Method for detecting target nucleic acid sequence

Abstract

The present invention provides a method of detecting a target nucleic acid sequence in a target nucleic acid molecule using a padlock probe and rolling circle amplification (RCA) to produce a second generation of RCA products in a 2-stage RCA reaction, i.e., so-called super RCA (Super RCA, sRCA), by which the target nucleic acid sequence can be detected and distinguished from other nucleic acid sequences. The methods utilize asymmetric PCR technology and can be multiplexed to detect a plurality of different target nucleic acid sequences in one or more target nucleic acid molecules. Kits for use in the methods are also provided.

Description

Technical Field

[0001] The present disclosure and invention relate to the field of nucleic acid detection. In particular, the present disclosure and invention relate to a method for detecting a target nucleic acid sequence in a target nucleic acid molecule, wherein the method uses a padlock probe and rolling circle amplification (RCA) in a 2-stage RCA reaction, i.e., a so-called super RCA (SuperRCA, sRCA) to produce a second generation RCA product, by which the target nucleic acid sequence can be detected and distinguished from other nucleic acid sequences. The method utilizes asymmetric PCR technology, and can be multiplexed to detect multiple target nucleic acid sequences different 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 can be applied in many different areas, particularly clinically for personalized medicine and diagnosis, prognosis and / or treatment of diseases, such as cancer, infectious diseases and hereditary or genetic diseases, as well as in research and biosafety.

[0003] The target nucleic acid sequence can be easily detected using labeled hybridization probes, but simple hybridization probes have a relatively high detection limit and cannot be easily used to distinguish similar nucleic acid sequences. In order to improve sensitivity, the target nucleic acid molecules containing the target sequence can usually be amplified to increase the amount of the target sequence that can be used for detection. Any of a variety of techniques known in the art can be used for amplification, including PCR and RCA.

[0004] PCR is of course a well-known nucleic acid detection technique for exponential amplification using two primers to produce a linear amplification product. Although PCR is widely used in the context of nucleic acid detection, it also has the following disadvantages: the amplicon contains the forward and reverse strands of the target nucleic acid molecule, wherein only the forward strand contains the desired target nucleic acid sequence, so the amplicon must be denatured to separate the double strands and allow the probe to hybridize with the target nucleic acid sequence. In addition, during PCR, errors introduced by the polymerase will be amplified, and as the number of PCR cycles increases, the polymerase error rate also increases. Although this is tolerable in many applications, it is a special problem when PCR is used to detect variant sequences, such as mutations, especially those that differ only in individual nucleotides, especially when these variants are low frequency or low abundance.

[0005] Asymmetric PCR is a variant of PCR, and is also well known in the art, and is generally used to preferentially amplify one chain of a nucleic acid molecule rather than another chain. Primers are added in unequal molar ratios, such as 10: 1, to favor the synthesis of the desired chain. Therefore, compared with the primers of the non-desired chain (restricted primers), the primers of the desired chain are very excessive, and this imbalance leads to the amplification of two stages. The first stage is exponential, and similar to conventional PCR, an amplicon comprising two chains is produced. This initial stage ends after consuming the primers of the non-desired chain, followed by a second stage, which uses the excess primers of the chain to selectively amplify the desired chain, constituting the linear stage of asymmetric PCR. This stage has a lower reaction efficiency, because the melting temperature of the restrictive primer is reduced to below the optimal reaction annealing temperature due to its lower concentration, and additional circulation is required to produce an amplicon of reasonable yield.

[0006] Linear-After-The-Exponential (LATE)-PCR was developed to address the efficiency and yield issues faced by asymmetric PCR, and because the limiting primer concentration decreases in the middle of the reaction, it uses a limiting primer with a higher melting temperature than the excess primer to maintain the reaction efficiency. WO2016 / 184902 describes a method in which a linear asymmetric incremental polymerase reaction (AIPR) is followed by an exponential PCR. This is to improve the accuracy of detecting low-abundance mutant DNA sequences by reducing the polymerase error rate in the early cycles of PCR, thereby reducing false positive signals. The initial AIPR occurs at a high annealing temperature, while the subsequent exponential PCR occurs at a lower annealing temperature, and both stages are performed in a partitioned reaction such as droplet digital PCR.

[0007] RCA uses 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 the amplification template. Such concatemers typically form a ball or "spot" that can be easily visualized and detected, and therefore RCA-based assays have been used for the detection of nucleic acids and, indeed, more generally as a reporter system for the detection of any target analyte. Both target nucleic acid analytes (which themselves can be directly circularized), probes or reporter nucleic acids can provide template nucleic acid circles for RCA.

[0008] The specificity of nucleic acid detection methods can be improved by using probes that require dual recognition or have two binding sites for the target nucleic acid sequence (e.g., padlock probes). Padlock probes are usually linear oligonucleotides with two independent target complementary binding regions connected by a middle "backbone" region. After the probe binds (hybridizes) to the target nucleic acid sequence, the two ends of the probe can be connected together to circularize the probe, with single nucleotide identification specificity within 6bp around the connection site. The circularized 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 assays currently in use. Therefore, padlock probes provide an additional layer of specificity because only probes that are correctly base paired at the connection site will be connected to produce a template for the molecule to be detected.

[0009] RCA-based detection has been described, which relies on secondary amplification of the initial RCA product to increase the amount of the detected product, thereby providing amplification of the signal in the detection. These include, for example, hyperbranched RCA, which produces many unclustered subsequent RCA products through strand displacement activity. Recently, the so-called "super RCA" (superRCA, sRCA) reaction has been developed, which includes two or more rounds of RCA amplification, in which the product of the second RCA reaction is connected to the product of the first RCA. A version of this sRCA method is described in WO2014 / 0796209. In this method, a probe that can provide a primer or a primer is hybridized with the initial RCA product and is used to initiate the amplification of the second RCA template ring hybridized with the "primer-probe". The second RCA template can be generated by the 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 the padlock probe is used to directly bind to the initial RCA product.

[0010] In many applications, the nucleic acid sequence to be detected is present at low levels, for example in rare mutations, or in cell-free DNA (cfDNA) in plasma or other clinical samples, or when limited sample amounts are available. In this case, very sensitive detection methods are required, in particular methods that enable high amplification of the target nucleic acid. PCT / EP2021 / 084061 describes an 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, which serve as the first RCA template for the sRCA reaction.

[0011] Given the ubiquity and convenience of PCR as an amplification method, we have been working to provide a robust and easily automated assay that combines PCR and sRCA for the detection of rare variants. However, the polymerase error rate in the PCR step remains an issue that needs to be addressed, as it negatively impacts the accuracy of the assay overall, for example, when a large number of exponential PCR cycles are performed to generate circular amplification templates for the RCA reaction. In addition, the undesired strands of the amplicon reduce the efficiency of the assay by competing with the desired strand to generate the RCA template.

[0012] There is a continuing need for further optimized assays with improved efficiency and sensitivity. The present disclosure and invention aim to provide such a method.

[0013] Brief description

[0014] In the present method, asymmetric PCR is used to generate the amplicon of the target nucleic acid sequence for subsequent superRCA (sRCA) reaction. The asymmetric PCR step is improved using primers with different melting temperatures, which include an exponential PCR stage at a first annealing temperature and a linear amplification stage at a second higher annealing temperature to amplify the desired chain. These two stages can be carried out in either order of the first exponential stage or the second exponential stage. In order to improve the accuracy of the present method, a low number of amplification cycles is maintained to reduce the occurrence of polymerase errors, particularly to maintain a low number of exponential cycles. The amplicon of the desired chain is connected into a ring, and then amplified by RCA to generate a first RCA product containing a plurality of repeated complementary copies of the target sequence. The first RCA product obtained is then detected with a padlock probe specific to the complementary copy of the target sequence. A further second RCA reaction is carried out using a circular padlock probe to generate a second (i.e., secondary) RCA product, which is detected to detect the target sequence.

[0015] The use of a low number of exponential PCR cycles, e.g., no more than 12, can reduce the accumulation of polymerase errors in the amplicon and improve the accuracy of the assay results by reducing the number of false positives or false negatives. In addition, when the linear phase is located after the exponential phase, the accumulation of single-stranded amplicons of the desired strand after the linear amplification phase makes denaturation of the double-stranded amplicons optional to allow hybridization with the ligation template or access to the ligase. In addition, the low yield of the undesired strand improves the efficiency of the amplicon ligation step due to reduced competition with the desired strand for the ligation template or ligase.

[0016] Therefore, the first aspect of the present invention provides a method for detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, the method comprising:

[0017] (a) performing an asymmetric PCR reaction using a set of primers to produce an amplicon of a target sequence, the primers 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, wherein the asymmetric PCR reaction comprises, in either order:

[0018] (i) an exponential PCR phase, comprising no more than 12 cycles, in which the primers are PCR reacted using the first and second primers.

[0019] annealing at a first annealing temperature; and

[0020] (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 only one strand is amplified, thereby preferentially accumulating (or in other words, amplifying) single-stranded amplicons of the target nucleic acid sequence;

[0022] (b) contacting the single-stranded amplicon from step (a) with a ligation mixture comprising a ligase and ligating the 5' and 3' ends of the amplicon to circularize the single-stranded amplicon;

[0023] (c) performing a first RCA reaction using the circularized amplicon as a first RCA template to produce a first RCA product (RCP) comprising a plurality of repeats of complementary copies of the target nucleic acid sequence in the amplicon;

[0024] (d) contacting the first RCP with a padlock probe specific for the target nucleic acid sequence and allowing the probe to hybridize to complementary copies of the plurality of repeats;

[0025] (e) directly or indirectly ligating the hybridized padlock probe to circularize the hybridized padlock probe;

[0026] (f) performing a second RCA reaction using the circularized padlock probe as a second RCA template to produce a second RCP comprising a plurality of repeated complementary copies of the circularized padlock probe;

[0027] wherein steps (d) to (f) are optionally repeated one or more times; and

[0028] (g) Detecting the second or final RCP to detect the circularized padlock probe and thereby the target nucleic acid sequence.

[0029] The second RCP (also referred to as secondary RCP) forms a superRCA (sRCA) product with the first RCP to which they are attached, which is the product detected for analysis. Similarly, if the padlock detection and RCA steps are repeated, more generations of RCP (until the last round of RCP (the "final RCP")) will be attached to the previous round of RCP, together forming the sRCA product.

[0030] In one embodiment, the exponential phase PCR reaction (i) is first performed, followed by a linear amplification reaction (ii). In such an 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.

[0031] It should be understood that the desired strand that is preferentially amplified and circularized into the first RCA template can be selected based on the corresponding strands to which the first and second primers bind. Therefore, the target sequence can be preferentially amplified in either direction based on the selection. Next, therefore, the target nucleic acid sequence mentioned in the present invention includes a sequence that is complementary to the target nucleic acid sequence.

[0032] Thus, in step (d), the padlock probe is hybridized to a plurality of repeated complementary copies of the target sequence. In this case, the complement of the target sequence bound by the padlock probe in the first RCP is the complement of the target sequence present in the amplicon. In this regard, the term "complement" is synonymous with "complementary sequence" or "complementary copy".

[0033] It will also be appreciated that the single-stranded amplicon in step (b) is the preferentially amplified strand.

[0034] In one embodiment, step (b) is a template-directed ligation, and the contacting of step (b) comprises contacting the amplicon with a ligation template. Conveniently, the ligation template is included in the ligation reaction mixture. However, this is not absolutely necessary, and in one embodiment, a template-free ligation may be performed. This method uses a ligase that does not require a ligation template, such as CircLigase TM Enzyme.

[0035] The target nucleic acid sequence may be an analyte for detection by the method, or a reporter for an analyte detected by the method.

[0036] The above method can be performed in a multiplex manner to detect multiple different target nucleic acid sequences that may be present in one or more nucleic acid molecules, wherein in step (a), multiple asymmetric PCR reactions are performed using different primer sets to produce amplicons of multiple different target nucleic acid molecules or amplicons of multiple different target nucleic acid sequences, wherein the multiple PCR reactions are performed individually in parallel or in multiplex together.

[0037] In one embodiment, prior to step (b), amplicons from multiple separate asymmetric PCR reactions are pooled. In another embodiment, multiple asymmetric PCR reactions are performed together in the same reaction mixture.

[0038] Said method can be used for detecting the variant target nucleic acid sequence in the target nucleic acid molecule.The target nucleic acid sequence can usually appear in the form of variants, such as allelic variants, or mutants and wild-type sequences, and may need to detect which variant exists.This shows that the target nucleic acid sequence can be one of the many different variants that the nucleic acid sequence may appear.

[0039] Thus, in one embodiment, the method is for detecting a variant target nucleic acid sequence in a target nucleic acid molecule in a sample, and steps (d) to (g) comprise:

[0040] (d) contacting the first RCP with two or more padlock probes, each padlock 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 plurality of repeats of the target sequence;

[0041] (e) directly or indirectly ligating a padlock probe that hybridizes to the complement of a variant target sequence (i.e., hybridizes to the complement of a variant sequence that serves as a target or corresponds to the padlock probe) to circularize the hybridized padlock probe;

[0042] (f) performing a second RCA reaction using the circularized padlock probe as a second RCA template to produce a second RCP comprising a plurality of repeated complementary copies of the circularized padlock probe;

[0043] (g) detecting the final (eg, second) RCP to detect the circularized padlock probe, thereby detecting the variant target nucleic acid sequence.

[0044] In step (e), correctly hybridized padlock probes, ie, those that correctly and specifically hybridize to the complement of the variant target sequence they are designed to detect (ie, padlock probes that hybridize to the complement of their respective target sequence), are ligated.

[0045] The method can be used for the detection of DNA or RNA and can be performed in homologous or heterologous form. It can be used for detecting target nucleic acid sequences in in situ samples or in separated form samples or liquid samples.

[0046] Advantageously, the above method can be carried out in a single temperature-controlled reaction vessel, the method comprising:

[0047] (i) providing a reaction mixture comprising the single-stranded amplicon from step (a), wherein the reaction mixture comprises a PCR polymerase and an excess of PCR primers relative to the amplicon,

[0048] (ii) reducing excess primers from the reaction mixture of (i) and / or removing primer sequences from the amplicon produced by (i);

[0049] (iii) 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 to circularize the amplicon, wherein the ligation reaction is performed under conditions where the ability of the PCR polymerase to extend the hybridized 3' end of the amplicon on the ligation template is inhibited;

[0050] (iv) adding an RCA mix comprising one or more RCA reagents, said RCA reagents comprising at least an RCA polymerase, to the reaction mixture from (iii) and performing a first RCA reaction using the circularised amplicon as a first RCA template to produce a first RCA product (RCP) comprising complementary copies of a plurality of repeats of the target nucleic acid sequence in the amplicon;

[0051] (v) heating to inactivate the RCA polymerase;

[0052] (vi) contacting the first RCP with a padlock probe specific for the target nucleic acid sequence and hybridizing the padlock probe to the complement of the target sequence in the plurality of repeats;

[0053] (vii) 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 hybridized 3' end of the padlock probe is inhibited;

[0054] (viii) removing or rendering inert any unattached padlock probes in the reaction mixture of (vii);

[0055] (ix) performing a second RCA reaction using the circularized padlock probe as a second RCA template to generate a second RCP comprising a plurality of repeated complementary copies of the circularized padlock probe;

[0056] wherein steps (i) to (ix) are carried out in a single reaction vessel and the temperature is controlled during the steps; wherein steps (vi) to (ix) are optionally repeated one or more times; and

[0057] (x) detecting the second or final RCP to detect the circularized padlock probe and thereby the target nucleic acid sequence.

[0058] In the above method, the padlock probe may be:

[0059] (i) a 1-part padlock probe in the form of a single circularisable oligonucleotide, the 5' and 3' ends of which contain target-binding regions; or

[0060] (ii) a 2-part padlock probe comprising a first oligonucleotide having a first binding region complementary to the target at its 3' end and a second binding region complementary to the ligation template at its 5' end; and a second oligonucleotide having a first binding region complementary to the target at its 5' end and a second binding region complementary to the ligation template at its 3' end; wherein the 3' and 5' ends of the first and second oligonucleotides are linked together, with the target sequence in the first RCP, and the ligation template as templates, respectively.

[0061] In a second aspect, a kit for detecting a target nucleic acid sequence in a target nucleic acid molecule is provided, the kit comprising:

[0062] (i) a primer set for an asymmetric PCR reaction, wherein the primers are capable of amplifying a target nucleic acid sequence and comprise a first primer having a first melting temperature (Tm) and a second primer having a second Tm that is at least 10° C. lower than the first Tm; and

[0063] (ii) a padlock probe comprising a target binding region specific for a target nucleic acid sequence; and optionally,

[0064] (iii) a ligation template comprising binding regions at or near the 5' and 3' ends that are capable of hybridizing to complementary binding sites in an amplicon of the target nucleic acid sequence;

[0065] In one embodiment, the kit is used to detect multiple different target nucleic acid sequences in one or more target nucleic acid molecules, and comprises multiple different primer sets (i) and padlock probes (ii), each padlock probe being specific to a different target nucleic acid sequence, and an optional ligation template (iii).

[0066] In another embodiment, the kit is for detecting different variants of a target nucleic acid sequence and comprises two or more padlock probes, each probe being specific for a different variant of the target sequence.

[0067] Detailed description

[0068] The present method provides a high fidelity and high sensitivity method for detecting a specific nucleotide sequence (the terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein). The method is particularly suitable for detecting variants of a target sequence that may be present in a sample, such as a mutant sequence. In particular, rare target sequences or sequence variants, or sequences or variants that are present in low abundance or at low levels, can be detected.

[0069] The method combines a primary amplification of the target sequence by an asymmetric PCR step with a sRCA reaction to produce a sRCA product (sRCP) comprising at least a first and a second RCP, which is detected to detect the target sequence.

[0070] Therefore, in the first step, the amplicon of the target nucleic acid sequence present in the target nucleic acid molecule in the sample is produced by asymmetric PCR. These amplicons include double-stranded amplicons produced in exponential PCR reactions (e.g., in the first stage, or the first reaction of asymmetric PCR), and single-stranded amplicons of the desired chain containing the target nucleic acid sequence produced in linear amplification reactions (e.g., in the second stage or the second reaction of the asymmetric PCR step). The number of cycles of the exponential PCR reaction remains low, no more than 12 times, particularly no more than 11 or 10 times. Therefore, the single-stranded amplicons of the desired chain are dominant. If necessary, the double-stranded amplicons can be denatured to release the desired single chain. The single-stranded amplicons are cyclized by connection, and RCA is performed to produce RCA products containing multiple repeated series of complementary copies of the target sequence. These amplified target sequences are then detected with target sequence-specific padlock probes, which, after hybridization with the target sequence-complementary sequence, are cyclized in sequence (forming the second RCA template, also referred to as the secondary RCA template) and amplified by RCA to produce the second (or secondary) RCA product, by which the target sequence can be detected.

[0071] The generation of RCA products, particularly sRCA products, provides a signal that is easily detected and counted. A digital count readout can be achieved. This allows digital detection of the reaction product of each detected target molecule. In addition, the product of the second (or further) RCA reaction is a large product that contains multiple (hundreds, and possibly up to 1000 or so) copies of the complement of the target sequence and has a significant size. This significant reaction product can be easily collected with minimal risk of mixing with any other material in the reaction.

[0072] The present method provides a high level of amplification of the target sequence, by which the sensitivity of the detection method is increased, enabling the detection and identification of 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 allows for accurate quantification of target nucleic acid sequences. For example, the ratio of different target nucleic acid sequences can be determined with high accuracy, such as in the case of determining chromosome copy number, where target sequences from 2 different chromosomes can be detected and compared. For example, in non-invasive prenatal testing (NIPT), this may be particularly useful for detecting trisomy, such as chromosome 21. The basic principle of the detection method is as follows Figure 1 shown.

[0073] As mentioned above, the use of asymmetric PCR to detect target nucleic acid sequences is known in the art, and various forms of asymmetric PCR protocols are known. In order to optimize the SuperRCA protocol, the present method provides a circularized RCA template from a specific asymmetric PCR protocol for the subsequent 2-stage RCA reaction. It is noteworthy that by including an asymmetric PCR step, the sensitivity of the method is increased due to the provision of high-yield amplicons of the target nucleic acid sequence, but the negative impact of polymerase errors on the accuracy of the method is limited.

[0074] In the asymmetric PCR protocol used in the method of the present invention, two PCR primers are designed to have different annealing temperatures I, and temperature control is used during the method to control which primers anneal and are therefore able to initiate 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 primer with the higher Tm anneals. An increase or decrease in temperature is used to switch between phases, depending on which phase is performed first. As described above, the two phases (i) and (ii) can be performed in either order, i.e., first the exponential phase (i) and then the linear phase (ii); or in an alternative embodiment, the linear phase (ii) is performed first, followed by the exponential phase (i).

[0075] In a preferred embodiment of the method, a first exponential reaction is first performed using both primers, and then a second linear amplification reaction is performed using only the first primer.

[0076] In reaction (i) of step (a), both primers are extended and both chains are amplified (e.g., a chain containing the target sequence and its complement (complementary chain)). In reaction (ii) of step (a), only the first primer is extended, and thus only one chain is amplified (e.g., only the chain containing the target sequence is amplified).

[0077] In other words, in a linear amplification reaction, there is unidirectional replication (in a single direction only), as opposed to bidirectional replication in an exponential reaction. In a linear amplification reaction, the copies synthesized are the complement of the sequence (or strand) to which the first primer anneals, and therefore the copies synthesized cannot serve as templates for further amplification. Thus, for each thermal cycle, only one complementary copy is synthesized per original single-stranded template.

[0078] The specificity of the method is also increased by reducing the number of exponential PCR cycles. In the present method, a small amount of exponential PCR cycles are performed in the exponential (e.g., initial) phase of the asymmetric PCR step, specifically no more than 12, or particularly no more than 11 or 10 cycles, which has the effect of reducing the accumulation of polymerase errors in the amplicon, reducing the ratio of false positives and false negatives in the assay results. In fact, in some embodiments, the number of exponential cycles may be no more than 9, 8, or 7 cycles. For example, 6 cycles may be performed.

[0079] In addition, the efficiency of the method can be improved because the double-stranded amplicon does not need to be denatured to generate a single strand for the ligation step. In one embodiment of the method, the second stage of the asymmetric PCR step includes a linear amplification reaction to generate a single-stranded amplicon of the target nucleic acid sequence, which can be circularized in the ligation step without the need for a further denaturation step.

[0080] The efficiency of the method is also improved by reducing the competition between the complementary strand and the target strand in the ligation step. In the present method, when the denaturation step is not performed, the complementary strand of the double-stranded amplicon remains hybridized with the desired strand and is less likely to compete with the single-stranded amplicon for the ligation template or ligase. In one embodiment, a denaturation step may be performed. However, in the present method, the accumulation of high-yield single-stranded amplicon advantageously exceeds the complementary strand of a relatively low number, so the efficiency of the method is maintained to a great extent.

[0081] In the present method, unlike conventional asymmetric PCR (e.g., LATE-PCR), the asymmetric PCR step does not rely on the use of a limiting concentration of one primer. Therefore, the asymmetric PCR step can use substantially equal proportions of the first and second primers, further improving the efficiency and simplicity of the method. To achieve this, the first primer has a higher melting temperature (Tm) than the second primer, and the second primer binds to a complementary strand (i.e., a strand that is complementary to the strand that the first primer binds to, or an extension product of the first primer).

[0082] In one embodiment of the method, the annealing temperature of the first exponential PCR phase is lower than the Tm of the two primers, so that the two primers can anneal. After, for example, no more than 12 (or no more than 11 or 10) exponential PCR cycles, the annealing temperature is increased to above the Tm of the second primer, but below the Tm of the first primer, to initiate a second linear amplification phase, wherein only the first primer is allowed to anneal, thereby generating only single-stranded amplicons of the desired strand.

[0083] Similarly, when linear amplification is performed first, the annealing temperature in the first stage is higher than the Tm of the second primer and lower than the Tm of the first primer, so that only the first primer can anneal. After a selected number of linear cycles, the annealing temperature is lowered to below the Tm of both primers, so that they are all annealed, and an exponential PCR reaction is performed, not exceeding 12 cycles.

[0084] However, as discussed further below, the annealing temperature need not be lower than the Tm of the primers in order to anneal the primers, as is indeed shown in Example 1 below. Thus, the annealing temperature may be several degrees (e.g., 1-7° C.) higher than the Tm of the primers to be annealed. It is important to use the difference in the two annealing temperatures to distinguish the annealing of the primers and the difference between the two stages.

[0085] It should be understood that the exponential PCR reaction will produce a double-stranded amplicon, wherein the second strand comprises a complementary copy (i.e., a reverse complement) in the opposite direction of the first strand sequence. The second linear amplification reaction will only produce a copy of one of the two strands, depending on which of the two strands is annealed by the first higher Tm primer. Therefore, according to primer design, one or the other strand can be preferentially selected for amplification, such as the sense strand (or positive (+) strand) or the antisense strand (or negative (-) strand). Therefore, as described above, the term "target nucleic acid sequence" includes two directions of the sequence, such as sense and antisense sequences. Therefore, when referring to the target nucleic acid sequence, the complement of the target sequence can be appropriately included according to the context, i.e., they can include a sequence complementary to the target sequence.

[0086] In one embodiment, the first primer is in conjunction with the complement of the chain comprising the target nucleic acid sequence, and the second primer is in conjunction with the chain comprising the target nucleic acid sequence.Therefore, the extension product of the first primer contains a copy of the target nucleic acid sequence.Usually but not necessarily, in the case of genomic sequence, the target nucleic acid sequence will be its sense strand of the target nucleic acid molecule, or the sequence that occurs in 5' to 3' chain.Therefore, in one embodiment, the target nucleic acid sequence can be in the first chain (or in other words, 5' to 3' chain) of a double-stranded nucleic acid molecule, and the first primer can be in conjunction with the second chain (or in other words, 3' to 5' chain).

[0087] In one embodiment of the method, unlike the AIPR method, an exponential PCR reaction is performed first, followed by a linear amplification reaction.

[0088] In the method, no matter how the order of exponential phase and linear phase is, primers can be advantageously provided in the amplification reaction mixture with similar or substantially the same concentration. In one embodiment, PCR primers are provided with the same concentration range or the same concentration. In one embodiment, two primers are provided with a concentration that will not be exhausted in the asymmetric PCR reaction. In other words, two primers are kept excessive during the asymmetric PCR step. In other words, both primers are not used with limiting concentrations. In other words, both primers are used with non-limiting concentrations. Conveniently, PCR primers can be used with the typical concentration of conventional or exponential PCR reactions, such as 100-1000nM.

[0089] Advantageously, the asymmetric PCR step can be performed by thermal cycling to maximize efficiency. This can be accomplished by procedures well known in the art. Therefore, in one embodiment, the method can include a heating step to denature the target nucleic acid molecule, and then the temperature can be lowered to allow the first and second primers to anneal (bind or hybridize) to their complementary binding sites in the target molecule, and the annealed primers are subsequently extended by the PCR polymerase. This may include further lowering and / or raising the temperature to optimize the conditions for primer extension, depending on the PCR polymerase used. The reaction is then heated again to denature the double-stranded molecule, and the cycle is started again, i.e., further primer annealing and extension are allowed.

[0090] In the reverse order, linear amplification is performed first, and after initial denaturation, the temperature is lowered to a temperature that allows only the first primer to anneal, and then the first primer is extended and the cycle is repeated, similar to the above method.

[0091] The number of exponential cycles can vary according to selection, and can depend on the properties of target nucleic acid, sample, etc. As long as no more than 12 cycles are performed, for example, 4, 5, 6, 7, 8, 9, 10, 11 or 12 cycles are performed. Therefore, in an embodiment, no more than 11, 10, 9 or 8 cycles can be performed, for example, from any one of 4, 5 or 6 to any one of 8, 9, 10, 11 or 12. This is much less than conventional PCR reactions. It has been observed that the accumulation of products is proportional to the number of repeated cycles. This results in an increase in the production of double-stranded amplicons of the target sequence, and therefore it is necessary to perform fewer linear amplification cycles to produce single-stranded amplicons of reasonable yield. The higher yield of double-stranded amplicons results in increased efficiency and sensitivity of the detection method, but due to the accumulation of polymerase errors, specificity is reduced. If necessary or suitable, more or less exponential cycles can be performed, and the number of linear amplification cycles can be adjusted accordingly. Considering the impact of sample size and cycle number on the efficiency of the method and the accuracy of the results, those skilled in the art have the ability to determine how many exponential and linear cycles are needed. In one embodiment, at least 7 cycles of linear amplification are performed, for example 7, 8, 9, 10, 11 or 12 cycles are performed. As described above, more or fewer cycles of linear amplification may be performed if necessary or appropriate.

[0092] The method can be advantageously automated. Thus, in one embodiment, the method is designed to allow a 2-stage RCA reaction (i.e., sRCA) to be performed on a single-stranded amplicon of a target nucleic acid sequence in a single reaction vessel without any manual input, and can be extended for commercial applications in a robotic workflow.

[0093] Most generally, the above method can be used to detect the target nucleic acid sequence in the target nucleic acid molecule. The term "detection" is widely used in this article, including 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 amount of the sRCA product (e.g., the second RCA product) produced, and may include simply detecting whether it exists or any form of measurement of the CA product. In this regard, it should be understood that by detecting the second or additional or final RCP, because it is attached to the first or previous RCP, the entire sRCA product is actually detected. Therefore, the RCA product of step (f) can be detected as a "signal" of the target nucleic acid sequence. Therefore, detecting the second (or additional) RCA product in step (g) includes determining, measuring, evaluating or assaying the presence or absence or quantity or position of the second (or additional) RCA product in any way. The presence of the second (or further) RCA product (i.e., confirmation of its presence or amount) indicates or identifies the presence of the target nucleic acid sequence, because the successful generation of the RCA product ultimately depends on the presence of the target nucleic acid molecule, more specifically on the presence of the target nucleic acid sequence therein.

[0094] Including quantitative and qualitative determination, measurement or evaluation, including semi-quantitative. Such determination, measurement or evaluation can be relative, for example, when detecting two or more different target nucleic acid sequences or target molecules in a sample, it can also be absolute. Therefore, in one embodiment, the method can be used to quantify or determine the amount of target nucleic acid sequences present. The term "quantitative" can refer to absolute or relative quantification when used to quantify the target nucleic acid sequence in a sample. Absolute quantification can be achieved by adding one or more control nucleic acid molecules of known concentration and / or by referring to the detected target nucleic acid sequence level with known control nucleic acid molecules or sequences (for example, by generating a standard curve). Alternatively, relative quantification can be achieved by comparing the detection level or amount between two or more different target nucleic acid molecules or different target sequences, so as to provide relative quantification of each of two or more different nucleic acid molecules or sequences, i.e. relative quantification 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 (e.g., chromosome) can be compared.

[0095] The target nucleic acid sequence is a sequence in any nucleic acid molecule that is desired to be detected or identified, or in other words, a target for determination. 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.

[0096] Typically, the target sequence is an analyte to be detected, such as a nucleic acid present in a sample, such as a cell or tissue sample or any biological sample, etc. Therefore, the target sequence can be a naturally occurring sequence, or a derivative, copy or amplicon thereof. However, this is not necessary, and the target sequence can alternatively be a reporter of the analyte to be detected. The reporter nucleic acid can be used or generated in the process of detecting any analyte, such as a protein or other biomolecule or small molecule in a sample. Therefore, the reporter nucleic acid can be provided as a label or marker for an analyte-binding probe, and can be detected to detect the analyte, such as in an immunoassay, such as in an immunoPCR (immunoPCR) or an immunoRCA (immunoRCA) reaction. The reporter nucleic acid can be generated during the detection process, for example, by a ligation reaction in a proximity ligation detection, or an extension reaction in a proximity extension determination, or by a cleavage reaction, etc. Therefore, such a reporter target nucleic acid can be a synthetic or artificial sequence.

[0097] 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 sub-portion 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 sub-portion thereof, or an amplicon or a copy thereof, etc.

[0098] In another embodiment, the target nucleic acid molecule is a target RNA molecule. It can be RNA or other nucleic acid molecules in a nucleic acid molecule pool, such as genomic nucleic acid, whether human or from any source, from a transcriptome, or any other nucleic acid (e.g., organelle nucleic acid, i.e., mitochondrial or plastid nucleic acid), whether naturally occurring or synthetic. Therefore, the target RNA molecule can be or can be derived from a coding (i.e., 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 (micro RNA, miRNA). In another embodiment, the target RNA molecule is 16S RNA, for example, wherein 16S RNA comes from and identifies a microorganism (e.g., a pathogenic microorganism) in a sample. Alternatively, the target RNA molecule can be a genomic RNA, such as ssRNA or dsRNA of a virus with RNA as its genetic material. These viruses mainly include Ebola virus, HIV, SARS, SARS-CoV2, influenza virus, hepatitis C virus, West Nile virus, polio virus, and measles virus. 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.

[0099] When the target molecule is an RNA molecule, the method may include an initial step of generating a cDNA copy of the target RNA molecule. The cDNA molecule is then amplified.

[0100] An asymmetric PCR reaction is performed using a set of primers including 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 to generate an amplicon of the target sequence.

[0101] The term "amplicon" as used herein is defined as the amplification product of an asymmetric PCR reaction. The amplicon used in the present invention generally refers to a single-stranded amplicon produced in the linear amplification stage, which is ligated to the first RCA template for the SuperRCA reaction, but may also include a double-stranded amplicon produced in the exponential PCR stage, depending on the context and where appropriate.

[0102] A pair of PCR primers can be expressed as forward and reverse primers, wherein the forward primer is combined with the complementary strand of the target sequence to be amplified. Therefore, in one embodiment of the present method, the first primer (forward primer) has a sequence complementary to the second strand (negative strand) and therefore combines (anneals) with it, and then extends the first primer to produce a copy of the desired first strand (positive strand). The second strand can also be referred to as the antisense strand or template strand, which is the complement of the first strand, and vice versa. The second primer (reverse primer) has a sequence complementary to the first strand, and therefore combines (anneals) with the first strand, and then extends to produce a copy of the second strand. The first strand can also be referred to as the desired strand, sense strand or non-template strand. Alternatively, the first primer anneals with the second strand and extends on the second strand to produce the amplicon of the desired first strand, while the second primer anneals with the first strand and extends on the first strand to produce the complement of the desired strand. The first primer also anneals with the extension product of the second primer and the complement of the second strand, and the second primer also anneals with the extension product of the first primer and the complement of the first strand. Thus, in such an embodiment, the first primer may be referred to as a negative strand primer and the second primer may be referred to as a positive strand primer. Similarly, in another embodiment, the first primer may be a reverse primer, etc. (i.e., it may bind to the first strand (i.e., the positive strand / 5' to 3' strand), etc.).

[0103] The melting temperature of a primer is the temperature at which half of the primers are bound to the nucleic acid strand. In other words, it is the temperature at which equilibrium is reached between bound and unbound primers. Depending on various factors of primer design, primers may have different Tms. This may include, among other things, the nucleotide composition and length of the primer. Thus, a primer may be designed to have sequence mismatches, or it may contain modified nucleotides, such as locked DNA (LNA) bases, or it may be modified by the addition of a minor groove binder.

[0104] The primer set used in the method includes a first primer whose Tm is higher than the second primer, specifically at least 10°C higher than the second primer. Alternatively, the first primer has a first Tm and the second primer has a second Tm that is at least 10°C lower than the first Tm. In one embodiment, the second Tm is at least 11, 12, 13, 14, 15, 20, 25 or 30°C lower than the first Tm. The first primer may have a first Tm of 60-72°C, such as 65-70°C, such as 65, 66, 67, 68, 69 or 70°C. In one embodiment, the first primer may have a first Tm of 67°C. The second primer may have a second Tm of 50-62°C, such as 54-60°C, such as 54, 55, 56, 57, 58, 59 or 60°C. In one embodiment, the second primer may have a second Tm of 50°C or 55°C.

[0105] Due to the difference of primer Tm, different annealing temperatures can be used to control the amplification of target sequence. The annealing temperature of PCR reaction is the temperature at which primer is combined with or annealed to nucleic acid chain. Specifically, considering the second lower Tm, the first annealing temperature can be used so that two primers are annealed in the exponential PCR stage, thereby causing two primers to extend on their respective chains and produce double-stranded amplicon. In other words, both chains of target nucleic acid molecule are amplified. In other words, the first chain and its complement and the second chain and its complement are amplified. The second annealing temperature can be used to anneal only the primer (first primer) with a higher first Tm to the desired chain, such as template (or antisense) chain, to produce the single-stranded amplicon of the desired nucleic acid sequence. Therefore, for example, in the linear amplification stage of asymmetric PCR method, the first primer with a higher first Tm anneals to the template (second) chain to amplify the desired (first) chain. In other words, in the linear amplification stage, only the first chain is amplified.

[0106] The annealing temperature of one or more primers can be the same as the Tm of the primer to be annealed or the primer with the lowest Tm. However, as understood in the art, it can be different. For example, the annealing temperature can be 4-10°C higher or lower than the primer Tm, for example 4-10°C higher, or 4-8°C higher, for example 4, 5, 6, 7 or 8°C higher. For example, the first annealing temperature can be 5-7°C higher than the second Tm of the second primer, for example 6°C higher, and the second annealing temperature can be 6-8°C higher than the first Tm of the first primer, for example 7°C higher.

[0107] In order to anneal the primers in each PCR cycle, any double-stranded nucleic acid molecule must first be denatured. Therefore, when the target nucleic acid molecule appears or is provided in a double-stranded form at the beginning of each cycle, the temperature is raised to at least 90°C, such as 90-100°C, or 94-98°C, such as 98°C, but the appropriate denaturation temperature can vary according to the Tm of the double-stranded nucleic acid molecule. The Tm of the molecule is affected by factors such as size, complexity, and GC content, and the appropriate denaturation temperature can be determined by those skilled in the art. The denaturation of the double-stranded nucleic acid molecule separates the chain into a single-stranded nucleic acid molecule to which the primer can anneal. The denaturation step can be carried out for at least 10 seconds to 3 minutes, such as 10 seconds to 1 minute, such as 15 seconds to 30 seconds. In one embodiment, the initial denaturation step of separating the double-stranded target nucleic acid molecule is carried out for 30 seconds, and the subsequent denaturation step of separating the extension product from the original chain or denaturing the amplicon is carried out for 15 seconds.

[0108] After the denaturation step of the PCR cycle, the temperature can be fully reduced as described above so that the first and second primers for exponential reaction are annealed, or the first primer that is only used for linear amplification reaction is annealed. Once the primers are annealed, the temperature can be maintained so that the primers are extended by the PCR polymerase in the primer extension step of the PCR cycle. Or, the extension temperature can be different from the annealing temperature, for example, higher than the annealing temperature. In one embodiment, the extension temperature is the same as the annealing temperature. In another embodiment, the extension temperature is different from the annealing temperature. The annealing and extension steps of the PCR cycle can last at least 60-180 seconds, for example 100-140 seconds, for example 120 seconds in total. In one embodiment, the total annealing and extension time in each cycle is 120 seconds.

[0109] Thus, the asymmetric PCR reaction of step (a) may include contacting the sample (or more specifically the target nucleic acid molecule) with the first and second PCR primers and PCR reagents (which will include at least a polymerase and dNTPs), and in one embodiment, the exponential reaction of step (a)(i) may include: denaturing the target nucleic acid molecule if it is double-stranded; incubating the PCR reaction mixture at a first annealing temperature to anneal the two primers; incubating the PCR reaction at a first extension temperature, which may be the same or different from the first annealing temperature, to extend the primers; and optionally repeating the denaturation, primer annealing and extension steps. Subsequently, the linear reaction of step (a)(ii) may include: denaturing the double-stranded amplicon of step (a)(i); incubating the PCR reaction mixture at a second annealing temperature to anneal the first primer; incubating the PCR reaction at a second extension temperature, which may be the same or different from the second annealing temperature, to extend the primers; and optionally repeating the denaturation, primer annealing and extension steps.

[0110] In another embodiment, linear amplification is performed first, and the linear reaction of step (a)(ii) may include: if the target nucleic acid molecule is double-stranded, denaturing it; incubating the PCR reaction mixture at a second annealing temperature to anneal only the first primer; incubating the PCR reaction mixture at a second extension temperature, which may be the same as or different from the second annealing temperature, to extend the first primer; and optionally repeating the denaturation, primer annealing and extension steps. Subsequently, the exponential reaction of step (a)(i) may include: subjecting the reaction mixture of step (a)(ii) to a denaturation step; incubating the PCR reaction mixture at a first annealing temperature to anneal both the first and second primers; incubating the PCR reaction at a first extension temperature, which may be the same as or different from the first annealing temperature, to extend both primers; and optionally repeating the denaturation, primer annealing and extension steps.

[0111] The first primer may be defined as a "high" or "high temperature" primer, and the second primer may be defined as a "low" or "low temperature" primer. Thus, the first annealing temperature at which both primers anneal may be defined as "low" accordingly, and the second annealing temperature at which only the first (high) primer binds may be defined as "high" accordingly. Thus, the first annealing temperature is lower than the second annealing temperature.

[0112] In general, the second annealing temperature ("high" annealing temperature) is typically at least about 8-12°C higher than the first annealing temperature ("low" annealing temperature). In one embodiment, it is at least 10°C higher than the first annealing temperature. Ideally, the difference between the first (low) and second (high) annealing temperatures is about 10°C.

[0113] As mentioned above, the advantage of this method is to reduce the number of exponential PCR cycles to reduce the accumulation of polymerase errors. Advantageously, in order to minimize polymerase errors, no more than 12, 11 or 10 cycles (i.e. 12, 11 or 10 or less) are performed. In one embodiment, no more than 5-8 cycles are performed, for example, no more than 8, 7, 6 or 5 cycles are performed. In another embodiment, 6 cycles are performed.

[0114] In the present method, the number of linear amplification cycles performed is selected based on the number of exponential cycles performed and the expected yield of single-stranded amplicons of the target sequence. Typically, no more than 40, 39, 35, 30, 25 or 20 cycles are performed. In one embodiment, at least 6-12 cycles are performed, such as at least 6, 7, 8, 9, 10, 11 or 12 cycles are performed. In another embodiment, 8 cycles are performed.

[0115] According to principles well known in the art, PCR primers are designed to amplify the target sequence and therefore anneal at sites within the target sequence or flanking the target sequence. This may depend on the nature of the target sequence, such as whether a different target sequence is detected, or whether a different variant (e.g., mutant or allele) of a gene is detected. In general, a forward primer may anneal near or at the 3' end of a complementary target sequence. A reverse primer may anneal near or at the 3' end of a target sequence.

[0116] In the case of multiple reactions, where there is more than one target molecule or more than one target sequence, the amplification of each target may be performed separately in parallel, i.e. a separate amplification reaction may be performed, each reaction using a set of PCR primers. In this case, the resulting individual asymmetric PCR reaction mixtures, or more specifically, portions or aliquots thereof, may be pooled to provide the reaction mixture of step (a). Thus, in one embodiment, a plurality of separate asymmetric PCR reactions are performed and the amplicons therefrom are pooled prior to step (b).

[0117] Alternatively, the amplification of different target sequences / molecules can be multiplexed in the same reaction mixture. Therefore, asymmetric PCR reactions can be performed using multiple (or multiple) primer sets (or in other words, primer pools). It should be understood in this regard that when studying different variants of a target sequence, the same PCR primer set can be used to produce its amplicon (in other words, the amplicon can include one or more of many different variants, which can be amplified using the same primer set; primers can be designed to be incorporated in the site flanking the variant site). In the case of multiplexed amplification in the same reaction mixture, the number of sequences amplified can be 2 to 10,000 or more.

[0118] Therefore, in order to detect target sequences in two or more different target molecules, multiple PCR primer sets (e.g., primer pairs) can be used, each primer set being specific to different target sequences, i.e., having target binding regions complementary to primer binding sites in different target sequences or flanking different target sequences in the target molecule. In this regard, it should be understood that the flanking binding sites in different target molecules will be different for different target sequences, so that the padlock probes are specifically bound. Alternatively or additionally, different and unrelated target sequences in the same target molecule can be detected, for example, different sequences in different genes on a chromosome, again using multiple different PCR primer sets, each primer set being specific to different target sequences. However, as described above, in a specific embodiment, the method can be used to detect which of a plurality of possible different variant sequences is present in a given target molecule, for example, whether there is a wild-type or mutant sequence, or which of a plurality of possible mutants, or different allelic variants or polymorphisms, etc. In this experimental scheme, a universal primer set is used to generate an amplicon, and then which variant is present is determined by using a plurality of different padlock probes, each probe being specific to a different variant of the target sequence.

[0119] The term "multiple" or "multiplicity" as used herein refers to two or more, such as 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 sets of primers (and subsequent ligation templates or padlock probes) can be used. The number that can be used is not limited and can vary. This will depend on the purpose of the method, the nature of the sample, the target nucleic acid sequence to be detected, and the number of possible variants, etc. Therefore, in order to detect, for example, wild-type and mutant variants, the number of different padlocks will depend on the number of possible different mutants, and can be, for example, 2-6, 2-5, 2-4 or 2-3 different padlocks. It should be understood that different aspects can be combined to increase the overall multiplicity of detection. For example, in a given sample, the method can be used to detect different variants of different target sequences.

[0120] When the amplicon is produced by different primers, for example, produced by different target molecules, or by different target sequences amplified by different primers of the same molecule, they may have different terminal sequences. PCR primers can introduce different or selected terminal sequences. Therefore, the PCR primers used to amplify different target sequences can have 5' terminal sequences that are not complementary to the target sequence / molecule, and can be used to provide a common or universal terminal sequence for all amplicons. In this way, it is possible to obtain amplicons all having the same terminal sequence. This makes it possible to use the same connection template to cyclize all amplicons (for example, or the amplicons produced from the same sample) in the reaction mixture. In some cases, if necessary, all amplicons from a group or a group can have the same (i.e. common) terminal sequence, and different groups or groups can have different terminal sequences. For example, all amplicons from a given sample can have the same terminal sequence, but amplicons from different samples (they are pooled together) can have different terminal sequences, thereby the same target sequence from different samples can be distinguished. Therefore, in one embodiment, the terminal sequence can be defined as the binding site of the connection template or the binding site comprising the connection template.

[0121] Thus, in one embodiment, the PCR primers (and the subsequent ligation templates) can be considered universal primers, or universal primers for a group of target sequences or a group of variant target sequences. Thus, the PCR primers can have target binding regions that are complementary to binding sites (e.g., flanking regions) shared by different target sequences in the target molecule (i.e., shared binding sites flanking different target sequences or different variants of a target sequence). Alternatively, the amplification primers can have target binding regions that are common or universal for different target sequences or different variants, or common for a group of target sequences or a group of variants. That is, the amplification primers can have target binding regions that are capable of hybridizing to complementary binding sites shared by different target sequences or different variants of a target sequence in the target molecule. However, padlock probes are specific for target sequences or different variants of a target sequence, as further described below.

[0122] In various embodiments, PCR primers can be specific for a particular target nucleic acid sequence. Thus, for example, primer sets can be used, each primer set being specific for a different target sequence. This can be used for diagnostic tests, such as NIPT, where different sequences can be detected, for example, to detect different chromosomes (and determine their copy number, e.g., to detect trisomy), or for any situation where detection of one or more specific target sequences is desired.

[0123] In one embodiment, the PCR primers can be designed in a manner that contributes to the subsequent connection step (b) of the method. Therefore, the primers may include one or more uracil bases so that the primers are degraded. In particular, the amplicon can be subjected to a digestion step with uracil-DNA-glycosylase (UDG) to digest the primer portion (extension product) of the amplicon. This is particularly directed to the first primer ("high Tm" primer). This contributes to the connection efficiency; the connection template can be designed to hybridize with the 5' end sequence of the extended portion of the amplicon sequence. After the asymmetric PCR step, the PCR primers will be present in excess in the amplification reaction mixture, and in many cases are very excessive, so this modification helps to reduce or minimize the competition of the unextended primers to the connection template. Similar effects can be achieved by adding phosphorothioate bonds in the primers in combination with 5' single-stranded nucleic acid exonucleases.

[0124] In order to carry out the asymmetric PCR step, the target nucleic acid is contacted with PCR primers and other reagents required for amplification. A sample containing the target nucleic acid molecule or its part or component or aliquot can be contacted with the reagent. For example, a cell or tissue sample, or any sample containing cells, or a cell extract, can be directly contacted with the reagent. The sample can be pre-treated or processed before contact. In other embodiments, the target nucleic acid can be first separated or removed from the sample. The procedure for extracting or purifying nucleic acid such as DNA from various types of samples is well known in the art. For example, nucleic acid can be separated from cells, or separated from cell-free samples such as blood plasma. In some cases, it may also be necessary to fragment the nucleic acid molecules. The procedures for this are known in the art, and include specific digestion, for example using nucleases, including restriction enzymes, or by non-specific methods, such as shearing.

[0125] As described above, for ease of operation and automation, it is generally desirable to perform multiple tests (i.e., multiplexed tests) in one container, particularly for the sRCA step. To achieve this, in the case where a separate parallel asymmetric PCR amplification has been performed, after the asymmetric PCR reaction of (a), a portion or portions of the resulting reaction mixture of the asymmetric PCR reaction for producing amplicons may be introduced or added to the reaction container. For example, one or more amplification reaction mixtures in a volume of 1-5 μL may be added to the container. However, in another embodiment, the amplification may be performed in the container.

[0126] Therefore, the method can be advantageously automated. Therefore, in one embodiment, the method is designed to allow a 2-stage (or more-stage) RCA reaction (i.e., sRCA) of a single-stranded amplicon of a target nucleic acid sequence in a single reaction vessel without any manual input, and can be extended for commercial applications in a robotic workflow.

[0127] "Single reaction vessel" refers to a single physical or structural compartment, i.e. a physical structure that holds the volume of the reaction mixture. The term "compartment" as used in the present invention does not include droplets or emulsions or the like. Likewise, this does not exclude the use of droplets or emulsions in any step of the "single container embodiment" of the method (i.e. within the reaction vessel in steps (i) to (x)). It should also be understood that for the purpose of detection in step (x), the second (or more) RCA product can be removed from the reaction vessel, for example, to transfer it to a detection instrument, such as a flow cytometer or a microscope slide or the like. Thus, the container can be any container suitable for carrying out the steps of the method. Typically, this will be a reaction tube, but it can be a container of any form or construction, including, for example, a reaction well or part of a microfluidic circuit.

[0128] As mentioned above, a limited number of exponential PCR cycles are performed, so that the resulting mixture contains an excess of PCR primers relative to the amplicon. Although a low cycle number has the advantage of improving the specificity of detection, the result is that there is an excess of PCR primers in the reaction mixture. Even so, a limited number of cycles may also result in a very large excess of primers, such as 1000 times of excess or more. Therefore, the primer excess may be at least 1000, 2000, 5000, 7000 or 10,000 times. In some cases, it may be higher, exceeding tens or hundreds of thousands of times. For example, in a representative amplification reaction, the resulting reaction mixture may contain an amplicon (such as a full-length PCR product) of the order of 100 fM, and the order of magnitude of the primer is 500 nM. This excess primer may interfere with the downstream steps of the method.

[0129] Although in some embodiments the amplicons may be isolated from the PCR amplification reaction mixture, it may be convenient to address this issue by performing a clean-up step to remove excess primers. This is particularly true in a single vessel format of the method.

[0130] Thus, in one embodiment, it may be convenient to include a cleanup step to remove or reduce at least the PCR primers and optionally the PCR polymerase. This may be accomplished by enzymatic digestion and / or dilution.

[0131] 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.

[0132] If there is PCR polymerase in the reaction mixture in a subsequent step, the PCR polymerase may inhibit the connection of the amplicon by extending the hybridized 3' end of the amplicon on the ligation template (if used). Therefore, one way to reduce this interference is to perform a non-template ligation step of the single-stranded amplicon. However, in another embodiment, the polymerase can be inactivated by enzymatic digestion. In another embodiment, the hybridization of the amplicon to the ligation template can be controlled by temperature so that the 5' end hybridizes first, followed by the 3' end. These solutions can also be used to prevent the extension of the 3' end of the padlock probe hybridized in the second ligation step (step (e)).

[0133] In the case of enzymatic digestion of the primers, this can be achieved by contacting the reaction mixture of (a) with a digestive enzyme capable of degrading the primers. Conveniently, this can be an enzyme with exonuclease activity, in particular an enzyme with exonuclease activity as its main catalytic activity (as opposed to a polymerase with exonuclease activity or a part thereof with exonuclease activity). Thus, an exonuclease can be used (this term does not include a polymerase or a part thereof in the present invention).

[0134] The term "contacting" is used broadly in the present invention to include contacting the reagents in question. Thus, one may be added to the other, vice versa, or they may be introduced into each other, etc. However, in the case of a single container procedure carried out in steps (i) to (ix), contacting conveniently includes adding the reagent in question to a reaction mixture present in a single container, or in other words, adding one or more reagents or reagent mixtures in question to the container. Thus, in the case of step (ii), reducing the primer excess may include adding a reagent or reagent mixture containing a digestive enzyme to the reaction mixture of (i), particularly to the container containing the reaction mixture of (i).

[0135] Exonuclease can be any suitable exonuclease. Exonucleases for removing non-desired nucleic acids are known in the art, including, for example, exonucleases I, III or λ or mixtures thereof. The selected exonucleases should have strict single-strand specific activity, such as ExoI or RecJf.

[0136] Other digestion systems may also be employed. For example, if the amplification primers contain uracil, they may be conveniently digested using another well-known digestion system, uracil DNA glycosylase (UDG), as described above.

[0137] In another embodiment, amplicon can be generated 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.

[0138] The removal step may further include an enzyme digestion of the amplification polymerase. However, this is not absolutely necessary, because the interference from the polymerase can be resolved by other means. Nevertheless, in one embodiment, in addition to the enzyme with exonuclease activity, the removal reagent mixture may also include an enzyme with protease activity. This may be any suitable protease, including serine proteases, cysteine ​​proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteinases and asparagine peptide lyases, but usually this will be proteinase K. Advantageously, a thermolabile or non-thermophilic protease is used, because it can be subsequently heated and inactivated. Therefore, a heating step may be included to inactivate the protease after use, as further discussed below. It is advantageous to use a protease in the step, because the protease also degrades the exonuclease. In this regard, a balance may occur between the enzymatic action of the exonuclease and the digestion of the protease. However, it has been found that the sufficient degradation of the amplification primer may occur before the exonuclease inactivation, and is completely within the scope of the conventional skills of those skilled in the art to find and optimize the reaction conditions.

[0139] The clearing agent, 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 reaction mixture (e.g. a reaction vessel in the form of a single vessel) may be incubated under conditions suitable for the clearing reaction to occur. As a representative example, the incubation may be 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.

[0140] If the method is used to detect variants, such as mutations, then a clean-up step by enzymatic digestion of the primers is generally optimal. Preferably, a protease digestion of the amplification polymerase is also included.

[0141] However, as an alternative to enzymatic digestion, a dilution of the amplification reaction mixture can be used to dilute the primers. This may be sufficient to reduce their interference to an acceptable level. In general, this may require a large dilution factor, which can be determined by routine experiments. For example, a dilution factor of at least × 200 can be used. This can be achieved by contacting the reaction mixture with a diluent, for example, by adding a diluent to the reaction mixture, or by adding a portion or aliquots of the amplification reaction mixture to the diluent. The diluent can conveniently be 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 case of a dilution step, a smaller aliquot of a single amplification reaction mixture or its mixture pool can be contacted with a diluent, for example, a 0.1-0.2L aliquot.

[0142] Thus, in one embodiment, steps (i) and (ii) of the single container protocol can be combined and can be performed by adding a portion or aliquot of one or more amplification reaction mixtures to a volume of a removal reagent mixture present in the container. The removal reagent mixture can be a simple diluent, such as a buffer.

[0143] After the asymmetric PCR step, and optionally after any cleanup step (e.g., step (ii) of the single container protocol), there is an optional step of denaturing the double-stranded amplicon into a single strand. This is not necessary, as the amplicon is substantially prepared in a single-stranded form, but it may be advantageous to include such a step in some embodiments. If included, the method may include a heating step to denature the amplicon, for example, heating to 95°C for 1-2 minutes, or similar conditions. Such a heating step may also inactivate any protease included in the cleanup reaction. In general, however, it has been found to be beneficial to include a separate, milder heating step, for example, incubating at 50-60°C for 5-20 minutes, for example, incubating at 55°C for 10 minutes to inactivate the protease (if used). The method may then proceed to the ligation step (b), or to a denaturation step followed by a ligation step.

[0144] In this regard, if a denaturation step is included, it is convenient to add the ligation mixture in step (b), heat to denature, and then reduce the temperature to carry out the ligation. Thus, the denaturation and contacting of step (b) can be carried out simultaneously or in any order. Alternatively, if there is no denaturation step at this time, the ligation mixture can be added and the reaction mixture can then be heated to inactivate the protease (if used).

[0145] The actual ligation reaction can be controlled by controlling the reaction temperature (i.e., the ligation reaction can be initiated). Annealing of the ligation template (if used) and / or the activity of the ligase can be included. In other words, if denaturation is included, the ligation reaction occurs after denaturation, but the reagents, ligase, and, in some embodiments, the ligation template for ligation can be added to the reaction mixture before or simultaneously with the denaturation of step (b). As described above, a ligation template will usually be included because it tends to give better results, but this is not necessary, and non-templated ligation can also be selected. This may depend on the specific method used, as well as the amplicon used and the ligation conditions, etc. In one embodiment, the ligation template is included in the ligation reaction mixture.

[0146] After denaturation, when a ligation template is used, the temperature is lowered to allow the amplicon to hybridize (or in other words anneal) to the ligation template and for the ligase to catalyze the ligation reaction. If there is no denaturation step, this temperature reduction may not be necessary, and the method may instead include increasing the temperature of the ligation step (e.g., for annealing of the ligation template and / or for ligase activity).

[0147] The ligation template comprises a sequence or region complementary to the single-stranded amplicon terminal sequence of preferential amplification. The end of the amplicon chain is hybridized with the ligation template, thereby making the end of the hybridized amplicon 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 a specific single amplicon (in other words, the amplicon comprising a specific target sequence can have a homologous ligation template, which is designed to hybridize with it, but not with other amplicons). 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 terminal sequence provided to the amplicon.

[0148] In this regard, it is usually the case that all amplicons (present in the reaction mixture) are ligated. Thus, in the case of a reaction mixture containing a plurality of different amplicons (representing a plurality of different target sequences), all different types of amplicons that may be present or to be detected are ligated into rings. Thus, the step of circularizing the amplicons by ligation can be considered a step of sample preparation, or a step of preparing a library of molecules to be amplified and probed by padlock probes.

[0149] The ligation is performed by a ligase. It can 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, E. coli ligase, and thermostable ligases such as Taq ligase, Tth ligase, Pfu ligase and 9°N TMDNA ligase. Advantageously, a thermostable or thermophilic ligase is used because it can remain in active form in the reaction mixture for connection of the padlock probe in the subsequent step (e) (step (vii) of the single container method). Suitable conditions for connection are known in the art, and any necessary and / or desired reagents can be combined with the reaction mixture and maintained under conditions sufficient to connect. Obviously, the connection conditions can depend on the ligase used in the method of the present invention. Thus, for example, Ampligase can be used, which allows the ligation reaction to be carried out at, for example, 50-60°C (e.g., 55-60°C, e.g., 58°C).

[0150] When making 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., cyclization) of ssDNA templates having a 5'-phosphate and a 3'-hydroxyl group. In one embodiment, it is desirable to design the ligation reaction in an asymmetric ligation structure such that there is stronger hybridization between the amplicon (extended primer product) and the ligation template than between the unextended primer and the ligation template. As described above, this can be achieved by primer design.

[0151] Also as mentioned above, in some embodiments, it may be necessary to carry out ligation under conditions where the ability of the amplicon chain 3' end that extends and is hybridized to the ligation template is suppressed. It should be understood that if the amplicon has been digested with protease in the previous removal step, such conditions will be reached. However, this can also be achieved in other ways, most notably by controlling the hybridization of the amplicon with the ligation template so that the 5' end of the amplicon is first hybridized. This enables ligation to be carried out before any significant extension occurs at the 3' end of the hybridization. Therefore, a 2-step hybridization can be carried out, where the 5' end of the amplicon is annealed to the ligation template at a first temperature, and then the 3' end of the amplicon is annealed to the ligation template by lowering the temperature.

[0152] 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 particularly 55-60°C.

[0153] The annealing temperature can be lowered for annealing of the 3' end. Similarly, suitable conditions can be selected based on the known knowledge in the art and the specific reagents (e.g., enzymes used). 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.

[0154] A suitable ligase can be selected to achieve this 2-step annealing. In this regard, thermophilic ATP-dependent ligases are available, which are active in the temperature range of 37-95°C. Thus, this allows the use of a second / annealing temperature of about 37-45°C, which is 10°C lower than the first annealing temperature.

[0155] Those skilled in the art know how to design the connection template and / or amplicon end to realize the differential annealing of 5' and 3' amplicon ends, for example, to realize the different Tm of each end annealing.Therefore, by appropriate design and selecting appropriate conditions, including temperature, balance can be obtained, which makes the stable hybridization of 5' end, followed by the hybridization of 3' end.For example, the sequence of 3' end of the amplicon hybridized with the connection template may be shorter than the 5' end sequence.As representative embodiment, 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.

[0156] Once the amplicons are circularized by ligation, they are subjected to RCA in step (c). The RCA reaction is well known in the art, so the conditions of this step can be designed or selected according to the experimental schemes 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 serves 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.

[0157] To ensure that no unwanted polymerase-catalyzed extension reactions occur, the reagents required for RCA can be conveniently added to the reaction mixture after the ligation step. In a single container method, it will be added 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 in general, it is convenient to add an RCA reagent mixture comprising an RCA polymerase and dNTPs. The actual RCA mixture typically includes a suitable buffer and other ingredients to assist the RCA reaction, which is well known in the art. As a representative embodiment, using Phi29 polymerase, the RCA reaction can be carried out at 37°C, for example 20-40 minutes.

[0158] After the first RCA reaction, and prior to subsequent steps (e.g., prior to the addition of the padlock probe, or prior to its ligation), there may be a step to inactivate the RCA polymerase used, e.g., by heating. Such a heat inactivation step may, for example, comprise heating at 55-65°C for a period of time, e.g., 5-15 minutes, e.g., 10 minutes. Such an experimental protocol is included in the single container protocol above.

[0159] Therefore, after this heat inactivation step, the temperature can be lowered, if necessary, to allow the first RCP of the subsequent step to contact and hybridize with the padlock probe.

[0160] The RCA reaction produces a first RCA product (RCP) in series, which comprises multiple repeated copies of the complement of the connected amplicon. Therefore, it comprises 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 combined with the padlock probe-it is enough for some or more binding sites in the first RCP to be combined with the padlock probe. More specifically, the target sequence in at least one monomer of the first RCP in series is combined with the padlock probe, but more specifically, 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.

[0161] The method relies on a variety of 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, in which the RCA product is detected by hybridization of a probe (e.g., a detection probe) with the product, which 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 under conditions that are conducive to hybridization.

[0162] The padlock probe is specific to the target sequence and can therefore be used to identify or detect the target sequence or its variant. Therefore, the padlock probe comprises a target-specific binding region 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 distinguish different target sequences or variants. In this respect, it should be 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 in the present invention to refer to target sequences present in the target molecule and its complement, wherein which of them is actually bound by the padlock probe will depend on the accuracy of the method and which chain is amplified, etc. The design of variant-specific or allele-specific probes is 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 the case of detecting a variant sequence, the padlock probe genotypes the target sequence in the first RCP accordingly, and can be referred to as a genotyping padlock probe.

[0163] As described above, in the case of detecting variant target sequences, two or more padlock probes can be used, each specific for a different variant sequence, and it can be detected which of them produces a second RCP to detect or identify which variant is present.

[0164] Padlock probes can also be defined as circularizable probes. The use of padlocks or circularizable probes is well known in the art, including in RCA reactions. Circularizable probes comprise one or more linear oligonucleotides that can be linked together to form a ring. Padlock probes are well known and widely used, and are widely reported and described in the literature. Therefore, the principle of padlock detection is well known, and the design and application of padlock probes are known and described in the art. Padlock probes are typically linear circularizable oligonucleotides that hybridize with a target nucleic acid sequence or molecule in such a way that the 5' and 3' connectable ends of the probe are juxtaposed to be directly or indirectly linked together, with a gap in the middle. By connecting the hybridized 5' and 3' ends of the probe, the probe is circularized. It should be understood that in order for circularization (connection) to occur, the connectable 5' end of the padlock probe has a free 5' phosphate group.

[0165] To enable juxtaposition of 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.

[0166] To achieve ligation, the 3' and 5' ends to be ligated ("ligatable" 3' and 5' ends) are hybridized to the target sequence in the first RCP, which serves as a ligation template. Depending on the probe design, the ligatable ends of the padlock probe can be juxtaposed in a variety of ways for ligation. When the target binding site is located at the end of the padlock probe, the binding of the padlock probe can cause the ends to become juxtaposed as described. 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 ligated to each other. Therefore, in this case, the ligatable 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-continuous) binding site in the target sequence. Under this arrangement, the 5' ligatable end of the probe is provided by the actual 5' end of the probe. However, the ligatable 3' end of the probe is generated by the extension of the 3' end of the probe hybridization, using the target sequence as an extension template to fill the gap between the hybridization ends of the probe. The extension reaction brings the extended 3' ends of the probe into juxtaposition for ligation. In this case, the ligatable 3' end of the probe is therefore the extended 3' end of the probe.

[0167] In other embodiments, the 3' and / or 5' ends that can be connected can be made or generated by cutting. Therefore, when the target binding site is not located at the end of the padlock probe, but is located inside the end, close to (rather than at) the end of the probe, the probe will hybridize with the target in a manner that there are unhybridized nucleotides at the end of the probe. In other words, after the probe hybridizes, there are overhangs, flaps, or unhybridized additional sequences at one or both ends of the probe. If the unhybridized sequence is at the 3' end, this will prevent the connection of the hybridization probe, or actually prevent extension. When the probe hybridizes with its target (i.e., by cutting of the hybridization probe), these unhybridized regions or nucleotides can be removed by cutting, particularly by enzymatic cutting.

[0168] Hybridization of the padlock probe to the internal 5' target binding site will produce a structure with a so-called 5' flap. Padlock probes designed in this way, as well as procedures and enzymes for cleaving these probes, are known in the art. Any enzyme capable of performing a reaction that removes the 5' flap may be used in this step, i.e. any enzyme capable of cleaving, degrading or digesting 5' single-stranded sequences that are not hybridized to the target nucleic acid molecule, but typically this will be an enzyme with 5' nuclease and / or structure-specific cleavage activity.

[0169] Structure-specific cleavage enzymes are enzymes that can recognize the connection between a single-stranded 5' overhang and a DNA duplex and cleave the single-stranded overhang. Such enzymes are known in the art and include flap-like endonucleases (FENS), which are enzymes with endonuclease activity that can catalyze the hydrolytic cleavage of phosphodiester bonds at the junction of single-stranded and double-stranded DNA. For example, the enzyme can be a natural or recombinant archaeal FEN1 enzyme from P. Furiosus (Pfu), A. Fulgidus (Afu), M. Jannaschii (Mja), or M. Thermoautotrophicum (Mth).

[0170] Enzymes with 5' nuclease activity include enzymes with 5' exonuclease and / or 5' endonuclease activity, and again, such enzymes are known in the art, such as Taq DNA polymerase and its 5' nuclease domain, or exonuclease VIII. Other examples are RecJf and T5 exonucleases.

[0171] In order to cut the unhybridized 3' end (or 3' flap), an enzyme with 3' nuclease activity can be used. This can be a 3' exonuclease or a 3' endonuclease activity. It can be provided by a polymerase with 3' exonuclease activity or its 3' exonuclease domain, or by a separate exonuclease, such as exonuclease I, or by an endonuclease. As representative embodiments, the enzyme can be T4 DNA polymerase, T7 DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, Pyrococcus furiosus (Pfu) DNA polymerase and / or Pyrococcus woesei (Pwo) DNA polymerase.

[0172] In particular, polymerases with 3' exonuclease activity can be used in the step of extending the hybridized 3' end of the gap-filling padlock probe - such enzymes will remove the unhybridized 3' nucleotides before the extension reaction occurs, leaving the hybridized 3' end. Polymerases with 3' exonuclease activity but without strand displacement activity are ideal. In the case of a cycling protocol, thermophilic polymerases should be used. These include: Q5 / Q5U DNA polymerase, Phusion / Phusion U DNA polymerase, Taq DNA polymerase, Stoffel DNA polymerase, Pwo DNA polymerase, Kappa DNA polymerase, and SuperFi DNA polymerase.

[0173] The position of the target binding site away from the 3' or 5' end of the padlock probe will determine the length of the 3' or 5' flap. In general, it is preferred that the 3' target binding site is reasonably close to the 3' end, such as within 7 or 6 or less nucleotides of the 3' end, such as 5, 4, 3, 2 or 1 nucleotides of the end. For the 5' target binding site, it can be a longer distance. In one embodiment, "close to" the 5' or 3' end of the probe refers to within 12 nucleotides or less nucleotides of the end, such as within 10, 9, 8, 7 or 6 nucleotides of the end. In the case of the 3' end, it can be, for example, within 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides of the end or less, such as within 6 nucleotides or less.

[0174] In another embodiment, the 3' end of the first padlock probe may comprise a hairpin structure, which comprises a 3' target binding region. In other words, the 3' target binding region may be at least partially contained in 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.

[0175] The padlock probe may be provided in 2 or more parts that are ligated together. This may involve providing an additional ligation template, for example, in the case of a 2-part probe where each part contains only one target binding region and the other end of each part hybridizes to a common ligation template. In another embodiment, the 2-part padlock may take the form of a "linker" oligonucleotide, having two target binding regions at or near the 5' and 3' ends, respectively, which hybridize to the target, with a gap between them, and a gap oligonucleotide that hybridizes in the gap between the ends. The gap oligonucleotide may partially or completely fill the gap.

[0176] However, in a typical embodiment, the padlock is provided in the form of a single circularisable oligonucleotide, whether a gap-filling padlock or not.

[0177] In a specific embodiment, the padlock probe does not have a secondary structure, more specifically, does not contain an intramolecular double-stranded region or a stem-loop structure. However, a dumbbell probe with a secondary structure is a special subtype of a padlock probe. The dumbbell probe contains two stem-loop structures, the stems are connected to each other, and one of the "loops" is not closed but open, with free 5' and 3' ends that can be used to connect to each other. This "open loop" serves as the target binding domain of the probe. The function of the closed loop is only as a spacer to connect the ends of the duplex (stem). In other words, it can be regarded as a padlock probe, forming a duplex region between the complementary sequences (regions) of the padlock. The region of the duplex serves as a signal domain to which the intercalator can bind. Therefore, the "open loop" of the dumbbell probe can contain a complementary target binding region.

[0178] In another embodiment, as described above, the padlock probe may include a hairpin structure comprising the 3' end of the probe.

[0179] Although padlock probes can be designed to have target-specific binding regions that are specific for a particular target sequence, it should be understood that non-specific hybridization may occur, particularly in the case of variant target sequences that are similar in sequence (e.g., when the variant is a single nucleotide variant, such as SNPs, etc.). However, the high specificity of padlock probes stems from the fact that such 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 are ligated (i.e., padlock probes that have correctly hybridized to the complement of the target sequence they want to detect, or in other words, their corresponding or homologous, or respective target sequences).

[0180] The second ligation (of the padlock probe) can be performed under similar conditions to the first. In general, the padlock probe can be added to the ligation mixture of the reaction mixture, i.e., to the reagent mixture containing the ligation reagent. This may be similar or identical to the ligation mixture / reagent used in the previous ligation step. In one embodiment, the addition of a ligase may be included. However, if a thermostable ligase is used for the first ligation, further addition of a ligase may not be required. Obviously, a separate ligation template is not required in this step, as the ligation is templated by the first RCP bound by the padlock probe. Therefore, the second ligation is a template ligation.

[0181] For the aforementioned amplicon connection in step (b), in certain embodiments, the connection in step (e) can be carried out under conditions where the ability of the polymerase to extend the 3' end of the hybridized padlock probe is suppressed. This includes amplification polymerase and RCA polymerase from the original reaction mixture. RCA polymerase can be inactivated in the previous heating step. In particular, this can ensure that any hybridized but unconnected padlock probe will not perform any unwanted extension of its hybridized 3' end. It should be understood that this applies to the expected connectable 3' end of the padlock probe. Therefore, if the connectable 3' end is generated by a gap filling extension reaction, the conditions of the connection reaction are such that the connectable 3' end is no longer extended further. The strategy to ensure these conditions 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. In the case where the desired sequence needs to be incorporated into the second RCA template, for example, for further amplification, the gap filling padlock may be useful. In one embodiment, this can be used for the generation of, for example, NGS colonies.

[0182] In the case of padlock probes where gap-filling extension is not required, any amplification polymerase may be inactivated by enzymatic digestion in a prior first cleanup step, if such step is included in the method. If such enzymatic digestion is not included in the first cleanup step, or the first cleanup step is not performed, then undesired extensions may similarly be avoided by controlling hybridization of the padlock probes as described above, i.e., using different annealing temperatures, with a 2-step hybridization of the padlock probes.

[0183] After the ligation step, it may be desirable or advantageous to remove or render inert any unattached probes in the reaction mixture. By inert is meant that the padlock probes are unable to participate in any unwanted hybridization or ligation reactions. In particular, the 3' end of the probe is unable to hybridize with any nucleic acid present in the mixture and initiate an extension reaction. In this regard, it may be particularly desirable to ensure that unattached padlock probes remaining in the mixture after the ligation reaction are unable to hybridize with a second RCP when the second RCP is produced. Therefore, a cleanup step may be performed at this stage in practice (this may be the first or second cleanup, depending on whether the first cleanup has been performed). In the case of a single container method, two cleanup steps will be included. A cleanup reagent mixture may be added to achieve this.

[0184] The clearing agent may be a digestive enzyme that degrades the unreacted probe, such as an enzyme having exonuclease activity as described above. When such an enzyme is used, it is inactivated by heating before the subsequent second RCA step (step (f)). For example, if lambda exonuclease is used, the reaction mixture may be incubated at 30-37°C for 10-20 minutes and then heat-inactivated at about 70-75°C for 10-30 minutes. It may be appropriately varied depending on the specific reagent used, and determining and optimizing the digestion and inactivation conditions is within the routine skill of those skilled in the art.

[0185] Alternatively or additionally, a dilution step may be performed to perform the removal. Thus, a volume of diluent may be added to the reaction mixture. In particular, the reaction mixture may be diluted at least 4×. As described above, when the method is used to detect sequence variants, removal by enzymatic digestion is generally preferred.

[0186] Alternatively or additionally, a padlock probe forming a hairpin can be used. This type of probe comprises a self-complementary sequence that can hybridize to form a hairpin structure comprising the 3' end of the probe. The stem of the hairpin can be designed to have thermal stability, which does not prevent the formation of a hybrid between the loop of the hairpin structure and the target sequence in the first RCP, nor does it inhibit RCA. However, the stem is sufficiently stable for initiating extension, and therefore the unconnected padlock probe is converted into a complete hairpin, which cannot hybridize with the second RCA product or use the second RCA product as an extension template to initiate extension. This padlock probe that can be inactivated is referred to as a suicide padlock probe. Suicide padlock probes are also described in US 6,573,051.

[0187] The second RCA reaction is carried out using the connected padlock probe as the second RCA template. In order to trigger the second RCA, the second RCA reagent mixture is contacted with (e.g., added to) the reaction mixture containing the connected padlock. In general, this will involve the addition of an RCA polymerase, which can be as described above, and can be provided in a reagent mixture as described above. Although all reagents for the second RCA can be added together in a reagent mixture, it may be advantageous to add them individually or stepwise. The second RCA is initiated by a separate RCA primer. It can be pre-hybridized with the padlock probe, or can be added to the reaction mixture individually, or included in the RCA reagent mixture. The binding site of the RCA primer can 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).

[0188] In one embodiment, the RCA primer for the second RCA can be included in the removal reagent mixture. In another embodiment, it can be added separately with the removal reagent mixture at the same time. In another embodiment, it can be added to the reaction mixture after the exonuclease to be removed is inactivated. The second RCA can be initiated by adding an RCA reagent mixture comprising RCA polymerase and dNTP at this time. In another embodiment, RCA polymerase is added in the first step, and then dNTP and RCA primer are added separately.

[0189] When the RCA primer is included in the cleanup mixture, the presence of the phosphorothioate bond can protect it from digestion. Alternatively, exonucleases can be used to degrade double-stranded DNA and cleanup of single-stranded DNA with a 5' phosphate group; padlock probes have a 5' phosphate group and are therefore degraded by such exonucleases, and RCA primers may not have a 5' phosphate group and may therefore be protected from digestion by exonucleases. An example of such an exonuclease is lambda exonuclease.

[0190] In another embodiment, the removal of the unconnected padlock probe can be carried out by RCA polymerase. Therefore, Phi29 has 3' exonuclease activity, which can be used to digest the unconnected padlock. In such an embodiment, an RCA polymerase with 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 carried out. In this case, the RCA reaction can be carried out by adding primers and nucleotides. Therefore, in this embodiment, before the second RCA reaction is carried out, for the purpose of removal, an RCA polymerase can be added before the second RCA step.

[0191] As described above, reagents, procedures and protocols for performing RCA reactions are known in the art and can be used herein.

[0192] The method of the invention requires a first RCA step using a circularized amplicon as a template, and at least a second RCA step using a circularized padlock probe as a template. The method may include a further RCA step to produce a third or further RCA product using a second, third or fourth padlock probe, etc., each RCA product targeting a target nucleic acid sequence. In other words, steps (d) to (f) may be repeated if necessary. The final generation of RCA products may be detected.

[0193] It should be understood that the further generation of this RCA can increase the signal finally detected. This may correspondingly lead to an increase in signal amplification. However, it should also be understood that when the first and additional padlock probes are gap-filling padlocks, this will lead to an increase in the synthesis of copies 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, the method may lead to a large number of cloned products of the filling sequence. In other words, the filling sequence (i.e., the target nucleic acid sequence or a portion thereof) can be amplified. This is useful for preparing the reaction. If multiple continuous (i.e., two or more) first and additional padlock probe connection steps involve gap filling of the same or overlapping sequences, more copies of the specific target sequence can be produced.

[0194] Detect the second or other RCP product as described above to detect the target nucleic acid sequence. To achieve this, the RCP can be made detectable. For example, it can have a detectable label or a method that can make it detectable. 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 or other RCP can be included with one or more RCA reagents added to the reaction mixture, or the detection reagent can be added to the reagent mixture after the second or other RCP has been produced. Alternatively, the label can be incorporated into the RCP when the RCP is produced, for example, using labeled nucleotides.

[0195] In an advantageous embodiment, the detection agent is a detection oligonucleotide that hybridizes to RCP, as further described below. RCP is detected by detecting said detection oligonucleotide. In this regard, the detection oligonucleotide may have a label or some other detectable portion.

[0196] For the actual detection, the reaction mixture or a portion thereof can be removed from the container and moved to a detection instrument, or further processed before detection. Thus, in one embodiment, contact with the detection reagent can occur outside the reaction vessel in which the preceding steps of the method are performed.

[0197] The method can be carried out in a heterogeneous or homogeneous form. That is, it can be carried out on a solid phase (or support), or in a solution or suspension (i.e., without a solid phase or support), or indeed on both, since the solid phase can be introduced at a later stage, for example, at a step of detecting a second RCP, or at a stage of producing a second RCP, etc. In this regard, in a single container embodiment of the method, until the second RCP is produced, it should be understood that any solid phase will be 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).

[0198] The format of the method may be chosen based on the nature of the sample or target nucleic acid molecule, or the desired readout or detection technology used. In one embodiment, the method is an in-solution method, ie a method performed in a liquid phase contained in a container.

[0199] It will be noted from the above description that the method involves temperature changes within and between the different steps. In fact, a feature of the method is that it is carried out under temperature control, at least for the asymmetric PCR step. Conveniently, the method can be carried out in a thermal cycler. This allows for rapid control of temperature changes.

[0200] As mentioned above, described method comprises the multiple steps that reagent contacts with target nucleic acid molecule or reaction mixture subsequently.Easy, they can join in the container containing initial reaction mixture.Reagent can join in buffer conveniently.In other words, the various reagent mixtures used in the step of said method generally comprise buffer.Particularly in the case of single-step method, the step of described method is carried out in the same reaction vessel, without intermediate washing steps.In this case, even or in fact not in single container form, the buffer for adding various reagents also needs to be compatible.In one embodiment, all various reagents or reagent mixtures use identical buffer.

[0201] The target nucleic acid molecule can be present in the sample, or obtained from the sample. The sample can be any sample, which comprises any amount of nucleic acid from any source or any 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.

[0202] The sample can be any sample containing the target nucleic acid molecule, including natural samples and synthetic samples, i.e., naturally occurring materials or prepared preparations. Naturally occurring samples can be treated or processed before being subjected to the method of the present invention. All biological and clinical samples are included, such as any cell or tissue sample of an organism, or any body fluid or preparation derived therefrom, and samples such as cell cultures, cell preparations, cell lysates, etc. Environmental samples are also included, such as soil and water samples or food samples. The sample can be freshly prepared, or can be pre-treated in any convenient manner, such as for storage.

[0203] Thus, representative samples include any material that may contain target nucleic acid molecules, including, for example, food and related products, clinical and environmental samples. The sample may contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles. Thus, such biological materials may include all types of mammalian and non-mammalian cells, plant cells, algae (including blue-green algae), fungi, bacteria, protozoa, etc., or viruses. The cell may be, for example, a human cell, a bird cell, a reptile cell, etc., without limitation.

[0204] 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 samples may be pre-processed in any convenient or desired manner in preparation for use in the methods, for example, by cell lysis or purification, nucleic acid isolation, etc.

[0205] In one embodiment, the sample comprises a microbial cell or virus isolated from a clinical sample or a clinical sample culture. In such a sample, the target nucleic acid molecule can be a nucleotide sequence present in a microbial cell, such as a characteristic, or distinguishing, or identifying nucleotide sequence of a microbial cell or virus at any level, such as at the type, group, class, genus, species, or strain level.

[0206] In another embodiment, the sample may comprise cell-free DNA. The sample may be a sample directly containing cell-free DNA, such as plasma or serum, or may be isolated cell-free DNA.

[0207] In another embodiment, the sample may comprise exosomes.

[0208] Since the target nucleic acid molecule itself need not be the target analyte of analysis, but can be, for example, a reporter molecule used or produced in the analytical process of any desired analyte, the sample need not be a natural sample containing nucleic acid, or the source of nucleic acid (e.g., cell or virus, or biological or clinical material, etc.). As mentioned above, the sample can be a synthetic or artificial sample. Therefore, it can be a sample in which the analyte in which the target nucleic acid has been produced or the target nucleic acid molecule has been added has been detected and measured. It can be a reaction mixture or a reaction product, such as a product produced by an immunoassay that detects the target analyte, such as immune PCR, immune RCA or adjacent detection (e.g., proximity connection detection (PLA) or adjacent extension detection (PEA)).

[0209] The target analyte can be any analyte that needs to be detected. As described above, in embodiments, the target nucleic acid molecule of the method of the present invention is a 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 sugar groups on proteins. Therefore, the target analyte can be a modified protein, for example, with a post-translational modification detected in the detection of the analyte.

[0210] 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 bodies have been proposed as biomarkers for prostate cancer, and specific or selected prostatic bodies or other extracellular vesicles can be detected and distinguished by detecting one or more surface proteins thereon.

[0211] The padlock probe may contain one or more additional sequences that can be used to introduce sequences into the ligation product and thus into the RCP (as a complementary copy). This can be, for example, a label or detection sequence, such as a barcode or recognition motif, or a binding site for a detection probe or primer. This is particularly true for padlock probes. This additional sequence can be found, for example, in a portion of the backbone region of the padlock probe, i.e., the region between the target binding regions. In a dumbbell probe, it can be 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 universal or shared sequences so that different ligation probes in a multiplex setting can be processed or detected together, such as sample indexing, etc., or introducing binding sites for universal or shared amplification primers. This will enable different ligation probes to be amplified together, for example in library amplification by PCR or RCA.

[0212] In particular, the padlock probe may comprise a detection sequence that can be detected. The complement of the detection sequence will be integrated into the second (or further) RCP and can be detected, for example by binding of the detection probe thereto, or by sequencing. The detection sequence may be specific to the padlock probe and therefore specific to the target sequence or sequence variant to be detected. Therefore, each padlock probe may have a different detection sequence. The detection sequence may be detected to detect or identify which padlock probe is amplified in the RCA, and thus which target sequence is present. For example, the experimental scheme may be applied to the context of a method for detecting target sequence variants, wherein each padlock is provided with a detection sequence specific to a particular variant. Therefore, the detection sequence may be regarded as a marker or recognition sequence. The term "detection sequence" used in the present invention includes a detection sequence present in a padlock probe and a complementary copy present in an RCP.

[0213] Thus, label / barcode sequences, in particular including detection sequences, can be used to "label" different padlock probes so that they or their ligations or amplification products can be easily distinguished from each other. In addition, such sequences can be used to label different samples, etc., for example, so that they can be pooled (i.e., "sample" labels or markers). Thus, in a multiplex setting, different probes (i.e., probes for different target nucleic acid sequences or different variants) can have different label sequences (e.g., different labels or detection sequences) and / or they can have the same label sequence, for example for the introduction of a common or universal sequence. These 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, for example, for multiplexed detection of multiple target nucleic acids in a sample.

[0214] The term "hybridization" used in the present invention 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 share base pair organizational homology, they are "complementary" to each other. Therefore, a complementary region in a molecule, probe or sequence refers to a portion of a molecule, 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 complementarity between sequences, although this is not excluded. Therefore, the complementary region may include one or more mismatches. Therefore, "complementary" as used herein refers to "functionally complementary", i.e., a degree of complementarity sufficient to mediate high-yield hybridization, including a degree of complementarity less than 100%. The degree of mismatch allowed can be controlled by appropriately adjusting 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 multiple variables, including, for example, the length and base pair composition, ionic strength and incidence of mismatched base pairs of each molecule or probe oligonucleotide. Therefore, the design of appropriate probes, ligation templates or primers and their binding regions, as well as the conditions under which they hybridize to their respective targets, are well within the routine skills of those skilled in the art.

[0215] The complementary region, for example, to the target sequence of the binding region in the padlock probe, or the complementary region between the detection sequence and the detection probe, or the RCA primer of the circularized padlock probe, or the PCR primer of the target nucleic acid molecule / sequence, or the connection template of the amplicon, etc. can be at least 6 nucleotides long to ensure the specificity of the binding, or more specifically, at least 7, 8, 9 or 10 nucleotides long. The upper limit of the length of this region is not important, but can be, for example, up to 50, 40, 35, 30, 25, 20 or 15 nucleotides. The length of the complementary region can therefore be between any of the above-mentioned lower length limit and upper length limit. In the case of a padlock probe, the length of a single target binding region can be in a lower range, so that when hybridized with its target, the total length of the two binding regions is within the range of the upper limit. For example, a single target binding region can be 8-15, for example 10-12 nucleotides, so that the total hybridization length is 16-30 nucleotides long, for example 20-24. Under the constraints of probe conformation, spacing of domains and desired or favourable hybridisation, it may be desirable to minimise the overall length of the padlock probe in order to minimise the size of the loop affected by RCA and thereby minimise the length of the complementary region.

[0216] The second RCP (and / or any further generation RCP) may 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 procedures used in the method, the detection protocol employed may detect the RCP non-specifically or specifically.

[0217] For example, RCPs can be detected directly, for example, concatemers can be cut to produce monomers, which can be detected using gel electrophoresis, or more typically by hybridization of labeled detection oligonucleotides that hybridize to the RCP neutralization and detection sequence (which can alternatively be referred to as a detection probe), as described above. However, the detection oligonucleotides do not need to be directly labeled. For example, the detection oligonucleotides can be unlabeled probes used as sandwich probes. The concept of sandwich probes is well known in the art and can be applied according to any convenient experimental scheme. Sandwich probes can bind to RCPs, but are not directly labeled themselves; instead, they contain sequences to which labeled secondary oligonucleotides can bind, thereby forming a "sandwich" structure between the RCP and the labeled secondary oligonucleotides.

[0218] Non-sequence specific nucleic acid labeling methods can be used, such as DNA binding stains or dyes well known in the literature, or by incorporating RCPs using labeled nucleotides. Alternatively, RCPs can be detected indirectly, for example the product can be amplified by PCR, and the amplified product can be detected.

[0219] 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 by turbidity, magnetic, particle counting, electrical, surface sensing or weight-based detection techniques. In general, these techniques are associated with in-solution analysis.

[0220] The labeled detection oligonucleotide can be labeled with any detectable label that can directly or indirectly emit a signal. For example, the label can be detectable by spectroscopy or microscopy, for example, it can be a fluorescent or colorimetric label, microparticle or enzyme label. Any label used in immunohistochemical techniques can be used.

[0221] In multiplex procedures for detecting different target sequences and / or variant target sequences, different second or additional RCPs can be detected and distinguished by in situ sequencing, including, for example, synthetic sequencing, hybridization sequencing and ligation sequencing, next generation sequencing and / or sequential barcode decoding technology, including synthetic sequencing, ligation or hybridization, and / or by using detection probes. According to well-known techniques in the art, a combined labeling method can be used according to the level of multiplexing. For example, a large number of repetitive sequences in sRCA products can be distinguished by using ratio labeling of fluorescent or other spectrophotometrically detectable probes. Such ratio-labeled detection probes can be used for flow cytometry or microscopic detection techniques (such as imaging) to detect a large number of sequences, for example, at least two fluorophores can be combined in different ratios to produce multiple fluorescent label groups. For example, it has been found that using a combination of two fluorophores in different ratios, 7 different populations can be produced. This can be expanded using 3 or 4 color combinations.

[0222] In the method involving the use of detection oligonucleotides, the detection oligonucleotide or any secondary labeling oligonucleotide can be labeled with a directly or indirectly detectable label. A directly detectable label is a label that can be directly detected without using additional reagents, while an indirectly detectable label is a label that can be detected by using one or more additional reagents, for example, wherein the label is a member 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: a fluorescent label, a radioisotope label, a chemiluminescent label, etc. In many embodiments, the label is a fluorescent label, wherein the labeling agent used in these embodiments is a fluorescently labeled nucleotide, such as a fluorescently labeled CTP (such as Cy3-CTP, Cy5-CTP), etc. The fluorescent moiety that can be used to label 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 the above-mentioned labels, can also be used.

[0223] Although various detection methods can be used, the second or additional RCP can be detected by microscopy or flow cytometry. In both cases, a directly or indirectly labeled detection oligonucleotide can be used, for example, with a fluorescent label that is easy to detect. In particular, in microscopy-based methods, the RCP can be detected by imaging.

[0224] The use of such a detection technique advantageously enables the second or further RCP to be digitally recorded.Indeed, due to the degree of signal amplification provided by the present method, the second or further RCP is visualized and they can be detected by a camera or any device including a camera (eg a mobile phone).

[0225] In order to detect the second or further RCP produced in a homogenous form, it can be captured or brought to a solid support or surface for imaging or more extensive microscopic detection. The second RCP is a second generation RCA product, larger and heavier, and can therefore be easily brought to the surface by centrifugation. Thus, for example, the test tube or plate can be easily rotated to allow the second RCP to fall to the bottom of the test tube or well for detection by microscopy, particularly imaging.

[0226] Variant sequence to be detected in the described method can comprise 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 comprise two or more bases.Therefore, the variant sequence can comprise a section of nucleotides, wherein 2 or more bases can be variants.Variant bases can be continuous or non-continuous.

[0227] The length of the target sequence is not important and can vary according to the environment, the properties of the target molecule, the target sequence or the variant position or site. Therefore, as a representative example only, the target sequence can be 1 to 10, such as 1-15, 1-12, 1-10, 1-8, 1-7 or 1-6 nucleotides long. However, in such an embodiment, a target sequence longer than a single nucleotide may be beneficial to improve specificity, and in such an embodiment, the target sequence can 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 can be, for example, 4-10, 4-8, 4-7, 4-6, 5-10, 5-8, 5-7 or 6-8 nucleotides long.

[0228] As described above, the present invention also provides a kit for implementing the method. The kit may include PCR primers and padlock probes as described above, optionally together with one or more ligation templates, and / or one or more reagents and / or kit instructions for use. Such reagents include dNTPs, polymerases and ligases, and RCA primers for the first and / or second RCA reactions. In addition, components may include buffers or other reaction components for one or more different reactions. Other optional components may include tools 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, those described above. Other optional components may include solid supports and / or tools 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 as a website address.

[0229] The advantages of the method of the present invention are discussed above. These advantages are particularly beneficial in 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 discover and detect tumor-derived mutant DNA sequences in patient samples, especially including cell-free DNA in plasma, such as circulating tumor DNA. Therefore, the method can be used to diagnose or monitor cancer, or, for example, reveal the recurrence of cancer. The method can be used for any cell-free DNA and can also be used for prenatal testing, especially including NIPT. The technology is fast, has low instrument requirements, and multiple analyses of sequence variants can be performed to increase sensitivity.

[0230] Detection of low frequency or rare sequence variants or mutations also requires high specificity of detection, as well as minimizing the risk of introducing artificial sequence changes that could be mistaken for variant sequences. The present method also combines limited PCR cycles with RCA amplification and locks the target sequence by padlock probes that require dual target recognition.

[0231] In the present method, a first RCP is generated in a highly specific manner, and the production of a highly amplified second RCP is dependent on the presence of the first RCP.

[0232] Each RCA product typically contains hundreds, or in some cases about thousands, of complements of the RCA template ring (e.g., circularized amplicon or padlock probe) used to generate it. The second RCA product is obtained by performing RCA on the circularized padlock probe on the first RCA product, and can therefore contain, for example, 1000×1000 monomer sequences, and therefore has a large size and can have a molecular weight of up to tens of gigadaltons. In addition, as described above, further 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 can be easily visualized as a separate product, such as by a microscope. Every second or further, the RCA product can be detected as a clonal product, generated from a single target nucleic acid molecule, without the need for RCA reactions to distinguish. For example, when labeled with a fluorescent detection probe, the prominent bright fluorescent reaction product enables the counting and distinction of individual amplification products of single molecules in a wide field of view at low magnification (e.g., 20 times). The second or further RCA reaction product is large and bright enough to be recorded with a standard flow cytometer. This allows counting and digital scoring to be performed rapidly within a few minutes using conventionally available instruments, thereby providing excellent quantitative accuracy over a wide dynamic range. As described above, the repetitive sequences present in the second or further RCA products in series allow products labeled with different combinations of fluorophores to be analyzed by ratiometric labeling techniques, thereby allowing increased multiplicity. Due to the large volume and molecular weight of the sRCA products, they can also be enriched by centrifugation in a common desktop centrifuge or similar equipment. In fact, low-speed centrifugation or unit gravity may be sufficient. Therefore, the new method of the present invention achieves multiple advantages.

[0233] As described above, the strong signal amplification provided by the second RCA reaction enables rapid and easy visualization of the signal, for example under a microscope at low magnification or on a digitally scanned image, and thus enables rapid and easy visualization of examination results in clinical situations, such as examination of pathology results in routine use. Therefore, the method of the present invention is particularly suitable for clinical analysis procedures.

[0234] The method is useful for identifying rare integrated copies of viral genomes in human tissues, or for detecting rare RCA products, for example in inefficient mutation detection in tissues. Another example when easy identification of rare events may be helpful is screening for the presence of circulating tumor cells (CTCs) in the overwhelming majority of non-CTC cells. The strong signal produced by the method allows for rapid and easy identification of events (detection of CTCs) at low magnification.

[0235] The method enables speeding up of detection assays, which may be valuable at the point of care, such as a doctor's office, etc. In this regard, the second RCA may be performed in a relatively short time.

[0236] A significant advantage of being able to perform the method in a single reaction vessel is that it allows for automation.

[0237] In addition, the increase in signal intensity / speed can enable other detection methods other than traditional fluorescence-based methods, such as the use of turbidimetry, magnetism, particle counting, electricity, surface sensing and weight-based detection techniques. For example, a single sRCA product from the second RCA has a potential weight of several femtograms after 1 hour of amplification. This weight increase 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 size and weight of the second RCP increase enables it to be effectively positioned on the surface by centrifugation. Conveniently, the above positioning can be performed at a speed of 3000× within 15 minutes, which is in contrast to the first RCA product, which cannot be effectively captured by centrifugation using a desktop centrifuge.

[0238] The method is capable of generating an enhanced signal localized to the first RCA product, and it 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 colorimetric reagent such as HRP and imaged by a smartphone camera. Therefore, the method can have an equivalent reaction to digital PCR, but does not require emulsions, microstructures, or conditions where there is exactly one template in each compartment.

[0239] The prominent amplification products obtained by the method of the present invention will further enable cloning of individual RCPs, since the products obtained from the individual first RCA templates are visible. Thus, individual second RCA products can be identified and isolated. For example, by means of the amplification method of the present invention, visual separation can be achieved in low-melting agarose without the need for amplification, and the products can then be isolated, for example by observation with a toothpick, similar to the isolation of bacterial colonies.

[0240] The detection of rare mutations is very important in clinical diagnosis. For example, mutations in certain genes (such as KRAS mutations) may be important in diagnosis and can be used to identify the emergence of acquired resistance to specific therapies (such as anti-EGFR therapy). In recent years, many efforts have been focused on developing methods for detecting such mutations. This method can provide a useful supplement to such methods.

[0241] In addition to being able to detect point mutations present at low frequencies in a DNA sample, the methods of the present invention also provide a powerful means of screening a DNA sample for the presence of any and all very large numbers of different target sequences in a manner not possible with PCR alone or any other current method. However, unlike methods based on PCR alone, the use of padlock probes and RCA allows the application of hundreds of thousands of probes in parallel without loss of target selectivity.

[0242] Furthermore, the discrete nature of the sRCA products allows for minimal risk of mixing with any other material in the reaction by producing one reaction product for each target sequence detected and collecting these significant reaction products for digital detection. For example, a probe mixture can be produced 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 amplifying the tag sequence on the padlock probe and hybridizing the product to a tag array or the like.

[0243] Furthermore, as described above, wherein the second and optionally further ligation and RCA reactions are performed using gap-filling padlocks, preparative production of copies of the target nucleic acid sequence is possible.

[0244] The sRCA method of the present invention also improves the accuracy of genotyping by detecting repeat sequences of a single RCA product rather than a single target sequence. Therefore, occasional mistyping of padlock probes can be tolerated as long as they are much less than the correct results in a single RCA product and will not lead to erroneous results. This allows genotyping to be performed by a majority voting mechanism. This may also have an impact on how to perform padlock probe-based genotyping, in which case, as long as the ratio between correct and incorrect reactions is satisfactory and the padlock probe detects a sufficient number of repeats, it is possible to enhance the ability to distinguish sequences by using conditions that do not detect any variant with 100% efficiency.

[0245] The method will now be described in more detail with reference to the following figures and non-limiting examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0246] Figure 1Schematic diagram of the method for generating SuperRCA amplification products. A) The DNA sequence of interest in the sample is amplified by asymmetric PCR, including a first exponential PCR reaction (A1), followed by a second linear amplification reaction (A2), resulting in preferential accumulation of the desired strand. B) The amplified strand is converted into a single-stranded DNA circle by templated ligation of its 5' and 3' ends. C) The oligonucleotides from the templated circularization reaction are then used 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, wrapped around the RCA products, are then templated for a secondary RCA reaction using the added oligonucleotides as primers. F) For each starting DNA circle, the reaction produces a large cluster of predominantly single-stranded DNA objects, called SuperRCA products. Up to one million fluorescently labeled hybridization probes can bind to each mutant or wild-type specific product, allowing efficient counting by methods such as standard flow cytometry. DETAILED DESCRIPTION

[0247] Example 1 - Experimental protocol for an exemplary asymmetric PCR-sRCA method

[0248] The following PCR primers were designed for detecting KRAS sequences.

[0249] Primers:

[0250] KRAS upstream primer (Fwd): ATTATAAGGCCTGCTGAAAATGACTGAATATAAACTUG Tm = 66.7°C

[0251] KRAS downstream primer (Rev): TCGTCAAGGCACTCTT Tm = 55.1°C

[0252] The method is performed in a single tube format.

[0253] Materials and methods

[0254] Extraction of genomic DNA DNA was extracted from BM cells or whole blood using the QIAamp DNA Blood mini kit (Qiagen cat. 51104) and eluted in 50 μL elution buffer.

[0255] PCR-based library preparation :Tube A

[0256] Pre-amplification based on high-fidelity asymmetric PCRThe sequence of interest in genomic DNA was amplified using SuperFi DNA polymerase in a 25L PCR reaction system containing 1× SuperFi buffer, 0.2mM dNTPs, 100nM Fwd / Rev PCR primers, 330ng gDNA, and 0.0005U / L SuperFi DNA polymerase. The PCR program was as follows: 98°C for 30 seconds, 6 cycles of 98°C for 15 seconds, and 62°C for 120 seconds for the exponential phase reaction. Then 8 cycles of 98°C for 15 seconds and 72°C for 120 seconds were performed for the linear amplification reaction, in which only the forward primer was annealed and extended.

[0257] SuperRCA Analysis :Tube B

[0258] Removal of primers and PCR polymerase :

[0259] 1 μL of each target in the PCR-based library preamplification was mixed with 20 μL of a clearing solution containing 1× SuperRCA buffer (Rarity Bioscience AB), 0.125 μL UNG (Thermo Fisher), and 0.0006 U / μL thermolabile proteinase K. The resulting mixture was incubated at 37° C. for 10 minutes and then at 55° C. for 10 minutes.

[0260] Ligase-mediated circularization of one strand of the PCR product . 20 μL of ligation solution containing 1× SuperRCA buffer (RarityBioscience), 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 cleanup solution containing the amplified PCR product. The mixture was incubated at 95°C for 1 min and then at 58°C for 30 min.

[0261] Amplification of target sequences by first RCA The circularized strands of the PCR product containing the target nucleotide position were amplified by RCA. 5 μL of 1× SuperRCA buffer (Rarity Bioscience), 1.8 mM dNTPs (Invitrogen), 2.5 U Hi29 polymerase (New England Biolabs), and 0.5 μg / μL BSA were added to the circularized product. The reaction was incubated at 37°C for 30 minutes and then at 65°C for 10 minutes.

[0262] Genotyping of RCA products by padlock probe ligationThe padlock probes were hybridized to the first RCA product and ligated in a sequence-specific manner by adding 5 μL of ligation mix containing 1× 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.

[0263] Digestion of unreacted genotyping probes 5 μL of clearing solution containing 1× SuperRCA buffer (Rarity Bioscience AB), 1.2 M primers, and 1 U / L thermolabile exoI was added to the reaction mixture and incubated at 37°C for 15 min and then at 75°C for 20 min.

[0264] Secondary RCA templated by the binding of padlock probes to the primary RCA product 5 μL of RCA mixture containing 1× SuperRCA buffer (Rarity Bioscience), 0.6 mM dNTPs, and 6 U Phi29 DNA polymerase (New England Biolabs) was added to the mixture, and the system was incubated at 37° C. for 30 minutes.

[0265] Digital recording of SuperRCA products by flow cytometry The final reaction mixture containing the SuperRCA products was diluted into hybridization buffer containing 100 nM fluorescently labeled oligonucleotide probes specific for the different SuperRCA products in 1× SuperRCA buffer (Rarity Bioscience) to a final volume of 250 μL. The solution was applied to a CytoFlex flow cytometer (Beckman Coulter) and SuperRCA products were counted for 150 s per sample at “medium speed” (30 μL / min).

Claims

1. A method for detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, the method comprising: (a) performing an asymmetric PCR reaction using a set of primers to produce an amplicon of a target sequence, the primers 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, wherein the asymmetric PCR reaction comprises, in either order: (i) an exponential PCR phase, comprising no more than 12 cycles, in which the primers are annealed at a first annealing temperature using first and second primers for annealing; and (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 only one strand is amplified, thereby preferentially accumulating single-stranded amplicons of the target nucleic acid sequence; (b) contacting the single-stranded amplicon from step (a) with a ligation mixture comprising a ligase and ligating the 5' and 3' ends of the amplicon to circularize them; (c) performing a first RCA reaction using the circularized amplicon as a first RCA template to produce a first RCA product (RCP) comprising a plurality of repeats of complementary copies of the target nucleic acid sequence in the amplicon; (d) contacting the first RCP with a padlock probe specific for the target nucleic acid sequence and allowing the probe to hybridize to complementary copies in the plurality of repeats; (e) directly or indirectly ligating the hybridized padlock probe to circularize the hybridized padlock probe; (f) performing a second RCA reaction using the circularized padlock probe as a second RCA template to produce a second RCP comprising a plurality of repeated complementary copies of the circularized padlock probe; wherein steps (d) to (f) are optionally repeated one or more times; and (g) 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: The exponential reaction of step (a)(i) is carried out first, followed by the linear reaction of step (a)(ii).

3. The method according to claim 1 or claim 2, wherein: The target nucleic acid sequence is an analyte for detection by the method, or is a reporter for an analyte detected by the method.

4. The method according to any one of claims 1 to 3, wherein the method is performed in a multiplex manner to detect multiple different target nucleic acid sequences, wherein in step (a), multiple asymmetric PCR reactions are performed using different primer sets to produce multiple different target nucleic acid molecules or amplicons of multiple different target nucleic acid sequences, wherein the multiple PCR reactions are performed individually in parallel, or performed together in multiplexes.

5. The method of claim 4, wherein a plurality of separate asymmetric PCR reactions are performed and the amplicons therefrom are pooled prior to step (b).

6. The method according to any one of claims 1 to 5, wherein the method is used to detect variant target nucleic acid sequences in target nucleic acid molecules in a sample, and steps (d) to (g) comprise: (d) contacting the first RCP with two or more padlock probes, each padlock 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 their target sequence in multiple replicates; (e) directly or indirectly ligating the padlock probe that has hybridized to the complement of its variant target sequence to circularize the hybridized padlock probe; (f) performing a second RCA reaction using the circularized padlock probe as a second RCA template to produce a second RCP comprising a plurality of repeated complementary copies of the circularized padlock probe; (g) detecting the second or final RCP to identify the circularized padlock probe and thereby identify the variant target nucleic acid sequence.

7. The method according to any one of claims 1 to 6, wherein: The target nucleic acid molecule is genomic DNA.

8. The method according to any one of claims 1 to 6, wherein: The target nucleic acid molecule is RNA, and the method comprises generating a cDNA copy of the target RNA prior to performing the asymmetric PCR reaction of step (a).

9. The method according to any one of claims 6 to 8, wherein: The target nucleic acid sequence is a mutant target nucleic acid sequence or a wild-type sequence that may exist at a given position in a target nucleic acid molecule, or an allelic variant at a target position in a target nucleic acid molecule, or a polymorphism that may exist in a target nucleic acid molecule.

10. The method according to any one of claims 1 to 7 or 9, wherein The target nucleic acid molecule is a cell-free DNA molecule.

11. The method according to any one of claims 1 to 10, wherein: The sample is a liquid biopsy sample.

12. The method according to claim 11, wherein: The sample is plasma.

13. The method according to any one of claims 1 to 10, wherein: Detection of variant nucleic acid sequences in situ in cells or tissue samples.

14. The method according to any one of claims 1 to 13, wherein: The sample is a clinical sample or is prepared from a clinical sample.

15. The method according to any one of claims 1 to 14, wherein: The method is carried out in a single temperature-controlled reaction vessel and comprises: (i) providing a reaction mixture comprising the single-stranded amplicon from step (a), wherein the reaction mixture comprises a PCR polymerase and an excess of PCR primers relative to the amplicon; (ii) reducing excess primers from the reaction mixture of (i) and / or removing primer sequences from the amplicon produced by (i); (iii) 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 to circularize the amplicon, wherein the ligation reaction is performed under conditions where the ability of the PCR polymerase to extend the hybridized 3' end of the amplicon on the ligation template is inhibited; (iv) adding an RCA mix comprising one or more RCA reagents, said RCA reagents comprising at least an RCA polymerase, to the reaction mixture from (iii) and performing a first RCA reaction using the circularised amplicon as a first RCA template to produce a first RCA product (RCP) comprising complementary copies of a plurality of repeats of the target nucleic acid sequence in the amplicon; (v) heating to inactivate the RCA polymerase; (vi) contacting the first RCP with a padlock probe specific for the target nucleic acid sequence and hybridizing the padlock probe to the complement of the target sequence in the plurality of repeats; (vii) 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 hybridized 3' end of the padlock probe is inhibited; (viii) removing or rendering inert any unattached padlock probes in the reaction mixture of (vii); (ix) performing a second RCA reaction using the circularized padlock probe as a second RCA template to generate a second RCP comprising a plurality of repeated complementary copies of the padlock probe; wherein steps (i) to (ix) are carried out in a single reaction vessel and the temperature is controlled during the steps; wherein steps (vi) to (ix) are optionally repeated one or more times; and, (x) detecting the second or final RCP to detect the circularized padlock probe and thereby the target nucleic acid sequence.

16. The method according to any one of claims 1 to 15, wherein: The padlock probe is: (i) a 1-part padlock probe in the form of a single circularisable oligonucleotide, the 5' and 3' ends of which contain target-binding regions; or (ii) a 2-part padlock probe comprising a first oligonucleotide having a first binding region complementary to the target at its 3' end and a second binding region complementary to the ligation template at its 5' end; and a second oligonucleotide having a first binding region complementary to the target at its 5' end and a second binding region complementary to the ligation template at its 3' end; wherein the 3' and 5' ends of the first and second oligonucleotides are linked together, with the target sequence in the first RCP, and the ligation template as templates, respectively.

17. The method of claim 16, wherein: The ligated template was used as primer for a second RCA reaction.

18. The method according to any one of claims 1 to 17, wherein: The padlock probes each comprise a detection sequence specific for the padlock probe, and the second or additional RCP is detected by a detection probe that hybridizes to a complementary copy of the detection sequence in the second or additional RCP.

19. The method according to claim 18, wherein: The detection probe is labeled with a detectable label, preferably a fluorescent label.

20. The method according to any one of claims 1 to 19, wherein: The secondary or final RCP is detected by microscopy or flow cytometry.

21. The method according to any one of claims 1 to 20, wherein: Up to step (f), the process is a homogeneous process carried out in solution or suspension.

22. The method according to any one of claims 1 to 20, wherein: The method is performed on a solid support.

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

24. A kit for detecting a target nucleic acid sequence in a target nucleic acid molecule, the kit comprising: (i) a primer set for an asymmetric PCR reaction, wherein the primers are capable of amplifying a target nucleic acid sequence and comprise 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 (ii) a padlock probe comprising a target binding region specific for a target nucleic acid sequence; and optionally, (iii) a ligation template comprising binding regions at or near the 5' and 3' ends that are capable of hybridizing to complementary binding sites in the amplicon of the target nucleic acid sequence.

25. The kit according to claim 24, wherein The kit is used to detect multiple different target nucleic acid sequences in one or more target nucleic acid molecules and comprises multiple different primer sets (i) and padlock probes (ii), each padlock probe being specific to a different target nucleic acid sequence, and an optional ligation template (iii).

26. The kit according to claim 24 or 25, wherein The kit is used to detect different variants of a target nucleic acid sequence and comprises two or more different padlock probes, each probe being specific to a different variant of the target sequence.

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