sense RNA or double-stranded RNA from a viral genome, or positive-sense RNA from a retroviral RNA genome.

[0107] When the target molecule is an RNA molecule, the method may include a preliminary step of generating a cDNA copy of the target RNA molecule.

[0108] Methods for producing nucleic acid products used herein are well known in the art and extensively described in the literature, as are detection methods using them.

[0109] Thus, RCA is widely known as an amplification technique and many detection methods have mentioned and described the use of RCA to produce a detectable product. As mentioned above, for example, WO2015 / 071445 describes the basic principles of the sRCA reaction and its general use in detection assays, the disclosure of which is incorporated herein by reference.

[0110] According to principles well known in the art, the template ring for the RCA reaction or the first RCA reaction for the sRCA reaction can be generated by the cyclization of a probe (particularly a padlock probe) of the target nucleic acid sequence. The padlock probe can be in a variety of forms, either in a single-part form or in a multi-part (such as a two-part) form. They include gap-filling padlock probes (also known as molecular inversion probes (MIPs)). The RCA template can optionally be a preformed ring that forms part of a target-specific probe (e.g., hybridizes with a target-specific probe, or with a nucleic acid portion or a domain thereof), or is used together with a target-specific probe, such as in an immunoRCA reaction. Similarly, it can be a cyclizable oligonucleotide that is connected to form a ring during the analytical reaction. Thus, RCP (as a representative nucleic acid product) can be the product of an immunoRCA reaction or any type of detection reaction comprising an RCA step, for example, proximity probe assays that produce circular nucleic acid molecules (see, for example, DuolinkTM PLA from Sigma Aldrich), and modified PLAs using so-called “unfolded” proximity probes, which comprise opening or unfolding a hairpin by cleavage to release a nucleic acid domain that can circularize to form an RCA template (see Klaesson et al, 2018, Scientific Reports 8, 5400).

[0111] In more detail, padlock probes can also be defined as circularizable probes. The use of padlock probes or circularizable probes is well known in the industry, including use in RCA reactions. Circularizable probes include one or more linear oligonucleotides that can be linked together to form a ring. Padlock probes are well-known and widely used, and are reported and described in detail in the literature. Therefore, the principle of padlock detection is well understood, and the design and use of padlock probes are well-known in the art in the art. Padlock probes are generally linear circularizable oligonucleotides, and the way in which they hybridize with a target nucleic acid sequence or molecule is to juxtapose the 5' and 3' connectable ends of the probe so as to be directly or indirectly connected together, leaving a gap in the middle. By connecting the hybridized 5' end and 3' end of the probe, the circularized probe. It is understood that in order for circularization (connection) to occur, the connectable 5' end of the padlock probe has a free 5' phosphate group.

[0112] In order to juxtapose the ends of the padlock probe for ligation, the padlock probe is designed to have target binding sites at or near its 5' and 3' ends. That is, the complementary regions that allow the padlock probe to bind to its target are located at or near both ends of the padlock probe.

[0113] In order to achieve ligation, the 3' and 5' ends (the "ligatable" 3' and 5' ends) to be connected need to hybridize with the target sequence in the first RCP as a ligation template. Depending on the probe design, the ligatable ends of the padlock probe can be juxtaposed in various ways for ligation. When the target binding site is located at the end of the padlock probe, the binding of the padlock probe can place the ends in the juxtaposition state. When the complementary binding sites in the target molecule or sequence are directly adjacent to each other (or continuous), the ends of the padlock probe can hybridize directly adjacent to each other (i.e., there is no gap) and can be directly connected to each other. Therefore, in this case, the ligatable end of the probe is provided by the actual end of the probe. However, in an optional conformation, the padlock probe is a gap-filling padlock probe, so the binding site at the end of the padlock probe does not hybridize with the adjacent binding site, but hybridizes with the non-adjacent (non-continuous) binding site in the target sequence. In this setting, the 5' ligatable end of the probe is provided by the actual 5' end of the probe. However, the ligatable 3' end of the probe is generated by extending the hybridized 3' end of the probe, using the target sequence as an extension template to fill the gap between the hybridized ends of the probe. The extension reaction brings the extended 3' ends of the probe into juxtaposition for ligation. In this case, the ligatable 3' end of the probe is therefore the extended 3' end of the probe.

[0114] The padlock probe can be provided by two or more parts connected together. This may involve providing an additional connection template, for example, in the case of a two-part probe, each part contains only one target binding region, and the other end of each part hybridizes with a common connection template. In another embodiment, the two-part padlock (2-partpadlock) can take the form of a "connector" oligonucleotide, which has two target binding regions at or near the 5' and 3' ends, respectively, which hybridize with the target and have a gap between them, and a gap oligonucleotide, which hybridizes with the gap between the ends. The gap oligonucleotide can partially or completely fill the gap.

[0115] Alternatively, the RCA template for producing RCP (or the first RCA template of sRCA) can be a cyclized target nucleic acid sequence or amplicon, such as PCR or other copies thereof. The target nucleic acid molecule or the amplicon of the target sequence can be cyclized using a connection template hybridized to the molecular end. The cyclization adapter (also referred to as "selector") for the cyclization of the target nucleic acid molecule is described in WO99 / 049079, WO2003 / 012119 and WO2005 / 070630.

[0116] HCR is also well known and widely described, and HCR has been used as a signal amplification method in detection assays. An HCR-based proximity assay is described in WO2015 / 118029, which is incorporated herein by reference along with the other documents cited above.

[0117] WO2022 / 117769 describes a detection method based on sRCA using a gap-filling padlock probe, and is specifically suitable for detecting variant target nucleic acid sequences. In one embodiment, the method herein is particularly suitable for use in the context of such a method. In this method, a target-specific first padlock probe "captures" a target sequence by hybridizing with a target nucleic acid molecule, and generates a complementary copy of the target sequence by a gap-filling extension reaction. The extended 3' end of the first padlock probe is connected to the 5' end of the padlock probe to cyclize it, thereby generating a first RCA template. The cyclized first padlock probe containing a complementary copy is then amplified by RCA to generate a first RCA product containing multiple copies of the target sequence. The first RCA product obtained is then detected with a second padlock probe specific to the target sequence. A further second RCA reaction is performed on the second padlock probe of the cyclization, which is used to generate a second RCA product connected to the first RCA product, and the obtained sRCA product is detected to detect the target sequence. Therefore, the obtained sRCA product can represent the nucleic acid product in the method herein.

[0118] In this optional version of the sRCA-based analysis, padlock probes are not used, but amplicon of the target sequence can be conveniently prepared by PCR reaction to produce a copy of the target sequence, although any amplification method can be used. The amplicon is then circularized by ligation (convenient but necessary to use a ligation template) to generate a first RCA template. It is amplified by RCA, and thereafter the detection method continues as described above to produce a detected sRCA reaction product. The resulting sRCA product is another example of a nucleic acid product representing the method herein.

[0119] Therefore, in a specific embodiment, the detection method for detecting two or more target nucleic acid sequences herein may include:

[0120] (a) providing a reaction mixture comprising an amplicon of a target nucleic acid sequence;

[0121] (b) connecting the 5' end and the 3' end of the single-stranded amplicon in (a) to circularize it;

[0122] (c) performing a first RCA reaction using the circularized amplicon as a first RCA template to produce a first RCA product (RCP) comprising a plurality of repeats of a complementary copy of the target nucleic acid sequence in the amplicon;

[0123] (d) contacting the first RCP with two or more different padlock probes specific for two or more target nucleic acid sequences and hybridizing the probes to complementary copies in the plurality of repeats;

[0124] (e) directly or indirectly ligating the ends of the hybridized padlock probes, which have hybridized to their complementary target sequences (i.e., to the targets of the respective padlock probes or to the complement of the nucleic acid sequences corresponding to the respective padlock probes), to circularize the hybridized padlock probes;

[0125] (f) performing a second RCA reaction using the circular padlock probe as a second RCA template to generate a second RCP containing a plurality of repeated complementary copies of the circularized padlock probe, wherein a different second RCP is generated for each target nucleic acid sequence to be detected in the reaction mixture;

[0126] (g) providing multiple copies of one or more tags to each second RCP of (f), wherein each different second RCP is differentially labeled and distinguishable from another second RCP, wherein the tags are provided during or after the generation of the second RCP in (f), thereby generating a detection mixture comprising two or more differently labeled second RCPs, wherein a resolving agent is included in the detection mixture to facilitate the resolution of the differentially labeled RCPs, wherein the resolving agent is used to inhibit interactions between different labels;

[0127] (h) detecting the labeled second RCP in the reaction mixture, thereby detecting the target nucleic acid sequence.

[0128] In step (e), correctly hybridized padlock probes, ie those that have correctly and specifically hybridized to the complement of the target sequence they are designed to detect (ie padlock probes that have hybridized to their respective target sequence complements), are ligated.

[0129] It will therefore be appreciated that in step (d), the padlock probes will hybridise to their intended target (ie specifically) when that target sequence is present.

[0130] Although padlock probes can be designed to have target-specific binding regions that are specific for a particular target sequence, it will be appreciated that non-specific hybridization may occur, particularly in variant target sequences that are similar in sequence (e.g., the variant is a single nucleotide variant, such as a SNP, etc.). However, the high specificity of padlock probes is due to the failure of such non-specifically hybridized padlock probes to ligate (and therefore any such non-ligated padlock probes will not be amplified in the subsequent RCA step). Therefore, in the second ligation step of the above-described detection method, padlock probes that have hybridized to their target sequence complements (i.e., padlock probes that have correctly hybridized to the complement of the target sequence that they wish to detect, or in other words, their corresponding or homologous or respective target sequences) are ligated.

[0131] In one embodiment, the amplicon in (a) is a PCR amplicon. This includes any variant of PCR, such as asymmetric PCR.

[0132] In one embodiment, the method may include the step of performing an amplification reaction (such as PCR) to generate amplicons. The amplicons of the respective target sequences may be prepared together in a multiplex manner in a single reaction mixture, or they may be prepared separately in parallel and then pooled or mixed to provide the reaction mixture of (a).

[0133] When the detection method (such as the method discussed above) is carried out in a single reaction vessel, it is particularly advantageous to improve the detection by using an analytical agent according to the methods herein. This "single container" method has advantages from the perspective of ease of operation and automation, but requires a "clean" reaction to reduce or avoid any potential interference between the reaction components or other materials present in the reaction mixture.

[0134] sRCA methods such as those described above require a first RCA step and at least a second RCA step using a circular padlock probe as a template. The method may include a further RCA step to produce a third or further generation of RCA products using a third or fourth padlock probe, etc., each probe targeting a target nucleic acid sequence. The final generation of RCA products may be detected.

[0135] In order to carry out the step of detecting nucleic acid products, the reaction mixture or a portion of its aliquot can be removed from the container in which the reaction is carried out and moved to a detection instrument, or used for further processing before detection. In addition, detection can also be carried out directly, for example, the colored reaction product in the reaction container can be directly observed (for example, using colored magnetic beads to mark the reaction product in a test tube).

[0136] The method can be performed in a heterogeneous or homogeneous format. That is, it can be performed on a solid phase (or support), or in solution or suspension (i.e. without a solid phase or support) or indeed in both, since the solid phase can be introduced at a later stage.

[0137] The format of the method may be chosen based on the nature of the sample, or the target nucleic acid molecule, or the desired reads, or the detection technology used. In one embodiment, the method is an in-solution method, ie a method performed in a liquid phase contained in a reaction vessel.

[0138] The target nucleic acid molecule can be present in a sample, or obtained from a sample. A sample can be any sample containing any amount of nucleic acid from any source or any origin, wherein it is desired to detect a target nucleic acid sequence in the target nucleic acid molecule. Therefore, the sample can be any clinical or nonclinical sample, and can be any biological, clinical or environmental sample in which a target nucleic acid molecule may be present.

[0139] The sample can be any sample containing the target nucleic acid molecule, and includes natural samples and synthetic samples, i.e., naturally occurring materials or prepared preparations. Naturally occurring samples can be treated or processed before the method herein is performed. All biological and clinical samples are included, such as any cell or tissue sample of an organism, or any body fluid or preparation produced therefrom, and samples such as cell culture, cell preparations, cell lysates. Environmental samples, such as soil and water samples or food samples are also included. The sample can be freshly prepared, or can be pre-processed in any convenient manner, for example, for storage.

[0140] Thus, representative samples include any material that may contain target nucleic acid molecules, including, for example, food and related products, clinical and environmental samples. The sample may contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles. Thus, such biological materials may include various mammalian and non-mammalian cells, plant cells, algae (including blue-green algae), fungi, bacteria, protozoa, etc., or viruses. The cell may be, for example, a human cell, a bird cell, a reptile cell, etc., but is not limited thereto.

[0141] Thus, representative samples include whole blood and blood-derived products, such as plasma, serum and buffy coat, blood cells, urine, stool, cerebrospinal fluid or any other body fluid (e.g., respiratory secretions, saliva, milk, etc.), tissue, biopsy specimens, cell cultures, cell suspensions, conditioned medium or other samples of cell culture components, etc. The samples may be pre-treated in any convenient or desired manner in preparation for use in the method, such as by cell lysis or purification, nucleic acid isolation, etc.

[0142] In one embodiment, the sample comprises a microbial cell or virus isolated from a clinical sample or from a culture of a clinical sample. In such a sample, the target nucleic acid molecule can be a nucleotide sequence present in a microbial cell, for example, a nucleotide sequence that is characteristic or distinguishable or identifiable to a microbial cell or virus at any level (e.g., at the type, group, class, genus, species, or strain level).

[0143] In another embodiment, the sample may contain cell-free DNA. The sample may be a sample such as plasma or serum that directly contains cell-free DNA, or may be separated cell-free DNA.

[0144] Since the target nucleic acid molecule itself does not need to be the target analyte to be detected, but can be a reporter molecule used or produced in the process of detecting any desired analyte, the sample does not need to be a sample naturally containing nucleic acid, or a nucleic acid source (such as cells or viruses, or biological or clinical materials, etc.). As mentioned above, the sample can be a synthetic or artificial sample. Therefore, it can be a sample that has been subjected to analyte detection analysis, in which the target nucleic acid has been produced, or the target nucleic acid molecule has been added thereto. It can be a reaction mixture or a reaction product, for example, a product produced by an immunoassay for detecting the target analyte, such as immuno-PCR, immuno-RCA or proximity detection (such as proximity ligation detection (PLA) or proximity extension detection (PEA)).

[0145] The term "hybridization" as used herein refers to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form a duplex by Watson-Crick base pairing or any similar base pair interaction. Two nucleotide sequences are "complementary" to each other when these molecules share base pair organizational homology. Therefore, a complementary region in a molecule or probe or sequence refers to a portion of the molecule or probe or sequence that is capable of forming a duplex. Hybridization does not require 100% complementarity between sequences, so a complementary region does not require complete complementarity between sequences, but this is not excluded. Therefore, a complementary region may contain one or more mismatches. Therefore, "complementary" as used herein refers to "functionally complementary", i.e., a level of complementarity sufficient to mediate effective hybridization, which encompasses a degree of complementarity less than 100%. The degree of mismatch that can be tolerated can be controlled by appropriately adjusting the hybridization conditions. Personnel in the art who master nucleic acid technology can determine the stability of the duplex empirically according to the guidance provided in the art, and some variables considered include, for example, the length and base pair composition of the respective molecule or probe oligonucleotide, ionic strength, and the incidence of mismatched base pairs. Therefore, it is within the routine skills of those skilled in the art to design appropriate probes, ligation templates or primers, etc., as well as their binding regions and conditions for their hybridization with respective targets for any reaction step described herein.

[0146] For example, the complementary region to the target sequence in the padlock probe binding region, or between the detection sequence and the detection probe, or between the RCA primer and the circularized padlock probe, may be at least 6 nucleotides long to ensure the specificity of the binding, or more specifically, at least 7, 8, 9 or 10 nucleotides long. The upper limit of the length of this region is not important, for example, it can be up to 50, 40, 35, 30, 25, 20 or 15 nucleotides. Therefore, the complementary region can have a length within the range between any of the above-mentioned lower length limits and the upper length limit. With regard to padlock probes, the length of a single target binding region can be in a lower range, so that when hybridized with their targets, the total length of the two binding regions is in a higher range. For example, a single target binding region can be 8-15, such as 10-12 nucleotides, so the total hybridization length is 16-30 nucleotides long, such as 20-24. It may be necessary to minimize the total length of the padlock probe to minimize the size of the ring structure for the RCA reaction within the limits of the probe conformation, domain spacing, and desired or favorable hybridization, so as to minimize the length of the complementary region.

[0147] Any convenient protocol or detection means may be used to detect the labeled nucleic acid product. This may depend on the target sequence to be detected, the purpose of the method and / or the specific details of the procedures employed in the method and the label used.

[0148] For example, the labeled product can be detected using any well-established nucleic acid molecule analysis method known in the literature, including mass spectrometry, CyTOF, microscopy, real-time PCR, fluorescent probes, microarrays, colorimetric analysis such as ELISA, flow cytometry, mass spectrometry, or by turbidimetric, magnetic, particle counting, electrical, surface sensing, or weight-based detection techniques.

[0149] Depending on the multiplexing level, a combination of labeling methods well known in the art can be used. For example, a large number of repetitive sequences in sRCA products can be distinguished in a large number of such products by ratio-labeling with fluorescent or other spectrophotometric detectable probes. Such ratio-labeled detection probes can be used for flow cytometry or microscopic detection techniques (such as imaging) to detect a large number of sequences, for example, at least two fluorophores can be combined in different proportions to produce multiple fluorescent label populations. For example, it has been found that using a combination of two fluorophores in different proportions, 7 different populations can be produced. This can be expanded using 3 or 4 color combinations.

[0150] Although various detection methods can be used, the labeled nucleic acid product can be conveniently detected by microscopy or flow cytometry. In both cases, directly or indirectly labeled detection oligonucleotides can be used, for example, oligonucleotides with easily detectable fluorescent tags. Specifically, in microscopy-based methods, the labeled product (e.g., RCP) can be detected by imaging.

[0151] The use of such detection techniques advantageously allows digital recording of nucleic acid products. In fact, the degree of signal amplification provided by the high molecular weight nucleic acid products / NAAs in the methods herein allows them to be visualized and they can be detected by a camera or any device including a camera (e.g., a mobile phone).

[0152] In order to detect the products produced in a homogeneous form, they can be captured or brought to a solid support or surface to facilitate imaging or more general microscopic detection. Specifically for sRCA products, the second RCP, which is a second generation RCA product, is larger and heavier, and is therefore easily dropped to the surface by centrifugation. Therefore, for example, the second RCP can be easily dropped to the bottom of the test tube or plate by rotating the test tube or plate for microscopic detection, particularly imaging detection.

[0153] The method will now be described in more detail with reference to the following figures and non-limiting examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0154] Figure 1 : Schematic representation of the method for generating SuperRCA amplification products. A) The DNA sequence of interest in the sample is amplified by PCR. B) The amplified strands are converted into single-stranded DNA circles by template ligation at the 5' and 3' ends. C) The oligonucleotides that serve as templates for the circularization reaction can then be used as primers for the RCA reaction. D) The RCA products are interrogated with mutant or wild-type sequence-specific padlock probes. E) The ligated padlock probes are wrapped around the RCA products, which are then primed with templated secondary RCA reactions by the added oligonucleotides. F) For each starting DNA circle, the reaction produces large clusters of predominantly single-stranded DNA objects, called SuperRCA products. Up to one million fluorescently labeled hybridization probes can bind to each mutant or wild-type-specific product, allowing efficient counting by, for example, standard flow cytometry.

[0155] Figure 2 : Flow cytometric readout data of a single-tube SuperRCA assay showing (A) the IDH2p.R140Q mutation at 1% spike-in in a reaction containing BSA; (B) the IDH2p.R140Q mutation at 1% spike-in in a reaction without BSA.

[0156] Figure 3 : Flow cytometric readout data of single tube SuperRCA analysis showing IDH2R172K mutation in wild-type samples at (A) 1.024%, (B) 0.064%, (C) 0.001%, and (D) IDH2R172K mutation negative.

[0157] Figure 4: Fourfold serial dilutions of genomic DNA from cells with IDH2p.R172K point mutation were analyzed using the SuperRCA procedure or ddPCR. The genomic DNA sample containing mutant genomic DNA serially diluted with wild-type genomic DNA was divided into two parts and analyzed using SuperRCA or ddPCR. According to the manufacturer's instructions, 330ng of DNA (equivalent to 100,000 human haploid genomes) was analyzed for each sample in the SuperRCA analysis, while 66ng (equivalent to 20,000 human haploid genomes) was used for ddPCR. Each data point was calculated as the average of three biological replicates, and the limit of detection (LoD) of each analyte for ddPCR and SuperRCA detection methods was calculated (LoD = mean (wild type) + 3×SD (wild type)) and represented by dotted lines in the figure.

[0158] Figure 5 : Patient UPN126 was diagnosed with primary myelofibrosis two years before the diagnosis of AML. Bone marrow samples at the time of AML diagnosis (day 0) showed high levels of IDH2p.R140Q mutation as documented by NGS, SuperRCA, and ddPCR. Retrospective analysis of initial bone marrow samples during the myelofibrosis stage was negative for IDH2p.R140Q mutation by SuperRCA and ddPCR analysis. SCT was performed on day 139, at which time considerable levels of mutation remained. Approximately three months after SCT, low levels of IDH2p.R140Q mutation were detected by SuperRCA analysis, while ddPCR did not identify detectable levels of mutation at this time point. All subsequent blood and bone marrow samples were negative by SuperRCA and ddPCR analysis, indicating complete remission.

[0159] Figure 6 : Flow cytometric readout data of single tube SuperRCA assays showing (A) R132H mutation at 1.024% spike-in in an assay containing 0.002% BSA (by weight or w / v); (B) R132H mutation at 1.024% spike-in in an assay containing 0.01% BSA (by weight or w / v); (C) R132H mutation at 1.024% spike-in in an assay containing 0.01% BSA (by weight or w / v); (D) R132H mutation at 1.024% spike-in in an assay containing 0.02% BSA (by weight or w / v). DETAILED DESCRIPTION

[0160] Example

[0161] Materials and methods

[0162] Extraction of genomic DNA.

[0163] DNA was extracted from BM cells or whole blood using the QIAamp DNA Blood Mini Kit (Qiagen cat. 51104) and eluted in 50 μL elution buffer.

[0164] PCR-based library preparation: Tube A

[0165] High-fidelity PCR pre-amplification.

[0166] Sequences of interest in genomic DNA were amplified using SuperFi DNA polymerase (Thermo Scientific) in a 25 μL PCR reaction containing 1X SuperFi buffer, 0.2 mM dNTPs, 100 nM Fwd / Rev PCR primers, 330 ng gDNA, and 0.0005 U / μL SuperFi DNA polymerase. The PCR program was as follows: 98°C for 30 seconds, 10 cycles of 98°C for 15 seconds, 62°C for 120 seconds, and a final extension at 72°C for 5 minutes.

[0167] SuperRCA Analysis :B tube

[0168] Primer and PCR polymerase purification :

[0169] 1 μL of each target from PCR-based library preparation was mixed with 20 μL of purification solution containing 1X SuperRCA buffer (Rarity Bioscience AB), 0.125 μL ExoI (Thermo Fisher Inc.), and 0.0006 U / μL thermostable proteinase K. The mixture was incubated at 37°C for 10 minutes and then at 55°C for 10 minutes.

[0170] Ligase-mediated circularization of single-stranded PCR products .

[0171] 20 μL of ligation solution containing 1X SuperRCA buffer (Rarity Bioscience), 100 nM ligation template (complementary to both ends of the single-stranded amplification product), 0.5 mM NAD (Sigma) and 2 U Ampligase (Lucigen) were added to the purified solution containing the amplified PCR product. The mixture was incubated at 95°C for 1 minute and then at 58°C for 30 minutes.

[0172] Target sequence amplification by first RCA .

[0173] The PCR product chain containing the circularization of the target nucleotide position is amplified by RCA. 5 μL 1X SuperRCA buffer (Rarity Bioscience), 1.8mM dNTP (Invitrogen), 2.5U Phi29 polymerase (New England Biolabs) and 1.8μg / μL BSA are added to the circularized product. The resulting reaction mixture contains excessive BSA. The reactant is incubated at 37°C for 30 minutes, then at 65°C for 10 minutes.

[0174] Genotyping of RCA products by padlock probe ligation .

[0175] The padlock probes were hybridized to the first-generation RCA products and ligated in a sequence-specific manner by adding 5 μL of ligation mix (containing 1X SuperRCA buffer (Rarity Bioscience), 3 mM NAD (Sigma), 2.5 U Ampligase (Lucigen), and 60 nM genotyping padlock probe pairs) to the reaction mixture and incubated at 55 °C for 30 min.

[0176] Digestion of unreacted genotyping probes .

[0177] 5 μL of purification solution containing 1X SuperRCA buffer (Rarity Bioscience AB), 1.2 μM primers and 1 U / μL Lambda exo was added to the reaction mixture and incubated at 37°C for 15 min and then at 75°C for 20 min.

[0178] Second RCA templated by padlock probes bound to the primary RCA product .

[0179] 5 μL of RCA mix containing 1X SuperRCA buffer (Rarity Bioscience), 0.6 mM dNTPs, and 6 U Phi29 DNA polymerase (New England Biolabs) was added to the mixture, and the reaction was incubated at 37°C for 30 min.

[0180] Digital recording of SuperRCA products by flow cytometry .

[0181] The final reaction mixture containing SuperRCA products was diluted in 1X SuperRCA buffer (Rarity Bioscience) to hybridization buffer containing 100 nM fluorophore-labeled oligonucleotide probes specific for different SuperRCA products in a final volume of 250 μ L. The solution was added to a CytoFlex flow cytometer (Beckman Coulter) and SuperRCA products were counted at "medium" speed (30 μL / min) for 150 seconds per sample.

[0182] Example 1

[0183] The presence of BSA in the SuperRCA assay helps distinguish the mutant and wild-type populations by preventing them from aggregating into clusters in solution; the absence of BSA will result in an increased population in the upper right corner of the graph (the double-positive population). Figure 2 A) helps to include more mutation events into the gate, increase the detected mutation frequency, and reduce the double positive events in the upper right corner. The reaction without BSA ( Figure 2 B) will lead to an underestimate of the frequency of mutant alleles and a higher percentage of double positive events that do not fall into any one gate. Tables 1 to 2 below show the number of events detected by the flow cytometer for each reaction. P3 and P1 are probes for detecting mutant events, and P2 is a probe for detecting wild-type events. It can be seen that the percentage of all events captured by a single probe is higher in Table 1 compared to Table 2, indicating a reduction in clustering and therefore a reduction in double positives.

[0184] Tables 1 to 2 list the raw data of flow cytometric readouts showing the 1% IDH2p.R140Q mutation with BSA and the 1% IDH2p.R140Q mutation without BSA, respectively.

[0185] Table 1 Figure 2 A)

[0186]

[0187]

[0188] Table 2 Figure 2 B)

[0189]

[0190] Example 2

[0191] The single-tube SuperRCA assay was used to analyze samples spiked with different levels of the IDH2p.R172K mutation, ranging from 1% to 0% (including wild-type samples). The SuperRCA product was detected using a BD Fortessa flow cytometer ( Figure 3 ). Tables 3 to 6 show the number of events of IDH2p.R172K mutation with different abundance in each sample detected by flow cytometry. P3 is the probe for detecting mutant events, and P2 is the probe for detecting wild-type events. Figure 3 As can be seen from the flow chart and the data in Tables 3 to 6, as the level of mutation incorporation in the sample decreases from 1% to 0%, the signal from P3 also decreases. Even at a very low mutation level of 0.001%, SuperRCA analysis is still able to detect mutation signals ( Figure 3 C), demonstrating the sensitivity of the analysis.

[0192] Tables 3 to 6 show the raw data of flow cytometry readings, showing that the proportions of IDH2R172K mutation were 1%, 0.064%, 0.001% and 0% (WT), respectively.

[0193] Table 3 ( Figure 3 A)

[0194]

[0195] Table 4 ( Figure 3 B)

[0196]

[0197] Table 5 ( Figure 3 C)

[0198]

[0199] Table 6 ( Figure 3 D)

[0200]

[0201] Example 3: Detection of ultra-low frequency AML point mutation IDH2p.R172K using SuperRCA or ddPCR detection

[0202] A PCR amplicon targeting a DNA sequence containing the IDH2 p.R172K mutation was used in spike-in DNA samples for the SuperRCA preamplification step. The SuperRCA protocol was then applied to the amplified PCR products to estimate the mutant allele frequency in each spike-in sample, and the SuperRCA products were analyzed using a Cytoflex flow cytometer. In the corresponding ddPCR analysis, the same spike-in DNA samples were used in the Bio-Rad ddPCR platform, 66 ng per replicate, and two replicates were performed. The results are shown in Figure 2. Figure 4 As shown, SuperRCA analysis has a lower limit of detection (LoD) compared to ddPCR, successfully detecting mutant DNA up to 100,000-fold more than normal DNA, while ddPCR shows substantially greater variability and is not sensitive enough to detect very low frequency mutations.

[0203] Example 4: Monitoring AML patients carrying the IDH2 p.R140Q mutation using SuperRCA, NGS, and ddPCR analysis Disease progression

[0204] Samples from an AML patient (UPN126) carrying the IDH2 p.R140Q mutation were analyzed at several time points using SuperRCA, NGS, and ddPCR assays to explore whether SuperRCA detection is suitable for monitoring disease course ( Figure 5). Patient UPN126 was previously diagnosed with primary myelofibrosis with a JAK2 mutation, which transformed to secondary AML two years after the initial diagnosis. NGS at the AML diagnosis was performed by clinical collaborators at Uppsala University Hospital using the Trusight NGS panel and sequencing using the Illumina MiSeq platform, which revealed the presence of the IDH2p.R140Q mutation along with previously known JAK2 mutations. In retrospective analysis by SuperRCA and ddPCR analysis, initial bone marrow samples from the myelofibrosis stage were negative for the IDH2 p.R140Q mutation, suggesting that clonal evolution occurred during AML transformation. PCR amplicons targeting a DNA sequence containing the IDH2 p.R140Q mutation were applied to patient DNA samples for a SuperRCA preamplification step. The SuperRCA protocol was then applied to the amplified PCR products to estimate the mutant allele frequency in the samples at each time point, and the SuperRCA products were analyzed using a Cytoflex flow cytometer. In the corresponding ddPCR assay, the same DNA sample was used in the Bio-Rad ddPCR platform, 66 ng per replicate, and two replicates were performed using the Bio-Rad pre-validated IDH2 p.R140QddPCR assay. The patient achieved CR after the first course of intensive chemotherapy, but after 3 courses of intensive chemotherapy, the MRD positivity remained at approximately 2% (by comparing the expression of CD34, CD117, CD13, and DR + Allogeneic SCT was performed on day 139. The patient was subsequently monitored by serial blood and BM samples analyzed by ddPCR and NGS, and all six samples collected after SCT showed undetectable levels of IDH2 p.R140Q mutation in both analyses, indicating that the patient remains in remission at more than two years of follow-up after SCT. A blood sample collected 3 months after SCT tested positive by SuperRCA but was below the detection limit of ddPCR ( Figure 5 ), indicating that SuperRCA analysis has higher sensitivity.

[0205] Example 5: Determination of the effective concentration of BSA as a secondary analytical agent

[0206] Samples containing 1.024% R132H mutation were analyzed using different concentrations of BSA in a single tube SuperRCA assay. SuperRCA products were detected using a BD Fortessa flow cytometer ( Figure 6 ). Figure 6Flow cytometric results are shown for 0.002% BSA, 0.005% BSA, 0.01% BSA or 0.02% BSA (by weight or w / v) in the assay. Tables 7 to 10 list the raw data for flow cytometric readings at each BSA concentration.

[0207] from Figure 6 As can be seen from the data in Tables 7 to 10, when the concentration of BSA in the SuperRCA analysis is in the range of 0.002% to 0.02% (by weight or w / v), it can prevent the mutant and wild-type populations from clustering in the solution, thereby helping to distinguish the mutant and wild-type populations; otherwise, the absence of BSA will result in an increase in the population in the upper right corner of the figure (double-positive population), as exemplified by Figure 2 As shown in B. Figure 6 As shown, the presence of BSA helps to put more mutation events into the gate, improve the detected mutation frequency, and reduce the double positive events in the upper right corner. Tables 7 to 10 below list the number of events detected by flow cytometry for each reaction.

[0208] Tables 7 to 10 list the raw data of flow cytometer readings using 0.002% BSA, 0.005% BSA, 0.01% BSA and 0.02% BSA (by weight or w / v) as the resolving agent, respectively.

[0209] Table 7 Figure 6 A)

[0210]

[0211]

[0212] Table 8 Figure 6 B)

[0213]

[0214] Table 9 Figure 6 C)

[0215]

[0216] Table 10 Figure 6 D)

[0217]

Claims

1. A method for detecting two or more differently labeled high molecular weight nucleic acid products in a mixture, wherein: The products are detected by detecting one or more tags directly or indirectly attached to each product, and the method includes adding a resolving agent to the detection mixture to facilitate the resolution of the differently labeled products, wherein the function of the resolving agent is to inhibit the interaction between the different tags.

2. A method for detecting two or more target nucleic acid sequences in one or more nucleic acid molecules in a sample, the method comprising: (a) for each target nucleic acid sequence, generating a high molecular weight nucleic acid product as a detection analysis reaction product of the sequence, and generating a reaction mixture comprising the generated nucleic acid products for two or more target nucleic acid sequences; (b) providing multiple copies of one or more labels for each nucleic acid product in (a), wherein each nucleic acid product is differently labeled and can be distinguished from another nucleic acid product, wherein the labels are provided during the production of the nucleic acid products in (a) or thereafter, thereby producing a detection mixture comprising two or more differently labeled nucleic acid products, wherein a resolving agent is included in the detection mixture to facilitate the resolution of the differently labeled products, wherein the function of the resolving agent is to inhibit the interaction between the different labels; (c) detecting the labeled nucleic acid product in the detection reaction mixture, thereby detecting the target nucleic acid sequence.

3. The method according to claim 1 or claim 2, wherein: The analytical agent is or comprises a protein.

4. The method according to any one of claims 1 to 3, wherein: Protein is an inert protein.

5. The method according to any one of claims 1 to 4, wherein: The analytical agent is selected from skimmed milk powder or milk protein, serum or serum protein, gelatin, SSB or random sequence peptides.

6. The method according to any one of claims 1 to 5, wherein: The resolving agent is casein, serum albumin or globulin, optionally an immunoglobulin.

7. The method according to any one of claims 1 to 6, wherein: The analytical reagent is porcine serum albumin, bovine serum albumin, human serum albumin or bulk IgG.

8. The method according to claim 1 or claim 2, wherein: The analytical reagent is not or does not contain a protein.

9. The method according to any one of claims 1, 2 or 8, wherein: The desorbent is a detergent or a non-amino acid polymer, optionally polyvinyl alcohol.

10. The method according to claim 9, wherein: The desorbent is selected from polyvinylpyrrolidone (PVP) and Tween-20.

11. The method according to any one of claims 1 to 10, wherein: The high molecular weight nucleic acid product is a nucleic acid amplification product, a nucleic acid polymer or a nucleic acid hybridization product.

12. The method according to claim 11, wherein: The nucleic acid product is an RCA product or an HCR product, or a product of a branched DNA amplification reaction.

13. The method according to any one of claims 1 to 12, wherein: The label is a spectrophotometrically detectable label.

14. The method according to claim 13, wherein: The label is a fluorescent label, a colored label or a colorimetric label.

15. The method according to any one of claims 1 to 14, wherein: The nucleic acid product is labeled by hybridization to a labeled detection oligonucleotide.

16. The method according to any one of claims 1 to 15, wherein: The resolving agent is included in or present in one or more steps of generating the nucleic acid product.

17. The method according to claim 16, wherein: The nucleic acid product is a sRCA product, and an excess of a resolving agent is included in the reaction mixture of the first RCA for the sRCA reaction and is retained in the reaction mixture for subsequent steps.

18. The method according to any one of claims 15 to 17, wherein: The interrogating agent is included in a labeling mixture that includes a mixture of labeled detection oligonucleotides.

19. The method according to any one of claims 1 to 18, wherein: The interrogating agent is included in the labeling mixture that is contacted with the mixture of nucleic acid products to label the nucleic acid products, or the interrogating agent is added to the nucleic acid products before, simultaneously with, or after the labeling mixture but before detecting the labeled nucleic acid products.

20. The method according to any one of claims 1 to 19, wherein: The nucleic acid product is a super RCA product generated using a target-specific padlock probe, which hybridizes to a first RCA product, the first RCA product comprising multiple repeats of the target nucleic acid sequence or its complementary copy, wherein the padlock probe hybridized to the multiple repeats is circularized by ligation and used for RCA to produce a second RCA product.

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