Method for detecting target nucleic acid sequence in single reaction vessel

Abstract

The present disclosure and invention relate to the field of nucleic acid detection. Specifically, the present disclosure and invention relates to a method for detecting a target nucleic acid sequence in a target nucleic acid molecule in which the target sequence is amplified and cyclized, then using rolling circle amplification (RCA) and a lock-on probe in a 2-stage RCA reaction, i.e., so-called SuperRCA (sRCA), to produce a second round of RCA product, target nucleic acid sequences can be detected and distinguished from other nucleic acid sequences by the second round of RCA products. The methods have been optimized, can be carried out in a single reaction vessel, and can be carried out multiplexed to detect different multi-target nucleic acid sequences in one or more target nucleic acid molecules. A kit for use in the method is also provided.

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 a method for detecting a target nucleic acid sequence in a target nucleic acid molecule, wherein the target sequence is amplified and cyclized, and then a rolling circle amplification (RCA) and a padlock probe are used in a 2-stage RCA reaction (i.e., the so-called SuperRCA (sRCA)) to produce a second round of RCA products to detect the target nucleic acid sequence and distinguish it from other nucleic acid sequences. The method has been optimized and can be performed in a single reaction vessel and can be performed in multiples to detect different multi-target nucleic acid sequences in one or more target nucleic acid molecules. A kit for the method is also provided. Background Art

[0002] Detection of target nucleic acid sequences has applications in many different areas, including, in particular, personalized medicine in the clinic and diagnosis, prognosis and / or treatment of diseases, such as cancer, infectious diseases and genetic disorders, as well as in research and biosafety.

[0003] The target nucleic acid sequence can be easily detected using a labeled hybridization probe, but a simple hybridization probe has a relatively high detection limit and is not 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, including PCR and RCA.

[0004] As is well known, PCR is an exponential amplification technique that uses two primers to produce a linear amplification product. Although PCR is widely used, its disadvantage is that errors introduced by the polymerase are amplified, and the incidence of polymerase errors increases as the number of PCR cycles increases. While this is tolerable in many applications, it is a significant problem when PCR is used to detect variant sequences, such as mutations, especially those with only a few nucleotide differences, especially when these are low-frequency or low-abundance variants.

[0005] RCA utilizes a strand displacement polymerase and requires a circular amplification template. Amplification of the circular template provides a tandem RCA product containing multiple copies of a sequence complementary to that of the amplification template. Such concatemers typically form a ball or "mass" that can be easily visualized and detected, and therefore RCA-based assays have been used for the detection of nucleic acids and, in fact, more generally, for the detection of any target analyte reporter system. Either the target nucleic acid (which itself can be directly circularized) or the probe or more generally the reporter nucleic acid can provide the template nucleic acid ring for RCA.

[0006] The specificity of nucleic acid detection methods can be improved by using probes (e.g., padlock probes) that require dual recognition or have two binding sites for the target nucleic acid sequence. Padlock probes are typically linear oligonucleotides with two independent target complementary binding regions connected by a "backbone" region in the middle. After the probe binds (hybridizes) to the target nucleic acid sequence, the two ends of the probe can be connected together to cyclize the probe, with single nucleotide identification specificity within 6bp around the connection site. The circular padlock probe can then be used as a template for the RCA reaction, and the RCA product can be detected. This forms the basis of many detection methods currently used. Therefore, padlock probes provide additional specificity because only probes with correct base pairing at the connection site will be connected to generate a template for the molecule to be detected.

[0007] RCA-based assays have been described, which rely on secondary amplification of the initial RCA product to increase the amount of the detected product, thereby providing amplification of the signal in the assay. These include, for example, hyperbranched RCA, which produces many unclustered subsequent RCA products by strand displacement activity. The so-called "super RCA" (sRCA) reaction recently developed includes two or more rounds of RCA amplification, in which the product of the second round of RCA reaction is connected to the product of the first round of reaction. A sRCA method is described in WO2014 / 0796209. In this method, a probe that can provide or act as a primer is hybridized with the initial RCA product and 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 circularization of a padlock probe hybridized with the "primer-probe". In WO 2015 / 071445, an alternative sRCA method called "padlock sRCA" is described, in which a padlock probe is used to directly bind to the initial RCA product.

[0008] In many applications, the nucleic acid sequence to be detected is present in very low levels, for example in rare mutations, or cell-free DNA (cfDNA) in plasma or other clinical samples, or when sample quantities are limited. In this case, very sensitive detection methods are required, in particular methods that can achieve high amplification of the target nucleic acid. PCT / EP2021 / 084061 describes a sRCA-based detection method that is particularly useful in this regard, in which a padlock probe binds to a target nucleic acid sequence and undergoes gap-filling extension and ligation to produce many circular copies of the target nucleic acid sequence as the first RCA template for the sRCA reaction.

[0009] Although the above-mentioned sRCA methods provide improved and sensitive detection to a certain extent, they contain many steps that require a certain degree of human involvement. These methods have not yet been developed into commercial detection methods, so there are still human errors and they are limited to small-scale use. The optimization of the protocol of the automated workflow performed in a single container faces difficulties related to the interference of reagents added in previous steps or during the experimental protocol, such as PCR primers and polymerase, which remain in the reaction mixture and cannot be washed out. Usually, excess PCR primers can be avoided by ensuring that they are completely consumed after a large number of PCR cycles. However, this approach can lead to the accumulation of polymerase errors in the amplicon, which is not conducive to the accuracy of the assay.

[0010] There is a continuing need for sRCA-based nucleic acid detection methods that are amenable to automation and can achieve high throughput and result reliability without compromising sensitivity. The present disclosure and inventions are directed to providing such methods. Summary of the invention

[0011] In the present method, an optimized protocol was derived that allows the steps of generating the sRCA product for detection to be performed in a single reaction vessel.

[0012] For example, a linear amplicon of a target nucleic acid sequence is produced by PCR, and is cyclized by connection to form the first RCA template for sRCA reaction. In order to improve the sensitivity and accuracy of the method, the number of amplification cycles is maintained at a low level to reduce the incidence of polymerase errors. Therefore, the resulting amplification reaction mixture contains an excess of amplicon primers and amplification polymerase relative to the amplicon. Since the method is carried out in a single container, and the amplicon cannot be simply separated from the other parts of the reaction mixture, a cleaning step is required before the amplicon is connected into a ring to prevent primers and polymerase from interfering with the subsequent steps of the method. In this regard, it is important to connect under conditions that prevent the amplification polymerase from interfering. As described below, this can be achieved in a variety of ways, including degrading primers and / or polymerases, diluting the reaction mixture, and / or controlling the connection reaction to ensure that the connection is performed before any unnecessary extension occurs at the 3' end to be connected. In this way, the extension of the amplicon hybridized with the connection template and the unwanted extension of the amplification (such as PCR) primer can be avoided.

[0013] Subsequently, the circularized amplicon is subjected to an RCA reaction to produce a first RCA product (RCP) containing multiple complementary copies of the target sequence. The first RCP is then probed with a padlock probe specific to the target sequence (particularly its complementary sequence), and the circularized padlock probe is subjected to a second RCA reaction to produce a second RCA product, i.e., the detected sRCA product, to detect the target sequence.

[0014] As with the first ligation and RCA reaction, it is important that the padlock ligation be performed under conditions that inhibit interference from any polymerase present in the reaction mixture and further reduce interference from any unligated padlock probe. This can be achieved similarly to the above strategy as well as by temperature control of the process.

[0015] Therefore, in a first aspect, the present invention provides a method for detecting a target nucleic acid sequence in a target nucleic acid molecule, the method comprising:

[0016] (a) providing a reaction mixture comprising an amplicon of a target nucleic acid sequence, an amplification polymerase, and an excess of amplification primers relative to the amplicon, wherein the amplicon is a single-stranded or double-stranded linear molecule and comprises a single copy of the target nucleic acid sequence;

[0017] (b) reducing the excess primer in the reaction mixture in (a);

[0018] (c) denaturing the double-stranded amplicon into a single-stranded amplicon if necessary;

[0019] (d) contacting the reaction mixture with a ligation mixture comprising a ligase, and performing a ligation reaction to ligate the 5' and 3' ends of the amplicon, thereby circularizing the amplicon, wherein the ligation reaction is performed under conditions where the ability of the amplification polymerase to extend the 3' end of the hybridized amplicon on the ligation template is inhibited;

[0020] (e) adding an RCA mix comprising one or more RCA reagents, the RCA reagents including at least an RCA polymerase, to the reaction mixture from (d) and performing a first RCA reaction using the circular amplicon as a first RCA template to produce a first RCA product (RCP), the first RCA product comprising a plurality of repeats of a complementary copy of the target nucleic acid sequence in the amplicon;

[0021] (f) heating to inactivate the RCA polymerase;

[0022] (g) contacting the first RCP with a padlock probe specific for the target nucleic acid sequence, allowing the padlock probe to hybridize to complementary copies in the plurality of repeats (or more specifically to the complement of the target sequence);

[0023] (h) directly or indirectly ligating the hybridized padlock probe to circularize the hybridized padlock probe, wherein the ligation reaction is performed under conditions in which the ability of a polymerase to extend the 3' end of the hybridized padlock probe is inhibited;

[0024] (i) removing unligated padlock probes from the reaction mixture of (h) or rendering them inert;

[0025] (j) performing a second RCA reaction using the circularized padlock probe as a second RCA template to generate a second RCP containing multiple repeated complementary copies of the padlock probe;

[0026] wherein steps (a) to (j) are carried out in a single reaction vessel and the temperature is controlled during the steps; wherein steps (g) to (j) are optionally repeated one or more times; and

[0027] (k) detecting the second or final RCP to detect the circularized padlock probe, thereby detecting the target nucleic acid sequence.

[0028] The second RCP (also referred to as secondary RCP) forms the superRCA (sRCA) product with the first RCP to which they are attached, which is the product to be analyzed. Similarly, if the padlock detection and RCA steps are repeated, the subsequent rounds of RCP will be attached to the previous round of RCP until the last round of RCP ("final RCP"), together forming the sRCA product.

[0029] Specifically, in step (b) of the method, excess primer is reduced by enzymatic digestion of the primer and / or by diluting the reaction mixture. More specifically, in one embodiment, enzymatic digestion of the primer is carried out by contacting the reaction mixture with an exonuclease, or, in the case where the primer contains uracil residues, with uracil DNA glycosylase (UDG). Dilution can be carried out by adding a certain volume of diluent to the reaction mixture. In one embodiment, the reaction mixture is diluted at least 200X.

[0030] In a more specific embodiment, the method may include a pre-step of performing an amplification reaction to produce an amplicon of the target nucleic acid sequence. In other words, the method may include a step of performing an amplification reaction to provide a reaction mixture of step (a). Alternatively, step (a) may include performing an amplification reaction to provide a reaction mixture.

[0031] In another more specific embodiment, said amplification reaction is a PCR reaction.

[0032] In one embodiment, step (i) of the method may comprise removing or rendering inert the unligated padlock probe in the reaction mixture in (h) by enzymatic digestion, diluting (e.g., at least 4X dilution) or extending the 3' end of the padlock probe that forms a hairpin structure to form a hairpin.

[0033] Typically, a ligation template will be included in the ligation reaction mixture to serve as a template for ligating the amplicons. However, this is not absolutely necessary, and in one embodiment, non-templated ligation can be performed. This uses a ligase that does not require a ligation template, such as CircLigase TM Enzyme.

[0034] Said method can be used for detecting the variant target nucleic acid sequence in the target nucleic acid molecule.The target nucleic acid sequence may exist in a variant form usually, such as an allelic variant or a mutant and a wild-type sequence, and may need to detect which variant exists.This shows that the target nucleic acid sequence can be one of many different variants of the nucleotide sequence that may occur.

[0035] Thus, in one embodiment, the method is for detecting variant target nucleic acid sequences in a target nucleic acid molecule in a sample, and steps (g) to (k) comprise:

[0036] (g) contacting the first RCP with two or more padlock probes, each probe comprising a target binding region specific for a different variant of the target nucleic acid sequence, and allowing the probes to hybridize to the complement of the target sequence in the plurality of repeats;

[0037] (h) directly or indirectly ligating a padlock probe that has hybridized to its variant target sequence complement (i.e., to the target as the padlock probe or to the variant sequence complement corresponding to the padlock probe) to circularize the hybridized padlock probe;

[0038] (i) removing unligated padlock probes from the reaction mixture of (h) or rendering them inert;

[0039] (j) performing a second RCA reaction using the circularized padlock probe as a second RCA template to generate a second RCP containing multiple repeated complementary copies of the circularized padlock probe;

[0040] (k) detecting the final (eg, second) RCP to identify circularized padlock probes, thereby identifying variant target nucleic acid sequences.

[0041] In step (h), correctly hybridized padlock probes, ie probes that correctly and specifically hybridize to the complement of the variant target sequence designed to be detected (ie padlock probes that hybridize to the complement of the respective target sequence) are ligated.

[0042] It will therefore be appreciated that in step (g), the padlock probe will hybridise to (ie specifically hybridise to) the intended target where that target sequence is present.

[0043] When using the method to detect variant sequences, it is usually the case that step (b) of the method comprises reducing the excess primers in the reaction mixture in (a) by enzymatic digestion rather than by dilution. It is further preferred to avoid using dilution in the subsequent step (i) as a means of reducing unligated padlock probes in the reaction mixture in (h).

[0044] Therefore, it can be seen that the method can use multiple different padlock probes to carry out multiple detections to detect multiple different variants (or its content) that may exist in the sample. However, advantageously, further multiple detections are possible. Therefore, the above method can be carried out multiple times to detect multiple different target nucleic acid sequences in one or more target nucleic acid molecules, including detecting multiple variants of target nucleic acid sequences in one or more target nucleic acid molecules.

[0045] In the above method, the padlock probe may be:

[0046] (i) a 1-part padlock probe in the form of a single circularisable oligonucleotide comprising target binding regions at its 5' and 3' ends; or

[0047] (ii) A 2-part padlock probe comprising a first oligonucleotide having a first target complementary binding region at its 3' end and a second binding region complementary to a ligation template at its 5' end, and a second oligonucleotide having a first target complementary binding region at its 5' end and a second binding region complementary to a ligation template at its 3' end; wherein the 3' and 5' ends of the first and second oligonucleotides are linked together and are templated by the target sequence in the first RCP and the ligation template, respectively.

[0048] Other configurations of 2-part lock probes may also be used.

[0049] In a second aspect, a kit for detecting a target nucleic acid sequence in a target nucleic acid molecule is provided, the kit comprising:

[0050] (i) one or more different ligation templates comprising an amplicon binding region capable of hybridizing to a complementary binding site in an amplicon of a target nucleic acid sequence, thereby ligating the 3' and 5' ends of the amplicon;

[0051] (ii) one or more different padlock probes comprising a target binding region specific for a target nucleic acid sequence; and optionally one or more:

[0052] (iii) exonucleases;

[0053] (iv) proteinase K;

[0054] (v) dilution buffer;

[0055] (vi) ligase;

[0056] (vii) RCA polymerase;

[0057] (viii) RCA primer;

[0058] (ix) dNTPs;

[0059] (x) a detection oligonucleotide capable of hybridizing to an RCP generated using the circularized padlock probe as RCA template.

[0060] In one embodiment, the kit is used to detect variant target nucleic acid sequences in target nucleic acid molecules in a sample, and may comprise two or more padlock probes in part (ii), each probe comprising a target binding region specific for a different variant target nucleic acid sequence. In one embodiment, each padlock probe comprises a detection sequence by which they can be distinguished from each other (i.e., the detection sequence is different from the detection sequence of another padlock probe).

[0061] Detailed description

[0062] The present method provides a high-fidelity, high-sensitivity method for detecting specific nucleotide sequences (the terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein). The method is particularly useful for detecting target sequence variants, such as mutant sequences, that may be present in a sample. Specifically, rare target sequences or sequence variants, or sequences or variants that are present in low abundance or at low levels, can be detected.

[0063] An amplicon of a target nucleic acid sequence present in a target nucleic acid molecule in a sample is produced. It is circularized by ligation and then subjected to RCA to generate RCA products containing multiple repeated tandem complementary copies of the target sequence. These amplified target sequences are then detected with a target sequence-specific padlock probe, which, after hybridization with a complementary sequence to the target sequence, is in turn circularized (to form a second RCA template, also referred to as a secondary RCA template) and amplified by RCA to produce a second (or secondary) RCA product, by which the target sequence can be detected. The generation of the RCA product provides a signal that is easily detected and counted. A digital counting readout can be achieved. This enables digital detection of the reaction product of each detected target molecule. In addition, the product of the second RCA reaction is a large product containing multiple (hundreds, and possibly up to a thousand or so) copies of the complement of the target sequence and having a significant size. These significant reaction products can be easily collected with minimal risk of mixing with any other material in the reaction.

[0064] The method of the present invention provides a high level amplification of the target sequence, by which the sensitivity of the detection method is increased, making it possible to detect and identify very rare target sequences. Thus, rare sequence variants or sequences or variants present at low levels ("low abundance") can be detected, identified or distinguished. In addition, the method is also capable of accurately quantifying the target nucleic acid sequence. For example, the ratio of different target nucleic acid sequences can be determined with high precision, for example, in the case of determining chromosome copy number, target sequences from 2 different chromosomes can be detected and compared. This may be particularly useful in detecting trisomy syndromes (such as chromosome 21) in situations such as non-invasive prenatal testing (NIPT).

[0065] The basic principle diagram of the detection method is shown in Figure 1 As shown. The optimization function included in the present method can be automated. Therefore, the present method can perform a two-stage RCA reaction (i.e., sRCA) on the amplicon of the target nucleic acid sequence in a single reaction vessel without any manual work, and can be extended to commercial use in a robotic workflow. In order to keep fewer polymerase errors in the amplicon, it is best to perform fewer amplification cycles, such as less than 12, 11 or 10 cycles. Although this has the advantage of improving analytical sensitivity, the result is that there are excessive amplification primers in the reaction mixture. If this problem is not solved, the excess primers will compete with the amplicon for binding to the ligation template, thereby reducing the yield of the circularized amplicon required for the first RCA reaction. In addition, even if the ligation template is not used, although this may reduce interference with the primers, they may still interfere with downstream reactions. Therefore, the method of the present invention is designed to include a cleaning step to remove or otherwise reduce at least the amplification primers, and optionally the amplification polymerase.

[0066] In one embodiment, excess primer is reduced by enzymatic digestion. In another embodiment, a volume of diluent is added to the reaction mixture to dilute the reaction mixture.

[0067] Another reagent still present in the reaction mixture of the amplification step is an amplification polymerase. This can prevent the connection of the amplicon by extending the hybridization 3' end of the amplicon on the ligation template. In one embodiment, the polymerase can be inactivated by enzymatic digestion. In another embodiment, the hybridization of the amplicon and the ligation template can be controlled by temperature so that the 5' end is first hybridized, and then the 3' end. These schemes can also be used to prevent the hybridization 3' end of the padlock probe from extending during the second ligation step.

[0068] After the second ligation step, there is an excess of padlock probes which, if allowed to remain, may hybridize to the newly generated second RCP and cause multiple strand displacement or interfere with the detection of the second RCP. Therefore, it is desirable to remove these unligated or unhybridized padlock probes or render them inert so that they cannot participate in unwanted hybridization or extension reactions.

[0069] In one embodiment, the unattached padlock probe is degraded by enzymatic digestion. In another embodiment, the reaction mixture is diluted, ideally at least 4X. In another embodiment, the padlock probe is in a so-called "hairpin-forming" format as described further below, where the 3' end can be extended to form a hairpin, thereby rendering the probe inert. Different embodiments may be suitable or preferred for different applications of the method, and may allow for efficient sRCA detection, maintaining high sensitivity.

[0070] Most commonly, the method is used to detect variant target nucleic acid sequences in target nucleic acid molecules. The term "detection" widely used herein includes any method for determining the presence of a target nucleic acid sequence in a target nucleic acid molecule. In the present method, the target nucleic acid is detected by detecting the presence or quantity of the second RCA product produced, and may include simply detecting whether it exists, or measuring the RCA product in any form. In this regard, it should be understood that by detecting the second or further or final RCP (because it is connected to the first or previous RCP), the entire sRCA product is actually detected. Therefore, the RCA product of step (j) can be detected as a "signal" of the target nucleic acid sequence. Therefore, detecting the second RCA product in step (k) includes determining, measuring, evaluating or detecting the presence or absence or quantity or position of the second RCA product in any way. The presence of the second RCA product (i.e., confirmation of its presence or quantity) indicates or identifies the presence of the target nucleic acid sequence, because the successful generation of the RCA product ultimately depends on the presence of the target nucleic acid molecule, especially the presence of the target nucleic acid sequence therein.

[0071] Including quantitative and qualitative determination, measurement or assessment, including semi-quantitative.Such determination, measurement or assessment may be relative, for example, when two or more different target nucleic acid sequences or target molecules in a sample are detected, or absolute.Therefore, in one embodiment, the method can be used to quantify or determine the amount of the target nucleic acid sequence present.When the target nucleic acid sequence in a quantitative sample, the term "quantitative" can refer to absolute quantification or relative quantification.Absolute quantification can be completed (for example, by generating a standard curve) by comprising one or more control nucleic acid molecules of known concentration and / or by referring to the detection level of the target nucleic acid sequence and known control nucleic acid molecules or sequences. Alternatively, relative quantification can be completed by comparing the detection level or amount between two or more different target nucleic acid molecules or different target sequences, to provide the relative quantification of each nucleic acid molecule or sequence 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 (for example chromosome) can be compared.

[0072] The target nucleic acid sequence is a sequence that needs to be detected or identified in any nucleic acid molecule, or in other words, a target for detection. 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.

[0073] 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 it can be a derivative, copy or amplicon thereof. However, this is not necessary, and the target sequence can alternatively be a reporter of the analyte to be measured. The reporter nucleic acid can be used or produced in the determination process of any analyte in the sample, such as a protein or other biomolecule or small molecule. Therefore, the reporter nucleic acid can be provided as a label or marker for a binding probe for the analyte, and can be detected to detect the analyte, for example in an immunoassay, such as in an immunoPCR or immunoRCA reaction. The reporter nucleic acid can be produced during the detection process, for example, by a ligation reaction in a proximity ligation detection, or an extension reaction in a proximity extension detection, or by a cleavage reaction, etc. Therefore, such a reporter target nucleic acid can be a synthetic or artificial sequence.

[0074] 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 or an amplicon thereof, or it can be a cDNA or a subfraction thereof, or an amplicon or a copy thereof, etc.

[0075] In another embodiment, the target nucleic acid molecule is a target RNA molecule. It can be an RNA molecule or other nucleic acid molecule in an RNA library, such as a genomic nucleic acid (whether human or from any source), a transcriptome nucleic acid or any other nucleic acid (such as an organelle nucleic acid, i.e., a mitochondrial or plastid nucleic acid), whether naturally occurring or artificially synthesized. Therefore, the target RNA molecule can be or can be derived from a coding (i.e., pre-mRNA or mRNA) or non-coding RNA sequence (e.g., tRNA, rRNA, snoRNA, miRNA, siRNA, snRNA, exRNA, piRNA, and long ncRNA). In one embodiment, the target nucleic acid molecule is a microRNA (miRNA). In another embodiment, the target RNA molecule is a 16S RNA, for example, wherein the 16S RNA 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 an ssRNA or dsRNA of a virus with RNA as its genetic material. It is noteworthy that such viruses include Ebola virus, HIV, SARS, SARS-CoV2, influenza, hepatitis C, West Nile fever, poliomyelitis, and measles. Thus, the target RNA molecule can be sense RNA, antisense RNA or double-stranded RNA from a viral genome, or sense RNA from a retroviral RNA genome.

[0076] 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. The cDNA molecule is then amplified.

[0077] A key feature of the method is that the steps of forming the sRCA product from the target amplicon are all carried out in a single reaction vessel. This refers to a single physical or structural compartment, i.e. a physical structure that contains the volume of the reaction mixture. The term "compartment" as used herein does not itself include droplets or emulsions, but this does not exclude the use of droplets or emulsions in any step of the method (i.e. within the reaction vessel in steps (a) to (j)). It will also be understood that for the purpose of detection in step (k), the second (or further) RCA product may be removed from the reaction vessel, for example, by transferring it to a detection instrument such as a flow cytometer or microscope slide. Thus, the container may be any container suitable for carrying out the steps of the method. Typically, this will be a reaction tube, but it may also be a container of any form or construction, for example including a reaction well or part of a microfluidic circuit.

[0078] The amplification is performed to produce a target amplicon to be detected by the method, or more specifically, the amplification reaction mixture of preparation (a) can be any amplification method that produces a linear amplicon containing a single copy of the target nucleic acid sequence. Alternatively, the amplicon in step (a) can be defined as an amplicon produced by an amplification method using a linear template. It can be seen that this includes known amplification methods other than RCA (which uses a circular template and produces a plurality of repeated amplicons (RCP) containing complementary copies of the template). Therefore, in the above method, in step (a), the amplicon can be optionally defined as having been produced by an amplification method other than RCA, or does not include or is not an RCA product.

[0079] Typically, amplification methods use two or more amplification primers, typically two primers.

[0080] Thus, the amplification method may be any known amplification method other than RCA. This may include, for example, PCR (the term is used broadly herein to include any variant of PCR), LAMP, NASB, SMAP, SDA, helicase-dependent amplification (HDA) or recombinase polymerase amplification (RPA).

[0081] A convenient amplification method is PCR. In one embodiment, the PCR is a conventional PCR reaction using two primers in the whole reaction process. In another embodiment, the method is asymmetric PCR. Generally speaking, asymmetric PCR is a 2-stage reaction, including a stage (exponential amplification stage) using two primers and a stage (linear amplification) using only one primer. Usually, asymmetric PCR can be achieved by providing one of the primers of a limited concentration so that it becomes reduced or exhausted, and linear amplification is carried out only with non-restrictive primers. However, this can also be achieved by designing primers so that they have different annealing temperatures (Tm), and in the method, temperature control is used to control which primers anneal, so that an amplification reaction can be initiated.

[0082] Thus, in a specific embodiment, an asymmetric PCR reaction is performed to produce an amplicon of a target sequence using a primer set comprising a first primer having a first melting temperature (Tm) and a second primer having a second Tm at least 10°C lower than the first Tm, and the asymmetric PCR reaction comprises, in either order:

[0083] (i) an exponential PCR phase comprising no more than 12 cycles (e.g. no more than 11 or 10 cycles) in which the primers are annealed at a first annealing temperature that allows annealing of the first and second primers; and

[0084] (ii) a linear amplification phase, in which the primers are annealed at a second, higher annealing temperature that allows only the first primer to anneal and amplify only one strand, thereby preferentially accumulating single-stranded amplicons of the target nucleic acid sequence.

[0085] In one embodiment, the exponential phase PCR reaction (i) is first performed, followed by a linear amplification reaction (ii). In this embodiment, it can be said that the asymmetric PCR reaction comprises (i) a first exponential phase and (ii) a second linear amplification phase, wherein each phase is as defined above.

[0086] In the above-described asymmetric PCR scheme, two PCR primers are designed to have different annealing temperatures (Tm), and temperature control is used during the method to control which primers anneal and thus enable the amplification reaction (i.e., there is a temperature "switch" in the method). Thus, the exponential reaction is carried out at a lower temperature, at which both primers anneal, while the linear amplification is carried out at a higher temperature, at which only the higher Tm primer anneals. The increase or decrease in temperature is used to switch between the different phases, depending on which phase is carried out first.

[0087] As mentioned above, in the usual method, it is advantageous to carry out a limited number of amplification cycles, so that the resulting mixture comprises an excessive amount of amplification primers relative to the amplicon. Typically, no more than 40, 39, 35, 30, 25 or 20, for example no more than 18, 16, 15, 14, 13, 12, 11 or 10 cycles are carried out. Advantageously, in order to minimize polymerase errors, no more than 10 cycles (i.e. 10 or less) are carried out.

[0088] Even such a limited number of cycles will result in a very large excess of primers, such as 1000-fold excess or more. Thus, the primer excess can be at least 1000, 2000, 5000, 7000, or 10,000-fold. In some cases, it may be much higher, on the order of tens of thousands or hundreds of thousands of times excess. For example, in a representative amplification reaction, the resulting reaction mixture may contain about 100 fM of the amplicon (e.g., full-length PCR product) and about 500 nM of primers.

[0089] In the case of multiple reactions with more than one target molecule or more than one target sequence, the amplification of each target can be performed separately and in parallel, i.e., a separate amplification reaction can be performed, each reaction using a single set of primers (e.g., a single PCR primer pair). In this case, each amplification reaction mixture, especially a portion or aliquot mixture thereof, can be combined to provide the reaction mixture of step (a). Alternatively, the amplification of different target sequences / molecules can be performed multiple times in the same reaction mixture. Therefore, the amplification reaction can be performed using multiple sets (or multiple) of primers (or in other words, a primer pool). In this regard, it should be understood that when studying different variants of a target sequence, the same set of amplification primers can be used to generate its amplicon (in other words, the amplicon may contain one or more of many different variants, which may have been amplified using the same primer set; the primers can be designed to bind to sites on both sides of the variant position). When multiple amplification is performed in the same reaction mixture, the number of sequences amplified may be 2 to 10,000 or more.

[0090] Therefore, in order to detect the target sequence in two or more different target molecules, multiple amplification primer sets (for example primer pairs) can be used, each primer has specificity to different target sequences, i.e. has a target binding region, these target binding regions are complementary to the primer binding sites in the target molecule, and these primer binding sites are located at different target sequence interiors or flanks. In this respect, it should be understood that for different target sequences, the flank binding sites in different target molecules will be different, to allow the specific binding of the first padlock probe. Alternatively or additionally, multiple different amplification primer sets can be used again to detect different and independent target sequences in the same target molecule, for example, different sequences in different genes on the chromosome, and each primer set has specificity to different target sequences. However, as mentioned above, in a specific embodiment, the method can be used to detect which one of many possible different variant sequences exists in a given target molecule, for example, whether there is a wild-type or mutant sequence, or which one of many possible mutants, or different allele variants, or polymorphism etc. In such a scheme, a common primer set is used to generate an amplicon, which variant is then determined by using multiple different padlock probes, each specific for a different variant of the target sequence.

[0091] As used herein, the term "multiple" or "multiplicity" means 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, thousands or tens of thousands of primer sets (and corresponding connection templates or padlock probes) can be used. The usable quantity is not limited and can be varied. This will depend on the purpose of the method, the nature of the sample, the target nucleic acid sequence to be detected, the quantity of possible variants, etc. Therefore, taking the detection of wild-type and mutant variants as an example, the number of different padlock probes depends on the number of different mutants that may exist, for example, it may be 2-6, 2-5, 2-4 or 2-3 different padlock probes. It should be understood that different aspects can be combined to improve the overall multiplicity of detection. For example, in a given sample, the method can be used to detect different variants of different target sequences.

[0092] Where the amplicons are produced by different primers, for example, from different target molecules, or different target sequences amplified by different primers of the same molecule, they may have different terminal sequences. Different or selected terminal sequences can be introduced by amplification primers. Therefore, the amplification primers used to amplify different target sequences can be provided with a 5' terminal sequence that is not complementary to the target sequence / molecule, and it can be used to provide a common or universal terminal sequence to all amplicons. In this way, amplicons with the same terminal sequence can be obtained. In this way, all amplicons in the reaction mixture (or, for example, amplicons produced from the same sample) can be cyclized using the same ligation template. In some cases, if necessary, the same (i.e., common) terminal sequence can be provided for all amplicons in a group, while different terminal sequences can be provided for different groups. For example, all amplicons from a given sample can have the same terminal sequence, but amplicons from different samples pooled together can have different terminal sequences, so that the same target sequence from different samples can be distinguished. Therefore, the terminal sequence can be defined as a binding site for a ligation template or a binding site comprising a ligation template.

[0093] Therefore, in one embodiment, the amplification primer (and the subsequent connection template) can be regarded as a universal primer, or a universal primer for a group of target sequences, or a universal primer for a group of variant target sequences. Therefore, the amplification primer can have a target binding region (i.e., a common binding site for different target sequences or different variants of target sequences) complementary to the binding site (e.g., flanking region) shared by different target sequences in the target molecule. In other words, the amplification primer can have a common or universal target binding region for different target sequences or different variants, or a common binding region for a group of target sequences or a group of variants. In other words, the amplification primer can have a target binding region, which can hybridize with the complementary binding site shared by different target sequences or different variants of target sequences in the target molecule. However, as described below, the padlock probe has specificity to the target sequence or the different variants of the target sequence.

[0094] In this regard, it should be understood that PCR primers bind to different strands, and therefore, the reverse primer will bind to the complementary strand of the strand to which the forward primer binds. In other words, the second primer or reverse primer will bind to the extension product of the first primer, or to the first of the two strands of the double-stranded molecule. The extension product of the second primer will contain the complementary sequence of the target sequence. Therefore, the target sequence mentioned herein may include the complementary sequence of the target sequence, i.e., the sequence complementary to the target sequence, as appropriate in the context.

[0095] In various embodiments, the amplification primers may be specific for a particular target nucleic acid sequence. Thus, for example, primer sets may be used, each primer set being specific for a different target sequence. This may be useful in diagnostic tests, such as NIPT, where different sequences may be detected, such as to detect different chromosomes (and determine their copy number, such as to detect trisomy), or in any situation where detection of one or more specific target sequences is desired.

[0096] In order to amplify, the target nucleic acid is contacted with an amplification primer and other reagents necessary for amplification. A sample containing a target nucleic acid molecule or a portion or fraction or aliquot thereof can be contacted with a reagent. For example, a cell or tissue sample, or any sample containing a cell or cell extract, can be directly contacted with a reagent. The sample can be pre-treated or processed before contact. In other embodiments, the target nucleic acid can first be separated or removed from the sample. The procedure for extracting or purifying nucleic acid (e.g., DNA) from various types of samples is well known in the art. For example, nucleic acid can be separated from cells or cell-free samples such as blood plasma. In some cases, nucleic acid molecule fragmentation may also be desirable. Its procedure is known in the art, including specific digestion, such as using a nuclease including a restriction enzyme, or by non-specific methods, such as shearing.

[0097] To provide the amplification reaction mixture of (a), all or a portion of the amplification reaction used to produce the amplicon or a portion of the resulting amplification reaction mixture can be introduced or added to a reaction vessel (i.e., a single reaction vessel used in the method). For example, a volume of 1-5 μl of one or more amplification reaction mixtures can be added to a vessel. However, in another embodiment, the amplification can be performed in a vessel, i.e., the reaction mixture of (a) can be produced in a vessel.

[0098] In step (b) of the method herein, excess amplification primers present in the reaction mixture in (a) are reduced. This is the first clean-up step of the method. This can be accomplished by enzymatic digestion and / or dilution.

[0099] In the case of enzymatic digestion, this can be achieved by contacting the reaction mixture in (a) with a digestive enzyme capable of degrading the primer. Conveniently, this may be an enzyme having exonuclease activity, in particular an enzyme having exonuclease activity as its main catalytic activity (as opposed to a polymerase having exonuclease activity, or a portion thereof having exonuclease activity). Thus, an exonuclease may be used (this term does not include a polymerase or a portion thereof in this context).

[0100] The term "contacting" is used broadly herein to include contacting the reagents in question. Thus, one reagent may be added to the other reagent, or vice versa, or two reagents may be introduced into each other, etc. However, in the case of single-container procedures carried out in steps (a) to (j), contacting refers to adding the relevant reagent to the reaction mixture in a single container, or in other words, adding the relevant reagent or reagent mixture to the container. Thus, in the case of step (b), this may involve adding a reagent or reagent mixture containing a digestive enzyme to the reaction mixture of (a), particularly to the container containing the reaction mixture of (a).

[0101] The exonuclease can be any suitable exonuclease. Exonucleases for removing unwanted nucleic acids are known in the art, for example including exonucleases I, III or λ or mixtures thereof. The selected exonucleases should have strict single-strand specific activity, for example ExoI or RecJf.

[0102] Other digestion systems can also be used. For example, if the amplification primer contains uracil, then uracil DNA glycosylase (UDG) can be used for digestion, which is another well-known digestion system.

[0103] In another embodiment, the amplicon can be produced using nucleotides resistant to exonuclease digestion (e.g., comprising modified bases that can inhibit exonuclease activity) and primers consisting of nucleotides sensitive to exonuclease digestion (e.g., normal or conventional nucleotides). In this way, the primer portion of the amplicon can be digested while retaining the newly synthesized sequence in the amplicon.

[0104] This cleaning step may further include enzymatic digestion of the amplification polymerase. However, this is not absolutely necessary, because interference from the polymerase can be resolved by other means. However, in one embodiment, a cleaning reagent mixture is added to the container, and the mixture includes an enzyme with protease activity in addition to an enzyme with exonuclease activity. This can be any suitable protease, including serine proteases, cysteine ​​proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases and asparagine peptide lyases, but is typically proteinase K. It is best to use a thermolabile or non-thermophilic protease because it may be inactivated by heating later. Therefore, a heating step may be included to inactivate the protease after use, as discussed further below. It is advantageous to use a protease in this step because the protease also degrades the exonuclease. In this regard, a balance will occur between the enzymatic action of the exonuclease and its digestion by the protease. However, it has been found that before the exonuclease is inactivated, sufficient degradation of the amplification primer can occur, and it is completely within the routine skills of those skilled in the art to find and optimize the reaction conditions.

[0105] The clean-up reagent (such as an enzyme) may be provided in any suitable buffer or diluent. Advantageously, this may be a buffer suitable for the subsequent ligation and RCA reactions. The container may be incubated under conditions suitable for carrying out the clean-up reaction. As a representative example, this may be incubated at 37°C for 10 minutes, but this may of course be varied or optimised, depending on the exact nature of the enzyme used etc., and may include leaving the reaction mixture at room temperature for a period of time.

[0106] If the method is used to detect variants, such as mutations, then it is generally preferred to perform the clean-up step (b) by enzymatic digestion of the primers. Preferably, this also includes protease digestion of the amplification polymerase.

[0107] However, as an alternative to enzyme digestion, a method of diluting the amplification reaction mixture can be used to dilute the primers. This may be enough to reduce their interference to an acceptable level. Generally speaking, this may require enough dilution factors, which can be determined by routine experiments. For example, a dilution factor of at least X200 can be used. This can be achieved by contacting the reaction mixture with a diluent, for example, adding a diluent to the reaction mixture, or adding a portion or aliquots of the amplification reaction mixture to the diluent. The diluent can be conveniently a suitable buffer, for example, a buffer suitable for subsequent steps of the method. In another embodiment, the diluent can be a reagent mixture for the connection step. In the dilution step, a smaller portion of aliquots of a single amplification reaction mixture or its pool can be contacted with a diluent, for example, a 0.1-0.2 μl aliquot.

[0108] Thus, in one embodiment, steps (a) and (b) may be combined and may be performed by adding a portion or aliquot of one or more amplification reaction mixtures to a volume of a cleaning reagent mixture in a container. Such a cleaning reagent mixture may be a simple diluent, such as a buffer.

[0109] After step (b), there is a step of denaturing the double-stranded amplicon into a single strand. This may not be necessary if the amplicon is essentially prepared in single-stranded form, for example by an asymmetric PCR reaction. In the case of conventional PCR (i.e., fully exponential phase PCR), the amplicon is typically present in double-stranded form. In this case, the method includes a heating step to denature the amplicon, for example, heating to 95°C for 1-2 minutes, or similar conditions. Such a heating step can also inactivate any protease included in the clean-up reaction. However, in general, it has been found that if a protease is used, it is best to include a separate, milder heating step, such as incubating at 50-60°C for 5-20 minutes, for example incubating at 55°C for 10 minutes, to inactivate the protease, and then proceed to the denaturation and ligation steps.

[0110] In this regard, in step (d), it is convenient to add a ligation mixture, heat denaturation, and then cool down for ligation. Therefore, the contacting of step (c) and step (d) can be carried out simultaneously or in any order. The actual ligation reaction can be controlled by controlling the reaction temperature (i.e., the ligation reaction can be triggered). This may include annealing of the ligation template (when used) and / or the activity of the ligase. In other words, if necessary, the ligation reaction occurs after denaturation, but the ligation reagent, ligase, and in some embodiments, the ligation template can be added to the reaction mixture of step (b) before or simultaneously with the denaturation of step (c). As described above, a ligation template is usually included because this often results in better results, but this is not necessary, and non-template ligation can also be selected. This may depend on the specific method used, as well as the amplicon and ligation conditions used. In one embodiment, the ligation template is included in the ligation reaction mixture.

[0111] After denaturation, the temperature is lowered to allow the amplicon to hybridize (or in other words anneal) to the ligation template used, and to allow the ligase to catalyze the ligation reaction. If there is no denaturation step, this temperature reduction may not be necessary, and instead the method may involve increasing the temperature of the ligation step (e.g., for annealing of the ligation template and / or ligase activity).

[0112] The ligation template comprises a sequence or a region complementary to the end sequence of the amplicon. More specifically, the ligation template comprises a sequence complementary to the end of a chain of the amplicon. The end of the amplicon chain is hybridized with the ligation template, thereby the end of the amplicon of the hybridization is juxtaposed to connect (i.e. adjacent to each other, so that they can connect). As mentioned above, the ligation template can be designed to be complementary to specific individual amplicon (in other words, the amplicon comprising the specific target sequence can have a homology ligation template, and this template is designed to hybridize with it, but not with other amplicon hybridizations). In other embodiments, the ligation template can be hybridized with the group or subset of the amplicon, or with all amplicons in the reaction mixture, depending on the end sequence provided to the amplicon.

[0113] In this respect, it is generally the case that all amplicons present in the reaction mixture (or more specifically a chain representing a specific amplicon) are connected. Therefore, in the case where the reaction mixture comprises a plurality of different amplicons (representing a plurality of different target sequences), all different types of amplicons that may be present or to be determined are connected to form a ring. Therefore, the step of cyclizing the amplicon by connection can be considered as a sample preparation step, or a step of preparing a molecular library, so as to amplify and detect with a padlock probe.

[0114] The ligation is performed by a ligase. This may be any suitable ligase known in the art. Representative ligases of interest include, but are not limited to, temperature-sensitive ligases, such as bacteriophage T4 DNA ligase, bacteriophage T7 ligase, and E. coli ligase, and thermostable ligases, such as Taq ligase, Tth ligase, Pfu ligase and 9°N TM DNA ligase. Advantageously, a thermostable or thermophilic ligase is used because it can be retained in the reaction mixture in an active form so that the padlock probe is subsequently connected in step (h). Suitable ligation conditions are known in the art, and any necessary and / or suitable reagents can be combined with the reaction mixture and maintained under conditions sufficient to connect. Obviously, the ligation conditions may depend on the ligase used in the method of the invention. Therefore, for example, Ampligase can be used, which allows ligation to be performed at, for example, 50-60°C (e.g., 55-60°C, e.g., 58°C).

[0115] To make connections without a connection template, CircLigase can be used TM ssDNA ligase. This is a thermostable ATP-dependent ligase that catalyzes the intramolecular ligation (i.e., circularization) of ssDNA templates that have a 5'-phosphate group and a 3'-hydroxyl group.

[0116] As mentioned above, ligation is carried out under the condition that the ability of the 3' end of the amplicon chain that amplification polymerase extends and connects the template hybridization is suppressed.It should be appreciated that if amplification polymerase has been digested with protease in the previous cleaning step, such conditions will be reached.However, this can also be achieved in other ways, the most important being that the 5' end of the amplicon is hybridized first by controlling the hybridization of the amplicon and the connection template.This enables ligation to proceed before any significant extension occurs at the hybridization 3' end.Therefore, two-step hybridization can be carried out, so that the 5' end of the amplicon is annealed with the connection template at the first temperature, and then the temperature is reduced so that the 3' end of the amplicon is annealed with the connection template.

[0117] For example, the first annealing temperature can be selected to promote or optimize annealing of the 5' end of the amplicon.For example, an annealing temperature of 50-75°C can be used, such as 53-60°C, or more specifically, 55-60°C.

[0118] The annealing temperature can be lowered for annealing of the 3' end. Again, appropriate conditions can be selected based on knowledge in the art and specific reagents (such as enzymes used, etc.). For example, after the first annealing step, the temperature can be lowered to 28-40°C, such as 28-35, 30-35, 28-33, 30-33, 28-32 or 30-32°C, etc., for the second annealing step.

[0119] A suitable ligase can be selected to achieve this two-step annealing. In this regard, thermophilic ATP-dependent ligases can be used, which are active in the temperature range of 37-95°C. Thus, a second / annealing temperature of about 37-45°C can be used, which is 10°C lower than the first annealing temperature.

[0120] Those skilled in the art will know how to design the connection template and / or the amplicon end to be able to achieve differential annealing of the 5' and 3' amplicon ends, for example, to achieve different Tm for the annealing of the respective ends. Therefore, by appropriate design and selection of appropriate conditions (including temperature), a balance can be obtained to stably hybridize the 5' end and then hybridize the 3' end. For example, the sequence at the 3' end of the amplicon hybridized with the connection template may be shorter than the 5' end sequence. As a representative example, the 3' end sequence can be at least 6 nucleotides long, for example at least 7 or 8 nucleotides long, for example 6-20, 6-18, 6-15 or 6-12 nucleotides long. The 5' end sequence can be at least 12 nucleotides long, for example at least 13, 14 or 15 nucleotides long, for example 12-30, 12-25, 12-20, 12-18 or 12-15 nucleotides long.

[0121] Once the amplicons are circularized by ligation, they are subjected to RCA in step (e). The RCA reaction is well known in the art, and the conditions of this step can therefore be designed or selected according to protocols and principles known and described in the literature. A strand displacement polymerase such as Phi29 or a derivative thereof is used. Conveniently, the ligation template acts as a primer for this first RCA reaction (i.e. as an "RCA primer"). However, this is not necessary, and a separate RCA primer can be used. In addition, if a ligation template is not used, an RCA primer needs to be provided.

[0122] To ensure that no unwanted polymerase-catalyzed extension reactions occur, the reagents required for RCA are added to the reaction mixture after the ligation step. Therefore, an RCA mixture containing at least an RCA polymerase is added. Other RCA reagents, such as dNTPs, may be added before this, but generally speaking, it is convenient to add an RCA reagent mixture comprising an RCA polymerase and dNTPs. As known in the art, an RCA reagent mixture typically includes an appropriate buffer and other components to aid the RCA reaction. As a representative example, using Phi29 polymerase, the RCA reaction can be carried out at 37°C, for example, for 20-40 minutes.

[0123] After the first RCA reaction, and prior to subsequent steps (e.g. prior to adding the padlock probe, or prior to ligation thereof), there is a step of inactivating the RCA polymerase used, e.g. by heating. For example, such a heat inactivation step may comprise heating at 55-65°C for a period of time, e.g. 5-15 minutes, e.g. 10 minutes.

[0124] Therefore, after this heat inactivation step, the temperature can be lowered, if necessary, in order to contact the first RCP with the padlock probe and allow it to hybridize in a subsequent step.

[0125] The RCA reaction produces a concatemer first RCA product (RC) comprising multiple repeated copies of the complementary sequence of the connected amplicons. Therefore, it contains multiple repeated complementary copies of the target sequence present in the amplicon. In this way, the target sequence is amplified. The target sequence provides a binding site for the padlock probe. Therefore, the first RCP provides multiple binding sites for the padlock probe. Therefore, more specifically, multiple copies of a given padlock probe can hybridize with the first RCP. However, it should be understood that not every available binding site in the first RCP needs to be occupied or bound by the padlock probe-it is sufficient for a certain number or multiple binding sites in the first RCP to be bound by the padlock probe. More specifically, a target sequence in at least one monomer of the concatameric first RCP is bound by a padlock probe, but more specifically, a target sequence in at least 2, 4, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, 80, 100, 150, 200, 300, 400, 500, 700, 1000 or more monomers is bound by a padlock probe.

[0126] The method relies on multiple probes that can hybridize with the first RCA product. Therefore, it should be understood that the first RCA product needs to be available for probe hybridization. This requirement is a feature of all RCA-based detection methods, i.e., the RCA product is detected by hybridizing a probe (e.g., a detection probe) with the product, and is well known in the art. Therefore, it may be advantageous for the first RCA product to have a low secondary structure. However, according to principles well known in the art, this feature can be compensated by carrying out the method under conditions that are conducive to hybridization.

[0127] The padlock probe is specific to the target sequence and is therefore used to identify or detect the target sequence or its variant. Therefore, the padlock probe comprises a target-specific binding region that is specific to a specific target sequence or a specific variant. Therefore, the target binding region is designed to be complementary to a specific sequence in the target sequence, or to distinguish or discern different target sequences or variants. In this respect, it is understood that the padlock probe binds to the complement of the target sequence because it appears in the amplicon subjected to RCA. Therefore, the term "target specificity" is widely used herein to refer to the target sequence that appears in the target molecule and its complement, and which one of them the padlock probe actually binds to will depend on the exact nature of the method, and which amplicon chain is connected, etc. The design of variant-specific probes or allele-specific probes is well known in the art, so the binding region of the padlock probe can be easily designed to detect or identify the desired target sequence or variant. In variant sequence detection, the padlock probe can be genotyped to the target sequence in the first RCP accordingly and can be referred to as a genotyping padlock probe.

[0128] As described above, where variant target sequences are detected, two or more padlock probes may be used, each specific for a different variant sequence, and which of these probes produces the second RCP may be detected in order to detect or identify which variant is present.

[0129] The padlock probe can be alternatively defined as a circularizable probe. The use of padlock probes or circularizable probes is well known in the art, 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 known and described in the art. Padlock probes are typically linear circularizable oligonucleotides, and the mode of its hybridization with a target nucleic acid sequence or molecule is to juxtapose the 5' end and 3' end of the probe, so as to be directly or indirectly linked together, leaving a gap in the middle. By connecting the hybridized 5' and 3' ends of the probe, the probe is cyclized. It should be understood that in order for cyclization (connection) to occur, the connectable 5' end of the padlock probe has a free 5' phosphate group.

[0130] 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 the ends of the padlock probe.

[0131] In order to connect, the 3' and 5' ends to be connected ("connectable" 3' and 5' ends) are hybridized with the target sequence in the first RCP, which serves as a connection template. According to the probe design, the connectable ends of the padlock probe can be juxtaposed in various ways for connection. When the target binding site is located at the end of the padlock probe, the combination of the padlock probe can cause the end to be in the described juxtaposed state. When the complementary binding sites in the target molecule or sequence are directly adjacent to each other (or adjacent), the ends of the padlock probe will 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 connectable end of the probe is provided by the actual end of the probe. However, in another configuration, 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-adjacent) binding site in the target sequence. In this arrangement, the 5' connectable end of the probe is provided by the actual 5' end of the probe. However, the connectable 3' end of the probe is generated by the extension of 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 juxtaposes the extended 3' ends of the probe for ligation. In this case, the ligatable 3' end of the probe is the extended 3' end of the probe.

[0132] In another embodiment, the 3' end of the first padlock probe may include a hairpin structure, which includes the 3' target binding region. In other words, the 3' target binding region may be at least partially contained within the hairpin structure at the 3' end of the padlock probe. When the probe hybridizes to the target molecule, strand displacement will cause the hairpin to open and allow the 3' end of the probe to hybridize to the target molecule.

[0133] The padlock probe can be provided by two or more parts connected together. This may involve providing an additional connection template, for example, in a probe consisting of two parts, each of which contains only one target binding region, and the other end of each part is hybridized with a common connection template. In another embodiment, the padlock consisting of two parts can take the form of a "connector" oligonucleotide, which has two target binding regions at or near the 5' and 3' ends, respectively, the two target binding regions hybridize with the target, there is a gap between the target binding regions, and a gap oligonucleotide hybridized in the gap between the ends. The gap oligonucleotide can partially or completely fill the gap. In another embodiment, the padlock consisting of two parts can include two linear probes, which are included in the target binding region at the 5' end of one probe and the target binding region at the 3' end of another probe. The target binding region of the probe hybridizes with their respective target sequences on the RCA product, thereby template-connecting the probe into a single linear reporter molecule. Then the second RCA primer templates the connection of the remaining 5' and 3' ends to circularize the probe.

[0134] However, in typical embodiments, the padlock probe is provided as a single circularizable oligonucleotide, whether or not as a gap-filling padlock probe.

[0135] In a specific embodiment, the padlock probe does not have a secondary structure, especially does not include an intramolecular double-stranded region or a stem-loop structure. However, a dumbbell probe with a secondary structure is a specific subtype of a padlock probe. The dumbbell probe includes two stem-loop structures, the stems are connected to the stems, and one of the "loops" is not closed, but open, with free 5' and 3' ends that can be connected to each other. This "open loop" serves as the target binding domain of the probe. The closed loop is only used as a spacer to connect the duplex ends (stems). In other words, it can be viewed as a padlock probe that forms a duplex region between the complementary sequences (regions) of the padlock. The region of the duplex is used as a signaling domain that an intercalator can bind to. Therefore, the "open loop" of the dumbbell probe can include a complementary target binding region.

[0136] In another embodiment, as described above, the padlock probe may include a hairpin structure comprising the 3' end of the probe.

[0137] 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 the case of variant target sequences that are similar in sequence (e.g., where the variant is a single nucleotide variant, such as a SNP, etc.). However, the high specificity of padlock probes is due to the fact that these non-specifically hybridized padlock probes cannot be ligated (and therefore any such unligated padlock probes will not be amplified by the subsequent RCA step). Therefore, in the second ligation step of the method herein, padlock probes that have hybridized to the complement of their target sequence (i.e., have correctly hybridized to the complement of the target sequence they are intended to detect, or in other words, their corresponding or homologous or respective target sequence) are ligated.

[0138] The second connection (of the padlock probe) can be carried out under conditions similar to the first connection. Generally speaking, the padlock probe can be added to the connection mixture of the reaction mixture, i.e., added to the reagent mixture consisting of the connection reagent. This may be similar or identical to the connection mixture / reagent used in the previous connection step. In one embodiment, this may involve the addition of a ligase. However, if a thermostable ligase is used for the first connection, it may not be necessary to further add a ligase. Obviously, a separate connection template is not needed in this step because the connection is templated by the first RCP bound by the padlock probe. Therefore, the second connection is a templated connection.

[0139] As with the amplicon connection in the aforementioned step (d), the connection in step (h) is carried out under conditions that inhibit the ability of the polymerase to extend the hybridized 3' end of the padlock probe. This includes the amplification polymerase and RCA polymerase from the original reaction mixture. The RCA polymerase is inactivated in the previous step. In particular, this is to ensure that any hybridized but unconnected padlock probe hybridized 3' end will not undergo any unwanted extension. It should be understood that this requirement applies to the expected connectable 3' end of the padlock probe. Therefore, if the connectable 3' end is produced by a gap filling extension reaction, the condition of the connection reaction is that the connectable 3' end will not be further extended. The strategy to ensure this condition is as described above. Therefore, this may involve enzymatic degradation of any polymerase added for the gap filling extension step after the gap filling extension has occurred. Alternatively, the extension polymerase can be inactivated by heating. The gap filling padlock may be useful in the case where the desired sequence needs to be incorporated into the second RCA template (e.g., for further amplification). In one embodiment, this can be used for, for example, NGS colony generation.

[0140] In the case of padlock probes where gap-filling extension is not required, any amplification polymerase may have been inactivated by enzymatic digestion in the first cleanup step. If such enzymatic digestion is not included in the first cleanup step, then similarly, unwanted extension can be avoided by controlling the hybridization of the padlock probe as described above, i.e., performing a two-step hybridization of the padlock probe using different annealing temperatures.

[0141] After the ligation step, any unattached probes in the reaction mixture are removed or rendered inert. By inert is meant that the padlock probes cannot participate in any unwanted hybridization or ligation reactions. In particular, the 3' end of the probe cannot hybridize with any nucleic acid present in the mixture and initiate an extension reaction. As mentioned above, it is particularly desirable to ensure that the unattached padlock probes remaining in the mixture after the ligation reaction cannot hybridize with the second RCP when the second RCP is produced. Therefore, in practice, a second clean-up step is performed at this stage. A clean-up reagent mixture can be added to achieve this.

[0142] The cleaning reagent may be a digestive enzyme for degrading unreacted probes, such as an enzyme with exonuclease activity as discussed above. If such an enzyme is used, it is inactivated by heating prior to the subsequent second RCA step (step (j)). For example, if lambda exonuclease is used, the reaction mixture may be incubated at a temperature of 30-37°C for 10-20 minutes and then heat-inactivated at about 70-75°C for 10-30 minutes. This may be appropriately varied depending on the exact reagents used, and the digestion and inactivation conditions are determined and optimized within the routine skill of those skilled in the art.

[0143] Alternatively or additionally, a dilution step may be performed to perform the cleanup of step (i). Thus, an amount of diluent may be added to the reaction mixture. In particular, the reaction mixture may be diluted at least 4X. As described above, when the method is used to detect sequence variants, cleanup by enzymatic digestion is generally preferred.

[0144] Alternatively, or additionally, a hairpin-shaped padlock probe can be used. Such a probe comprises a self-complementary sequence, which can hybridize to form a hairpin structure comprising the 3' end of the probe. The stem of the hairpin can be designed to have a thermal stability that does not prevent the formation of a hybrid between the target sequence in the loop of the hairpin structure and the first RCP, nor does it inhibit RCA. However, the stem is sufficiently stable to induce extension, so that the unconnected padlock probe is converted into a complete hairpin, which can not hybridize with the second RCA product or use the second RCA product as an extension template to induce extension. This padlock probe that can be inactivated is referred to as a suicide padlock probe. The suicide padlock probe is also described in US 6,573,051.

[0145] After the cleaning step (i), a second RCA reaction is performed using the connected padlock probe as a second RCA template. In order to trigger the second RCA, a second RCA mixed reagent is added to the reaction mixture, at which time the mixture contains the connected padlock probe. In general, this will involve the addition of an RCA polymerase, which may be as described above and may be provided in a reagent mixture as described above. Although all reagents for the second RCA can be added together to a reagent mixture, it may be advantageous to add them individually or stepwise. The second RCA is initiated by a separate RCA primer. It may be pre-hybridized with the padlock probe, may be added to the reaction mixture alone, or may be included in the RCA reagent mixture. The binding site for the RCA primer may be provided in a region of the padlock probe that is different from the target binding region (e.g., in the backbone region of the padlock, between the target binding ends).

[0146] In one embodiment, the RCA primer for the second RCA may be included in the cleaning reagent mixture. In another embodiment, it may be added separately at the same time as the cleaning reagent mixture. In another embodiment, it may be added to the reaction mixture after the exonuclease used for cleaning is inactivated. The second RCA may then be initiated by adding an RCA reagent mixture comprising an RCA polymerase and dNTPs. In another embodiment, the first step is to add the RCA polymerase, followed by the addition of dNTPs and RCA primers, respectively.

[0147] When the RCA primer is included in the clean-up mixture, it can be protected from digestion by having a modification that resists digestion (e.g., an exonuclease-resistant modification, including, for example, a phosphorothioate bond). Alternatively, an exonuclease can be used in the clean-up that degrades double-stranded DNA and single-stranded DNA having a 5' phosphate group; the padlock probe has a 5' phosphate group and is therefore degraded by such an exonuclease, and an RCA primer can be provided without a 5' phosphate group and is therefore protected from digestion by the exonuclease. An example of such an exonuclease is lambda exonuclease.

[0148] In another embodiment, the cleaning of the unconnected padlock probe can be performed by RCA polymerase. Therefore, Phi29 has 3' exonuclease activity and can be used to digest the unconnected padlock. In such an embodiment, an RCA polymerase with a 3' exonuclease can be added, and the enzyme can be digested (i.e., the reaction mixture can be incubated). Subsequently, the RCA reaction can be performed. In this case, this can be continued by adding primers and nucleotides to allow the RCA reaction to proceed. Therefore, in this embodiment, the RCA polymerase can be added before the second RCA step to achieve the purpose of cleaning (i.e., step (i), before performing the second RCA reaction).

[0149] As described above, reagents, procedures and protocols for conducting RCA reactions are known in the art and can be used herein.

[0150] The method herein requires a first RCA step with a circularized amplicon as a template and at least a second RCA step with a circularized padlock probe as a template. The method may include a further RCA step to generate a third or more rounds of RCA products using a second, third or fourth padlock probe, etc., each padlock probe targeting a target nucleic acid sequence. In other words, steps (g) to (j) may be repeated if necessary. The final generated RCA product may be detected.

[0151] It should be understood that this further generated RCA can play a role in increasing the signal finally detected. This can accordingly amplify the increased signal. However, it should also be understood that when the first and further padlock probes are gap-filling padlocks, this will result in an increase in the synthesis of a copy of the target nucleotide sequence or a portion thereof (depending on whether the continuous gap-filling reactions are completely overlapping or partially overlapping). In other words, this method may result in a large number of clones of filled sequences being produced. In other words, the filled sequence (i.e., the target nucleic acid sequence or a portion thereof) can be amplified. This is very useful for preparing a reaction. If multiple continuous (i.e., two or more) first and further padlock probe connection steps involve gap-filling of identical or overlapping sequences, then more copies of specific target sequences can be produced.

[0152] Detection of the second round or further RCP product as described above is to detect the target nucleic acid sequence. To achieve this, the RCP can be made detectable. For example, it can be provided with a detectable label or a means for detecting it. Conveniently, in one embodiment, a detection reagent can be used to achieve this. This will be described in more detail below. However, in short, the detection reagent for detecting the second round or further RCP can be included in one or more RCA reagents added to the reaction mixture, or the detection reagent can be added to the reagent mixture after the second round or further RCP is produced. Alternatively, the label can be incorporated into the RCP when the RCP is produced, for example, using labeled nucleotides.

[0153] In an advantageous embodiment, the detection agent is a detection oligonucleotide that hybridizes to the RCP, as further described below. The RCP is detected by detecting the detection oligonucleotide. In this regard, the detection oligonucleotide may have a label or some other detectable moiety.

[0154] For the actual detection, a portion of the reaction mixture or its aliquot may be removed from the container and moved to a detection instrument, or further processed prior to detection. Thus, in one embodiment, contact with the detection reagent may occur outside the reaction vessel in which the preceding steps of the method are performed.

[0155] The method can be performed in a heterogeneous or homogeneous form. That is, the method can be performed on a solid phase (or support), or in a solution or suspension (i.e. without a solid phase or support) or indeed both, since the solid phase can be introduced at a later stage, for example, in the step of detecting the second RCP, or at the stage of producing the second RCP, etc. In this regard, assuming that the method is performed in a single container until the second RCP is produced, it should be understood that any solid phase is a solid phase that can be added to the container, such as a particulate solid phase such as beads, or the solid phase will be provided by the container itself (e.g. the wall and / or bottom of the container).

[0156] The format of the method may be chosen depending on the nature of the sample, or the target nucleic acid molecule, or the desired read-out or detection technique.In one embodiment, the method is an in-solution method, ie a method performed in a liquid phase in a container.

[0157] It can be noted from the above description that the method involves temperature variations within and between different steps. In fact, the method is characterized in that it is carried out under temperature control. Conveniently, the method can be carried out in a thermal cycler. In this way, the temperature variation can be controlled at any time.

[0158] As mentioned above, these methods involve multiple steps, wherein reagents are added to a container containing the initial reaction mixture. Reagents can be added conveniently in a buffer. In other words, the various reagent mixtures used in the steps of the above method generally include a buffer. Since steps (a) to (j) are carried out in the same reaction vessel, there is no need to add a cleaning step, so the buffer for adding various reagents should be compatible. In one embodiment, all reagents or reagent mixtures use the same buffer.

[0159] The target nucleic acid molecule can be present in a sample, or can be obtained from a sample. The sample can be any sample containing any amount of nucleic acid, can be from any source or origin, and it is necessary to detect the target nucleic acid sequence in the target nucleic acid molecule therein. Therefore, the sample can be any clinical or non-clinical sample, and can be any biological, clinical or environmental sample in which the target nucleic acid molecule may be present.

[0160] 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 samples 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 preparation, cell lysate. Environmental samples are also included, such as soil and water samples or food samples. The sample can be freshly prepared, or can be pre-processed in any convenient manner, such as storage.

[0161] Therefore, 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. Therefore, such biological materials may include all types of mammalian and non-mammalian cells, plant cells, algae (including blue-green algae), fungi, bacteria, protozoa, etc., or viruses. For example, the cell may be a human cell, a bird cell, a reptile cell, etc., but is not limited thereto.

[0162] 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, cell culture, cell suspension, conditioned medium or other samples of cell culture components, etc. The sample may be pre-treated in any convenient or desired manner in preparation for use in the method, such as by cell lysis or purification, isolation of nucleic acids, etc.

[0163] In one embodiment, the sample comprises a microbial cell or virus isolated from a clinical sample or 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, discriminative or identifiable to a microbial cell or virus at any level (e.g., type, group, class, genus, species or strain level).

[0164] 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.

[0165] In another embodiment, the sample may comprise exosomes.

[0166] Since the target nucleic acid molecule itself is not necessarily the target analyte detected, for example, it can be a reporter molecule used or produced in the process of detecting any desired analyte, so the sample is not necessarily a sample containing nucleic acid naturally, nor is it necessarily 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. It can be a sample that has been subjected to analyte detection and determination, wherein 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 immune PCR, immune RCA or proximity detection (such as proximity ligation detection (PLA) or proximity extension detection (PEA)).

[0167] The target analyte can be any analyte that needs to be detected. As mentioned above, in embodiments, the target nucleic acid molecule of the method herein is the target analyte. In other embodiments, wherein the target nucleic acid molecule is a reporter molecule, 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 can appear as glycosyl groups on proteins. Therefore, the target analyte can be a modified protein, for example, with a post-translational modification detected in the analyte assay.

[0168] In one embodiment, the target analyte can be a component of a protein or protein molecule detected on the surface of a cell or vesicle or other cell or subcellular compartment. For example, extracellular vesicles or exosomes can be detected and distinguished by the presence of different proteins on their surface. Prostatic vesicles have been proposed as biomarkers for prostate cancer, and specific or selected prostatic vesicles or other extracellular vesicles can be detected and distinguished by detecting one or more surface proteins thereon.

[0169] The padlock probe may comprise one or more further sequences which can be used to introduce a sequence into the ligation product, thereby introducing an RCP (as a complementary copy). For example, this can be a label or detection sequence, such as a barcode or identification motif, or a binding site for a detection probe or primer. This is particularly true for padlock probes. For example, such further sequences can be found in a portion of the padlock probe backbone region (i.e., the region between the target binding regions). In a dumbbell probe, it can be located in the duplex region of the probe. Labels such as barcodes or probe / primer binding sites can be designed according to different needs / purposes, for example, introducing a universal or common sequence so that different connected probes in a multiplex setting can be processed or detected together, such as sample indexing, etc., or introducing a binding site for a universal or common amplification primer. This will enable different connection probes to be amplified together, such as library amplification by PCR or RCA.

[0170] In particular, the padlock probe may comprise a detection sequence that can be detected. The complementary sequence of the detection sequence will be incorporated into the second (or further) RCP, and can be detected, for example, by combining the detection probe with it or by sequencing. The detection sequence can be specific to the padlock probe, and therefore specific to the target sequence or sequence variant that needs to be detected. Therefore, each padlock probe can have different detection sequences. The detection sequence can be used to detect or identify which padlock probe is amplified in the RCA, thereby determining which target sequence is present. Such a scheme can, for example, be applied to a method for detecting a target sequence variant, wherein each padlock is provided with a detection sequence specific to a particular variant. Therefore, the detection sequence can be considered as a marker or identification sequence. The term "detection sequence" used herein includes the detection sequence that occurs in the padlock probe, and the complementary copy that occurs in the RCP.

[0171] Thus, label / barcode sequences, including in particular detection sequences, can be used to "label" different padlock probes so that they or their ligations or amplification products can be easily distinguished from one another. In addition, such sequences can be used to label different samples, etc., for example, so that they can be combined (i.e., "sample" labels or markers). Thus, in a multiplex setup, different probes (i.e., probes for different target nucleic acid sequences or different variants) can be provided with different label sequences (e.g., different markers or detection sequences) and / or they can be provided with the same label sequence, for example, for the introduction of a common or universal sequence. Such methods can be used in combination with specific detection methods, including the use of detection probes or sequencing methods, such as sequencing by hybridization, sequencing by ligation, or other next-generation sequencing chemistries, such as multiplex detection of multiple target nucleic acids in a sample.

[0172] As used herein, the term "hybridization" or "hybridization" 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. When two nucleotide sequences have base pair organizational homology, the two nucleotide sequences are "complementary" to each other. Therefore, a complementary region in a molecule or probe or sequence refers to a portion of the molecule or probe or sequence that can form a duplex. Hybridization does not require 100% complementarity between sequences, so the regions that are complementary to each other do not require complete sequence complementarity, although this is not excluded. Therefore, a complementary region may contain one or more mismatches. Therefore, as used herein, "complementary" refers to "functionally complementary", i.e., a degree of complementarity sufficient to mediate fruitful hybridization, including hybridization with a degree of complementarity less than 100%. The degree of mismatch tolerance can be controlled by appropriately adjusting the hybridization conditions. A person skilled in the art of nucleic acid technology can determine duplex stability empirically based on the guidance provided in the art, taking into account many variables, including, for example, the length and base pair composition of each molecule or probe oligonucleotide, ionic strength, and the incidence of mismatched base pairs. Therefore, the design of suitable probes or ligation templates or primers and their binding regions and the conditions under which they hybridize to their respective targets are well within the routine skills of those skilled in the art.

[0173] The complementary region, for example, the complementary region to the target sequence in the padlock probe binding region, or the complementary region between the detection sequence and the detection probe, or the complementary region between the RCA primer and the circularized padlock probe, etc., can be at least 6 nucleotides long to ensure the specificity of the binding, especially at least 7, 8, 9 or 10 nucleotides long. The upper limit of the length of this region is not important, but can be, for example, up to 50, 40, 35, 30, 25, 20 or 15 nucleotides. Therefore, the length of the complementary region can be between the above-mentioned lower length limit and the upper length limit. With regard to the padlock probe, the length of a single target binding region can be in a lower range, so when hybridizing with the target, the total length of the two binding regions is in a higher range. For example, a single target binding region can be 8-15 nucleotides, such as 10-12 nucleotides, so the total hybridization length is 16-30 nucleotides, such as 20-24 nucleotides. Under the constraints of the conformation of the probe, the spacing of the domains, and the required or favorable hybridization, it may be necessary to minimize the total length of the padlock probe to minimize the size of the ring subjected to RCA, therefore, reducing the length of the complementary region as much as possible.

[0174] The second RCP (and / or any subsequent round of RCP) can be detected using any convenient protocol. Depending on the target sequence to be detected, the purpose of the method and / or the specific details of the procedure used in the method, the detection protocol used can detect RCP non-specifically or specifically.

[0175] For example, the RCP can be detected directly, for example, the concatemer can be cut to produce monomers, which can be detected using gel electrophoresis, or more typically by hybridization with a labeled detection oligonucleotide (also referred to as a detection probe) that hybridizes to a detection sequence in the RCP, as described above. However, the detection oligonucleotide does not need to be directly labeled. For example, the detection oligonucleotide can be an unlabeled probe that acts as a "sandwich" probe. The concept of "sandwich" probes is well known in the art and can be applied according to any convenient scheme. Sandwich probes can bind to the RCP, but are not directly labeled themselves; instead, they contain a sequence to which a labeled second oligonucleotide can bind, thereby forming a "sandwich" between the RCP and the labeled second oligonucleotide.

[0176] RCP can also be detected using non-sequence specific nucleic acid labeling methods widely known in the literature, such as DNA binding stains or dyes, or by incorporating RCP using labeled nucleotides. Alternatively, RCP can be detected indirectly, for example, the RCP product can be amplified by PCR, and the amplified product can be detected.

[0177] RCP can be detected using any well-established nucleic acid molecule analysis method known in the literature, including liquid chromatography, electrophoresis, mass spectrometry (including CyTOF), microscopy, real-time PCR, fluorescent probes, chip methods, colorimetric analysis (such as ELISA), flow cytometry, mass spectrometry, or using turbidity, magnetic, particle counting, electrical, surface sensing, weight detection technology. Generally speaking, such methods are related to solution determination.

[0178] The detection oligonucleotide of labeling can be with any detectable labeling, and these labels can send signal directly or indirectly.For example, labeling can be by spectral or microscopic detection, for example it can be fluorescent or colorimetric labeling, particle or enzyme labeling.Any labeling used in immunohistochemical technique can be used.

[0179] In multiplex procedures for detecting different target sequences and / or variant target sequences, different second or further RCPs can be detected and distinguished by in situ sequencing, including, for example, by synthetic sequencing, by hybridization sequencing and by ligation sequencing, next generation sequencing and / or sequential barcode decoding technology, including by synthetic sequencing, ligation sequencing or hybridization sequencing, and / or by using detection probes. Depending on the level of multiplexing, a combined labeling method can be used according to techniques well known in the art. For example, a large number of repetitive sequences in sRCA products can be distinguished by ratio labeling of probes detectable by fluorescence or other spectrophotometry. Such ratio-labeled detection probes can be used in 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 ratios to produce multiple fluorescently labeled groups. For example, it has been found that 7 different groups can be created using a combination of two fluorophores in different ratios. This can be extended to using 3-color or 4-color combinations.

[0180] In the method involving the use of detection oligonucleotides, the detection oligonucleotide or any second labeling oligonucleotide can be labeled with a directly or indirectly detectable marker. A directly detectable label is a label that can be directly detected without the use of additional reagents, and an indirectly detectable label is a label that can be detected by using one or more additional reagents, for example, a label is a component of a signal generation system consisting of two or more components. In many embodiments, the label is a directly detectable label, wherein the directly detectable label of interest includes but is not limited to: fluorescent labeling, radioisotope labeling, chemiluminescent labeling, etc. In many embodiments, the label is a fluorescent label, wherein the labeling agent used in such embodiments is a fluorescently labeled nucleotide, such as fluorescently labeled CTP (such as Cy3-CTP, Cy5-CTP), etc. The fluorescent moiety that can be used for labeling nucleotides to produce labeled probe nucleic acids (i.e., detection probes) includes but is not limited to: fluorescein, cyanine dyes such as Cy3, Cy5, Alexa 555, Bodipy 630 / 650, etc. Other labels known in the art, such as those mentioned above, can also be used.

[0181] Although various detection methods can be used, conveniently the second or further RCP can be detected by microscopy or flow cytometry. In both cases, a detection oligonucleotide directly or indirectly labeled can be used, for example using an easily detectable fluorescent label. In particular, in microscopy-based methods, the RCP can be detected by imaging.

[0182] The benefit of using this detection technique is that the second or further RCPs can be digitally recorded. In fact, due to the degree of signal amplification provided by the method, the second or further RCPs can be visualized and thus detected by a camera or any device including a camera, such as a mobile phone.

[0183] In order to detect the second or further RCPs produced in a homogenous form, they can be captured or brought to a solid support or surface for imaging or more generally microscopic detection. The second RCP is the product of the second round of RCA, is larger and heavier, and therefore easily falls to the surface by centrifugation. Thus, for example, the tube or well plate can be easily rotated to bring the second RCP to the bottom of the tube or well for detection by microscopy, particularly imaging detection.

[0184] Variant sequence to be detected in the method can include one or more variant bases. Therefore, it can be a single nucleotide variant, such as a single nucleotide polymorphism (SNP) or a sudden change, or it can include two or more bases. Therefore, the variant sequence can include a section of nucleotides, wherein two or more bases can be variants. The variant base can be continuous or discontinuous.

[0185] The length of the target sequence is not critical and may vary according to the environment, the properties of the target molecule, the target sequence or the variant position or locus. Therefore, as a representative example only, the target sequence may be 1 to 10, such as 1-15, 1-12, 1-10, 1-8, 1-7 or 1-6 nucleotides long. However, in certain embodiments, a target sequence longer than a single nucleotide may be beneficial for improving specificity, and in such embodiments, the target sequence may be any one of 2, 3, 4, 5 or 6 nucleotides to any one of 6, 7, 8, 9, 10, 12, 15 or 20 nucleotides long. Therefore, an exemplary target sequence may be, for example, 4-10, 4-8, 4-7, 4-6, 5-10, 5-8, 5-7 or 6-8 nucleotides long.

[0186] Also provided is a kit for implementing the method as described above. The kit may include a ligation template and a padlock probe as described above, optionally together with instructions for use of one or more reagents and / or the kit. Such reagents include dNTPs, polymerases and ligases, and RCA primers for the first and / or second RCA reactions. In addition, the components may include buffers or other reaction components for one or more different reactions. Further optional components may include devices or reagents for detecting a second or further RCP. This may include, for example, detection oligonucleotides and any necessary secondary labeling reagents, including, for example, as described above. Further optional components may include solid supports and / or devices for capturing and / or fixing target nucleic acid molecules or reaction components. The instructions may be, for example, in printed form, or on a computer-readable medium, or may be a website address.

[0187] The advantages of the methods herein are discussed above. These advantages are particularly beneficial when detecting target sequences or variants in complex samples or when they are present in low abundance. As described above, the present method provides a high degree of signal amplification, making the method very sensitive. Therefore, the method is particularly suitable for detecting or identifying very rare sequence variants. For example, the method can be used to find and detect tumor-derived mutant DNA sequences in patient samples, particularly including cell-free DNA in plasma. Therefore, the method may be helpful in the diagnosis or monitoring of cancer, or, for example, revealing the recurrence of cancer. The method can be used for any cell-free DNA and can also be used for prenatal testing, particularly NIPT. The technology is fast, has minimal instrumentation requirements, and can perform multiple analyses of sequence variants, thereby increasing sensitivity.

[0188] Detection of low frequency or rare sequence variants or mutations also requires highly specific detection and minimizes the risk of introducing artificial sequence changes that may be mistaken for variant sequences. This is also provided by the combination of limited PCR cycles used in the present method with RCA amplification and targeting of the target sequence by padlock probes requiring dual target recognition.

[0189] In the present method, a first RCP is generated in a highly specific manner, while the production of a highly amplified second RCP is dependent on the presence of the first RCP.

[0190] Each RCA product typically contains hundreds or thousands of complements of RCA template circles (such as circularized amplicons or padlock probes) that are used to generate RCA products. The second round of RCA products are obtained by RCA of the circularized padlock probes on the first round of RCA products, and therefore can contain, for example, 1000X1000 monomer sequences, and thus have a larger volume and a molecular weight of up to billions of Daltons. In addition, as described above, more copies of the monomer sequence can be produced by further RCA reactions. This reaction product is easily detected. The size of the second RCA product can reach several microns, and they can be easily visualized as a single product, such as by a microscope. Each second or further RCA product can be detected as a cloning product, and they are produced by a single target nucleic acid molecule without compartmentalizing the RCA reaction. For example, when labeled with a fluorescent detection probe, the prominent fluorescent reaction product can count and distinguish the single amplification products of a single molecule in a wide field of view at a low magnification (such as 20 times). The second or further RCA reaction product is large and bright enough to be recorded by standard flow cytometry. This allows counting and digital scoring to be performed in minutes using existing instrumentation, providing excellent quantitative accuracy over a wide dynamic range. As described above, the repetitive sequences present in the second or further RCA products of the concatemer can be analyzed by ratiometric labeling techniques using products labeled with different combinations of fluorophores, thereby increasing multiplexing. Due to their large size and considerable molecular weight, sRCA products can also be enriched by centrifugation in a common benchtop centrifuge or similar. In practice, low speed centrifugation or unit gravity may be sufficient. Therefore, the new method described here has multiple advantages.

[0191] As described above, the strong signal amplification provided by the second RCA reaction allows the signal to be readily and easily visualized, for example, under a low-power microscope or on a digital scanned image, so that the test results can be quickly and easily visualized in clinical situations, for example, in routine use to check pathological results. Therefore, the method of the present invention is particularly suitable for clinical analysis procedures.

[0192] The method can be helpful for identifying rare integrated copies of viral genomes in human tissues, or for detecting rare RCA products, for example, in the case of inefficient mutation detection in tissues. Another example of easy identification of rare events is screening for the presence of circulating tumor cells (CTCs) in the vast majority of non-CTC cells. The strong signal generated by the method allows for quick and easy identification of events (detection of CTCs) at low magnification.

[0193] The method described could speed up assay analysis, which could be valuable at the point of care, such as a doctor's office, where a second RCA could be performed in a relatively short period of time.

[0194] Being able to perform the method in a single reaction vessel is a significant advantage, allowing for automation.

[0195] In addition, the increase in signal intensity / speed can also use other detection methods other than traditional fluorescence detection methods, such as turbidity, magnetic, particle counting, electrical, surface sensing and weight detection technology. For example, a single sRCA product from the second round of RCA has a potential weight of several femtograms after 1 hour of amplification. This increase in weight can be detected by methods and means known in the art, such as cantilevers, surface plasma methods and microbalances, such as quartz crystal microbalances. In addition, as described above, the increase in size and weight of the second RCP enables it to be effectively positioned to the surface by centrifugation. Conveniently, this can be performed at 3000X in 15 minutes, while the first round of RCA products cannot be effectively captured by centrifugation using a desktop centrifuge.

[0196] The present method is able to generate an enhanced signal localized to the first RCA product, and it also confers the ability to count individual reaction products (second RCA products) using a standard flow cytometer or distributed on a flat surface, etc., for high-precision digital detection. In particular, the second RCP can be stained with a chromogenic reagent such as HRP and imaged by a smartphone camera. Therefore, the method can achieve a reaction equivalent to digital PCR, but does not require emulsions or microfabricated structures, nor does it require the condition that only one template is present in each compartment.

[0197] The presence of distinct amplification products derived from the present method will further allow for single RCP cloning, as the products obtained from a single first RCA template can be visualised. Individual second RCA products can therefore be identified and isolated. For example, with the current amplification method, visualisation can be achieved in low melting point agarose, separation can be performed without amplification and then the isolated products can be obtained, for example using a toothpick, similar to the isolation of bacterial colonies.

[0198] The detection of rare mutations is very important for diagnosis in the clinic. For example, mutations in certain genes (e.g., KRAS mutations) may have important diagnostic significance and may help identify the emergence of acquired resistance to specific therapies (e.g., anti-EGFR therapy). In recent years, a lot of effort has been focused on developing methods to detect such mutations. The present method may provide a useful supplement to such methods.

[0199] In addition to being able to detect point mutations that occur at low frequencies in DNA samples, this method also provides a powerful means to screen DNA samples for the presence of any or all of a large number of different target sequences, which is not possible with PCR alone or any other existing method. However, unlike PCR-based methods alone, the use of padlock probes and RCA allows the use of hundreds of thousands of probes simultaneously without compromising target selectivity.

[0200] Furthermore, the discrete nature of sRCA products allows for digital detection by generating one reaction product for each target sequence detected and collecting these outstanding reaction products, thereby minimizing the risk of mixing with any other material in the reaction. For example, a probe mix can be created for all types of bacteria or all species of insects or fungi. This can then be used to identify positive reaction products, for example, by PCR amplification of the tag sequence on the padlock probe and hybridization of the product to a tag array or the like.

[0201] Furthermore, as described above, where the second and optionally further ligation and RCA reactions are carried out using a gap-filling lock format, preparative production of copies of the target nucleic acid sequence becomes possible.

[0202] Current sRCA methods also improve genotyping precision by detecting repeat sequences rather than individual target sequences in a single RCA product. Therefore, occasional false inputs of padlock probes can be tolerated without leading to erroneous results, as long as they are much less common than the correct results in a single RCA product. This allows genotyping to be performed by a majority voting mechanism. This may also have implications for how padlock probe-based genotyping is performed, as long as the ratio of correct to false reactions is satisfactory and the padlock probe detects a sufficient number of repeat sequences, sequence discrimination can be enhanced by using conditions in which neither variant is detected with 100% efficiency.

[0203] The method will now be described in more detail with reference to the following figures and non-limiting examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0204] Figure 1: Schematic diagram of the method for generating SuperRCA amplification products. A) The target DNA sequence in the sample is amplified by PCR. B) The amplified strand is converted into a single-stranded DNA circle by templated ligation at its 5' and 3' ends. C) The oligonucleotides that serve as templates for the circularization reaction can then serve as primers for the RCA reaction. D) The RCA products are then detected using padlock probes specific for the mutant or wild-type sequence. E) The ligated padlock probes are wrapped around the RCA product and then used as templates for a second RCA reaction, initiated by the added oligonucleotides. F) For each starting DNA circle, the reaction produces large aggregates consisting mainly of single-stranded DNA objects, called SuperRCA (sRCA) 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.

[0205] Figure 2 : Flow cytometry readout data of single-tube SuperRCA assay showing the proportion of IDH2 R172K mutation in samples: (a) 1.024%, (b) 0.064%, (c) 0.001% and (d) wild-type sample, which is negative for IDH2 R172K mutation.

[0206] Figure 3 : Fourfold serial dilutions of genomic DNA from cells harboring the IDH2 p.R172K point mutation were analyzed using the SuperRCA procedure or ddPCR. Genomic DNA samples with mutant genomic DNA serially diluted in wild-type genomic DNA were divided into two parts and analyzed using either the SuperRCA or ddPCR assays. According to the manufacturer's instructions, 330 ng of DNA (equivalent to 100,000 human haploid genomes) was analyzed per sample in the SuperRCA assay, while 66 ng (equivalent to 20,000 human haploid genomes) was used for ddPCR. Each data point was calculated as the mean of three biological replicates, and the limit of detection (LoD) for each analyte was calculated for ddPCR and SuperRCA assays, respectively (LoD = Mean (Wildtype) + 3 × SD (wildtype)) and is represented by dashed lines in the figures.

[0207] Figure 4: Patient UPN126 was diagnosed with primary myelofibrosis two years prior to the diagnosis of acute myeloid leukemia (AML). Bone marrow samples at the time of AML diagnosis (day 0) showed high levels of the IDH2p.R140Q mutation as documented by NGS, SuperRCA, and ddPCR. Retrospective analysis of initial bone marrow samples during the myelofibrosis phase was negative for the IDH2 p.R140Q mutation by SuperRCA and ddPCR. SCT was performed on day 139, at which time fairly high levels of the mutation were still present. Approximately three months after SCT, the SuperRCA assay detected low levels of the IDH2 p.R140Q mutation, which was not detected by ddPCR at this time point. All subsequent blood and bone marrow samples were negative by SuperRCA and ddPCR analysis, indicating complete remission. DETAILED DESCRIPTION

[0208] Materials and methods

[0209] Extraction of genomic DNA.

[0210] DNA was extracted from BM cells or whole blood using the QIAamp DNA Blood Mini Kit (Qiagen Cat. No. 51104) and eluted in 50 μL elution buffer.

[0211] PCR-based library preparation :A tube

[0212] High-fidelity PCR pre-amplification :

[0213] SuperFi DNA polymerase (ThermoScientific) was used to amplify the target sequence in genomic DNA in a 25 μl PCR reaction containing 1X SuperFi buffer, 0.2 mM dNTP, 100 nM Fwd / Rev PCR primer, 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.

[0214] SuperRCA assay :B tube

[0215] Primer and PCR polymerase cleanup :

[0216] 1 μL of each target from the PCR-based library preparation was mixed with 20 μL of a clean-up solution containing 1X SuperRCA buffer (RarityBioscience 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.

[0217] Ligase-mediated circularization of one strand of the PCR product :

[0218] 20 μl of ligation solution containing 1X SuperRCA buffer (Rarity Bioscience), 100 nM ligation template (complementary to both ends of one strand of the amplified product), 0.5 mM NAD (Sigma) and 2 U Ampligase (Lucigen) were added to the clean-up solution containing the amplified PCR product. The mixture was incubated at 95°C for 1 minute and then at 58°C for 30 minutes.

[0219] Target sequence amplification by first RCA:

[0220] The circular strand of the PCR product containing the target nucleotide site was amplified by RCA. 5 μl 1X SuperRCA buffer (Rarity Bioscience), 1.8 mM dNTP (Invitrogen) and 2.5 U Phi29 polymerase (New England Biolabs) were added to the circularized product. The reaction was incubated at 37°C for 30 minutes and then at 65°C for 10 minutes.

[0221] Genotyping of RCA products by padlock probe ligation:

[0222] The padlock probes were hybridized to the first-round 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.

[0223] Digestion of unreacted genotyping probes:

[0224] 5 μL of clean-up solution containing 1X SuperRCA buffer (Rarity Bioscience AB), 1.2 μM primers, and 1 U / μL Lambdaexo was added to the reaction mixture and incubated at 37°C for 15 min and then at 75°C for 20 min.

[0225] Templated secondary RCA by padlock probe binding to primary RCA product:

[0226] 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.

[0227] Digital documentation of SuperRCA products by flow cytometry:

[0228] The final reaction mixture containing the SuperRCA products was diluted into hybridization buffer containing 100 nM fluorophore-labeled oligonucleotide probes specific for the different SuperRCA products in 1X SuperRCA buffer (Rarity Bioscience) to a final volume of 250 μL. The solution was applied to a CytoFlex flow cytometer (Beckman Coulter) and the SuperRCA products were counted at a “medium” speed (30 μl / min) for 150 seconds per sample.

[0229] Example 1

[0230] The single-tube SuperRCA assay was used to analyze samples spiked with varying levels of the IDH2 p.R172K mutation, ranging from 1% to 0% (including wild-type samples). SuperRCA products were detected using a BD Fortessa flow cytometer ( Figure 2 ). For each sample with different abundance of IDH2 p.R172K mutation, the number of events detected by flow cytometry is shown in Tables 1 to 4 below. P3 is the probe that detects mutant events and P2 is the probe that detects wild-type events. Figure 2 As can be seen from the flow chart and the data in Tables 1 to 4, as the mutation incorporation level in the sample decreases from 1% to 0%, the P3 signal also decreases. Even at an extremely low level of only 0.001%, the SuperRCA assay can still detect the mutation signal ( Figure 2 C), which demonstrates the sensitivity of the assay.

[0231] Tables 1-4 list the raw data of flow cytometry readings, showing that the proportions of IDH2 R172K mutation were 1%, 0.064%, 0.001% and 0% (WT), respectively.

[0232] Table 1 Figure 2 A)

[0233]

[0234] Table 2 Figure 2 B)

[0235]

[0236] Table 3 Figure 2 C)

[0237]

[0238] Table 4 Figure 2 D)

[0239]

[0240] Example 2 Detection of ultra-low frequency AML point mutation IDH2p.R172K using SuperRCA or ddPCR detection

[0241] PCR amplicons targeting a DNA sequence containing the IDH2 p.R172K mutation were spiked into 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 spiked sample, and the SuperRCA products were analyzed using a Cytoflex flow cytometer. For the corresponding ddPCR assays, the same spiked DNA samples were used in the Bio-Rad ddPCR platform, 66 ng per replicate, and performed in duplicate. Figure 3 The results shown demonstrate that the SuperRCA assay has a lower limit of detection (LoD) and is able to successfully detect mutant DNA at up to 100,000-fold excess in normal DNA compared to ddPCR, which exhibits greater variability and is not sensitive enough to detect very low frequency mutations.

[0242] Example 3 Monitoring of AML patients with IDH2p.R140Q mutation using SuperRCA, NGS and ddPCR assays Disease progression

[0243] Samples from an AKL patient (UPN126) with the IDH2 p.R140Q mutation were analyzed at multiple time points using SuperRCA, NGS, and ddPCR assays to explore whether SuperRCA detection is suitable for monitoring disease progression ( Figure 4). Patient UPN126 was previously diagnosed with primary myelofibrosis associated with a JAK2 mutation, which had 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 NGSpanel and sequenced using the Illumina MiSeq platform, and the results showed the presence of the IDH2 p.R140Q mutation together with the previously known JAK2 mutation. In a retrospective analysis of the initial bone marrow sample during the myelofibrosis stage, both SuperRCA and ddPCR assays were negative for the IDH2 p.R140Q mutation, suggesting that clonal evolution occurred at the time of AML transformation. PCR amplicons targeting a DNA sequence containing the IDH2 p.R140Q mutation were applied to the patient DNA sample for the SuperRCA pre-amplification 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. For the corresponding ddPCR assay, the same DNA sample was used in the Bio-Rad ddPCR platform, 66 ng per replicate, and was performed in duplicate using the Bio-Rad pre-validated IDH2P.R 140QddPCR assay. The patient achieved CR after the first course of intensive chemotherapy but remained MRD positive (measured by flow cytometry of bone marrow cells with CD34, CD117, CD13, and DR+ markers) after 3 courses of intensive chemotherapy. An allogeneic SCT was performed on day 139. The patient was subsequently monitored by ddPCR and NGS analysis of serial blood and BM samples, and all six samples collected after SCT showed undetectable levels of IDH2 pR 140Q mutation by both detection methods, indicating that the patient is still in remission after more than two years of follow-up after SCT. A blood sample collected 3 months after SCT was positive by SuperRCA, but was below the detection limit of ddPCR ( Figure 4 ), demonstrating that the SuperRCA assay has a high sensitivity.

Claims

1. A method for detecting a target nucleic acid sequence in a target nucleic acid molecule, the method comprising: (a) providing a reaction mixture comprising an amplicon of a target nucleic acid sequence, an amplification polymerase, and an excess of amplification primers relative to the amplicon, wherein the amplicon is a single-stranded or double-stranded linear molecule and comprises a single copy of the target nucleic acid sequence; (b) reducing the excess primer in the reaction mixture in (a); (c) denaturing the double-stranded amplicon into a single-stranded amplicon if necessary; (d) contacting the reaction mixture with a ligation mixture comprising a ligase, and performing a ligation reaction to ligate the 5' and 3' ends of the amplicon, thereby circularizing the amplicon, wherein the ligation reaction is performed under conditions where the ability of the amplification polymerase to extend the 3' end of the hybridized amplicon on the ligation template is inhibited; (e) adding an RCA mix comprising one or more RCA reagents, the RCA reagents including at least an RCA polymerase, to the reaction mixture from (d) and performing a first RCA reaction using the circular amplicon as a first RCA template to produce a first RCA product (RCP), the first RCA product comprising a plurality of repeats of a complementary copy of the target nucleic acid sequence in the amplicon; (f) heating to inactivate the RCA polymerase; (g) contacting the first RCP with a padlock probe specific for the target nucleic acid sequence, allowing the padlock probe to hybridize to complementary copies in the plurality of repeats; (h) directly or indirectly ligating the hybridization padlock probe to circularize the hybridization padlock probe, wherein the ligation reaction is performed under conditions in which the ability of a polymerase to extend the 3' end of the hybridized padlock probe is inhibited; (i) removing unligated padlock probes from the reaction mixture of (h) or rendering them inert; (j) performing a second RCA reaction using the circularized padlock probe as a second RCA template to generate a second RCP containing multiple repeated complementary copies of the circularized padlock probe; wherein steps (a) to (j) are carried out in a single reaction vessel and the temperature is controlled during the steps; wherein steps (g) to (j) are optionally repeated one or more times; (k) Detecting the second or final RCP to detect the circularized padlock probe and thereby the target nucleic acid sequence.

2. The method according to claim 1, wherein: In step (b), excess primers are reduced by enzymatic digestion and / or by diluting the reaction mixture.

3. The method according to claim 1 or claim 2, wherein: The method comprises a preliminary step of performing an amplification reaction to produce amplicons of the target nucleic acid sequence.

4. The method according to any one of claims 1 to 3, wherein: The amplicon is or has been generated by PCR.

5. The method according to any one of claims 1 to 4, wherein: The method is for detecting variant target nucleic acid sequences in target nucleic acid molecules in a sample, and steps (g) to (k) comprise: (g) contacting the first RCP with two or more padlock probes, each probe comprising a target binding region specific for a different variant of the target nucleic acid sequence, and allowing the probes to hybridize to the complement of the target sequence in the plurality of repeats; (h) directly or indirectly ligating the padlock probe hybridized to its variant target sequence complement to circularize the hybridized padlock probe; (i) removing unligated padlock probes from the reaction mixture of (h) or rendering them inert; (j) performing a second RCA reaction using the circularized padlock probe as a second RCA template to generate a second RCP containing multiple repeated complementary copies of the circularized padlock probe; (k) detecting the second or final RCP to identify circularized padlock probes, thereby identifying variant target nucleic acid sequences.

6. The method according to any one of claims 1 to 5, wherein: The method is performed in a multiplexed fashion to detect multiple different target nucleic acid sequences in one or more target nucleic acid molecules.

7. The method according to any one of claims 1 to 6, wherein: Step (b) also includes enzymatically digesting the amplification polymerase.

8. The method according to any one of claims 2 to 7, wherein: Enzymatic digestion of the primers is performed by contacting the reaction mixture with an exonuclease or, in the case where the primers contain uracil residues, with uracil DNA glycosylase (UDG).

9. The method according to any one of claims 2 to 8, wherein: After the enzymatic digestion, the digestive enzymes are inactivated by enzymatic digestion or by heating.

10. The method according to any one of claims 7 to 9, wherein: Proteinase K is added to degrade the amplification polymerase and the exonuclease used, and wherein the method further comprises heating to inactivate the proteinase K.

11. The method according to any one of claims 1 to 4 and 6 to 10, wherein Step (b) comprises adding a volume of diluent to the reaction mixture to dilute it at least 200X.

12. The method according to any one of claims 1 to 11, wherein: In step (d), the reaction mixture is also contacted with a ligation template.

13. The method according to claim 12, wherein: In step (d), a two-step hybridization of the ligation template is performed to anneal the 5' end of the amplicon to the ligation template at a first temperature, and then the temperature is lowered to anneal the 3' end of the amplicon to the ligation template.

14. The method according to any one of claims 1 to 13, wherein: (i) in step (e), the RCA mixture comprises an RCA polymerase and dNTPs; and / or (ii) in step (e), a first RCA is initiated by said ligation template; and / or (iii) The ligase in step (d) is thermophilic.

15. The method according to claim 14, wherein in (iii), the denaturation in step (c) occurs simultaneously with or subsequent to the addition of the ligation mixture in step (d), and then the temperature for the ligation reaction is reduced; and / or wherein, The ligation in step (h) is performed by the ligase from step (d).

16. The method according to any one of claims 1 to 15, wherein: In step (g), a two-step hybridization of the padlock probe is performed to anneal the 5' end of the padlock probe to the first RCP at a first temperature, and then the temperature is lowered to anneal the 3' end of the padlock probe to the first RCP.

17. The method according to any one of claims 1 to 15, wherein in step (i): (a) removing unligated padlock probes by exonuclease enzymatic digestion, and the method further comprises heating to inactivate the exonuclease prior to the second RCA in step (j); or (b) Before starting the second RCA reaction, the unligated padlock probe is removed by enzymatic digestion with an RCA polymerase with 3' exonuclease activity.

18. The method according to any one of claims 1 to 4 and 6 to 17, wherein Step (i) comprises adding a volume of diluent to the reaction mixture to dilute it at least 4X.

19. The method according to any one of claims 1 to 18, wherein: The padlock probe is a hairpin-forming padlock probe comprising a hairpin loop structure comprising the 3' end of the padlock probe, and in step (i), the step of rendering the unligated and unhybridised padlock probe inert comprises extending the 3' end in the hairpin loop structure.

20. The method according to any one of claims 1 to 19, wherein: A detection reagent for detecting the second RCP is added to the reaction mixture together with the one or more RCA reagents, or wherein the detection reagent is added after the second RCP is produced.

21. The method according to any one of claims 1 to 20, wherein: The detection oligonucleotide hybridizes to the second RCP, and wherein, in step (k), the second RCP is detected by detecting the detection oligonucleotide.

22. The method according to claim 21, wherein: The detection oligonucleotide is labeled with a detectable tag.

23. The method according to any one of claims 1 to 22, wherein: The second RCP is detected by microscopy or by flow cytometry.

24. The method of any one of claims 1 to 23, wherein: The reagents added to the reaction mixture of step (a) are provided in the same buffer.

25. A kit for detecting a target nucleic acid sequence in a target nucleic acid molecule, the kit comprising: (i) one or more ligation templates comprising an amplicon binding region capable of hybridizing to a complementary binding site in an amplicon of a target nucleic acid sequence, thereby ligating the 3' and 5' ends of the amplicon; (ii) one or more padlock probes comprising a target binding region specific for a target nucleic acid sequence; and optionally one or more: (iii) exonucleases; (iv) proteinase K; (v) dilution buffer; (vi) ligase; (vii) RCA polymerase; (viii) RCA primer; (ix) dNTPs; (x) a detection oligonucleotide capable of hybridizing to an RCP generated using the circularized padlock probe as RCA template.

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