Patent Description:
The detection of target nucleic acid molecules has applications in many different fields, including notably clinically, for personalized medicine and in the diagnosis, prognosis and/or treatment of diseases, such as cancer, infectious diseases and inherited or genetic disorders, as well as in research and biosecurity.

Target nucleic acid molecules may be detected using labelled hybridization probes, but hybridization probes have relatively high detection limit, and cannot readily be used to discriminate between similar nucleic acid sequences. To increase sensitivity, target nucleic acid molecules are typically amplified, to increase the amount of target nucleic acid sequence available for detection. Any of a variety of techniques known in the art may be used for the amplification, including rolling circle amplification (RCA).

RCA utilizes a strand displacement polymerase enzyme, and requires a circular amplification template. Amplification of the circular template provides a concatenated RCA product (RCP) comprising multiple copies of a sequence complementary to that of the amplification template. Such a concatemer typically forms a ball or "blob", which may be visualized and detected, and thus RCA-based assays have been adopted for the detection of nucleic acid molecules.

The specificity of nucleic acid molecule detection may be improved by the use of probes requiring dual recognition, or two binding sites for a target nucleic acid molecule, such as padlock probes. Padlock probes are linear oligonucleotides with two separate target-complementary binding regions, connected by an intervening "backbone" region. When the padlock probe has bound (hybridized) to its target nucleic acid sequence, the ends of the padlock probe may be ligated together to circularize the padlock probe. The circularized padlock probe may then be used as the template for a RCA reaction, and the RCP may be detected. The usage of padlock probes in detection of target nucleic acid molecules was initially described in <CIT>.

<CIT> relates to multiplexed methods of detecting an analyte in a sample using two or more padlock probes each specific to a different target sequence. The target sequence is either part of an analyte or indicative of the presence of an analyte in the sample. Each padlock probe comprises an analyte-specific reporter sequence, and either a restriction cleavage site located <NUM>' of the analyte-specific reporter sequence or a first amplification primer binding site for an amplification reaction.

Padlock probes and RCA have been suggested as detection of nucleic acid molecules of pathogens, such as viruses or bacteria, such as in <CIT> and <CIT>.

<NPL>) discloses the introduction of a cross-hybridizing DNA oligonucleotide during rolling circle amplification to obtain smaller fluorophore-labeled RCPs. The reduced size of the RCPS increases the local concentration of fluorophores and thereby the signal intensity increases together with the signal-to-noise ratio.

<NPL>) discloses an assay for detection of pathogens. The assay uses a pair of universal primers for identifying target genes per reach tube. Ring padlock probes and corresponding universal primers start hyperbranched rolling circle amplification (HRCS) under the action of the polymerase to obtain branched chain amplification products, which are entangled with magnetic particles to form aggregated magnetic particle clusters.

A limitation of the prior art technology is that RCA needs to be run for a relative long period of time in order to produce RCPs of sufficient length in order to form a ball or "blob", which may be visualized and detected.

There is, thus, a need for a technology that speeds up the detection of target nucleic acid molecules.

It is a general objective to enable detection of target nucleic acid molecules in a shorter period of time as compared to prior art protocols based on padlock probes and rolling circle amplification.

This and other objectives are met by embodiments as disclosed herein.

Further embodiments of the invention are defined in the dependent claims.

An aspect of the invention relates to a method for detecting at least one target nucleic acid molecule in a sample. The method comprises contacting the sample with padlock probes comprising at their <NUM>' and <NUM>' ends target-binding regions complementary to probe-binding regions in the at least one target nucleic acid molecule. The method also comprises joining the <NUM>' and <NUM>' ends of the padlock probes while the target-binding regions are hybridized to the probe-binding regions to form circular padlock probes. The method further comprises rolling circle amplifying the circular padlock probes with labelled amplification primers comprising a detectable label and a probe-binding region complementary to a primer-binding region of the padlock probes to generate labelled rolling circle products. The method further comprises contacting the labelled rolling circle products with oligonucleotides comprising a plurality of binding regions having a nucleic acid sequence corresponding to at least a portion of the padlock probes outside of the primer-binding region to form an agglutinate of labelled complexes between the oligonucleotides and the labelled rolling circle products. The method additionally comprises detecting the agglutinate of the labelled complexes to detect the at least one target nucleic acid molecule.

Another aspect of the invention relates to kit for detecting at least one target nucleic acid molecule in a sample. The kit comprises padlock probes comprising at their <NUM>' and <NUM>' ends target-binding regions complementary to probe-binding regions in at least one target nucleic acid molecule. The kit also comprises labelled amplification primers comprising a detectable label and a probe-binding region complementary to a primer-binding region of the padlock probes. The kit further comprises oligonucleotides comprising a plurality of binding regions having a nucleic acid sequence corresponding to at least a portion of the padlock probes outside of the primer-binding region.

A further aspect of the invention relates to a microfluidic chip comprising a sample input configured to receive a sample comprising at least one target nucleic acid molecule and a microfluidic channel in fluid connection with the sample input. The microfluidic channel comprises a reagent section comprising i) padlock probes comprising at their <NUM>' and <NUM>' ends target-binding regions complementary to probe-binding regions in the at least one target nucleic acid molecule, ii) a ligase adapted to ligate the <NUM>' and <NUM>' ends of the padlock probes while the target-binding regions are hybridized to the probe-binding regions to form circular padlock probes, iii) labelled amplification primers comprising a detectable label and a probe-binding region complementary to a primer-binding region of the padlock probes, and iv) a polymerase adapted to rolling circle amplify the circular padlock probes with the labelled amplification primers to generate labelled rolling circle products. The microfluidic channel also comprises a detection section comprising oligonucleotides comprising a plurality of binding regions having a nucleic acid sequence corresponding to at least a portion of the padlock probes outside of the primer-binding region. The microfluidic chip also comprises a detection window aligned with at least a portion of the detection section and arranged to enable visual access to the at least a portion of the detection section.

The agglutinate-promoting oligonucleotides contacted with the labelled rolling circle products (RCPs) promote formation of a detectable ball or agglutinate even for comparatively short RCPs, thereby only requiring a short duration of rolling circle amplification to get a detectable signal. Experimental data as presented herein indicate that the RCA can be reduced by at least a factor of four and still producing a more easily detectable signal as compared to prior art protocols lacking the usage of agglutinate-promoting oligonucleotides.

The present invention generally relates to detection of target nucleic acid molecules, and in particular to detection of such target nucleic acid molecules using padlock probes and rolling circle amplification.

Padlock probes and rolling circle amplification (RCA) can be used to detect the presence of target nucleic acid molecules in a sample at a high specificity since the padlock probes require dual recognition and ligation to form a circular padlock probe that can be amplified by RCA into an RCA product (RCP), which is a concatemeric product comprising multiple repeats of a complementary copy of the circularized padlock probe. In the art, such an RCP can be detected using labelled amplification primers during RCA so that the resulting RCPs will incorporate the label. If the RCPs have sufficient length, i.e., contain a sufficient high number of complementary copies of the circular padlock probe, the RCPs will become entangled with each other forming a labelled ball that could, due to the presence of the labels, be detected. In the art, the RCA must therefore be run for a comparatively long period of time in order to produce RCPs of sufficient length in order to form the ball or "blob", which may be visualized and detected. As a consequence, the RCA reaction time constitutes the major portion of the total time for detecting target nucleic acid molecules in a sample in the prior art.

The present invention significantly speeds up such detection by merely requiring comparatively shorter RCPs, and thereby shorter RCA reaction times, and still being able to detect the labelled RCPs. This is possible according to the invention by using oligonucleotides comprising a plurality of binding regions, to which the labelled RCPs can bind. The oligonucleotides thereby promote entanglement of also shorter labelled RCPs to form a detectable ball or "blob", i.e., an agglutinate or aggregate, at a significant shorter period of time as compared to prior art techniques. This can be seen by comparing <FIG> showing detectable agglutinates or aggregates of labelled RCPs bound to oligonucleotides according to the invention following <NUM> of RCA and <FIG> showing prior art, i.e., no oligonucleotides, after <NUM> (<FIG>), <NUM> (<FIG>) and <NUM> (<FIG>) of RCA. Hence, the present invention provides more easily detectable labelled complexes already after merely <NUM> of RCA as compared to the prior art with <NUM> of RCA.

An aspect of the invention thereby relates to a method for detecting at least one target nucleic acid molecule <NUM> in a sample, see <FIG> and <FIG>. The method comprises contacting the sample in step S1 with padlock probes <NUM> comprising at their <NUM>' and <NUM>' ends <NUM>, <NUM> target-binding regions <NUM>, <NUM> complementary to probe-binding regions <NUM>, <NUM> in the at least one target nucleic acid molecule <NUM>, see <FIG>. A next step S2 comprises joining the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM> while the target-binding regions <NUM>, <NUM> are hybridized to the probe-binding regions <NUM>, <NUM> to form circular padlock probes <NUM>', see <FIG>. The method also comprises rolling circle amplifying the circular padlock probes <NUM>' in step S3 with labelled amplification primers <NUM> comprising a detectable label <NUM> and a probe-binding region <NUM> complementary to a primer-binding region <NUM> of the padlock probes <NUM> to generate labelled rolling circle products <NUM>', see <FIG>. The labelled rolling circle products <NUM>' are contacted in step S4 with oligonucleotides <NUM>, 30A, see <FIG>, comprising a plurality of binding regions <NUM> having a nucleic acid sequence corresponding to at least a portion of the padlock probes <NUM> outside of the primer-binding region <NUM> to form an agglutinate <NUM> of labelled complexes <NUM> between the oligonucleotides <NUM>, 30A and the labelled rolling circle products <NUM>', see <FIG>. The agglutinate <NUM> of the labelled complexes <NUM> are then detected in step S5 to detect the at least one target nucleic acid molecule <NUM>.

The method as shown in <FIG> thereby produces an agglutinate <NUM> of labelled complexes <NUM> if the sample contained the at least one target nucleic acid molecule <NUM>. This means that presence of the agglutinate <NUM> of the labelled complexes <NUM> in step S5 of <FIG> is an indication of presence of the at least one target nucleic acid molecule <NUM> in the sample <NUM>.

The at least one target nucleic acid molecule <NUM> detected by the method as shown in <FIG> could be any nucleic acid molecule, such as a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or a nucleic acid molecule comprising synthetic nucleotides or nucleotide analogues, such as locked nucleic acid (LNA), peptide nucleic acid (PNA), etc. The at least one target nucleic acid molecule <NUM> could be single-stranded or double-stranded.

Illustrative, but non-limiting, examples of target DNA molecules include genomic DNA molecules, cell-free DNA (cfDNA) molecules, circulating tumor DNA (ctDNA) molecules, complementary DNA (cDNA) molecules, or indeed any other DNA molecule from any source, such as viral, bacterial or fungal DNA molecules.

Illustrative, but non-limiting, examples of target RNA molecules include viral, bacterial or fungal RNA molecules, or indeed RNA molecules from any source, such as RNA genomic molecules, complementary RNA (cRNA) molecules, RNA molecules from virions (vRNA), micro RNA (miRNA) molecules, small interfering RNA (siRNA) molecules, messenger RNA (mRNA) molecules, transfer RNA (tRNA) molecules, ribosomal RNA (rRNA) molecules, such as <NUM> rRNA molecules, small nuclear RNA (snRNA) molecules, small nucleolar RNA (snoRNA) molecules, extracellular RNA (exRNA) molecules, piwi-interacting RNA (piRNA) molecules and long non-coding RNA molecules.

The target nucleic acid molecule is preferably present in a sample, such as a biological sample, for instance, from a subject, such as an animal subject, preferably a mammal subject, and more preferably a human subject. In such a case, the biological sample could be a body fluid sample, such as a blood sample, a blood plasma sample, a blood serum sample, a saliva sample, a sputum sample, a mucosal sample, such as a nasal mucosal lining fluid sample, an oropharyngeal sample, a nasopharyngeal sample, a nasal sample, a bronchoalveolar lavage fluid (BALF) sample, a breast milk sample, a cerebrospinal fluid sample, a urine sample, a feces sample, a tear fluid sample or an endometrial fluid sample, or a body tissue sample, such as a biopsy sample, or a cell sample.

The sample may contain any pathogen or cellular material, including prokaryotic cells, eukaryotic cells, viruses, bacteria, fungi, bacteriophages, mycoplasmas, protoplasts and organelles. For instance, the sample may contain pathogens, such as bacteria, fungi and/or viruses, isolated from a clinical sample taken from a subject. In such a sample, the at least one target nucleic acid molecule may be at least one fungal nucleic acid molecule, at least one bacterial nucleic acid molecule and/or at least one viral nucleic acid molecule.

Other examples include environmental samples including, but not limited to, soil samples, water samples, food samples, etc..

In an embodiment, the at least one target nucleic acid molecule is indicative of the presence of an analyte. For instance, the at least one target nucleic acid molecule could be a reporter for the analyte. Such a reporter can then be used or generated during an assay for an analyte, such as a target molecule, which is further described herein in connection <FIG>. The target nucleic acid molecule may then act as a reporter and be in the form of a tag or label attached to or forming part of one or more target-binding molecules. A target nucleic acid molecule as a reporter is optionally generated during an assay or in connection with detection of such a target molecule, for example by a ligation reaction in a proximity ligation assay, an extension reaction in a proximity extension assay, or by a cleavage reaction. In such a case, the at last one target nucleic acid molecule could be at least one synthetic or artificial nucleic acid molecule.

The sample may optionally be treated or processed prior to contacting the sample with the padlock probes <NUM> in step S1 of <FIG>. For instance, any target nucleic acid molecules <NUM> could be isolated, separated or removed from an original sample to get an enriched sample that is used in step S1. Other examples of sample processing include fragmenting larger nucleic acid molecules present in the sample into shorter nucleic acid fragments, at least a portion of which are used as target nucleic acid molecules <NUM>.

The method of the invention uses padlock probes <NUM> that are contacted in step S1 with the sample. The padlock probes <NUM> of the invention comprise, from their <NUM>' end <NUM> towards their <NUM>' end <NUM>, a first target-binding region <NUM>, a connecting bridge <NUM>, and a second target-binding region <NUM>. The connecting bridge <NUM>, also referred to as backbone region, connects the two target-binding regions <NUM>, <NUM> present at the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM>, see <FIG>. This connecting bridge <NUM> comprises the primer-binding region <NUM>, to which the labelled amplification primers <NUM> can bind in step S3.

Padlock probe <NUM> as used herein refers to any probe capable being circularized following hybridization to a target nucleic acid molecule <NUM>.

The target-binding regions <NUM>, <NUM> of the padlock probes <NUM> are complementary to probe-binding regions <NUM>, <NUM> in the at least one target nucleic acid molecule <NUM>.

Complementary as used herein refers both to complete complementarity of nucleotide sequences, in some cases referred to as an identical sequence, as well as complementarity sufficient to achieve the desired binding of nucleotide sequences. Complementary refers to the standard base pairing rules between G-C, A-T and A-U. Certain nucleotides not commonly found in natural nucleotide sequences or chemically synthesized as mentioned in the foregoing may be included in the nucleotide sequences described herein. Complementarity need not be perfect. In clear contrast, stable duplexes may contain mismatched base pairs, degenerative, or unmatched nucleotides. Complementary is characterized by the capacity for precise pairing of purine and pyrimidine bases of one sequence (strand, oligonucleotide) of a nucleic acid molecule to another sequence of a nucleic acid molecule, such that the order of purine and pyrimidine bases matches and binds to (hybridizes with) the other, complementary sequence.

The complementarity between the target-binding regions <NUM>, <NUM> and the probe-binding regions <NUM>, <NUM> allows the target-binding regions <NUM>, <NUM> of the padlock probes <NUM> to hybridize to the probe-binding regions <NUM>, <NUM> of the at least one target nucleic acid molecule <NUM>. Thus, the at least one target nucleic acid molecule <NUM> is contacted with the padlock probes <NUM> under hybridization conditions, during which the first target-binding region <NUM> complementary to a first probe-binding region <NUM> of the at least one target nucleic acid molecule <NUM> hybridizes to this first probe-binding region <NUM> and the second target-binding region <NUM> complementary to a second probe-binding region <NUM> of the at least one target nucleic acid molecule <NUM> hybridizes to this second probe-binding region <NUM> as shown in <FIG>. First and second as used herein for the first and second probe-binding region <NUM>, <NUM> and the first and second target-binding region <NUM>, <NUM> do not denote any sequence of order, at which these regions <NUM>, <NUM>; <NUM>, <NUM> hybridize to each other.

Hybridization or hybridization condition denotes the process in which single-stranded nucleotide sequences anneal to complementary nucleotide sequences. Such annealing between complementary nucleotide sequences is dependent on several parameters including, for instance, ionic strength, temperature, length of the target-binding regions <NUM>, <NUM>, and G-C-nucleotides content of the target-binding regions <NUM>, <NUM>.

Step S1 in <FIG> could be performed according to various embodiments. For instance, the padlock probes <NUM> could be added to the sample, which is then present in a suitable vessel, such as a well of a multi-well plate, a reaction tube, for instance an Eppendorf® tube, etc. Alternatively, the padlock probes <NUM> could be pre-loaded in such a well or reaction tube and then the sample is added thereto. It is also possible to use a microfluidic chip <NUM>, see <FIG> pre-loaded with, among others, the padlock probes <NUM> and comprising a sample input or inlet <NUM>, to which the sample is added. A further embodiment is to mix the sample with the padlock probes <NUM> and then adding the mixture to the well, reaction tube or microfluidic chip <NUM>.

The next step S2 in <FIG> comprises joining the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM> while the target-binding regions <NUM>, <NUM> are hybridized to the probe-binding regions <NUM>, <NUM> to form the circular padlock probes <NUM>', see <FIG>. The joining of the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM> could be a direct join of the <NUM>' and <NUM>' ends <NUM>, <NUM> or an indirect join of the <NUM>' and <NUM>' ends <NUM>, <NUM>.

In an embodiment, step S2 in <FIG> comprises ligating the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM> while the target-binding regions <NUM>, <NUM> are hybridized to the probe-binding regions <NUM>, <NUM> to form the circular padlock probes <NUM>'. In such an embodiment, a ligating agent is used to ligate the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM>. In an embodiment, the ligating agent is a ligase, and in particular a DNA ligase. A ligase is an enzyme that facilitates the joining of nucleotide strands together by catalyzing the formation of a phosphodiester bond. Any ligase capable of ligating together the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM> while the target-binding regions <NUM>, <NUM> are hybridized to the probe-binding regions <NUM>, <NUM> can be used according to the embodiments.

In a particular embodiment, the ligating agent is a thermostable ligating agent, preferably a thermostable ligase, and in particular a thermostable DNA ligase. For instance, the thermostable ligase could be selected from the illustrative group comprising Ampligase® DNA ligase, Taq DNA ligase, Pfu DNA ligase and <NUM>°N™ DNA ligase. Other non-limiting, but illustrative, examples of DNA ligases include Chlorella virus DNA ligase, also referred to as Paramecium bursaria Chlorella virus <NUM> (PBCV-<NUM>) DNA ligase or SplintR ligase, Escherichia coli DNA ligase encoded by the lig gene; T4 or T7 DNA ligase from bacteriophage T4 or T7; DNA ligase I, II, III or IV. An illustrative, but non-limiting, example of RNA ligases is T4 RNA ligase <NUM>, also referred to as T4 Rnl2 or gp24.

Optionally, step S2 of <FIG> comprises adding a ligating agent, preferably a ligase, more preferably a DNA ligase, to the sample or a mixture of the sample and the padlock probes <NUM> to ligate together the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM>. Alternatively, the ligating agent could be pre-mixed with the padlock probes <NUM>.

In an embodiment, the <NUM>' end <NUM> of the padlock probes <NUM> comprises a <NUM>' phosphate group to facilitate ligation of the <NUM>' and <NUM>' ends <NUM>, <NUM> using the ligating agent.

In the above-described embodiments, the padlock probes <NUM> hybridize to the at least one target nucleic acid molecule <NUM> with the <NUM>' and <NUM>' ends <NUM>, <NUM> directly adjacent to each other to facilitate direct ligation of the <NUM>' and <NUM>' end <NUM>, <NUM>.

In other embodiments, the target-binding regions <NUM>, <NUM> of the padlock probes <NUM> hybridize to the probe-binding regions <NUM>, <NUM> of the at least one target nucleic acid molecule <NUM> with a gap between the <NUM>' and <NUM>' ends <NUM>, <NUM>. The joining of the <NUM>' and <NUM>' ends <NUM>, <NUM> in step S2 is then an indirect joining of these ends <NUM>, <NUM>. In such a case, the gap between the <NUM>' and <NUM>' ends <NUM>, <NUM> is filled with at least one gap-filling oligonucleotide or molecular inversion probe that is capable of binding to the target nucleic acid molecule <NUM> at one or more regions in between the probe-binding regions <NUM>, <NUM>. Alternatively, or in addition, the gap can be filled by extension of the <NUM>' end <NUM> of the padlock probes <NUM> while hybridized to the at least one target nucleic acid molecule <NUM>. In such a case, the target nucleic acid molecule <NUM> acts as a template for the extension with the padlock probe <NUM> acting as primer. Such an extension is performed by a polymerase and nucleotides (dNTPs) added to the sample or a mixture of the sample and padlock probes <NUM>, or pre-mixed with the padlock probes <NUM>. In the above-described embodiments, a ligating agent, such as the above-described ligases, could join the <NUM>' and <NUM>' ends <NUM>, <NUM> and the gap-filling oligonucleotide(s) or the <NUM>' end and the extended <NUM>' end <NUM>.

Examples of polymerases that can be used for such an extension include polymerases used in step S3 and further discussed below.

Optionally, the polymerase used for <NUM>' end extension could be inactivated prior to rolling circle amplification in step S3. Such a polymerase inactivation can be performed according to well-known techniques including, but not limited to, heat inactivation.

The formed circular single stranded padlock probes <NUM>' are then amplified in step S3 by so-called RCA using labelled amplification primers <NUM> to generate the labelled RCPs <NUM>'. RCA uses a strand-displacing polymerase to extend the labelled amplification primers <NUM> hybridized to the primer-binding region <NUM> of the circular padlock probes <NUM>', see <FIG>. The strand displacing activity of the polymerase displaces the extended labelled amplification primers <NUM> effectively causing the circular padlock probes <NUM>' to "roll" during RCA.

Illustrative, but non-limiting, examples of strand-displacing polymerases that could be used in step S3 include Phi29 DNA polymerase, Bst polymerase, Klenow fragment, and derivatives thereof.

The strand-displacing polymerase and nucleotides (dNTPs) needed for RCA could be added to sample or the mixture of the sample and the padlock probes <NUM>, or are pre-mixed with the padlock probes <NUM>.

The labelled amplification primers <NUM> used in the RCA of step S3 in <FIG> comprise a probe-binding region <NUM> complementary to the primer-binding region <NUM> of the padlock probes <NUM>. Accordingly, the labelled amplification primers <NUM> are capable of hybridizing to this primer-binding region <NUM> of the circular padlock probes <NUM>'. The labelled amplification primers <NUM> could be added together with strand-displacing polymerase to the sample or the mixture of the sample and the padlock probes <NUM>, or could be pre-mixed with the padlock probes <NUM>. Thus, the labelled amplification primers <NUM> do not significantly interfere with the joining of the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM> and could thereby be present in the reaction mixture during such a joining in step S2.

The labelled amplification primers <NUM> comprise a detectable label <NUM>, preferably at or in connection with their <NUM>' ends. The detectable label <NUM> enables detection of the labelled complexes <NUM> produced in step S4 if the sample contained the at least one target nucleic acid molecule <NUM>. The label <NUM> could be any detectable label <NUM>. Illustrative, but non-limiting, examples of such detectable labels <NUM> include beads, microparticles, nanoparticles, fluorophores, radiolabels, metal-containing labels, colorimetric labels, etc..

In an embodiment, the detectable labels <NUM> are detectable beads <NUM>. In this embodiment, step S3 comprises RCA the circular padlock probes <NUM> with the labelled amplification primers <NUM> comprising a detectable bead <NUM> and the probe-binding region <NUM> to generate bead-labelled RCPs <NUM>'.

The detection of the agglutinate <NUM> of the complexes <NUM> incorporating the detectable label <NUM> in step S5 can then be performed according to various embodiments depending on the particular detectable label <NUM>. For instance, agglutinates <NUM> of complexes <NUM> incorporating beads, microparticles, nanoparticles, metal-containing labels or colorimetric labels could be detected by visible inspection or light microscopy, whereas fluorescence microscopy could be used to detect agglutinates <NUM> of complexes <NUM> comprising fluorophores <NUM> and autoradiography could be used for detection of agglutinates <NUM> of radiolabeled complexes <NUM>. Further, colorimetric readout by image analysis of images captured by a camera could be used to detect colorimetric labels.

Step S4 of <FIG> comprises contacting the labelled RCPs <NUM>' with oligonucleotides <NUM>, 30A comprising a plurality of binding regions <NUM> having a nucleic acid sequence corresponding to at least a portion of the padlock probes <NUM> outside of the primer-binding region <NUM> to form an agglutinate <NUM> of labelled complexes <NUM> between the oligonucleotides <NUM>, 30A and the labelled RCPs <NUM>', see <FIG>. This means that the oligonucleotides <NUM>, 30A comprises a plurality of binding regions <NUM> each capable of binding to a respective labelled RCP <NUM>'. Accordingly, the oligonucleotides <NUM>, 30A capture the labelled RCPs <NUM>' produced in the RCA in step S3.

The binding regions <NUM> of the oligonucleotides <NUM>, 30A correspond to at least a portion of the padlock probes <NUM> outside of the primer-binding region <NUM>. This at least a portion of the padlock probes <NUM> is preferably a portion of the connecting bridge <NUM> outside of the primer-binding region <NUM>. The at least a portion of the padlock probes <NUM> corresponds to the binding regions <NUM> and thereby enables the binding regions <NUM> to hybridize to the labelled RCPs <NUM>' at a respective portion of the repeats that is complementary to the at least a portion of the padlock probes <NUM>. This means that this at least a portion of the padlock probes <NUM> is different than and preferably non-overlapping with the primer-binding region <NUM>. The at least a portion of the padlock probes <NUM> may, though, at least partly overlap into the primer-binding region <NUM> with a short number of nucleotides as long as the binding regions <NUM> preferentially bind to the labelled RCPs <NUM>' over the labelled amplification primers <NUM>. This means that the oligonucleotides <NUM>, 30A bind the labelled RCPs <NUM>' but not the labelled amplification primers <NUM> or at least has preferential binding of the labelled RCPs <NUM>' over the labelled amplification primers <NUM>.

In an embodiment, the oligonucleotides <NUM> are linear single-stranded oligonucleotides <NUM> comprising the plurality of binding regions <NUM> as shown in <FIG>. In another embodiment, the oligonucleotides 30A are circular single-stranded oligonucleotides 30A comprising the plurality of binding regions <NUM> as shown in <FIG>. It is also possible to use a combination of linear oligonucleotides <NUM> and circular oligonucleotides 30A in step S4.

In an embodiment, the oligonucleotides 30A are circular oligonucleotides 30A. In such an embodiment, step S4 comprises contacting the labeled RCPs <NUM>' with circular oligonucleotides 30A comprising the plurality of binding regions <NUM> to form the agglutinate <NUM> of the labelled complexes <NUM> between the circular oligonucleotides 30A and the labelled RCPs <NUM>'.

The oligonucleotides <NUM>, 30A with captured labelled RCPs <NUM>' become entangled forming a ball or "blob", i.e., an agglutinate <NUM> as indicated in <FIG>. Such a ball or blob is detectable due to the presence of a plurality of labels <NUM> carried by the labelled RCPs <NUM>' hybridized to the oligonucleotides <NUM>, 30A'. Accordingly, the formed ball or "blob" can be detected as mentioned in the foregoing.

In an embodiment, step S4 in <FIG> comprises contacting the labelled RCPs <NUM>' with the oligonucleotides <NUM>, 30A to form an agglutinate <NUM> of multiple, i.e., at least two, labelled complexes <NUM>, see <FIG>. In such an embodiment, step S5 comprises detecting the agglutinate <NUM> of the multiple labelled complexed <NUM> to detect the at least one target nucleic acid molecule <NUM>. Hence, in this embodiment, multiple labelled complexes <NUM> are entangled to form an agglutinate <NUM> comprising multiple such labelled complexes <NUM>. <FIG> illustrates such agglutinates <NUM> comprising colored beads as labels <NUM>.

<FIG> is a flow chart illustrating an additional, optional step of the method shown in <FIG>. The method continues from step S2 in <FIG> and continues to step S10. This step S10 comprises removing any non-joined padlock probes <NUM> following joining the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM> in step S2 but prior to RCA the circular padlock probes <NUM> in step S3. The method then continues to step S3 in <FIG>.

<FIG> is a flow chart illustrating an embodiment of the removing step S10 in <FIG>. In this embodiment, the method comprises adding an exonuclease to the sample in step S11 to enzymatically degrade the non-joined padlock probes <NUM>. The method also comprises inactivating the exonuclease in step S11.

The exonuclease added to the sample in step S11 enzymatically cleaves nucleotides one at a time from the <NUM>' end <NUM> or the <NUM>' end <NUM> of the non-circularized padlock probes <NUM>. The exonuclease thereby catalyzes a hydrolyzing reaction that breaks phosphodiester bonds at either the <NUM>' end <NUM> or the <NUM>' end <NUM>. The exonuclease will not degrade the circular padlock probes <NUM>' following joining of the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM>.

Illustrative, but non-limiting, examples of exonucleases that could be used in the embodiment shown in <FIG> include exonuclease I, II, III, IV, V, VI, VII, and VIII.

The inactivation of the exonuclease in step S12 can be performed according to well-known techniques, including heat inactivation.

In this embodiment, the labelled amplification primers <NUM> are preferably added to the sample following inactivation of the exonuclease to prevent the exonuclease from degrading the labelled amplification primers <NUM>. Alternatively, if the labelled amplification primers <NUM> comprises the detectable label <NUM> at their <NUM>' ends, then the labelled amplification primers <NUM> could be present when the exonuclease is added if the exonuclease is a <NUM>' → <NUM>' exonuclease.

In another embodiment, the non-joined padlock <NUM> could be removed in step S10 by washing. In such an embodiment, a solid phase is used to capture the circular padlock probes <NUM>'. For instance, capturing oligonucleotides that will bind only circular padlock probes <NUM>' could be immobilized on the solid phase, such as by streptavidin-biotin binding. In such a case, the capturing oligonucleotides <NUM>, <NUM> will not capture free padlock probes <NUM>, i.e., non-circular padlock probes <NUM>. These free padlock probes <NUM> will be present in the solution and can then be washed away, such as by using a washing buffer. The streptavidin-biotin binding could, following the washing step, be released via competition reaction to thereby release the circular padlock probes <NUM>' and RCA can be performed.

In an embodiment, the padlock probes <NUM> could be so-called self-destruction padlock probes <NUM>. In such an embodiment, the <NUM>' arm of the padlock probes <NUM> is designed to enable hybridization to the padlock probe <NUM> with only a few nucleotides. This means that if the padlock probe <NUM> is not ligated, the padlock probe <NUM> will form a hairpin structure once the polymerase is added.

Removal of non-joined padlock probes <NUM> in step S10 could be beneficial to reduce the risk of non-joined padlock probes <NUM> binding to the RCPs <NUM>' forming a double-stranded product. Such a binding of non-joined padlock probes <NUM> to the RCPs <NUM>' could compete with binding of the RCPs <NUM>' to the oligonucleotides <NUM>, 30A. Non-joined padlock probes <NUM> could also be extended by the polymerase during RCA, and thereby using up a portion of the polymerase, thereby lowering the amount of formed RCPs <NUM>'.

In an embodiment, RCA in step S3 comprises RCA the circular padlock probes <NUM>' with the labelled amplification primers <NUM> to generate the labelled RCPs <NUM>' comprising up to <NUM> repeats of a nucleic acid sequence complementary to a nucleic acid sequence of the padlock probes <NUM>. Hence, in this embodiment, the labelled RCPs <NUM>' are concatemers comprising up to <NUM> copies of a nucleic acid sequence complementary to the circular padlock probes <NUM>'. In a preferred embodiment, the labelled RCPs <NUM>' comprise up to <NUM> repeats, and preferably up to <NUM> repeats of the nucleic acid sequence complementary to the nucleic acid sequence of the padlock probes <NUM>. In a currently preferred embodiment, the labelled RCPs <NUM>' comprise from <NUM> up to <NUM> repeats, preferably from <NUM> up to <NUM> repeats, and more preferably from <NUM> up to <NUM> repeats of the nucleic acid sequence complementary to the nucleic acid sequence of the padlock probes <NUM>.

Hence, the labelled RCPs <NUM>' generated according to these embodiments require comparatively short RCA times to thereby produce labelled RCPs <NUM>' with merely one or a few repeats. The present invention still enables detection of the labelled RCPs <NUM>' even though they merely contain such few repeats by using the oligonucleotides <NUM>, 30A that capture multiple such labelled RCPs <NUM>' in step S4. Hence, the present invention enables visual detection of the labelled RCPs <NUM>' with merely <NUM> of RCA (<FIG>), whereas visual detection of the labelled RCPs <NUM>' without usage of the oligonucleotides <NUM>, 30A of the invention require <NUM> of RCA to generate a high number of repeats of the nucleic acid sequence complementary to the nucleic acid sequence of the padlock probes <NUM>, and thereby sufficiently long labelled RCPs <NUM>'.

In an embodiment, step S4 of <FIG> comprises contacting the labelled RCPs <NUM>' with the oligonucleotides <NUM>, 30A comprising at least <NUM> repeats of the binding region <NUM>. In a preferred embodiment, the oligonucleotides <NUM>, 30A comprise at least <NUM> repeats of the binding region <NUM>, preferably from <NUM> up to <NUM> repeats of the binding region <NUM>.

Hence, the oligonucleotides <NUM>, 30A comprise a comparatively high number of such binding regions <NUM> to thereby enable binding of a high number of labelled RCPs <NUM>' per such an oligonucleotide <NUM>, 30A. This means that each oligonucleotide <NUM>, 30A will typically bind a plurality of labelled RCPs <NUM>' and thereby comprise a plurality of detectable labels <NUM>, which facilitates detection of the labelled complexes <NUM> between the oligonucleotides <NUM>, 30A and the labelled RCPs <NUM>' even though the labelled RCPs <NUM>' merely comprise few repeats of the nucleic acid sequence complementary to the nucleic acid sequence of the padlock probes <NUM>.

The oligonucleotides <NUM>, 30A of the invention could be regarded as being agglutinate-promoting oligonucleotides <NUM>, 30A as they promote formation of detectable labelled complexes <NUM> and agglutinates <NUM> comprising the labelled RCPs <NUM>'. Hence, the oligonucleotides <NUM>, 30A compensate for the reduced length of the labelled RCPs <NUM>' by binding to multiple labelled RCPs <NUM>' that together cause formation of detectable complexes <NUM> and agglutinates <NUM> even though the labelled RCPs <NUM>' as such are too small or short to form any detectable agglutinates <NUM> without the oligonucleotides <NUM>, 30A.

In an embodiment, multiple sets of labelled amplification primers <NUM>, 20A that are complementary to different primer binding regions <NUM>, 11A in the padlock probes <NUM>. This is schematically shown in <FIG>. In such an embodiment, step S3 of <FIG> preferably comprises RCA the circular padlock probes <NUM>' with multiple sets of labelled amplification primers <NUM>, 20A to generate the labelled RCPs <NUM>'. The labelled amplification primers <NUM>, 20A in each set of the multiple sets comprise the same probe-binding region <NUM>, 21A. The probe-binding region <NUM> of the labelled amplification primers <NUM> in one set of the multiple sets is complementary to another primer-binding region <NUM> of the padlock probes <NUM> than the primer-binding region 21A of the labelled amplification primers 20A in another set of the multiple sets. The labelled amplification primers <NUM>, 20A in the multiple sets have or comprise the same detectable label <NUM>.

In <FIG>, two sets of different amplification primers <NUM>, 20A are used. In such a case, the amplification primers <NUM> in the first set comprise a first probe-binding region <NUM> complementary to a first primer-binding region <NUM> of the padlock probes <NUM> and a detectable label <NUM>. Correspondingly, the amplification primers 20A in the second set comprise a second probe-binding region 21A complementary to a second primer-binding region 11A of the padlock probes <NUM> and the detectable label <NUM>. This means that the connecting bridge <NUM> comprises, in this embodiment, two primer-binding regions <NUM>, 11A.

The embodiments are not limited to using merely two sets of labelled amplification primers <NUM>, 20A but could include more than two different sets.

In some embodiments, it could be beneficial to use multiple sets of labelled amplification primers <NUM>, 20A to increase the number of labelled RCPs <NUM>' produced in the RCA of step S3. As a consequence, a high number of labelled RCPs <NUM>' can be produced even though the RCA of step S3 is run for a short period of time. This in turn means that a high number of labelled RCPs <NUM>' are available to bind to the oligonucleotides <NUM>, 30A in step S4, which in turn facilitates formation of detectable labelled complexes <NUM>. This is possible since the labelled amplification primers <NUM>, 20A in the multiple sets comprise the same type of detectable label <NUM>, thereby producing the same detectable signal.

Another embodiment of increasing the detectable signal is to use multiple sets of padlock probes <NUM>, 10A as shown in <FIG>. In such an embodiment, step S1 in <FIG> comprises contacting the sample with multiple sets of padlock probes <NUM>, 10A. The padlock probes <NUM>, 10A in each set of the multiple sets comprise the same target-binding regions <NUM>, <NUM>; 12A, 13A. The target-binding regions <NUM>, <NUM> of the padlock probes <NUM> in one set of the multiple sets are complementary to other probe-binding regions <NUM>, <NUM> in the at least one target nucleic acid molecule <NUM> than the target-binding regions 12A, 13A of the padlock probes 10A in another set of the multiple sets. Step S3 comprises, in this embodiment, RCA the circular padlock probes <NUM>', 10A' with multiple sets of labelled amplification primers <NUM>, 20A to generate multiple sets of labelled rolling circle products <NUM>'. The labelled amplification primers <NUM>, 20A in each set of the multiple sets comprise the same probe-binding region <NUM>, 21A. The probe-binding region <NUM> of the labelled amplification primers <NUM> in one set of the multiple sets is complementary to another primer-binding region <NUM> of the padlock probes <NUM>, 10A than the primer-binding region 21A of the labelled amplification primers 20A in another set of the multiple sets. The labelled amplification primers <NUM>, 20A in the multiple sets have or comprise the same detectable label <NUM>.

In <FIG>, two sets of different padlock probes <NUM>, 10A are used. In such a case, the padlock probes <NUM> in a first set comprises a first set of target-binding regions <NUM>, <NUM> that are complementary to a first set of probe-binding regions <NUM>, <NUM> in the at least one target nucleic acid molecule <NUM>. Correspondingly, the padlock probes 10A in a second set comprises a second set of target binding regions 12A, 13A that are complementary to a second set of probe-binding regions 2A, 3A in the at least one target nucleic acid molecule <NUM>. This means that padlock probes <NUM>, 10A in the different sets hybridize to different regions <NUM>, <NUM>; 2A, 3A in the at least one target nucleic acid molecule <NUM>, see <FIG>. Furthermore, the labelled amplification primers <NUM> in a first set comprise a first probe-binding region <NUM> complementary to a first primer-binding region <NUM> of the padlock probes <NUM> in the first set and the detectable label <NUM>. Correspondingly, the labelled amplification primers 20A in a second set comprise a second probe-binding region 21A complementary to a second primer-binding region 11A of the padlock probes 10A in the second set and the detectable label <NUM>.

In this embodiment, two species or sets of labelled RCPs <NUM>' will be produced in the RCA of step S3. The two species of labelled RCPs <NUM>' comprise different nucleic acid sequences since they are produced by RCA of different padlock probes <NUM>, 10A having at least partly different nucleic acid sequences <NUM>, <NUM>, <NUM>; 2A, 3A, 11A.

In an embodiment, the padlock probes <NUM>, 10A in the multiple sets comprise a respective portion having the same nucleic acid sequence as the nucleic acid sequence of the binding regions <NUM> of the oligonucleotides <NUM>, 30A. This same portion corresponds to at least a portion of the connecting bridge <NUM>, 16A outside of the primer-binding region <NUM>, 11A. In such an embodiment, the oligonucleotides <NUM>, 30A can capture the different species or sets of labelled RCPs <NUM>' since at least a portion of the nucleic acid sequence of labelled RCPs <NUM>' is the same for the different sets of padlock probes <NUM>, 10A and therefore for the labelled RCPs <NUM>'. Hence, a single species of oligonucleotides <NUM>, 30A could thereby be used to capture the multiple sets of labelled RCPs <NUM>'.

In another embodiment, step S4 of <FIG> comprises contacting the multiple sets of labelled RCPs <NUM>' with multiple sets of oligonucleotides <NUM>, 30A to form an agglutinate <NUM> of multiple sets of labelled complexes <NUM> between the oligonucleotides <NUM>, 30A and the labelled RCPs <NUM>'. In this embodiment, the oligonucleotides <NUM>, 30A in each set of the multiple sets comprise the same binding regions <NUM>. The binding region <NUM> of the oligonucleotides <NUM>, 30A in one set of the multiple sets has a nucleic acid sequence different than the nucleic acid sequence of the binding region <NUM> of the oligonucleotides in another set of the multiple sets. In this embodiment, different species of oligonucleotides <NUM>, 30A are used to capture the different species of labelled RCPs <NUM>'. This means that multiple sets of labelled complexes <NUM> are formed but all these labelled complexes <NUM> comprise the same type of detectable label <NUM> and thereby produce the same detectable signal in the form of a detectable agglutinate <NUM>.

In a further embodiment, the labelled amplification primers <NUM>, 21A of the two sets have different detectable labels <NUM>. In such a case, a target nucleic acid molecule <NUM> comprising the first and second sets of probe-binding regions <NUM>, <NUM>; 2A, 3A will produce two detectable signals in the form of differently labelled agglutinates <NUM> or an agglutinate <NUM> comprising labelled complexes <NUM> with different labels <NUM>. Hence, in such a case a correct detection of the target nucleic acid molecule <NUM> in the sample would imply detection of two different labelled complexes <NUM>. In such an embodiment, step S1 in <FIG> comprises contacting the sample with multiple sets of padlock probes <NUM>, 10A. The padlock probes <NUM>, 10A in each set of the multiple sets comprise the same target-binding regions <NUM>, <NUM>; 12A, 13A. The target-binding regions <NUM>, <NUM> of the padlock probes <NUM> in one set of the multiple sets are complementary to other probe-binding regions <NUM>, <NUM> in the at least one target nucleic acid molecule <NUM> than the target-binding regions 12A, 13A of the padlock probes 10A in another set of the multiple sets. Step S3 comprises, in this embodiment, RCA the circular padlock probes <NUM>', 10A' with multiple sets of labelled amplification primers <NUM>, 20A to generate multiple sets of labelled rolling circle products <NUM>'. The labelled amplification primers <NUM>, 20A in each set of the multiple sets comprise the same probe-binding region <NUM>, 21A and the same detectable label <NUM>. The probe-binding region <NUM> of the labelled amplification primers <NUM> in one set of the multiple sets is complementary to another primer-binding region <NUM> of the padlock probes <NUM>, 10A than the primer-binding region 21A of the labelled amplification primers 20A in another set of the multiple sets. Furthermore, the detectable label <NUM> of the labelled amplification primers <NUM> in one set of the multiple sets is different than the detectable label of the labelled amplification primers 20A in another set of the multiple sets. Step S4 of <FIG> comprises contacting the multiple sets of labelled RCPs <NUM>' with multiple sets of oligonucleotides <NUM>, 30A to form at least one agglutinate <NUM> of multiple sets of labelled complexes <NUM> between the oligonucleotides <NUM>, 30A and the labelled RCPs <NUM>'. In this embodiment, the oligonucleotides <NUM>, 30A in each set of the multiple sets comprise the same binding regions <NUM>. The binding region <NUM> of the oligonucleotides <NUM>, 30A in one set of the multiple sets has a nucleic acid sequence different than the nucleic acid sequence of the binding region <NUM> of the oligonucleotides in another set of the multiple sets. In this embodiment, different species of oligonucleotides <NUM>, 30A are used to capture the different species of labelled RCPs <NUM>'. This means that an agglutinate <NUM> of multiple sets of labelled complexes <NUM> or multiple agglutinates <NUM>, each of a respective set of labelled complexes <NUM>, are formed and produce the different detectable signals.

This embodiment could increase the specificity of the method by requiring multiple different detectable signals to confirm presence of the at least one target nucleic acid molecule <NUM> in the sample.

A further embodiment is shown by <FIG>. In this embodiment, step S1 of <FIG> comprises contacting the sample with multiple sets of padlock probes <NUM>, 10A. The padlock probes <NUM>, 10A in each set of the multiple sets comprise the same target-binding regions <NUM>, <NUM>; 12A, 13A. The target-binding regions <NUM>, <NUM> of the padlock probes <NUM> in one set of the multiple sets are complementary to other probe-binding regions <NUM>, <NUM> in the at least one target nucleic acid molecule <NUM> than the target-binding regions 12A, 13A of the padlock probes 10A in another set of the multiple sets, see <FIG>. However, in this embodiment, the padlock probes <NUM>, 10A in the multiple sets comprise the same primer binding region <NUM>.

This means that only a single set of labelled amplification primers <NUM> are needed in the RCA of step S3 since the labelled amplification primers <NUM> can bind to the circular padlock probes <NUM>', 10A' even though these circular padlock probes <NUM>', 10A' partly comprise different nucleic acid sequences <NUM>, <NUM>; 12A, 13A since the primer-binding region <NUM> is the same in the multiple sets of padlock probes <NUM>, 10A, see <FIG>.

The present invention can be used to detect the presence of multiple different target nucleic acid molecules <NUM>, 1B in a single sample, see <FIG>. In such an embodiment, step S1 of <FIG> comprises contacting a sample comprising multiple different target nucleic acid molecules <NUM>, 1B with multiple sets of padlock probes <NUM>, 10B, see <FIG>. The padlock probes <NUM>, 10B in each set of the multiple sets comprise the same target-binding regions <NUM>, <NUM>; 12B, 13B. The target-binding regions <NUM>, <NUM> of the padlock probes <NUM> in one set of the multiple sets are complementary to other probe-binding regions <NUM>, <NUM> in the multiple different target nucleic acid molecules <NUM>, 1B than the target-binding regions 12B, 13B of the padlock probes 10B in another set of the multiple sets.

Step S2 comprises, in this embodiment, joining the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM>, 10B while the target-binding regions <NUM>, <NUM>; 12B, 13B are hybridized to the probe-binding regions <NUM>, <NUM>; 2B, 3B to form circular padlock probes <NUM>', 10B'.

Step S3 comprises, in this embodiment, RCA the circular padlock probes <NUM>', 10B' with multiple sets of labelled amplification primers <NUM>, 20B to generate the labelled RCPs <NUM>', 20B', see <FIG> and <FIG>. The labelled amplification primers <NUM>, 20B in each set of the multiple sets comprise the same probe-binding region <NUM>, 21B and the same detectable label <NUM>, 22B. The probe-binding region <NUM> of the labelled amplification primers <NUM> in one set of the multiple sets is complementary to another primer-binding region <NUM> of the padlock probes <NUM>, 10B than the primer-binding region 21B of the labelled amplification primers 20B in another set of the multiple sets. The detectable label <NUM> of the labelled amplification primers <NUM> in one set of the multiple sets is different than the detectable label 22B of the labelled amplification primers 20B in another set of the multiple sets.

The method also comprises, in this embodiment, contacting the labelled RCPs <NUM>', 20B' with multiple sets of oligonucleotides <NUM>, 30B in step S4 to form at least one agglutinate <NUM> of the labelled complexes <NUM>, 40B between the oligonucleotides <NUM>, 30B and the labelled RCPs <NUM>', 20B', see <FIG>. In this embodiment, the oligonucleotides <NUM>, 30B in each set of the multiple sets comprise the same binding regions <NUM>, 31B. The binding regions <NUM> of the oligonucleotides <NUM> in one set of the multiple sets have different nucleic acid sequences than the binding regions 31B of the oligonucleotides 30B in another set of the multiple sets.

Step S5 comprises detecting at least one agglutinates <NUM> of multiple labelled complexes <NUM>, 40B with different labels <NUM>, 22B to detect the multiple target nucleic acid molecules <NUM>, 1B.

This step S5 could, thus, involve detecting one agglutinate 50that is a mixture of the multiple labelled complexes <NUM>, 40B with different labels <NUM>, 22B or detecting multiple agglutinates <NUM>, each being an agglutinate <NUM> of multiple complexed <NUM>, 40B with a respective label <NUM>, 22B.

The above-described and in <FIG> shown embodiment thereby enables detection of the presence of multiple different target nucleic acid molecules <NUM>, 1B in the same sample. As an example, such an embodiment could be used to detect the presence of multiple pathogens, such as bacteria and/or viruses, in a sample if the padlock probes <NUM>, 10B of the different sets are designed to bind to target nucleic acid molecules <NUM>, 1B from different bacteria and/or viruses.

This embodiment thereby provides a multiplex detection allowing identification of one or more different species of target nucleic acid molecules in a single sample, which thereby could indicate the presence of one or more pathogens.

The embodiments described in the foregoing in connection with <FIG> can be applied to the embodiment discussed above and in connection with <FIG>. In such a case, multiple sets of padlock probes could be targeting one nucleic acid molecule <NUM> and one or multiple sets of padlock probes could be targeting another nucleic acid molecule 1B in the same sample.

As mentioned in the foregoing, the embodiments described in connection with <FIG> are not limited to using two sets of padlock probes <NUM>, 10A, 10B implying that more than two sets could be used.

In an embodiment, the at least one target nucleic acid molecule is not originally present in the sample to be tested. Rather the sample comprises at least one target molecule <NUM>, see <FIG>. Such a target molecule <NUM> could then be a molecule other than a nucleic acid molecule or be a complex of one or more nucleic acid molecules and one or more other molecules. As an illustrative example, the target molecule <NUM> could be a protein, a polypeptide or at least comprise at least one protein or polypeptide domain.

In such a case, target-binding molecules <NUM>, <NUM> having affinity for, and preferably binding specifically to, the target molecule <NUM> and comprising a respective target nucleic acid molecule <NUM>', <NUM>" could be used. This means that the target nucleic acid molecules <NUM>', <NUM>" are thereby introduced by the target-binding molecules <NUM>, <NUM>. In such a case, the target-binding molecules <NUM>, <NUM> bind to different regions, such as epitopes, of the target molecule <NUM> to bring the target nucleic acid molecules <NUM>', <NUM>" attached to, or forming part of, the target-binding molecules <NUM>, <NUM> in close proximity of each other allowing a padlock probe <NUM> to bind to respective probe-binding regions <NUM>, <NUM> in the respective target nucleic acid molecules <NUM>', <NUM>" as shown in <FIG>.

The detection of the target nucleic acid molecules <NUM>', <NUM>" in the sample is then indicative of the presence of the target molecule <NUM> in the sample.

In an embodiment, the target-binding molecules <NUM>, <NUM> are antibodies, or antigen-binding fragments thereof.

The antibodies <NUM>, <NUM>, or the antigen-binding fragments thereof, specifically bind to the target molecule <NUM>, and preferably to different epitopes on the target molecule <NUM>.

The specificity of an antibody, or an antigen-binding fragment thereof, can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation (Kd) of an antigen with the antibody, or the antigen-binding fragment thereof, is a measure for the binding strength between an antigenic determinant, i.e., epitope, and an antigen-binding site on the antibody, or the antigen-binding fragment thereof. The lesser the value of Kd, the stronger the binding strength between the antigenic determinant and the antibody, or the antigen-binding fragment thereof. Alternatively, the affinity can also be expressed as the affinity constant (Ka), which is <NUM>/Kd. As will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest.

Avidity is the measure of the strength of binding between an antibody, or an antigen-binding fragment thereof, and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antibody, or the antigen-binding fragment thereof, and the number of pertinent binding sites present on the antibody, or the antigen-binding fragment thereof.

Typically, antibodies, or antigen-binding fragments thereof, will bind to their antigen with a dissociation constant (Kd) of <NUM>-<NUM> to <NUM>-<NUM> moles/liter (M) or less, and preferably <NUM>-<NUM> to <NUM>-<NUM> M or less and more preferably <NUM>-<NUM> to <NUM>-<NUM> M, i.e., with an association constant (Ka) of <NUM><NUM> to <NUM><NUM> M-<NUM> or more, and preferably <NUM><NUM> to <NUM><NUM> M-<NUM> or more and more preferably <NUM><NUM> to <NUM><NUM> M-<NUM>.

Typically, any Kd value greater than <NUM>-<NUM> M (or any Ka value lower than <NUM><NUM> M-<NUM>) is generally considered to indicate non-specific binding.

Specific binding of an antibody, or an antigen-binding fragment thereof, to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art.

In an embodiment, the antibodies <NUM>, <NUM> are monoclonal antibodies. In other embodiments, the antibodies <NUM>, <NUM> are polyclonal antibodies, or a combination of monoclonal and polyclonal antibodies.

An antigen-binding fragment of an antibody as used herein can be selected from a group consisting of a single chain antibody, a Fv fragment, a scFv fragment, a Fab fragment, a F(ab')<NUM> fragment, a Fab' fragment, a Fd fragment, a single-domain antibody (sdAb), a scFv-Fc fragment, and a di-scFv fragment.

In such embodiment, step S1 of <FIG> comprises contacting the sample with multiple target-binding molecules <NUM>, <NUM> binding specifically to a target molecule <NUM>, and padlock probes <NUM>. Each target-binding molecule <NUM>, <NUM> of the multiple target-binding molecules <NUM>, <NUM> comprise a respective target nucleic acid molecule <NUM>', <NUM>". The padlock probes <NUM> comprise at their <NUM>' and <NUM>' ends target-binding regions <NUM>, <NUM> complementary to probe-binding regions <NUM>, <NUM> in the respective target nucleic acid molecules <NUM>', <NUM>". Step S2 comprises, in this embodiment, joining the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM> while the target-binding regions <NUM>, <NUM> are hybridized to the probe-binding regions <NUM>, <NUM> and the multiple target-binding molecules <NUM>, <NUM> are binding to the target molecule <NUM> to form circular padlock probes <NUM>'. In this embodiment, step S5 comprises detecting the agglutinate <NUM> of the labelled complexes <NUM> to detect the respective target nucleic acid molecules <NUM>', <NUM>" and thereby the target molecule <NUM>.

In an embodiment, steps S1-S4 of <FIG> are performed in a reaction tube <NUM>, such as an Eppendorf® tube, see <FIG>, or in a well of a multi-well plate. The detection step S5 can also be performed with the labelled complexes <NUM> present in the reaction tube <NUM> or well.

In an embodiment, the above-described steps S1-S4 and optionally also S5 are performed in solution, i.e., without the need for any solid phase.

In another embodiment, the method of the invention is performed using a microfluidic chip <NUM>, which will be further described below in connection with <FIG>.

Another aspect of the invention relates to a kit for detecting at least one target nucleic acid molecule <NUM> in a sample. The kit comprises padlock probes <NUM> comprising at their <NUM>' and <NUM>' ends <NUM>, <NUM> target-binding regions <NUM>, <NUM> complementary to probe-binding regions <NUM>, <NUM> in at least one target nucleic acid molecule <NUM>. The kit also comprises labelled amplification primers <NUM> comprising a detectable label <NUM> and a probe-binding region <NUM> complementary to a primer-binding region <NUM> of the padlock probes <NUM>. The kit further comprises oligonucleotides <NUM>, 30A comprising a plurality of binding regions <NUM> having a nucleic acid sequence corresponding to at least a portion of the padlock probes <NUM> outside of the primer-binding region <NUM>.

In an embodiment, the kit further comprises a ligase adapted to ligate the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM> while the target-binding regions <NUM>, <NUM> are hybridized to the probe-binding regions <NUM>, <NUM> to form circular padlock probes <NUM>'.

In an embodiment, the kit also comprises a polymerase adapted to rolling circle amplify the circular padlock probes <NUM>' with the labelled amplification primers <NUM> to generate labelled RCPs <NUM>'.

Examples of ligases and polymerases that could be included in the kit are presented in the foregoing.

The kit may optionally also comprise other components required by the ligase and/or the polymerase, such as nucleotides (dNTPs), buffer, etc..

The kit may also comprise a reaction vessel, such as a reaction tube <NUM>, well, or a microfluidic chip <NUM>, in which to perform the detection of at least one target nucleic acid molecule <NUM>.

The kit of the invention is adapted to perform the method for detecting at least one target nucleic acid molecule <NUM> as discussed above in connection with <FIG>.

The various embodiments of the padlock probes <NUM>, labelled amplification primers <NUM>, and oligonucleotides <NUM>, 30A discussed in the foregoing in connection with <FIG> also apply to the kit of the invention.

The invention also relates to a microfluidic chip or device <NUM>, see <FIG>. The microfluidic chip <NUM> comprises a sample input or inlet <NUM> configured to receive a sample comprising at least one target nucleic acid molecule <NUM>.

The microfluidic chip <NUM> also comprises a microfluidic channel <NUM> in fluid connection with the sample input <NUM>. The microfluidic channel <NUM> comprises a reagent section <NUM> comprising padlock probes <NUM> comprising at their <NUM>' and <NUM>' ends <NUM>, <NUM> target-binding regions <NUM>, <NUM> complementary to probe-binding regions <NUM>, <NUM> in the at least one target nucleic acid molecule <NUM>. The reagent section <NUM> also comprises a ligase adapted to ligate the <NUM>' and <NUM>' ends <NUM>, <NUM> of the padlock probes <NUM> while the target-binding regions <NUM>, <NUM> are hybridized to the probe-binding regions <NUM>, <NUM> to form circular padlock probes <NUM>". The reagent section <NUM> further comprises labelled amplification primers <NUM> comprising a detectable label <NUM> and a probe-binding region <NUM> complementary to a primer-binding region <NUM> of the padlock probes <NUM>. The reagent section <NUM> additionally comprises a polymerase adapted to rolling circle amplify the circular padlock probes <NUM>' with the labelled amplification primers <NUM> to generate labelled RCPs <NUM>'.

The microfluidic channel <NUM> also comprises a detection section <NUM> comprising oligonucleotides <NUM>, 30A comprising a plurality of binding regions <NUM> having a nucleic acid sequence corresponding to at least a portion of the padlock probes <NUM> outside of the primer-binding region <NUM>.

The microfluidic chip <NUM> also comprises a detection window <NUM>, <NUM>, <NUM> aligned with at least a portion of the detection section <NUM> and arranged to enable visual access to the at least a portion of the detection section <NUM>.

The microfluidic chip <NUM> is thereby a device pre-loaded with all or at least a majority of the reagents and components needed to enable detection of a target nucleic acid molecule <NUM> in a sample input into the sample input <NUM>. In other words, the reagents and components are preferably pre-loaded to be present in the reagent section <NUM> but with the oligonucleotides <NUM>, 30A preferably present at the detection section <NUM>. In such case, the reagents and components could all be provided as a mixture in the same part of the microfluidic channel <NUM>. Alternatively, the reagents and components could be distributed at different portions of the microfluidic channel <NUM>. In such a case, the reagent section <NUM> typically extends along a comparatively longer part of the microfluidic channel <NUM> as compared to providing all the reagents and components at the same part of the microfluidic channel <NUM>. For instance, the reagents and components could be distributed in the following order, from an upstream position in connection with the sample input <NUM> in a downstream direction towards the detection section <NUM>, the padlock probes <NUM>, the ligase, the labelled amplification primers <NUM> and the polymerase, or the padlock probes <NUM>, the labelled amplification primers <NUM>, the ligase and the polymerase as illustrative, but non-limiting, examples.

For instance, the reagent section <NUM> could comprise an upstream or ligase reagent section comprising the padlock probes <NUM> and the ligase and a downstream or RCA reagent section comprising the labelled amplification primers <NUM> and the polymerase.

The sample added to the sample input <NUM> is a liquid sample potentially comprising the at least one target nucleic acid molecule <NUM>. The liquid part of the sample will then carry the target nucleic acid molecule <NUM> up to the reagents and components in the reagent section <NUM> and further carry the resulting circular padlock probes <NUM>', and the labelled RCPs <NUM>' towards the end of the microfluidic channel <NUM> and thereby past the detection section <NUM>. The joining reaction catalyzed by the ligase and the RCA catalyzed by the polymerase will preferably thereby take place as the liquid is transported along the microfluidic channel <NUM>. The microfluidic channel <NUM> is then preferably designed to allow sufficient time for the joining and RCA reactions to take place so that a sufficient number of labelled RCPs <NUM>' are produced when reaching the detection section <NUM>. As an example, the microfluidic channel <NUM> could comprise a matrix or a membrane, such as a nitrocellulose membrane, through which the liquid is migrating. Optionally, wick could be arranged in connection with the end of the microfluidic channel <NUM> to draw the liquid from the sample input <NUM> towards the end of the microfluidic channel <NUM>.

The oligonucleotides <NUM>, 30B are preferably provided in the portion of the microfluidic channel <NUM> aligned with the detection window <NUM>, <NUM>, <NUM>. For instance, the oligonucleotides <NUM>, 30B could be immobilized, such as attached, directly or indirectly, to the wall(s) of the microfluidic channel <NUM> or to the matrix or membrane if present in the microfluid channel <NUM>. As a consequence, any labelled RCPs <NUM>' transported along the microfluidic channel <NUM> will be captured by the, preferably immobilized, oligonucleotides <NUM>, 30B to form labelled complexes <NUM> at the detection section <NUM>.

Such labelled complexes <NUM> or agglutinates <NUM> of multiple labelled complexes <NUM> could then be detected through the detection window <NUM>, <NUM>, <NUM>. In such a case, the detectable label <NUM> of the labelled amplification primers <NUM> is preferably visibly detectable, such as in the form of colored beads or microparticles, or a colorimetric label. The presence of the labelled complexes <NUM> or agglutinates <NUM> in the detection section <NUM> could then be verified through the detection window <NUM>, <NUM>, <NUM>. If such labelled complexes <NUM> or agglutinates <NUM> become visible through the detection window <NUM>, <NUM>, <NUM> the sample contained the at least one target nucleic acid molecule <NUM>.

<FIG> illustrate a microfluidic chip <NUM> with a single detection window <NUM>. Such a microfluidic chip <NUM> could then be used to detect the presence of a single target nucleic acid molecule <NUM> in the sample. <FIG> illustrate a microfluidic chip <NUM> with multiple detection windows <NUM>, <NUM>, <NUM>. In such a case, the detection section <NUM> comprises multiple portions with oligonucleotides <NUM>, 30B designed to capture different species of labelled RCPs <NUM>', 20B' as discussed above in connection with <FIG>, to thereby produce different labelled complexes or agglutinates <NUM>, <NUM>', <NUM>". In such a case, each detection window <NUM>, <NUM>, <NUM> is aligned with a respective such portion of the detection section <NUM> with oligonucleotides <NUM>, 30B. Such a microfluidic chip <NUM> could then be used to detect presence of multiple different target nucleic acid molecules <NUM>, 1B in a single sample.

In an embodiment, the microfluidic channel <NUM> of the microfluidic chip <NUM> also comprises a control section <NUM> comprising oligonucleotides comprising a plurality of binding regions having a nucleic acid sequence complementary to the probe-binding region <NUM> of the labelled amplification primers <NUM>. The microfluidic chip <NUM> comprises, in this embodiment, a control window <NUM> aligned with the control section <NUM> and arranged to enable visual access to the control section <NUM>. In such a case, the oligonucleotides present, preferably immobilized, at the control section <NUM> can capture any labelled amplification primers <NUM> to form a labelled complex or agglutinate <NUM> between the oligonucleotides and the labelled amplification primers <NUM>. Such a labelled complex or agglutinate <NUM> visible through the control window <NUM> verifies sufficient transport of the liquid with reagents and components from the sample input <NUM> and the reagent section <NUM> and up to the control section <NUM>. This control section <NUM> is preferably arranged downstream of the detection section <NUM>, i.e., with the detection section <NUM> present between the reagent section <NUM> and the control section <NUM>.

A labelled complex or agglutinate <NUM> visible through the control window <NUM> but no labelled complex or agglutinate <NUM> visible through any of the detection window(s) <NUM>, <NUM>, <NUM> indicate a negative result, i.e., the sample does not contain the at least one target nucleic acid molecule <NUM>. However, labelled complexes or agglutinates <NUM>, <NUM>', <NUM>", <NUM> visible both through the control <NUM> and through at least one detection window <NUM>, <NUM>, <NUM> indicate a positive result, i.e., the sample contained the at least one target nucleic acid molecule <NUM>.

The various embodiments of the padlock probes <NUM>, labelled amplification primers <NUM>, and oligonucleotides <NUM>, 30A discussed in the foregoing in connection with <FIG> also apply to the microfluidic chip <NUM> of the invention.

This example investigated the feasibility of the present invention showing the production of a visible signal indicating presence of a target nucleic acid molecule in a sample already following <NUM> minutes of rolling circle amplification, whereas traditional padlock probe and RCA based detection required <NUM> minutes of rolling circle amplification to produce a visible signal.

<NUM> padlock probes with <NUM>' phosphate group (SEQ ID NO: <NUM>-<NUM>, Table <NUM>) were mixed with 1U/µl SplintR ligase (New England Biolabs) and 1X SplintR ligation buffer (New England Biolabs) and mixed with <NUM> synthetic target nucleic acid molecules (SEQ ID NO: <NUM>-<NUM>, Table <NUM>). The hybridization and ligation were allowed to proceed for <NUM> at room temperature (<NUM>-<NUM>). Any unbound padlock probes were removed by exonuclease treatment by adding 1U/µl Exol and Exolll to the ligation mixture and incubating at <NUM> for <NUM> followed by heat inactivation of the exonuclease enzymes at <NUM> for <NUM>. The ligation mixture was mixed with <NUM> color-coded microparticle-coupled amplification primers (amplification primers had <NUM>' biotin, SEQ ID NO: <NUM>, Table <NUM>) mixed with <NUM>/ml bovine serum albumin (BSA), <NUM> dNTP, 1X phi29 polymerase buffer and <NUM> U/µl phi29 polymerase and the rolling circle amplification was performed at <NUM> for <NUM>. The RCA mixture was mixed with pre-made oligonucleotides (<NUM> repeats of the sequences in SEQ ID NO: <NUM>-<NUM>, Table <NUM>) resulting in instant agglutination.

The results are shown in <FIG> showing that the oligonucleotides promoted the labelled RCPs to agglutinate and collapse into a visual, dyed aggregate that could be seen with the naked eye.

The reaction process presented above was repeated using <NUM>, <NUM> or <NUM> minutes of RCA but without the last step of mixing the RCA mixture with the pre-made oligonucleotides. In this case, the color-coded microparticles were coupled to detection oligonucleotides having <NUM>' biotin (SEQ ID NO: <NUM>, Table <NUM>). The results are presented in <FIG> (<NUM> RCA), 23B (<NUM> RCA) and 23C (<NUM>). As is shown in these figures, even after <NUM> of RCA, hardly any visual, dyed aggregates are visible.

The reaction process presented above was repeated. Half of the samples were contacted with oligonucleotides comprising binding regions having a nucleic acid sequence corresponding to at least a portion of the padlock probes and half of the samples were contacted with oligonucleotides comprising binding regions having nucleic sequences not corresponding to at least a portion of the padlock probes. As is shown in <FIG>, visual, dyed aggregates were only formed in the samples with correct sequence in the pre-made oligonucleotides. This indicates that the present invention has high specificity in producing labelled complexes.

Claim 1:
A method for detecting at least one target nucleic acid molecule (<NUM>, <NUM>', <NUM>") in a sample, the method comprising:
contacting (S1) the sample with padlock probes (<NUM>) comprising at their <NUM>' and <NUM>' ends (<NUM>, <NUM>) target-binding regions (<NUM>, <NUM>) complementary to probe-binding regions (<NUM>, <NUM>) in the at least one target nucleic acid molecule (<NUM>, <NUM>', <NUM>");
joining (S2) the <NUM>' and <NUM>' ends (<NUM>, <NUM>) of the padlock probes (<NUM>) while the target-binding regions (<NUM>, <NUM>) are hybridized to the probe-binding regions (<NUM>, <NUM>) to form circular padlock probes (<NUM>');
rolling circle amplifying (S3) the circular padlock probes (<NUM>') with labelled amplification primers (<NUM>) comprising a detectable label (<NUM>) and a probe-binding region (<NUM>) complementary to a primer-binding region (<NUM>) of the padlock probes (<NUM>) to generate labelled rolling circle products (<NUM>');
contacting (S4) the labelled rolling circle products (<NUM>') with oligonucleotides (<NUM>, 30A) comprising a plurality of binding regions (<NUM>) having a nucleic acid sequence corresponding to at least a portion of the padlock probes (<NUM>) outside of the primer-binding region (<NUM>) to form an agglutinate (<NUM>) of labelled complexes (<NUM>) between the oligonucleotides (<NUM>, 30A) and the labelled rolling circle products (<NUM>'); and
detecting (S5) the agglutinate (<NUM>) of the labelled complexes (<NUM>) to detect the at least one target nucleic acid molecule (<NUM>, <NUM>', <NUM>").